Polymethylated γ-carbolines with potent anti-bovine viral diarrhea virus (BVDV) activity

Article abstract of DOI:10.3987/COM-08-S(F)68

Several anti-BVDV agents with a polymethylated γ-carboline skeleton were synthesized, and their anti-BVDV activity was evaluated. The most potent antiviral agent, SK3M4M5M (20), was synthesized by Pd-catalyzed Buchwald-Hartwig amination reaction followed

Full text of DOI:10.3987/COM-08-S(F)68

HETEROCYCLES, Vol. 77, No. 2, 2009
779
HETEROCYCLES, Vol. 77, No. 2, 2009, pp. 779 - 785. © The Japan Institute of Heterocyclic Chemistry
Received, 28th July, 2008, Accepted, 9th September, 2008, Published online, 11th September, 2008
DOI: 10.3987/COM-08-S(F)68
POLYMETHYLATED γ-CARBOLINES WITH POTENT ANTI-BOVINE
VIRAL DIARRHEA VIRUS (BVDV) ACTIVITY
Hiroshi Aoyama,a,*,¶ Kumiko Sako,a, ¶ Shinichi Sato,a Masahiko Nakamura,a
Hiroyuki Miyachi,a Yukinori Goto,b Mika Okamoto,b Masanori Baba,b,* and
Yuichi Hashimotoa
aInstitute of Molecular and Cellular Biosciences, University of Tokyo, 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. Sakuragaoka, Kagoshima 890-8544, Japan, E-mail:
aoyama@iam.u-tokyo.ac.jp
Abstract – Several anti-BVDV agents with a polymethylated γ-carboline skeleton
were synthesized, and their anti-BVDV activity was evaluated. The most potent
antiviral agent, SK3M4M5M (20), was synthesized by Pd-catalyzed
Buchwald-Hartwig amination reaction followed by annulation reaction as key
steps. The structure-activity relationship was analyzed.
INTRODUCTION
Human hepatitis C virus (HCV: a member of the Flaviviridae family) infection is thought to be a major
cause of human hepatitis globally.1,2 The WHO estimates that approximately 170 million people are
infected by this virus and the infection persists in more then 80% of the infected population.3 Moreover,
about 60% of cases progress to chronic liver disease, which in turn, can lead to development of cirrhosis,
hepatocellular carcinoma, and liver failure.4,5 Currently, the most effective treatment of chronic hepatitis
C is the use of pegylated interferon-α in combination with the nucleoside analogue ribavirin. However,
the virus cannot be eliminated from approximately 50-60% of the infected patients treated with these
agents.6 In addition, this therapy is associated with serious side-effects.7,8 Therefore, alternative agents
for the treatment and prevention of HCV infection are urgently needed.
Bovine viral diarrhea virus (BVDV) is a member of Pestivirus, which also belongs to the Flaviviridae
family.9 Since the anti-HCV agent rivabirin showed activity against BVDV replication in vitro, BVDV is
This paper is dedicated to Professor Emeritus Keiichiro Fukumoto on the occasion of his 75th birthday.

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thought to be a good model for human HCV (HCV does not replicate efficiently in cell cultures or
animals).10-12 Recently, we found that 6-5-6 fused heteroaromatic compounds such as γ-carboline (1) and
its mono-methylated analogs, including SK5M (2), exhibit rather potent anti-BVDV activity in antiviral
screening using Madin–Darby bovine kidney (MDBK) cells infected with BVDV (Figure 1).13
Preliminary structure-activity relationship studies indicated that the introduced methyl group is quite
critical for the activity, depending on its position.13
N
N
N
N
H
γ-carboline (1; SK00)
EC50: 2.0 μM, CC50: 44 μM
SI = 22
SK5M (2)
EC50: 0.26 μM, CC50: 29 μM
SI = 111
Figure 1. Structures of γ-carboline (1; SK00) and SK5M (2). EC50: anti-BVDV activity, CC50:
cytotoxicity toward the host cells, SI: the selectivity index (SI = CC50/EC50).
In this paper, we disclose syntheses of di- and tri-methylated γ-carboline analogs and the results of
evaluation of their anti-viral activity against BVDV.
RESULTS AND DISCUSSION
Our previous report indicates that the introduction of a methyl group at the 1, 4, or 5 position of
γ-carboline (1) efficiently enhanced the anti-BVDV activity of γ-carboline (1).13 Among the
mono-methylated γ-carboline analogs, 5-methyl-γ-carboline (2, SK5M) exhibits the most potent
anti-BVDV activity.13 On the basis of this result, we expected that additional methylation of SK5M (2) at
various positions might lead to the development of more potent antiviral agents.
9
6
1
8
7
N
N
R'
R
3
N
N
4
SK5M (2)
R = Me, R' = H or R = H, R' = Me
Figure 2. Structural development of dimethylated γ-carbolines based on SK5M (2).
We planned to synthesize dimethylated γ-carbolines 12-18, and trimethylated γ-carbolines 19, 20 from
3-11 by N-methylation using dimethyl carbonate as a methylating agent in the final step. Monomethylated

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γ-carbolines 3-9 were prepared according to the method described in our previous report, and
1,4-dimethyl-γ-carboline (10, SK1M4M) was synthesized from 1-aminopropan-2-one ethylene acetal (21)
by a similar procedure to that used for the synthesis of 5 (Scheme 1).13 Although synthesis of
3,4-dimethyl-γ-carboline (11, SK3M4M) was expected to be feasible via a similar procedure, the
corresponding starting material was difficult to obtain. Thus, we attempted to prepare 11 from
commercially available 2,3-dimethyl-4-nitropyridine N-oxide (23) by employing Pd-catalyzed amination
and subsequent annulation reaction as key steps. The nitro group and N-oxide of 23 were reduced by iron
powder under an acidic condition, followed by Pd-catalyzed Buchwald-Hartwig amination reaction with
1-bromo-2-iodobenzene to give intermediate 25.14 Finally, the annulation reaction of 25 with Pd(OAc)2
afforded 3,4-dimethyl-γ-carboline, SK3M4M (11).14
N-Methylation of γ-carbolines 3-11 was performed as summarized in Scheme 1. As expected,
N-methylation of 3-6, 8-11 proceeded smoothly to give 12-15, 17-20 in moderate yields. However,
compound 16 could not be obtained from 7 under similar conditions. Therefore we tried to synthesize 16
by Mitsunobu reaction using 7, TMAD, P(n-Bu)3, and MeOH. Although the yield was not high, 16 was
obtained.
The anti-BVDV activity of the prepared compounds was evaluated by the method described in the
previous report.13 The EC50 values for anti-BVDV activity, the CC50 values for cytotoxicity toward the
host cells, and the values of the selectivity index (CC50/EC50: SI) of the polymethylated γ-carbolines
10-20 are summarized in Table 1. Among the dimethylated compounds, SK3M4M (11), SK3M5M (13)
and SK4M5M (14) exhibited anti-BVDV activity one order of magnitude higher than that of the
monomethyl compound SK5M (2): the EC50 values of 11, 13 and 14 were 0.072, 0.062 and 0.043 μM,
respectively. Other dimethylated compounds showed similar potency of anti-BVDV activity to 2, but
their cytotoxicity seemed to be increased except in the case of SK5M7M (16). The trimethylated
compound SK1M4M5M (19) exhibited favorable anti-viral activity, but also showed the highest
cytotoxicity. Interestingly, SK3M4M5M (20),15 in which one methyl group of 19 is switched from
the1-position to the 3-position, showed the most potent anti-BVDV activity with the EC50 value of 3.5
nM. Moreover, the SI value of SK3M4M5M (20, SI = 2057) was about 20-fold higher than that of the
lead compound SK5M (2, SI = 111), indicating great improvement of the selectivity. These results
suggest that the best position of SK5M (2) for methyl group substitution, in terms of enhancement of
anti-BVDV activity, is the 3 or 4 position. In addition, methylation at the 1, 6, 8, or 9 position seems to
enhance the cytotoxicity. The relationship between anti-BVDV activity and introduction position of
methyl group is unclear. However, enhancement of the basicity of pyridine moiety in association with
electron-donating character of methyl group(s) might be one of the factor(s) of the improved anti-BVDV
activity, because introduction of methyl group(s) on the pyridine ring caused enhancement of the

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anti-BVDV activity whereas those on the indole ring are not effective.
N
N
a
b
H2N
O
O
N
H
N
O
O
H
21
22
SK1M4M (10)
O
Br
e
c
d
N
N
N
N
O2N
H2N
N
H
N
H
23
24
25
SK3M4M (11)
f
N
N
R'
R
R'
R
N
N
H
SK1M (3): R = 1-Me, R' = H
SK3M (4): R = 3-Me, R' = H
SK4M (5): R = 4-Me, R' = H
SK6M (6): R = H, R' = 6-Me
SK8M (8): R = H, R' = 8-Me
SK9M (9): R = H, R' = 9-Me
SK1M5M (12): R = 1-Me, R' = H
SK3M5M (13): R = 3-Me, R' = H
SK4M5M (14): R = 4-Me, R' = H
SK5M6M (15): R = H, R' = 6-Me
SK5M8M (17): R = H, R' = 8-Me
SK5M9M (18): R = H, R' = 9-Me
SK1M4M5M (19): R = 1,4-Me2, R' = H
SK3M4M5M (20): R = 3,4-Me2, R' = H
SK1M4M (10): R = 1,4-Me2, R' = H
SK3M4M (11): R = 3,4-Me2, R' = H
g
N
N
N
H
N
SK7M (7)
SK5M7M (16)
Scheme 1. Synthetic route to di- and tri-methylated γ-carbolines 10-20.
Reagents and comments. a) 3-Acetylindole, benzene; b) 90% H3PO4, 4% (2 steps); c) Fe, AcOH, 76%; d)
1-bromo-2-iodobenzene, Pd2(dba)3, DPPF, NaOtBu, toluene, 99%; e) Pd(OAc)2, Na2CO3, DMF, 20%; f)
DMC, K2CO3, DMF, 8-73%; g) MeOH, TMAD, P(n-Bu)3, THF, toluene, 11%.
The carboline skeleton generally has the ability to intercalate into DNA, so we next evaluated the
interaction of polymethylated γ-carbolines with double-stranded DNA. As expected, all of the
polymethylated γ-carbolines showed affinity for double-stranded DNA, with association constants of the
order of 104 M-1. However, no clear correlation between the anti-BVDV activity or cytotoxicity and the

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783
DNA affinity was observed (data not shown). This result suggests that the anti-BVDV activity and
cytotoxicity of polymethylated γ-carbolines are not attributable to interaction with DNA.
Table 1. Anti-viral activity, cytotoxicity, and selectivity index of polymethylated γ-carbolines (10-20).
9
1
8
7
N
R'
R
3
N
6
4
R"
R
H
R'
H
R"
EC50 (μM)
CC50 (μM)
SIa
Me
H
0.26
29
111
SK5M (2)
1,4-Me2
3,4-Me2
1-Me
3-Me
4-Me
H
H
H
0.12
5.2
25
43
347
26
SK1M4M (10)
SK3M4M (11)
SK1M5M (12)
SK3M5M (13)
SK4M5M (14)
SK5M6M (15)
H
0.072
0.20
H
Me
Me
Me
Me
Me
5.2
21
H
0.062
0.043
0.30
339
223
37
H
9.6
11
6-Me
7-Me
H
0.30
40
133
SK5M7M (16)
SK5M8M (17)
SK5M9M (18)
SK1M4M5M (19)
SK3M4M5M (20)
H
8-Me
9-Me
H
Me
Me
Me
Me
0.35
7.7
6.4
2.2
7.2
22
22
H
0.29
1,4-Me2
3,4-Me2
0.063
0.0035
35
H
2057
aSI = CC50/EC50
To interpret the qualitative structure-activity relationship (SAR) mentioned above, we examined the
quantitative structure-activity relationship (QSAR) for anti-BVDV activity of the compounds shown in
Table 1 and the mono-methylated γ-carbolines described in our previous report.13 The QSAR analysis
was performed by using the QSAR applications of the molecular operating environment (MOE 2006)16
with the genetic algorithm analysis applied to all descriptors of MOE (184 kinds). The correlation plot
between pEC50 values obtained from observed EC50 values and calculated pEC50 values according to eq. 1
is displayed in Figure 3.
pEC50 (μM) = -0.30177+0.40904×(wienerPol)-0.07763×(vdw_area)+0.04366×(PEOE_VSA_HYD)
-0.0691×(PEOE_VSA+1)
(1)

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The meanings of the parameters of eq. 1 are as follows: (i) wienerPol17: Wiener polarity number, (ii)
vdw_area: van der Waals surface area, (iii) PEOE_VSA_HYD18: total hydrophobic van der Waals surface
area, (iv) PEOE_VSA+118: total positive 1 van der Waals surface area.
The observed pEC50 values were positively correlated with calculated pEC50 values with the correlation
coefficient value (R2) of ca. 0.84. In addition, the positive coefficients of wienerPol and
PEOE_VSA_HYD in Eq. 1 indicate that the anti-BVDV activity tends to be enhanced when the molecule
has a hydrophobic functional group and/or becomes more polarized. This result is broadly consistent with
our qualitative SAR. In fact, EC50 values of γ-carboline derivatives with one or more methyl groups on
the pyridine moiety were found to decrease in proportion to the number of methyl groups possibly due to
the hydrophobic and electron-donating property of methyl group.
Figure 3. Correlation of observed and calculated (with eq. 1) pEC50 values for polymethylated
γ-carbolines.
In conclusion, we have synthesized several polymethylated γ-carbolines, and found that
3,4,5-trimethyl-γ-carboline (SK3M4M5M: 20) exhibits two-orders-of-magnitude higher anti-viral activity
than the lead compound SK5M (2), with the EC50 value of 3.5 nM. The result of QSAR analysis were

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consistent with our SAR data. Further structural development based on the QSAR analysis is in progress,
together with biological studies.
ACKNOWLEDGEMENTS
We are grateful to Seiichi Kobayashi of Ryoka Systems Inc. for performing the QSAR analysis. The work
described in this paper was partially supported by Grants-in Aid for Scientific Research from the Japan
Science and Technology Agency (JST) The Ministry of Education, Culture, Sports, Science and
Technology, Japan, the Japan Society for the Promotion of Science.
REFERENCES (AND NOTES)
¶The first two authors contributed equally.
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