Design, synthesis, and evaluation of new noncross-linking pyrrolobenzodiazepine dimers with efficient DNA binding ability and potent antitumor activity

Article abstract of DOI:10.1021/jm020124h

New sequence selective mixed imine-amide pyrrolobenzodiazepine (PBD) dimers have been developed that are comprised of DC-81 and dilactam of DC-81 subunits tethered to their C8 positions through alkanedioxy linkers (comprised of three to five and eight car

Full text of DOI:10.1021/jm020124h

J . Med. Chem. 2002, 45, 4679-4688
4679
Design , Syn th esis, a n d Eva lu a tion of New Non cr oss-Lin k in g
P yr r oloben zod ia zep in e Dim er s w ith Efficien t DNA Bin d in g Ability a n d P oten t
An titu m or Activity
Ahmed Kamal,*,† G. Ramesh,† N. Laxman,† P. Ramulu,† O. Srinivas,† K. Neelima,‡ Anand K. Kondapi,‡
V. B. Sreenu,§ and H. A. Nagarajaram§
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology,
Hyderabad 500 007, India, Department of Biochemistry, School of Life Sciences, University of Hyderabad,
Hyderabad 500 046, India, and Laboratory of Computational Biology and Bioinformatics, Centre for
DNA Finger Printing and Diagnostics, Hyderabad 500 076, India
Received March 19, 2002
New sequence selective mixed imine-amide pyrrolobenzodiazepine (PBD) dimers have been
developed that are comprised of DC-81 and dilactam of DC-81 subunits tethered to their C8
positions through alkanedioxy linkers (comprised of three to five and eight carbons). Thermal
denaturation studies show that after 18 h of incubation with calf thymus DNA at a 5:1 DNA/
ligand ratio, one of them (5c) increases the ∆Tm value by 17.0 °C. Therefore, these
unsymmetrical molecules exhibit significant DNA minor groove binding affinity and 5c linked
through the pentanedioxy chain exhibits efficient DNA binding ability that compares with the
cross-linking DSB-120 PBD dimer (∆Tm ) 15.4 °C). Interestingly, this imine-amide PBD dimer
has been linked with a five carbon chain linker unlike DSB-120, which has two DC-81 subunits
with a three carbon chain linker, illustrating the effect of the noncross-linking aspect by
introducing the noncovalent subunit. The binding affinity of the compounds has been measured
by restriction endonuclease digestion assay based on inhibition of the restriction endonuclease
BamHI. This study reveals the significance of noncovalent interactions in combination with
covalent bonding aspects when two moieties of structural similarities are joined together. This
allows the mixed imine-amide PBD dimer with a five carbon chain linker to achieve an isohelical
fit within the DNA minor groove taking in to account both the covalent bonding and the
noncovalent binding components. This has been supported by molecular modeling studies, which
indicate that the PBD dimer with a five carbon chain linker gives rise to maximum stabilization
of the complex with DNA at the minor groove as compared to the other PBD dimers with three,
four, and eight carbon chain linkers. The energy of interaction in all of the complexes studied
is correlated to the ∆Tm values. Furthermore, this dimer 5c has significant cytotoxicity in a
number of human cancer cell lines.
In tr od u ction
species.5 Their interactions with DNA are unique since
they bind within the minor groove of DNA forming a
covalent aminal bond between the C11 position of the
central B-ring and the N2 amino group of a guanine
base.5,6 The cytotoxic and antitumor activity of PBDs
are attributed to their ability to form covalent DNA
adducts. Molecular modeling, solution NMR, fluorim-
etry, and DNA footprinting experiments have shown
that these molecules have a preferred selectivity for Pu-
G-Pu sequences7,8 and can be oriented with their A-rings
pointed either toward the 3′- or the -5′ end of the
covalently bonded DNA strand as in the case of anthra-
mycin and tomaymycin. The PBDs have been shown to
inhibit endonuclease enzyme cleavage of DNA9 and to
block transcription by inhibiting DNA polymerase in a
sequence specific manner,10 processes which may be
relevant for the biological activity.
There has been considerable interest in the design
and development of DNA interactive ligands that are
capable of binding to DNA in a sequence selective
manner.1-3 However, in spite of rational design of
synthetic DNA intercalaters, few such compounds that
are in current clinical use exhibit sequence selectivity.
These compounds with the ability to target and then
down regulate individual genes have potential use as
drugs for therapy of genetic-based diseases including
some cancers4 and as research tools for using functional
genomic studies. Therefore, the synthesis of small
molecules, which exhibit DNA sequence selectivity, is
of importance for the targeting of rapidly growing tumor
cells.
The pyrrolo[2,1-c][1,4]benzodiazepine (PBD) anti-
tumor antibiotics are a well-known class of sequence
selective DNA binding agents derived from Streptomyces
The PBDs have also been used as a scaffold to attach
ethylenediaminetetraacetic acid (EDTA),11 epoxide,12
polyamide,13 and oligopyrrole14 moieties leading to novel
hybrids of PBD, which have exhibited sequence selective
DNA-cleaving and cross-linking properties. Thurston
and co-workers have synthesized homologous series of
* To whom correspondence should be addressed. Tel: +91-40-
7193157. Fax: +91-40-7160512 or +91-40-7160757. E-mail:
ahmedkamal@iict.ap.nic.in.
† Indian Institute of Chemical Technology.
‡ University of Hyderabad.
§ Centre for DNA Finger Printing and Diagnostics.
10.1021/jm020124h CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/17/2002

4680 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Kamal et al.
Sch em e 1a
a
Reagents and conditions: (i) SOCl2, L-proline methylester hydrochloride, Et3N, H2O, 0 °C, 85%. (ii) BF3‚OEt2-EtSH, CH2Cl2, room
temperature, 88%. (iii) Pd/C, H2, 40 psi, 62%.
C8 diether-linked PBD dimers (DSB-120) that span
approximately six base pairs of DNA and in which
sequence selectivity has also increased (e.g., purine-
GATC-pyrimidine).15,16 DSB-120 exhibits potent in vitro
cytotoxicity and enhances DNA binding affinity and
sequence specificity as compared to the natural product
DC-81. This improvement in biological activity has been
attributed to the ability of these compounds to link to
DNA irreversibly via guanine residues on opposite
strands because of the presence of two active sites (i.e.,
two imine functionalities).17 The in vivo studies of this
compound were not encouraging, and the low therapeu-
tic index observed was partly due to the reaction of this
compound with cellular thiol-containing molecules be-
fore reacting at the tumor site.18 Recently, another new
cross-linking PBD dimer (SJ G-136) having C2/C2′-exo
unsaturation (that exhibits high DNA binding affinity)
has been prepared by the same group.19 This investiga-
tion has highlighted the effect of C2 unsaturation on
the in vitro and in vivo cytotoxic activity. Interestingly,
the comparison of this PBD dimer with its tetralactam
analogue demonstrates that for maximum cytotoxicity
an electrophilic imine or carbinolamine moiety is es-
sential at the N10-C11 position of the PBD units. We
have been interested in the structural modifications of
the PBD ring system and development of new synthetic
strategies.20-29 It has been observed in the literature
that no effort has been made to prepare and investigate
PBD dimers with one imine functionality alone for
exploring their cytotoxicity as noncross-linking agents.
Therefore, it was of interest to synthesize and evaluate
mixed dimers of PBD that contain an imino functional-
ity in one of the PBD rings and an amido group in the
other, which are linked at the C8 position by a suitable
alkane spacer. Herein, we report the synthesis and
biological evaluation of potent mixed imine-amide PBD
dimers.
alkanes. Nitration of 10 followed by ester hydrolysis and
coupling with (2-S)-pyrrolidinecarbaxaldehyde diethyl
thioacetal gives 13. One key intermediate (14) has been
prepared by linking 8 and 13 while the alternative key
intermediate (15) has been prepared by linking 13 and
2. Compounds 14 and 15 upon reduction with tin
chloride provide 16, which has a dilactam moiety at one
end and the amino functionality on the other. Depro-
tection of the thioacetal group by using the method of
Thurston and co-workers16 affords the target molecules
5a -d that contain both the imino and the amido
functionalities (Scheme 2).
Th er m a l Den a tu r a tion Stu d ies. The DNA binding
affinity of the novel noncross-linking imine-amide PBD
dimers (5a -d ) was investigated by thermal denatur-
ation using calf thymus (CT)-DNA.9 The studies for
these compounds (5a -d ) were carried out at DNA/
ligand molar ratios of 5:1. The increase in melting
temperature (∆Tm) for each compound was examined
at 0 and 18 h incubation at 37 °C (Table 1). Data for
DC-81 and DSB-120 are included in Table 1 for com-
parison. It is observed from the data that compound 5c
is an efficient stabilizing agent for double-stranded CT-
DNA. This compound elevates the helix melting tem-
perature of the CT-DNA by a remarkable 17 °C after
incubation at 37 °C for 18 h. In the same experiment,
the DC-81 dimer (DSB-120) gives a ∆Tm of 15.4 °C. This
result illustrates the significant effect of introducing an
amide functionality for one of the PBD rings with an
increase in the size of the linker spacer from three
carbons in DSB-120 to five carbons in 5c. However, the
naturally occurring DC-81 having one imine group
exhibits a ∆Tm of 0.7 °C. It is interesting to note that
as the linker chain increases from three to five carbons,
there is an enhancement in the DNA binding affinity
and with further increase of this chain from five to eight
carbons the DNA stabilization is reduced as in the case
of 5d . As generally observed for the PBD dimers,
compounds 5a -d exert most of the effect upon the GC-
rich or high-temperature regions of the DNA melting
curves. In a manner similar to DSB-120, compound 5
provides about 75% of the stabilizing effect without prior
incubation, suggesting the kinetic effect in the PBD
reactivity profile.
Resu lts a n d Discu ssion
Ch em istr y. Synthesis of the imine-amide mixed
dimers of PBD30 has been carried out employing the
commercially available vanillin. Oxidation of vanillin
followed by benzylation and nitration by a literature
method31 provides the starting material 6. L-Proline
methyl ester has been coupled with 6 to give compound
7, which upon debenzylation with BF3.OEt2-EtSH gives
compound 8. Compound 2 has been prepared from
compound 7 by hydrogenation over Pd/C at 40 psi
(Scheme 1). The other precursor has been prepared from
vanillic acid methyl ester 9 by its etherification with
dibromoalkanes to afford 10. The monoalkylation of 9
has been achieved by using 3 molar equiv of the dibromo
These results clearly demonstrate that replacement
of one of the electrophilic N10-C11 imine moieties
present in DSB-120 with a lactam functionality and
increasing from three to five the carbon linker length,
although abolishing one of the covalent DNA interaction
sites, presumably enhance the noncovalent component
of interaction. Interestingly, a further increase in the
length to an eight carbon linker dramatically reduces
the DNA binding affinity.

Pyrrolobenzodiazepine Dimers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4681
Sch em e 2a
a
Reagents and conditions: (i) Br (CH2)nBr, K2CO3, CH3COCH3, reflux, 82-86%. (ii) SnCl4-HNO3, CH2Cl2, -25 °C, 88-91%. (iii) 1 M
LiOH, THF, MeOH, H2O (3:1:1), room temperature, 89-93%. (iv) SOCl2, DMF, THF, H2O, 2(S)-pyrrolidinecarbaldehyde diethyl thioacetal,
Et3N, 89-92%. (v) Compound 8, K2CO3, CH3COCH3, reflux, 85-90%. (vi) Compound 2, K2CO3, CH3COCH3, reflux, 72-76%. (vii) SnCl2,
2H2O, MeOH, reflux, 80-85%. (viii) HgCl2, CaCO3, CH3CN-H2O (4:1), 55-61%.
Ta ble 1. Thermal Denaturation Data for DC-81 (1), DSB-120
(3), and Compounds 5a -d with Calf Thymus DNA
Ta ble 2. log GI50, log TGI, and log LC50 MG MIDs of In Vitro
Cytotoxicity Data for Compounds 5a -c against Human Tumor
Cell Linesa
induced ∆Tm (°C)a
after incubation at 37 °C
compd
log GI50
log TGI
log LC50
[PBD]:[DNA]
compd
molar ratiob
0 h
18 h
5a
-4.88
-5.14
-6.92
-6.94
-4.37
-4.69
-5.95
-5.65
-4.08
-4.30
-4.56
-4.35
5b
5c
5a
5b
5cc
5d
1
1:5
1:5
1:5
1:5
1:5
1:5
6.5
5.0
14.0
4.2
0.3
10.2
7.0
8.5
17.0
5.0
0.7
15.4
5cb
a
GI50, drug molar concentration causing 50% cell growth
inhibition; TGI, drug concentration causing total cell growth
inhibition (0% growth); LC50, drug concentration causing 50% cell
death (-50%); MG MID, mean graph midpoints, the average
3
a
For CT-DNA alone at pH 7.00 ( 0.01, ∆Tm ) 66.5 °C ( 0.01
(mean value from 30 separate determinations); all ∆Tm values are
b
sensitivity of all cell lines toward the test agent. Repeat testing
in the primary screen values.
b
( 0.1-0.2 °C. For a 1:5 molar ratio of [ligand]/[DNA], where CT-
DNA concentration ) 100 µM and ligand concentration ) 20 µM
in aqueous sodium phosphate buffer [10 mM sodium phosphate
+ 1 mM EDTA, pH 7.00 ( 0.01]. c The ∆Tm for 5c at a [ligand]:
[DNA] molar ratio of 1:5 increased to a value of 17.0 °C after 18
h of incubation.
causing 50% cell growth inhibition (GI50), total cell
growth inhibition (TGI, 0% growth), and 50% cell death
(LC50, -50% growth) as compared with the control was
calculated. The mean graph midpoint (MG MID) values
of log TGI and log LC50 as well as log GI50 for 5a -c are
listed in Table 2. As demonstrated by mean graph
pattern, compound 5c exhibits an interesting profile of
activity and selectivity for various cell lines. The MG
MID of log TGI and log LC50 showed a pattern similar
to the log GI50 MG MIDs.
The comparison of the data in Table 3 reveals the
importance of an alkane spacer. As the alkane spacer
increased from three to five, the cytotoxic activity was
moderately enhanced. The five carbon spacer of com-
pound 5c confers a suitable fit in the minor groove of
Cytotoxicity. Compounds 5a -c were evaluated in
vitro against sixty human tumor cells derived from nine
cancer types (leukemia, nonsmall-cell lung cancer, colon
cancer, central nervous system (CNS) cancer, mela-
noma, 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 of continuous drug exposure was used, and a
sulforhodamine B (SRB) protein assay was used to
estimate cell viability or growth. The concentration

4682 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Kamal et al.
Ta ble 3. Log LC50 (Concentration in mol/L Causing 50%
Lethality) Values for Compounds 5a -ca
cancer
5a
5b
5c
5cb
leukemia
nonsmall-cell lung
colon
-4.48
-4.00
-4.11
-4.00
-4.08
-4.02
-4.04
-4.00
-4.02
-4.62
-4.23
-4.24
-4.34
-4.38
-4.23
-4.26
-4.17
-4.27
-5.32
-4.10
-4.52
-4.43
-5.48
-4.22
-4.35
-4.00
-4.44
-4.00
-4.23
-4.46
-4.44
-5.00
-4.06
-4.29
-4.00
-4.42
CNS
melanoma
ovarian
renal
prostate
breast
a
Each cancer type represents the average of 6-8 different
b
cancer cell lines. Repeat testing in the primary screen values.
F igu r e 2. Lane 1, control plasmid DNA; lane 2, BamHI
linearized DNA; lane 3-10, PBD-plasmid DNA complexes
digested by BamHI with PBD (5a ) concentrations of 1.8, 3.7,
5.5, 7.3, 9.2, 11.0, 12.8, and 14.6 µM, respectively.
Ta ble 4. Stoichiometric Ratios of Drug to DNA Used for
RED100 Assay
concn of
concn of
drug (µM) drug:DNA ratio
drug (µM) drug:DNA ratio
1.8
3.7
5.5
7.3
16.6:1
8.1:1
5.4:1
4.1:1
9.2
11.0
12.8
14.6
3.2:1
2.7:1
2.3:1
2.05:1
F igu r e 1. Structures of the PBD monomer DC-81 (1), PBD
dilactam (2), and PBD dimers DSB-120 (3), SJ G-136 (4), and
5.
Because the drug was preincubated with PBD, the
DNA-drug complexes were subjected to BamHI diges-
tion, and the protection of cleavage of the GVGATCC
sequence by PBD suggests that these molecules selec-
tively interact with G-rich sequences in DNA. The
inability of PBD in protecting cleavage of AATVATT by
SspI suggests that these PBD have low affinity to AT-
rich sequences. Therefore, PBD prefers G-rich sequences
in DNA binding; 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.9
Molecu la r Mod elin g Stu d ies. Energetically favor-
able models of the DNA-PBD-dimer complexes for
5a -d molecules were built using the systematic proce-
dure as described in the methods section, involving
molecular modeling and docking followed by detailed
molecular dynamic simulations. The energy of interac-
tion (Eint) between the DNA and the PBD-dimer
molecule in a complex was calculated as a measure of
stability of that complex. Table 5 gives the values of Eint
obtained for the four complexes. The lowest value of Eint
corresponds to the DNA-5c complex (Figure 3) indicat-
ing that the molecule 5c forms energetically the most
stable complex with DNA. It is interesting to note that
the Eint progressively decreases indicating increase in
stability as the number of alkane spacer units increases
from three to five. However, further increase in the
double helix DNA and shows slightly higher activity in
this series of compounds 5a -c. This observation was
supported by molecular modeling and thermal dena-
turation studies. Furthermore, melanoma, leukemia,
colon, and CNS are the more sensitive cancers to these
compounds 5a -c.
In h ibition of Restr iction En d on u clea se Ba m HI.
The melting temperature study indicated that PBD
possesses DNA binding affinity. To confirm their inter-
action with DNA restriction endonuclease, a protection
assay was performed. The RED100 assay is based upon
the ability of PBD to inhibit the cleavage activity of
restriction endonuclease BamHI. The inhibition of
BamHI cleavage activity by the ligand bound to the
DNA suggests that PBD binding sites overlap with the
BamHI cleavage site. The study was carried out to
determine the stoichiometric ratio at which PBD inhib-
its the DNA linearization by BamHI. The results of this
experiment for a representative compound 5a as shown
in Figure 2 suggest that the PBD inhibits BamHI
digestion in a dose-dependent manner with a stoichio-
metric ratio and are illustrated in Table 4. It is observed
that compound 5a binds to DNA at a stoichiometric ratio
of 2.05:1.

Pyrrolobenzodiazepine Dimers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4683
Ta ble 5. Values of Energy of Interactions Calculated for the
DNA-PBD Dimer Complexes
complex
energy of interaction (Eint) in kcal mol-1
DNA-5a
DNA-5b
DNA-5c
DNA-5d
-123.4
-129.2
-139.7
-137.5
F igu r e 4. Plot of the calculated values of energy of interac-
tions (Eint in kcal mol-1, 9) and the melting temperatures (4Tm
in °C, [ (0 h) and 2 (18 h)) measured from thermal denatur-
ation studies for the four DNA-PBD dimer complexes vs the
number of spacer units.
tribute to the stability in all of the complexes, in
addition to the covalent linkage formed between the
imine PBD subunit and the G8. Only in the cases of
5c,d , the complexes are further stabilized by a hydrogen
bond formed between the carbonyl oxygen of the C10-
N11 amide functionality of the PBD subunit and the
amino group of the 12th guanine nucleotide. It is worth
noting that the amide PBD subunits in both the
complexes of 5c,d occupy the same site in the minor
groove. The complex formed by 5c is energetically more
stable than the complex formed by 5d because the chain
of alkane spacer units in 5c forms a better isohelical fit
(and thus gives rise to more favorable nonbonded
interactions) within the minor groove than the longer
chain in 5d .
Con clu sion s
Introduction of an amide functionality in one of the
PBD rings and an increase of the dimer’s linker length
to a five carbon chain significantly enhance the DNA
binding ability of the noncross-linking PBD dimer as
evaluated by thermal denaturation studies. Modeling
studies suggest that apart from the covalent linkage
formed between the imine PBD subunit and the G8,
there are a number of favorable van der Waals and
Coulombic interactions that are formed between the
DNA and the mixed imine-amide PBD dimer. Further-
more, the complex formed by compound 5c (with a five
carbon chain linker) is energetically more stable than
the complexes formed by 5a ,b,d as it forms a better
isohelical fit within the minor groove of DNA. The
restriction endonuclease studies suggest that these
molecules selectively interact with G sequences in DNA
and have low affinity to AT-rich sequences. In addition,
compound 5c exhibits an interesting profile of in vitro
cytotoxic activity and selectivity for various cell lines.
This compound is being evaluated in the hollow fiber
assay and a number of human tumor xenografts and is
likely to be taken up for preclinical development.
F igu r e 3. Projection diagram showing the DNA-5c complex.
(a) Side-on view and (b) down the helix axis.
number of spacer units from five to eight does not lead
to an increase in the Eint; in fact, Eint is lowered by about
2 kcal mol-1. The Eint values are, in fact, correlated to
the ∆Tm values measured from thermal denaturation
experiments (Figure 4).
In general, a number of favorable van der Waals and
Coulombic interactions are formed between the DNA
and the PBD-dimer molecules and they together con-

4684 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Kamal et al.
4.06 (m, 1H), 6.50 (s 1H), 7.45 (s,1H), 8.10 (br s, 1H). MS (EI)
m/z 262 [M]+‚.
Exp er im en ta l Section
Syn th etic Ch em istr y. Reaction progress was monitored
by thin-layer chromatography (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 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 (THF) (sodium benzophenone ketyl), methanol (magne-
sium methoxide), acetonitrile (calcium hydride).
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 TMS. Spin multiplicities are described
as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet), and 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 a trap current of 200 µA and 4 kV of
acceleration voltage. High resolution mass spectra (HRMS)
were recorded on VG Autospec N mass spectrometer at 200
°C, 70 eV, with a trap current of 200 µA and 7 kV of
acceleration voltage.
Meth yl-4-(3-br om op r op oxy)-3-m eth oxyben zoa te (10a ).
To a solution of vanillin methyl ester 9 (182 mg, 1 mmol) in
acetone (30 mL) were added anhydrous K2CO3 (553 mg, 4
mmol) and 1,3-dibromo propane (605 mg, 3 mmol), and the
mixture was refluxed in an oil bath for 48 h. The reaction was
monitored by TLC using EtOAc-hexane (2:8), K2CO3 was
removed by filtration, and the solvent was evaporated under
the vacuum and was purified by column chromatography (10%
EtOAc-hexane) to afford compound 10a as white solid (260
mg, 86%); mp 117-118 °C. 1H NMR (CDCl3): δ 2.35-2.45 (m,
2H), 3. 64 (t, 2H, J ) 6.3 Hz), 3.94 (s, 3H), 3.98 (s, 3H), 4.20
(t, 2H, J ) 6 Hz), 6.88 (d, 1H, J ) 8.2 Hz), 7.50 (s, 1H), 7.65
(d, 1H, J ) 8.8 Hz). MS (EI) m/z 303 [M]+‚.
Meth yl-4-(4-br om obu toxy)-3-m eth oxyben zoa te (10b).
The compound 10b was prepared according to the method
described for the compound 10a employing vanillin methyl
ester 9 (182 mg, 1 mmol) and 1,4-dibromobutane (648 mg, 3
mmol), and the crude product was purified by column chro-
matography (10% EtOAc-hexane) to afford compound 10b as
1
white solid (260 mg, 82%); mp 122-124 °C. H NMR (CDCl3):
δ 2.0-2.2 (m, 4H), 3.55 (t, 2H, J ) 6.3 Hz), 3.94 (s, 3H), 3.97
(s, 3H), 4.15 (t, 2H, J ) 6.1 Hz), 6.88 (d, 1H, J ) 8.2 Hz), 7.55
(s, 1H), 7.65 (d, 1H, J ) 8.8 Hz). MS (EI) m/z 317 [M]+‚.
Meth yl-4-(5-Br om open tyloxy)-3-m eth oxyben zoate (10c).
The compound 10c was prepared according to the method
described for the compound 10a employing vanillin methyl
ester 9 (182 mg, 1 mmol) and 1,5-dibromopentane (689 mg, 3
mmol), and the crude product was purified by column chro-
matography (10% EtOAc-hexane) to afford compound 10c as
a white solid (275 mg, 83%); mp 125-127 °C. 1H NMR
(CDCl3): δ 1.8-2.0 (m, 6H), 3.48 (t, 2H, J ) 6.4 Hz), 3.93 (s,
3H), 3.96 (s, 3H), 4.15 (t, 2H, J ) 5.9 Hz), 6.85 (d, 1H, J ) 8.2
Hz), 7.55 (s, 1H), 7.65 (d, 1H, J ) 8.8 Hz). MS (EI) m/z 331
[M]+‚.
Meth yl-(2S)-N-[4-ben zyloxy-5-m eth oxy-2-n itr oben zoyl]-
p yr r olid in e-2-ca r boxyla te (7). To a stirred suspension of
compound 6 (303 mg, 1 mmol) and thionyl chloride (476 mg,
4.0 mmol) in dry benzene (15 mL) was added dimethylforma-
mide (DMF) (4-5 drops), and the stirring was continued for 6
h. The benzene was evaporated in a vacuum, and the resultant
oil was dissolved in dry THF (20 mL) and added dropwise over
a
period of 30 min to a stirred suspension of L-proline
methylester hydrochloride (248 mg 1.5 mmol), triethylamine
(303 mg, 3 mmol), and ice water (20 mL) cooled in an ice bath.
After the addition was completed, the reaction mixture was
brought to ambient temperature and stirred for an additional
hour. The THF was evaporated in a 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 a vacuum and was purified by column
chromatography (30% EtOAc-hexane) to afford compound 7
as yellow oil (352 mg, 85%). 1H NMR (CDCl3): δ 1.80-2.10
(m, 3H), 2.12-2.35 (m, 1H), 3.10-3.18 (m, 1H),3.20-3.30 (m,
1H), 3.70 (s, 3H), 3.92 (s, 3H), 4.60-4.70 (m, 1H), 5.10 (s, 2H),
6.80 (s, 1H), 7.25-7.42 (m, 5H), 7.60 (s, 1H). MS (EI) m/z 414
[M]+‚.
Meth yl-(2S)-N-[4-h yd r oxy-5-m eth oxy-2-n itr oben zoyl]-
p yr r olid in e-2-ca r boxyla te (8). To a stirred solution of EtSH
(1.20 g, 19 mmol) and BF3‚OEt2 (1.419 g, 10 mmol) was added
dropwise compound 7 (414 mg, 1 mmol) in dichloromethane
(15 mL) at room temperature. Stirring was continued until
the reaction was completed by TLC, and then, the solvent was
evaporated in a vacuum. The residue was quenched with 5%
NaHCO3 solution (20 mL) and then extracted with dichlo-
romethane (2 × 20 mL). The combined organic phases were
washed with saturated brine (10 mL) and dried over Na2SO4,
and the solvent was removed by vacuum and purified by
column chromatography (70% EtOAc-hexane) to afford com-
pound 8 as pale yellow oil (285 mg, 88%). 1H NMR (CDCl3): δ
1.87-2.50 (m, 4H), 3.15-3.40 (m, 2H), 3.80 (s, 3H), 4.0 (s, 3H),
4.55-4.70 (m, 1H), 6.80 (s, 1H), 7.68 (s, 1H). MS (EI) m/z 265
[M-COOCH3]+‚.
Meth yl-4-(8-br om ooctyloxy)-3-m eth oxyben zoa te (10d ).
The compound 10d was prepared according to the method
described for the compound 10a employing vanillin methyl
ester 9 (182 mg, 1 mmol) and 1,8-dibromooctane (816 mg, 3
mmol), and the crude product was purified by column chro-
matography (10% EtOAc-hexane) to afford compound 10c as
1
an oil (275 mg, 83%). H NMR (CDCl3): δ 1.30-1.60 (m, 8H),
1.85-2.00 (m, 4H) 3.40 (t, 2H, J ) 6.2 Hz), 3.93 (s, 3H), 3.96
(s, 3H), 4.15 (t, 2H, J ) 6 Hz), 6.85 (d, 1H, J ) 8.2 Hz), 7.55
(s, 1H), 7.65 (d, 1H, J ) 8.8 Hz). MS (EI) m/z 373 [M]+‚.
Me t h yl-4-(3-b r om op r op oxy)-5-m e t h oxy-2-n it r ob e n -
zoa te (11a ). A freshly prepared mixture of stannic chloride
(260 mg, 1.156 mmol) and fuming nitric acid (98 mg, 1.56
mmol) in dichloromethane was added dropwise over 5 min with
stirring to a solution of 10a (303 mg, 1 mmol) in dichlo-
romethane (30 mL) at -25 °C (dry ice/carbon tetrachloride).
The mixture was maintained at -25 °C for a further 5 min,
quenched with water (20 mL), and then allowed to return to
room temperature. The organic layer was separated, and the
aqueous layer was extracted with dichloromethane (2 × 20
mL). The combined organic phase was dried (Na2SO4) and
evaporated in a vacuum, and it was purified by column
chromatography (20% EtOAc-hexane) to give 11a as a yellow
1
oily liquid (313 mg, 90%). H NMR (CDCl3): δ 2.30-2.48 (m,
2H), 3.55-3.65 (t, 2H, J ) 6.2 Hz), 3.89 (s, 3H), 3.95 (s, 3H),
4.25 (t, 2H, J ) 6.1 Hz), 7.05 (s, 1H), 7.45 (s, 1H). MS (EI) m/z
348 [M]+‚.
Me t h y l-4-(4-b r o m o b u t o x y )-5-m e t h o x y -2-n it r o b e n -
zoa te (11b). Compound 10b (317 mg, 1 mmol) was nitrated
by the similar method described earlier for 11a to get 11b as
(11aS)-8-Hydr oxy-7-m eth oxy-1,2,3,10,11,11a-h exah ydr o-
5H-p yr r olo[2,1-c][1,4]ben zod ia zep in e-5,11-d ion e (2). The
compound 7 (414 mg, 1.0 mmol) was dissolved in methanol
(10 mL), and 10% Pd-C (200 mg) was added. The mixture
was hydrogenated at room temperature under atmospheric
pressure for 10 h. The catalyst was removed by filtration
through Celite, and then, the solvent was evaporated under
vacuum and purified by column chromatography (80% EtOAc-
hexane) to afford 2 as a pluffy solid (162 mg, 62%); mp 278-
280 °C. 1H NMR (CDCl3): δ 1.90-2.10 (m, 3H), 2.75-2.85
(m,1H), 3.50-3.67 (m, 1H), 3.70-3.85 (m, 1H), 3.94 (s, 3H),
1
a pale yellow oily liquid (218 mg, 88%). H NMR (CDCl3): δ
1.95-2.15 (m, 4H), 3.45 (t, 2H, J ) 6.34 Hz), 3.85 (s, 3H), 3.95
(s, 3H), 4.10 (t, 2H, J ) 6.12 Hz), 7.02 (s, 1H), 7.38 (s, 1H).
MS (EI) m/z 362 [M]+‚.
Met h yl-4-(5-b r om op en t yloxy)-5-m et h oxy-2-n it r ob en -
zoa te (11c). Compound 10c (331 mg, 1 mmol) was nitrated
by the similar method described earlier for 11a to get 11c as
1
a light yellow oily liquid (345 mg, 91%). H NMR (CDCl3): δ

Pyrrolobenzodiazepine Dimers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4685
1.75-2.0 (m, 6H), 3.40 (t, 2H, J ) 6.31 Hz), 3.85 (s, 3H), 3.95
(s, 3H), 4.0-4.1 (t, 2H, J ) 6 Hz), 7.0 (s,1H), 7.4 (s,1H). MS
(EI) m/z 376 [M]+‚.
3H), 4.10 (t, 2H, J ) 6 Hz), 4.60-4.71 (m, 1H), 4.85 (d, 1H, J
) 4.3 Hz), 6.80 (s, 1H), 7.65 (s, 1H). MS (FAB) 535 [M]+‚.
(2S)-N-[4-(5-Br om op en t yloxy)-5-m et h oxy-2-n it r ob en -
zoyl]p yr r olid in e-2-ca r boxa ld eh yd e Dieth yl Th ioa ceta l
(13c). The compound 13c was prepared according to the
method described for the compound 13a by employing 12c (362
mg, 1 mmol) and (2S)-pyrrolidine-2-carboxaldehyde diethyl
Me t h yl-4-(8-b r om ooct yloxy)-5-m e t h oxy-2-n it r ob e n -
zoa te (11d ). Compound 10d (373 mg, 1 mmol) was nitrated
by the similar method described earlier for 11a to get 11d as
1
a light yellow oily liquid (376 mg, 90%). H NMR (CDCl3): δ
1
thioacetal, which gave 13c as a oily liquid (505 mg, 92%). H
1.30-1.60 (m, 8H), 1.85-2.00 (m, 4H) 3.40 (t, 2H, J ) 6.38
Hz), 3.93 (s, 3H), 3.96 (s, 3H), 4.15 (t, 2H, J ) 6.21 Hz), 6.85
(s, 1H), 7.60 (s, 1H).
NMR (CDCl3): δ 1.21-1.40 (m, 8H), 1.60-2.40 (m, 8H), 2.65-
2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.45 (t, 2H, J ) 6.34 Hz),
3.95 (s, 3H), 4.10 (t, 2H, J ) 6 Hz), 4.60-4.71 (m, 1H), 4.85
(d, 1H, J ) 4.33 Hz), 6.80 (s, 1H), 7.65 (s, 1H). MS (FAB) 549
[M]+‚.
(2S)-N-[4-(5-Br om ooctyloxy)-5-m eth oxy-2-n itr oben zoyl]-
p yr r olid in e-2-ca r boxa ld eh yd e Dieth yl Th ioa ceta l (13d ).
The compound 13d was prepared according to the method
described for the compound 13a by employing 12d (404 mg, 1
mmol) and (2S)-pyrrolidine-2-carboxaldehyde diethyl thioac-
etal, which gave 13d as an oily liquid (532 mg, 90%). 1H NMR
(CDCl3): δ 1.21-1.60 (m, 14H), 1.80-2.20 (m, 8H), 2.65-2.85
(m, 4H), 3.15-3.30 (m, 2H), 3.45 (t, 2H, J ) 6.2 Hz), 3.95 (s,
3H), 4.10 (t, 2H, J ) 6.1 Hz), 4.60-4.71 (m, 1H), 4.85 (d, 1H,
J ) 4.3 Hz), 6.80 (s, 1H), 7.70 (s, 1H).
4-(3-Br om op r op oxy)-5-m et h oxy-2-n it r ob en zoic a cid
(12a ). An amount of 2 N lithium hydroxide monohydrate (1.22
mL) was added to a solution of 11a (348 mg, 1 mmol) in THF-
H2O-MeOH (4:1:1), and the mixture was stirred at room
temperature for 12 h. After most of the THF and methanol
was evaporated, the aqueous phase was acidified with 12 N
HCl to pH 7 and reextracted with EtOAc to give a 12a as a
1
white solid (297 mg, 89%); mp 118-120 °C. H NMR (CDCl3):
δ 2.28-2.48 (m, 2H), 3.6 (t, 2H, J ) 6.4 Hz), 3.98 (s, 3H), 4.25
(t, 2H, J ) 6.2 Hz), 7.20 (s, 1H), 7.40 (s, 1H).
4-(4-Br om ob u t oxy)-5-m et h oxy-2-n it r ob en zoic
Acid
(12b). The compound (12b) was prepared by the same method
described earlier for 12a employing 11b (362 mg, 1 mmol),
which gave 12b as a white solid (324 mg, 93%); mp 176-178
°C. 1H NMR (CDCl3): δ 2.0-2.15 (m, 4H), 3.50 (t, 2H, J )
6.35 Hz), 3.96 (s, 3H), 4.15 (t, 2H, J ) 6.11 Hz), 7.18 (s, 1H),
7.38 (s, 1H).
(2S)-N-{4-[Meth yl-(2S)-N-(5-m eth oxy-2-n itr oben zoyl)-
p yr r olid in e-ca r b oxyla t e-4-yloxy]p r op oxy-5-m et h oxy-2-
n itr oben zoyl}pyr r olidin e-2-car boxaldeh yde Dieth yl Th io-
a ceta l (14a ). To a solution of 13a (521 mg, 1 mmol) in dry
acetone (30 mL) was added anhydrous K2CO3 (552 mg, 4 mmol)
and 8 (324 mg, 1 mmol). The reaction mixture was refluxed
in an oil bath for 48 h. The reaction was monitored by TLC
using EtOAc-hexane (6:4) as a solvent system. K2CO3 was
removed by filtration, and the solvent was removed under
vacuum. The crude product was purified by column chroma-
tography (60%EtoAc-hexane) to afford a yellow oil (688 mg,
4-(5-Br om op en tyloxy)-5-m eth oxy-2-n itr oben zoic Acid
(12c). The compound (12c) was prepared by the same method
described earlier for 12a employing 11c (376 mg, 1 mmol),
which gave 12c as a white solid (330 mg, 91%); mp 166-168
1
°C. H NMR (CDCl3): δ 1.9-2.1 (m, 6H), 3.45 (t, 2H, J ) 6.3
Hz), 3.95 (s, 3H), 4.1-4.15 (t, 2H, J ) 6.2 Hz), 7.18 (s, 1H),
7.38 (s, 1H).
1
90%). H NMR (CDCl3): δ 1.20-1.40 (m, 6H), 1.50-2.40 (m,
4-(8-Br om ooct yloxy)-5-m et h oxy-2-n it r ob en zoic Acid
(12d ). The compound (12d ) was prepared by the same method
described earlier for 12a employing 11d (418 mg, 1 mmol),
10H), 2.60-2.90 (m, 4H), 3.10-3.40 (m, 4H), 3.80 (s 3H), 3.90
(s, 3H), 3.98 (s, 3H), 4.18-4.38 (m, 4H), 4.60-4.75 (m, 2H),
4.85 (d, 1H, J ) 4.4 Hz), 6.70 (s, 1H), 6.80 (s, 1H), 7.6 (s, 1H),
7.70 (s, 1H). MS (FAB) 765 [M + H]+‚.
1
which gave 12d as an oil (355 mg, 88%). H NMR (CDCl3): δ
1.30-1.60 (m, 8H), 1.85-2.00 (m, 4H) 3.40 (t, 2H, J ) 6.32
Hz), 3.96 (s, 3H), 4.15 (t, 2H, J ) 6 Hz), 6.85 (s, 1H), 7.60 (s,
1H). MS (EI) m/z 404 [M]+‚.
(2S)-N-{4-[Meth yl-(2S)-N-(5-m eth oxy-2-n itr oben zoyl)-
p yr r olid in e-ca r b oxyla t e-4-yloxy]b u t oxy-5-m et h oxy-2-
n itr oben zoyl}pyr r olidin e-2-car boxaldeh yde Dieth yl Th io-
a ceta l (14b). The compound 14b was prepared according to
the method described for 14a by employing 8 and 13b (535
(2S)-N-[4-(3-Br om opr opoxy)-5-m eth oxy-2-n itr oben zoyl]-
p yr r olid in e-2-ca r boxa ld eh yd e Dieth yl Th ioa ceta l (13a ).
DMF was added to a stirred suspension of compound 12a (334
mg, 1 mmol) and thionyl chloride (476 mg, 4 mmol) in dry
benzene (15 mL), and the stirring was continued for 6 h. The
benzene was evaporated in vacuo, and the resultant oil was
dissolved in dry THF (20 mL) and added dropwise over a
period of 30 min to a stirred suspension of (2S)-pyrrolidine-
2-carboxaldehyde diethyl thioacetal (205 mg, 1 mmol), triethyl-
amine (303 mg, 3 mmol), and ice water (20 mL) cooled in an
ice bath. After the addition was completed, the reaction
mixture was brought to ambient temperature and stirred for
an additional hour. The THF was evaporated in a 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 a vacuum, and the crude
product was purified by column chromatography (30%EtoAc-
hexane) to afford 13a as a oily liquid (464 mg, 89%). NMR
(CDCl3): δ 1.20-1.40 (m, 6H), 1.70-2.50 (m, 6H), 2.60-2.90
(m, 4H), 3.20-3.35 (m, 2H), 3.65 (t, 2H, J ) 6.22 Hz), 3.95
(s, 3H), 4.25(t, 2H, J ) 5.98 Hz), 4.63-4.75 (m, 1H), 4.85 (d,
1H, J ) 4.3 Hz), 6.80 (s, 1H), 7.70 (s, 1H). MS (FAB) 521
[M]+‚.
1
mg, 1 mmol) to afford 14b as a yellow oil (662 mg, 85%). H
NMR (CDCl3): δ 1.20-1.40 (m, 6H), 1.50-2.40 (m, 12H), 2.60-
2.90 (m, 4H), 3.10-3.40 (m, 4H), 3.80 (s 3H), 3.88 (s, 3H), 3.98
(s, 3H), 4.10-4.20 (m, 4H), 4.60-4.80 (m, 2H), 4.85 (d, 1H, J
) 4.3 Hz), 6.70 (s, 1H), 6.80 (s, 1H), 7.6 (s, 1H), 7.70 (s, 1H).
MS (FAB) 779 [M + H]+‚.
(2S)-N-{4-[Meth yl-(2S)-N-(5-m eth oxy-2-n itr oben zoyl)-
p yr r olid in e-ca r boxyla te-4-yloxy]p en tyloxy-5-m eth oxy-2-
n itr oben zoyl}pyr r olidin e-2-car boxaldeh yde Dieth yl Th io-
a ceta l (14c). The compound 14c was prepared according to
the method described for 14a by employing 8 and 13c (549
mg, 1 mmol) to afford 14c as a yellow oil (697 mg, 88%). 1H
NMR (CDCl3): δ 1.20-1.40 (m, 8H), 1.50-2.40 (m, 12H), 2.60-
2.90 (m, 4H), 3.10-3.40 (m, 4H), 3.80 (s 3H), 3.88 (s, 3H), 3.98
(s, 3H), 4.10-4.20 (m, 4H), 4.60-4.80 (m, 2H), 4.85 (d, 1H, J
) 4.2 Hz), 6.70 (s, 1H), 6.80 (s, 1H), 7.6 (s, 1H), 7.70 (s, 1H).
MS (FAB) 794 [M + H]+‚.
(2S)-N-{4-[Meth yl-(2S)-N-(5-m eth oxy-2-n itr oben zoyl)-
p yr r olid in e-ca r boxyla te-4-yloxy]octyloxy-5-m eth oxy-2-
n itr oben zoyl}pyr r olidin e-2-car boxaldeh yde Dieth yl Th io-
a ceta l (14d ). The compound 14d was prepared according to
the method described for 14a by employing 8 and 13d (591
1
mg, 1 mmol) to afford 14d as a yellow oil (697 mg, 88%). H
(2S)-N-[4-(4-Br om obu toxy)-5-m eth oxy-2-n itr oben zoyl]-
p yr r olid in e-2-ca r boxa ld eh yd e Dieth yl Th ioa ceta l (13b).
The compound 13b was prepared according to the method
described for the compound 13a by employing 12b (348 mg, 1
mmol) and (2S)-pyrrolidine-2-carboxaldehyde diethyl thio-
acetal, which gave 13b as a oily liquid (476 mg, 89%). 1H NMR
(CDCl3): δ 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, 2H, J ) 6.25 Hz), 3.95 (s,
NMR (CDCl3): δ 1.20-1.60 (m, 14H), 1.80-2.40 (m, 12H),
2.60-2.90 (m, 4H), 3.10-3.40 (m, 4H), 3.80 (s 3H), 3.88 (s,
3H), 3.98 (s, 3H), 4.10-4.20 (m, 4H), 4.60-4.80 (m, 2H), 4.85
(d, 1H, J ) 4.38 Hz), 6.70 (s, 1H), 6.80 (s, 1H), 7.6 (s, 1H),
7.70 (s, 1H). MS (FAB) 835 [M]+‚.
(2S)-N-{4-[3-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H-p yr r olo[2,1-c]-[1,4]ben zod ia zep in e-5,11-d ion e-8-

4686 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Kamal et al.
yloxy)p r op oxy]-5-m eth oxy-2-n itr oben zoyl}p yr r olid in e-
2-ca r boxa ld eh yd e Dieth yl Th ioa ceta l (15a ). To a solution
of 13a (521 mg, 1 mmol) in dry acetone (30 mL) was added
anhydrous K2CO3 (552 mg, 4 mmol) and 2 (262 mg, 1 mmol).
The reaction mixture was refluxed in an oil bath for 48 h. The
reaction was monitored by TLC using EtOAc-hexane (9:1) as
a solvent system. K2CO3 was removed by filtration, and the
solvent was removed under vacuum. The crude product was
purified by column chromatography (90%EtoAc-hexane) to
yloxy)bu t oxy]-5-m et h oxy-2-a m in oben zoyl}p yr r olid in e-
2-car boxaldeh yde Dieth yl Th ioacetal (16b). The compound
16b was prepared according to the method described for the
compound 16a employing the compound 14b/15b (778 mg/716
mg, 1 mmol) to afford the amino diethyl thioacetal 16b as a
yellow liqiud (549 mg, 80%/562 mg, 82%). 1H NMR (CDCl3):
δ 1.20-1.40 (m, 6H), 1.50-2.40 (m, 11H), 2.50-2.85 (m, 5H),
3.45-3.70 (m, 4H), 3.75 (s, 3H), 3.90 (s, 3H), 4.0-4.20 (m, 5H),
4.55-4.75 (m, 2H), 6.20 (s, 1H), 6.50 (s, 1H), 6.85 (s, 1H), 7.40
(s, 1H), 8.45 (br s, NH exchangeable).
1
afford a yellow oil (688 mg, 90%). H NMR (CDCl3): δ 1.20-
1.65 (m, 6H), 1.70-2.60 (m, 9H), 2.65-2.98 (m, 5H), 3.20-
3.40 (m, 2H), 3.50-3.75 (m, 1H), 3.75-3.90 (m, 1H) 3.95 (s
3H), 4.0 (s, 3H), 4.06 (m, 1H), 4.20-4.45 (m, 4H), 4.65-4.85
(m, 1H), 4.90 (d, 1H, J ) 4.28 Hz), 6.55 (s, 1H), 6.85 (s, 1H),
7.48 (s, 1H), 7.75 (s, 1H), 7.90 (br s, NH exchangeable). MS
(FAB) 703 [M + H]+‚.
(2S)-N-{4-[5-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H -p yr r olo[2,1-c][1,4]b en zod ia zep in e-5,11-d ion e-8-
yloxy)pen tyloxy]-5-m eth oxy-2-am in oben zoyl}pyr r olidin e-
2-car boxaldeh yde Dieth yl Th ioacetal (16c). The compound
16c was prepared according to the method described for the
compound 16a employing the compound 14c/15c (792 mg/730
mg, 1 mmol) to afford the amino diethyl thioacetal 16c as a
yellow liquid (574 mg, 80%/588 mg, 84%). 1H NMR (CDCl3):
δ 1.20-1.40 (m, 8H), 1.50-2.40 (m, 11H), 2.50-2.85 (m, 5H),
3.45-3.70 (m, 4H), 3.75 (s, 3H), 3.90 (s, 3H), 4.0-4.20 (m, 5H),
4.55-4.75 (m, 2H), 6.20 (s, 1H), 6.50 (s, 1H), 6.85 (s, 1H), 7.40
(s, 1H), 8.52 (br s, NH exchangeable).
(2S)-N-{4-[4-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H-p yr r olo[2,1-c]-[1,4]ben zod ia zep in e-5,11-d ion e-8-
yloxy)bu toxy]-5-m eth oxy-2-n itr oben zoyl]p yr r olid in e-2-
ca r boxa ld eh yd e Dieth yl Th ioa ceta l (15b). The compound
15b was prepared according to the method described for 15a
by employing 2 and 13b (535 mg, 1 mmol) to afford 15b as a
1
yellow oil (662 mg, 85%). H NMR (CDCl3): δ 1.20-1.60 (m,
(2S)-N-{4-[8-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H -p yr r olo[2,1-c][1,4]b en zod ia zep in e-5,11-d ion e-8-
yloxy)octyloxy]-5-m eth oxy-2-a m in oben zoyl]p yr r olid in e-
2-car boxaldeh yde Dieth yl Th ioacetal (16d). The compound
16d was prepared according to the method described for the
compound 16a employing the compound 14d /15d (834 mg/772
mg, 1 mmol) to afford the amino diethyl thioacetal 16d as a
yellow liquid (593 mg, 80%/623 mg, 84%). 1H NMR (CDCl3):
δ 1.20-1.60 (m, 14H), 1.80-2.30 (m, 11H), 2.50-2.85 (m, 5H),
3.45-3.70 (m, 4H), 3.75 (s, 3H), 3.90 (s, 3H), 4.0-4.20 (m, 5H),
4.55-4.75 (m, 2H), 6.20 (s, 1H), 6.50 (s, 1H), 6.85 (s, 1H), 7.40
(s, 1H), 8.40 (br s, NH exchangeable).
6H), 1.60-2.20 (m, 11H), 2.50-3.0 (m, 5H), 3.15-3.40 (m, 2H),
3.50-3.80 (m, 2H), 3.85 (s 3H), 3.95 (s, 3H), 4.0-4.30 (m, 5H),
4.60-4.75 (m, 1H), 4.85 (d, 1H, J ) 4.4 Hz), 6.40 (s, 1H), 6.78
(s, 1H), 7.40 (s, 1H), 7.65 (s, 1H), 8.20-8.40 (br s, NH
exchangeable). MS (FAB) 717 [M + H]+‚.
(2S)-N-{4-[5-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H-p yr r olo[2,1-c]-[1,4]ben zod ia zep in e-5,11-d ion e-8-
yloxy)p en tyloxy]-5-m eth oxy-2-n itr oben zoyl]p yr r olid in e-
2-car boxaldeh yde Dieth yl Th ioacetal (15c). The compound
15c was prepared according to the method described for 15a
by employing 2 and 13c (549 mg, 1 mmol) to afford 15c as a
1
yellow oil (697 mg, 88%). H NMR (CDCl3): δ 1.20-1.50 (m,
7-Me t h o x y -8-{3-[7-m e t h o x y -(11a S )-1,2,3,10,11,11a -
h exa h yd r o-5H-P BD-5,11-d ion e-8-yloxy]p r op oxy}-(11a S)-
1,2,3,11a-tetr ah ydr o-5H-pyr r olo[2,1-c][1,4]ben zodiazepin -
5-on e (5a ). A solution of 16a (672 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
6H), 1.50-2.50 (m, 13H), 2.70-2.90 (m, 5H), 3.20-3.40 (m,
2H), 3.50-3.90 (m, 2H), 3.95 (s 3H), 4.0 (s, 3H), 4.0-4.25 (m,
5H), 4.65-4.80 (m, 1H), 4.90 (d, 1H, J ) 4.38 Hz), 6.45 (s,
1H), 6.85 (s, 1H), 7.48 (s, 1H), 7.70 (s, 1H), 8.40 (br s, NH
exchangeable). MS (FAB) 731 [M + H]+‚.
((2S)-N-{4-[8-(7-Meth oxy-(11aS)-1,2,3,10,11,11a -h exa h y-
d r o-5H-p yr r olo[2,1-c]-[1,4]ben zod ia zep in e-5,11-d ion e-8-
yloxy)octyloxy]-5-m eth oxy-2-n itr oben zoyl]p yr r olid in e-
2-car boxaldeh yde Dieth yl Th ioacetal (15d). The compound
15d was prepared according to the method described for 15a
by employing 2 and 13d (591 mg, 1 mmol) to afford 15d as a
a
Celite bed. The clear yellow organic supernatant was
extracted with saturated 5% NaHCO3 (20 mL) and brine (20
mL), and the combined organic phase was dried (Na2SO4). The
organic layer was evaporated in a vacuum and purified by
column chromatography (90% CH2Cl2-MeOH) to give com-
pound 5a as pale yellow oil (318 mg, 58%). This material was
repeatedly evaporated from CHCl3 in vacuo to generate the
imine form. 1H NMR (DMSO-d6 + CDCl3): δ 1.80-2.18 (m,
7H), 2.20-2.85 (m, 3H), 3.45-3.85 (m, 5H), 3.85-4.0 (m, 7H),
4.08-4.35 (m, 4H), 6.65 (s, 1H), 6.80 (s, 1H); 7.30 (s, 1H), 7.50
(s, 1H), 7.65 (d, 1H, J ) 4.8 Hz), 9.95 (br s, NH exchangeable).
1
yellow oil (687 mg, 89%). H NMR (CDCl3): δ 1.20-1.60 (m,
12H), 1.80-2.20 (m, 13H), 2.70-2.90 (m, 5H), 3.20-3.40 (m,
2H), 3.50-3.90 (m, 2H), 3.95 (s 3H), 4.0 (s, 3H), 4.0-4.25 (m,
5H), 4.65-4.80 (m, 1H), 4.80 (d, 1H, J ) 4.32 Hz), 6.45 (s,
1H), 6.75 (s, 1H), 7.40 (s, 1H), 7.60 (s, 1H), 8.80 (br s, NH
exchangeable).
(2S)-N-{4-[3-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H-p yr r olo[2,1-c]-[1,4]ben zod ia zep in e-5,11-d ion e-8-
yloxy)p r op oxy]-5-m eth oxy-2-a m in oben zoyl}p yr r olid in e-
2-car boxaldeh yde Dieth yl Th ioacetal (16a). The compounds
14a /15a (764 mg/702 mg, 1 mmol) were dissolved in methanol
(20 mL) and added SnCl2‚2H2O (2.256 g, 10 mmol/1.125 g, 5
mmol) was refluxed for 1.5 h or until the TLC indicated that
the 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 a yellow
oil, which because of potential stability problems,32 was briefly
MS (FAB) 549 [M + H]+‚. HRMS [M + H]+‚ calcd for
25
C
29H33N4O7 m/z 549.234925; obsd (FAB) m/z 549.235223; [R]D
+202.6° (c ) 0.5, CHCl3); reverse phase HPLC (C4 stationary
phase, 75% MeOH/H2O mobile phase, 254 nm) Rt ) 4.85 min,
% peak area ) 98.54%. Calcd for C29H32N4O7: C, 63.49; H,
5.87; N, 10.21.
7-Me t h o x y -8-{4-[7-m e t h o x y -(11a S )-1,2,3,10,11,11a -
h exa h yd r o-5H-p yr r olo[2,1-c][1,4]ben zod ia zep in e-5,11-d i-
on e-8-yloxy]bu toxy}-(11a S)-1,2,3,11a -tetr a h yd r o-5H-p yr -
r olo[2,1-c][1,4]ben zod ia zep in -5-on e (5b). The compound 5b
was prepared according to the method described for the
compound 5a employing 16b (686 mg, 1 mmol) to afford 5b
1
as a pale yellow oil (344 mg, 61%). 1H NMR (DMSO-d6
+
characterized by H NMR and then used directly in the next
1
step (537 mg, 80%/571 mg, 85%). H NMR (CDCl3): δ 1.20-
CDCl3): δ 1.80-2.18 (m, 9H), 2.20-2.80 (m, 3H), 3.45-3.85
(m, 5H), 3.85 (s, 3H), 3.90 (s, 3H), 3.98-4.20 (m, 5H), 6.55 (s,
1H), 6.75 (s, 1H); 7.30 (s, 1H), 7.41 (s, 1H), 7.60 (d, 1H, J ) 5
Hz), 9.85 (br s, NH exchangeable). MS (FAB) 563 [M + H]+‚.
HRMS [M + H]+‚ calcd for C30H35N4O7 m/z 563.250575; obsd
1.40 (m, 6H), 1.50-2.40 (m, 9H), 2.50-2.85 (m, 5H), 3.45-
3.70 (m, 4H), 3.75 (s, 3H), 3.90 (s, 3H), 4.0-4.20 (m, 5H), 4.55-
4.75 (m, 2H), 6.20 (s, 1H), 6.50 (s, 1H), 6.85 (s, 1H), 7.40 (s,
1H), 8.50 (br s, NH exchangeable).
25
(2S)-N-{4-[4-(7-Meth oxy-(11a S)-1,2,3,10,11,11a -h exa h y-
d r o-5H -p yr r olo[2,1-c][1,4]b en zod ia zep in e-5,11-d ion e-8-
(FAB) m/z 563.251967; [R]D +294.33° (c ) 0.5, CHCl3);
reverse phase HPLC (C4 stationary phase, 75% MeOH/H2O

Pyrrolobenzodiazepine Dimers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4687
mobile phase, 254 nm) Rt ) 4.99 min, % peak area ) 98.8%.
(a ) Mod elin g of DNA Du p lex a n d P BD Dim er Str u c-
tu r es. The 15-mer sequence GGGGCGAGAGAGGGG with
central AGA triplet representing the preferred binding site Pu-
G-Pu for the PBD molecule was chosen for modeling the
B-DNA duplex structure. The model was constructed using the
BIOPOLYMER module. Models for the PBD dimers 5a -d
were built using the BUILDER module. First, the PBD
molecules were “sketched” in two dimensions (2D) and then
converted into three dimensional (3D) entities using the 2D
to 3D conversion tool in the BUILDER module. During
modeling, it was assumed that both of the PBD units are
having (S)-stereochemistry at the chiral C11 atom.
(b) Dock in g Stu d ies. Docking of a PBD dimer into the
minor groove of the DNA duplex was carried out manually,
such that the N10-C11 imine functionality and the exocyclic
C2-amino group of G8 are nearly at a bonding distance from
each other so that a covalent bond can be formed. After the
covalent bond was created, the PBD dimer in the minor groove
was manually oriented in such a way that the PBD with amide
functionality is oriented toward the 3′ end of a covalently
linked DNA strand. Some of the dihedral angles about the
C-C bonds of the spacer units were manually adjusted such
that the entire PBD dimer has an isohelical fit within the
minor groove of the DNA duplex. The potentials and the
charges were fixed using the CVFF force field. The complex
so formed was subjected to energy minimization using a
conjugate gradient technique until full convergence (energy
gradient e 0.001) was reached. During energy minimization
and MD simulations, constraints were applied to fix the DNA
duplex structure.
Calcd for C30H34N4O7: C, 64.04; H, 6.09; N, 9.96.
7-Me t h o x y -8-{5-[7-m e t h o x y -(11a S )-1,2,3,10,11,11a -
h exa h yd r o-5H-p yr r olo[2,1-c][1,4]ben zod ia zep in e-5,11-d i-
on e-8-yloxy]p en tyloxy}-(11a S)-1,2,3,11a -tetr a h yd r o-5H-
pyr r olo[2,1-c][1,4]ben zodiazepin -5-on e (5c). The compound
5c was prepared according to the method described for the
compound 5a employing 16c (700 mg, 1 mmol) to afford 5c as
a pale yellow oil (317 mg, 55%). 1H NMR (DMSO-d6
+
CDCl3): δ 1.50-2.18 (m, 11H), 2.20-2.80 (m, 3H), 3.45-3.85
(m, 5H), 3.85 (s, 3H), 3.90 (s, 3H), 3.98-4.20 (m, 5H), 6.58 (s,
1H), 6.75 (s, 1H); 7.25 (s, 1H), 7.38 (s, 1H), 7.65 (d, 1H, J )
5.1 Hz), 9.85 (br s, NH exchangeable). MS (FAB) 577 [M +
H]+‚. HRMS [M + H]+‚ calcd for C31H37N4O7 m/z 577.266225,
25
obsd (FAB) m/z 577.264803; [R]D +303.00° (c ) 0.5, CHCl3);
reverse phase HPLC (C4 stationary phase, 75% MeOH/H2O
mobile phase, 254 nm) Rt ) 5.17 min, % peak area ) 99%.
Calcd for C31H36N4O7: C, 64.57; H, 6.29; N, 9.71.
7-Me t h o x y -8-{8-[7-m e t h o x y -(11a S )-1,2,3,10,11,11a -
h exa h yd r o-5H -p yr r olo[2,1-c]-[1,4]b en zod ia zep in e-5,11-
d ion e-8-yloxy]octyloxy}-(11a S)-1,2,3,11a -tetr a h yd r o-5H-
pyr r olo[2,1-c][1,4]ben zodiazepin -5-on e (5d). The compound
5d was prepared according to the method described for the
compound 5a employing 16d (742 mg, 1 mmol) to afford 5d
as a pale yellow oil (346 mg, 56%). 1H NMR (DMSO-d6
+
CDCl3): δ 1.20-2.40 (m, 17H), 2.60-2.80 (m, 3H), 3.45-3.85
(m, 5H), 3.85 (s, 3H), 3.90 (s, 3H), 3.98-4.20 (m, 5H), 6.65 (s,
1H), 6.80 (s, 1H); 7.38 (s, 1H), 7.45 (s, 1H), 7.60 (d, 1H, J ) 5
Hz), 10.05 (br s, NH exchangeable). MS (FAB) 619 [M + H]+‚;
25
[R]D +435.20° (c ) 0.25, CHCl3); reverse phase HPLC (C4
(c) Molecu la r Dyn a m ics Sim u la tion s. The following
protocol was used to carry out the molecular dynamics stud-
ies: heating phase (equilibration) ) 0-10 ps and length of
MD simulation after the heating phase ) 100 ps.
stationary phase, 75% MeOH/H2O mobile phase, 254 nm) Rt
) 5.78 min, % peak area ) 96.07%. Calcd for C34H42N4O7: C,
66.00; H, 6.84; N, 9.06.
Th er m a l Den a tu r a tion Stu d ies. Compounds were sub-
jected to thermal denaturation studies with duplex form CT-
DNA using an adaptation of a reported procedure.33 Working
solutions in aqueous buffer (10 mM NaH2PO4/Na2HPO4, 1 mM
Na2EDTA, pH 7.00 + 0.01) containing CT-DNA (100 µM in
phosphate) and the PBD (20 µM) were prepared by addition
of concentrated PBD solutions in MeOH to obtain a fixed
[PBD]/[DNA] molar ratio 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 Hitachi 150-20 spectropho-
tometer fitted with KPC-6 thermoprogramer and SPR-7 tem-
perature controller, and heating was applied at 1 °C min-1 in
the 40-90 °C range. DNA helix f coil transition temperatures
(Tm) were obtained from the maxima in the d(A260)/dT deriva-
tive plots. Results are given as the mean ( standard deviation
from three determinations and are corrected for the effects of
MeOH cosolvent using a linear correction term.34 Drug-induced
During simulations, all of the intermittent structures of the
complexes formed at every successive 5 ps were saved and later
they were subjected to energy minimization. The minima
obtained for each complex were screened to identify the lowest
energy minimum, and that was taken as a representative of
energetically favorable complex for further studies. Energy of
interaction between the DNA and the PBD dimer molecule in
a complex was calculated as follows:
Eint ) Ecomplex - (EDNA + EPBD
)
where Eint ) energy of interaction of a complex, Ecomplex ) total
energy of the complex, and EDNA and EPBD are the individual
total energies of the DNA and the PBD dimer molecules
calculated after they are separated from each other.
Ack n ow led gm en t. We thank the National Cancer
Institute, Maryland, for in vitro anticancer assay in
human cell lines. G.R., N.L., P.R., and O.S. are thankful
to CSIR, New Delhi, for the award of research fellow-
ships.
alterations in DNA melting behavior are given by ∆Tm
)
Tm(DNA + PBD) - Tm(DNA alone), where the Tm value for
the PBD-free CT-DNA is 66.50 ( 0.01. The fixed [PBD]/[DNA]
ratio used did not result in binding saturation of the host DNA
duplex for any compound examined.
RED100 Restr iction En d on u clea se Digestion Assa y.
PBD-DNA complexes were made by preincubating plasmid
DNA (0.5 µg) in concentration ranges of 1.8-14.6 µM of PBD
in restriction buffer (150 mM NaCl, 10 mM tris HCl, 1 mM
dithithreitol, 100 µg BSA (bovine serum albumin)) in a reaction
volume of 49 µL for 1 h at 37 °C. The PBD-DNA com-
plexes were added with 20 units of BamHI (1 µL) to
initiate the restriction digestion and incubated at 37 °C. The
reaction was stopped by addition of loading buffer (60%
sucrose, 0.25% bromophenol blue, 0.5% xylene cyanol, 10 mM
Tris HCl). The DNA was separated by 1.0% agarose gel
electrophoresis in Tris-acetate EDTA buffer (TAE-40 mM Tris
base, pH 8.0, 18 mM acetic acid, 1 mM EDTA) at 100 V for 2
h. The gels were stained with ethidium bromide and photo-
graphed.9
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