Potent antitumor bifunctional DNA alkylating agents, synthesis and biological activities of 3a-aza-cyclopenta[a]indenes

Article abstract of DOI:10.1016/j.bmc.2009.06.018

A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-8H-3a-azacyclopenta[a]indene-1-yl derivatives were synthesized for antitumor evaluation. The preliminary antitumor studies revealed that these agents ex

Full text of DOI:10.1016/j.bmc.2009.06.018

Bioorganic & Medicinal Chemistry 17 (2009) 5614–5626
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Potent antitumor bifunctional DNA alkylating agents, synthesis
and biological activities of 3a-aza-cyclopenta[a]indenes
Rajesh Kakadiya a,f, Huajin Dong b, Pei-Chih Lee a,e, Naval Kapuriya a,f, Xiuguo Zhang b, Ting-Chao Chou b,
Te-Chang Lee a,d, Kalpana Kapuriya a, Anamik Shah f, Tsann-Long Su a,c,
*
a Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
b Preclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
c Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
d National Research Institute of Chinese Medicine, Taipei 112, Taiwan
e Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan
f Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-
8H-3a-azacyclopenta[a]indene-1-yl derivatives were synthesized for antitumor evaluation. The prelimin-
ary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and antitumor
therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little
or no cross-resistance to either Taxol or Vinblastine. Remarkably, complete tumor remission in nude mice
bearing human breast carcinoma MX-1 xenograft by 13a,b and 14g,h and significant suppression against
prostate adenocarcinoma PC3 xenograft by 13b were achieved at the maximum tolerable dose with rel-
atively low toxicity. In addition, these agents induce DNA interstrand cross-linking and substantial G2/M
phase arrest in human non-small lung carcinoma H1299 cells. The current studies suggested that these
agents are promising candidates for preclinical studies.
Received 18 May 2009
Revised 9 June 2009
Accepted 11 June 2009
Available online 16 June 2009
Keywords:
Cyclopenta[a]indene
Structure–activity relationships
Interstrand cross-linking
Cell cycle
Ó 2009 Elsevier Ltd. All rights reserved.
Antitumor agents
1. Introduction
O
O
O
O
OCONH2
OMe
OH
H2N
Me
N
A large number of synthetic compounds or naturally occurring
products contain two reactive nucleophilic centers in the molecule.
These compounds often exhibit potent antitumor activity since
they can covalently bind to DNA. For example, many synthetic N-
mustards1 bearing a N,N-bis(2-chloroethyl)amine active pharma-
cophore and cis-platinum complexes2,3 are currently used clini-
cally for cancer chemotherapy.4 The antibiotic anticancer
mitomycin C (MMC, 1, Fig. 1)5–7 and its analogue, indoloquinone
EO9 (2),8 have been demonstrated to be able to cross-link to
DNA double strands after bioreductive activation.9 Another class
of compounds, such as thioimidazoles (i.e., carmethizole, 3),10,11
bis(hydroxymethyl)-pyrrolidines (i.e., 412,13 and 514), and 2,3-dihy-
droxy-6,7-bis(hydroxymethyl)-1H-pyrrolizines [i.e., 6 (IPP)],15,16
have been also demonstrated to have DNA interstrand cross-link-
ing activity, that have certain similarities with MMCs.14 The halo-
substituted pyrrolizine bis(carbamate) 6 was shown to have signif-
icant reproducible antitumor activity against a broad range of
experimental murine neoplasias and human tumor xenografts in
nude athymic mice and was selected for more extensive preclinical
N
NH
N
Me
OH
2 EO9
1 Mitomycin C (MMC)
OAc
OAc
OH
OH
OCONHMe
Me
OCONHMe
N
N
Me
N
N
Me
S
Me
5
3
4
OCONHCHMe 2
OCONHCHMe 2
R1
OR3
OR3
N
N
R2
Cl
Cl
7
6 (IPP)
Figure 1. Chemical structure of some DNA bifunctional alkylating agents.
* Corresponding author. Tel.: +886 2 2789 9045; fax: +886 2 2782 5573.
E-mail address: tlsu@ibms.sinica.edu.tw (T.-L. Su).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.018

R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
5615
studies.17 However, no further information has been published
whether this agent was in clinical trials.
inhibitory properties in a panel of tumor cell lines are included
in this study. Furthermore, we also describe the capability of
DNA interstrand cross-linking and influence on cell cycle of the
newly synthesized derivatives.
Studies on the mechanism of action showed that DNA inter-
strand cross-linking by bis(carbamate)pyrroles or pyrrolizines
was probably via a SN1 electrophilic reaction.14,16 These derivatives
are capable of forming an DNA interstrand cross-link with the
short oligonucleotide 50-ACGT at the 50-CG residues at the minor
groove region.14 The potential electrophilic reactivity of these
agents (the carbamates moieties are leaving group in an alkyl-oxy-
gen cleavage mechanism) would be modulated by the degree of
electronic perturbation in participating of the pyrrole. Thus, the
electronic effects induced from the substituted phenyl groups at
C2 of pyrroles or substituent at C5 of pyrrolizines would affect
the cytotoxicity of these congeners. However, it was reported that
the in vivo antileukemic activity and the host toxicity were not al-
tered to considerable degree as a function of electronic properties
of phenyl substituent probably due to the insignificance of the
electronic effects.18,19 In contrast with pyrrole derivatives, the
C5-phenyl and the pyrrole ring in 5-phenylpyrrolizines are
coplanar. It was suggested that the planarity of the molecule
may be required for their antitumor activity. Studies on the struc-
ture–activity relationships (SAR) of 5-phenylpyrrolizines revealed
that compounds having electron-donating substituent(s) (OMe)
on the phenyl ring were generally more toxic than those
compounds bearing electron-withdrawing substituent(s) (halo-
gen). While, the in vivo antitumor activities were comparable or
slightly less potent in compounds having an electron-donating
substituent.20,21 These studies also suggested that the lipophilicity
of compound might affect its antitumor potency.
2. Results and discussion
2.1. Chemistry
The (2-hydroxymethyl-3-phenyl-8H-3a-azacyclopenta[a]indene-
1-yl derivatives (13a–l) and their bis(methylcarbamate) (14a–l) were
synthesized by following the method developed by Anderson et al.18
with modification (Scheme 2). Treatment of indoline-2-carboxylic
acid (8) with appropriate acid chlorides afforded 1-acyl-indoline-2-
carboxylic acids (11b–k), which were reacted with dimethyl acety-
lenedicarboxylate (DMAD) in acetic anhydride by heating at 120 °C
to yield diester 12b–k. The diester 12a was prepared directly from 8
by reacting with acetic anhydride and DMAD. While, the diester 12l
was synthesized starting from 5-methoxyindole 9 via N-acylation
by treating with 4-methoxybenzoyl chloride in the presence of trieth-
ylamine followed bycatalytichydrogenation usingPtO2/H2 in EtOH to
give intermediate 11l. Reaction of 11l with acetic anhydride and
DMAD afforded compound 12l. The diester functions of12a–l werere-
duced to bis-alcohol derivatives 13a–l by treating with LiAlH4 in a
mixture of ether/CH2Cl2 in an ice bath. Treatment of 13a–l with
methyl isocyanate afforded the desired bis(carbamate) derivatives
14a–l in good yield.
2.2. Biological results
Based on the potent antitumor activities and mechanism of
action of MMC and bis(carbamates)pyrrolizidines, we have synthe-
sized a series of bis(hydroxymethyl) of 3a-azacyclopenta[a]indene-
1-yl (7, R3 = OH, Fig. 1) and their bis(methylcarbamate) derivatives
(7, R3 = OCONHMe) for antitumor studies. These agents can be con-
sidered as ‘benzologues’ of pyrrolizines. One can expect that the
newly synthesized compounds might be able to cross-link to the
macromolecular DNA via a similar mechanism of action as that
of pyrroles or pyrrolizines (Scheme 1). We have introduced an alkyl
or substituted phenyl moiety at C3 of the 3a-azacyclopen-
ta[a]indene-1-yl to investigate their effects on antitumor activity.
The results revealed that the newly synthesized compounds exhib-
ited significant in vitro cytotoxicity and potent in vivo therapeutic
efficacy. The synthesis of 3a-azacyclopenta[a]indene-1-yl deriva-
tives and their structure–activity relationships for the growth
2.2.1. In vitro cytotoxicity
Table 1 shows the cytotoxicity of 1,2-bis(hydroxylmethyl) of
cyclopenta[a]indenes (13a–l) and their counterparts 1,2-bis(meth-
ylcarbamate) derivatives (14a–l) against human lymphoblastic
leukemia CCRF/CEM cell line and its subcell lines resistant to Vin-
blastine and Taxol. It shows that the size and the electron property
of the substituent at C3 altered the cytotoxicity of these agents. In
the series of bis(hydroxymethyl) derivatives (13a–l), the C3 alkyl
substituted derivatives (13a,b) are more cytotoxic than the phenyl
substituted derivatives (13c–l). In the series of C3 alkyl substituted
compounds, the C3-Me derivative (13a) is more potent than 13b
(C3-Et), whereas the order of cytotoxic effects for the series of C3
phenyl substituted derivatives is as follows: Ph > OMe-Ph > halo-
Ph, with the exception of C20,60-diOMe derivative (13j; the least
R1
R1
-X
R1
X
X
X
X
X
N
N
N
dG
R2
R2
R2
R1
R1
R1
dG
-X
X
X
dG
N
N
N
dG
dG
R2
R2
R2
R1
dG
dG
N
R2
Scheme 1. The proposed mechanism of DNA bis-alkylation by cyclopenta[a]indenes.

5616
R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
d (R2 = Me)
R1
R1
R1
a
d
COOMe
COOMe
COOH
COOH
N
N
H
N
O
R2
11 b-l
R2
12 a-l
8
e
c
R1
MeO
MeO
CH2OH
CH2OH
b
COOH
COOH
N
N
N
H
O
R2
13 a-l
9
MeO
f
10
R1
a: R1 = H, R2 = Me
g: R1 = H, R2 = 4'-OMePh
h: R1 = H, R2 = 2'-OMePh
i: R1 = H, R2 = 3',4'-OMePh
j: R1 = H, R2 = 2',6'-OMePh
k: R1 = H, R2 = 3',4',5'-triOMePh
l: R1 = OMe, R2 = 4'-OMePh
CH2OCONHMe
CH2OCONHMe
b: R1 = H, R2 = Et
c: R1 = H, R2 = Ph
N
d: R1 = H, R2 = 4'-FPh
e: R1 = H, R2 = 4'-ClPh
f: R1 = H, R2 = 3',4'-diFPh
R2
14 a-l
Scheme 2. Reagents and conditions: (a) acid chloride/Et3N/dry CH2Cl2, 0–25 °C, 3 h; (b) 4-methoxybenzoyl chloride/Et3N/dry CH2Cl2/DMAP, 0–25 °C, 5 h; (c) PtO2/H2/EtOH,
14 h; (d) dimethyl acetylenedicarboxylate/AC2O, 120 °C; (e) LiAlH4/CH2Cl2/EtO2, 0 °C, 15 min; (f) MeNCO/CH2Cl2/Et3N, rt, 4–5 h.
potent). It clearly demonstrated that the electron property of the
substituent at the C3 phenyl ring affected the cytotoxic effects of
these agents; compounds having an electron-donating substituent
were generally more potent than compounds bearing electron-
withdrawing functionality. But, this effect was not shown in
bis(methylcarbamate) derivatives. Among the OMe substituted
derivatives, 13g, 13h, and 13i, bearing OMe function(s) at C40,
C20, and C30,40, respectively, were more cytotoxic than those deriv-
atives having the same substituent at C20,60 and C30,40,50 (13j and
13k, respectively), indicating that introduction of a bulky substitu-
ent at C3 decreased the cytotoxicity of cyclopent[a]indenes. Similar
observations were found in the series of bis(methylcarbamate)
derivatives.
We used CCRF-CEM/Taxol and CCRF-CEM/VBL, which are sub-
cell lines of CCRF-CEM cells that are 330-fold resistant to Taxol,
and 680-fold resistant to Vinblastine, respectively, when compar-
ing with the IC50 of the parent cell line, to study whether the newly
synthesized compounds process multi-drug resistance toward Tax-
ol or Vinblastine. As shown in Table 1, the newly synthesized
cyclopenta[a]indenes have little or no cross-resistance to either
Taxol or Vinblastine. This suggests that all newly synthesized com-
pounds are neither a good substrate of membrane multidrug resis-
tance transporters (i.e., p-glycoprotein) nor mutated tubulin. Thus,
the newly synthesized compounds are effective against multi-drug
resistant tumors although both Taxol and Vinblastine are more
cytotoxic than the newly synthesized derivatives.
The antiproliferative activity of cyclopenta[a]indenes in inhibit-
ing human solid tumors such as breast carcinoma MX-1 and colon
carcinoma HCT-116 (Table 1) cell growth in vitro were also inves-
tigated. To explore further, compounds 13a, 13b, 13g, 13l, 14a,
14b, 14g, and 14l were selected to study their cell growth inhibi-
tory effect against other solid tumor such as lung carcinoma
H1299, oral tumor OECM1, prostate tumor PC3, and brain glioma
U87 (Table 2). Generally, the bis(methylcarbamate) compounds
were more cytotoxic than the corresponding parent bis(hydroxy-
methyl) derivatives with the exception of compounds 13a, 13c,
13e, and 13k. These agents were more effective against CCRF/
CEM, MX-1, and OECM1 cell growth among the tumor cell lines
tested.
2.2.2. In vivo therapeutic efficacy
Based on the results of the in vitro cytotoxicity, solubility and
toxicity to the host, we selected compound 13a,b and 14g,h for fur-
ther antitumor evaluation in animal model. Figure 2A and B shows
the therapeutic efficacy of 13a,b and 14g,h in nude mice bearing
human breast carcinoma MX-1 xenograft. At the maximal tolerate
doses [13b/50 mg/kg and 14g/5 mg/kg, daily for 3 times (QD ꢀ 3);
for 13a/18 mg/kg (Q2D ꢀ 2) and 14h/15 mg/kg (Q2D ꢀ 4), via
intravenous (iv) injection], these agents induce total tumor remis-
sion (complete remission, CR) with relatively low toxicity. In hu-
man prostate adenocarcinoma PC3 xenograft in nude mice
(Fig. 3A and B), compound 13b produced complete tumor suppres-
sion at the dose of 50 mg/kg, QD ꢀ 4, iv inj. Although, 15% of body
weight loss was observed during the treatment, it recovered soon
after the cessation of drug administration. The antitumor efficacy
of 13a and 13b in nude mice bearing human ovarian adenocarci-
noma SK-OV-3 xenograft was also studied (Fig. 4A and B). We
found that 13a and 13b yielded 53% and 78% tumor suppression,
respectively, on day 22 at the dose of 20 mg/kg, QD ꢀ 4, and
50 mg/kg, QD ꢀ 3), respectively, via iv injection. These studies sug-
gest that the newly synthesized cyclopent[a]indenes possess supe-
rior therapeutic efficacy against human breast MX-1 and human
prostate adenocarcinoma PC3 over human ovarian adenocarci-
noma SK-OV-3 xenograft.
2.2.3. DNA cross-linking study by alkaline agarose gel shift
assay
Early works on the study of DNA interstrand cross-linking by
IPP (6) and related compounds demonstrated that these agents
preferentially cross-link deoxyguanosine residues at the duplex
nucleotide (a short oligonucleotide 50-ACGT) at the sequence
50-CG residues, a common target site in duplex DNA.14 To deter-
mine whether the newly synthesized derivatives are able to
cross-link to DNA double-strands, we adopted alkaline agarose
gel shift assay25 to investigate the DNA cross-linking activity of
several representative bis(hydroxymethyl) derivatives (13a, 13b,
and 13l) and bis(carbamate) derivatives (14g, 14h, and 14l).
We first mixed the pEGFP-N1 plasmid DNA with various concen-
trations of tested compounds (1, 10, and 20 lM) or melphalan

R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
5617
Table 1
The cytotoxicity of 1,2-bis(methanol) derivatives (13a–l)a and 1,2-bis(methylcarbamate) derivatives (14a–l)b of cyclopenta[a]indenes against human lymphoblastic leukemia
(CCRF-CEM) and its drug-resistant sublines (CCRF-CEM/Taxol and CCRF-CEM/VBL) and human solid tumor (breast carcinoma MX-1 and colon carcinoma HCT-116) cell growth
in vitroc
R1
OR3
N
OR3
R2
Compd
R1
R2
Cell growth inhibition (IC50
CCRF-CEM/Taxold
, l
M)c
CCRF/CEM
CCRF-CEM/VBLd
MX-1
HCT-116
13a
14a
13b
14b
13c
14c
13d
14d
13e
14e
13f
14f
13g
14g
13h
14h
13i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
Me
Me
Et
Et
0.010 0.0004
0.12 0.07
0.36 0.1
0.07 0.005
0.25 0.004
0.36 0.01
4.5 0.3
0.14 0.004
4.3 0.004
5.7 0.6
0.067 0.0002 [0.88ꢀ]e
0.063 0.005 [0.53ꢀ]
0.23 0.004 [0.64ꢀ]
0.1 0.0003 [1.5ꢀ]
0.25 0.03 [1.0ꢀ]
0.55 0.004 [1.6ꢀ]
11 0.1 [2.5ꢀ]
0.032 0.002 [0.39ꢀ]
0.12 0.004 [0.67ꢀ]
0.14 0.009 [0.39ꢀ]
0.08 0.003 [1.2ꢀ]
0.22 0.02 [0.90ꢀ]
1.7 0.05 [4.7ꢀ]
0.36 0.03
0.34 0.01
1.1 0.03
0.66 0.009
1.3 0.011
5.9 0.02
22 0.1
0.74 0.04
6.1 0.1
9.9 0.4
0.34 0.0008
0.3 0.001
0.83 0.002
0.71 0.001
1.9 0.1
0.75 0.01
8.7 0.6
0.40 0.007
5.7 0.09
8.9 0.4
C6H5
C6H5
40-FC6H4
7.2 0.2 [1.6ꢀ]
40-FC6H4
0.28 0.004 [2.0ꢀ]
4.5 0.2 [1.0ꢀ]
0.30 0.008 [2.2ꢀ]
2.4 0.1 [0.55ꢀ]
40-ClC6H4
40-ClC6H4
2.3 0.2 [0.40ꢀ]
3.3 0.07 [0.58ꢀ]
7.5 0.2 [1.4ꢀ]
30,40-Di-FC6H3
30,40-Di-FC6H3
40-MeOC6H4
40-MeOC6H4
20-MeOC6H4
20-MeOC6H4
30,40-Di-MeOC6H3
30,40-Di-MeOC6H3
20,60-Di-MeOC6H3
20,60-Di-MeOC6H3
30,40,50-Tri-MeOC6H2
30,40,50-Tri-MeOC6H2
40-MeOC6H4
40-MeOC6H4
Taxol
5.2 0.2
7.5 0.3 [1.4ꢀ]
7.0 0.02
8.2 0.3
11 0.4
0.90 0.02
6.1 0.2
0.64 0.007
1.2 0.005
0.049 0.003
1.0 0.2
0.14 0.029
1.0 0.3
0.35 0.07
8.1 0.2
0.64 0.2
2.2 0.1
12 0.3
0.12 0.005
0.062 0.0001
0.0012 0.0001
0.00073 0.0009
0.65 0.02 [1.0ꢀ]
1.6 0.02 [1.4ꢀ]
0.61 0.02 [0.97ꢀ]
1.7 0.03 [1.5ꢀ]
11 0.005
1.9 0.01
8.7 0.04
1.3 0.01
11 0.05
1.8 0.001
26 0.5
5.3 0.3
0.063 0.007 [1.3ꢀ]
2.7 0.03 [2.6ꢀ]
0.070 0.004 [1.4ꢀ]
1.9 0.007 [1.9ꢀ]
0.25 0.005 [1.8ꢀ]
1.1 0.02 [1.1ꢀ]
2.6 0.1
12
2
0.36 0.003 [2.7ꢀ]
1.9 0.07 [1.8ꢀ]
0.15 0.004
4.3 0.3
0.66 0.02
43 0.3
14i
13j
14j
13k
14k
13l
0.50 0.007 [1.4ꢀ]
7.5 0.09 [0.92ꢀ]
0.62 0.01 [0.97ꢀ]
0.99 0.04 [0.45ꢀ]
14 0.04 [1.2 ꢀ]
0.14 0.004 [1.2ꢀ]
0.098 0.01 [1.6ꢀ]
0.40 0.04 [333ꢀ]
0.078 0.01 [107ꢀ]
0.39 0.02 [1.1ꢀ]
6.2 0.1 [0.76ꢀ]
0.72 0.05 [1.1ꢀ]
1.1 0.004 [0.52ꢀ]
8.4 0.003 [0.73ꢀ]
0.062 0.001 [0.53ꢀ]
0.062 0.002 [1.0ꢀ]
1.2 0.05 [1000ꢀ]
0.50 0.1 [685ꢀ]
1.1 0.01
17 0.4
29
2
7.2 0.07
1.5 0.001
0.87 0.004
0.046 0.0007
0.0022 0.0002
1.9 0.08
0.67 0.03
0.44 0.02
0.0011 0.0003
0.0011 0.0003
14l
VBT
a
R3 = H.
b
c
R3 = CONHMe.
Cell growth inhibition was measured by the XTT assay28 for leukemic cells and the SRB assay29 for solid tumor cells after 72-h incubation using a microplate spectro-
photometer as described previously.30 IC50 values were determined from dose–effect relationship at six or seven concentrations of each drug by using the CompuSyn software
by Chou and Martin32 based on the median-effect principle and plot.33,34 Ranges given for Taxol and Vinblastine were mean SE (n = 4).
d
CCRF-CEM/Taxol and CCRF-CEM/VBL are subcell lines of CCRF-CEM cells that are 333-fold resistant to Taxol, and 685-fold resistant to Vinblastine, respectively, when
comparing with the IC50 of the parent cell line.
e
Numbers in the brackets are fold of cross-resistant determined by comparison with the corresponding IC50 of the parent cell line.
Table 2
As shown in Figure 5, all the tested compounds were able to
interact with the plasmid DNA and resulted in interstrand
cross-links, which could not separated into single strand under
the alkaline condition. These results demonstrated that the new-
ly synthesized 3a-azacyclopenta[a]indenes are capable of induc-
ing DNA cross-linking formation.
The cytotoxicity of 1,2-bis(methanol) derivatives (13a–l) and 1,2-bis(methylcarba-
mate) derivatives (14a–l) of cyclopenta[a]indenes against human solid tumor (lung
carcinoma H1299, oral carcinoma OECM1, prostate carcinoma PC3, glioma U87) cell
growth in vitroa
Compound
Cell growth inhibition (IC50, l
M)b
H1299
OECM1
PC3
U87
2.2.4. DNA interstrand cross-linking in human non-small lung
carcinoma H1299 cell lines
13a
14a
13b
14b
13g
14g
13l
15.16 3.22
>50
14.14 2.69
>50
50.3 1.98
42.55 2.47
19 3.01
20.89 2.45
0.86 1.21
1.45
19.68 2.34
27.18
16.56 4.10
>50
55.5 3.18
38.17 0.93
23.2 1.12
21.97 3.25
19.32 3.56
>50
18.20 1.33
>50
45.49 3.11
34.35 2.83
24.36 1.83
38.28 2.98
1.24 2.51
11.8 0.21
9.26 2.85
4.63 1.25
1.54 0.72
1.27 1.13
To determine whether the newly synthesized derivatives are
able to form DNA cross-linking in cells, bis(hydroxymethyl) 13l
and its bis(methylcarbamate) derivative 14l were selected as the
representatives compounds for modified single cell gel electropho-
resis assay (modified comet assay).22,23 It is based on the property
of negatively charged DNA fragments migration when electric field
is applied to the gel after cell lysis.24 DNA cross-linking agent mito-
mycin C (MMC) was chosen as a positive control although this
agent was more effectively cross-link with DNA under anaerobic
conditions.5 Human non-small lung carcinoma H1299 cells were
treated with 13l, 14l, and MMC for 1 h at various concentrations
under aerobic conditions and then irradiated with a dose of
20 Gy to induce DNA strand breaks. Afterward, the irradiated cells
were subjected for modified comet assay. Figure 6 shows the dis-
14l
a
Cell growth inhibition was measured by the WST-1 assay31 after a 72 h-incu-
bation using a microplate spectrophotometer as described in Section 4.
b
The values are averages of at least two separate determinations.
(1 mM) in a tube and kept at 37 °C for 2 h. Afterward, the pEG-
FP-N1 plasmid DNA were linearized by digestion with BamHI
and then subjected to electrophoresis on a 0.8% alkaline agarose
gel. Ethidium bromide staining was used to visualize the DNA.

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R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
Figure 2. Therapeutic effect of 13a (18 mg/kg, Q2D ꢀ 2, iv inj., n = 5, p < 0.0001 on
day 14–24, no relapse on day 40), 13b (50 mg/kg, QD ꢀ 3, iv inj., n = 5, p < 0.0001 on
day 14–24, 3/5 CR on day 22; 5/5 CR on day 24, no relapse on day 40), 14g (5 mg/kg,
QD ꢀ 3, iv inj., n = 5, p < 0.0001, on day 16–24, 4/5 CR on day 20, 5/5 CR on day 22, 2/
5 relapse on D28, 3/5 no relapse on day 40), and 14h (15 mg/kg, Q2D ꢀ 4, iv inj.,
n = 4, p < 0.0001 on day 12–24, 3/4 CR on day 16, 4/4 CR on day 18, 1/4 relapse on
D28, 3/4 no relapse on day 40) in nude mice bearing human mammary carcinoma
MX-1 xenograft, control (n = 5); control (d), 13a (j), 13b (h), 14g (ꢀ), 14h (
D); (A)
average tumor size changes; (B) average body weight changes.
tribution of tail moment in H1299 cells. Since ionic radiation re-
sulted DNA fragments in cells treated with DNA cross-linking
agents would reduce their electrophoretic migration rate under
alkaline condition, the length and fragment content of the tail is in-
versely proportional to the amount of DNA interstrand cross-link-
ing. Since interstrand cross-link formation can be represented as
percent decrease in tail moment, Figure 7 shows the levels of
cross-linked DNA varied with compound concentrations. Under
Figure 3. The therapeutic effects of 13b (50 mg/kg, QD ꢀ 4, iv injection) in nude
mice bearing human prostate adenocarcinoma PC3 xenograft; n = 5; control (Ç),
13b (j); (A) average tumor size changes; (B) average body weight changes; the p
values for the treated versus the untreated group from day 20 to 28 is p < 0.001.
3. Conclusions
We have designed and synthesized a series of DNA bifunctional
alkylating agents, bis(hydroxymethyl)-8H-3a-azacyclopenta[a]indene-
1-yl derivatives (13a–l) and their bis(carbamate) derivatives
(14a–l) for antitumor evaluation. These agents can be considered
as ‘benzologues’ of pyrrolizines and may have a similar mechanism
of action to bis-carbamoyloxymethyl derivatives of pyrroles and
pyrrolizines or MMC. The present studies demonstrated that these
newly synthesized compounds exhibited potent cytotoxicity
against human leukemia and various solid tumor cell growths
in vitro and potent antitumor efficacy in vivo with a relatively
low toxicity. Detailed SAR studies demonstrated that the size and
electron properties of the substituent at C3 affected the cytotoxic-
ity of these agents. Compounds 13a,b and 14g,h were found to
have potent therapeutic efficacy against human breast MX-1 xeno-
graft in animal model. Complete tumor remission was achieved in
nude mice bearing human breast carcinoma MX-1 xenograft by
these derivatives. Interestingly, we also found that compound
13b was able to significantly suppress human prostate adenocarci-
noma PC3 xenograft in nude mice. Studies on the DNA interstrand
cross-linking suggested that the newly synthesized derivatives are
potent bifunctional DNA cross-linking agents. Furthermore, both
derivatives induced substantial G2/M phase arrest. On the basis
of the present studies, these agents should be considered as prom-
ising lead compounds for preclinical studies.
the same experimental conditions (100 lM), 14l, MMC, and 13l in-
duce 97.9%, 76.5%, and 35.5% DNA interstrand cross-linking,
respectively. The results showed that the order of potency for
DNA cross-linking induction by these agents is 14l > MMC > 13l.
2.2.5. Cell cycle inhibition
DNA interacting/damaging agents are known to induce cell
cycle perturbations and arrest the cell cycle progression predomi-
nantly at the G2/M boundary.26 Compounds 13l, a bis(hydroxy-
methyl) derivative, and 14l, a bis(methylcarbamate) derivative,
were selected as the representatives compounds to investigate
their influence on cell cycle progression. We treated human non-
small lung carcinoma H1299 cells with these compounds at the
concentrations of 10 lM. At the time indicated (24, 48, and 72 h),
the cells were harvested, stained with propidium iodide (PI) and
analyzed with a flowcytometer. As shown in Figure 8, both 13l
and 14l remarkably delayed the cell cycle progression. As com-
pared to the control culture, we observed the accumulation of
the S phase cells at 24 h after treatment with 13l and 14l and fol-
lowed by the accumulation of the G2/M phase at 48 and 72 h. Fur-
thermore, increased sub-G1 population were noticed in cells
treated with these 2 compounds for 72 h (Fig. 8).

R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
5619
4.2. 5-Methoxy-1-(4-methoxybenzoyl)-1H-indole-2-carboxylic
acid (10)
To a solution of commercially available 5-methoxyindole-2-
carboxylic acid 9 (6.69 g, 35 mmol) and triethylamine (10 mL,
71.74 mmol) in anhydrous CH2Cl2 (70 mL) containing catalytic
amount of 4-dimethylaminopyridine (DMAP) was added dropwise a
solutionof4-methoxybenzoylchloride(6.20 g,36.34 mmol)inCH2Cl2
(15 mL) at 0 °C. The reaction mixture was stirred at this temperature
for 15 min and then at roomtemperaturefor4 h. The reactionmixture
was washed successively with 5% aqueous KHSO4 solution
(2 ꢀ 20 mL), brine (25 mL), dried over Na2SO4, and evaporated under
reduced pressure todryness. The solidresiduewas recrystallizedfrom
methanol to yield 10 as a white solid (7.4 g, 65%); mp 185–187 °C; 1H
NMR (400 MHz, DMSO-d6) d = 13.2 (br s, 1H), 7.59 (d, J = 8.9 Hz, 2H),
7.38 (d, J = 9.0 Hz, 1H), 7.33 (s, 1H), 7.28 (d, J = 2.5 Hz, 1H), 7.07 (d,
J = 8.9 Hz, 2H), 7.02 (dd, J = 9.0, 2.5 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H).
Anal. Calcd for C18H15NO5ꢁ0.1H2O: C, 65.73; H, 4.65; N, 4.25. Found:
C, 65.77; H, 4.61; N, 4.24.
4.3. 1-Propionyl-2,3-dihydro-1H-indole-2-carboxylic acid (11b)
To a solution of indoline-2-carboxylic acid 8 (8.16 g, 50 mmol)
and triethylamine (10 mL, 71.74 mmol) in anhydrous CH2Cl2
(60 mL) containing catalytic amount of DMAP was added dropwise
a solution of propionyl chloride (5.55 g, 60 mmol) in CH2Cl2
(20 mL) at 0 °C. The reaction mixture was stirred at this tempera-
ture for 15 min and then at room temperature for 2.5 h. The reac-
tion mixture was washed successively with 5% aqueous KHSO4
solution (2 ꢀ 20 mL), brine (25 mL), dried over Na2SO4, and evapo-
rated under reduced pressure to dryness. The residue was crystal-
lized from methanol to yield 11b (9.19 g, 83%); mp 173–175 °C;
d = 13.26 (br s, 1H), 8.09–8.07 (m, 1H), 7.23–7.15 (m, 2H), 7.01–
6.98 (m, 1H), 5.15 (d, J = 16.0 Hz, 1H), 3.57 (dd, J = 16.0, 11.0 Hz,
1H), 3.17 (d, J = 16.0 Hz, 1H), 2.51 (q, J = 7.6 Hz, 2H), 1.05 (t,
J = 7.6 Hz, 3H). Anal. Calcd for C12H13NO3: C, 65.74; H, 5.98; N,
6.39. Found: C, 65.73; H, 6.07; N, 6.34.
Figure 4. The therapeutic effects of 13a (20 mg/kg, QD ꢀ 4, iv injection) and 13b
(50 mg/kg, QD ꢀ 3, iv injection) in nude mice bearing human ovarian adenocarci-
noma SK-OV-3 xenograft; n = 5; control (d), 13a (D), 13b (h); (A) average tumor
size changes; (B) average body weight changes; the values for the treated versus the
untreated group from day 20 to 28 is p < 0.001.
By following the same procedure as describe for 11b, the fol-
lowing compounds 11c–k were synthesized.
4.3.1. 1-Benzoyl-2,3-dihydro-1H-indole-2-carboxylic acid (11c)
Compound 11c was synthesized from indoline-2-carboxylic
acid 8 (6.52 g, 40 mmol), triethylamine (11 mL, 78 mmol), benzoyl
chloride (5.62 g, 40 mmol); yield (10.66 g, 81%); mp 209–210 °C
(lit. mp 191–193 °C);27 1H NMR (400 MHz, DMSO-d6) d = 13.01
(br s, 1H), 8.17–8.14 (m, 1H), 7.49 (m, 5H), 7.26–7.25 (m, 2H),
7.06 (m, 1H), 4.97 (s, 1H), 3.63–3.61 (m, 1H), 3.09 (d, J = 16.3 Hz,
1H); MS: m/z 268.0 (M+H)+.
Figure 5. Representative DNA cross-linking gel shift assay for 13a, 13b, 13l, 14h,
14g, and 14l at the concentrations of 1, 10, and 20
lM. Control lane shows single-
stranded DNA (SS), while CL shown in all tested lanes is DNA double-stranded
cross-linking. Melphalan at 1 lM was used as a positive control.
4.3.2. 1-(4-Fluorobenzoyl)-2,3-dihydro-1H-indole-2-carboxylic
acid (11d)
Compound 11d was synthesized from indoline-2-carboxylic
acid 8 (6.52 g, 40 mmol), triethylamine (11 mL, 78 mmol), and 4-
fluorobenzoyl chloride (6.34 g, 40 mmol); yield (9.28 g, 81%); mp
215–216 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.98 (br s, 1H),
8.16–7.03 (m, 8H), 5.01 (d, J = 9.3 Hz, 1H), 3.63 (dd, J = 16.3,
11.1 Hz, 1H), 3.10 (d, J = 16.5 Hz, 1H). Anal. Calcd for C16H12FNO3:
C, 67.36; H, 4.24; N, 4.91. Found: C, 67.22; H, 4.58; N, 4.72.
4. Experimental
4.1. General methods and materials
Melting points were determined on a Fargo melting point appa-
ratus and are uncorrected. Column chromatography was carried
out over Silica Gel G60 (70–230 mesh). Thin-layer chromatography
was performed on Silica Gel G60 F254 plates with short wavelength
UV light for visualization. Elemental analyses were done on a
Heraeus CHN–O Rapid instrument. 1H NMR spectra were recorded
on 400 MHz spectrometer with Me4Si as an internal standard. The
chemical shifts were reported in ppm (d) relative to TMS and cou-
pling constants (J) hertz (Hz).
4.3.3. 1-(4-Chlorobenzoyl)-2,3-dihydro-1H-indole-2-carboxylic
acid (11e)
Compound 11e was synthesized from indoline-2-carboxylic
acid (8) (6.52 g, 40 mmol), triethylamine (11 mL, 78 mmol), and
4-chlorobenzoyl chloride (5 g, 40 mmol); yield (9.35 g, 77%); mp
184–187 °C; 1H NMR (MHz, DMSO-d6) d = 12.91 (br s, 1H), 7.81–
7.53 (m, 4H), 7.27–7.25 (m, 2H), 7.18–7.03 (m, 2H), 5.02 (d,

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R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
No drug, no irradiation
No drug, 20 Gy irradiation
10 µM
25 µM
25 µM
25 µM
50 µM
50 µM
50 µM
100 µM
100 µM
100 µM
10 µM
10 µM
Figure 6. Typical comet images of DNA interstrand cross-linking study by modified comet assay under the aerobic conditions in H1299 cells. Cells were treated with various
concentrations of 13l and 14l for 1 h prior to 20 Gy X-ray irradiation. Mitomycin C (MMC) was used as a positive control.
luorobenzoyl chloride (7.06 g, 40 mmol); yield (8.53 g, 70%); mp
248–250 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.99 (br s, 1H),
8.15 (m, 1H), 7.64 (t, J = 8.7 Hz, 1H), 7.60–7.55 (m, 1H), 7.41 (s, 1H),
7.28–7.06 (m, 3H), 5.02 (dd, J = 10.9, 2.9 Hz, 1H), 3.63 (dd, J = 16.4,
11.2 Hz, 1H), 3.12 (d, J = 16.2 Hz, 1H). Anal. Calcd for C16H11F2NO3:
C, 63.37; H, 3.66; N, 4.62. Found: C, 63.12; H, 3.97; N, 4.83.
4.3.5. 1-(4-Methoxybenzoyl)-2,3-dihydro-1H-indole-2-
carboxylic acid (11g)
Compound 11g was synthesized from indoline-2-carboxylic acid
(8) (6.52 g, 40 mmol), triethylamine (11 mL, 78 mmol), and 4-
methoxybenzoyl chloride (6.82 g, 40 mmol); yield (10.88 g, 92%);
mp 196–198 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.91 (br s, 1H),
7.50–7.46 (m, 2H), 7.24 (d, J = 7.3 Hz, 1H), 7.09–6.97 (m, 5H), 5.01
(dd, J = 11.0, 3.3 Hz, 1H), 3.82 (s, 3H), 3.61 (dd, J = 16.6, 11.0 Hz,
1H), 3.08 (dd, J = 16.6, 3.2 Hz, 1H). Anal. cacld. for C17H15NO4: C,
68.68; H, 5.09; N, 4.71. Found: C, 68.10; H, 5.06; N, 4.62.
Figure 7. Percentages of DNA interstrand cross-linking in H1299 cells induced by
13l and 14l at various concentrations. Interstrand cross-link formation represented
as percent of decreased tail moment. Mitomycin C (MMC) was served as a positive
control. Data were presented as mean of 3 independent experiments. Bars, SD.
4.3.6. 1-(2-Methoxybenzoyl)-2,3-dihydro-1H-indole-2-
carboxylic acid (11h)
Compound 11h was synthesized from indoline-2-carboxylic acid
(8) (8 g, 49 mmol) and triethylamine (14 mL, 100 mmol) and 2-
methoxybenzoyl chloride (6.82 g, 49 mmol); yield (13.17 g, 91%);
mp 189–192 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.91 (br s, 1H),
7.49–7.47 (m, 2H), 7.24–7.23 (m, 2H), 7.08 (m, 1H), 7.02–6.98 (m,
3H), 5.02 (dd, J = 7.3, 2.2 Hz, 1H), 3.82 (s, 3H), 3.61 (dd, J = 10.9,
7.5 Hz, 1H), 3.08 (dd, J = 11.0, 1.6 Hz, 1H). Anal. Calcd for
C17H15NO4: C, 68.68; H, 5.09; N, 4.71. Found: C, 68.25; H, 5.04; N,
4.61.
J = 9.8 Hz, 1H), 3.63 (dd, J = 16.6, 11.2 Hz, 1H), 3.13 (d, J = 16.6 Hz,
1H). Anal. Calcd for C16H12ClNO3: C, 63.69; H, 4.01; N, 4.64. Found:
C, 62.15; H, 3.96; N, 4.51.
4.3.4. 1-(3,4-Difluorobenzoyl)-2,3-dihydro-1H-indole-2-
carboxylic acid (11f)
Compound 11f was synthesized from indoline-2-carboxylic acid
(8) (6.52 g, 40 mmol), triethylamine (11 mL, 78 mmol) and 3,4-dif-

R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
5621
Figure 8. Effects of 13l and 14l on cell cycle progress in H1299 cells. Cells were treated with 13l and 14l for 24, 48, and 72 h and cell cycle phases were analyzed by flow
cytometric technique. The distribution of cells at the sub-G1 (apoptotic cells), G0/G1, S, and G2/M phases was estimated with ModFit LT 2.0 software.
4.3.7. 1-(3,4-Dimethoxybenzoyl)-2,3-dihydro-1H-indole-2-
carboxylic acid (11i)
3,4,5-trimethoxybenzoyl chloride (8.49 g, 36.81 mmol); yield
(9.92 g, 75%); mp 224–227 °C; 1H NMR (400 MHz, DMSO-d6)
d = 13.01 (br s, 1H), 8.31 (s, 1H), 7.25 (d, J = 7.49 Hz, 1H), 7.15 (m,
1H), 7.03–7.02 (m, 1H), 6.77 (s, 2H), 4.97–4.95 (m, 1H), 3.77 (s, 6H),
3.71 (s, 3H), 3.65–3.59 (m, 1H), 3.10–3.05 (m, 1H). Anal. Calcd for
C19H19NO6: C, 63.86; H, 5.36; N, 3.92. Found: C, 62.27; H, 5.29; N, 3.76.
Compound 11i was synthesized from indoline-2-carboxylic acid
(8) (5.03 g, 30.82 mmol), triethylamine (8.6 mL, 61.64 mmol), and
3,4-dimethoxybenzoyl chloride (6.18 g, 30.82 mmol); yield (8.20 g,
81%); mp 113–115 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.92 (br
s, 1H), 7.26–7.24 (m, 1H), 7.10–6.98 (m, 6H), 5.00 (dd, J = 10.9,
3.1 Hz, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.62 (dd, J = 16.6, 11.1 Hz,
1H), 3.07 (dd, J = 16.6, 2.9 Hz, 1H). Anal. Calcd for C18H17NO5: C,
66.05; H, 5.23; N, 4.28. Found: C, 65.47; H, 5.74; N, 4.28.
4.3.10. 5-Methoxy-1-(4-methoxybenzoyl)-2,3-dihydro-1H-
indole-2-carboxylic acid (11l)
To a solution of 10 (7.02 g, 21.57 mmol) in ethanol (200 mL)
was added platinum oxide (0.7 g). The mixture was hydrogenated
for 6 h at 32 psi. The reaction mixture was filtered through a pad of
Celite. The filter cake was washed with ethanol. The combined fil-
trate and washings was evaporated in vacuo to dryness. The solid
residue was recrystallized from ethanol to yield 11l (6.35 g, 90%);
mp 151–152 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.92 (br s,
1H), 7.45 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.86 (m, 2H),
6.67 (s, 1H), 5.01–4.97 (m, 1H), 3.81 (s, 3H), 3.70 (s, 3H), 3.63–
3.56 (m, 1H), 3.05 (m, 1H). Anal. Calcd for C18H17NO5: C, 66.05;
H, 5.23; N, 4.28. Found: C, 65.69; H, 5.47; N, 4.14.
4.3.8. 1-(2,6-Dimethoxybenzoyl)-2,3-dihydro-1H-indole-2-
carboxylic acid (11j)
Compound 11j was synthesized from indoline-2-carboxylic acid
(8) (7.88 g, 48.28 mmol), triethylamine (8.6 mL, 61.64 mmol), and
2,6-dimethoxybenzoyl chloride (9.69 g, 48.28 mmol); yield
(12.77 g, 81%); mp 84–85 °C; 1H NMR (400 MHz, DMSO-d6)
d = 12.88 (br s, 1H), 7.25–7.23 (m, 1H), 7.10–6.97 (m, 6H), 5.00
(dd, J = 10.9, 3.2 Hz, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 3.61 (dd,
J = 16.6, 11.1 Hz, 1H), 3.07 (dd, J = 16.6, 2.9 Hz, 1H). Anal. Calcd
for C18H17NO5?H2O: C, 62.60; H, 5.54; N, 4.05. Found: C, 62.54;
H, 5.25; N, 3.97.
4.4. 3-Methyl-8H-3a-aza-cyclopenta[a]indene-1,2-dicarboxylic
acid dimethyl ester (12a)
4.3.9. 1-(3,4,5-Trimethoxybenzoyl)-2,3-dihydro-1H-indole-2-
carboxylic acid (11k)
Compound 11k was synthesized from indoline-2-carboxylic acid
(8) (6.01 g, 36.81 mmol), triethylamine (10.4 mL, 74.61 mmol), and
A mixture of indoline-2-carboxylic acid 8 (10.60 g, 65 mmol)
and dimethyl acetylenedicarboxylate (9.24 mL, 80 mmol) in acetic
anhydride (110 mL) was stirred in a flask equipped with a reflux

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R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
condenser and a gas bubbler to monitor carbon dioxide evolution
during the reaction. The mixture was heated to 120 °C where car-
bon dioxide evolution was most intense. The temperature was
maintained until gas evolution had ceased. The dark solution was
concentrated in vacuo and the solid residue was crystallized from
methanol to yield 12a (14.40 g, 78%); mp 162–164 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.53–7.46 (m, 2H), 7.37–7.33 (m, 1H),
7.27–7.20 (m, 1H), 4.01 (s, 2H), 3.89 (s, 3H), 3.85 (s, 3H), 2.74 (s,
3H). Anal. Calcd for C16H15NO4: C, 67.36; H, 5.30; N, 4.91. Found:
C, 67.30; H, 5.31; N, 4.85.
7.41–7.40 (m, 1H), 7.25–7.22 (m, 2H), 6.68–6.65 (m, 1H), 4.16 (s,
2H), 3.80 (s, 3H), 3.62 (s, 3H). Anal. Calcd for C21H15F2NO4: C,
65.80; H, 3.94; N, 3.65. Found: C, 65.83; H, 4.03; N, 3.62.
4.4.6. 3-(4-Methoxyphenyl)-8H-3a-aza-cyclopenta[a]indene-
1,2-dicarboxylic acid dimethyl ester (12g)
Compound 12g was synthesized from 11g (7.42 g, 25 mmol)
and dimethyl acetylenedicarboxylate (4.7 mL, 37.3 mmol) in acetic
anhydride (40 mL); yield (8.42 g, 89%); mp 157–159 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.59–7.58 (m, 1H), 7.44 (d, J = 8.3 Hz,
2H), 7.21–7.20 (m, 2H), 7.09 (d, J = 8.3 Hz, 2H), 6.67–6.65 (m,
1H), 4.15 (s, 2H), 3.85 (s, 3H), 3.78 (s, 3H), 3.60 (s, 3H). Anal. Calcd
for C22H19NO5: C, 70.02; H, 5.07; N, 3.71. Found: C, 69.80; H, 6.08;
N, 3.62.
4.4.1. 3-Ethyl-8H-3a-aza-cyclopenta[a]indene-1,2-dicarboxylic
acid dimethyl ester (12b)
A mixture of 11b (4.49 g, 20.5 mmol) and dimethyl acetylenedi-
carboxylate (5.68 mL, 40 mmol) in acetic anhydride (40 mL) was
stirred in a flask equipped with a reflux condenser and a gas bub-
bler to monitor carbon dioxide evolution during the reaction. The
mixture was heated to 120 °C where carbon dioxide evolution
was most intense. The temperature was maintained until gas evo-
lution had ceased. The dark solution was concentrated in vacuo to
dryness and the solid residue was recrystallized from methanol to
yield 12b (4.61 g, 75%); mp 86–88 °C; 1H NMR (400 MHz, DMSO-
d6) d = 7.64–7.59 (m, 2H), 7.47–7.43 (m, 1H), 7.31–7.27 (m, 1H),
4.07 (s, 2H), 3.76 (s, 3H), 3.75 (s, 3H), 3.08 (q, J = 7.4 Hz, 2H), 1.22
(t, J = 7.4 Hz, 3H). Anal. Calcd for C17H17NO4: C, 68.21; H, 5.72; N,
4.68. Found: C, 68.22; H, 5.74; N, 4.65.
4.4.7. 3-(2-Methoxy-phenyl)-8H-3a-aza-cyclopenta[a]indene-
1,2-dicarboxylic acid dimethyl ester (12h)
Compound 12h was synthesized from 11h (9.07 g, 30.5 mmol)
and dimethyl acetylenedicarboxylate (5 mL, 40.81 mmol) in acetic
anhydride (40 mL); yield (8.90 g, 77%); mp 162–164 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.58–7.53 (m, 2H), 7.36–7.34 (m, 1H),
7.21–7.19 (m, 3H), 7.11–7.07 (m, 1H), 6.53–6.51 (m, 1H), 4.23–
4.09 (m, 2H), 3.79 (s, 3H), 3.65 (s, 3H), 3.58 (s, 3H). Anal. Calcd
for C22H19NO5: C, 70.02; H, 5.07; N, 3.71. Found: C, 70.03; H,
5.06; N, 3.69.
By following the same procedure as describe for 12b, the fol-
lowing compounds 12c–l were synthesized.
4.4.8. 3-(3,4-Dimethoxyphenyl)-8H-3a-aza-
cyclopenta[a]indene-1,2-dicarboxylic acid dimethyl ester (12i)
Compound 12i was synthesized from 11i (9.01 g, 27.53 mmol)
and dimethyl acetylenedicarboxylate (4 mL, 32 mmol) in acetic
anhydride (40 mL); yield (8.9 g, 79%); mp 186–187 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.60–7.58 (m, 1H), 7.24–7.22 (m, 2H),
7.12 (s, 1H), 7.10 (s, 1H), 7.08–7.05 (m, 1H), 6.77–6.75 (m, 1H),
4.16 (s, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 3.75 (s, 3H), 3.63 (s, 3H). Anal.
Calcd for C23H21NO6: C, 67.80; H, 5.20; N, 3.44. Found: C, 67.57; H,
5.22; N, 3.39.
4.4.2. 3-Phenyl-8H-3a-aza-cyclopenta[a]indene-1,2-
dicarboxylic acid dimethyl ester (12c)
Compound 12c was synthesized from 11c (7.48 g, 28 mmol)
and dimethyl acetylenedicarboxylate (4 mL, 32 mmol) in acetic
anhydride (40 mL); yield (7.5 g, 77%); mp 149–151 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.67–7.51 (m, 6H), 7.23–7.18 (m, 2H),
6.67–6.60 (m, 1H), 4.15 (s, 2H), 3.79 (s, 3H), 3.62 (s, 3H). Anal. Calcd
for C21H17NO4ꢁ0.9H2O: C, 69.40; H, 5.21; N, 3.85. Found: C, 69.42;
H, 4.65; N, 3.83.
4.4.9. 3-(2,6-Dimethoxyphenyl)-8H-3a-aza-
cyclopenta[a]indene-1,2-dicarboxylic acid dimethyl ester (12j)
Compound 12j was synthesized from 11j (12.11 g, 37 mmol)
and dimethyl acetylenedicarboxylate (5.5 mL, 44.90 mmol) in ace-
tic anhydride (55 mL); yield (10.53 g, 70%); mp 84–86 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.58–7.55 (m, 1H), 7.49 (t, J = 8.3 Hz, 1H),
7.22–7.18 (m, 2H), 6.82 (s, 1H), 6.80 (s, 1H), 6.50–6.55 (m, 1H), 4.15
(s, 2H), 3.79 (s, 3H), 3.63 (s, 6H), 3.54 (s, 3H). Anal. Calcd for
C23H21NO6: C, 67.80; H, 5.20; N, 3.44. Found: C, 67.12; H, 5.20; N,
3.41.
4.4.3. 3-(4-Fluorophenyl)-8H-3a-aza-cyclopenta[a]indene-1,2-
dicarboxylic acid dimethyl ester (12d)
Compound 12d was synthesized from 11d (7.13 g, 25 mmol)
and dimethyl acetylenedicarboxylate (4 mL, 32 mmol) in acetic
anhydride (40 mL); yield (6.63 g, 72%); mp 152–155 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.54–7.51 (m, 2H), 7.47 (d, J = 7.4 Hz,
1H), 7.21–7.15 (m, 3H), 6.65 (d, J = 7.93 Hz, 1H), 7.12 (t,
J = 7.4 Hz, 1H), 4.12 (s, 2H), 3.89 (s, 3H), 3.72 (s, 3H). Anal. Calcd
for C21H16FNO3ꢁ0.67H2O: C, 66.84; H, 4.63; N, 3.71. Found: C,
66.94; H, 4.46; N, 3.65.
4.4.10. 3-(3,4,5-Trimethoxyphenyl)-8H-3a-aza-
cyclopenta[a]indene-1,2-dicarboxylic acid dimethyl ester (12k)
Compound 12k was synthesized from 11k (9 g, 25.18 mmol)
and dimethyl acetylenedicarboxylate (4.2 mL, 34.28 mmol) in ace-
tic anhydride (45 mL); yield (9.62 g, 87%): mp 205–207 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.59 (d, J = 6.9 Hz, 1H), 7.29–7.21 (m, 2H),
6.87 (d, J = 7.6 Hz, 1H), 6.80 (s, 2H), 4.16 (s, 2H), 3.99 (s, 3H), 3.77 (s,
3H), 3.76 (s, 6H), 3.65 (s, 3H). Anal. Calcd for C24H23NO7: C, 65.90;
H, 5.30; N, 3.20. Found: C, 65.81; H, 5.35; N, 3.12.
4.4.4. 3-(4-Chlorophenyl)-8H-3a-aza-cyclopenta[a]indene-1,2-
dicarboxylic acid dimethyl ester (12e)
Compound 12e was synthesized from 11e (7.54 g, 25 mmol)
and dimethyl acetylenedicarboxylate (4 mL, 32 mmol) in acetic
anhydride (40 mL); yield (7.32 g, 79%); mp 166–168 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.64–7.54 (m, 5H), 7.25–7.21 (m, 2H),
6.68–6.64 (m, 1H), 4.16 (s, 2H), 3.79 (s, 3H), 3.62 (s, 3H). Anal. Calcd
for C21H16ClNO4: C, 66.06; H, 4.22; N, 3.67. Found: C, 65.92; H,
4.22; N, 3.64.
4.4.11. 6-Methoxy-3-(4-methoxyphenyl)-8H-3a-aza-
cyclopenta[a]indene-1,2-dicarboxylic acid dimethyl ester (12l)
Compound 12l was synthesized from 11l (4.24 g, 12.9 mmol)
and dimethyl acetylenedicarboxylate (2.4 mL, 19.5 mmol) in acetic
anhydride (24 mL); yield (3.75 g, 71%); mp 240–242 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.42 (d, J = 6.9 Hz, 2H), 7.19 (d,
J = 2.4 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.77 (dd, J = 8.8, 2.6 Hz,
1H), 6.55 (d, J = 8.8 Hz, 1H), 4.09 (s, 2H), 3.85 (s, 3H), 3.77 (s, 3H),
4.4.5. 3-(3,4-Difluoro-phenyl)-8H-3a-aza-cyclopenta[a]indene-
1,2-dicarboxylic acid dimethyl ester (12f)
Compound 12f was synthesized from 11f (8.14 g, 26.82 mmol)
and dimethyl acetylenedicarboxylate (4.7 mL, 37.3 mmol) in acetic
anhydride (40 mL); yield (8.37 g, 81%); mp 156–158 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.70–7.67 (m, 1H), 7.64–7.59 (m, 2H),

R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
5623
3.73 (s, 3H), 3.60 (s, 3H). Anal. Calcd for C23H21NO5: C, 67.80; H,
5.20; N, 3.44. Found: C, 67.83; H, 5.19; N, 3.39.
7.16–7.13 (m, 1H), 7.08–7.04 (m, 1H), 6.78 (d, J = 7.8 Hz, 1H),
4.80 (t, J = 5.3 Hz, 1H), 4.73 (t, J = 5.3 Hz, 1H), 4.55 (d, J = 5.3 Hz,
2H), 4.26 (d, J = 5.3 Hz, 2H), 3.93 (s, 2H). Anal. Calcd for
4.5. (3-Methyl-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13a)
C19H16ClNO2: C, 70.05; H, 4.95; N, 4.30. Found: C, 69.58; H, 5.05;
N, 4.20.
A solution of 12a (3.14 g, 11 mmol) in anhydrous dichlorometh-
ane (30 mL) was added dropwise to a stirred suspension of LiAlH4
(1.04 g, 27.5 mmol) in anhydrous ether (45 mL) at 0 °C. The mix-
ture was stirred for 15 min after the addition was complete. The
excess LiAlH4 was carefully decomposed by the slow, sequential
addition of water (1.3 mL), 15% NaOH aqueous solution (1.3 mL),
and water (3.6 mL). The precipitated solid inorganic salt was re-
moved by filtration and washed with dichloromethane. The com-
bined filtrate and washings was concentrated in vacuo to
dryness. The residue was triturated with ether, the solid product
was collected by filtration, and dried to give 13a (1.95 g, 77%);
mp 123–125 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.48–7.46 (m,
2H), 7.33–7.29 (m, 1H), 7.10–7.07 (m, 1H), 4.61 (t, J = 5.2 Hz, 1H),
4.47 (t, J = 5.2 Hz, 1H), 4.44 (d, J = 5.2 Hz, 2H), 4.35 (d, J = 5.2 Hz,
2H), 3.80 (s, 2H), 2.53 (s, 3H). Anal. Calcd for C14H15NO2: C,
73.34; H, 6.59; N, 6.11. Found: C, 73.08; H, 6.59; N, 6.04.
4.5.5. (3-(3,4-Difluoro-phenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13f)
Compound 13f was synthesized from 12f (3.83 g, 10 mmol) in
anhydrous dichloromethane (30 mL) and LiAlH4 (0.95 g, 25 mmol)
in anhydrous ether (40 mL); yield 2.25 g (68%): mp 146–147 °C; 1H
NMR (400 MHz, DMSO-d6) d = 7.62–7.57 (m, 2H), 7.49 (d,
J = 4.89 Hz, 1H), 7.36–7.34 (m, 1H), 7.16 (t, J = 5.23 Hz, 1H), 7.07
(t, J = 4.92 Hz, 1H), 6.78 (d, J = 5.27 Hz, 1H), 4.77 (t, J = 3.5 Hz,
1H), 4.73 (t, J = 3.5 Hz, 1H), 4.56 (d, J = 3.5 Hz, 2H), 4.26 (d,
J = 3.5 Hz, 2H), 3.93 (s, 2H). Anal. Calcd for C19H15F2NO2: C,
69.72; H, 4.62; N, 4.28. Found: C, 69.39; H, 4.68; N, 4.13.
4.5.6. (3-(4-Methoxyphenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13g)
Compound 13g was synthesized from 12g (2 g, 8.35 mmol) in
anhydrous dichloromethane (30 mL) and LiAlH4 (0.76 g, 20 mmol)
in anhydrous ether (30 mL); yield (1.33 g, 78%): mp 143–144 °C; 1H
NMR (400 MHz, DMSO-d6) d = 7.46 (d, J = 7.5 Hz, 1H), 7.49–7.42
(m, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 7.04 (t,
J = 7.4 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 4.74 (t, J = 5.2 Hz, 1H), 4.60
(t, J = 5.2 Hz, 1H), 4.55 (d, J = 5.2 Hz, 2H), 4.25 (d, J = 5.2 Hz, 2H),
3.90 (s, 2H), 3.84 (s, 3H). Anal. Calcd for C20H19NO3: C, 74.75; H,
5.96; N, 4.36. Found: C, 74.53; H, 6.05; N, 4.18.
By following the same procedure as describe for 13a, the follow-
ing compounds 13b–l were synthesized.
4.5.1. (3-Ethyl-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13b)
Compound 13b was synthesized from 12b (2.5 g, 8.35 mmol) in
anhydrous dichloromethane (30 mL) and LiAlH4 (0.793 g,
20.87 mmol) in anhydrous ether (30 mL); yield (1.31 g, 64%); mp
131–133 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.46–7.45 (m, 1H),
7.39–7.37 (m, 1H), 7.34–7.30 (m, 1H), 7.09–7.06 (m, 1H), 4.63 (t,
J = 5.2 Hz, 1H), 4.47 (t, J = 5.2 Hz, 1H), 4.45 (d, J = 5.2 Hz, 2H), 4.36
(d, J = 5.2 Hz, 2H), 3.81 (s, 2H), 2.89 (q, J = 7.3 Hz, 2H), 1.18 (t,
J = 7.3 Hz, 3H). Anal. Calcd for C15H17NO2: C, 74.05; H, 7.04; N,
5.76. Found: C, 73.22; H, 6.94; N, 5.71.
4.5.7. (3-(2-Methoxyphenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13h)
Compound 13h was synthesized from 12h (2.54 g, 6.73 mmol)
in anhydrous dichloromethane (25 mL) and LiAlH4 (0.8 g,
21 mmol) in anhydrous ether (40 mL); yield 1.65 g (76%): mp
132–134 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.50–7.42 (m, 2H),
7.34 (dd, J = 7.4, 1.68 Hz, 1H), 7.17–7.15 (d, J = 8.1 Hz, 1H), 7.11–
7.05 (m, 2H), 6.99 (t, J = 7.4 Hz, 1H), 6.47 (d, J = 7.81 Hz, 1H), 4.77
(t, J = 5.3 Hz, 1H), 4.57–4.55 (m, 1H), 4.54 (t, J = 5.2 Hz, 1H), 4.33
(d, J = 5.2 Hz, 2H), 4.10 (d, J = 5.2 Hz, 2H), 3.96–3.81 (m, 1H), 3.63
(s, 3H). Anal. Calcd for C20H19NO3: C, 74.75; H, 5.96; N, 4.36. Found:
C, 74.45; H, 6.01; N, 4.34.
4.5.2. (2-Hydroxymethyl-3-phenyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13c)
Compound 13c was synthesized from 12c (2.08 g, 6 mmol) in
anhydrous dichloromethane (35 mL) and LiAlH4 (0.568 g,
15 mmol) in anhydrous ether (50 mL); yield (1.24 g, 71%); mp
169–171 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.55–7.44 (m, 6H),
7.12–7.09 (m, 1H), 7.05–7.03 (m, 1H), 6.74–6.73 (m, 1H), 4.80 (t,
J = 4.8 Hz, 1H), 4.68 (t, J = 4.8 Hz, 1H), 4.55 (d, J = 4.8 Hz, 2H), 4.27
(d, J = 4.8 Hz, 2H), 3.93 (s, 2H). Anal. Calcd for C19H17NO2: C,
78.33; H, 5.88; N, 4.81. Found: C, 78.50; H, 5.98; N, 4.72.
4.5.8. (3-(3,4-Dimethoxyphenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13i)
Compound 13i was synthesized from 12i (3.26 g, 8 mmol) in
anhydrous dichloromethane (20 mL) and LiAlH4 (1 g, 26.35 mmol)
in anhydrous ether (40 mL); yield (2.17 g, 77%); mp 160–162 °C; H
NMR (400 MHz, DMSO-d6) d = 7.28 (d, J = 7.2 Hz, 1H), 7.15–7.01 (m,
5H), 6.80 (d, J = 8.0 Hz, 1H), 4.73 (t, J = 5.2 Hz, 1H), 4.61 (t,
J = 5.2 Hz, 1H), 4.55 (d, J = 5.2 Hz, 2H), 4.26 (d, J = 5.2 Hz, 2H),
3.91 (s, 2H), 3.84 (s, 3H), 3.75 (s, 3H). Anal. Calcd for C21H21NO4:
C, 71.78; H, 6.02; N, 3.99. Found: C, 70.65; H, 6.02; N, 3.82.
4.5.3. (3-(4-Fluorophenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13d)
Compound 13d was synthesized from 12d (2.78 g, 7.6 mmol) in
anhydrous dichloromethane (26 mL) and LiAlH4 (0.72 g, 19 mmol)
in anhydrous ether (40 mL); yield (1.69 g, 72%); mp 164–166 °C; 1H
NMR (400 MHz, DMSO-d6) d = 7.56–7.54 (m, 1H), 7.46 (d,
J = 7.31 Hz, 1H), 7.38–7.34 (m, 2H), 7.12 (t, J = 7.31 Hz, 1H), 7.04
(t, J = 7.31 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 4.79 (t, J = 3.9 Hz, 1H),
4.68 (t, J = 3.9 Hz, 1H), 4.57 (d, J = 4.5 Hz, 2H), 4.27 (d, J = 4.5 Hz,
2H), 3.92 (s, 2H). Anal. Calcd for C19H16FNO2: C, 73.77; H, 5.21;
N, 4.53. Found: C, 73.30; H, 5.48; N, 4.32.
4.5.9. (3-(2,6-Dimethoxyphenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13j)
Compound 13j was synthesized from 12j (2.5 g, 6.13 mmol) in
anhydrous dichloromethane (30 mL) and LiAlH4 (0.83 g,
21.87 mmol) in anhydrous ether (50 mL); yield (1.72 g, 80%); mp
146–147 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.46–7.40 (m, 2H),
7.05 (t, J = 7.8 Hz, 1H), 6.97 (t, J = 7.7 Hz, 1H), 6.80 (s, 1H), 6.78 (s,
1H), 6.39–6.37 (m, 1H), 4.81 (t, J = 5.3 Hz, 1H), 4.55 (d, J = 5.3 Hz,
2H), 4.28 (t, J = 4.8 Hz, 1H), 4.15 (d, J = 5.0 Hz, 2H), 3.87 (s, 2H),
3.62 (s, 6H). Anal. Calcd for C21H21NO4: C, 71.78; H, 6.02; N, 3.99.
Found: C, 71.19; H, 6.01; N, 3.92.
4.5.4. (3-(4-Chlorophenyl)-2-hydroxymethyl-8H-3a-aza-
cyclopenta[a]inden-1-yl)methanol (13e)
Compound 13e was synthesized from 12e (1.91 g, 5 mmol) in
anhydrous dichloromethane (20 mL) and LiAlH4 (0.47 g,
12.5 mmol) in anhydrous ether (35 mL); yield (1.16 g, 71%): mp
213–215 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.62–7.45 (m, 5H),

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R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
4.5.10. (3-(3,4,5-Trimethoxyphenyl)-2-hydroxymethyl-8H-3a-
aza-cyclopenta[a]inden-1-yl)methanol (13k)
(br s, 1H), 3.94 (s, 2H), 2.80 (d, J = 4.7 Hz, 3H), 2.78 (t, J = 4.7 Hz,
3H). Anal. Calcd for C23H23N3O4: C, 68.13; H, 5.72; N, 10.36. Found:
C, 67.69; H, 5.75; N, 10.31.
Compound 13k was synthesized from 12k (2.62 g, 6 mmol) in
anhydrous dichloromethane (35 mL) and LiAlH4 (0.83 g,
21.87 mmol) in anhydrous ether (40 mL); yield (1.71 g, 75%); mp
132–134 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.47 (d, J = 7.3 Hz,
1H), 7.17 (t, J = 7.5 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.92 (d,
J = 7.9 Hz, 1H), 6.79 (s, 2H), 4.73 (t, J = 4.7 Hz, 1H), 4.66 (t,
J = 4.7 Hz, 1H), 4.55 (d, J = 4.4 Hz, 2H), 4.30 (d, J = 4.4 Hz, 2H),
3.91 (s, 2H), 3.78 (s, 6H), 3.76 (s, 3H). Anal. Calcd for C22H23NO5:
C, 69.28; H, 6.08; N, 3.67. Found: C, 68.33; H, 6.04; N, 3.60.
4.6.3. Methylcarbamic acid 3-(4-fluorophenyl)-2-
methylcarbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-
yl-methyl ester (14d)
Compound 14d was synthesized from 13d (0.77 g, 2.5 mmol),
triethylamine (2.5 mL), and methyl isocyanate (1.47 mL, 25 mmol);
yield (0.92 g, 87%); mp 199–200 °C; 1H NMR (400 MHz, DMSO-d6)
d = 7.55–7.53 (m, 2H), 7.50 (d, J = 7.3 Hz, 1H), 7.38 (t, J = 8.7 Hz,
2H), 7.15 (t, J = 7.44 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.95 (br s,
1H), 6.92 (br s, 1H), 6.66 (d, J = 7.8 Hz, 1H), 5.05 (s, 2H), 4.79 (s,
2H), 3.96 (s, 2H), 2.58 (d, J = 4.5 Hz, 3H), 2.55 (t, J = 4.5 Hz, 3H).
Anal. Calcd for C23H22FN3O4: C, 65.24; H, 5.24; N, 9.92. Found: C,
65.00; H, 5.35; N, 9.71.
4.5.11. (2-Hydroxymethyl-6-methoxy-3-(4-methoxyphenyl)-
8H-3a-aza-cyclopenta[a] inden-1-yl)methanol (13l)
Compound 13l was synthesized from 12l (1.73 g, 3.56 mmol)
in anhydrous dichloromethane (20 mL) and LiAlH4 (0.45 g,
11.85 mmol) in anhydrous ether (40 mL); yield (1.35 g, 91%); mp
186–187 °C; 1H NMR (400 MHz, DMSO-d6) d = 7.40 (d, J = 8.7 Hz,
2H), 7.10 (d, J = 2.3 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H), 6.69 (dd,
J = 8.7, 2.5 Hz, 1H), 6.64 (d, J = 8.7 Hz, 1H), 4.71 (t, J = 4.4 Hz, 1H),
4.56 (t, J = 4.4 Hz, 1H), 4.53 (d, J = 4.4 Hz, 2H), 4.24 (d, J = 4.4 Hz,
2H), 3.87 (s, 2H), 3.84 (s, 3H), 3.71 (s, 3H). Anal. Calcd for
C21H21NO4: C, 71.78; H, 6.02; N, 3.99. Found: C, 71.59; H, 5.90;
N, 3.78.
4.6.4. Methylcarbamic acid 3-(4-chlorophenyl)-2-
methylcarbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-
yl-methyl ester (14e)
Compound 14e was synthesized from 13e (0.81 g, 2.5 mmol),
triethylamine (2.5 mL), and methyl isocyanate (1.47 mL, 25 mmol);
yield (1.06 g, 96%); mp 204–206 °C; 1H NMR (600 MHz, CDCl3)
d = 7.51–7.38 (m, 5H), 7.08–7.04 (m, 2H), 6.77–6.76 (m, 1H), 5.19
(s, 2H), 4.98 (s, 2H), 4.71 (br s, 1H), 4.67 (br s, 1H), 3.94 (s, 2H),
2.73 (m, 6H). Anal. Calcd for C23H22ClN3O4: C, 62.80; H, 5.04; N,
9.55. Found: C, 62.72; H, 5.14; N, 9.41.
4.6. Methylcarbamic acid 3-methyl-2-
methylcarbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-
yl-methyl ester (14a)
4.6.5. Methylcarbamic acid 3-(3,4-difluoro-phenyl)-2-methyl-
carbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-yl-
methyl ester (14f)
To a solution of 13a (0.80 g, 3.5 mmol) and triethylamine
(2.5 mL) in anhydrous dichloromethane (10 mL) was added drop-
wise methyl isocyanate (2 mL, 35 mmol) in anhydrous dichloro-
methane (5 mL). The mixture was stirred at room temperature
for additional 5 h. The mixture was concentrated under reduced
pressure to dryness. The residue was triturated with a mixture of
ether/methanol (15:1), the solid product was collected by filtra-
tion, washed with ether (10 mL), and dried in vacuo to yield 14a
(1.18 g, 98%); mp 86–88 °C; 1H NMR (400 MHz, DMSO-d6)
d = 7.52–7.47 (m, 2H), 7.34–7.30 (m, 1H), 7.14–7.10 (m, 1H), 6.86
(br s, 2H), 4.95 (s, 2H), 4.92 (s, 2H), 3.84 (s, 2H), 2.55 (d,
J = 2.0 Hz, 3H), 2.54 (d, J = 2.0 Hz, 3H), 2.51 (s, 3H). Anal. Calcd for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.70; H, 6.35;
N, 11.99.
Compound 14f was synthesized from 13f (0.81 g, 2.5 mmol), tri-
ethylamine (2.5 mL) and methyl isocyanate (1.47 mL, 25 mmol);
yield (0.93 g, 84%); mp 151–152 °C; 1H NMR (400 MHz, DMSO-
d6) d = 7.63–7.57 (m, 2H), 7.52 (d, J = 4.5 Hz, 1H), 7.36 (s, 1H),
7.18 (d, J = 5.0 Hz, 1H), 7.12 (d, J = 5.1 Hz, 1H), 6.96 (br s, 1H),
6.95 (br s, 1H), 6.73 (d, J = 5.2 Hz, 1H), 5.05 (s, 2H), 4.82 (s, 2H),
3.97 (s, 2H), 2.58 (d, J = 4.4 Hz, 3H), 2.55 (t, J = 4.4 Hz, 3H). Anal.
Calcd for C23H21F2N3O4: C, 62.58; H, 4.80; N, 9.52. Found: C,
62.49; H, 4.97; N, 9.33.
4.6.6. Methylcarbamic acid 3-(4-methoxy-phenyl)-2-methyl-
carbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-yl-
methyl ester (14g)
By following the same procedure as describe for 14a, the follow-
ing compounds 14b–l were synthesized.
Compound 14g was synthesized from 13g (1 g, 3 mmol), triethyl-
amine (3.5 mL), and methyl isocyanate (1.76 mL, 30 mmol); yield
(0.73 g, 70%); mp 152–153 °C; 1H NMR (400 MHz, DMSO-d6)
d = 7.49 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.10–7.07 (m,
4H), 6.92 (br s, 1H), 6.90 (br s, 1H), 6.69 (d, J = 7.6 Hz, 1H), 5.04 (s,
2H), 4.78 (s, 2H), 3.95 (s, 2H), 3.85 (s, 3H), 2.58 (d, J = 4.8 Hz, 3H),
2.56 (t, J = 4.8 Hz, 3H). Anal. Calcd for C24H25N3O5: C, 66.19; H,
5.79; N, 9.65. found: C, 66.13; H, 5.61; N, 9.41.
4.6.1. Methylcarbamic acid 3-ethyl-2-methyl
carbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-yl-
methyl ester (14b)
Compound 14b was synthesized from 13b (0.85 g, 3.5 mmol),
triethylamine (2 mL), and methyl isocyanate (2 mL, 35 mmol);
yield (1.24 g, 99%); mp 165–166 °C; 1H NMR (400 MHz, DMSO-
d6) d = 7.50–7.48 (m, 1H), 7.45–7.42 (m, 1H), 7.37–7.33 (m, 1H),
7.15–7.11 (m, 1H), 6.90 (br s, 1H), 6.84 (br s, 1H), 4.96 (s, 2H),
4.93 (s, 2H), 3.85 (s, 2H), 2.93 (q, J = 7.3 Hz, 2H), 2.55 (d,
J = 2.8 Hz, 3H), 2.54 (d, J = 2.8 Hz, 3H), 1.18 (t, J = 7.3 Hz, 3H). Anal.
Calcd for C19H23N3O4: C, 63.85; H, 6.49; N, 11.76. Found: C, 63.67;
H, 6.71; N, 11.71.
4.6.7. Methylcarbamic acid 3-(2-methoxy-phenyl)-2-methyl-
carbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-yl-
methyl ester (14h)
Compound 14h was synthesized from 13h (1.25 g, 3.89 mmol),
triethylamine (2.5 mL) and methyl isocyanate (1 mL, 18.12 mmol);
yield (1.47 g, 87%); mp 200–203 °C; 1H NMR (400 MHz, DMSO-d6)
d = 7.53–7.46 (m, 2H), 7.32–7.30 (m, 1H), 7.19 (d, J = 8.12 Hz, 1H),
7.14–7.04 (m, 3H), 6.95 (br s, 1H), 6.86 (br s, 1H), 6.47 (d,
J = 7.7 Hz, 1H), 5.04 (s, 2H), 4.86 (d, J = 11.9 Hz, 1H), 4.60 (d,
J = 11.9 Hz, 1H), 3.94 (s, 2H), 3.64 (s, 3H), 2.58 (d, J = 4.6 Hz, 3H),
2.54 (t, J = 4.6 Hz, 3H). Anal. Calcd for C24H25N3O5: C, 66.19; H,
5.79; N, 9.65. Found: C, 65.71; H, 5.87; N, 9.53.
4.6.2. Methylcarbamic acid 2-methylcarbamoyloxymethyl-3-
phenyl-8H-3a-aza-cyclopenta[a]inden-1-yl-methyl ester (14c)
Compound 14c was synthesized from 13c (0.73 g, 2.5 mmol),
triethylamine (2.5 mL) and methyl isocyanate (1.47 mL, 25 mmol);
yield (0.87 g, 86%); mp 196–198 °C; 1H NMR (600 MHz, CDCl3)
d = 7.48–7.42 (m, 5H), 7.38–7.368 (m, 1H), 7.05–7.02 (m, 2H),
6.77–6.76 (m, 1H), 5.20 (s, 2H), 5.01 (s, 2H), 4.73 (br s, 1H), 4.69

R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
5625
4.6.8. Methylcarbamic acid 3-(3,4-dimethoxy-phenyl)-2-
methyl-carbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-
yl-methyl ester (14i)
(IC50–IC90) of paclitaxel for six months. OECM1 (human gingival
squamous carcinoma cells) was obtained from Dr. T. Y. Liu of the
National Yang-Ming University, Taiwan.
Compound 14i was synthesized from 13i (1.5 g, 4.27 mmol), tri-
ethylamine (2.5 mL) and methyl isocyanate (2.5 mL, 42.60 mmol);
yield (1.80 g, 91%); mp 165–168 °C; 1H NMR (400 MHz, DMSO-d6)
d = 7.50–7.49 (m, 1H), 7.18–7.07 (m, 3H), 7.03–7.00 (m, 2H), 6.92
(br s, 2H), 6.80–6.79 (m, 1H), 5.04 (s, 2H), 4.81 (s, 2H), 3.95 (s,
2H), 3.85 (s, 3H), 3.75 (s, 3H), 2.58 (d, J = 4.6 Hz, 3H), 2.55 (t,
J = 4.6 Hz, 3H). Anal. Calcd for C25H27N3O6: C, 64.50; H, 5.85; N,
9.03. Found: C, 64.28; H, 5.86; N, 8.95.
4.7.2. Cytotoxicity assays
The cytotoxic effects of the newly synthesized compounds were
determined in T-cell acute lymphocytic leukemia (CCRF-CEM) and
their resistant subcell lines (CCRF-CEM/Taxol and CCRF-CEM/VBL)
by the XTT assay28 and human solid tumor cells (i.e., breast carci-
noma MX-1 and colon carcinoma HCT-116) the SRB assay29 in a
72 h incubation using a microplate spectrophotometer as de-
scribed previously.30 After the addition of phenazine methosul-
fate-XTT solution, incubated at 37 °C for 6 h and absorbance at
450 and 630 nm was detected on a microplate reader (EL 340).
The cytotoxicity of the newly synthesized compounds against
non-small cell lung cancer (H1299), human prostate cancer cell
line (PC3), human glioma (U87), and oral cancer cell line (OECM1)
were determined by the WST-1 assay31 in a 72 h incubation using a
microplate spectrophotometer as described previously. After the
addition of WST-1 solution, it was incubated at 37 °C for 4 h.
Absorbance at 460 and 630 nm was detected on a microplate read-
er. IC50 values were determined from dose–effect relationship at
six or seven concentrations of each drug using the CompuSyn soft-
ware by Chou and Martin32 based on the median-effect principle
and plot.33,34
4.6.9. Methylcarbamic acid 3-(2,6-dimethoxyphenyl)-2-
methyl-carbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-
yl-methyl ester (14j)
Compound 14j was synthesized from 13j (0.75 g, 2.13 mmol),
triethylamine (1.5 mL), and methyl isocyanate (0.8 mL,
13.56 mmol); yield (0.89 g, 90%); mp 154–156 °C; 1H NMR
(400 MHz, DMSO-d6) d = 7.48–7.45 (m, 2H), 7.10–7.08 (m, 1H),
7.04–7.01 (m, 1H), 7.00–6.97 (m, 1H), 6.81 (s, 1H), 6.80 (br s,
1H), 6.79 (br s, 1H), 6.41–6.39 (m, 1H), 5.03 (s, 2H), 4.66 (s, 2H),
3.91 (s, 2H), 3.62 (s, 6H), 2.59 (d, J = 3.6 Hz, 3H), 2.52 (t,
J = 3.6 Hz, 3H). Anal. Calcd for C25H27N3O6: C, 64.50; H, 5.85; N,
9.03. Found: C, 63.18; H, 5.74; N, 8.63.
4.6.10. Methylcarbamic acid 3-(3,4,5-trimethoxyphenyl)-2-
methylcarbamoyloxymethyl-8H-3a-aza-cyclopenta[a]inden-1-
yl-methyl ester (14k)
4.7.3. In vivo studies
Athymic nude mice bearing the nu/nu gene were obtained from
NCI, Frederick, MD and used for all human tumor xenografts. Male
nude mice 6 weeks or older weighing 20–24 g or more were used.
Compounds were administered via the tail vein for iv injection as
described previously.30 Tumor volume was assessed by measuring
length ꢀ width ꢀ height (or width) by using a caliper. Vehicle used
Compound 14k was synthesized from 13k (1.03 g, 2.7 mmol),
triethylamine (1.5 mL) and methyl isocyanate (1 mL, 18.12 mmol);
yield (1.27 g, 95%); mp 204–205 °C; 1H NMR (400 MHz, DMSO-d6)
d= 7.52–7.50 (d, J = 7.5 Hz, 2H), 7.22–7.18 (t, J = 7.2 Hz, 1H), 7.13–
7.10 (t, J = 7.2 Hz, 1H), 6.94 (s, 1H), 6.93 (br s, 1H), 6.91 (br s, 1H),
6.77 (s, 2H), 5.05 (s, 2H), 4.83 (s, 2H), 3.96 (s, 2H), 3.79 (s, 6H), 3.78
(s, 3H), 2.57 (d, J = 4.6 Hz, 3H), 2.55 (t, J = 4.6 Hz, 3H). Anal. Calcd
for C26H29N3O7: C, 63.02; H, 5.90; N, 8.48. Found: C, 62.74; H,
5.92; N, 8.42.
was 50 lL DMSO and 40 lL Tween 80 in 160 lL saline. The maxi-
mal tolerate dose of the tested compound was determined and ap-
plied for the in vivo therapeutic efficacy assay. For tumor-bearing
nude mice during the course of the experiment, the body weight
refers to total weight minus the weight of the tumor. All animal
studies were conducted in accordance with the guidelines for the
National Institute of Health Guide for the Care and Use of Animals
and the protocol approved by the Institutional Animal Care and
Use Committee.
4.6.11. Methylcarbamic acid 6-methoxy-3-(4-methoxyphenyl)-
2-methylcarbamoyl-oxymethyl-8H-3a-aza-cyclopenta[a]inden-
1-yl-methyl ester (14l)
Compound 14l was synthesized from 13l (0.954 g,
2.71 mmol), triethylamine (1.5 mL), and methyl isocyanate
(0.95 mL, 16.24 mmol); yield (1.17 g, 93%); mp 110–112 °C; 1H
NMR (400 MHz, DMSO-d6) d = 7.38 (d, J = 8.6 Hz, 2H), 7.13 (d,
J = 2.5 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.92 (br s, 1H), 6.88 (br
s, 1H), 6.71 (dd, J = 8.7, 2.5 Hz, 2H), 6.60 (d, J = 8.7 Hz, 1H),
5.02 (s, 2H), 4.80 (s, 2H), 3.92 (s, 2H), 3.84 (s, 3H), 3.71 (s,
3H), 2.58 (d, J = 4.5 Hz, 3H), 2.55 (t, J = 4.5 Hz, 3H). Anal. Calcd
for C25H27N3O6: C, 64.50; H, 5.85; N, 9.03. Found: C, 63.41; H,
5.80; N, 8.84.
4.7.4. Alkaline agarose gel shift assay
Formation of DNA cross-linking was analyzed by alkaline aga-
rose gel electrophoresis.25 In brief, purified pEGFP-N1 plasmid
DNA (1500 ng) was mixed with various concentrations (0.1–
20 lM) of 13a, 13b, 13l, 14h, 14g, and 14l in 40 lL binding buffer
(3 mM sodium chloride/1 mM sodium phosphate, pH 7.4, and
1 mM EDTA). The reaction mixture was incubated at 37 °C for
2 h. At the end of reaction, the plasmid DNA was linearized by
digestion with BamHI and followed by precipitation with ethanol.
The DNA pellets were dissolved and denatured in alkaline buffer
4.7. Biological experiments
(0.5 N NaOH–10 mM EDTA). An aliquot of 20
lL of DNA solution
4.7.1. Tumor and cell lines
(1000 ng) was mixed with a 4
l
L of 6 ꢀ alkaline loading dye and
Human colon carcinoma HCT-116 cells, human prostate adeno-
carcinoma PC3, ovarian adenocarcinoma SK-OV-3, human lung
large cell carcinoma H1299, and human glioma cells U87 were ob-
tained from American Type Culture Collection (ATCC, Rockville,
MD). Human mammary carcinoma (MX-1) tumor cells were ob-
tained from MSKCC cell bank. The CCRF-CEM human lymphoblastic
leukemia cells and their Vinblastine resistant subline (CCRF-CEM/
VBL, 680-fold resistance in vitro) were obtained from Dr. William
Beck of the University of Illinois, Chicago, and CCRF-CEM/Taxol
(330-fold resistance in vitro) resistant cells were produced by
exposing the parent cells to increasing sublethal concentration
then electrophoretically resolved on a 0.8% alkaline agarose gel
with NaOH–EDTA buffer at 4 °C. The electrophoresis was carried
out at 18 V for 22 h. After staining the gels with an ethidium bro-
mide solution, and the DNA was then visualized under UV light.
4.7.5. Modified single cell gel electrophoresis (comet) assay
Intracellular DNA interstrand cross-linking was analyzed using
a modified comet assay.22,23 All steps were carried out under sub-
dued lighting. Briefly, H1299 cells (2 ꢀ 105 cells) were plated in a
60 mm dish and incubated at 37 °C with 5% CO2 for 24 h. The grow-
ing cells were treated with tested compounds (13l, 14l, and mito-

5626
R. Kakadiya et al. / Bioorg. Med. Chem. 17 (2009) 5614–5626
mycin C) for 1 h, and then irradiated with 20 Gy of X-ray to induce
DNA strand breaks. Afterward, an aliquot of 5 ꢀ 105 cells were
quickly embedded in 1.2% low melting point agarose that was lay-
ered on a 1% agarose-precoated microscope slide. The cells in aga-
rose gel were treated with in lysis buffer [100 mmol/L sodium
EDTA, 2.5 mol/L NaCl, 10 mmol/L Tris–HCl (pH 10)] containing 1%
Triton X-100 and 10% dimethyl sulfoxide (immediately before
use) for 1 h and followed with alkali buffer [300 mmol/L NaOH,
1 mmol/L sodium EDTA (pH 13.5)] for 20 min. Electrophoresis
was then conducted at 25 V and 300 mA in the same buffer for
20 min. The slides were neutralized with neutralizing buffer
[250 mmol/L Tris–HCl (pH 7.5)] for at least, and then stained with
the Institute of Biological Chemistry (Academia Sinica) for provid-
ing the NMR service and the National Center for High-performance
computing for computer time and facilities.
Supplementary data
Supplementary data (elemental analysis data of compounds
11b–l, 12a–l, 13a–l, and 14a–l) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2009.06.018.
References and notes
20 lM YOYO-1 dye (Molecular Probes). The comet image was ta-
ken under fluorescence microscope (shortwave pass filter 450–
490 nm, chromatic beam splitter 510 nm, and longwave pass filter
520 nm) with a digital camera (DCS-420; Kodak, Rochester, NY,
USA). For each treatment, the tail moment of 100 cells was deter-
mined by aid of Comet Assay III software (available from www.per-
ceptive.co.uk). The levels of interstrand cross-linking are
proportional to the decrease in the tail moment in the irradiated
drug treated sample compared to the irradiated untreated control.
The decrease in tail moment is calculated by the following
formula:
1. Palmer, B. D.; Wilson, W. R.; Pullen, S. M.; Denny, W. A. J. Med. Chem. 1990, 33,
112.
2. Kasparkova, J.; Novakova, O.; Vrana, O.; Farrell, N.; Brabec, V. Biochemistry
1999, 38, 10997.
3. Kloster, M.; Kostrhunova, H.; Zaludova, R.; Malina, J.; Kasparkova, J.; Brabec, V.;
Farrell, N. Biochemistry 2004, 43, 7776.
4. Hansson, J.; Lewensohn, R.; Ringborg, U.; Nilsson, B. Cancer Res. 1987, 47, 2631.
5. Tomasz, M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73.
6. Doll, D. C.; Weiss, R. B.; Issell, B. F. J. Clin. Oncol. 1985, 3, 276.
7. Sartorelli, A. C. Cancer Res. 1988, 48, 775.
8. Oostveen, E. A.; Speckamp, W. N. Tetrahedron 1987, 43, 255.
9. Tomasz, M.; Lipman, R.; Chowdary, D.; Pawlak, J.; Verdine, G. L.; Nakanishi, K.
Science 1987, 235, 1204.
10. Anderson, W. K.; Bhattacharjee, D.; Houstion, D. M. J. Med. Chem. 1989, 32, 119.
11. Elliot, W. L.; Fry, D. W.; Anderson, W. K.; Nelson, J. M.; Hook, K. E.; Hawkins, P.
A.; Leopold, W. R. Cancer Res. 1991, 51, 4581.
% of DNA with interstrand cross-linking
ꢂ
ꢀ
ꢁ
ꢃ
TMdi ꢂ TMcu
TMci ꢂ TMcu
¼
1 ꢂ
ꢀ 100
12. White, I. N. H.; Mattocks, A. R. Biochem. J. 1972, 128, 291.
13. Weidner, M. F.; Sigurdsson, S. T.; Hopkins, P. B. Biochemistry 1990, 29, 9225.
14. Woo, J.; Sigurdsson, S. T.; Hopkins, P. B. J. Am. Chem. Soc. 1993, 115, 3407.
15. Andeson, W. K.; New, J. S.; Corey, P. F. Arzneim. Forsch. 1980, 30, 765.
16. Anderson, W. K.; Corey, P. F. J. Med. Chem. 1977, 20, 812.
17. (a) Anderson, W. K.; Chang, C. P.; Corey, P. F.; Halat, M. J.; Jones, A. N.;
McPherson, H. L., Jr.; New, J. S.; Rick, A. C. Cancer Treat. Rep. 1982, 66, 91; (b)
Anderson, W. K. Cancer Res. 1982, 42, 2168.
where TMdi = mean tail moment of drug treated, irradiated sample,
TMcu = mean tail moment of unirradiated control sample, TMci = -
mean tail moment of irradiated control sample.
4.7.6. Flow cytometric analysis
18. Anderson, W. K.; McPherson, H. L.; New, J. S.; Rick, A. C. J. Med. Chem. 1984, 27,
1321.
The effects of compounds 13l and 14l on cell cycle progression
were analyzed with a flow cytometer as described previously.35
Briefly, human non-small cell lung carcinoma H1299 cells were
treated with these compounds at 10 lM. At the time indicated,
the attached cells were trypsinized, washed with phosphate buffer
saline (PBS), fixed with ice-cold 70% ethanol for 30 min. The cells
were stained with 4 lg/mL propidium iodide (PI) in PBS containing
1% Triton X-100 and 0.1 mg/mL RNase A. The stained cells were
then analyzed using the FACS SCAN flow cytometer (Becton Dick-
inson, San Joes, CA, USA). The percentage of the cells in each cell
cycle phase was determined using the ModFit LT 2.0 software
based on the DNA histograms.
19. Anderson, W. K.; Halat, M. J. J. Med. Chem. 1979, 22, 977.
20. Anderson, W. K.; Chang, C. P.; McPherson, H. L., Jr. J. Med. Chem. 1983, 26, 1333.
21. Anderson, W. K.; McPherson, H. L. J. Med. Chem. 1982, 25, 84.
22. Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L. Exp. Cell Res. 1988, 175,
184.
23. Friedmann, B. J.; Caplin, M.; Savic, B.; Shah, T.; Lord, C. J.; Ashworth, A.; Hartley,
J. A.; Hochhauser, D. Mol. Cancer Ther. 2006, 5, 209.
24. Moinet-Hedin, V.; Tabka, T.; Poulain, L.; Godard, T.; Lechevrel, M.; Saturnino,
C.; Lancelot, J. C.; Talaer, J. Y. L.; Gauduchon, P. Anti-Cancer Drug Des. 2000, 15,
109.
25. Hartley, J. A.; Berardini, M. D.; Souhami, R. L. Anal. Biochem. 1991, 193, 131.
26. O’Connell, M. J.; Walworth, N. C.; Carr, A. M. Trends Cell Biol. 2000, 10, 296.
27. Walter, G. Chem. Ber. 1892, 25, 1261.
28. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger,
T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827.
29. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
Acknowledgments
30. Chou, T.-C.; O’Connor, O. A.; Tong, W. P.; Guan, Y.; Zhang, Z.-G.; Stachel, S. J.;
Lee, C.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8113.
31. Ngamwongsatit, P.; Banada, P. P.; Panbangred, W.; Bhunia, A. K. J. Microbiol.
Methods 2008, 73, 211.
32. Chou, T.-C.; Martin, N. CompuSyn software; ComboSyn: Paramus, NJ, 2005.
33. Chou, T.-C.; Talalay, P. Adv. Enzyme Regul. 1984, 22, 27.
34. Chou, T.-C. Pharmacol. Rev. 2006, 58, 621.
We are grateful to Academia Sinica (Grant No. AS-96-TP-B06)
for financial support. T.-C.C. was supported by the Sloan-Kettering
Institute General Fund. The NMR spectra of synthesized com-
pounds were obtained at High-Field Biomolecular NMR Core Facil-
ity supported by the National Research Program for Genomic
Medicine (Taiwan). We would like to thank Dr. Shu-Chuan Jao in
35. Lee, T. C.; Ko, J. L.; Jan, K. Y. Toxicology 1989, 56, 289.