2
A1B2
120
member
library
HO
A2B2
OH O
BL1
BL2
A2B2
A3B2
MIC = 0.2 mg/mL
Set 2
BL1-BL20
BL3
BL4
A4B2
A5B2
BL5
A6B2
BL6
BL7
BL8
BL9
BL10
BL11
BL12
BL13
BL14
BL15
BL16
BL17
BL18
BL19
BL20
Results &
Discussion
Results and Discussion
The present work describes the design and combinatorial synthesis of small parallel
libraries of chalcones, peptidyl chalcones and peptidyl heterocycles, either in solution
or on solid phase. The chalcone libraries, both parallel and indexed, were synthesized
under Claisen-Schmidt conditions in solution phase. Peptidyl chalcones were
synthesized on solid phase using phosphorane bound resin, while the derived
heterocycles were synthesized in solution phase. The characterization of all the
synthesized compounds was based on their physicochemical and spectroscopic data.
Combinatorial chemistry is an exciting approach to chemical synthesis that enables
the creation of a large number of organic compounds by linking chemical blocks in all
possible combinations. Fig 2.1 shows a comparison of the conventional versus
combinatorial synthesis.
Conventional synthesis
One compound only
Combinatorial synthesis
A library of nine compounds
Fig 2.1. Comparison of conventional versus combinatorial synthesis.
Classical synthesis i.e. the conversion of a reactant A to a final product D usually
involves a multistep sequence, followed by purification and complete characterization
of the products before screening. Guided by the biological activity of the previous
compound, the next analogue is then designed, prepared and screened again. This
process is repeated to optimize both activity and selectivity. On the contrary,
combinatorial synthesis involves the synthesis of a large number of compounds; this
collection may be a chemical mixture, a physical mixture, or individual pure
Chapter 2 ♦Results and Discussion
- 36 -
compounds. The collection is then tested for biological activity and the active
compound is identified finally by deconvolution and is made in quantity as a single
compound.1
2.1. Combinatorial synthesis
Parallel solution phase synthesis is the most straightforward method for library
preparation due to its close resemblance to traditional synthesis. It involves the
reaction of a single compound A with multiple reactants (B1, B2, …Bn), which gives
rise to a compound library of n individual products AB1, AB2…ABn. The library is
then evaluated, usually without purification and with only minimal characterization of
the individual compounds, by means of a rapid-throughput screening methodology.
Individual compounds are synthesized in different vessels and are available directly
for analysis after purification. Parallel chemistry has the advantage that doing all the
reactions at a time in different vessels saves time. However, the work-up has to be
done separately for each NCE.
2.1.1. Synthesis of a parallel library of amino chalcones (1-20)
A small parallel library of twenty different substituted amino chalcones (1-20) was
synthesized in solution phase under Claisen Schmidt conditions using different
substituted benzaldehydes and 4′-aminoacetophenone (Scheme 2.1). The selection of
substituents on benzaldehyde (B ring) was made on the basis of their size,
lipophilicity and electronic properties and they were changed systematically, one at a
time at 2-, 3- and 4- positions to get a set of regioisomers. The reaction was carried
out at room temperature and the reaction time varied from 3-8 h.146
H2N
R
H
O
NaOH/ EtOH
B
H2N
O
R
A
O
1-20
No.
R
No.
R
No.
R
No.
R
1
H
6
3-OCH3
11
4-NO2
16
2-Br
2
2-OH
7
4-OCH3
12
2-Cl
17
3-Br
3
3-OH
8
3,4-(OCH3)2
13
3-Cl
18
4-CH3
4
4-OH
9
2-NO2
14
4-Cl
19
3-OH, 4-OCH3
5
2-OCH3
10
3-NO2
15
4-F
20
4-N(CH3)2
Scheme 2.1. Parallel synthesis of a library of amino chalcones (1-20).
Chapter 2 ♦Results and Discussion
- 37 -
The synthesized chalcones were either purified by recrystallization or by flash
chromatography using n-hexane and ethyl acetate as eluent. The purity was assessed
through multiple TLC and the Rf values were recorded. Melting points were found to
vary from 55 to 200 oC, while the yield varied from 60-87%. The characterization was
made on the basis of physical parameters and spectral analysis as given in Chapter 4.
In IR, amino chalcones, being the primary amines, gave two bands of medium
intensity in the range of 2847-3474 cm–1 and 2555-3385 cm–1. The C=O and C=C
stretching vibrations appeared in the range of 1600-1690 cm–1 and 1508-1600 cm–1,
respectively. A weak peak of aromatic C–H stretching was often observed at 30413246 cm–1. A broad peak due to –OH group appeared at 3065-3600 cm–1. The
stretching frequencies for different substituents on ring B e.g. C-OCH3, N-CH3, C-Br,
C-Cl, C-F in different chalcones were observed in the range of 3041-3239, 2904, 736752, 842-869, 1092 cm–1. The two bands of unsymmetrical and symmetrical
stretching in chalcones 9-11 for C-NO2 appeared at 1404-1514 cm–1. In an inspection
of the IR stretching frequencies of different chalcones, it was observed that electron
withdrawing substituents particularly at ortho and para positions on ring B shifted the
stretching frequency corresponding to an amino group on ring A by a higher value
e.g. the nitro group at ring B was found to shift the stretching frequencies of the 4NH2 on ring A by about 10 cm-1 i.e. p-NO2 substituted B ring resulted in the highest
shift of 4-NH2 group; at 3481 cm-1 and 3385 cm-1; o-NO2 resulted in a lower shift;
3472 cm-1, 3382 cm-1, whereas the m-NO2 was found to shift these frequencies to
3469 cm-1 and 3380 cm-1.
The HR-MS spectra of chalcones 1-20 showed the molecular ion peak in each case
confirming the identity of these compounds. Halogen substituted chalcones (12-14
and 16-17) showed isotopic peaks corresponding to molecular ion and respective,
fragments in almost 3:1 ratio for Cl35: Cl37 (12-14) and 1:1 ratio for Br79: Br81 (16-17),
respectively. The mass fragmentation pattern of chalcone 1 is given in Scheme 2.2.
Chapter 2 ♦Results and Discussion
- 38 +
H2N m/z = 66
m/z = 91
+
-C2H2
+
O
H2N
H2N
H2C
m/z = 222
m/z = 92
+
-H
H2N
H2N
O
m/z = 223
+
O
m/z = 131
-CO
CH
m/z = 103
m/z = 120
+
-CO
+
+
C O
-H
-H
+
Scheme 2.2. Mass fragmentation pattern of amino chalcone 1.
The 1H NMR data of all compounds 1-20 were recorded and a few generalizations
could be made e.g. the aromatic protons of ring A H-2′, H-6′ and H-3′, H-5′ always
appeared as two distinct doublets with an integration of two protons. The vinylic
protons appeared as two distinct doublets, each with an integration of one proton with
a coupling constant of ~14-16 Hz in all cases. 1H NMR data of chalcone 2 is given in
Table 2.1 as a representative example.
The most characteristic chemical shifts are those of α- and β- olefinic protons (vinylic
protons). α Proton appeared upfield at 6.98 ppm, while β proton appeared at 7.78 ppm
with a coupling constant of 14.8 Hz indicating a trans orientation of the double bond
and hence an E configuration of the compound. The –NH2 protons appeared at 5.98
ppm. The two doublets at 8.01 ppm and 7.31 ppm (J = 8.3 Hz) were assigned to H-2′,
6′ and H-3′, 5′, respectively.
Chapter 2 ♦Results and Discussion
- 39 -
Table 2.1. 1H NMR data of 1-(4′-aminophenyl)-3-(2-hydroxyphenyl)-2-propen-1-one (2).
HO
3'
H2N
4'
β
2'
3
1
4
5
6
1'
α
5'
6'
2
O
2
δ ppm
Multiplicity
Integration
Coupling constant
Assignment
J (Hz)
5.98
bs
2H
-
NH2
6.98
d
1H
14.8
H−C(α)
7.75
d
1H
14.8
H−C(β)
8.01
d
2H
8.3
H−C(2′,6′)
7.31
d
2H
8.3
H−C(3′,5′)
7.41
d
1H
8.0
H−C(3)
7.04
d
1H
8.1
H−C(6)
6.98
t
1H
8.0
H−C(5)
6.95
t
1H
8.0
H−C(4)
9.88
s
1H
-
OH
2.1.2. Biological screening of a chalcone library
Amino chalcones have been reported in the literature with potential antitumor, antiproliferative, antiparasitic and bactericidal activities.138-139 The synthesized amino
chalcones were tested for their inhibitory potential as phosphatase inhibitors,
antibacterial and cytotoxic agents, as discussed in the following sections.
2.1.2.1. Phosphatase inhibition
A phosphatase is an enzyme that removes a phosphate group from its substrate by
hydrolyzing phosphoric acid monoesters into a phosphate ion and a molecule with a
free hydroxyl group. They act opposite to the kinases/phosphorylases, which add a
phosphate group to proteins by using higher energy molecules such as ATP. The
addition of a phosphate group may activate or deactivate an enzyme (e.g. Kinase
signaling pathways) or enable a protein-protein interaction to occur; therefore,
phosphatases are integral to many signal transduction pathways e.g. in diabetes. Their
role in signal transduction is because they regulate the proteins to which they are
Chapter 2 ♦Results and Discussion
- 40 -
attached. In order to reverse the regulatory effect, the phosphate group is removed,
which occurs on its own by hydrolysis, or is mediated by protein phosphatases.
Insulin is the major hormone responsible for lowering blood sugar level. The lack of
insulin as a result of the degeneration of special insulin producing cells (called beta
cells) in pancreas,170 or the lack of response of target cells to normal circulating
insulin levels, are the key pathological features of type I and type II diabetes,171
respectively. Insulin binding to its cell surface transmembrane receptor stimulates
autophosphorylation and activation of intrinsic protein tyrosinase kinase (PTK)
activity and subsequent phosphorylation of insulin receptor substrates.
The control of insulin receptor signaling involves the coordinated action of both
positive and negative regulatory proteins. Among the negative regulatory proteins,
protein tyrosinase phosphatases (PTPs) play a prominent role and their inhibition
mimics several actions of insulin including the stimulation of glucose uptake.172-174
The development of drugs capable of inhibiting PTPs may allow the enhanced or
prolonged activation of the insulin receptor and have therapeutic use in the treatment
of type II diabetes.175-177
With the aim of finding some potential antidiabetic compounds, the synthesized
amino chalcones 1-20 were tested against a large variety of fifteen protein
phosphatases, e.g. Mycobacterium tuberculosis inosit phosphatase (Mtb Inosit
Pptase),
Mycobacterium
tuberculosis
phosphoglyceromutase
(Mtb
PGM),
Mycobacterium tuberculosis aroA (Mtb aroA), Mycobacterium tuberculosis aroK
(Mtb aroK), Mycobacterium tuberculosis protein tyrosinase phosphatase A (Mtb
ptpA), human protein tyrosinase phosphatase 1B (hum ptp1B), human protein
tyrosinase phosphatase N3 (hum ptpN3), human protein tyrosinase phosphatase N5
(hum ptpN5), human protein tyrosinase phosphatase N7 (hum ptpN7), human protein
tyrosinase phosphatase RJ (hum ptpRJ), human protein tyrosinase phosphatase RK6
(hum ptpRK6), human protein tyrosinase phosphatase ROG7 (hum ptpROG7), human
protein tyrosinase phosphatase RR (hum ptpRR), human protein tyrosinase
phosphatase RS (hum ptpRS) and Mycobacterium tuberculosis protein tyrosinase
phosphatase SHP2 (ptp SHP2). All compounds were tested at a concentration of 0.1
mM. They were found weakly active against Mtb Inosit Pptase, but were found to be
very potent when tested against (Mtb PGM). The data of the activity of the tested
Chapter 2 ♦Results and Discussion
- 41 -
compounds 1-20 represented as % inhibition against Mtb inosit Pptase is given in
Table 2.2. Compounds 1, 3-9 and 13 inhibited the target enzyme only to some extent.
Table 2.2. Mtb inosit Pptase inhibitory activity of chalcones 1-20.
No.
% Inh.
No.
% Inh.
No.
% Inh.
No.
% Inh.
1
21
6
4
11
-
16
-
2
-
7
26
12
-
17
-
3
32
8
3
13
7
18
-
4
21
9
14
14
-
19
-
5
8
10
-
15
-
20
-
PGM is the key enzyme for isomerization of 3-phosphoglycerate to 2phosphoglycerate in the glycolysis pathway during respiration and PGM inhibitors are
expected to maintain blood sugar levels for routine tasks in ischemic patients.178 The
phosphoglycerate kinase step follows after isomerization step involving PGM and this
results in the production of ATP, synthesis of pyruvic acid for aerobic production of
energy in Krebs cycle and chimiosmosis. PGM is also involved in gluconeogenic
transformation of phosphoenolpyruvate into glycogen and results in weakness after
strenuous exercise. Overacting PGM may result in lower blood sugar levels in
diabetic patients. PGM is found in all species but uses different variants. Chalcones 120 when tested against Mtb PGM, were found to be quite potent. The data is
represented as % inhibition in Table 2.3. Chalcones 8 and 19 showed 60 % inhibition,
while chalcone 3, 13 and 15 did not show any activity. Chalcones 4 and 11 with
substituents 4-OH and 4-NO2 groups were found to be the lead structures of the
library with 96 and 99 % inhibition, respectively.
Table 2.3. Mtb PGM inhibitory activity of chalcones 1-20.
No.
% Inh.
1
44
2
No.
% Inh.
No.
% Inh.
No.
% Inh.
6
43
11
99
16
24
17
7
44
12
47
17
17
3
-
8
60
13
-
18
48
4
96
9
45
14
41
19
60
5
15
10
34
15
-
20
41
It may be concluded that the suitable competitive reversible inhibitors of PGM e.g.
the lead structures 4 and 11 are expected to be potential candidates for developing into
antidiabetic drugs. Molecular docking studies on these chalcones in the active site of
Chapter 2 ♦Results and Discussion
- 42 -
PGM providing further insight into the mechanism of their inhibition have been
carried out as discussed in Chapter 3.
2.1.2.2. Antibacterial activity
Chalcones are well known to have antibacterial properties.140 The members of the
library (1-20) were, therefore, tested for their bactericidal activity against six bacterial
strains, namely Escherichia coli, Bacillus subtilis, Enterobacter aerogenes,
Staphylococcus aureus, Pseudomonas pickitti and Salmonella setubal. Each
compound and the controls were tested at a concentration of 0.1 mg/mL.
Roxithromycin and Cefixim were used as positive control, whereas DMSO was used
as negative control.
Table 2.4. Bactericidal activity of amino chalcones (1-20).
No.
S.aureus
E.aerogens
1
0
0
0
11
2
0
10
0
11
3
0
0
0
11
4
0
0
0
11
5
0
0
0
11
6
0
0
0
11
7
0
0
0
11
8
0
0
0
0
9
0
0
9
0
10
0
0
9.5
0
11
0
0
0
0
12
0
0
0
0
13
0
0
0
11
14
0
10
0
0
15
0
0
9
0
16
0
0
9
0
17
0
0
9
0
18
0
0
9
0
19
11
12
13
0
20
0
0
0
0
Ra
26
26
19
21
b
24
26
32
21
C
a
Zone of Inhibition (mm)
B.subtilis
E.coli
R= Roxithromycin (= Rulid®); b C= Cefixim®.
Chapter 2 ♦Results and Discussion
- 43 -
It is evident from the data in Table 2.4 that all the tested chalcones did not show any
significant activity against any of the tested bacterial strains. Compounds 1-7 and 13
showed weak activity against E. aerogenes, while 2 and 14 showed weak activity
against B.subtilis. Compounds 9, 10, 15, 16, 17 and 18 showed very weak activities
against E.coli, while chalcone 19 showed moderate activity against E.coli. The same
compound was found weakly active against B.subtilis and S.aureus.
2.1.2.3. Brine shrimp lethality studies
Chalcones, in general, are known to have cytotoxic potential179-180 and amino
chalcones have been reported as cytotoxic agents.138-140 Studies on the cytotoxicity of
chalcones 1-20 was carried out through brine shrimp lethality (BSL) assay, which is a
rapid and economic benchtop assay employing the use of easily available brine
shrimps; Artimisia salina as test animals.
Artimisia salina
The test is a pre-screen of antitumor properties.181-182 BSL testing was performed with
different concentrations (150, 100 & 50 µg/mL) of the test samples prepared in
DMSO and LD50 (lethal dose to kill 50% of test animals) values were recorded in µM
quantities as shown in Table 2.5.
Table 2.5. BSL studies on amino chalcones (1-20).
No.
LD50
No.
LD50
No.
LD50
No.
LD50
1
6.68
6
3.79
11
130.56
16
8.34
2
46.50
7
0.24
12
16.22
17
83.71
3
60.54
8
13
52.30
18
150.11
4
7.10
9
160.63
14
14.61
19
-
5
2.72
10
557.02
15
614.42
20
2.59
-
Chalcone 7 with methoxy substituent on ring B was found to be the LEAD structure
with an LD50 value of 0.24 µM, while chalcones 1, 4-6, 16 and 20 showed strong
cytotoxic behavior as reflected from their LD50 values ranging from 2.59-8.34 µM.
Chalcones 2, 3, 12-14 and 17 showed good cytotoxic activity with LD50 < 100.
Chapter 2 ♦Results and Discussion
- 44 OCH3
H2N
O
7 (LD50 = 0.24 µM)
2.2. Indexed combinatorial synthesis
The synthesis of positional scanning library also called indexed libraries represents
one of the most useful protocols for mixture synthesis. It is much less time intensive
as compared to parallel synthesis of individual compounds and produces depository
libraries for use in multiple screens with immediate deconvolution.183 Deconvolution
is a multistep process where smaller libraries are successively prepared and tested to
identify the individual active members of a combinatorial library. Positional scanning
libraries provide lead identities in a single round of testing and the method is ideally
suited for cellular assays that involve membrane-bound targets.184 The synthesis of
positional scanning libraries represents one of the most useful protocols for mixture
synthesis and can easily be conducted in solution phase but it is not easily adaptable
to solid phase synthesis.185
Indexed combinatorial synthesis is a mixture synthesis, which involves the reaction of
two components (building blocks) at a time such that one component is kept constant,
while the other is used as an equimolar mixture. Alternatively, the synthesis is carried
out through keeping second building block constant and varying the first one. These
efforts result in the synthesis of two daughter libraries (two sets of indexed libraries,
also called sub-libraries or pools). The two libraries (product mixtures) are tested and
their activities are used as “indices” to the rows or columns of a 2D matrix reflecting
the activities of individual compounds. Libraries can be preceded with the third
building block for getting 3D indexed libraries as well and the process may
continue.65
Two indexed combinatorial libraries (120 and 175 member) of chalcones were
synthesized in solution phase and tested for their antibacterial, cytotoxic and
antitumor properties followed by deconvolution, which led to the identification of
potent antibacterial and antitumor chalcones as leads of two libraries. The reliability
and success of the process was assessed by confirming the presence of all component
chalcones in the indexed library (product mixture) through GC-MS analysis.
Chapter 2 ♦Results and Discussion
- 45 -
2.2.1. A 120-member chalcone library147
A 120 member chalcone library was designed and synthesized with a view of finding
potential antibacterial chalcones through deconvolution.
2.2.1.1. Synthesis. A 120 member indexed chalcone library was designed by using
six acetophenones (A1-A6) and 20 benzaldehydes (B1-B20) under Claisen Schmidt
conditions (Scheme 2.3).
R
R'
R
O
H
NaOH/ EtOH
R'
O
Acetophenones
O
Benzaldehydes
R′
Benzaldehydes
R
R
A1
C6H5
B1
Ph
B11
4-NO2C6H4
A2
2′-OHC6H4
B2
2-OHC6H4
B12
2-ClC6H4
A3
4′-NH2C6H4
B3
3-OHC6H4
B13
3-ClC6H4
A4
2′,4′,5′-(OCH3)3C6H2
B4
4-OHC6H4
B14
4-ClC6H4
A5
3′,4′,5′-(OCH3)3C6H2
B5
2-OCH3C6H4
B15
4-FC6H4
A6
2′,4′-(Br)2C6H3
B6
3-OCH3C6H4
B16
2-BrC6H4
B7
4-OCH3C6H4
B17
3-BrC6H4
B8
3,4-(OCH3)2C6H3
B18
4-CH3C6H4
B9
2-NO2C6H4
B19
3-OH,4-OCH3C6H3
B10
3-NO2C6H4
B20
4-N(CH3)2C6H4
Scheme 2.3. Synthesis of a 120 member chalcone library.
The library was synthesized in the form of two sets of daughter libraries (Set 1 and Set
2). Set 1 contained six daughter libraries, each containing 20 compounds, whereas Set
2 contained twenty daughter libraries, each containing 6 compounds (Fig. 2.2).
Parent indexed library
n=6
n = 20
∑ R´COCH3
+
∑ RCHO
R
n = 120
∑
R'
O
Indexed daughter libraries (Set 1) (6 libraries, each containing 20 compounds)
n = 20
R´COCH3
+
R
n = 20
∑ RCHO
∑
R'
O
Indexed daughter libraries (Set 2) (20 libraries, each containing 6 compounds)
n=6
∑
R
n=6
R´COCH3 +
RCHO
∑
R'
O
Fig. 2.2. Synthesis of indexed libraries of Sets 1 and 2.
Chapter 2 ♦Results and Discussion
- 46 -
The presence of all the library members in each library was confirmed through GCMS analysis. The identification of some small molecular libraries through GC-MS has
already been reported.186-187 For indexed library, the daughter libraries were run
through a DB-5ms column containing a stationary phase of 5% phenyl and 95%
dimethylarylenesiloxane with He gas. A 5973 inert MS selective detector was used.
The GC-MS spectrum of AL1 library is given in Fig 2.3, whereas the mass fragments
contained in each peak are given in Fig. 2.4 (a-c) and Fig. 2.5 (d-l). The GC-MS of
the daughter library AL1 is discussed in detail. The library was analyzed using
different GC-MS programs and the best results were found when the temperature was
kept from 120 to 300 °C at a rate of 10°C/min, whereas inlet was kept at 250 °C. The
20 members of the library were found separated as 12 peaks, but the relative peak
areas indicated the presence of all 20 compounds. All the compounds were eluted in
17 minutes. The first peak separated at 11.2 min with M+. peak of 226 indicating the
presence of fluorochalcone. The second peak separated at 11.4 min, which contained
four chalcones as indicated through the relative peak area. The presence of these
chalcones was confirmed through the M+. and fragment ion peaks. The M+. peaks at
208 indicated the presence of unsubstituted chalcone. A hydroxychalcone was
detected through a weak [M + H]+ peak at 226. A strong peak at 207 indicated the
presence of M+. – NO2 from 2- nitrochalcone or 4-nitrochalcone. The third peak
appeared at 12.75 min with a weak M+. peak at 238 and [M – CH3]+ peak at 223
indicating a methoxy chalcone. The peaks at 12.93 min, 13.10 min and 13.28 min
corresponded to chlorochalcones, each giving a molecular ion peak at 242 and M+.+2
peak at 244 with relative abundance of 3:1. The 3-hydroxy-4-methoxychalcone
appeared at 13.71 min giving [M – O]+ peak at 238, [M – OH]+ at 237 and [M –
OCH3]+ at 223. A bromochalcone was eluted at 14.09 min giving M+. and M+.+2
peaks at 286 and 288 in 1:1 ratio. At 14.22 min, a peak eluted with a peak area
indicating the presence of four chalcones. A careful analysis indicated the presence of
two hydroxy- and two methoxy-chalcones. The molecular ion peaks were observed as
M+. at 238 and [M + H]+ at 239 for methoxy chalcones, whereas as M+. at 224 and [M
– H]+ at 223 for hydroxy chalcone. The 238 – 28 and 239 – 28 were the peaks found
at 210 and 211 after CO. removal from methoxy chalcones. Similarly, 224 – CO. and
223 – CO. were observed at 196 and 195 for hydroxy chalcones. At 14.28 min,
Chapter 2 ♦Results and Discussion
- 47 -
Fig. 2.3. The GC-MS spectrum of the library AL1 containing 20 compounds.
Fig. 2.4. The mass spectra for each GC peaks (a-c).
Chapter 2 ♦Results and Discussion
- 48 -
Fig. 2.5. The mass spectra for each GC peaks (d-l).
Chapter 2 ♦Results and Discussion
- 49 -
a bromochalcone appeared with M+. and M+. + 2 peaks at 286 and 288. The spectrum
showed very weak peaks at 256 and 258 after CO. removal from M+. and M+. + 2. The
methyl chalcone also appeared in the same peak as indicated by [M + H]+ peak at 223.
Furthermore, a peak at 207 indicated the [M – CH3]+ for methyl chalcone or the M+. –
Br. for bromochalcone. Dimethoxychalcone and a nitrochalcone at 15.79 min were
identified through the M+. peaks at 268 and 253, respectively. The 253 – O. was
observed at 237 whereas 253 – NO. appeared at 223 and 253 – NO2 at 207 confirming
the presence of a nitro chalcone. The last peak appeared at 16.7 min containing the
N,N-dimethylaminochalcone with a molecular ion peak at 251. The M+.– CO.
appeared at 223.
The synthesized indexed library can be represented as a 2D matrix, wherein the x-axis
has six structural variants (acetophenones), and the y-axis has 20 structural variants
(benzaldehydes) leading to a 6 × 20 grid (Table 2.6).
Table. 2.6. Conceptual matrix for an indexed 120 member chalcone library.
Acetophenones
A1
A2
A3
A4
A5
A6
B1
A1 B1
A2 B1
A3 B1
A4 B1
A5 B1
A6 B1
B2
A1 B2
A2 B2
A3 B2
A4 B2
A5 B2
A6 B2
B3
A1 B3
A2 B3
A3 B3
A4 B3
A5 B3
A6 B3
B4
A1 B4
A2 B4
A3 B4
A4 B4
A5 B4
A6 B4
B5
A1 B5
A2 B5
A3 B5
A4 B5
A5 B5
A6 B5
B6
A1 B6
A2 B6
A3 B6
A4 B6
A5 B6
A6 B6
B7
A1 B7
A2 B7
A3 B7
A4 B7
A5 B7
A6 B7
B8
A1 B8
A2 B8
A3 B8
A4 B8
A5 B8
A6 B8
B9
A1 B9
A2 B9
A3 B9
A4 B9
A5 B9
A6 B9
B10
A1 B10
A2 B10
A3 B10
A4 B10
A5 B10
A6B10
B11
A1 B11
A2 B11
A3 B11
A4 B11
A5 B11
A6B11
B12
A1 B12
A2 B12
A3 B12
A4 B12
A5 B12
A6B12
B13
A1 B13
A2 B13
A3 B13
A4 B13
A5 B13
A6B13
B14
A1 B14
A2 B14
A3 B14
A4 B14
A5 B14
A6B14
B15
A1 B15
A2 B15
A3 B15
A4 B15
A5 B15
A6B15
B16
A1 B16
A2 B16
A3 B16
A4 B16
A5 B16
A6B16
B17
A1 B17
A2 B17
A3B17
A4 B17
A5 B17
A6B17
B18
A1 B18
A2 B18
A3 B18
A4 B18
A5 B18
A6B18
B19
A1 B19
A2 B19
A3 B19
A4 B19
A5 B19
A6B19
B20
A1 B20
A2 B20
A3 B20
A4 B20
A5 B20
A6B20
Benzaldehydes
Chapter 2 ♦Results and Discussion
- 50 -
The number of the building blocks was chosen such that the library was large enough
to demonstrate the principle but small enough to verify the composition of library by
analytical methods. Besides, the size of the library was kept such that it was soluble
in an assay mixture for its effective screening. The chemistry of the reaction used in
the preparation of library was investigated on a single compound. The choice of the
reagents reflects the chemical diversity in terms of hydrophobic and lipophilic
substituents. All library components share a common α,β-unsaturated carbonyl
functionality.
Synthesis of such a library in indexed fashion resulted in 26 daughter libraries with
six members in Set 1, while 20 members in Set 2. Both sets were subjected to
screening experiments followed by deconvolution and subsequent LEAD
identification. The assay value of each cell is contained in the combination AxBy (x =
1–6, y = 1–20) (Table 2.6). Obviously, the examination of all the pure compounds
would have required 120 experiments. Since only one cell out of 120 may possess the
maximum response function, the next step involves its identification without looking
at all 120 cells. The best way of doing this is to choose any B for testing with all Atype compounds; conversely, any A for testing with all B-type members.
120 Member Parent
Library
Indexed Libraries
Set 1: Pools 1-6
Set 2. Pools 1-20
1. A1 + B1-20
2. A2 + B1-20
:
6. A6 + B1-20
1. B1 + A1-6
2. B2 + A1-6
:
20. B20 + A1-6
Screening
LEAD
Fig. 2.6. A general schematic diagram for library synthesis in search of the LEAD in a
bioassay.
Chapter 2 ♦Results and Discussion
- 51 -
By screening six columns and 20 rows, which are indices to the cells at their
intersections, as mixtures, only 26 reactions (instead of 120) need to be carried out to
find the maximum response. Therefore, a combinatorial synthesis of 26 sub-libraries
was carried out. This method has the advantages over the parallel approach in that it
is fast and relatively inexpensive. However, the number of compounds prepared is
much greater than the number of chemical steps required
2.2.1.2. Antibacterial studies. All 26 indexed libraries were subjected to antibacterial
studies against six bacterial strains; three Gram positive (B. subtillis ATTCC 6633,
M. leuteus and S. aureus ATCC 6538) and three Gram negative (E. coli AATCC
1522, E. aerogenes AATCC 13048 and S. setubal ATCC 19196) using the Agar Well
Diffusion method.188 Each compound was tested twice, once as a component of sublibraries of Set 1 and then as a member of sub-libraries of Set 2. Thus, a total of 26
assays were required to reveal the antibacterial activity of 120 compounds, which
corresponded to a factor of 4.6 in terms of time improvement in the synthesis and data
collection efficiency.
The antibacterial activities of the sub-libraries AL1-AL6 and BL1-BL20 are shown in
Tables 2.7 and 2.8, respectively. All libraries were tested at a concentration of 0.1
mg/mL. The libraries were found to be active against S. aureus and B. subtilis, very
weekly active against E. coli, while inactive towards the other three bacterial strains
i.e. M. leuteus, E. aerogenes and S. setubal. Roxithromycin (Rulid®) and Cefixime®
were used as standard positive controls in these studies.
Table 2.7. Antibacterial activities of Set 1 sub-libraries (pools AL1-AL6).
Zone of inhibition(mm)a
S. aureus
B. subtilis
E. coli
AL1
15.1±0.15
16.0±0.55
11.5±0.50
AL2
10.9±0.15
12.0±0.26
0
AL3
AL4
a
Zone of inhibition(mm) a
8.8±0.12
0
10.8±0.11
0
0
0
S. aureus
B. subtilis
E. coli
AL5
10.7±0.20
10.7±0.16
0
AL6
0
17.8±0.20
15.1±0.05
b
26.5±1.30
32.1±0.31
11.6±0.10
c
21.3±0.70
12.4±0.18
31.9±0.17
R
C
mean value of three replicates, b R = Roxithromycin, c C =Cefixime®.
After having synthesized the sub-libraries and carrying out their antibacterial
screening, the next step is the synthesis of the library corresponding to the most active
column and the most active row, and to identify the most active library and the lead
structures therein by deconvolution.
Chapter 2 ♦Results and Discussion
- 52 -
Table 2.8. Antibacterial activities of Set 2 sub-libraries (pools BL1-BL20).
Zone of inhibition (mm)a
S. aureus
Zone of inhibition (mm)a
B. subtilis
E. coli
S. aureus
B. subtilis
E. coli
BL1
9.4±0.32
12±0.20
0
BL12
0
0
8.8±0.15
BL2
18.6±0.11
0
15.3±0.05
BL13
11.3±0.05
0
8.6±0.11
BL3
10.9±0.05
0
0
BL14
11.1±0.23
12.2±0.15
0
BL4
13.7±0.05
0
0
BL15
0
12.1±0.05
0
BL5
0
0
0
BL16
10.8±0.10
14.1±0.17
0
BL6
8.6±0.17
0
0
BL17
16.2±0.15
16.2±0.05
0
BL7
8.5±0.00
0
0
BL18
11.1±0.05
13.1±0.15
0
BL8
0
0
0
BL19
0
0
0
BL9
0
0
0
BL20
BL10
11.5±0.05
BL11
0
10.9±0.10
0
0
10.5±0.20
10.5±0.05
0
b
26.5±1.3
32.1±0.31
11.6±0.1
c
21.3±0.70
12.4±0.18
31.9±0.17
R
0
C
2.2.1.3. Deconvolution. Deconvolution is the process by which active compounds
are identified in library mixtures. The antibacterial activities of the libraries were used
as indices to the cells of columns and rows of the conceptual matrix (Table 2.9).
Table 2.9. Calculated zone of inhibition (mm) of chalcones against S. aureus.
Set 1
Set 2
AL1
AL2
AL3
AL4
AL5
AL6
BL1
12.2
10.1
9.1
4.7
10.0
13.6
BL2
16.9
14.8
13.7
9.3
14.7
18.2
BL3
13.0
10.9
9.8
5.4
10.8
14.3
BL4
14.4
12.3
11.2
6.8
12.2
15.7
BL5
7.6
5.4
4.4
0
5.3
8.9
BL6
11.8
9.7
8.7
4.3
9.6
13.2
BL7
11.8
9.7
8.6
4.25
9.6
13.1
BL8
7.5
5.4
4.4
0
5.3
8.9
BL9
7.5
5.4
4.4
0
5.3
8.9
BL10
13.3
11.2
10.1
5.75
11.1
14.6
BL11
7.5
5.4
4.4
0
5.3
8.9
BL12
7.5
5.4
4.4
0
5.3
8.9
BL13
13.2
11.1
10.0
5.65
11
14.5
BL14
13.1
11
9.95
5.55
10.9
14.4
BL15
7.5
5.4
4.4
0
5.3
8.9
BL16
12.9
10.8
9.0
5.4
10.7
14.3
BL17
15.6
13.5
12.5
8.1
13.4
17
BL18
13.1
11
9.9
5.55
10.9
14.4
BL19
7.5
5.4
4.4
0
5.3
8.9
BL20
12.8
10.7
9.6
5.25
10.6
14.1
Chapter 2 ♦Results and Discussion
- 53 -
The data was expanded on 120 cells of chalcones on the matrix against S. aureus,
since the libraries were found to be the most active against this strain only. This data
expansion was carried out for each cell by taking the average of the activities of the
respective column and row cell, and the data were used as indices to the particular
cell. This resulted in calculated antibacterial activity against S. aureus for all 120
chalcones of the matrix. For the identification of the lead compounds, the individual
members of the most active column and row needed to be identified. A careful
examination of the data revealed that the members of the column AL1 and the row
BL2 showed maximum inhibition. Hence, parallel synthesis of the individual
members of these two sub-libraries was accomplished.
Parallel synthesis of active libraries AL1 and BL2. Parallel solution phase synthesis
of a total of twenty five chalcones belonging to the active libraries AL1 and BL2 was
carried out under standard Claisen-Schmidt conditions using a 24 parallel reactions
workstation, GreenHouse Synthesizer (Scheme 2.4).
R
R'
R
H
O
NaOH/ EtOH
O
Synthesizer
O
R
R
O
AL1 (A1B1-A1B20)
(21 - 40)
GreenHouse
R'
R
R
21
H
28
3,4-(OCH3)2
35
4-F
22
2-OH
29
2-NO2
36
2-Br
23
3-OH
30
3-NO2
37
3-Br
24
4-OH
31
4-NO2
38
4-CH3
25
2-OCH3
32
2-Cl
39
3-OH,4-OCH3
26
3-OCH3
33
3-Cl
40
4-N(CH3) 2
27
4-OCH3
34
4-Cl
HO
R′
R'
O
BL2 (A1B2-A6B2)
(2, 22, 41 – 44)
R′
R′
2*
4′-NH2
41
2′-OH
43
3′,4′,5′ -(OCH3)2
22**
H
42
2′,3′,4′- (OCH3)2
44
2′,4′-(Br)2
*already mentioned in scheme 2.1.
** already mentioned above.
Scheme 2.4. Synthesis of individual chalcones of active libraries AL1 and BL2.
Chapter 2 ♦Results and Discussion
- 54 -
All the compounds were recrystallized from EtOH and identified through IR, GCMS
and 1H NMR data. The purity of the compounds was checked through multiple TLC
(solvent system, n-Hex: EtOAc; 9:1, 3:1) and the Rf values were recorded. The yield
of chalcones varied from 61-85 % and the data is given in the Experimental Section.
FT-IR spectral data of chalcones 21-44 is given in the Experimental Section. The
C=O and C=C stretching vibrations appeared in the range of 1631-1690 cm–1 and
1508-1606 cm–1, respectively. A weak peak of Ar-H was observed at 3041-3246 cm–
1
. The stretching frequencies for different substituents on the B ring, derived from
benzaldehydes i.e. C-OH, C-OCH3, N-CH3, C-Br, C-Cl, C-F and C-NO2 were also
observed.
1
H NMR data of chalcones 21-44 has been reported in Experimental Part in detail.
Generally, protons on ring A appeared as a downfield doublet for H-2′, 6′, then a
triplet for H-4′ followed by a triplet for H-3′, 5′ with a coupling constant in the range
of 7-8 Hz. The α-proton at the vinylic position appeared in the range of 7.39-7.74
ppm, whereas β-proton appeared at 7.73-8.17 ppm, each with a coupling constant of
14-16 Hz. 1H-F coupling was observed in compound 35 (Fig. 2.7). The 1H-F coupling
for ortho position was found in the range of 6-11 Hz, whereas for meta position, it
was 3-9 Hz. In 35, protons 3 and 5 appeared as a doublet of doublets (dd) at 7.98 ppm
(J = 9.0, 7.0 Hz), whereas protons 2 and 6 appeared as an apparent triplet (app t) at
7.30 ppm (J = 8.5 Hz) due to H-F coupling.
Fig. 2.7. 1H NMR of 1-(phenyl)-3-(4′-fluorophenyl)-2-propen-1-one (35).
Chapter 2 ♦Results and Discussion
- 55 -
Screening of libraries AL1 and BL2. The active libraries AL1 and BL2 were tested
against six bacterial strains; however, they were found active only toward two strains
i.e. S. aureus and B. subtilis.
Table 2.10. Antibacterial activities of individual chalcones of library AL1.
Zone of inhibition (mm)a
Compound
a
Zone of inhibition (mm)a
S. aureus
B. subtilis
Compound
S. aureus
B. subtilis
A1B1
10.5±0.10
9.2±0.10
A1B12
0
9.5±0.10
A1B2
10.9±0.01
9.7±0.20
A1B13
11.1±0.02
9.2±0.05
A1B3
16.6±0.05
12.5±0.10
A1B14
0
0
A1B4
10.1±0.20
11.7±0.02
A1B15
0
0
A1B5
0
0
A1B16
0
0
A1B6
0
10.7±0.03
A1B17
10.23±0.06
9.5±0.10
A1B7
0
10.4±0.07
A1B18
0
0
A1B8
0
0
A1B19
0
0
A1B9
15.1±0.10
14.9±0.10
A1B20
0
0
b
A1B10
0
9.5±0.10
R
26.5±1.3
32.1±0.31
A1B11
0
0
Cc
21.3±0.70
12.4±0.18
mean value of three replicates. b R = Roxithromycin (positive control). c C =Cefixime® (positive control).
Table 2.11. Antibacterial activities of individual chalcones of library BL2.
Zone of inhibition (mm)a
Compound
a
Zone of inhibition (mm)a
S. aureus
B. subtilis
Compound
A1B2
10.9±0.01
12.5±0.20
A5B2
13.2±0.05
A2B2
18.2±0.10
17.6±0.05
A6B2
9.4±0.10
b
S. aureus
B. subtilis
13.4±0.10
0
A3B2
9.6±0.10
11.5±0.01
R
26.5±1.3
32.1±0.39
A4B2
13.1±0.10
13±0.10
Cc
21.3±0.70
12.4±0.18
mean value of three replicates. b R = Roxithromycin (positive control). c C =Cefixime® (positive control).
The results of the antibacterial studies of chalcones of sub-libraries AL1 and BL2 are
given in Tables 2.10 and 2.11, respectively. The minimum inhibitory concentration
(MIC) values of the members of AL1 and BL2 are given in Table 2.12. The
concentration of each library member and of the control was 0.1 mg/mL in DMSO. It
is evident from the data in Table 2.10 and 2.11 that chalcones A1B3 and A2B2 were
the most active members of the libraries AL1 and BL2. Based on the MIC values,
chalcone A1B2 and A2B2 (MIC = 0.2 mg/mL) may be regarded as the LEAD of the
designed 120-member library.
Chapter 2 ♦Results and Discussion
- 56 -
Table 2.12. Minimum inhibitory concentration (MICs)a of active chalcones (mg/mL).
a
Compound
S.aureus
B.subtilis
Compound
S.aureus
B.subtilis
A1B1
0.5
0.4
A1B9
0.4
0.3
A1B2
0.2
-
A1B17
0.5
-
A1B3
0.4
-
A2B2
0.2
0.2
A1B4
-
0.5
A4B2
0.3
0.4
A1B6
-
0.6
A5B2
0.4
0.6
A1B7
-
0.6
MIC = minimum concentration at which no colony was observed after incubation.
HO
HO
OH O
O
A1B2 (22)
A2B2 (41)
LEADS
It is evident that starting from designing of the combinatorial library to a sequential
logical identification of the lead through positional scanning protocol led to a
reduction in the number of steps from 120 to 26, thereby, reducing the cost in terms
of time and resources in the identification of a lead. Hence a positional scanning
protocol can be considered as a cost effective technique for rapid lead discovery. This
identification of the lead from the designed library through deconvolution has been
shown schematically in Figs. 2.8 and 2.9.
2.2.1.4. Structure activity relationship. Based on the antibacterial activities, it is
also possible to deduce some trends in structure activity relationship (SAR). The
following order of decreasing activity was noticed:
A2B2
>
A4B2
A5B2
HO
HO
O
O
HO
>
A1B2
O
O
>
A3B2
>
A6B2
HO
HO
HO
Br
H2 N
O
O
OH O
>
O
O
O
O
Br
O
The activity of the chalcones was found to be dependent on the substitution pattern on
ring A. Chalcones with electron donors at ortho position of A ring were found as the
most active as indicated from 2-OH and 2-OCH3 substituted chalcones A2B2 and
Chapter 2 ♦Results and Discussion
- 57 -
A5B2. A steric bulk at ortho position of ring A appeared to be detrimental for activity
as indicated from the least potent chalcone A6B2.
A1B1
HO
A1B2
A1B3
120
member
library
AL1
AL2
Set 1
AL1-AL6
AL3
O
A1B2
A1B4
A1B5
MIC = 0.2 mg/mL
A1B6
A1B7
AL4
AL5
AL6
A1B8
A1B9
A1B10
A1B11
A1B12
A1B13
A1B14
A1B15
A1B16
A1B17
A1B18
A1B19
A1B20
Fig. 2.8. Identification of lead through deconvolution of a 120 member library (Set 1) against
S. aureus.
Chapter 2 ♦Results and Discussion
- 58 -
A1B2
120
member
library
A2B2
BL1
BL2
Set 2
BL1-BL20
BL3
BL4
BL5
A3B2
A4B2
HO
OH O
A2B2
MIC = 0.2 mg/mL
A5B2
A6B2
BL6
BL7
BL8
BL9
BL10
BL11
BL12
BL13
BL14
BL15
BL16
BL17
BL18
BL19
BL20
Fig. 2.9. Identification of lead through deconvolution of a 120 member library (Set 2) against
S. aureus.
Chapter 2 ♦Results and Discussion
- 59 -
2.2.1. 175 Member chalcone library148
Chalcones are well known to act as antitumor agents by interacting and
depolymerizing tubulin dimers in living cells.189 Tubulin is a globular protein of
molecular weight 100,000, which exists in dynamic equilibrium with microtubules.
Microtubules are vital components of the cell and are responsible for intracellular
transport, mechanical stabilization and formation of the mitotic spindle during cell
division. Vinblastine, vincristine, epithilone and taxol are known as tubulin binders
that work by depolymerization of microtubules.190 Some chalcones are known to bind
to tubulin dimer and halt mitosis at G2/M phase.191 Using chalcones as antitumor
agents may prove quite successful in cancer chemotherapy, as spindle poisons exert
their influence when mitosis is in the metaphase. Furthermore, a number of α,βunsaturated ketones have been reported to demonstrate preferential reactivity towards
thiols in contrast to amino and hydroxy groups.192 The chalcones, therefore, are
expected to be free from problems of mutagenecity and carcinogenecity associated
with a number of alkylating agents used in cancer chemotherapy.193
Based on the well known antitumor properties of a chalcone template,106-114 the
studies were expanded for antitumor evaluation of a diverse set of chalcones.
Therefore, a 175 member chalcone library was designed, synthesized and evaluated
for antitumor potency through potato-disc tumor inhibition (PDT) assay.148
Fig. 2.10. Mechanism of tubulin depolymerization (courtesy from Calbiochem).194
Chapter 2 ♦Results and Discussion
- 60 -
2.2.2.1. Synthesis. Having two chalcones as potential candidates to be developed as
antibacterials and two other chalcones as LEADS in BSL bioassay in a 120 member
library, the studies were extended to a still larger library. The building blocks were
chosen with a view to have diversity in terms of hydrophobicity, hydrophilicity and
electronic effects. Seven acetophenones A1-A7 and 25 aldehydes B1-B25 were used to
design this library.
R
R'
R
O
H
NaOH / EtOH R'
O
O
Aldehyde
R
Aldehyde
R
B1
C6H5
B14
3-NO2C6H4
R′
B2
2-OHC6H4
B15
3-OH,4-OCH3C6H4
A1
C6H5
B3
3-OHC6H4
B16
4-N(CH3)2C6H4
A2
2΄-OHC6H4
B4
4-OHC6H4
B17
4-CH3C6H4
A3
3΄-OHC6H4
B5
2-OCH3C6H4
B18
2-Thienyl
A4
4΄-OHC6H4
B6
3-OCH3C6H4
B19
5-Methyl-2-thienyl
A5
2΄-NH2C6H4
B7
4-OCH3C6H4
B20
5-Bromo-2-thienyl
A6
3΄-NH2C6H4
B8
3,4-(OCH3)2C6H3
B21
5-Nitro-2-thienyl
A7
4΄-NH2C6H4
B9
2-ClC6H4
B22
2-Pyrrolyl
B10
3-ClC6H4
B23
2-Pyridyl
B11
4-ClC6H4
B24
3-Pyridyl
B12
3-BrC6H4
B25
4-Pyridyl
B13
4-FC6H4
Acetophenone
Scheme 2.5. Building blocks for the synthesis of a 175 member library.
The testing of this indexed library has been conceptually represented as a 2-D matrix,
wherein x-axis has 7 structural variants (acetophenones), while y-axis has 25
structural variants (aldehydes) leading to a 7 × 25 grid, as discussed earlier (Fig.
2.11).
The synthesis of these chalcone libraries was carried out using standard ClaisenSchmidt conditions.148 Each of the 32 row and column reaction was carried out with
one specific acetophenone An and a mixture of 25 aldehydes leading to daughter
libraries of Set 1. The libraries (BL1-BL25) of Set 2 were obtained likewise by the
reaction of an aldehyde Bn with a mixture of seven acetophenones. To eliminate
kinetics effects, all reactions were forced to completion by conducting them with a
stoichiometric quantity of the unitary reagent relative to the total of the mixed
Chapter 2 ♦Results and Discussion
- 61 -
reagents. The synthesis of library BL7 was carried out independently both under
microwave irradiation and conventional heating. The presence of all the components
of the mixture in BL7 was indicated by means of TLC (thin-layer chromatography).
The composition of the library was then compared in two experiments and found to
be the same under both conventional heating and microwave irradiation. The
successful synthesis of indexed libraries was confirmed through GC-MS analysis.
Set 1
Set 2
BL1
AL1
AL2
AL3
AL4
AL5
AL6
AL7
A1 B1
A2 B1
A3 B1
A4 B1
A5 B1
A6 B1
A7 B1
BL2
A1 B2
A2 B2
A3 B2
A4 B2
A5 B2
A6 B2
A7 B2
BL3
A1 B3
A2 B3
A3 B3
A4 B3
A5 B3
A6 B3
A7 B3
BL4
A1 B4
A2 B4
A3 B4
A4 B4
A5 B4
A6 B4
A7 B4
BL5
A1 B5
A2 B5
A3 B5
A4 B5
A5 B5
A6 B5
A7 B5
BL6
A1 B6
A2 B6
A3 B6
A4 B6
A5 B6
A6 B6
A7 B6
BL7
A1 B7
A2 B7
A3 B7
A4 B7
A5 B7
A6 B7
A7 B7
BL8
A1 B8
A2 B8
A3 B8
A4 B8
A5 B8
A6 B8
A7 B8
BL9
A1 B9
A2 B9
A3 B9
A4 B9
A5 B9
A6 B9
A7 B9
BL10
A1 B10
A2 B10
A3 B10
A4 B10
A5 B10
A6 B10
A7B10
BL11
A1 B11
A2 B11
A3 B11
A4 B11
A5 B11
A6 B11
A7B11
BL12
A1 B12
A2 B12
A3 B12
A4 B12
A5 B12
A6 B12
A7B12
BL13
A1 B13
A2 B13
A3 B13
A4 B13
A5 B13
A6 B13
A7 B13
BL14
A1 B14
A2 B14
A3 B14
A4 B14
A5 B14
A6 B14
A7 B14
BL15
A1 B15
A2 B15
A3 B15
A4 B15
A5 B15
A6 B15
A7 B15
BL16
A1 B16
A2 B16
A3 B16
A4 B16
A5 B16
A6 B16
A7 B16
BL17
A1 B17
A2 B17
A3B17
A4 B17
A5 B17
A6 B17
A7 B17
BL18
A1 B18
A2 B18
A3 B18
A4 B18
A5 B18
A6 B18
A7 B18
BL19
A1 B19
A2 B19
A3 B19
A4 B19
A5 B19
A6 B19
A7 B19
BL20
A1 B20
A2 B20
A3 B20
A4 B20
A5 B20
A6 B20
A7 B20
BL21
A1 B21
A2 B21
A3 B21
A4 B21
A5 B21
A6 B21
A7 B21
BL22
A1 B22
A2 B22
A3 B22
A4 B22
A5 B22
A6 B22
A7 B22
BL23
A1 B23
A2 B23
A3 B23
A4 B23
A5 B23
A6 B23
A7 B23
BL24
A1 B24
A2 B24
A3 B24
A4 B24
A5 B24
A6 B24
A7 B24
BL25
A1 B25
A2 B25
A3 B25
A4 B25
A5 B25
A6 B25
A7 B25
Fig. 2.11. Conceptual matrix for designing a 175 member chalcone library.
2.2.2.2. Antitumor studies. Crown gall is a neoplastic disease of plants, which occurs
in more than 60 families of dicotyledons and many gymnosperms. Crown galls cause
Chapter 2 ♦Results and Discussion
- 62 -
the bulging of a mass of tissues from stems and roots of woody and herbaceous
plants. These tumors may be spongy or hard, and may or may not have deleterious
effects on the plant. Histologically, crown-gall tumors are similar to those found in
humans and animals. The causative agents of this disease are specific strains of the
Gram-negative Agrobacterium tumefaciens (AT). The inhibition of crown-gall tumor
(induced by A. tumefaciens in potato-disc tissue) is an assay based on antimitotic
activity. This assay is capable of detecting a broad range of known and novel
antitumor effects. During infection of plant material with A. tumefaciens, a tumorproducing plasmid, found in bacterial DNA, is incorporated in the chromosomal plant
DNA. When plant tissue is wounded, it releases phenols and other compounds that
activate the Ti-plasmid in A. tumefaciens. The Ti-plasmid causes the plant’s cells to
multiply rapidly without going through apoptosis, resulting in tumor formation that
are similar in nucleic acid content and histology to human and animal cancers. The
relevance of the crown-gall-tumor system to the general cancer problem has been
thoroughly reviewed.195-197
The validity of this bioassay is based on the observation that certain tumorigenic
mechanisms are similar in plants and animals. For example, it has been observed that
the inhibition of crown-gall tumor on potato discs and their subsequent growth shows
good correlation with compounds and extracts active in the 3PS leukemic mouse
assay.198 It has also been reported that the potato disc tumor assay is statistically more
predictive in terms of 3PS activity than either the 9KB or the 9PS cytotoxicity
assays199 and can be used as a fairly rapid, inexpensive, and reliable prescreen for
antitumor activity.200
PDT, being a simple bench top assay has been used as a pre-screen for evaluation of
antitumor potential of different compounds obtained either from nature or from
synthetic world. The designed chalcone libraries of Sets 1 and 2 were, therefore,
subjected to this bioassay. The screening of two sets of libraries enabled the testing of
each compound twice, once as a component of the sub-libraries of Set 1, and then as a
member of the sub-libraries of Set 2. Thus, a total of 32 assays were required to find
the antitumor activity of 175 compounds which corresponded to a factor of ~ 5.5 in
terms of time improvement in the synthesis and data collection efficiency.
Chapter 2 ♦Results and Discussion
- 63 -
The sub-libraries of two sets, AL1-AL7 and BL1-BL25, were screened initially at a
concentration of 1000 ppm and their antitumor activities were determined as shown in
Table 2.13.
Table 2.13. PDT inhibition studies of libraries of Sets 1 and 2 at 1000 ppm concentration.
Library
% Inh.
Library % Inh.
Library
% Inh.
Library
% Inh.
AL1
100
BL3
95
BL12
100
BL21
80
AL2
96
BL4
100
BL13
79
BL22
66
AL3
100
BL5
100
BL14
57
BL23
95
AL4
95
BL6
96
BL15
66
BL24
33
AL5
71
BL7
100
BL16
65
BL25
74
AL6
78
BL8
100
BL17
64
AL7
48
BL9
100
BL18
69
BL1
40
BL10
77
BL19
79
BL2
85
BL11
100
BL20
64
All the libraries showed significant antitumor activity. Libraries AL1, AL3, BL4-BL5,
BL7-BL9 and BL11-BL12 showed 100% tumor inhibition. Further short listing was
done by screening these nine libraries at lower concentrations, e.g. at 100 and 10
ppm, as shown in Table 2.14. As a result, the libraries AL1 and BL9 (corresponding to
column 1 and row 9, respectively) were found to be the most active, both at 10 and
100 ppm.
Table 2.14. PDT inhibition studies on the active libraries at 10 and 100 ppm concentration.
Library
% Inh.
Library
% Inh.
Library
% Inh.
AL1
64 (88)
BL5
33 (54)
BL9
73 (95)
AL3
60 (84)
BL7
15 (29)
BL11
14 (35)
BL4
11 (78)
BL8
23 (45)
BL12
22 (41)
Values in parentheses correspond to inhibition at 100 ppm.
2.2.2.3. Deconvolution. Having synthesized and screened all the sub-libraries, the
identification of the lead(s) was carried out by deconvolution. This required the
calculation of activities of all the members of the library using the experimentally
determined values at 1000 ppm, as indices to the cells of columns and rows of the 2D
matrix. The assay value of each cell is contained in the combination AxBy (x = 1-7
and y = 1-25). Since only one cell out of 175 may possess the maximum response
function (Rxy), deconvolution is the identification of this particular combination
Chapter 2 ♦Results and Discussion
- 64 -
without looking at all 175 cells. Therefore, each B (aldehyde) was tested with all A’s,
(acetophenones) and each A was tested with all B’s.
The data was expanded in 175 cells of chalcones on the matrix by taking the average
of the activity of the respective column and row, which were used as indices to the
particular cells. This resulted in calculated antitumor activities for all 175 chalcones
in the 2D matrix as shown in Table 2.15.
Table 2.15. Calculated antitumor activities of designed library at a concentration of 1000 ppm.
Set 1
Set 2
BL1
AL1
AL2
AL3
AL4
AL5
AL6
AL7
70
69
70
67
56
59
44
BL2
92
92
92
90
78
82
67
BL3
97
96
97
95.
83
86
71
BL4
99
BL5
100
100
97
85
89
74
99
100
100
97
85
89
74
BL6
98
97
98
96
84
87
72
BL7
99
85
89
74
97
85
89
74
99
100
100
100
97
BL9
100
100
100
97
85
89
74
BL10
88
87
88
86
74
77
62
BL11
99
85
89
74
99
100
100
97
BL12
100
100
97
85
89
74
BL13
89
89
89
87
75
78
64
BL14
78
78
78
76
64
67
53
BL15
83
82
83
80
68
72
57
BL16
82
82
82
80
68
71
57
BL17
82
81
82
79
68
71
56
BL18
84
83
84
82
70
73
58
BL19
89
88
89
87
75
78
63
BL20
82
81
82
79
67
71
56
BL21
90
89
90
87
76
79
64
BL22
83
82
83
80
68
72
57
BL23
97
96
97
95
83
86
71
BL24
66
66
66
64
52
55
41
BL25
87
86
87
84
73
76
61
BL8
99
A number of chalcones showed 100% inhibition at 1000 ppm concentration (Table
2.13). The activities of nine short-listed sub-libraries (inhibiting 100% at 1000 ppm)
were screened at lower concentrations i.e. 100 and 10 ppm values. The inhibitory
potentials at 10 ppm concentrations were used as indices to the cells of the 2D matrix
Chapter 2 ♦Results and Discussion
- 65 -
comprising two columns and seven rows and the data was spread over 14 cells, as
shown in Table 2.16.
Table 2.16. Calculated antitumor activities of the most active library members at 10 ppm.
Set 1
Set 2
AL1
AL3
BL4
37.5
35.5
BL5
48.5
46.5
BL7
39.5
37.5
BL8
43.5
41.5
BL9
50.5*
48.5
BL11
39.0
37.0
BL12
43.0
41.0
*
most active member.
It is evident that both sub-libraries AL1 and BL9 may be predicted to be the mostactive. Thus, for the identification of the most active chalcone, parallel synthesis of
the members of the most active column AL1, and the most active row BL9, was
carried out by means of microwave assisted organic synthesis.
Synthesis of members of active libraries AL1, AL3 and BL9. Members of the active
columns AL1 and AL3 as well as active row BL9 were synthesized in parallel under
microwave irradiation. The structures of the members of AL1 and AL3 are given in
Schemes 2.6, 2.7, whereas those of BL9 are given in Scheme 2.8.
H
O
R
R
NaOH/ EtOH
M.w.
O
O
R
R
R
21
Ph
32
2-ClC6H4
46
5-Methyl-2-thienyl
22
2-OHC6H4
33
3-ClC6H4
47
5-Bromo-2-thienyl
23
3-OHC6H4
34
4-ClC6H4
48
5-Nitro-2-thienyl
24
4-OHC6H4
35
4-FC6H4
49
2-Pyrrolyl
25
2-OCH3C6H4
37
3-BrC6H4
50
2-Pyridyl
A1B1-A1B25
26
3-OCH3C6H4
38
4-MeC6H4
51
3-Pyridyl
(21-30, 3235,37-40,
45-52)
27
4-OCH3C6H4
39
3-OH,4-OCH3C6H3
52
4-Pyridyl
28
3,4(OCH3)C6H3
40
4-N(CH3)2C6H4
30
3-NO2C6H4
45
2-Thienyl
R
O
Scheme. 2.6. Parallel synthesis of chalcones of active column AL1.
Chapter 2 ♦Results and Discussion
- 66 -
R
53
R
C6H5
62
3-ClC6H4
71
5-Methyl-2-thienyl
R 54
2-OHC6H4
63
4-ClC6H4
72
5-Bromo-2-thienyl
55
3-OHC6H4
64
3-BrC6H4
73
5-Nitro-2-thienyl
56
4-OHC6H4
65
4-FC6H4
74
2-Pyrrolyl
57
2-OCH3C6H4
66
3-NO2C6H4
75
2-Pyridyl
58
3-OCH3C6H4
67
3-OH,4-OCH3C6H3
76
3-Pyridyl
59
4-OCH3C6H4
68
4-N(CH3)2C6H4
77
4-Pyridyl
60
3,4(OCH3)C6H3
69
4-MeC6H4
61
2-ClC6H4
70
2-Thienyl
OH
O
A3B1-A3B25
(53-77)
R
Scheme. 2.7. Parallel synthesis of chalcones of active column AL3.
R′
Cl
R'
O
A1B9-A7B9 (12, 32, 62, 78-81)
R′
12
4′-NH2C6H4
79
4′-OHC6H4
32
C6H5
80
2′-NH2C6H4
62
3′-OHC6H4
81
3′-NH2C6H4
78
2′-OHC6H4
Scheme. 2.8. Parallel synthesis of chalcones of active row BL9.
FT-IR spectral analysis of all the chalcones A1B1-A1B25, A3B1-A3B25 and A1B9-A7B9
was carried out. The C=O and C=C stretching vibrations for the prepared compounds
appeared in the range of 1631-1690 cm–1 and 1508-1606 cm–1, respectively. Aryl C-H
stretches are often observed at 3041-3246 cm–1.
1
H NMR spectra of the synthesized chalcones were recorded. A detailed spectral
analysis of 1-(phenyl)-3-(5-methyl-2-thienyl)-2-propen-1-one (46) has been given in
Table 2.17. The most characteristic chemical shifts were those of vinylic protons H
(α) and H (β); the former appeared upfield at 7.21 ppm, while the later appeared at
7.88 ppm. The coupling constant was 15.3 Hz indicating a trans orientation of the
double bond. The methyl protons appeared as a singlet at 2.53 ppm. In thienyl ring,
doublet (d) at 6.76 ppm with a coupling constant 3.6 Hz was assigned to H–C(3), a
doublet at 7.18 ppm with 3.6 Hz was assigned to H–C(4). A doublet at 8.01 ppm with
a coupling constant 7.2 Hz was assigned to H–C(2′, 6′). A triplet at 7.51 ppm with
coupling constant 7.2 Hz was assigned to the H–C(3′, 5′), whereas a triplet at 7.59
ppm, again with a coupling constant 7.2 Hz was assigned to H–C(4′).
Chapter 2 ♦Results and Discussion
- 67 -
Table 2.17. 1H NMR data of 1-(phenyl)-3-(5-methyl-2-thienyl)-2-propen-1-one (46).
Me
1
3'
4'
1'
2
3
α
5'
6'
4
β
2'
5
S
O
46
δ ppm
Multiplicity
Integration
J (Hz)
Assignment
2.53
s
3H
-
H−C(CH3)
6.76
d
1H
3.6
H−C(3)
7.18
d
1H
3.6
H−C(4)
7.21
d
1H
15.3
H-C(α)
7.51
t
2H
7.2
H−C(3′, 5′)
7.59
t
2H
7.2
H−C(4′)
7.88
d
1H
15.3
H−C(β)
8.01
d
2H
7.2
H−C(2′, 6′)
Antitumor screening. The purified and characterized compounds were assessed for
their antitumor potencies through PDT assay. The individual members of the active
libraries AL1, AL3 and BL9 were tested for their antitumor potential at 10, 100 and
1000 ppm concentrations and the data is shown in Tables 2.18-2.20.
Table 2.18. Antitumor activities of chalcones of library AL1.
Chalcone
a
Inhibitiona
Chalcone
Inhibitiona
Chalcone
Inhibitiona
A1B1
17.0, 34.1, 62.1
A1B10
43.1, 47.5, 76.8
A1B19
26.2, 37.7, 45.9
A1B2
35.3, 40.2, 80.5
A1B11
71.9, 75.7, 96.3
A1B20
54.1, 56.4, 78.7
A1B3
53.6, 58.5, 100
A1B12
64.6, 74.4, 78.2
A1B21
44.3, 54.1, 54.1
A1B4
34.1, 56.1, 64.6
A1B13
32.9, 53.6, 93.9
A1B22
40.9, 75.4, 80.3
A1B5
19.5, 52.6, 57.5
A1B14
37.8, 58.5, 69.5
A1B23
67.2, 80.3, 83.6
A1B6
34.1, 48.8, 54.8
A1B15
53.6, 54.9, 74.4
A1B24
63.9, 98.4, 90.2
A1B7
35.4, 47.7, 53.6
A1B16
10.6, 29.5, 31.1
A1B25
42.6, 49.2, 68.8
A1B8
56.1, 68.7, 70.7
A1B17
14.7, 14.7, 36.1
A1B9
100, 100, 100
A1B18
11.5, 32.7, 49.2
Percent inhibition at 10, 100 and 1000 ppm, respectively. All the chalcones were tested on twelve
potato-discs at each concentration.
Chapter 2 ♦Results and Discussion
- 68 -
Table 2.19. Antitumor activities of chalcones of library AL3.
Chalcone
a
Inhibitiona
Chalcone
Inhibitiona
Inhibitiona
Chalcone
A3B1
72.8, 100, 100
A3B10
51.0, 79.7, 100
A3B19
75.6, 80.2, 100
A3B2
100, 100, 100
A3B11
33.2, 63.6, 100
A3B20
65.4, 66.7, 82.0
A3B3
57.8, 100, 100
A3B12
53.2, 77.9, 100
A3B21
65.4, 70.9, 73.1
A3B4
35.4, 70.5, 100
A3B13
37.1, 76.6, 100
A3B22
73.8, 81.8, 100
A3B5
76.8, 100, 100
A3B14
75.6, 91.0, 96.1
A3B23
57.7, 100, 100
A3B6
25.9, 49.3, 100
A3B15
64.9, 72.7, 100
A3B24
76.9, 77.9, 83.3
A3B7
31.2, 33.8, 100
A3B16
72.3, 80.8, 100
A3B25
80.8, 89.5, 94.9
A3B8
60.2, 83.6, 100
A3B17
78.2, 84.2, 100
A3B9
38.9, 60.2, 100
A3B18
52.5, 100, 100
Percent inhibition at 10, 100 and 1000 ppm, respectively. All the chalcones were tested on twelve
potato-discs at each concentration.
Table 2.20. Antitumor activities of the chalcones of library BL9.
a
Chalcone
Inhibitiona
Chalcone
Inhibitiona
A1B9
100, 100, 100
A5B9
49.4, 59.1, 100
A2B9
100, 100, 100
A6B9
55.8, 70.1, 100
A3B9
61.0, 78.6, 98.0
A7B9
52.6, 66.8, 86.4
A4B9
70.7, 77.9, 100
Percent inhibition at 10, 100 and 1000 ppm, respectively. All the chalcones were tested on twelve
potato-discs at each concentration.
It is evident from the data that the most active members of the library A1B9 and A2B9
and A3B2 (cf. for structures, Scheme 2.5) displayed 100% tumor inhibition at a
concentration of 10 ppm, and thus, can be regarded as leads of the designed library. It
may be recalled that the calculated deconvolution led to the identification of the same
chalcone, A1B9, as lead structure. However, the experimental data showed that A2B9
and A3B2 also exhibited 100% tumor inhibition at 10 ppm and, therefore, are equally
important candidates for developing into highly effective chemotherapeutic agents.
Cl
OH
HO
O
O
A1B9 (32)
A3B2 (61)
Cl
OH O
A2B9 (78)
Chapter 2 ♦Results and Discussion
- 69 -
The identification of the lead structure in 175 member library through positional
scanning method is shown in Figures. 2.12 and 2.13.
AL1
A1B1
A3B1
175
member
library
A1B2
A3B2
AL2
A1B3
A3B3
A1B4
A3B4
AL3
A1B5
A3B5
A1B6
A3B6
Set 1
AL1-AL7
A1B7
A3B7
AL4
A1B8
A3B8
A1B9
A3B9
AL5
A1B10
A3B10
A1B11
A3B11
A1B12
A3B12
AL6
A1B13
A3B13
A1B14
A3B14
AL7
A1B15
A3B15
A1B16
A3B16
A1B17
A3B17
A1B18
A3B18
A1B19
A3B19
A1B20
A3B20
A1B21
A3B21
A1B22
A3B22
A1B23
A3B23
A1B24
A3B24
A1B25
A3B25
OH
HO
Cl
O
O
A3B2
A1B9
LEADS
Fig. 2.12. Identification of antitumor leads through deconvolution of 175 member library (Set 1).
Chapter 2 ♦Results and Discussion
- 70 -
BL1
BL2
175
member
library
BL3
BL4
BL5
A1B9
BL6
A2B9
BL7
Set 2
BL1-BL25
BL8
A3B9
BL9
BL9
BL10
A4B9
BL11
A5B9
BL12
BL13
A6B9
BL14
A7B9
BL15
BL16
BL17
BL18
BL19
BL20
BL21
BL22
BL23
BL24
BL25
Cl
Cl
OH O
O
A2B9
A1B9
LEADS
Fig. 2.13. Identification of antitumor leads through deconvolution of 175 member library (Set 2).
Chapter 2 ♦Results and Discussion
- 71 -
Therefore, deconvolution by means of positional scanning is a quite cost-effective
protocol capable of identifying a lead compound; however subtle differences in
activity within the library cannot be detected. This disadvantage, however, is more
than compensated by the advantage of the ease of library synthesis.
2.2.2.4. Structure activity relationship. Following the identification of hits of the
designed library by deconvolution, it is essential to highlight the structural features
that contribute to tumor inhibition. Such a correlation may be performed at the library
level as well as at the level of individual compounds. The order of activity within
these seven sub-libraries, in terms of substituent is
H > OH > NH2
or more specifically,
H > 3-OH > 2-OH > 4-OH > 3-NH2 > 2-NH2 > 4-NH2
Evidently, the library derived from unsubstituted acetophenone (AL1) shows 100%
tumor inhibition, while all the three libraries derived from isomeric amino
acetophenones (AL5-AL7) show weak tumor inhibitory potential (Table 2.16).
Moreover, the substituents at para position of ring B lower the activity to a greater
extent as compared to those at ortho or meta positions. Among the libraries of Set 2
(BL1-BL25), seven sub-libraries (BL4, BL5, BL7, BL8, BL9, BL11 and BL12) showed
100% tumor inhibition at a concentration of 1000 ppm. All the sub-libraries of
various analogues of methoxy benzaldehydes (BL5, BL7 and BL8) showed 100%
activity at 1000 ppm, reflecting the significance of a OCH3 substituent at the aromatic
ring. Regarding the tumor inhibitory potential of the individual chalcones, it is
interesting to note that A1B9, A2B9 and A3B2 were found to be the lead structures,
since this further confirms the above conclusion that an unsubstituted A-ring is
important for tumor inhibition, whereas ortho substitution was found to be important
on ring B (o-chloro and o-hydroxy substituents in lead structures). Moreover,
chalcones substituted with an electron donor substituent on ring A, e.g., N(CH3)2 or
3CH3 group were found to be the least active. Varieties of chalcones were derived
from heteroaryl aldehydes, but the pyridyl chalcones A1B23-A1B25 and A3B23-A3B25
were found to be significantly active. Furthermore, A3B19 and A3B22 containing
methyl substituted thienyl and pyrrolyl moieties as ring B resulted in significant
antitumor potencies.
Chapter 2 ♦Results and Discussion
- 72 -
2.3. Peptidyl chalcones and peptidyl heterocycles
The standard approach of combining heterocycles and peptides is the fusion of a
small-molecule fragment in the side chain of amino acid building blocks or to
integrate heterocycles via standard acylation of amino groups of the peptide and the
heterocycles to create hybrid molecules.201-206 Only few examples have been reported
where heterocycles are integrated in the peptide backbone itself, as this requires in
most cases specific, C-terminal derivatization of peptides.207-212 Current strategy
describes the synthesis of peptidyl chalcones on solid phase by the use of 2phosphoranylideneacetate
developed
as
a
linker
reagent
on
polymeric
triphenylphosphine resin. The use of triphenylphosphine resin has been reported in the
Wittig and Mitsunobu reactions.213-214 Polymer-supported C-acylation was reported
for the first time, employing triphenylphosphine resin for the synthesis of polymeric
2-phosphoranylidene acetonitrile as a polymer reagent and an anchoring group
allowing subsequent derivatization of the immobilized product.215-216 Furthermore, Cacylation of polymer-supported 2-phosphoranylidene acetates was developed as a
0linker reagent with protected amino acids yielding 2-acyl-2-phosphoranylidene
acetates as flexible intermediates for the C-terminal variation of carboxylic acids. As
a result, several novel peptidyl bis- and tris-electrophiles have become accessible,
namely peptidyl diketoesters, peptidylketoaldehydes and peptidyl vinyl ketones (Fig.
2.14).217
Pep HN
Ph
Ph
R1 P
OR*
O
R1
Pep HN
∗
O
O
R1
∗
R″
Pep HN
R1
Pep HN
∗
∗ ∗ ∗
O
O
∗
O
OR*
O
H
O
Fig. 2.14. Peptidyl bis- and tris- electrophiles obtained from 2-acyl-2-phosphoranylidene
acetate.
Chapter 2 ♦Results and Discussion
- 73 -
On the basis of above findings, a small library of peptidyl chalcones was designed,
synthesized and then converted to different classes of heterocyles, namely oxazoles,
pyrazolines, pyrazoles, thiazepines and diazepines.218
2.3.1. Synthesis of peptidyl chalcones
The conjugation of peptides with different heterocycles based on C-acylation of
peptides led to peptidyl chalcones, which are also known as peptidyl vinyl ketones.
The peptidyl chalcones were synthesized on phosphorane bound polystyrene
divinylbenzene (PS) support using polymer-supported carbanion equivalents.
Developing carbanion equivalents on solid support is an ideal approach for
establishing complex reaction sequences including C-C coupling steps in polymerassisted synthesis. Ideally, the successful method should allow reactions with easily
available building blocks, smooth reaction conditions and further derivatization after
the C-C coupling step. Lithiated dithioacetals have been reported as polymersupported carbanion equivalents.219-221 These acetals are strongly basic and, therefore,
are not a convincing approach for the synthesis of sensitive products. The integration
of a C-acylation step into the standard protocol of peptide synthesis would be
especially attractive for the synthesis of peptide mimetics and in the attempts to
reduce the peptide character of inhibitors, which is a common challenge in drug
development programs.
Polymer-supported acyl anion equivalents were first established using phosphoranePS support for synthesizing a library of norstatine isosteres, active as aspartic protease
inhibitors.215 Furthermore, C-acylations of polymer-supported 2-phosphoranylidene
acetates (linker reagents) with different protected amino acids yielded 2-acyl-2phosphoranylidene acetates, which are flexible intermediates for the C-terminal
variation of carboxylic acids: peptidyl-2,3-diketoesters, peptidyl vinyl ketones,
peptidyl-2-ketoaldehydes and 1,3-diamino-2-hydroxy-propanes.217 Based on the
previous studies, a parallel library of peptidyl chalcones has been synthesized on
phosphorane-PS support and then subsequently converted to a library of peptidic
heterocycles. This integration of heterocycles in the peptide backbone with specific Cterminal derivatization may lead to novel peptidomimetics.218
The synthetic strategy involved the following main steps:
1. Synthesis of linker reagent.
Chapter 2 ♦Results and Discussion
- 74 -
2. Protection of triphenylphosphin-PS support.
3. Deprotonation leading to Wittig ylide.
4. Acylation of Wittig ylide.
5. Amide couplings leading to peptides.
6. N-Acetylation of peptides.
7. Deprotection of phosphorane.
8. Synthesis of peptidyl chalcones.
2.3.1.1. Synthesis of linker reagent
DCC/ DMAP
OH
Br
O
O
Br
Si
Si
O
OH
82
As a first step toward polymer-supported C-acylation, linker reagent has been
introduced as a tool in solid phase synthesis combining the functions of a polymer
reagent and those of an anchoring group allowing for subsequent derivatization of the
immobilized product.217 2-Trimethylsilylethyl-2-bromoacetate (82) was used as linker
reagent on phosphorane supported polystyrenedivinylbenzene resin. This ester could
be synthesized by pyridine catalyzed esterification of an acid halide with
trimethylsilyl ethanol or through Steglich esterification. In the second method, the
acid part is activated using dicyclohexylcarbodiimide (DCC) and catalytic amount of
4-N,N-dimethylaminopyridine (DMAP) and then reacted with the desired alcohol.222
This method has the advantage of giving only the desired ester under controlled
conditions. The synthesis was achieved in good yield by activating bromoacetic acid
with DCC/DMAP followed by esterification with 2-trimethylsilylethanol. The crude
product after vacuum distillation gave the desired ester, which was further used as a
facile linker reagent for C-C acylations on phosphorane support. The product was
characterized by 1H NMR and 13C NMR analysis.
1
H NMR analysis of 2-trimethylsilylethyl-2-bromoacetate (82) showed a singlet of
nine protons at 0 ppm for trimethylsilyl group, two triplets at 1.10 and 4.21 ppm, each
of two protons for two methylenes attached to Si(CH3)3 and an ester moiety
respectively. The two methylene protons attached to Br appeared as singlet at 3.90
ppm (Fig. 2.15).
Chapter 2 ♦Results and Discussion
- 75 -
Fig. 2.15. 1H NMR analysis of 2-trimethylsilylethyl-2-bromoacetate 82.
2.3.1.2. Protection of triphenylphosphin-PS support
Ph
O
Br
O
P
Ph
Ph
P
Ph
Br
Si
Toluene,
Mw,15 min
O
O
Si
83
The linker reagent [2-trimethylsilylethyl-2-bromoacetate 82] was loaded on
triphenylphosphin-PS support for synthesizing a Wittig salt. The commercially
available triphenylphosphin-PS support was alkylated with the synthesized linker
under microwave irradiation at 100 °C for 15 min in toluene (successful alkylation
can also be carried out through stirring at room temperature for 72 h). The alkylation
was confirmed after recording ATR-FT-IR spectrum of the intermediate obtained
(Fig. 2.16).
A
b
s
o
r
b
a
n
c
e
Wave number cm
-1
Fig. 2.16. ATR-FT-IR of triphenylphosphorane support after alkylation with trimethylsilylethyl
bromoacetate.
Chapter 2 ♦Results and Discussion
- 76 -
The infrared spectrum of alkylated support 83 showed C-H and Ar-H absorbance of
polystyrene at 2851 cm-1 and 3056 cm-1, while a characteristic absorbance at 1735
cm-1 corresponded to the ester carbonyl functionality. The resultant protected
triphenyl phosphin-PS support was accessible for the synthesis of a Wittig ylide
through base catalyzed elimination of an acidic proton at methylene directly attached
to the phosphorous atom.
2.3.1.3. Deprotonation leading to Wittig ylide
Ph
P
Ph
Br
O
Si
Ph
Ph
TEA / DCM P
O
O
Si
O
84
The phosphonium salt obtained after alkylation has highly acidic proton and was
easily deprotonated using triethylamine as a base. The resulting Wittig ylide
(polymer-bound phosphoranylidene acetate) enjoys greater stability due to resonance
stabilization with the adjacent carbonyl moiety of the ester group. The product, after
thorough drying, was stored at room temperature. IR analysis of product 84 showed a
peak at 1740 cm-1 that was assigned to ester C=O and this confirmed the identity of
Wittig ylide.
2.3.1.4. Acylation of Wittig ylide
Polymer-bound phosphoranylidene acetate i.e. Wittig ylide can serve as an equivalent
analogue of polymer bound acyl anion. The acylation of the Wittig ylide 84 with
different F-moc protected amino acids serves as the starting point for standard peptide
synthesis. However, polymer-bound phosphoranylidene acetate, being a weak
nucleophile, posed a major challenge in finding efficient acylation conditions. Some
early attempts of acylation remained unsuccessful using different activating agents for
enhancing the electrophilicity of the
carbonyl
of
diisopropylcarbodiimide (DIC) with catalytic amount of
the
amino
acid
e.g.
DMAP, N-ethyl-N′-(3-
dimethylaminopropyl)-carbodiimide hydrochloride (EDC) with DMAP and 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophos
phate-3-oxide (HATU) with diisopropylethylamine (DIPEA). Successful acylations
could be performed with 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT),
whereas usage of fluoro-N,N,N′,N′-bis(tetramethylene)formamidiniumhexafluorophos
phate (TFFH) as acylation agent destroyed labile trimethylsilyl group on linker
Chapter 2 ♦Results and Discussion
- 77 -
reagent. Therefore, for synthesizing a library of peptidyl chalcones, MSNT and
lutidine (as a base) were used for racemization-free acylation of phosphoranylidene
acetate. MSNT catalysed C-acylation of phosphorane was performed with a broad
choice of carboxylic acids. This reagent tolerated the standard side-chain protecting
groups used in peptide synthesis such as Boc, tert-butyl, and trityl groups.
Ph
Ph
P
Ph
R1
P
Ph
Fmoc-AA-OH
O
O
FmocHN
Si
MSNT, Lutidine, DCM
O
O
Si
O
85, R1 =
86, R1 =
The mechanism of this C-acylation is shown in Scheme 2.9. Lutidine (as a base)
deprotonates OH of an amino acid and results in a nucleophile. This nucleophile
attacks the sulphone of MSNT. As a result, triazole departs whereas the amino acid
now contains a better leaving group (mesitylenesulfonate). In the following step,
phosphoranylidene acetate (a weak nucleophile) attacks the carbonyl sulphonate ester
resulting in the removal of sulphonate while the amino acid is loaded on 84 through
acylation (Scheme 2.9).
R1
Fmoc
N
H
O
O
O
N
R1
S N
N
O
NO2 Fmoc N
H
O
S
O O
O
N
N
N
NO2
Ph
P
Ph
RO
O
O
S O
O
Fmoc
N
H
Ph
Ph
R1 P
OR
O
O
Scheme 2.9. MSNT/Lutidine mediated acylation on phosphorane support.
The basicity of the phosphorane reagent did not allow cleavage of detectable amounts
of the Fmoc-protecting group. This was verified by the negative Kaiser test
subsequent to all acylations. The coupling yield of the acylated products 85 and 86
Chapter 2 ♦Results and Discussion
- 78 -
was determined by spectrophotometric quantification of Fmoc group that was cleaved
off a dried resin sample ~ 5 mg (Table 2.21).
Table 2.21. % Yield of acetylated products obtained on solid support.
Entry
Amino acid
Yield (%)a
Conditions
O
FmocHN
5 equiv Fmoc-Phe, 5 equiv MSNT,
OH
85
88
4.9 equiv 2,6-lutidine, DCM, RT.
O
FmocHN
86
5 equiv Fmoc-Val, 5 equiv MSNT,
OH
84
4.9 equiv 2,6-lutidine, DCM, RT.
a
Calculated after standard Fmoc cleavage of ~ 5 mg sample.
The IR spectrum of the acylated support 85 showed great similarity to the
corresponding polymeric Wittig salt 83. Important differences were the absorption
due to Si-C bond, which after alkylation could be seen at 837 cm-1, while Fmoc
moiety appeared as a double peak at 742-760 cm-1.
After successful acylation, the protecting group Fmoc a was removed from small
samples ~ 5 mg of resin, using 20% piperidine/DMF and the cleaved adducts c and d
were quantified through measuring absorbance λ1 = 267 nm, λ2 = 289 nm and λ3 =
301 nm, as shown in Scheme 2.10. The concentration of the cleaved Fmoc moiety
was calculated by Beer Lambert Law (Section 4A.3.4.).
HN R
HN R
O
O
O
O
H
H
a
N H
b
N H
HN R
O
H2N R
N
O
CO2
NH
d
c
Scheme 2.10. Elimination of base-labile Fmoc moiety demasking amino functionality.
Chapter 2 ♦Results and Discussion
- 79 -
2.3.1.5. Amide coupling leading to peptide synthesis
Ph
Ph
R1
Ph
P
O
FmocHN
O
Fmoc
Si
R1
O
H
N
amide coupling
Rn
O
Ph
P
O
N
H
O
n
Si
O
n = 1, 2
87-91
The acylated products 85-86 were subjected to Fmoc deprotection using 20%
piperidine/DMF (2 x 6 min). The resulting free N-terminus amino group was used as
the attachment site for adding amino acid monomers thus yielding peptidyl 4-amino3-oxo-2-phosphoranylidene butanoates 87-91. The activating agents were first
developed for racemization-free-amide coupling on phosphorane support through test
reactions. Various coupling methods using different coupling reagents e.g., DIC, N[(1H-benzotriazol-1-yloxy)(dimethylamino)methylene]-N-methylmethanaminiumtetra
fluoroborate (TBTU) with DIPEA and DIC with N-hydroxybenzotriazole (HOBT)
were tested for the coupling of Fmoc amino acids. The reaction of DIC with a
carboxylic acid yields a highly reactive O-acyl urea. In order to enhance the
electrophilicity of the carboxylate group, the negatively charged oxygen must first be
"activated" to a better leaving group.
R2
Fmoc
N
H
N C N
OH
R2
Fmoc
O
H
N
O
N
H
O HN
a
b
R2
Fmoc
N
H
R1
H
N
N
O
O
c
f
Fmoc
N
H
O
R2
O
e
OR
H2N
N
H
d
N
H
O
H
N
O
OR
R1
Scheme 2.11. Amide coupling through DIC.
When DIC is used for this purpose, the negatively charged oxygen acts as a
nucleophile, attacking the central carbon in DIC. Nucleophilic attack by an amino
group (of amino acid loaded onto support) to the former C-terminus (carbonyl group
of incoming amino acid now present as an ester b) results into amide coupling
(Scheme 2.11). However, intramolecular rearrangement of b proceeds without H+
Chapter 2 ♦Results and Discussion
- 80 -
uptake, together with the migration of an amine moiety to carbonyl carbon of ester
functionality resulting into a by product f, which was stuck to resin and was not easily
removed through washings. As a result, the desired product was contaminated with
incomplete peptide sequence and urea derivative f, as indicated through the LC-MS of
the cleaved product obtained in the test reaction.
O
N
N
N
N
N
N
O
O-Form
N
N
N
N
Fmoc
O
H
N
Fmoc
O
O
H
N
O
R2
R2
N
N
N
O
N-Form
Fmoc
O
H
N
N
O
base H
N
BF4
R2
O
N
N
N
N
N
N
O
O
O-Form
R2
O
R2
NHFmoc
N-Form
NHFmoc
O
HN
OR base H
R1
R2
Fmoc
N
H
O
H
N
O
N
OR
R1
N
N
O
base H
Scheme 2.12. Amide coupling through TBTU/base.
TBTU containing non-nucleophilic tetrafluoroborate anion and DIPEA were tested for
effective amide coupling. TBTU exists in two isomeric forms i.e. O-TBTU and NTBTU, existing in dynamic equilibrium under basic conditions. The equilibrium shifts
towards N- Form in strong basic conditions such as in the presence of Et3N of DIPEA.
Under basic reaction conditions, the carboxylic acid part of amino acid deprotonates.
The resulting carboxylate anion initially attacks the α-carbon atom of the immonium
salt (uronium (O-form) or the guanidinium N-oxide (N-form)) to form an unstable
acyloxyimmonium intermediate with the release of benzotriazolyloxy anion (BtO−).
Chapter 2 ♦Results and Discussion
- 81 -
The BtO− nucleophile, thus generated, attacks the acyloxyimmonium intermediate
generating benzotriazolyl ester, which rearranges into the corresponding N-acylated
isomer. The two forms of the active intermediates reacted with the free amine ending
of phosphorane support resulting in the synthesis of the corresponding amide (Scheme
2.12).223
When the cleaved product was analyzed through LC-MS, the product had low
enantiopurity that was expected to result from intramolecular rearrangement of
acyloxyimmonium intermediate to 4H-5-oxazolone and subsequent epimerization in
the desired product (Scheme 2.13).
R
O
HN
O
O
N
BF4
N
O
R
H
O
N
R O
N
R'
R'
O
N
R
O
N
R'
O
H O
O
R'
N
Scheme 2.13. Mechanism of 4H-5-oxazolone mediated epimerization.
Another coupling reagent HOBT was used after pre-activation of Fmoc amino acids
with DIC. HOBT together with DIC was found to be the safest with the phosphorane
support and Fmoc chemistry. The reaction proceeds through nucleophilic attack of the
negatively charged oxygen on the central carbon in DIC (Scheme 2.14). The yields of
the amide coupling reactions using DIC/HOBT are given in Table 2.22.
Fmoc
R2
N C N
R2
Fmoc
OH
N
H
O
O
N
H
N
H
HN
O
N
H
OR
H2N
O
H
N
O
OR
H
N
R2
R1
O
R2
N
H
O
OH
N
N
N
O
N
N
N
Fmoc
N
H
O
N
N
N
Fmoc
O
R1
Scheme 2.14. Mechanism of DIC/HOBT mediated amide coupling.
The yields of amide couplings were determined after the Fmoc deprotection of small
resin samples.
Chapter 2 ♦Results and Discussion
- 82 -
Table 2.22. Yields of the standard amide coupling using DIC/HOBT.
No.
Amino acid
Ph
O
87
FmocHN
Yield (%)a
Product
OH
Ph
P
O
FmocHN
86
O
N
H
O
Si
O
Ph
O
FmocHN
O
OH
FmocHN
N
H
88
OH
FmocHN
N
H
Ph
O
O
OH
84
Ph
P
O
N
H
84
O
O
Si
O
Ph
OH
H
N
FmocHN
O
a
Si
O
O
91
82
Ph
FmocHN
FmocHN
Si
O
P
O
90
O
O
89
FmocHN
O
Ph
O
FmocHN
Ph
P
P
O
N
H
Ph
O
O
83
Si
O
Calculated after standard Fmoc cleavage of ~ 5 mg sample.
2.3.1.6. N-Terminal acetylation of peptides (92-95)
The amino terminus of the peptides 87-89 and 91 was deprotected from Fmoc moiety
using standard F-moc cleavage conditions. The resulting unprotected peptides were
subjected to N-terminal amine acetylation using acetic anhydride (5 eq) in DMF
resulting in acetylated peptide sequences 92-95. The reaction was repeated two times
to ensure complete acetylation. The complete conversion of N-terminal amino group
to N-acetyl derivatives was confirmed through the Kaiser test (Section 4A.3.11).
Chapter 2 ♦Results and Discussion
- 83 -
Ph
H
R1
O
H
N
Rm
N
H
n
Ph
Ph
P
Ac2O/DMF
O
O
Si
O
O
R1
O
H
N
Rm
O
N
H
n
n = 1-2
Ph
P
O
Si
O
n = 1-2
92, R1 =
R2 =
93, R1 =
R2 =
94, R1 =
R2 =
95, R1 =
R2 =
R3 =
2.3.1.7. Deprotection of phosphorane support
After having accomplished a successful synthesis of the desired peptide sequence, the
final step was the removal of trimethylsilylethyl group. Tetrabutylammonium fluoride
(TBAF) is well known for TMSE removal.224 However, it was found to destroy
peptide part under basic condition. By controlling the pH in buffer medium, the
removal of tetrabutylammonium ions from the resin could not be achieved even after
repeated washings and always resulted in contaminated products of successive steps.
Furthermore, tris(dimethylamino)sulfonium difluorotrimethylsilicate (TAS-F) was
found as a safe desilylating reagent by removing TMSE without any washing
problems (Fig 2.17).225 TAS-F, a mild neutral agent is an in situ source of fluoride
ion. It successfully removed trimethyl silyl moiety and resulted in subsequent
decarboxylation of supported peptides (Scheme 2.12).
N
N
S
N
F
Si
F
Fig. 2.17. tris(Dimethylamino)sulfonium difluorotrimethylsilicate (TAS-F).
R2
N
H
Ph
Ph
R1 P
O
O
O
F
Si
R2
C2H4
(CH3)3SiF
N
H
Ph
Ph
R1 P
O
O
O
Ph
Ph
R1 P
R2
CO2
N
H
H
O
H
Scheme 2.12. Hydrolysis of protected 4-amino-3-oxo-2-phosphoranylidene butanoates with
TAS-F.
Chapter 2 ♦Results and Discussion
- 84 -
It was, therefore, used for deprotection of all the peptides sequences 92-95.
Ph
H
N
O
R1
O
N
H
Rm
n
Ph
Ph
P
TAS-F/DMF
O
O
Si
O
O
R1
O
H
N
N
H
Rm
n
n = 1, 2
Ph
P
H
O
n = 1, 2
96-99
92-95
The successful removal of TMSE was confirmed through ATR-FT-IR of the resulting
supports 96-99 by the disappearance of Si-C vibration at 837 cm-1 in the resulting
products (Fig 2.18).
Fig. 2.18. ATR FT-IR of 96 after removal of TMSE protection with TAS-F.
2.3.1.8. Synthesis of peptidyl chalcones (100-107)
Peptidyl-3-amino-2-oxo-1-phosphoranylidene
propanes
96-99
obtained
were
subjected to Wittig cleavage using a variety of aliphatic and aromatic aldehydes that
led to the formation of peptidyl chalcones. All the reactions were performed in THF.
In case of aliphatic aldehydes, the reactions were complete in 4-5 h by stirring at room
temperature, whereas with aromatic aldehydes, the reaction mixture had to be heated
at 50oC for 5-6 h.
Ph
H
N
O
R1
O
Rm
N
H
n
Ph
P
H
N
R'CHO/THF
H
O
n = 1, 2
O
R1
O
Rm
N
H
n
R'
O
n = 1, 2
100-107
The resulting peptidyl chalcones were purified through preparative HPLC in some
cases. The yields were calculated for the pure compounds, whereas the purity of the
crude products was assessed through LC analysis (Table 2.23).
Chapter 2 ♦Results and Discussion
- 85 -
Table 2.23. Synthesis of peptidyl chalcones 100-107.
Entry
100
O
H
N
O
101
N
H
O
102
106
N
H
a
74
85
65
80
72
64
78
90
74
80
71
Br
Br
Br
O
F
O
N
H
O
N
H
O
H
N
Cl
O
H
N
O
61
S
O
N
H
O
O
Cl
O
H
N
N
H
68
Cl
O
N
H
104
105
80
O
H
N
O
70
F
N
H
103
O
O
O
H
N
O
62
F
N
H
H
N
Yield (%)b
O
O
H
N
O
107
Purity (%)a
Product
O
O
N
H
O
Purity of crude products assessed using LCMS; b isolated yield.
Chapter 2 ♦Results and Discussion
- 86 -
The structures of the synthesized peptides 100-107 were confirmed through HR-MS,
1
H NMR and
13
C NMR analysis. In the HR-MS analysis of chalcone 100, the
molecular ion peak (M+H+) was found as 421.2456.
Fig. 2.19. 1H NMR (300 MHz, CDCl3) spectrum of chalcone 100.
Regarding 1H NMR analysis, the methyl protons of the aliphatic chain appeared as a
doublet at 0.91 ppm with a coupling constant of 7.8 Hz and an integration of six
protons, while the methine CH proton appeared as a multiplet at 1.65-1.81 ppm. A
three proton singlet at 1.94 ppm was assigned to protons of the acetyl group. A broad
triplet at 2.07 ppm with a coupling constant of 6.1 Hz was assigned to allylic CH2
protons. Benzylic protons from two phenylalanine units were found as multiplets at
2.91-3.20 ppm with an integration of four protons. α-Protons of phenylalanine units
were found as multiplets at 4.64-4.78 ppm and 4.85-5.05 ppm, each with one proton
integration. The two doublets at 6.15 ppm and 6.56 ppm, each with a coupling
constant of 7.3 Hz were assigned to two NH protons of the amide linkage. The
doublet at 6.15, was assigned to α-vinylic protons. A large coupling constant of 15.9
Hz confirmed the trans configuration of the double bond in the synthesized chalcone.
The β-vinylic proton appeared downfield as a multiplet along with aromatic protons
of phenyl rings at 6.89-7.06 ppm, with an integration of 11 H′s.
13
C NMR spectrum further confirmed the identity of the product as shown in Fig.
2.20. Important signals were observed at 21.9, 22.7, 27.3 ppm for methyl, methine
and methylene protons, respectively. Chiral carbons appeared at 53.9 and 56.6 ppm,
respectively, whereas the three carbonyls were found at 169.3, 169.9 and 195.6 ppm.
Chapter 2 ♦Results and Discussion
Fig. 2.20.
- 87 -
13
C NMR (100 MHz, CDCl3) spectrum of chalcone 100.
2.3.2. Synthesis of peptidyl heterocycles
Molecular combinations of peptides and heterocycles have been investigated in order
to blend the desirable properties of peptides with those of heterocycles to obtain
bioactive
and
bioavailable,
metabolically
stable
and
membrane-permeable
molecules.226 Peptidyl chalcone derivatives are accessible for converting into a wide
variety of heterocycles. As mentioned earlier (Chapter 1), chalcones may undergo a
(3+2) annulation with a variety of reagents leading to HC’s of varying size e.g.,
reaction of enone moiety with o-aminothiophenol and o-phenylenediamine leads to 7membered heterocycles i.e. 1,5-benzothiazepines and 1,5-benzodiazepines. Likewise,
the reaction of enone with PhNHNH2 or NH2OH led to 5-membered pyrazoles and
oxazoles respectively.218
N O
H
N
O
R2
O
Rn
N
H
n
N NR
R'
N NR
O
N
S
n = 1, 2
N
O
Fig. 2.21. Variety of heterocycles accessible through α,β-unsaturated ketone template.
Following the same strategy, peptidyl chalcones were reacted with different reagents
to yield different peptidyl heterocycles, as discussed in following sections.
Chapter 2 ♦Results and Discussion
- 88 -
2.3.2.1. Synthesis of peptidyl oxazoles
Oxazoles are the rigid scaffolds containing the elements of amide bond.227 They are
therefore astute choice for developing drug-like characteristics in desired peptide
sequences. They are known as adrenergic receptor agonists and bind to the dopamine
receptor.228-229 Peptidyl oxazoles were prepared by reacting peptidyl chalcones with
hydroxylamine (3 eq) in ethanol in the presence of AcOH and NaOAc as catalysts,
whereby the products were separated as white solids.151 The structures of peptidyl
oxazoles were confirmed through HR-MS, 1H NMR and
13
C NMR analysis. The
purity and yield of oxazoles 108-109 are shown in Table 2.24.
Table 2.24. Synthesis of peptidyl oxazoles 108-109.
Entry
108
Purity (%)a
Product
O
H
N
Yield (%)b
O
N
N
H
O
90
85
80
68
F
Cl
109
a
O
H
N
O
N
H
N
O
Purity of crude products assessed using LCMS; b isolated yield.
The synthesis of 108, for example, was confirmed through HRMS analysis where the
molecular ion peak (M+H+) was found at 507.1830 as expected (507.1833). In the 1H
NMR spectrum (Fig. 2.22), a singlet at 1.84 ppm with an integration of 3 protons was
assigned to the acetyl group. Three signals, a dd, a multiplet and another dd with an
integration of one, two and one proton, respectively, appeared in the range of 2.453.45 ppm indicating the presence of a CH2 group. These multiplets were assigned to
benzylic protons of phenylalanines. A dd appeared at 2.73 showing geminal coupling
(12 Hz) and a vicinal coupling (9 Hz). Similarly, a dd at 3.14 ppm appeared with the
coupling constants of 15 Hz and 8 Hz, respectively. The two dd’s at 5.39 and 4.52,
each with an integration of one proton were assigned to two α- hydrogens at two
Chapter 2 ♦Results and Discussion
- 89 -
chiral centers; dd’s showed a vicinal coupling constant of 6 Hz and 15 Hz,
respectively, indicating the cis and trans placement of two vicinal protons with
Fig. 2.22. 1H NMR (300 MHz, CDCl3) spectrum of oxazole 108.
respect to the chiral hydrogen. Two amide NH protons appeared as singlets at 6.70
and 6.90 ppm, whereas a multiplet in the range of 7.03-7.22 ppm with an integration
of 14 protons was assigned to aromatic protons of phenyl and oxazole rings.
Fig. 2.24.
In
13
13
C NMR (100 MHz, CDCl3) spectrum of 108.
C NMR spectrum of oxazole 108 (Fig. 2.24), peaks for all carbons were
observed. The most upfield signal at 22.58 ppm was assigned to the methyl carbon of
terminal acetyl group. The peaks at 36.98 ppm and 37.64 ppm were assigned to
methylene carbons of two benzyl groups. The methine carbons of the chiral centers
appeared downfield at 43.57 ppm and 53.90 ppm. The other important signals
Chapter 2 ♦Results and Discussion
- 90 -
corresponded to the carbons of oxazole ring. C-5 attached to nitrogen of oxazole
appeared at 170.90 ppm, whereas the one attached to oxygen (C-3) appeared at 157.56
ppm. The other C-4 was found at 115.4 ppm. The two carbonyl carbons appeared at
169.15 and 171.05 ppm, respectively, while the aromatic carbons appeared in the
range of 115.35 ppm to 159.15 ppm.
13
C-F19 couplings were observed in the cases
where peaks were separated. The carbon as a doublet at 157.65 ppm and a 1J of 296
Hz was assigned to one attached to fluorine atom. The ipso carbon of 2-chloro-6fluorophenyl ring attached to oxazole ring appeared as a doublet at 139.64 ppm with a
2
J coupling as 96 Hz. The carbon attached to chlorine atom appeared at 129.45 with a
3
J coupling of ~ 12 Hz.
2.3.2.2. Synthesis of peptidyl pyrazolines
Pyrazolines are important N-containing five-membered heterocyclic compounds used
as antitumor, antibacterial and antituberculotic agents.230 Peptidyl pyrazolines were
prepared by reacting peptidyl chalcone with hydrazines (Table 2.25).153
Table 2.25. Synthesis of peptidyl pyrazolines 110-114.
Entry
110
O
N
H
111
H
N
H
N
N
H
114
N
H
H
N
95
65
98
61
95
66
65
53
89
67
N N
O
N
H
N N
O
N
H
O
H
N
Yield (%)b
N NH
O
O
O
N
H
a
H
N
O
N
H
N
H
O
O
112
O
O
O
N
H
113
Purity (%)a
Product
N N
O
O
Purity of crude products; b isolated yield.
N
H
N N
H
Chapter 2 ♦Results and Discussion
- 91 -
A mixture of AcOH and NaOAc was used as catalyst in pyrazoline synthesis. The
synthesized compounds were separated as white solids. The peptidyl pyrazolines 110114 were oxidized to the respective pyrazoles without further confirmation.
2.3.2.3. Synthesis of peptidyl pyrazoles
Peptidyl pyrazolines 110-114 were oxidized to the corresponding pyrazoles 115-118
by their reaction with dichlorodicyanoquinone (DDQ). The residue was subjected to
purification by preparative HPLC and pyrazoles were separated as white solids. The
resulting products were characterized through NMR analysis. Yield and purity of the
synthesized products are given in Table 2.26, while the characterization was done on
the basis of LC-MS and 1H NMR data.
Table 2.26. Synthesis of peptidyl pyrazoles 115-118.
Entry
115
O
N
H
116
H
N
H
N
N
H
a
H
N
N
H
H
N
Yield (%)b
89
>95
84
>95
85
>95
60
>95
N NH
O
N N
O
N
H
O
O
N
H
N
H
O
O
117
O
O
O
N
H
118
Purity (%)a
Product
N N
O
O
N
H
N N
Purity of crude products; b isolated yield.
In 1H NMR analysis of peptidyl pyrazole 115 (Table 2.27), the most upfield doublet at
1.22 ppm with an integration of 3 Hs and a coupling constant of 7.9 Hz, was assigned
to CH3 group of alanine, whereas the singlet at 1.73 ppm was assigned to methyl of
acetyl group. A singlet of 3 Hs appeared at 1.98 ppm was assigned to methyl group at
C-5 of the pyrazole. Two multiplets at 2.65-2.73 ppm and 2.91-2.95 ppm, each with
Chapter 2 ♦Results and Discussion
- 92 -
an integration of two protons were assigned to the benzylic protons of two
phenylalanines. The three chiral centers present in the peptide part of the molecule
appeared in the range of 4.20-4.24 ppm, 4.46-4.49 ppm and 4.99-5.05 ppm, each with
a multiplicity of one proton. The aromatic hydrogen of pyrazole ring (H-4) was found
as a singlet at 5.31 ppm, whereas the NH hydrogen of pyrazole appeared as a singlet
at 5.86 ppm. The protons of three amide NH were found at 6.64 ppm, 8.02 ppm and
8.20 ppm as doublets, each with a coupling constant of ~ 8 Hz. The aromatic protons
corresponding to two phenyl rings appeared at 7.12-7.25 ppm as multiplet of ten
protons.
Table 2.27. 1H-NMR analysis (300 MHz, DMSO-d6) of peptidyl pyrazole 115.
δ ppm
Multiplicity
Integration
Coupling constant
J (Hz)
Assignment
1.22
d
3H
7.9
H-Cβ(Ala)
1.73
s
3H
-
H-C(CH3CO)
1.98
s
3H
-
H-C(CH3 pyrazole)
2.65-2.73
m
2H
-
H-Cβ1,β1′(Phe,
Benzyl)
2.91-2.95
m
2H
-
H-Cβ2,β2′(Phe,
Benzyl)
4.20-4.24
m
1H
-
H-Cα(Ala)
4.46-4.49
m
1H
-
H-Cα(Benzyl)
4.99-5.05
m
1H
-
H-Cα(Phe)
5.31
s
1H
-
H-C(CH pyrazole)
5.86
s
1H
-
NH(pyrazole)
6.64
d
1H
8.0
NHI
7.12-7.25
m
10H
-
H-C(arom.)
8.02
d
1H
8.1
NHII
8.20
d
1H
8.0
NHIII
2.3.2.4. Synthesis of peptidyl benzothiazepines
Benzothiazepines exhibit diverse biological activities. They are known as calcium
antagonist,231
anticonvulsant,232
anticancer,233
antihypertensive234
and
antithrombotic.235 Peptidyl benzothiazepines 119-123 were synthesized by reacting
peptidyl chalcones with o-aminothiophenol at reflux temperatures under an inert N2
Chapter 2 ♦Results and Discussion
- 93 -
atmosphere.146 The residue was subjected to purification by preparative HPLC to
afford benzothiazepines as off-white to pale yellow solids. The resulting products
were confirmed through 1H NMR,
13
C NMR and HRMS analysis. The data of the
purity and yield of peptidyl benzothiazepines is given in Table 2.28.
Table 2.28. Synthesis of peptidyl benzothiazepines 119-123.
Entry
Purity (%)a
Product
Yield (%)b
F
O
119
O
120
O
H
N
H
N
N
H
S
N
Cl
80
52
60
63
78
75
64
71
50
55
O
N
H
S
N
F
O
H
N
O
N
H
121
S
N
Cl
Cl
O
H
N
O
N
H
122
S
S
N
Cl
F
O
123
H
N
O
N
H
S
N
Cl
Cl
a
Purity of crude products assessed through LCMS. b Isolated yield.
Spectroscopic analysis of 119 is discussed as a representative example. Its synthesis
was confirmed through HRMS analysis, wherein molecular ion peak was found as
(M+H+) at 600.1842. In the 1H NMR spectrum (Fig. 2.25), a singlet for acetyl protons
appeared at 1.95 ppm. One of the methylene protons of benzothiazepine ring appeared
Chapter 2 ♦Results and Discussion
- 94 -
as a multiplet along with β protons of the benzyl rings with an integration of five
protons at 2.84-3.14 ppm. The second methylene proton of benzothiazepine ring
appeared as a dd at 3.55 ppm with an integration of one proton and coupling constant,
9.7 Hz and 6.0 Hz, respectively. The methine proton of benzothiazepine moiety
appeared as a dd at 4.65 ppm with coupling constants of 10.2 Hz and 6.0 Hz. The two
chiral carbons of the peptide part were found as doublet of doublet at 5.04 and 5.39
ppm, each with an integration of one proton and coupling constants of ~ 13 Hz and ~
6 Hz, respectively. Aromatic protons of phenyl rings gave a multiplet of 16 protons in
the range of 7.06-7.58 ppm, whereas one aromatic proton appeared as a doublet at
7.58 ppm with a coupling constant of 7.3 Hz. The two amide hydrogens were found as
singlets at 6.18 and 8.08 ppm, respectively.
Fig. 2.25. 1H NMR (300 MHz, CDCl3) spectrum of 119.
In
13
C NMR spectrum (Fig. 2.26), three protons of acetyl group appeared at 22.22
ppm. The important peaks of methane and methylene carbons of benzothiazepine ring
were found at 35.31 ppm and 37.73 ppm, respectively. Furthermore, methylene
protons corresponding to two phenylalanine moieties were found at 45.02 ppm and
48.91ppm. The two peaks for chiral α- carbons appeared at 54.92 and 59.47 ppm. The
characteristic peaks for the two carbonyl carbons were found at 169.53 ppm and
173.23 ppm. The carbon corresponding to C=N part of benzothiazepine ring appeared
at 163.14 ppm, while the other peaks in a range of 114.45 ppm to 158.54 ppm were
assigned to aromatic carbons. 13C-19F coupling was observed for the ipso C attached
to F, resulting in a doublet at 156.9 ppm with a coupling constant of 320 Hz.
Chapter 2 ♦Results and Discussion
Fig. 2.26.
- 95 -
13
C NMR (100 MHz, CDCl3) spectrum of 119.
The identity of other benzothiazepines 120-123 was also confirmed on the basis of
their spectral data.
2.3.2.5. Synthesis of peptidyl benzodiazepines
Benzodiazepine is a potent scaffold for the treatment of cardiovascular disorders,236
epilepsy237 and HIV.238 Peptidyl benzodiazepines 124-125 were synthesized through a
base catalyzed reaction of peptidyl chalcones with 1,2-phenylenediamine under an
atmosphere of nitrogen. Crude products were subjected to purification by preparative
HPLC. Yield and purity of the isolated peptidyl benzodiazepines are given in Table
2.29.
Table 2.29. Synthesis of peptidyl benzodiazepines 124-125.
Entry
Purity (%)a
Product
Yield (%)b
F
O
H
N
O
N
H
124
NH Cl
N
80
48
62
50
F
125
a
O
H
N
O
N
H
N
NH Cl
Purity of crude products assessed through LCMS; b isolated yield
Chapter 2 ♦Results and Discussion
- 96 -
The identity of synthesized benzodiazepines was confirmed spectroscopically. The
HRMS analysis of the peptidyl benzodiazepine 124 showed a M+H+ peak at
533.2140. In the 1H NMR (Table 2.30), a doublet at 1.15 ppm with an integration of
six protons for two methyl protons of i-Pr group, whereas a multiplet at 1.21 ppm1.30 ppm with an integration of one proton was assigned to methine proton of i-Pr
group. A three proton singlet at 1.90 ppm was assigned to N-terminal acetyl group. A
multiplet of two protons at 2.75 ppm-2.80 ppm was assigned to methylene protons of
phenylalanine. A multiplet at 2.95-3.10 ppm with an integration of two protons was
assigned to methylene protons of diazepine ring, whereas a one proton multiplet at
3.40-3.43 ppm was assigned to NH of diazepine ring. The methine proton at the chiral
center of phenylalanine part appeared as a multiplet with one proton integration at
4.17 ppm-4.24 ppm. Another multiplet at 4.75-4.81 ppm with an integration of one
proton was assigned to the chiral carbon attached to isopropyl group. Furthermore, the
methine proton of diazepine ring was assigned to a one proton multiplet at 4.98-5.04
ppm. The aromatic protons of benzene rings and the two amide NHs appeared as a
multiplet at 6.80-7.91 ppm showing an integration of fourteen protons.
Table 2.30. 1H-NMR analysis (300 MHz, CDCl3) of peptidyl benzodiazepine 124.
δ ppm
Multiplicity
Integration
Coupling constant
J (Hz)
Assignment
1.15
d
6H
7.9
CH3 isopropyl
1.21-1.30
m
1H
-
CH isopropyl
1.90
s
3H
-
H-C(CH3CO)
2.75-2.80
m
2H
-
H-Cβ1,β2(Phe)
2.95-3.10
m
2H
-
CH2 (diazepin)
3.40-3.43
m
1H
-
NH (diazepin)
4.17-4.24
m
1H
-
H-Cα(Phe)
4.75-4.81
m
1H
-
H-Cα(CH isopropyl)
4.98-5.04
m
1H
-
H-C(CH diazepin)
6.8-7.91
m
14H
-
H-Carom., NHII,III
13
C NMR (Table 2.31) of benzodiazepine 124 showed characteristic peaks for all
carbons where 13C-19F coupling was observed for carbon attached to F atom, resulting
in the splitting of the parent peak into two in its appearance as a doublet at 152.3 ppm
Chapter 2 ♦Results and Discussion
- 97 -
with a JCF of 311 Hz. Similarly, the ipso carbon attached to diazepine ring appeared
as a doublet at 140.7 ppm with 2J as 80 Hz.
Table 2.31.
δ ppm
13
C-NMR analysis (100 MHz, CDCl3) of peptidyl benzodiazepine 124.
Assignment
δ ppm
Assignment
δ ppm
Assignment
18.6
CH3
36.0
CH2
111.6-141.1
aromatic
18.7
CH3
37.1
CH
151.8, 152.7
Aromatic C-F
22.9
CH3 (acetyl)
42.6
CH (chiral)
165.1
C=N
28.6
CH (isopropyl)
54.2
CH (chiral)
172.8, 184.4
C=O
The present work led to establishment of a new strategy for construction of Cterminal derivatized peptidyl heterocycles using novel bis-electrophiles (peptidyl
chalcones) established after successful C-acylation of phosphorane support. The
results are being compiled in the form of a publication.218 All the synthesized peptidyl
chalcones and derived heterocycles have been submitted for bioevaluation against
different targets.