Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. 4. Structure-activity relationships of N-alkyl substituted pyrrole fused at the 7,8-positions

Article abstract of DOI:10.1021/jm7010657

In our continuing effort to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the structure-activity relationship (SAR) of alkyl substituted pyrrole fused at the 7,8-positions. A methyl group substituted a

Full text of DOI:10.1021/jm7010657

J. Med. Chem. 2008, 51, 417–423
417
Discovery of 4-Aryl-4H-chromenes as a New Series of Apoptosis Inducers Using a Cell- and
Caspase-Based High Throughput Screening Assay. 4. Structure–Activity Relationships of
N-Alkyl Substituted Pyrrole Fused at the 7,8-Positions
William Kemnitzer,† John Drewe,† Songchun Jiang,† Hong Zhang,† Candace Crogan-Grundy,† Denis Labreque,‡
Monica Bubenick,‡ Giorgio Attardo,‡ Real Denis,‡ Serge Lamothe,‡ Henriette Gourdeau,‡ Ben Tseng,† Shailaja Kasibhatla,† and
Sui Xiong Cai*,†
Epicept Corporation, 6650 Nancy Ridge DriVe, San Diego, California 92121, and Shire-Biochem Inc., 275 Armand-Frappier BouleVard, LaVal,
Quebec, Canada H7V 4A7
ReceiVed August 28, 2007
In our continuing effort to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer
agents, we explored the structure-activity relationship (SAR) of alkyl substituted pyrrole fused at the 7,8-
positions. A methyl group substituted at the nitrogen in the 7-position of the pyrrole ring led to a series of
potent apoptosis inducers with potency in the low nanomolar range. These compounds were also found to
be low nanomolar or subnanomolar inhibitors of cell growth, and they inhibited tubulin polymerization,
indicating that methylation of the 7-position nitrogen does not change the mechanism of action of these
chromenes. Compound 2d was identified as a highly potent apoptosis inducer with an EC50 value of 2 nM
and a highly potent inhibitor of cell growth with a GI50 value of 0.3 nM in T47D cells.
Chart 1
Introduction
Recent advances in our understanding of apoptosis, or
programmed cell death, have indicated that excessive or
improper inhibition of apoptosis plays a critical role in tumor
development and metastasis.1 As a result, chemotherapeutic
agents that target the apoptosis pathways could have broad
ramifications for the treatment of cancer. In fact, many com-
monly used chemotherapeutic drugs induce tumor cell apopto-
sis.2 The proapoptotic chemotherapeutic agents that target
tubulin, including paclitaxel and vinblastine, are among the most
successful and commonly prescribed anticancer therapies. The
colchicine binding site located on the monomeric unpolymerized
R/ꢀ-tubulin represents another potential tubulin target for the
development of apoptosis inducing chemotherapeutic agents.
Clinical trials are currently in progress for combretastatin A-4
phosphate prodrug 1a (CA4P) and amide prodrug 1b (AVE-
8062) (Chart 1).3 Both compounds inhibit tubulin polymerization
by binding at the colchicine site. In addition, these compounds
have been identified as vascular-disrupting agents (VDA) for
their ability to target and disrupt tumor vasculature.3–5
We have been interested in the discovery and development
of apoptosis inducers as potential anticancer agents and have
developed a cell- and caspase-based high throughput screening
(HTS) system for the discovery of apoptosis inducers.6 We have
reported the discovery of 4-aryl-4H-chromenes as a new series
of potent apoptosis inducing agents possessing vascular-
disrupting activity.7 The 4-aryl-4H-chromenes inhibit tubulin
polymerization and bind at or close to the binding site of
colchicine. They are also active in the multidrug resistant MES-
SA/DX5 tumor cells and are highly active as single agents and
in combination with other anticancer agents in several tumor
models.8 We have previously reported the structure-activity
relationship (SAR) of the 4-aryl group of chromene and the
Chart 2
identification of 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-
cyano-7-dimethylamino-4H-chromene (2a) (Chart 2) as a lead
compound.9 More recently, we have reported the SAR of a fused
ring at the 7,8-positions and identified 2-amino-4-(3-bromo-4,5-
dimethoxyphenyl)-3-cyano-4H-pyrrolo[2,3-h]chromene (2b) as
a low nanomolar potent lead compound (Chart 2).10 Comparison
of structure 2a with structure 2b suggests that introduction of
an alkyl group at the nitrogen of 2b such as shown in 2d might
increase activity. Herein, we report the discovery and SAR of
7-methyl-4H-pyrrolo[2,3-h]chromenes as highly potent apoptosis
inducers and inhibitors of cell growth.
Results and Discussion
Chemistry. The substituted 4-aryl-chromenes 2d-2j and
3a-3f were prepared by a one-pot reaction of substituted aryl
aldehyde and aryl alcohol with malononitrile in good yield
according to methods previously described (Scheme 1).9 The
intermediate 4-hydroxy-1-methyl-1H-indole (6a) was synthe-
sized from methylation of 4-benzyloxy-1H-indole (4a) to give
5a followed by deprotection, and 7-hydroxy-1-methyl-1H-indole
* To whom correspondence should be addressed. Telephone: 858-202-
4006. Fax: 858-202-4000. E-mail: scai@epicept.com.
†
Epicept Corporation.
Shire-Biochem Inc.
‡
10.1021/jm7010657 CCC: $40.75
2008 American Chemical Society
Published on Web 01/16/2008

418 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Kemnitzer et al.
Scheme 1a
In comparison, compound 2g from methylation of the 9-position
nitrogen of compound 2c was about 10-fold less active than
2c, suggesting that there is a space-limiting pocket at the
9-position. Similar to 2g, the 8-methyl analogue 2h10 was 7-fold
and 17-fold less active than 2b and 2d, respectively, indicating
that the position of the methyl group is critical for activity.
Replacing the methyl group at the 7-position with a hydroxy-
methyl group gave the analogue 2e, which has a potency
comparable to that of 2d, with an EC50 value of 3 nM in T47D
cells. Moreover, the hydroxy group in 2e might improve the
solubility profile. It also provides a site for the preparation of a
phosphate prodrug, similar to what has been successfully done
in the case of the combretastatin A-4 phosphate prodrug.14 In
addition, the hydroxy group also provides a site for potential
bioconjugation such as attachment of 2e to a protein. In
comparison, the 7-ethyl analogue 2f was almost 20-fold less
active than 2d, suggesting that the pocket around the 7-position
is size limited. The importance of having a methyl substitution
at the 7-position was further highlighted by the saturated ring
analogue 2i, which was found to have an EC50 value comparable
to that of 2d. In addition, the 7-methylimidazolo[5,4-h]chromene
analogue 2j was also highly active, with a potency approaching
that of 2d.
a Conditions: (a) EtOH, piperidine, room temperature (rt).
Scheme 2a
a Conditions: (a) NaH, DMF, iodomethane, rt; (b) dimethyl oxalate,
KOBu-t; (c) 5% Pd/C, H2(g) at 40 psi, MeOH.
Scheme 3a
We next explored the SAR of the 4-aryl group of 7-methyl-
4H-pyrrolo[2,3-h]chromenes (Table 2). A 3,4,5-trisubstituted
phenyl group, such as the 3,4-methylenedioxo-5-methoxyphenyl
analogue (3a) led to a >8-fold decrease in potency relative to
2d. However, the trisubstituted 3,4,5-trimethoxyphenyl analogue
(3b) was very potent, only 2-fold less potent than 2d. The 3,5-
dimethoxyphenyl analogue (3c) and the 3-methoxy analogue
(3d) were both highly potent analogues, with EC50 values only
2–3-fold less potent than those of 2d. Interestingly, the potency
difference between 3,4,5-trisubstituted analogue 2d, 3,5-
dimethoxy analogue 3c, and 3-methoxy analogue 3d is relatively
small for the 7-methyl-4H-pyrrolo[2,3-h]chromenes compared
to what we observed earlier for 4H-pyrrolo[2,3-h]chromenes,10
which had potency differences of 6–10-fold. Similar to the
reported SAR for 7-NMe2, 7,8-disubstituted, and 7,8-fused
chromenes,9,10,12 the 5-substituted 3-pyridyl analogues 3e and
3f were also highly potent and about as active as 2d, with EC50
values of 3 nM each in T47D cells.
a Conditions: (a) HC(O)H, 2 N NaOH, EtOH, rt; (b) EtBr, NaH, THF;
(c) 5% Pd/C, H2(g) at 50 psi, MeOH.
Scheme 4a
a Conditions: (a) 5% Pd/C, H2(g) at 40 psi, MeOH, HCl(conc)
.
(6b) was synthesized similarly from methylation of 7-benzyloxy-
1H-indole (4b) followed by deprotection (Scheme 2). Similarly,
the intermediate 4-hydroxy-1-hydroxymethyl-1H-indole (8a)
was prepared from the reaction of 4a with formaldehyde under
basic conditions to give 7a followed by deprotection, and
1-ethyl-4-hydroxy-1H-indole (8b) was synthesized from reaction
of 4a with bromoethane in the presence of sodium hydride
followed by deprotection (Scheme 3). Hydrogenation of 6a
under acidic conditions produced the saturated intermediate
4-hydroxy-2,3-dihydro-1-methyl-1H-indole (9) (Scheme 4).
4-Hydroxy-1-methyl-1H-benzimidazole, the intermediate for the
synthesis of compound 2j, was prepared from 2-amino-3-nitro-
phenol in four steps according to reported procedure.11 Substi-
tuted aryl aldehydes for the synthesis of compounds 3e and 3f
are not commercially available and were synthesized according
to published procedures.9,12
Structure–Activity Relationship (SAR) Studies. The apop-
tosis inducing activities of 4-aryl-4H-chromenes were measured
by our proprietary cell- and caspase-based HTS assay13 in
human breast cancer cells T47D, human colon cancer cells
HCT116, and hepatocellular carcinoma cancer cells SNU398.
By maintaining the 2-amino, the 3-cyano, and the preferred 4-(3-
bromo-4,5-dimethoxyphenyl) groups, the SAR of the alkyl
substituted pyrrole ring was explored. Compound 2d, with a
methyl substitution at the 7-position nitrogen, was one of the
most potent compounds in this series, having an EC50 value of
2 nM in T47D cells, 3 nM in HCT116 cells, and 2 nM in
SNU398 cells (Table 1). Compound 2d was almost 10-fold more
potent than 2a and >2-fold more potent than 2b in T47D cells.
We also tested four known anti-tubulin apoptosis inducers
in our assays, and the results are included in Table 2 for
comparison. Vinblastine and vincristine were found to have EC50
values of 43 nM against T47D cells, which are >20-fold less
active than compound 2d in the same assay. Colchicine was
found to have an EC50 value of 9 nM, and Taxol was found to
have an EC50 value of 36 nM. Overall, these data indicted that
2d and several of its related analogues are more potent than
these reference compounds in our apoptosis activation assay.
The activities of these compounds toward the colon cancer
cell line HCT116 and the hepatocellular carcinoma cancer cell
line SNU398 were roughly parallel to their activity toward T47D
cells. In general, HCT116 cells were slightly less sensitive (about
1.2-fold less sensitive as indicated by the EC50 value) to the
compounds than T47D cells in this assay. SNU398 cells
displayed a tendency to be slightly more sensitive to the
compounds than T47D cells in this assay.
Selected compounds were also tested in the standard growth
inhibition assay. The growth inhibition assays in T47D,
HCT116, and SNU398 cells were run in a 96-well microtiter
plate as described previously.13 In brief, T47D, HCT116, and
SNU398 cells were exposed to the test compound for 48 h at
37 °C. CellTiter-Glo reagent (Promega) was added, and the

DiscoVery of 4-Aryl-4H-chromenes as Apoptosis Inducers
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 419
Table 1. SAR of 2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4H-chromenes with an Alkyl Substituted Fused Ring at the 7,8-Positions in the
Caspase Activation Assay
a Data are the mean of three or more experiments and are reported as mean ( standard error of the mean (SEM). b Data from ref 9. c Data from ref 10.
NA, not applied.
samples were mixed by agitation, incubated at room temperature
for 10–15 min, and then read using a luminescent plate reader
(Model Spectrafluor Plus Tecan Instrument). The GI50 is defined
as 50% inhibition of cell growth. GI50 values, in comparison
with their EC50 values, are summarized in Table 3.
Among those tested, compounds 2d and 2e were found to be
the most potent inhibitors of tumor cell growth in T47D cells.
Compound 2d had a GI50 value of 0.3 nM while 2e had a GI50
value of 2 nM in T47D cells. Compounds 2d and 2e were
significantly more potent in the growth inhibition assay in T47D

420 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Kemnitzer et al.
Table 2. SAR of 2-Amino-4-aryl-3-cyano-7-methyl-4H-pyrrolo[2,3-h]chromenes in the Caspase Activation Assay
a Data are the mean of three or more experiments and are reported as mean ( standard error of the mean (SEM). b NA, not applied.
cells than compound 2b. Compounds 2d and 2e were respec-
tively >26-fold and 4-fold more potent than 2b in T47D cells.
Interestingly, compounds 3e and 2i were more potent inhibitors
of tumor cell growth in SNU398 cells than in T47D cells and

DiscoVery of 4-Aryl-4H-chromenes as Apoptosis Inducers
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 421
Table 3. Inhibition of Cell Growth Activity of Substituted
2-Amino-4-aryl-3-cyano-4H-pyrrolo[2,3-h]chromenes
glassware. Thin-layer chromatography (TLC), usually using ethyl
acetate/hexane as the solvent system, was used to monitor reactions.
Solvents were removed by rotary evaporation under reduced
pressure; where appropriate, the compound was further dried using
a vacuum pump. The 1H NMR spectra were recorded at 300 MHz.
All samples were prepared as dilute solutions in either deuterio-
chloroform (CDCl3) with 0.05% v/v tetramethylsilane (TMS) or
dimethyl-d6-sulfoxide (CD3SOCD3) with 0.05% v/v TMS. Chemical
shifts are reported in parts per million (ppm) downfield from TMS
(0.00 ppm), and J coupling constants are reported in hertz.
Elemental analyses were performed by Numega Resonance Labs,
Inc. (San Diego, CA). Melting points were determined on a glass
capillary melting point apparatus. Human breast cancer cells T47D,
human colon cancer cells HCT116, and hepatocellular carcinoma
cancer cells SNU398 were obtained from the American Type
Culture Collection (Manasas, VA). Tubulin was obtained from
Cytoskeleton (Boulder, CO).
4-Benzyloxy-1-methyl-1H-indole (5a). To a white suspension
of sodium hydride (0.089 g, 2.2 mmol) in DMF (4.50 mL) was
added 4-benzyloxy-1H-indole (4a) (0.500 g, 2.2 mmol) in portions
over 5 min. The resulting brown mixture was allowed to warm to
room temperature over 1 h, and then iodomethane (0.28 mL, 4.5
mmol) was added dropwise. After stirring at room temperature for
5 h, an additional 2 equiv of iodomethane (0.28 mL, 4.5 mmol)
was added to the brown mixture. The reaction mixture was stirred
for 1 h, quenched with water (10 mL), and then extracted with
ethyl acetate (2 × 20 mL). The extracts were dried over Na2SO4,
filtered through sintered glass, and concentrated to yield 0.607 g
of a brown oil residue. The residue was purified by flash column
chromatography (elution with EtOAC/hexanes, 1:4) to yield 0.253
g (47%) of 5a as a brown oil: 1H NMR (CDCl3, δ) 7.52–7.48 (m,
2H), 7.41–7.31 (m, 3H), 7.13–7.03 (m, 2H), 6.98–6.95 (m, 1H),
6.63–6.57 (m, 2H), 5.22 (s, 2H), 3.77 (s, 3H).
GI50 (µM)a
compound
2b
2d
2e
2i
T47D
0.008 ( 0.001b
0.0003 ( 0.0001 0.0014 ( 0.0003 0.0002 ( 0.0001
0.002 ( 0.001
0.007 ( 0.002
0.008 ( 0.002
0.007 ( 0.002
0.007 ( 0.001
HCT116
SNU398
0.020 ( 0.004
0.014 ( 0.001
0.004 ( 0.001
0.004 ( 0.0004
0.003 ( 0.001
NDc
0.001 ( 0.0003
0.001 ( 0.0002
0.002 ( 0.0002
NDc
3e
Vinblastine
Colchicine
NDc
NDc
a Data are the mean of three or more experiments and are reported as
mean ( standard error of the mean (SEM). b Data from ref 10. c ND, not
determined.
were comparable to 2e. We also tested two reference com-
pounds, and the data are included in Table 3. Compound 2d
was found to be >10-fold more potent than the known apoptosis
inducers vinblastine and colchicine in the growth inhibition assay
in T47D cells.
Similar to compound 2b (MX-76747), 2d (MX-126303) was
found to be a tubulin inhibitor that binds at or close to the
colchicine site of ꢀ-tubulin.7 Compound 2d was found to inhibit
tubulin polymerization with an IC50 value of 900 nM. In
addition, compound 2d was found to arrest MCF-7 breast
carcinoma cells in the G2/M phase of the cell cycle followed
by apoptosis as measured by cell cycle analysis,7 confirming
that 2d and related compounds induce apoptosis through similar
pathways to those of other chromenes. Therefore, 4-aryl-7-
methyl-4H-pyrrolo[2,3-h]chromenes have the same mechanism
of action as our previously reported chromenes.
4-Hydroxy-1-methyl-1H-indole (6a). To a brown solution of
5a (0.250 g, 1.12 mmol) and methanol (2.25 mL) in a hydrogenation
apparatus par shaker flask was added 5% Pd/C (0.119 g) to give a
black mixture. The flask was attached to a hydrogenation apparatus,
degassed (3×), and then filled with hydrogen gas. After the final
degassing, the par shaker flask was filled with hydrogen gas at 40
psi and shaken for 16 h. The reaction product was diluted with
methanol (15 mL), filtered through a layer of Celite (2.5 in d × 2
in h), and washed with additional warm methanol (75 mL). The
organic filtrate was concentrated to yield 0.160 g (97%) of 6a as
Conclusion
We have explored the SAR of the apoptosis inducing 4-aryl-
4H-chromenes via introducing an alkyl group on the pyrrole
ring fused at the 7,8-positions. A methyl substitution at the
nitrogen in the 7-position of the pyrrole ring led to the discovery
of compound 2d with low nanomolar potency. In comparison,
a methyl group substituted at either the 8- or 9-positions led to
a decrease in potency relative to the nonsubstituted analogues.
Significantly, reducing the pyrrole ring to the saturated analogue
2i maintained high potency and indicates the importance of the
N-methyl substitution. The 7-hydroxymethyl analogue 2e is also
highly active and has additional possibilities for the preparation
of prodrugs or bioconjugation. Interestingly, the 7-ethyl analogue
2f was about 20-fold less active than 2d, suggesting a size
limited pocket at the 7-position. Further exploration of the SAR
of the 4-aryl group of 2d led to a group of highly potent
7-methyl-4H-pyrrolo[2,3-h]chromenes. In addition to 2d, the
analogues 3b-3f all had <10 nM potency in the caspase
activation assay in T47D, HCT116, and SNU398 cells. These
analogues were also found to be highly active in the growth
inhibition assay and to inhibit tubulin polymerization, similar
to the case of chromenes with a pyrrole fused ring at the 7,8-
positions. Compound 2d is significantly more potent than the
previous lead compound 2b, with an EC50 value of 2 nM and
a GI50 value of 0.3 nM in T47D cells. Additional SAR and in
vivo studies of 4-aryl-4H-chromenes will be reported in future
publications.
1
black oil: H NMR (CDCl3, δ) 7.07 (t, J ) 8.2 Hz, 1H), 6.96 (d,
J ) 2.7 Hz, 1H), 6.92 (d, J ) 8.4 Hz, 1H), 6.54–6.51 (m, 2H),
4.60 (br s, 1H), 3.75 (s, 3H).
7-Hydroxy-1-methyl-1H-indole (6b). A mixture of 7-benzy-
loxyindole (4b) (0.300 g, 1.34 mmol), dimethyl oxalate (0.317 g,
2.68 mmol), and potassium tert-butoxide (0.302 g, 2.68 mmol) in
DMF (5.0 mL) was stirred at 110 °C overnight. The solution was
poured into a saturated solution of NaHCO3 (20 mL) and extracted
with EtOAc. The organic layer was separated, washed with brine,
and dried over Na2SO4; the solvent was removed by rotary
evaporation to give 0.200 g (63%) of 7-benzyloxy-1-methyl-1H-
indole (5b). Compound 5b was reacted under hydrogenation
conditions similar to those of 5a to produce 0.090 g (46%) of 6b:
1H NMR (CDCl3, δ) 7.19–7.16 (m, 1H), 6.94 (d, J ) 3.0 Hz, 1H),
6.86 (t, J ) 7.5 Hz, 1H), 6.48–6.46 (m, 1H), 6.40 (d, J ) 3.0 Hz,
1H), 5.05 (s, 1H), 4.07 (s, 3H).
4-Hydroxy-1-hydroxymethyl-1H-indole (8a). A solution of 4a
(1.00 g, 4.48 mmol), formaldehyde (2.00 mL, 26.8 mmol), and 2
N NaOH (2.24 mL, 4.48 mmol) in EtOH (10 mL) was stirred at
room temperature for 4 h, and the solvent was removed in vacuo.
The crude material was purified by flash column chromatography
(EtOAc/hexanes, 1:3) to yield 1.13 g of 4-benzyloxy-1-hydroxy-
methyl-1H-indole (7a). Compound 7a was hydrogenated under
conditions similar to those of compound 5a to give 0.580 g (80%)
of 8a: 1H NMR (CDCl3, δ) 7.15–7.07 (m, 3H), 6.60–6.55 (m, 2H),
5.62 (d, J ) 7.5 Hz, 2H), 4.93 (s, 1H), 2.37 (t, J ) 7.2 Hz, 1H).
1-Ethyl-4-hydroxy-1H-indole (8b). To a brown solution of 4a
(1.00 g, 4.48 mmol) in THF (22 mL) at 0 °C was added sodium
Experimental Section
General Methods and Materials. Commercial-grade reagents
and solvents obtained from Acros, Aldrich, Chem-Impex, Lancaster,
TCI, or VWR were used without further purification, except as
indicated. All reaction mixtures were stirred magnetically; moisture-
sensitive reactions were performed under argon in oven-dried

422 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Kemnitzer et al.
hydride (60%, 0.269 g, 11.2 mmol) in portions over 5 min. The
resulting suspension was stirred at 0 °C for 5 min, and then
bromoethane (0.330 mL, 4.48 mmol) was added dropwise. The
reaction mixture was equilibrated to room temperature and then
stirred overnight. It was quenched with water (10 mL) and extracted
with ethyl acetate (2 × 70 mL), and the organic extracts were then
washed with brine, dried over MgSO4, filtered, and concentrated.
The brown residue was purified by flash column chromatography
(EtOAC/hexanes, 1:20) to yield 0.585 g (52%) of 4-benzyloxy-1-
ethyl-1H-indole (7b) as a white solid. Compound 7b was hydro-
genated under conditions similar to those of compound 5a to yield
8b (100%) as a black oil: 1H NMR (CDCl3, δ) 7.08–7.02 (m, 2H),
6.94 (d, J ) 8.3 Hz, 1H), 6.54–6.48 (m, 2H), 4.70 (br s, 1H), 4.13
(q, J ) 7.3 Hz, 2H), 1.43 (t, J ) 7.4 Hz, 3H).
4-Hydroxy-2,3-dihydro-1-methyl-1H-indole (9). Compound 6a
(0.410 g, 2.79 mmol) was hydrogenated with 5% Pd/C in methanol
(20 mL) and 0.28 mL of concentrated aqueous HCl under H2 (40
psi) to yield 0.120 g (29%) of 9: 1H NMR (CDCl3, δ) 6.98 (t, J )
8.5 Hz, 1H), 6.19–6.12 (m, 2H), 4.51 (s, 1H), 3.34 (t, J ) 8.4 Hz,
2H), 2.90 (t, J ) 8.7 Hz, 2H), 2.75 (s, 3H).
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,7-dihy-
dro-7-methyl-pyrano[2,3-e]indole (2d). To a yellow solution of
5-bromoveratraldehyde (2.50 g, 10.2 mmol), ethanol (34.0 mL),
malononitrile (0.673 g, 10.2 mmol), and 6a (1.50 g, 10.2 mmol)
was added piperidine (0.500 mL, 5.10 mmol), and the solution was
stirred at room temperature for 12 h. The resulting precipitate was
collected by filtration, washed with ethanol (50 mL), and dried in
vacuo to yield 3.00 g (66%) of 2d as a yellow solid: mp 207–209
°C (dec); 1H NMR (CDCl3, δ) 7.07–7.02 (m, 2H), 6.91 (d, J ) 2.1
Hz, 1H), 6.77–6.75 (m, 2H), 6.57 (dd, J ) 0.9 Hz, 1H), 4.78 (s,
1H), 4.70 (br s, 2H), 3.82 (s, 6H), 3.78 (s, 3H). Anal. (C21H18
BrN3O3) C, H, N.
The following compounds were prepared from the corresponding
aryl aldehyde, aryl alcohol, and malononitrile with piperidine in
ethanol by a procedure similar to that described for the preparation
of compound 2d.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,7-dihy-
dro-7-(hydroxylmethyl)pyrano[2,3-e]indole (2e). Compound 2e
was prepared from 5-bromoveratraldehyde, malononitrile, and 8a
and was isolated as a white solid (42%): mp 196–198 °C; 1H NMR
(CDCl3, δ) 7.22–7.18 (m, 2H), 6.90 (d, J ) 1.8 Hz, 1H), 6.81 (d,
J ) 8.4 Hz, 1H), 6.75 (d, J ) 1.8 Hz, 1H), 6.64 (d, J ) 3.3 Hz,
1H), 5.61 (s, 2H), 4.77 (s, 1H), 4.70 (br s, 2H), 3.83 (s, 3H), 3.82
(s, 3H), 2.47 (br s, 1H). Anal. (C21H18BrN3O4) C, H, N.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,7-dihy-
dro-7-ethylpyrano[2,3-e]indole (2f). Compound 2f was prepared
from 5-bromoveratraldehyde, malononitrile, and 8b and was isolated
as a white solid (67%): mp 156–158 °C; 1H NMR (CDCl3, δ) 7.13
(d, J ) 3.0 Hz, 1H), 7.06 (d, J ) 8.5 Hz, 1H), 6.92 (d, J ) 1.9 Hz,
1H), 6.76 (d, J ) 1.9 Hz, 1H), 6.74 (d, J ) 8.8 Hz, 1H), 6.58 (d,
J ) 3.0 Hz, 1H), 4.77 (s, 1H), 4.69 (br s, 2H), 4.14 (q, J ) 7.4 Hz,
2H), 3.83 (s, 3H), 3.82 (s, 3H), 1.46 (t, J ) 7.4 Hz, 3H). Anal.
(C22H20BrN3O3) C, H, N.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,7-dihy-
dro-7-methylpyrano[2,3-e]benzimidazole (2j). Compound 2j was
prepared from 5-bromoveratraldehyde, malononitrile, and 4-hy-
droxy-1-methyl-1H-benzimidazole and was isolated as a light brown
1
solid (48%): mp 243–245 °C (dec); H NMR (CDCl3, δ) 7.88 (s,
1H), 7.11 (d, J ) 8.4 Hz, 1H), 6.92 (d, J ) 1.8 Hz, 1H), 6.90 (d,
J ) 8.7 Hz, 1H), 6.72 (d, J ) 2.1 Hz, 1H), 4.84 (br s, 2H), 4.80 (s,
1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H). Anal. (C20H17BrN4O3)
C, H, N.
2-Amino-3-cyano-4-(3,4-methylenedioxo-5-methoxyphenyl)-
4,7-dihydro-7-methylpyrano[2,3-e]indole (3a). Compound 3a was
prepared from myristicin aldehyde, malononitrile, and 6a and was
1
isolated as a white solid (55%): mp 186–188 °C (dec); H NMR
(CDCl3, δ) 7.05–7.01 (m, 2H), 6.79 (d, J ) 8.4 Hz, 1H), 6.55 (d,
J ) 3.0 Hz, 1H), 6.46 (s, 1H), 6.33 (d, J ) 1.2 Hz, 1H), 5.92–5.90
(m, 2H), 4.75 (s, 1H), 4.64 (br s, 2H), 3.88 (s, 3H), 3.77 (s, 3H).
Anal. (C21H17N3O4) C, H, N.
2-Amino-3-cyano-4-(3,4,5-trimethoxyphenyl)-4,7-dihydro-7-
methylpyrano[2,3-e]indole (3b). Compound 3b was prepared from
3,4,5-trimethoxybenzaldehyde, malononitrile, and 6a and was
isolated as a white solid (66%): mp 182–184 °C; 1H NMR (CDCl3,
δ) 7.06–7.02 (m, 2H), 6.80 (d, J ) 8.1 Hz, 1H), 6.57 (d, J ) 2.4
Hz, 1H), 6.42 (s, 2H), 4.79 (s, 1H), 4.66 (s, 2H), 3.81 (s, 3H), 3.79
(s, 6H), 3.78 (s, 3H). Anal. (C22H21N3O4) C, H, N.
2-Amino-3-cyano-4-(3,5-dimethoxyphenyl)-4,7-dihydro-7-me-
thylpyrano[2,3-e]indole (3c). Compound 3c was prepared from
3,5-dimethoxybenzaldehyde, malononitrile, and 6a and was isolated
as a white solid (60%): mp 196–198 °C; 1H NMR (CDCl3, δ)
7.04–7.00 (m, 2H), 6.81 (d, J ) 8.4 Hz, 1H), 6.55 (d, J ) 3.0 Hz,
1H), 6.37–6.31 (m, 3H), 4.77 (s, 1H), 4.64 (br s, 2H), 3.77 (s, 3H),
3.74 (bs, 6H). Anal. (C21H19N3O3) C, H, N.
2-Amino-3-cyano-4-(3-methoxyphenyl)-4,7-dihydro-7-meth-
ylpyrano[2,3-e]indole (3d). Compound 3d was prepared from
3-methoxybenzaldehyde, malononitrile, and 6a and was isolated
as a white solid (69%): mp 189–192 °C; 1H NMR (CDCl3, δ)
7.23–7.18 (m, 1H), 7.05–7.00 (m, 2H), 6.83–6.74 (m, 4H), 6.56
(d, J ) 3.0 Hz, 1H), 4.81 (s, 1H), 4.64 (br s, 2H), 3.763 (s, 3H),
3.757 (s, 3H). Anal. (C20H17N3O2) C, H, N.
2-Amino-3-cyano-4-(5-methylpyridin-3-yl)-4,7-dihydro-7-me-
thylpyrano[2,3-e]indole (3e). Compound 3e was prepared from
5-methylpyridine-3-carbaldehyde, malononitrile, and 6a and was
1
isolated as a brown solid (73%): mp 222–226 °C (dec); H NMR
(CDCl3, δ) 8.34 (d, J ) 2.1 Hz, 1H), 8.31 (m, 1H), 7.29 (s, 1H),
7.06 (d, J ) 3.0 Hz, 1H), 7.02 (m, 1H), 6.71 (d, J ) 8.4 Hz, 1H),
6.57 (m, 1H), 4.85 (s, 1H), 4.77 (br s, 2H), 3.77 (s, 3H), 2.26 (s,
3H). Anal. (C19H16N4O) C, H, N.
2-Amino-3-cyano-4-(5-methoxypyridin-3-yl)-4,7-dihydro-7-
methylpyrano[2,3-e]indole (3f). Compound 3f was prepared from
5-methoxypyridine-3-carbaldehyde, malononitrile, and 6a and was
1
isolated as a white solid (12%): mp 212–216 °C (dec); H NMR
(CDCl3, δ) 8.19–8.15 (m, 2H), 7.07–6.99 (m, 3H), 6.73 (d, J )
8.4 Hz, 1H), 6.57 (d, J ) 3.0 Hz, 1H), 4.89 (s, 1H), 4.75 (br s,
2H), 3.81 (s, 3H), 3.78 (m, 3H). Anal. (C19H16N4O2) C, H, N.
Supporting Information Available: Caspase activation assay
(EC50), cell growth inhibition assays (GI50), the tubulin inhibition
assay, and a table of elemental analysis data for the targeted
compounds 2d-2g, 2i-2j, and 3a-3f. This material is available
free of charge via the Internet at http://pubs.acs.org.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,9-dihy-
dro-9-methylpyrano[3,2-g]indole (2g). Compound 2g was pre-
pared from 5-bromoveratraldehyde, malononitrile, and 6b and was
1
isolated as a white solid (52%): mp 206–207 °C (dec); H NMR
(CDCl3, δ) 7.27 (d, J ) 8.4 Hz, 1H), 6.99 (d, J ) 3.3 Hz, 1H),
6.90 (d, J ) 1.8 Hz, 1H), 6.77 (d, J ) 2.4 Hz, 1H), 6.61 (d, J )
8.1 Hz, 1H), 6.42 (d, J ) 3.0 Hz, 1H), 4.79 (s, 1H), 4.62 (br s,
2H), 4.09 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H). Anal. (C21H18BrN3O3)
C, H, N.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-4,7,8,9-
tetrahydro-7-methylpyrano[2,3-e]indole (2i). Compound 2i was
prepared from 5-bromoveratraldehyde, malononitrile, and 9 and was
isolated as a white solid (26%): mp 199–201 °C; 1H NMR (CDCl3,
δ) 6.88 (s, 1H), 6.72 (s, 1H), 6.67 (d, J ) 8.4 Hz, 1H), 6.20 (d, J
) 8.4 Hz, 1H), 4.60–4.57 (m, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 3.40
(t, J ) 8.1 Hz, 2H), 2.98 (t, J ) 8.7 Hz, 2H), 2.74 (s, 3H). Anal.
(C21H20BrN3O3) C, H, N.
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