Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b] isoquinoline derivatives, synthesis and biological activity

Article abstract of DOI:10.1016/j.bmc.2010.11.030

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.

Full text of DOI:10.1016/j.bmc.2010.11.030

Bioorganic & Medicinal Chemistry 19 (2011) 275–286
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline
derivatives, synthesis and biological activity
Ravi Chaniyara a,d, Naval Kapuriya a, Huajin Dong b, Pei-Chih Lee a, Sharda Suman a, Bhavin Marvania a,d
,
Ting-Chao Chou b, Te-Chang Lee a, Rajesh Kakadiya a, Anamik Shah d, 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 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 linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The
preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate)
derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro.
1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitu-
mor studies in animal models. The results show that this agent can induce complete tumor remission or
significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3)
xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with
DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase.
The results warrant further antitumor investigation against other human tumor growth in animal
models.
Received 14 September 2010
Revised 9 November 2010
Accepted 9 November 2010
Available online 6 December 2010
Keywords:
Pyrrolo[1,2-b]isoquinoline
Structure–activity relationships
DNA interstrand cross-linking
Cell cycle
Ó 2010 Elsevier Ltd. All rights reserved.
Antitumor agents
1. Introduction
et al. synthesized bis(carbamoylmethyl) derivatives of pyrrolo
[2,1-a]isoquinolines (9) and pyrrolo[1,2-a]quinolines (10), which
Among DNA alkylating agents, the naturally occurring antitu-
mor alkylating agent, mitomycin C (MMC, 1, Fig. 1) is a clinically
useful chemotherapeutic agent for treating various cancer
patients.1,2 MMC and its synthetic analogue indoloquinone EO9
(2),3 which bears two reactive nucleophilic centers in the molecule,
were reported to be capable of cross-linking with DNA. The qui-
none moiety of these agents plays an important role in their anti-
tumor activity. It requires bioreductive activation to switch on the
nucleophilic centers on the pyrrole ring to allow interaction with
DNA, which forms biadducts.4
Another class of bifunctional DNA alkylators, such as thioimidaz-
oles (i.e., carmethizole, 3, Fig. 1),5 bis(hydroxymethyl)pyrrole
derivatives (i.e., 46, 57 and 68), 2,3-dihydroxy-6,7-bis(alkylcarba-
mates)pyrrolizines [e.g., 7 (IPP) and 8]9 were developed originally
from the pyrrolizine alkaloids. Of the bis(alkylcarbamates)pyrroli-
zine analogues, compound 7 was found to have significant antitu-
mor activity against a broad range of experimental human tumor
xenografts.10 It was demonstrated that the electronic properties of
the substituent(s) on the phenyl ring as well as the lipophilicity
and planarity of the molecule may affect the antitumor activity
and toxicity of compounds belonging to this class.10 Later, Anderson
bear angular tricyclic structures to limit the deviation from co-pla-
narity of the phenyl and pyrrolo rings.11 The results showed that
these agents exhibited a broad spectrum of antitumor activity
against a wide range of tumors.
Recently, we have synthesized
a series of bis(hydroxy-
methyl)azacyclopenta[a]indenes and their bis(methylcarbamate)
derivatives.12 These agents can be considered as ‘benzologues’ of
bis(hydroxymethyl)pyrrolizines and were able to cross-link to
DNA double strands. These analogues exhibited potent cytotoxicity
and antitumor activity against human lymphoblastic leukemia and
various solid tumors.12 Remarkably, complete tumor remission
(CR) in nude mice bearing human breast carcinoma MX-1 xenograft
by bis(hydroxymethyl) derivatives (11 and 12, Fig. 1) and bis(meth-
ylcarbamate) derivatives (13 and 14) and significant suppression
against prostate adenocarcinoma PC3 xenograft by 12 were
achieved.
One of the drawbacks of using DNA alkylating agents is that these
drugs may lose antitumor activity because tumor cells possess DNA
repair mechanisms to fix DNA damage. More recently, we found that
the combination treatment of 13 and arsenic trioxide (ATO, DNA
repair inhibitor) significantly suppressed human large cell lung
carcinoma H460 xenograft (>82%) and cisplatin-resistant NTUB1/P
human bladder carcinoma xenografts (>92%) in nude mice.13
These exciting results prompted us to continue designing and
⇑
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 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.030

276
R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
Figure 1. Chemical structure of some DNA bifunctional alkylating agents.
synthesizing new bis(hydroxymethyl)pyrrolizine analogues for
antitumor studies.
various acid chlorides (R1COCl) in presence of 2 N NaOH to give
N-acyl-3-carboxy-1,2,3,4-tetrahydroisoquinolines (18b–j) by fol-
lowing the literature procedure.15,16 Compounds 18b–j were then
reacted with dimethyl acetylenedicarboxylate (DMAD) in acetic
anhydride at 60–70 °C yielded 5,10-dihydropyrrolo[1,2-b]isoquin-
oline-1,2-dicarboxylic acid dimethyl esters (19b–j). The methyl
ester derivative 19a (R1 = Me) was prepared in good yields directly
from 17 by reacting with acetic anhydride and DMAD.17 The ester
function of 19a–j was reduced to bis(hydroxymethyl) derivatives
20a–j by treating with LiAlH4 in a mixture of ether/CH2Cl2 at
0–5 °C. Compounds 20a–j were then further treated with
methyl-, ethyl- or iso-propylisocyanate in presence of triethyl-
amine (TEA) to furnish the desired bis(alkylcarbamate)-5,10-dihy-
dropyrrolo[1,2-b]isoquinoline-1-yl derivatives (21–23) in good to
high yields.
As mentioned previously, pyrrolo[2,1-a]isoquinolines (9) and
pyrrolo[1,2-a]quinolines (10) bear an angular tricyclic ring system.
To investigate whether derivatives with a linear tricyclic ring sys-
tem also possess potent antitumor activity and/or have superior
potency than the corresponding bis(hydroxymethyl)azacyclopen-
ta[a]indenes, we have synthesized a series of linear 5,10-dihydro-
pyrrolo[1,2-b]isoquinolines and their bis(alkylcarbamates) (15),
all which were subjected to antitumor evaluation. The results show
that the newly synthesized analogues exhibit significant antitumor
activity and are able to induce DNA interstrand cross-linking. Here-
in, we report the antitumor activity of these agents against various
human tumor cell growths both in vitro and in vivo and mecha-
nism of action studies.
2. Results and discussion
2.1. Chemistry
2.2. Biological results
2.2.1. In vitro cytotoxicity
Table 1 shows the antiproliferative activities of the newly syn-
thesized bis(hydroxylmethyl)-5,10-dihydropyrrolo[1,2-b]isoquin-
oline derivatives (20) and their bis(alkylcarbamate) derivatives
(21–23) against human lymphoblastic leukemia (CCRF-CEM) and
its drug-resistant sublines resistant to Taxol (CCRF-CEM/Taxol)
and Vinblastine (CCRF-CEM/VBL) cell growth in vitro. It demon-
strated that the newly synthesized conjugates possess significant
cytotoxicity with IC50 in submicro molar range. In the series of
The synthetic route for bis(hydroxymethyl)-5,10-dihydropyr-
rolo[1,2-b]isoquinolin-1-yl derivatives (20) and their bis(alkylcar-
bamate) derivatives (21–23) is shown in Scheme 1. The known
3-carboxy-1,2,3,4-tetrahydroisoquinoline (17) was synthesized by
treating the commercially available
D,L-phenylalanine (16) with
formaldehyde and concd HCl by following the procedure devel-
oped by Dean.14 Compound 17 was N-acylated by treating with

R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
277
Scheme 1. Reagents and conditions: (i) 37% formalin/concd HCl, reflux; (ii) acid chloride, 2 N NaOH/acetone, room temperature; (iii) DMAD/Ac2O, 60–70 °C; (iv) LiAlH4,
CH2Cl2/Et2O, 0–5 °C; (v) R2NCO/Et3N, room temperature.
bis(hydroxymethyl) derivatives, it showed that 3-alkyl (Me or Et)
substituted derivatives are more cytotoxic than the 3-phenyl
substituted compounds. The order of their potency, for example,
is 20a (3-Me) > 20b (3-Et) > 20c (3-Ph), indicating that the com-
pounds having a smaller size of substituent at C3 have greater
cytotoxicity. A similar observation is found in 3-phenyl deriva-
tives: the cytotoxicity decreases when the number of the methoxy
functions increases (20h vs 20i vs 20j). Furthermore, the cytotox-
icity decreases when the number of the halo function increases
in the halo substituted 3-phenyl derivatives (20d > 20f and
20e > 20g). In this study, one can observe that the C3-methoxy-
phenyl derivatives are somewhat more cytotoxic than the corre-
sponding halophenyl compounds. This suggests that the electron
properties of the substituent(s) on the phenyl ring have very little
influence over their potency.
In the series of bis(alkylcarbamates) derivatives, the alkylcarba-
mate moiety may serve as a better leaving group than that of the
OH group. Thus, the bis(alkylcarbamates) derivatives may easily
generate nucleophilic cations on both methylene functions that al-
low the cations to become more favorable targets of DNA. In the
series of 3-phenyl substituted derivatives, the bis(alkylcarbamates)
are generally more potent than their corresponding bis(hydroxy-
methyl) derivatives, except for compound bis(ethylcarbamate)
22i, which is as potent as the corresponding bis(hydroxymethyl)
20i and more cytotoxic than bis(iso-propylcarbamates) 23i. How-
ever, in the series of C3-alkyl derivatives, the bis(hydroxymethyl)
derivatives (20a and 20b) are more potent than their correspond-
ing bis(alkylcarbamates) (21a, 22a, and 23a; 22b and 23b,
respectively).
Our previous research on the SAR studies of 1,2-bis(hydroxylm-
ethyl)cyclopenta[a]indenes and their counterparts 1,2-bis(meth-
ylcarbamate) derivatives (11–14, Fig. 1) demonstrated that the
size and the electron property of the substituents at the C3 position
affected the cytotoxicity of these agents.12 However, we found that
the cytotoxicity of pyrrolo[1,2-b]isoquinolines is mainly affected
by the size of the substituents at the C3 position rather than the
electron property in the current studies. In comparison with the
potency of both these series, the bis(hydroxymethyl) derivatives
of pyrrolo[1,2-b]isoquinoline are more cytotoxic than the corre-
sponding cyclopenta[a]indenes. In contrast, the bis(alkylcarba-
mate) derivatives of cyclopenta[a]indenes are more potent than
the corresponding pyrrolo[1,2-b]isoquinolines.
Our previous report demonstrated that cyclopenta[a]indenes
have no multi-drug resistance toward antitumor agents such as
Taxol and Vinblastine. To realize whether the newly synthesized
pyrrolo[1,2-b]isoquinoline derivatives also have no cross-resis-
tance to these two agents, we evaluated their cytotoxicity against
CCRF-CEM/Taxol and CCRF-CEM/VBL, which are subcell lines of
CCRF-CEM cells that are 330-fold resistant to Taxol, and 680-fold
resistant to Vinblastine, respectively. As shown in Table 1, the
newly synthesized pyrrolo[1,2-b]isoquinolines have no cross-resis-
tance to either Taxol or Vinblastine. This suggests that all deriva-
tives are neither a good substrate of membrane multi-drug
resistance transporters (i.e., p-glycoprotein) nor mutated tubulin.
The antiproliferative activity of the selected pyrrolo[1,2-b]iso-
quinoline derivatives in inhibiting human solid tumors such as
breast carcinoma MX-1, colon carcinoma HCT-116, non-small cell
lung carcinoma H1299, prostate PC3, oral carcinoma OECM1 and
glioma U87 cell growth in vitro (Table 2) were also investigated.
Of these compounds, C3-Me derivative (20a) was found to have
potent cytotoxicity in inhibiting MX-1 cell growth in vitro with
IC50 values of 0.66
tent inhibitory activity against human colon carcinoma HCT-116
with IC50 values of 0.29, 0.04 and 0.36 M, respectively. The tested
lM. Compounds 20a, 21e, and 21f exhibited po-
l
compounds have good to moderate effects against H1299, PC3,
OECM1 and U87 cell growth in vitro.
2.2.2. In vivo antitumor activity
To investigate the antitumor activity of pyrrolo[1,2-b]isoquino-
line derivatives in animal models, we selected compound 20a for
evaluating its therapeutic efficacy in animal models since this
agent has the most potent cytotoxicity among all of the com-
pounds tested and exhibits a broad spectrum of antitumor activity
in inhibiting both CCRF/CEM and other solid tumors in vitro. Nude
mice implanted with human breast carcinoma MX-1 xenograft
were given 30 mg/kg, every two days for two times (Q2D ꢀ 2),
intravenous injection (iv injection) on day 8 and 10 (Fig. 2).
Remarkably, it shows that complete tumor remission against
human breast carcinoma MX-1 in nude mice was achieved
(Fig. 2A). This concentration was established from tolerability
studies. Under this dosage, one can see that nude mice’s body
weights recovered after cessation of the treatment, indicating the
low toxicity of the compound to the host (Fig. 2B). In another
experiment, we found that 20a was able to effectively suppress

278
R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
Table 1
The cytotoxicity of newly synthesized bis(hydroxymethyl)-5,10-dihydropyrrolo[1,2-b]isoquinolin-1-yl derivatives (20) and their bis(alkylcarbamate) derivatives (21–23) against
human lymphoblastic leukemia (CCRF-CEM) and its drug-resistant sublines (CCRF-CEM/Taxol and CCRF-CEM/VBL)a
Compd
R1
R2
Cell growth inhibition (IC50
CCRF-CEM/Taxolb
lM)
CCRF-C EM
CCRF-CEM/VBLb
20a
20b
20c
20d
20e
20f
20g
20h
20i
Me
Et
H
H
H
H
H
H
H
H
0.08 0.02
0.18 0.01
0.51 0.01
0.51 0.01
0.60 0.002
1.14 0.22
8.44 0.06
0.23 0.03
1.10 0.04
1.97 0.46
0.13 0.01
0.19 0.01
0.49 0.09
0.18 0.03
0.24 0.002
0.44 0.01
0.26 0.01
0.31 0.01
0.59 0.01
2.08 0.02
2.37 0.02
0.37 0.02
1.07 0.001
1.41 0.02
0.27 0.004
0.57 0.01
0.45 0.01
0.76 0.03
0.54 0.003
1.33 0.01
3.55 0.02
0.34 0.002
1.29 0.10
0.89 0.01
0.003 0.0003
0.0007 0.001
0.10 0.001 [1.22ꢀ]c
0.21 0.003 [1.15ꢀ]
0.50 0.04 [0.99ꢀ]
0.88 0.03 [1.72ꢀ]
0.77 0.17 [1.28ꢀ]
1.26 0.26 [1.11ꢀ]
4.82 0.01 [0.57ꢀ]
0.25 0.001 [1.02ꢀ]
0.76 0.02 [0.69ꢀ]
2.88 0.05 [1.45ꢀ]
0.11 0.002 [0.86ꢀ]
0.22 0.004 [1.18ꢀ]
0.45 0.20 [0.91ꢀ]
0.13 0.03 [0.72ꢀ]
0.11 0.001 [0.48ꢀ]
0.21 0.01 [0.48ꢀ]
0.26 0.03 [1.01ꢀ]
0.43 0.05 [1.39ꢀ]
0.35 0.01 [0.59ꢀ]
1.25 0.02 [0.60ꢀ]
0.90 0.002 [0.38ꢀ]
0.22 0.003 [0.58ꢀ]
0.81 0.01 [0.75ꢀ]
2.14 0.08 [1.52ꢀ]
0.23 0.002 [0.84ꢀ]
0.32 0.01 [0.56ꢀ]
0.23 0.02 [0.50ꢀ]
0.23 0.01 [0.30ꢀ]
0.63 0.01 [1.16ꢀ]
0.78 0.003 [0.58ꢀ]
2.47 0.07 [0.69ꢀ]
0.19 0.002 [0.56ꢀ]
0.60 0.02 [0.46ꢀ]
1.20 0.01 [1.35ꢀ]
0.43 0.05 [330ꢀ]
0.08 0.01 [106.2ꢀ]
0.09 0.01 [1.1ꢀ]
0.19 0.02 [1.03ꢀ]
0.29 0.02 [0.56ꢀ]
0.49 0.002 [0.95ꢀ]
0.72 0.18 [1.2ꢀ]
0.95 0.21 [0.84ꢀ]
6.42 0.05 [0.76ꢀ]
0.11 0.001 [0.48ꢀ]
0.62 0.01 [0.56ꢀ]
2.78 0.09 [1.40ꢀ]
0.08 0.001 [0.59ꢀ]
0.16 0.05 [0.87ꢀ]
0.47 0.05 [0.97ꢀ]
0.09 0.02 [0.49ꢀ]
0.12 0.003 [0.51ꢀ]
0.24 0.01 [0.53ꢀ]
0.23 0.001 [0.90ꢀ]
0.57 0.01 [1.85ꢀ]
0.57 0.06 [0.97ꢀ]
0.67 0.003 [0.32ꢀ]
1.19 0.04 [0.50ꢀ]
0.35 0.01 [0.93ꢀ]
0.65 0.02 [0.60ꢀ]
3.13 0.03 [2.23ꢀ]
0.14 0.003 [0.50ꢀ]
0.18 0.0002 [1.42ꢀ]
0.25 0.01 [0.55ꢀ]
0.23 0.001 [0.31ꢀ]
0.57 0.04 [1.06ꢀ]
0.61 0.05 [0.46ꢀ]
1.35 0.04 [0.38ꢀ]
0.16 0.001 [0.47ꢀ]
0.77 0.05 [0.59ꢀ]
1.42 0.05 [1.59ꢀ]
1.27 0.05 [980ꢀ]
0.50 0.12 [679.5ꢀ]
C6H5
40-F-C6H4
40-Cl-C6H4
30,40-Di-F–C6H3
30,40-Di-Cl–C6H3
40-MeO–C6H4
30,40-Di-MeO–C6H3
30,40,50-Tri-MeO–C6H2
Me
H
H
20j
21a
21e
21f
21h
22a
22b
22c
22d
22e
22f
CONHMe
CONHMe
CONHMe
CONHMe
CONHEt
CONHEt
CONHEt
CONHEt
CONHEt
CONHEt
CONHEt
CONHEt
CONHEt
CONHEt
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
CONH-i-Pr
40-Cl–C6H4
30,40-Di-F–C6H3
40-MeO–C6H4
Me
Et
C6H5
40-F–C6H4
40-Cl–C6H4
30,40-Di-F–C6H3
30,40-Di-Cl–C6H3
40-MeO–C6H4
30,40-Di-MeO–C6H3
30,40,50-Tri-MeO–C6H2
Me
22g
22h
22i
22j
23a
23b
23c
23d
23e
23f
23g
23h
23i
Et
C6H5
40-F–C6H4
40-Cl–C6H4
30,40-Di-F–C6H3
30,40-Di-Cl–C6H3
40-MeO–C6H4
30,40-Di-MeO–C6H3
30,40,50-Tri-MeO–C6H2
23j
Taxol
Vinblastine
a
Cell growth inhibition was measured by the XTT assay20 for leukemic cells after 72-h incubation using a microplate spectrophotometer as described previously.22 Similar
in vitro results were obtained by using the Cell Counting Kit-8 for the CCK-8 assays as described by technical manual of Dojindo Molecular Technologies, Inc. (Gaithersburg,
MD; Website: www.dojindo.com). IC50 values were determined from dose-effect relationship at six or seven concentrations of each drug by using the CompuSyn software by
Chou and Martin24 based on the median-effect principle and plot using the serial deletion analysis.25,26 Ranges given for Taxol and vinblastine were mean SE (n = 4).
b
CCRF-CEM/Taxol and CCRF-CEM/VBL are subcell lines of CCRF-CEM cells that are 330-fold resistant to Taxol, and 680-fold resistant to vinblastine, respectively, when
comparing with the IC50 of the parent cell line.
c
Numbers in the brackets are fold of cross-resistant determined by comparison with the corresponding IC50 of the parent cell line.
human ovarian tumor SK-OV-3 implanted in nude mice on day 22
at the dose of 20 mg/kg, every day for four times (QD ꢀ 4), iv injec-
tion (Fig. 3A and B). The results of MX-1 and SK-OV-3 xenografts
studies show the potential utility of compound 20a in inhibiting
the growth of both tumors.
suggesting that DNA cross-linking may be the main mechanism
of action for these agents.
2.2.4. Cell cycle inhibition
It is well known that DNA interacting agents can alter the cell cy-
cle progression by arresting the cell cycle at the G2/M phase.19 Pre-
viously, we have demonstrated that 3a-aza-cyclopenta[a]indene
derivatives were able to induce G2/M arrest.12 We therefore studied
the inhibitory effect of 20a on cell cycle distribution (Table 3). The
human non-small lung carcinoma H1299 cells were treated with
2.2.3. DNA cross-linking study
To realize whether the newly synthesized compounds are capa-
ble of cross-linking with DNA double strands, pEGFP-N1 plasmid
DNA was treated with bis(hydroxymethyl) derivatives (20a and
20h) and their corresponding bis(alkylcarbamate) derivatives
(22a and 23h, respectively) at various concentrations as indicated
20a at the concentrations of 1.25, 2.5, and 5 lM for 24 h. The cells
were harvested, stained with propidium iodide (PI) and analyzed
with a flow cytometer. It clearly shows that 20a remarkably accu-
mulated the cells at G2/M phase. Furthermore, increased sub-G1
populations were noticed in cells treated with 20a at each
concentration.
(1, 10, and 20
l
M) using alkaline agarose gel shifting assay
(Fig. 4).18 Melphalan (1, 5, and 10
lM) was used as the positive
control. As revealed in Figure 4, one can see that all of the tested
compounds were able to induce DNA interstrand cross-linking,

R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
279
Table 2
The cytotoxicity of newly synthesized bis(hydroxymethyl)-5,10-dihydropyrrolo[1,2-b]isoquinolin-1-yl derivatives (20) and their bis(alkylcarbamate) derivatives (21–23) against
human solid tumors (breast carcinoma MX-1, colon carcinoma HCT-116, lung carcinoma H1299, prostate carcinoma PC3, oral carcinoma OECM1 and glioma U87) cell growth
in vitro
Compd
Cell growth inhibition (IC50 lM)
MX-1a
HCT-116a
H1299b
PC3b
OECM1b
U87b
20a
20d
20e
20f
20h
21a
21e
21f
21h
22a
22d
22e
22h
23a
23d
23e
0.66 0.03
4.01 0.05
3.28 0.01
6.99 0.35
1.99 0.07
1.68 0.01
1.18 0.01
2.73 0.05
1.33 0.02
2.27 0.01
1.51 0.03
1.69 0.06
1.27 0.05
1.28 0.01
1.17 0.19
1.70 0.09
0.98 0.03
0.035 0.00514
0.0029 0.0002
ND
0.29 0.01
2.46 0.08
3.06 0.46
3.21 0.21
1.13 0.07
1.17 0.02
0.04 0.002
0.36 0.001
1.07 0.05
0.90 0.03
0.79 0.05
0.62 0.10
0.75 0.004
1.38 0.02
0.63 0.001
0.69 0.01
0.97 0.04
0.0013 0.0005
0.0018 0.0004
ND
2.48 1.22
8.76 1.15
24.73 8.16
NDc
13.94 0.83
ND
ND
ND
ND
19.42 0.42
3.94 1.13
37.25 0.89
27.61 1.65
9.77 3.75
8.13 1.34
25.15 4.05
13.16 1.94
ND
6.07 1.70
5.93 1.52
9.85 0.79
ND
10.93 1.21
ND
ND
ND
ND
15.45 1.84
9.59 1.31
10.98 1.14
18.54 0.82
16.87 7.42
12.29 1.34
9.97 1.32
6.79 1.01
ND
6.10 1.11
6.99 0.12
12.05 0.67
ND
6.12 0.21
ND
ND
ND
ND
11.34 2.07
12.52 1.70
25.32 0.18
10.48 0.13
11.80 2.58
11.05 2.61
26.10 7.86
8.83 0.74
ND
27.91 3.90
23.25 0.26
26.68 5.79
ND
10.57 0.23
ND
ND
ND
ND
13.89 4.90
28.36 4.86
33.59 3.91
17.06 1.78
26.61 4.21
36.87 5.98
22.72 2.03
21.04 0.67
ND
23h
Taxol
Vinblastine
Cisplatin
ND
4.95 0.60
ND
26.65 4.19
ND
ND
ND
ND
a
b
c
Cell growth inhibition was measured by the SRB assay21 for solid tumor cells after 72-h incubation using a microplate spectrophotometer as described previously.22
Cell growth inhibition was determined by the Alamar blue assay23 in a 72 h incubation using a microplate spectrophotometer as described previously.
Not determined.
Figure 2. Therapeutic effects of 20a in nude mice bearing MX-1 human mammary
xenograft (iv injection, n = 4). (A) Average tumor size changes. (B) Average body
weight changes.
Figure 3. Therapeutic effects of 20a in nude mice bearing ovarian adenocarcinoma
SK-OV-3 xenograft (iv injection, n = 4). (A) Average tumor size changes. (B) Average
body weight changes.

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R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
Figure 4. Representative DNA cross-linking gel shift assay for bis(hydroxymethyl) derivatives (20a and 20h) and their corresponding bis(alkylcarbamate) derivatives (22a
and 23h, respectively) at various concentrations as indicated. Control lane shows single-stranded DNA (SS), while CL shown in all tested lanes is DNA double-stranded cross-
linking. Melphalan (1, 5, and 10 lM) was used as a positive control.
Table 3
Effects of compound 20a on cell cycle progress in human non-small cell lung adenocarcinoma H1299
Concentration 0
M)
1.25
2.5
5
(
l
Sub G1
G1
S
14.1 0.7
42.9 0.8
22.5 0.3
20.5 0.9
21.6 4.2
5.6 0.6
32.7 5.9
40.1 6.5
26.6 0.7
7.7 0.7
18.3 8.4
47.3 2.1
26.5 3.0
0.5 0.3
24.7 4.6
48.4 2.2
G2/M
chromatography or crystallization. Elemental analyses were done
on a Heraeus CHN-O Rapid instrument. 1H NMR spectra were
recorded on a 600 MHz, Brucker AVANCE 600 DRX and 400 MHz,
Brucker Top-Spin spectrometers in the indicated solvent. The
chemical shifts were reported in ppm (d) relative to TMS and cou-
pling constants (J) in Hertz (Hz) and s, d, t, m, br s, refer to singlet,
doublet, triplet, multiplet, broad, respectively. High performance
liquid chromatography analysis for checking purity of synthesized
compounds were recorded on a Hitachi D-2000 Elite instrument:
3. Conclusion
In the present studies, we have synthesized a series of linear
pyrrolo[1,2-b] isoquinoline derivatives for antitumor evaluation.
Both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives
show potent antitumor activity in inhibiting various human tumor
xenografts in vitro. Among these analogues, we discovered com-
pound 20a, which was selected for antitumor studies in animal
models, exhibits potent therapeutic efficacy against human breast
MX-1 xenograft in nude mice, as complete tumor remission was
observed. This agent is also able to significantly suppress human
ovarian tumors implanted in nude mice. The results reported here-
in warrant further investigation to optimize the schedule and dos-
age to get greater suppression of other human tumor growth in
animal models. Additional, the evaluation of the antitumor activity
of 20a in combination with DNA repair inhibitor (e.g., ATO) is cur-
rently undergoing in our laboratory.
column, Mightysil RP-18 GP 250–4.6 (5
MeCN/THF (50:50 v/v); flow rate, 1 mL/min; injected sample
10 L, column temp, 27 °C; wavelength, 254 nm. The purity of all
lm); mobile phase,
l
tested compounds was P95% based on analytical HPLC.
4.2. 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid
hydrochloride (17)
A mixture of D,L-phenylalanine (16, 50 g, 3.03 mol), concd HCl
(325 mL) and 37% formalin (110 mL) was heated to a gentle reflux
with vigorous stirring. After 30 min, another portion of formalin
(50 mL) and concd HCl (110 mL) was added. The reaction mixture
was further stirred and heated for 4 h and cooled to room temper-
ature. The white solid separated out was filtered and washed with
methanol (30 mL) to give 17 64.1 g, yield 98%; mp >280 °C (lit.14
mp 286–290 °C). 1H NMR (DMSO-d6) d 3.15 (1H, m, CH), 3.32
(1H, m, CH), 4.30 (2H, m, CH2), 4.43 (1H, m, CH), 7.20–7.35 (4H,
m, 4 ꢀ ArH), 9.89 (1H, br s, exchangeable, NH); 10.06 (1H, br s,
exchangeable, COOH).
4. Experimental section
4.1. General methods and materials
All commercial chemicals and solvents were reagent grade and
were used without further purification unless otherwise specified.
Melting points were determined on a Fargo melting point appara-
tus and are uncorrected. Thin-layer chromatography was per-
formed on Silica Gel G60 F254 (Merck) with short-wavelength UV
light for visualization. All reported yields are isolated yields after

R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
281
4.3. 2-Propionyl-1,2,3,4-tetrahydroisoquiniline-3-carboxylic
acid (18b)
4.45 (2H, m, CH2), 4.99 (1H, m, CH), 7.08–7.13 (4H, m, 4 ꢀ ArH),
7.44–7.46 (1H, m, ArH), 7.66–7.70 (2H, m, 2 ꢀ ArH), 12.67 (1H, br
s, exchangeable, COOH). Anal. Calcd for (C17H17Cl2NO3): C, 58.31;
H, 3.74; N, 4.00. Found: C, 58.19; H, 4.04; N, 3.87.
To a suspension of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic-
acid hydrochloride (17, 10 g, 46.8 mmol) in acetone (60 mL) was
added 2 N NaOH (40 mL) solution at room temperature. The clear
solution obtained then added dropwise into a solution of propionyl
chloride (5.2 g, 57 mmol) in acetone (20 mL) at room temperature,
simultaneously 2 N NaOH was added dropwise and pH maintained
above 10. The reaction mixture was stirred at room temperature
for 2 h, the solvent was evaporated under reduce pressure. The
solution was acidified to pH 5–6 with 3 N HCl. The white solid sep-
arated, filtered it and dried to give 18b 9 g, yield 87%; mp 173–
174 °C. 1H NMR (DMSO-d6) d 1.03 (3H, m, Me), 2.45 (2H, m, CH2),
3.13 (2H, m, CH2), 4.52 (2H, m, CH2), 5.09 (1H, m, CH), 7.16–7.20
(4H, m, 4 ꢀ ArH), 12.63 (1H, br s, exchangeable, COOH). Anal. Calcd
for (C13H15NO3): C, 66.94; H, 6.48; N, 6.00. Found: C, 66.64; H, 6.48;
N, 5.89.
4.3.6. 2-(4-Methoxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (18h)
Compound 18h was prepared from 17 (10.0 g, 46.8 mmol) and
4-methoxybenzoyl chloride (9.8 g, 57.0 mmol). Yield, 12.6 g
(86%); mp 178– 180 °C; 1H NMR (DMSO-d6) d 3.28 (2H, m, CH2),
3.85 (3H, s, MeO), 4.60 (2H, m, CH2), 5.02 (1H, m, CH), 6.90–6.94
(3H, m, 3 ꢀ ArH), 7.18–7.22 (3H, m, 3 ꢀ ArH), 7.42–7.48 (2H, m,
2 ꢀ ArH), 12.66 (1H, br s, exchangeable, COOH). Anal. Calcd for
(C18H17NO4): C, 69.44; H, 5.50; N, 4.50. Found: C, 69.12; H, 5.62;
N, 4.27.
4.3.7. 2-(3,4-Dimethoxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (18i)
By following the same synthetic procedure as that for 18b, the
following compounds were synthesized.
Compound 18i was prepared from 17 (10.0 g, 46.8 mmol) and
3,4-dimethoxybenzoyl chloride (12.5 g, 57 mmol). Yield, 11.9 g
(61%); mp 235–236 °C; 1H NMR (DMSO-d6) d 3.19 (2H, m, CH2),
3.77 (6H, s, 2 ꢀ MeO), 4.56 (2H, m, CH2), 5.04 (1H, m, CH), 6.99–
7.04 (4H, m, 4 ꢀ ArH), 7.17–7.23 (3H, m, 3 ꢀ ArH), 12.88 (1H, br
s, exchangeable, COOH). Anal. Calcd for (C19H19NO5): C, 66.85; H,
5.61; N, 4.10. Found: C, 66.65; H, 5.66; N, 3.86.
4.3.1. 2-Benzoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid (18c)
Compound 18c was prepared from 17 (8.0 g, 37.4 mmol) and
benzoyl chloride (6.43 g, 45.6 mmol). Yield, 9.9 g (77%); mp 168–
169 °C (lit.15 mp 168–169 °C). 1H NMR (DMSO-d6) d 3.18 (2H, m,
CH2), 4.52 (2H, m, CH2), 5.08 (1H, m, CH), 7.14–7.22 (4H, m,
4 ꢀ ArH), 7.41–7.49 (5H, m, 5 ꢀ ArH), 12.85 (1H, br s, exchange-
able, COOH).
4.3.8. 2-(3,4,5-Trimethoxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (18j)
Compound 18j was prepared from 17 (7.0 g, 32.7 mmol) and
3,4,5-trimethoxybenzoyl chloride (9.3 g, 39.9 mmol). Yield, 12.0 g
(98%); mp 186–189 °C; 1H NMR (DMSO-d6) d 3.19 (2H, m, CH2),
3.70 (3H, s, MeO), 3.80 (6H, s, 2 ꢀ MeO), 4.59 (2H, m, CH2), 5.02
(1H, m, CH), 6.70 (2H, s, 2 ꢀ ArH), 7.13–7.28 (4H, m, 4 ꢀ ArH),
12.72 (1H, br s, exchangeable, COOH). Anal. Calcd for (C20H21NO6):
C, 64.68; H, 5.70; N, 3.77. Found: C, 64.44; H, 5.50; N, 3.44.
4.3.2. 2-(4-Fluorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (18d)
Compound 18d was prepared from 17 (10.0 g, 46.8 mmol) and
4-fluorobenzoyl chloride (9.91 g, 57.0 mmol). Yield, 13.5 g (97%);
mp 179–180 °C; 1H NMR (DMSO-d6) d 3.20 (2H, m, CH2), 4.52
(2H, m, CH2), 5.06 (1H, m, CH), 7.19–7.33 (6H, m, 6 ꢀ ArH), 7.48–
7.52 (2H, m, 2 ꢀ ArH), 12.76 (1H, br s, exchangeable, COOH). Anal.
Calcd for (C17H14FNO3): C, 68.22; H, 4.71; N, 4.68. Found: C, 68.08;
H, 4.57; N, 4.42.
4.4. 3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
dicarboxylic acid dimethyl ester (19a)
Dimethyl acetylenedicarboxylate (6.39 g, 45.0 mmol) was
added into a mixture of 1,2,3,4-tetrahydroisoquinoline-3-carbox-
ylic acid (17, 10 g, 46.8 mmol) in acetic anhydride (70 mL) and
the reaction mixture was heated at 70 °C with stirring for 1.5 h.
The reaction mixture was evaporated to dryness in vacuo. The res-
idue was recrystallized from MeOH to give 19a, 11.0 g (74%); mp
152–154 °C (lit.17 mp 140–142 °C). 1H NMR (DMSO-d6) d 2.40
(3H, s, Me), 3.70 (3H, s, COOMe), 3.72 (3H, s, COOMe), 4.18 (2H,
s, CH2), 5.06 (2H, s, CH2), 7.21–7.32 (2H, m, 2 ꢀ ArH), 7.35–7.39
(2H, m, 2 ꢀ ArH).
4.3.3. 2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (18e)
Compound 18e was prepared from 17 (10.0 g, 46.8 mmol) and
4-chloroenzoyl chloride (10.1 g, 57.0 mmol). Yield, 13.8 g (93%),
mp 77–79 °C; 1H NMR (DMSO-d6) d 3.29 (2H, m, CH2), 4.49 (2H,
m, CH2), 5.02 (1H, m, CH), 7.01–7.17 (4H, m, 4 ꢀ ArH), 7.40–7.58
(4H, m, 4 ꢀ ArH), 12.81 (1H, br s, exchangeable, COOH). Anal. Calcd
for (C17H14ClNO3): C, 64.67; H, 4.47; N, 4.44. Found: C, 64.52; H,
4.56; N, 4.33.
4.3.4. 2-(3,4-Difluorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (18f)
4.4.1. 3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
dicarboxylic acid dimethyl ester (19b)
Compound 18f was prepared from 17 (10.0 g, 46.8 mmol) and
3,4-difluorobenzoyl chloride (10.2 g, 57.0 mmol). Yield, 14.2 g
(95%); mp 89–91 °C; 1H NMR (DMSO-d6) d 3.18 (2H, m, CH2),
4.48 (2H, m, CH2), 5.02 (1H, m, CH), 7.09–7.15 (4H, m, 4 ꢀ ArH),
7.33 (1H, s, ArH), 7.46–7.54 (2H, m, 2 ꢀ ArH), 12.84 (1H, br s,
exchangeable, COOH). Anal. Calcd for (C17H13F2NO3): C, 64.35; H,
4.13; N, 4.41. Found: C, 64.21; H, 4.26; N, 4.56.
A mixture of dimethyl acetylenedicarboxylate (DMAD) (6.39 g,
45.0 mmol) and 18b (7.0 g, 30.0 mmol) in acetic anhydride
(50 mL) was heated at 65 °C with stirring for 1.5 h. The reaction
mixture was evaporated to dryness in vacuo and the residue was
recrystallized from MeOH to give 19b, 8.77 g (93%); mp 88–
89 °C. 1H NMR (DMSO-d6) d 1.15 (3H, t, J = 7.6 Hz, Me), 2.85 (2H,
q, J = 7.6 Hz, CH2), 3.71 (6H, s, 2 ꢀ COOMe), 4.19 (2H, s, CH2),
5.12 (2H, s, CH2), 7.29–7.33 (2H, m, 2 ꢀ ArH), 7.38–7.43 (2H, m,
2 ꢀ ArH). Anal. Calcd for (C18H19NO4): C, 68.99; H, 6.11; N, 4.47.
Found: C, 68.96; H, 6.05; N, 4.38.
4.3.5. 2-(3,4-Dichlorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (18g)
Compound 18g was prepared from 17 (10.0 g, 46.8 mmol) and
3,4-dichlorobenzoyl chloride (12.1 g, 57.0 mmol). Yield, 14.5 g
(89%); mp 101–102 °C; 1H NMR (DMSO-d6) d 3.27 (2H, m, CH2),
By following the same synthetic procedure as that for 19b, the
following compounds were synthesized.

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R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
4.4.2. 3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
dicarboxylic acid dimethyl ester (19c)
ArH), 7.07 (1H, d, J = 8.0 Hz, ArH), 7.22–7.25 (1H, m, ArH), 7.25–
7.32 (2H, m, 2 ꢀ ArH), 7.40–7.42 (1H, m, ArH). Anal. Calcd for
(C24H23NO6): C, 68.40; H, 5.50; N, 3.32. Found: C, 68.02; H, 5.53;
N, 2.94.
Compound 19c was prepared from DMAD (6.0 g, 41.0 mmol)
and 18c (8.0 g, 28.4 mmol). Yield, 7.0 g (68%); mp 137–138 °C. 1H
NMR (DMSO-d6) d 3.58 (3H, s, COOMe), 3.76 (3H, s, COOMe),
4.33 (2H, s, CH2), 4.98 (2H, s, CH2), 7.21–7.32 (3H, m, 3 ꢀ ArH),
7.41–7.53 (6H, m, 6 ꢀ ArH). Anal. Calcd for (C22H19NO4): C,
73.12; H, 5.30; N, 3.88. Found: C, 72.91; H, 5.30; N, 3.53.
4.4.9. 3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19j)
Compound 19i was prepared from DMAD (5.7 g, 40.0 mmol)
and 18j (10.0 g, 26.9 mmol). Yield, 6.2 g (51%); mp 165–166 °C.
1H NMR (DMSO-d6) d 3.63 (3H, s, COOMe), 3.74 (6H, s, COOMe
and MeO), 3.80 (6H, s, 2 ꢀ MeO), 4.32 (2H, s, CH2), 5.00 (2H, s,
CH2), 6.75 (2H, s, 2 ꢀ ArH), 7.22–7.26 (1H, m, ArH), 7.28–7.32
(1H, m, ArH), 7.35–7.37 (1H, m, ArH), 7.40–7.42 (1H, m, ArH). Anal.
Calcd for (C25H25NO7): C, 66.51; H, 5.58; N, 3.10. Found: C, 66.25;
H, 5.59; N, 3.01.
4.4.3. 3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19d)
Compound 19d was prepared from DMAD (10.7 g, 65.0 mmol)
and 18d (15.0 g, 50.1 mmol). Yield, 11.1 g (58%); mp 149–150 °C.
1H NMR (DMSO-d6) d 3.58 (3H, s, COOMe), 3.76 (3H, s, COOMe),
4.32 (2H, s, CH2), 4.96 (2H, s, CH2), 7.23–7.30 (1H, m, ArH), 7.32–
7.36 (4H, m, 4 ꢀ ArH), 7.40–7.42 (1H, m, ArH), 7.49–7.52 (2H, m,
2 ꢀ ArH). Anal. Calcld for (C22H18FNO4): C, 69.65; H, 4.78; N,
3.69. Found: C, 69.54; H, 4.82; N, 3.78.
4.5. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]dimethanol (20a)
4.4.4. 3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-
A solution of 19a (8.0 g, 26.7 mmol) in anhydrous dichloro-
methane (20 mL) was added dropwise in to a stirred suspension
of LiAlH4 (2.5 g, 66.0 mmol) in anhydrous diethyl ether (50 mL)
at 0 to ꢁ5 °C. The reaction mixture was further stirred for 15 min
after the addition was completed. The excess hydride was de-
stroyed by the sequential addition of water (2.5 mL), 15% aqueous
NaOH (2.5 mL), and water (2.5 mL) at 0 °C. The mixture was fil-
tered through a pad of Celite, the solid residue was washed with
dichloromethane. The combined filtrate and washings were evap-
orated to dryness in vacuo. The residue was recrystallized from
ether to give 20a, 4.2 g (64.0%); mp 99–101 °C. 1H NMR (DMSO-
d6) d 2.22 (3H, s, Me), 3.96 (2H, s, CH2), 4.33 (2H, m, CH2), 4.38
(4H, m, CH2 and exchangeable, 2 ꢀ OH), 4.93 (2H, s, CH2), 7.24–
7.28 (2H, m, 2 ꢀ ArH), 7.34–7.36 (2H, m, 2 ꢀ ArH). Anal. Calcd for
(C15H17NO2): C, 74.05; H, 7.04; N, 5.76. Found: C, 74.24; H, 6.97;
N, 5.71.
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19e)
Compound 19e was prepared from DMAD (6.8 g, 47.0 mmol)
and 18e (10.0 g, 31.6 mmol). Yield, 8.0 g (64%); mp 164–166 °C.
1H NMR (DMSO-d6) d 3.60 (3H, s, COOMe), 3.76 (3H, s, COOMe),
4.32 (2H, s, CH2), 4.98 (2H, s, CH2), 7.20–7.26 (1H, m, ArH), 7.27–
7.33 (2H, m, 2 ꢀ ArH), 7.39–7.43 (1H, m, ArH), 7.48 (2H, d,
J = 8.5 Hz, 2 ꢀ ArH), 7.57 (2H, d, J = 8.5 Hz, 2 ꢀ ArH). Anal. Calcd
for (C22H18ClNO4): C, 66.75; H, 4.58; N, 3.54. Found: C, 66.52; H,
4.85; N, 3.66.
4.4.5. 3-(3, 4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19f)
Compound 19f was prepared from DMAD (6.71 g, 47.0 mmol)
and 18f (10.0 g, 31.5 mmol). Yield, 8.4 g (67%); mp 142–144 °C.
1H NMR (DMSO-d6) d 3.61 (3H, s, COOMe), 3.76 (3H, s, COOMe),
4.31 (2H, s, CH2), 5.00 (2H, s, CH2), 7.22–7.42 (5H, m, 5 ꢀ ArH),
7.54–7.61 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C22H17F2NO4): C,
66.50; H, 4.31; N, 3.52. Found: C, 66.15; H, 4.44; N, 3.36.
By following the same synthetic procedure as that for 20a, the
following compounds were synthesized.
4.5.1. [3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]dimethanol (20b)
4.4.6. 3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19g)
Compound 19e was prepared from DMAD (8.5 g, 60.0 mmol)
and 18g (14.0 g, 40.0 mmol). Yield, 11.0 g (63%); mp 169–170 °C.
1H NMR (DMSO-d6) d 3.61 (3H, s, COOMe), 3.77 (3H, s, COOMe),
4.31 (2H, s, CH2), 5.02 (2H, s, CH2), 7.22–7.33 (3H, m, 3 ꢀ ArH),
7.40–7.47 (2H, m, 2 ꢀ ArH), 7.76–7.78 (2H, m, 2 ꢀ ArH). Anal. Calcd
for (C22H17Cl2NO4): C, 61.41; H, 3.98; N, 3.26. Found: C, 61.33; H,
3.95; N, 2.89.
Compound 20b was prepared from 19b (5.0 g, 15.9 mmol) and
LiAlH4 (1.5 g, 39.0 mmol). Yield, 3.15 g (76%) as syrup; 1H NMR
(DMSO-d6) d 1.10 (3H, t, J = 7.6 Hz, Me), 2.66 (2H, q, J = 7.6 Hz,
CH2), 3.95 (2H, s, CH2), 4.33 (3H, m, CH2 and exchangeable, OH),
4.39 (3H, m, CH2 and exchangeable, OH), 4.96 (2H, s, CH2), 7.23–
7.28 (2H, m, 2 ꢀ ArH), 7.33–7.39 (2H, m, 2 ꢀ ArH).
4.5.2. [3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]dimethanol (20c)
4.4.7. 3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-
Compound 20c was prepared from 19c (7.0 g, 19.4 mmol) and
LiAlH4 (1.8 g, 48.0 mmol). Yield, 4.7 g (80%); mp 150–152 °C. 1H
NMR (DMSO-d6) d 4.06 (2H, s, CH2), 4.28 (2H, d, J = 4.8 Hz, CH2),
4.50 (2H, d, J = 4.8 Hz, CH2), 4.54 (2H, br s, exchangeable OH),
4.98 (2H, s, CH2), 7.18 (1H, t, J = 7.2 Hz, ArH), 7.26 (2H, t,
J = 7.2 Hz, 2 ꢀ ArH), 7.36–7.39 (2H, m, 2 ꢀ ArH), 7.45–7.51 (4H,
m, 4 ꢀ ArH). Anal. Calcd for (C20H19NO2): C, 78.66; H, 6.27; N,
4.59. Found: C, 78.48; H, 6.42; N, 4.41.
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19h)
Compound 19h was prepared from DMAD (7.5 g, 53.0 mmol)
and 18h (10.0 g, 35.1 mmol). Yield, 8.4 g (66%); mp 155–158 °C.
1H NMR (DMSO-d6) d 3.58 (3H, s, COOMe), 3.75 (3H, s, COOMe),
3.82 (3H, s, MeO), 4.31 (2H, s, CH2), 4.95 (2H, s, CH2), 7.05–7.07
(2H, m, 2 ꢀ ArH), 7.21–7.24 (1H, m, ArH), 7.28–7.31 (2H, m,
2 ꢀ ArH), 7.37–7.42 (3H, m, 3 ꢀ ArH). Anal. Calcd for (C23H21NO5):
C, 70.58; H, 5.41; N, 3.58. Found: C, 70.25; H, 5.44; N, 3.49.
4.5.3. [3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-
4.4.8. 3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19i)
Compound 19i was prepared from DMAD (5.6 g, 39.0 mmol)
and 18i (9.0 g, 26.4 mmol). Yield, 9.0 g (81%); mp 202–203 °C. 1H
NMR (DMSO-d6) d 3.60 (3H, s, COOMe), 3.66 (3H, s, MeO), 3.78
(3H, s, COOMe), 3.82 (3H, s, MeO), 4.32 (2H, s, CH2), 5.00 (2H, s,
CH2), 6.99 (1H, dd, J = 2.0 and 8.0 Hz, ArH), 7.02 (1H, d, J = 2.0 Hz,
b]isoquinoline-1,2-diyl]dimethanol (20d)
Compound 20d was prepared from 19d (10.0 g, 26.4 mmol) and
LiAlH4 (2.5 g, 65.0 mmol). Yield, 6.36 g (74%); mp 135–136 °C. 1H
NMR (DMSO-d6) d 4.05 (2H, s, CH2), 4.26 (2H, d, J = 4.8 Hz, CH2),
4.49 (2H, d, J = 4.8 Hz, CH2), 4.53 (2H, t, J = 4.8 Hz, exchangeable,
OH), 4.96 (2H, s, CH2), 7.17–7.21 (1H, m, ArH), 7.26–7.34 (4H, m,
4 ꢀ ArH), 7.38 (1H, m, ArH), 7.48–7.52 (2H, m, 2 ꢀ ArH). Anal.

R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
283
Calcd for (C20H18FNO2): C, 74.29; H, 5.61; N, 4.33. Found: C, 74.38;
H, 5.68; N, 3.98.
7.27 (2H, m, 2 ꢀ ArH), 7.33–7.38 (2H, m, 2 ꢀ ArH). Anal. Calcd for
(C23H25NO5): C, 69.86; H, 6.37; N, 3.54. Found: C, 69.71; H, 6.37;
N, 3.45.
4.5.4. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]dimethanol (20e)
4.6. General procedure for preparing bis(alkylcarbamate)
derivatives (21–23)
Compound 20e was prepared from 19e (7.0 g, 17.7 mmol) and
LiAlH4 (1.63 g, 44 mmol). Yield, 3.1 g (52%); mp 170–172 °C. 1H
NMR (DMSO-d6) d 4.06 (2H, s, CH2), 4.28 (2H, d, J = 4.8 Hz, CH2),
4.50 (2H, d, J = 4.8 Hz, CH2), 4.55 (2H, m, exchangeable, OH), 4.99
(2H, s, CH2), 7.18–7.20 (1H, m, ArH), 7.21–7.27 (2H, m, 2 ꢀ ArH),
7.29 (1H, d, J = 7.6 Hz, ArH), 7.38 (2H, d, J = 7.2 Hz, 2 ꢀ ArH), 7.48
(2H, d, J = 7.2 Hz, 2 ꢀ ArH). Anal. Calcd for (C20H18ClNO2): C,
70.69; H, 5.34; N, 4.12. Found: C, 70.42; H, 5.37; N, 4.05.
To
a solution of bis(hydroxymethyl) derivatives (20a–20j,
1.0 equiv) and triethylamine (2–3 equiv) in anhydrous dichloro-
methane was added alkylisocyanate (5 equiv). The reaction mix-
ture was stirred at ambient temperature (for 3–20 h) under an
argon atmosphere. After the completion of the reaction, the reac-
tion mixture was evaporated to dryness in vacuo. The residue
was triturated with ether, and solid separated was collected by fil-
tration. The desired product was either obtained by recrystalliza-
tion or liquid chromatography.
4.5.5. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]dimethanol (20f)
Compound 20f was prepared from 19f (7.5 g, 18.0 mmol) and
LiAlH4 (1.73 g, 47 mmol). Yield, 5.3 g (83%); mp 145–146 °C. 1H
NMR (DMSO-d6) d 4.05 (2H, s, CH2), 4.27 (2H, d, J = 5.2 Hz, CH2z),
4.48 (2H, d, J = 5.2 Hz, CH2), 4.53 (1H, t, J = 5.2 Hz, exchangeable,
OH), 4.60 (1H, t, J = 5.2 Hz, exchangeable, OH), 5.01 (2H, s, CH2),
7.18–7.33 (4H, m, 4 ꢀ ArH), 7.37–7.39 (1H, m, ArH), 7.52–7.54
(2H, m, 2 ꢀ ArH). Anal. Calcd for (C20H17F2NO2): C, 70.37; H,
5.02; N, 4.10. Found: C, 70.08; H, 5.01; N, 3.76.
4.6.1. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]bis(methylene) bis(methylcarbamate) (21a)
Compound 21a was synthesized from 20a (0.80 g, 3.2 mmol),
Et3N (1 mL) and methylisocyanate (1.14 g, 20 mmol). Yield, 0.53 g
(44%); mp 174–176 °C. 1H NMR (DMSO-d6) d 2.23 (3H, s, Me),
2.53 (6H, m, 2 ꢀ Me), 3.99 (2H, s, CH2), 4.88 (2H, s, CH2), 4.92
(2H, s, CH2), 4.95 (2H, s, CH2), 6.74 (2H, br s, exchangeable, NH),
7.21–7.32 (2H, m, 2 ꢀ ArH), 7.34–7.38 (2H, m, 2 ꢀ ArH). Anal. Calcd
for (C19H23N3O4): C, 63.85; H, 6.49; N, 11.76. Found: C, 63.54; H,
6.57; N, 11.64.
4.5.6. [3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]dimethanol (20g)
Compound 20g was prepared from 19g (9.0 g, 20.9 mmol) and
LiAlH4 (1.9 g, 52.0 mmol). Yield, 5.7 g (72%); mp 150–151 °C. 1H
NMR (DMSO-d6) d 4.05 (2H, s, CH2), 4.26 (2H, d, J = 4.8 Hz, CH2),
4.48 (2H, d, J = 4.8 Hz, CH2), 4.53 (1H, t, J = 4.8 Hz, exchangeable
OH), 4.63 (1H, t, J = 4.8 Hz, exchangeable, OH), 5.03 (2H, s, CH2),
7.18–7.21 (1H, m, ArH), 7.23–7.27 (2H, m, 2 ꢀ ArH), 7.28–7.30
(1H, m, ArH), 7.38–7.40 (1H, m, ArH), 7.46–7.48 (2H, m, 2 ꢀ ArH).
Anal. Calcd for (C20H17Cl2NO2): C, 64.18; H, 4.58; N, 3.74. Found:
C, 63.89; H, 4.40; N, 3.87.
4.6.2. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate)
(21e)
Compound 21e was synthesized from 20e (1.0 g, 3.0 mmol),
Et3N (1 mL), and methylisocyanate (0.85 g, 15 mmol). Yield, 1.1 g
(82%); mp 194–196 °C. 1H NMR (DMSO-d6)
d 2.54 (6H, m,
2 ꢀ Me), 4.09 (2H, s, CH2), 4.80 (2H, s, CH2), 4.97 (2H, s, CH2),
5.03 (2H, s, CH2), 6.81 (2H, br s, exchangeable, NH), 7.17–7.23
(1H, m, ArH), 7.24–7.30 (2H, m, 2 ꢀ ArH), 7.36–7.40 (1H, m, ArH),
7.45–7.47 (2H, m, 2 ꢀ ArH), 7.55–7.57 (2H, m, 2 ꢀ ArH). Anal. Cacld
for (C24H24ClN3O4): C, 63.50; H, 5.33; N, 9.26. Found: C, 63.33; H,
5.35; N, 9.20.
4.5.7. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]dimethanol (20h)
Compound 20h was prepared from 19h (7.5 g, 19.0 mmol) and
LiAlH4 (1.77 g, 47 mmol). Yield, 4.3 g (67%); mp 174–176 °C. 1H
NMR (DMSO-d6) d 3.82 (3H, s, MeO), 4.04 (2H, s, CH2), 4.26 (2H,
s, CH2), 4.48 (4H, m, CH2 and exchangeable, OH), 4.93 (2H, s,
CH2), 7.04–7.06 (2H, m, 2 ꢀ ArH), 7.17–7.21 (1H, m, ArH), 7.24–
7.27 (2H, m, 2 ꢀ ArH), 7.36–7.39 (3H, m, 3 ꢀ ArH). Anal. Calcd for
(C21H21NO3): C, 75.20; H, 6.31; N, 4.18. Found: C, 75.36; H, 6.30;
N, 4.13.
4.6.3. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate)
(21f)
Compound 21f was synthesized from 20f (1.0 g, 2.9 mmol),
Et3N (1 mL), and methylisocyanate (0.85 g, 15 mmol). Yield, 1.0 g
(77%); mp 190–192 °C; 1H NMR (DMSO-d6)
d 2.54 (6H, m,
2 ꢀ Me), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 4.99 (2H, s, CH2),
5.03 (2H, s, CH2), 6.83 (2H, br s, exchangeable, NH), 7.18–7.23
(1H, m, ArH), 7.25–7.31 (3H, m, 3 ꢀ ArH), 7.36–7.40 (1H, m, ArH),
7.52–7.61 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C24H23F2N3O4): C,
63.29; H, 5.09; N, 9.23. Found: C, 63.00; H, 5.12; N, 9.16.
4.5.8. [3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]dimethanol (20i)
Compound 20i was prepared from 19i (8.0 g, 18.9 mmol) and
LiAlH4 (1.75 g, 47 mmol). Yield, 5.34 g (77%); mp 145–146 °C. 1H
NMR (DMSO-d6) d 3.79 (6H, s, 2 ꢀ MeO), 4.04 (2H, s, CH2), 4.29
(2H, s, CH2), 4.52 (4H, br s, CH2 and exchangeable, OH), 4.97 (2H,
s, CH2), 6.96–6.98 (1H, m, ArH), 7.05–7.07 (2H, m, 2 ꢀ ArH),
7.19–7.28 (3H, m, 3 ꢀ ArH), 7.37–7.39 (1H, m, ArH). Anal. Calcd
for (C22H23NO4): C, 72.31; H, 6.34; N, 3.83. Found: C, 72.25; H,
6.34; N, 3.95.
4.6.4. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate)
(21h)
Compound 21h was synthesized from 20h (1.0 g, 2.9 mmol),
Et3N (1 mL), methylisocyanate (1.6 g, 29 mmol). Yield, 1.2 g
(87%); mp 182–184 °C. 1H NMR (DMSO-d6)
d 2.53 (6H, m,
4.5.9. [3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]dimethanol (20j)
2 ꢀ Me), 3.85 (3H, s, MeO), 4.08 (2H, s, CH2), 4.78 (2H, s, CH2),
4.93 (2H, s, CH2), 5.01 (2H, s, CH2), 6.81 (2H, br s, exchangeable,
NH), 7.06–7.08 (2H, m, 2 ꢀ ArH), 7.17–7.23 (1H, m, ArH), 7.24–
7.30 (2H, m, 2 ꢀ ArH), 7.32–7.40 (3H, m, 3 ꢀ ArH). Anal. Calcd for
(C25H27N3O5): C, 66.80; H, 6.05; N, 9.35. Found: C, 66.72; H, 6.15;
N, 9.24.
Compound 20j was prepared from 19j (6.0 g, 13.3 mmol) and
LiAlH4 (1.2 g, 33.0 mmol). Yield, 3.1 g (58%); mp 146–147 °C. 1H
NMR (DMSO-d6) d 3.74 (3H, s, MeO), 3.82 (6H, s, 2 ꢀ MeO), 4.05
(2H, s, CH2), 4.31 (2H, s, CH2), 4.50 (2H, s, CH2), 4.53 (2H, s,
exchangeable, OH), 5.04 (2H, s, CH2), 6.73 (2H, s, 2 ꢀ ArH), 7.21–

284
R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
4.7. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
(70%); mp 189–190 °C. 1H NMR (DMSO-d6)
d 0.98 (6H, t,
diyl]bis(methylene)bis (ethylcarbamate) (22a)
J = 6.8 Hz, 2 ꢀ Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 4.09 (2H, s, CH2),
4.81 (2H, s, CH2), 5.00 (2H, s, CH2), 5.02 (2H, s, CH2), 6.95 (2H, br
s, exchangeable, NH), 7.19–7.21 (1H, m, ArH), 7.25–7.39 (4H, m,
4 ꢀ ArH), 7.53–7.60 (2H, m, 2 ꢀ ArH). Anal. Calcd for
(C26H27F2N3O4): C, 64.59; H, 5.63; N, 8.69. Found: C, 64.50; H,
5.60; N, 8.69.
Compound 22a was synthesized from 20a (1.0 g, 4.1 mmol),
Et3N (0.5 mL), and ethylisocyanate (1.1 g, 16 mmol). Yield, 0.83 g
(53%); mp 208–209 °C; 1H NMR (DMSO-d6) d 0.97 (6H, t, J = 8 Hz,
2 ꢀ Me), 2.25 (3H, s, Me), 2.95 (4H, q, J = 8 Hz, CH2), 4.00 (2H, s,
CH2), 4.89 (2H, s, CH2), 4.93 (2H, s, CH2), 4.96 (2H, s, CH2), 6.86
(2H, br s, exchangeable, NH), 7.23–7.29 (2H, m, 2 ꢀ ArH), 7.34–
7.35 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C21H27N3O4): C, 65.44; H,
7.06; N, 10.90. Found: C, 65.29; H, 7.14; N, 10.89.
4.7.6. [3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl)bis(methylene]bis (ethylcarbamate)
(22g)
Compound 22g was synthesized from 20g (1.0 g, 2.6 mmol),
Et3N (1.0 mL), and ethylisocyanate (0.85 g, 10.7 mmol). Yield,
1.15 g (83%); mp 208–209 °C. 1H NMR (DMSO-d6) d 0.98 (6H, t,
J = 6.8 Hz, 2 ꢀ Me), 2.97 (4H, q, J = 6.8 Hz, CH2), 4.09 (2H, s, CH2),
4.82 (2H, s, CH2), 5.02 (4H, s, 2 ꢀ CH2), 6.95 (2H, br s, exchangeable,
NH), 7.19–7.22 (1H, m, ArH), 7.26–7.31 (2H, m, 2 ꢀ ArH), 7.39–7.45
(2H, m, 2 ꢀ ArH), 7.70–7.72 (2H, m, 2 ꢀ ArH). Anal. Calcd for
(C26H27Cl2N3O4): C, 60.47; H, 5.27; N, 8.14. Found: C, 60.34; H,
5.13; N, 8.10.
4.7.1. [3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]bis(methylene)bis (ethylcarbamate) (22b)
Compound 22b was synthesized from 20b (0.5 g, 1.9 mmol),
Et3N (0.5 mL), ethylisocyanate (0.55 g, 7.7 mmol). Yield, 0.39 g
(50%); mp 127–129 °C. 1H NMR (DMSO-d6)
d 0.95 (6H, t,
J = 7.2 Hz, 2 ꢀ Me), 1.10 (3H, t, J = 7.2 Hz, Me), 2.69 (2H, q,
J = 7.2 Hz, CH2), 2.97 (4H, m, CH2), 3.99 (2H, s, CH2), 4.89 (2H, s,
CH2), 4.93 (2H, s, CH2), 4.99 (2H, s, CH2), 6.84 (2H, br s, exchange-
able, NH), 7.23–7.29 (2H, m, 2 ꢀ ArH), 7.34–7.39 (2H, m, 2 ꢀ ArH).
Anal. Calcd for (C22H29N3O4): C, 66.14; H, 7.32; N, 10.52. Found: C,
66.35; H, 7.53; N, 10.74.
4.7.7. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate)
(22h)
4.7.2. [3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]bis(methylene)bis (ethylcarbamate) (22c)
Compound 22h was synthesized from 20h (1.0 g, 2.9 mmol),
Et3N (1.0 mL), and ethylisocyanate (0.84 g, 11.8 mmol). Yield,
0.92 g (65%); mp 165–166 °C. 1H NMR (DMSO-d6) d 0.99 (6H, t,
J = 6.8 Hz, 2 ꢀ Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 3.83 (3H, s, MeO),
4.09 (2H, s, CH2), 4.78 (2H, s, CH2), 4.94 (2H, s, CH2), 5.02 (2H, s,
CH2), 6.92 (2H, br s, exchangeable, NH), 7.05–7.08 (2H, m, 2 ꢀ ArH),
7.19–7.22 (1H, m, ArH), 7.27–7.29 (2H, m, 2 ꢀ ArH), 7.35–7.39 (3H,
m, 3 ꢀ ArH). Anal. Calcd for (C27H31N3O5): C, 67.91; H, 6.54; N,
8.80. Found: C, 67.73; H, 6.86; N, 8.57.
Compound 22c was synthesized from 20c (1.0 g, 3.2 mmol),
Et3N (1.0 mL), and ethylisocyanate (1.2 g, 16.0 mmol). Yield,
1.17 g (81%); mp 170–171 °C; 1H NMR (DMSO-d6) d 0.97 (6H, t,
J = 7.2 Hz, 2 ꢀ Me), 2.98 (4H, q, J = 7.2 Hz, CH2), 4.10 (2H, s, CH2),
4.80 (2H, s, CH2), 4.97 (2H, s, CH2), 5.03 (2H, s, CH2), 6.94 (2H, br
s, exchangeable, NH), 7.18–7.20 (1H, m, ArH), 7.26–7.29 (2H, m,
2 ꢀ ArH), 7.36–7.40 (4H, m, 4 ꢀ ArH), 7.42–7.44 (2H, m, 2 ꢀ ArH).
Anal. Calcld for (C26H29N3O4): C, 69.78; H, 6.53; N, 9.39. Found:
C, 69.64; H, 6.54; N, 9.37.
4.7.8. [3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate)
(22i)
4.7.3. [3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate)
(22d)
Compound 22i was synthesized from 20i (1.0 g, 2.7 mmol), Et3N
(1 mL), and ethylisocyanate (0.77 g, 10.9 mmol). Yield, 0.96 g
Compound 22d was synthesized from 20d (1.0 g, 3.0 mmol),
Et3N (1.2 mL), and (1.1 g, 15.0 mmol). Yield, 1.08 g (76%); mp
183–184 °C. 1H NMR (DMSO-d6) d 0.98 (6H, t, J = 6.8 Hz, 2 ꢀ Me),
2.97 (4H, q, J = 6.8 Hz, CH2), 4.09 (2H, s, CH2), 4.79 (2H, s, CH2),
4.95 (2H, s, CH2), 5.02 (2H, s, CH2), 6.93 (2H, br s, exchangeable,
NH), 7.20–7.22 (1H, m, ArH), 7.27–7.41 (5H, m, 5 ꢀ ArH), 7.46–
7.48 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C26H28FN3O4): C, 67.08; H,
6.06; N, 9.03. Found: C, 67.24; H, 6.02; N, 8.88.
(69%); mp 124–125 °C. 1H NMR (DMSO-d6)
d 0.98 (6H, t,
J = 6.8 Hz, 2 ꢀ Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 3.77 (3H, s, MeO),
3.82 (3H, s, MeO), 4.08 (2H, s, CH2), 4.79 (2H, s, CH2), 4.98 (2H, s,
CH2), 5.02 (2H, s, CH2), 6.93 (2H, br s, exchangeable NH), 6.96–
6.98 (2H, m, 2 ꢀ ArH), 7.07–7.09 (1H, m, ArH), 7.18–7.22 (1H, m,
ArH), 7.25–7.28 (2H, m, 2 ꢀ ArH), 7.30–7.37 (1H, m, ArH). Anal.
Calcd for (C28H33N3O6): C, 66.26; H, 6.55; N, 8.28. Found: C,
66.34; H, 6.45; N, 8.11.
4.7.4. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate)
(22e)
4.7.9. [3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]
isoquinoline-1,2-diyl]bis (methylene)bis(ethylcarbamate) (22j)
Compound 22j was synthesized from 20j (0.79 g, 2.0 mmol),
Et3N (0.8 mL), and ethylisocyanate (0.57 g, 8.0 mmol). Yield,
0.94 g (99%); mp 170–171 °C. 1H NMR (DMSO-d6) d 0.97 (6H, t,
J = 6.8 Hz, 2 ꢀ Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 3.73 (3H, s, MeO),
3.80 (6H, s, 2 ꢀ MeO), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 5.02
(2H, s, CH2), 5.05 (2H, s, CH2), 6.70 (2H, s, 2 ꢀ ArH), 6.95 (2H, br
s, exchangeable, NH), 7.19–7.21 (1H, m, ArH), 7.23–7.26 (1H, m,
ArH), 7.27–7.29 (1H, m, ArH), 7.33–7.35 (1H, m, ArH). Anal. Calcd
for (C29H35N3O7): C, 64.79; H, 6.56; N, 7.82. Found: C, 64.68; H,
6.42; N, 7.97.
Compound 22e was synthesized from 20e (1.0 g, 2.9 mmol),
Et3N (1.0 mL), and ethylisocyanate (0.84 g, 18.0 mmol). Yield,
0.92 g (64%); mp 191–192 °C. 1H NMR (DMSO-d6) d 0.98 (6H, t,
J = 7.2 Hz, 2 ꢀ Me), 2.97 (4H, q, J = 7.2 Hz, CH2), 4.09 (2H, s, CH2),
4.80 (2H, s, CH2), 4.97 (2H, s, CH2), 5.02 (2H, s, CH2), 6.92 (2H, br
s, exchangeable, NH), 7.18–7.22 (1H, m, ArH), 7.25–7.27 (2H, m,
2 ꢀ ArH), 7.28–7.30 (1H, m, ArH), 7.37–7.39 (2H, m, 2 ꢀ ArH),
7.44–7.46 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C26H28ClN3O4): C,
64.79; H, 5.86; N, 8.72. Found: C, 64.54; H, 5.84; N, 8.82.
4.7.5. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis (ethyl carbamate)
(22f)
4.8. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]bis(methylene)bis(iso-propylcarbamate) (23a)
Compound 22f was synthesized from 20f (1.0 g, 2.9 mmol),
Et3N (1 mL), and ethylisocyanate (0.8 g, 11.7 mmol). Yield, 1.0 g
Compound 23a was synthesized from 20a (0.5 g, 2.0 mmol),
Et3N (0.4 mL), and isopropylisocyanate (0.69 g, 8.0 mmol). Yield,

R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
285
0.55 g (65%); mp 215–218 °C. 1H NMR (DMSO-d6) d 0.99 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 2.25 (3H, s, Me), 3.54 (2H, m, CH), 3.99 (2H, s,
CH2), 4.89 (2H, s, CH2), 4.93 (2H, s, CH2), 4.96 (2H, s, CH2), 6.78
(2H, br s, exchangeable, NH), 7.25–7.29 (2H, m, 2 ꢀ ArH), 7.34–
7.35 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C23H31N3O4): C, 66.81; H,
7.56; N, 10.16. Found: C, 66.69; H, 7.66; N, 10.10.
for (C28H31F2N3O4): C, 65.74; H, 6.11; N, 8.21. Found: C, 65.51; H,
6.05; N, 8.38.
4.8.6. [3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-
propylcarbamate) (23g)
Compound 23g was synthesized from 20g (0.5 g, 1.3 mmol),
Et3N (0.5 mL), and isopropylisocyanate (0.45 g, 5.3 mmol). Yield,
0.6 g (82%); mp 192–193 °C. 1H NMR (DMSO-d6) d 1.01 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 3.57 (2H, m, CH), 4.09 (2H, s, CH2), 4.81 (2H,
s, CH2), 5.02 (4H, s, 2 ꢀ CH2), 6.87 (2H, br s, exchangeable, NH),
7.19–7.21 (1H, m, ArH), 7.25–7.27 (2H, m, 2 ꢀ ArH), 7.30–7.37
(1H, m, ArH), 7.43–7.45 (1H, m, ArH), 7.69 (1H, s, ArH) 7.73–7.75
(1H, m, ArH). Anal. Calcd for (C28H31N3O4Cl2): C, 61.77; H, 5.74;
N, 7.72. Found: C, 61.84; H, 5.67; N, 7.56.
4.8.1. [3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]bis(methylene)bis(iso-propylcarbamate) (23b)
Compound 23b was synthesized from 20b (0.26 g, 1.0 mmol),
Et3N (0.4 mL), and isopropylisocyanate (0.34 g, 4.0 mmol). Yield,
0.25 g (58%); mp 155–158 °C. 1H NMR (DMSO-d6) d 1.00 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 1.10 (3H, t, J = 7.6 Hz, Me), 2.69 (2H, q,
J = 7.6 Hz, CH2), 3.55 (2H, m, CH), 3.99 (2H, s, CH2), 4.89 (2H, s,
CH2), 4.93 (2H, s, CH2), 4.99 (2H, s, CH2), 6.79 (2H, br s, exchange-
able, NH), 7.25–7.29 (2H, m, 2 ꢀ ArH), 7.33–7.39 (2H, m, 2 ꢀ ArH).
Anal. Calcd for (C24H33N3O4): C, 67.42; H, 7.78; N, 9.83. Found: C,
67.11; H, 7.55; N, 9.71.
4.8.7. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-
propylcarbamate) (23h)
4.8.2. [3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-
diyl]bis(methylene)bis(iso-propylcarbamate) (23c)
Compound 23h was synthesized from 20h (0.5 g, 1.4 mmol),
Et3N (0.5 mL), and isopropylisocyanate (0.5 g, 5.9 mmol). Yield,
0.49 g (65%); mp 199–200 °C. 1H NMR (DMSO-d6) d 1.00 (12H, d,
J = 6.0 Hz, 4 ꢀ Me), 3.57 (2H, m, CH), 3.82 (3H, s, MeO), 4.07 (2H,
s, CH2), 4.77 (2H, s, CH2), 4.92 (2H, s, CH2), 5.01 (2H, s, CH2), 6.85
(2H, br s, exchangeable, NH), 7.04–7.06 (2H, m, 2 ꢀ ArH), 7.17–
7.21 (1H, m, ArH), 7.25–7.28 (2H, m, 2 ꢀ ArH), 7.34–7.36 (3H, m,
3 ꢀ ArH). Anal. Calcd for (C29H35N3O5): C, 68.89; H, 6.98; N, 8.31.
Found: C, 68.93; H, 6.96; N, 8.14.
Compound 23c was synthesized from 20c (0.5 g, 1.6 mmol),
Et3N (0.5 mL), and isopropylisocyanate (0.54 g, 6.4 mmol). Yield,
0.52 g (74%); mp 175–176 °C. 1H NMR (DMSO-d6) d 0.99 (12H, d,
J = 6.8 Hz, 4 ꢀ Me), 3.62 (2H, m, CH), 4.10 (2H, s, CH2), 4.80 (2H,
s, CH2), 4.98 (2H, s, CH2), 5.03 (2H, s, CH2), 6.86 (2H, br s, exchange-
able, NH), 7.18–7.22 (1H, m, ArH), 7.26–7.28 (2H, m, 2 ꢀ ArH),
7.38–7.42 (4H, m, 4 ꢀ ArH), 7.49–7.51 (2H, m, 2 ꢀ ArH). Anal. Calcd
for (C28H33N3O4): C, 70.71; H, 6.99; N, 8.84. Found: C, 70.59; H,
6.92; N, 8.67.
4.8.8. [3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene) bis(iso-
4.8.3. [3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-
propylcarbamate) (23d)
propylcarbamate) (23i)
Compound 23i was synthesized from 20i (0.37 g, 1.0 mmol),
Et3N (0.4 mL), and isopropylisocyanate (0.34 g, 4 mmol). Yield,
0.30 g (56%); mp 188–189 °C. 1H NMR (DMSO-d6) d 1.01 (12H, d,
J = 6.0 Hz, 4 ꢀ Me), 3.57 (2H, m, CH), 3.77 (3H, s, MeO), 3.82 (3H,
s, MeO), 4.08 (2H, s, CH2), 4.78 (2H, s, CH2), 4.98 (2H, s, CH2),
5.01 (2H, s, CH2), 6.84 (2H, br s, exchangeable, NH), 6.95–6.97
(2H, m, 2 ꢀ ArH), 7.06–7.08 (1H, m, ArH), 7.18–7.21 (1H, m, ArH),
7.25–7.29 (2H, m, 2 ꢀ ArH), 7.30–7.37 (1H, m, ArH). Anal. Calcd
for (C30H37N3O6): C, 67.27; H, 6.96; N, 7.84. Found: C, 67.27; H,
7.06; N, 7.62.
Compound 23d was synthesized from 20d (0.5 g, 1.5 mmol),
Et3N (0.5 mL), and isopropylisocyanate (0.51 g, 6.0 mmol). Yield,
0.62 g (81%); mp 190–191 °C. 1H NMR (DMSO-d6) d 0.99 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 3.60 (2H, m, CH), 4.09 (2H, s, CH2), 4.79 (2H,
s, CH2), 4.95 (2H, s, CH2), 5.02 (2H, s, CH2), 6.86 (2H, br s, exchange-
able, NH), 7.18–7.22 (1H, m, ArH), 7.26–7.29 (5H, m, 5 ꢀ ArH),
7.31–7.33 (2H, m, 2 ꢀ ArH). Anal. Calcd for (C28H32FN3O4): C,
68.14; H, 6.53; N, 8.51. Found: C, 68.31; H, 6.40; N, 8.60.
4.8.4. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-
4.8.9. [3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis (methylene)bis(iso-
propylcarbamate) (23e)
propylcarbamate) (23j)
Compound 23e was synthesized from 20e (0.45 g, 1.3 mmol),
Et3N (0.5 mL), and isopropylisocyanate (0.42 g, 5.0 mmol). Yield,
0.43 g (64%); mp 191–192 °C. 1H NMR (DMSO-d6) d 1.01 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 3.57 (2H, m, CH), 4.09 (2H, s, CH2), 4.80 (2H,
s, CH2), 4.97 (2H, s, CH2), 5.02 (2H, s, CH2), 6.86 (2H, br s, exchange-
able, NH), 7.18–7.22 (1H, m, ArH), 7.25–7.30 (2H, m, 2 ꢀ ArH),
7.37–7.39 (1H, m, ArH), 7.46 (2H, d, J = 8.0 Hz, 2 ꢀ ArH), 7.55 (2H,
d, J = 8.0 Hz, 2 ꢀ ArH). Anal. Calcd for (C28H32ClN3O4): C, 65.94; H,
6.32; N, 8.24. Found: C, 65.76; H, 6.17; N, 8.35.
Compound 23j was synthesized from 20j (0.39 g, 1.0 mmol),
Et3N (0.4 mL), and isopropylisocyanate (0.34 g, 4 mmol). Yield,
0.32 g (57%); mp 189–190 °C. 1H NMR (DMSO-d6) d 1.01 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 3.57 (2H, m, CH), 3.73 (3H, s, MeO), 3.80 (6H,
s, 2 ꢀ MeO), 4.08 (2H, s, CH2), 4.79 (2H, s, CH2), 5.01 (2H, s, CH2),
5.05 (2H, s, CH2), 6.69 (2H, s, 2 ꢀ ArH), 6.87 (2H, br s, exchangeable,
NH), 7.19–7.29 (2H, m, 2 ꢀ ArH), 7.33–7.39 (2H, m, 2 ꢀ ArH). Anal.
Calcld for (C31H39N3O7): C, 65.82; H, 6.95; N, 7.43. Found: C, 65.68;
H, 6.74; N, 7.11.
4.8.5. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-
4.9. Biological experiments
propylcarbamate) (23f)
4.9.1. Cytotoxicity assays
Compound 23f was synthesized from 20f (0.5 g, 1.4 mmol),
Et3N (0.5 mL), and isopropylisocyanate (0.49 g, 5.8 mmol). Yield,
0.62 g (83%); mp 176–178 °C. 1H NMR (DMSO-d6) d 1.02 (12H, d,
J = 6.4 Hz, 4 ꢀ Me), 3.58 (2H, m, CH), 4.09 (2H, s, CH2), 4.81 (2H,
s, CH2), 5.00 (2H, s, CH2), 5.02 (2H, s, CH2), 6.87 (2H, br s, exchange-
able, NH), 7.19–7.21 (1H, m, ArH), 7.26–7.30 (3H, m, 3 ꢀ ArH),
7.37–7.39 (1H, m, ArH), 7.51–7.59 (2H, m, 2 ꢀ ArH). Anal. Calcld
The effects of the newly synthesized compounds on cell growth
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 assay20 and human solid tumor cells (i.e., breast car-
cinoma MX-1 and colon carcinoma HCT-116) the SRB assay21 in a
72 h incubation using a microplate spectrophotometer as described
previously.22 After the addition of phenazine methosulfate-XTT

286
R. Chaniyara et al. / Bioorg. Med. Chem. 19 (2011) 275–286
solution at 37 °C for 6 h, absorbance at 450 and 630 nm was detected
on a microplate reader (EL 340; Bio-Tek Instruments Inc., Winooski,
VT). The cytotoxicity of the newly synthesized compounds against
non-small cell lung cancer H1299, human prostate cancer PC3, oral
carcinoma OECM1 and human glioma U87 were determined by the
Alamar blue assay23 in a 72 h incubation using a microplate spectro-
photometer as described previously. After the addition of Alamar
blue solution, it was incubated at 37 °C for 6 h. Absorbance at 570
and 600 nm was detected on a microplate reader. IC50 values were
determined from dose-effect relationship at six or seven concentra-
tions of each drug by using the CompuSyn software by Chou and
Martin24 based on the median-effect principle and plot.25,26 Ranges
given for Taxol and vinblastine were mean SE (n = 4).
trypsinized, washed with phosphate buffer saline (PBS), and 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 Dickinson, 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.
Acknowledgments
We are grateful to the National Science council (Grant No. NSC
99-2320-B-001-014) and Academia Sinica (Grant No. AS-96-TP-
B06) for financial support. T.-C. Chou was supported by the
Sloan-Kettering Institute GeneralFund. The NMR spectra of synthe-
sized compounds were obtained at High-Field Biomolecular NMR
Core Facility supported by the National Research Program for
Genomic Medicine (Taiwan). We would like to thank Dr. Shu-
Chuan Jao in the Institute of Biological Chemistry (Academia
Sinica) for providing the NMR service and the National Center for
High-performance computing for computer time and facilities.
4.9.2. In vivo studies
Athymic nude mice bearing the nu/nu gene were used for human
breast tumor MX-1 and human ovarian adenocarcinoma SK-OV-3
xenograft. Outbred Swiss-background mice were obtained from
the National Cancer Institute (Frederick, MD). Male mice 8 weeks
old or older weighing about 22 g were used for the experiments.
Drug was administrated via the tail vein by iv injection.22 Tumor vol-
umes were assessed by measuring length ꢀ width ꢀ height (or
width) by using caliper. Vehicle used was DMSO (50
lL) and Tween
80 (40 L) in saline (160 L). The maximal tolerable dose of the
l
l
References and notes
tested compound was determined and applied for the in vivo antitu-
mor activity assay. All animal studies were conducted in accordance
with the guidelines of the National Institutes of Health Guide for the
Care and Use of Animals and the protocol approved by the Memorial
Sloan-Kettering Cancer Center’s Institutional Animal Care and Use
Committee.
1. Crooke, S. T. In Mitomycin C. Current Status and New Development; Carter, S. K.,
Crooke, S. T., Alder, N. A., Eds.; Academic Press: New York, 1979.
2. Satorelli, A. C. Cancer Res. 1988, 48, 775.
3. Oostveen, E. A.; Speckmap, W. N. Tetrahedron 1987, 43, 255.
4. Tomasz, M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73.
5. 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.
6. Weidner, M. F.; Sigurdsson, S. T.; Hopkins, P. B. Biochemistry 1990, 29, 9225.
7. Woo, J.; Sigurdsson, S. T.; Hopkins, P. B. J. Am. Chem. Soc. 1993, 115, 3407.
8. Anderson, W. K.; Halat, M. J. J. Med. Chem. 1979, 22, 977.
9. Andeson, W. K.; New, J. S.; Corey, P. F. Arzneim.-Forsch. 1980, 30, 765.
10. Anderson, W. K. Cancer Res. 1982, 42, 2168.
11. Anderson, W. K.; McPherson, H. L.; New, J. S.; Rick, A. C. J. Med. Chem. 1984, 27,
1321.
12. Kakadiya, R.; Dong, H.; Lee, P.-C.; Kapuriya, N.; Zhang, X.; Chou, T.-C.; Lee, T.-C.;
Kapuriya, K.; Shah, A.; Su, T.-L. Bioorg. Med. Chem. 2009, 17, 5614.
13. Lee, P.-C.; Kakadiya, R.; Su, T.-L.; Lee, T.-C. Neoplasia 2010, 12, 376.
14. Dean, R. T.; Rapoport, H. J. Org. Chem. 1978, 43, 2115.
4.9.3. Alkaline agarose gel shift assay
Formation of DNA cross-linking was analyzed by alkaline aga-
rose gel electrophoresis. In brief, purified pEGFP-N1 plasmid DNA
(1.5
lg) was mixed with various concentrations (1–20
lM) of
20a, 22a, 20h and 23h in 40
lL binding buffer (3 mM sodium chlo-
ride/1 mM sodium phosphate, pH 7.4, and 1 mM EDTA). The reac-
tion mixture was incubated at 37 °C for 2 h. At the end of reaction,
the plasmid DNA was linearized by digestion with BamHI and fol-
lowed by precipitation with ethanol. The DNA pellets were dis-
solved and denatured in alkaline buffer (0.5 N NaOH–10 mM
15. Shinkai, H.; Toi, K.; Kumashiro, I.; Seto, Y.; Fukuma, M.; Dan, K.; Toyoshima, S. J.
Med. Chem. 1988, 31, 2092.
16. Grunewald, G. L.; Romero, F. A.; Criscione, K. R. J. Med. Chem. 2005, 48, 134.
17. Anderson, W. K.; McPherson, H. L.; New, J. S. J. Heterocycl. Chem. 1980, 17, 513.
18. Hartley, J. A.; Berardini, M. D.; Souhami, R. L. Anal. Biochem. 1991, 193, 131.
19. O’Connell, M. J.; Walworth, N. C.; Carr, A. M. Trends Cell Biol. 2000, 10, 296.
20. 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.
21. 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.
EDTA). An aliquot of 20
lL of DNA solution (1 lg) was mixed with
a 4 L of 6 X alkaline loading dye and then electrophoretically
l
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 bromide solution, and the
DNA was then visualized under UV light.
22. 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.
23. Al-Nasiry, S.; Hanssens, M.; Luyten, C.; Pijnenborn, R. Hum. Reprod. 2007, 22,
1304.
24. Chou, T.-C.; Martin, N. Compusyn Software; ComboSyn, Inc.: Paramus, NJ, 2005.
25. Chou, T.-C.; Talalay, P. Adv. Enzyme Regul. 1984, 22, 27.
26. Chou, T.-C. Pharmacol. Rev. 2006, 58, 621.
4.9.4. Flow cytometric analysis
The effects of 20a on cell cycle distribution were analyzed with
a
flow cytometer as previously described.12 Briefly, human
non-small cell lung carcinoma H1299 cells were treated with 20a
at 1.25, 2.5, and 5 M for 24 h. The attached cells were then
l