A novel achiral seco-cyclopropylpyrido[e]indolone (CPyI) analog of CC-1065 and the duocarmycins: Synthesis, DNA interactions, in vivo anticancer and anti-parasitic evaluation

Article abstract of DOI:10.1016/j.bmc.2010.05.078

The synthesis of an achiral seco-hydroxy-aza-CBI-TMI analog (8) of the duocarmycins is reported. Its specificity for the DNA minor groove of AT-rich sequences and covalent bonding to adenine-N3 was ascertained by a thermal cleavage assay. Compound 8 was found to be cytotoxic in the nanomolar range against murine and human cancer cells. It was further demonstrated that compound 8 was active against murine melanoma (B16-F0) grown in C57BL/6 mice. Compound 8 was also shown to inhibit the growth of the protozoan parasites Leishmania donovani, Leishmania mexicana, Trypanosoma brucei, and Plasmodium falciparum in culture.

Full text of DOI:10.1016/j.bmc.2010.05.078

Bioorganic & Medicinal Chemistry 18 (2010) 5016–5024
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel achiral seco-cyclopropylpyrido[e]indolone (CPyI) analog of CC-1065
and the duocarmycins: Synthesis, DNA interactions, in vivo anticancer and
anti-parasitic evaluation
Sameer Chavda a, Balaji Babu a, Stephanie K. Yanow b,c, Armando Jardim c, Terry W. Spithill c,d
,
Konstantinos Kiakos e, Jerome Kluza e, John A. Hartley e, Moses Lee a,f,
*
a Division of Natural Sciences and Department of Chemistry, Hope College, 35 East 12th Street, Holland, MI 49423, USA
b Department of Public Health Sciences, University of Alberta, 8440-112th Street, Edmonton, Canada AB T6G 2J2
c Institute of Parasitology, 21, 111 Lakeshore Road, Ste. Ann-de-Bellevue HX9 3V9, Quebec, Canada
d School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga 2678, Australia
e UCL Cancer Institute, University College London, Paul O’ Gorman Building, 72 Huntley Street, London WC1E 6BT, UK
f Department of Chemistry, Furman University, Greenville, SC 29613, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of an achiral seco-hydroxy-aza-CBI-TMI analog (8) of the duocarmycins is reported. Its
specificity for the DNA minor groove of AT-rich sequences and covalent bonding to adenine-N3 was
ascertained by a thermal cleavage assay. Compound 8 was found to be cytotoxic in the nanomolar range
against murine and human cancer cells. It was further demonstrated that compound 8 was active against
murine melanoma (B16-F0) grown in C57BL/6 mice. Compound 8 was also shown to inhibit the growth of
the protozoan parasites Leishmania donovani, Leishmania mexicana, Trypanosoma brucei, and Plasmodium
falciparum in culture.
Received 29 April 2010
Revised 26 May 2010
Accepted 31 May 2010
Available online 4 June 2010
Keywords:
Duocarmycins
Centanamycin
Achiral
Ó 2010 Elsevier Ltd. All rights reserved.
Minor groove
DNA
Alkylation
Adenine-N3
Anticancer
Anti-parasite
Antimalaria
Antitrypanosoma
Antileishmania
1. Introduction
yatakemycin 3, isolated from Streptomyces sp., represents the latest
and most potent member of this class of compounds.9
The potent cytotoxic compounds (+)-CC-10651,2 (1) and (+)-
duocarmycin SA (DUMSA, 2)2,3 as shown in Figure 1 are DNA minor
groove and AT sequence selective alkylating agents isolated from
the fermentation broth of Streptomyces zelensis4 and Streptomyces
sp.,5 respectively. Both 1 and 2 exert biological activity by the cova-
lent modification of the cyclopropylpyrrolo[e]indolone (CPI) moi-
ety with the adenine-N3 of DNA. The mechanism of action was
confirmed by the isolation of adenine-CC-10656 and adenine-duo-
carmycin SA adducts7 from thermally cleaved DNA-substrate ad-
ducts. (+)-CC-1065 and (+)-DUMSA preferentially bind to the
sequences 50-PuNTTA-30 and 50-AAAAA-30 reacting with the N3-po-
sition of the underlined adenine residue.6–8 Most recently, (+)-
Although (+)-CC-1065 exhibits potent cytotoxicity, its potential
as an anticancer drug is hampered by delayed lethal toxicity to mi-
ce.4b DUMSA was found to be devoid of this toxicity, however tox-
icity towards bone marrow was observed instead.5 As a result,
several analogs of CC-1065 and the duocarmycins have been devel-
oped for clinical evaluation, including adozelesin,1b,4c,10 carzele-
sin,11 bizelesin,12 and KW-2189.13 All of these compounds, aside
from bizelesin and KW-2189, failed to progress beyond Phase I
clinical evaluation due to severe toxicity towards the bone mar-
row.10–12 On the other hand, bizelesin and KW-2189 have been
investigated in Phase II clinical trials against several different types
of cancer.14 Over the past two decades, there has been a continued
interest in the development of novel analogs of CC-1065 and the
duocarmycins that show effective anticancer activity whilst main-
taining limited toxicity towards host cells. Numerous analogs of
* Corresponding author. Tel.: +1 616 395 7190; fax: +1 616 395 7923.
E-mail address: lee@hope.edu (M. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.078

S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
5017
NH2
N
OH
O
N
OCH3
OH
N
H
OCH3
N
O
OCH3
N
H
H3C
OCH3
OCH3
N
N
O
OH
N
N
H
N
H
N
H
MeS
CH3O2C
O
OCH3
O
O
N
H
O
N
H
OH
N
H
O
OCH3
O
O
(+)-Yatakemycin, 3
(+)-CC-1065, 1
(+)-DUMSA, 2
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
Cl
Cl
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
N
H
N
N
N
N
H3C
H3CO2C
N
H
N
N
H
OCH3
H
H
O
O
O
O
N
N
R
OH
OH
OH
4
7
5, R = OH
6, R = NH2
8
Figure 1. Structures of (+)-CC-1065 (1), (+)-duocarmycin SA (2), (+)-yatakemycin (3), racemic seco-hydroxy-CBI-TMI (4), which is a previously reported chiral variant of 8.16b
Also depicted are the structures of achiral molecules seco-hydroxy-CBI-TMI (5), seco-amino-CBI-TMI (6), and seco-hydroxy-aza-CBI-TMI (8).
CC-1065 and the duocarmycins with reduced toxicity towards
bone marrow have been reported1 according to the principle that
the cytotoxic potency of the CPI moiety-containing compounds
bears a direct relation to their solvolytic stability. DUMSA, one of
the most cytotoxic analogs, is also the most solvolytically stable
member of this class of compounds.2,3,15 Examples of such analogs
include cyclopropylbenzo[e]indolone (CBI),16 cyclopropylpyrazol-
o[e]indolone (CPzI),17 cyclopropylfurano[e]indolone (CFI),18 and
cyclopropylindolone (CI).19 seco-Hydroxy-CBI-TMI 4 is an example
of a CBI analog that has been widely studied by many groups
including our research group.16,20
We have reported the design, synthesis and biological activity
of the achiral analog represented by seco-hydroxy-CBI-TMI 5 as
part of a study to establish whether the chiral center present in
the natural products is required for DNA sequence recognition
and biological activity. As a result, 5, which lacks a stereocenter,
was found to retain DNA sequence selectivity as well as having
comparable in vitro cytotoxicity potency against B16 murine mel-
specific alkylation along with in vitro and in vivo anticancer and
anti-parasitic properties of compound 8 are reported.
2. Results and discussion
2.1. Synthesis
The synthesis of the hydroxyl CPyI derivative 8 (Scheme 1) was
based on a modified Skraup quinoline synthesis to provide the core
structure 10.21a Thus methacrolein was treated with 9 in the pres-
ence of bromine (1.0 equiv, AcOH, 100 °C, 1 h) to provide 10.24 Pro-
tection of phenol 10 (1.2 equiv of BnBr, 1.1 equiv of NaH, DMF 25 °C,
overnight) gave 11 (19% yield over two steps based on 9), which
was followed by reaction of 11 with di-tert-butyl malonate
(1.1 equiv), in DMF, in the presence of a stoichiometric amount of
NaH (overnight, 60 °C) to furnish diester 12 in 67% yield. Subse-
quent acid-promoted removal of the tert-butyl groups (trifluoro-
acetic acid in CH2Cl2, 3 days) followed by decarboxylation of the
diacid intermediate afforded acid 13 which was immediately ester-
ified to give the compound 14 in 28% yield based on 12 over two
steps. Reduction of ester 14 with DIBAL-H (2 equiv, 0 °C, 10 min)
followed by treatment of the resultant alcohol 15 (formed in 47%
yield) with acetic anhydride gave acetate 16 in 17% yield. Selective
reduction of the aromatic nitro group (SnCl2ꢀ2H2O, 5 equiv, 30 min)
yielded the resulting amine 17 in 76% yield which was immediately
coupled with 5,6,7-trimethoxyindole-2-carboxylic acid in the pres-
ence of benzotriazol-1-yloxy-tripyrrolidinophosphonium hexaflu-
orophoshate (PyBop) and N,N-diisopropylethylamine (Hunig’s
base) to give amide 18 in 55% yield after 10 days. Methanolysis of
the acetate group in 18 gave alcohol 19 in 67% yield. Mesylation
of 19 (4.0 equiv methanesulfonyl chloride in presence of 4.0 equiv
triethylamine, 30 min, 0 °C) followed by chlorination (55 equiv LiCl,
dry DMF, 5 days, 25 °C), furnished 21 (via 20 which was used
directly in its crude form). Removal of the protecting group on 21
by catalytic hydrogenation over 10% Pd/C provided the target achi-
ral compound 8 in overall 50%. The compounds described in the
paper were characterized by 500 MHz 1H NMR, FT-IR, mass spec-
trometry, and accurate mass measurements. The >96% purity of
compound 8 was ascertained by 1H NMR and TLC analyses.
anoma to its racemic counterpart 4 (IC50 0.027
lM for 5 vs
0.078
l
M for 4).20 Moreover, at the highest dosage, 5 and its achiral
amino congener seco-amino CBI-TMI 6 (centanamycin) were found
to exhibit activity against murine melanoma (B16-F0) grown in
C57BL/6 mice as a solid tumor whilst maintaining minimal sys-
temic toxicity.20 This prompted us to refine the CBI pharmacophore
present in achiral seco-hydroxy-CBI-TMI 5 and to further enhance
its solubility in aqueous media, DNA binding and anticancer prop-
erties hence the development of seco-amino-CBI-TMI 6 within our
laboratory.20 Boger and co-workers synthesized 1,2,9,9a-tetrahy-
drocyclopropa[c]pyrido[3,2-e]indol-4-one-7-carboxylate 7 (CPyI)
as a pharmacophore that was expected to enhance reactivity.21
The presence of the fused pyridine functionality in 7 was shown
to allow for the chelation of metal cations such as Zn2+ to the 8-
ketoquinoline functionality on the CPyI pharmacophore which, in
turn would provide a suitable means to selectively activate the
agent upon the addition of a suitable Lewis acid. It has been re-
ported that Zn is found in breast carcinoma at levels 700% higher
than normal breast cells of the same type. It is hypothesized that
analog 7 might exhibit an enhanced activity against breast carci-
noma attributable to this difference in Zn levels.22 For these rea-
sons, it was logical to synthesize and study an achiral version of
7, namely seco-cyclopropylpyrido[e]indolone (CPyI) analog
8
2.2. Thermal cleavage analysis
(seco-aza-CBI-TMI) of seco-hydroxy-CBI-TMI 5. In addition to
ascertaining the anticancer and DNA binding properties of 8, it
was interesting to investigate 8 for anti-parasitic activity as its
amino congener 6 has been shown to have potent antimalarial
and transmission-blocking activity.23 Herein the DNA sequence
The sequence specificity of seco-hydroxy-aza-CBI-TMI 8 was as-
sessed by a thermally induced DNA strand cleavage experiment,
which is commonly used to probe the sequence specific covalent
bonding with the purine-N3 in the minor groove.20 The 208 base

5018
S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
CO2tBu
CO2tBu
Cl
tBuO2C CO2tBu
Cl
Cl
Me
CHO
Br2, AcOH
NO2
Me
NO2
Me
NO2
PhCH2Br, K2CO3
DMF, overnight
, 60% NaH
Me
NO2
H2N
N
N
DMF, overnight
N
OH
OCH2Ph
OH
OCH2Ph
10
11
12
9
OH
NO2
CO2Et
NO2
CO2H
Ac2O
Me
TFA
CH2Cl2
Me
NO2
c. H2SO4
EtOH
DIBAL-H
THF
Me
N
TFA
Pyridine
N
N
OCH2Ph
OCH2Ph
OCH2Ph
13
14
15
OMe
OAc
NH2
OAc
NO2
OAc
OMe
OMe
K2CO3
MeOH
H
N
TMI, PyBOP
DIEA, CH2Cl2
Me
SnCl2
AcOEt
Me
Me
N
H
O
N
N
N
OCH2Ph
OCH2Ph
OH
OCH2Ph
16
17
18
OMe
OMe
OMe
OMe
OMe
OMe
OMs
Cl
OMe
OMe
OMe
LiCl
H
N
H
N
MsCl, Et3N
CH2Cl2
H
N
Me
Me
N
H
N
H
Me
DMF
N
O
O
H
N
N
O
N
OCH2Ph
OCH2Ph
OCH2Ph
OMe
OMe
OMe
19
20
21
Cl
H
N
Me
N
H
HCO2NH4
10% Pd/C, THF
O
N
OH
8
Scheme 1. Synthesis of seco-hydroxy-aza-CBI-TMI (8).
pair DNA fragment used for these studies was obtained by PCR
amplification from the pUC18 plasmid that was linearized with
Hind III. A 50-32P labeled primer was used as the forward primer
so that each final probe copy was singly end labeled. A representa-
tive result from the thermally induced DNA strand break experi-
ment on the previously reported seco-amino-CBI-TMI analog 620b
is depicted in Figure 2 along with seco-hydroxy-aza-CBI-TMI 8.
The results indicate that both 6 and 8 have sequence selectivity
for the 30-A(865)AAAA cluster and in addition, display a dose-
dependent response DNA alkylation effect. However, seco-hydro-
xy-aza-CBI-TMI analog 8 was about 10-fold less reactive than 6.
It is also worthy to note that 8 appeared to be more sequence dis-
criminating than 6 which produced additional minor bands on the
gel mainly at A(772) and A(843). However, in comparison to achi-
ral seco-hydroxy-CBI-TMI, 5 (thermal cleavage gel not shown), 8
appears to be 10-fold more reactive.20
cell lines with both IC50 values (180 and 150 nM, respectively). In
relation to the activity of achiral seco-hydroxy-CBI-TMI 5, 8 is
about 10-fold more active in B16 cells (IC50 2.3 nM for 8 vs
27 nM for 5) and roughly 75-fold (IC50 0.72 nM for 8 vs 55 nM
for 5; Table 1) in L1210 cells. In addition, analog 8 possesses com-
parable cytotoxicity to CC-1065, 1 against L1210 cells, (IC50
30 pM).27 Furthermore, this is consistent with the observation that
the cytotoxicity correlates with the length of the molecule, which,
in turn, reflects its capacity for non-covalent interactions with the
minor groove of DNA.20b In this case, the methyl group present on
the aza-CBI moiety of 8 adds the extra length to the molecule over
its non-methylated congeners 5 and 6.
Compound 8 was subjected to further cytotoxicity testing at the
National Cancer Institute against a panel of 54 human cancer cell
lines.28 Cell viability after 48 h of continuous exposure was ascer-
tained using a sulforhodamine B assay. Compound 8 gave compa-
rable cytotoxicity results against some cancers when compared to
the NCI data for the previously reported seco-amino-CBI-TMI 6.16
Figure 3 shows the LC50 values (the concentration for killing 50%
of the cells) for compound 8 against the panel of 54 different tumor
cell lines. Bars extending to the right indicate cells that are more
sensitive than average to 8, whereas bars to the left indicate less
sensitive cells. Several observations were made on this result. First,
8 exhibited activity in the nanomolar range for certain skin, central
nervous system (CNS) and lung cancers. Second, 8 was selectively
toxic towards a certain type of ovarian cancer over 6 whilst unlike
6, 8 showed no toxicity against any cell lines associated with breast
cancer (Fig. 3). Third, both 6 and 8 showed limited activity against
leukemic cells. Fourth, both compounds showed activity against a
number of solid tumors including melanoma, lung, colon and CNS.
The specific reasons for the trends in cytotoxicity are not clear, and
efforts are ongoing in our laboratories to address these issues.
2.3. Cytotoxicity studies
An MTT-based growth inhibition assay was used to determine
the cytotoxicity of achiral seco-hydroxy-aza-CBI-TMI 8.24,25 In
addition to 8 the concentrations required for inhibiting the growth
of murine L1210 lymphoma and B16-F0 melanoma cells by 50%
(IC50 values) for compounds 4, 5 and 6 were determined and re-
ported previously following 3 days continuous exposure.20,26 The
IC50 values are given in Table 1. For comparison, the reported
IC50 value CC-1065, 1, against L1210 (3 days exposure) was re-
ported as 30 pM.27 Remarkably, 8 inhibited the growth of the tu-
mor cells at sub-nanomolar concentrations, being approximately
10-fold more active against L1210 cells than against B16 cells
(IC50 0.72 nM for L1210 vs 2.3 nM for B16 cells; Table 1). In com-
parison to 8, 6 exhibits decreased activity in both L1210 and B16

S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
5019
by day 16. At this point a decrease in tumor size followed until
the death of the last mouse on day 21, with the average size of
the tumor being ꢁ2000 mm3. As part of this study, the weights
of the animals were also monitored at regular intervals. It was
found that under the dose administered, compound 8 did not in-
duce any significant weight loss following drug injection, com-
pared to animals treated with the vehicle only, thus indicating
no noticeable toxicity. This could be further corroborated by the
fact that the mice in all groups had comparable red blood cell
counts along with weights of the liver, kidney and spleen. The
moderate reduction in tumor size induced by 8 along with minimal
systemic toxicity encouraged us to consider exploring the anti-tu-
mor activity at a higher dosage.
2.5. Anti-parasitic activity of compound 8
With the information in hand that compound 6, an AT sequence
specific-adenine-N3 alkylating agent, has potent antimalarial
activity in vivo and in vitro against Plasmodium falciparum,23 and
attenuates Plasmodium berghei30 sporozoites we assessed the abil-
ity of 8 to inhibit growth of the protozoan parasites, Leishmania
donovani, Leishmania mexicana, Trypanosoma brucei, and Plasmo-
dium falciparum in culture. The results provide evidence that com-
pounds 6 and 8 show significant activity against P. falciparum with
IC50 in low nanomolar range (Table 2). Overall, compound 6 was
more active than 8 against L. donovani promastigotes and T. brucei
procyclic life cycle stages. However, the aza-compound 8 gave
comparable activity against P. falciparum indicating that this com-
pound or the chemical scaffold possesses pharmacological poten-
tial for development as an antimalarial agent. Surprisingly,
compounds 6 appeared to be far less potent against the kinetoplas-
tid parasite L. mexicana whereas compound 8 is less potent against
L. donovani, L. mexicana and T. brucei (Table 2). This difference may
not be surprising since compounds 6 and 8 are postulated to exert
their cytotoxic activity through their AT-sequence specific DNA le-
sions. The abundance of adenine-thymine nucleotides in the Plas-
modium genome31 is 80.6% making this parasite more susceptible
to these drugs in comparison to the Leishmania and Trypanosoma
genomes which have an adenine-thymine nucleotide content of
41%.32 It should be emphasized that in contrast to the genomic
DNA, the mitochondrial (kinetoplastid) DNA of Leishmania and Try-
panosoma has a high AT codon bias which may be the target of
compounds 6 and 8 in these parasites.33
Figure 2. Thermal cleavage gel on compounds 6 and 8 showing adenine-N3 lesions
on the bottom strand of a 50-32P labeled 208-bp fragment of pUC18 at the drug
concentrations indicated. Arrows indicate the position and sequence context of the
alkylated adenines (underlined). Drug/DNA incubations were for 5 h at 37 °C.
Table 1
Cytotoxicity of compounds 4–6 and 8
Compd
Cytotoxicity (IC50 given in nM)
L1210
B16
4
5
6
8
8.4
180
55
0.72( 0.1)
78
150
27
2.3( 1)
3. Conclusion
2.4. In vivo anticancer studies of compound 8 on B16-F0 murine
melanoma
In conclusion, the favorable DNA interaction properties, cyto-
toxicity against tumor cell lines, activity against murine melanoma
grown in mice as a solid tumor and in vitro activity against
P. falciparum strongly indicate that achiral seco-hydroxy-aza-CBI-
TMI warrants further investigation as an anticancer agent and as
a potential anti-parasitic agent. Efforts are ongoing within our
laboratory to gain further insight into the selective activity against
P. falciparum as well as the sensitivity profiles observed in the NCI
cytotoxicity screen. The results from this study effectively demon-
strate that analogs of CC-1065 and the duocarmycins, which lack a
stereocenter would still exhibit DNA sequence specificity and anti-
tumor activity.
This study was based according to a similar protocol disclosed
by Li et al.29 A total of eight animals within each group, 5–7 week
old female C57/BL/6J mice were inoculated in the left flank with
cultured B16-F0 murine melanoma cells on day 0. On days 1, 5,
and 9 mice were treated with a 10 mg/kg dose of 8 in vehicle solu-
tion. Compound 8 was formulated in 1:2 PET/5% glucose solution
and administered via an intraperitoneal route. The volume of the
tumor was monitored every other day. These measurements were
ceased when four of the animals in the test group (50%) had died.
The in vivo anticancer effects for compound 8 were encouraging
(data not shown). For the control (untreated) group, four of the
mice had died by day 16, with the tumor volume steadily increas-
ing from day 9 from a barely visible volume of ꢁ100 mm3, reaching
ꢁ2300 mm3 by the time 50% of the mice had died (day 16). In com-
parison, in the treated group, the fourth mice had died on day 21,
thus indicating an increase in lifespan within this group of mice.
After the final 10 mg/kg injection (day 9), the average tumor size
increased steadily from a volume of ꢁ100 mm3 to ꢁ1700 mm3
4. Experimental
4.1. General
Solvents and organic reagents were purchased from Aldrich or
Fisher and used without further purification. Dichloromethane

5020
S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
Figure 3. NCI cytotoxicity screen data (LC50 values) for compound 8 against a panel of 54 different human cancer cell lines.
(DCM) and dimethylformamide (DMF) were distilled over P2O5 and
BaO, respectively, prior to use. Melting points (mp) were per-
formed using a Mel-temp instrument, and the results were uncor-
rected. Infrared (IR) spectra were recorded using a Perkin Elmer
Paragon 500 FT-IR spectrophotometer as films on KBr disks. 1H
are reported at 20 °C in parts per million (ppm) downfield from
internal tetramethylsilane. High-resolution mass spectra (HRMS)
and low-resolution mass spectra (LRMS) were provided by the
Mass Spectrometry Laboratory, University of South Carolina,
Columbia, SC. Reactions were monitored using thin-layer chroma-
tography (TLC) using commercially available precoated plates
(Merck Kieselgel 60 F254 silica). Visualization was achieved with
NMR spectra were obtained using
a Varian Unity INOVA
500 MHz instrument unless otherwise stated. Chemical shifts (d)

S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
5021
Table 2
was washed with Et2O to yield 14 (7.2 g, 28%) as a brown solid.
Mp 192–194 °C; 1H NMR (CDCl3, 500 MHz) 8.95 (d, 2.0, 1H), 8.17
(d, 2.0, 1H), 7.54 (d, 7.5, 2H), 7.52 (s, 1H), 7.40 (t, 7.0, 2H), 7.34
(t, 7.5, 1H), 5.47 (s, 2H), 4.24 (s, 2H), 4.20 (q, 7.0, 2H), 2.60 (s,
3H), 1.26 (t, 7.5, 3H); EIMS m/z 380 (M+, 45%), EIHR m/z
380.1376 (M+, C21H20N2O5 requires 380.1372); IR (neat) mmax
1724, 1584, 1519, 1501, 1457, 1426, 1382, 1373, 1357, 1324,
1243, 1207, 1118, 1018, 974, 916, 881, 845, 796, 754, 734,
In vitro cytotoxicity data of compounds
Trypanosoma brucei, Plasmodium falciparum and Leishmania mexicana
6 and 8 against Leishmania donovani,
Compd
Cytotoxicitya (nM)
T. brucei P. falciparum
1.9
56
L. donovani
L. mexicana
6
8
13
340
1.8b
1.8
1024
119
a
The error for these studies is 10%.
Previously reported within our research group.23
699 cmꢂ1
.
b
4.1.4. Compound 15
To a suspension of 14 (15.2 g, 40.0 mmol) in dry THF (300 mL)
at 0 °C, DIBAL-H (1.5 M, 53.3 mL, 79.9 mmol) was added slowly
and the solution was stirred at the same temperature for 10 min.
The mixture was poured into water (0.6 L), then CHCl3 (1 L) was
added to the mixture and the gel was filtered over Celite. The or-
ganic layer was washed with saturated aqueous NaCl (50 mL),
dried (Na2SO4) and concentrated under reduced pressure. The res-
idue was triturated with CHCl3 to yield 15 (6.2 g, 47%) as an orange
solid. Mp 186–188 °C; 1H NMR (CDCl3, 500 MHz) 8.93 (d, 2.0, 1H),
8.35 (s, 1H), 7.52 (d, 7.0, 2H), 7.39 (t, 7.0, 2H), 7.36 (s, 1H), 7.33 (t,
7.5, 1H), 5.44 (s, 2H), 4.03 (q, 7.0, 2H), 3.41 (t, 7.0, 2H), 2.60 (s, 3H),
1.85 (t, 5.5, 1H); EIMS m/z 338 (M+, 40%), EIHR m/z 338.1271 (M+,
UV light at 254 nm or I2 vapor staining. In addition to NMR and ele-
mental analysis, HPLC analysis was used to determine the purity
(>95%) of the compounds.
4.1.1. Synthesis of 11
To a solution of methacrolein (3.0 mL, 37.1 mmol) in AcOH
(140 mL), bromine (1.9 mL, 37.1 mmol) was added and the result-
ing solution was stirred at room temperature until the appearance
of a faint red color. Compound 9 was then added (7.0 g, 37.1 mmol)
and the resulting mixture was heated at 100 °C for 1 h. The reac-
tion mixture was filtered, and the solid was washed with water
and dried in the hood to yield 10 (4.7 g, mixture with 9) as a brown
solid. To a solution of 10 (4.7 g, 19.7 mmol) in DMF (47 mL), K2CO3
(3.81 g, 27.6 mmol) and benzyl bromide (2.80 mL, 23.6 mmol)
were added and the resulting mixture was stirred overnight at
room temperature. The mixture was filtered, concentrated and ex-
tracted with ethyl acetate (50 ml). The solution was washed with
water (50 mL) and saturated aqueous NaCl (50 mL), then dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by silica gel column (AcOEt/Petroleum ether = 1:2)
and washing with Et2O to yield 11 (2.3 g, 19% from 9) as a yellow
solid. Mp 140–141 °C; 1H NMR (CDCl3, 500 MHz) 8.97 (d, 2.0, 1H),
8.51 (d, 2.0, 1H), 7.52 (d, 7.5, 2H), 7.40 (t, 7.0, 2H), 7.38 (s, 1H), 7.34
(t, 7.0, 1H), 5.46 (s, 2H), 2.63 (s, 3H); EIMS m/z 238 (M+, 100%); IR
C19H18N2O4 requires 338.1267); IR (neat) mmax 3232, 1521, 1504,
1454, 1374, 1363, 1335, 1312, 1241, 1202, 1123, 1052, 980, 913,
886, 838, 798, 782, 743, 720, 701 cmꢂ1
.
4.1.5. Compound 16
To a solution of 15 (6.2 g, 18.9 mmol) in dry pyridine (16 mL),
98% acetic anhydride (2.2 mL, 22.7 mmol) was added and the
resulting mixture stirred overnight at room temperature. The mix-
ture was then concentrated and dissolved in AcOEt (200 mL). The
organic layer was washed with H2O (50 mL), saturated aqueous
NaCl (10 mL), organic layer dried (over Na2SO4) and concentrated
under reduced pressure to give a brown residue which was washed
with i-Pr2O to yield 16 (1.2 g, 17%) as a pale brown solid. Mp 130 °C
(decomp.); 1H NMR (CDCl3, 500 MHz) 8.95 (s, 1H), 8.48 (s, 1H), 7.53
(d, 7.5, 2H), 7.40 (t, 7.0, 2H), 7.38 (s, 1H), 7.33 (t, 8.0, 1H), 5.46 (s,
2H), 4.42 (t, 8.0, 2H), 3.47 (t, 8.0, 2H), 2.63 (s, 3H), 2.05 (s, 3H);
EIMS m/z 380 (M+, 30%), EIHR m/z 380.1385 (M+, C21H20N2O5 re-
quires 380.1372); IR (neat) 3060, 2971, 2897, 1732, 1582, 1523,
1499, 1454, 1358, 1333, 1310, 1239, 1140, 1043, 898, 860, 838,
(neat) mmax 3034, 1594, 1531, 1498, 1454, 1374, 1346, 1325, 1255,
1126, 1007, 970, 913, 884, 839, 784, 747, 697 cmꢂ1
.
4.1.2. Compound 12
To a suspension of 60% NaH washed with petroleum ether (4.5 g,
0.11 mol) in DMF (200 mL), a solution of di-tert-butyl malonate
(20.4 mL, 89.4 mmol) in DMF (50 mL) and 11 (24.5 g, 74.5 mmol)
were added sequentially followed by heating at 60 °C overnight.
The mixture was quenched slowly by adding saturated aqueous
NH4Cl until hydrogen gas evolution stopped. The mixture was di-
luted with water (300 mL). The resulting precipitate was filtered
and dried in the hood to yield 12 (34.2 g, 67%) as a brown solid.
Mp 150–152 °C; 1H NMR (CDCl3, 500 MHz) 8.92 (d, 2.0, 1H), 8.32
(d, 2.0, 1H), 7.53 (d, 7.0, 2H), 7.40 (s, 1H), 7.40 (t, 8.0, 2H), 7.34 (t,
7.5, 1H), 5.47 (s, 2H), 5.25 (s, 1H), 2.56 (s, 3H), 1.45 (s, 18H); EIMS
m/z 508 (M+, 20%), EIHR m/z 508.2211 (M+, C28H32N2O7 requires
508.2210); IR (neat) mmax 2979, 2927, 1728, 1584, 1529, 1499,
804, 778, 734, 694, 668 cmꢂ1
.
4.1.6. Compound 17
To a solution of 16 (3.0 g, 7.89 mmol) in AcOEt (60 mL),
SnCl2ꢀ2H2O (8.90 g, 39.4 mmol) was added and the resulting mix-
ture stirred for 30 min at room temperature. The reaction was
quenched with saturated aqueous NaHCO3 (100 mL) and extracted
with CHCl3 (300 mL). The CHCl3 layer was washed with saturated
aqueous NaCl (10 mL), dried (over Na2SO4) and evaporated to dry-
ness to yield 17 (2.1 g, 76%) as an orange foam. Mp 116 °C (de-
comp.); 1H NMR (CDCl3, 500 MHz) d 8.67 (s, 1H), 7.96 (s, 1H), 7.52
(d, 7.0, 2H), 7.35 (t, 7.0, 2H), 7.28 (t, 7.5, 1H), 7.20 (s, 1H), 5.42 (s,
2H), 4.21 (t, 7.5, 2H), 3.14 (t, 7.5, 2H), 2.52 (s, 3H), 2.07 (s, 3H),
1.62 (br s, 2H); EIMS m/z 350 (M+, 80%); IR (neat) mmax 3308,
3172, 1954, 2883, 1734, 1613, 1505, 1457, 1417, 1386, 1365,
1457, 1391, 1368, 1316, 1252, 1160, 1136, 847, 736, 699, 668 cmꢂ1
.
4.1.3. Compound 14
To a solution of 12 (34.2 g, 67.3 mmol) in CH2Cl2 (680 mL), tri-
fluoroacetic acid (68 mL) was added and the resulting solution stir-
red for 3 days at room temperature. The reaction mixture was
concentrated under reduced pressure to give carboxylic acid 13,
which was immediately dissolved in ethanol containing 5% con-
centrated H2SO4 (34 mL) and refluxed overnight. The resulting
mixture was concentrated and extracted with AcOEt (500 mL).
The solution was washed with water (100 mL), saturated aqueous
NaHCO3 (100 mL), saturated aqueous NaCl (100 mL), dried
(Na2SO4) and concentrated under reduced pressure. The residue
1237, 1200, 1133, 1076, 1045, 1028, 912, 845, 734, 697, 668 cmꢂ1
.
4.1.7. Compound 18
To a solution of 17 (1.0 g, 2.85 mmol) in CH2Cl2 (10 mL), 5,6,7-
trimethoxyindole-2-carboxylic acid (TMI) (0.86 g, 3.42 mmol),
N,N-diisopropylethylamine (1.21 mL, 6.85 mmol) and ben-
zotriazol-1-yloxy-tripyroridinophosphonium hexafluorophosphate
(PyBOP) (1.78 g, 3.42 mmol) were added under an N2 atmosphere
and stirred for 10 days at room temperature. The reaction mixture

5022
S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
was extracted with CH2Cl2 (100 mL) and washed with saturated
aqueous NaHCO3 (20 mL), saturated aqueous NaCl (20 mL), dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (AcOEt/Petro-
leum ether = 4:1) to yield 18 (0.91 g, 55%) as a brown foam. Mp
80–81 °C; 1H NMR (CDCl3, 500 MHz) 9.35 (s, 1H), 8.79 (d, 1.5,
1H), 8.64 (s, 1H), 8.00 (s, 1H), 7.91 (s, 1H), 7.58 (d, 7.5, 2H), 7.37
(t, 7.5, 2H), 7.29 (t, 7.5, 1H), 7.19 (s, 1H), 6.87 (s, 1H), 5.45 (s,
2H), 4.35 (t, 7.0, 2H), 4.10 (s, 3H), 3.95 (s, 3H), 3.93 (s, 3H), 3.36
(t, 7.0, 2H), 2.56 (s, 3H), 2.17 (s, 3H); TOF MS ES+ m/z 584 (M++H,
100%), TOFHR m/z 584.2401 (M++H, C33H34N3O7 requires
584.2397); IR (neat) mmax 3307, 2936, 1736, 1652, 1606, 1582,
1535, 1501, 1465, 1411, 1366, 1305, 1237, 1122, 1047, 998, 912,
8.62 (s, 1H), 8.47 (s, 1H), 7.94 (s, 1H), 7.61 (s, 1H), 6.96 (s, 1H),
6.86 (s, 1H), 4.09 (s, 3H), 3.95 (s, 3H), 3.95 (t, 7.0, 2H), 3.92 (s,
3H), 3.52 (t, 6.0, 2H), 2.58 (s, 3H), 1.60 (br s, 1H); TOFMS ES+ m/z
470 (M++H, 100%), TOFHR m/z 470.1485 (M++H, C24H25ClN3O5 re-
quires 470.1483); IR (neat) mmax 3310, 2936, 2833, 2361, 1636,
1583, 1536, 1500, 1464, 1408, 1383, 1365, 1303, 1241, 1113,
1048, 997, 909, 833, 731, 669 cmꢂ1
.
4.2. Cytotoxicity studies on murine leukemia and melanoma
cell lines
The P815, B16-F0, and L1210 cell lines were obtained from
American Type Tissue Culture Collection (ATCC) and the studies
833, 736, 698 cmꢂ1
.
were conducted exactly according to
a recently published
procedure.20a
4.1.8. Compound 19
A solution of 18 (0.91 g, 1.56 mmol) in MeOH (18 mL) was trea-
ted with K2CO3 (0.26 g, 1.89 mmol) and stirred for 3 h at room tem-
perature. The reaction mixture was extracted with CHCl3 (100 mL)
and washed with water (20 mL), saturated aqueous NaCl (20 mL),
dried (Na2SO4) and concentrated under reduced pressure to yield
19 (0.57 g, 67%) as a brown solid. Mp 110 °C (decomp.); 1H NMR
(CDCl3, 500 MHz) 10.03 (s, 1H), 9.24 (s, 1H), 8.67 (d, 1.5, 1H),
7.85 (s, 1H), 7.80 (s, 1H), 7.56 (d, 7.5, 2H), 7.35 (t, 7.0, 2H), 7.28
(t, 8.0, 1H), 7.06 (d, 2.0, 1H), 6.82 (s, 1H), 5.35 (s, 2H), 4.13 (t, 5.5,
2H), 4.08 (s, 3H), 3.95 (s, 3H), 3.90 (s, 3H), 3.21 (t, 5.0, 2H), 2.49
(s, 3H); TOF MS ES+ m/z 542 (M++H, 100%), TOF HRMS m/z
542.2307 (M++H, C31H32N3O6 requires 542.2291); IR (neat) mmax
3271, 1936, 1652, 1607, 1586, 1536, 1501, 1462, 1412, 1368,
4.3. NCI in vitro cytotoxicity screen
Compound 8 was also tested against a panel of 60 human can-
cer cell lines at the National Cancer Institute, Bethesda, MD.28 The
cytotoxicity studies were conducted using a 48 h exposure proto-
col and a sulforhodamine B assay. Dose–response curves were used
to generate the GI50 (concentration of drug needed to inhibit the
growth by 50%), TGI (total growth inhibition), and LC50 values (con-
centration needed to kill 50% of cells).
4.4. Thermal cleavage assay
The thermally-induced cleavage assay was used to probe
purine-N3 alkylations on the same region of the pUC18 plasmid
and under the same experimental conditions as the ones previously
reported for compound 6.20b Briefly, the 208-bp probe subjected to
drug modification was PCR amplified and defined by the primers 50-
CTCACTCAAAGGCGGTAATAC-30 and 50-TGGTATCTTTATAGTCCTGTC
1305, 1257, 1127, 1048, 996, 909, 831, 730, 699 cmꢂ1
.
4.1.9. Compound 21
A solution of 19 (0.57 g, 1.05 mmol) in CH2Cl2 (8 mL) was treated
with triethylamine (0.6 mL, 4.21 mmol) and methanesulfonyl chlo-
ride (0.33 mL, 4.21 mmol) at 0 °C and stirred for 0.5 h. The reaction
mixture was extracted with CHCl3 (50 mL) and washed with H2O
(10 mL), saturated aqueous NaHCO3 (10 mL), saturated aqueous
NaCl (20 mL), dried (Na2SO4) and concentrated under reduced pres-
sure to yield 20 (0.9 g, quant.) as a crude black oil which was imme-
diately dissolved in dry DMF (20 mL). LiCl (2.46 g, 58.1 mmol) was
added and the reaction mixture was stirred for 5 days at room tem-
perature. The reaction was quenched with H2O (100 mL). The
resulting solid was filtered and dried in the hood to yield 21
(0.65 g, quant.) as a pale brown solid. Mp 150 °C (decomp.); 1H
NMR (CDCl3, 500 MHz) 9.49 (br s, 1H), 8.89 (br s, 1H), 8.82 (s,
1H), 8.05 (s, 1H), 7.69 (s, 1H), 7.51 (d, 7.5, 2H), 7.32 (t, 6.5, 2H),
7.28 (m, 1H), 7.05 (s, 1H), 6.85 (s, 1H), 5.33 (s, 2H), 4.10 (s, 3H),
3.95 (t, 7.0, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.55 (t, 6.0, 2H), 2.56 (s,
3H); TOFMS ES+ m/z 560 (M++H, 100%), TOFHR m/z 560.1967
(M++H, C31H31ClN3O5 requires 560.1952); IR (neat) mmax 3244,
2936, 1654, 1577, 1542, 1506, 1465, 1410, 1369, 1305, 1238,
G-30, the latter being 50-end radiolabeled with [ 32P] ATP (5000 Ci/
c-
mmol, Amersham) using T4 polynucleotide kinase (New England
Biolabs). Oligonucleotides were synthesised by Eurofins MWG Op-
eron. 4 pmol of each primer were used for the exponential amplifi-
cation of the 749–956 region of 0.4 ng of the pUC18 plasmid
template DNA. The resulting PCR product was detected by agarose
gel electrophoresis, isolated and purified using the Geneclean III kit
(MP Biomedicals) following the manufacturer’s standard protocol.
Drug–DNA incubations were carried out in TEOA buffer
[25 mmol/L triethanolamine, 1 mmol/L EDTA (pH, 7.2)] at 37 °C
for 5 h, in a total volume of 50 lL, using 10 lL of the purified probe
per reaction, enough to yield an activity of at least 200 counts/s. Fol-
lowing precipitation and lyophilisation, dried DNA pellets were
heated at 90 °C for 30 min in a total volume of 100
lL of sodium cit-
rate buffer [1.5 mmol/L sodium citrate, 15 mmol/L NaCl (pH, 7.2)].
Samples were finally resuspended in 5
and subjected to electrophoresis.
lL formamide loading buffer
1200, 1118, 1049, 998, 910, 835, 752, 730, 699, 668 cmꢂ1
.
4.5. In vivo anticancer study of compound 8 on the growth of
B16-F0 murine melanoma in C57/BL/6 mice
4.1.10. Achiral aza-CBI-TMI 8
A solution of 21 (0.65 g, 1.16 mmol) in THF (13 mL) was added
to 10% Pd/C (0.65 g) and cooled to ꢂ78 °C. The suspension was de-
gassed under vacuum and under N2 atmosphere. The mixture was
warmed to room temperature and treated with a solution of
ammonium formate (0.75 g, 11.6 mmol) in water (3.0 mL), and
the reaction mixture was stirred for 3 days. The suspension was fil-
tered over Celite and the filtrate was extracted with AcOEt
(100 mL). The organic layer was washed with H2O (10 mL), satu-
rated aqueous NaCl (20 mL), dried (Na2SO4) and concentrated un-
der reduced pressure to yield 8 as a crude orange solid. The solid
was washed with Et2O to yield 15 (0.27 g, 50%) as a pure orange so-
lid. Mp 220 °C (decomp.); 1H NMR (CDCl3, 500 MHz) 9.25 (s, 1H),
Female C57/BL/6J mice were obtained from the Jackson Labora-
tory and were 5–7 weeks old upon arrival. Groups of eight mice
were inoculated with B16 cells that came directly from a culture
flask without first being passaged through a mouse. The cultured
cells were washed three times with PBS and resuspended in PBS
at a concentration of 4.0 ꢃ 106 cells/mL. The cell suspensions
(50 lL) were delivered subcutaneously (sc) to the left flank at
day 0. At this concentration, each mouse received 200,000 tumor
cells. The length and width of the tumors were measured almost
every other day using calipers (Bel-Art Products, Vernier Type H

S. Chavda et al. / Bioorg. Med. Chem. 18 (2010) 5016–5024
5023
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(mm3) = length (mm) ꢃ [width (mm)]2.
The drug solutions were formulated by mixing the appropriate
amounts for each drug with 1.6 mL of PET (poly(ethylene glycol)
400, absolute ethanol, and Tween 80 in 6:3:1 portions) and
3.2 mL of 5% glucose in water. In each case clear, colorless solutions
were obtained. These solutions were stored at 20 °C and the com-
pound’s stability was monitored by RP-HPLC analysis. Each mouse
received 100 lL of the drug solution via an intraperitoneal (ip)
administration route on days 1, 5, and 9. Each injection delivered
10 mg/kg of compound 8. Animal weights were taken every other
day. The control group of mice was treated on a similar schedule
with 100 lL of the vehicle.
4.6. Plasmodium growth inhibition studies on compound 8
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4.7. Growth inhibition of Leishmania and Trypanosoma brucei
by compound 8
Leishmania donovani and Leishmania mexicana promastigotes
(5 ꢃ 104 cells/well) were cultured in 200
lL of DME-L media supple-
mented with 10% heat inactivated fetal bovine serum, hemin, xan-
thine, and penicillin-streptomycin.34 Trypanosoma brucei 427
procyclic forms were grown in SDM 79 media. The parasites were
cultured for 72 h at 26 °C in media containing in the presence of
0.03 nM to 64
lM compound 8. Cell proliferation was assessed by
adding 20 L of Alamar blue dye solution (Trek Diagnositics, Cleve-
l
land, OH) and measuring the levels of dye reduction by fluorescence
(excitation530 nm and emission590 nm) after a 15 h incubation. All
experiments were performed at least twice using triplicate samples.
Data was analyzed using the MicroCal Origins 7.0 software package.
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Support (to M.L.) from Taiho Pharmaceutical Co. of Japan,
National Science Foundation and National Cancer Institute are
gratefully acknowledged. J.A.H. thanks Cancer Research UK
(C2259/A9994) for support. The authors also thank Tetsuji Asao
and Atsushi Sato of Taiho Pharmaceutical Co. for their support. Car-
ly Price and Stephen Hudson are acknowledged for their assistance
with the in vivo anticancer experiments. A.J. and T.S. acknowledge
financial support from Natural Science and Engineering Research
Council of Canada and Fonds québécois de la recherche sur la nat-
ure et les technologies. T.S. acknowledges support from the Canada
Research Chair program. T. Spithill holds a Canada Research Chair
in Immunoparasitology.
Supplementary data
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