Synyhesis and anti-hepatitis C virus activity of morpholino triazine derivatives

Article abstract of DOI:10.3987/COM-10-11961

A series of morpholino triazines was synthesized and evaluated for anti-hepatitis C virus (HCV) activity. Incorporation of OMe, CN and F into the phenyl moiety afforded analogues with moderate potency and good selectivity, as assessed with the subgenomic HCV RNA replicon assay.

Full text of DOI:10.3987/COM-10-11961

HETEROCYCLES, Vol. 81, No. 6, 2010
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HETEROCYCLES, Vol. 81, No. 6, 2010, pp. 1419 - 1426. © The Japan Institute of Heterocyclic Chemistry
Received, 8th April, 2010, Accepted, 23rd April, 2010, Published online, 26th April, 2010
DOI: 10.3987/COM-10-11961
SYNYHESIS AND ANTI-HEPATITIS
C
VIRUS ACTIVITY OF
MORPHOLINO TRIAZINE DERIVATIVES
Takashi Misawa,a Mohammed T. A. Salim,b Mika Okamoto,b Masanori
Baba,*,b Hiroshi Aoyama,a Yuichi Hashimoto,a and Kazuyuki Sugita*,a
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. (*Corresponding authors: K. Sugita for chemical part and M. Baba for
biological part)
E-mail : sugitakazu@iam.u-tokyo.ac.jp; E-mail : m-baba@vanilla.ocn.ne.jp
Abstract – A series of morpholino triazines was synthesized and evaluated for
anti-hepatitis C virus (HCV) activity. Incorporation of OMe, CN and F into the
phenyl moiety afforded analogues with moderate potency and good selectivity, as
assessed with the subgenomic HCV RNA replicon assay.
Hepatitis C virus (HCV) infection is a principle cause of chronic liver disease1,2 leading to cirrhosis,
hepatocellular carcinoma or liver failure in humans.3,4 HCV is estimated to infect over 170 million people
worldwide.5 The best current treatment option is a pegylated interferon / ribavirin combination, but this
has only limited efficacy against the most prominent HCV genotype (1a/1b), and has significant side
effects.6 Chronic HCV infection has been associated with liver fibrosis, liver cirrhosis, heptocellular
carcinoma, and other forms of liver dysfunction. In industrialized nations, HCV infection has become a
major reason for orthotopic liver transplantation. Given the widespread impact of this disease, there is a
substantial medical need for new anti-HCV agents to complement current therapies.
We have previously found extremely versatile mother nuclei7–11 so-called multi-templates12-16 that act as
highly efficient scaffolds for the creation of biologically active compounds. This time, we focused on a
diphenylmethane template, which has already yielded several potent compounds.8,9,11 Along this line, we

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have already reported some anti-HCV compounds with good activity.17 The benzyl triazine unit was
chosen
as
an
extension
of
the
diphenylmethane
was
template.
A
series
of
2-benzyl-4-(morpholin-4-yl)-1,3,5-triazine
derivatives
synthesized,
together
with
bismorpholino-1,3,5-triazine derivatives, and their anti-HCV activity was examined.
The compounds described in this paper were prepared according to usual organic synthetic methods.18
The general synthesis of morpholino triazine compounds (1) – (18) is outlined in Chart 1. Starting from
cyanuric trichloride, the benzyl unit, morpholine ring and phenyl substituent were introduced.
Chart 1. Synthesis of morpholino triazine compounds
The anti-HCV activity of morpholino triazine compounds was determined in the established HCV RNA
replicon cells.19 Briefly, #50-1 cells carrying sub-genomic HCV RNA replicons were cultured in the
presence of various concentrations of the test compounds for 3 days. The intracellular HCV RNA and

HETEROCYCLES, Vol. 81, No. 6, 2010
1421
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA levels were determined by real-time reverse
transcription (RT)-PCR. The anti-HCV activity and cytotoxicity of test compounds were expressed as
50% effective concentration (EC50) and 50% cytotoxic concentration (CC50), defined in terms of decrease
of HCV RNA and GAPDH RNA levels to 50% of the respective control levels. The results are shown in
Figure 1 and Figure 2.
Table 1. Structures of synthesized morpholino triazine compounds
As shown in Figure 1, dimorpholiono-hydroxyphenyl triazine derivatives (1〜3) showed relatively strong
cytotoxicity. Among these compounds, the 3-hydroxyl analog (2) was the most cytotoxic. Substitution of

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the hydroxyphenyl group with a benzyl group, i.e., compound (4), resulted in decreased cytotoxicity,
though (4) was completely inactive toward HCV RNA transcription. Then, we replaced one molpholino
group with a phenyl group to obtain (5), which showed moderate anti-HCV activity (approximately 60%
inhibition) with moderate cytotoxicity (approximately 55% inhibition). This led us to choose a
2-benzyl-4-morpholino-6-phenyl-1,3,5-triazine skeleton as a lead scaffold, and the effects of a substituent
introduced into the phenyl moiety were examined. Introduction of a hydroxyl group into position 2
(ortho) or 4 (para) (6 and 8, respectively) resulted in the appearance of selective anti-HCV activity
(Figure 1). In constrast, the 3-hydroxy analog (7) showed strong cytotoxicity. As was the case in the
dimorpholino-hydroxypehyl triazine derivatives (1〜3), a meta-hydroxyl group seems to induced potent
cytotoxicity. In the case of methoxybenzyl derivatives (9〜11), the 2-methoxy analog (9) was inactive,
and the 3- and 4-methoxy derivatives (10 and 11, respectively) showed selective anti-HCV activity. It is
noteworthy that compound (11) showed approximately 60% inhibition of HCV RNA transcription at 10
μM, with almost no cytotoxicity.
Figure 1. Anti-HCV activity of morpholino triazine compounds at 10 μM
(Percent inhibition of HCV gene and host cell gene (GAPDH) expression at 10 μM)
180
160
140
120
100
80
inhibition of HCV RNA production
inhibition of GAPDH RNA production
60
40
20
0
1: m‐ 2: m‐ 3: m‐ 4: m 5: Ph 6: 2‐ 7: 3‐ 8: 4‐ 9: 2‐ 10: 3‐ 11: 4‐
2OH 3OH 4OH
OH
OH
OH OMe OMe OMe
compounds
Based on the results shown in Figure 1, replacement of the phenyl group with a pyridyl group, and
introduction of an electron-withdrawing group into the phenyl group were carried out for further
structural development (Figure 2). Replacement of the phenyl ring with a 3-pyridine (12; Py) did not
improve the anti-HCV activity or selectivity. Among the cyanophenyl derivatives (13〜15), only the
3-cyano analog (14) showed selective anti-HCV activity (Figure 2). In the case of fluorophenyl

HETEROCYCLES, Vol. 81, No. 6, 2010
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derivatives, the 3-fluoro and 4-fluoro analogs (17 and 18, respectively) showed selective anti-HCV
activity at 10 μM.
Figure 2. Anti-HCV activity of morpholino triazine compounds at 5 and 10 μM
(Percent inhibition of HCV gene and host cell gene (GAPDH) expression at 5 and 10 μM)
180
160
140
inhibition of HCV RNA production at
5μM of compound
120
100
inhibition of GAPDH RNA production at
5μM of compound
80
60
inhibition of HCV RNA production at
40
20
0
10μMof compounds
inhibition of GAPDH RNA production at
10μMof compound
12: Py 13: 2‐CN 14: 3‐CN 15: 4‐CN 16: 2‐F 17: 3‐F 18: 4‐F 8: 4‐OH
compounds
In summary, compounds which bear a hydroxyl group (8; 4-OH), a cyano moiety (14; 3-CN) or a fluorine
atom (17; 3-F), (18; 4-F) on the phenyl ring showed moderate anti-HCV activity (approximately 45-65%
inhibition of HCV RNA transcription at 10 μM) and good selectivity (almost no cytotoxicity or less than
15% inhibition of GAPDH RNA transcription) in subgenomic HCV RNA replicon assay. Although the
values for biological data vary from experiment to experiment, they are intrinsically reproducible. At this
stage, we cannot fully characterize the structure-activity relationship. Even though, our substituted
triazine derivatives appear to have potential for the development of anti-HCV agents, and further
extensive investigation of compounds based on the benzyl triazine nucleus is expected to provide
promising drug candidates.
ACKNOWLEDGEMENTS
The work described in this paper was partially supported by Grants-in-Aid for Scientific Research from
the Science and Technology Incubation Program in Advanced Regions, Japan Science and Technology
Agency (JST), and The Ministry of Education, Culture, Sports, Science and Technology, Japan, and a
grant from the Japan Society for the Promotion of Science.

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Goodman, M. H. Ling, S. Cort, and J. K. Albrecht, N. Engl. J. Med., 1998, 339, 1485.
7. Y. Hashimoto, Arch. Pharm. Life Sci., 2008, 341, 536.
8. S. Hosoda, D. Matsuda, H. Tomoda, and Y. Hashimoto, Mini-Rev. Med. Chem., 2009, 9, 572.
9. S. Hosoda, H. Aoyama, Y. Goto, M. T. A. Salim, M. Okamoto, M. Hashimoto, M. Baba, and Y.
Hashimoto, Bioorg. Med. Chem. Lett., 2009, 19, 3157.
10. M. Nakamura, T. Hamasaki, M. Tokitou, M. Baba, Y. Hashimoto, and H. Aoyama, Bioorg. Med.
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11. S. Hosoda, D. Matsuda, H. Tomoda, M. Hashimoto, H. Aoyama, and Y. Hashimoto, Bioorg. Med.
Chem. Lett., 2009, 19, 4228.
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13. Y. Hashimoto, Bioorg. Med. Chem., 2002, 10, 461.
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16. Y. Hashimoto, Cell Biol. Rev., 1991, 25, 209.
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Bioorg. Med. Chem., 2010, doi: 10.1016/j.bmc.2010.02.057.
18. Data for the compounds:
1
2-(2-Hydroxyphenyl)-4,6-bis(morpholin-4-yl)-1,3,5-triazine (1): H-NMR (500 MHz, CDCl3) δ:
3.75–3.80 (8H, m), 3.80–3.88 (8H, m), 6.87–6.92 (1H, m), 6.92–6.96 (1H, m), 7.35–7.41 (1H, m),
8.31–8.34 (1H, m), 13.6 (1H, s). HR-FAB-MS m/z: 343.1611 (Calcd for C17H21N5O3: (M)+,
343.1644).
1
2-(3-Hydroxyphenyl)-4,6-bis(morpholin-4-yl)-1,3,5-triazine (2): H-NMR 500MHz, CDCl3) δ:
3.73–3.80 (8H, m), 3.80–4.10 (8H, m), 6.95–6.98 (1H, m), 7.26–7.29 (1H, m), 7.82–7.84 (1H, m),
7.87–7.91 (1H, m). Anal. Calcd for C17H21N5O3 · 0.5H2O: C, 57.94; H, 6.29; N, 19.87. Found: C,
57.53; H, 5.89; N, 19.73.
1
2-(4-Hydroxyphenyl)-4,6-bis(morpholin-4-yl)-1,3,5-triazine (3): H-NMR (500MHz, CDCl3) δ:
3.72–3.79 (8H, m), 3.79–4.02 (8H, m), 5.13 (1H, s), 6.86 (2H, d, J=18.3 Hz), 8.30 (2H, d, J=18.3
Hz). HR-FAB-MS m/z: 343.1692 (Calcd for C17H21N5O3: (M)+, 343.1644).
2-Benzyl-4,6-dimorpholino-1,3,5-triazine (4): 1H-NMR (500 MHz, CDCl3) δ: 3.68–3.78 (16H, m),
3.80 (2H, s), 7.21 (1H, t, J=7.3 Hz), 7.28 (2H, t, J=7.3 Hz), 7.36 (2H, d, J=7.3 Hz). MS (FAB) m/z
342 (M+H)+.

HETEROCYCLES, Vol. 81, No. 6, 2010
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2-Benzyl-4-morpholino-6-phenyl-1,3,5-triazine (5): 1H-NMR (500 MHz, CDCl3) δ: 3.75–3.78 (4H,
m), 3.89–4.01 (4H, m), 4.04 (2H, s), 7.23 (1H, t, J=7.3 Hz), 7.31 (2H, t, J=7.3 Hz), 7.45 (4H, t,
J=7.3 Hz), 7.51 (1H, t, J=7.3 Hz), 8.41 (2H, d, J=7.3 Hz). HR-FAB-MS m/z: 332.1620 (Calcd for
C20H20N4O: (M) +, 332.1637).
1
2-Benzyl-4-(2-hydroxyphenyl)-6-morpholino-1,3,5-triazine (6): H-NMR (500 MHz, CDCl3) δ:
3.72–3.76 (4H, m), 3.88–3.91 (4H, m), 6.98 (1H, d, J=7.3 Hz), 7.02 (1H, t, J=7.3 Hz), 7.21 (1H, t,
J=7.3 Hz), 7.30 (2H, t, J=7.3 Hz), 7.40 (1H, td, J=7.3, 1.8 Hz), 7.48 (2H, d, J=7.3 Hz), 7.73 (1H, dd,
J=7.3, 1.8 Hz). MS (FAB) m/z 349 (M+H)+. Anal. Calcd for C20H20N4O2·0.2H2O: C, 68.24; H, 5.84:
N, 15.92. Found: C, 68.12; H, 5.97; N, 16.18.
1
2-Benzyl-4-(3-hydroxyphenyl)-6-morpholino-1,3,5-triazine (7): H-NMR (500 MHz, CDCl3) δ:
3.72–3.78 (4H, m), 3.88–3.98 (4H, m), 4.03 (2H, s), 6.99 (1H, d, J=7.3 Hz), 7.23 (1H, t, J=7.3 Hz),
7.31 (2H, t, J=7.3 Hz), 7.32 (1H, t, J=7.3 Hz), 7.42 (2H, d, J=7.3 Hz), 7.87–7.88 (1H, m), 7.99 (1H,
d, J=7.3 Hz). MS (FAB) m/z 349 (M+H)+. Anal. Calcd for C20H20N4O2·0.1H2O: C, 68.59; H, 5.81:
N, 16.00. Found: C, 68.52; H, 5.80; N, 16.21.
1
2-Benzyl-4-(4-hydroxyphenyl)-6-morpholino-1,3,5-triazine (8): H-NMR (500 MHz, CDCl3) δ:
3.74–3.76 (4H, m), 3.87–3.99 (4H, m), 4.01 (2H, s), 6.87 (2H, dt, J=7.3, 2.0 Hz), 7.22 (1H, t, J=7.3
Hz), 7.30 (2H, t, J=7.3 Hz), 7.42 (2H, d, J=7.3 Hz), 8.33 (2H, dt, J=7.3, 2.0 Hz). MS (FAB) m/z 349
(M+H)+. Anal. Calcd for C20H20N4O2·0.2H2O: C, 68.24; H, 5.84: N, 15.92. Found: C, 68.08; H,
5.92; N, 15.94.
1
2-Benzyl-4-(2-methoxyphenyl)-6-morpholino-1,3,5-triazine (9): H-NMR (500 MHz, CDCl3) δ:
3.71–3.75 (4H, m), 3.84 (3H, s), 3.83–3.95 (4H, m), 4.04 (2H, s), 7.00 (1H, d, J=7.3 Hz), 7.02 (1H, t,
J=7.3 Hz), 7.23 (1H, t, J=7.3 Hz), 7.31 (2H, t, J=7.3 Hz), 7.40 (1H, td, J=7.3, 1.2 Hz), 7.44 (2H, d,
J=7.3 Hz), 7.71 (1H, dd, J=7.3, 1.2 Hz). MS (FAB) m/z 363 (M+H+)+. Anal. Calcd for
C21H22N4O2·0.4H2O: C, 68.24; H, 6.22: N, 15.16. Found: C, 68.48; H, 6.11; N, 14.98.
2-Benzyl-4-(3-methoxyphenyl)-6-morpholino-1,3,5-triazine (10): 1H-NMR (500 MHz, CDCl3) δ:.
MS (FAB) m/z 363 (M+H)+. Anal. Calcd for C21H22N4O2: C, 69.59; H, 6.12: N, 15.46. Found: C,
69.31; H, 6.14; N, 15.54.
1
2-Benzyl-4-(4-methoxyphenyl)-6-morpholino-1,3,5-triazine (11): H-NMR (500 MHz, CDCl3) δ:
3.73–3.81 (4H, m), 3.88 (3H, s), 3.91–3.98 (4H, m), 4.04 (2H, s), 7.05 (1H, dd, J=7.3, 2.0 Hz), 7.23
(1H, t, J=7.9 Hz), 7.31 (2H, t, J=7.3 Hz), 7.36 (1H, t, J=7.9 Hz), 7.43 (2H, d, J=7.9 Hz), 7 96 (1H,
d, J=2.0 Hz), 8.38 (1H, td, J=7.9, 2.0 Hz). MS (FAB) m/z 363 (M+H)+. Anal. Calcd for C21H22N4O2:
C, 69.59; H, 6.12: N, 15.46. Found: C, 69.76; H, 6.09; N, 15.62.
2-Benzyl-4-morpholino-6-(3-pyridinyl)-1,3,5-triazine (12): 1H-NMR (500 MHz, CDCl3) δ:
3.74–3.80 (4H m), 3.90–4.00 (4H, brs), 4.04 (2H, s), 7.24 (1H, t, J=7.5 Hz), 7.31 (2H, t, J=7.5 Hz),
7.37 (1H, t, J=7.5 Hz), 7.43 (2H, d, J=7.5 Hz), 7.64 (1H, dt, J=7.5, 1.5 Hz), 7.72 (1H, dd, J=7.5, 1.5
Hz), 8.58 (1H, d, J=1.5 Hz). HR-FAB-MS m/z: 333.1613 (Calcd for C19H19N5O: (M) +, 333.1590).
1
2-Benzyl-4-(2-cyanophenyl)-6-morpholino-1,3,5-triazine (13): H-NMR (500 MHz, CDCl3) δ:
3.70–3.76 (4H, m), 3.86–3.94 (4H, m), 4.02 (2H, s), 7.23 (1H, t, J=8.0 Hz), 7.31 (2H, t, J=7.0 Hz),
7.40 (2H, d, J=7.0 Hz), 7.48–7.53 (2H, m), 7.79 (1H, d, J=7.0 Hz), 8.09 (1H, t, J=7.0 Hz).
HR-FAB-MS m/z: 357.1635 (Calcd for C21H19N5O: (M) +, 357.1590).
1
2-Benzyl-4-(3-cyanophenyl)-6-morpholino-1,3,5-triazine (14): H-NMR (500 MHz, CDCl3) δ:
3.74–3.80 (4H, m), 3.90–4.00 (4H, m), 4.03 (2H, s), 7.24 (1H, t, J=8.0 Hz), 7.56 (1H, t, J=8.0 Hz),
7.77 (1H, d, J=8.0 Hz), 8.64 (1H, d, J=8.0 Hz), 8.72 (1H, s). HR-FAB-MS m/z: 357.1629 (Calcd for
C21H19N5O: (M) +, 357.1590).
1
2-Benzyl-4-(4-cyanophenyl)-6-morpholino-1,3,5-triazine (15): H-NMR (500 MHz, CDCl3) δ:
3.75–3.78 (4H, m), 3.90–4.00 (4H, m), 4.04 (2H, s), 7.24 (1H, t, J=7.5 Hz), 7.32 (2H, t, J=7.5 Hz),
7.42 (2H, d, J=7.5 Hz), 7.73 (2H, d, J=8.5), 8.51 (2H, d, J=8.5 Hz). HR-FAB-MS m/z: 357.1570
(Calcd for C21H19N5O: (M) +, 357.1590).
1
2-Benzyl-4-(2-fluorophenyl)-6-morpholino-1,3,5-triazine (16): H-NMR (500 MHz, CDCl3) δ:
3.73–3.75 (4H, m), 3.88–3.97 (4H, m), 4.05 (2H, s), 7.14 (1H, dd, J=7.5, 2.5 Hz), 7.21–7.24 (2H, m),

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7.31 (2H, d, J=7.5 Hz), 7.42–7.47 (3H, m), 8.09 (1H, td, J=7.5, 2.5 Hz). HR-FAB-MS m/z:
350.1527 (Calcd for C20H19FN4O: (M) +, 350.1543).
2-Benzyl-4-(3-fluorophenyl)-6-morpholino-1,3,5-triazine (17): H-NMR (500 MHz, CDCl3) δ:
1
3.73–3.79 (4H, m), 3.90–4.00 (4H, m), 4.03 (2H, s), 7.18–7.25 (3H, m), 7.31 (2H, td, J=8.0, 2.0 Hz),
7.39–7.44 (3H, m), 8.01–8.09 (1H, m), 8.21 (1H, dd, J=8.0, 2.0 Hz). HR-FAB-MS m/z: 350.1521
(Calcd for C20H19FN4O: (M) +, 350.1543).
1
2-Benzyl-4-(4-fluorophenyl)-6-morpholino-1,3,5-triazine (18): H-NMR (500 MHz, CDCl3) δ:
3.73–3.79 (4H, m), 3.86–3.99 (4H, m), 4.06 (2H, s), 7.15 (2H, t, J=7.5 Hz), 7.23 (1H, td, J=8.0, 2.0
Hz), 7.31 (2H, td, J=8.0, 2.0 Hz), 7.42 (2H, d, J=7.5 Hz), 8.43 (2H, td, J=8.0, 2.0 Hz). HR-FAB-MS
m/z: 350.1578 (Calcd for C20H19FN4O: (M) +, 350.1543).
2-Benzyl-4,6-dichloro-1,3,5-triazine (20): 1H-NMR (500 MHz, CDCl3) δ: 4.18 (2H, s), 7.29 (1H, d,
J=7.0 Hz), 7.33–7.41 (4H, m). MS (FAB) m/z 240 (M+H)+.
1
2-Benzyl-4-chloro-6-morpholino-1,3,5-triazine (21): H-NMR (500 MHz, CDCl3) δ: 3.71–3.73
(4H, m), 3.81–3.87 (4H, m), 3.93 (2H, s), 7.24, (1H, t, J=7.3 Hz), 7.31 (2H, t, J=7.3 Hz), 7.35 (2H,
d, J=7.3 Hz). MS (FAB) m/z 291 (M+H)+.
2-Chloro-4,6-bis(morpholin-4-yl)-1,3,5-triazine (22): 1H-NMR (500 MHz, CDCl3) δ:
3.65–3.85(16H, m).
19. N. Ishii, K. Watashi, T. Hishiki, K. Goto, D. Inoue, M. Hijikata, T. Wakita, N. Kato, and K.
Shimotohno, J. Virol., 2006, 80, 4510.