Synthesis and DNA binding affinity of novel A-C8/C-C2-exo unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers.

Article abstract of DOI:10.1016/j.bmc.2004.06.013

The synthesis of novel A-C8/C-C2-exo unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers is reported and these dimers show significant DNA binding affinity and they also exhibit moderate anticancer activity.

Full text of DOI:10.1016/j.bmc.2004.06.013

Bioorganic & Medicinal Chemistry 12 (2004) 4337–4350
Synthesis and DNA binding affinity of novel A-C8/C-C2-exo
unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine
dimers
Ahmed Kamal,* O. Srinivas, P. Ramulu, G. Ramesh, P. Praveen Kumar and
M. Shiva Kumar
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India
Received 17 May 2004; revised 10 June 2004; accepted 11 June 2004
Available online 3 July 2004
Abstract—The synthesis of novel A-C8/C-C2-exo unsaturated alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers is re-
ported and these dimers show significant DNA binding affinity and they also exhibit moderate anticancer activity.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Thurston and co-workers7 have developed new pyr-
rolobenzodiazepine dimers with remarkable DNA cross-
linking ability. Moreover, the same group has reported
the first example of A-C8/C-C2 amido-linked PBD
dimer8 (4) and recently we have reported A-C8/C-C2
alkoxyamido-linked tail to head type PBD dimer (5)
with significant DNA binding ability and low anticancer
activity.9 Therefore, introduction of C2-exo unsatura-
tion is an important component that probably is causing
the flattening of the C-ring to produce an improved
isohelical fit within the DNA minor groove.10 In view of
these findings and in conjunction with our pursuit
towards the design and synthesis of PBD hybrids,11 we
have considered the synthesis of A-C8/C-C2-exo unsat-
urated alkoxyamido linked PBD dimers (6a–b and 7a–b)
as a significant and important aspect in the development
of novel PBD dimers with potential anticancer activity
and DNA binding ability. Further, we have also pre-
pared such PBD dimers (8a–b) with amide functionality
at N10–C11position of A-C8 component to investigate
the effect of the absence of one of the imine functionality
on the DNA binding profile.
In the last decade there has been increasing interest-
towards the synthesis of DNA sequence selective agents,
particularly low molecular weight antitumour antibio-
tics. Pyrrolo[2,1-c][1,4]benzodiazepines are naturally
occurring antitumour antibiotics that interact within the
minor groove of DNA forming monocovalent adducts.1
Recently, there has been tremendous interest in the de-
sign and synthesis of DNA interstrand cross-linking
agents that are likely to enhance the sequence selectivity
and in turn could increase selectivity for tumour cells.2
In this context a large number of PBD dimers have been
designed and synthesized with a view to explore their
DNA cross-linking ability and their sequence selectiv-
ity.3 These PBD dimers have been joined through dif-
ferent positions such as A-C7/A-C70, A-C8/A-C80 (3) and
C-C2/C-C20 (2), amongst these A-C8/A-C80 linked PBD
dimers have shown promising cytotoxicity and efficient
cross-linking property.4 Further it is observed from the
literature that C2-exo unsaturation as in case of tom-
aymycin significantly enhances the anticancer potential
and DNA binding ability5 in comparison to non-C2-exo
unsaturation (DC-81) and this feature has been exten-
sively investigated for many synthetic analogues.6
2. Results and discussion
2.1. Synthesis
Keywords: Pyrrolobenzodiazepine dimers; DNA binding affinity;
Cytotoxicity.
Synthesis of A-C8/C-C2-exo unsaturated alkoxyamido-
linked PBD dimers 6a–b has been carried out by
* Corresponding author. Tel.: +91-40-27193157; fax: +91-40-271931-
89; e-mail: ahmedkamal@iict.res.in
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.013

4338
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
amidation of the A-C8 alkoxyamine components (14a–b)
and the C-C2-exo unsaturated acid component (22a).
The C8 alkoxyamine has been synthesized in the fol-
lowing manner; the precursor methyl 4-benzyloxy-5-
acetal group affords the desired compounds 8a–b
(Scheme 3).
methoxy-2-nitrobenzoate
9
has been prepared by
2.2. DNA interactions: thermal denaturation studies
employing the literature method,12 which upon deben-
zylation with BF3ÆOEt2–EtSH gives compound 10 and
etherification with Boc-protected bromoalkylamines
provide 11a–b. These compounds have been hydrolyzed
and coupled with (2-S)-pyrrolidinecarboxaldehyde di-
ethyl thioacetal to give 13a–b, which upon deprotection
provide the desired intermediate amines 14a–b (Scheme
1). The other precursor C2-exo unsaturated acid (22a)
has been prepared by employing 4-benzyloxy-5-meth-
oxy-2-nitrobenzoic acid 15a as the starting material,
which has been obtained by the procedure described in
The DNA binding ability for these A-C8/C-C2-exo
unsaturated alkoxyamido-linked PBD dimers has been
determined by thermal denaturation studies using calf
thymus (CT) DNA. Interestingly, in this assay four of
these PBD dimers that is, 6a,b, 7a and 7b elevate the
helix melting temperature of CT-DNA by a 5.7, 8.3, 5.6
and 6.4 °C, respectively, after incubation for 18 h at
37 °C. On the other hand, the A-C8/A-C80 PBD dimer,
DSB-120 exhibits a DTm of 15.3 °C after 18 h of incu-
bation. Further, the values of Tm enhances with the in-
crease of incubation time. This demonstrates that these
PBD dimers show significant DNA binding affinity but
not as high as DSB-120. On the contrary, the imine–
amide dimers (8a and 8b) do not exhibit significant
DNA binding ability (Table 1).
the literature.12 trans-4-Hydroxy-
L-proline methyl ester
hydrochloride has been coupled to compound 15a to
give the nitro ester 16a. The hydroxy group is protected
with TBDMS-Cl followed by reduction with DIBAL-H
to produce the corresponding aldehyde, which is pro-
tected with EtSH/TMS-Cl. Interestingly, in this reaction
protection of aldehyde to diethyl thioacetal and depro-
tection of TBDMS takes place in the same step to afford
compound 19a. Then C2-hydroxy group is oxidized
with TPAP/NMO to give compound 20a, which upon
Horner–Emmons olefination with methyl diethyl-
phosphonoacetate affords compound 21a. In this
reaction, (E) ester3d (21a) has been obtained exclusively,
which upon hydrolysis affords the corresponding acid
22a. The key intermediates 23a–b have been prepared by
amidation of compound 22a with 14a–b. The com-
pounds 23a–b have been reduced with SnCl2Æ2H2O to
afford the corresponding amino diethyl thioacetal (25a–
b). Deprotection of amino diethyl thioacetal with HgCl2/
CaCO3 provides the target molecules 6a–b (Scheme 2).
The compounds 7a–b have been prepared in the
same manner by employing with commercially avail-
able 4,5-dimethoxy-2-nitrobenzoic acid (Scheme 2).
The compounds 30a–b have been prepared by amida-
tion of compounds 22a and 29a–b, then subsequent
reduction followed by deprotection of diethyl thio-
2.3. Cytotoxicity
Compounds 6a and 8b have been evaluated for their in
vitro cytotoxicity against 60 human tumour cell lines
derived from nine cancer types (leukaemia, nonsmall cell
lung cancer, colon cancer, CNS cancer, melanoma,
ovarian cancer, renal cancer, prostate cancer and breast
cancer). According to the in vitro screening data from
the National Cancer Institute (NCI) initial primary test,
the compounds 6a and 8b have cytotoxic potency
against many cell lines. Compound 6a exhibits a wide
spectrum activity against 32 cell lines in nine cell panels,
with GI50 values of <10 lM, whereas compound 8b
exhibits activity against 24 cell lines in seven cancer cell
panels, with GI50 values of <50 lM. A reference com-
pound of the same family DRG-16 ranges from 0.001 to
7.94 nM (mean 0.12 nM).7 The GI50 values of com-
pounds 6a and 8b for different cell lines have been
illustrated in Table 2.
H
BnO
MeO
NO2
OMe
HO
NO2
OMe
N
O
NO2
OMe
ii
i
BOC
n
MeO
MeO
O
O
O
10
9
11a-b
H
N
H
N
CH(SEt)2
O
NO2
N
O
NO2
iii
iv
BOC
n
BOC
n
OH
MeO
MeO
12 a-b
O
O
13 a-b
CH(SEt)2
H2N
O
NO2
N
v
n
MeO
O
14 a-b n = 2, 3
Scheme 1. Reagents and conditions: (i) EtSH–BF3OEt2, CH2Cl2, 12 h, rt, 75%; (ii) Boc-protected bromo alkylamine, K2CO3, DMF, rt, 24 h, 85–
88%; (iii) 1 N LiOH, THF–H2O–MeOH, 2 h, rt, 80–83%; (iv) 2(S)-pyrrolidinecarboxaldehyde diethyl thioacetal, EDCl–HOBt, CH2Cl2–H2O, 24 h, rt,
60–65%; (v) TFA, CH2Cl2, 8 h, rt.

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
4339
16a-b
17a-b
18a-b
ii
NO2
NO2
OH
NO2
RO
RO
RO
iii
CHO
COOCH3
OH
i
N
N
H3CO
H3CO
H3CO
OTBDMS
18 a-b
O
O
O
15 a-b
16 a-b
iv
NO2
NO2
RO
RO
CH(SEt)2
CH(SEt)2
OH
v
N
N
H3CO
H3CO
O
O
O
20 a-b
19 a-b
vi
NO2
NO2
RO
RO
CH(SEt)2
O
CH(SEt)2
O
vii
N
N
H3CO
H3CO
OCH3
OH
O
O
22 a-b
21 a-b
viii
NO2
N
RO
CH(SEt)2
O
CH(SEt)2
NO2
O
H3CO
N
H
n
O
N
H3CO
O
ix
23 a-b
24 a-b
NH2
RO
CH(SEt)2
O
CH(SEt)2
NH2
N
O
n
H3CO
N
H
O
N
H3CO
O
25 a-b
26 a-b
x
6 a-b; R = Bn n = 1, 2
7 a-b; R = Me
Scheme 2. Reagents and conditions: (i) SOCl2, C6H6, trans-4-hdroxy- -proline methylester hydrochloride, Et3N, H2O, THF, 0 °C, 1 h, 75–80%; (ii)
L
TBDMS-Cl, CH2Cl2, 12 h, rt, 90–92%; (iii) DIBAL-H, CH2Cl2, ꢀ78 °C, 45 min, 65–68%; (iv) EtSH, TMS-Cl, CHCl3, 18 h, rt, 72–75%; (v) TPAP–
NMO, CH2Cl2–MeCN, 2.5 h, rt, 76–78%; (vi) methyl diethylphosphonoacetate, NaH, THF, 0 °C, 2 h, 80–84%; (vii) 1 N LiOH, THF–H2O–MeOH,
2 h, rt, 83–87%; (viii) EDCl–HOBt, compound 14a–b, CH2Cl2–H2O, 24 h, rt, 54–60%; (ix) SnCl2–2H2O, MeOH, reflux, 3 h, 70–75%; (x) HgCl2–
CaCO3, CH3CN–H2O, 12 h, rt, 48–55%.
2.4. RED100-restriction endonuclease digestion assay
alkoxyamido-linked PBD dimers, which inhibit the
DNA linearization by BamH1. The results of this
experiment for compounds 6a,b, 7a and 7b shown in
Figure 2 suggest that the PBD dimers inhibit BamH1.
There are differences in the inhibitory activity exhibited
by PBDs evaluated in this assay. It is observed that the
ranking order is 6b > 7b > 6a > 7a for inhibition of
BamH1 cleavage is in agreement with the DNA bind-
ing affinity as determined by thermal denaturation.
These results clearly demonstrate that as the linker
chain increases from two to three carbon spacer as in
case of 6b there is an enhancement in the inhibitory
activity (Fig. 1).
Many studies have employed restriction endonuclease
inhibition to confirm the relative binding affinity of
DNA-interactive small molecule ligands.13–16 A quan-
titative restriction enzyme digest (RED100) assay was
developed in which the inhibition of DNA cleavage by
BamH1 was used to probe the DNA binding capability
of PBD monomers.17 We have earlier investigated this
assay for preferences of base pair selectivity of the
imine–amide PBD dimers.18 Recently this technique
has also been used to study the covalent DNA inter-
action of PBD dimers and it is capable to distinguish
between the monomeric and dimeric families.7 The
BamH1 cleavage sequence G#GATCC overlaps with
several favoured PBD binding sites suggesting ligand
binding has the potential to inhibit the BamH1 cleav-
age activity. The study has been carried out to deter-
mine the ability of A-C8/C-C2-exo unsaturated
2.5. Conclusions
The increase of alkane spacer from two to three carbons
in the newly designed A-C8/C-C2-exo unsaturated

4340
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
H
COOMe
COOMe
COOMe
N
O
NO2
N
H2N
O
NO2
N
HO
NO2
N
BOC
n
n
i
ii
H3CO
H3CO
H3CO
O
O
27
O
28 a-b
29 a-b n = 2, 3
iii
NO2
N
BnO
CH(SEt)2
O
COOMe
O
NO2
H3CO
n
N
O
H
N
O
H3CO
30 a-b
iv
NH2
BnO
CH(SEt)2
O
H
N
O
H
N
O
n
H3CO
N
H
O
N
H3CO
O
31 a-b
v
8a-b n = 1, 2
Scheme 3. Reagents and conditions: (i) Boc-protected bromo alkylamine, K2CO3, DMF, rt, 24 h, 80–82%; (ii) TFA, CH2Cl2, 8 h, rt; (iii) EDCl–
HOBt, compound 22a, CH2Cl2–H2O, 24 h, rt, 65–67%; (iv) SnCl2–2H2O, MeOH, reflux, 3 h, 76–78%; (v) HgCl2–CaCO3, CH3CN–H2O, 12 h, rt, 51–53%.
Table 1. Thermal denaturation data for A-C8/C-C2-exo unsaturated
alkoxyamido-linked PBD dimers with CT-DNA
3. Experimental
DTm (°C)a After incubation at 37 °C
3.1. Synthetic chemistry
PBD
dimers
[PBD]/
[DNA]
molar ratiob
0 h
18 h
36 h
48 h
Reaction progress was monitored by thin-layer chro-
matography (TLC) using GF254 silica gel with fluores-
cent indicator on glass plates. Visualization was
achieved with UV light and iodine vapour unless
otherwise stated. Chromatography was performed using
Acme silica gel (100–200 and 60–120 mesh). The
majority of reaction solvents were purified by distillation
under nitrogen from the indicated drying agent and used
fresh: dichloromethane (calcium hydride), tetrahydro-
furan (sodium benzophenone ketyl), methanol (magne-
sium methoxide) and acetonitrile (calcium hydride).
6a
6b
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1.2
5.7
8.3
5.6
6.4
0.6
0.9
0.7
15.3
7.1
10.0
6.8
8.4
12.6
7.7
2.3
1.5
7a
7b
2.0
0.3
7.8
0.7
9.0
1.0
8a
8b
0.5
0.3
1.1
1.4
DC-81
DSB-120 1:5
10.1
a For CT-DNA alone at pH 7.00 ꢁ 0.01, Tm ¼ 69:0 °C ꢁ 0.01 (mean
value from 30 separate determinations), all DTm values are ꢁ0:1–
0.2 °C.
b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion ¼ 100 lM and ligand concentration ¼ 20 lM in aqueous sodium
phosphate buffer [10 mM sodium phosphateþ1 mM EDTA,
pH 7.00 ꢁ 0.01].
1H NMR spectra were recorded on Varian Gemini
200 MHz spectrometer using tetramethylsilane (TMS) as
an internal standard. Chemical shifts are reported in
parts per million (ppm) downfield from tetramethyl-
silane. Spin multiplicities are described as s (singlet), br s
(broad singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). Coupling constants are reported in Hertz
(Hz). Low resolution mass spectra (LRMS) were
recorded on VG-7070H Micromass mass spectrometer
at 200 °C, 70 eV with trap current of 200 lA and 4 kV
acceleration voltage. Optical rotations are measured on
Horiba, high sensitive polarimeter, SEPA-300.
alkoxyamido-linked PBD dimers considerably enhances
the DNA melting temperatures. The restriction endo-
nuclease study also demonstrates this aspect and sug-
gests these molecules selectively interact with
G
sequences in DNA and low affinity with AT-rich
sequences. However, the DNA binding potential of these
PBD dimers is comparatively less than the A-C8/A-C80
PBD dimers. The in vitro anticancer activities for the
representative compounds 6a and 8b are not that sig-
nificant.
3.2. Methyl-4-hydroxy-5-methoxy-2-nitrobenzoate (10)
To a stirred solution of EtSH (1.20 g, 19 mmol) and
BF3ÆOEt2 (1.419 g, 10 mmol) was added dropwise to the

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
4341
hexane) to afford compound 10 (170 mg, 75%). 1H
NMR (CDCl3): d 3.89 (s, 3H), 3.95 (s, 3H), 6.25 (br s,
1H), 7.05 (s, 1H), 7.45 (s, 1H). MS (EI) m=z 227 [M]þÅ.
Table 2. In vitro cytotoxicity of compound 6a and 8b in selected
human cancer cell lines
Cancer panel/cell line
GI50 (lM)
6a
8b
Leukaemia
CCRF-CEM
K-562
3.3. Methyl-4-(N-t-butoxycarbonyl)aminoethoxy-5-
methoxy-2-nitrobenzoate (11a)
1.1
2.7
1.1
1.4
1.2
40.8
––
MOLT-4
SR
RPMI-8226
29.6
17.5
43.6
To a solution of compound 10 (227 mg, 1 mmol) in
dimethylformamide (10 mL) was added, anhydrous
K2CO3 (553 mg, 4 mmol) and Boc-protected bromo
ethylamine (269 mg, 1.2 mmol) and the mixture was
stirred for 24 h at room temperature. The reaction was
monitored by TLC using EtOAc–hexane (1:1) and
K2CO3 was removed by filtration and the solvent was
evaporated under the vacuum. The residue was dissolved
in ethyl acetate (2 ꢂ 20 mL) and washed with water. The
combined organic phases were dried over Na2SO4 and
the solvent was removed by vacuum and purified by
column chromatography (25% EtOAc–hexane) to afford
compound 11a (315 mg, 85%). 1H NMR (CDCl3): d 1.45
(s, 9H), 3.89 (s, 3H), 3.60 (t, 2H, J ¼ 6:22 Hz), 3.95 (s,
3H), 4.10 (t, 2H, J ¼ 5:98 Hz), 5.0 (br s, 1H), 7.05 (s, 1H),
7.45 (s, 1H). MS (EI) m=z 370 [M]þÅ.
Nonsmall cell lung
HOP-62
HOP-92
4.2
––
41.3
47.2
––
NCI-H23
NCI-H460
NCI-H522
9.2
––
38.9
31.3
1.6
Colon
HCT-116
HT-29
2.9
––
45.2
47.5
33.3
KM-12
SW-620
9.3
5.3
CNS
SF-268
SF-539
SNB-19
SNB-75
U251
2.1
––
39.6
32.4
43.3
32.7
33.3
8.6
––
3.4. Methyl-4-(N-t-butoxycarbonyl)aminopropoxy-5-
methoxy-2-nitrobenzoate (11b)
2.8
Melanoma
LOX IMVI
SK-MEL-5
MALME-3M
SK-MEL-2
UACC-62
2.7
1.3
2.2
1.5
2.6
43.3
22.5
––
The compound 11b was prepared according to the
method described for the compound 11a employing
compound 10 (227 mg, 1 mmol) and Boc-protected
bromopropylamine (286 mg, 1.2 mmol) to afford crude
compound, which was purified by column chromato-
graphy (30% EtOAC–hexane) to give compound 11b
––
––
Ovarian
IGROV1
OVCAR-3
OVCAR-5
OVCAR-8
1.4
2.1
4.0
3.8
––
––
––
––
1
(338 mg, 88%). H NMR (CDCl3): d 1.45 (s, 9H), 1.90–
2.05 (m, 2H), 3.89 (s, 3H), 3.60 (t, 2H, J ¼ 6:22 Hz), 3.95
(s, 3H), 4.10 (t, 2H, J ¼ 5:98 Hz), 5.0 (br s, 1H), 7.05 (s,
1H), 7.45 (s, 1H). MS (EI) m=z 384 [M]þÅ.
Prostate
PC-3
3.5
––
3.5. 4-(N-t-Butoxycarbonyl)aminoethoxy-5-methoxy-2-
nitrobenzoicacid (12a)
Renal
RXF-393
SN12C
786-0
1.9
2.6
3.6
7.0
33.6
18.4
––
Lithium hydroxide monohydrate (2 N, 1.22 mL) was
TK-10
––
added to
bonyl)aminoethoxy-5-methoxy-2-nitrobenzoate
a
solution of methyl-4-(N-t-butoxycar-
11a
Breast
(370 mg, 1 mmol) in THF–H2O–MeOH (4:1:1) and the
mixture stirred at room temperature for 2 h. After most
of the THF and methanol were evaporated, the aqueous
phase was acidified with 12 N HCl to pH 7 and re-ex-
tracted with EtOAc to give a 4-(N-t-butoxycarbonyl)-
aminoethoxy-5-methoxy-2-nitrobenzoic acid 12a (285
mg, 80%). 1H NMR (CDCl3): d 1.45 (s, 9H), 3.60 (t, 2H,
J ¼ 6:4 Hz), 3.95 (s, 3H), 4.20 (t, 2H, J ¼ 6:2 Hz), 5.0 (br
s, 1H), 7.20 (s, 1H), 7.40 (s, 1H). MS (EI) m=z 356 [M]þÅ.
MCF-7
2.6
5.6
2.9
––
32.0
34.4
44.2
23.9
––
HS-578T
MDA-MB-435
BT-549
T-47D
2.7
compound 9 (317 mg, 1 mmol) in dichloromethane
(15 mL) at room temperature. Stirring was continued
until TLC completed the reaction and then the solvent
was evaporated in vacuum. The residue was quenched
with 5% NaHCO3 solution (20 mL) and then extracted
with dichloromethane (2 ꢂ 20 mL). The combined or-
ganic phases were washed with saturated brine (10 mL),
dried over Na2SO4 and the solvent removed by vacuum
and purified by column chromatography (30% EtOAc–
3.6. 4-(N-t-Butoxycarbonyl)aminopropoxy-5-methoxy-2-
nitrobenzoicacid (12b)
The compound 12b was prepared according to
the method described for the compound 12a

4342
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
10
11
9
6
N
N
N
HO
H
H
H
3
8
O
O
11a
1
N
N
N
7
H3CO
2
N
H
N
H
5
O
4
n
O
O
2
n = 1-3
1 (DC-81)
N
N
O
O
H
H
N
n
N
OCH3
H3CO
O
O
3 n = 1, 3
N
H3CO
H3CO
H
O
H
N
N
N
N
H
O
N
O
R
N
BnO
4
R = Me, H
H
O
O
N
O
H3CO
H
N
n
O
H
N
H3CO
O
5
n = 1, 3, 4
N
RO
H
N
O
N
H3CO
H
n
N
H
O
N
H3CO
O
6a, n = 1, R = Bn; 6b, n = 2, R = Bn
7a, n = 1, R = Me; 7b, n = 2, R = Me
N
BnO
H
O
H
N
O
H
O
N
H3CO
n
N
H
O
N
H3CO
O
8a, n = 1
8b, n = 2
Figure 1. Structures of pyrrolobenzodiazepines and their dimers.
Figure 2. RED100-restriction endonuclease digestion assay for A-C8/C-C2-exo unsaturated alkoxyamido-linked PBD dimers with CT-DNA inhib-
itory activity of 6a,b, 7a and 7b on the cleavage of plasmid pBR322 by restriction endonuclease BamH1 (20 units in 2 lL) for 1 h at 37 °C. The cut (C)
and uncut (UC) products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining under UV illumination. Lane 1:
control pBR322; lane 2: complete digest of pBR322 by BamH1.
employing compound 11b (384 mg, 1 mmol) to afford
1
3.7. (2S)-N-[4-(N-t-Butoxycarbonyl)aminoethoxy-5-
methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal (13a)
compound 12b (409 mg, 87%). H NMR (CDCl3): d
1.45 (s, 9H), 1.95–2.05 (m, 2H), 3.60 (t, 2H,
J ¼ 6:4 Hz), 3.98 (s, 3H), 4.25 (t, 2H, J ¼ 6:2 Hz), 5.0
(br s, 1H), 7.20 (s, 1H), 7.45 (s, 1H). MS (EI) m=z 370
[M]þÅ.
To a solution of 12a (356 mg, 1 mmol) in dichlorome-
thane–water (20:20 mL), were added EDCI (384 mg,

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
4343
2 mmol), HOBt (270 mg, 2 mmol) and (2S)-pyrrolidine-
2-carboxaldehyde diethyl thioacetal (205 mg, 1 mmol)
and the mixture was stirred for 24 h at room tempera-
ture. The dichloromethane was evaporated in vacuum,
the residue was dissolved in ethyl acetate and washed
with saturated sodium bicarbonate solution and then
with brine solution. The combined organic phases were
dried over Na2SO4, the solvent was removed under
vacuum and purified by column chromatography (50%
EtOAc–hexane) to afford compound 13a (524 mg, 60%).
1H NMR (CDCl3): d 1.20–1.40 (m, 6H), 1.45 (s, 9H),
1.80–2.10 (m, 4H), 2.60–2.90 (m, 4H), 3.20–3.35 (m,
2H), 3.60 (t, 2H, J ¼ 6:22 Hz), 3.95 (s, 3H), 4.10 (t, 2H,
J ¼ 5:98 Hz), 4.63–4.73 (m, 1H), 4.80 (d, 1H, J ¼
4:3 Hz), 5.0 (br s, 1H), 6.75 (s, 1H), 7.60 (s,1H). MS
(FAB) 544 [MþH]þÅ.
cooled in an ice bath. After the completion of addition,
the reaction mixture was brought to ambient tempera-
ture and stirred for an additional 1 h. The THF was
evaporated in vacuum and the aqueous layer was
washed with ethyl acetate (10 mL). The aqueous phase
was then adjusted to pH 3 using 6 N HCl and extracted
with ethyl acetate and washed with brine, dried over
Na2SO4 and evaporated in vacuum and was purified by
column chromatography (70% EtOAc–hexane) to afford
compound 16a (323 mg, 75%). 1H NMR (CDCl3): d
2.10–2.22 (m, 1H), 2.30–2.45 (m, 1H), 2.95–3.20 (m,
2H), 3.42–3.50 (m, 1H), 3.80 (s, 3H), 4.0 (s, 3H), 4.40–
4.50 (m, 1H), 4.75–4.85 (m, 1H), 5.20 (s, 2H), 6.82 (s, 1H),
7.3–7.50 (m, 5H) 7.70 (s, 1H). MS (FAB) 431 [MþH]þÅ.
3.12. Methyl-(2S,4R)-N-[4,5-dimethoxy-2-nitrobenzoyl]-
4-hydroxypyrrolidine-2-carboxylate (16b)
3.8. (2S)-N-[4-(N-t-Butoxycarbonyl)aminopropoxy-5-
methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal (13b)
The compound 16b was prepared according to the
method described for the compound 16a employing
compound 4,5-dimethoxy nitro benzoic acid (227 mg,
1 mmol) to give crude product, which was purified by
column chromatography (80% EtOAc–hexane) to afford
compound 16b (283 mg, 80%). 1H NMR (CDCl3): d 2.0–
2.20 (m, 1H), 2.25–2.45 (m, 1H), 2.80–3.20 (m, 2H),
3.50–3.60 (m, 1H), 3.80 (s, 3H), 3.98 (s, 3H), 4.0 (s, 3H),
4.40–4.50 (m, 1H), 4.75–4.85 (m, 1H), 6.82 (s, 1H), 7.70
(s, 1H); MS (EI) 354 [M]þÅ.
The compound 13b was prepared according to the
method described for the compound 13a by employing
12b (370 mg, 1 mmol) to afford crude compound, which
was purified by column chromatography (55% EtOAC–
hexane) to give compound 13b (362 mg, 65%). 1H NMR
(CDCl3): d 1.20–1.40 (m, 6H), 1.45 (s, 9H), 1.70–2.35
(m, 6H), 2.65–2.90 (m, 4H), 3.18–3.30 (m, 2H), 3.40 (t,
2H, J ¼ 6:20 Hz), 3.95 (s, 3H), 4.18 (t, 2H, J ¼ 6:10 Hz),
4.63-4.75 (m, 1H), 4.80 (d, 1H, J ¼ 4:29 Hz), 5.20 (br s,
1H), 6.80 (s, 1H), 7.62 (s, 1H). MS (FAB) 558 [MþH]þÅ.
3.13. Methyl-(2S,4R)-N-[4-benzyloxy-5-methoxy-2-
nitrobenzoyl]-4-(t-butyldimethylsilyl)-oxypyrrolidine-2-
carboxylate (17a)
3.9. (2S)-N-[4-(2-Aminoethoxy)-5-methoxy-2-nitrobenz-
oyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (14a)
Solid TBDMS-chloride (226 mg, 1.5 mmol) was added in
one portion to a solution of compound 16a (430 mg,
1 mmol) and imidazole (136 mg, 2 mmol) in anhydrous
dichloromethane (20 mL) and allowed to stir at room
temperature for 12 h under a nitrogen atmosphere. The
reaction mixture was poured into water and then ex-
tracted with dichloromethane (2 ꢂ 20 mL). The com-
bined organic phases were dried over Na2SO4 and the
solvent was removed by vacuum and purified by column
chromatography (30% EtOAc–hexane) to afford com-
To a solution of Boc-compound 13a (543 mg, 1 mmol) in
dry dichloromethane was added trifluoroacetic acid
(1 mL) at 0 °C and stirred under nitrogen for 8 h, the
reaction mixture was then concentrated in vacuum and
then it was used directly in the next step.
3.10. (2S)-N-[4-(3-Aminopropoxy)-5-methoxy-2-nitrobenz-
oyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (14b)
1
pound 17a (490 mg, 90%). H NMR (CDCl3): d 0.18 (s,
The compound 14b was prepared according to the
method described for the compound 14a by employing
13b (557 mg, 1 mmol) and TFA.
6H), 0.85 (s, 9H), 2.08–2.20 (m, 1H), 2.25–2.40 (m, 1H),
3.0–3.10 (m, 1H), 3.40–3.55 (m, 1H), 3.80 (s, 3H), 4.00
(s, 3H), 4.40–4.50 (m, 1H), 4.75–4.85 (m, 1H), 5.20 (s,
2H), 6.80 (s, 1H), 7.30–7.50 (m, 5H) 7.70 (s, 1H). MS
(FAB) 545 [MþH]þÅ.
3.11. Methyl-(2S,4R)-N-[4-benzyloxy-5-methoxy-2-
nitrobenzoyl]-4-hydroxypyrrolidine-2-carboxylate (16a)
3.14. Methyl-(2S,4R)-N-[4,5-dimethoxy-2-nitrobenzoyl]-
4-(t-butyldimethylsilyl)oxypyrrolidine-2-carboxylate (17b)
To a stirred suspension of compound 15a (303 mg,
1 mmol) and thionyl chloride (476 mg, 4.0 mmol) in dry
benzene (15 mL) was added DMF (four–five drops) and
the stirring was continued for 6 h. The benzene was
evaporated in vacuum and the resultant oil dissolved in
dry THF (20 mL) and added drop wise over a period of
30 min to a stirred suspension of trans-4-hydroxy-L-
proline methylester hydrochloride (270 mg 1.5 mmol),
triethylamine (303 mg, 3 mmol) and ice water (20 mL)
The compound 17b was prepared according to the
method described for the compound 17a employing
compound 16b (354 mg, 1 mmol) to give the crude
product, which was purified by column chromatography
(40% EtOAc–hexane) to afford the compound 17b
(430 mg, 92%). 1H NMR (CDCl3): d 0.18 (s, 6H), 0.85 (s,
9H), 2.10–2.20 (m, 1H), 2.25–2.40 (m, 1H), 3.0–3.10 (m,

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A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
1H), 3.40–3.50 (m, 1H), 3.80 (s, 3H), 3.98 (s, 3H), 4.0 (s,
3H), 4.40–4.50 (m, 1H), 4.70–4.80 (m, 1H), 6.80 (s, 1H),
7.65 (s, 1H). MS (FAB) 469 [MþH]þÅ.
3.10–3.20 (d 1H, J ¼ 11:15 Hz), 3.41–3.49 (dd, 1H,
J ¼ 3:7, 3.7 Hz), 3.95 (s, 3H), 4.25–4.38 (m, 1H), 4.80–
4.90 (m, 2H), 5.20 (s, 2H), 6.80 (s, 1H), 7.30–7.50 (m,
5H), 7.70 (s, 1H). MS (FAB) 507 [MþH]þÅ.
3.15. (2S,4R)-N-[4-Benzyloxy-5-methoxy-2-nitro-
benzoyl]-4-(t-butyldimethylsilyl)oxypyrrolidine-2-carbox-
aldehyde (18a)
3.18. (2S,4R)-N-[4,5-Dimethoxy-2-nitrobenzoyl]-4-hydro-
xypyrrolidine-2-carboxaldehyde diethyl thioacetal (19b)
Diisobutyl alminium hydride solution (2.5 mL of 1.0 M
solution in hexane) was added drop wise to a vigorously
stirred solution of the compound 17a (544 mg, 1 mmol)
in anhydrous dichloromethane (10 mL) under dry
nitrogen at ꢀ78 °C (dry ice–acetone). After the mixture
was stirred for an additional 45 min, excess of reagent
was decomposed by careful addition of methanol (2 mL)
followed by 5% HCl (2 mL). The resulting mixture was
allowed to warm room temperature and the organic
layer was removed, the aqueous layer was extracted with
ethylacetate (4 ꢂ 10 mL). The organic layers were com-
bined, dried over Na2SO4 and the solvent was evapo-
rated in vacuum to afford the crude aldehyde 18a
(334 mg, 65%). 1H NMR (CDCl3): d 0.16 (s, 6H), 0.80 (s,
9H) 2.10–2.30 (m, 1H), 2.42–2.56 (m, 1H), 3.20–3.30 (m,
1H), 3.40–3.52 (m, 1H), 4.0 (s, 3H), 4.40–4.50 (m, 1H),
4.75–4.90 (m, 1H), 5.20 (s, 2H), 6.75 (s, 1H), 7.30–7.50
(m, 5H), 7.75 (s, 1H), 9.78–9.80 (d, 1H, J ¼ 2:0 Hz). MS
(FAB) 515 [MþH]þÅ.
The compound 19b was prepared according to the
method described for the compound 19a employing
compound 18b (438 mg, 1 mmol) to afford crude com-
pound, which was purified by column chromatography
(70% EtOAc–hexane) to give compound 19b (323 mg,
75%). 1H NMR (CDCl3): d 1.20–1.40 (m, 6H), 1.80–2.35
(m, 3H), 2.62–2.90 (m, 4H), 3.20 (d, 1H, J ¼ 10 Hz),
3.40–3.50 (dd, 1H, J ¼ 8:5, 3.5 Hz), 3.95 (s, 3H), 3.98 (s,
3H), 4.25–4.38 (m, 1H), 4.80–4.90 (m, 2H), 6.80 (s, 1H),
7.65 (s, 1H). MS (FAB) 431 [MþH]þÅ.
3.19. (2S)-N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-4-
oxo-pyrrolidine-2-carboxaldeyde diethyl thioacetal (20a)
A solution of 19a (506 mg, 1 mmol) in dichloromethane
(15 mL) was treated with acetonitrile (2 mL), molecular
ꢀ
sieves (4 A, 500 mg) and NMO (176 mg, 1.5 mmol).
After the mixture was stirred for 15 min at room tem-
perature, TPAP (18 mg, 0.05 mmol) was added to the
reaction mixture and it was stirred for further 2.5 h at
room temperature until TLC (50% EtOAc–hexane)
indicated complete consumption of starting material.
Concentration of the reaction mixture in vacuum fol-
lowed by column chromatography (40% EtOAc–hex-
3.16. (2S,4R)-N-[4,5-Dimethoxy-2-nitrobenzoyl]-4-
(t-butyldimethylsilyl)oxypyrrolidine-2-carboxaldehyde
(18b)
1
The compound 18b was prepared according to the
method described for the compound 18a employing
compound 17b (468 mg, 1 mmol) to afford compound 18b
(298 mg, 68%). 1H NMR (CDCl3): d 0.18 (s, 6H), 0.85 (s,
9H) 2.10–2.20 (m, 1H), 2.25–2.40 (m, 1H), 3.0–3.10 (m,
1H), 3.40–3.50 (m, 1H), 3.98 (s, 3H), 4.0 (s, 3H), 4.40–
4.50 (m, 1H), 4.70–4.80 (m, 1H), 6.80 (s, 1H), 7.65 (s, 1H),
9.78–9.80 (d, 1H, J ¼ 2:2 Hz). MS (FAB) 439 [MþH]þÅ.
ane) gave the pure ketone 20a (393 mg, 78%). H NMR
(CDCl3): d 1.20–1.40 (m, 6H), 2.600–2.90 (m, 6H), 3.55
(d, 1H, J ¼ 17:0 Hz), 3.78–3.95 (d, 1H, J ¼ 17:0 Hz), 4.0
(s, 3H), 4.60 (d, 1H, J ¼ 4:26 Hz), 5.15–5.25 (m, 3H),
6.75 (s, 1H), 7.30–7.50 (m, 5H), 7.75 (s, 1H). MS (FAB)
505 [MþH]þÅ.
3.20. (2S)-N-[4,5-Dimethoxy-2-nitrobenzoyl]-4-oxo-
pyrrolidine-2-carboxaldeyde diethyl thioacetal (20b)
3.17. (2S,4R)-N-[4-Benzyloxy-5-methoxy-2-nitrobenz-
oyl]-4-hydroxypyrrolidine-2-carboxaldehyde diethyl thio-
acetal (19a)
The compound 20b was prepared according to the
method described for the compound 20a employing
compound 19b (430 mg, 1 mmol) to give crude com-
pound, which was purified by column chromatography
(50% EtOAc–hexane) to afford compound 20b (325 mg,
Ethanethiol (278 mg, 4.4 mmol) was added to a stirred
solution of nitroaldehyde 18a (514 mg, 1 mmol) in dry
chloroform (15 mL) under nitrogen atmosphere. The
mixture was stirred for a further 30 min followed by the
addition of trimethylsilyl chloride (540 mg, 5 mmol).
After a further 18 h of stirring of the mixture, when TLC
indicated that reaction was completed, the reaction
mixture was carefully neutralized with sodium bicar-
bonate solution and extracted with chloroform
(2 ꢂ 10 mL). The combined organic phase was dried
over Na2SO4 and evaporated in vacuum to give the
crude diethyl thioacetal, which was purified by column
chromatography (60% EtOAc–hexane) to afford com-
1
76%). H NMR (CDCl3): d 1.20–1.40 (m, 6H), 2.70–3.0
(m, 6H), 3.55 (d, 1H, J ¼ 15:0 Hz), 3.85 (d, 1H,
J ¼ 15:0 Hz), 3.95 (s, 3H), 4.0 (s, 3H), 4.65 (d, 1H,
J ¼ 4:40 Hz), 5.15–5.25 (m, 1H), 6.75 (s, 1H), 7.75 (s,
1H). MS (FAB) 429 [MþH]þÅ.
3.21. (2S)-N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-4-
(methoxycarbonylmethylidene)-pyrrolidine-2-carboxalde-
hyde diethyl thioacetal (21a)
1
pound 19a (364 mg, 72%). H NMR (CDCl3): d 1.20–
1.40 (m, 6H), 2.20–2.35 (m, 2H), 2.62–2.90 (m, 4H),
Dry THF (10 mL) was added to the sodium hydride
(80 mg of 60% dispersion in oil, 2 mmol) under a nitrogen

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
4345
atmosphere and the mixture was cooled to 0 °C. The cool
solution was treated drop wise with a solution of methyl
diethylphosphonoacetate (420 mg, 2 mmol) in THF
(5 mL) under nitrogen and allowed to stir for 1 h at room
temperature. The reaction mixture was cooled 0 °C and
treated drop wise with a solution of the ketone 20a
(504 mg, 1 mmol) in THF (2 ꢂ 5 mL) under nitrogen. The
reaction mixture was stirred for a further 2 h until TLC
(50% EtOAc–hexane) indicated complete consumption
of starting material. The THF was evaporated in vacuum
and the mixture was washed with sodium bicarbonate
solution and then extracted with ethyl acetate
(2 ꢂ 20 mL). The combined organic phases were washed
with saturated brine and dried over Na2SO4. The solvent
was removed under vacuum and purified by column
chromatography (30% EtOAc–hexane) to afford com-
compound 21b (484 mg, 1 mmol) to afford compound
1
22b (409 mg, 87%). H NMR (CDCl3): d 1.25–1.45 (m,
6H), 2.65–2.90 (m, 4H), 3.10–3.25 (m, 2H), 3.90–5.10
(m, 8H), 4.78 (d, 1H, J ¼ 4:28 Hz), 5.35–5.40 (m, 1H),
5.92 (s, 1H), 6.80 (s, 1H), 7.68 (s, 1H). MS (FAB) 471
[MþH]þÅ.
3.25. (2S)-N-{4-[(2S)-N-(4-Benzyloxy-5-methoxy-2-
nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thio-
acetal-4-(carboxamidomethylidene)-N-ethyl]oxy-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal (23a)
To a solution of 22a (544 mg, 1 mmol) in dichlorome-
thane–water (20:20 mL), were added EDCI (384 mg,
2 mmol), HOBt (270 mg, 2 mmol) and compound 14a
(443 mg, 1 mmol) and the mixture was stirred for 24 h at
room temperature. The dichloromethane was evapo-
rated in vacuum, the residue was dissolved in ethyl
acetate and washed with saturated sodium bicarbonate
solution and then with brine solution. The combined
organic phases were dried over Na2SO4, the solvent was
removed under vacuum and purified by column chro-
matography (80% EtOAc–hexane) to afford compound
1
pound 21a (448 mg, 80%). H NMR (CDCl3): d 1.25–
1.45 (m, 6H), 2.65–2.90 (m, 4H), 3.05–3.30 (m, 2H), 3.65
(s, 3H), 3.95 (s, 3H), 4.20–4.30 (m, 1H), 4.45–4.60 (m,
1H), 4.80 (d, 1H, J ¼ 4:2 Hz), 4.90–5.0 (m, 1H), 5.20 (s,
2H), 5.35–5.40 (m, 1H), 5.90 (s, 1H), 6.80 (s, 1H), 7.30–
7.50 (m, 5H), 7.75 (s, 1H). MS (FAB) 561 [MþH]þÅ.
3.22. (2S)-N-[4,5-Dimethoxy-2-nitrobenzoyl]-4-
(methoxycarbonylmethylidene)pyrrolidine-2-carbox-
aldehyde diethyl thioacetal (21b)
1
23a (524 mg, 54%). H NMR (CDCl3): d 1.20–1.45 (m,
12H), 1.75–2.35 (m, 4H), 2.60–2.90 (m, 8H), 3.0–3.30
(m, 4H), 3.55–3.65 (m, 2H), 3.90–4.10 (m, 8H), 4.15 (t,
2H, J ¼ 5:98 Hz), 4.63–4.72 (m, 1H), 4.80 (m, 2H), 5.20
(s, 2H), 5.30–5.35 (m, 1H), 5.90 (s, 1H), 6.50–6.55 (m,
1H), 6.75 (s, 1H), 6.82 (s, 1H), 7.30–7.50 (m, 5H), 7.65
(s, 1H), 7.75 (s, 1H). MS (FAB) 971 [MþH]þÅ.
The compound 21b was prepared according to the
method described for the compound 21a employing
compound 20b (428 mg, 1 mmol) to give crude product,
which was purified by column chromatography (40%
EtOAc–hexane) to afford compound 21b (402 mg, 83%).
1H NMR (CDCl3): d 1.25–1.45 (m, 6H), 2.65–2.90 (m,
4H), 3.05–3.30 (m, 2H), 3.60–3.75 (m, 3H), 3.96 (s, 3H),
3.98 (s, 3H), 4.20–4.30 (m, 1H), 4.45–4.60 (m, 1H), 4.80
(d, 1H, J ¼ 4:2 Hz), 5.35–5.40 (m, 1H), 5.90 (s, 1H), 6.80
(s, 1H), 7.68 (s, 1H). MS (FAB) 485 [MþH]þÅ.
3.26. (2S)-N-{4-[(2S)-N-(4-Benzyloxy-5-methoxy-2-
nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thio-
acetal-4-(carboxamidomethylidene)-N-propyl]oxy-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal (23b)
3.23. (2S)-N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-4-
(carboxymethylidene)pyrrolidine-2-carboxaldehyde
diethyl thioacetal (22a)
The compound 23b was prepared according to the
method described for the compound 23a by employing
22a and 14b (457 mg, 1 mmol) to afford crude com-
pound, which was purified by column chromatography
(85% EtOAC–hexane) to give compound 23b (562 mg,
Lithium hydroxide monohydrate (2 N, 1.22 mL) was
added to a solution of 21a (560 mg, 1 mmol) in THF–
H2O–MeOH (4:1:1) and the mixture stirred at room
temperature for 2 h. After most of the THF and meth-
anol were evaporated, the aqueous phase was acidified
with 12 N HCl to pH 7 and extracted with EtOAc to give
1
57%). H NMR (CDCl3): d 1.20–1.45 (m, 12H), 1.75–
2.35 (m, 6H), 2.65–2.90 (m, 8H), 2.95–3.30 (m, 4H),
3.40–3.50 (m, 2H), 3.80–4.05 (m, 8H), 4.15 (t, 2H,
J ¼ 5:80 Hz), 4.60–4.70 (m, 1H), 4.75–4.82 (m, 2H), 5.20
(s, 2H), 5.35–5.40 (m, 1H), 5.92 (s, 1H), 6.55 (m, 1H),
6.75 (s, 1H), 6.80 (s, 1H), 7.30–7.50 (m, 5H), 7.60 (s,
1H), 7.70 (s, 1H). MS (FAB) 986 [MþH]þÅ.
1
22a (452 mg, 83%). H NMR (CDCl3): d 1.25–1.45 (m,
6H), 2.65–2.90 (m, 4H), 3.10–3.25 (m, 2H), 3.90–4.10
(m, 5H), 4.78 (d, 1H J ¼ 4:28 Hz), 5.20 (s, 2H), 5.35–
5.40 (m, 1H), 5.95 (s, 1H), 6.80 (s, 1H), 7.35–7.55 (m,
5H), 7.75 (s, 1H). MS (FAB) 545 [MþH]þÅ.
3.27. (2S)-N-{4-[(2S)-N-(4,5-Dimethoxy-2-nitrobenzoyl)-
pyrrolidine-2-carboxaldehyde diethyl thioacetal-4-(car-
boxamidomethylidene)-N-ethyl]oxy-5-methoxy-2-nitro-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(24a)
3.24. (2S)-N-[4,5-Dimethoxy-2-nitrobenzoyl]-4-
(carboxymethylidene)pyrrolidine-2-carboxaldehyde
diethyl thioacetal (22b)
The compound 24a was prepared according to the
method described for the compound 23a employing
compound 22b (470 mg, 1 mmol) and 14a (443 mg,
The compound 22b was prepared according to the
method described for the compound 22a employing

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A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
1 mmol) to afford crude product, which was purified by
column chromatography (90% EtOAc–hexane) to give
compound 24a (501 mg, 56%). 1H NMR (CDCl3): d
1.20–1.45 (m, 12H), 1.75–2.35 (m, 4H), 2.60–2.90 (m,
8H), 2.95–3.30 (m, 4H), 3.55–3.70 (m, 2H), 3.90–4.10
(m, 11H), 4.05–4.15 (m, 2H), 4.60–4.70 (m, 1H), 4.75–
4.85 (m, 2H), 5.30–5.40 (m, 1H), 5.90 (s, 1H), 6.50 (br s,
1H), 6.75 (s, 1H), 6.82 (s, 1H) 7.60 (s, 1H), 7.65 (s, 1H).
MS (FAB) 896 [MþH]þÅ.
compound 23b (984 mg, 1 mmol), to afford the amino
diethyl thioacetal 25b (693 mg, 75%). 1H NMR (CDCl3):
d 1.15–1.45 (m, 12H), 1.60–2.35 (m, 6H), 2.55–2.85 (m,
8H), 2.90–3.20 (m, 2H), 3.35–3.65 (m, 6H), 3.75 (s, 3H),
3.77 (s, 3H), 4.05 (t, 2H, J ¼ 6:0 Hz), 4.52–4.70 (m, 3H),
5.10 (s, 2H), 5.35–5.45 (m, 1H), 5.85 (s, 1H), 6.15–6.25
(m, 2H), 6.60–6.80 (m, 3H) 7.30–7.50 (m, 5H).
3.31. (2S)-N-{4-[(2S)-N-(4,5-dimethoxy-2-aminobenzoyl)-
pyrrolidine-2-carboxaldehyde diethyl thioacetal-4-(carb-
oxamidomethylidene)-N-ethyl]oxy-5-methoxy-2-amino-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(26a)
3.28. (2S)-N-{4-[(2S)-N-(4,5-Dimethoxy-2-nitrobenzoyl)-
pyrrolidine-2-carboxaldehyde diethyl thioacetal-4-(car-
boxamidomethylidene)-N-propyl]oxy-5-methoxy-2-nitro-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(24b)
The compound 26a was prepared according to the
method described for the compound 25a employing
compound 24a (895 mg, 1 mmol) to afford compound
The compound 24b was prepared according to the
method described for the compound 24a employing
compound 22b (470 mg, 1 mmol) and 14b (457 mg,
1 mmol) to afford crude product, which was purified by
column chromatography (95% EtOAc–hexane) to give
compound 24b (545 mg, 60%). 1H NMR (CDCl3): d
1.20–1.45 (m, 12H), 1.72–2.35 (m, 6H), 2.65–2.90 (m,
8H), 2.95–3.30 (m, 4H), 3.55–3.70 (m, 2H), 3.90–4.05
(m, 11H), 4.02–4.15 (m, 2H), 4.60–4.70 (m, 1H), 4.75–
4.82 (m, 2H), 5.30–5.40 (m, 1H), 5.92 (s, 1H), 6.55 (br s,
1H), 6.75 (s, 1H), 6.80 (s, 1H) 7.60 (s, 1H), 7.68 (s, 1H).
MS (FAB) 910 [MþH]þÅ.
1
26a (593 mg, 71%). H NMR (CDCl3): d 1.20–1.45 (m,
12H), 1.70–2.40 (m, 4H), 2.60–2.90 (m, 8H), 2.95–3.25
(m, 2H), 3.45–3.70 (m, 4H), 3.80 (s, 6H), 3.85 (s, 3H),
4.0–4.20 (m, 3H), 4.55–4.75 (m, 4H), 5.40–5.50 (m, 1H),
5.90 (s, 1H), 6.18 (s, 1H), 6.22 (s, 1H), 6.70–6.80 (m, 3H).
3.32. (2S)-N-{4-[(2S)-N-(4,5-Dimethoxy-2-aminobenzoyl)-
pyrrolidine-2-carboxaldehyde diethyl thioacetal-4-(carb-
oxamidomethylidene)-N-propyl]oxy-5-methoxy-2-amino-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(26b)
3.29. (2S)-N-{4-[(2S)-N-(4-Benzyloxy-5-methoxy-2-
aminobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thio-
acetal-4-(carboxamidomethylidene)-N-ethyl]oxy-5-meth-
oxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal (25a)
The compound 26b was prepared according to the
method described for the compound 25a employing
compound 24b (909 mg, 1 mmol) to afford compound
1
26b (620 mg, 73%). H NMR (CDCl3): d 1.20–1.45 (m,
12H), 1.70–2.45 (m, 6H), 2.60–2.90 (m, 8H), 2.95–3.25
(m, 2H), 3.45–3.70 (m, 4H), 3.80 (s, 6H), 3.85 (s, 3H),
4.0–4.20 (m, 3H), 4.55–4.75 (m, 4H), 5.40–5.50 (m, 1H),
5.90 (s, 1H), 6.18 (s, 1H), 6.22 (s, 1H), 6.70–6.80 (m, 3H).
The compound 25a (970 mg, 1 mmol) was dissolved in
methanol (20 mL) and added SnCl2Æ2H2O (2.256 g,
10 mmol) was refluxed for 3 h or until the TLC indicated
that reaction was complete. The methanol was evapo-
rated under vacuum and the aqueous layer was then
carefully adjusted to pH 8 with 10% NaHCO3 solution
and then extracted with ethyl acetate (2 ꢂ 30 mL). The
combined organic phase was dried over Na2SO4 and
evaporated under vacuum to afford the amino diethyl
thioacetal, 25a, which due to potential stability problems,
3.33. 7-Methoxy-8-{2-[8-benzyloxy-7-methoxy-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo-[2,1-c][1,4]benzodiaze-
pine-5-one-2-(carboxamidomethylidene)ethyl]oxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-
5-one (6a)
1
was briefly characterized by H NMR and then used di-
A solution of 25a (910 mg, 1 mmol), HgCl2 (1.226 g,
4.52 mmol) and CaCO3 (492 mg, 4.92 mmol) in aceto-
nitrile–water (4:1) was stirred slowly at room tempera-
ture until TLC indicates complete loss of starting
material. The reaction mixture was diluted with EtOAc
(30 mL) and filtered through a Celite bed. The clear
yellow organic supernatant was washed with saturated
5% NaHCO3 (20 mL), brine (20 mL) and the combined
organic phase is dried over Na2SO4. The organic solvent
was evaporated in vacuum and purified by column
chromatography (92% EtOAc–MeOH) to give com-
pound 6a (318 mg, 48%). This compound was repeatedly
evaporated from chloroform in vacuum to generate the
rectly in the next step (655 mg, 72%). 1H NMR (CDCl3):
d 1.15–1.45 (m, 12H), 1.70–2.35 (m, 4H), 2.55–2.85 (m,
8H), 2.95–3.25 (m, 4H), 3.45–3.85 (m, 10H), 3.98–4.05 (t,
2H, J ¼ 6:0 Hz), 4.45–4.50 (m, 1H), 4.62–4.70 (m, 2H),
5.10 (s, 2H), 5.25–5.35 (m, 1H), 5.85 (s, 1H), 6.15–6.25
(m, 2H), 6.65-6.85 (m, 3H), 7.35–7.50 (m, 5H).
3.30. (2S)-N-{4-[(2S)-N-(4-Benzyloxy-5-methoxy-2-
aminobenzoyl)pyrrolidine-2-carboxaldehyde diethyl
thioacetal-4-(carboxamidomethylidene)-N-propyl]oxy-5-
methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal (25b)
25
1
imine form. ½aꢃ þ290.333 (c 0.5, CH2Cl2). H NMR
D
The compound 25b was prepared according to the
method described for the compound 25a employing the
(CDCl3): d 2.03–2.16 (m, 3H), 2.25–2.35 (m, 3H), 3.15–
3.82 (m, 6H), 3.85 (s, 3H), 3.90 (s, 3H), 4.20 (t, 2H,

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
4347
J ¼ 4:8 Hz), 4.40–4.50 (m, 2H), 5.20 (s, 2H), 5.88 (s,
1H), 6.60–6.65 (m, 1H), 6.80 (s, 1H), 6.85 (s, 1H), 7.25–
7.75 (m, 7H), 7.65 (d, 2H, J ¼ 4:46 Hz). MS (FAB) 664
[MþH]þÅ. Anal. Calcd for (C37H37N5O7): C, 66.96, H,
5.62, N, 10.55. Found: C, 66.85, H, 5.69, N, 10.32.
3.85 (m, 6H), 3.90–4.0 (m, 9H), 4.05–4.30 (m, 3H), 4.35–
4.55 (m, 2H), 5.90 (s, 1H), 6.80 (s, 1H), 6.83 (s, 1H),
6.91–6.93 (m, 1H), 7.50 (s, 1H), 7.54 (s, 1H), 7.66 (d, 1H,
J ¼ 4:30 Hz), 7.70 (d, 1H, J ¼ 3:80 Hz). MS (FAB) 602
[MþH]þÅ. Anal. Calcd for (C32H35N5O7): C, 63.88, H,
5.86, N, 11.64. Found: C, 63.92, H, 5.94, N, 11.56.
3.34. 7-Methoxy-8-{3-[8-benzyloxy-7-methoxy-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo-[2,1-c][1,4]benzodiaze-
pine-5-one-2-(carboxamidomethylidene)propyl]oxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]ben-
zodiazepin-5-one (6b)
3.37. Methyl-(2S)-N-[4-(N-t-butoxycarbonyl)aminoeth-
oxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxylate
(28a)
The compound 28a was prepared according to the
method described for the compound 11a employing
compound 27 (324 mg, 1 mmol) and Boc-protected
bromo ethyl amine (269 mg, 1.2 mmol) to give crude
product, which was purified by column chromatography
(60% EtOAc–hexane) to afford compound 28a (373 mg,
The compound 6b was prepared according to the
method described for the compound 6a employing 25b
(924 mg, 1 mmol) to give crude product, which was
purified by column chromatography (90% EtOAc–
25
MeOH) to afford compound 6b (338 mg, 52%). ½aꢃ
1
D
80%). H NMR (CDCl3): d 1.40 (m, 9H), 1.85–2.15 (m,
1
þ454.0 (c 0.5, CH2Cl2). H NMR (CDCl3): d 2.0–2.20
4H), 2.20–2.40 (m, 1H), 3.15–3.22 (m, 1H), 3.23–3.38 (m,
1H), 3.60 (t, 2H, J ¼ 6:20 Hz), 3.80 (s, 3H), 4.0 (s, 3H),
4.15 (t, 2H, J ¼ 5:98 Hz), 4.65–4.78 (m, 1H), 5.0 (br s,
1H), 6.82 (s, 1H), 7.62 (s, 1H). MS (FAB) 468 [MþH]þÅ.
(m, 4H), 2.25–2.40 (m, 2H), 3.20 (d, 1H, J ¼ 4:8 Hz),
3.50–3.90 (m, 7H), 3.95 (s, 6H), 4.10–4.30 (m, 2H), 4.40–
4.50 (m, 2H), 5.20 (s, 2H), 5.89 (s, 1H), 6.80 (s, 1H), 6.85
(s, 1H), 6.88–6.92 (m, 1H), 7.30–7.50 (m, 5H), 7.52 (s,
1H), 7.55 (s, 1H), 7.66 (d, 2H, J ¼ 4:46 Hz). MS (FAB)
678 [MþH]þÅ. Anal. Calcd for (C38H39N5O7): C, 67.34,
H, 5.80, N, 10.33. Found: C, 67.42, H, 5.77, N, 10.21.
3.38. Methyl-(2S)-N-[4-(N-t-butoxycarbonyl)aminoprop-
oxy-5-methoxy-2-nitrobenzoyl]-pyrrolidine-2-carboxylate
(28b)
The compound 28b was prepared according to the
method described for the compound 11a employing
compound 27 (324 mg, 1 mmol) and Boc-protected
bromo propyl amine (286 mg, 1.2 mmol) to give crude
product, which was purified by column chromatography
(65% EtOAc–hexane) to afford compound 28b (395 mg,
3.35. 7-Methoxy-8-{2-[7,8-dimethoxy-(11aS)-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepine-5-one-
2-(carboxamidomethylidene)-ethyl]oxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiaze-
pin-5-one (7a)
1
82%). H NMR (CDCl3): d 1.40 (m, 9H), 1.85–2.18 (m,
The compound 7a was prepared according to the
method described for the compound 6a employing 26a
(835 mg, 1 mmol) to give crude product, which was
5H), 2.20–2.40 (m, 1H), 3.15–3.40 (m, 4H), 3.80 (s, 3H),
3.95 (s, 3H), 4.16 (t, 2H, J ¼ 5:96 Hz), 4.65–4.72 (m,
1H), 5.22 (br s, 1H), 6.82 (s, 1H), 7.62 (s, 1H). MS
(FAB) 482 [MþH]þÅ.
purified by column chromatography (87% EtOAc–
25
MeOH) to afford compound 7a (294 mg, 50%). ½aꢃ
D
1
þ430.417 (c 0.4, CHCl3). H NMR (CDCl3): d 2.0–2.15
(m, 2H), 2.25–2.40 (m, 2H), 3.20–3.25 (m, 1H), 3.45–
3.60 (m, 2H), 3.65–3.85 (s, 4H), 3.90–4.0 (m, 9H), 4.05–
4.20 (m, 3H), 4.40–4.55 (m, 2H), 5.90 (s, 1H), 6.40–6.55
(m, 1H), 6.80 (s, 1H), 6.83 (s, 1H), 7.50 (s, 1H), 7.53 (s,
1H), 7.66 (d, 1H, J ¼ 4:46 Hz), 7.70 (d, 1H,
J ¼ 4:46 Hz). MS (FAB) 588 [MþH]þÅ. Anal. Calcd for
(C31H33N5O7): C, 63.36, H, 5.66, N, 11.92. Found: C,
63.45, H, 5.74, N, 11.83.
3.39. Methyl-(2S)-N-[4-(2-aminoethoxy)-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxylate (29a)
The compound 29a was prepared according to the
method described for the compound 14a by employing
28a (467 mg, 1 mmol) and TFA.
3.40. Methyl-(2S)-N-[4-(3-aminopropoxy)-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxylate (29b)
3.36. 7-Methoxy-8-{3-[7,8-dimethoxy-(11aS)-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepine-5-one-
2-(carboxamidomethylidene)-propyl]oxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiaze-
pin-5-one (7b)
The compound 29b was prepared according to the
method described for the compound 14a by employing
28b (481 mg, 1 mmol) and TFA.
The compound 7b was prepared according to the
method described for the compound 6a employing 26b
(849 mg, 1 mmol) to give crude product, which was
3.41. Methyl-(2S)-N-{4-[(2S)-N-(4-benzyloxy-5-methoxy-
2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thio-
acetal-4-(carboxamidomethylidene)-N-ethyl]oxy-5-meth-
oxy-2-nitrobenzoyl}pyrrolidine-2-carboxylate (30a)
purified by column chromatography (85% EtOAc–
25
MeOH) to afford compound 7b (331 mg, 55%). ½aꢃ
D
þ480.333 (c 0.4, CH2Cl2). 1H NMR (CDCl3): d 2.0–2.15
The compound 30a was prepared according to the
method described for the compound 23a by employing
(m, 4H), 2.25–2.40 (m, 2H), 3.10–3.25 (m, 1H), 3.50–

4348
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
22a and 29a (367 mg, 1 mmol) to give crude product,
which was purified by column chromatography (90%
EtOAc–hexane) to afford compound 30a (582 mg, 65%).
1H NMR (CDCl3): d 1.22–1.42 (m, 6H), 1.55–2.45 (m,
6H), 2.65–2.90 (m, 4H), 3.0–3.40 (m, 4H), 3.60 (t, 2H,
J ¼ 6:2 Hz), 3.80 (s, 3H), 4.0 (s, 6H), 4.15 (t, 2H, J ¼
5:98 Hz), 4.65–4.75 (m, 1H), 4.80 (d, 1H, J ¼ 4:4 Hz),
5.20 (s, 2H), 5.35–5.40 (m, 1H), 5.92 (s, 1H), 6.55–6.60
(m, 1H), 6.75 (s, 1H), 6.85 (s, 1H), 7.30–7.50 (m, 5H),
7.60 (s, 1H), 7.75 (s, 1H). MS (FAB) 896 [MþH]þÅ.
3.85 (s, 3H), 3.90–4.0 (m, 3H), 4.45–4.60 (m, 2H), 5.0 (s,
2H), 5.30–5.40 (m, 1H), 5.82 (s, 1H), 6.20 (s, 1H), 6.45
(s, 1H), 6.55–6.65 (m, 1H), 6.80 (s, 1H), 7.25–7.40 (m,
6H), 8.60 (br s, NH exchangeable).
3.45. 7-Methoxy-8-{2-[8-benzyloxy-7-methoxy-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo-[2,1-c][1,4]benzodiaze-
pine-5-one-2-(carboxamidomethylidene)ethyl]oxy}-(11aS)-
1,2,3,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-
diazepin-5,11-dione (8a)
The compound 8a was prepared according to the
method described for the compound 6a employing 31a
(803 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and
CaCO3 (246 mg, 2.46 mmol) to give crude product,
which was purified by column chromatography (87%
EtOAc–MeOH) to afford compound 8a (346 mg, 51%).
3.42. Methyl-(2S)-N-{4-[(2S)-N-(4-benzyloxy-5-methoxy-
2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thio-
acetal-4-(carboxamidomethylidene)-N-propyl]oxy-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxylate (30b)
The compound 30b was prepared according to the
method described for the compound 23a by employing
22a and 29b (381 mg, 1 mmol) to give crude product,
which was purified by column chromatography (87%
EtOAc–MeOH) to afford compound 30b (609 mg, 67%).
1H NMR (CDCl3): d 1.22–1.42 (m, 6H), 1.85–2.20 (m,
5H), 2.35–2.40 (m, 1H), 2.65–2.85 (m, 4H), 2.90–3.40
(m, 5H), 3.50–3.60 (m, 2H), 3.80 (s, 3H), 4.0 (s, 6H),
4.05–4.20 (m, 3H), 4.65–4.75 (m, 1H), 4.85 (d, 1H,
J ¼ 4:38 Hz), 5.20 (s, 2H), 5.30–5.40 (m, 1H), 5.92 (s,
1H), 6.55–6.60 (m, 1H), 6.75 (s, 1H), 6.85 (s, 1H), 7.30–
7.50 (m, 5H), 7.60 (s, 1H), 7.75 (s, 1H). MS (FAB) 910
[MþH]þÅ.
25
1
½aꢃ þ352.667 (c 0.5, CH2Cl2). H NMR (CDCl3): d
D
1.90–2.20 (m, 4H), 2.75–3.80 (m, 8H), 3.85 (s, 3H), 3.95
(s, 3H), 4.0 (d, 2H, J ¼ 4:8 Hz), 4.35–4.55 (m, 2H), 5.20
(s, 2H), 5.90 (s, 1H), 6.55 (s, 1H), 6.80 (s, 1H), 6.95–7.0
(m, 1H), 7.20–7.50 (m, 6H),7.55 (s, 1H), 7.66 (d, 1H,
J ¼ 4:40 Hz), 8.7 (br s, NH exchangeable). MS (FAB)
680 [MþH]þÅ. Anal. Calcd for (C37H37N5O8): C, 65.38,
H, 5.49, N, 10.30. Found: C, 65.23, H, 5.53, N, 10.35.
3.46. 7-Methoxy-8-{3-[8-benzyloxy-7-methoxy-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo-[2,1-c][1,4]benzodiaze-
pine-5-one-2-(carboxamidomethylidene)propyl]oxy}-
(11aS)-1,2,3,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-
benzodiazepin-5,11-dione (8b)
3.43. 7-Methoxy-8-{2-[(2S)-N-(4-benzyloxy-5-methoxy-
2-aminobenzoyl)pyrrolidine-2-carboxaldehyde diethyl
thioacetal-4-(carboxamidomethylidene)-ethyl]oxy}-
(11aS)-1,2,3,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-
benzodiazepin-5,11-dione (31a)
The compound 8b was prepared according to the
method described for the compound 8a by employing
31b (817 mg, 1 mmol) to give crude product, which was
purified by column chromatography (87% EtOAc–
25
D
MeOH) to afford compound 8b (368 mg, 53%). ½aꢃ
The compound 31a was prepared according to the
method described for the compound 25a employing the
compound 30a (895 mg, 1 mmol) to afford the amino
diethyl thioacetal 31a (610 mg, 76%). 1H NMR (CDCl3):
d 1.15–1.40 (m, 6H), 1.60–2.10 (m, 6H), 2.50–2.80 (m,
4H), 2.95–3.20 (m, 2H), 3.35–3.60 (m, 4H), 3.75 (s, 3H),
3.82 (s, 3H), 4.0 (t, 2H, J ¼ 5:98 Hz), 4.50–4.60 (m, 2H),
5.0 (s, 2H), 5.35–5.40 (m, 1H), 5.82 (s, 1H), 6.20 (s, 1H),
6.45–6.55 (m, 1H), 6.80 (s, 1H), 6.90 (s, 1H), 7.25–7.42
(m, 6H), 9.10 (br s, NH exchangeable).
þ410.256 (c 0.325, CH2Cl2). 1H NMR (CDCl3): d 1.90–
2.20 (m, 6H), 2.75–3.80 (m, 8H), 3.85 (s, 3H), 3.95 (s,
3H), 4.0 (d, 2H, J ¼ 4:6 Hz), 4.35–4.50 (m, 2H), 5.20 (s,
2H), 5.90 (s, 1H), 6.60 (s, 1H), 6.65–6.70 (m, 1H), 6.85
(s, 1H), 7.20–7.50 (m, 6H), 7.55 (s, 1H), 7.65 (d, 1H,
J ¼ 4:40 Hz), 8.82 (br s, NH exchangeable). MS (FAB)
694 [MþH]þÅ. Anal. Calcd for (C38H39N5O8): C, 65.79,
H, 5.67, N, 10.09. Found: C, 65.84, H, 5.60, N, 10.03.
3.47. Thermal denaturation studies
3.44. 7-Methoxy-8-{3-[(2S)-N-(4-benzyloxy-5-methoxy-
2-aminobenzoyl)pyrrolidine-2-carboxaldehyde diethyl
thioacetal-4-(carboxamidomethylidene)propyl]oxy}-
(11aS)-1,2,3,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-
benzodiazepin-5,11-dione (31b)
Compounds were subjected to thermal denaturation
studies with duplex-form calf thymus DNA (CT-DNA)
using an adaptation of a reported procedure.19 Working
solutions in aqueous buffer (10 mM NaH2PO4/
Na2HPO4, 1 mM Na2EDTA, pH 7.00þ0.01) containing
CT-DNA (100 lm in phosphate) and the PBD (20 lm)
were prepared by addition of concentrated PBD solu-
tions in MeOH to obtain a fixed [PBD]/[DNA] molar
ratio of 1:5. The DNA–PBD solutions were incubated at
37 °C for 0, 18, 36 and 48 h prior to analysis. Samples
were monitored at 260 nm using a Beckman DU-7400
spectrophotometer fitted with high performance tem-
The compound 31b was prepared according to the
method described for the compound 25a employing the
compound 30b (895 mg, 1 mmol) to afford the amino
diethyl thioacetal 31b (629 mg, 78%). 1H NMR (CDCl3):
d 1.15–1.40 (m, 6H), 1.85–2.10 (m, 6H), 2.50–2.80 (m,
4H), 2.95–3.20 (m, 3H), 3.35–3.60 (m, 4H), 3.75 (s, 3H),

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 4337–4350
4349
Molecular Aspects of Anticancer Drug–DNA Interactions;
Neidle, S., Waring, M. J., Eds.; Macmillan: UK, 1993;
Vol. 1, p 54; (c) Kamal, A.; Rao, M. V.; Laxman, N.;
Ramesh, G.; Reddy, G. S. K. Curr. Med. Chem. Anti-
Cancer Agents 2002, 2, 215.
perature controller, and heating was applied at
1 °C minꢀ1 in the 40–90 °C range. DNA helix coil tran-
sition temperatures (Tm) were obtained from the maxima
in the (dA260Þ=dT derivative plots. Results are given as
the mean ꢁ standard deviation from three determina-
tions and are corrected for the effects of MeOH co-sol-
vent using a linear correction term.20 Drug-induced
alterations in DNA melting behaviour are given by:
DTm ¼ TmðDNA þ PBDÞ ꢀ Tm (DNA alone), where the
Tm value for the PBD-free CT-DNA is 69.0 ꢁ 0.01. The
fixed [PBD]/[DNA] ratio used did not result in binding
saturation of the host DNA duplex for any compound
examined.
2. (a) Woynarowski, J. M.; Napier, C.; Trevino, A. V.;
Arnett, B. Biochemistry 2000, 39, 9917; (b) Woynarowski,
J. M.; Trevino, A. V.; Rodriguez, K. A.; Hardies, S. C.;
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3.48. In vitro evaluation of cytotoxic activity
In routine screening, compounds have been tested
against 60 human tumour cell lines derived from nine
cancer types (leukaemia, nonsmall cell lung cancer,
colon cancer, CNS cancer, melanoma, ovarian cancer,
renal cancer, prostate cancer and breast cancer). For
each compound, dose response curves for each cell line
were measured at a minimum of five concentrations at
10-fold dilutions. A protocol of 48 h continuous drug
exposure was used, and a sulforhodamine B (SRB)
protein assay was used to estimate cell viability or
growth. The concentration causing 50% cell growth
inhibition (GI50), total cell growth inhibition (TGI, 0%
growth) and 50% cell death (LC50, ꢀ50% growth)
compared with the control was calculated.
6. Gregson, S. J.; Howard, P. W.; Corcoran, K. E.; Barcella,
S.; Yasin, M. M.; Hurst, A. A.; Jenkins, T. C.; Kelland, L.
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3.49. Restriction endonuclease inhibition
7. Gregson, S. J.; Howard, P. W.; Gullick, D. R.; Hamag-
uchi, A.; Corcoran, K. E.; Brooks, N. A.; Hartley, J. A.;
Jenkins, T. C.; Patel, S.; Guille, M. J.; Thurston, D. E.
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8. Gregson, S. J.; Howard, P. W.; Thurston, D. E. Bioorg.
Med. Chem. Lett. 2003, 13, 2277.
9. Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G. Bioorg.
Med. Chem. Lett. 2003, 13, 3955.
10. Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N.
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11. (a) Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.;
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Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G.
Bioorg. Med. Chem. Lett. 2002, 12, 1933; (c) Kamal, A.;
Ramesh, G.; Ramulu, P.; Srinivas, O.; Rehana, T.; Sheelu,
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Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med.
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Stock solutions of each PBD (100 lM) were prepared by
dissolving each compound in DMSO (Sigma). These
were stored at ꢀ20 °C. Plasmid (pBR322) containing
single BamH1 site was used in this assay. Restriction
endonuclease and the relevant buffer were obtained from
NEB. The DNA fragment (500 ng) was incubated with
each PBD (see Fig. 2 for PBD concentrations) in a final
volume of 16 lL for 16 h at 37 °C. Next 10 ꢂ BamH1
buffer (2 lL) was added, and the reaction mixture was
made up to 20 lL with BamH1 (20 units) and then
incubated for 1 h at 37 °C. Then loaded on to a 1%
agarose gel electrophoresis in Tris–acetate EDTA buffer
at 80 V for 2 h. The gels were stained with ethidium
bromide and photographed.
Acknowledgements
We thank the National Cancer Institute, Maryland for
the in vitro anticancer assay in human cancer cell lines.
We are also grateful to CSIR, New Delhi for the award
of research fellowships to O.S., P.R., G.R. and P.P.K.
12. Thurston, D. E.; Murty, V. S.; Langley, D. R.; Jones, G.
B. Synthesis 1990, 81.
13. Balcarova, Z.; Mrazek, J.; Kleinwachter, V.; Brabec, V.
Gen. Physiol. Biophys. 1992, 11, 579.
14. Brabec, V.; Balcarova, Z. Eur. J. Biochem. 1993, 216,
183.
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