γ-Carboline derivatives with anti-bovine viral diarrhea virus (BVDV) activity

Article abstract of DOI:10.1016/j.bmc.2008.01.052

Based on anti-viral screening of our heteroaromatics derived from thalidomide, the γ-carboline skeleton has been identified as a superior scaffold structure for compounds with potent anti-bovine viral diarrhea virus (BVDV) activity. Structural development studies led to a potent anti-viral agent, SK5M (5-methyl-γ-carboline), with the EC₅₀ of 0.26 μM.

Full text of DOI:10.1016/j.bmc.2008.01.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3780–3790
c-Carboline derivatives with anti-bovine viral diarrhea
virus (BVDV) activity
Kumiko Sako,a Hiroshi Aoyama,a,* Shinichi Sato,a
Yuichi Hashimotoa and Masanori Babab,*
aInstitute of Molecular & Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bDivision of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences,
Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
Received 7 January 2008; revised 25 January 2008; accepted 27 January 2008
Available online 31 January 2008
Abstract—Based on anti-viral screening of our heteroaromatics derived from thalidomide, the c-carboline skeleton has been iden-
tified as a superior scaffold structure for compounds with potent anti-bovine viral diarrhea virus (BVDV) activity. Structural devel-
opment studies led to a potent anti-viral agent, SK5M (5-methyl-c-carboline), with the EC50 of 0.26 lM.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Therefore, alternative agents for the treatment and pre-
vention of HCV infection are urgently needed.
Viruses belonging to the Flaviviridae family cause clini-
cally significant diseases in humans and animals. This
virus family includes three genera, that is, Pestiviruses
[including bovine viral diarrhea virus (BVDV)], Flavivi-
ruses [including yellow fever (YFV), dengue, and West
Nile viruses], and Hepaciviruses [including hepatitis C
virus (HCV)].1
We have been engaged in structural development studies
of thalidomide (1: N-a-phthalimidoglutarimide),8–11
which was launched as a sedative/hypnotic agent, but
subsequently withdrawn because of its teratogenicity.12
Nevertheless it has since been found to be effective
against various diseases, including multiple myeloma
(MM), AIDS, and Hansen’s disease.8–12 Our structural
development studies based on anti-viral activity-related
bioassay systems/molecular targets, including tumor
necrosis factor (TNF)-a production inhibition, a-gluco-
sidase inhibition, etc.,8–11 led us develop compounds
with a 6-5-6 fused heteroaromatic ring system (Fig. 1).
Based on the structural development scheme shown in
Figure 1, we screened commercially available 6-5-6 fused
heteroaromatic compounds, that is, fluorene (2), carba-
zole (3) and dibenzofuran (4), for anti-viral activity
(Fig. 2).
HCV infection is thought to be a major cause of human
hepatitis globally.2,3 The World Health Organization
(WHO) estimates that at least 170 million people world-
wide are chronically infected with this virus.4 Most
infections become persistent, and about 60% of cases
progress to chronic liver disease, which in turn, can lead
to development of cirrhosis, hepatocellular carcinoma,
and liver failure.5,6 With the exception of YFV, no vac-
cine exists against the members of Flaviviridae. Cur-
rently, the standard treatment for chronic hepatitis C
consists of pegylated interferon (IFN)-a in combination
with the nucleoside analog ribavirin (1-b-D-ribofurano-
syl-1,2,4-triaxole-3-carboxamide). However, the virus
cannot be eliminated from approximately half of the in-
fected patients treated with these agents.7 In addition,
the side effects of these agents are sometimes serious.
Because BVDV is thought to be a good model for hu-
man HCV (HCV does not replicate efficiently in cell cul-
tures or animals),13–15 we performed the anti-viral
screening of typical 6-5-6 fused heteroaromatic com-
pounds using Madin–Darby bovine kidney (MDBK)
cells infected with BVDV (Nose strain), as described
previously.15,16 We found that all three compounds (2–
4) examined possess anti-BVDV activity without appar-
ent cytotoxicity, though they are not so potent, having
Keywords: Anti-BVDV agents; c-Carboline; Thalidomide.
*
Corresponding authors. E-mail: aoyama@iam.u-tokyo.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.052

K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
3781
Figure 1. Armchair structural development of thalidomide (1) to a 6-5-6 fused heteroaromatic ring system.
pared, basically as described by other researchers. The
synthetic routes to a- (5), c- (7), and d-carboline (8)
are summarized in Scheme 1.17 Briefly, Pd-catalyzed
cross-coupling reaction of iodobenzenes and aminopyri-
dines with Pd2(dba)3 and DPPF, followed by ortho-cou-
pling reaction using Pd(OAc)2 gave the three carboline
isomers, 5, 7, and 8.
The anti-BVDV activity of the prepared carboline iso-
mers is illustrated in Figure 3. As shown in the figure,
the introduction of a nitrogen atom into one of the phe-
nyl rings of carbazole (3) had a dramatic effect. Intro-
duction of a nitrogen atom at the a (a-carboline: 5) or
d position (d-carboline: 8) caused decrease or disappear-
ance of anti-BVDV activity, with no apparent effect on
cytotoxicity. On the other hand, introduction of a nitro-
gen atom at the b (b-carboline: 6) or c position (c-carb-
oline: 7) greatly enhanced the anti-BVDV activity,
affording EC50 values of 8.8 and 2.0 lM, respectively.
Though the cytotoxicity was also enhanced by the deriv-
atization, the CC50 values of b-carboline (6) and c-carb-
oline (7) are 8.8 and 20 times larger than the
corresponding EC50 values, respectively, which seems
permissible for lead compounds that are to be structur-
ally developed. Since c-carboline (7) possessed more po-
tent anti-BVDV activity, it was selected for further
structural development.
Figure 2. Anti-viral activity and cytotoxicity of commercially available
6-5-6 fused heteroaromatic compounds. EC50: 50% effective concen-
tration, based on the reduction of BVDV replication-induced cell
destruction. CC50: 50% cytotoxic concentration, based on the reduc-
tion of viable cell number.
EC50 values of 58–71 lM (Fig. 2). Among these com-
pounds, we focused on carbazole (3) because of its po-
tential for easy derivatization, and we searched for
derivatives with potent anti-BVDV activity. Thalido-
mide (1) itself did not show the activity.
In this article, we describe the identification of the c-
carboline skeleton as a scaffold for potent anti-BVDV
agents, its structure–activity relationship, and the crea-
tion of a potent anti-BVDV agent, SK5M (5-methyl-c-
carboline).
2. Carboline isomers
3. c-Carbolines
Based on our preliminary investigation mentioned
above, we examined the anti-BVDV activity of carboline
isomers (Fig. 3). All of the carboline isomers were pre-
Next, we examined the effect of methylation of c-carbo-
line (7) on its anti-BVDV activity and cytotoxicity. The
methylated analogs were synthesized as shown in
Figure 3. Anti-viral activity and cytotoxicity of carboline isomers (5–8). EC50: 50% effective concentration, based on the reduction of BVDV
replication-induced cell destruction. CC50: 50% cytotoxic concentration, based on the reduction of viable cell number.

3782
K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
Scheme 1. Reagents and conditions: (a) Pd2(dba)3, DPPF, t-BuONa, toluene, 100–115 ꢁC; (b) Pd(OAc)2, Na2CO3, DMF, 165 ꢁC.
Schemes 2 and 3. 1-Methyl-c-carboline (9, SK1M) and
2-methyl-c-carboline (10, SK2M) were synthesized from
Trp-P-2ÆAcOH18 and c-carboline (7), respectively.
Methylated c-carboline analogs 11–17 were synthesized
from the 3-formylindole analogs 29–33 by means of
acid-supported cyclization via the imine derivatives. As
Scheme 2. Reagents and conditions: (a) NaNO2, KBr, aq HBr, 0–80 ꢁC; (b) H2, 10% Pd/C, MeOH, rt; (c) MeI, 2-PrOH, 100 ꢁC; (d) KOH, hot H2O,
rt; (e) NCS, PPh3, DMF, THF, H2O, reflux; (f) R-NH2, benzene, 110 ꢁC; (g) H3PO4, 165 ꢁC; (h) DMC, K2CO3, DMF, 160 ꢁC; (i)
(MeO)2CHCH2NH2, benzene, 110 ꢁC.

K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
3783
Scheme 3. Reagents and conditions: (a) RI, NaH, DMF, THF, rt; (b) ROH, TMAD, P(n-Bu)3, THF, toluene, 50 ꢁC.
shown in Scheme 2, bromination of Trp-P-2ÆAcOH with
NaNO2 and KBr under acidic conditions, followed by
debromination with Pd/C under an H2 atmosphere,
afforded SK1M (9). The methylpyridinium iodide inter-
mediate 28 was prepared from c-carboline and MeI, and
then treatment of 28 with KOH aqueous solution gave
SK2M (10). The formylation of indole and its methyl
analogs with NCS, PPh3, and DMF by Sugimoto’s
method19 gave the 3-formylindole derivatives 29–33, fol-
lowed by condensation with acetalamine and cyclization
with H3PO4 to afford SK3M (11), SK4M (12), SK6M
(14), SK7M (15), SK8M (16), and SK9M (17), respec-
tively. N-Methylation of c-carboline at the 5-position
was carried out using dimethyl carbonate with K2CO3
to afford SK5M (13).
The anti-BVDV activity and cytotoxicity of the pre-
pared methylated c-carboline derivatives (9–17) are
shown in Figure 4. The effect of methylation on the
activity seems to be dependent on the position at which
Figure 4. Anti-viral activity, cytotoxicity, and selectivity index of methylated c-carboline derivatives (9–17). EC50: 50% effective concentration, based
on the reduction of BVDV replication-induced cell destruction. CC50: 50% cytotoxic concentration, based on the reduction of viable cell number. SI:
CC50/EC50
.

3784
K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
the methyl substituent is introduced. Introduction of a
methyl substituent at positions 2, 3, and 8 decreased
the anti-BVDV activity, whereas introduction at other
sites, that is, positions 1, 4, 5, 6, 7, and 9, increased
the activity. Broadly speaking, methylation at sites that
cause the resulting structure to become longer (positions
2, 3, and 8: that is, the b and c positions) seems to de-
crease the activity, with the exception of SK7M (15),
while methylation at sites that cause the resulting struc-
ture to become wider (positions 1, 4, 5, 6, and 9: that is,
the nitrogen atom at position 1, and the a and d posi-
tions) seems to enhance the activity. The 5-methylated
analog, SK5M (13), showed the most potent anti-BVDV
activity among this series of the compounds, with an
EC50 value of 0.26 lM.
methyl substituent effects on anti-BVDV activity and
cytotoxicity showed only a limited correlation, and as
a result, the values of the selectivity index (SI va-
lue = CC50/EC50) varied from 4.0 to 111.5. From the
standpoints of both potent anti-BVDV activity and high
selectivity index, SK5M (13) seems to be the best lead
compound, having EC50 and SI values of 0.26 lM and
111.5, respectively. The dose–response curves of the
anti-BVDV activity and the cytotoxicity elicited by
SK5M (13) are shown in Figure 5.
Because the nitrogen atom at the 5 position appeared to
be the most appropriate position at which to introduce a
substituent (vide supra), other substituents were intro-
duced at this position (compounds 18–24, Fig. 6). Syn-
thesis of these N-alkylated derivatives of c-carboline
18–24 is summarized in Scheme 3. Compounds 18–22
were prepared by general N-alkylation using NaH and
the corresponding alkyl iodide. Compounds 23 and 24
were synthesized by using the corresponding alkyl alco-
hol with TMAD and P(n-Bu)3.
As for cytotoxicity, the introduction of a methyl group
at positions 2 and 3 decreased the cytotoxicity, while
introduction at other positions increased it. Thus, the
As shown in Figure 6, introduction of an ethyl group at
the nitrogen atom at the 5-position (SK5E: 18) did not
affect the anti-BVDV activity of c-carboline (7), but en-
hanced the cytotoxicity. Substitution at the same posi-
tion with a larger group decreased the anti-BVDV
activity compared with that of c-carboline (7). The re-
sult suggests that introduction of a bulky group at the
nitrogen atom at position 5 is unfavorable for the activ-
ity of the derivatives. The substituent effect seems to be
larger for cytotoxicity than for anti-BVDV activity, be-
cause the anti-BVDV activities of compounds 19–24 are
similar, with EC50 values of 8.2–11 lM [except SK5H
(22) whose EC50 value is larger than its CC50 value],
whereas CC50 of these compounds range from 7.4 to
30 lM. In the case of derivatives with a normal alkyl
chain pendant, a clear structure–activity relationship
for cytotoxicity was observed, that is, cytotoxicity in-
creased in the order of: SK5M (methyl: 13) > SK5E
(ethyl: 18) > SK5nP (n-propyl: 19) > SK5Bu (n-butyl:
21) > SK5H (n-hexyl: 22). The potency order of anti-
BVDV activity of these compounds is just the reverse,
Figure 5. Anti-viral activity and cytotoxicity elicited by SK5M (13).
Figure 6. Anti-viral activity and cytotoxicity of N-substituted c-carboline derivatives (18–24). EC50: 50% effective concentration, based on the
reduction of BVDV replication-induced cell destruction. CC50: 50% cytotoxic concentration, based on the reduction of viable cell number.

K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
3785
except for SK5H (22), whose EC50 value could not be
6. Experimental
6.1. General comments
determined (vide supra). Thus, SK5M (13) was consid-
ered to be the best lead compound, having the most po-
tent anti-BVDV activity and the largest SI value among
the compounds prepared. Since SK5M could suppress
viral RNA synthesis at 6 h after virus infection in a
dose-dependent fashion (data not shown), it is assumed
that the compound inhibits an early step of the replica-
tion cycle of BVDV.
Melting points were determined by using a Yanagimoto
hot-stage melting point apparatus and are uncorrected.
1H NMR spectra were recorded on a JEOL JNM-
GX500 (500 MHz) spectrometer. Chemical shifts are ex-
pressed in d (ppm) values with tetramethylsilane (TMS)
as an internal reference. The following abbreviations are
used: s = singlet, d = doublet, t = triplet, m = multiplet,
dd = double doublet, dt = double triplet, br = broad.
Mass spectra (MS) and high-resolution mass spectra
(HRMS) were recorded on a JEOL JMS-DX303
spectrometer.
4. Growth inhibition of HL-60 cells by c-carbolines
To explore the mechanism of cytotoxicity of c-carbo-
lines, we examined the effect of c-carboline (7) (low cyto-
toxicity) and SK1M (9) (high cytotoxicity) on apoptosis
involving the caspase cascade. First, we investigated the
cell growth-inhibitory activity of c-carboline (7) and
SK1M (9) toward HL-60 cells. Both compounds inhib-
ited HL-60 cell growth, with IC50 values of 11.4 lM
for c-carboline (7) and 15.3 lM for SK1M (9). Next,
we examined the effect of Z-VAD fmk, a caspase-family
inhibitor, on the HL-60 cell growth-inhibitory activity
elicited by c-carboline (7) and SK1M (9). As shown in
Figure 7, the cell growth inhibition elicited by c-carbo-
line (7) was completely reversed by Z-VAD fmk (closed
column), although the growth inhibition elicited by
SK1M (9) was only partially reversed by addition of
Z-VAD fmk (closed column). These results suggest that
the cytotoxicity elicited by c-carboline (7) and SK1M (9)
is at least partly due to the induction of apoptosis via the
caspase cascade.
6.2. Materials
Fluorene (2) was purchased from Sigma–Aldrich, Inc.
Carbazole (3) and dibenzofuran (4) were purchased
from Kanto Chemical Co., Inc. b-Carboline (6) was pur-
chased from Tokyo Chemical Industry Co., Ltd. All
other compounds were of the best available commercial
grade.
6.2.1. 2-Anilino-3-bromopyridine (25)17. A mixture of 2-
amino-3-bromopyridine (204 mg, 1.18 mmol), iodoben-
zene (0.160 mL, 1.43 mmol), t-BuONa (162 mg,
1.68 mmol),
tris(dibenzylideneacetone)-dipalladium
(54.7 mg, 0.060 mmol), and 1,10-bis(diphenylphos-
phino)ferrocene (66.7 mg, 0.120 mmol) in dry toluene
(15 mL) was stirred for 14 h at 100 ꢁC. After cooling
to room temperature, the mixture was diluted with
Et2O, filtered through a Celite pad, and washed with
Et2O. The filtrate was evaporated, and the residue was
purified by silica-gel column chromatography using n-
hexane/AcOEt (20:1) as the eluent to give 204 mg
(69.3%) of the title compound as a yellow oil. 1H
NMR (500 MHz, CDCl3) d 8.16 (br, 1H), 7.75 (d,
J = 7.3 Hz, 1H), 7.61 (d, J = 7.9 Hz, 2H), 7.36 (t,
J = 7.9 Hz, 2H), 7.09–7.05 (m, 2H), 6.64 (br, 1H). MS
(FAB, M+) m/z 249.
5. Conclusion
Based on a 6-5-6 fused heteroaromatic system derived
from thalidomide, we developed a potent lead com-
pound for anti-BVDV agents, SK5M (13), which has
an EC50 value of 0.26 lM and an SI value of 111.5. Fur-
ther structural development should yield highly potent
and selective drug candidates.
6.2.2. a-Carboline (5)17. A mixture of 2-anilino-3-bro-
mopyridine (109 mg, 0.437 mmol), Pd(OAc)2 (10.5 mg,
0.047 mmol), and Na2CO3 (67.8 mg, 0.640 mmol) in
dry DMF (5 mL) was refluxed at 165 ꢁC for 67 h. After
cooling to room temperature, the mixture was diluted
with AcOEt and filtered through a Celite pad. The fil-
trate was washed successively with water and brine,
dried over MgSO4, and evaporated. The residue was
purified by silica-gel column chromatography using n-
hexane/AcOEt (10:1) as the eluent to give 13.5 mg
(18.4%) of the title compound as a yellow powder. Mp
210–211 ꢁC. 1H NMR (500 MHz, DMSO-d6) d 11.91
(s, 1H), 8.56 (d, J = 7.7 Hz, 1H), 8.43 (d, J = 4.7 Hz,
1H), 8.18 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H),
7.47 (t, J = 8.1 Hz, 1H), 7.26–7.23 (m, 2H). HRMS
(FAB) calcd for C11H9N2 169.0766; found: 169.0816
(M+H)+.
Figure 7. Growth inhibition of HL-60 cells by 10 lM c-carboline (7)
and SK1M (9) in the presence (filled column) or absence (open
column) of Z-VAD fmk (100 lM).
6.2.3. 4-(20-Bromoanilino)pyridine (26)17. A mixture of 4-
aminopyridine (944 mg, 10.0 mmol), 1-bromo-2-iodo-

3786
K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
benzene (1.35 mL, 10.5 mmol), t-BuONa (1.14 g,
11.9 mmol), tris(dibenzylideneacetone)-dipalladium
trate was washed successively with water and brine,
dried over MgSO4, and evaporated. The residue was
purified by silica-gel column chromatography using n-
hexane/AcOEt (10:1) as the eluent to give 30.2 mg
(41.2%) of the title compound as a yellow powder. Mp
189–190 ꢁC. 1H NMR (500 MHz, DMSO-d6) d 11.42
(s, 1H), 8.44 (d, J = 4.3 Hz, 1H), 8.17 (d, J = 7.7 Hz,
1H), 7.86 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H),
7.49 (t, J = 7.7 Hz, 1H), 7.38 (dd, J = 8.1, 4.3 Hz, 1H),
7.23 (t, J = 7.7 Hz, 1H). HRMS (FAB) calcd for
C11H9N2 169.0766; found: 169.0794 (M+H)+.
(137 mg, 0.150 mmol), and 1,10-bis(diphenylphos-
phino)ferrocene (200 mg, 0.361 mmol) in dry toluene
(30 mL) was stirred for 18 h at 115 ꢁC. After cooling
to room temperature, the mixture was diluted with
Et2O and filtered through a Celite pad. The filtrate
was evaporated, and purified by silica-gel column chro-
matography using CH2Cl2/MeOH (29:1 to 9:1) as the
eluent to give 2.50 g (99.9%) of the title compound as
1
a gray solid. H NMR (500 MHz, CDCl3) d 8.34 (dd,
J = 2.0, 6.5 Hz, 2H), 7.60 (dd, J = 1.5, 8.0 Hz, 1H),
7.44 (dd, J = 1.5, 8.0 Hz, 1H), 7.29 (ddd, J = 1.5, 8.0,
8.0 Hz, 1H), 6.96 (ddd, J = 1.5, 8.0, 8.0 Hz, 1H), 6.87
(dd, J = 2.0, 6.5 Hz, 1H), 6.18 (s, 1H). MS (FAB,
[M+H]+) m/z 249, 251.
6.2.7. 1-Methyl-c-carboline (9, SK1M). A solution of
Trp-P-2ÆAcOH18 (103 mg, 0.400 mmol) in a mixture of
concd HBr aq/H2O = 4:1 (5.0 mL) was added to a solu-
tion of NaNO2 (55.2 mg, 0.800 mmol) in H2O (0.5 mL)
at 0 ꢁC and the mixture was stirred for 5 min at the same
temperature. To this was added a solution of KBr
(191 mg, 1.60 mmol) in H2O (1.0 mL) and the whole
was stirred at 80 ꢁC for 3 h. After cooling to room tem-
perature, the reaction was quenched by adding 10 N
NaOH aq. and saturated aqueous NaHCO3 solution un-
til the solution became basic. The aqueous solution was
then extracted with AcOEt and the organic phase was
washed with water, dried over MgSO4, and evaporated.
The residue was purified by silica-gel column chroma-
tography using n-hexane/AcOEt (6:1) as the eluent to
give 19.1 mg (11.4%) of tribrominated 1-methyl-c-carb-
oline as a pale-yellow powder. This material was used
in the next step without further examination. 1H
NMR (500 MHz, DMSO-d6) d 12.28 (s, 1H), 8.25 (d,
J = 1.5 Hz, 1H), 7.69 (dd, J = 1.5, 9.0 Hz, 1H), 7.59 (d,
J = 9.0 Hz, 1H), 2.88 (s, 3H). MS (FAB, [M+H]+) m/z
419, 422.
6.2.4. c-Carboline (7)17. A mixture of 4-(20-bromoanili-
no) pyridine (2.50 g, 10.0 mmol), Pd(OAc)2 (112 mg,
0.500 mmol), and Na2CO3 (1.48 g, 14.0 mmol) in
DMF (20 mL) was stirred for 20 h at 165 ꢁC. After cool-
ing to room temperature, the mixture was diluted with
AcOEt and washed successively with water. The organic
phase was extracted with 2 N HCl aq. and the aqueous
phase was neutralized with NaOH pellets. The resulting
brown precipitate was removed by filtration and addi-
tional NaOH pellets were added to the pale-yellow fil-
trate until it became basic. The resulting precipitate
was collected in a funnel by suction, washed with water,
and dried in vacuo. The crude product was purified by
recrystallization from toluene to give 1.21 g (72.0%) of
the title compound as a pale-yellow powder. Mp 230–
231 ꢁC. 1H NMR (500 MHz, DMSO-d6) d 11.70 (s,
1H), 9.32 (s, 1H), 8.41 (dd, J = 1.0, 5.5 Hz, 1H), 8.22
(d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.46 (dd,
J = 8.0, 8.0 Hz, 1H), 7.45 (d, J = 5.5 Hz, 1H), 7.25 (dd,
J = 8.0, 8.0 Hz, 1H). HRMS (FAB) calcd for C11H9N2
169.0766; found: 169.0785 (M+H)+.
A solution of the tribrominated 1-methyl-c-carboline
(19.1 mg, 0.046 mmol) in a mixture of MeOH (1.5 mL)
and AcOEt (1.0 mL) was added to Et3N (25.0 lL,
0.179 mmol) and 10% Pd/C (0.8 mg), and the mixture
was stirred at room temperature under an H2 atmo-
sphere. After 18 h, the reaction mixture was filtered
through a Celite pad and the filtrate was evaporated.
To the residue was added saturated aqueous NaHCO3
solution and the aqueous phase was extracted with
AcOEt. The organic phase was dried over MgSO4 and
evaporated. The residue was purified by silica-gel col-
umn chromatography using acetone/MeOH (20:1) as
the eluent to give 7.7 mg (92.6%) of the title compound
6.2.5. 3-Anilino-2-bromopyridine (27)17. A mixture of 3-
amino-2-bromopyridine (205 mg, 1.19 mmol), iodoben-
zene (0.160 mL, 1.43 mmol), t-BuONa (161 mg,
1.67 mmol),
tris(dibenzylideneacetone)-dipalladium
(57.3 mg, 0.0626 mmol), and 1,10-bis(diphenylphos-
phino)ferrocene (68.9 mg, 0.124 mmol) in dry toluene
(15 mL) was stirred at 100 ꢁC for 14 h. After cooling
to room temperature, the mixture was diluted with
Et2O and filtered through a Celite pad. The filtrate
was evaporated, and the residue was purified by silica-
gel column chromatography using n-hexane/AcOEt
(20:1) as the eluent to give 109 mg (36.6%) of the title
compound as a yellow oil. 1H NMR (500 MHz, CDCl3)
d 7.85 (dd, J = 4.7 1.7 Hz, 1H), 7.44 (dd, J = 8.1, 1.7 Hz,
1H), 7.36 (dt, J = 7.3, 0.86 Hz, 2H), 7.16 (dd, J = 7.3,
0.86 Hz, 2H), 7.14–7.08 (m, 2H), 6.16 (s, 1H). MS
(FAB, M+) m/z 249.
as
a
white powder. Mp 229–230 ꢁC. 1H NMR
(500 MHz, DMSO-d6) d 11.72 (s, 1H), 8.27 (d,
J = 5.5 Hz, 1H), 8.13(d, J = 8.5 Hz, 1H), 7.56 (d,
J = 8.5 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.32 (d,
J = 5.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 2.92 (s, 3H).
HRMS (FAB) calcd for C12H11N2 183.0922; found:
183.0953 (M+H)+.
6.2.8. 2-Methyl-c-carbolinium iodide (28)19. A mixture of
c-carboline (7) (173 mg, 1.03 mmol) and iodomethane
(0.100 mL, 1.61 mmol) in 2-propanol (8 mL) was re-
fluxed at 100 ꢁC for 3 h. Then the excess iodomethane
and 2-propanol were removed under reduced pressure,
and the residue was recrystallized from EtOH to give
268 mg (83.7%) of the title compound as a white pow-
6.2.6. d-Carboline (8)17. A mixture of 3-anilino-2-bromo-
pyridine (109 mg, 0.436 mmol), Pd(OAc)2 (12.3 mg,
0.055 mmol), and Na2CO3 (66.5 mg, 0.627 mmol) in
dry DMF (5 mL) was refluxed at 165 ꢁC for 67 h. After
cooling to room temperature, the mixture was diluted
with AcOEt and filtered through a Celite pad. The fil-

K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
3787
der. 1H NMR (500 MHz, DMSO-d6) d 9.61 (s, 1H), 8.55
(d, J = 7.3 Hz, 1H), 8.34 (d, J = 7.9 Hz, 1H), 7.92 (d,
J = 7.3 Hz, 1H), 7.77–7.71 (m, 2H), 7.53 (dd, J = 7.9,
8.1 Hz, 1H), 4.43 (s, 3H). MS (FAB, [M+H]+) m/z 183.
1H), 8.18 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 3.0 Hz, 1H),
7.23 (s, 1H), 7.16 (d, J = 8.3 Hz, 1H), 2.48 (s, 3H). MS
(FAB, [M+H]+) m/z 160.
6.2.14. 3-Formyl-7-methylindole (33). According to the
typical procedure, 3-formyl-7-methylindole (33) was ob-
tained from 7-methyl-1H-indole as a yellow powder,
which was used in the next step without purification.
1H NMR (500 MHz, DMSO-d6) d 10.1 (s, 1H), 9.25
(br, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 3.1 Hz,
1H), 7.23 (dd, J = 7.9, 7.3 Hz, 1H), 7.12 (d, J = 7.3 Hz,
1H), 2.53 (s, 3H). MS (FAB, [M+H]+) m/z 160.
6.2.9. 2-Methyl-c-carboline (10, SK2M)19. A solution of
2-methyl-c-carbolinium iodide (163 mg, 0.526 mmol) in
hot water (5 mL) was added to 50% aqueous KOH solu-
tion (2.6 mL). The resulting light yellow precipitate was
separated by filtration, washed with cold water, and
dried in vacuo to give 39.0 mg (52.6%) of the title com-
1
pound as a light yellow powder. Mp 172–173 ꢁC. H
NMR (500 MHz, DMSO-d6) d 8.67 (s, 1H), 8.02 (d,
J = 7.7 Hz, 1H), 7.87(d, J = 7.9 Hz, 1H), 7.66–7.62 (m,
2H), 7.57 (dd, J = 7.3, 8.1 Hz, 1H), 7.29 (dd, J = 7.3,
7.9 Hz, 1H), 4.24 (s, 3H). HRMS (FAB) calcd for
C12H11N2 183.0922; found: 183.0967 (M+H)+.
6.2.15. Benzyl 1-(N-methoxy-N-methylcarbamoyl)ethyl-
carbamate (34). A mixture of N-CBZ-L-alanine (230 mg,
1.03 mmol), N,O-dimethylhydroxylamine hydrochloride
(204 mg, 2.09 mmol), 1-hydroxybenzotriazole monohy-
drate (169 mg, 1.25 mmol), 1-ethyl-3-(3-dimethylamino-
6.2.10. 3-Formylindole (29): typical procedure for the
formylation of indoles. N-Chlorosuccinimide (1.07 g,
8.01 mmol) was added little by little to a solution of
PPh3 (2.09 g, 7.98 mmol) in dry THF (80 mL) with stir-
ring for 30 min at room temperature. N,N-Dimethylform-
amide (1.24 mL, 16.0 mmol) was added to the suspension,
and the mixture was refluxed for 1 h. Then to this was
added 1H-indole (313 mg, 2.67 mmol), and the mixture
was refluxed for 1 h. The volatile solvent was removed,
and to the remaining solution was added H2O (30 mL).
The mixture was refluxed for an additional 1 h. The reac-
tion was quenched by adding NaOH aqueous solution
and the mixture was extracted with AcOEt. The organic
phase was washed with brine, dried over MgSO4 and
evaporated. The residue was purified by silica-gel column
chromatography using n-hexane/AcOEt (1:1) as the elu-
ent to give 3-formylindole (292 mg, 75.3%) as a yellow
propyl)
carbodiimide
hydrochloride
(246 mg,
1.59 mmol), and diisopropylethylamine (0.220 mL,
1.26 mmol) in dry CH2Cl2 (10 mL) was stirred for
30 min at room temperature. The reaction was quenched
by adding water and the organic phase was washed with
water, dried over MgSO4, and evaporated. The residue
was purified by silica-gel column chromatography using
n-hexane/AcOEt (1:1) as the eluent to give 239 mg
1
(87.0%) of the title compound as a white powder. H
NMR (500 MHz, CDCl3) d 7.37–7.28 (m, 5H), 5.53
(br s, 1H), 5.10 (q, J = 12.4 Hz, 2H), 4.76–4.73 (m,
1H), 3.77 (s, 3H), 3.21 (s, 3H), 1.35 (d, J = 6.8 Hz,
3H). MS (FAB, [M+H]+) m/z 267.
6.2.16. Benzyl 1-formylethylcarbamate (35). A mixture of
34 (239 mg, 0.896 mmol) and lithium aluminum hydride
(38.7 mg, 1.02 mmol) in dry THF (5 mL) was stirred for
15 min at 0 ꢁC. The reaction was quenched by adding
Na2SO4Æ10H2O and MgSO4. The resulting precipitate
was removed by filtration. The filtrate was evaporated
and the residue was purified by silica-gel column chro-
matography using n-hexane/AcOEt (3:1) as the eluent
to give 77.3 mg (41.6%) of the title compound as a col-
1
powder. H NMR (500 MHz, CDCl3) d 11.1 (s, 1H),
8.66 (br, 1H), 8.33 (dd, J = 3.0, 6.0 Hz, 1H), 7.85 (d,
J = 3.0 Hz, 1H), 7.47–7.43 (m, 1H), 7.36–7.31 (m, 2H).
MS (FAB, [M+H]+) m/z 146.
6.2.11. 3-Formyl-4-methylindole (30). According to the
typical procedure, 3-formyl-4-methylindole (30) was ob-
tained from 4-methyl-1H-indole as a yellow powder,
which was used in the next step without purification.
1H NMR (500 MHz, CDCl3) d 10.2 (s, 1H), 8.73 (br,
1H), 7.95 (d, J = 3.4 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H),
7.21 (dd, J = 7.1, 8.1 Hz, 1H), 7.09 (d, J = 7.1 Hz, 1H),
2.86 (s, 3H). MS (FAB, [M+H]+) m/z 160.
1
orless oil. H NMR (500 MHz, CDCl3) d 9.64 (s, 1H),
7.27–7.22 (m, 5H), 5.35 (br s, 1H), 5.02 (s, 2H), 4.24–
4.16 (m, 1H), 1.27 (d, J = 7.7 Hz, 3H). MS (FAB,
[M+H]+) m/z 208.
6.2.17. Benzyl 1-(1,3-dioxolan-2-yl)ethylcarbamate (36).
A mixture of 35 (73.7 mg, 0.356 mmol), pyridinium p-
toluenesulfonate (4.3 mg, 17.1 lmol), and ethylene gly-
col (64 lL, 1.15 mmol) in dry benzene (6 mL) was re-
fluxed for 4 h at 110 ꢁC. The reaction was quenched
by adding water, and the organic phase was washed with
water, saturated aqueous NaHCO3 solution, and brine,
dried over MgSO4, and evaporated. The residue was
purified by Chromatorexꢂ column chromatography
usingn-hexane/AcOEt (4:1) as the eluent to give
56.9 mg (63.6%) of the title compound as a colorless
6.2.12. 3-Formyl-5-methylindole (31). According to the
typical procedure, 3-formyl-5-methylindole (31) was ob-
tained from 5-methyl-1H-indole as a yellow powder,
which was used in the next step without purification.
1H NMR (500 MHz, CDCl3) d 10.0 (s, 1H), 8.73 (br,
1H), 8.13 (s, 1H), 7.81 (d, J = 3.0 Hz, 1H), 7.33 (d,
J = 8.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 2.49 (s, 3H).
MS (FAB, [M+H]+) m/z 160.
1
oil. H NMR (500 MHz, CDCl3) d 7.36–7.30 (m, 5H),
5.11 (s, 2H), 4.85 (s, 1H), 4.02–3.86 (m, 4H), 1.16 (d,
6.2.13. 3-Formyl-6-methylindole (32). According to the
typical procedure, 3-formyl-6-methylindole (32) was ob-
tained from 6-methyl-1H-indole as a pale red powder,
which was used in the next step without purification.
1H NMR (500 MHz, CDCl3) d 10.0 (s, 1H), 8.59 (br,
J = 7.3 Hz, 3H). MS (FAB, [M+H]+) m/z 252.
6.2.18. 3-Methyl-c-carboline (11, SK3M). A mixture of
36 (397 mg, 1.58 mmol) and 20% Pd(OH)2/C (404 mg)

3788
K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
in AcOEt (10 mL) was stirred under an H2 atmosphere
for 15 h at room temperature. The reaction mixture
was filtered through a Celite pad and the filtrate was
evaporated. To this was added a solution of 29
(188 mg, 1.30 mmol) in dry benzene (10 mL) and the
mixture was refluxed for 5 h at 110 ꢁC. After the mixture
had cooled to room temperature, the benzene was re-
moved under reduced pressure. Then 90% phosphoric
acid, which had been stirred for 2 h at 165 ꢁC, was
added to the residue and stirring was continued for an-
other 30 min at 165 ꢁC. To the reaction mixture was
added ice and the resulting precipitate was filtered
through a Celite pad. The filtrate was basified with dil
NH3 and extracted with CH2Cl2. The organic phase
was extracted with dil. HCl, and the HCl layer was basi-
fied with dil. NaOH aqueous solution. The resulting pre-
cipitate was collected in a funnel and purified by PLC
using chloroform/EtOH (10:1) as the eluent to give
4.3 mg (3.1%, three steps) of the title compound as a
pale brown powder. Mp 225–226 ꢁC. 1H NMR
(500 MHz, DMSO-d6) d 11.4 (s, 1H), 8.75 (s, 1H), 8.17
(d, J = 7.7 Hz, 1H), 7.93 (s, 1H), 7.54 (d, J = 8.1 Hz,
1H), 7.50 (dd, J = 6.8, 8.1 Hz, 1H), 7.19 (dd, J = 6.8,
7.7 Hz, 1H), 2.60 (s, 3H). HRMS (FAB) calcd for
C12H11N2 183.0922; found: 183.0952 (M+H)+.
ethylene glycol (0.660 mL, 11.8 mmol) in dry benzene
(10 mL) was refluxed for 4 h at 110 ꢁC. The reaction
was quenched by adding water, and the organic phase
was washed with water, saturated aqueous NaHCO3
solution and brine, dried over MgSO4, and evaporated.
The residue was purified by Chromatorexꢂ column
chromatography using n-hexane/AcOEt (3:1) as the elu-
ent to give 791 mg (85.8%) of the title compound as a
1
colorless oil. H NMR (500 MHz, CDCl3) d 7.27–7.20
(m, 5H), 5.02 (s, 1H), 4.89 (br, 1H), 3.89–3.82 (m,
4H), 3.26 (d, J = 6.0 Hz, 1H), 1.24 (s, 3H). MS (FAB,
[M+H]+) m/z 252.
6.2.22. 4-Methyl-c-carboline (12, SK4M). A mixture of
39 (791 mg, 3.15 mmol) and 20% Pd(OH)2/C (811 mg)
in AcOEt (20 mL) was stirred under an H2 atmosphere
for 5 h at room temperature. The reaction mixture was
filtered through a Celite pad and the filtrate was evapo-
rated. To this was added a solution of 29 (458 mg,
3.91 mmol) in dry benzene (10 mL) and the mixture
was refluxed for 5 h at 110 ꢁC. After the mixture had
cooled to room temperature, the benzene was removed
under reduced pressure. Then 90% phosphoric acid,
which had been treated for 2 h at 165 ꢁC, was added
to the residue and stirring was continued for another
30 min at 165 ꢁC. To the reaction mixture was added
ice, and the resulting precipitate was removed by filtra-
tion through a Celite pad. The filtrate was basified with
dil. NH3 and extracted with CH2Cl2. The organic phase
was extracted with dil. HCl and the HCl layer was basi-
fied with dil NaOH aqueous solution. The resulting pre-
cipitate was collected in a funnel and purified by silica-
gel column chromatography using chloroform/EtOH
(10:1) as the eluent to give 32.6 mg (5.7%, three steps)
of the title compound as a pale brown powder. Mp
6.2.19. Benzyl 2-hydroxypropylcarbamate (37). To a mix-
ture of 1-amino-2-propanol (0.780 mL, 9.97 mmol), 4-
(dimethylamino)pyridine (250 mg, 2.05 mmol), and tri-
ethylamine (6.0 mL, 42.9 mmol) in dry DMSO (10 mL)
was added benzyl chloroformate (6.42 mL, 45.0 mmol)
little by little, and the mixture was stirred for 5 h at
50 ꢁC. The reaction was quenched by adding saturated
aqueous NaHCO3 solution, and the whole was extracted
with AcOEt. The organic phase was washed with dil.
HCl and brine, dried over MgSO4, and evaporated.
The residue was purified by silica-gel column chroma-
tography using n-hexane/AcOEt (5:1) as the eluent to
give 1.70 g (81.5%) of the title compound as a pale-yel-
1
197–198 ꢁC. H NMR (500 MHz, DMSO-d6) d 11.7 (s,
1H), 9.17 (s, 1H), 8.24 (s, 1H), 8.19 (d, J = 7.5 Hz,
1H), 7.56 (d, J = 8.1 Hz, 1H), 7.46 (dd, J = 7.1, 8.1 Hz,
1H), 7.25 (dd, J = 7.1, 7.5 Hz, 1H), 2.51 (s, 3H). HRMS
(FAB) calcd for C12H11N2 183.0922; found: 183.0875
(M+H)+.
1
low oil. H NMR (500 MHz, CDCl3) d 7.28–7.19 (m,
5H), 5.06 (br, 1H), 5.02 (s, 2H), 3.86–3.81 (m, 1H),
3.28–3.23 (m, 1H), 3.00–2.94 (m, 1H), 1.09 (d,
J = 6.4 Hz, 3H). MS (FAB, [M+H]+) m/z 210.
6.2.23. 5-Methyl-c-carboline (13, SK5M). A mixture of
c-carboline (7) (83.0 mg, 0.493 mmol), K2CO3
(32.3 mg, 0.234 mmol) and dimethyl carbonate
(0.850 mL, 1.01 mmol) in dry DMF (5 mL) was refluxed
for 7 h at 160 ꢁC. After cooling to room temperature,
the reaction mixture was poured into water and ex-
tracted with AcOEt. The organic layer was washed with
brine, dried over MgSO4, and evaporated. The residue
was purified by silica-gel column chromatography using
chloroform/EtOH (15:1) as the eluent to give 54.3 mg
(60.4%) of the title compound as a pale-yellow powder.
Mp 84–85 ꢁC. 1H NMR (500 MHz, DMSO-d6) d 9.34 (s,
1H), 8.48 (d, J = 5.6 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H),
7.67 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 5.6 Hz, 1H), 7.55
(dd, J = 7.9, 8.1 Hz, 1H), 7.31 (dd, J = 7.9, 8.1 Hz,
1H), 3.89 (s, 3H). HRMS (FAB) calcd for C12H11N2
183.0922; found: 183.0898 (M+H)+.
6.2.20. Benzyl 2-oxopropylcarbamate (38). A mixture of
37 (1.70 g, 8.15 mmol), K2CO3 (11.4 g, 8.21 mmol), N-
chlorosuccinimide (1.21 g, 9.06 mmol), N-tert-butylben-
zenesulfenamide (74.0 lL, 0.470 mmol), and molecular
˚
sieves 4 A (8.15 g) in dry CH2Cl2 was stirred for 2 h at
0 ꢁC. The resulting precipitate was removed by filtration
through a Celite pad, and the filtrate was washed with
saturated aqueous NaHCO3 solution and brine, dried
over MgSO4, and evaporated. The residue was purified
by silica-gel column chromatography using n-hexane/
AcOEt (3:1) as the eluent to give 761 mg (45.0%) of
the title compound as a white powder. 1H NMR
(500 MHz, CDCl3) d 7.28–7.19 (m, 5H), 5.37 (br, 1H),
5.02 (s, 2H), 4.01 (d, J = 5.1 Hz, 2H), 2.09 (s, 3H). MS
(FAB, [M+H]+) m/z 208.
6.2.21. Benzyl (2-methyl-1,3-dioxolan-2-yl)methylcarba-
mate (39). A mixture of 38 (761 mg, 3.67 mmol), pyrid-
inium p-toluenesulfonate (38.5 mg, 0.153 mmol), and
6.2.24. 6-Methyl-c-carboline (14, SK6M): typical proce-
dure for the synthesis of methyl analogs of c-carboline at
the 6–9 positions. A mixture of 33 and aminoacetalde-

K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
3789
hyde dimethylacetal (0.550 mL, 4.84 mmol) in dry ben-
zene (10 mL) was refluxed for 5 h at 110 ꢁC. After the
solution had cooled to room temperature, the benzene
was evaporated. Then 90% phosphoric acid (10 mL),
which had been treated for 2 h at 165 ꢁC, was added
to the residue and stirring was continued for another
30 min at 165 ꢁC. To the reaction mixture was added
ice, and the resulting precipitate was removed by filtra-
tion through a Celite pad. The filtrate was basified with
dil. NH3 and extracted with CH2Cl2. The organic phase
was extracted with dil. HCl and the HCl layer was basi-
fied with dil NaOH aqueous solution. The resulting pre-
cipitate was collected and purified by silica-gel column
chromatography using chloroform/EtOH (5:1) as the
eluent to give 5.7 mg (0.8%, three steps) of SK6M as a
low powder. Mp 64–65 ꢁC. 1H NMR (500 MHz,
DMSO-d6) d 9.34 (s, 1H), 8.47 (d, J = 5.8 Hz, 1H),
8.25 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.62
(d, J = 5.8 Hz, 1H), 7.53 (dd, J = 7.3, 8.1 Hz, 1H), 7.30
(dd, J = 7.3, 7.5 Hz, 1H), 4.46 (q, J = 7.3 Hz, 2H), 1.32
(t, J = 7.3 Hz, 3H). HRMS (FAB) calcd for C13H13N2
197.1079; found: 197.1116 (M+H)+.
6.2.29. 5-n-Propyl-c-carboline (19, SK5nP). According
to the typical procedure using 1-iodopropane
(0.16 mL, 1.60 mmol), 98.8 mg (86.7%) of 5-n-propyl-
c-carboline (19) was obtained as a pale brown powder.
1
Mp 65–66 ꢁC. H NMR (500 MHz, DMSO-d6) d 9.34
(s, 1H), 8.46 (d, J = 5.6 Hz, 1H), 8.25 (d, J = 7.7 Hz,
1H), 7.70 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 5.6 Hz, 1H),
7.52 (dd, J = 7.3, 8.3 Hz, 1H), 7.30 (dd, J = 7.3,
7.7 Hz, 1H), 4.38 (t, J = 7.3 Hz, 2H), 1.80 (dt, J = 7.3,
7.3 Hz, 2H), 0.85 (t, J = 7.3 Hz, 3H). HRMS (FAB)
calcd for C14H15N2 211.1235; found: 211.1203 (M+H)+.
1
brown powder. Mp 219–220 ꢁC. H NMR (500 MHz,
DMSO-d6) d 9.32 (s, 1H), 8.52 (d, J = 5.6 Hz, 1H),
8.46 (br, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.40 (d,
J = 5.6 Hz, 1H), 7.32–7.23 (m, 2H), 2.60 (s, 3H). HRMS
(FAB) calcd for C12H11N2 183.0922; found: 183.0909
(M+H)+.
6.2.30. 5-i-Propyl-c-carboline (20, SK5iP). According to
the typical procedure using 2-iodopropane (0.16 mL,
1.60 mmol), 64.9 mg (55.7%) of 5-i-propyl-c-carboline
(20) was obtained as a white powder. Mp 100–101 ꢁC.
1H NMR (500 MHz, DMSO-d6) d 9.34 (s, 1H), 8.43
(d, J = 5.8 Hz, 1H), 8.26 (d, J = 7.7 Hz, 1H), 7.78 (d,
J = 8.1 Hz, 1H), 7.69 (d, J = 5.8 Hz, 1H), 7.50 (dd,
J = 8.1, 8.2 Hz, 1H), 7.28 (dd, J = 7.7, 8.2 Hz, 1H),
5.16–5.10 (m, 1H), 1.62 (d, J = 6.8 Hz, 6H). HRMS
(FAB) calcd for C14H15N2 211.1235; found: 211.1217
(M+H)+.
6.2.25. 7-Methyl-c-carboline (15, SK7M). According to
the typical procedure, 106 mg (30.1%, two steps) of 7-
methyl-c-carboline (15) was obtained from 32 (307 mg,
1.93 mmol) as a pale-yellow powder. Mp 200–201 ꢁC.
1H NMR (500 MHz, DMSO-d6) d 11.6 (s, 1H), 9.24
(s, 1H), 8.36 (d, J = 5.8 Hz, 1H), 8.07 (d, J = 7.9 Hz,
1H), 7.40 (d, J = 5.8 Hz, 1H), 7.34 (s, 1H), 7.07 (d,
J = 7.9 Hz, 1H), 2.48 (s, 3H). HRMS (FAB) calcd for
C12H11N2 183.0922; found: 183.0925 (M+H)+.
6.2.26. 8-Methyl-c-carboline (16, SK8M). According to
the typical procedure, 15.6 mg (2.1%, three steps) of 8-
methyl-c-carboline (16) was obtained from 31 as a
6.2.31. 5-n-Butyl-c-carboline (21, SK5Bu). According to
the typical procedure using 1-iodobutane (0.16 mL,
1.60 mmol), 80.8 mg (65.6%) of 5-n-butyl-c-carboline
(21) was obtained as a pale brown powder. Mp 58–
1
brown powder. Mp 207–208 ꢁC. H NMR (500 MHz,
1
DMSO-d6) d 11.6 (s, 1H), 9.27 (br, 1H), 8.37 (br, 1H),
8.00 (s, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.41 (br, 1H),
7.28 (d, J = 8.1 Hz, 1H), 2.47 (s, 3H). HRMS (FAB)
calcd for C12H11N2 183.0922; found: 183.0935 (M+H)+.
59 ꢁC. H NMR (500 MHz, DMSO-d6) d 9.34 (s, 1H),
8.46 (d, J = 5.6 Hz, 1H), 8.25 (d, J = 7.9 Hz, 1H), 7.68
(d, J = 8.1 Hz, 1H), 7.61 (d, J = 5.6 Hz, 1H), 7.52 (dd,
J = 7.1, 8.1 Hz, 1H), 7.29 (dd, J = 7.1, 7.9 Hz, 1H),
4.40 (t, J = 7.3 Hz, 2H), 1.77–1.71 (m, 2H), 1.30–1.23
(m, 2H), 0.85 (t, J = 7.3 Hz, 3H). HRMS (FAB) calcd
for C15H17N2 225.1392; found: 225.1366 (M+H)+.
6.2.27. 9-Methyl-c-carboline (17, SK9M). According to
the typical procedure, 83.1 mg (53.6%, two steps) of 9-
methyl-c-carboline (17) was obtained from 30 (135 mg,
0.851 mmol) as a white powder. Mp 180–181 ꢁC. 1H
NMR (500 MHz, DMSO-d6) d 11.7 (s, 1H), 9.28 (s,
1H), 8.41 (d, J = 5.6 Hz, 1H), 7.46 (d, J = 5.6 Hz, 1H),
7.40–7.34 (m, 2H), 7.06 (d, J = 6.8 Hz, 1H), 2.81 (s,
3H). HRMS (FAB) calcd for C12H11N2 183.0922;
found: 183.0919 (M+H)+.
6.2.32. 5-n-Hexyl-c-carboline (22, SK5H). According to
the typical procedure using 1-iodohexane (0.23 mL,
1.56 mmol), 22.4 mg (16.9%) of 5-n-hexyl-c-carboline
1
(22) was obtained as a brown oil. H NMR (500 MHz,
DMSO-d6) d 9.33 (s, 1H), 8.46 (d, J = 5.6 Hz, 1H),
8.25 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.61
(d, J = 5.6 Hz, 1H), 7.53 (d, J = 7.3, 8.3 Hz, 1H), 7.30
(d, J = 7.3, 7.7 Hz, 1H), 4.40 (t, J = 7.3 Hz, 2H), 1.26–
1.18 (m, 8H), 0.79 (t, J = 7.3 Hz, 3H). HRMS (FAB)
calcd for C17H21N2 253.1705; found: 253.1735 (M+H)+.
6.2.28. 5-Ethyl-c-carboline (18, SK5E): typical procedure
for N-alkylation of c-carboline. A mixture of c-carboline
(7) (101 mg, 0.603 mmol), NaH (42.2 mg, 0.967 mmol),
and iodoethane (0.14 mL, 1.75 mmol) in dry THF
(5 mL) and dry DMF (5 mL) was stirred for 30 min at
room temperature. The volatile materials were removed
under reduced pressure and the residue was poured into
water. The mixture was extracted with AcOEt. The or-
ganic phase was washed with brine, dried over MgSO4,
and evaporated. The residue was purified by silica-gel
column chromatography using chloroform as the eluent
to give 79.6 mg (67.3%) of the title compound as a yel-
6.2.33. 5-Benzyl-c-carboline (23, SK5Bn). A mixture of
c-carboline (7) (86.0 mg, 0.511 mmol), P(n-Bu)3
(0.250 mL, 1.03 mmol), and benzyl alcohol (0.105 mL,
1.01 mmol) in dry THF (3 mL) and dry toluene (3 mL)
was added to N,N,N0,N0-tetramethylazodicarboxamide
(177 mg, 1.03 mmol) at 0 ꢁC. The mixture was stirred
for 15 h at 50 ꢁC, then poured into water, and the whole
was extracted with AcOEt. The organic phase was

3790
K. Sako et al. / Bioorg. Med. Chem. 16 (2008) 3780–3790
washed with water and brine, dried over MgSO4, and
evaporated. The residue was purified by silica-gel col-
umn chromatography using chloroform/EtOH (50:1)
as the eluent to give 127.5 mg (96.6%) of the title com-
bated with c-carboline (7) (10 lM final concentration,
DMSO final concentration 0.5%, v/v) or SK1M (9)
(10 lM final concentration, DMSO final concentration
0.5%, v/v) in RPMI-1640 containing 10% FBS with or
without Z-VAD fmk (100 lM final concentration,
DMSO final concentration 0.1%, v/v) at 37 ꢁC under a
5% CO2 atmosphere. After 62 h in culture, the viability
of the cells was evaluated by cell counting under a
microscope. The viability was estimated by comparison
with that of DMSO (final concentration 0.6%, v/v)-trea-
ted cells.
1
pound as a yellow powder. Mp 109–110 ꢁC. H NMR
(500 MHz, DMSO-d6)
d 9.37 (s, 1H), 8.47 (d,
J = 5.8 Hz, 1H), 8.28 (d, J = 7.7 Hz, 1H), 7.69–7.67
(m, 2H), 7.50 (dd, J = 6.8, 8.1 Hz, 1H), 7.31 (dd,
J = 7.7, 8.1 Hz, 1H), 7.29–7.17 (m, 5H), 5.69 (s, 2H).
HRMS (FAB) calcd for C18H15N2 259.1235; found:
259.1270 (M+H)+.
6.2.34.
5-(N,N-Diethylaminoethyl)-c-carboline
(24,
SK5DAE). A mixture of c-carboline (7) (104 mg,
0.615 mmol), P(n-Bu)3 (0.300 mL, 1.24 mmol), and
N,N-diethylethanolamine (0.16 mL, 1.20 mmol) in dry
THF (5 mL) and dry toluene (5 mL) was added to
References and notes
1. Tan, S. L.; Pause, A.; Shi, Y.; Sonenberg, N. Nat. Rev.
Drug. Disc. 2002, 1, 867.
2. Liang, T. J.; Rehermann, B.; Seeff, L. B.; Hoofnagle, J. H.
Ann. Intern. Med. 2000, 132, 296.
N,N,N0,N0-tetramethylazodicarboxamide
(214 mg,
1.24 mmol) at 0 ꢁC. The mixture was stirred for 15 h
at 50 ꢁC, then poured into water, and the whole was ex-
tracted with AcOEt. The organic phase was washed with
water and brine, dried over MgSO4, and evaporated.
The residue was purified by silica-gel column chroma-
tography using chloroform/EtOH (30:1) as the eluent
to give 46.8 mg (28.5%) of the title compound as a yel-
low oil. 1H NMR (500 MHz, DMSO-d6) d 9.32 (s,
1H), 8.45 (d, J = 5.8 Hz, 1H), 8.24 (d, J = 7.5 Hz, 1H),
7.66 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 5.8 Hz, 1H), 7.52
(dd, J = 7.3, 7.7 Hz, 1H), 7.28 (dd, J = 7.3, 7.5 Hz,
1H), 4.43 (t, J = 6.4 Hz, 2H), 2.73 (t, J = 6.4 Hz, 2H),
2.44 (q, J = 7.3 Hz, 4H), 0.74 (t, J = 7.3 Hz, 6H). HRMS
(FAB) calcd for C17H22N3 268.1814; found: 268.1783
(M+H)+.
3. Hayashi, P. H.; Di Bisceglie, A. M. Med. Clin. North Am.
2005, 89, 371.
4. Memon, M. I.; Memon, M. A. J. Viral Hepat. 2002, 9, 84.
5. Echevarria-Mayo, J. M. Enferm. Infecc. Microbiol. Clin.
2006, 24, 45.
6. Bosch, F. X.; Ribes, J.; Cleries, R.; Diaz, M. Clin. Liver
Dis. 2005, 9, 191.
7. McHutchison, J. G.; Gordon, S. C.; Schiff, E. R.;
Shiffman, M. L.; Lee, W. M.; Rustgi, V. K.; Goodman,
Z. D.; Ling, M. H.; Cort, S.; Albrecht, J. K. N. Eng. J.
Med. 1998, 339, 1485.
8. Hashimoto, Y. Curr. Med. Chem. 1998, 5, 163.
9. Hashimoto, Y. Bioorg. Med. Chem. 2002, 10, 461.
10. Hashimoto, Y. Mini-Rev. Med. Chem. 2002, 2, 543.
11. Hashimoto, Y.; Tanatani, A.; Nagasawa, K.; Miyachi, H.
Drugs Future 2004, 29, 383.
12. Bartlett, J. B.; Dredge, K.; Dalglish, A. G. Nat. Rev.
Cancer 2004, 4, 314.
6.3. Bioassays15,16
13. Buckwold, V. E.; Beer, B. E.; Donis, R. O. Antiviral Res.
2003, 60, 1.
14. Buckwold, V. E.; Wei, J.; Wenzel-Mathers, M.; Russell, J.
Antimicrob. Agents Chemother. 2003, 47, 2293.
15. Yanagida, K.; Baba, C.; Baba, M. Antiviral Res. 2004, 64,
195.
16. Baba, C.; Yanagida, K.; Kanzaki, T.; Baba, M. Antiviral.
Chem. Chemother. 2005, 16, 33.
17. Iwai, T.; Yasuhara, A.; Sakamoto, T. J. Chem. Soc.,
Perkin Trans. 1 1999, 1505.
MDBK cells (1 · 104 cells/well) were infected with
BVDV (Nose strain) at a multiplicity of infection of
0.01 and incubated at 37 ꢁC in the presence of various
concentrations of test compounds. After 3 days, cell via-
bility of virus-infected and mock-infected cells were
determined by the MTT and LDH methods,
respectively.
HL-60 cells were cultured in RPMI-1640 containing
10% heat-inactivated fetal bovine serum (FBS; Gibco
BRL) at 37 ꢁC under a 5% CO2 atmosphere. HL-60 cells
(1 · 105/mL) were seeded on a 96-well plate and incu-
18. Takeda, K.; Ohta, T.; Shudo, K.; Okamoto, T.; Tsuji, K.;
Kosuge, T. Chem. Pharm. Bull. 1977, 25, 2145.
19. Sugimoto, O.; Mori, M.; Moriya, K.; Tanji, K. Helv.
Chim. Acta 2001, 84, 1112.