SYNOPSIS
The work carried out during my research tenure has been compiled in
the
form
of
a
thesis
entitled
"Combinatorial
Synthesis
of
Pyrrolobenzodiazepine Antibiotics". The main aim of this work is to generate
libraries
of
large
number
pyrrolobenozodiazepines,
which
of
biologically
are
known
active
to
be
molecules
like
DNA-binding
and
potentially anticancer molecules. Efforts have been made to find out their
activity against bacteria like mycobacterium tuberculosis. The thesis has
been divided into four chapters.
CHAPTER I: This chapter covers the general introduction about drug
discovery, combinatorial chemistry, pyrrolobenzodiazepines, the aim and
objectives of the present work.
CHAPTER II: This chapter deals with the generation of 210 compound library
of pyrrolo[2,1-c][1,4]benzodiazepine-5,11-diones on solid-phase and their
screening
against
Mycobacterium
tuberculosis,
which
is
the
causes
tuberculosis.
CHAPTER III: This chapter describes the development of solid-phase synthetic
strategies for pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics and
this chapter has been divided in to three sections. SECTION A: This section
consists of the reduction of aromatic nitro/azido functionality on solidsupport employing Al/NiCl2.6H2O and Al/NH4Cl and these reagents have
been utilized for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepines on solidsupport.
SECTION B: This section deals with a new traceless approach for the solidphase
synthesis
intramolecular
of
aza-Wittig
pyrrolo[2,1-c][1,4]benzodiazepines
cyclization
through
reductive
involving
cleavage
by
employing DIBAL-H.
SECTION C: This section comprises of the solid phase synthesis of DNAinteractive pyrrolo[2,1-c][1,4]benzodiazepines by using chloromethyl Wang
I
SYNOPSIS
resin attached to isatoic anhydride as the starting material and synthetic
route involving cyclocondensation followed by reductive cleavage.
CHAPTER IV: This chapter comprises of polymer-assisted solution phase
strategy for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepine anticancer
agents, including naturally occurring DC-81 antibiotic.
CHAPTER I
INTRODUCTION
In this chapter deals with the general introduction about Drug
Discovery, Combinatorial Chemistry and Pyrrolobenzodiazepines. Since the
beginning of modern synthetic organic chemistry, the goal of chemists has
been to produce single compounds in as pure a form as possible. In this
way many new organic molecules were prepared as drug candidates and
submitted to biological tests. Generally, thousands of new materials had to
be prepared to find a single new drug entity. The one-by-one synthesis of
thousands of new compounds followed by their one-by-one testing made the
drug discovery process a very tedious, time consuming and expensive task.
Finding of novel drug is a complex process. Historically, the main source of
biologically active compounds used in drug discovery programs has been
natural products, isolated from plant, animal or fermentation sources.
COMBINATORIAL CHEMISTRY
Combinatorial
chemistry
is
a
new
methodology
developed
by
researchers in the pharmaceutical industry to reduce the time and costs
associated with producing effective and competitive new drugs. By
accelerating the process of chemical synthesis, this method is having a
profound effect on all branches of chemistry, especially on drug discovery.
Through the rapidly evolving technology of combinatorial chemistry, it is
II
SYNOPSIS
now possible to produce libraries of small molecules to screen for novel
bioactivities.
This
powerful
new
technology
has
begun
to
help
pharmaceutical companies to find new drug candidates quickly, save
significant money in preclinical development costs and ultimately change
their fundamental approach to drug discovery.
Combinatorial chemistry is used to synthesize large number of
chemical compounds by combining sets of building blocks. Each newly
synthesized compound’s composition is slightly different from the previous
one. In this way the bench chemists can single handedly prepare many
hundreds or thousands of compounds in the time usually taken to prepare
only a few by orthodox methodologies. Over the last few years, the
combinatorial chemistry has emerged as an exciting new paradigm for the
drug discovery. In a very short time the topic has become the focus of
considerable scientific interests and research efforts.
PYRROLO[2,1-c][1,4]BENZODIAZEPINE ANTIBIOTICS
Cancer is a diseases characterized by uncontrolled growth or spread
of abnormal cells. Since it involves the conversion of any normal cells to a
cancerous cell showing tandem replication and cell division at much faster
rate in comparison to the normal cells and thus provides a potential target
area for the development of chemotherapeutic agents. It is now clear that
chemotherapy’s most effective role in solid tumours is as an adjuvant to
initial therapy by surgical or radiotherapeutic procedures. Chemotherapy
becomes critical to effective treatment because only systemic therapy can
attack micrometastases. These agents can be categorized into functional
subgroups
like
alkylating
agents,
antimetabolites,
antibiotics,
and
antimitotics. The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) belonging to the
class of DNA-interactive antitumour antibiotics have the potential as
regulators of gene expression with possible therapeutic application in the
III
SYNOPSIS
treatment of genetic disorders including cancer. The first PBD antitumour
antibiotic anthramycin has been described by Leimgruber et. al. in 1963, and
since then a number of compounds have been developed on PBD ring
system leading to DNA binding ligands.
H3C
8
OH H
9
N
11
10
OCH 3
H
11a
7
N
5
6
2
3
O
Tomaymycin
Anthramycin
N
H
H
N
H3CO
CONH 2
4
O
N
HO
1
O
N
O
N
N
OCH 3 H3CO
O
SJG-136
H
O
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of potent
naturally occurring low molecular weight antitumour antibiotics originally
isolated from various Streptomyces species. Their common interaction with
DNA has been extensively investigated and it is considered unique since
they bind within the minor groove of DNA forming a covalent aminal bond
between the C11-position of the central B-ring and the N2 amino group of
guanine base. A number of naturally occurring and synthetic compounds
based on PBD ring system, such as anthramycin, tomaymycin, DC-81 and
its dimmers (presently, SJG-136 is under clinical evaluation), have shown
varying degrees of DNA binding affinity and anti-cancer activity.
O
O
N
HN
H2N
N
H
N
HN
H
N
HH N
N
H
N
DNA
N
N
O
11R/S aminal
O
N10-C11 imine
PBD-DNA interaction
IV
N
N
DNA
SYNOPSIS
CHAPTER II
SOLID-PHASE SYNTHESIS OF A LIBRARY OF PYRROLOBENZODIAZEPINE-5,11DIONES WITH POTENTIAL ANTITUBERCULAR ACTIVITY
The benzodiazepine moiety has gained much attention in the
synthetic community, mainly because for its representation as a member of
the family of ‘privileged scaffolds’. In fact, the first heterocyclic templates
prepared on a solid-support has been that of 1,4-benzodiazepines followed
by a large number of reports on the synthesis of similar skeleton.
Pyrrolo[2,1-c][1,4]benozodiazepine-5,11-dione is merely the proline fused
1,4-benzodiazepine-2,5-dione scaffold. However, not much efforts have been
made for the development of solid-phase synthesis of such pyrrolo[2,1c][1,4]benozodiazepine-5,11-diones. This tricyclic ring system has been used
for a number of pharmaceutical applications, such as template for design
and assembly of peptidomimetic agents, anxiolytic drugs, anticonvulsants
and
herbicides.
Interestingly,
N(10)-substituted
pyrrolo[2,1-
c][1,4]benozodiazepine-5,11-diones have shown enhanced biological activity
compared to the corresponding hydrogen analogues particularly in the
preclinical study of anxiolytic agents
A versatile combinatorial approach has been developed and utilized
for the rapid synthesis of pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione (PBD5,11-dione) libraries 10, 15 and 19 containing 210 compounds with varied
substitutions in A, B and C rings. The key aspect of the synthetic strategy
includes Staudinger, intermolecular aza-Wittig reaction followed by imine
reduction and base mediated cyclative cleavage results in the formation of
final resin free compounds. The synthetic design includes, the preparation
of a large array of resin bound 2-azidobenzoyl proline acids, the generation
of iminophosphoranes from these corresponding proline acids, and the
solid-phase aza-Wittig reaction of the iminophosphorane intermediates to
V
SYNOPSIS
imino derivatives followed by their reduction to the corresponding amino
compounds, which, undergo intramolecular cyclization by the cleavage of
the resin. Moreover, the N-alkylation and esterification at C2 position has
been achieved by employing this synthetic sequence.
The Merrifield resin (1) is coupled to the Boc protected L-proline (2) in
presence of potassium fluoride to give the corresponding resin-bound
proline acid (3). The Boc group of 3 was deprotected upon treatment with
trifluoroacetic acid (TFA/CH2Cl2, 3:7) followed by its washing with
triethylamine (1% Et3N/THF) to give the resin-bound proline. The Bocdeprotected resin bound proline acid (4) is coupled with different 2azidobenzoic acids (5{1-11}), employing EDCI and HOBt to afford the desired
resins (6). (Scheme 1).
Scheme 1
O
O
O
O
ii
i
Cl
COOH
1
O
iii
Boc N
X
3
Boc N
R1
HN
X
4
X
O
N3
N
N3
R1
COOH
6
O
X
5
2
Reagents and conditions: (i) KF, DMF, 50 oC, 24 h; (ii) 30% TFA/CH2Cl2, 45 min; (iii) 2-azidobenzoic acids 5,
EDCI, HOBt, DMF, rt, 15-24 h.
The compounds 6 thus obtained are treated with a five fold excess
triphenylphosphine in dry toluene at room temperature to produce the
iminophosphorane intermediate resins (6a) and condensed with different
aldehydes (7) to yield the corresponding imines (8). This step is repeated in
CH2Cl2 under reflux condition to ensure that the entire iminophosphorane
intermediate was converted to the imino compound. These imines (8) are
later reduced with NaCNBH3 in 1% AcOH-DMA to give the amino
VI
SYNOPSIS
substituted products (9), and this step is once again repeated for complete
reduction. Resin bound amino compounds (9) are finally cyclized by treating
with lithiated 5-phenyl-2-oxazolidinone to afford the desired PBD-5,11diones 10 as illustrated in Scheme 2.
Additionally, the hydroxyl group of resin-bound N-(2-azidobenzoyl)-4hydroxypyrrolidine-2-carboxylic acids 6 is activated to obtain different types
of esters 12 by treatment with respective chlorides 11. These compounds (12)
are treated with a five fold excess of triphenylphosphine in dry toluene at
room temperature to produce the iminophosphoranes (12a) and this upon
condensation with different aldehydes (7) provide the corresponding imines
(13). The imines are reduced with NaCNBH3 in 1% AcOH-DMA to give the
amino resins (14). Finally the PBD-5,11-diones (15) with diversity in the Cring have been obtained by cyclative cleavage of amino resins (14) employing
lithiated 5-phenyl-2-oxazolidinone.
Further, the mesyl group of N-(2-azidobenzoyl)-4-methylsulfonyl
oxypyrrolidine-2-carboxylic acid resins 12 is treated with NaN3 to give the
bis-azido resins (16). These compounds are then reacted with a ten fold
excess of triphenylphosphine in dry toluene at room temperature to produce
the
iminophosphoranes
(16a)
and
these
upon
condensation
with
benzaldehyde 7 provide the corresponding imine resins (17). These imine
resins are reduced with NaCNBH3 in 1% AcOH-DMA to give the amino
resins (18), which upon cyclative cleavage with lithiated 5-phenyl-2oxazolidinone afford the desired C-ring 2-amino substituted PBD-5,11diones (19).
The building blocks for the library synthesis (prolines, 2-azidobenzoic
acids, aldehydes, carbonyl and sulfonyl chlorides) have been selected and
used for the generation of 210 compound library with satisfactory yields of
the final products 10, 15 and 19, comprising of 3 sub-libraries (126 + 81 + 3).
VII
SYNOPSIS
Scheme 2
O
O
O
N3
R1
v
N
6
N
6
O
O
R2
O
N
16
16a
ii
O
O
R1
N
OR 3
O
17
13
iii
O
N
N
X
O
8
ii
Ph
O
R1
N
i
17
N
R1
N3
16
i
12a
13
ii
8
N
O
R3 = Ac, Ms, Ts
12
i
6a
R1
OR 3
12
X = H, OH
O
N3
vi
R1
X
O
R2
O
O
N3
N
O
Ph
iii
iii
Ph
R2
O
NH
R1
R2
O
R1
N
9
O
NH
R1
14
R2
Ph
O
N
H
R1
O
N
H
R1
N
Ph
iv
R2
O
N
H
R1
N
X
O
10
18
iv
iv
N
H
O
OR 3
O
O
N
N
X
O
O
NH
O
O
N
OR 3
O
N
H
19
15
Reagents and conditions: (i) TPP, toluene, rt, 3 h; ii) aldehydes 7, CH2Cl2, 4 h; (iii) NaCNBH3, 1% AcOH-DMA,
rt, 4 h; (iv) lithiated oxazolidinone, THF, 0 oC, 2 h; (v) chlorides 11, Et3N, DMAP, CH2Cl2, 0 oC, overnight; (vi)
NaN3, DMF, 50 oC, overnight.
VIII
Ph
SYNOPSIS
These compounds have been screened for in vitro activity against
Mycobacterium tuberculosis H37Rv at 50 g/mL concentration by using agar
diffusion assay procedure. From the generated library of 210 compounds,
142 compounds have been screened and amongst these 25 compounds
have shown to completely inhibit the growth of Mycobacterium tuberculosis
(H37Rv ATCC 27294) at 50 g/mL concentrations (Figure 1).
aldehyde (7)
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
H
7
8
9
acid (5)
10
11
C2- proline
1
2
3
1
OH
OAc
2
3
1
2
3
1
OM s
2
OTs
active compound
3
tested compound
under testing
Figure 1. Antimycobacterium activity data for the PBD- 5,11-diones of 10, 15 and 19.
A versatile approach for the solid-phase synthesis of PBD-5,11-diones
has been developed. The diversity at N(10)-position can be created in a facile
manner by employing this methodology. Moreover, the N-alkylation and
esterification at C(2)-position has been achieved by employing this synthetic
sequence. The reaction conditions used in this protocol are mild and
IX
SYNOPSIS
compounds are obtained in good yields. This method can be potentially used
for
the
generation
compounds
using
of
large
automated
number
of
synthesizer.
pyrrolobenzodiazepine-based
(Journal
of
Combinatorial
Chemistry, 2006, in press)
CHAPTER III
DEVELOPMENT OF SOLID PHASE SYNTHETIC STRATEGIES
PYRROLOBENZODIAZEPINE ANTITUMOUR ANTIBIOTICS
FOR
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of potent,
naturally
occurring,
antitumour
antibiotics
produced
by
various
Streptomyces species. These compounds bind selectively in the minor groove
of DNA while a covalent aminal bond between the electrophilic C11-position
of the PBD and the nucleophilic N2-amino group of a guanine base, possibly
result in the biological activity. A number of naturally occurring and
synthetic compounds based on this PBD ring system, such as anthramycin,
chicamycin, abbeymycin, DC-81 and its dimers have shown varying degrees
of DNA binding affinity and anticancer activity. Moreover, pyrrolo[2,1c][1,4]benzodiazepine-5,11-diones (PBD-5,11-diones) are known as noncovalent interactive minor groove binders. These are also intermediates for
the synthesis of structurally modified PBD-imines via oxidation of secondary
amines or by the reduction of N-protected dilactams. Further, these are
known to exhibit different type of biological properties such as antiphage
activity, analgesic antagonist, anti-inflammatory, psychomotor depressant
activity and herbicidal properties. There are many methods known for the
solution phase synthesis of PBD imines. However, there are only few reports
on the solid phase synthesis of these PBD antitumour antibiotics.
This chapter has been divided into three sections. Section A consists
of the reduction of aromatic nitro/azido functionality on solid-support
X
SYNOPSIS
employing Al/NiCl2.6H2O and Al/NH4Cl and these reagents have been
applied for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepines on solidsupport. Section B deals with a new traceless approach for the solid-phase
synthesis of pyrrolo[2,1-c][1,4]benzodiazepines involving intramolecular azaWittig cyclization through reductive cleavage by employing DIBAL-H. Section
C comprises of the solid phase synthesis of DNA-interactive pyrrolo[2,1c][1,4]benzodiazepines by using chloromethyl Wang resin attached to isatoic
anhydride
as
the
starting
material
and
synthetic
route
involving
cyclocondensation followed by reductive cleavage.
SECTION A:
REDUCTION OF AROMATIC NITRO/AZIDO FUNCTIONALITY ON SOLID
SUPPORT EMPLOYING Al/NICl2.6H2O AND Al/NH4Cl: SYNTHESIS OF
PYRROLOBENZODIAZEPINES
The preparation of small molecule libraries on solid phase is emerging
as an expedient method and is being utilized towards generating
compounds for screening against biological systems, and enhances the drug
discovery effort. Since many combinatorial libraries are generated on solid
support, adapting standard synthetic transformations to the solid phase is
an essential part of increasing the range of compounds, which are
accessible by this technique.
The reduction of aromatic nitro/azido functionalities to corresponding
amines is an important transformation in synthetic organic chemistry, used
to construct a variety of biologically active molecules, especially in
heterocyclic and medicinal chemistry. In solution-phase chemistry, the
reduction of aromatic nitro/azido functionalities is readily accomplished
with a wide variety of reagents. Many of these methods, however, require
heavy metal catalysts, acid conditions, high temperatures or pressure,
which renders most of them not suitable for application to solid phase
XI
SYNOPSIS
organic chemistry. However, relatively few reagents are known for this
synthetically and industrially important reaction on a solid support.
In recent years, aluminium based reagents have been found wide
applications in synthetic chemistry because of their ready availability, easy
handling and low costs. In the present work in search of a solid-phase
compatible method a process by employing Al/NiCl2·6H2O and Al/NH4Cl
reagent systems has been developed for the reduction of aromatic
nitro/azido functionality (1) to the amino functionality (2) (Scheme 1).
Scheme 1
O
X
O
i
R
NH 2
R
1
2
X = NO2, N3
Reagents and conditions: (i) Al/NiCl2.6H2O, THF, rt, Al/NH4Cl, DMF or EtOH, reflux.
SOLID PHASE SYNTHESIS OF PYRROLOBENZODIAZEPINES EMPLOYING Al/NICl2·6H2O
AND Al/NH4Cl
In continuation of these efforts towards the development of solidphase synthetic methodologies for PBD ring systems, a neutral, mild and
efficient method has been employed using Al/NiCl2·6H2O or Al/NH4Cl
reagent system. In this investigation the solid phase procedure has been
applied for the synthesis of biologically important compounds, particularly
C2-hydroxy imine-containing PBD ring systems and their 5,11-diones. As
reported in the literature, that C-ring hydroxy substitution plays an
important role in executing the biological activities as seen in case of
naturally occurring PBDs such as chicamycin A and B, neothramycin A and
B, and abbemycin.
XII
SYNOPSIS
Fmoc-protected 4-hydroxy proline methylester (5) has been obtained
from commercially available trans-4-hydroxy proline (3) by employing thionyl
chloride in methanol to give the trans-4-hydroxy proline methylester
hydrochloride (4). This upon protection with Fmoc-Cl using triethylamine
affords the Fmoc protected 4-hydroxy proline methylester (5) (Scheme 2).
Scheme 2
COOCH 3
COOCH 3
COOH
i
ii
Fmoc-N
HCl.HN
HN
3
OH
OH
OH
5
4
Reagents and conditions: (i) SOCl2, MeOH; (ii) Fmoc-Cl, triethylamine, CH2Cl2, 0 oC.
The Wang resin (6) is activated as trichloroacetimidate (7) by
Hanessian protocol and linked to the hydroxyl group of the N-Fmocprotected trans-4-hydroxy proline methyl ester (5) to obtain the resin-bound
N-Fmoc-protected trans-4-hydroxy proline methyl ester (8). The product,
after cleavage of the Fmoc group using 20% piperidine/DMF, is coupled
with 2-azidobenzoic acid (10) in the presence of DCC and DMAP to provide
the amide resin 11. The reduction of ester group of 11 by DIBAL-H followed
by reductive cyclization using Al/NiCl2·6H2O or Al/NH4Cl and cleavage
affords
the
2-hydroxy-7,8-substituted
PBD
imine
(14).
Furthermore,
reductive cyclization of 11 using Al/NiCl2·6H2O or Al/NH4Cl affords the PBD5,11-dione (15), which upon cleavage from the resin yields 2-hydroxy-7,8substituted PBD-5,11-dione (16) (Scheme 3).
An efficient and cost effective solid phase synthesis of substituted
arylamines from their corresponding nitro and azido substrates has been
demonstrated. This procedure has been further extended towards the
synthesis of pyrrolo[2,1-c][1,4]benzodiazepines and their dilactams in good
XIII
SYNOPSIS
yields. This method is expected to generate combinatorial libraries for
pyrrolo[2,1-c][1,4]benzodiazepines based compounds particularly those
having a 2-hydroxy substituent on the C-ring (Tetrahedron Letters, 2003, 44,
4741).
COOCH 3
Scheme 3
OH
O
i
7
6
CCl 3
+ Fmoc-N
NH
OH
5
ii
COOCH 3
HN
COOCH 3
iii
O
Fmoc-N
O
9
8
X
iv
R
COOH
10 X = NO2, N3
X
COOCH 3
R
X
v
CHO
R
N
11
N
O
O
vi
H
N
vi
O
N
H
R
N
15
O
O
12
N
O
O
H
R
O
O
13
vii
vii
H
N
O
H
N
R
N
O
H
R
N
OH
16
14
O
OH
Reagents and conditions: (i) Cl3CCN, DBU, CH2Cl2; (ii) BF3.OEt2 or CF3SO3H, CH2Cl2;
(iii) 20% piperidine/DMF; (iv) DCC, DMAP, CH2Cl2, 0oC; (v) DIBAL-H, CH2Cl2, -78 oC;
(vi) Al/NiCl2.H2O, THF, rt or Al/NH4Cl, DMF or EtOH, reflux, 3 h; (vii) TFA/CH2Cl2 (1:3).
XIV
SYNOPSIS
SECTION B:
SOLID-PHASE SYNTHESIS OF PYRROLOBENZODIAZEPINES
INVOLVING REDUCTIVE CLEAVAGE
One of the challenges of the solid-phase combinatorial synthesis of
heterocyclic compounds is developing chemical routes that provide access to
the target compounds without leaving any trace of the linker used for
tethering the starting building blocks to the solid support. The imine
containing biologically significant pyrrolobenzodiazepine ring system is a
reactive moiety and requires extremely mild conditions for the cleavage from
the resin during its solid-phase synthesis. In the present investigation a new
traceless
approach
for
the
solid-phase
synthesis
of
pyrrolo[2,1-
c][1,4]benzodiazepines based on reductive cleavage followed by cyclization
employing DIBAL-H has been developed.
Treatment of Wang resin (6) with thionylchloride gives the chloro
Wang resin (17). Chloro Wang resin (17) on treating with potassium
thioacetate in DMF has provided the thioester resin 18. The reduction of 18
using LiBH4 in THF at room temperature gives the thiol Wang resin 19
(Scheme 4).
XV
SYNOPSIS
Scheme 4
i
O
O
Cl
OH
chloro Wang resin (17)
Wang resin (6)
SH
ii
iii
O
O
O
SH
S
18
thiol Wang resin (19)
Reagents and conditions: (i) SOCl2, CH2Cl2, rt, 1 h; (ii) CH3COSK, DMF, rt, 12 h; (iii) LiBH4, THF, rt, 8 h.
The precursor Boc protected proline acid chloride (22) has been
prepared by treating L-prolines (20) with Boc anhydride in presence of 2N
NaOH solution followed by polystyrene triphenylphosphine (PS-TPP) in CCl4
(Scheme 5). This method is advantageous as it avoids the use of acid
liberating reagents like SOCl2, (COCl)2 that leads to BOC-deprotection.
Scheme 5
COOH
COOH
i
HN
20
COCl
ii
Boc N
21
Boc N
22
Reagents and conditions: (i) Boc anhydride, 2N NaOH, THF, rt, 2 h; (ii)
PS-TPP, CCl4, reflux, 4 h.
The resin 19 is attached to the Boc protected proline acid chloride (22)
using triethylamine in dichloromethane to afford Boc protected proline
thioester resin 23. The intermediate (24), after the deprotection of the Boc
group using TFA, is coupled to the corresponding 2-azidobenzoic acid (10) in
the presence of TBTU and DIPEA to provide the required resins (25).
XVI
SYNOPSIS
Treatment of 25 with excess of TPP in dry toluene at room temperature
produced the corresponding resins of iminophosphoranes (26). Finally, the
resins 26 have been treated with DIBAL-H in dry dichloromethane at -78 oC
for 12 h to afford the desired PBD imines (27) (Scheme 6) in good yields (57–
65%). Further, amino resins (28) have been obtained from reduction of 25
with TPP and THF-water. Finally, the dilactams (29) were obtained from
cleavage of resin 28 with K2CO3 and MeOH.
Scheme 6
Boc N
S
O
SH
19
COCl
ii
HN
Boc N
i
S
O
24
23
22
N3
iii
R
COOH
10
N
H
R
PPh3
N
v
R
N
S
O
R
N
O
26
27
S
O
N3
iv
O
N
O
25
vi
H
N
O
H
R
N
O
O
NH 2
vii
R
S
N
O
28
29
Reagents and conditions: (i) triethylamine, CH2Cl2, 0 oC, 6 h; (ii) TFA, CH2Cl2, rt, 1 h; (iii) 2-azidobenzoic acid
(10), TBTU, DIPEA, DMF, rt, 6 h; (iv) TPP, anhydrous toluene, rt, 3 h; (v) DIBAL-H, CH2Cl2, -78 oC, 12 h; (vi)
TPP, THF-water, 2 h; (vii) K2CO3, MeOH-water, 1 h.
A new traceless solid-phase strategy for imine-containing pyrrolo[2,1c][1,4]benzodiazepine ring systems has been demonstrated. This is an
interesting process involving intramolecular aza-Wittig cyclization through
XVII
SYNOPSIS
reductive cleavage by employing DIBAL-H. These reaction conditions are
readily amenable for generating a PBD combinatorial library with diversity
in A and C rings (Tetrahedron Letters, 2004, 45, 7667).
SECTION C:
SOLID-PHASE SYNTHESIS
CYCLO CONDENSATION
OF
PYRROLOBENZODIAZEPINES INVOLVING
The traceless solid-phase synthesis, in which the group used for the
attachment to solid supports is not left with the final target molecule, holds
great importance because it is established fact that the functional group has
a dramatic effect on the biological efficacy of the molecule. In some cases,
the functional group left on the target molecule may not be desired for
bioactivity and may limit the final structural optimization.
The interesting biological activity exhibited by imine containing PBDs
and their dilactam analogues prompted for the development of a new
expeditious solid-phase synthetic methodology for the preparation of these
compounds. In the present investigation, imine containing PBDs have been
prepared on solid-phase by altogether a new and practical approach that
involves the use of polymer-bound isatoic anhydrides as starting materials.
This in turn is coupled with corresponding prolines in DMF at 100-110 ºC
to afford the polymer-bound PBD-5,11-diones. It is observed that by use of
ultrasound in this step it not only reduces the time of the reaction but also
maintains temperature around 50 oC instead of the usual high temperature
(>100 oC). These resin bound PBD-5,11-diones upon mild reduction with
lithium borohydride or sodium borohydride provide the polymer-bound
carbinolamines and finally cleavage of the resin affords the desired PBD
imines. This is one of shortest route for the solid-phase synthesis of imine
containing PBDs that involves four steps with good overall yields (60-66%).
XVIII
SYNOPSIS
The chloromethyl Wang resin (17) and various substituted isatoic
anhydrides (30) have been prepared by known literature methods. The
chloro Wang resin was attached to the isatoic anhydrides to produce the
resin bound isatoic anhydrides (31). These isatoic anhydrides (31) attached
to chloromethyl Wang resin are treated with L-proline or trans-4hydroxyproline to afford the corresponding polymer-bound PBD-5,11-diones
(32). These are reduced with lithium borohydride or sodium borohydride to
give carbinolamine PBD resins (33). Finally, the resin is cleaved by TFA/H2O
to provide the crude products (34) (Scheme 7). Furthermore, these polymerbound PBD-5,11-diones (32) upon cleavage by TFA/H2O gives PBD
dilactams (35) and are further purified by column chromatography.
Scheme 7
N
R
ii
COOH
O
31 O
O
N
O
HN
H
R
N
32 O
iv
OH
H
N
R
N
X
X
33 O
X
Cl
iii
iv
Chloromethyl Wang resin
i
H
N
R
O
30 O
H
N
O
O
H
R
N
H
R
N
35 O
N
X
34 O
X
Reagents and conditions: (i) NaH, anhydrous DMF, rt, 24 h; (ii) DMF, 50 oC, )))), 1 h or DMF, 100-110
o
C, 8 h; (iii) LiBH4, THF, -10 oC, 6 h or NaBH4, MeOH:THF (7:3), 0 oC, 6 h; (iv) TFA:H2O ( 9:1).
An efficient, cost-effective and practical solid-phase synthesis of
pyrrolo[2,1-c][1,4]benzodiazepines
and
XIX
their
dilactams
have
been
SYNOPSIS
demonstrated
by
employing
substituted
isatoic
anhydrides.
This
methodology is highly suitable for the generation of a combinatorial library,
not only of PBD imines but also of PBD-5,11-diones with diversity in both
the A and C rings (Synlett, 2004, 1841)
CHAPTER IV
POLYMER ASSISTED SOLUTION PHASE STRATEGY
PYRROLOBENZODIAZEPINES ANTICANCER AGENTS
FOR THE
SYNTHESIS
OF
In the last few years, the use of solution-phase methods has received
a considerable amount of attention for the parallel synthesis of low
molecular weight compound libraries. Moreover, in the field of solution
phase library generation, the use of polymer-supported reagents is emerging
as a leading strategy that not only gives the advantage of product isolation
and purification of solid phase chemistry but also provides the benefits of
the traditional solution-phase reactions.
The
imine
or
carbinolamine-containing
pyrrolo[2,1-
c][1,4]benzodiazepines are a family of low molecular weight natural products
originally isolated from Streptomyces species, that are known to exhibit
antitumour activity. These antibiotics bind selectively in the minor groove of
DNA while a covalent aminal bond is formed between the electrophilic C11position of the PBD and the nucleophilic N2-amino group of a guanine base,
resulting in biological activity. The S-configuration at the chiral C11aposition provides the PBD structure with the necessary right handed twist
to fit snugly within the minor groove. In conjunction with these efforts and
to the best of my knowledge, for the first time polymer-supported reagents
has been used for the synthesis of pyrrolo[2,1-c][1,4]benzodiazepines.
Further, dilactams are known to exhibit different type of biological
properties
such
as
antiphage
activity,
analgesic
antagonist,
anti-
inflammatory, psychomotor depressant activity and herbicidal properties.
XX
SYNOPSIS
Starting materials prolinol (3) and proline methyl ester (5) have been
prepared from the commercially available L-proline by employing polymer
supported reagents. L-Prolinol (3) has been obtained from L-proline (1) by
reduction with borohydride exchange resin (2) (BER). Proline esterification
has been carried out on L-proline employing amberlyst 15 (4) in MeOH. By
this method water-soluble prolinol and proline methyl ester isolation
becomes easier as this avoids water work-up unlike the conventional
methods (Scheme 1).
Scheme 1
COOCH 3
HN
Amberlyst 15
4
COOH
+
NMe 3-BH4
CH 2OH
2
HN
i
HN
ii
1
5
3
Reagents and Conditions: i) 4, MeOH, rt, 96%; ii) 2, MeOH, rt, 94%.
The synthetic route consists of the coupling of L-prolinol (3) with the
corresponding 2-azidobenzoic acids (6). Interestingly, in the coupling
reaction of prolinol (3) to the azido benzoic acid (6) by using polymersupported cyclohexylcarbodiimide (7), the excess of acid and urea byproducts can be simply filtered from the azido alcohols 8. Upon oxidation by
modified Swern procedure using polymer-supported sulfoxide (9a) (PSS),
compounds 8 afford the azido aldehydes 10, which, upon intramolecular
reductive cyclization with polymer-supported TPP (11), afford the desired
imines 12 containing the pyrrolobenzodiazepine ring system. The oxidation
of
8
have
also
been
carried
out
by
employing
polymer-supported
perruthenate (9b) (PSP) as an alternative reagent. The use of PSS (9a) and
PSP
(9b)
makes
this
method
devoid
of
the
unpleasant
smell
of
dimethylsulfide and further the formation of polymer-linked phosphine
oxide can be easily filtered out from the PBD imines. In the case of 12,
XXI
SYNOPSIS
debenzylation by employing Pd/C provides the naturally occurring DC-81
(13). The PBD dilactams (15) have also been obtained by polymer-supported
reagents employing L-proline methyl ester (5) instead of L-prolinol to yield
PBD dilactams 15 (Scheme 2).
Scheme 2
R
7
R
COOH
CH 2OH
N3
N C N
N3
N
i
O
6
8
O
O
9a
or
+
NMe 3-RuO 4
N C N
v
O
S
5
7
ii
9b
COOCH 3
N3
R
N
14 O
N
O
10
PPh2
PPh2
iii
iii
11
11
H
N
CHO
N3
R
O
H
N
H
R
R
N
N
iv
15 O
N
HO
12
O
H
N
H3CO
O
13
Reagents and conditions: (i) 3, CH2Cl2, rt, 96-99%; (ii) (COCl)2, TEA, CH2Cl2, -50 oC -rt, 93-96%;
(iii) CH2Cl2, rt, 94-97%; (iv) 10% Pd/C, 1,4-Cyclohexadiene; (v) 5, CH2Cl2, rt, 97-99%.
An efficient procedure has developed for the clean preparation of
imine-containing PBDs starting from L-proline and azido benzoic acids
using polymer-supported reagents. It is noteworthy in the entire process,
XXII
SYNOPSIS
the work-up has been simplified to filtration and evaporation for all the
steps. Application of this protocol has generated an array of iminecontaining PBDs and their dilactam derivatives, and further, in this
methodology all the reagents could be reused thus addressing the problems
of environmental and economical sustainability (‘green’ chemistry) (Synlett,
2004, 2523)
XXIII