CHAPTER ONE
1.0
Introduction
The Human Immunodeficiency Virus (HIV) belongs to the class of viruses called
retroviruses. This virus causes a weakness of the immune system which results in a
disease called Acquired Immunodeficiency Syndrome (AIDS). The global epidemic of
AIDS has become one of the most pressing public health emergencies of this century.
The first reports of AIDS date back to 1981.1 However, current data suggest that AIDS
has existed for at least several decades.2
While both forms of the human
immunodeficiency virus (HIV), type 1 and type 2, are retroviruses capable of causing
fatal AIDS, a longer incubation period is generally associated with infection with the
latter together with a more indolent course of disease.3 Maternal-foetal transmission of
HIV-2 is limited, and infection with HIV-2 seems to provide natural protection, estimated
to be approximately 70%, against infection with HIV-1 in certain high-risk groups. 2 HIV2, initially endemic to West Africa, also is spreading worldwide.
Over the years, a variety of chemotherapeutic approaches towards the treatment of HIV-1
have been developed; the strategies employed target the key biomolecules which have
been identified to play important roles in the life-cycle of HIV-1 virus. The first class of
HIV-1 drugs approved for used by the United States Food Drugs and Administration
Agency (FDA), were Nucleoside Reverse Transcriptase Inhibitors (NRTIs), for example
AZT(zidovudine), d4T(stavudine). These drugs were found to exert their effect by
inhibiting the reverse transcription process whereby the HIV-1 viral RNA is converted
into viral double-stranded DNA. This is catalysed by the reverse transcriptase (RT)
enzymes. However as resistance of the HIV-1 to these NRTIs emerged, so other drugs
were required. The next generation that were developed also targeted the HIV-1 RT
enzymes.
These compounds are known as Non-nucleoside Reverse Transcriptase
Inhibitors (NNRTIs), example of these compounds are delavirdine, efavirenz, nevirapine.
The difference between NNRTIs and NRTIs is that they bind to the RT enzyme at
1
different binding sites. Unfortunately, the HIV-1 virus has also developed resistance
towards the NNRTIs over time.
Another target which has been exploited in the development of anti-HIV drugs is viral
entry into the host cell (i.e. virus fusion). The fusion inhibitor, enfuvirtide is currently
approved for HIV-1 treatment.4 Disappointingly, the HIV-1 virus developed resistance
towards these drugs. Another drug that targets HIV-1 virus entry is moraviroc developed
by Pfizer Ltd, UK. This drug was approved for use as anti-HIV therapeutic in August
2007.
Another biological target that has been extensively investigated for the development of
anti-HIV drugs is the protease enzyme. This enzyme is responsible for the cleavage of
the viral proteins (Gag and Gag-pol) produced at the end of the HIV-1 life cycle. Thus
several protease inhibitors (PIs) have been prepared and marketed to date, for example,
ritonavir, indinavir, lopinavir and atazanavir. However, like the other drug classes, HIV1 has been shown to develop resistance towards PIs.5
Since drug resistance was commonly observed in patients when taking the individual
anti-HIV-1 drug, another strategy to combat the HIV-1 virus was implemented, the
Highly Active Anti-Retrovirus Therapy (HAART). This is a combination therapy
involving the patient receiving a cocktail of three anti-HIV drugs, typically one PI and
two NRTI or one NNRTI and two NRTI. Although this approach has proved successful
with little side effects,6 HIV-1 viral resistance has been observed and there are
compliance issues for some patients.7
The emerging resistance and side effects produced through the constant administration of
the currently approved HIV-1 drugs means that there is a continuing need for the
development of new anti-HIV drugs, especially those acting on novel biological targets.
Thus, the principal objective of this research project was to prepare and evaluate a series
of novel guanine compounds for potential use as either NNRTIs or integrase (IN)
inhibitors.
HIV-1 integrase is the enzyme that enhances the integration of HIV-1
2
genomic DNA into the host cell. IN has appeared a valid new biological target for antiHIV drug intervention. Although, there are currently no integrase inhibitors on the market
which inhibit this enzyme, there are a few in clinical trials, for example, diketo aryl
(DKA) and catechols. 8 Furthermore, IN is a valid biological target as it has no human
equivalent like RT.
1.1
Global Epidemic of Human Immunodeficiency Syndrome
Since the initial report of HIV-1 in 1981, the United Nation (UN), World Health
Organization (WHO) and other public bodies have intensified their efforts towards
eradicating this deadly disease.
This has been approached from two angles, that is the
preventive approach (involving health education) and the curative approach (through
active research into a cure). The latest statistics on the world epidemic of AIDS and HIV
were published by UNAIDS/WHO in November 2006,9 and are summarised in Table 1
Estimate
Range
(million)
(million)
People living with HIV/AIDS in 2006
39.5
34.1-47.1
Adults living with HIV/AIDS in 2006
37.2
32.1-44.5
Women living with HIV/AIDS in 2006
17.7
15.1-20.9
Children living with HIV/AIDS in 2006
2.3
1.7-3.5
People newly infected with HIV/AIDS in 2006
4.3
3.6-6.6
Adults newly infected with HIV in 2006
3.8
3.5-5.7
Children newly infected with HIV in 2006
0.53
0.41-0.66
AIDS deaths in 2006
2.9
2.5-3.5
Adult AIDS deaths in 2006
2.6
2.2-3.0
Child AIDS deaths in 2006
0.38
0.29-0.50
Table 1: Global HIV/AIDS estimates, end of 20069
3
Figure 1 shows the global trend relative to reported cases of HIV-1 infection since 1990
till the end of 2005.10 From this, it can be seen that the number of people in the world
living with HIV-1 has risen from ca. 8 million in 1990 to 40 million by the end of
2005/early 2006 and this number is still growing. Furthermore, of these 40 million
people, 25 million live in sub-Saharan Africa (that is 62.5% of these people reside in
Africa).
(Year)
Figure 1
Global Trend of HIV-110
More than 25 million people have died of AIDS worldwide since 1981. Young people
(under 25 years old) make up half of all new HIV infections reported worldwide with
approximately 6,000 infected with HIV-1 every day. In developing and transitional
countries, 7.1 million people are in immediate need of life-saving AIDS drugs; however,
only 2.0 million (28%) are receiving the appropriate treatments.9
In the UK, the number of reported cases of HIV-1 infection per country between 1991
and June 2007 can be seen in Table 2. The striking feature here is that there was a
marked increase in the number of people diagnosed with HIV-1 between 2000 and 2005.
The reason for this was thought to be due to the fact that an effective health education
programme was no longer being pursued. Thus, a programme of prevention awareness
4
was undertaken. Although there was a decrease in the number of people diagnosed with
HIV/AIDS recorded the following year, it is difficult to ascertain whether this was solely
as a result of the improved prevention education strategy.
Year of
country
diagnosis
England
Wales
N.
Scotland
UK
Ireland
Channel
Isles/Isle
Total
1991
and 17,461
of Man
276
96
1,703
19,536
28
earlier
1992
2,547
50
12
138
2,747
1
1993
2,408
40
12
181
2,641
2
1994
2,373
45
15
157
2,590
8
1995
2,460
45
12
148
2,665
1
1996
2,503
35
17
170
2,725
6
1997
2,542
46
9
167
2,764
8
1998
2,661
29
9
164
2,863
6
1999
2,943
35
15
156
3,149
0
2000
3,661
45
19
159
3,884
1
2001
4,837
63
19
182
5,101
5
2002
5,938
76
27
225
6,266
7
2003
6,916
108
32
279
7,335
4
2004
7,041
107
62
346
7,556
4
2005
7,115
122
63
362
7,662
4
2006
6,595
154
57
287
7,093
2
Until
June 1,849
64
13
124
2,050
0
1,340
489
4,948
88,627
87
2007
Total
81,850
Table 2: New cases of HIV-1 infection reported in UK per annum 9
5
1.2
Viruses
Viruses are tiny infectious agents, which were first noticed by Dmitri Iwanowski on 12th
February 1892 while he was studying a mosaic tobacco disease.11 Extensive research
work in the culture of live cells and some microscopy observation led scientists in the
20th century to eventually observe the structure of virus. A representative structure of
viruses is illustrated in Figure 2.12 Viruses can be viewed as living as they have
reproductive abilities, although only with the assistance of a host organism. Since viruses
themselves do not contain ribosomes, they are unable to synthesise proteins. Thus in
order to translate its viral messenger RNA into viral proteins, the virus makes use of the
ribosomes from the host cells. Furthermore, in general, a virus cannot store or generate
energy; all their energy and metabolic functions have to be derived from the host cell.12
Bilipid layer
Nucleic Acid
Glycoproteins
Envelope
Capsid
Capsomer
Figure 2: General Structure of Viruses.12
6
As shown in Figure 2, the structure of a virus is made up of several components:
(i)
Capsid: The capsid is the viral protein coat, composed of capsomers, which
enclose the viral nucleic acids. The capsid together with its enclosed nucleic acid
is generally referred to as the nucleocapsid. The capsid has three functions: (a) to
protect the viral nucleic acid from enzymatic degradation. (b) to enable viral
binding through special sites on its surface (c) and to facilitate the virion to
penetrate the host cell membrane in order to inject the viral nucleic acids into the
cytoplasm of the host cell. Under the right conditions, viral RNA in a liquid
suspension of protein molecules will self-assemble into a capsid and become a
functional and infectious virus.12
(ii)
Capsomer: This is the morphological unit, one or more of which constitutes the
viral capsid. The capsomer consists of one or more structural unit called
protomers or monomers. 12
(iii)
Envelope: A glycoprotein envelope surrounding the nucleocapsid is common in
many viruses. The envelope is composed of two lipid layers interspersed with
protein molecules (lipoprotein bilayer) and it may contain material from the
membrane of the host cell as well as that of the virus. The virus obtains the lipid
molecules from the membrane of the host cell during the viral budding process.
However, the virus replaces the proteins from the host cell membrane with its
own proteins, creating a hybrid structure of host cell-derived lipids and virusderived proteins. Many viruses also develop spikes made of glycoprotein on their
envelopes which help them to attach to specific host cell surfaces. 12
(iv)
Glycoprotein: This substance is composed of a protein which is covalently linked
to a carbohydrate unit. The main function of the viral glycoprotein is for immune
cell recognition. The viral glycoprotein has three main parts: (1) the external
domain which interacts with the host cell, thereby leading to viral binding to the
host; (2) transmembrane segment, which is the viral envelope membrane, and (3)
the endodomain, which is mainly the internal part of glycoprotein.12
7
(v)
Nucleic Acid: This substance in the virus encodes all the genetic information that
is needed for the biosynthesis of the viral proteins. While double-stranded DNA
is responsible for this in prokaryotic and eukaryotic cells, only a few groups of
viruses actually use DNA. Most viruses house all their genetic information within
single-stranded RNA. There are two types of RNA-based viruses. These are the
plus strand RNA viruses in which the viral m-RNA ultimately produced has the
same base sequence as the original viral RNA The second class type are the minus
strand RNA viruses in which the base sequence of the messenger RNA is
complimentary to the original viral RNA. The latter viruses contain the virion
enzyme, RNA-dependent RNA transcriptase.
This enzyme catalyses the
production of complementary messenger RNA from the virion genomic RNA.12
1.3
Classification of Human Immunodeficiency Virus type 1 Strains
HIV-1 viruses can be sub-divided into three main groups denoted, M, N, and O. Group O
is restricted to West Africa, whereas N was discovered in Cameroon in 1998, although it
has subsequently been found to be very rare. The major group is Group M and this group
has been further sub-divided into nine classes (clades) as shown in Figure 3.13
HIV-1
Group M
A
B
HIV-2
Group N
C
D
Group O
F
G
H
J
K
CRFs
Figure 3: Diagram illustrating the different levels of HIV classifications13
When viruses of different subclasses meet in the cell of an infected person, they share
their genetic materials resulting in the formation of a new strain.
8
Although many of
these strains do not survive very long, those that do and go on to infect other people are
referred to as “circulating recombinant forms” (CRFs).14
The most widely spread CRFs and HIV sub-types are not evenly distributed over the
world. Sub-types B and C are the commonest strains worldwide. Sub-type C is found
mainly in eastern Africa, India, and Nepal, while sub-type B is found in Europe, the
Americas, Japan and Australia. Sub-type A and CRF A/G are common in West and
Central Africa while D is limited to east and central Africa. CRF A/E has been located in
south-east Asia, G and CRF A/G have been observed in western and eastern Africa and
central Europe. Sub-type A has only been reported in Central Africa, J only in Central
America and K only in the Democratic Republic of Congo and Cameroon. 15,16
The geographic preferences of each of these sub-types may have major applications in
the development of suitable drug regimens designed for the treatment of HIV-1
infections. Currently, most of the marketed anti- HIV-1 drugs target sub-type B. Thus, it
has been proposed that the efficacies may be compromised in regions where the strain is
not predominant. This suggestion has not yet been confirmed. To date, it is not known if
the different sub-types respond to antiviral drugs in different ways.
1.3.1 Origin of HIV-1
In 1999, a group of researchers led by Professor Beatrice Hansat the University of
Alabama at Birmingham provided an answer to the mystery surrounding the origin of this
deadly virus.17 During the course of their studies, they sequenced the genome of a new
SIVcpz (Simian Immunodeficiency Virus from chimps) strain. This sequencing was
carried out on the sub-species of all known SIVcpz infected chimpanzees. It revealed
that two chimpanzee sub-species found in Central Africa (P.t troglodytes) and Eastern
Africa (P.t. Schweinfurthii) were the reservoir of SIVcpz. Furthermore, it was deduced
that all HIV-1 strains known to infect man, including the HIV-1 groups M, N, and O
were related to those SIVcpz lineage found in P.t. troglodytes.
9
Therefore, it was
concluded that the primary source of HIV-1 is from chimpanziee sub-species the P.t.
troglodyte, which reside in Central Africa.18
The virus responsible for HIV was first isolated in 1983 by Robert Gallo of the United
States together with the French scientist, Luc Montagnier.19 Since that time, a tremendous
amount of research focusing upon the causative agent of AIDS has been carried out and
much has been learned about the structure of the virus and its typical course of action.
HIV is one of the groups of a typical virus called retroviruses that maintain their genetic
information in the form of ribonucleic acid (RNA). Through the use of a viral enzyme
known as reverse transcriptase (RT), HIV like other retroviruses, produces viral
deoxyribonucleic acid (DNA) from its RNA, upon internalization into a host cell RNA.
The viral DNA is then integrated into the host DNA the genetic information of HIV to
become permanently incorporated into the genome of the host cell.
High-resolution electron microscopy has illustrated that HIV-1 [Figure 4] is an enveloped
virus of about 100 nm in diameter. 20 It contains an outer lipid bilayer, derived from the
host cell during maturation, which consists of two major viral glycoprotein; the external
gp120 and the transmembrane gp41 protein (gp stands for glycoprotein and the number
refers to the mass of protein in kilo Daltons). Immediately inside the outer envelope is
the membrane-associated protein p18, which provides a matrix for the viral structure and
is vital for the integrity of the virion. The matrix surrounds a characteristic dense
cylindrical nucleiod, containing capsid protein p24. Inside the nucleiod are two identical
RNA strands together with the viral RNA-dependent DNA polymerase (pol) p66/p55
(that is RT), nucleoprotein p9, integrase protein p12 and protease p15.
10
Figure 4: Structure of HIV-121
1.3.2
HIV-1 Life Cycle
Figure 5 highlights the various stages in the life cycle of the HIV virus.
Figure 5: HIV life cycle22
11
a.
Viral Entry
The HIV life cycle [Figure 5] begins with high-affinity binding of the gp120 envelope
protein of HIV-1 to the receptor CD4 on the surface of the host cell.23 The CD4 receptor
is a protein molecule which is found predominantly on a subset of T-lymphocytes
responsible for the helper, or inducer function in the immune response. The binding of
the viral glycoprotein to the CD4 receptor of the host is followed by the binding of the
HIV-1 gp120 to one of the host cell surface co-receptors. There are two main types of
co-receptors on the cell surface of the host,24 the CCR-5 co-receptor, a C-C chemokine
(Chemoattractant cytokines) co-receptor, which mediates the entry of non-syncytiuminducing (NSI), or monocytotropic, strains of HIV-1 and the CXCR-4 co-receptor. This is
a C-X-C chemokine co-receptor, also known as fusin, and is expressed on the surface of
T-lymphocytes.
The complete binding of the HIV-1 glycoprotein to both the CD4
receptor and either CCR-5 or CXCR-4 co-receptor triggers conformational changes in the
viral envelope.
This exposes the hydrophobic fusion domain of gp41 and enables
complete fusion of the viral envelope with the cell membrane of the host [Figure 6].
Figure 6: HIV-1 entry mechanisms25
12
CCR-5 using viruses are the early viruses isolated in an HIV infected individual. This
isolation suggested that HIV may be selective in its choice of binding to the co-receptor,
but this does not rule the other co-receptor out completely. This might be another source
of resistance in future, should HIV-1 develop a strain that binds to the CXCR-4 co
receptor.
b.
Reverse Transcription
Following binding, the fusion of virus with the host cell membrane occurs via the gp41
envelope molecules, and the HIV-1 genomic RNA is uncoated and internalised into the
host cell. Subsequently, the viral enzyme RT copies the viral genomic RNA into doublestranded viral DNA. Human immunodeficiency virus type 1 reverse transcriptase (RT)
was discovered in 1970 by Baltimore et al.26 This enzyme [Figure 7] is a heterodimer
composed of a 560 residue subunit (p66) and a smaller subunit (p51) that contains the Nterminal 440 residues of p66. The C-terminal portion of p66 forms the RNase H domain,
while the N-terminal portion is the polymerase domain. Only p66 has a functional
polymerase action site and a DNA-binding cleft formed by the p66 fingers, palm and
thumb sub-domains. The role of p51 appears to be primarily structural.27,28
Figure 7: HIV-1 Reverse Transcriptase Structure
13
RT is used by retroviruses to transcribe viral single-stranded RNA genomes into singlestranded DNA, to construct the Watson-Crick complementary DNA strand, and
ultimately yield the double-stranded DNA required for integration into the host’s
chromosomes. Production of double-stranded DNA is primed by the host cell lysinetRNA which partly unfolds and anneals to the 5'-end of the viral genomic RNA. This is
extended by the polymerase function of the RT to give a DNA-RNA hybrid. The RNA
component of this hybrid is degraded by the RNase H function of RT once it has been
copied. The polymerase function of RT is then able to synthesise the second strand of
DNA, possibly primed by a rump of the viral RNA.29
c.
Viral Integration
The viral DNA migrates to the host cell nucleus where it is integrated into the
chromosome of the host cell through the action of another virally-encoded enzyme,
integrase (IN). The incorporation of this “provirus” into the cell genome is permanent.
The provirus may either remain transcriptionally inactive (latent) or manifest a high level
of gene expression with active production of the HIV-1 virus.30
Incorporation of viral double-stranded DNA into the genome of the host cell forms the
basis of life-long infection. Therefore, this biochemical event, catalysed by IN, is a
pivotal step in the HIV-1 life cycle and as such is worthy of investigation in the
development of new anti-HIV chemotherapies. Analysis of the structure if HIV-1 IN has
revealed that it is composed of three domains [Figure 8] and the structures of each of
these domains have been individually determined by X-ray crystallography and NMR
studies.31
14
Core
N-terminus
Catalyses
Polynucleotidyl
Transfer
Multimerization
212
50
1
HTH Fold
HH CC
Dimer(NMR)
Binds zinc
DNA binding
RNAseH
D
D
Fold
35 E
Dimer (X-Ray)
Binds Mg2+
288
SH3 Fold
Dimer (NMR)
Binds DNA
Figure 8: Three domains of HIV-1 IN
The catalytic core domain contains the invariant triad of acidic residues, Asp64, Asp116
and Glu152 (the DDE moiety).32 These catalytic residues are in close proximity and coordinated to a divalent metal ion typically magnesium. Approximately 40-100 IN are
packaged within each HIV-1 particle.28 The main function of IN is to catalyse the
integration of the viral cDNA into the genome of the infected cells. However, it has also
been postulated that it may function as a co-factor for reverse transcription.30
Although the core domain of IN is clearly responsible for catalysis, the functional roles of
the other two domains are less clear. The structure of the catalytic core domain (CCD) of
HIV-1 integrase consists of a central five-stranded β-sheet with six surrounding helices.
Three amino acids in the CCD are highly conserved among retrotransposon and retroviral
integrase.33 Mutation of these residues generally leads to a loss of all catalytic activities
of these proteins, and they are therefore thought to be essential components of the
integrase active site. Two of these amino acids in HIV-1 Integrase are Asp64 and
Asp116. The third conserved residue, Glu152, lies near the other two in a 13-residue
disordered region (not visualized by X-ray crystallography) bordered by residues 140 and
154.33
15
Studies in vitro have indicated that functional HIV-1 integrase is a multimeric protein,
but the number of monomers in a functional enzyme has not been determined with
certainty. Examination of the crystal structure shows two contacts between CCD
monomers, one of which suggests a dimeric model for functional integrase. In this
contact, the two monomers are related by a dyad axis, with a large, solvent-excluded
surface (1300 Å2/monomer).
The interaction between core domain monomers can be
compared to the interactions involved in antibody binding of protein antigens, which
generate solvent excluded interfaces of ~800 Å2 for each molecule.34
The C-terminal domain of IN binds DNA non-specifically. The N-terminal domain of
HIV-1 contains a conserved pair of His and Cys residues, a motif similar to zinccoordinating residues of zinc fingers. Although this domain does indeed bind zinc35 its
structure is totally different from that of conventional zinc finger proteins. It consists of a
bundle of three α-helices36 and has an SH3 fold, although there is no known functional
responsibility with SH3 domains of other proteins.
Retroviral integration occurs through a series of DNA cleavage and joining reactions.37,38
The integration process is illustrated in Figure 9. In the first step (3'-end processing), IN
removes 2 nucleotides from the 3'-end of each strand of linear viral DNA so that the viral
3'-ends terminate with a conserved cystine-adenosine (CA) dinucleotide. The second step
(3'-end joining) is a concerted cleavage-ligation reaction during which IN makes a
staggered cut in target DNA at the cleavage site. The product of this reaction is a gapped
intermediate. The last step is 5'-end joining. The integration process is completed by
removal of the 2 unpaired nucleotides at the 5'-ends of the viral DNA and repair of the
gaps between the viral and target DNA. This interaction produces the short direct repeats
that flank the provirus. The latter step is likely performed by host DNA repair enzymes;
thus, inhibition of either of the first 2 steps is likely to inhibit viral integration and
subsequent replication.
16
Figure 9: Schematic illustration of IN-mediated 3'- end processing of viral DNA and
nucleophilic attack on host DNA. HIV IN first removes 2 nucleotides from each 3'-end of
long terminal repeat (LTR) leaving terminal CA dinucleotide. This processed genome
undergoes nucleophilic attack on host chromosomal DNA (strand transfer) catalyzed by
IN. The resulting product is repaired, likely mediated by host repair machinery.39
d.
Viral Maturation/ Cleavages
Once the viral DNA enters the nucleus, where it is integrated into the genetic material of
the cell by HIV-1 IN, the activation of the host cells results in transcription of the viral
DNA into messenger RNA, which is then translated into viral proteins. HIV-1 protease
(PR), the third virally-encoded enzyme is now required at this stage to cleave the viral
polyprotein precursor into viral proteins.40 Protease enzymes are responsible for the
cleavages of gag protein (the protein of the nucleocapsid envelope around the RNA of the
retrovirus) and gag-pol proteins to produce structural and functional viral proteins.
17
HIV-1 PR is a 99 amino acid aspartyl protease which functions as a homodimer with only
one active site which has C2-symmetry in the free form.40,41 PR carries out its catalytic
process by:
(a)
making use of activated water molecules to attack the amide bond carbonyl of the
scissile bond of the substrate [Figure 10], the water molecule can be activated by
either the zinc cation (zinc metalloproteinase) or by the two aspartyl β-carboxyl
groups at the active site (the aspartate proteases).40
(b)
using the nucleophillic atom of an amino acid side chain to initiate amide
hydrolysis. This is done by the activation of the hydroxyl group or thiol by
another amino acid side chain. This activated nucleophile attacks the carbonyl of
the scissile amide bond [Figure 10] to form an ester or a thioester acyl
intermediate. Eventually, this acyl enzyme intermediate is hydrolysed by a water
molecule to the corresponding hydrolysis products.40
Scissile bond
S1
S3
N
H
Figure 10:
P1
P3
O
S'2
P2'
O
H
N
N
H
O
O
H
N
O
N
H
P2
P1'
S2
S'1
H
N
O
N
H
O
P 3'
S'3
The nomenclature P1…Pn, P1’…Pn’ designate amino acids residues of
peptide substrates. The corresponding binding sites on the protease are
referred to as S1…..Sn; S1’……Sn’
18
Based on the fact that HIV-1 PR enzyme can be classified as an aspartic protease,
Suguma et al. [Figure 11]42 proposed a mechanism for the action of HIV-1 PR in the life
cycle of HIV-1 virus. They proposed that the Asp. group that is closer to the nucleophilic
water molecule was assigned negative charge [Figure 11], and the water molecule that is
held between the catalytic aspartates is activated by the negative aspartate side chain. The
activated water molecule then attacks the carbonyl group in the substrate scissile bond to
generate a tetrahedral intermediate. The protonation of the scissile amide N atom and
rearrangement result in the break down of the tetrahedral intermediate to the hydrolysis
products.43
P1
P1
H
N
H
N
O
O
H H
H O
P 1'
O
H
H
O
O
O
O
O
O
D
O
D
D
D
T
G
P 1'
OH
T
T
T
G
G
G
P1'
O
+ H 2N
OH
P1
O
H
O
O
O
D
D
T
T
G
G
Figure 11: Catalytic mechanism for aspartic protease proposed by Suguma et al.42
19
Furthermore, Jaskolski et al.44 proposed a new model for HIV-1 PR; in this mechanism
[Figure 12], the hydrolysis reaction is viewed as a one-step process during which the
nucleophillic water molecule and the acidic proton attack the scissile peptide bond in a
concerted manner.44
Figure 12: Concerted catalytic mechanism for HIV-1 protease enzyme suggested by
Jaskolski et al.44
The activation of the provirus from the latent state by selective and constitutive host
transcription factors, notably from the NF-Кβ family of DNA enhancer binding proteins,
leads to the sequential production of various viral m-RNAs. These m-RNAs are
subsequently translated into the regulatory proteins, Tat, Rev and Nef. The viral core is
formed by assembly of these proteins, enzymes, and genomic RNA at the plasma
membrane of the cells. Budding of the progeny virion occurs through the membrane of
the host cell, where the core acquires its external envelope. During the final budding
process, cleavage of the gag-pol polyprotein precursor by HIV protease occurs, which
leads to morphological maturation of virions.30
20
1.4
Common Chemotherapy of Human Immunodeficiency Virus Type 1
Infection
As shown above in section 1.3, the replicative cycle [Figure 5] of HIV-1 presents several
viable biological targets that have been and still are being exploited in the development of
current and future anti-HIV drugs. These are:
(i)
Viral entry
(ii)
Reverse transcription
(iii)
Viral Integration
(iv)
Gene expression
(v)
Cleavage agents
(vi)
Virion maturation.
By inhibiting any of these stages, HIV-1 can be prevented from spreading throughout the
cell. 45
1.4.1 Viral Entry Inhibitors
A number of compounds have been designed to block the interaction of HIV-1 virus with
the CCR-5 co-receptors. Examples include: vicriviroc 146, aplaviroc 2 and moraviroc 3
[Figure 13]. The subsequent problem of high liver toxicity47 has since led to the
termination of the use of aplaviroc and vicriviroc. Moraviroc, which was developed by
Pfizer, was reviewed on April 24, 2007 by United States Food Drug and Administrations
and it was recommended for approval, on August 6, 2007.48
21
Figure 13: HIV-1 viral entry inhibitors
Another compound which targets the entry mechanism of HIV-1 is fuzeon 4 (Enfuvirtide,
or T-20), Fuzeon is a peptide-based drug [Figure 14]. Fuzeon inhibits formation of the
initial entry interaction between the HIV-1 gp41 and the CD4 receptor of the host.
Fuzeon blocks the virus before the cell fusion process, therefore it has no effect on the
intracellular process of the host.
Gp41 of the HIV-1 virus consists of cytoplasm,
transmembrane and extracellular domains.49 There are four major functional domains in
the extracellular domain of gp41. These regions are: fusion peptide (FP), N-terminal
heptad repeat (NHR1 or NHR2), the C-terminal heptad repeat (CHR2 or CHR1) and a
22
triptophan-rich (TR) region. These regions were considered as potential targets for
designing HIV-1 drugs.
CH3CO-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-LysAsn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2
4
Figure 14: The amino acid sequence of Fuzeon
Fuzeon exerts its anti-viral effect by preventing changes in the conformation of gp41 that
are required by the HIV-1 virion to enter the host cell.50,51 Figure 15 illustrates this
inhibition process in more detail.
NH2
HR1
HR2
COOH
2
1
Fuzeon
Figure 15: Inhibition process of fuzeon
Fuzeon binds to Heptad Repeat (HR1) of the gp41 glycoprotein, thereby preventing HR1
from interacting with Heptad Repeat (HR2,) and so inhibiting the formation of the coiledcoil bundle necessary to initiate fusion of HIV-1 to the host cell.52,53 Liu et al. 25 observed
that the inhibition of fusion takes place by interacting with the gp41 NHR to form stable
six-helix bundles (6-HB) and blocking gp 41 from forming a fusogenic core between
viral gp41 NHR(N-terminal Heptad repeat) and CHR(C-terminal Heptad Repeat). The
principal problem associated with the use of Fuzeon is that HIV-1 has found a way to
produce mutations in the heptad repeat 1(HR1).
23
1.4.2
Reverse Transcriptase Inhibitors
HIV-1 reverse transcriptase inhibitors can be divided into two major classes: The
nucleoside (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).
1.4.2.1
Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
Nucleoside reverse transcriptase inhibitors (NRTIs) are nucleoside analogues and they
exert their effects by binding to RT and inhibiting viral DNA synthesis, thus preventing
the virus from replicating.54 Examples of some common NRTIs are AZT (3′-azido-2′, 3′dideoxythimidine, or retrovir zidovudine) 5, d4T (stavudine) 6, and ddc (zalcitabine) 7
[Figure 16]. During the inhibition process, the NRTIs are first converted into their active
triphosphate forms by a process known as phosphorylation (Scheme 1). This
biotransformation is an enzyme-catalysed process. The enzymes commonly employed
are thymidine kinase, deoxycytidine kinase or inosine phosphotransferase. These
enzymes initially convert the NRTIs into their corresponding monophosphates which are
subsequently triphosphorylated. Once in their triphosphate form, they can be incorporated
into the ss-DNA by RT during the reverse transcription process. However, since the
NRTIs lack the 3′-OH present in natural nucleotides, subsequent elongation of the chain
is prevented and thus DNA synthesis terminates.55
O
CH3
HN
HO
CH3
HN
N
O
NH2
O
O
HO
N
O
N
HO
N
O
O
N3
5
AZT(Zidovudine)
7
6
d4T(Stavudine)
ddC(Zalcitabine)
Figure 16: Structure of some current NRTIs
24
Scheme 1: Incorporation of nucleoside analogues into elongating viral component
DNA47
25
1.4.2.2 Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
The other major family of small ligand inhibitors of HIV-1 RT are the non-nucleoside
reverse transcriptase inhibitors (NNRTIs).
Non-nucleoside reverse transcriptase
inhibitors are non-competitive inhibitors; this is because their binding position is quite
different from the DNA binding site. The NNRTIs share a number of properties which
distinguish them from the previously described NRTIs. They are highly specific, they
present little cytotoxicity and they do not require activation through intracellular
metabolism and phosphorylation to exert their effect.35 NNRTIs bind allosterically to the
enzyme, thereby leading to an alteration in the conformation and functions of the
enzyme. NNRTIs interact with the RT enzyme at a hydrophobic pocket located near to
the polymerase active site of the enzyme. This binding site is called the non-nucleoside
binding site (NNBS).56
There are four key amino acid residues present in the
hydrophobic pocket, Phe 227, Trp 229, and Leu 234 from the “palm” region of the
binding site and Tyr 319 from the “thumb” region [Figure 7, page 13].
These four
residues are conserved throughout the HIV-1 strains (wild-type and mutants).57
The first generation of NNRTIs introduced in 1990s and approved by USA FDA for
clinical use are nevirapine 12, delavirdine 13, and efavirenz 14 [Figure 17].58
26
Efavirenz
14
Figure 17: Structures of some NNRTIs
The NNRTIs primarily bind to hydrophobic pocket in the RT which is located in the p66
subunit.59 Although efavirenz, nevirapine and delavirdine bind in the same general area
of the hydrophobic pocket, some differences exist in their specific interactions. As a
result, on virologic failure some NNRTI mutations are more likely to occur with certain
NNRTIs. K103N, for example, is typically associated with initial efavirenz resistance,
while Y181C is associated with initial nevirapine resistance. These mutations, although
considered to be drug specific, can still lead to significant cross-resistance among the
three first generation NNRTIs.
These first generation NNRTIs suffered set backs because the HIV-1 virus was able to
develop resistance to these compounds.
A single mutation in the amino acids in the
binding site eliminates the effectiveness of these compounds. In addition to the multidrug resistance developed by the virus, some side effects such as deadly toxicities have
27
been reported for efavirenz 14 and nevirapine 12 and this is a major limitation to the use
of first generation NNRTIs.
In order to overcome the multi-drug resistance problem of the first generation NNRTIs,
rilpivirine 15 and etravirine 16 [Figure 18] were developed by Tibotech Pharmaceuticals
and are currently in their different stages of clinical trials. These compounds are classed
as second generation NNRTIs. They were designed in order to overcome the resistance
mechanisms through “conformational flexibility”; both etravirine and rilpivirine are able
to alter their shape and position in order to bind to the NNBS in the enzyme RT.60,61
Figure 18: Structures of some second generations NNRTIs
Structural studies of etravirine, a diarylpyrimidine (DAPY) analogue,59,62 indicate that it
possesses several characteristics that enable it to bind in the hydrophobic pocket of the
reverse transcriptase enzyme using multiple conformations.
These characteristics include:
*
the ability to bind in at least two conformationally distinct modes,
*
the ability to exercise torsional flexibility and develop alternative conformational
variants, and
*
the ability to reposition itself within the hydrophobic pocket to promote better
binding, this is as a result of its compact nature.63
28
Ripilvirine is well tolerated with less CNS disturbance than efavirenz, and has nonteratogenic potential.64
1.4.3
Resistance
of
HIV-1
Reverse
Transcriptase
Enzymes
to
Reverse
Transcriptase Inhibitors
The tolerance of HIV-1 RT to non-standard base pairs and modified ribose units is very
high. The high tolerance made it easier for the reverse transcriptase enzymes to develop
a great rate mutation in the HIV-1 genome. It has been observed that about 10 bases
mutate in the HIV-1 genome per replication cycle.65 Numerous toxic effects such as
lactic acidosis and hepatic steatosis have been associated with the use of NRTIs. The
reason for their toxicity has been linked to the inhibition of mitochondrial DNA
synthesis.66,67
The emergence of one or more HIV-1 strains with altered RT genes leads to resistance of
the HIV-1 to nucleoside analogue drugs. A novel NNRTI resistance-conferring mutation,
M230L, which was identified in NNRTI-experienced patients, has been associated with
dose-dependent stimulation of HIV replication.68
This mutation can dramatically
decrease susceptibilities to NNRTI drugs. Pharmaceutical companies are still working on
new categories of NNRTIs such as benzophenones and diaryl pyrimidines (DAPYs).
1.4.4 Highly Active Anti-Retroviral Therapy
As highlighted earlier, due to resistance of HIV-1 towards the current approved FDA
drugs when administered individually, there was a need for the investigation of a multidrug or combination therapy (this type of drug therapy is known as HAART). HAART,
which was developed in 1996, is now the standard treatment for HIV-1. Typically,
HAART consists of one protease or non-nucleoside reverse transcriptase inhibitor
(NNRTIs) being given in combination with two nucleoside reverse transcriptase
inhibitors (NRTIs). This combination regime has been found to effectively suppress HIV-
29
1 replication thereby leading to the restoration of the immune system of an HIV-1
infected patient.69
However, there are several limitations to HAART. Firstly, it is often not well tolerated
by the patients. Furthermore, long term exposure of patients to PI-containing HAART
has been shown to result in several side effects such as hyperbilirubinaemia, insulin
resistance, hyperlipidaemia, body fat redistributions, osteopenia, and osteoporosis.70
Thus, HAART requires discipline, is expensive and can lead to the development of multidrug resistance if the regimen is not adhered to correctly.71,72 Based on these problems,
additional therapies are needed especially those which act on another site in the HIV-1
virus life cycle. The requirement to find alternative drugs has led to the evaluation of
another target in the HIV-1 virus for drug interaction. This target is the third viral
enzyme, HIV-1 integrase.
1.4.5 Integrase Inhibitor Compounds
The first IN inhibitor was reported about ten years ago.73 Although there are no IN
inhibitors yet on the market, some have now reached the early stages of clinical trials.
The IN inhibitors are discussed under the following headings.
1.4.5.1 The Diketo Aryl Compounds
The most clinically advanced IN inhibitor recorded to date is compound 17 [Figure 19]
developed by Merck and GSK-Shionogi. This compound belongs to the diketo aryl
(DKA) class of compounds which act by selectively inhibiting the strand-transfer process
catalysed by IN.
30
17
Figure 19: Structure of a DKA
The characteristic of DKA is selective inhibition of the integrase strand-transfer step at
nanomolar concentrations.74 Inspite of the selective strand-transfer properties of DKAs,
they have also been found to have some remarkable effects on 3′-processing at 30-70 fold
higher concentrations.75 Another of Shionogo’s DKA compounds 5ClTEP 18 proved to
be important because it was co-crystallised with the CCD of IN in close association with
the catalytic DDE triad.76 Further development in the use of DKA IN inhibitors led to the
discovery of naphthyridine carboxamides 22. This is considered to be a DKA-like
molecule because it contains a β-hydroxyl ketone and it shares a similar IN inhibitory
mechanism to DKAs (selectively inhibits the strand transfer process). Another DKA
derivative, 19 [Figure 20], has also reached clinical trials.
For adequate binding of DKA to HIV-1 integrase, a divalent metal ion is necessary. For
example, it was found that 17, through the carboxylate functionality, bound to both Mg2+
and Mn2+ effectively, but 5ClTEP, 18 [Figure 20], showed better inhibitory properties in
the presence of Mn2+ than
Mg2+.
Therefore, the replacement of tetrazole by a
carboxylate markedly increases the inhibitory activity of the hybrid compound in Mg2+.77
The carboxylate, therefore, might be important for metal chelation.78 The carboxylate
portion is, however, not required for binding to the integrase complex. SAR studies have
revealed that the presence of an aromatic ring in the drug is an important requirement for
the potency and for strand-transfer selectivity of the IN inhibitors. It has been shown that
the aromatic groups can be functionalised with a wide range of substituents including an
azido group 23.79 The azido functionality of azido-DKAs contributes to reduced toxicity
and has a direct role in metal chelation. 79
31
Figure 20: Chemical structures of antiviral integrase inhibitors
The functional diketo or β-hydroxy-keto groups are known to have metal-chelating
functions and metal-dependent inhibition by DKAs and DKA-like compounds has been
32
interpreted as indicating a direct interaction of these drugs with the divalent metal in the
enzyme catalytic site.79 The metal coordination could also be important for shaping the
catalytic pocket of integrase and therefore the DKA-binding site.79, 74
Figure 21 shows the interaction of the integrase (IN) 3′- processing inhibitor, 5CITEP,
bound to HIV-1 IN. 5CITEP is drawn with an orange carbon backbone while the donor
DNA-interacting amino acid residues are represented as sticks. The putative metal ions
within the IN active site are shown as yellow spheres.
Finally, the possible hydrogen
bonding interactions between 5ClTEP and the active site amino acid residues are
indicated as dashed lines.80
Figure 21 Possible binding mode of integrase inhibitor 1880
The binding of DKAs to integrase has been a focus for researchers because of the
importance of DKAs and DKA-like derivatives as antiviral lead compounds and their
unique mechanism of action. The proposed mechanism of action of DKAs is illustrated
in Figure 22.
33
Figure 22: Proposed mechanism of action of DKAs
The model above [Figure 22] shows that DKAs inhibits integrase at the interface with
viral DNA and divalent metal ion. The model shows that:79
34
(a)
integrase has two binding sites, namely the donor site of the viral DNA (blue
colour) and the acceptor site for the host DNA (red colour).
(b)
after 3′-processing, the integrase-DNA undergoes structural changes that render
the acceptor-site competent for binding the host DNA.
(c)
binding of the host (acceptor) DNA to the donor site leads to strand transfer.
(d)
the DKA inhibitor (grey colour) can only bind to the acceptor site after 3′processing.
(e)
the hypothetical binding of DKAs, for example MA-DKA, at the interface of the
integrase-DNA-divalent metal complex. The processed viral 3′-DNA end (blue
colour) is bound to integrase (the three acidic catalytic residues are shown in red),
ready to attack a host DNA phosphodiester bond.
A proposition has been made that DKAs chelate the metal in the integrase catalytic site
and stabilize the macromolecular integrase-DNA complex at the 3′-processing step of the
reaction. 79
Recently, raltegravir 24, a β-diketo acid compound, has been reported to inhibit HIV-1
integrase.81 This compound was approved by the FDA on October 12, 2007 for use with
another anti-HIV agent in the treatment of HIV-1 infection; it is the first IN inhibitor to
be approved by the FDA. Raltegravir has been found to inhibit the strand-transfer
process of HIV-1 IN and to exhibit a potent in vitro activity against both wild type and
more importantly, a wide range of drug-resistant HIV-1 clinical trials isolates. 82
35
24
Figure 23: Structure of raltegravir
1.4.5.2 Catechol Derivatives
Catechol derivatives are another class of possible HIV-1 integrase inhibitors that have
been reported.83 The biological activities of catechol derivatives have been attributed to
the presence of bis-aryl moieties in their scaffold, and this is a very significant structural
component in many of the potent catechols derivatives. These compounds are made up
of two aryl units, one of which contains 1,2-dihydroxy substitution and separated from
the other aryl unit by the linker.
Among this class of compounds is caffeic acid
83,84,85
[Figure 24]. Compound 25 was reported to exhibit
phenethyl ester (CAPE) 25
good inhibition against isolated HIV-1 IN, but due to dose limiting toxicities in in vitro
studies, it could not be further investigated.
This, therefore led to the investigation of
other IN inhibitors such as, compounds 26 (1-methoxyoxalyl-3,5-dicaffeoyl quinic acid,
DCQAs) 83 and compound 27 L-chicoric acid (7-decaffeoyltartaric acid) in an attempt to
overcome the problem of toxicities. These compounds were found to inhibit HIV-1
replication and integration. In another studies to overcome the toxic effects of catechols
in cell culture,83 which might be related to the cross reactivity of metal-dependent
requiring enzymes or covalent protein modification, a new class of HIV-1 IN 28 was
synthesised. These catechol isosteres inhibit HIV-1 IN enzymes in vitro. 83
36
Figure 24: Structures of some catechol derivatives
However, it has been postulated that catechol derivatives elicit their effects by interfering
with the co-ordination of metal ions required for phosphoryl transfer rather than directly
binding to IN itself.83
37
1.4.5.3 Other Integrase Inhibitors
29
30
Figure 25: Structures of other IN inhibitors
Compounds 29 and 30 [Figure 25] are promising anti-integrase agents because they have
a favourable pharmacokinetic profile. This is because their oral bioavailability is >60%
and half life of about 5 h in rhesus macaques.86 Compound 29 and 30 have reached
clinical trials.87
Other potential IN-specific inhibitors [Figure 26] have been investigated. The antiviral
activity of compound 31 has been demonstrated to reduce the effect of viraemia as well
as chronic infections in rhesus macaques infected with simian immunodeficiency virus.
Compound 32 has been shown to inhibit HIV-1 IN in vitro, but its antiviral target has not
yet been established in HIV-1 infected cells.79
Figure 26: Structures of other potential IN inhibitors
38
1.4.6
Protease Inhibitors
It has been reported that budding immature viral particles that contain a catalytically
inactive form of HIV-1 PR cannot undergo maturation to afford an infective form of the
HIV-1 virus.40 Based on this premise, a number of HIV-1 PR inhibitors (PIs) have been
designed to make sure that PR enzymes are rendered inactive.
Therefore, PIs were
designed in such a way that the hydrolysable peptide linkage has replaced by the nonhydrolysable hydroxyethylene group.
HIV-1 PIs act by blocking HIV-1 aspartyl
protease, a viral enzyme that cleaves the HIV-1 gag and gag-polyprotein at nine specific
cleavage sites to produce shorter, functional proteins.
Three of the nine cleavage
reactions occur between phenylalanine or tyrosine and a proline residue.40
In general, PIs show only slight side effects and a tolerable toxicity profile. 40 However,
one of the main complications of PIs is that there is a risk of lipodydstrophy. This
symptom encompasses a wide range of manifestations such as insulin resistance,
hyperlipidaemia and body fat redistribution. This manifested in PIs interfering with the
metabolism of fat through cytochrome P450 enzymes in the liver.
There is also an increased risk of HIV patients developing cardiovascular diseases with
PI therapy. Some widely used PIs [Figure 27 and 28] include saquinavir88 33 which was
developed by Hoffman-La Roche (kί = 0.12 nM), amprenavir 34 was discovered by
Vertex pharmaceuticals (kί = 0.6 nM),89 Lilly and Agouron reported nelfinavir 35 (kί = 2
nM),90 indinavir 36 (kί = 0.56 nM)91 developed by the Merck group, while Abbott
laboratories marketed ritonavir 37 (kί = 0.01 nM)92and lopinavir 38 (kί = 0.003 nM).
Unfortunately, due to the high polymorphism of HIV-1, the virus rapidly selects for
variant PI-resistant strains. This kind of resistance has often been observed for HIV-1
positive patients after treatment with PIs approved for clinical use.40
39
Figure 27 FDA approved protease inhibitors
40
Figure 28 Other FDA approved protease inhibitors
1.5
Guanine-Derived Compounds as Novel HIV-1 Therapeutics
The research reported in this thesis is based on the investigation of novel guanine
compounds as HIV-1 therapeutics. Therefore, this section will focus on the various
attempts that have been reported to develop HIV-1 drugs bearing guanine scaffolds.
1.5.1 Guanine-Derived Compound as RT Inhibitors
The first carbocylic guanine compound to show potential as a therapeutic agent for
HIV/AIDS was carbovir (carbocyclic-2′,3′-didehydro-2′,3′-dideoxyguanine) 39 [Figure
29].93
41
39
Figure 29: Structure of carbovir
Both the racemic form of carbovir and its (-)-enantiomer have been synthesised and
evaluated in various cell lines.94 (-)-Carbovir (EC50 = 0.31 μg/mL in MT-4 cells) was
found to be approximately two-fold more potent than the racemic version (EC50 = 0.52
μg/mL in MT-4 cells), indicating that most of the anti-HIV activity of racemic carbovir
resides in the (-)-carbovir.95 Carbovir is believed to exert its anti-HIV-1 effect by the
same mechanism as other dideoxy nucleosides, such as AZT and ddC.96
Analogues of carbovir, compounds 40 and 41 [Figure 30], have recently been synthesised
and their anti-HIV-1 activities have been evaluated.97 Both of these compounds were
synthesised in their racemic forms. The carbocyclic oxetanocin98 40, and the adenosine
analogue 41 were found to have an EC50 of 0.2 μg/mL in the MT4-cell line; however, the
IC50 of 40 was 40 μg/mL. The oxetanocin hypoxanthine 40 was found to have the highest
selectivity index of all the oxetanocin-purine tested.
42
40
41
Figure 30: Structure of analogues of carbovir
Cyclobutyl G, compound 42 [Figure 31], is another guanine-containing compound that
has been found to be very active against HIV-1 as well as other viral diseases such as
herpes simplex virus (HSV-1 and HSV-2) and hepatitis B. This compound has been
found to be resistant to phosphorolytic cleavage by pyrimidine nucleoside phosphorylase
enzymes due to the absence of a furanose ring oxygen.97,99 The therapeutic activity of
carbocyclic nucleosides has been attributed to their conformational mobility, the distance
separating the hydroxymethyl oxygen and pseudo-glycosidic nitrogen, the presence of
lipophilic groups and the absolute and relative configurations of these molecules.100
42
Figure 31: Structure of Cyclobutyl G
Guanosine prodrugs have been successfully used in improving the pharmacokinetics of
already potently-active, guanosine NRTIs.101
For example, the 6-oxopurine ring
substituent in dioxolane guanosine (DXG) 43 and carbovir [Figure 32] has been replaced
with an amino group to afford diaminopurine dioxolane 44 and abacavir 45 respectively.
43
The presence of the amino functionality in these guanosine analogues improves their
lipophilicity, solubility and oral bioavailability. The presence of the cyclopropyl
substituted secondary amine in abacavir 45 improved its absorption into the central
nervous system. 102
Figure 32: Structures of guanine prodrugs
Compound 45 is currently in Phase I/II of clinical development for the treatment of HIV1. Compound 45 exerts its biological effect by first being deaminated by adenosine
deaminase. The resulting dioxolane guanine (DXG) is then further metabolised to its 5′triphosphate form (DXG-TP) by host cell enzymes.103 Subsequently, DXG-TP acts as a
potent substrate inhibitor of the HIV-1 RT.104 The incorporation of DXG 5′-triphosphate
(DXG-TP) into nascent DNA results in chain termination. It has been shown that a virus
harbouring HIV-1 RT mutations which are resistant to AZT, abacavir and efavirenz
remained sensitive to inhibition by DXG. 105
44
Acyclovir 46 [Figure 33] is another example of a guanine-containing compound that has
antiviral activity.106 Acyclovir is able to carry out its inhibitory activity because it has
greater affinity for the HSV viral form of thymidine kinase than the human equivalent.
Thymidine kinase converts acyclovir into acyclovir monophosphate which is then further
transformed into its corresponding diphosphate by another cellular enzyme, guanylate
kinase. Finally, upon being transformed into its triphosphate form, it binds to HSV RT
and terminates the replication cycle of the HSV virus. 107
46
Figure 33: Structure of acyclovir
Furthermore, guanine derivative 47 [Figure 34] has been found to show comparable
activity with that of PMEA 48 against HIV-1 and HIV-2 in MT4-cell cultures and against
clinical HIV-1 isolates from peripheral blood mononuclear cells.108 The anti-retroviral
activities of PMEG 49 and compound 47 in cell culture were found to have EC50 values
of 0.26 μM and 17.7 μM respectively.
45
Figure 34: Structures of other antiviral purine compounds
1.5.2 Guanine-Derived Compounds as IN Inhibitors
Recently, a family of G-tetrad-forming oligonucleotides has been developed as potential
anti-HIV therapeutic drugs.109
The G-tetrad (G-quartet) DNA structure was first
determined by Gellert et al.110 G-tetrads are multi-stranded structures held together by
square planes of four guanines interacting by forming Hoogsteen hydrogen bonds.111
Each G-base makes two hydrogen bonds to its neighbouring G-bases (N1 to O6 and N2
to N7) [Figure 35].112 G-tetrad formation is the fundamental driving force for a large
number of G-rich DNA and RNA oligomers to form highly ordered structures. These
oligomers have been found to be involved in human genomes and telomerases.113 They
have also shown promising applications in nanotechnology. Inhibition of integrase
activity in vitro strongly depends on the stability of the G-quartet. When associated with
K+ ions [Figure 36], they form symmetrical and compact intramolecular G-tetrads and the
folded loop domains greatly increase their structural stability.114 This highly stable and
46
compact structure enhances the permeability of the guanosine tetrad-forming
oligonucleotides into cells and their ability to resist nuclease digestion. The structural
features of the G-tetrad induced by K+ ions are essential for the inhibition of the HIV-1
integrase activity.114
The most potent inhibitors of HIV-1 integrase were found to be T30695,
5′g*ggtgggtgggtggg*t-3′ and T30177, 5′-g*tggtgggtgggtggg*t-3′(where * shows that
there is a phosphorothioate linkage at the two end g residues.).109
IC50 values of
inhibition for HIV-1 integrase 3′-processing and strand transfer, obtained from a gelbased method, were 47 and 24 nM for T30695 and 79 and 49 nM for T30177.109 NMR
and kinetic data have been used to demonstrate that, in response to K+ binding, T30695
folded into a stable and symmetric G-tetrad. The folding was shown to be a two-step
process, and is dependent on the nature of the alkali metal ion added. The first step of the
process involved the coordination of one K+ ion, which competes with a Li+ ion to bind
within the core of two G-tetrads. The second step involves the binding of two additional
K+ ions to the loop domains. NMR and optical analysis have shown that the second
binding step is associated with substantial ordering of the oligonucleotides fold.109 NMR
data and molecular modelling have determined that T30695, in the absence of K+ and
presence of Li+ ions, forms an intramolecular G-tetrad with opened loop structures. Upon
coordination with three K+ ions, the loop structure is rearranged, and the bases of loops
are folded onto the underlying G-quartets. The structure of T30695 in the presence of K+
becomes symmetric and compact. The inhibition of HIV-1 integrase activity was found to
greatly increase upon K+ binding to the loops. Thus, the folding of the loop domains of
these oligonucleotides plays an important role in the function of G-tetrad forming
oligonucleotides.115
47
dR
H
N
N
H
N
H
H
N
N
H
O
O
H
H N
N
N
H
H
N
O
N
N H
O
N
dR
N dR
N
H
H
N
H
N
N
N
N
N
dR
H
H
Figure 35: Four G-bases associate through Hoogsteen hydrogen bonding to form a cyclic
structure 115
dR
H
N
N
N
H
N
N
H
H N
K+
O
O
N
N
H
N
dR
N
H
N dR
N
N
H
N
H
O
O
H
H
H
H
N
N H
H
N
N
N
N
dR
H
Figure 36: Assembly of four guanine nucleobases into a G-tetrad stabilized by a central
potassium ion.116
1.6
Peptide Nucleic Acids
Peptide nucleic acids [PNAs] [Figure 37] are DNA mimics which were originally
developed by Nielsen et al. in 1991.117 In order to overcome some of the limitations
associated with natural or other oligonucleotides analogues which had been developed at
the time. These include poor cellular uptake and nuclease stability. PNAs contains an
uncharged pseudo-peptide backbone in place of the normal sugar-phosphate backbone,
which is isomorphous with the DNA backbone [Figure 38].118,119,120,121 The nucleobases
48
of peptide nucleic acids are attached to the pseudo-peptide backbone via a methylene
carbonyl linker. Peptide nucleic acids are able to mimic DNA and RNA perfectly,
because they contain an achiral backbone and they are relatively flexible.117
Figure 37: Peptide Nucleic Acid
49
NH2
A
N
N
N
N
O
U
HO
O
O
NH
N
OH
O P
O
O
NH2
O
N
O5'
NH2
N
N
HO
O
OH
N
P O
O
3'
T
O
P
N
OH
O
G
O
N
O
O
NH
O
O
O-
N
N
O
A
C
NH
N
NH2
O
-
O
N
O
O
P
O
O
O
O
N
P
O
O
P
O
N
N
O
O
O
P
OH
O-
O-
O
O3'
O
N
-
O
O
C
O
5'
RNA
NH2
G
NH
N
NH2
O
-
O
DNA
O
O
P
O-
O-
Figure 38: Structures of DNA and RNA
1.6.1 Properties of Peptide Nucleic Acids (PNAs)
PNAs and DNA have no functional groups in common except the nucleobases and as a
result of this they have completely different chemical stability. For example, DNA
depurinates on treatment with strong acids while PNAs are acid stable. This is the major
reason why it is possible to synthesise PNAs using the standard protecting groups from
50
peptide chemistry which requires cleavage with trifluoromethanesulphonic acid or
anhydrous HF.122
PNAs bind with higher affinities to complementary nucleic acids than their natural
counterparts. A mixed purine-pyrimidine PNA hybridizes to complementary ssDNA and
RNA oligomers in a sequence specific manner by the formation of anti-parallel double
helices in compliance with the Watson-Crick base pairing rule (A binding to T and C
binding to G).123 PNA backbones, unlike DNA and RNA, are uncharged. The presence
of the uncharged PNA backbone gives relative stability to the complexes formed by
PNAs, either with itself or with DNA or RNA, because there is no electrostatic repulsion
when PNA hybridizes to its target nucleic acid sequence.122,124,125
For example,
PNA:ssDNA hybrids are more stable than the corresponding DNA:DNA complexes and
this results in an increase in the melting temperature (Tm) of approximately 1 °C/base.
The formation of PNA: DNA duplexes have been investigated using the pentadecamer HTGTACGTCACAACTA-NH2.126 This oligomer forms a duplex with complementary
antiparallel DNA with a Tm of 69.5 °C, whereas the corresponding DNA: DNA duplexes
have a Tm of 56.1 °C.
PNA: RNA complexes are even more stable showing an increase
in about 1.5 °C/base compared to DNA: RNA hybrids. Homopyrimidine PNAs form
extremely stable (PNA)2/RNA triplexes with target RNA and shown similar Tm values to
their corresponding (PNA)2/DNA triplexes.
The most interesting aspect of the PNA: DNA duplex is its dependence on ionic strength.
According to Tomac et al.127 the Tm of DNA: DNA hybrids were shown to increase by
more than 20 °C for a 10-mer with increasing salt concentrations, whereas the Tm of
PNA: DNA duplexes decreased. The contrasting effect of ionic strength on duplex
formation can be explained by the association of counter ions in the case of DNA: DNA
duplex formation and by displacement of counter ions in the case of PNA: DNA duplex
formation.128
Furthermore the formation of (PNA)2/DNA triplexes has been observed when
homopyrimidine PNAs or PNAs with a high pyrimidine:purine ratio bind to
51
complementary DNA using both Watson-Crick and Hoogsteen base pairing in a 2:1
stoichiometry [Figure 39].
The thermal melting (Tm) obtained for these types of
complexes has been found to be greater than 70.0 °C for decamer hybrids. However, the
length of the oligomer has been observed to be an additional contributory factor to the
stability of the triplex formed. For example, an increase in Tm of ca. 10 °C per base pair
has been observed.129,130
Figure 39
These triple helices are much more stable than their corresponding DNA duplexes. For
example the Tm value of the hybrid of the PNA “10-mer” H-(T)10 with its Watson-Crick
complementary deoxyribose (dA)10 was measured as 73 °C whereas the corresponding
(dT)10:(dA)10 is less than 23 °C.
The high stability of (PNA) 2/ DNA triplexes enables strand displacement to occur upon
targeting double-stranded DNA. During strand displacement, the homopyrimidine PNA
oligomers displace the pyrimidine strand of the complementary dsDNA targets to form a
(PNA) 2/DNA triplex with the homopurine strand. This type of displacement gives rise
to a P-loop.
The formation of the P-loop during strand invasion has been visualized
using electron microscopy.133
For example when a linear dsDNA target containing
d(A) 98/d(T) 98 insert was challenged with PNA H-T10-LysNH2, a P-loop of 90-100 bases
in length were was observed.131 ,132 ,133 Homopurine PNAs also invade dsDNA but fail to
form triplexes and their invasion complexes are less stable.134 With a few exceptions,135
52
strand displacement using mixed sequence PNAs has not been observed; presumably this
is because their strand invasion complexes are even less stable. Figure 40 below gives a
summary of the three binding modes of PNA with dsDNA.136, 126 ,137
D loop
PNA
PNA
PNA
dsDNA
Nucleobases
dsDNA
Nucleobases
A
Triplex
dsDNA
Nucleobases
PNA
B
C
Triplex
Duplex
invasion
Invasion
Figure 40: Complexes of PNA with dsDNA128
Finally, PNAs can also form extremely stable duplexes with another strand of
complementary PNA. For example a duplex formed by the PNA decamer H-gtagatcact(L)-Lys-NH2 and the complementary sequence H-agtgatctac-(L)-Lys-NH2 has a Tm of 67
°C compared to a Tm of 33.5 °C reported for the corresponding DNA/DNA duplex.138
1.6.2
PNAs as a Potential Anti-HIV-1 Therapeutics
Peptide nucleic acids manifest high biological stabilities.139 They have high resistance
against cell nucleases, proteases and peptidases.140 Another advantage of PNA is that they
generally exhibit low toxicity and they are not prone to non-specific binding to cellular
proteins.130 However, a slight draw back to their use is that PNA oligomers may undergo
very slow enzymatic hydrolysis in both cell extracts and in vivo.133
53
Peptide nucleic acids can inhibit the transcriptional process because of their ability to
form a stable triplex structure with DNA.
This type of complex formed creates a
structural hindrance which affects the working of the RNA polymerase.141,142
However, despite their numerous advantages, PNAs possess a number of limitations.
Since pure PNAs are neutral compounds with a tendency for self-aggregation and limited
water solubility, they show poor bioavailability.143 In addition, they cannot be delivered
into the cell by conventional cationic formulations such as liposome (as well as
lipofectin, lipofectamine, e.t.c) or microspheres.137 However, the introduction of charged
groups, for instance a C-terminal lysine amide, can greatly improve their solubility.
Another difficulty is that PNA aqueous solubility drops with increasing length and
purine: pyrimidine ratio.144
The higher the purine content, the higher the aqueous
solubility of the PNA. Homoadenine PNA polymer has been found to be highly watersoluble.
Positive charges have also been introduced by modifying the PNA backbone,
for instance by replacement of the glycines by lysines.137 The incorporation of only two
such groups was found to greatly increase the solubility of the oligomers. Alternatively,
negative charges can be introduced into the oligomers, such as by preparing PNA-DNA
chimeras. These too showed enhanced water solubility.134
1.7
Aims and Objectives of the Research
Currently, there are about twenty one antiretroviral drugs approved for the treatment of
HIV-1 infections,145 but the problems of cost, toxicity, viral suppression and drug
resistance still persist. Therefore, there is a need to synthesise new compounds with
antiretroviral activity and to exploit novel drug targets.
From section 1.4, it is clear that two major classes of RT inhibitors have been
investigated (the NRTIs and NNRTIs). It has been shown that NNRTIs have a number of
specific properties which distinguish them from the NRTIs. The NNRTIs are highly
specific, present little cytotoxicity and do not require activation through intracellular
metabolism and phosphorylation to exert their effect. Furthermore, it has also been
54
shown that the first generation of NNRTIs suffered a set back because the HIV-1 virus
was able to develop resistance to these compounds. The resistance of HIV-1 virus to the
first generation NNRTIs was as a result of the way they bind to the amino acids in
NNBS. A single mutation in the amino acids in the NNBS affects the binding of the first
generation NNRTIs to the NNBS. Hence, there is a need to discover another generation
of NNRTIs which will be able to overcome this resistance mechanism. Based on this
resistance problem, another generation of NNRTIs is being investigated; notable among
these second generation NNRTIs are etravirne and riplivirine. These compounds are able
to overcome the resistance mechanism through their conformational flexibility.
Upon closely examining the structure of the second generation NNRTIs, it was clearly
seen that, these compounds bear desirable features (aromatic substituents and hydrogen
bond donor) which are required for binding in NNBS. Based on these desirable features,
it was reasoned that the proposed guanine compounds were worthy of examination as
potential NNRTIs.
Since guanine-rich natural oligonucleotides T30695, 5′g*ggtgggtgggtggg*t-3′ and
T30177, 5′-g*tggtgggtgggtggg*t-3′ have been shown to be potent HIV-1 integrase
inhibitors, it was reasoned that PNA analogues of T30695 and T30177 would also be
potential HIV-1 integrase inhibitors. The stability advantages of PNAs towards nucleases
and proteases mean that these guanine-rich PNA compounds will be more stable than the
natural oligonucleotides in the cell.
In order to synthesise these guanine-rich PNA oligomers using conventional peptide
synthesis, the synthesis of protected guanine-PNA monomers 48 and 49 [Figure. 41] in
high yield was first required.
55
Figure 41: Guanine PNA monomers
The challenges in the synthesis of 48 and 49 were that guanine molecules are notoriously
insoluble in aprotic organic solvents and there is a problem of non-reactivity of the 2amino group on the purine ring. Therefore, one of our aims was to design a high yielding
route to the synthesis of Cbz- and Boc-protected guan-9-ylacetic acids. The classical
carbamate peptide protecting groups Cbz and Boc were the groups of choice since it is
known that these moieties would remain intact during the peptide synthesis protocol but
could be readily removed at the end of solid phase synthesis of PNA oligomers.
Therefore, the aims of research work were:
1
To develop a high yielding route for the synthesis of Cbz-and Boc-protected
guan-9-ylacetic acids.
2.
To synthesise guanine PNA monomers 48 and 49 [Figure 41], which would be
used subsequently in the construction of guanine-rich PNA oligomers.
3
To biologically evaluate the guanine derivatives synthesised as HIV-1 reverse
transcriptase and integrase inhibitors.
56
4
To initiate structure-activity studies on active compounds.
5
To explore the solution phase synthesis of guanine dimers.
6
To investigate solid phase synthesis of guanine-rich PNA oligomers i.e the PNA
analogues of T30695 and T30177.
57
CHAPTER TWO
RESULTS AND DISCUSSION
CHEMICAL SYNTHESIS OF GUANINE-CONTAINING COMPOUNDS
As outlined in section 1.7, one of the aims of this research was to design reproducible and
high yielding routes to the synthesis of N2-Cbz- and N2-Boc-protected guan-9-ylacetic
acid. These compounds, once synthesised, would be coupled to the “traditional” PNA
backbone to form guanine PNA monomers. The high-yielding synthesis of N2-Cbz and
N2-Boc-protected guanine-PNA monomers from 2-amino-6-chloropurine has been an
ongoing challenge faced in this group. Synthesis of guanine PNA monomers in a high
yield is very important for the successful synthesis of guanine-rich PNA oligomers using
either solid phase or solution phase synthetic approaches. The following sections will
discuss the established literature routes to the synthesis of N2-Cbz- and N2-Bocprotected guan-9-ylacetic acids presenting both the advantages and limitations. Finally
our approaches to the development of novel routes for the synthesis of N2-Cbz- and N2Boc-protected guan-9-ylacetic acids will be described.
2.1
Established Routes for the Synthesis of PNA Monomers
2.1.1 Traditional Route to all PNA Monomers
Thymidinyl, adenyl and cytidinyl-PNA monomers 50 are typically prepared according to
the following retrosynthetic analysis outlined in Scheme 2.
58
BP
P
B
O
O
+
R
R
N
H
N
COOH
OH
50
H
N
N
H
51
CO2R'
52
R= Fmoc; Boc
B= A; C;T
P= Protecting group for any
exocyclic amino functionality
R"= R' = alkyl group
B
O
P
56
+
BrCH2CO2R"
+
H
RHN
55
H2N
54
CO2R'
53
H2 N
OH
OH
57
Scheme 2
The retrosynthetic route [Scheme 2] illustrates that PNA monomers are comprised of a
peptide molecule (aminoethylglycine unit, always referred to as the PNA peptide
backbone) 52 and a nucleobase acetic acid 51. The synthesis of 52 from the precursor 57
can be achieved by treating compound 57 with excess di-tert-butyl dicarbonate followed
by the oxidation of the diol with NaIO4 to give an aldehyde 54. The next reaction
involves reductive amination of 54. This can be accomplished by treating 54 with
ethylglycine hydrochloride 53 in the presence of sodium cyanoborohydride. This reaction
results in the formation of 52.
The preparation of 51 [Scheme 2] involves the initial protection of the exocyclic amino
functionality of the nucleobase (adenine and cytosine).
Examples of the common
protecting groups are Mmt, acyl, Cbz and Boc. The protection of the nucleobase by a
suitable protecting agent is then followed by alkylation of the nucleobase. This is
59
achieved by treating 56 with 55. This reaction affords N9-alkylated adenine or N4alkylated cytosine. Subsequently, the ester functionality is hydrolysed by treating the
esters with a base. This affords 51 after work-up. The synthesis of thymidinyl acetic acid
is relatively easy because the thymine moiety does not have any exocyclic amino groups
which require protection.
The final step in the traditional route is the coupling together of 51 and 52 to give 50
using commercially available coupling agents. The purine nucleobase acetic acids 51 are
coupled
to
the
backbone
52
using
BOP
(O-benzotriazol-1-yl-N,N,N′,N′-
tetramethyluroniumhexafluorophosphate) and HOBT (1-hydroxybenzotriazole) or EDC
(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) as the coupling reagents.
The guanine-PNA monomer is more challenging because the guanine base cannot be
derivatised in three simple steps as for the others. In order to prepare the guanine
analogue of 51, it was necessary to start from the commercially-available 2-amino-6chloropurine. The reasons are because the guanine molecule is not regioselective, it has
poor solubility in aprotic organic solvents and low reactivity of its N2-amino group.
For the successful synthesis of PNA oligomers using either a solid or solution phase
synthetic strategy, the choice of the protecting groups for both the N-(2-aminoethyl
glycine unit) and the nucleobase (C, G, A) is very important. These protecting groups
must be orthogonal to each other in order to achieve their selective cleavage during the
oligomerization process. For example in Scheme 2, if R= Fmoc, P=Boc or if R=Boc,
P=Cbz.
2.1.2 Guan-9-ylacetic Acid Synthetic Routes
Preparation of protected guanine PNA monomers using the established route discovered
by Nielsen et al.117 is very difficult. Therefore, various routes have been investigated in
order to synthesise N2-protected guan-9-ylacetic acids.
60
Thompson et al. [Scheme 3]146 investigated an approach to the synthesis of the N2-Cbz
protected guan-9-ylacetic acid. It was observed that the synthesis of N2-protected guan9-ylacetic acid from guanine proved to be most challenging. They reported that the
alkylation of guanine with methyl bromoacetate followed by attempted protection of the
exocyclic amino group under very strongly basic conditions gave complex product
mixtures.
In order to overcome this problem, the reactions shown Scheme 3 were
investigated.
This route started with the alkylation of commercially available 2-amino-
6-chloropurine 58. The alkylation reaction was carried out by treating 58 in DMF with
allyl bromide and K2CO3.
After work-up and purification by flash column
chromatography 59 was obtained as a major product in 59% yield.
Subsequently, 59
was converted into the guanine derivative by treating 59 with aqueous HCl at reflux for 2
h. After work-up, 60 was afforded as a white solid in a quantitative yield. The next step
in Scheme 3 involved the protection of the exocyclic amino group with Cbz. Therefore,
60 in THF was treated with potassium hydride/18-crown-6 and N-(benzyloxycarbonyl)
imidazole. After work-up, 61 was obtained in 91% yield. The final step is the conversion
of the N9-allyl group of 61 to the corresponding acetic acid. This was achieved by the
ozonolysis of 61 followed by oxidation to give the desired product 62 in 75% yield.
61
Cl
N
Cl
N
N
H
N
N
i
NH2
N
58
O
ii
N
N
N
N
NH2
NH
N
NH2
59
60
iii
O
N
O
NH
N
O
NH
iv
N
O
OH
N
N
H
O
N
62
N
N
H
O
O
61
Scheme 3
Reagents and conditions: (i) Allyl bromide, K2CO3, DMF; (ii) 1 M(aq) HCl, reflux; (iii)
Cbz-imidazole, 18-crown-6, KH, THF; (iv) (a) O3, CH2Cl2, MeOH (b) Me2S (c) NaClO2,
NaH2PO4, H2O, THF, (CH3)3COH, 2-methyl-2-butene.
Howarth et al.147 have reported the synthesis of peptide-based nucleic acids derived from
α-amino acids [designated as α-PNAs]. All the nucleobase α-amino acids were prepared
starting from L-homoserine.
In this approach, the Cbz-protected guanyl α-PNA
monomer proved to be the most problematic to synthesise due to the reasons described on
page 60. Therefore they opted to replace the Cbz-protecting group with an acetyl group.
Their approach to the synthesis of this guanine-α-PNA monomer is shown in Scheme 4.
62
O2N
Cl
Cl
N
N
H
N
H2N
i
N
O
N
N
ii
O
NH2
O
58
H2N
N
H
H3CO
N
N
N
N
N
N
O
O
O
63
64
N
H
H3CO
iii
O
HN
O
N
H
O2N
N
iv
N
N
O
O
O
N
H
H3CO
O
N
N
O
N
H
N
N
O
O
66
N
H
H3CO
O
65
Scheme 4
Reagents and conditions: (i) (S)-2-(N-Boc-amino)-4-bromobutyric acid methyl ester,
K2CO3, DMF, RT; (ii) 2-nitrophenol, DABCO, Et3N, 1,2-dichloroethane, RT; (iii)
CH3COCl, pyridine, RT; (iv) 1,1,3,3-tetramethylguanidine, o-nitrobenzaldoxime,
acetonitrile, RT, overnight.
The first step in Scheme 4 was the alkylation of commercially-available 58.
The
alkylation was achieved by treating 58 in anhydrous DMF with (S)-2-(N-Boc-amino)-4-
63
O
bromobutyric acid methyl ester, in the presence of anhydrous K2CO3, at room
temperature.
After work-up, 63 was obtained in 90% yield. Subsequently, this was
followed by the replacement of the 6-chloro group with the desired oxygen functionality.
Howarth et al.147 did not used sodium alkoxide for the replacement of 6-chloro group
because its use could lead to the racemization of the amino acid. Therefore, they used an
established approach developed by Reese et al.148 This involved the replacement of the
6-chloro group of 63 with a 2-nitrophenoxy group. Therefore, a solution of 63 in 1,2dicholoromethane was treated with 2-nitrophenol, DABCO, Et3N, at room temperature
overnight. After work-up and purification by column chromatography 64 was obtained
in a high yield.
The next step in Scheme 4 was the protection of exocyclic 2-amino
group of 64. However, because of their failure to successfully protect the exocyclic
amino end of the purine with a Cbz group, they opted for the use of an acyl group.
Compound 64 was treated with acetyl chloride in anhydrous pyridine at room
temperature. After work-up, 65 was obtained in 94% yield. Finally, the nitrophenoxy
moiety was removed by reacting 65 with 1,1,3,3-tetramethylguanidine and 2nitrobenzladoxime in anhydrous acetonitrile. After work-up and purification by flash
column chromatography, 66 was obtained in 44% yield.
The first synthetic approach to the synthesis of N2-Boc-protected guan-9-ylacetic acid
was developed by Sugiyama et al.149 [Scheme 5]. The first approach in Scheme 5
involved the conversion of 58 into 67. This conversion was carried out over three steps.
The first step involved the treatment of 58 in DMF with allylbromide and K2CO3. After
work-up, the resulting product was treated with di-tert-butyl dicarbonate in order to
protect the exocyclic amino group of the purine ring.
order to overcome the solubility problem of guanine.
The third step was designed in
Therefore, the Boc-protected
intemediate was further protected at the O6- position with a trimethylsilyethyl (TMSE)
group. After work-up and purification, 67 was obtained.
The next approach in Scheme 5 was the conversion of 67 into 68; this transformation was
achieved in three steps. This approach is similar to that reported by Thompson et al.146
[Scheme 3] the only difference being is that Thompson et al.146 used ozonolysis followed
64
by oxidation to generate the N9-acetic acid, whereas Sugiyama et al.149 made use of
oxygen-rich OsO4 followed by oxidation.
Cl
N
OTMSE
N
N
H
N
i,ii,iii
NH2
N
N
N
N
OTMSE
O
N
H
N
iv,v,vi
O
N
O
58
OH
67
N
N
O
N
H
68
Scheme 5
Reagents and conditions: (i) Allylbromide, DMF; (ii) Boc2O, DMAP, DMF; (iii) NaH,
TMSCH2CH2OH; (iv) OsO4, Me3NO; (v) NaIO4 (vi) NaClO2.
Another approach to the synthesis of N2-carbamate-protected purine compound was
developed by Dey and Garner.150
They reported the successful synthesis of both N2-
Boc-protected guanine 72 [Scheme 6] and N2-Boc-6-chloropurine 75 [Scheme 7],
starting from guanine 69 and 2-amino-6-chloropurine 58 respectively. In Scheme 6, the
first step involved the formation of glyoxal adduct 70. This was achieved by treating 69
with glyoxal and water at 60 °C for 24 h. Subsequently, 70 was treated with excess
Boc2O in the presence of catalytic DMAP in THF for 5 days. This gave tetra-Boc
derivative 71 in 75% yield after filtration through silica gel. Finally, the Boc-guanine 72
was obtained by exposing 71 to ammonium hydroxide in aqueous THF. After work-up, it
was obtained in 55% yield.
65
O
O
N
N
H
O
NH
N
i
NH2
N
N
H
O
OH
N
OH
N
H
N
ii
N
N
OBoc
N
N
Boc
69
70
OBoc
N
Boc
71
iii
O
N
N
H
NH
N
NHBoc
72
Scheme 6
Reagents and conditions: (i) Glyoxal, water, 60 °C, 24 h; (ii) Boc2O, DMAP, THF, RT,
120 h; (iii) NH4OH, water, THF, pH 11.
Furthermore, Scheme 7 involved protection of the exocyclic amino group of 2-amino-6chloropurine 58 with a Boc group. The first step in this approach involved the treatment
of 58 in dry THF with excess (4.5 eq) Boc2O, a catalytic amount of DMAP at room
temperature overnight.
After work-up and purification, a single tris-Boc-protected
product 73 was obtained in 97% yield. The next step in the scheme was the selective
cleavage of the N9-Boc group of 73.
This was achieved by treating the tris-Boc-
protected compound 73 with NaHCO3 in methanol at 50 °C. Purification of the crude
product by column chromatography afforded a di-Boc-protected compound 74 in 87%
yield. The final step in the pathway was the conversion of the di-Boc compound 74 into
a mono-Boc compound 75. This conversion involved the treatment of 74 with NaOH and
ethanol for 3 days. After work-up and purification, the desired mono-Boc product 75 was
obtained in 71% yield.
66
Cl
Cl
N
N
N
N
N
N
O
i
NH2
N
O
ii
N
N
H
Cl
O
N
O
58
N
O
N
H
O
N
N
O
O
73
O
O
74
iii
Cl
N
N
H
N
N
O
N
H
75
Scheme 7
Reagents and conditions: (i) Boc2O, DMAP, THF, RT, 10 h; (ii) NaHCO3, MeOH, H2O,
50 °C; (iii) NaOH, CH3CH2OH, 3 days
Breiphol et al.151 have also reported the synthesis of Mmt-protected guan-9-ylacetic acid
[Scheme 8]. Their justification for investigating the use Mmt instead of the carbamate
protection (Cbz or Boc) was based on the fact that the repetitive cleavage of the
carbamate intermediates during the chain elongation process and the final cleavage of the
PNAs from resin involved the use of harsh conditions (such as TFA or trifluoromethane
sulfonic acid).
In their approach, they developed a milder method which involved the
use of the acid-labile Mmt group as the protecting group for the guanine nucleobase and
the base labile Fmoc group for protection of the PNA backbone. This work was later
repeated by Bialy et al.152
The first step in the synthesis of Mmt-protected guan-9-
ylacetic acid involved the alkylation of the N9-nitrogen of 58 in DMF with methyl
bromoacetate and sodium hydride.
After work-up and purification by column
67
O
chromatography, 76 was obtained in 75% yield. The next stage concerned the protection
of the exocyclic amino group of 76 with a Mmt group. This protection was achieved by
treating 76 with Mmt chloride, pyridine and triethylamine.
After work-up and
purification by flash chromatography, 77 was afforded in 60% yield. Finally
saponification of compound 77 with hot 10% (w/v) aqueous NaOH carried out two
conversions; the hydrolysis of the ester to an acid and the replacement of the 6-chloro
group with a 6-hydroxy function.
These two reactions afforded compound 78 in an
overall yield of 72%.
Cl
Cl
N
N
H
N
N
i
NH2
N
Cl
N
N
N
ii
N
NH2
N
N
N
O
Mmt
O
O
58
N
H
O
76
77
iii
O
N
NH
N
N
N
H
Mmt
O
HO
78
Scheme 8
Reagents and Conditions: (i) 1 equiv. NaH, 1 equiv. methyl-2-bromoacetate, DMF, 4 h;
(ii) 1.5 equiv. Mmt-Cl, 1 equiv. DIPEA, THF overnight; (iii) 10% NaOH(aq), reflux, 2 h.
Will et al.153 have also developed a scheme for the synthesis of N2-acyl-protected guan9-ylacetic acid [Scheme 9].
They were of the opinion that this approach was an
alternative strategy which will open the way to a combination of PNA and
68
oligonucleotide synthesis. In this preparation, guanine 69 was treated with isobutanoyl
chloride in DMF and triethylamine as the base, at high temperature. After work-up, 79
was obtained in a quantitative yield. This was followed by the alkylation of 79 at the N9position using methyl bromoacetate in the presence of sodium hydride. After work-up
and purification by column chromatography, 80 was obtained in 40% yield. The final
stage in the pathway involved the hydrolysis of the ester functionality of 80. The
hydrolysis of 80 was carried out by treating it with aqueous NaOH to give the desired N2acyl-protected guan-9-yl acetic acid 81 in 62% yield.
N
NH
N
H
N
O
O
O
NH2
i
N
N
H
NH
N
N
H
O
ii
N
O
NH
N
N
N
H
O
69
79
80
O
iii
O
N
NH
N
N
O
N
H
O
HO
81
Scheme 9
Reagents and conditions: (i) Isobutanoyl chloride, Et3N, DMF; (ii) Methyl bromoacetate,
NaH, DMF; (iii) NaOH, H2O, dioxane.
From the work of the various researchers reviewed, it was obvious that most of the
researchers did not use either Boc or Cbz to protect the exocyclic amino group of
guanine. Bialy et al.152 and Breiphol et al.151 [Scheme 8, page 68] successfully prepared
69
N2-Mmt-protected guan-9-ylacetic acid in a high yield but this is different from the target
of this thesis which was to use Cbz- or a Boc-group to protect the exocyclic amino
function. The problem of solubility in organic solvents, the choice of the protecting
group and non-regioselectivity in the alkylation of guanine are setbacks in the approach
of Will et al.153 [Scheme 9, page 69].
Thompson et al146. [Scheme 3, page 62] reported an approach for Cbz protection of
guanine nucleobase.
However, previous attempts to repeat this work in our group had
not been successful.
The Dey and Garner approach150 [Scheme 7, page 67] was a
promising route but they had not taken their approach to the synthesis of the desired
guan-9-ylacetic acid. However we deemed that their approach [Scheme 7] was worthy
of further investigation.
It was reasoned that Scheme 7 could be adapted for the preparation of both N2-Cbz- and
N2-Boc-protected guan-9-ylacetic acids and this is shall be discussed in the following
section.
70
2.2
An Attempt to Synthesise N2-Carbamate Protected Guan-9-ylacetic Acids
Cl
Cl
N
N
H2N
N
N
i
R1
N
H
N
Cl
N
R1
N
N
R3
R2
N
N
H
N
R2
74: R1=R2=Boc
83: R1=R2=Cbz
73: R1=R2=R3=Boc
82: R1=R2=R3=Cbz
58
N
N
ii
iii
O2N
O
N
HN
R1
N
N
N
R2
Cl
O
O
v
N
N
R1
N
R1
N
N
R2
OCH2CH3
88: R1=R2=Boc
89: R1=R2=Cbz
N
R2
O
N
N
iv
N
N
O
OCH2CH3
OCH2CH3
86: R1=R2=Boc
87: R1=R2=Cbz
84: R1=R2=Boc
85: R1=R2=Cbz
vi
O
N
HN
R1
N
R2
N
N
O
OH
90: R1=R2=Boc
91: R1=R2=Cbz
Scheme 10
Reagents and Conditions: i (a)Boc2O, DMAP, DMF, RT, overnight; (b) Cbz2O, DMAP,
DMF, RT overnight; (ii) NaHCO3, CH3OH, 50 °C for 1 h then RT overnight; (iii)
BrCH2COOCH2CH3, K2CO3, DMF, RT, 5 h; (iv) o-Nitrophenol, DABCO, acetonitrile,
Et3N, RT, overnight; (v) o-nitobenzaldoxime, N,N,N,N-tetramethylguanidine, 1,2dichloroethane, RT, overnight; (vi) 1M(aq) NaOH, dioxane.
71
Initially the synthesis of the Boc-protected compound 90 was investigated. The first two
steps in this pathway [Scheme 10] involved repeating the procedure of Dey and Garner148
[Scheme 7] directly. Thus, 2-amino-6-chloro purine 58 was treated in dry THF with an
excess of di-tert-butyl dicarbonate and catalytic amount of DMAP according to their
procedure.
Unfortunately,
upon
work-up
and
purification
by
flash
column
chromatography, the desired tris-Boc-protected compound 73 was only afforded in a 26%
yield rather than the 97% yield quoted in the literature.
In an attempt to optimise this reaction, the solvent was changed from THF to dry DMF.
The reason for changing the solvent was to study the effect of a change in polarity on the
progress of the reaction. It was observed that using DMF resulted in the formation of a
more homogeneous solution. Furthermore, DMF, being a more dipolar solvent, is better
able to stabilize charge separation and the transition state of the reaction.
This
modification resulted in a drastic improvement in the yield of 73. After work-up and
purification by column chromatography, 73 was afforded in 97% yield. All spectra
recorded for 73 were consistent with that reported by Dey and Garner.150 In the 1H NMR
spectrum, the singlet at δ 1.42 corresponded to the 18 protons in the di-tertbutoxycarbonyl group attached to the N2–amino group of the purine ring. The remaining
9 protons from the N9-tert-butoxycarbonyl group of 73 produced a singlet at δ 1.65. The
singlet at δ 8.56 was assigned to the purine C8-H.
The next step in our synthetic pathway involved the selective hydrolysis of the N9-Boc
protecting group of 73 to give the di-Boc compound 74 [Scheme 10].
This was
successfully achieved using Dey and Garner’s procedure under identical conditions.150
This involved treatment of 73 in methanol with sodium hydrogen carbonate, first at 50 °C
for 1 h before being left at room temperature overnight. After work-up, compound 74
was obtained in an 84% yield and it was deemed sufficiently pure by 1H NMR and
NMR analysis to be used in the subsequent step.
72
13
C
Having successfully synthesised compound 74, alkylation of the N9-position of the
purine ring was subsequently investigated applying the method reported by Dueholm et
al.154 This involved treating a solution of 74 in DMF with ethyl bromoacetate in the
presence of sodium hydride at room temperature overnight. This reaction afforded 84 in
a reproducible yield of 75%, after purification by flash column chromatography using
ethyl acetate as the eluting solvent. The N9-alkylated product was the only regioisomer
detected. The spectral analysis of 84 confirmed its formation. In the 1H NMR spectrum,
a triplet at δ 1.3 was observed with a coupling constant of 7.2 Hz which corresponded to
the three protons in the ethyl ester. The quartet at δ 4.3 corresponded to the other two
protons in the ethyl ester. The 1H NMR and
13
C NMR spectra of 84 were comparable
with the results reported for N9-alkylated-2-aminopurines by Green et al.155and Kjellberg
et al.156 This supported the claim that the product isolated was the N9-subtituted
regioisomer.
The next step in this pathway involved the conversion of the 6-chloro substituent of
compound 84 into a moiety that upon cleavage would afford the desired 6-oxo group of
guanine [Scheme 10]. This conversion was carried out using the approach described by
Howarth et al.147 This involved first substitution of the 6-chloro function with a 2nitrophenoxy group to afford intermediate 86 followed by hydrolysis of the 2nitrophenoxy moiety to give the guanine compound 88.
Compound 84 was then
transformed into 86 upon treatment with 2-nitrophenol in the presence of DABCO and
triethylamine.
Following work-up and purification by flash column chromatography, 86
was obtained in a 70% yield. Subsequently, the 2-nitrophenoxy function of 86 was
cleaved using, 1,1,3,3-tetramethylguanidine and o-nitrobenzaldoxime.
Unfortunately,
upon work-up and purification of the crude reaction residue afforded by flash column
chromatography, none of the desired product 88 could be isolated. It was reasoned that
this may be due to the fact that the desired product had been lost at the purification stage
as guanine compounds are polar and insoluble in organic solvents.
Based on this observed problem, it was decided to investigate other methods for purifying
the crude residue which avoided the use of column chromatography. The best result was
73
achieved by partitioning in different organic solvents. The crude residue was mixed with
water to afford slurry and then this was washed sequentially with petroleum ether,
followed by diethyl ether and finally ethyl acetate. Subsequently, evaporation of the
ethyl acetate phase gave the desired product 88 in a 20% yield. The main difficulty
encountered with this reaction was separation of the desired product from the crude
reaction mixture as it contained a large amount of unreacted o-nitrobenzaldoxime and
1,1,3,3-tetramethylguanidine starting materials together with their side products.
In an attempt to optimize this reaction, it was decided to explore the effect of using a
smaller number of equivalents of, o-nitrobenzaldoxime and 1,1,3,3-tetramethylguanidine
on the yield of 88. The reaction was repeated with 3 equivalents of both reagents.
However, unfortunately, no significant improvement was obtained and the purification of
88 remained problematic. Therefore, this approach was abandoned and an alternative
route to the synthesis compound 90 was sought.
2.2.1 Alternative Routes Investigated Towards the Synthesis of Boc-Protected N9Guanylacetic Acid
This method was investigated based on the established literature approach reported by
Linn et al.157 The approach was based on substituting the 6-chloro group of the purine
ring with a DABCO moiety, followed by the alkaline hydrolysis of the DABCO moiety
to give the 6-oxo functionality of guanine [Scheme 11].
74
N
N
N
O
O
N+
Cl
i
O
N
O
N
N
O
O
N
N
O
ii
O
O
N
O
N
N
OCH2CH3
N
O
O
O
O
92
Scheme 11
Reagents and conditions: (i) DABCO, DMF, RT, 48 h; (ii) 1 M (aq) NaOH
Therefore, 84 was treated with DABCO at room temperature in anhydrous DMF, for 48
h. Subsequently, after the work-up, and purification, the desired product 92 was afforded
in 60% yield. The 1H NMR spectra of 92 gave a singlet at δ 1.45, which corresponded to
eighteen protons of the Boc-group attached to the N2-amino group. The multiplets at δ
2.51 corresponded to 12 protons of the DABCO group. With 92 in hand, its hydrolysis
using 1 M (aq) NaOH was next investigated. Two reaction conditions were studied. The
first involved treatment of 92 with 1 M (aq) NaOH at 50 °C for 1 h and the second
method involved leaving the reaction mixture at room temperature overnight.
Unfortunately, neither of these two methods gave 90.
All attempts to synthesise a N2-diBoc-guan-9-ylacetic acid discussed in section 2.2 and
2.2.1 were unsuccessful. The main problems identified from the various approaches tried
were: (i) the inability to separate compound 88 [Scheme 10] from the column, and (ii) the
lability of the di-Boc functionality under the basic conditions required to hydrolyse the
ethyl ester and DABCO [Scheme 11]. Therefore, another pathway to N2-Boc-protected
guan-9-ylacetic acid was sought.
75
N
N
O
O
OH
OCH2CH3
84
N
HN
90
2.2.2 Synthesis of N2-Boc-Guan-9-ylacetic Acid
The synthetic strategy investigated for the preparation of the desired mono-Boc-protected
guanine derivative 97 is shown in Scheme 12.
Cl
Cl
Cl
N
N
H2N
O
i
O
N
H
N
N
N
O
O
N
ii
N
O
N
O
O
O
58
O
N
N
N
N
H
N
O
73
74
iii
N
N
Cl
N
O
O
N
N
N
H
O
v
O
N
N
N
N
N
O
95
O
O
N
N
N
N
H
O
97
N
OH
Scheme 12
Reagents and conditions: (i) Boc2O, DMAP, DMF, RT, overnight; (ii) NaHCO3,CH3OH,
50 °C for 1 h then for RT overnight; (iii) CH3CH2OH, NaOH, RT, 70 h; (iv)
BrCH2COOEt, DMF, K2CO3, RT, 5 h; (v)1,2,4-triazole, DMF, K2CO3, 50 °C, 5 h;
(vi) 1M(aq) NaOH, dioxane, 50 °C,1 h.
76
N
H
OCH2CH3
OCH2CH3
HN
N
H
vi
O
N
N
iv
O
H
O
96
Cl
N
N
O
75
The procedures for the first three steps (step i, ii and iii) are identical to those reported by
Dey and Garner,150 which have been discussed earlier in Scheme 7. These reactions gave
73, 74 and 75 in 97%, 85% and 75% yield, respectively.
Subsequently, 75 was
alkylated, by treating it with ethyl bromoacetate and K2CO3 in DMF. After work-up and
purification by flash column chromatography, 95 was obtained as a glassy white solid in
80% yield. The 1H NMR spectrum showed a singlet at δ 1.43 which corresponded to the
presence of the nine protons of the tert-butoxycarbonyl group at the N2-position, and a
triplet at δ 1.2 with a coupling constant of 7.2 Hz corresponding to the three protons of
the methyl group of the ethyl ester. The quartet at δ 4.12 corresponded to the two protons
of the methylene group of the ethyl ester and the singlet δ 8.01 corresponded to the C8-H
of the purine ring.
The next step in the pathway involved the conversion of the 6-chloro substituent in 95
into a moiety which upon cleavage would afford the desired 6-oxo group of guanine.
This time it was decided to explore the use of 1,2,4-triazole for this purpose, since our
attempt to use 2-nitrophenoxy or DABCO groups for the di-Boc compound [Schemes 10
and 11] had not given any of the desired product. Therefore, 95 was converted into 96 by
treating 95 with 1,2,4-triazole in DMF in the presence of K2CO3 at 50 °C for 7 h. After
work-up and purification by flash column chromatography, 96 was afforded as a white
foam in a 60% yield. The 1H NMR spectrum of 96 showed a singlet at δ 8.32 which
corresponded to the two protons at C3 and C5 of the triazole moiety. Both of them
appear at the same δ value because they are in the same environment.
The final step in this pathway involved the conversion of 96 into 97.
This was
successfully achieved by treating a solution of compound 96 in dioxane with 1 M (aq)
solution of sodium hydroxide at 50 °C. After work-up, 97 was afforded in a 55% yield.
The disappearance of the two proton singlet at δ 8.32 confirmed that the triazole group
had been hydrolysed; also the disappearance of the ethyl ester protons at δ 1.29 and 4.22
confirmed that the ester had been hydrolysed to the acid. The observed 13C NMR peak at
δ 176.9 also corresponds to the acidic carbonyl functionality. The HRMS (ES, H+)
recorded an m/z 310.2915 which also confirmed the synthesis of compound 97.
77
2.2.3 Optimization of the Route to the Synthesis of Compound 95
The selective cleavage of the di-Boc to mono-Boc derivative in the Dey and Garner
approach [Scheme 7]150 had been achieved over a long period of time (70 h). Therefore
it was decided to investigate alternative conditions for selectively converting a di-Boc
compound into a mono-Boc compound [Scheme 13].
Cl
O
O
O
N
N
N
Cl
O
N
N
O
84
N
OCH2CH3
N
N
H
O
O
N
N
O
95
OCH2CH3
Scheme 13
Reagents and conditions: LiCl, ACN, 65 °C, 18 h.
Scheme 14 was based on selective cleavage of a di-Boc compound using LiCl. This
approach was reported by Hernandez et al.158 who found that the selective cleavage of a
di-Boc compound with LiCl took place within 18 h. Therefore, 84 was treated with LiCl
at 65 °C for 18 h. After work-up, 95 was obtained in 60% yield. The success of this
approach reduced drastically the time taken to synthesise 95.
2.3
Alternative Route Independent of Dey and Garner’s Approach
The alternative route was based on the work reported by Braverman et al.159 and Toshio
et al.160 [Scheme 14]. In their approaches, it was stated that an amino group could be
protected with a carbamate functionality by first generating an isocyanate in situ which
was then easily captured by variety of alcohols.
78
O
H
R N C CX3
98
i
R
N C O + CHX3
ii
O
H
R N C OR'
100
99
R = R' = Alkyl group
Scheme 14
Reagents and conditions: (i) Base, DMF; (ii) ROH.
The approaches of Braverman et al.159 and Toshio et al.160 were similar because their first
step involved the conversion of the amino group into trihaloacetamide 98 [Scheme 14].
This was followed by the conversion of the trihaloacetamide into isocyanate 99 in situ.
Finally, a variety of alcohols were added to the isocyanate generated to obtain the desired
carbamates 100. The slight differences in the two approaches were: (i) in the type of base
used in step i [Scheme 14]. Braverman et al.159 used DBU as a base while Toshio et
al.160 used Na2CO3; (ii) Braverman et al.159 used neat alcohol while Toshio et al.160 added
the alcohol in the presence of CuCl and n-Bu4NCl. Toshio et al.160 claimed that the
addition of alcohol in the absence of n-Bu4NCl gave a lower yield of the carbamate.
Furthermore, for the generation of isocyanate, we investigated the approach of Eckert et
al.161 [Scheme 15].
This approach involved the use of triphosgene to generate the
isocyanate in situ.
In their investigation, 101 was treated with triphosgene in 1,2-
dichlorobenzene under nitrogen at 130 °C for 2 h. After work-up, compound 102 was
obtained in 92% yield. They also found out that this type of reaction could be achieved at
a faster rate if the reaction was conducted at a higher temperature (180 °C).
79
O
C
N
H2N
N
NH2
C
O
101
102
Scheme 15
Reagents and conditions: Triphosgene, 1,2-dichlorobenzene, 130 °C, 2 h.
Based on the established literature approaches presented in Schemes 14 and 15, a new
route [Scheme 16] to N2-Boc-protected guan-9-ylacetic acid was designed.
80
H2N
ii
i
N
H2N
N
H
N
N
O
N
N
N
N
Cl
Cl
Cl
O
N
N
N
H
O
O
58
93
N
N
OCH2CH3
OCH2C
H3
95
iii
N
N
O
O
O
N
N
HN
N
H
O
iv
N
N
N
N
O
N
H
O
TM
N
N
O
OH
97
OCH2
CH3
96
Scheme 16
Reagents and conditions: (i) BrCH2COOEt, DMF, K2CO3, RT, 5 h; (ii)(a) triphosgene,
130 °C; (b) tert-butyl alcohol, 70 °C, RT, overnight; (iii) 1,2,4-triazole, DMF, K2CO3, 50
°C, 7 h; (iv) 1 M(aq) NaOH , dioxane, 50 °C, 1 h.
Scheme 16 involved the treatment of compound 58 in DMF with bromoacetate, K2CO3,
at room temperature for 5 h. After work-up, compound 93 was obtained in 75% yield
[Scheme 16].
Subsequently, the exocyclic amino group was activated by treating 93
with triphosgene in a mixture of anhydrous 1,2-dichlorobenzene and dichloromethane at
130 °C for 3 h. The temperature of the reaction mixture was then reduced to 70 °C and
tert-butanol was added. The reaction temperature had to be reduced to 70 °C because at a
high temperature of around 120 °C, the Boc group can be readily cleaved. This reaction
was successful and 95 was obtained in 75% yield. The 1H NMR and
13
C NMR spectra
corresponded to those already reported for 95 on page 78. The success of this reaction
enabled the synthetic approach to 97 to be modified as shown in Scheme 16. Steps iii
81
and iv in Scheme 16 followed the same procedure as for step v and vi already discussed
in Scheme 12 on page 76.
Finally, a novel synthetic approach to mono-Boc-N2-guan-9-ylacetic acid [Scheme 16]
has been successfully designed. This approach is plausible compared with Scheme 12
based on the following observations: (i) the total number of steps in the synthesis of
mono-Boc-N2-protectedguan-9-ylacetic acid 97 has been reduced from six to four; (ii)
the time taken to complete the whole route was greatly reduced, i.e one step in Scheme
12 required 3 days for completion whereas the entire pathway outlined in Scheme 16
could be accomplished within 3 days; (iii) Scheme 16 produced mono-Boc-N2-protected
guan-9-ylacetic acid 97 in a high yield; and (iv) Scheme 16 was reproducible.
2.4
Synthesis of N2-Benzyloxycarbonyl Guan-9-ylacetic Acid
Following the success in developing a viable synthetic route for the preparation of N2tert-butoxycarbonylguan-9-ylacetic acid 97 from 2-amino-6-chloropurine 58 [Scheme
16], attention was shifted to the synthesis of the corresponding N2-benzyloxycarbonyl
analogue.
The initial routes proposed were based on the approach presented in Scheme 10 (page
71). The first step involved treatment of 2-amino-6-chloropurine 58 in anhydrous DMF
with benzyl dicarbonate in the presence of a catalytic amount of DMAP.
this reaction did not work.
Surprisingly,
Therefore, a variety of alternative reagents and conditions
were tried. These included: (i) treatment of compound 58 with benzyl chloroformate in
the presence of different amounts of DMAP (this method was based on an established
literature procedure)162; (ii) treatment of compound 58 with excess benzyl dicarbonate in
the presence of DMAP; (iii) treatment of compound 58 with Rapoport’s reagent;163and
(iv) treatment of compound 58 with benzyl chloroformate in the presence of zinc at 50
°C.
82
However, it was observed that when excess dibenzyl dicarbonate or benzyl chloroformate
[Scheme 17] were used, a very distinct single product 103 was obtained in yields ranging
from 55-64%. The spectral analysis of 103 by 1H NMR showed a singlet at δ 5.49, which
corresponded to the two protons of the exocyclic amino group, a singlet at δ 5.71 which
corresponded to two protons of the benzyl methylene group and a singlet at δ 8.01 which
corresponded to C8-H of the purine ring (which agreed with the data reported by Green et
al.155 for an N9-subtituted purine ring). There was also a multiplet at δ 7.32-7.61 which
corresponded to the five aromatic protons of the benzyloxycarbonyl group. Finally, Xray crystallography revealed that the benzyloxycarbonyl group was actually attached to
the N9-position of the purine ring [Figure 42].
Cl
Cl
N
N
H2N
N
N
N
H2N
N
H
N
N
O
O
58
103
Scheme 17
Reagents and conditions: (i) DMAP, Cbz2O, DMF, RT, 18 h; or (ii) CbzCl, DMAP,
DMF, RT, 18 h.
83
Figure 42: X-ray crystallographic structure of 103
This X-ray structure of 103 revealed a striking feature in the behaviour of hydrogen
bonding which existed within the crystal. It was found that 103 crystallized as two
crystallographically independent molecules (A and B) [Figure 42].
The hydrogen
bonding links between molecules A and B resulted in a special arrangement which led to
the formation of two six-membered rings.
2.4.1
Synthesis of N2-Cbz-Protected Guan-9-ylacetic Acid via an Isocyanate
Intermediate
Due to the inability to successfully protect the exocyclic amino group of 2-amino-6chloro purine 58 with the benzyloxycarbonyl group, the route outlined in Scheme 10 was
abandoned.
It was then decided to explore the method reported by Braverman et al.159
and Toshio et al.160 [Scheme 14].
In order to achieve this, the reactions shown in
Scheme 18 were investigated.
84
Cl
Cl
Cl
N
N
N
N
i
H 2N
H2N
N
Cl
ii
N
N
N
H
Cl
Cl
O
N
N
N
H
O
O
58
OCH2CH3
93
N
N
OCH2CH3
104
iii
iv
Cl
Cl
Br
Br
Br
O
N
N
O
v
N
H
N
N
N
N
O
O
N
H
N
N
O
OCH2CH3
OCH2CH3
106
105
Scheme 18
Reagents and conditions: (i) BrCH2COOEt,DMF, K2CO3, RT, 5 h; (ii) trichloroacetic
anhydride, CH2Cl2, DMAP, RT, overnight; (iii) tribromoacetyl chloride, pyridine, RT,
overnight; (iv) (a) DBU, CH2Cl2, 80 °C, 8 h; (b) benzyl alcohol, RT, overnight; (v) (a)
DBU, CH2Cl2, RT, 4 h (b) benzyl alcohol, RT, overnight.
The first step in Scheme 18 involved treatment of 2-amino-6-chloropurine 58 in DMF
with ethyl bromoacetate and K2CO3 at room temperature for 5 h. Purification of the
crude product by column chromatography afforded 93 in 78% yield. Subsequently, the
exocyclic amino group of 93 was activated by treating 93 in DCM with trichloroacetic
anhydride, in the presence of catalytic amount of DMAP at room temperature overnight.
This afforded the desired N2-trichloroacetamide purine 104 in 45% yield.
Finally, 104
was converted into 105 by treatment with DBU at 80 °C for 8 h following an established
literature procedure.159,160 Subsequently, benzyl alcohol was added and the reaction
mixture left to stir at room temperature overnight.
After work-up, the desired N2-
benzyloxycarbonyl-protected purine compound 105 was obtained in 20% yield.
85
An alternative route to compound 105 was also investigated [Scheme 18]. This involved
the treatment of 93 with tribromoacetyl chloride. It was decided to explore this approach
because Braverman et al.159 reported that the reaction of an N2-tribromoacetyl compound
with DBU to form the corresponding isocyanate intermediate occurred more readily at
room temperature than with the trichloro derivative. Thus 93 was converted into the
tribromoacetyl purine analogue 106 [Scheme 18] by treating 93 with tribromoacetyl
chloride in the presence of anhydrous pyridine. After work-up and purification, 106 was
afforded in 75% yield. Finally, 106 in DCM was treated with DBU at room temperature
for 4 h, followed by the addition benzyl alcohol. The reaction mixture was left to stir at
room temperature overnight. However, after work-up and purification 105 was obtained
in the same yield 20%.
Since both the routes described above gave 105 in a disappointing 20% yield, another
reaction was investigated [Scheme 19]. The alternative approach was based on the use of
triphosgene to activate the exocyclic amino group of 93. Thus, a solution 93 in 1,2dichlorobenzene/dichloromethane [Scheme 19] was treated with triphosgene in the
presence of catalytic amount of DMAP, at 130 °C for 3 h. After 3 h, the temperature of
the reaction was reduced to 100 °C before triethylamine and excess benzyl alcohol were
added. The reaction mixture was left to stir at 100 °C for a further 5 h. After work-up
and purification, compound 105 was obtained in 60% yield. The 1H NMR spectra of 105
gave a singlet at δ 5.25 which corresponded to the two protons of the benzyl methylene
group, a multiplet at δ 7.25-7.42 which corresponded to the five aromatic protons of the
benzyloxycarbonyl group, a singlet at δ 8.02 corresponding to the carbamate NH. This
proton appears at lower field because of the possibility of hydrogen bonding between the
carbonyl functionality of the carbamate and the NH proton.
86
Cl
Cl
N
N
H2N
N
N
O
N
N
O
N
H
O
N
N
O
OCH2CH3
OCH2CH3
105
93
Scheme 19
Reagents and conditions: (i) Triphosgene, 1,2-dichlorobenzene, CH2Cl2, DMAP,
pyridine, 100 °C, 3 h; (ii) Et3N, benzyl alcohol, 100 °C, 5 h.
Therefore, a relatively reproducible approach to the synthesis of 105 [Scheme 19] had
been successfully developed.
The next step in the synthetic pathway involved
replacement of the 6-chloro group with a group which could be easily converted into the
required 6-oxo functionality of guanine [Scheme 20]. In order to achieve this, a solution
of compound 105 in dry DMF was treated with 1,2,4-triazole and K2CO3 at 50 °C for 7 h.
After work-up and purification, 107 was obtained as a glassy solid in 65% yield [Scheme
20].
N
Cl
N
N
N
O
N
O
O
N
H
N
N
N
N
O
N
H
O
N
N
O
OCH2CH3
105
107
Scheme 20
Reagents and conditions:1,2,4-Triazole, DMF, K2CO3, 50 °C, 7 h.
87
OCH2
CH3
The final step in the synthetic pathway involved the conversion of 107 into N2benzyloxycarbonylguan-9-ylacetic acid 108 [Scheme 21]. The optimum conditions for
accomplishing this conversion were found to be the treatment of 107 in dioxane with a
solution of 1 M(aq) NaOH, at 50 °C for 1 h. After work-up, 108 was obtained in 55%
yield. The 1H NMR spectrum of 108 showed that the singlet peak at δ 8.32, which had
been attributed to the two triazole protons in the 1H NMR spectrum recorded for 107, had
disappeared as well as the peaks associated with the ethyl ester protons. The
13
C NMR
spectra showed a signal δ 170.0, which corresponded to the carbonyl carbon of the
carboxylic acid.
Finally, the accurate mass spectral analysis found a peak at m/z
344.0994 which further confirmed the synthesis of 108.
N
N
O
N
O
N
N
O
N
N
N
H
O
O
H
N
O
OH
OCH2CH3
107
N
N
H
O
N
N
108
Scheme 21
Reagents and conditions: 1M (aq) NaOH:dioxane (1:1), 50 °C, 1 h.
Therefore, by combining Schemes 19, 20 and 21, the overall reproducible route for the
synthesis N2-benzyloxycarbonylguan-9-ylacetic acid 108 from 2-amino-6-chloropurine
58 in high yield is summarised in Scheme 22 below.
88
Cl
Cl
Cl
N
N
H2N
N
i
H2N
N
H
N
N
ii
O
N
H
93
N
N
O
O
58
N
N
O
N
N
OCH2CH3
105
OCH2CH3
iii
N
O
O
O
N
N
N
N
HN
H
O
O
N
H
OH
108
N
N
O
iv
N
N
107
N
N
O
OCH2CH3
Scheme 22
Reagents and conditions: (i) BrCH2CO2Et, DMF, K2CO3, RT, 5 h; (ii) (a) triphosgene,
1,2-dichlorobenzene, CH2Cl2, DMAP, pyridine, 100 °C, 3 h; (b) Et3N, benzyl alcohol,
100 °C, 5 h; (iii) 1,2,4-triazole, DMF, K2CO3, 50 °C, 7 h; (iv) 1 M(aq) NaOH:dioxane
(1:1), 50 °C, 1 h.
2.4.2 Effect of Ring Deactivation on the Rate of Reaction of Benzyl Alcohol with
an Isocyanate
It was decided to investigate the effect of ring deactivation on the rate of reaction of an
alcohol with an isocyanate [Scheme 23]. Therefore, 93 was first treated with triphosgene
in 1,2-dichlorbenzene at 130 °C for 3 h. Subsequently, p-nitrobenzyl alcohol was added
at 90 °C and the mixture was left to stir at this temperature for a further 15 min. After
work-up, 109 was obtained in 78% yield.
89
Cl
Cl
N
N
H2N
O
N
N
i,ii
O
N
N
H
O2N
O
N
N
N
O
OCH2CH3
OCH2CH3
93
109
Scheme 23
Reagents and conditions: (i) triphosgene,1,2-dichlorobenzene, 90 °C, 3 h; (ii) pnitrobenzyl alcohol, 90 °C, 15 min.
Scheme 23 showed that the presence of an electron-withdrawing group on the phenyl ring
of the alcohol deactivated the phenyl ring. Thus, the acidity of the phenoxy proton was
increased which led to an increase in the reactivity of the benzyl alcohol with the
isocyanate generated in situ.
2.4.3
Effect of Increasing Lipophilicity of the Ester at the N9-Position on the
Purification and Yield of the desired N2-Benzyloxycarbonyl Protected Guan9-ylacetic Acid.
The major focus of this research work was to design a high yielding (greater than or equal
to 50%) route to the synthesis of Cbz-protected guan-9-ylacetic acid. Therefore, it was
reasoned that it may be possible to improve the yield of of N2-Cbz guan-9-ylacetic acid
108 by increasing the lipophilicity of the group attached at the N9-position of the purine
ring. The basis for this assumption was that this strategy may enhance the solubility of
the purine derivatives in organic solvents. This would allow for easier purification of
both the intermediates and final product by flash column chromatography.
In order to
achieve this aim, the ethyl ester group at the N9-position of the purine ring was replaced
with a tert-butyl ester [Scheme 24].
90
Cl
Cl
N
N
H2N
N
N
Cl
i
H2N
N
H
N
N
N
N
HN
ii
N
N
N
O
O
O
O
58
110
O
111
O
iii
NO2
O
O
HN
O
O
iv
v
HN
N
N
N
HN
N
HN
O
O
N
O
O
N
N
N
O
HN
N
N
O
OH
O
O
O
O
108
113
112
Scheme 24
Reagents and conditions: (i) BrCH2COOC(CH3)3, K2CO3, DMF, RT, 5 h; (ii) (a)
triphosgene, THF,
reflux, 3 h; (b) benzyl alcohol, reflux, 3 h: (iii) o-nitrophenol,
DABCO,
ACN,
Et3N,
RT,
overnight;
(iv)
o-nitrobenzaldoxime,
1,1,3,3
tetramethylguanidine, ACN, RT overnight ; (v) TFA, DCM, RT , 3 h.
The first step in the optimization route [Scheme 24] involved the treatment of
commercially-available 2-amino-6-chloropurine 58 in dry DMF with K2CO3 and tertbutyl bromoacetate at room temperature for 5 h. After work-up and purification, 110 was
obtained in 75% yield. The 1H NMR spectrum showed a singlet at δ 1.41 which
corresponded to the nine protons of the tert-butyl ester group while the singlet at δ 5.38
corresponded to the two protons of the exocyclic 2-amino group. The 1H NMR and 13C
91
NMR spectra again agreed with the data reported by Green et al.155 for N9-substituted 2aminopurine.
Subsequently, 110 in dry THF was treated with triphosgene in the presence of
triethylamine at reflux for 3 h. After addition of benzyl alcohol, the resulting mixture
was allowed to stir at reflux for a further 3 h.
Solvent evaporation followed by
purification afforded 111 in 80% yield. This yield is an improvement over that which
had been obtained for the ethyl ester analogue 105 reported earlier on page 87.
The next step was the conversion of the 6-chloro group of 111 into a functionality which
could be easily converted into the 6-oxo group of guanine.
This was achieved by
treating a solution of 111 in dry acetonitrile with o-nitrophenol in the presence of
DABCO and Et3N, at room temperature, according to the established literature
procedure.147 After work-up and purification by flash chromatography, 112 was obtained
in 90% yield.
The displacement of the 6-nitrophenoxy moiety of 112 to give the desired 6-oxo guanine
compound was achieved as described in the literature.147 This involved treating 112 with
o-nitrobenzaldoxime and 1,1,3,3-tetramethyl guanidine in anhydrous acetonitrile. After
work-up and purification by column chromatography, 113 was obtained in 75% yield.
The success of this reaction, confirmed the theory that, by increasing the lipophilicity of
the substituent at the N9-position it might be possible to successfully separate the product
of the hydrolysis of 6-nitrophenoxy moiety by flash column chromatography.
Finally, the last step was the hydrolysis of the tert-butyl group at the N9- position to give
the desired N2-Cbz-guan-9-ylacetic acid 108. This was achieved by treating 113 in
dichloromethane with TFA at room temperature for 3 h.
After work-up, 108 was
obtained as a pure white solid in 90% yield.
In conclusion, Scheme 24 is a novel reaction route to N2-Cbz-guan-9-yl acetic acid 108
which is reproducible, fast and high yielding (36.5% cf 16.7% from Scheme 22).
92
2.5
Synthesis of PNA Backbone
Following the successful synthesis of the desired Cbz-and Boc-protected guan-9-ylacetic
acids; the next step was to synthesise a suitable PNA backbone that would be coupled to
the guanine nucleobase to generate the required guanine-PNA monomers. Two different
types of PNA backbones were synthesised; these were Fmoc-PNA backbone and BocPNA backbone. The Fmoc-PNA backbone was coupled with the N2-Boc-protected guan9-ylacetic acid to form a guanine-PNA monomer, because Boc- and Fmoc- are
orthogonal. Also, the Boc-PNA backbone was coupled with the N2-Cbz protected guan9-ylacetic acid to form a guanine-PNA monomer.
2.5.1
Synthesis of Fmoc-PNA Backbone
The peptide molecule of the PNA backbone had to be protected at both the amino end
and the carboxyl end. This would allow gradual and systematic coupling during the
oligomerization process when using either a solid phase or solution phase approach.
Therefore, Scheme 25 was designed for the synthesis of N-Fmoc-protected PNA
backbone. The choice of the allyl group for the protection of the carboxyl function was
based on the fact that it should be possible to cleave this group when needed without
affecting the Fmoc protecting group.
93
H
NH2
H2N
i
O
N
ii
N
H2N
O
H
O
OH
O
OH
N
H
116
115
114
iii
O
H
O
N
O
OCH2CH=CH2
N
H
117
Scheme 25
Reagents
and
conditions:
(i)
ClCH2COOH,
RT,
overnight;
(ii)
Fmoc
succinimidylcarbonate, 10% Na2CO3, dioxane, RT, 4 h; (iii) (a) dicyclohexylamine,
ethanol (b) ally bromide, dioxane, RT, overnight.
The first step in the synthesis of Fmoc PNA backbone involved the treatment of 1,2diaminoethane 114 with chloroacetic acid (this was based on the method employed by
Murray.164 After work-up, 115 was afforded in 80% yield. The next step involved the
introduction of Fmoc group to protect the free amino end of 115. This was achieved by
treating 115 in dioxane with fluorenylmethyl succinimidyl carbonate in the presence of
Na2CO3 at room temperature for 4 h. After work-up, 116 was obtained in 63% yield.
Finally, the free carboxylic group of 116 was protected with an allyl group.
The
protection of the carboxylic group was carried out over a two step reaction. First, the
carboxylic acid functionality of 116 was activated with dicyclohexylamine, this involved
the treatment of 116 with dicyclohexylamine, at room temperature. The reaction was
94
monitored until the mixture became basic. Subsequently, diethyl ether was added to the
basic solution.
precipitate salt.
The addition of diethyl ether resulted in the formation of a white
This was the dicyclohexylammonium salt of 116 and this was
subsequently dissolved in dioxane and treated with allyl bromide at room temperature
overnight. After work-up and purification, 117 was obtained in 75% yield.
2.5.2
Synthesis of Boc-PNA Backbone
Two schemes were investigated for the synthesis of the Boc-PNA peptide backbone. The
first scheme was based on an established literature procedure [Scheme 26].128 This
involved the treatment of 3-aminopropane-1,2-diol 118 in water with Boc2O. The pH of
the mixture was adjusted to 10.5, before being left for 4 h. Subsequently, the solution
was extracted into dichloromethane and the dichloromethane extract was evaporated to
give 119 in 94% yield. The next reaction in the scheme involved the oxidation of 119.
The oxidation was achieved by treating 119 in water with NaIO4, at room temperature for
1 h.
After work-up, aldehyde 120 was obtained in 85% yield.
Finally, 120 was
converted into the PNA backbone by reductive amination. This reaction was carried out
by treating 120 with NaCNBH3, methanol and glycine methyl ester hydrochloride. After
work-up, 121 was obtained in 59% yield. When glycine ethyl ester hydrochloride was
used in place of glycine methyl ester hydrochloride, 122 was obtained in 60% yield.
95
O
OH
H2N
i
OH
118
O
OH
O
N
H
ii
O
O
N
H
OH
H
120
119
iii
O
O
N
H
H
N
O
121 R = OMe
122 R = OEt
Scheme 26
Reagents and conditions: (i) Boc2O, water, 2 M(aq) NaOH, pH 10.5, RT; (ii) NaIO4,
water, RT, 1 h; (iii) NaCNBH3, CH3OH, glycine methyl ester.HCl or glycine ethyl
ester.HCl, RT, overnight.
The second scheme investigated [Scheme 27] was designed for the synthesis of 122. This
approach was also based on an established literature procedure.128 This was investigated
in order to reduce the number of steps required to synthesise the target Boc-PNA peptide
backbone 122 because the fewer the steps of a reaction, the better the overall yield of
guanine PNA monomer. This approach involved first the treatment of 1,2-diaminoethane
114 with excess di-tert-butoxydicarbonate at 0 °C, for 4 h. After work-up, 123 was
obtained in 60% yield. Finally, 123 was alkylated by treating 123 in dichloromethane
with triethylamine, ethyl bromoacetate and NaI at room temperature overnight. After
work-up, 122 was obtained in 65% yield.
96
R
O
NH2
H2N
i
O
N
H
NH2
ii
122
123
114
Scheme 27
Reagents and conditions: (i) Boc2O, 0 °C; (ii) Et3N, BrCH2COOCH2CH3, NaI, RT,
overnight.
2.6
Synthesis of Guanine-PNA Monomers
The chemistry part of this research work was to synthesise guanine-PNA monomers in a
high yield. The guanine-PNA monomers would then be used in the synthesis of guaninerich PNA oligomers, using either a solid or solution phase synthetic strategy. The
synthesis of guanine PNA monomers is presented in the following subsections.
2.6.1
Synthesis of Guanine-PNA Monomers for Fmoc Solid Phase or Solution
Phase Strategy
This approach involved the treatment of solution of 97 in dry DMF with coupling agent
HBTU and DIPEA.
Subsequently, the Fmoc-PNA backbone 117 was added and the
reaction mixture was left to stir at room temperature overnight. The completion of the
reaction was judged by using TLC analysis. After work-up, the desired guanine-PNA
monomer 48 was obtained in 50% yield [Scheme 28]. The 1H NMR spectroscopic
analysis of the compound showed a singlet at δ 1.4, which corresponded to the nine
protons of the N2- tert-butoxycarbonyl group attached to the guanyl nucleobase, and a
multiplet at δ 7.2-7.6 corresponding to the eight protons of the two aromatic rings of the
Fmoc group. A singlet at δ 7.8 was also observed which corresponded to the purine C8H and the multiplet peak between δ 3.2-3.5 corresponded to the four ethylene protons of
the PNA backbone.
97
O
O
O
N
HN
N
H
+
N
N
H
O
N
O
N
O
OH
O
OCH2CH=CH2
H
117
97
O
O
O
H
N
H
N
N
O
O
O
N
N
OCH2CH=CH2
N
N
H
O
48
Scheme 28
Reagents and conditions: HBTU, DIPEA, DMF, RT, overnight.
2.6.2
Synthesis of Guanine-PNA Monomer for Boc Solid Phase or Solution Phase
Strategy.
The synthesis of a guanine-PNA monomer suitable for use in a Boc-solid phase strategy
is shown in Scheme 29. This involved the treatment of 108 in DMF with HBTU, DIPEA
and 121. The mixture was left to stir at room temperature overnight. After work-up, 49
was obtained in 55% yield. The 1H NMR spectral analysis of 49 showed a singlet peak
at δ 1.40 which corresponded to the nine proton of the Boc-group, and a multiplet at δ
3.21-3.45 corresponding to the four ethylene protons of the PNA backbone.
Furthermore, the multiplet peak at δ 7.29-7.52 corresponded to the five aromatic protons
of the benzyloxycarbonyl group and the singlet at δ 7.91 corresponded to C8-H of the
98
purine ring. In order to prevent loss of material, compound 49 was not purified further
using column chromatography. It was believed that the work-up in the next stage of the
pathway would definitely remove any impurities that may have been left.
O
O
O
O
H
N
N
H
H
N
N
O
O
OCH3
+
N
H
108
121
N
N
O
OH
O
O
O
H
N
H
O
O
N
N
N
H
N
N
O
OCH3
N
O
49
Scheme 29
Reagents and conditions: HBTU, DIPEA, DMF, RT, overnight.
An analogue of 49, compound 125, was also synthesised [Scheme 30]. This approach
involved the hydrolysis of the ester functionality at the N9-position of 112. Therefore,
112 in dichloromethane was treated with TFA at room temperature for 3 h. After workup, 124 was obtained in 90% yield. Finally, the coupling of 124 with the PNA backbone
was achieved by treating 124 in DMF with HBTU, DIPEA and 121. After work-up and
purification by flash column chromatography, the PNA monomer 125 was obtained in
70% yield.
99
O2N
O2N
O
O
112
i
N
HN
O
N
N
ii
O
N
N
O
O
N
N
H
O
O
O
OH
O
124
N
N
N
H
O
N
125
Scheme 30
Reagents and conditions: (i) TFA, DCM, RT, 3 h; (ii) 121, HBTU, DIPEA, DMF, RT,
overnight.
2.7
Solution Phase Synthesis of Guanine-PNA Dimer
In order to achieve the solution phase synthesis of guanine-PNA dimers using the Bocstrategy, the sample of compound 49 was divided into two portions. To the first portion
was added TFA in dichloromethane. This reaction was carried out in order to cleave the
Boc group so that the amino end of the PNA backbone would be free for amide coupling.
This reaction afforded compound 127 in 85% yield [Scheme 31].
The second portion
was treated with LiOH in THF to give acid 126 in 82% yield [Scheme 31].
100
OCH3
O
O
i
O
N
HN
N
H
O
O
O
N
N
O
N
N
H
OH
126
49
O
O
O
ii
N
HN
N
H
N
N
O
O
N
H2N
OCH3
127
Scheme 31
Reagents and conditions: (i) LiOH, THF, RT, 3 h; (ii) TFA, DCM, RT, 3 h.
The next step in the scheme was to couple the two deprotected guanine-PNA monomers
126 and 127 together to form a guanine-PNA dimer. The dimerization was carried out in
DMF using HOBT and DCC as the coupling agent. Unfortunately, upon purification by
flash column chromatography, the desired product 128 [Figure 43] was not recovered. It
was reasoned that the dimer had been lost upon purification due to its high polarity.
Therefore, it was decided to investigate purification of the crude product afforded from
this reaction by trituration several times with diethyl ether. This approach was successful
but dimer 128 was obtained in a disappointing yield of 20%.
101
O
O
HN
CbzHN
N
N
N
N
O
O
H3CO
N
NHCbz
O
O
N
NH
N
N
H
O
N
H
O
128
Where Cbz = -CO2CH2Ph
Figure 43: Guanine PNA dimer
In order to try and improve the yield of dimer 128, it was decided to investigate the
coupling of the o-nitrophenoxylguanine-PNA monomer derivative 125 whose synthesis
has been described previously on page 100. It was reasoned that the resulting dimer
could be more amenable to purification by flash column chromatography due to the
presence of O6-nitrophenoxyl moiety which may increase the solubility of the guanine
compound in organic solvent. Therefore, the reactions outlined in Scheme 32 were
investigated. Again the sample of compound 125 was divided into two portions. The
first portion was treated with TFA in dichloromethane and, upon work-up and
purification, 129 was obtained in 80% yield. The other portion of 125 was treated with
LiOH in THF. After work-up, 130 was obtained in 67% yield [Scheme 32].
Finally,
compounds 129 and 130 were coupled in DMF with DCC in the presence of HOBT at
room temperature overnight. After work-up and purification by column chromatography,
the desired product 131 [Figure 44] was obtained in 80%. This result showed that the
presence of the o-nitrophenoxyl group at the 6-position of the purine ring reduced the
polarity of the compound, making it more soluble in organic solvents and allowing for
easier purification.
102
NO2
O
O
i
O
N
N
N
H
N
N
O
O
N
H2N
125
OCH3
129
NO2
O
ii
O
O
N
H
N
N
O
O
O
N
N
O
N
N
H
OH
130
Scheme 32
Reagents and conditions: (i) TFA, DCM, RT, 3 h; (ii) LiOH, THF, RT, 3 h.
103
O2N
O2N
O
O
N
CbzHN
N
N
N
N
N
O
O
H3CO
N
N
H
N
NHCbz
O
O
N
N
O
N
H
O
131
Where Cbz = -CO2CH2Ph
Figure 44: O6-nitrophenoxyl guanine-PNA dimer
However, the subsequent removal of the O6-protecting group from 131 using onitrobenzaldoxime and 1,1,3,3-tetramethylguanidine in acetonitrile only gave the dimer
128 in a disappointing yield of 22%, as it too required purification by column
chromatography. Unfortunately no other route for successfully purifying dimer 128 could
be identified.
2.8
Synthesis of Thymine-PNA Monomer
It was decided to synthesise the thymine-PNA monomer as it was hoped that, if time
permitted, it may be possible to investigate synthesis of guanine-rich PNA oligomers
using solid phase synthesis. As described earlier in the introduction, it was reasoned that
a 16-mer PNA oligomer containing 12 guanine nucleobases and 4 thymine nucleobases,
could be a potential HIV-1 integrase inhibitor based on a literature report.114 Therefore,
Scheme 33 was investigated in order to synthesise a thymine-PNA monomer. This
scheme was based on an established procedure.164 The first step in Scheme 33 involved
alkylation of the thymine molecule 132 at the N4-position. Two methods to achieve this
were investigated.
The first involved treatment of 132 with aqueous KOH and
bromoacetic acid. After work-up and purification, 133 was afforded in 65% yield. The
104
second method involved alkylation of 132 at the N4-position using tert-butyl
bromoacetate at 82 °C for 22 h. After work-up, the crude product was hydrolysed using
TFA in dichloromethane at room temperature for 3 h. After work-up, 133 was afforded
in 97% yield.
Subsequently, 133 was coupled to the Boc-PNA backbone 122 in
anhydrous DMF using DIPEA and HBTU as the coupling agent, at room temperature
overnight.
After work-up and purification by flash column chromatography, the desired
thymine-PNA monomer 134 was obtained in 55% yield. Finally, 134 in dioxane was
treated with LiOH at room temperature for 4 h. After work-up, 135 was afforded as a
white foam in 70% yield.
Scheme 33
Reagents and conditions: (i) KOH, H2O, BrCH2COOH, 40 °C, pH 5.5; (ii) (a) K2CO3,
BrCH2COOC(CH3)3, DMF, 82 °C, 22 h; (b) TFA, DCM, RT, 3 h; (iii) DIPEA, DMF,
HBTU, RT, overnight; (iv) LiOH, dioxane, RT, 4 h.
105
2.9
Solid Phase Synthesis of Guanine-rich PNA Oligomers: H-TGGG-Lys-NH2
An attempt was made to construct a oligomer of sequence H-[TGGG]4-Lys-NH2 The
reason for selecting this particular sequence was due to the fact that it has been found that
the analogous 16-mer guanine-rich oligonucleotides, containing guanine and thymine
nucleobases, had the ability to inhibit HIV-1 integrase.120 It was therefore decided that it
would be interesting to compare these findings with the PNA analogue. It was reasoned
that a lysine residue would need to be incorporated at the C-terminus in order to increase
solubility of the resulting oligomer in aqueous media. As a model for the synthesis of H[TGGG]4-Lys-NH2, it was decided to first study H-TGGG-Lys-NH2 using the solid phase
strategy. If the solid phase synthesis of this model tetramer was successful, the solid
phase strategy of the desired 16-mer PNA would then be investigated. The synthesis of
the required Boc-T-OH PNA monomer 135 has been described earlier [see scheme 33,
page 105]. Likewise, the synthesis of the Boc-GCbz-OH PNA monomer 126 has been
discussed in scheme 31 page 101.
The solid phase synthesis was carried out by first swelling 200 mg of MBHA resin in
DMF: DCM (1:1) for 24 h. Subsequently, the solvent was removed and resin was rewashed twice with fresh DMF: DCM solvent mixture. The swollen resin was then
neutralized using 5% DIPEA in DCM.
The resin was then derivatised with Boc-
protected lysine by treating this solid support with a pre-activated coupling mixture of
Boc-Lys-OH, HBTU and DIPEA in DMF. After agitating the resulting heterogeneous
mixture for 5 h, the solvent was removed and the resin was washed with a mixture DMF:
DCM (1:1). Any remaining underivatised free amino functions were then capped by
treating the resin with a mixture of acetic anhydride: DMF: collidine (1:8:1) for 1 h.
After this time, a Kaiser test165 was performed and this gave a negative result (yellow
colour), showing that all amino functions had now been derivatised. This fully loaded
resin was then ready for use in the in the solid phase synthesis.
106
Thus, the Boc-Lys-functionalised MBHA resin was first re-swollen in DMF:DCM (1:1).
The derivatised resin was then treated with a mixture of 95% TFA and 5% m-cresol to
cleave the Boc-protecting group of the solid support bound lysine. The m-cresol acted as
a tert-butyl cation scavenger. The success of this deprotection was monitored again by
using a Kaiser test.165 This time formation of a blue colour confirmed that the Boccleavage had been successful. Subsequently, the deprotection mixture was removed and
the resin was washed with fresh DMF:DCM (1:1). The Boc-protected guanine PNA
monomer 126 was next coupled again using HBTU as the activating reagent, in the
presence of a slight excess of N,N-diisopropylethylamine (DIPEA). Another Kaiser
test165 was then carried out which gave a negative result (yellow colour). Subsequently,
the coupling mixture was removed and the resin was washed with DMF before being
capped again using the mixture of acetic anhydride: DMF: collidine (1:8:1).
The
capping mixture was removed and the resin was washed with DMF:DCM (1:1).
This cycle of Boc-deprotection, coupling and capping was repeated until the required
protected oligomer of Boc-[TGCbzGCbzGCbz]-Lys had been assembled on the MBHA
resin. Once this stage had been reached, the oligomer was cleaved from the solid support
and concomitantly deprotected using a cocktail containing a mixture of thioanisole,
TFMSA and TFA. The crude oligomer was precipitated from the cleavage mixture by
the addition of a ten-fold excess of anhydrous diethyl ether.
The resulting suspension
was centrifuged and the supernatant was decanted off from the resulting pellet. The
pellet was then re-suspended in anhydrous diethyl ether and re-centrifuged to remove
residual acid and scavengers. This “washing” process was repeated 5 times. Finally, the
pellet was dried using a gentle stream of nitrogen gas to afford the crude product as a
white solid. As the yield of this PNA oligomer was very low (4.8 mg), it could not be
further purified by reverse phase HPLC.
However, a mass spectrum recorded on the
crude product gave a m/z peak which corresponded to the mass of the desired oligomer,
confirming that it had been successfully prepared. Unfortunately, due to time constraints,
application of this solid phase synthesis protocol for the preparation of the 16-mer PNA
oligomer was not possible.
107
2.10
Conclusion
In conclusion, reproducible schemes for the synthesis of N2-Boc-protected and N2-Cbzprotected-guan-9ylacetic acids, 97 [Scheme 16] and 108 [Scheme 24] respectively, from
commercially available 2-amino-6-chloropurine 58 have been successfully developed. In
both cases, the carbamate protecting group was introduced via treatment of the
appropriate 2-amino-6-chloropurin-9ylacetic acid derivative with triphosgene, to form the
isocyanate, followed by addition of either tert-butanol or benzyl alcohol, respectively.
Using this approach, compound 97 was afforded in 55% yield whilst 108 was obtained in
90% yield.
Furthermore, both 97 and 108 have been successfully coupled to the Fmoc-protected
PNA backbone 117 and Boc-protected PNA backbone 121, respectively, using HBTU
and DIPEA to give the desired guanyl-PNA monomers 48 and 49 in 50% and 55% yields,
respectively. An attempt was made to construct guanine-rich PNA oligomers using solid
phase synthesis, taking H-TGGG-Lys-NH2 as a model.
Although the various Kaiser
tests167 conducted after each coupling step showed that coupling had been successful, the
yield of the crude oligomer afforded after cleavage and deprotection was very low.
Therefore it was not possible to carry out any further purification or full characterization.
Several of the intermediates, monomers and dimers reported in this chapter have been
subsequently submitted for biological evaluation as potential novel HIV-1 NNRTIs and /
or integrase inhibitors. The biological findings are presented in chapter three of this
thesis.
108
CHAPTER THREE
EXPLORATION OF GUANINE-CONTAINING COMPOUNDS FOR ANTIREVERSE TRANSCRIPTASE AND ANTI-INTEGRASE ACTIVITIES
3.1
HIV-1 Therapeutic studies of the purine compounds
The second part of this thesis explores the bioassays of the various compounds
synthesised for HIV-1 reverse transcriptase and integrase inhibiting properties.
Therefore, a variety of guanine-PNA intermediates and guanine-PNA monomers, in
various states of deprotection, were sent to both Dr. G. Maga at the IGM-CNR, Pravia,
Italy to screen for their inhibitory properties towards HIV-1 reverse transcriptase; the
same set of compounds was sent to Dr. J.F. Mouscadet of ENSC, Cachan, Paris for
screening for anti-HIV-1 integrase activity.
The list of all compounds sent for analysis is shown Figure 44 below:
Figure 45: Guanine compounds submitted for bioassay
73: R=Cl, R1=R2=R3=Boc;
74: R=Cl, R1=H, R2=R3=Boc
75: R=Cl, R1=R2=H, R3=Boc
84: R=Cl, R1=-CH2CO2Et; R2=R3=Boc
86: R= o- NO2C6H4O-, R1= -CH2CO2Et, R2=R3=Boc;
93: R=Cl, R1= -CH2CO2Et, R2=R3=H
94: R=o- NO2C6H4O-, R1= -CH2CO2Et, R2=R3=H
95: R=OH, R1= -CH2CO2Et, R2=H, R3= Boc,
109
96: R=C2H2N3-, R1= -CH2CO2Et, R2=H, R3=Boc.
103: R=Cl, R1= -CO2CH2C6H5, R2=R3=H
105: R=Cl, R1= -CH2CO2Et, R2=H, R3= -CO2CH2C6H5;
106: R=Cl, R1= -CH2CO2Et, R2=H, R3= -COCBr3
108: R=OH, R1= -CH2COOH, R2=H, R3= -CO2CH2C6H5
109: R=Cl, R1= -CH2CO2Et, R2=H, R3=p-NO2-C6H5CH2CO2,
112: R=o-NO2-C6H4O-, R1= -CH2CO2C (CH3) 3, R2=H, R3= -C6H5CH2CO2
113: R=OH, R1= -CH2CO2C (CH3) 3, R2=H, R3= -C6H5CH2CO2
124: R= o-NO2-C6H4O-, R1= -CH2COOH, R2=H, R3= C6H5CH2CO2-
3.1.2 Non-nucleoside Reverse Transcriptase Inhibitors
As has already been discussed in the literature review, non-nucleoside reverse
transcriptase inhibitors NNRTIs are different from NRTIs because; (i) NNRTIs are noncompetitive inhibitors, that is they have a different binding site from that of the NTP
substrate; (ii) they do not require activation through intracellular metabolism and
phosporylation to exert their effect; (iii) NNRTIs bind allosterically to the enzyme and
their binding interaction with reverse transcriptase takes place in the hydrophobic pocket
of the enzyme. This binding site is called the non-nucleoside binding site (NNBS).
The first generation of this class of compounds was introduced in 1990 and they are
nevirapine 12 and delavirdine 13 and efavirenz 14. These first generation NNRTIs were
discovered to have the following advantages over NRTIs: long half life, low pill burden
and general tolerability.
These advantages are offset by the problem of multi-drug
resistance because they have a low genetic barrier to resistance. The reason for this
problem was that the amino acids units in the enzyme to which the compound binds in
the NNBS can easily be mutated. Once mutation occurs, the binding of the compounds
to the NNBS is easily affected, thereby leading to loss of potency. Furthermore, a serious
toxic effect has been reported in patients using efavirenz and nevirapine.
110
The strength and weakness of the first generation serve as the basis for an urgent need to
investigate further next generation drug candidates. These new generations of NNRTIs
(second generation NNRTIs) were designed to overcome the resistance mechanism
through conformational flexibility. The new class of NNRTIs that have been investigated
to overcome this resistance mechanism are etravirine 15 and rilpivirine 16 (page 28).
Looking at the structures of the second generation NNRTIs, they have two aromatic
groups for non-hydrogen bond binding in NNBS (that is, they can be used for
hydrophobic interactions in the NNBS).
There is an NH, which can be used to form
hydrogen bonds with lysine. These compounds are also flexible, therefore, they can
adapt to the minor changes in the NNBS site. Other structurally similar NNRTIs include
N-phenthyl-N’-thiazolylthiourea derivatives PETT 136, PETT-1 137, trovirdine 138, Osubstituted N-acyl-N-arylthiocarbamates (ACTs) 139, thiocarbamates C-TCs 140 and OTCs 141 and thiocarbamate UC-38 (NSC 629243) 142 [Figure 46].
111
Figure 46 NNRTIs which are structurally similar to guanine scaffolds
Based on the structural features of the new generation of NNRTIs, it was believed that
the guanine scaffolds [Figure 45, page 109] would be able to exhibit similar inhibitory
properties as those reported for the second generation NNRTIs. To the best of our
knowledge, these compounds had not been investigated in this respect to date. Therefore,
they presented a novel scaffold for NNRTIs.
112
3.1.3 Integrase Inhibitors
Integrase inhibitors are a class of drug compounds which inhibit the process of
integration of the viral DNA into the host genomic DNA. Examples of such compounds
that are currently being investigated by different researchers have been discussed in
Section 1.4.7 of the literature review.
A pharmacophore for anti-HIV-1 integrase active compounds has been proposed based
on the molecular framework of DKAs and DKA-like molecules. The pharmacophore is a
term used to describe the key binding groups and their spatial arrangements which are
responsible for the biological activity of a compound. The pharmacophore of DKAs,
which contributes to their HIV-1 integrase inhibitory activity, contains an aromatic
moiety, a carbonyl group (which can serve as the hydrogen-bond acceptor) and either NH
or OH groups (which can serve as hydrogen bond donors). These structural features are
also present in the guanine scaffold [Figure 45, page 109]. It was therefore reasoned that
the guanine compounds prepared during the course of these studies were worthy of
investigation as potential anti HIV-1 integrase inhibitors.
3.2
Evaluation of Guanyl Compounds as Potential NNRTIs
The compounds described in Figure 44 above were all evaluated as potential NNRTIs.
These studies were carried out against wild-type reverse transcriptase. From the various
compounds investigated, compounds 108, 112, 113 and 124 [Figure 47] were deemed
worthy of further investigation because their IC50 values (Table 3) fell within the range
proposed (i.e IC50 ≤ 100 μM).
113
Figure 47: Guanine scaffolds that exhibit NNRTIs properties
Compound No
RT Inhibition(IC50)
108
5μM
112
20μM
113
100 μM
124
22μM
Table 3: Guanine compounds that exhibit NNRTIs properties
The following biological parameters were investigated for 108, 112, 113 and 124;
(i)
Time–dependent inhibition of HIV-RT
The time-dependent experiment was used to investigate the rate of association between
enzyme and the compounds synthesised (i.e 108, 112, 113 and 124). This investigation
was achieved by studying the rate of association over time using the koff, kon [Figure 48]
and the concentration of the ligand.
kon
E
Enzyme
+
L
EL
koff
Enzyme . Ligand complex
Ligand
where Ligand = 108, 112, 113 and 124
Figure 48: Enzyme- Ligand binding association
114
koff is the dissociation rate constant, which measures the “off rate” for the ligand
dissociating from the enzyme per second. While the kon measures the association binding
constant. Therefore the overall equilibrium binding constant for the enzyme – ligand
association, Ki, is given as:
Ki = koff / kon
During the investigation, it was found out that the guanyl derivatives 108,112,113 and
124 were characterised by slow apparent association rates (kon). These were of the order
of 103 -104 slower when compared with nevirapine and efavirenz (Table 4). However,
once formed, the corresponding enzyme-inhibitor complexes were found to be extremely
stable. This property was confirmed by their very low dissociation rates (koff), which
were of the order 102 fold slower when compared with nevirapine and efavirenz (Table
4).
Compound
Ki μM
kon (M-1S-1)
koff(S-1)
113
400(±30)
n.d
n.d
108
4.7(± 0.8)
17.6(±0.8)
8.3(±0.3) x10-5
112
3.7(±0.8)
15.1(±0.7)
5.5 (±0.5) x 10-5
124
3.0(±0.5)
8.3(±0.2)
2.5 (±0.1) x10-5
Nevirapine
0.4 (± 0.03)
1.5(±0.2) x 103
0.6(±0.1) x 10-3
Efavirenz
0.03(± 0.005)
34 (± 1) x 103
1.0(±0.4) x 10-3
Table 4: Kinetic parameters for N2-Cbz-guanyl acetic acid derivatives binding to wild
type HIV-1 RT.
(ii)
Type of inhibition of HIV-1 RT
In order to determine whether guanyl compounds 108, 112, 113 and 124 were either
reversible or irreversible inhibitors, equilibrium dilution experiments were conducted.
115
Compound 108 was used for the purpose of this study. It was found that RT activity
increased with increasing dilution. These results showed that 108 acted as a reversible
inhibitor of RT.
Figure 49: Effect of dilution on the inhibition of HIV-1 RT by compound
(iii)
Activity against mutant strains of HIV-1 RT
Resistance to RT inhibition by compounds 124 and 108 was further investigated and this
was found to be due to mutation-specific effects. This effect mainly caused an increase
in their dissociation (koff) rates [Figure 50A-D].
The association (kon) rate of 108 was
largely unaffected by mutants Y181I and L100I [Figure 50B and D] and only marginally
decreased by mutations K103N and Y188L [Figure 50A and C]. Furthermore, the kon
value of 124 was affected only by the mutation K103N. When these values were
compared to the prototype NNRTIs, nevirapine and efavirenz [Figure 50E], a number of
differences were noted. First, all the mutations decreased the nevirapine association (kon)
116
rate, contrary to what was observed for the guanyl derivatives. In addition, the increase
in the koff values induced by the mutations was much greater for nevirapine than for 108,
112 and 113, with the exception of 124 against the K103N mutant RT [Figure 50A]. In
the case of efavirenz, mutations Y181I and Y188L affected both the kon rate and koff rate,
whereas the K103N mutation had a more marked effect on the kon rate than the koff rate.
Overall compound 124 showed a better activity profile in terms of relative resistance,
than nevirapine and efavirenz against all the mutants tested.
124
108
124
108
108
124
12
124
4
108
124
108
124
108
108
124
12
4
108
124
Figure 50 : Resistance profiles for compounds 108, 124, nevirapine and efavirenz
117
3.3
Evaluation of Guanyl Compounds as Potential IN inhibitors
The compounds described in Figure 45 were also submitted for investigation of their
inhibitory activities against HIV-1 integrase.
The results of these antiviral assays
compared to the standard of 17 at different concentrations are shown in Table 5 below.
Unfortunately, none of these guanyl compounds showed any significant anti-viral activity
within the cut off range.
Compound
IN activity (% of control)
No
CM21
500 μM
50 μM
5 μM
0.5 μM
-0.1
8.3
11.3
37.1
86
34.2
59.3
75.2
116.8
105
46.7
51.5
90.4
136.1
103
10.0
64.1
94.2
95.2
75
124.5
105.4
90.0
111.8
109
127.7
150.4
139.8
115.8
106
66.1
146.0
120.2
122.2
108
28.9
80.6
111.2
121.1
113
25.9
264.1
127.8
119.8
124
28.5
108.5
97.3
127.4
112
137.7
109.8
141.1
133.3
Table 5: Anti viral assays of the Guanine Scaffolds
118
CHAPTER FOUR
STRUCTURAL ACTIVITY STUDIES OF THE BIOACTIVE COMPOUNDS
In order to further understand the inhibition properties of the active compounds and
improve their activities, analogues of lead compounds 108, 112, 113, and 124 identified
from both the RT and IN bioassays were prepared. It was reasoned that variation of the
substituents may affect the electronic properties of lead compounds 108, 112, 113, and
124 and this may contribute towards either the understanding of the activity of the leads
and /or improvement in activity.
One modification that we proposed to investigate was the effect of an electronwithdrawing group on the phenyl ring of the Cbz-group. Thus, analogues of 108, 112,
113, and 124 were prepared as shown in Scheme 35. It has been observed that the
reduction of nitro groups to hydroxylamines or amines represents a very large change and
can generate a “switch” which can generate cytotoxins. This approach has been used to
target delivery of toxic alkylating species to hypoxic tumor cells.166,167 The nitro group is
a strong electron withdrawing group (Hammett electronic parameter σρ =0.78) and it can
be converted to an electron donating hydroxylamino group (σρ=-0.34) upon NTRreduction. This large difference in electronic effect (∆σρ=1.12) is exploited to effect the
formation of highly cytotoxic reactive species. The “switch” properties of nitro groups
led to the investigation of a design [Scheme 34] for the synthesis of possible pro-drug
compounds.
119
NO2
Cl
H2N
Cl
N
N
O
i
70%
N
N
O
O
N
N
N
H
N
N
OtBu
O
110
143
OtBu
O2N
ii
80%
O
O
O
O
N
HN
N
H
iv
O
75%
N
N
N
N
O
N
H
N
N
O
O2N
OtBu
O
O2N
OtBu
146
144
90% iii
90% v
O2N
O
O
O
O
N
HN
N
H
O
N
N
O
O
O2N
N
N
N
H
O
O2N
OH
147
N
N
145
OH
Scheme 34
Reagents and conditions: (i) (a) (Cl3CO)2CO, Et3N, THF, reflux, 3 h; (b) pNO2PhCH2OH, reflux, 3 h; (ii) o-nitrophenol, DABCO, Et3N, ACN, RT, overnight; (iii)
TFA,DCM, RT, 3 h; (iv) 1,1,3,3,-TMG, o-nitrobenzaldoxime, ACN, RT, overnight; (v)
TFA,DCM, RT, 3 h.
120
Steps i-v of Scheme 34 were undertaken following similar procedures already discussed
for preparation of 108 [Scheme 24 on page 91 and 92].
It was reasoned that extension of the chain length could be another useful tactic for
designing drugs in order to optimise drug-target interactions. This is because the chain
length in the lead compound may not be sufficient to allow for maximum binding
interactions to occur.
Thus, it was deemed that the effect of increasing the methylene
chain of the N9-carboxylic acid on binding was worthy of study. Therefore, synthesis of
compounds 163, 164, 165 and 166 were explored as shown in scheme 35.
121
O2N
Cl
N
N
i
58
H2N
N
O
n=2=ethyl-3-bromopropionate
n=3=ethyl-4-bromobutyrate
n=4=ethyl-5-bromovalerate
n=5=ethyl-6-bromohexanoate
O
ii
148;. n= 2(78%)
149; n=3(70%)
150; n=4(75%)
151; n=5(72%)
N
N
N
( )n
H2N
OCH2CH3
N
O
152; n= 2(80%)
153; n=3(85%)
154; n=4(82%)
155; n=5(90%)
N
( )n
OCH2CH3
iii
O
O
N
HN
CbzHN
N
HN
N
O
v
CbzHN
N
N
( )n
O
N
( )n
O2N
iv
O
N
N
OCH2CH3
OH
163: n = 3(55%)
164: n = 4(66%)
165: n = 5(70%)
160; n=3( 62%)
161; n=4(60%)
162, n=5(60%)
N
( )n
CbzHN
N
156; n=2(80%)
O
157; n=5(80%)
158; n=3(not purified)
159; n=4(not purified)
OCH2CH3
v
O2N
O
N
N
CbzHN
N
O
N
( )n
166; n=2(66%) OH
Scheme 35
Reagents and conditions: (i) K2CO3, DMF, Br(CH2)nalkanoate; (ii) o-nitrophenol,
DABCO, Et3N, DMF, RT, overnight; (iii)(a) C3Cl6O3, Et3N, 1,2-dichlorobenzene, 130
°C, 3 h (b) PhCH2OH, 130 °C, 3 h; (iv) o-nitrobenzaldoxime, 1,1,3,3-TMG, ACN, RT,
overnight; (v) LiOH, dioxane, RT, 3 h.
122
The first step in Scheme 35 was the alkylation of compound 58. This was achieved by
treating a solution of 58 in anhydrous DMF separately with ethyl-3-bromopropionate,
ethyl-4-bromobutyrate, ethyl-5-bromovalerate or ethyl-6-bromohexanoate.
After work-
up and purification of each of these reactions, compounds 148, 149, 150, and 151 were
obtained in yields of 75%, 85%, 77% and 82%, respectively. The next step involved
replacement of the 6-chloro group of 148, 149, 150 and 151 with the o-nitrophenoxy
moiety, using o-nitrophenol, DABCO, Et3N in DMF. After work-up, compounds 152,
154 and 155 were obtained in an 80% yield, whilst 153 was received in a 75% yield. The
third step in the synthetic pathway concerned protection of the exocyclic amino group on
the purine ring of 152, 153, 154 and 155 with a carbamate group. This reaction was
carried out by employing the novel procedure developed for the synthesis of N2benzyloxycarbonyl-protected guan-9-ylacetic acid 108. (see pages 91 and 92). Thus,
solutions of 152, 153, 154 and 155 in THF were treated with triphosgene at 130 °C for 3
h. This was followed by the addition of benzyl alcohol and the reaction at 130 °C for a
further 3 h. Subsequently, the products of the reactions with 152 and 155 were purified
by column chromatography to afford 156 and 157 both in a yield of 80%. The products
of the reactions with 153 and 154 were not purified. Instead they were taken through to
the next stages (i.e steps iv and v, Scheme 35) in the pathway as crude products.
Subsequently, the o-nitrophenoxy group of 157, 158 and 159 was cleaved to give the 6oxo functionality of guanine.
This was achieved by treating solutions of either pure 159
or crude 157 or 158 in acetonitrile with 1,1,3,3-tetramethylguanidine and onitrobenzaldoxine at room temperature overnight. After work-up and purification by
flash column chromatography, 160 and 161 were afforded in 62% and 60% yields over
the two step, whilst 162 was obtained in a yield of 60% from this single step. An attempt
to convert the O6-nitrophenoxy group of 156 into the oxo functionality was not
successful. It was believed that the product was lost on the column during purification.
The final step involved the hydrolysis of the ethyl esters of 156,160, 161, and 162. This
was achieved by treating these compounds with LiOH in dioxane at room temperature for
3 h. After work-up, the desired products 163, 164,165 and 166 were obtained as white
solids in yields ranging from 55% to 70%.
123
Another modification that was considered worthy of investigation concerned variation of
the functional group at the N9-position of the purine ring in order to understand the
binding properties of the lead compounds.
Therefore it was proposed to explore the
effect of replacing the carboxylic acid moiety with an alcohol 170 on NNRTI activity
[Scheme 36]. The first step in the synthesis of compound 170 involved treatment of a
solution of 58 in anhydrous DMF with 2-bromoethanol at room temperature for 3 h.
After work-up, compound 167 was obtained in 80% yield.
Subsequently, the 6-chloro
group of 167 was converted into the o-nitrophenoxy group following the procedure
presented on page 90. After work-up, compound 168 was obtained as a yellow foam in
80% yield.
Before the N2-amino group of the purine ring of 168 could be protected
with a Cbz group, it was necessary to next protect the –OH function in order to prevent it
from interfering with the triphosgene. Thus, a solution of 166 in anhydrous DMF was
treated with TBDMS-Cl and imidazole at room temperature overnight. After work-up
and purification by column chromatography 169 was obtained in 60% yield.
The next
stage was to protect the N2-amino group of 169 with a Cbz group. This protection was
successfully carried out following the procedure previously developed [page 92 and 93].
From the 1H NMR and
13
C NMR spectra recorded for the purified product, it was
observed that in addition to the protection of N2-amino function, silyl deprotection of the
hydroxyl group had occurred. Therefore, the desired alcohol 170 was obtained in 80%
yield.
124
O2N
Cl
58
i
80%
H2N
O
N
N
N
N
ii
H2N
HOH2C
N
N
80%
N
N
HOH2C
168
167
60% iii
O2N
O2N
O
O
80%
N
N
CbzHN
iv
N
N
N
N
H2N
N
N
OH
170
169
O Si
Scheme 36
Reagents and conditions: (i) K2CO3, BrCH2CH2OH; (ii) o-nitrophenol, DABCO, Et3N,
ACN, RT, overnight; (iii) TBDMS-Cl, imidazole, DMF, RT, overnight; (iv)(a) C3Cl6O3,
Et3N, THF, reflux, 3 h (b) PhCH2OH, reflux, 3 h.
Tetrazoles are rather acidic with a pKa of 5.0. This is exactly the same as that of a
carboxylic acid and because of this, tetrazoles have been used widely as a replacement
for carboxylic acids in order to increase the bioavailability of a drug. Therefore, it was
reasoned that it would be interesting to study the tetrazole analogue 174 of the NNRTI
lead compound 108 reported in chapter three. Compound 174 was prepared as outlined
in Scheme 37.
125
O2N
Cl
Cl
N
H2N
O
N
N
N
H
i
78%
N
N
H2N
N
ii
90%
N
H2N
CN
58
N
N
N
N
171
172
CN
80% iii
O2N
O2N
O
iv
O
65%
N
N
N
N
CbzHN
N
N
N
CbzHN
N
NH
N N
174
N
CN
173
Scheme 37
Reagents and conditions: (i) BrCH2CN, K2CO3, DMF, RT, 5 h; (ii) o-nitrophenol,
DABCO, Et3N, ACN, RT, overnight; (iii) (a) triphosgene, THF, Et3N, reflux, 3 h (b)
benzyl alcohol, reflux, 2 h; (iv) NaN3, Et3N.HCl, DMF, 120 °C, 48 h.
The first step in Scheme 37 involved treatment of commercially-available 58 with
bromoacetonitrile in DMF at room temperature for 5 h. After work-up and purification
by column chromatography, 171 was afforded as a white solid in 78% yield.
Subsequently, the 6-chloro group of 171 was converted into the o-nitrophenoxy moiety.
These conversion was achieved by treating 171 with DABCO, o-nitrophenol and Et3N in
acetonitrile at room temperature overnight. After work-up and purification by column
chromatography, 172 was obtained as a yellow solid in 90% yield. The protection of the
N2-exocyclic amino group of 172 with Cbz was carried out by first treating 172 with
triphosgene at reflux for 3 h, followed by benzyl alcohol at reflux for 2 h. Evaporation of
126
the solvent followed by purification by column chromatography afforded the desired
product 173 as a yellow foam in 80% yield.
The final step in this pathway involved
conversion of the nitrile group 173 into the desired tetrazole group.
This reaction
involved treatment of 173 with sodium azide and triethylammonium hydrochloride in
toluene at reflux overnight. After work-up and purification, the desired product 174 was
obtained as a yellow foam in 65% yield.
In summary, a range of analogues of the guan-9-ylacetic acid NNRTI lead compound 108
have been successfully prepared. Their biological activities are currently undergoing
evaluation.
127
CHAPTER FIVE
CONCLUSION AND FUTURE WORK
5.1
Conclusion
The synthesis of N2-Boc- and N2-Cbz-guan-9-ylacetic acids in high yield was necessary
for the synthesis of guanine PNA monomers. The protected guanine-PNA monomers
were needed for the synthesis of guanine-rich PNA oligomers. There was an established
literature report that a 16-mer guanine-rich DNA oligomer could inhibit the integration of
HIV-1 viral DNA into the DNA of the host. Furthermore, the structural features of
guanine scaffolds synthesised made them worthy of investigation as novel second
generation NNRTIs.
Prior to the commencement of this research work, Thompson et al.146 and Sugiyama et
al.149 have investigated a synthetic approach to the synthesis of Cbz-protected guan-9-yl
acetic acids and Boc-protected guan-9-ylacetic acid respectively. The Thompson et al.146
approach was not investigated further here because previous attempts in our group to
employ this approach did not meet with any success. The Sugiyama et al.149 approach
was also not investigated because it deemed to be too complicated and required many
steps.
The novel method highlighted in Scheme 12, was inspired by the idea proposed by Dey
and Garner.150 This approach was successful and the desired N2-Boc-protected guan-9ylacetic acid 97 was prepared in six steps and overall yield of 16.3%. An attempt to
design a high-yielding synthesis of N2-diBoc-protected guan-9-ylacetic acid 90 was not
successful. The best yield recovered for N2-diBoc-guan-9-ylacetic acid 90 was 20%
[Scheme 10]. The reason for this low yield was because it proved to be very difficult to
purify the crude product by column chromatography.
128
The selective transformation of di-Boc compound 84 into mono-Boc compound 95
[Scheme 13] has been successfully achieved.
This strategy was an improvement over
Dey and Garner’s approach [Scheme 8],150 because, by changing the reagents from
NaOH in ethanol to LiCl in acetonitrile, this reaction could be accomplished within 18 h
rather than the reported 70 h. By using the approach outlined in Scheme 14, the time
taken to synthesise mono-Boc-N2 guan-9-ylacetic acid 97 from commercially-available
2-amino-6-chloropurine 58 was greatly reduced (from 5 days to 3 days).
Further investigation into the design of a completely novel route for the synthesis of N2mono-Bocguan-9-ylacetic acid 97 was carried out. This involved the use of triphosgene
to activate the exocyclic 2-amino group of the purine ring to generate an isocyanate. This
intermediate was subsequently treated with tert-butanol to give the desired Boccarbamate 95 [Scheme 16]. An attempt was made to generate the 6-oxo function of
guanine by converting the 6-chloro group of 95 into the o-nitophenoxy moiety which
could later be removed according the method reported by Howarth et al.147
This
approach was not successful. Therefore, the 6-chloro substituent was replaced with
1,2,4-triazole which, following hydrolysis of the triazolyl group, gave the required
guanine derivative 97 in an overall yield of 18.6%.
Dey and Garner’s approach was applied to the synthesis of N2-Cbz-protected guan-9ylacetic acid compound 90. This route was not successful because the required tri-Cbzprotected intermediate could not be generated by treatment of 2-amino-6-chloropurine 58
with Cbz2O or Cbz-chloroformate or Rapoport’s reagent.
The only compound isolated
from any of these reactions was the N9-Cbz-protected derivative 103 [Scheme 18]. The
production of 103 was confirmed by X-ray crystallography. The most striking feature of
103 was that it showed two crystallographic structures joined together by hydrogen bonds
to form six-membered rings [Figure 42].
Due to the inability to successfully apply Dey and Garner’s approach to the synthesis of
the desired N2-Cbz-protected guan-9-ylacetic acid, the use of triphosgene to activate the
exocyclic 2-amino group on the purine ring to form an isocyanate was investigated. The
129
addition of benzyl alcohol to the isocyanate generated the desired N2-Cbz-protected
derivative 105 [Scheme 22]. Following triazolyl substitution and hydrolysis the required
N2-Cbz-guan-9-ylacetic acid 108 was obtained in an overall yield of 16.7%. Although
this route had been successful, it was reasoned that an alternative approach may lead to
an overall improvement in the yield of 108. It was recognized that if the lipophilicity of
the group at the N9-position of the purine ring were increased, then purification of the
intermediates to 108 would be easier. Therefore, the N9-ethyl ester was replaced with a
tert-butyl ester. This strategy was successful and afforded 108 in an overall yield of
36.5% [Scheme 24].
The
synthesis
of
the
Fmoc-N-(2-aminoethyl)
glycine
117
and
Boc-N-(2-
aminoethyl)glycine 121 PNA backbones were successfully achieved following
established literature procedures [Schemes 25 and 27].
Subsequently, the protected
guan-9-ylacetic acids 108 and 97 were coupled to the appropriate PNA backbone (121 or
117, respectively) to afford the corresponding orthogonally-protected guanine-PNA
monomers 48 [Scheme 29] and 49 [Scheme 30] in the same yield of 55%.
An attempt was made to synthesise guanine-PNA dimers. This was disappointing as the
guanine-PNA dimers could only be obtained in a 20% yield. The solid phase synthesis of
H-TGGG-Lys-NH2 was studied for the future preparation of the 16-mer guanine-rich
PNA oligomer. The model tetramer was successfully prepared, but the yield was low.
Due to time constraints, further investigations into the synthesis of these oligomers could
not be conducted.
The various monomers and intermediates synthesised in this thesis were tested for their
inhibitory activities towards HIV-1 IN and RT enzymes. Compounds 108, 112, 113 and
124 all showed inhibitory activities towards wild-type HIV-1 RT with IC50 values of
5μM, 20 μM, 100 μM and 22 μM, respectively. To the best of our knowledge, this is the
first report of guanyl derivatives that directly target HIV-1 RT. Even though their
absolute potencies of inhibition are in the micromolar range, and thus are much higher
130
than those of other NNRTIs currently under development, these results indicate that the
derivatives exhibit less sensitivity to mutations associated with drug-resistance.
5.2
Recommendation
The solid phase synthesis of 16-mer guanine-rich PNA oligomer was not tried because of
the problems associated with preparation of the model tetramer H-TGGG-Lys-NH2. This
single attempt resulted in the product being afforded in a very low yield. Therefore,
further studies on this will be required in the future.
Although the results of the bioassay of the compounds synthesized were not in the
nanomolar range, the novel scaffold of this class of compounds and their interesting
resistance profile towards drug-resistant RT mutants make them worthy of further
development. It is also possible that the relatively weak affinities of our compounds
might affect their resistance profiles and that more potent compounds would show
different sensitivities to drug-resistance mutations. Thus, QSAR studies on an expanded
set of compounds are presently underway in order to improve the binding rate (and hence
the absolute potency of inhibition) of these derivatives and to assess their drug-resistance
profile.
131
CHAPTER SIX
EXPERIMENTAL
6.1
Materials ad Methods
6.1.1 Reagents and Solvents
All reagents were purchased from Alfa Aesar, Fluka, Novabiochem or Sigma Aldrich
Chemical Company Limited. Unless otherwise stated, reagents were used without further
purification.
All solvents used were of analytical grade and purchased from standard commercial
companies. No further purification was carried out prior to use except for petroleum
ether (40-60 °C) which was purified by distillation. 1,2-Dichloroethane, N,Ndimethylformamide and acetonitrile were dried by standing over activated 3Å molecular
sieves. Tetrahydrofuran was dried over sodium using benzophenone as an indicator
(solution turns blue/purple) and freshly distilled prior to each reaction under nitrogen gas.
6.1.2 Chromatography
Analytical thin layer chromatography (TLC) was carried out on aluminium pre-coated
silica gel 60PF254 plates (Merck). Visualization was generally achieved under UV ( =
254 nm).
Additionally, the plates were stained with either a solution of
Phosphomolybdic acid (PMA) in methanol (70% w/v) or a potassium permanganate
solution (3 g KMnO4, 20 g K2CO3, 5 mL 5% NaOH (w/v), 300 mL distilled water).
Column Chromatography was carried out using silica gel 60Å, particle size 35-70
micron, purchased from Fisher.
132
6.1.3 Instrumentation
Melting points were measured using a melting point SMP10 instrument from Stuart
Scientific (Bibby Sterling Ltd). Infra-red spectra were recorded on Perkin Elmer RX FTIR spectrophotometer. Due to contamination with KOH, the recorded IR spectra in KBr
always showed an O-H stretch at approximately 3450cm-1.
Elemental analyses were carried on a CEC 400 Elemental analyser (Central Equipment
Corporation) by Mrs. Christina Graham at the Heriot Watt University. Single X-ray
diffraction was carried out by Dr. Georgina M.Rosair at the Heriot Watt University on a
Bruker Nonius X8 Apex CCD single crystal diffractometer. The NMR AC 200 and AC
400 MHz were carried out by Dr. Alan Boyd at Heriot Watt University.
Mass spectra were recorded courtesy of the EPSRC Mass Spectrometry Service Centre at
the University of Wales, Swansea.
6.2
Experimental Procedures
2-[bis(tert-Butoxycarbonyl)amino]-6-chloro-N-9 tert-butoxycarbonyl purine (73) 150
Cl
N,N-dimethylaminopyrimidine (0.037 g, 0.30 mmol) was
N
N
added to a stirred solution of 2-amino-6-chloropurine
N
(0.51g, 3.00 mmol) in dry THF (30 mL) at RT.
O
Subsequently, a solution of di-tert-butyl dicarbonate
O
N
O
N
O
O
O
(2.62 g, 12.00 mmol) in dry THF (30 mL) was added
dropwise. The reaction mixture was left to stir for further
73
22 h. After this time TLC analysis indicated that all the starting material had been
consumed. The solvent was removed under reduced pressure and the crude yellow oil
afforded was purified by column chromatography. The desired product 73 (0.46g, 26%)
was afforded as a white solid. Rf = 0.35 (ethyl acetate: petroleum ether, 2:8); δH
(200MHz, CDCl3) 1.42 (18H, s, 2x (CH3)3C), 1.65 (9H, s, (CH3)3C), 8.56 (1H, s, C8-H);
133
δC (50.3MHz, CDCl3) 27.8 (CH3), 83.7 (C ), 130.6 (C), 138.4 (CH), 140.7 (C), 144.5 (C),
145.2 (C), 149.4 (C), 150.4 (C), 151.6 (C), 153.5 (C)
2-[N,N-Di-tbutoxycarbonylamino] -6-chloropurine (74) 150
A saturated aqueous solution of sodium hydrogen
carbonate (5 mL) was added to a stirred solution of purine
73 (0.10 g, 0.21 mmol) in methanol (20 mL). The turbid
mixture was stirred at 50 °C for 1 h and then overnight at
Cl
N
N
N
H
N
O
N
O
RT. The bulk of the solvent was removed under reduced
O
O
74
pressure and distilled water (30 mL) was added to the
resulting residue. The aqueous layer solution was extracted with chloroform (30 x 50
mL) and the combined organic extracts were dried over anhydrous MgSO4. Filtration
followed by solvent evaporation gave a crude yellow oil. This was re-dissolved in ethyl
acetate (40 mL) and filtered through silica gel (3 cm x 10 cm) using ethyl acetate (200
mL) as the eluent. The solvent was removed under reduced pressure to give the desired
compound 74 (0.065 g, 84%) as a white solid. Rf = 0.59 (Ethyl acetate). δH (200MHz,
CDCl3) 1.42 (18H, s, 2x (CH3)3C), 8.56 (1H, s, C8-H); δC (50.3MHz, CDCl3) 27.8
(CH3), 84.4 (C), 125.0 (C), 132.7 (C), 145.6 (CH), 150.5 (C), 151.3 (C), 53.1 (C)
Ethyl-2-[N, N-Di-tbutoxycarbonyl amino]-6-chloropurin-9-ylacetate (84)
METHOD A
Cl
A heterogeneous mixture of purine 74 (0.59 g, 1.58
mmol), anhydrous K2CO3 (0.48 g, 3.48 mmol) and
N
ethyl bromoacetate (0.35 mL, 3.16 mmol) in anhydrous
N
DMF (30 mL) was stirred at RT for 5 h. Subsequently,
the precipitate was removed and the filtrate was
concentrated under reduced pressure. The crude residue
N
N
O
O
N
O
O
O
H3CH2CO
84
afforded was re-dissolved in chloroform (50 mL), and the resulting organic solution was
134
washed with water (3 x 20 mL) and dried over anhydrous MgSO4. Filtration followed by
solvent evaporation gave a viscous yellow oil which was purified by column
chromatography [ethyl acetate]. This oil afforded was triturated with cold diethyl ether to
give the desired compound 84 (0.480 g, 75%) as a white solid.
METHOD B
Sodium hydride (0.07 g, 3.00 mmol) was added to a suspension of purine 74 (0.59 g, 1.58
mmol) in dry DMF (60 mL) at RT under argon. The resulting mixture was left to stir for 2
h. Subsequently, ethyl bromoacetate (0.33 mL, 3.00 mmol) was added at RT and the
reaction mixture was left to stir overnight. The solvent was evaporated in vacuo and the
crude residue was purified using column chromatography (ethyl acetate). The desired
compound 84 (0.55g, 77%) was obtained as a white solid. Rf = 0.72 (ethyl acetate). mp
202-204 °C. δH (200 MHz, CDCl3) 1.42 (18H, s, 2 x (CH3)3C), 1.32 (3H, t, J = 7.2,
OCH2CH3), 4.3 (2H, q, J = 7.2, OCH2CH3,), 5.0 (2H, s, N9-CH2COOCH2CH3), 8.56 (1H,
s, C8-H); δC (50.3MHz, CDCl3) 14.3 (CH3), 28.0 (CH3), 45.0 (CH2), 62.0 (CH2), 83.0 (C),
131.4 (C), 143.4 (CH), 144.0 (C), 149.4 (C), 150.4 (C), 152.4 (CO), 170.0 (C). vmax(KBr,
cm-1): 3196(w), 2977(w), 1744(s), 1626(s), 1447(m), 1375(m), 1156(s), 1092(w). LRMS
(EI) (m/z)(%): 456[(10),(M+)], 355(28), 298(48), 282(52), 255(78), 182(78), 146(70),
83(59), 57(100); Elemental Analysis for C19H27N5O6Cl: found %C= 50.22, H= 5.77,
N=15.39. requires: %C=50.06, H= 5.75, N=15.36;
Ethyl-2-[bis-(tert-butoxycarbonyl)amino]-6-(2-nitrophenol)purin-9-ylacetate (86)
NO2
Compound 84 (0.20 g, 0.44 mmol) was suspended in dry
1,2-dichloroethane (10 mL). A solution of 2-nitrophenol
(0.18 g, 1.32 mmol), 1,4-diazabicyclo[2,2,2]octane (0.049
g, 0.44 mmol) and triethylamine (184 μL, 1.32 mmol) in
O
N
N
N
N
O
N
O
dry 1,2-Dichloroethane (5 mL) was added dropwise to the
O
reaction mixture with stirring under nitrogen.
The
resulting mixture was left to stir at RT overnight.
135
O
H3CH2CO
86
O
Subsequently, the reaction mixture was diluted with dichloromethane (50 mL) and
washed with a saturated solution of NaHCO3 (30 mL). The aqueous layer was reextracted with dichloromethane (3 x 50 mL) and the combined organic extracts were
dried over anhydrous MgSO4. Solvent evaporation in vacuo afforded a crude product,
which was chromatographed on silica (ethyl acetate) to give the desired product 86 (0.17
g, 70%) as an off-white solid. Rf = 0.6 [ethyl acetate] mp 220-221 °C. δH (200 MHz,
CDCl3) 1.42 (18H, s, 2x(CH3)3C), 1.39 (3H, t, J= 7.2, OCH2CH3), 4.29 (2H, q, J= 7.2,
OCH2CH3,), 5.15 (2H, s, N9-CH2COOCH2CH3) 8.56 (1H, s, C8-H) 7.21-7.89(4H, m, ArH); δC (50.3 MHz, CDCl3) 14.3(CH3), 28.0 (CH3), 45.0 (CH2), 62.0 (CH2), 83.0 (C),
117.8 (C), 121.0 (C), 125.0 (C), 126.0 (CH), 128.0 (CH), 135.0 (CH), 138.0 (C), 143.0
(CH), 145.0 (C), 150.0 (C),151.4 (CH), 156.0 (CO), 167.0 (C); vmax(KBr, cm-1): 3451(w),
3340(m), 3124(w), 2950(m), 1744(w), 1526(w), 1301(s), 1239(w), 1156(w).); LRMS
(EI) (m/z) (%): 558 [(4), (M+)], 551(6), 537(4), 513(4), 471(2), 458(100), 444(48),
414(6), 402(78), 385(88), 371(42); Elemental Analysis for C25H30N6O9 found %C=
53.67, H= 5.40, N= 14.94. requires %C= 53.71, H= 5.41, N= 15.05.
Ethyl-2-[bis-(tert-Butoxycarbonyl)amino]guan-9-ylacetate (88)
Compound 86 (0.21 g, 0.37 mmol) and 2O
nitrobenzaldoxime (0.61g, 3.70 mmol) were dissolved
N
NH
in dry ACN (10 mL). 1,1,3,3-tetramethyl guanidine
(418 µL, 3.33 mmol) was added under argon and the
resulting solution was left to stir overnight.
N
O
The
solvent was evaporated in vacuo and the residue
afforded was dissolved in ethyl acetate (50 mL). The
N
O
N
O
O
O
H3CH2CO
88
resulting organic solution was washed with a saturated solution of NaHCO3 (2 x 50 mL).
The organic layer was separated and dried over anhydrous MgSO4. Filtration followed by
solvent evaporation gave a crude oil which was re-dissolved in methanol (20 mL) and
water (80 mL). The mixture was then extracted sequentially with petroleum ether (2 x 50
mL), diethyl ether (2 x 50mL), and finally ethyl acetate (2 x 50 mL). The combined ethyl
acetate extracts were dried over anhydrous MgSO4.
136
Filtration followed by solvent
evaporation gave a colourless oil, which solidified on the addition of water.
The
precipitate was collected by filtration and dried in vacuo. The desired product 88 (0.032
g, 20%) was afforded as a white solid mp 234-236 °C; δH (400 MHz, CDCl3) 1.42 (18H,
s, 2x (CH3)3C), 1.28 (3H, t, J = 7.2, OCH2CH3), 4.29 (2H, q, J = 7.2, OCH2CH3), 4.71
(2H, s, N9-CH2COOCH2CH3) 7.29 (1H, s, C8-H) ; δC (100.6 MHz, CDCl3) 14.2 (CH3),
28.0 (CH3), 44.4 (CH2), 60.4 (CH2), 119.2 (C), 133.6 (C), 135.6 (C), 147.2 (CH), 150.9
(C), 155.6 (C), 166.8 (C), 171.2 (C); vmax(KBr,cm-1): 3418(m), 2978(m), 1733(s),
1626(s), 1434(m), 1333(m), 1156(s), 1120.7(w). LRMS (EI) (m/z) (%): 437 [(71), (M+)],
380 (63), 256(77), 115(100), 84(38), 57(91),
HRMS (ES, H+): Calculated for
C19H27N5O7 [M+H+] is 438.4635; Found 438.4634; Elemental analysis found is %C=
52.17, %H= 6.22, %N=15.99. requires: %C=52.17; %H= 6.22; %N= 16.01.
2-[bis(tert-Butoxycarbonyl)amino]guan-9-ylacetic acid (90)
O
Compound 88 (0.10 g, 0.22 mmol) was dissolved in 1,4dioxane (5 mL) and a 1 M (aq) solution of sodium hydroxide
N
(4 mL) was added. The resulting mixture was stirred at RT
N
for 1 h. Subsequently, dichloromethane (6 mL) was added
and the aqueous layer was separated. The aqueous phase was
NH
N
O
O
N
O
O
O
HO
extracted further with fresh dichloromethane (3 x 5 mL). The
90
pH of the aqueous solution was then adjusted to pH 3 using
2M (aq) citric acid solution and extracted with ethyl acetate (3 x 6 mL). The combined
organic layers were dried over anhydrous MgSO4. Filtration followed by solvent
evaporation afforded a crude oil which solidified on trituration with diethyl ether to give
90 (0.08g, 87%) as a white solid. mp 250-252 °C; δH (400 MHz, CD3OD) 1.42 (18H, s,
2x (CH3)3C), 4.89 (2H, s, N9-CH2COOH), 8.51 (1H, s, C8-H); δC (100.6 MHz, CD3OD)
28.0 (CH3), 45.7 (CH2), 86.3 (C), 125.6 (C), 129.4 (C), 131.2 (C), 134.4 (CH), 146.5 (C),
150.4 (C), 170.2 (C); vmax(KBr,cm-1) 3548(bm), 2977(w), 1735(s), 1626(s), 1614(s),
1479(m), 1412(m), 1371(m), 1064(w). LRMS (EI) (m/z) (%): 409 [(21), (M+)], 367(49),
296(68), 115(55), 134(100), 45(85), HRMS (ES, H+): calculated for C17H23N5O7 [M+H+]
137
is 410.4094; found 410.4096; Elemental analysis found %C=50.02; H= 5.67; N= 16.90.
requires %C=49.88; H=5.66; N= 17.11.
2-[(tert-Butoxycarbonyl)amino]-6-chloropurine 75 150
Crude compound 74 (0.60 g, 1.62 mmol) was dissolved in
ethanol (15 mL) and 1 M(aq) solution of sodium hydroxide
(10 mL) and the resulting mixture was left to stir at RT for
70 h. The solvent was removed in vacuo and water (30 mL)
Cl
N
N
H
was added to the crude residue afforded. The pH of the
N
N
O
N
H
O
75
solution was adjusted to 4-5 using 2 M (aq) citric acid and extracted with chloroform (3 x
70 mL). The combined organic layers were washed with brine (40 mL), dried over
anhydrous MgSO4, filtered and concentrated in vacuo. Trituration of the crude viscous
oil with diethyl ether (100 mL) initiated immediate crystallization. The mixture was
cooled to -20 °C and left for an hour before the precipitate was collected by filtration and
dried in vacuo. The desired compound 75 (0.28 g, 65%) was obtained as a white
crystalline solid. δH (200MHz, CDCl3) 1.51 (9H, s, (CH3)3CO), 7.82 (1H s, N-H), 8.31
(1H, s, C8-H); δC (100.6MHz, CDCl3) 27.8 (CH3), 133.6 (C), 135.6(C), 144.9 (CH),
145.3 (C), 150.1 (C), 151.6 (C), 155.4 (C)
Ethyl-2-amino-6-chloropurin-9-ylacetate (93)154
Anhydrous K2CO3 (1.09 g, 7.91 mmol) was added to a solution
Cl
of compound 58 (0.61 g, 3.60 mmol) in DMF (50 ml) and the
N
mixture was stirred under argon at RT for 10 mins. Ethyl
N
bromoacetate (0.80 mL, 7.19 mmol) was added and the
resulting mixture was stirred at RT for 5 hr. Filtration followed
N
N
NH2
O
H3CH2CO
93
by solvent evaporation in vacuo gave a crude yellow residue, which was
chromatographed on silica (ethyl acetate) to give the desired product 93 (0.78 g, 87%)
as a white solid. Rf=0.57 (ethyl acetate).
δH (200MHz, CDCl3):1.32 (3H, t, J=7.2,
CH3CH2), 4.28 (2H, q, J=7.2, CH3CH2), 4.81 (2H, s, N9-CH2CO2Et ), 5.12 (2H, brs,
138
NH2), 7.81 (1H, s, C8-H); δC (50.3MHz, CDCl3): 14.0 (CH3), 44.0 (CH2), 62.4 (CH2),
142.4 (CH), 149.6 (C), 159.1 (C), 162.0 (C), 166.7 (C)
Ethyl-2-[(tert-butoxycarbonyl)amino]-6-chloropurin-9-ylacetate (95)
Method 1
Cl
Anhydrous K2CO3 (0.14 g, 1.03 mmol) was added to a
N
stirred solution of compound 75 (0.12 g, 0.47 mmol) in
N
anhydrous DMF (20 ml) and the heterogeneous mixture
was left to stir at RT under argon for 20 min.
O
N
N
N
H
O
O
H3CH2CO
95
Subsequently, ethyl bromoacetate (0.10 ml, 0.93 mmol) was added and the mixture was
stirred at RT for 5 h. Filtration followed by solvent evaporation afforded a crude residue.
This was re-dissolved in water (100 mL) and the resulting aqueous solution was extracted
with dichloromethane (2 x 50 mL). The combined organic extracts were dried over
anhydrous MgSO4. Filtration followed by solvent evaporation afforded a crude product
which was chromatographed on silica (ethyl acetate) to give the desired compound 95
(0.26 g, 80%) as a glassy white solid.
Method 2
Lithium chloride (0.16 g, 3.76 mmol) was added to a solution of compound 84 (0.20 g,
0.44 mmol) in dry acetonitrile and the resulting mixture was stirred at 65 °C overnight.
Filtration followed by solvent evaporation afforded a crude product which was
chromatographed on silica as in method 1. This gave the desired compound 95 (0.07 g,
60%) as a glassy white solid.
Method 3
Triphosgene (0.78 g, 2.94 mmol) was added to a stirred solution of compound 93 (0.50 g,
1.96 mmol) in 1,2-dichlorobenzene: dichloromethane (1:1) (30 mL). The resulting
139
mixture was heated at 130 °C for 3 h. Subsequently, the reaction temperature was
reduced to 70 °C and a solution of tert-butanol (0.58 g, 7.84 mmol) dissolved in dry
dichloromethane (30 ml) was added. The reaction was left to stir at 70 °C for 30 min,
before being allowed to stir at RT overnight. The solvent was removed by evaporation in
vacuo and the crude product obtained was chromatographed on silica as described in
method 1. This afforded the desired product 95 (0.56 g, 75%) as a glassy white solid. Rf
0.53 [ethyl acetate]. δH (200 MHz, CDCl3) : 1.43 (9H, s, (CH3)3C), 1.21 (3H, t, J = 7.2,
OCH2CH3), 4.15 (2H, q, J = 7.2, OCH2CH3), 4.92 (2H, s, N9-CH2COOEt), 8.01 (1H, s,
C8-H); δC (50.3 MHz, CDCl3) 14.1 (CH3), 27.9 (CH3), 44.2 (CH2), 62.5 (CH2), 81.0 (C),
125.2 (C), 127.2 (C), 144.8 (CH), 148.2 (C), 150.1 (C), 152.5 (C), 166.5 (C); vmax(KBr,
cm-1): 3196(w), 2977(w), 1735(s), 1626(s), 1450(m), 1380(m), 1210(s), 1156(s),
1093(w); LRMS (EI) (m/z) (%): C14H18N5O4Cl:
355 (12) [M+], 319(4), 268(24),
147(100), 101(54), 73(32), 57(22).
Ethyl-2-[(tert-butoxycarbonyl) amino]-6-triazolpurin-9-ylacetate( 96)
Triazole (0.25 g, 3.60 mmol), and K2CO3 (0.55 g, 3.96
N
N
mmol) were added to a solution 95 (0.64 g, 1.80 mmol)
N
in anhydrous DMF (25 mL) and the resulting mixture
N
was stirred at 50 °C for 7 h. Subsequently, the solvent
N
was removed in vacuo and the residue was purified by
column chromatography (ethyl acetate) to afford the
O
N
N
N
H
O
O
H3CH2CO
96
desired compound 96 (0.42 g, 60%) as a white foam. Rf 0.45(ethyl acetate); δH (200
MHz, CDCl3): 1.29 (3H, t, OCH2CH3, J= 7.18Hz), 1.41 (9H, s, (CH3)3CO), 4.22 (2H, q,
OCH2CH3, J=7.18Hz), 4.88 (2H, s, N9-CH2COOEt), 7.61 (1H, s, C8-H), 8.32 (2H, s,
C3’-H and C5’-H); δC (50.3 MHz, CDCl3): 14.1 (CH3), 28.7 (CH3), 57.4 (CH2), 62.3
(CH2), 80.2 (C), 127.3 (C), 139.4 (CH), 140.3 (CH), 145.1 (C), 148.5 (C), 150.2 (C),
155.5 (C), 170.4 (C); vmax(KBr, cm-1): 3196(w), 2977(w), 1744(s), 1626(s), 1452(m),
1370(m), 1146(s), 1089.7(w); LRMS (EI) (m/z) (%):388((6) [M+]), 330(12), 319(64),
299(100), 212(10), 74(6), 68(45), 55(36); Elemental analysis for C16H20N8O4 : requires
C=49.48%, H= 5.19%, N= 28.85%, found C= 49.74%, H= 5.19%, N=29.12%.
140
2-[(tert-Butoxycarbonyl)amino]guan-9-yl acetic acid (97)
Compound 96 (0.30 g, 0.77 mmol) was dissolved in 1, 4-
O
dioxane (5 mL) and 2 M (aq) sodium hydroxide (4 mL)
N
was added. The resulting mixture was stirred 50 °C for 1
N
h. Subsequently, the solvent was removed in vacuo and
water (20 mL) was added. The resulting aqueous solution
NH
N
O
N
H
O
O
HO
97
was extracted with dichloromethane (3 x 5 mL). The pH of the aqueous solution was
adjusted to 3 using 2M (aq) solution of citric acid. The aqueous layer was extracted with
ethyl acetate (3 x 6 mL). The combined organic extracts were dried over anhydrous
MgSO4. Filtration followed by solvent evaporation afforded a crude viscous oil which on
trituration with diethyl ether (3 x 40 mL), afforded the desired compound 97 (0.16 g,
55%) as white foam. δH (200 MHz, CD3OD) 1.58(s, 9H, (CH3)3C), 4.67 (s, 2H, N9CH2COOH), 7.98(s, 1H, NH) 8.39(s, 1H, C8-H); δC (50.3 MHz, CD3OD) 28.3((CH3)3C),
45.2(CH2), 82.6(C(CH3)3), 129.3(C), 142.2(CH), 147.9(C), 151.6(C), 155.1(CO),
170.8(CO), 176.9(CO2H); vmax(KBr, cm-1): 3340(bm), 3196(w), 2977(m), 1735(s),
1624(s), 1450(m), 1379(m), 1156(s),1092(w); LRMS (EI) (m/z) (%): 309 ((16) [M+]),
264 (44), 252(24), 126(75), 57(100); HRMS (ES, H+): calculated for C12H15N5O5 [M+H+]
is 310.2913; found 310.2915
Ethyl-2-amino-6-(2-nitrophenoxyl)purin-9-ylacetate (94)
NO2
To a solution of 93 (0.50 g, 1.96 mmol) in 1,2-dichloroethane
(20 mL) was added 1,4-diazabicyclo [2, 2, 2] octane (0.22 g,
1.96 mmol), o-nitrophenol (0.82 g, 5.88 mmol) and
O
N
N
triethylamine (0.81 mL, 5.88 mmol). The reaction mixture was
N
N
stirred at RT overnight. Subsequently, dichloromethane (10
O
mL) was added and the resulting organic phase was washed
with a saturated solution of NaHCO3 (20 mL). The combined
NH2
H3CH2CO
94
organic phase was dried over anhydrous MgSO4. Solvent evaporation in vacuo, afforded
141
a crude product which was chromatographed on silica (ethyl acetate) to give the desired
product 94 (0.60 g, 85%) as a yellowish solid. Rf = 0.67 (ethyl acetate), mp 198-200 °C;
δH (200 MHz, CDCl3): 1.29 (3H, t, J =7.2, CH2CH3), 4.31 (2H, q, J =7.2, CH2CH3), 4.69
(2H, s, N9-CH2CO2Et), 4.95 (2H, s, NH2), 7.34-7.81 (4H, m, Ar-H) 8.19 (1H, s, C8-H);
δC (50.3 MHz, CDCl3): 14.0 (CH3), 44.0 (CH2), 62.2 (CH2), 125.3 (C), 114.5 (CH), 126.0
(CH), 134.5 (C), 140.8 (C), 142.4 (C), 145.4 (CH), 155.4 (CH), 158.9 (C), 162.44 (C),
167.0 (C); vmax(KBr,cm-1): 3496(m), 3350(m), 3195(w),2978(m), 1733(s), 1626(s),
1420(m), 1379(m), 1350(s), 1156(s), 1095(w); LRMS (EI) (m/z) (%): 358(38, [M+]), 271
(66), 221(100), 138(68), 87(78); HRMS (ES+ H+): calculated for C15H14N6O5 [M+H+] is
359.3288; found 359.3287; Elemental analysis found C= 49.95%; H= 3.92%; N=
23.45%; requires C=50.28%; H= 3.94%; N= 23.45%
Ethyl- 2-[bis-(tert-butoxycarbonyl)amino]-6-(1, 4-diazabicyclo) undecanylpurin-9ylacetate (92)
To a solution of 84 (0.20 g, 0.44 mmol) in DMF was
N
added 1,4-diazabicyclo[2,2,2]octane (0.20 g, 1.76
N
mmol) and the resulting mixture was stirred under
N
N
argon at RT for 48 h. Subsequently, the solvent was
removed in vacuo to afford a crude oil. The crude oil
N
O
was dissolved in dichloromethane and the organic
solution was washed with water (3 x 50 mL). The
resulting organic solution was dried over anhydrous
N
O
N
O
O
O
H3CH2CO
92
MgSO4. Filtration followed by solvent evaporation afforded the desired product 92 (0.14
g, 60%) as a white foam. δH (200M Hz, CDCl3): 1.40 (18H, s, 2x(C(CH3)3C), 1.29 (3H,
t, J =7.2 Hz, CH2CH3), 4.12 (2H, q, J =7.2 Hz, CH2CH3), 2.61 (4H, bm, DABCO),
3.22(4H, bm, DABCO), 3.79 (4H, bm, DABCO) 4.89 (2H, s, N9-CH2COOEt), 7.92
(1H, s, C8-H); δC (50.3MHz, CDCl3):13.8 (CH3), 27.9 (CH3), 43.8 (CH2), 52.9 (CH2),
61.8 (CH2), 59.5 (CH2), 65.6 (CH2), 82.4(C), 117.4 (C), 139.1 (C), 144.5 (CH), 151.1
(C), 152.2 (C), 153.7 (C), 167.1 (C); vmax(KBr,cm-1): 3332(w), 2966(m), 1740(s),
1630(s), 1430(m), 1339(m), 1301(w), 1140.6(s); LRMS (EI) (m/z) (%): 532 ((9) [M+]),
142
443(84), 330(100), 314(62), 300(4), 225(42), 204(91), 88(60), 53(12). Elemental analysis
for C25H38N7O6: required C= 56.38%, H= 7.19%, N = 18.41%, found C=56.04%, H=
7.31%, N= 18.78%.
Benzyl-2-amino-6-chloropurin-9yl-carboxylate (103)
To a stirred solution of 2-amino-6-Chloropurine 58 (0.40 g, 2.36
Cl
mmol) and N,N-dimethyl amino pyridine (0.029g, 0.2 mmol) in
N
anhydrous dimethylformamide (50 mL) under argon was added
N
dibenzyl dicarbonate (2.4 mL, 9.42 mmol) and the reaction
O
N
N
NH2
O
mixture was stirred under argon for 18 h at RT. A TLC of the
reaction showed completion of the reaction.
The reaction
103
mixture was evaporated to dryness in vacuo. The crude residue
was purified by column chromatography (ethyl acetate: petroleum ether, 2:1). This
resulted in partial purification of the product; however, the solid afforded was still
contaminated with brown impurities. This was finally purified triturating the solid with
fresh quantities of diethyl ether (3 x 20 mL). The solid was subsequently dried in vacuo
to give the desired compound 103 (0.08 g, 26%) as a white solid. Rf = 0.35 (ethyl
acetate: Petroleum ether, 2:1). (mp 144-145 °C); δH (400 MHz, CDCl3)
5.49 (2H, s,
NH2), 5.71 (2H, s, Ph-CH2), 7.32-7.61 (5H, m, Ar-H), 8.19 (1H, s, C8-H); δC (100.6
MHz, CDCl3) 70.1 (CH2), 128.8 (C), 129.1 (CH), 133.6 (CH), 139.6 (CH), 140.5 (CH),
147.2 (C), 150.6 (C), 152.4 (C), 153.0 (C), 160.4 (C); vmax(KBr, cm-1): 3497(m),
3313(m), 1775(m), 1742(m), 1626(s), 1561(w), 1106.1(w); Elemental analysis found
C=51.18%, H= 3.32%, N=22.95%; requires C=51.14 %, H=3.32%, N=23.06%,
143
Ethyl-2-[Benzyloxycarbonyl]amino- 6-chloropurin-9-ylacetate(105)
Cl
Triphosgene (0.37 g, 1.26 mmol) was added to a
N
stirred solution of compound 93 (0.21 g, 0.84
N
mmol) in 1,2-dichlorobenzene/dichloromethane
(4:1) solvent mixture (50 mL).
The resulting
mixture was stirred at 130 °C for 3 h.
N
N
O
N
H
O
O
H3CH2CO
105
Subsequently, the reaction temperature was reduced to 100 °C and benzyl alcohol (0.26
mL, 2.52 mmol) was added. The reaction was left to stir at 100 °C for 15 min and then at
RT overnight. Solvent evaporation gave a crude brown coloured oil which was
chromatographed on silica (ethyl acetate) to give the desired compound 105 (0.20 g,
60%) as a white solid. Rf=0.38 (ethyl acetate), mp 180-182 °C; δH (200 MHz, CDCl3)
1.24 (3H, t, J = 7.2, CH2CH3), 4.22 (2H, q. J = 7.2,CH2CH3), 4.92 (2H, s, N9CH2COOEt), 5.25 (2H, s, Ph-CH2), 7.25-7.42 (5H, m, Ar-H). 7.65 (1H, s, C8-H), 8.02
(1H, s, NH); δC (50.3 MHz, CDCl3) 14.0 (CH3), 44.3 (CH2), 62.6 (CH2), 67.5 (CH2),
127.5 (C), 128.2 (CH), 128.6 (C), 128.6 (CH), 135.5 (CH), 144.7 (CH), 151.1 (C), 151.5
(C), 152.1 (C), 153.0 (C), 166.6 (C); vmax(KBr, cm-1): 2962(s), 1742(m), 1485(w),
1300(m), 1106(m) 991 (w); LRMS (EI) (m/z) (%):389(32) (M+), 344(36), 310(22),
281(32), 255(44), 208(66), 146(24), 91(100), 79(68), 65(70), 51(28); Elemental analysis
(C17H16N5O4Cl): found C=52.14%, H= 4.06%, N=17.97%; requires C=52.38%, H=
4.14%, N= 17.97%,
Ethyl-2-[(trichloro acetyl)amino]-6-chloropurin-9ylacetate (104)
Trichloroacetic anhydride was added to a stirred
solution of 93 (0.30 g, 1.17 mmol) and N, N-dimethyl
Cl
N
N
aminopyridine (0.29 g, 2.35 mmol) in anhydrous
DMF (40 mL) at RT.
The resulting mixture was
N
Cl
N
H
Cl
O
stirred at 50 °C for 4 h. Subsequently, the solvent
was evaporated in vacuo to afford a crude residue,
N
O
H3CH2CO
Cl
104
which was chromatographed on silica (ethyl acetate) to give the desired product 104
144
(0.21g, 45%) as a cream coloured solid. Rf = 0.51 (ethyl acetate) δH (400 MHz, CDCl3)
:1.32 (3H, t, J = 7.2 CH2CH3); 4.31 (2H, q. J = 7.2 ,CH2CH3), 5.01 (2H, s, N9CH2COOEt), 8.02 (1H, s, C8-H); δC (100.6 MHz, CDCl3) 14.0 (CH3), 44.5 (C), 50.4 (C),
62.7 (CH2), 128.7 (C), 145.8 (CH), 150.7 (C), 151.5 (C), 152.8 (C), 157.8 (C), 166.5 (C);
vmax(KBr, cm-1): 3325(m), 3105(m), 2961(s), 1732(m), 1430(m), 1370.2(m); LRMS (EI)
(m/z) (%) C11H9N5O3Cl4: 401(42) (M+), 324(32), 255(100), 224(82), 169(62), 92(4),
45(52).
Ethyl-N-[(2-Tribromo acetyl)amino]-6-chloro purine-9-ylacetate (106)
Cl
To a solution of 106 (0.50 g, 1.96 mmol) in anhydrous
N
pyridine (7 mL) was added tribromoacetyl chloride (1
N
N
mL) dropwise at 0 °C. The reaction was allowed to warm
N
slowly to RT before being left to stir overnight.
Subsequently, the reaction mixture was poured onto ice
O
Br
N
H
Br
O
H3CH2CO
Br
106
(15 mL) and the resulting aqueous solution extracted with
ethyl acetate (6 x 20 mL). The combined organic extracts were dried over anhydrous
MgSO4. Filtration followed by solvent evaporation gave a crude viscous orange oil
which was chromatographed on silica (ethyl acetate) to give the desired product 106
δH (200 MHz,
(0.78g, 75%) as a brown coloured solid. Rf = 0.49 (ethyl acetate
CDCl3):1.35 (3H, t, J = 7.2, CH2CH3), 4.12 (2H, q, J = 7.2, CH2CH3), 5.23 (2H, s, N9CH2CO2Et), 8.23 (1H, s, C8-H); δC (50.3 MHz, CDCl3): 14.2 (CH3), 36.0 (C), 44.6
(CH2), 62.9 (CH2), 116.2 (C), 145.9 (CH), 149.4 (C), 150.8 (C), 152.9 (C), 158.1 (C),
166.7 (C)
Ethyl-2-[(benzyloxycarbonyl)amino]-6-triazolpurin-9-ylacetate (107)
N
Anhydrous K2CO3 (0.51 g, 3.67 mmol) was
added to the stirred solution of 105 (0.65 g, 1.67
mmol)
in
anhydrous
DMF
(50
N
N
N
N
mL).
Subsequently, triazole (0.23 g, 3.34 mmol) was
N
N
O
N
H
O
H3CH2CO
145
107
O
added to the mixture and the resulting mixture was stirred at 50 °C for 7 h. Filtration
followed by solvent evaporation in vacuo afforded a residue which was chromatographed
on silica (ethyl acetate) to give the desired product 107 (0.46 g, 65%) as a glassy white
solid. Rf = 0.54 (ethyl acetate), δH (200 MHz, CDCl3): 1.32 (3H, t, J = 7.2, CH2CH3),
4.29 (2H, q, J = 7.2, CH2CH3), 4.59 (2H, s, N9- CH2OCH2CH3), 5.22 (2H , s, Ph-CH2),
7.22-7.48 (5H, m, Ar-H), 8.12 (1H, s, C8-H), 9.56 (2H, s, C3’-H and C5’-H); δC (50.3
MHz, CDCl3) 14.1 (CH3), 46.4 (CH2), 61.5 (CH2), 65.5 (CH2), 121.6 (C), 124.5 (CH),
128.5 (CH), 129.0 (CH), 141.2 (CH), 142.5 (C), 144.6 (CH), 145.2 (C), 147.8 (C), 151.2
(C), 155.6 (C), 166.9 (C); vmax(KBr,cm-1): 2964(s), 1745(m), 1485(w), 1310(m),
1106(w), 1012.6 (w); LRMS (EI) (m/z)(%): C19H18N8O4: 422 (8) (M+), 375(22),
256(100), 221(10), 91(82), 68(52), 65(42).
Ethyl-2-[(p-nitrobenzyloxy carbonyl)amino]-6-chloropurin-9-ylacetate (109)
Triphosgene (0.87 g, 2.94 mmol) was added
Cl
to solution of 93 (0.50 g, 1.96 mmol) in 1, 2-
N
dichlorobenzene (30 mL). The resulting
N
mixture was stirred at 130 °C for 3 h.
Subsequently, the reaction temperature was
reduced to 100 °C and a solution of p-
N
N
O
N
H
O
O
H3CH2CO
NO2
109
nitrobenzyl alcohol (1.20 g, 7.84 mmol) dissolved in dry 1, 2-dichlorobenzene (10 mL)
was added, the reaction was left to stir at 90˚C for 15 mins and then left to stir at RT
overnight. Solvent evaporation afforded a crude product which was chromatographed on
silica (ethyl acetate) to give the desired product 109 (0.66 g, 78%) as creamy coloured
foam. Rf = 0.38 (ethyl acetate). δH (200 MHz, CDCl3): 1.31 (3H, t, J= 7.2, CH2CH3),
4.30 (2H, q, J= 7.2, CH2CH3), 4.62 (2H, s, CH2), 5.21 (2H, s, Ph-CH2), 7.22 (2H, d, ArH), 7.4 (2H, d, Ar-H), 8.34(1H, s, C8-H); δC (50.3 MHz, CDCl3): 14.4 (CH3), 48.4 (CH2),
62.4 (CH2), 67.8 (CH2), 121.6 (CH), 127.8 (CH), 128.2 (C), 130.2 (C), 142.1 (CH), 144.1
(C), 145.4 (C),148.5 (C), 150.1 (C), 155.4 (C), 167.8 (C); vmax(KBr, cm-1): 3321(m),
3121(w), 2962(m), 1734(s), 1591(m), 1421(m), 1336(m), 1116(w), 845.4(s). LRMS (EI)
(m/z) (%): 435 ((15) [M+]), 398(42), 335(52), 255(15), 225(52), 137(100), 112(8),
146
73(72). Elemental analysis (C17H15N6O6Cl): required: C= 46.96%, H = 3.48%, N =
19.33%, found C= 47.16%, H = 3.62%, N = 19.68%.
Synthesis of Fmoc protected PNA backbone
N-(2-Aminoethyl)glycine (115)128,164
Cloroacetic acid (7.0 g, 74.5 mmol) was added dropwise to
H
N
ethylenediamine 114 (50 ml, 74.8 mmol) at 4 °C with stirring.
The resulting mixture was left to stir for 17 h at RT. The excess
H2N
O
OH
115
solvent was removed under reduced pressure to yield a paste. This was triturated with
DMSO (20 mL), followed by diethyl ether (2 x 30 mL) and the resulting solid crystallised
from EtOH: H2O (2:1) to yield the desired product 115 (5.7 g, 80%) as a white solid. δH
(200 MHz, D2O): 2.85-3.05 (4H, m, unresolved, CH2CH2), 3.25 (2H, s, CH2COOH); δC
(50.3 sMHz, D2O) 37.8 (CH2), 45.7 (CH2), 50.8 (CH2), 177.6 (C).
[(2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}ethyl) amino]acetic acid (116)
9H-Fluoren-9-ylmethoxycarbonyl
succinimidyl
carbonate (1.43 g, 4.24 mmol) in 1,4-dioxane (22
mL) was added to a solution of N-2-aminoethyl
glycine 115 (0.50 g, 4.24 mmol) in 10% aqueous
O
O
H
N
N
H
116
O
OH
Na2CO3 (10 mL). The reaction mixture was stirred at RT for 4 h. Subsequently, water
(200 mL) was added to the suspension. The aqueous solution was acidified to pH 2 using
concentrated HCl. The white solid which precipitated and the aqueous solution were
extracted with ethyl acetate (4 x 100 mL). The combined organic extracts were dried
over anhydrous MgSO4. Filtration followed by solvent evaporation in vacuo afforded a
colourless oil which solidified on trituration with diethyl ether (2 x 30 mL). Compound
116 (0.90 g, 63 %) was afforded as a white foam. δH (200 MHz, d6-DMSO): 3.51-3.62
(m, 4H, CH2CH2), 3.75(m, 1H, NHCH2COOH,), 3.81(s, 2H, NHCH2COOH), 4.46(d, 2H,
147
J=6.6 Hz, fluorenyl -CH2), 4.51(t, 1H, J=6.7 Hz, CHCH2), 7.04-7.51(m, 8H, Ar-H);
δC(50.3 MHz, d6-DMSO): 42.0(CH2), 46.5(CH2), 64.0(CH2), 67.1(CH2), 120.0(CH),
124.0(Ar-CH), 127.1(Ar-CH), , 128.2(Ar-CH), 140.0(Ar-C), 142.1(Ar-CH), 156.0(CO),
171.5(COOH); vmax(KBr, cm-1): 3305(bm), 3102(w), 2935(s), 1746(m), 1621(w),
1583(s), 1340(m), 1213(s), 866(s), 844.4(s); LRMS (EI) (m/z) (%): 340 ((28) [M+]),
223(44), 296(68), 117 (100); HRMS (ES+H+): calculated for C19H20N2O4 [M+H+]
341.3896; found 341.3898.
Propen-2-yl [(2-{[(9H-fluoren-9-ylmethoxy) carbonyl] amino} ethyl) amino] acetate
(117)
Formation of Dicyclohexylammonium Salt of N-(2 Fluorenyl ethyl) glycine acid
N-2-Fmoc ethyl glycine acid 116 (0.50 g, 1.47 mmol) was dissolved in ethanol (10 mL),
and dicyclohexylamine was added at RT with stirring, until the solution was basic.
Subsequently, diethyl ether (20 mL) was added and a white precipitate formed. The
white precipitate was collected by filtration and re-dissolved in dichloromethane (30 mL).
Evaporation under reduced pressure afforded the dicyclohexylammonium salt as white
foam. This was used without further purification for the preparation of 117.
Formation of Allyl compound
To
a
solution
of
dicyclohexylammonium salt (0.67 g,
O
O
1.31 mmol) in dioxane (20 mL) was
added allyl bromide (0.17 mL, 1.97
H
N
N
H
117
mmol) and the resulting mixture was
O
OCH2CH=CH2
left to stir at RT overnight. Subsequently, water (150 mL) and the aqueous solution was
extracted with ethyl acetate (6 x 30 mL). The combined organic extracts were dried over
anhydrous MgSO4.
Filtration followed by evaporation in vacuo gave compound 117
(0.37 g, 75%) as a white foam. δH (400 MHz, CDCl3): 2.61-3.52 (4H, m, CH2CH2), 4.11
148
(2H, s, CH2CO2CH2CH=CH2), 4.32 (2H, d, J =6.6 Hz , CH2CH=CH2), 4.50 (1H, t, J= 6.7
Hz, CHCH2), 4.62 (2H, d, J= 6.6 Hz, Fluorenyl CH2), 5.31 (2H, d, J=7.6 Hz, CH=CH2),
5.82 (1H, m, CH2CH=CH2), 7.22-7.81 (8H m, Ar-H); δC (100.6 MHz, CDCl3) 34.6
(CH2), 50.1 (C), 51.2 (CH2), 61.8 (CH2), 66.1 (CH2), 91.2 (CH2), 124.0(CH), 127.8 (CH),
130.2 (CH), 140.1 (C), 142.3 (C), 143.4 (C), 155.4 (C), 168.2 (C); vmax(KBr, cm-1):
3204(m), 3133(w), 2791(m), 1739(m), 1641(w), 1522(m), 1431(m), 965(s), 861(s),
831(s). LRMS (EI) (m/z) (%): 380 ((48) [M+]); 351(62), 337 (24), 296(14), 203(36),
178(24), 85(66), 28(100) HRMS (ES, H+): calculated for C22H24N2O4 [M+H+] is
381.4548; found 381.4550.
2-[(tert Butoxycarbonyl Protected Guanine PNA monomer (48)
Compound 117 (0.099 g, 0.29
O
mmol), HBTU (0.11 g, 0.29 mmol)
N
NH
O
and DIPEA (0.15 mL, 0.87 mmol)
N
were added to a solution of 97
(0.09 g, 0.29 mmol) in dry DMF
(30 mL).
was
O
The resulting mixture
stirred
at
RT
N
N
H
O
O
O
N
H
overnight.
N
48
O
OCH2CH=CH2
Subsequently, the solvent was removed in vacuo and the residue was re-dissolved in ethyl
acetate (100 mL). The resulting organic solution was washed with brine (2 x 40 mL) and
a saturated solution of NaHCO3 (3 x 40 mL). The organic solution was dried over
anhydrous MgSO4. Filtration followed by solvent evaporation afforded a brown coloured
oil which was chromatographed on silica (10% methanol/dichloromethane) to give the
desired product 48 (0.09 g, 50%) as a cream coloured foam. Rf = 0.39 δH (400 MHz,
CDCl3): 1.41(9H, s, (CH3)3), 3.12 (s, 2H, N-CH2COOCH2CH=CH2), 3.21-3.50( 4H, m,
CH2CH2 of ethyl glycine), 4.42 (1H, t, J=6.7 Hz, CHCH2), 4.61 (2H, s, NCH2CON), 4.78
(2H, d, J= 6.6 Hz, CHCH2O), 5.34(2H, d, J=7.6 Hz,
CH=CH2), 5.82 (1H, m,
CH2CH=CH2), 7.22-7.61 (8H, m, Ar-H), 7.81(1H, s, C8-H); δC (100.6 MHz, CDCl3):
27.9((CH3), 38.4 (CH2), 39.5 (CH2), 45.2 (CH2), 68.3 (CH2), 69.3 (CH2), 80.1 (C), 116.4
(C), 119.4 (CH2), 126.7 (CH), 128.4 (CH), 130.4 (C), 133.6 (CH), 143.1 (C), 145.4 (CH),
149
147.1 (C), 153.9(C), 156.9 (C), 157.1 (C), 158.2 (C), 169.2 (C), 171.2 (C); vmax(KBr, cm1
): 3200(m), 3116(w), 2891(m),1736(m), 1645(w), 1541(m), 1433(m), 1346(m), 1071(s),
866(m), 835(s); HRMS (ES, H+): calculated for C34H36N7O8[M+H+] is 670.7070; found
670.7072.
tert-Butoxycarbonylamino-1, 2-propandiol (119)164
A solution of 3-amino-1,2-propanediol 118 (10.00 g, 110
mmol) in water (250 mL) was cooled in an ice bath. di-tert-
O
O
N
H
butyl dicarbonate (25.00 g, 115.00 mmol) was added and the
OH
OH
119
temperature was allowed to warm to RT. During this time,
the pH was maintained at 10.5 by the addition of 2M (aq) NaOH. The resulting mixture
was concentrated to a paste in vacuo and triturated with dichloromethane (350 mL). The
suspension was filtered and the organic phase was dried over anhydrous MgSO4. After
filtration, the solvent was removed under reduced pressure to afford the desired
compound 119 (19.8 g, 94%) initially as oil which solidified on standing to form a white
solid. δH (200 MHz, CDCl3): 1.40 (9H, bs, (CH3)3C), 3.20 (2H, m, unresolved, NCH2),
3.55 (2H, m, unresolved, CH2OH,), 3.70 (1H, m, unresolved, CH), 4.20 (1H, br s, OH),
4.35 (1H, br s, OH), 5.20 (1H, br s, NH); δC (50.3MHz, CDCl3): 28.6 (CH3), 43.9 (CH2),
64.2 (CH2), 71.1 (CH), 78.0 (C), 156.2 (C).
tert-Butoxycarbonylaminoacetaldehyde (120)164
Sodium periodate (10.20 g, 40.70 mmol) was added to a
solution of 119 (7.60 g, 39.70 mmol) in water (75 mL) and the
solution stirred at RT for 1 h. The mixture was filtered and the
O
O
N
H
O
120
filtrate extracted with dichloromethane (5 x 100 mL). The
combined organic phases were dried over anhydrous MgSO4 and after filtration; the
solvent was removed under reduced pressure. The desired compound 120 (5.34 g, 85%)
was obtained as oil which solidified on standing to afford a white solid. δH (200 MHz,
CDCl3): 1.40 (9H, s, (CH3)3C), 4.00 (2H, d, J=5.2 Hz, CH2), 5.30 (1H, br s, NH), 9.55
150
(1H , t, J= 5.4 Hz, CHO); δC (50.3 MHz, CDCl3): 28.4 (CH3), 50.7 (CH2), 78.7 (C), 156.3
(C), 200.8 (CH).
N-tert butoxycarbonyl amino ethane (123)128
To a solution of ethylene-1, 2- diamine 114 (8.00 mL, 119.67
O
mmol) in anhydrous dichloromethane (20 mL), was slowly
O
added di-tertbutoxy dicarbonate (4.62 g, 21.18 mmol) at 0 °C.
NH2
N
H
123
This mixture was allowed to stir at RT overnight. Subsequently, dichloromethane (10
mL) was added and the mixture was washed with water (2 x 50 mL). The organic layer
was dried over anhydrous MgSO4. Solvent evaporation afforded the desired product 123
(11.40 g, 60%) as a white solid. δH (200 MHz, CDCl3): 1.37 (9H, s, (CH3)3C), 2.73 (2H,
m, CH2CH2NH2), 3.15 (2H, m, CH2CH2NH2), 5.02(1H, b, CONH); δC (50.3 MHz,
CDCl3) 28.8 (CH3), 40.5 (CH2), 41.6 (CH2), 79.1 (C), 156.1(C).
Alkyl-N-(2-tert-butoxycarbonylaminoethyl) glycinate (121 and 122)164
Method A
To a solution of t-butoxycarbonylaminoacetaldehyde
O
120 (5.23 g, 32.90 mmol) in methanol (100 mL) was
added either glycine methyl ester hydrochloride (10.32
g, 82.20 mmol) or glycine ethyl ester hydrochloride
O
N
H
H
N
O
OR
121: R=Me
122: R=Et
(11.47 g, 82.20 mmol), together with NaBH3CN (2.06 g, 32.90 mmol). Subsequently, the
mixture was stirred at RT for 17 h. The solvent was evaporated under reduced pressure
and the residue dissolved in water (100 mL). The pH of the resulting solution was
adjusted to 8.0 by the addition of 2M (aq) NaOH. The aqueous solution was extracted
with dichloromethane (5 x 150 mL). The combined organic extracts were washed with
brine (2 x 30mL), dried over anhydrous MgSO4.
Followed by filtration, solvent
evaporation under reduced pressure gave a crude colourless oil which was
chromatographed on silica (dichloromethane: methanol, 19:1) to give the desired product
151
(121, 4.5g, 59%; when R= methyl; and for 122 when R= ethyl, 4.8 g, 60%).as colourless
oil.
Method B
To a solution of compound 123 (8.00 g, 50.00 mmol), in anhydrous DMF (50 ml), was
added triethylamine (22.5 mL, 200.00 mmol,) , ethylbromoacetate (5.54 mL, 50.00
mmol) and sodium iodide (6.70 g,). The resulting mixture was allowed to stir at RT for
24 h. The solvent was removed in vacuo, the crude white residue was dissolved in
dichloromethane (100 mL), and the resulting organic solution was washed with water (2
x 50 mL). The combined organic extracts were dried over anhydrous MgSO4. Solvent
evaporation gave a crude product which chromatographed on silica (dichloromethane:
methanol, 9.5:0.5). Solvent evaporation afforded the title compound 122 (7.90 g, 65%);
as colourless oil. Rf= 0.61. Spectra analysis for 121, δH (200 MHz, CDCl3): 1.40 (9H, s,
(CH3)3C), 2.70 (2H, t, J=6.0, NHCH2), 3.15 (2H, q, J=6.0, CH2CH2NH), 3.35 (2H, s,
CH2CO2Me), 5.01 (1H, br s, ((CH3)3C)NH); δC(50.3 MHz, CDCl3) 14.5 (CH3), 28.7
((CH3), 41.2 (CH2), 48.6 (CH2), 51.4 (CH2), 61.2 (CH2), 79.2 (C ), 156.0 (C), 172.4 (C).
Spectra analysis for 122 δH (200 MHz, CDCl3): 1.23 (3H, t, J=7.0 Hz, CH2CH3), 1.42
(9H, (CH3)3C), 2.72(2H,t, J=6.0Hz, NHCH2), 3.21(2H, t, J=6.0 Hz, CH2NH), 3.35 (2H, s,
CH2CO2Et), 4.18 (2H, q, J=7.0 Hz, CH2CH3), 5.09 (1H, brs, NH). δC(50.3 MHz, CDCl3)
14.5 (CH3), 28.7 (CH3), 41.2 (CH2), 48.6 (CH2), 51.4 (CH2), 61.2 (CH2), 79.2 (C), 156.0
(C), 172.4 (C).
tertbutyl-2-amino-6-chloropurin-9-ylacetate (110)
Cl
Anhydrous K2CO3 (0.90 g, 6.49 mmol) was added to a solution of 58
N
N
(0.50 g, 2.95 mmol) in anhydrous DMF (50 mL) and the mixture was
stirred at RT for 10 min. Subsequently, tert-butyl bromoacetate
N
152
NH2
O
(0.87 mL, 5.90 mmol) was added and the resulting mixture was
stirred at RT for 5 h. Filtration followed by solvent evaporation in
N
O
110
vacuo gave a yellow residue.
The crude residue was chromatographed on silica (ethyl
acetate) to give the desired product 110 (0.63 g, 75%) as a white solid. Rf = 0.42, mp 198199 °C, δH (200 MHz, CDCl3): 1.41 (9H , s, (CH3)3C), 4.64 (2H, s, CH2), 5.38 (2H s,
NH2), 7.64 (1H, s, C8-H); δC (50.3 MHz, CDCl3) 27.8 (CH3), 44.7 (CH2), 83.6 (C), 124.6
(C), 142.5 (CH), 151.2 (C), 153.8 (C), 159.1 (C), 165.7 (C); vmax(KBr, cm-1): 3470(m),
3323(m), 3205(w), 2941(m), 1729(s), 1612(s), 1563(s), 1469(m), 1412(m), 1313(m),
1175(m); LRMS (EI) (m/z) (%): 283 ( (28) [M+]), 227(100), 210(12), 182(48), 146(14),
97(8), 84(28), 69(12), 57(54); Elemental analysis for C11H14N5O2Cl: found C= 46.37%;
H=5.03%; N=24.48%; requires C =46.57%; H=4.97%; N=24.68%.
Tert-butyl-2-[(benzyloxycarbonyl)amino]-6-chloropurin-9-ylacetate (111)
Triphosgene (1.60 g, 5.29 mmol) was added to a
solution of 110 (1.00 g, 3.52 mmol) and triethylamine
Cl
N
N
O
(1.5 mL, 10.60 mmol) in dry THF (50 mL) and, the
N
resulting mixture heated at
reflux
for 3
was added to the reaction mixture and the mixture
N
H
O
O
Subsequently, benzyl alcohol (1.10 mL, 10.6 mmol)
was heated at reflux for a further 3 h.
N
h.
O
111
Filtration
followed by solvent evaporation gave a brown coloured oil which was chromatographed
on silica (93:7 ethyl acetate: methanol) to give the desired product 111 (1.18 g, 80%) as a
cream coloured solid. Rf = 0.62, mp 210-212 °C, δH (200 MHz, d6-DMSO): 1.32 (9H,
s, (CH3)3C), 4.91 (2H, s, CH2), 5.12 (2H, s, Ph-CH2), 7.21-7.39 (5H, m, Ar-H), 8.39 (1H,
s, C8-H), 10.39 (1H, s, NH); δC (50.3 MHz, d6-DMSO) 27.9 (CH3), 45.8 (CH2), 67.8
(CH2), 83.9 (C3), 127.6 (CH), 128.9 (CH), 129.0 (CH), 129.2 (CH), 137.2 (CH), 147.7
(C), 151.2 (C), 153.7 (C), 160.0 (C), 167.6 (C); vmax(KBr,cm-1): 3446(m), 2971(m),
1729(s), 1612(s), 1563(s), 1469(m), 1412(m), 1323(m), 1075(m), 865.5(m); LRMS (EI)
(m/z) (%): 417((48) [M+]), 361(38), 316(82), 272(54), 254(60), 227(82), 208(100),
91(38); Elemental analysis for C19H20N5O4Cl: found C= 53.88%, H= 4.73% N=16.58%;
requires C=54.03%; H= 4.90%, N= 16.59%.
153
tertbutyl-2-[(benzyloxycarbonyl)amino]-6-(2-nitrophenoxy)purin-9-ylacetate (112)
To solution of 111 (0.60 g, 1.44 mmol) in acetonitrile
NO2
(30 mL), was added 1, 4-diazabicyclo [2, 2, 2] octane
O
(0.16 g, 1.44 mmol,), followed by o-nitrophenol (0.61
g, 4.31 mmol) and triethylamine (0.6 mL, 4.31
mmol).
overnight.
The reaction mixture was stirred at RT
N
N
O
N
N
H
O
O
Solvent evaporation gave a brown
coloured oil which was chromatographed on silica
N
O
112
(ethyl acetate: methanol, 90:10) to give the desired
product 112 (0.67 g, 90%) as yellow foam. Rf = 0.67. δH (200 MHz, d6-DMSO):1.45
(9H, s, ((CH3)3C), 4.81 (2H, s, CH2), 5.19 (2H, s, Ph-CH2), 7.12-7.29 (5H, m, Ar-H), 7.31
(2H, m, Ar-H), 7.59-7.71 (1H, m, Ar-H), 7.91-8.01 (1H, m, Ar-H), 8.12 (1H, s, C8-H); δC
(50.3 MHz, d6-DMSO): 27.9 (CH3), 45.8 (CH2), 67.5 (CH2), 83.8 (C), 117.3 (C), 126.4
(CH), 126.6 (CH), 127.6 (CH), 128.5 (CH), 128.8 (CH), 129.0 (CH), 136.0 (CH), 143.2
(CH), 145.6 (C), 146.3 (C), 153.5 (C), 153.8 (C),155.5 (C), 160.0 (C), 167.9 (C);
vmax(KBr, cm-1): 3425(w), 2978(w), 1747(s), 1618(m), 1529(s), 1452(w), 14109M0,
1348(m), 1239(s), 1181.6(s); LRMS (EI) (m/z) (%): 520((68) [M+]), 488( 32), 399(16),
136(44), 122(100), 90(56), 33 (46), HRMS (ES, H+): calculated for C25H24N6O7: [M+H+]
is 521.1779; found 521.1777.
tertbutyl-2-[(benzyloxycarbonyl) amino]guan-9-ylacetate (113)
To a solution of 112 (1.10 g, 2.16 mmol) and 2nitrobenzaldoxime (3.50 g, 21.60 mmol) in anhydrous
acetonitrile (20 mL) was added 1,1, 3,3-tetramethyl
O
N
NH
N
N
guanidine (2.30 mL, 19.00 mmol) under argon. The
resulting solution was left to stir at RT overnight.
The solvent was evaporated in vacuo and the residue
O
N
H
O
O
O
113
was purified using flash column chromatography
applying a gradient elution [starting from diethyl ether as the eluting solvent, followed by
154
90:10, ethyl acetate: methanol]. The desired product 113 (0.67 g, 75%) was afforded as a
white solid. Rf = 0.32, δH (200 MHz, d6-DMSO): 1.48 (9H, s, ((CH3)3C), 4.92 (2H, s,
NCH2CO), 5.31 (2H ,s, Ph-CH2), 7.41-7.54 (5H, m, Ar-H), 7.99 (1H, s, C8-H)), 10.51
(1H, s, NH); δC (50.3 MHz, d6-DMSO): 27.6 (CH3), 44.7 (CH2), 67.1 (CH2), 82.3 (C),
119.2 (C), 128.0 (CH), 128.3 (CH), 128.5 (CH), 135.5 (CH), 140.1 (CH), 147.6 (C),
149.3 (C), 154.8 (C), 155.1 (C), 166.6 (C); vmax(KBr, cm-1): 3259(w), 2989(w), 1721(s),
1612(s), 1566(s), 1509(w), 1409(w), 1368(w), 1238.7(s); LRMS (EI) (m/z) (%): 399
((24) [M+]), 346 (18), 310 (72), 212 (22), 90 (48), 54 (100), HRMS (ES, H+): calculated
for C19H21N5O5:[M+H+] is 400.1615; found 400.1617.
2-[(benzyloxycarbonyl) amino] guan-9-yl acetic acid (108)146
Method A
O
A solution of 1 M (aq) NaOH (5 mL) was added to a
solution of 107 (0.20 g, 0.47 mmol) in dioxane (5 mL),
and the mixture was stirred at 50 °C for 1 h.
N
NH
N
Subsequently, the solvent was removed in vacuo and
N
N
H
O
O
O
water (10 mL) was added. The pH of the aqueous HO
solution was reduced to 3 by the addition of 2 M (aq)
108
solution of citric acid. The resulting aqueous solution was extracted with ethyl acetate (4
x 50 mL). The combined organic extracts were dried over anhydrous MgSO4. Filtration
followed by solvent evaporation afforded the desired compound 108 (0.09 g, 55%) as a
white solid.
Method B
To a suspension of 113 (0.50 g, 1.22 mmol) in dichloromethane (10 mL) was added
trifluoroacetic acid (4 mL). The resulting solution was stirred at RT for 3 h. Solvent
evaporation under reduced pressure gave a brown coloured oil. This crude product was
triturated with diethyl ether and the white precipitate afforded was washed with water and
155
collected by filtration. The precipitate was dried in vacuo over P2O5 over night.
The
desired product 108 (0.418 g, 90%) was obtained as a white solid. (mp 216-217 °C
literature mp. 214-216 °C; δH (400 MHz, d6-DMSO):4.82 (2H, s, CH2), 5.25 (2H, s, PhCH2), 7.31-7.49 (5H, m, Ar-H), 7.95 (1H, s, C8-H), 11.31 (1H, s, NH), 11.41 (1H, s,
NH), 13.19 (1H, bs, CO2H); δC (100.6 MHz, d6-DMSO): 44.3 (CH2), 67.1 (CH2), 119.3
(C), 127.9 (CH), 128.3 (CH), 128.5 (CH), 135.5 (CH), 140.2 (CH), 147.3 (C), 149.3 (C),
154.5 (C), 155.1 (C), 170.0 (C); vmax(KBr, cm-1): 3383(bm), 1752(m), 1586(m), 1529(m),
1371(s), 1202(m),1093.4(w); HRMS (ES, H+): calculated for C15H13N5O5 [M+H+] is
344.0989; found 344.0994.
2[-(Benzyloxycarbonyl) amino]-6-(2-nitrophenoxy) guan-9-yl acetic acid (124)
O2 N
To a suspension of 112 (0.60 g, 1.17 mmol) in
dichloromethane was added trifluoroacetic acid (4
O
mL). The resulting solution was stirred at RT for 3 h.
N
N
Subsequently, solvent evaporation under reduced
pressure gave a brown coloured oil.
This crude
N
N
H
O
O
product was triturated with diethyl ether (2 x 30 mL)
and the white precipitate afforded was washed with
N
O
HO
124
water, before being collected by filtration. This gave
the desired compound 124 (0.49., 90%) as a white solid. mp 199-201 °C; δH (400 MHz,
d6-DMSO): 4.99 (2H, s, CH2), 5.25 (2H, s, Ph-CH2), 7.31-7.34 (5H, m, Ar-H), 7.51-7.69
(2H, m, Ar-H), 7.61-7.70 (1H, m, Ar-H), 7.80-7.89 (1H, m, Ar-H), 8.34 (1H, s, C8-H);
δC (100.6 MHz, d6-DMSO): 44.3 (CH2), 65.6 (CH2), 116.3 (C), 125.4 (CH), 125.7 (CH),
126.9 (CH), 127.5 (CH), 127.6 (CH), 127.8 (CH),128.3 (CH), 135.6 (CH), 136.5(CH),
141.7 (CH), 144.8 (C), 151.7 (C), 151.8 (C), 154.6 (C),158.1 (C), 159.5 (C), 168.9(C);
vmax(KBr, cm-1): 3373(bm), 3124(w), 2947(w), 2366(w), 1758(m), 1623(m), 1529(s),
1457(m), 1415(s), 1353(s), 1239(s); LRMS (EI) (m/z) (%) 464 ((66) [M+]), 420 (16), 342
(44), 245 (18), 123 (34), 91(100), 45 (56), HRMS (ES, H+): calculated for C21H16N6O7
[M+H+] is 465.1153; found 465.1159.
156
N-[(tbutoxycarbonyl)amino]ethyl-N-([2-(benzyloxycarbonyl)amino]-6-[(2nitrophenoxy)] guan-9-ylglycine methyl acetate (125)
Compound 124 (0.40 g, 0.86
O2 N
mmol), HBTU (0.33 g, 0.86 mmol)
and DIPEA (0.45 ml, 2.59 mmol)
O
were added to a solution of
N
compound 121 (0.20 g, 0.86 mmol)
N
in anhydrous DMF (30 mL). The
resulting mixture was stirred at RT
overnight.
Subsequently,
the
N
O
O
O
N
N
H
O
N
H
O
O
N
OCH3
125
solvent was removed in vacuo and
the crude brown oil was chromatographed on silica (ethyl acetate: methanol, 90:10) to
give the desired product 125 (0.348 g, 70%) as off white foam. Rf = 0.55. δH (400 MHz,
CD3OD) :1.41 (9H, s, ((CH3)3C), 3.24 (2H, t, J= 6.0 Hz, CH2N), 3.41 (2H, t, J = 6.0 Hz,
NCH2), 3.49 (3H, s, CH3), 4.12 (2H, s, CH2COOCH3) 4.87 (2H, s, CH2), 5.21 (2H, s, PhCH2), 7.21-7.39 (5H, m, Ar-H)), 7.29 (2H, m, Ar-H), 7.59-7.71 (1H, m, Ar-H), 7.89-8.01
(1H, m, Ar-H), 8.11 (1H, s, C8-H); δC(100.6 MHz, CD3OD): 15.6 (CH3), 28.8 (CH3)),
30.6 (CH2), 38.2 (CH2), 45.6 (CH2), 65.8 (CH2), 68.0 (CH2), 81.0 (C), 116.2 (C), 117.6
(C), 118.8 (CH), 125.6 (C), 126.8 (CH), 129.0 (CH), 137.0 (CH), 140.4 (Ar-C), 143.4
(CH), 146.6 (C), 153.8 (C) 156.1 (C), 158.9(C), 169.8(C), 171.6(C); vmax(KBr, cm-1):
3128(w), 3051(m), 2947(m), 2366(w), 1758(m), 1629(m), 1531(s), 1457(m), 1423(s),
1358(s), 1240(s), 966(m), 866(m); HRMS (ES, Na+): calculated for C31H34N8O10
[M+Na+] is 701.6530; found 701.6532; Elemental analysis required C=54.86%, H =
5.05%, N = 16.51%, found C=55.10%, H = 5.37%, N = 17.89%.
157
2-[2-(2(benzyloxycarbonyl)-6-(2-nitrophenoxy)-9H-purin-9yl)-N-(2-(tert
butoxycarbonyl) ethyl) acetyl] glycine acetic acid (130)
O2N
To a solution of 125 (0.30 g, 0.52
mmol) in dry THF (10 mL) was
O
added 2M (aq) LiOH (5 mL) and the
N
N
resulting solution was stirred at RT
for 3 h.
N
Subsequently, the solvent
was removed in vacuo and the
resulting residue was dissolved in
water (20 ml).
N
H
O
O
O
O
O
N
O
N
H
The pH of the
N
OH
130
aqueous solution was adjusted to 3 by the addition of a 1M (aq) solution of KHSO4,
where upon a white precipitate formed. The aqueous suspension was extracted with ethyl
acetate (6 x 20 mL) and the combined organic extracts were dried over anhydrous
MgSO4. Solvent evaporation under reduced pressure gave the desired product 130 as a
white solid.(0.23 g, 67%).mp. 218-219 °C. δH (400 MHz, CD3OD):1.42 (9H, s, (CH3)3C),
3.10 (2H, s, J=6.0 Hz, NHCH2 ), 3.46 (2H, t, J=6.1 Hz, CH2NH), 4.12 (2H, s,
CH2COOH), 4.69 (2H, s, CH2), 5.22 (2H, s, Ph-CH2), 7.21-7.39 (5H, m, Ar-H), 7.29
(2H, m, Ar-H), 7.61-7.69(1H, m, Ar-H), 7.89-8.02 (1H, m, Ar-H), 8.12 (1H, s, C8-H); δC
(100.6 MHz, CD3OD): 29.3 ((CH3), 38.0 (CH2), 46.0 (CH2), 48.2 (CH2), 68.3 (CH2), 80.8
(C), 127.2 (CH), 128.4 (CH), 129.4 (CH), 129.6 (CH), 130.4 (C), 133.1 (C), 133.5 (C),
136.7 (C), 138.2 (C), 140.4 (C), 143.4 (C)144.0 (CH), 147.1 (C), 149.6 (C), 150.5 (C),
154.2 (C), 156.3 (C) 160.7 (C), 169.6 (C), 170.0 (C); vmax(KBr,cm-1): 3305(bm),
3226(m), 3128(w), 2947(m), 2366(w), 1758(m), 1628(m), 1531(s), 1457(m), 1430(s),
1342(s), 1238(s), 967(m), 868(m); HRMS (ES, H+): Calculated for C30H32N8O10 [M+H+]
is 665.6441; Found 665.6444. Elemental analysis Required C = 54.22%, H = 4.85%, N =
16.86%, Found C = 54.56%, H = 5.11%, N = 17.21%.
158
2-(N-(2-aminoethyl)-2-(2-benzyloxycarbonyl)-6-(2-nitrophenoxy)-9H-purin-9yl)
acetyl) glycine methyl ester (129)
Trifluoroacetic acid (5 mL) was added to a
O2N
suspension of 125 (0.15 g, 0.22 mmol) in
dichloromethane (15 mL) and the reaction
O
A 10 fold
N
excess of diethyl ether was added and a
N
was left to stir at RT for 4 h.
precipitate formed. The ethereal solution
N
O
N
H
O
O
O
was decanted off and the precipitate was
triturated with further quantities of fresh
N
H2N
N
OCH3
129
diethylether (2 x 30 mL). The precipitate
was dried in vacuo to afford the desired product 129 (0.10 g, 80%) as a yellow foam. δH
(400 MHz, CD3OD): 2.89 (2H, t, J= 6.0 Hz, CH2), 3.42 (2H, t, J = 6.0 Hz, CH2), 3.56
(3H, s, CH3), 4.87 (2H, s, CH2), 4.61 (2H, s, CH2COOCH3), 5.68 (2H, s, Ph-CH2), 7.017.89 (9H, m, Ar-H), 8.14 (1H, s, C8-H); δC (100.6 MHz, CD3OD): 15.9 (CH3), 22.0
(CH2), 29.2 (CH2), 43.9 (CH2), 65.6 (CH2), 67.4 (CH2), 116.5 (C), 127.2 (CH), 128.4
(CH), 129.4 (CH), 129.6 (CH), 130.4 (C), 133.5 (C), 136.7 (C), 138.2 (C), 141.2 (C),
143.6 (C), 144.0 (CH), 147.1 (C), 150.6 (C), 160.7 (C), 169.6 (C), 170.0 (C); vmax(KBr,
cm-1): 3226(m), 3127(w), 2947(m), 2366(w), 1758(m), 1628(m), 1531(s), 1457(m),
1430(s), 1342(s), 1238(s), 946(m), 877(m); LRMS (EI) (m/z) (%) 578 ((14) [M+]), 455
(56), 431 (64), 324 (14), 297 (15), 224 (26), 147 (18), 123 (26), 118 (100), 91 (44),
HRMS (ES, H+): calculated for C26H26N8O8 [M+H+] is 579.5530; found 579.5533.
159
N-[2-(N-9-tertbutoxycarbonyl) amino ethyl]-N-[(2-N-(benzyloxycarbonyl) guan-9yl)
acetyl] glycine methyl acetate (49)
O
Compound 122 (0.13 g, 0.58 mmol),
was added to a stirred solution of
N
HBTU (0.22 g, 0.58 mmol), DIPEA
N
(0.30 mL, 1.74 mmol) and 108 (0.20
g, 0.58 mmol) in DMF (30 mL), the
resulting mixture was stirred at RT
N
O
O
N
H
O
O
O
O
NH
N
H
N
OCH3
49
overnight. Subsequently, the solvent
was removed in vacuo and ethyl acetate (100 mL) was added to the residue. The organic
solution was washed with brine (2 x 40 mL) and saturated solution of NaHCO3 (3 x 40
mL).
The combined organic extracts were dried over anhydrous MgSO4. Filtration
followed by solvent evaporation afforded the desired compound 108 (0.18g, 55%) as a
cream coloured foam. This was used in the next step without any further purification. δH
(400 MHz, d6-DMSO): 1.40 (9H, s, ((CH3)3C), 2.98 (2H, s, J=6.0 Hz, NHCH2 ), 3.34
(2H, t, J=6.1 Hz, CH2NH), 3.87 (3H, s, OCH3), 4.16 (2H , s, CH2), 4.69 (2H, s, N9CH2CO), 5.41 (2H, s, Ph-CH2), 7.29-7.51(5H, m, Ar-H), 7.91(1H, s, C8-H); δC (100.6
MHz, d6-DMSO): 14.1(CH3), 27.9 (CH3), 38.2 (CH2), 46.4 (CH2), 48.5 (CH2), 65.7
(CH2), 80.4 (C), 116.7 (C), 127.1 (CH), 128.5 (CH), 135.6 (CH), 141.2 (CH), 142.4
(CH), 142.4 (C), 150.3 (C), 155.7 (C), 156.4 (C), 156.9 (C), 166.6 (C), 168.9 (C),
171.8(C).
Methyl-2-(N-(2-aminoethyl)-2-(2-(benzyloxycarbonyl)-6-oxo-1,
6-dihydroxypurin-
9yl) acetamido) acetate (127)
O
Trifluoroacetic acid (5 mL) was added to a
N
suspension of 49 (0.15 g, 0.26 mmol) in
N
dichloromethane (15 mL) and the reaction was
H2N
N
127
160
N
N
H
O
O
left to stir at RT for 4 h. A 10 fold excess
diethyl ether was added and a white precipitate
NH
OCH3
O
O
was formed. The ethereal solution was decanted off and the precipitate was triturated
with further quantities of fresh diethyl ether (2 x 20 mL). The precipitate was dried in
vacuo to afford the desired product 127 (0.10 g, 85%), as a white solid. δH (400 MHz,
CD3OD): 3.42 (2H, t, J= 6.1 Hz, CH2), 3.62 (2H, t, J = 6.1 Hz, CH2), 3.99 (3H, s, CH3),
4.41 (2H, s, CH2), 4.62 (2H, s, CH2), 5.09 (2H, s, Ph-CH2), 7.10-7.61 (5H, m, Ar-H), 7.91
(1H, s, C8-H); δC (100.6 MHz, CD3OD): 21.6 (CH3), 38.4 (CH2), 58.5 (CH2), 45.6 (CH2),
66.4 (CH2), 125.4 (C), 117.6 (C), 120.8 (C), 127.6 (CH), 128.5 (CH), 132.9 (C), 142.1
(CH), 144.8 (C), 147.3 (C), 150.3 (C), 154.3 (C), 155.6 (C), 166.9 (C), 171.4 (C);
vmax(KBr, cm-1): 3226(m), 3128(w), 3015(m), 2857(m), 1749(m), 1618(m), 1533(s),
1457(m), 1436(s), 1332(s), 1018(s); LRMS (EI) (m/z) (%) 457 ((78) [M+]), 324 (52), 247
(64), 134 (58), 90 (48), 69 (100), 18 (21) HRMS (ES, H+): calculated for C20H23N7O6
[M+H+] 458.4568; found 458.4569.
2-(2-(benzyloxycarbonyl)-6-oxo-1, 6-dihydropurin-9-yl)-N-(2-(tert- butoxycarbonyl)
ethyl) acetamido) acetic acid (126)
O
To a solution of 49 (0.15 g, 0.26
N
mmol) in THF (10 mL) was added 2
N
M (aq) LiOH (5 mL) and the resulting
solution was stirred at RT for 3 h.
Subsequently,
the
solvent
was
N
N
H
O
O
O
O
O
O
NH
N
H
N
OH
126
removed in vacuo and the resulting
residue was dissolved in water (20 mL). The pH of the aqueous solution was adjusted to
3 by the addition of a solution of KHSO4 whereupon a white precipitate formed. The
aqueous suspension was extracted with ethyl acetate (6 x 20 mL) and the combined
organic extracts were dried over anhydrous MgSO4. Solvent evaporation under reduced
pressure gave the desired product 126 (0.15 g, 82%), as cream foam. δH (400 MHz, d6DMSO): 1.42 (9H, s, (CH3)3C), 3.21 (2H, m, CH2), 4.25 (2H, m, CH2), 4.92 (2H, s,
CH2) 5.22 (2H, s, Ph-CH2), 7.21-7.50 (5H, m, Ar-H), 7.91 (1H, s, C8-H), 11.5 ( 1H,
bs,CO2H); δC (100.6 MHz, d6-DMSO): 36.1 (CH2), 44.4 (CH2), 47.7 (CH2), 56.0 (CH2),
67.0 (CH2), 107.7 (C), 117.1 (C), 126.4 (CH), 128.0 (CH), 128.3 (CH), 135.6 (CH),
161
140.9 (CH), 142.5 (C), 145.6 (C), 147.5 (C), 149.8 (C), 154.8 (C), 154.9 (C), 155.4 (C),
155.6 (C), 170.0 (C); vmax(KBr, cm-1): 3128(w), 3035(bm), 2837(m), 1716(s), 1608(m),
1543(s), 1497(s), 1436(s), 1296(s), 1098(s); LRMS (EI) (m/z) (%) 543 ((18) [M+]), 498
(57), 396 (14), 327 ( 24), 254 (48), 218 (34), 148 (100), 90 (25), 78 (48), HRMS (ES,
H+): calculated for C24H29N7O8 [M+H+] is 544.5479; found 544.5481.
Methyl-2-(2-(2-(benzyloxycarbonyl)-6-(2-nitrophenoxy)-1.6-dihydropurin-9-yl)-N(2-(2-(2-(2-(2-(benzyloxycarbonyl)-6-(2-ntrophenoxy)-1,6-dihydropurin-9-yl)-N-(2tertbutoxycarbonyl) ethyl) acetamido)acetamido) ethyl acetamido) acetate (131)
Compound 130 (0.10 g,
0.17 mmol) and 129
(0.04 g, 0.17 mmol) and
HOBt (0.028 g, 0.21
mmol) were dissolved
in
DMF
(20
Subsequently,
amine
(48µL,
mL).
triethyl
0.35
mmol) was added to the
stirred mixture, this was
followed
by
the
addition of DCC (0.039 g, 0.19 mmol) before being left to stir at RT overnight. Solvent
evaporation gave crude yellow oil which was chromatographed on silica (90:10 ethyl
acetate: methanol) to give the desired product 131 (0.16 g, 80%) as a yellow foam. Rf =
0.54. δH (400 MHz, d6-DMSO) 1.42 (CH3), 3.22 (2H, m unresolved), 3.42 (2H,m,
unresolved CH2), 3.46 (2H, m, unresolved, CH2 ), 3.56 (3H, s, OCH3), 4.09 (2H, s, CH2),
4.12 (2H, s, CH2), 4.62 (4H, s, N9-CH2CO), 5.12 (4H, s, Ph-CH2), 7.19-7.21 (10H, m,
Ar-H), 8.11 (2H, s, C8-H); δC (100.6 MHz, d6-DMSO):28.5 (CH3)3, 36.4, 46.7, 47.8,
50.2, 65.6, 80.4, 116.7, 125.2, 126.6, 127.4, 128.7, 133.5, 145.3, 150.6, 156.5, 167.5,
169.6, 170.5; vmax(KBr, cm-1): 3126(w), 3047(m), 2973(m), 2368(w), 1740(s), 1628(s),
162
1553(s), 1455(m), 1426(m), 1343(m), 1065(m), 870.4(m); HRMS (ES, Na+): calculated
for C56H56N16O17 [M+Na+] is 1248.1576; found 1248.1578.
Methyl-2(2-(2-(benzyloxycarbonyl)-6-oxo-1,6-dihydropurin-9-yl)-N-(2-(2-(2-(2benzyloxycarbonyl)-6-oxo-1,6-dihydropurin-9-yl)-N-(-(tertbutoxycarbonyl)ethyl)
acetamido) acetamido) ethyl) acetamido) acetate (128)
METHOD A
O
The procedure for the
synthesis
N
of
N
compound 131 was
followed
when
coupling compounds
127(0.10
g,
O
O
0.18
0.18
mmol)
Subsequently
H
N
O
O
together.
N
H
O
N
H
N
O
O
mmol) and 126 (0.084
g,
N
N
O
O O
O
N
H
NH
N
N
N
HN
O
128
the
solvent was evaporated and the crude product obtained was triturated first with ethyl
acetate (2 x 40 mL), followed by diethyl ether (2 x 40 mL). Solvent evaporation in vacuo
afforded the desired product 128 (0.036g, 20%) as a cream solid.
METHOD B
The same procedure for the synthesis of compound 113 was followed. Using compound
131 (0.22 g, 0.18 mmol).
After the completion of the reaction, the solvent was
evaporated in vacuo, this gave a crude residue which was chromatographed on silica
using gradient elution, starting with diethyl ether first, followed by 90:10 ethyl acetate:
methanol. The ethyl acetate: methanol fraction gave the desired product (0.035 g, 20%),
Rf = 0.32 as a cream coloured foam. δH (400 MHz, d6-DMSO): 1.42 (CH3)3, 3.22 (2H,m
unresolved (CH2)), 3.42 (2H,m, unresolved, CH2), 3.46 (2H, m, unresolved, CH2), 3.56
163
(3H, s, CH3), 4.09 (2H, s, CH2), 4.12 (2H, s, CH2), 4.62 (4H, s), 5.12 (4H, s, Ph-CH2),
7.19-7.21 (10H, m, Ar-H), 8.11 (2H, s, C8-H); δC(100.6MHz, d6-DMSO): 28.5 ((CH3),
36.4 (CH2), 46.7 (CH2), 47.8 (CH2), 50.2 (CH2), 65.6 (CH2), 80.4 (C), 126.6 (CH), 127.4
(CH), 128.7 (CH), 145.3 (CH), 149.2 (C) 150.6 (C), 156.5 (C), 167.5 (C), 169.6(C),
170.5 (C); vmax(KBr, cm-1): 3321(m), 3148(w), 3025(bm), 2848(m), 1736(s), 1618(m),
1543(s), 1497(s), 1431(s), 1293(s), 1198(s); HRMS (ES, Na+): calculated for
C44H50N14O13 [M+Na+] is 1005.9634; found 1005.9636.
Ethyl-2-[(4-nitrobenzyloxycarbonyl)amino]-6-chloropurin-9-ylacetate (143)
Triphosgene (1.10 g, 3.77 mmol) was
Cl
added to a solution of 110 (0.71 g, 2.51
N
mmol) and triethylamine (1.2 mL, 7.53
O
N
N
N
mmol) in THF (40 mL) and the mixture
N
H
O
O
was heated at reflux for 5 h. Subsequently,
NO2
(H3C)3CO
p-nitrobenzyl alcohol (1.15 mL, 7.53
143
mmol) was added to the reaction mixture
and the mixture heated at reflux for a further 3 h. The solvent was removed in vacuo and
the crude yellow solid chromatographed on silica (7% methanol in ethyl acetate). The
desired product 143 (0.63 g, 70%) was obtained as a cream foam. .Rf = 0.68. δH (200
MHz, CD3OD):1.45 (9H, s, ((CH3)3C), 4.91 (2H, s, CH2), 5.34 (2H, s, Ph-CH2), 7.62
(2H, m, Ar-H), 8.24 (2H, m, Ar-H), 8.35 (1H, s, C8-H); δC (50.3 MHz, CD3OD): 28.3
(CH3), 46.3 (CH2), 66.7 (CH2), 84.5 (C ), 124.6 (CH), 124.8 (CH), 128.3 (C), 129.5 (C),
145.4 (CH), 148.1 (C), 149.1 (C), 150.1 (C), 168.1 (C); vmax(KBr, cm-1): 3336(m),
2879(m), 2368(w), 1740(s), 1632(s), 1567(s), 1470(m), 1426(m), 1336(m), 1239(m),
1175(m), 966(m); LRMS (EI) (m/z) (%): C19H19N6O6Cl: 462(26) (M+), 425(8), 329(48),
267(12), 200(4), 197(22), 132(100), 101(20), 89(42). Elemental analysis requires C=
49.31%, H = 4.14%, N = 18.16%, found C = 49.65%, H = 4.51%, N = 18.48%.
164
Tert-butyl-2-[(4-nitrobenzyloxycarbonyl)amino]-6-(2-nitrophenoxy)purin-9ylacetate(144)
NO2
To a solution of 143 (0.26 g, 0.56 mmol) in
acetonitrile (30 mL) was added DABCO
O
(0.06 g, 0.56 mmol), this was followed by
N
the addition of o-nitrophenol (0.23 g, 1.68
N
mmol) and triethyl amine (0.32 μL, 1.68
mmol). The resulting mixture was allowed
N
N
O
N
H
O
O
NO2
(H3C)3CO
to stir at RT overnight. Subsequently, after
144
work-up and purification by column chromatography, the desired product 144 (0.33 g,
80%), was obtained as yellow foam. Rf = 0.45 (ethyl acetate). δH (200 MHz, CD3OD):
1.62 (9H, s, (CH3)3C), 5.02 (2H, s, CH2), 5.42 (2H, s, Ph-CH2), 7.09-7.19(1H, m, Ar-H),
7.32 (1H, m, Ar-H), 7.62 (2H, m, Ar-H), 7.92 (2H, m, Ar-H), 8.21 (1H, m, Ar-H), 8.44
(1H, s, C8-H); δC (50.3 MHz, CD3OD): 27.9 (CH3), 45.8 (CH2), 66.0 (CH2), 83.8 (C),
119.5 (C),120.5 (CH), 124.2 (CH), 125.8 (CH), 126.4 (CH), 127.6(Ar-CH), 129.0 (CH),
130.8 (C) 136.0 (C), 143.2 (CH), 145.0 (C), 145.7 (C), 146.2 (C), 149.6 (C), 153.3 (C),
155.5 (C), 167.9 (C); vmax(KBr, cm-1): 3236(m), 3106(w), 2890(m), 2381(m), 1743(m),
1643(s), 1558(s), 1486(s), 1436(s), 1356(w), 1249(m), 1080(m), 868(m); HRMS (ES,
H+): calculated for C15H23N7O7 [M+H+] is 566.5108; found 566.5108.
2-[(4-nitrobenzyloxycarbonyl)amino]-6-(2-nitrophenoxy)purin-9-ylacetic acid (145)
NO2
To a suspension of 144 (0.23 g, 0.40 mmol) in
dichloromethane was added Trifluoroacetic acid
(4 mL). The resulting solution was stirred at RT
O
N
N
for 3 h. Subsequently, solvent evaporation under
reduced pressure gave a brown coloured oil. The
N
N
H
O
crude product was triturated with diethyl ether (2
x 30 mL) and the white precipitate afforded was
N
O
NO2
HO
145
165
O
washed with water (3 x 30 mL), before being collected by filtration and dried over P 2O5.
This afforded the desired product 145 (0.18 g, 90%) as a yellow solid. mp 212-213 ˚C: δH
(200 MHz, d6-DMSO): 48.21 (2H, s, CH2), 5.21 (2H, s, Ph-CH2) 7.01-7.42(1H, m, ArH), 7.69 (1H, m, Ar-H), 7.59 (2H m, , Ar-H), 8.21 (2H, m, Ar-H), 8.29 (1H, m, C8-H),
8.39 (1H, s, NH), 13.21 (1H, br s, CO2H); δC (50.3 MHz, d6-DMSO): 45.8 (CH2), 66.0
(CH2), 120.5 (CH), 124.2 (CH), 125.8 (CH), 126.4 (CH), 127.6 (CH), 129.0 (CH), 134.5
(C) 136.0 (C), 143.2 (CH), 145.0 (C), 145.7 (C), 146.2 (C), 148.4 (C), 150.5 (C), 153.3
(C), 155.5 (C), 173.1 (C); vmax(KBr, cm-1)
3436(bm), 3125(w), 2979(s), 2369(w),
1750(s), 1622(m), 1577(s), 1467(m), 1416(m), 1241(m), 1075(m); LRMS (EI) (m/z) (%)
509 ((16) [M+]), 464 (22), 387 (21), 371 (48), 245 (57), 139 124 (16), (100) HRMS (ES,
H+) : calculated (C21H15N7O7) 510.4027; found 510.4030.
Tert-butyl-2-[(4-nitrobenzyloxycarbonyl) amino]guan-9-ylacetate (146)
O
To a solution 145 (0.23 g, 0.40 mmol), and
2-nitrobenzaldoxime (0.64 g, 4.0 mmol) in
N
anhydrous acetonitrile (30 mL), was added
N
1,1, 3,3-tetramethylguanine (0.45 μL, 3.6
mmol) under argon. The resulting mixture
was left to stir at RT overnight. The solvent
O
NH
N
N
H
O
O
NO2
(H3C)3CO
146
was evaporated in vacuo and the crude residue was purified using flash column
chromatography, applying gradient elution (starting from diethyl ether as the eluting
solvent, followed by 90:10 ethyl acetate: methanol). The desired product 146 (0.13 g,
75%) was afforded as a yellow powder. Rf = 0.56. δH (200 MHz, d6-DMSO): 1.39 (9H,
s, (CH3)3C), 4.82 (2H, s, CH2), 5.38 (2H, s, Ph-CH2), 7.65 (2H, m, Ar-H), 7.89 (1H, s,
C8-H), 8.25 (2H, m, Ar-H), 11.49 (1H, br s, NH); δC (50.3 MHz, d6-DMSO): 27.7
(CH3), 44.8 (CH2), 65.7 (CH2), 82.3 (C), 119.2 (C), 123.3 (CH), 123.6 (CH), 127.0 (CH),
128.0 (CH), 128.3 (C), 140.1 (C), 143.5 (CH), 147.2 (C), 155.2 (C), 166.7 (C); vmax(KBr,
cm-1): 3436(m), 2979(s), 1746(m), 1622(s), 1573(s),1469(m), 1411(w), 1351(w),
1226(m), 895(m); LRMS (EI) (m/z) (%): C19H20N6O7: 444 (46) (M+), 400(12), 370(8),
356(22), 218(10), 147(26), 132(100), 97(14), 101(34), 101(34). Elemental analysis
166
required C = 51.35%, H = 4.54%, N = 18.91%. found C = 51.64%, H = 4.81%, N =
19.11%.
2-[Benzyloxycarbonyl]amino-6-(2-nitrophenoxy)purin-9-ylacetonitrile (173)
Triphosgene (0.96 g, 3.22 mmol) was added to a
NO2
solution of 172 (0.50 g, 1.61 mmol) and
O
triethylamine (0.91 μL, 4.83 mmol) in dry THF (50
mL), and the resulting mixture was heated at reflux
for 3 h. Subsequently, benzyl alcohol (0.5 μL, 4.83
mmol) was added and the mixture was heated
further at reflux for a further 5 h. The solvent was
N
N
N
N
O
N
H
O
CN
173
evaporated and the crude extract was chromatographed on silica using gradient elution,
with first, diethyl ether and followed by 10% methanol in ethyl acetate, Solvent
evaporation of the ethyl acetate: methanol fraction gave the desired product 173 (0.57 g,
80%) as a yellow foam. Rf = 0.64, δH (200 MHz, d6-DMSO): 5.12 (2H, s, NCH2CN), 5.51
(2H, s, Ph-CH2), 7.12-7.92 (9H, m, Ar-H), 8.52 (1H, s, C8-H); δC (50.3 MHz, d6-DMSO):
31.6 (CH2), 65.9 (CH2) 115.6 (C), 116.5 (CN), 125.6 (CH), 126.0 (CH), 127.2 (CH),
127.8 (CH), 128.0 (CH), 128.3 (CH), 128.5 (CH), 129.0 (C), 130.8 (C), 135.8 (CH),
136.6 (CH), 141.8 (CH), 143.6 (C), 151.7 (C), 152.4 (C), 154.1 (C); vmax(KBr, cm-1):
3150(w), 2850(s), 1633(m), 1578(m), 1518(m), 1483(m), 1382(s), 1246(m),1175(w),
1082(w), 1022(w); LRMS (EI) (m/z) (%) 445 ((28), [M+]), 419 (34), 354 (62), 254 (15),
226 (100), 123 (18), 91 (54), 27 (68), HRMS (ES, Na+): calculated for
C21H15N7O5:[M+Na+] 468.1027: found 468.1030.
167
2-[(4-nitrobenzyloxycarbonyl) amino]guan-9-ylacetic acid (147)
O
To a suspension of 146 (0.10 g, 0.23 mmol) in
dichloromethane
(10
mL)
Trifluoroacetic acid (4 mL).
was
added
The resulting
N
NH
N
mixture was stirred at RT for 3 h. Solvent
N
N
H
O
O
O
evaporation under reduced pressure gave a HO
NO2
147
brown coloured oil. The crude product was triturated with diethyl ether (3 x 20 mL).
This afforded a white precipitate which was washed with water (3 x 20 mL) and collected
by filtration. The precipitate was dried in vacuo over P2O5 overnight. This afforded the
desired product 147 (0.78 g, 90%) as a white solid. mp 224-226 °C; δH (200 MHz, d6DMSO): 4.89 (2H, s, CH2), 5.29 (2H, s, Ph-CH2), 7.62 (2H, m, Ar-H), 7.84 (1H, s, C8H), 8.14 (2H, m, Ar-H); δC (50.3 MHz, d6-DMSO): 44.8 (CH2), 65.5 (CH2), 119.2 (C),
123.3 (CH), 128.0 (CH), 128.3 (C), 140.1 (C), 143.5 (CH), 145.5 (C), 147.2 (C), 156.2
(C), 174.7 (C); vmax(KBr, cm-1): 3346(bm), 3212(m), 3011(w), 2876(s), 1736(m),
1616(s), 1567(s), 1457(m), 1421(w), 1346(w), 1006(m), 968(m); LRMS (EI) (m/z)
(%):C15H12N6O7: 388(24) (M+), 345(38), 317(14), 301(12), 287(18), 244(66), 131(36),
102(100), 91(46); Elemental Analysis: found: %C= 46.12, H= 3.13, N=22.01; requires
%C= 46.40, H= 3.12, N= 21.64.
General procedure for the synthesis of compounds 148,149,150, 151 and 165
To a solution of compound each of the compounds (0.50g, 1eq) in anhydrous DMF was
added anhydrous K2CO3 (2.2eq), followed by the addition of the bromoesters (2eq) (for
compounds 148, 149, 150 and 151) and bromoethanol (2eq) for compound 167, the
mixture was stirred at RT for 5 h. The completion of the reaction was determined by the
TLC. Solvent evaporation gave yellow oil. Water (100 mL) was added to the residue to
give a white precipitate. The precipitate was filtered and washed with diethyl ether (4 x
30 mL). This afforded the desired products as a white solid.
168
2-amino-6-chloropurin-9-ylethanol (167)
Compound 58 (0.50 g, 2.95mmol), anhydrous K2CO3 (0.90 g, 6.49
Cl
mmol) and bromoethanol (0.5 mL, 5.90 mmol) gave the desired
N
product 167 (0.50 g, 80%) as a white solid. mp 186-187 °C; δH
N
(200 MHz, d6-DMSO): 3.82 (2H , b, NCH2CH2OH), 4.21 (2H, m,
N
N
NH2
CH2OH
NCH2CH2OH), 6.92 (2H, s, NH2), 8.12 (1H, s, C8-H); δC (50.3
167
MHz, d6-DMSO): 46.6 (CH2), 59.6 (CH2) 120.5 (C), 124.2 (C), 144.6 (CH), 154.9 (C),
160.5 (C); vmax(KBr, cm-1): 3350(bm), 3205(w), 3105(w), 2981(m), 1620(w), 1519(m),
1437(m), 1357(m),1105(m); LRMS (EI) (m/z)(%): C7H8N5OCl: 213(89) (M+), 182(66),
169(98), 146(63), 134(100), 119(14), 106(14), 92(30), 86(16), 67(20), 53(30), 43(68).
Elemental analysis required C = 39.36%, H = 3.77%, N = 32.78%, found C = 39.64%, H
= 4.14%, N = 33.11%.
Ethyl-2-amino -6-chloropurin-9-ylpropanoate(148)
Cl
Compound 58 (0.50 g, 2.95 mmol), K2CO3 (0.89 g, 6.49 mmol),
ethyl-3-bromopropionate (0.75 mL, 5.90 mmol), gave the desired
N
product 148 (0.59 g, 75%) as a white solid. mp 179-180 °C; δH
(200 MHz, d6-DMSO): 1.15 (3H, t, OCH2CH3), 2.92 (2H, t,
N
( )2
NCH2CH2CO), 4.12 (2H, q, OCH2CH3), 4.34 (2H, t,
O
N
N
NH2
O
148
NCH2CH2CO), 6.95 (2H, s, NH2), 8.10 (1H, s, C8-H); δC (50.3
MHz, d6-DMSO): 14.4 (CH3), 33.2 (CH2), 58.8 (CH2), 60.3 (CH2), 123.3 (C), 143.3
(CH), 149.3 (C), 154.0 (C), 159.7 (C), 170.4 (C), vmax(KBr, cm-1): 3428(s), 3324(s),
3200(s), 2940(m), 2857(w), 1733(s), 1635(s), 1609(s), 1562(s), 1464(s); LRMS (EI)
(m/z) (%): 269 (62) (M+), 224(15), 196(22), 169(82), 146(12), 134(64), 97(16), 81(28),
69(72), 55(78).
169
Ethyl-2-amino -6-chloropurin-9-ylbutanoate (149)
Compound 58 (0.50 g, 2.95mmol), K2CO3 (0.89 g, 6.49 mmol)
ethyl-4-bromobutyrate (0.87 mL, 5.90 mmol), gave the desired
product 149 (0.71g, 85%) as a white solid. mp 199-200 °C; δH
Cl
N
N
(200 MHz, d6-DMSO): 1.22 (3H, t, OCH2CH3), 2.15 (2H, m,
N
( )3
NCH2CH2CH2CO), 2.35 (2H, t, NCH2CH2CH2CO), 3.98 (2H, t,
O
N
NH2
O
149
NCH2CH2CH2CO), 4.19 (2H, q, OCH2CH3), 6.92 (2H, s, NH2),
8.22 (1H, s, C8-H); δC (50.3 MHz, d6-DMSO) 14.1 (CH3), 24.5 (CH2), 26.2 (CH2), 30.8
(CH2), 60.1 (CH2), 123.6 (C), 143.5 (CH), 149.5 (C), 154.4 (C), 159.9 (C), 172.3 (C);
vmax(KBr, cm-1):
3428(s), 3324(s), 3200(s), 2940(m), 2857(w), 1733(s), 1635(s),
1609(s), 1562(s), 1464(s); LRMS (EI) (m/z) (%): 283 (59) (M+), 238(54), 210(50),
196(57), 183(74), 169(54), 134(58), 115(51), 92(38), 87(68), 69(47), 55(52), 43(100).
Ethyl-2-amino -6-chloropurin-9-ylpentanoate (150)
Cl
Compound 58 (0.5 g, 2.95 mmol), K2CO3 (0.89 g, 6.49 mmol) ethyl5-bromovalerate (0.93 mL, 5.90mmol), gave the desired product 150
N
(0.67 g, 77%) as a white solid. mp 181-183 °C: δH (200 MHz, d6m,
N
( )4
NCH2CH2CH2CH2CO), 1.96 (2H, m, NCH2CH2CH2CH2CO), 2.49
O
DMSO)
1.31
(3H,
t,
OCH2CH3),
1.65
(2H,
N
N
NH2
O
150
(2H, t, NCH2CH2CH2CH2CO), 3.72 (2H, t, NCH2CH2CH2CH2CO),
4.24 (2H, q, OCH2CH3), 7.15 (2H, s, NH2), 8.43 (1H, s, C8-H); δC (50.3 MHz, d6DMSO) :14.4 (CH3), 21.7 (CH2), 30.3 (CH2), 34.2 (CH2), 46.2 (CH2), 60.1 (CH2), 114.8
(C), 143.6 (CH), 149.8 (C), 154.5 (C), 160.4 (C), 172.9 (C); vmax(KBr, cm-1): 1463.5(s),
1562.1(s), 1608.8(s), 1634.7(s), 1733.4(s), 2857.1(w), 2940.2(m), 3199.6(s), 3324.1(s),
3427.9(s); LRMS (EI) (m/z) (%): 297(79) (M+), 252(78), 224(71), 196(78), 183(100),
169(82), 146(57), 134(73), 101(44), 55(54).
170
Ethyl-2-amino -6-chloropurin-9-yl hexanoate (151)
Cl
Compound 58 (0.5 g, 2.95 mmol), K2CO3 (0.89 g, 6.49 mmol)
ethyl-6-bromohexanoate (1.04 mL, 5.90 mmol), gave the
desired product 151 (0.75 g, 82%) as a white solid. δH (200
MHz, CD3OD)
1.22 (3H, t, OCH2CH3), 1.32 (2H, m,
NCH2CH2CH2CH2CH2CO),
1.72
(2H,
m,
N
N
( )5
N
N
NH2
O
O
151
NCH2CH2CH2CH2CH2CO), 1.92 (2H, m, NCH2CH2CH2CH2CH2CO), 2.35 (2H, t,
NCH2CH2CH2CH2CH2CO) 3.64 (2H, m, NCH2CH2CH2CH2CH2CO), 4.22 (2H, q,
OCH2CH3), 8.12 (1H, s, C8-H); δC (50.3 MHz, CD3OD): 14.5 (CH3), 25.4 (CH2), 28.6
(CH2), 30.2 (CH2), 34.7 (CH2), 44.5 (CH2), 61.4 (CH2), 144.7 (C), 150.2 (CH), 155.3 (C),
161.6 (C), 175.2 (C); vmax(KBr, cm-1): 3428(s), 3324(s), 3200(s), 2940(m), 2857(w),
1733(s), 1635(s), 1609(s), 1562(s), 1463(s); LRMS (EI) (m/z) (%) : 311(91) (M+),
266(77), 238(48), 224(100), 210(41), 196(65), 183(98), 169(46), 134(14), 88(53), 69(29),
51(91).
Ethyl-2-amino-6-(2-nitrophenoxy)purin-9-ylhexanoate (155)
NO2
To a stirred solution of compound 151 (0.50 g, 1.61 mmol), in dry
Acetonitrile (40 mL) was added 1, 4-Diazabicyclo [2,2,2] octane
O
(0.18 g, 1.61 mmol), o-nitrophenol (0.67 g, 4.82 mmol), and Triethyl
amine (0.6 mL, 4.82 mmol).
N
N
The mixture was at RT overnight.
Subsequently the solvent was evaporated and the crude extract was
N
( )5
chromatographed on silica (methanol: ethyl acetate, 5:95) to give the
desired product 155 (0.55 g, 80%) as a yellow foam. Rf = 0.5. δH
O
N
NH2
O
155
(200 MHz, CD3OD): 1.15 (3H, t, OCH2CH3), 1.35 (2H, m, NCH2CH2CH2CH2CH2CO),
1.63 (2H, m, NCH2CH2CH2CH2CH2CO), 1.83 (2H, m, NCH2CH2CH2CH2CH2CO), 2.3
(2H, t, NCH2CH2CH2CH2CH2CO) 3.52 (2H, m, NCH2CH2CH2CH2CH2CO), 4.12 (2H, q,
OCH2CH3), 7.21-7.32 (3H, m, Ar-H), 7.92 (1H, s, C8-H), 8.02 (1H, dd, Ar-H); δC (50.3
MHz, CD3OD): 14.1 (CH3), 24.9 (CH2), 26.6 (CH2), 30.0 (CH2), 34.4 (CH2), 44.1 (CH2),
171
61.0 (CH2), 114.4 (C), 120.7 (CH), 125.7 (CH), 126.2 (CH), 135.6 (CH), 137.6 (C),
142.2 (CH), 143.5 (C), 146.3 (C), 155.1 (C), 160.8 (C), 174.9 (C). vmax (KBr, cm-1):
3324(m). 3220(m), 3086(w), 2941(m), 2795(m), 1965(m), 1735(m), 1614(m), 1527(s),
1464(s), 1346.8(m); LRMS (EI) (m/z)(%): 414(52) (M+), 369(46), 327(44), 286(26),
226(46), 206(40), 164(28), 139(76), 122(56), 109(64), 81(74), 65(84), 53(100).
Ethyl-2-amino-6-(2-nitrophenoxy)purin-9-ylpentanoate (154)
To a stirred solution of compound 150 (0.50 g, 1.68 mmol), in dry
NO2
Acetonitrile (40 mL) was added 1,4-Diazabicyclo [2, 2,2] octane
O
(0.19 g, 1.68 mmol), o-nitrophenol (0.70 g, 5.05 mmol), and triethyl
amine (0.7 mL, 5.05 mmol).
The mixture was at RT overnight.
Subsequently the solvent was evaporated and the crude extract was
chromatographed on silica (methanol: ethyl acetate, 5:95 to give the
desired product 154 (0.538 g, 80%) as a yellow foam Rf = 0.6; δH
N
N
( )4
O
N
N
NH2
O
154
(200 MHz, CD3OD): 1.01 (3H, t, OCH2CH3), 1.52 (2H, m, NCH2CH2CH2CH2CO), 1.82
(2H, m, NCH2CH2CH2CH2CO), 2.22 (2H, t, NCH2CH2CH2CH2CO), 3.42 ( 2H, m,
NCH2CH2CH2CH2CO), 4.09 (2H, q, OCH2CH3), 7.08-7.95 (m, Ar-H), 8.10 (1H, s, C8H); δC (50.3 MHz, CD3OD) 14.4 (CH3), 22.9 (CH2), 30.1 (CH2), 34.2 (CH2), 45.5 (CH2),
61.4 (CH2), 114.7 (C), 121.7 (CH), 126.6 (CH), 127.8 (CH), 130.1 (C), 136.1 (CH),
142.8 (C), 143.8 (CH), 146.6 (C), 156.5 (C), 160.3 (C), 174.8 (C); vmax(KBr, cm-1):
3324(m), 3220(m), 3200(m), 3086(w), 2941(m), 2795(m), 1965(m), 1735(m), 1614(m),
1527(s)m 1464(s), 1347(m); LRMS (EI) (m/z) (%): 400 (90) (M+), 355(58), 297(66),
286(50), 252(40), 226(68), 206(42), 196(60), 164(52), 146(24), 134(46), 101(52), 69(70),
55(100).
172
Ethyl-2-amino-6-(2-nitrophenoxy)purin-9-ylbutanoate (153)
NO2
To a stirred solution of compound 149 (0.50 g, 1.77 mmol), in
dry Acetonitrile (40 mL) was added 1, 4-Diazabicyclo [2,2,2]
octane (0.20 g, 1.77mmol), o-nitrophenol (0.74 g, 5.30 mmol),
and triethyl amine (0.74 mL, 5.30 mmol). The mixture was left
O
N
Subsequently the solvent was
N
( )3
evaporated and the crude extract was chromatographed on silica
O
to stir at RT overnight.
N
N
NH2
O
153
(methanol: ethyl acetate, 10:90) to give the desired product 153
(0.511 g, 75%) as a yellow foam. Rf = 0.49 δH (200 MHz, d6-DMSO) 1.01(3H, t,
OCH2CH3), 1.94 (2H, m, NCH2CH2CH2CO), 2.26 (2H, t, NCH2CH2CH2CO), 3.81 (2H, t,
NCH2CH2CH2CO), 4.12 (2H, q, OCH2CH3), 6.32 (2H, s, NH2), 7.21-7.32 (3H, m, Ar-H),
7.92 (1H, s, C8-H), 8.02 (1H, dd, Ar-H); δC (50.3 MHz, d6-DMSO) :13.9 (CH3), 24.5
(CH2), 30.7 (CH2), 42.2 (CH2), 59.9 (CH2), 113.4 (C), 125.6 (CH), 126.5 (CH), 135.3
(CH), 141.3 (C), 142.1 (CH), 145.0 (C), 150.6 (C), 155.6 (C), 159.3 (C), 172.0 (C);
vmax(KBr, cm-1): 3324(m), 3220(m), 3200(m), 3086(w), 2941(m), 2795(m), 1965(m),
1735(m), 1614(s), 1527(s), 1464(s),1347(m); LRMS (EI) (m/z) (%): 386 (100) (M+),
341(33), 286(20), 265(22), 238(10), 226(40), 178(24), 164(34), 139(20), 115(20), 87(62),
69(25), 55(18), 43(40).
Ethyl-2-amino-6-(2-nitrophenoxy)purin-9-yl propionate(152)
NO2
To a stirred solution of compound 148 (0.50 g, 1.86 mmol), in dry
Acetonitrile (40 mL) was added 1, 4-Diazabicyclo [2, 2, 2] octane
O
(0.21 g, 1.86 mmol), o-nitrophenol (0.78 g, 5.58 mmol), and triethyl
N
N
amine (0.75 mL, 5.58 mmol). The mixture was left to stir at RT
overnight. Subsequently the solvent was evaporated and the crude
N
( )2
extract was chromatographed on silica (methanol: ethyl acetate,
10:90) to give the desired product 152 (0.55 g, 80%) R f= 0.4 as a
O
N
NH2
O
152
yellow foam. δH (200 MHz, CD3OD) 1.12 (3H, t, OCH2CH3), 2.93 (2H, t, NCH2CH2CO),
173
4.15 (2H, t, NCH2CH2CO), 4.45 (2H, q, OCH2CH3), 7.21-7.32 (3H, m, Ar-H), 8.01 (1H,
s, C8-H), 8.02 (1H, dd, Ar-H); δC (50.3 MHz, CD3OD) 14.4 (CH3), 34.7 (CH2), 45.6
(CH2), 61.9 (CH2), 114.7 (C), 121.1 (CH), 126.6 (CH), 127.7 (CH), 136.1 (CH), 138.0
(C), 142.9 (CH), 143.9 (C), 146.7(C), 155.5 (C), 160.6 (C), 172.5 (C) ; vmax(KBr, cm-1):
3324(m), 3220(m), 3200(m), 3086(w), 2941(m), 2795(m), 1965(m), 1735(m), 1614(m),
1527(s), 1464(s), 1347(m); LRMS (EI) (m/z) (%): 372(59) (M+), 326(57), 298(30),
251(74), 226(78), 206(30), 178(50), 134(48), 122(30), 106(18), 92(24), 81(18), 73(42),
55(100).
2-amino-6-(2-nitrophenoxy)purin-9-ylethanol (168)
To a stirred solution of compound 167 (0.50 g, 2.35 mmol), in dry
NO2
Acetonitrile (40 mL) was added 1, 4-Diazabicyclo [2, 2, 2] octane
(0.26 g, 2.35 mmol), o-nitrophenol (0.98 g, 7.04 mmol), and
O
triethyl amine (0.98 mL, 7.04 mmol). The mixture was left to stir
N
at RT overnight. Subsequently the solvent was evaporated and the
N
N
N
NH2
crude extract was chromatographed on silica (methanol: ethyl
acetate, 10:90) to give the desired product 168 (0.593 g, 80%) as a
CH2OH
168
yellow foam. Rf = 0.39 δH (200 MHz, d6-DMSO): 3.52 (2H, t, NCH2CH2OH), 3.95 (2H,
m, NCH2CH2OH), 6.21 (2H, bs, NH2), 6.72-7.81 (4H, m, Ar-H), 7.83 (1H, s, C8-H); δC
(50.3 MHz, d6-DMSO): 45.9 (CH2), 59.3 (CH2), 113.7 (C), 125.4 (CH), 126.9 (CH),
130.3 (C), 135.6 (CH), 142.5 (CH), 145.4 (C), 152.4 (C), 155.9 (C), 158.7 (C), 159.6 (C);
vmax(KBr, cm-1): 3324(m), 3220(m), 3200(m), 3086(w), 2941(m), 2795(m), 1965(m),
1735(m), 1614(m), 1527(s), 1464(s), 1347(m); LRMS (EI) (m/z) (%): 316(34), (M+),
270(36), 239(18), 226(24), 195(24), 185(18), 167(18), 149(100), 137(58), 123(46),
109(48), 95(88).
174
Ethyl-2-[Benzyloxycarbonyl]amino-6-(2-nitrophenoxy)purin-9-ylpropionate (156)
NO2
To a solution of compound 152 (0.50 g, 1.34 mmol),
in THF (30 mL) was added triphosgene (0.80 g, 2.68
O
mmol), Et3N (0.56 mL, 4.03 mmol), the mixture was
reflux for 3 h, and benzyl alcohol (0.42 mL, 4.03
mmol) was added, the mixture was further stirred at
N
N
( )2
reflux for 2 h. Subsequently, the crude extract was
N
N
O
N
H
O
O
O
chromatographed on silica (methanol: ethyl acetate,
156
10:90), to give the desired product 156 (0.54 g, 80%)
as a yellow foam. Rf = 0.65. δH (200 MHz, CD3OD): 1.18 (3H, t, OCH2CH3), 2.82 (2H, t,
NCH2CH2COOEt), 4.12 (2H, q, OCH2CH3), 4.42 (2H, t, NCH2CH2COOEt), 5.01 (2H, s,
Ph-CH2), 7.09-8.02 (9H, m, Ar-H), 8.21 (s, 1H, C8-H); δC (50.3 MHz, CD3OD):
14.1(CH3), 35.1(CH2), 45.8 (CH2), 62.4 (CH2), 68.3 (CH2), 118.3 (C),126.4 (CH), 127.2
(CH), 127.6 (CH), 128.4 (CH), 129.3 (CH), 129.6 (CH), 129.9 (CH), 136.8 (CH), 137.3
(CH), 143.9 (CH), 146.2 (C), 147.0 (C), 150.1 (C), 154.1 (C), 154.5 (C), 155.9 (C), 160.1
(C), 177.9 (C); vmax(KBr, cm-1):
3425(m), 3240(m), 3106(w), 3088(w), 2961(s),
2795(m), 1965(m), 1746(m), 1619(s), 1518(m), 1454(s), 1235(s), 1077(w); LRMS (EI)
(m/z) (%): C24H22N6O7: 506 (26) (M+) , 460(20), 416(6), 384(12), 178(12), 172(8),
152(100), 123(20), 91(68), 43(42). Elemental analysis required C = 56.92%, H = 4.38%,
N = 16.59%; found C = 57.11%, H = 4.64%, N = 16.82%.
Ethyl-2-[Benzyloxycarbonyl]amino-6-(2-nitrophenoxy)purin-9-ylhexanoate (157)
To a solution of compound 155 (0.50 g, 1.21 mmol),
NO2
and Et3N (0.50 mL, 3.62 mmol), in THF (50 mL),
O
was added triphosgene (0.72 g, 2.42 mmol), and
reflux for 3 h, subsequently, benzyl alcohol (0.37 mL,
3.62 mmol) was added, and the reaction was left to
stir at reflux for 2 h. Subsequently, the solvent was
N
N
( )5
N
N
O
N
H
O
O
157
175
O
evaporated and the crude extract was chromatographed on silica (methanol: ethyl acetate,
10:90), to give the desired product 157 (0.50 g, 80%) as a yellow foam. Rf = 0.72. δH
(200 MHz, CD3OD):1.24 (3H, t, J=7.0, OCH2CH3), 1.29 (2H, m, NCH2CH2CH2CH2CH2
CO2Et),
1.72
(2H,
m,
NCH2CH2CH2CH2CH2CO2Et),
NCH2CH2CH2CH2CH2COOEt), 2.34 (2H, t, J=6.4,
2.04
(2H,
m,
NCH2CH2CH2CH2CH2 CO2Et),
3.24 (2H, t, NCH2CH2CH2CH2CH2 CO2Et), 4.12 (2H, q, OCH2CH3), 5.21 (2H, s, PhCH2), 7.32-8.32 (9H, m, Ar-H), 8.91 (1H, s, C8-H); δC (50.3 MHz, CD3OD): 14.5 (CH3),
25.18 (CH2), 26.8 (CH2), 29.9 (CH2), 34.4 (CH2), 53.8 (CH2), 61.4 (CH2), 68.0 (CH2),
116.6 (C), 125.9 (CH), 126.7 (CH), 127.2 (CH), 128.9 (CH), 129.2 (CH), 129.7 (CH),
130.3 (CH), 136.7 (CH), 137.6 (CH), 143.1 (CH), 144.2 (C), 145.8 (C), 150.2 (C), 153.7
(C), 154.2 (C), 155.5 (C), 158.9 (C), 175.2 (C); vmax(KBr, cm-1): 3446(m), 3241(m),
3119(w), 3088(w), 2963(m), 2799(s), 1965(m), 1620(s), 1524(m), 1341(m), 1227(s);
LRMS (EI) (m/z) (%): C27H28N6O7: 522(56) (M+), 440(17), 395(72), 353(62), 318(70),
272(42), 264(62), 252(90), 232(100), 190(88), 178(68), 152(42%); Elemental analysis
required C = 59.12%, H = 5.14%, N = 15.32%; found C = 59.45%, H = 5.46%, N =
15.66%.
Ethyl-2-[Benzyloxycarbonyl] amino guan-9-ylhexanoate (162)
O
To a solution of compound 157 (0.40 g, 0.77 mmol)
in Acetonitrile (40 mL)was added solution of onitrobenzaldoxime
(1.27
g,
7.66mmol)
in
N
Acetonitrile(10 mL), this was followed by the
N
( )
5
addition of solution of N,N,N,N-tetramethyl guanidine
O
(0.86 mL, 6.89 mmol) in acetonitrile (5 mL), the
NH
N
O
N
H
O
O
162
mixture was stirred at RT overnight. Subsequently the solvent was evaporated and the
resulting brownish residue was chromatographed on silica (ethyl acetate: methanol,
90:10) to give the desired product 162 (0.196 g, 60%) as a white solid. mp 215 – 217 °C;
Rf = 0.35. δH (200 MHz, CD3OD): 1.03 (3H, t, OCH2CH3), 1.16 (2H, m,
NCH2CH2CH2CH2CH2CO2Et), 1.42 (2H, m, NCH2CH2CH2CH2CH2CO2Et), 1.62 (2H, m,
NCH2CH2CH2CH2CH2CO2Et), 2.12 (2H, t, NCH2CH2CH2CH2CH2CO2Et), 3.32 (2H, t,
176
NCH2CH2CH2CH2CH2CO2Et), 4.01 (2H, q, OCH2CH3), 7.15-7.28 (5H, m, Ar-H), 7.68
(1H, s, C8-H); δC (50.3 MHz, CD3OD): 14.1 (CH3), 25.2 (CH2), 26.8 (CH2), 30.5 (CH2),
34.6 (CH2), 44.5 (CH2), 61.2 (CH2), 69.1 (CH2), 116.9 (C), 120.7 (CH), 129.5 (CH),
135.4 (CH), 136.5 (CH), 137.8 (CH), 141.1 (C), 143.5 (CH), 148.6 (C), 150.8 (C), 156.1
(C), 157.4 (C), 175.0 (C); vmax(KBr, cm-1): 3048(m), 2899(m), 1965(m), 1736(m),
1620(s), 1595(m), 1457(s), 1351(m), 1247(s), 968(s); LRMS (EI) (m/z) (%): C21H25N5O5:
427(44) (M+) , 383(42), 354(60), 321(4), 285(32), 253(4), 143(100), 91(66), 61(22),
56(18); Elemental analysis required C = 59.01%, H = 5.90%, N = 16.38%; found C =
59.38%, H = 6.01%, N = 16.64%.
Ethyl-2-[Benzyloxycarbonyl]amino guan-9-ylpentanoate (161)
O
To a stirring solution of compound 154 (0.50 g, 1.25
mmol) in dry THF (50 mL) was added triethyl amine
N
(0.5 mL, 3.75 mmol), followed by the addition of
N
( )
4
triphosgene (0.74 g, 2.50 mmol), the mixture was
refluxed for 3 h.
Subsequently, benzyl alcohol (0.39
mL, 3.75 mmol) was added, and the reaction was
NH
N
O
N
H
O
O
O
161
further stirred at reflux for 2 h, thin layer chromatography was used to determine the
completion of the reaction (Rf = 0.67). Subsequently, the solvent was evaporated and the
crude extract 159 was dissolved in ethyl acetate (50 mL), washed with water (5 x 50
mL), the combined organic layer was dried over anhydrous MgSO4. Solvent evaporation
afforded a yellow solid (0.60 g) which was used without any further purification for the
next step of the reaction.
To a solution of the crude product(0.60 g, 1.12 mmol) in
Acetonitrile (30 mL) was added o-nitrobenzaldoxime (1.87 g, 11.24 mmol), N,N,N,Ntetramethyl guanidine (1.26 mL, 10.08 mmol) and the resulting mixture was stirred at
RT overnight. Subsequently, the solvent was evaporated and the resulting brownish
residue was chromatographed (ethyl acetate: methanol, 90:10) to give the desired product
161 (0.31 g, 60%) as a cream foam. Rf = 0.43; δH (200 MHz, CD3OD) 1.01(3H, t,
OCH2CH3), 1.52 (2H, m, NCH2CH2CH2CH2CO), 1.82 (2H, m, NCH2CH2CH2CH2CO),
2.22 (2H, t, NCH2CH2CH2CH2CO), 3.42 (2H, m, NCH2CH2CH2CH2CO), 4.09 (2H, q,
177
OCH2CH3), 8.10 (1H, s, C8-H); δC (50.3 MHz, CD3OD): 14.4 (CH3), 22.9 (CH2), 30.1
(CH2), 34.2 (CH2), 45.5 (CH2), 61.4 (CH2), 114.7 (C), 121.7 (CH), 126.6 (CH), 127.5
(CH), 130.4 (CH), 136.1 (CH), 142.8 (CH), 143.8 (C), 146.6 (C), 150.6 (C) 156.5 (C),
160.3 (C), 174.8 (C); LRMS (EI) (m/z) (%): C20H23N5O5: 413 (24) (M+), 354(8), 323(4),
255(22), 165(10), 145(18), 91(48), 65(100), 55(22), 43(10); Elemental analysis required
C = 58.10%, H = 5.61%, N = 16.94%; found C = 58.45%, H = 5.96%, N = 17.14%.
Ethyl-2-[Benzyloxycarbonyl]amino guan-9-ylbutanoate (160)
To a solution of compound 153 (0.50 g, 1.30 mmol), in
O
THF (50 mL) was added triethyl amine (0.54 mL, 3.89
N
NH
mmol), this was followed by the addition of triphosgene
(0.77 g, 2.59 mmol), the resulting mixture was refluxed
for 3 h, subsequently, benzyl alcohol (0.40 mL, 3.89
mmol) was added, and the reaction was further stirred
N
( )
3
N
O
N
H
O
O
O
160
at reflux for 2 h, thin layer chromatography was used to
determine the completion of the reaction( Rf = 0.5).
Subsequently, the solvent was
evaporated and the crude extract 158 was dissolved in ethyl acetate (50 mL), washed with
water (5 x 50 mL). The organic extracts were dried over anhydrous MgSO4.
Solvent
evaporation afforded a yellow solid (0.55 g) which was used without any further
purification for the next step and of the reaction.
To a solution of the crude product
(0.50 g, 0.95 mmol) in Acetonitrile (30 mL) was added o-nitrobenzaldoxime (1.60 g, 9.50
mmol), N, N, N, N-tetramethyl guanidine (1.10 mL, 8.55 mmol), the mixture was stirred
at RT overnight.
Subsequently the solvent was evaporated and the resulting brownish
residue was chromatographed on silica (ethyl acetate: methanol, 90:10) to give the
desired product 160 (0.32 g, 62%) as a cream solid. Rf = 0.36. δH (200 MHz, CD3OD):
1.12 (3H, t, OCH2CH3), 2.08 (2H, m, NCH2CH2CH2CO2Et), 2.25 (2H, t,
NCH2CH2CH2CO2Et), 3.92 (2H, t, NCH2CH2CH2CO2Et), 4.09 (2H, q, OCH2CH3), 5.21
(2H, s, Ph-CH2), 7.21-7.34 (5H, m, Ar-H), 7.81 (1H, s, C8-H); δC (50.3 MHz,
CD3OD):14.8 (CH3), 26.6 (CH2), 32.3 (CH2), 44.6 (CH2), 62.1 (CH2), 69.6 (CH2), 115.7
(C), 120.9 (CH), 125.9 (CH), 129.8 (CH), 130.1 (CH), 137.1 (CH), 141.6 (CH), 149.3
178
(C), 150.2 (C), 151.4 (C), 153.4 (C). 156.6 (C), 174.6 (C); vmax(KBr, cm-1): 3305(w),
3223(w), 3115(m), 2850(m), 1646(m), 1523(s), 1475(m), 1366(m), 1105(w); LRMS (EI)
(m/z) (%): 399(46) (M+), 336(8), 291(22), 246(36), 218(16), 204(100), 191(56), 177(20),
164(10), 137(26), 108(22), 91(36), 69(38), 55(16), 43(24).
General method for the hydrolysis of the ester
To compound 160 (0.30 g, 0.75 mmol), 161 (0.30 g, 0.73 mmol), 162 (0.20 g, 0.47
mmol), or 156 (0.40 g, 0.79 mmol) was added, LiOH/THF (1:1), and the resulting
suspension was stirred at RT for 3 h.
Subsequently the solvent was evaporated and
water (10 mL) was added to the resulting residue. The pH of the solution was adjusted
to 4 and the aqueous solution was extracted by ethyl acetate (8 x 50 mL). The combined
organic extracts were evaporated to afford compound 163 (0.12 g, 60%), 164 (0.15 g,
55%), 165 (0.12 g, 66%), 166 (0.26 g, 70%) as white solids.
2-[Benzyloxycarbonyl] amino guan-9-ylpropionic acid (163)
(mp 214-216 °C), δH (200 MHz,CD3OD): 2.08 (2H,
m,
NCH2CH2CH2CO2H),
2.25
(2H,
O
t,
N
NCH2CH2CH2CO2H),
3.92
(2H,
NH
t,
NCH2CH2CH2CO2H), 5.21 (2H, s, Ph-CH2), 7.217.34 (5H, m, Ar-H), 7.81 (1H, s, C8-H); δC (50.3
MHz, CD3OD): 26.6 (CH2), 32.3 (CH2), 44.6 (CH2),
N
( )
3
HO
N
N
H
O
O
O
163
69.6 (CH2), 115.7 (C), 120.9 (CH), 125.9 (CH), 129.8 (CH), 130.1 (CH), 137.1 (CH),
141.6 (CH), 149.3 (C), 151.4 (C), 152.6 (C), 156.6 (C), 176.6 (C); vmax(KBr, cm-1):
3216(m), 3105(bm), 3010(w), 2851(m), 1725(s), 1610(m), 1436(m), 1015(m), 910.4(m);
LRMS (EI) (m/z) (%): 371(48) (M+), 328(8), 283(64), 247(4), 224(10), 91(100), 65(42),
52(40); Elemental analysis: for C17H17N5O5: found C= 55.10, H=4.60, N=19.2; requires:
%C= 54.98, H=4.61, N= 18.86.
179
2-[Benzyloxycarbonyl]amino guan-9-ylhexanoic acid (165)
O
(mp 218-210 °C),δH (200 MHz, CD3OD): 1.16 (2H, m,
NCH2CH2CH2CH2CH2CO2H),
1.42
NCH2CH2CH2CH2CH2CO2H),
(2H,
1.62
m,
(2H,m,
N
NCH2CH2CH2CH2CH2CO2H),
2.12
(2H,
t,
N
( )
5
NCH2CH2CH2CH2CH2CO2H),
3.32
(2H,
t,
HO
NH
N
O
N
H
O
O
165
NCH2CH2CH2CH2CH2CO2H), 7.15-7.28 (5H, m, ArH), 7.68 (1H, s, C8-H); δC (50.3 MHz, CD3OD): 25.2 (CH2), 26.8 (CH2), 30.5 (CH2),
34.6 (CH2), 44.5 (CH2), 69.1 (CH2), 116.9 (C), 120.7 (CH), 129.5 (CH), 135.4 (CH),
136.5 (CH), 137.8 (CH), 141.1 (CH), 148.6 (C), 150.8 (C), 154.2 (C), 156.1 (C), 157.4
(C), 178.2 (C); LRMS (EI) (m/z) (%) C19H21N5O5: 399 (42) (M+), 311(10), 220(100),
206(54), 170(4), 150(48), 91(80), 80(48), 66(60), 51(22); Elemental analysis: found
C=57.20, H=5.45, N=17.18; requires: %C= 57.14, H= 5.30, N= 17.53.
2-[Benzyloxycarbonyl]amino guan-9-ylbutanoic acid(164)
δH
(200MHz,CD3OD):
NCH2CH2CH2CH2CO),
1.52
1.82
(2H,
(2H,
O
m,
m,
NCH2CH2CH2CH2CO), 2.22 (2H t, J= 6.4 Hz,
N
m,
N
( )
4
NCH2CH2CH2CH2CO), 8.10 (1H, s, C8-H); δC
HO
NCH2CH2CH2CH2CO),
3.42
(2H,
NH
N
O
N
H
O
O
164
(50.3MHz, CD3OD): 22.9 (CH2), 30.1 (CH2), 34.2 (CH2), 45.5 (CH2), 61.4 (CH2), 114.7
(C), 121.7 (CH), 126.6 (CH), 127.5 (CH), 129.7 (CH), 136.1 (CH), 142.8 (CH), 143.8
(C), 146.6 (C), 152.4 (C), 156.5 (C), 160.3 (C), 174.8 (C); vmax(KBr,cm-1):
3208(s),
3111(bm), 3011(w), 2879(m), 1730(s), 1615(m), 1426(m), 1146(m); LRMS (EI) (m/z)
(%) C18H19N5O5: 385 (32) (M+), 297(44), 251(42), 247(88), 206(18), 180(100), 91(64),
65(22), 58(4), 43(22); Elemental Analysis: found %C=55.9, H=4.99, N=17.94; requires
%C= 56.1, H=4.97, N=18.17.
180
2-[Benzyloxycarbonyl]amino-6-(2-nitrophenoxy)purin-9-ylpropionic acid (166)
(mp 234-235 °C), δH (200 MHz, CD3OD): 2.82 (2H, t,
NO2
J= 6.2 Hz, NCH2CH2CO2H), 4.42 (2H, t, J=7.1 Hz,
O
NCH2CH2CO2H), 5.21 (2H, s, Ph-CH2), 7.10-8.21 (9H,
m, Ar-H), 8.42 (1H, s, C8-H); δC (50.3 MHz, CD3OD):
35.1 (CH2), 61.4 (CH2), 67.3 (CH2), 116.3 (C), 126.4
(CH), 127.2 (CH), 127.6 (CH), 128.6 (CH), 129.3
(CH), 129.6 (CH), 129.9 (CH), 135.8 (CH), 137.3
N
N
( )2
N
O
N
N
H
O
O
HO
166
(CH), 143.9 (CH), 146.2 (C), 147.0 (C), 154.1 (C),
150.2 (C), 154.5 (C), 155.9 (C), 160.1 (C), 180.9 (C); vmax(KBr, cm-1):
3216(m),
3116(bm), 3010(w), 2871(m), 2201(s), 1735(s), 1610(m), 1516(m), 1436(m), 1286(s),
1014.6(m); LRMS (EI) (m/z) (%): C22H18N6O7: 478 (22) (M+), 432(16), 404(8), 344(56),
341(80), 138(28), 122(66), 91(100), 88(50), 43(40); Elemental Analysis: found %C=
55.30; H= 3.80, N=17.80; requires %C= 55.23; H=3.79; N=17.57.
2-amino-6-(2-nitrophenoxy)purin-9-yl[2(-tertbutyldimethylsilyloxy)ethyl]ether (169)
To a solution of compound 168 (0.57 g, 1.80 mmol), in DMF (30
NO2
mL), was added tert-butyldimethyl silylchloride (0.33 g, 2.17
mmol), and imidazole (0.31 g, 5.41 mmol), the resulting mixture
was stirred at RT overnight.
O
N
N
Solvent evaporation gave a crude
extract which was chromatographed on silica (methanol: ethyl
acetate, 10:90) to give the desired product 169 (0.47 g, 60%) as a
yellow foam. Rf = 0.65. δH (200 MHz, CD3OD) 0.15 (6H, s,
N
N
NH2
C O
H2
Si
169
(CH3)2), 0.96 (9H, s, (CH3)3), 3.82 (2H, t, J=5.5 Hz,
NCH2CH2O), 4.34 (2H, t, J=6.0 Hz, NCH2CH2O), 7.12 (1H , m, Ar-H), 7.61-7.92 (2H,
m, Ar-H), 8.09(1H , s, C8-H), 8.22 (1H, m, Ar-H); δC (50.3 MHz, CD3OD): -5.6 (CH3),
18.9 (C), 26.2 (CH3), 49.6 (CH2), 62.1 (CH2), 114.9 (C), 120.9 (CH), 126.7 (CH), 127.6
(CH), 136.0 (CH), 137.9 (CH), 143.3 (CH), 143.5 (C), 144.0 (C), 146.8 (C), 156.6 (C),
166.4 (C); vmax(KBr, cm-1): 3311(m), 3101(w), 3075(w), 2841(m), 1611(m), 1576(s),
181
1436(s), 1346(m), 845.6(s); LRMS (EI) (m/z) (%): 430(46) (M+), 415(36), 373(100),
316(64), 284(26), 270(42), 252(78), 235(46), 226(68), 194(64), 170(54), 151(42),
122(38).
2-amino-6-chloropurin-9-ylacetonitrile (171)
Anhydrous K2CO3 (2.20 g, 6.91 mmol) was added to a solution of 58
Cl
(0.53 g, 3.14 mmol) in anhydrous DMF (50 mL). The resulting
N
mixture was stirred at RT for 10 min.
N
Subsequently, bromo
acetonitrile (0.42 mL, 6.28 mmol) was added and the mixture before
N
N
NH2
CN
171
being left to stir at RT for 5 h. Filtration, followed by solvent
evaporation in vacuo gave a crude product which was purified by column
chromatography (3% methanol in ethyl acetate). Evaporation of the solvent gave the
desired product 171 (0.509 g, 78%) as a white solid. (mp 200-201 °C), Rf 0.45 δH (200
MHz, d6-DMSO): 5.21 (2H, s, CH2), 6.89 (2H, br s, NH2), 8.11 (1H, s, C8-H); δC (50.3
MHz, d6-DMSO): 31.1 (CH2), 115.4 (CN), 122.8 (C), 142.1 (CH), 149.8 (C), 153.6 (C),
160.0 (C); vmax(KBr, cm-1): 3350(w), 3201(w), 2246(s), 1611(m), 1517(m), 847(m);
LRMS (EI) (m/z) (%): 208 (100) (M +), 173(52), 146(38), 119(6), 95(10), 81(12), 69(34),
55(34), 43(44); Elemental analysis: found: C= 40.22%, H= 2.24%, N= 39.87%; requires
C=40.3%, H=2.42%, N= 40.29%.
2-amino-6-(2-nitrophenoxy)purin-9-ylacetonitrile (172)
NO2
To a solution of 171 (.0.26 g, 1.25 mmol) in anhydrous acetonitrile
(30 mL) was added DABCO (0.14 g, 1.25 mmol), followed by onitrophenol (0.52 g, 3.75 mmol) and Et3N (0.71 mL, 3.75 mmol).
O
N
N
The mixture was left to stir at room temperature overnight.
Subsequently, after work-up, the crude residue was chromatographed
on silica (10% methanol in ethyl acetate) to give the desired product
N
N
NH2
CN
172
172 (0.35 g, 90%) as yellow solid. mp 198-200 °C; δH (200 MHz, d6DMSO): 5.29 (2H, s, CH2), 6.71 (2H, br s, NH2), 7.61 (2H, m, Ar-H), 7.89 (1H m, , Ar-
182
H), 8.12 (1H, s, C8-H), 8.21 (1H, m, Ar-H); δC (50.3 MHz, d6-DMSO): 30.9 (CH2), 115.4
(CN), 122.8 (C), 125.5 (CH), 125.7 (CH), 126.7 (CH), 135.5 (CH), 140.3 (C) 142.1 (CH),
149.8 (C), 153.61 (C), 158.7 (C), 160.0 (C); vmax(KBr, cm-1): 3401(w), 3292(w),
3150(w), 1633(m), 1578(m), 1518(m), 1485(m), 1382(s), 1246(m), 1175(w), 1082(w),
1022(w);LRMS (EI) (m/z) (%): 311 (100) (M+), 281(8), 265(98), 225(22), 198(12),
190(70), 146(16), 122(10), 97(8), 83(20), 69(38), 55(28), 43(38); Elemental Analysis: for
C13H9N7O3 found %C= 50.24, H=2.98, N= 31.8; requires %C=50.17, H= 2.91, N=31.5.
2-(2-[Benzyloxycarbonyl] amino -6-(2-nitrophenoxy)purin-9-yl)ethanol (168)
To a solution of compound 169 (0.50 g, 1.16 mmol), in
NO2
anhydrous THF (40 mL) was added Et3N (0.49 mL,
O
3.49 mmol), and triphosgene (0.69 g, 2.33 mmol), the
N
N
resulting mixture was reflux for 3 h. Subsequently,
benzyl alcohol (0.36 mL, 3.49 mmol) was added, and
the reaction further stirred at reflux for 2 h.
After
N
N
O
N
H
O
OH
170
work-up, the crude extract was chromatographed on
silica (methanol: ethyl acetate, 10:90) to give the desired product 170 (0.42 g, 80%) as a
yellow foam. Rf = 0.72; δH (200 MHz, CD3OD): 3.82 (2H, t, J=6.8 Hz, NCH2CH2OH),
3.99 (2H, m, NCH2CH2OH), 5.02 (2H, s, Ph-CH2), 7.12-8.11 (9H, m, Ar-H), 8.61 (1H, s,
C8-H); δC (50.3 MHz, CD3OD): 60.4 (CH2), 64.9 (CH2), 67.6 (CH2), 125.5 (C), 126.4
(CH), 126.7 (CH), 127.7 (CH), 127.9 (CH), 128.1 (CH), 128.6 (CH), 128.9 (CH), 129.0
(CH), 129.2 (CH), 136.2 (CH), 136.5 (C), 137.3 (C), 142.9 (C), 146.0 (C), 150.2 (C),
153.6(C); vmax(KBr, cm-1): 3561(bm), 3211(m), 3108(w), 3055(w), 2851(m), 1616(m),
1548(s), 1433(s), 1337(m), 1145(w); LRMS (EI)(m/z)(%) 450 ((22) [M+]), 434 (36), 360
(72), 328 (100), 243 (18)123 (44), 91 (68), HRMS (ES, H+): calculated for
C21H18N6O6:[M+H+] is 451.4216; found 451.4219.
183
Thymin-1-yl acetic acid (133)164
Thymine 132 (10.00 g, 79.30 mmol) was added to a solution of
O
potassium hydroxide (17.10 g, 305.00 mmol) in water (50 mL). The
NH
solution was warmed in a water bath to 40 C and bromoacetic acid
N
(16.50 g, 119.00 mmol) in water (25 mL) was added over 30 min and
O
O
the mixture was stirred for a further 30 min. The solution was then
OH
cooled to RT, the pH was adjusted to 5.5 using concentrated HCl; the
133
mixture was put in a fridge for 2 h. The solution was then filtered to remove any
unreacted thymine and the pH was further reduced to pH 2.0 again using concentrated
HCl and it was put in a freezer for 2 h. The ensuing precipitate was collected by filtration
and dried in vacuo over P2O5 to obtain the desired compound 133 (9.48 g, 65%) as a
white solid. δH (200 MHz, d6-DMSO): 1.74 (3H, s, CH3), 4.35 (2H, s, CH2), 7.45 (1H, s,
C6-H), 11.35 (1H, s, CO2H); δC (50.3 MHz, d6-DMSO) 12.3 (CH3), 48.9 (CH2), 108.8
(C5), 142.3 (C6), 151.4 (C), 164.9 (C), 170.1 (C).
N-(2-tert-Butoxyxarbonylaminoethyl)-N-(thymin-1-ylacetyl)-glycineethylester
(134)164
Compound 122(0.099 g, 0.29 mmol), was added
O
to a stirred solution of HBTU (0.11 g, 0.291
NH
mmol), DIPEA (0.15 mL, 0.87 mmol) and
N
compound 133 (0.09 g, 0.29 mmol) in DMF (30
O
O
mL), the resulting mixture was stirred at RT
overnight. Subsequently, the solvent was removed
in vacuo.
The residue was dissolved in ethyl
O
O
O
N
H
N
O
134
acetate (100 mL). The resulting organic solution was washed with brine (2 x 40mL), and
saturated solution of NaHCO3 (2 x 40 mL).
The organic solution was dried over
anhydrous MgSO4. Filtration followed by solvent evaporation afforded brown coloured
oil which was chromatographed on silica (7% methanol/Dichloromethane) to give the
desired product 134 (0.066 g, 55%) as a cream foam. δH (200 MHz, CDCl3): (2 rotational
184
isomers in a 2:1 ratio observed due to restricted rotation about the tertiary amide bond)
1.28 (ma) and 1.34 (min) (t, 3H, J=7.2 , CH2CH3), 1.42 (min) and 1.45 (ma) (s, 9H,
(CH3)3), 1.92 (s, 3H, C5-CH3), 3.35 (m, unresolved, 2H, CH2CH2NCO), 3.54 (t, 2H,
J=5.5 Hz, CH2CH2NCO), 4.04 (ma) and 4.16 (min) (s, 2H, NCH2CO2Et), 4.22 (ma) 4.24
(min) (q, 2H, J=7.2 Hz, CH2CH3), 4.42 (min) 4.58 (ma) (s, 2H, NCH2CON), 5.08 (min)
and 5.17 (ma) (t, 1H, J=6.2 Hz, (CH3)3NH), 6.95 (ma) and 7.05 (min) (s, 1H, C6-H), 8.58
(br s, 1H, N3-H). δC(50.3 MHz, CDCl3): (2 rotational isomers in a 2:1 ratio observed due
to restricted rotation about the tertiary amide bond) 12.3 (CH3), 14.0 (CH3), 28.3 ((CH3),
38.6 (CH2), 47.7 (CH2), 48.7 (CH2), 49.0 (CH2), 61.6 (CH2), 62.2 (CH2), 67.2 (CH2), 79.9
(C), 110.7 (C), 140.8 (C), 151.1 (C), 156.0 (C), 164.2 (C), 167.3 and 167.7 (C), 169.3 and
169.6 (C).
N-(2-tert-Butoxyxarbonylaminoethyl)-N-(thymin-1-ylacetyl)-glycineaceticacid
(135)164
N-(2-t-Butoxycarbonylaminoethyl)-N-(thymin-1-
O
ylacetyl)-glycine ethyl ester 134 (1.50 g, 3.64 mmol)
NH
was dissolved in THF (20 mL), to this solution was
N
added 1M (aq) LiOH (10 mL); the mixture was then
O
O
O
stirred at RT for 3 h. Water (10 mL) was added and the
aqueous layer was extracted with ethyl acetate (9 x 20
mL).
The combined organic phase was dried over
O
O
N
H
N
OH
135
anhydrous MgSO4 and filtered; the solvent was removed under reduced pressure to yield
the desired compound 135 (0.98 g, 70%) as a white foam. δH (200 MHz, d6-DMSO): (2
rotational isomers in a 2:1 ratio observed due to restricted rotation about the tertiary
amide bond) 1.37 (s, 9H, (CH3)3), 1.60 (s, 3H, CH3), 3.21-3.30 (unresolved, 4H, CH2,
NHCH2CH2N), 3.89 (ma) and 4.22 (min) (s, 2H, NCH2CO2H), 4.51 (min) and 4.72 (ma)
(s, 2H, NCH2CON), 6.79 (min) and 7.01(ma.) (br s, 1H, (CH3)3CNH), 7.32 (min) and
7.39 (ma) (s, 1H, C6-H), 12.01 (m, 1H, OH); δC(50.3 MHz, d6-DMSO): (2 rotational
isomers in a 2:1 ratio observed due to restricted rotation about the tertiary amide bond)
185
13.1(CH3), 27.9 ((CH3)3), 39.1 (CH2), 47.1 (CH2), 48.1 (CH2), 78.2 and 78.4 (C-O),
109.6 (C5), 143.4 (C6), 153.5 (CO), 157.1 (CONH), 165.2 (CO), 167.9 and 168.1
(CH2CON), 171.2 (CO2H).
2-[(Benzyloxycarbonyl)amino]-6-(2-nitrophenoxy)-N9-(1Htetrazol-5-yl)methyl
purine (174)
NO2
To a suspension of compound 173 (0.50 g, 1.12 mmol)
in toluene (40 mL) was added sodium azide (NaN3)
(0.22 g, 3.37 mmol), and triethyl amine hydrochloride
O
N
N
(Et3N.HCl) (0.46 g, 3.37 mmol), mixture was stirred
under reflux overnight. Subsequently the solvent was
N
N
N
H
O
NH
evaporated. Water (30 mL) was added to the residue
and filtered; the filtrate was extracted with ethyl acetate
O
N
N
N
174
(8 x 50 mL). The combined organic extracts were dried over anhydrous MgSO4. Solvent
evaporation afforded the desired product 174 (0.36 g, 65%) as yellow foam. δH (200
MHz, CD3OD): 4.42 (2H, s, NCH2), 5.72 (2H, s, Ph-CH2), 6.72-7.82 (9H, m, Ar-H), 8.12
(1H, s, C8-H); δC (50.3 MHz, CD3OD): 61.7 (CH2), 65.4 (CH2), 113.2 (C), 121.0 (CH),
121.3 (CH), 122.4 (CH), 126.3 (CH), 126.7 (CH), 128.2 (CH), 129.5 (CH), 130.1 (CH),
138.3 (CH), 139.4 (C), 141.7 (CH), 142.8 (C), 144.2 (C), 146.0 (C), 152.5 (C), 154.3
(C), 156.4 (C); vmax(KBr, cm-1):
3312(w), 3210(m), 3056(w), 2870(m), 1696(m),
1646(m), 1503(s), 1435(m), 1346(m), 1106(w); HRMS ES (nanospray) m/z 487(100%).
N-(Benzyloxycarbonyl) imidazole (175)163
O
Benzyl chloroformate (12.50 g, 73.2 mmol) was added to
imidazole (10.00 g, 166.00 mmol) in toluene (100 ml) and the
mixture was stirred for 17 h at RT. The mixture was then
O
N
N
175
filtered and the filtrate was evaporated to yield oil which
solidified on refrigeration to afford the desired product 175 (14.95 g 100%) as white
solid. δH (200 MHz, CDCl3): 5.40 (2H, s, CH2), 7.00 (1H, m, Ar-H), 7.21-7.52 (5H, m,
186
Ar-H), 8.12 (1H, m, Ar-H); δC(50.3MHz, CDCl3) : 69.7 (CH2), 117.0 (CH), 128.6 (CH),
128.8 (CH), 129.1, ( CH) 130.6 (CH), 133.9 (CH), 134.8 (CH) 137.1 (CH), 144.6 (C),
148.5 (C).
1-(Benzyloxycarbonyl)-3-ethylimidazolium Tetrafluoroborate(176)163
O
Triethyloxonium tetrafluoroborate (2.57 g, 13.53 mmol)
was added to N-(benzyloxycarbonyl) imidazole (2.60 g,
O
12.86 mmol) in dichloromethane (35 mL) and the mixture
176
N
N
BF4-
was stirred at RT for 2 h. This generated 176 which was
used insitu for the next reaction. δH (200 MHz, CDCl3):1.52 (3H, t, J=7.3, CH3), 4.35
(2H, q, J=7.3, CH2CH3), 5.50 (2H, s, CH2OCO), 7.34-7.54 (6H, m, Ar-H), 7.70 (1H, m,
Ar-H).
Peptide Nucleic Acid Synthesis164
General Method for loading MBHA Resin with first amino acid to Give a loading factor
of 0.15mm in a glass sinter tube (diameter ~1 cm) MBHA resin (100 mg) was swollen in
DMF:DCM 1:1 (1 mL) for 24 h. Subsequently, the solvent was removed by filtration and
the resin washed with DMF:DCM 1:1 (2 x 1 mL) neutralised with DIPEA 5% in DCM (1
mL) for 3 min. The solvent was removed and the resin was washed with DMF:DCM 1:1
(2 x 1 mL). Boc-protected Lysine (0.030 mmol) was dissolved in DMF, followed by the
addition of HBTU (10.8 mg, 0.029 mmol) and DIPEA (6 L, 0.035 mmol) in DMF (1
mL), which had been allowed to activate for 1 min. The glass sinter tube containing the
resin was then shaken in an orbital shaker for 5 h. After this time the solvent was
removed by filtration and the resin was washed with DMF:DCM 1:1 (2 x 1 mL). A
capping step was then carried out with a mixture of acetic anhydride:DMF:collidine 1:8:1
(1 mL) for 1 h. After this time a Kaiser test168 was carried out (see below). If negative
(yellow), the resin has been successfully loaded and can be used in solid phase synthesis.
187
Kaiser test165
A small amount of resin was removed from the glass sinter tube and placed in a
disposable test tube. To this was first added a few drops of ninhydrin solution (5 g of
ninhydrin in 100 mL of ethanol), followed by a few drops of phenol solution (80 g of
phenol in 20 mL ethanol) and finally a few drops of KCN solution (2 mL of 0.001 M (aq)
KCN in 98 mL pyridine). The mixture was then heated for 5 min at 120 °C. After this
time the colour of the solution indicated whether or not there were any free amino
functionalities present: purple or blue indicates there are and yellow indicates there are
not.
General Method for Solid Phase Synthesis of PNAs by the Boc/Cbz Strategy164
To MBHA resin (200 mg, loading = 0.15 mmol/g with the appropriate Boc-protected
amino acid) was added, firstly, a mixture of TFA (95% v/v and m-cresol 5% v/v) [x 3 for
the first amino acid and x 2 thereafter (1 mL)] and the mixture was allowed to stand for 4
min. After this time the solution was removed by filtration and the resin was washed
with DMF:DCM 1:1 (3 x 1 mL) followed by pyridine (2 x 1 mL). At this point a Kaiser
test was carried out to check the success of the de-protection (this was typically carried
out only every third amino acid). If the Kaiser test indicated this to have been successful,
coupling was carried out.
To Boc-protected PNA monomer (0.090 mmol) was added HBTU (32.4 mg, 0.086
mmol), DMF (750 L), pyridine (250 L) and DIPEA (19.2 L, 110 mmol) and this was
allowed to activate for 1 min. After this time the mixture was added to the resin and the
sinter tube was shaken in an orbital shaker for 20 min. After this time the solvent was
removed by filtration and the resin was washed with DMF (2 x 1 mL) before a Kaiser test
was carried out to check the success of coupling (if this showed the coupling to have been
incomplete, the coupling step was repeated). If successful, a capping step was carried out
with acetic anhydride: DMF: collidine 1:8:1 (1 mL) for 4 min; after this time the solution
was removed by filtration and the resin was then washed with DMF (3x 1 mL), piperidine
188
(5% v/v in DMF (1 mL)) for 4 min and finally with DMF:DCM 1:1 (5 x 1 mL) to
complete the cycle. This cycle was repeated for each addition of a PNA monomer unit.
In the case of the last PNA monomer unit, at the end of the cycle the resin was washed
with DCM (3 x 1 mL) and finally dried in a vacuum desiccator before cleavage.
General Method for Cleavage of a PNA from MBHA resin164
To dry MBHA resin with a PNA attached was added TFA (1 mL) and the mixture was
allowed to stand for 5 min before the solvent was removed by filtration and discarded.
Fresh cleavage solution comprising thioanisole (100 L), m-cresol (100 L), TFA (600
L) and TFMSA (200 L) was prepared. This solution was added to the resin and the
mixture was allowed to stand for 1 h. The mixture was then filtered under a slight
positive pressure of nitrogen. This step was repeated and the two aliquots of cleavage
mixture were combined. TFA (1 mL) was added, the mixture was allowed to stand for 10
min then the solution was removed by filtration and added to the combined aliquots of
cleavage mixture.
This mixture was then divided into two 10 mL centrifuge tubes and dry diethyl ether (10
mL) was added to each tube to precipitate the PNA oligomers. The mixture was then
cooled for a few minutes in ethanol/dry ice before centrifugation at 2500 rpm for 7 min.
Subsequently, the supernatant liquor was decanted and the solid PNA oligomer was resuspended in more diethyl ether (10 mL) in each tube and the mixture centrifuged. This
washing step was repeated 5 times after which the PNA oligomer was dried with a gentle
stream of dry nitrogen.
189
Appendix
X-ray data of Compound 103 (Figure 42)
Table 6. Crystal data and structure refinement for x80265prev
Identification code
x80265
Empirical formula
C13 H10 Cl N5 O2
Formula weight
303.71
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P21/c
Unit cell dimensions
a = 9.2724(5) Å
α= 90°.
b = 11.7943(6) Å
β= 99.180(2)°.
γ= 90°.
Volume
c = 24.4404(11) Å
2638.6(2) Å3
Z
8
Density (calculated)
Absorption coefficient
1.529 Mg/m3
0.302 mm-1
F(000)
1248
Crystal size
0.20 x 0.16 x 0.14 mm3
Theta range for data collection
2.42 to 28.23°.
Index ranges
-12<=h<=12, -15<=k<=15, -32<=l<=32
Reflections collected
90190
Independent reflections
6497 [R(int) = 0.0496]
Completeness to theta = 25.00°
99.9 %
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.9589 and 0.9420
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
Goodness-of-fit on F2
6497 / 0 / 440
Final R indices [I>2sigma(I)]
R1 = 0.0343, wR2 = 0.0883
1.075
190
R indices (all data)
R1 = 0.0482, wR2 = 0.0946
0.309 and -0.264 e.Å-3
Largest diff. peak and hole
Table 7: Bond lengths [Å] and angles [°] for x80265prev
_____________________________________________________
N(1A)-C(6A)
1.3153(18)
N(1A)-C(2A)
1.3730(17)
C(2A)-N(2A)
1.3350(17)
C(2A)-N(3A)
1.3620(17)
N(2A)-H(2A)
0.890(17)
N(2A)-H(2B)
0.861(17)
N(3A)-C(4A)
1.3292(17)
C(4A)-C(5A)
1.4003(18)
C(4A)-N(9A)
1.4036(17)
C(5A)-C(6A)
1.388(2)
C(5A)-N(7A)
1.3962(17)
C(6A)-Cl(6A)
1.7267(14)
N(7A)-C(8A)
1.2940(19)
C(8A)-N(9A)
1.4130(17)
C(8A)-H(8A)
0.950(17)
N(9A)-C(10A)
1.4000(18)
C(10A)-O(10A)
1.1994(17)
C(10A)-O(11A)
1.3336(16)
O(11A)-C(12A)
1.4525(18)
C(12A)-C(13A)
1.502(2)
C(12A)-H(12A)
0.995(18)
C(12A)-H(12B)
1.026(18)
C(13A)-C(18A)
1.392(2)
C(13A)-C(14A)
1.392(2)
C(14A)-C(15A)
1.384(2)
C(14A)-H(14A)
0.928(18)
191
C(15A)-C(16A)
1.384(2)
C(15A)-H(15A)
0.959(19)
C(16A)-C(17A)
1.384(2)
C(16A)-H(16A)
0.976(18)
C(17A)-C(18A)
1.403(2)
C(17A)-H(17A)
0.965(18)
C(18A)-H(18A)
0.943(17)
N(1B)-C(6B)
1.3195(17)
N(1B)-C(2B)
1.3600(17)
C(2B)-N(2B)
1.3403(17)
C(2B)-N(3B)
1.3513(17)
N(2B)-H(2C)
0.886(18)
N(2B)-H(2D)
0.836(18)
N(3B)-C(4B)
1.3240(17)
C(4B)-N(9B)
1.3986(17)
C(4B)-C(5B)
1.4017(18)
C(5B)-C(6B)
1.3863(19)
C(5B)-N(7B)
1.3973(17)
C(6B)-Cl(6B)
1.7271(13)
N(7B)-C(8B)
1.2988(18)
C(8B)-N(9B)
1.4062(17)
C(8B)-H(8B)
0.915(17)
N(9B)-C(10B)
1.4095(17)
C(10B)-O(10B)
1.2054(16)
C(10B)-O(11B)
1.3192(16)
O(11B)-C(12B)
1.4523(16)
C(12B)-C(13B)
1.505(2)
C(12B)-H(12C)
0.965(17)
C(12B)-H(12D)
0.975(17)
C(13B)-C(18B)
1.393(2)
C(13B)-C(14B)
1.395(2)
192
C(14B)-C(15B)
1.383(2)
C(14B)-H(14B)
0.980(18)
C(15B)-C(16B)
1.388(3)
C(15B)-H(15B)
0.93(2)
C(16B)-C(17B)
1.389(2)
C(16B)-H(16B)
0.93(2)
C(17B)-C(18B)
1.397(2)
C(17B)-H(17B)
0.984(19)
C(18B)-H(18B)
0.968(17)
C(6A)-N(1A)-C(2A)
117.19(12)
N(2A)-C(2A)-N(3A)
117.95(12)
N(2A)-C(2A)-N(1A)
115.74(12)
N(3A)-C(2A)-N(1A)
126.31(12)
C(2A)-N(2A)-H(2A)
117.7(10)
C(2A)-N(2A)-H(2B)
119.1(11)
H(2A)-N(2A)-H(2B)
121.2(15)
C(4A)-N(3A)-C(2A)
112.45(11)
N(3A)-C(4A)-C(5A)
126.78(12)
N(3A)-C(4A)-N(9A)
128.75(12)
C(5A)-C(4A)-N(9A)
104.47(11)
C(6A)-C(5A)-N(7A)
133.79(13)
C(6A)-C(5A)-C(4A)
114.64(12)
N(7A)-C(5A)-C(4A)
111.57(12)
N(1A)-C(6A)-C(5A)
122.61(12)
N(1A)-C(6A)-Cl(6A)
117.48(11)
C(5A)-C(6A)-Cl(6A)
119.90(11)
C(8A)-N(7A)-C(5A)
104.86(12)
N(7A)-C(8A)-N(9A)
113.52(12)
N(7A)-C(8A)-H(8A)
126.0(10)
N(9A)-C(8A)-H(8A)
120.5(10)
193
C(10A)-N(9A)-C(4A)
127.92(11)
C(10A)-N(9A)-C(8A)
126.30(11)
C(4A)-N(9A)-C(8A)
105.58(11)
O(10A)-C(10A)-O(11A) 126.10(13)
O(10A)-C(10A)-N(9A)
124.26(12)
O(11A)-C(10A)-N(9A)
109.64(11)
C(10A)-O(11A)-C(12A) 114.11(11)
O(11A)-C(12A)-C(13A) 109.77(12)
O(11A)-C(12A)-H(12A) 108.6(10)
C(13A)-C(12A)-H(12A) 110.5(10)
O(11A)-C(12A)-H(12B) 109.0(9)
C(13A)-C(12A)-H(12B) 108.8(9)
H(12A)-C(12A)-H(12B) 110.2(13)
C(18A)-C(13A)-C(14A) 119.79(13)
C(18A)-C(13A)-C(12A) 123.44(13)
C(14A)-C(13A)-C(12A) 116.77(13)
C(15A)-C(14A)-C(13A) 120.23(15)
C(15A)-C(14A)-H(14A) 122.7(11)
C(13A)-C(14A)-H(14A) 117.0(11)
C(16A)-C(15A)-C(14A) 120.40(15)
C(16A)-C(15A)-H(15A) 118.6(11)
C(14A)-C(15A)-H(15A) 120.9(11)
C(15A)-C(16A)-C(17A) 119.83(14)
C(15A)-C(16A)-H(16A) 119.3(10)
C(17A)-C(16A)-H(16A) 120.8(10)
C(16A)-C(17A)-C(18A) 120.31(14)
C(16A)-C(17A)-H(17A) 119.0(10)
C(18A)-C(17A)-H(17A) 120.6(10)
C(13A)-C(18A)-C(17A) 119.42(14)
C(13A)-C(18A)-H(18A) 118.7(10)
C(17A)-C(18A)-H(18A) 121.8(10)
194
C(6B)-N(1B)-C(2B)
117.47(11)
N(2B)-C(2B)-N(3B)
116.90(12)
N(2B)-C(2B)-N(1B)
116.33(12)
N(3B)-C(2B)-N(1B)
126.75(12)
C(2B)-N(2B)-H(2C)
118.6(11)
C(2B)-N(2B)-H(2D)
119.6(12)
H(2C)-N(2B)-H(2D)
120.9(16)
C(4B)-N(3B)-C(2B)
112.46(11)
N(3B)-C(4B)-N(9B)
128.77(12)
N(3B)-C(4B)-C(5B)
126.68(12)
N(9B)-C(4B)-C(5B)
104.54(11)
C(6B)-C(5B)-N(7B)
133.84(12)
C(6B)-C(5B)-C(4B)
114.68(11)
N(7B)-C(5B)-C(4B)
111.41(11)
N(1B)-C(6B)-C(5B)
121.94(12)
N(1B)-C(6B)-Cl(6B)
117.18(10)
C(5B)-C(6B)-Cl(6B)
120.85(10)
C(8B)-N(7B)-C(5B)
104.66(11)
N(7B)-C(8B)-N(9B)
113.53(12)
N(7B)-C(8B)-H(8B)
124.8(10)
N(9B)-C(8B)-H(8B)
121.6(10)
C(4B)-N(9B)-C(8B)
105.87(11)
C(4B)-N(9B)-C(10B)
129.56(11)
C(8B)-N(9B)-C(10B)
123.90(11)
O(10B)-C(10B)-O(11B) 127.68(13)
O(10B)-C(10B)-N(9B)
122.69(12)
O(11B)-C(10B)-N(9B)
109.62(11)
C(10B)-O(11B)-C(12B) 117.18(11)
O(11B)-C(12B)-C(13B) 107.83(11)
O(11B)-C(12B)-H(12C) 108.5(10)
C(13B)-C(12B)-H(12C) 112.4(10)
195
O(11B)-C(12B)-H(12D) 108.2(9)
C(13B)-C(12B)-H(12D) 111.3(10)
H(12C)-C(12B)-H(12D) 108.6(14)
C(18B)-C(13B)-C(14B) 118.88(14)
C(18B)-C(13B)-C(12B) 122.76(13)
C(14B)-C(13B)-C(12B) 118.36(13)
C(15B)-C(14B)-C(13B) 120.72(16)
C(15B)-C(14B)-H(14B) 121.2(10)
C(13B)-C(14B)-H(14B) 118.0(11)
C(14B)-C(15B)-C(16B) 120.47(15)
C(14B)-C(15B)-H(15B) 118.1(13)
C(16B)-C(15B)-H(15B) 121.4(12)
C(15B)-C(16B)-C(17B) 119.34(16)
C(15B)-C(16B)-H(16B) 121.1(12)
C(17B)-C(16B)-H(16B) 119.6(12)
C(16B)-C(17B)-C(18B) 120.34(17)
C(16B)-C(17B)-H(17B) 119.3(11)
C(18B)-C(17B)-H(17B) 120.4(11)
C(13B)-C(18B)-C(17B) 120.23(14)
C(13B)-C(18B)-H(18B) 119.0(10)
C(17B)-C(18B)-H(18B) 120.8(10)
_____________________________________________________________
Symmetry transformations used to generate equivalent atoms:
196
Table 8: Torsion angles [°] for x80265prev.
________________________________________________________________
C(6A)-N(1A)-C(2A)-N(2A)
178.30(12)
C(6A)-N(1A)-C(2A)-N(3A)
-1.07(19)
N(2A)-C(2A)-N(3A)-C(4A)
-177.89(12)
N(1A)-C(2A)-N(3A)-C(4A)
1.47(18)
C(2A)-N(3A)-C(4A)-C(5A)
-0.68(19)
C(2A)-N(3A)-C(4A)-N(9A)
178.64(12)
N(3A)-C(4A)-C(5A)-C(6A)
-0.4(2)
N(9A)-C(4A)-C(5A)-C(6A)
-179.85(11)
N(3A)-C(4A)-C(5A)-N(7A)
179.15(12)
N(9A)-C(4A)-C(5A)-N(7A)
-0.31(15)
C(2A)-N(1A)-C(6A)-C(5A)
-0.22(19)
C(2A)-N(1A)-C(6A)-Cl(6A)
178.70(9)
N(7A)-C(5A)-C(6A)-N(1A)
-178.53(14)
C(4A)-C(5A)-C(6A)-N(1A)
0.87(19)
N(7A)-C(5A)-C(6A)-Cl(6A)
2.6(2)
C(4A)-C(5A)-C(6A)-Cl(6A)
-178.02(10)
C(6A)-C(5A)-N(7A)-C(8A)
179.87(15)
C(4A)-C(5A)-N(7A)-C(8A)
0.45(15)
C(5A)-N(7A)-C(8A)-N(9A)
-0.41(16)
N(3A)-C(4A)-N(9A)-C(10A)
5.5(2)
C(5A)-C(4A)-N(9A)-C(10A)
-175.06(13)
N(3A)-C(4A)-N(9A)-C(8A)
-179.39(13)
C(5A)-C(4A)-N(9A)-C(8A)
0.06(14)
N(7A)-C(8A)-N(9A)-C(10A)
175.45(13)
N(7A)-C(8A)-N(9A)-C(4A)
0.23(16)
C(4A)-N(9A)-C(10A)-O(10A)
-5.0(2)
C(8A)-N(9A)-C(10A)-O(10A)
-179.14(14)
C(4A)-N(9A)-C(10A)-O(11A)
175.20(12)
C(8A)-N(9A)-C(10A)-O(11A)
1.04(19)
197
O(10A)-C(10A)-O(11A)-C(12A)
5.9(2)
N(9A)-C(10A)-O(11A)-C(12A)
-174.26(11)
C(10A)-O(11A)-C(12A)-C(13A)
-177.07(12)
O(11A)-C(12A)-C(13A)-C(18A)
-6.4(2)
O(11A)-C(12A)-C(13A)-C(14A)
173.23(12)
C(18A)-C(13A)-C(14A)-C(15A)
-0.5(2)
C(12A)-C(13A)-C(14A)-C(15A)
179.89(14)
C(13A)-C(14A)-C(15A)-C(16A)
1.2(2)
C(14A)-C(15A)-C(16A)-C(17A)
-1.1(2)
C(15A)-C(16A)-C(17A)-C(18A)
0.3(2)
C(14A)-C(13A)-C(18A)-C(17A)
-0.3(2)
C(12A)-C(13A)-C(18A)-C(17A)
179.33(14)
C(16A)-C(17A)-C(18A)-C(13A)
0.4(2)
C(6B)-N(1B)-C(2B)-N(2B)
177.65(12)
C(6B)-N(1B)-C(2B)-N(3B)
-0.7(2)
N(2B)-C(2B)-N(3B)-C(4B)
-177.94(13)
N(1B)-C(2B)-N(3B)-C(4B)
0.4(2)
C(2B)-N(3B)-C(4B)-N(9B)
177.97(13)
C(2B)-N(3B)-C(4B)-C(5B)
-0.8(2)
N(3B)-C(4B)-C(5B)-C(6B)
1.4(2)
N(9B)-C(4B)-C(5B)-C(6B)
-177.62(11)
N(3B)-C(4B)-C(5B)-N(7B)
178.68(13)
N(9B)-C(4B)-C(5B)-N(7B)
-0.33(15)
C(2B)-N(1B)-C(6B)-C(5B)
1.3(2)
C(2B)-N(1B)-C(6B)-Cl(6B)
-176.89(10)
N(7B)-C(5B)-C(6B)-N(1B)
-178.12(14)
C(4B)-C(5B)-C(6B)-N(1B)
-1.61(19)
N(7B)-C(5B)-C(6B)-Cl(6B)
0.0(2)
C(4B)-C(5B)-C(6B)-Cl(6B)
176.55(10)
C(6B)-C(5B)-N(7B)-C(8B)
176.41(15)
C(4B)-C(5B)-N(7B)-C(8B)
-0.18(15)
198
C(5B)-N(7B)-C(8B)-N(9B)
0.65(16)
N(3B)-C(4B)-N(9B)-C(8B)
-178.30(14)
C(5B)-C(4B)-N(9B)-C(8B)
0.67(14)
N(3B)-C(4B)-N(9B)-C(10B)
-7.6(2)
C(5B)-C(4B)-N(9B)-C(10B)
171.37(13)
N(7B)-C(8B)-N(9B)-C(4B)
-0.87(16)
N(7B)-C(8B)-N(9B)-C(10B)
-172.23(12)
C(4B)-N(9B)-C(10B)-O(10B)
-173.60(13)
C(8B)-N(9B)-C(10B)-O(10B)
-4.4(2)
C(4B)-N(9B)-C(10B)-O(11B)
5.50(19)
C(8B)-N(9B)-C(10B)-O(11B)
174.71(12)
O(10B)-C(10B)-O(11B)-C(12B)
2.0(2)
N(9B)-C(10B)-O(11B)-C(12B)
-177.00(11)
C(10B)-O(11B)-C(12B)-C(13B)
174.38(11)
O(11B)-C(12B)-C(13B)-C(18B)
-6.13(18)
O(11B)-C(12B)-C(13B)-C(14B)
173.70(12)
C(18B)-C(13B)-C(14B)-C(15B)
0.0(2)
C(12B)-C(13B)-C(14B)-C(15B)
-179.89(14)
C(13B)-C(14B)-C(15B)-C(16B)
-0.1(2)
C(14B)-C(15B)-C(16B)-C(17B)
0.0(2)
C(15B)-C(16B)-C(17B)-C(18B)
0.3(2)
C(14B)-C(13B)-C(18B)-C(17B)
0.3(2)
C(12B)-C(13B)-C(18B)-C(17B)
-179.87(14)
C(16B)-C(17B)-C(18B)-C(13B)
-0.4(2)
________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
199
Table 9: Hydrogen bonds for x80265prev [Å and °].
________________________________________________________________________
____
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
________________________________________________________________________
____
N(2A)-H(2A)...N(7B)#10.890(17)
2.197(17)
3.0771(17)
169.9(14)
N(2A)-H(2B)...N(1B) 0.861(17)
2.212(18)
3.0634(16)
169.9(15)
N(2B)-H(2D)...N(3A) 0.836(18)
2.302(19)
3.1378(17)
177.8(17)
N(2B)-H(2D)...O(10A) 0.836(18)
2.464(17)
2.7501(15)
101.1(13)
N(2B)-H(2C)...O(10A) 0.886(18)
2.432(17)
2.7501(15)
101.6(13)
C(8B)-H(8B)...N(7A)#2 0.915(17)
2.375(17)
3.2779(18)
169.1(14)
________________________________________________________________________
____
Symmetry transformations used to generate equivalent atoms:
#1 -x+2,y-1/2,-z+1/2 #2 x+1,y+1,z
200
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