Phosphonate-linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates: Synthesis, DNA-binding affinity and cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2008.01.040

Pyrrolobenzodiazepine-diethylphosphonate conjugates have been designed and synthesized that link through two different types of spacers that are simple alkane chain and also a piperazine moiety side-armed with the alkane chains. These pyrrolobenzodiazepin

Full text of DOI:10.1016/j.bmc.2008.01.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3895–3906
Phosphonate-linked pyrrolo[2,1-c][1,4]benzodiazepine
conjugates: Synthesis, DNA-binding affinity and cytotoxicity
Ahmed Kamal,a,* P. Praveen Kumar,a B. N. Seshadri,a O. Srinivas,a M. Shiva Kumar,a
Subrata Sen,b Nisha Kurian,b Aarti S. Juvekarb and Surekha M. Zingdeb
aBiotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology,
Hyderabad, Andhra Pradesh 500 007, India
bAdvanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Khargar, Navi Mumbai 410 208, India
Received 23 November 2007; revised 22 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—Pyrrolobenzodiazepine-diethylphosphonate conjugates have been designed and synthesized that link through two differ-
ent types of spacers that are simple alkane chain and also a piperazine moiety side-armed with the alkane chains. These pyr-
rolobenzodiazepine conjugates have exhibited remarkable DNA-binding affinity and improved solubility in water,
representative compound 7d showing promising in vitro cytotoxicity.
a
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
either synthetic or isolated from Streptomyces species.10
On the other hand, phosphorus-containing hybrids such
as coumarin-phosphonate11 and chromone-phospho-
nate12 hybrids are novel group of compounds possessing
remarkable cytotoxicity, alkylating, and anticancer
activity against selected tumour cell lines. Similarly,
9-(2-phosphonylmethoxyethyl)guanine (PMEG) is an
acyclic nucleoside phosphonate derivative that has dem-
onstrated significant anticancer activity in a number of
in vitro and in vivo animal model systems and is selec-
tively active against a panel of human leukemic cells.13
A series of new aminophosphonicacid derivatives of vin-
blastine (VLB) have been synthesized and evaluated for
their in vitro and in vivo for antitumor activity.14
In recent years, combination chemotherapy with differ-
ent mechanisms of action is one of the methods that
are being adopted to treat cancer. Therefore, a single
molecule containing more than one pharmacophore,
each with different mode of action could be beneficial
for the treatment of cancer. Organophosphorous com-
pounds are important substrates in the study of bio-
chemical processes,1 and the well-known class of
phosphoro-organic alkylating drugs (cyclophospha-
mide2 and thiotepa3) utilized in cancer chemotherapy
in the last decade. a-Aminophosphonates and their
derivatives (1) are important compounds possessing di-
verse and useful biological activities ranging from agri-
culture to medicine (anticancer agents,4 antibiotics,5
and enzyme inhibitors6). These are bioisosteres of natu-
ral amino acids, and acting as competitive inhibitors,
biological properties are mostly associated with the tet-
rahedral structure of the phosphonyl group acting as a
transition-state analogue. In addition, the alkylating
properties of phosphonic esters have been exhaustively
described in many studies7,8 and have been a subject
of many patents.9 Moreover, phosphonic esters are
The imine or carbinolamine-containing pyrrlo[2,1-c]
[1,4]benzodiazepines are a family of low molecular
weight antitumor antibiotics originally isolated from
various Streptomyces species15 and examples of which
include DC-81 (2), anthramycin, tomaymycin and sib-
iromycin. These antibiotics bind selectively in the minor
groove of DNA through a covalent aminal bond be-
tween the electrophilic C11 position of the PBD moiety
and the nucleophilic N2-amino group of a guanine
base,16 resulting in the possible biological activity. In
the literature, a large number of PBD conjugates and
their dimers have been synthesized and evaluated for
their biological activity, particularly for their antitu-
mour potential.17–19 Recently mixed imine–amide and
Keywords: Pyrrolobenzodiazepine; Diethylphosphonate; DNA-bind-
ing affinity; Cytotoxicity.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.040

3896
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
imine–amine PBD dimers have been designed and syn-
thesized in this laboratory for investigating the contribu-
tion from the non-covalent interactions by one of the
subunits in these dimers. It has been observed that the
contribution by such a non-covalent component plays
an important role in the overall DNA-binding affinity
in such mixed-type of PBD dimers.20,21 These findings
provided further impetus to explore the combination
of certain non-covalent interacting groups with a PBD
moiety that led to the design and synthesis of a variety
of PBD hybrids. The research in this direction in this
laboratory afforded a number of such PBD hybrids with
enhanced DNA-binding ability and significant antican-
cer potential.22
gave (2S)-N-(4-hydroxy)-5-methoxy-2-nitrobenzoyl)
pyr-rolidine-2-carboxaldehyde diethyl thioacetal (9).
Etherification of hydroxy compound 9 with diethyl(3-
bromopropyl)phosphonate in the presence of K2CO3
in acetone afforded the desired nitro precursor (2S)
-N-{4-[3-(diethoxyphosphoryl)propyl]oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehydediethylthio-
acetal (10). Further, reduction of nitro compound 10 by
using SnCl2. 2H2O reflux in methanol provided the
amine compound 11. The deprotection of thioacetal
group with the HgCl2/CaCO3 yielded formation of the
target compound 630 (Scheme 1).
Similarly, synthesis of compounds 7a–e has been carried
out by employing hydroxy compound (9) as the starting
material, which upon monoalkylation was achieved by
employing dibromoalkanes-yielded (2S)-N-[4-(n-bromo-
alkyl)oxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-car-
boxaldehyde diethyl thioacetals (12a–c). These coupled
with n-bocpiperazine provided the boc-protected com-
pounds 13a–c. Further, deprotection of boc-protected
compounds 13a–c employing triflouroaceticacid in
dichloromethane gave corresponding amine compounds
14a–c. The coupling of diethyl(n-bromoalkyl)phospho-
nates with amine compounds 14a–c provided the desired
other precursors (2S)-N-{4-[n-[4-[n-(diethoxyphosphoryl)-
alkyl]piperazin-1-yl]alkyl]-oxy-5-methoxy-2-nitrobenzoyl}-
pyrrolidine-2-carboxaldehyde diethyl thioacetals (15a–e).
A two-step pathway achieved the final assembly of the
desired target compounds 7a–e in the above conven-
tional manner (Scheme 2).
During the last decade, a number of piperazine deriva-
tives have been synthesized for their chemotherapeutic
use in the area of medicinal chemistry.23 Michejda and
co-workers24 reported symmetrical bifunctional agents
as a promising antitumour class of compounds with
remarkable selectivity against colon cancers that possess
a piperazine moiety in its linker spacer. Recently, trans-
diamine dichloroplatinum(II) complexes with piperazine
ligands have exhibited significant cytotoxicity.25
Recently, synthesized C-8 linked N-methyl piperazine-
PBD monomer (3)26 and in its linker spacer incorporat-
ing a piperazine moiety PBD dimers (4)27 has been
synthesized that exhibit promising anticancer activity
and increased DNA-binding ability (Fig. 1), in compar-
ison to DC-81 and PBD dimer (DSB-120), respectively.
Further, these PBD dimers when linked through a piper-
azine moiety in its alkane chain spacer provided better
hydrophobic interactions with the DNA to make them
more stable ligands. Therefore, these observations indi-
cate that incorporation of a piperazine moiety in the al-
kane spacer linker enhances the DNA-binding potential
significantly.28
2.2. DNA interactions: thermal denaturation studies
The DNA-binding ability for these C8-linked PBD con-
jugates has been examined by thermal denaturation
studies using calf thymus (CT) DNA. Thermal denatur-
ation studies have shown that these compounds stabi-
lized the thermal helix ! coil or melting stabilization
(DTm) for the CTDNA duplex at pH 7.0, incubated
for 18 h at 37 ꢁC, where PBD/DNA molar ratio is
1:5.31,32 Interestingly, in this study PBD conjugates have
shown significant melting temperature values (4.5–
10.0 ꢁC) that are included in (Table 1). It is interesting
to observe that the compounds 7a, 7b and 7e have
shown higher DTm values, i.e., 9.0, 9.9 and 10.0 ꢁC,
respectively, where the PBD ring system and phospho-
nate units separated with 5 or 6 or 8 (n + m) carbon
length of alkane chain spacers along with piperazine
moiety. Another aspect which has been seen from the
thermal denaturation data is that for compounds 7a
and 7e, the DTm values do not change even after incuba-
tion for 18 h. In case of compounds 7c and 7 d examined
after 18 h of incubation the DTm values are 7.1 and
7.2 ꢁC, where those two functionalities are separated
Recently, we investigated the synthesis of C-8 methane-
sulphonate substituted pyrrolobenzodiazepines as po-
tential antitumour agents (5).29 In continuation of
these efforts and the above-said aspects it is considered
interesting to design and synthesize molecules based
on PBD linked to phosphonate residue like diet-
hylphosphonate with or without incorporating a pipera-
zine moiety in the linker spacer. It is envisaged that such
an investigation could unravel the intricacies underlin-
ing the role of phosphonate as well as piperazine moie-
ties. Moreover it is well known that these moieties are
very important with respect to the enhancement of
DNA-binding ability as well as improvement in the bio-
availability of such conjugates based on PBD ring
system.
2. Results and discussion
2.1. Chemistry
with
7
carbon length of alkane chain spacer
(n + m = 7) along with piperazine moiety. Moreover,
simple phosphonate–PBD conjugate (6) has shown low-
er DNA-binding affinity. In the same experiment N-
methyl piperazine-PBD conjugate (3) and naturally
occurring DC-81 (2) exhibited the D Tm values 6.9 and
0.7 ꢁC, respectively, whereas piperazine-incorporated
PBD dimers have shown DTm values (15.1–20.0 ꢁC),27
The compound 6 was prepared from the (2S)-N-(4-ben-
zyloxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbox-
aldehyde diethylthioacetal (8), which has been prepared
by the literature method28 and, this upon debenzylation

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
3897
R1
P
10
N
R2
9
11
N
N
HO
8
N
O
NH2
H
H
A
B
5
1
2
O
11a
C
N
MeO
N
7
MeO
6
1a R1 = R2 = OH
1b R1 = OH, R2 = H, alkyl, aryl
1c R1 = R2 = alkyl, aryl
3
O
O
2 (DC 81)
3
O
O
N
O
N
N
N
O
H
( )2
N
Me
S
O
H
H
O
N
OMe
N
MeO
N
MeO
O
O
O
5
4
Me
Me
O
O
( )n
N
O
Me
O
N
N
Me
O
( )m
P
H
N
P
O
H
O
O
N
MeO
N
MeO
O
O
7a n = 1, m = 1
7b n = 1, m = 2
7c n = 2, m = 2
7d n = 3, m = 1
7e n = 3, m = 2
6
Figure 1. Chemical structures of aminophosphonic acid derivatives, DC-81, PBD-conjugates PBD dimer and phosphonate–PBD conjugates.
NO2
CH(SEt)2
NO2 CH(SEt)2
N
HO
BnO
MeO
(i)
N
MeO
O
O
9
8
(ii)
Me
Me
O
O
Me
O
Me
O
NH2
CH(SEt)2
NO2
CH(SEt)2
P
O
P
O
(iii)
O
O
N
N
MeO
MeO
O
O
11
10
(iv)
Me
O
P
Me
O
N
O
H
O
N
MeO
O
6
Scheme 1. Reagents and conditions: (i) EtSH-BF3OEt2, CH2Cl2, 12 h, rt, 75%; (ii) diethyl(3-bromopropyl)phosphonate, K2CO3, acetone, reflux,
48 h, 80%; (iii) SnCl2 2H2O, MeOH, reflux, 4 h, 80%; (iv) HgCl2–CaCO3, CH3CN: H2O (4:1), 8–12 h, 60%.

3898
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
NO2 CH(SEt)2
NO2
CH(SEt)2
( )n
( )n
O
Br
O
Boc
(ii)
N
N
(i)
N
O
N
9
MeO
MeO
O
13a-b
12a-c n = 1-3
(iii)
Me
O
NO2
CH(SEt)2
NO2
CH(SEt)2
( )n
O
O
HN
N
( )n
Me
O
N
N
( )m
P
(iv)
N
N
O
O
MeO
MeO
O
14a-b
15a n = 1, m = 1
15b n = 1, m = 2
15c n = 2, m = 2
15d n = 3, m = 1
15e n = 3, m = 2
(v)
Me
O
NH2
CH(SEt)2
( )n
O
N
N
Me
O
( )m
P
N
O
MeO
O
16a n = 1, m = 1
16b n = 1, m = 2
16c n = 2, m = 2
16d n = 3, m = 1
16e n = 3, m = 2
(vi)
Me
O
N
O
( )n
N
N
Me
O
( )m
H
P
O
N
MeO
O
7a n = 1, m = 1
7b n = 1, m = 2
7c n = 2, m = 2
7d n = 3, m = 1
7e n = 3, m = 2
Scheme 2. Reagents and conditions: (i) dibromoalkanes, K2CO3, acetone, reflux, 48 h, 90%; (ii) n-bocpiperazine, K2CO3, DMF, reflux, 36 h, 80%;
(iii) TFA; CH2Cl2, rt, 8 h, 80%; (iv) Diethyl(2-bromoethyl)phosphonate, diethyl (2-bromo propyl)phosphonate, DMF, rt, 24 h, 60 %; (v) SnCl2
2H2O, MeOH, reflux, 4 h, 75%; (vi) HgCl2–CaCO3, CH3CN: H2O (4:1), 8–12 h, 60%.
upon incubation for 18 h. However, piparazine spacer
plays major influence in these conjugates as good inter-
action with base pairs and proper snug fit in the minor
groove of double stranded DNA.
been developed in which the inhibition of DNA cleavage
by BamHI is used to probe the DNA-binding capability
of PBD monomers.36 We have earlier investigated this
assay for preferences of base pair selectivity of the
imine–amide PBD dimers.20 Recently, this study has
been carried out to determine the DNA-binding ability
of benzothiadiazine–PBD conjugates.37 Moreover, this
technique has also been used to study the covalent
DNA interaction of the PBD dimers and it is capable
of distinguishing between the monomeric and dimeric
families.38 The drug was preincubated with DNA, the
2.3. RED100-restriction endonuclease digestion assay
Several studies have employed restriction endonuclease
inhibition to confirm the relative-binding affinity of
DNA-interactive small molecule ligands.33–35 A quanti-
tative restriction enzyme digestion (RED100) assay has

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
3899
Table 1. Thermal denaturation data of C8-linked phosphonate–PBD
conjugates with CT-DNA
inhibition of BamHI cleavage. The BamHI inhibition
of these conjugates has not correlated much with the
DNA-binding affinity that may be due to the ability of
piperazine moiety to interact with phosphonate back-
bone of DNA rather than to interact with the base pairs.
PBD conjugates
[PBD]:[DNA]
molar ratioa
DTm (ꢁC)b after
incubation at
37 ꢁC for
0 h
18 h
2.4. Cytotoxicity
6
7a
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
2.1
4.5
9.0
9.0
9.0
Compounds 6, 7d and 7e have been evaluated for their
in vitro cytotoxicity in selected human cancer cell lines
of breast, cervix, lung, colon, oral, ovarian and prostate
by using Sulforhodamine B (SRB) method.35,39 The com-
pounds exhibiting GI50 6 10ꢀ5 M are considered to be
active on the respective cell lines. Table 2 reveals that
compounds 6, 7d and 7e have exhibited significant effect
against some of cell lines and the activity was comparable
with adriamycin (ADR). Interestingly, the piperazine
containing compound 7d exhibited a strong effect in
terms of GI50 values on SiHa (0.21 lM), GURAV
(0.17 lM) and A2780 (0.17 lM) cell lines and the com-
pound 7e exhibited GI50 value to the order of 0.18 lM
against DWD cell line. The comparison of the data in Ta-
ble 2 reveals that absence of piperazine spacer in these
PBD conjugates moderately decreases cytotoxic activity
or shows no activity in some of the cell lines (6). However,
the in vitro cytotoxicity (GI50) for naturally occurring
DC-8116 is 0.38 and 0.33 lM in L1210 and PC-3 cell lines.
7b
7c
9.9
7.1
6.2
6.1
7d
7e
7.2
10.0
4.7
10.0
6.9
3
DC-81 (2)
PBD dimmer (4)
0.3
21.8
0.7
24.0
a For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion = 100 lM and ligand concentration = 20 lM in aqueous
sodium-phosphate buffer [10 mM sodium phosphate + 1 mM EDTA,
pH 7.00 0.01].
b For CT-DNA alone at pH 7.00 0.01, Tm = 69.2 ꢁC 0.01 (mean
value from 10 separate determinations), all DTm values are 0.1–
0.2 ꢁC.
DNA–drug complexes were subjected to BamHI diges-
tion, and the protection of cleavage of the G#GATCC
sequence by PBD suggests that these molecules selec-
tively interact with G-rich sequences in DNA. The
inability of PBD in protecting cleavage of AAT#ATT
by SspI suggests that these PBDs have low affinity to
AT rich sequences. Therefore, PBD prefers G-rich se-
quences in DNA binding, and this could be due to the
affinity of PBDs in covalent interaction with the free
amino group attached to the N2 of guanine in the
DNA.36 The study has been carried out to determine
the ability of 7a, 7b, 7d and 7e, which inhibited the
DNA linearization by BamHI. The results of this exper-
iment for compounds 7a, 7b, 7d and 7e have been shown
in Fig. 2, suggesting that the phosphonate–PBDs inhibit
BamHI. There have been differences in the inhibitory
activity exhibited by PBDs evaluated in this assay. It is
observed that ranking order is 7e > 7b > 7a > 7d for
The compound 7d has been selected for NCI-60 cell line
panel, and comparison of the data in Table 3 reveals the
average log10GI50, log10 TGI and log10 LC50 values of
the cytotoxic activity in 60 human cancer cell lines in
nine panels and each panel containing average of six
to eight human cancer cell lines. Interestingly the com-
pound 7d possesses selective anticancer activity particu-
larly for leukemia. The leukemia cancer values (log10
GI50, log10 TGI and log10 LC50) for the compound
(7d) have been described in Table 4.
Moreover, the mean graph mid point values of log10
TGI and log10 LC50 as well as log10 GI50 for 5 and 7d
Control
UC
Concentration (μM)
Control Concentration (μM)
C
5
10 15 20 25 30
UC
C
5
10 15 20 25 30
7a
7b
Control
UC
Concentration (μM)
Control
UC
Concentration (μM)
C
5
10 15 20 25 30
C
5
10 15 20 25 30
7d
7e
Figure 2. RED100-restriction endonuclease digestion assay for PBD–phosphonate conjugates with CT-DNA inhibitory activity of 7a, 7b, 7d and 7e
on the cleavage of plasmid pBR322 by restriction endonuclease BamHI (10 U in 1 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 BamHI.

3900
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
Table 2. GI50 valuesa (in lM) for compounds 6, 7d and 7e tested in
selected human malignant cell lines
the cytotoxicity activities exhibited by these new PBD
conjugates are significant.
Cell lines
6
7d
7e
2.21
ADR
MCF7b
SiHac
A-549d
Colo205e
KBf
GURAVf
DWDf
A2780g
PC-3h
2.02
2.10
2.01
0.21
2.64
1.87
1.92
0.18
0.18
7.25
0.14
0.17
0.17
0.11
0.17
1.81
3. Conclusion
2.46
i
i
i
In conclusion, C8-linked piperazine-containing phospho-
nate–PBD conjugates have been synthesized that exhibit
remarkable DNA-binding ability. The restriction endo-
nuclease study also demonstrates this aspect and suggests
that these molecules selectively interact with G-sequences
in DNA and low affinity with AT-rich sequences. More
importantly, inclusion of a piperazine moiety in the linker
spacer not only enhances the potency of such PBD conju-
gates but also their solubility profile. Moreover, 7d exhib-
its significant cytotoxicity in some of the cancer cell lines
and these investigations will also be helpful in the design
of improved DNA targeted molecules.
2.39
i
2.11
i
0.17
i
2.18
0.18
2.02
2.20
i
0.17
1.98
i
30.5
a Obtained from three determinations.
b Breast cancer.
c Cervix cancer.
d Lung cancer.
e Colon cancer.
f Oral cancer.
g Ovarian cancer.
h Prostate cancer.
i GI50 value not attained at the concentrations used in the assay; ADR,
adriamycin.
4. Experimental
Reaction progress was monitored by thin-layer chroma-
tography (TLC) using GF254 silica gel with fluorescent
indicator on glass plates. Visualization was achieved
with UV light and iodine vapor 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 dichloro-
methane (calcium hydride), tetrahydrofuran (sodium
benzophenone ketyl), methanol (magnesium methox-
ide), and acetonitrile (calcium hydride). 1H NMR spec-
tra were recorded on Varian Gemini 200 MHz
spectrometer using tetramethyl silane (TMS) as an inter-
nal standard. Chemical shifts are reported in parts per
million (ppm) down field from tetramethyl silane. Spin
multiplicities are described as s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). Coupling con-
stants are reported in Hertz (Hz). EI mass spectra were
recorded on a VG-7070H Micromass mass spectrometer
at 200 ꢁC, 70 eV, with a trap current of 200 lA and 4 kV
of acceleration voltage. ESI spectra were recorded on
Micro mass, Quattro LC using ESI+ software with cap-
illary voltage 3.98 kV and ESI mode positive ion trap
detector. High-resolution mass spectra were recorded
on QSTAR XL Hybrid MS/MS mass spectrometer.
FAB mass spectra were recorded on a LSIMS-VG-
AUTOSPEC Micromass spectrometer. Micro analytical
data (C, H and N) agreed with the proposed structures
within 0.4% of the theoretical values.
Table 3. log10 GI50, log10 TGI and log10 LC50 (concentration in mol/L)
values for the representative compound 7d
Cancer panel
log10 GI50
log10 TGI
log10 LC50
Leukemia
Lung
Colon
ꢀ5.71
ꢀ5.00
ꢀ5.00
ꢀ5.52
ꢀ5.11
ꢀ4.86
ꢀ5.01
ꢀ4.75
ꢀ5.25
ꢀ4.91
ꢀ4.34
ꢀ4.33
ꢀ4.52
ꢀ4.52
ꢀ4.26
ꢀ4.27
ꢀ4.20
ꢀ4.48
ꢀ4.00
ꢀ4.00
ꢀ4.03
ꢀ4.05
ꢀ4.09
ꢀ4.00
ꢀ4.02
ꢀ4.00
ꢀ4.02
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
Each cancer type represents the average of six to nine different cancer
cell lines.
Table 4. The leukemia cancer values for the representative compound
7d
Leukemia
Log GI50
Log TGI
Log LC50
CCRF-CEM
HL-60(TB)
K-562
ꢀ5.72
ꢀ5.74
ꢀ5.63
ꢀ5.67
ꢀ5.64
ꢀ5.86
nda
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
ꢀ4.51
ꢀ5.08
>ꢀ4.00
ꢀ5.20
nd
MOLT-4
RPMI-8226
SR
ꢀ5.37
a nd = not determined.
Table 5. log10 GI50 log10 TGI and log10 LC50 mean graph midpoints
(MG_MID) of in vitro cytotoxicity data for the compounds 5 and 7d
against human tumour cell lines
5. General procedure
Compound
log10 GI50
log10 TGI
log10 LC50
5.1. Synthesis of (2S)-N-(4-hydroxy)-5-methoxy-2-nitro-
benzoyl)pyrrolidine-2-carboxal-dehyde diethyl thioac-
etal (9)
7d
5a
ꢀ5.14
ꢀ4.62
ꢀ4.42
ꢀ4.25
ꢀ4.04
ꢀ4.07
a See Ref. 26.
To a stirred solution of EtSH (1.91 g, 19.0 mmol) and
BF3.OEt2 (1.41 g, 10 mmol) was added dropwise to the
compound 8 (0.490 g, 1 mmol) in dichloromethane
(10 mL) at room temperature. Stirring was continued un-
are listed in Table 5. As demonstrated by mean graph
pattern compound 7d exhibits an interesting profile of
activity and selectivity for various cell lines. Therefore,

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
3901
til TLC indicated completion of the reaction. The solvent
was evaporated in vacuum. The residue was quenched
with bicarbonate solution (1 · 25 mL) and then extracted
with ethyl acetate (3 · 25 mL). The combined organic
phase was washed with saturated brine (1 · 25 mL) dried
over Na2SO4 and the solvent removed in vacuum to afford
the crude product. This was further purified by column
chromatography using ethyl acetate–hexane (7:3) as elu-
ent to afford compound 9 (0.30 g, 75%).
MeCN–water (4:1) was stirred slowly at room tempera-
ture until TLC indicated complete loss of starting mate-
rial. The reaction mixture was diluted with EtOAc
(30 mL) and filtered through a celite bed. The clear yel-
low organic supernatant was extracted with saturated
5% NaHCO3 (20 mL), brine (20 mL) and the combined
organic phase was dried (Na2SO4). The organic layer
was evaporated in vacuum and purified by column chro-
matography (80% CH2Cl2–MeOH) to give compound 6
(0.25 g, 60%). This material was repeatedly evaporated
from CHCl3 in vacuum to generate the imine form.
1H NMR (CDCl3): d 7.65 (s, 1H), 6.75 (s, 1H), 6.20–6.32
(br s, 1H), 4.85 (d, 1H), 4.60–4.70 (m, 1H), 3.95 (s, 3H),
3.20–3.32 (m, 2H), 2.70–2.88 (m, 4H), 1.75–2.35 (m,
4H), 1.20–1.40 (m, 6H); MS (EI) 400 [M]+; Anal. Calcd
for C17H24N2O5S2: C, 50.98; H, 6.04; N, 6.99. Found: C,
50.63; H, 5.98; N, 6.58.
1H NMR (CDCl3): d 7.70 (s, 1H); 7.65 (d, J = 4.4 Hz,
1H), 6.80 (s, 1H), 4.62–4.75 (m, 1H), 4.10–4.30 (m,
6H), 3.90 (s, 3H), 3.50–3.70 (m, 2H), 1.76–2.39 (m,
8H), 1.25–1.40 (m, 6H); MS (ESI) 424 [M]+; Anal. Calcd
for C20H29N2O6P: C, 49.81; H, 6.79; N, 4.84. Found: C,
49.86; H, 6.75; N, 4.82.
5.2. Synthesis of (2S)-N-{4-[3-(diethoxyphosphoryl)pro-
pyl]oxy-5-methoxy-2-nitrobenzo-yl}pyrrolidine-2-carbox-
aldehyde diethyl thioacetal (10)
5.5. General procedure for the synthesis of compounds
(12a–c)
To a solution of compound 9 (400 mg, 1 mmol) in dry
acetone (15 mL) were added anhydrous K2CO3
(553 mg, 4 mmol), and diethyl(3-bromopropyl)phospho-
nate (310 mg, 1.2 mmol) and the mixture was stirred at
reflux temperature for 48 h. The reaction was monitored
by TLC using (95% EtOAc–methanol), K2CO3 was re-
moved by filtration and the solvent was evaporated un-
der the vacuum, diluted with water and extracted with
ethyl acetate. The combined organic phase dried
(Na2SO4) and evaporated in vacuum was purified by
column chromatography (3% EtOAc–methanol) to af-
ford compound 10 as yellow liquid (0.47 g, 80%).
5.5.1. (2S)-N-[4-(3-Bromopropyl)oxy-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxal-dehyde diethyl thioacetal
(12a). To a stirred solution of (2S)-N-(4-hydroxy)-5-
methoxy-2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde
diethyl thioacetal (9) (0.40 g, 1 mmol) in acetone
(15 mL) were added anhydrous potassium carbonate
(0.69 g, 5.0 mmol) and compound 1,3-dibromopropane
(0.80 g, 4 mmol). The reaction mixture was refluxed in
an oil bath for 48 h and the reaction was monitored
by TLC using ethyl acetate hexane (8:2) as a solvent
system. The potassium carbonate was removed by suc-
tion filtration and solvent was removed under vacuum
to afford the crude product. This was further purified
by column chromatography using ethyl acetate–hexane
(8:2) as a solvent system to give product 12a (0.46 g,
90%).
1H NMR (CDCl3): d 7.63 (s, 1H), 6.77 (s, 1H), 4.82 (d,
J = 3.7 Hz, 1H), 4.60–4.70 (m, 1H), 4.05–4.20 (m, 6H),
3.94 (s. 3H), 3.16–3.32 (m, 2H), 2.65–2.85 (m, 4H),
1.80–2.20 (m, 8H), 1.20–1.40 (m, 12H); MS (ESI) 593
[M+1]+; Anal. Calcd for C24H39N2O8PS2: C, 49.81; H,
6.79; N, 4.84. Found: C, 49.86; H, 6.75; N, 4.82.
1H NMR (CDCl3): d, 7.70 (s, 1H), 6.80 (s, 1H), 4.85 (d,
J = 4.3 Hz, 1H), 4.63–4.75 (m, 1H), 4.25 (t, J = 5.98 Hz,
2H), 3.95 (s, 3H), 3.65 (t, J = 6.22 Hz, 2H), 3.20–3.35
(m, 2H), 2.60–2.90 (m, 4H), 1.70–2.50 (m, 6H), 1.20–
1.40 (m, 6H); MS (FAB) 521 [M]+; Anal. Calcd for
C20H29BrN2O5S2: C, 46.06; H, 5.61; N, 5.37. Found:
C, 46.00; H, 5.65; N, 5.35.
5.3. Synthesis of (2S)-N-4-[3-(diethoxyphosphoryl)pro-
pyl]oxy-5-methoxy-2-aminobenzo-yl]pyrrolidine-2-car-
boxaldehyde diethyl thioacetal (11)
The compound 10 (593 mg, 1 mmol) dissolved in metha-
nol (20 mL) and added SnCl2Æ2H2O (1.13 g, 5 mmol)
was refluxed for 4 h or until the TLC indicated that reac-
tion was complete. The methanol was evaporated under
vacuum and the aqueous layer was then carefully adjusted
to pH 8 with 10% NaHCO3 solution and then extracted
with ethyl acetate (2 · 30 mL). The combined organic
phase was dried over Na2SO4 and evaporated under vac-
uum to afford the amino diethyl thioacetal 11 as brown oil
(0.43 g, 80%) which due to potential stability problems
was directly used in the next step without isolation.
5.5.2. (2S)-N-[4-(4-Bromobutyl)oxy-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxalde-hyde diethyl thioacetal
(12b). The compound 12b was prepared according to the
method described for compound 12a employing (2S)-
N-(4-hydroxy)-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-
carboxaldehyde diethyl thioacetal (9) (535 mg, 1 mmol)
and 1,4-dibromobutane (0.86 g, 4 mmol) to afford com-
pound 12b (0.48 g, 90%).
1H NMR (CDCl3): d 1.21–1.45 (m, 6H), 1.70–2.40 (m,
8H), 2.60–2.90 (m, 4H), 3.15–3.30 (m, 2H), 3.50 (t,
J = 6.25 Hz, 2H), 3.95 (s, 3H), 4.10 (t, J = 6 Hz, 2H),
4.60–4.71 (m, 1H), 4.85 (d, J = 4.3 Hz, 1H), 6.80 (s,
1H), 7.65 (s, 1H); MS (FAB) 535 [M]+; Anal. Calcd
for C21H31BrN2O5S2: C, 47.10; H, 5.83; N, 5.23. Found:
C, 47.15; H, 5.82; N, 5.26.
5.4. Synthesis of 7-methoxy-8-[3-(diethoxyphosphoryl)pro-
pyl]oxy-(11aS)-1,2,3,11a-tetra- hydro-5H-pyrrolo[2,1-c][1,4]
benzodiazepin-5-one (6)
A solution of 11 (549 mg, 1 mmol), HgCl2 (613 mg,
2.26 mmol) and CaCO3 (246 mg, 2.46 mmol) in

3902
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
5.5.3. (2S)-N-[4-(5-Bromopentyl)oxy-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxal-dehyde diethyl thioacetal
(12c). The compound 12c was prepared according to the
method described for compound 12a employing (2S)-
N-(4-hydroxy)-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-
carboxaldehyde diethyl thioacetal (9) (535 mg, 1 mmol)
and 1,5-dibromopentane (0.92 g, 4 mmol) to afford com-
pound 12c (0.49 g, 90%).
J = 6.82 Hz, 2H), 2.42–2.50 (m, 4H), 2.32–2.38 (m,
4H), 2.10–2.20 (m, 4H), 1.47 (s, 9H), 1.30–1.42 (m,
6H); MS (ESI) 641 [M+1]+; Anal. Calcd for
C30H48N4O7S2: C, 56.23; H, 7.55; N, 8.74. Found: C,
56.27; H, 7.56; N, 8.70.
5.6.3. (2S)-N-4-[5-[(41-tert-Butoxycarbonyl)piperazin-1-
yl]pentyl]oxy-5-methoxy-2-nitro
benzoyl]pyrrolidine-2-
carboxaldehyde diethyl thioacetal (13c). The compound
13c was prepared according to the method described
for compound 13a employing n-bocpiperazine (186 mg,
1 mmol) and (2S)-N-[4-(5-bromopentyl)oxy-5-methoxy-
1H NMR (CDCl3): d 7.65 (s, 1H); 6.80 (s, 1H), 4.85 (d,
J = 4.33 Hz, 1H), 4.60–4.71 (m, 1H), 4.10 (t, J = 6 Hz,
2H), 3.95 (s, 3H), 3.45 (t, J = 6.34 Hz, 2H), 3.15–3.30
(m, 2H), 2.65–2.85 (m, 4H), 1.60–2.40 (m, 8H), 1.21–
1.40 (m, 8H); MS (FAB) 549 [M]+; Anal. Calcd for
C22H33BrN2O5S2: C, 48.08; H, 6.05; N, 5.10. Found:
C, 48.09; H, 6.02; N, 5.11.
2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl
thioacetal (12c) (0.65 g, 1.2 mmol) to afford compound
13c (0.52 g, 80%).
1H NMR (CDCl3): d 7.66 (s, 1H), 6.77 (s, 1H), 4.81 (d,
J = 6.51 Hz, 1H), 4.61–4.70 (m, 1H), 4.15 (t,
J = 6.51 Hz, 2H), 3.95 (s, 3H), 3.39–3.47 (m, 4H),
3.17–3.30 (m, 2H), 2.66–2.87 (m, 4H), 2.54 (t,
J = 6.84 Hz, 2H), 2.40–2.48 (m, 4H), 2.30–2.38 (m,
4H), 1.92–2.14 (m, 6H), 1.46 (s, 9H), 1.31–1.40 (m,
6H); MS (ESI) 655 [M+1]+; Anal. Calcd for
C31H50N4O7S2: C, 56.86; H, 7.70; N, 8.56. Found: C,
56.82; H, 7.74; N, 8.53.
5.6. General procedure for the synthesis of compounds
(13a–c)
5.6.1. (2S)-N-4-[3-[(41-tert-Butoxycarbonyl)piperazin-1-
yl]propyl]oxy-5-methoxy-2-nitro benzoyl]pyrrolidine-2-
carboxaldehyde diethyl thioacetal (13a). To a stirred
solution of n-bocpiperazine (186 mg, 1 mmol) in dry
DMF (10 mL) were added anhydrous potassium
carbonate (0.69 g, 5.0 mmol) and compound (2S)-N-
[4(3-bromopropyl)oxy-5-methoxy-2-nitrobenzoyl]pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (12a)
(0.62 g, 1.2 mmol). The reaction mixture was stirred at
room temperature for 36 h and the reaction was moni-
tored by TLC using (4% ethyl acetate–methanol) as a
solvent system. The potassium carbonate was removed
by suction filtration and solvent was removed under vac-
uum, diluted with water and extracted with ethyl ace-
tate. The combined organic phase was dried (Na2SO4)
and evaporated in vacuum and it was purified by col-
umn chromatography (3% EtOAc–methanol) to afford
compound 13a as brown oil (0.50 g, 80%).
5.7. General procedure for the synthesis of compounds
(14a–c)
5.7.1.
(2S)-N-{4-[3-piperazinopropoxy]-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioac-
etal (14a). To a solution of boc-protected compound 13a
(627 mg, 1 mmol) in dry dichloromethane was added tri-
fluoroacetic acid (1 mL) at 0 ꢁC and stirred under nitro-
gen for 8 h, the reaction mixture was concentrated in
vacuum and then it was used directly in the next step.
5.7.2. (2S)-N-{4-[4-piperazinobutoxy]-5-methoxy-2-nitro-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(14b). The compound 14b was prepared according to the
method described for compound 14a employing boc-
protected compound 13b (641 mg, 1 mmol) and trifluo-
roacetic acid (1 mL) to afford compound 14b.
1H NMR (CDCl3): d 7.71 (s, 1H), 6.83 (s, 1H), 4.87–4.89
(d, J = 3.7 Hz, 1H), 4.68–4.76 (m, 1H), 4.18 (t,
J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.40–3.50 (t, J = 4.5 Hz,
4H), 3.20–3.32 (m, 2H), 2.70–2.85 (m, 4H), 2.52–2.60
(t, J = 6.7 Hz, 2H), 2.38–2.46 (t, J = 4.5 Hz, 4H), 2.03–
2.15 (m, 4H), 1.60–1.70 (m, 2H), 1.46 (s, 9H), 1.30–
1.39 (q, 6H); MS (ESI) 627 [M+1]+; Anal. Calcd for
C29H46N4O7S2: C, 55.57; H, 7.40; N, 8.94. Found: C,
55.51; H, 7.43; N, 8.95.
5.7.3. (2S)-N-{4-[5-piperazinopentoxy]-5-methoxy-2-nitro-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(14c). The compound 14c was prepared according to
the method described for compound 14a employing
boc-protected compound 13c (655 mg, 1 mmol) and tri-
fluoroacetic acid (1 mL) to afford compound 14c.
5.6.2. (2S)-N-4-[4-[(41-tert-Butoxycarbonyl)piperazin-1-
yl]butyl]oxy-5-methoxy-2-nitro benzoyl]pyrrolidine-2-car-
boxaldehyde diethyl thioacetal (13b). The compound 13b
was prepared according to the method described for
compound 13a employing n-bocpiperazine (186 mg,
1 mmol) and (2S)-N-[4-(4-bromobutyl)oxy-5-methoxy-
2 nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thi-
oacetal (12b) (0.64 g, 1.2 mmol) to afford compound 13b
(0.51 g, 80%).
5.8. General procedure for the synthesis of compounds
(15a–e)
5.8.1. (2S)-N-{4-[3-[4-[2-(Diethoxyphosphoryl)ethyl]pip-
erazin-1-yl]propyl]-oxy-5-meth-oxy-2-nitrobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (15a). To a
stirred solution of (2S)-N-[4-(3-piperazinopropyl)oxy-
5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal (14a) (441 mg, 1 mmol) in dry DMF
(10 mL) were added anhydrous potassium carbonate
(0.69 g, 5.0 mmol) and compound diethyl(2-bromoeth-
yl)phosphonate (294 mg, 1.2 mmol). The reaction
1H NMR (CDCl3): d 7.66 (s, 1H), 6.77 (s, 1H), 4.80–4.82
(d, J = 6.82 Hz, 1H), 4.61–4.70 (m, 1H), 4.12–4.18 (t,
J = 6.82 Hz, 2H), 3.95 (s, 3H), 3.40–3.49 (m, 4H),
3.19–3.30 (m, 2H), 2.68–2.88 (m, 4H), 2.52–2.58 (t,

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
3903
mixture was stirred at room temperature for 24 h and
the reaction was monitored by TLC using (10%
CH2Cl2–methanol) as a solvent system. The potassium
carbonate was removed by suction filtration and solvent
was removed under vacuum, diluted with water and ex-
tracted with ethyl acetate. The combined organic phase
dried (Na2SO4) and evaporated in vacuum was purified
by column chromatography (5% CH2Cl2–methanol) to
afford compound 15a as brown oil (0.45 g, 60%).
1H NMR (CDCl3): d 7.62 (s, 1H), 6.75 (s, 1H), 4.80 (d,
J = 3.6 Hz, 1H), 4.60–4.71 (m, 1H), 4.04–4.16 (m, 6H),
3.92 (s, 3H), 3.15–3.30 (m, 2H), 2.65–2.85 (m, 4H),
2.35–2.60 (m, 12H), 1.42–2.33 (m, 10H), 1.20–1.42 (m,
12H), 0.82–0.92 (m, 2H); MS (ESI) 733 [M+1]+; Anal.
Calcd for C32H55N4O8PS2: C, 53.46; H, 7.71; N, 7.79.
Found: C, 53.41; H, 7.76; N, 7.81.
5.8.5. (2S)-N-{4-[5-[4-[3-(Diethoxyphosphoryl)propyl]pip-
erazin-1-yl]pentyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrol-
idine-2-carboxaldehyde diethyl thioacetal (15e). The
compound 15e was prepared according to the method
described for compound 15a employing (2S)-N-[4-(5-pip-
erazinopentyl)oxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-
2-carboxaldehyde diethyl thioacetal (14c) (469 mg, 1
mmol) and diethyl(3-bromopropyl)phosphonate (310 mg,
1.2 mmol) to afford compound 15e (0.48 g, 65%).
1H NMR (CDCl3): d 7.61 (s, 1H), 6.78 (s, 1H), 4.82 (d,
1H, J = 3.5 Hz), 4.62–4.72 (m, 1H), 4.00–4.20 (m, 6H),
3.94 (s, 3H), 3.15–3.31 (m, 2H), 2.50–2.90 (m, 16H),
1.44–2.40 (m, 8H), 1.15–1.40 (m, 12H); MS (ESI) 537
[M+1]+; Anal. Calcd for C30H51N4O8PS2: C, 52.16; H,
7.44; N, 8.11. Found: C, 52.12; H, 7.40; N, 8.09.
5.8.2. (2S)-N-{4-[3-[4-[3-(Diethoxyphosphoryl)propyl]pip-
erazin-1-yl]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrol-
idine-2-carboxaldehyde diethyl thioacetal (15b). The
compound 15b was prepared according to the method
described for compound 15a employing (2S)-N-[4-(3-
piperazinopropyl)oxy-5-methoxy-2-nitrobenzoyl]pyrrol-
idine-2-carboxaldehyde diethyl thioacetal (14a) (441 mg,
1H NMR (CDCl3): d 7.61 (s, 1H), 6.77 (s, 1H), 4.82 (d,
J = 3.7 Hz, 1H), 4.61–4.71 (m, 1H), 4.00–4.16 (m, 6H),
3.94 (s, 3H), 3.15–3.30 (m, 2H), 2.65–2.85 (m, 4H),
2.35–2.60 (m, 12H), 1.42–2.33 (m, 12H), 1.20–1.40 (m,
12H), 0.84–0.92 (m, 2H); MS (ESI) 747 [M+1]+; Anal.
Calcd for C33H57N4O8PS2: C, 54.08; H, 7.84; N, 7.64.
Found: C, 54.12; H, 7.88; N, 7.60.
1 mmol)
and
(310 mg, 1.2 mmol) to afford compound 15b (0.46 g,
diethyl(3-bromopropyl)phosphonate
65%).
5.9. General procedure for the synthesis of compounds
(16a–e)
1H NMR (CDCl3): d 7.66 (s, 1H), 6.77 (s, 1H), 4.82 (d,
J = 3.9 Hz, 1H), 4.63–4.70 (m, 1H), 4.00–4.20 (m, 7H),
3.94 (s, 3H), 3.16–3.30 (m, 2H), 2.65–2.90 (m, 4H),
2.40–2.65 (m, 12H), 1.40–2.40 (m, 10H), 1.28–1.38 (m,
12H); MS (ESI) 719 [M+1]+; Anal. Calcd for
C31H53N4O8PS2: C, 52.82; H, 7.58; N, 7.95. Found: C,
52.79; H, 7.60; N, 7.98.
5.9.1. (2S)-N-{4-[3-[4-[2-(Diethoxyphosphoryl)ethyl]pip-
erazin-1-yl]propyl]-oxy-5-methoxy-2-aminobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (16a). The
compound 15a (705 mg, 1 mmol) dissolved in methanol
(20 mL) and added SnCl2Æ2H2O (1.13 g, 5 mmol) was re-
fluxed for 4 h or until the TLC indicated that reaction was
complete. The methanol was evaporated under vacuum
and the aqueous layer was then carefully adjusted to pH
8 with 10% NaHCO3 solution and then extracted with
ethyl acetate (2 · 30 mL). The combined organic phase
was dried over Na2SO4 and evaporated under vacuum
to afford the amino diethyl thioacetal 16a as brown oil
(0.50 g, 75%) which due to potential stability problems
was directly used in the next step without isolation.
5.8.3. (2S)-N-{4-[4-[4-[3-(Diethoxyphosphoryl)propyl]pip-
erazin-1-yl]butyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrroli-
dine-2-carboxaldehyde diethyl thioacetal (15c). The
compound 15c was prepared according to the method
described for compound 15a employing (2S)-N-[4-(4-
piperazinobutyl)oxy]-5-methoxy-2-nitrobenzoyl]pyrroli-
dine-2-carboxaldehyde diethyl thioacetal (14b) (455 mg,
1 mmol) and diethyl(3-bromopropyl)phosphonate (310
mg, 1.2 mmol) to afford compound 15c (0.47 g, 65%).
5.9.2. (2S)-N-{4-[3-[4-[3-(Diethoxyphosphoryl)propyl]pip-
erazin-1-yl]propyl]-oxy-5-methoxy-2-aminobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (16b). The
compound 16b was prepared according to the method
described for compound 16a employing (2S)-N-{4-[3-
[4-[3-(diethoxyphosphoryl)propyl]piperazin-1-yl]propyl]
-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxal-
dehyde diethyl thioacetal (15b) (469 mg, 1 mmol) and
SnCl2Æ2H2O (1.13 g, 5 mmol) to afford compound 16b
(0.51 g, 75%), which due to potential stability problems
was directly used in the next step without isolation.
1H NMR (CDCl3): d 7.61(s, 1H), 6.77 (s, 1H), 4.82 (d,
J = 3.7 Hz, 1H), 4.61–4.71 (m, 1H), 4.00–4.16 (m, 6H),
3.94 (s, 3H), 3.15–3.30 (m, 2H), 2.65–2.85 (m, 4H),
2.35–2.60 (m, 12H), 1.40–2.40 (m, 12H), 1.20–1.40 (m,
12H); MS (ESI) 733 [M+1]+; Anal. Calcd for
C32H55N4O8PS2: C, 53.46; H, 7.71; N, 7.79. Found: C,
53.41; H, 7.76; N, 7.81.
5.8.4. (2S)-N-{4-[5-[4-[2-(Diethoxyphosphoryl)ethyl]pip-
erazin-1-yl]pentyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrol-
idine-2-carboxaldehyde diethyl thioacetal (15d). The
compound 15d was prepared according to the method
described for compound 15a employing (2S)-N-[4-(5-
piperazinopentyl)oxy-5-methoxy-2-nitrobenzoyl]pyrrol-
idine-2-carboxaldehyde diethyl thioacetal (14c) (469 mg,
1 mmol) and diethyl(2-bromoethyl)phosphonate (294
mg, 1.2 mmol) to afford compound 15d (0.47 g, 65%).
5.9.3. (2S)-N-{4-[4-[4-[3-(Diethoxyphosphoryl)propyl]pip-
erazin-1-yl]butyl]-oxy-5-methoxy-2-aminobenzoyl}pyrrol-
idine-2-carboxaldehyde diethyl thioacetal (16c). The
compound 16c was prepared according to the method
described for compound 16a employing (2S)-N-{4-[3-
[4-[4-(diethoxyphosphoryl)butyl]piperazin-1-yl]propyl]-
oxy-5-methoxy-2-nitro- benzoyl}pyrrolidine-2-carboxal-

3904
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
dehyde diethyl thioacetal (15c) (469 mg, 1 mmol) and
SnCl2Æ2H2O (1.13 g, 5 mmol) to afford compound 16c
(0.52 g, 75%), which due to potential stability problems
was directly used in the next step without isolation.
1H NMR (CDCl3): d 7.69 (d, J = 4.4 Hz, 1H), 7.56 (s,
1H), 6.88 (s, 1H), 4.60–4.88 (m, 1H), 4.00–4.28 (m,
6H), 3.96 (s, 3H), 3.48–3.92 (m, 2H), 2.24–2.70 (m,
12H), 2.00–2.24 (m, 2H), 1.60–1.96 (m, 8H), 1.20–1.40
(m, 6H); MS (ESI) 564 [M+1]+; Anal. Calcd for
C27H43N4O6P: C, 58.90; H, 7.87; N, 10.18. Found: C,
58.92; H, 7.85; N, 10.22.
5.9.4. (2S)-N-{4-[3-[4-[2-(Diethoxyphosphoryl)ethyl]pip-
erazin-1-yl]pentyl]-oxy-5-methoxy-2-aminobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (16d). The
compound 16d was prepared according to the method
described for 16a employing (2S)-N-{4-[3-[4-[4-
(diethoxyphosphoryl)pentyl]piperazin-1-yl]ethyl]-oxy-5-
methoxy-2-nitro-benzoy l}pyrrolidine-2-carboxaldehyde
diethyl thioacetal (15d) (469 mg, 1 mmol) and SnCl2Æ2-
H2O (1.13 g, 5 mmol) to afford compound 16d (0.52 g,
75%), which due to potential stability problems was di-
rectly used in the next step without isolation.
5.10.3. 7-Methoxy-8-[4-[4-[3-(diethoxyphosphoryl)propyl]
piperazin-1-yl]butyl]oxy-(11aS) 1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (7c). The com-
pound 7c was prepared according to the method
described for compound 7a employing 16c (703 mg,
1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3
(246 mg, 2.46 mmol) to afford compound 7 c (0.34 g,
68%).
5.9.5. (2S)-N-{4-[3-[4-[3-(Diethoxyphosphoryl)propyl]pip-
erazin-1-yl]pentyl]-oxy-5-methoxy-2-aminobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (16e). The
compound 16e was prepared according to the method
described for compound 16a employing (2S)-N-{4-[3-
[4-[4-(diethoxyphosphoryl)pentyl]piperazin-1-yl]propyl]-
oxy-5-methoxy-2-nitro-benzoyl}pyrrolidine-2-carboxal-
dehyde diethyl thioacetal (15e) (469 mg, 1 mmol) and
SnCl2Æ2H2O (1.13 g, 5 mmol) to afford compound 16e
(0.53 g, 75%), which due to potential stability problems
was directly used in the next step without isolation.
1H NMR (CDCl3): d 7.65 (d, J = 4.4 Hz, 1H), 7.55 (s,
1H), 6.88 (s, 1H), 4.58–4.88 (m, 1H), 4.04–4.28 (m,
6H), 3.95 (s, 3H), 3.48–3.92 (m, 2H), 2.25–2.72 (m,
12H), 2.02–2.24 (m, 2H), 1.60–1.96 (m, 10H), 1.20–
1.40 (m, 6H); MS (ESI) 578 [M+1]+; Anal. Calcd for
C28H45N4O6P: C, 59.56; H, 8.03; N, 9.92. Found: C,
59.58; H, 8.00; N, 9.92.
5.10.4. 7-Methoxy-8-[5-[4-[2-(diethoxyphosphoryl)ethyl]
piperazin-1-yl]pentyl]oxy-(11aS)-1,2,3,11a-tetrahydro-
5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (7d). The com-
pound 7d was prepared according to the method
described for compound 7a employing 16d (703 mg,
1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3
(246 mg, 2.46 mmol) to afford compound 7d (0.34 g,
68%).
5.10. General procedure for the synthesis of compounds
(7a–e)
5.10.1. 7-Methoxy-8-[3-[4-[2-(diethoxyphosphoryl)ethyl]pip-
erazin-1-yl]propyl]oxy-(11a-S)-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (7a). A solution of
16a (675 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and
CaCO3 (246 mg, 2.46 mmol) in MeCN–water (4:1) was
stirred slowly at room temperature until TLC indicated
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 ex-
tracted with saturated 5% NaHCO3 (20 mL), and brine
(20 mL) and the combined organic phase was dried
(Na2SO4). The organic layer was evaporated in vacuum
and purified by column chromatography (80% CH2Cl2–
MeOH) to give compound 7a (0.32 g, 60%). This mate-
rial was repeatedly evaporated from CHCl3 in vacuum
to generate the imine form.
1H NMR (CDCl3): d 7.67 (d, J = 3.9 Hz, 1H), 7.50 (s,
1H), 6.80 (s, 1H), 4.00–4.20 (m, 7H), 3.87 (s, 3H),
3.42–3.82 (m, 2H), 1.80–2.80 (m, 16H), 1.20–1.72 (m,
8H), 0.80–0.96 (m, 2H); MS (ESI) 578 [M+1]+; Anal.
Calcd for C28H45N4O6P: C, 59.56; H, 8.03; N, 9.92.
Found: C, 59.58; H, 8.00; N, 9.92.
5.10.5. 7-Methoxy-8-[5-[4-[3-(diethoxyphosphoryl)propyl]
piperazin-1-yl]pentyl]oxy-(11a-S)-1,2,3,11a-tetrahydro-
5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (7e). The com-
pound 7e was prepared according to the method de-
scribed for compound 7a employing 16e (717 mg,
1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246
mg, 2.46 mmol) to afford compound 7e (0.36 g, 68%).
1H NMR (CDCl3): d 7.57 (d, J = 4.4 Hz, 1H), 7.50 (s,
1H), 6.83 (s, 1H), 4.00–4.20 (m, 7H), 3.92 (s, 3H),
3.50–3.88 (m, 2H), 2.40–2.80 (m, 12H), 2.20–2.40 (m,
2H), 1.80–2.15 (m, 6H), 1.20–1.36 (m, 6H); MS (ESI)
536 [M+1]+; Anal. Calcd for C26H41N4O6P: C, 58.20;
H, 7.70; N, 10.44. Found: C, 58.25; H, 7.72; N, 10.40.
1H NMR (CDCl3): d 7.66 (d, J = 4.0 Hz, 1H,), 7.52 (s,
1H, Ar), 6.82 (s, 1H), 4.02–4.22 (m, 7H), 3.86 (s, 3H),
3.40–3.82 (m, 2H), 1.80–2.80 (m, 16H), 1.20–1.76 (m,
10H), 0.82-0.98 (m, 2H); MS (ESI) 592 [M+1]+; Anal.
Calcd for C29H47N4O6P: C, 60.19; H, 8.19; N, 9.68.
Found: C, 60.22; H, 8.25; N, 9.62.
5.10.2. 7-Methoxy-8-[3-[4-[3-(diethoxyphosphoryl)propyl]
piperazin-1-yl]propyl]oxy-(11a-S)-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (7b). The com-
pound 7b was prepared according to the method
described for compound 7a employing 16b (689 mg,
1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246
mg, 2.46 mmol) to afford compound 7b (0.33 g, 68%).
6. Biological activity
6.1. Thermal denaturation studies
The DNA-binding affinity of the novel phosphonate–
PBD conjugates (6 and 7a–e) and N-methylpiperazinyl–

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
3905
PBD conjugate (3) has been evaluated through thermal
denaturation studies with duplex-form calf thymus
DNA (CT-DNA) using modified reported procedure.31
Working solutions in aqueous buffer (10 mM NaH2PO4/
Na2HPO4, 1 mM Na2EDTA, pH 7.00 + 0.01) 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 ra-
tio of 1:5. The DNA–PBD solutions were incubated at
37 ꢁC for 0, and 18 h prior to analysis. Samples were
monitored at 260 nm using a Beckman DU-7400 spectro-
photometer fitted with high-performance temperature
controller, and heating was applied at 1 ꢁC minꢀ1 in the
40–90 ꢁC range. DNA helix coil transition temperatures
(Tm) were obtained from the maxima in the (dA260)/dT
derivative plots. Results are given as means standard
deviation from three determinations and are corrected
for the effects of MeOH co-solvent using a linear correc-
tion term.32 Drug-induced alterations in DNA melting
behaviour are given by DTm = Tm(DNA + PBD) ꢀ Tm
(DNA alone), where the Tm value for the PBD-free CT-
DNA is 69.2 0.01. The fixed [PBD]/[DNA] ratio used
did not result in binding saturation of the host DNA du-
plex for any compound examined.
Percentage growth was expressed as the (ratio of average
absorbance of the test well to the average absorbance of
the control wells) · 100.
6.3. Restriction endonuclease inhibition
Stock solutions of each PBD (100 lM) were prepared by
dissolving each compound in DMSO (Sigma). These
were stored at ꢀ20 ꢁC. Plasmid (pBR 322) containing
single BamHI 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 · BamHI
buffer (2 lL) was added, and the reaction mixture was
made up to 20 lL with BamHI (20 U) and then incu-
bated 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.
Acknowledgments
The authors P.P.K., B.N.S., O.S and M.S.K. thank
CSIR, New Delhi for the award of research fellowships.
We are also thankful to the Department of Biotechnol-
ogy (BT/PR/7037/Med/14/933/2006), New Delhi for
financial assistance.
6.2. In vitro evaluation of cytotoxic activity
In-routine compounds 6, 7d and 7e have been evaluated
for their in vitro cytotoxicity in selected human cancer
cell lines of breast (Zr-75-1, MCF7), cervix (SiHa), lung
(Hop62, A-549), colon (Colo205), oral (KB, GURAV,
DWD), ovarian (A2780), and prostate (PC-3) origin.
A protocol of 48 h continuous drug exposure has been
used and a sulforhodamine B (SRB) protein assay has
been used to estimate cell viability or growth.40,41
References and notes
1. Noyori, R. Asymmetric Catalysis in Organic Synthesis;
John Wiley & Sons: New York, 1994.
2. Broch, R. F.; Canute, G. W. J. Med. Chem. 1991, 34,
3044.
The cell lines were grown in RPMI 1640 medium con-
taining 10% fetal bovine serum and 2 mM L-glutamine
and were inoculated into 96-well microtiter plates in
90 lL at plating densities depending on the doubling
time of individual cell lines. The microtiter plates were
incubated at 37 ꢁC, 5% CO2, 95% air, and 100% relative
humidity for 24 h prior to addition of experimental
drugs. Aliquots of 10 lL of the drug dilutions were
added to the appropriate microtiter wells already con-
taining 90 lL of cells, resulting in the required final drug
concentrations.
3. Pawelczyk, E.; Zajac, M. Chemiczne mechanizmy dzialania
lek’ow, (Akademia Medyczna in Poznan, 1995, pp 364–
377.
4. Kafarski, P.; Lejczak, B. Curr. Med. Chem. Anticancer
Agents 2001, 1, 301.
5. Atherton, F. R.; Hassall, C. H.; Lambert, R. W. J. Med.
Chem. 1986, 29, 29.
6. Smith, W. W.; Bartlett, P. A. J. Am. Chem. Soc. 1998, 120,
4622.
7. Budzisz, E.; Wojciechowska, J. G.; Ziebab, R.; Nawrotc,
B. New J. Chem 2002, 26, 1799.
8. Troev, K.; Botisov, G. Phosphorus, Sulfur Silicon 1987, 29,
129.
Plates were incubated further for 48 h and assay was ter-
minated by the addition of 50 lL of cold trichloro acetic
acid (TCA) (final concentration, 10% TCA) and incu-
bated for 60 min at 4 ꢁC. The plates were washed five
times with tap water and air-dried. Sulforhodamine B
(SRB) solution (50 lL) at 0.4% (w/v) in 1% acetic acid
was added to each of the wells, and plates were incu-
bated for 20 min at room temperature. The residual
dye was removed by washing five times with 1% acetic
acid. The plates were air-dried. Bound stain was subse-
quently eluted with 10 mM trizma base, and the absor-
bance was read on an ELISA plate reader at a
wavelength of 540 nm with 690 nm reference wave-
length. Percent growth was calculated on a plate–by-
plate basis for test wells relative to control wells.
9. Zech, J. D.; U.S. Patent 2824113, 1958; Chem. Abstr. 1958,
52, 11110.
10. Chmiel, A.; Grudzinski, S. Biotechnologia i Chemia
Antybiotyk ow, WN PWN, Warszawa, Ed.; 1998, pp 280.
11. Budzisz, E.; Brzezinska, E.; Krajewsk, U.; Rozalski, M.
Eur. J. Med. Chem. 2003, 38, 597.
12. Kostka, K.; Pastuszko, S.; Porada, M. Phosphorus, Sulfur
Silicone 1992, 71, 67.
13. Kramata, P.; Downey, K. M. Mol. Pharmacol. 1999, 56,
1262.
14. Lavielle, G.; Hautefaye, P.; Schaeffer, C.; Boutin, J. A.;
Claude, A.; Cudennec; Pierr, A. J. Med. Chem. 1991, 34,
1998.
15. Thurston, D. E. Molecular Aspects of Anticancer Drug–
DNA Interactions; The Macmillan Press Ltd.: London,
UK, 1993, pp 54–80.

3906
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 3895–3906
16. Petrusek, R. L.; Uhlenhopp, E. L.; Duteau, N.; Hurley, L.
H. J. Biol. Chem. 1982, 257, 6207.
17. Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433.
18. Kamal, A.; Rao, M. V.; Laxman, N.; Ramesh, G.;
Reddy, G. S. K. Curr. Med. Chem. Anticancer Agents
2002, 2, 215.
19. Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, A.
A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston,
D. E. J. Med. Chem. 2001, 44, 737.
20. Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.;
Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.;
Nagarajaram, H. A. J. Med. Chem. 2002, 45, 4679.
21. Kamal, A.; Ramesh, G.; Srinivas, O.; Ramulu, P.;
Laxman, N.; Rehana, T.; Deepak, M.; Achary, M. S.;
Nagarajaram, H. A. Bioorg. Med. Chem. 2004, 12, 5427.
22. Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G.; Kumar,
P. P. Bioorg. Med. Chem. Lett. 2004, 14, 4791.
29. Bose, D. S.; Thompson, A. S.; Ching, J.; Hartley, J. A.;
Berardini, M. D.; Jenkins, T. C.; Neidle, S.; Hurley, L. H.;
Thruston, D. E. J. Am. Chem. Soc. 1992, 114, 4939.
30. Balcarova, Z.; Mrazek, J.; Kleinwachter, V.; Brabec, V.
Gen. Physiol. Biophysics 1992, 11, 579.
31. Jones, G. B.; Davey, C. L.; Jenkins, T. C.; Kamal, A.;
Kneale, G. G.; Neidle, S.; Webster, G. D.; Thurston, D. E.
Anti-Cancer Drug Des. 1990, 5, 249.
32. Connaughie, A. W. M.; Jenkins, T. C. J. Med. Chem.
1995, 38, 3488.
33. alcarova, Z.; Mrazek, J.; Kleinwachter, V.; Brabec, V.
Gen. Physiol. Biophysics 1992, 11, 579.
34. Brabec, V.; Balcarova, Z. Eur. J. Biochem. 1993, 216, 183.
35. Collier, D. A.; Thuong, N. T.; Helene, C. J. Am. Chem.
Soc. 1991, 113, 1457.
36. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston,
D. E. Nucleic Acids Res. 1993, 21, 3671.
23. Shchekotikhin, A. E.; Shtil, A. A.; Luzikov, Y. N.;
Bobrysheva, T. V.; Buyanov, V. N.; Preobrazhenskaya,
M. N. Biorg. Med.Chem. 2005, 13, 2285.
37. Kamal, A.; Khan, M. N. A.; Srinivas, R. K.; Kaleem, A.
S.; Shiva, K. M.; Juvekar, A.; Sen, S.; Zingde, S. Bioorg.
Med. Chem. Lett. 2007, 17, 5400.
24. Cholody, W. M.; Hernandez, L.; Hassner, L.; Scudiero, D.
A.; Djurickovic, D. B.; Michejda, C. J. J. Med. Chem.
1995, 38, 3043.
25. Najajreh, Y.; Perez, J. M.; Navarro-Ranninger, C.;
Gibson, D. J. Med. Chem. 2002, 45, 5189.
26. Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.;
Ramulu, P. Bioorg. Med. Chem. Lett. 2002, 12, 1917.
27. Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.; Laxman,
E. Bioorg. Med. Chem. 2006, 14, 385.
28. Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G. Bioorg.
Med. Chem. Lett. 2003, 13, 3517.
38. 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.; Thruston, D. E.
J. Med. Chem. 2004, 47, 1161.
39. Sumner, W.; Bennett, G. N. Nucleic Acids Res. 1981, 9,
2105.
40. Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112.
41. Skehn, P.; Storeng, R.; Scudiero, A.; Monks, J.; McMo-
han, D.; Vistica, D.; Jonathan, T. W.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.