Synthesis of pyrrolo[2,3-d]pyrimidine nucleoside derivatives as potential anti-HCV agents

Article abstract of DOI:10.1016/j.bioorg.2006.07.003

Several Toyocamycin (4) analogues were examined for their ability to inhibit HCV RNA in a replicon assay. Among the compounds examined 4-methylthio (18) and 5-carboxamide oxime derivatives (23 and 27) of Toyocamycin were found to have good activity and selectivity.

Full text of DOI:10.1016/j.bioorg.2006.07.003

Bioorganic Chemistry 35 (2007) 25–34
www.elsevier.com/locate/bioorg
Synthesis of pyrrolo[2,3-d]pyrimidine nucleoside
derivatives as potential anti-HCV agents
*
Chamakura V.N.S. Varaprasad , Kanda S. Ramasamy,
Jean-Luc Girardet, Esmir Gunic, Vicky Lai, Weidong Zhong,
1
Haoyun An , Zhi Hong
Drug Discovery, Valeant Pharmaceuticals Research and Development, 3300 Hyland Avenue, Costa Mesa,
CA 92626, USA
Received 15 June 2006
Available online 1 September 2006
Abstract
Several Toyocamycin (4) analogues were examined for their ability to inhibit HCV RNA in a rep-
licon assay. Among the compounds examined 4-methylthio (18) and 5-carboxamide oxime deriva-
tives (23 and 27) of Toyocamycin were found to have good activity and selectivity.
ꢀ
2006 Elsevier Inc. All rights reserved.
Keywords: HCV NS5B inhibitors; Toyocamycin; Pyrrolo[2,3-d]pyrimidine nucleosides
1
. Introduction
Hepatitis C virus (HCV) infection is highly persistent and lacks an effective and well tol-
erated antiviral therapy. This has spurred intensive efforts to discover novel anti-HCV
agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HCV, constitutes a valid
target for drug discovery as it is the centerpiece for viral replication. Compared to the host
RNA and DNA polymerases, NS5B RdRp has distinct sub cellular localization at the
interface of the endoplasmic reticulum membrane and cytoplasm, a novel catalytic
*
Corresponding author. Fax: +1 714 641 7233.
E-mail address: vchamakura@gmail.com (C.V.N.S. Varaprasad).
Present address: ACEA Biosciences, Inc., 11585 Sorrento Valley Rd., Ste.103, San Diego, CA 92121, USA.
1
0
045-2068/$ - see front matter ꢀ 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2006.07.003

26
C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
H
3
C
CH
3
N
N
R5
NH2
N
N
N
N
HO
N
N
O
R7
5
7
2
: R = H;R = H
OH
OH
5
7
3
: R = H;R = ribofuranosyl
5
7
4
: R = CN;R = ribofuranosyl
1
, EC : 7.0 μΜ
50
5
7
CC 300 μΜ
5: R = CONH ; R = ribofuranosyl
2
50
Fig. 1. Adenosine lead derivative (1) and Toyocamycin (4) analogues.
mechanism and many unique structural features, all of which make it an attractive target
for developing effective anti-HCV therapeutics [1].
Currently, combinations of pegylated interferons and ribavirin are the leading therapy [2]
for hepatitis C virus. However, even these combinations have limited efficacy. Many classes
of nucleoside and non-nucleoside inhibitors of NS5B RdRp have been identified and
appeared in literature and patent applications in pursuit of a better treatment for HCV.
Our continued interest [3] to develop a promising treatment for HCV infection has led
us to screen our nucleoside library against HCV polymerase in a replicon assay. This effort
6
6
identified N ,N -dimethyl adenosine (1, Fig. 1) as the lead compound (EC : 7 lM, CC :
5
0
50
3
00 lM). Whereas Toyocamycin [4] (4) has long been, along with its congener nucleosides
Tubercidin (3) and Sangivamycin (5), studied for its antiviral (human cytomegalovirus,
HCMV]) and antitumor potencies. Accordingly, Toyocamycin analogues comprising
[
nucleoside [5] and the related heterocycle (2), pyrrolo[2,3-d]pyrimidine [6], modifications
were examined against HCMV. The nucleoside modifications included both of sugar
and aglycone moieties while pyrrolo[2,3-d]pyrimidine 2 heterocycle modifications involved
C5 and N7 positions. As part of SAR studies in order to improve the activity of lead aden-
osine derivative 1 several pyrrolo[2,3-d]pyrimidine nucleoside analogues related to Toyoc-
amycin 4 were synthesized and screened for their potential in the inhibition of HCV
polymerase in replicon assay [7]. In this report, we disclose results of our study.
2
. Materials and methods
2
.1. General
1
H NMR spectra were obtained using Varian Gemini 300 MHz instrument. Majority of
reagents were procured from Aldrich Chemical Company, MI, USA and used as received.
All anhydrous reactions were carried out under Argon atmosphere. The reactions were
monitored either by thin layer chromatography (TLC), using TLC sheets coated with silica
gel 60 F254 (Merck) and UV light visualization, or by LC-MS (waters) Micromass). The
products were purified by flash chromatography on Merck silica gel 60 (230–400 mesh
ASTM). The replicon assay, in presence of the inhibitors, was carried out as described
in the literature [7].

C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
27
2
.2. 3,4-Dihydro-4-oxo-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile (6)
To a solution of Toyocamycin 4 (7.275 g, 25 mmol) in a mixture of distilled water
200 ml) and glacial acetic acid (50 ml) solid NaNO (17.25 g, 250 mmol) was slowly add-
(
2
ed at 50 ꢁC. The reaction mixture was heated at 70 ꢁC for 1 h. The volatiles were evapo-
rated under reduced pressure to dryness and the residue was extracted with hot acetone
(
4· 100 ml). The combined acetone extracts were evaporated and the residue was purified
1
over flash silica gel chromatography to obtain the title product (6.13 g, 84%). H NMR
CD OD): d 8.18 (s, 1H), 8.00 (s, 1H), 6.14 (d, J = 5.4 Hz, 1H), 4.43 (t, J = 5.4 Hz,
(
3
1
1
H), 4.27 (t, J = 3.9, 5.4 Hz, 1H), 4.10 (dd, J = 3.0, 6.6 Hz, 1H), 3.80 (ddd, J = 2.7,
2.0 and 32 Hz, 2H). ES-MS (m/z+1) : 293.
+
2.3. 3,4-Dihydro-4-oxo-7-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-
carbonitrile (7)
To a solution of 3,4-dihydro-4-oxo-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-
carbonitrile 6 (5.84 g, 20 mmol) in pyridine (50 ml) acetic anhydride (8.46 ml, 90 mmol)
was added and stirred at room temperature for 16 h. The volatiles evaporated and the res-
idue was dissolved in a mixture of CH Cl (200 ml) and water (200 ml). The organic layer
2
2
was separated and washed with water (200 ml), aq. HCl (1 N, 2· 200 ml), and water
200 ml), dried (MgSO ) and evaporated over a rotary evaporator. The residue was puri-
(
4
fied over flash silica gel chromatography (CH Cl /MeOH using as eluants with increasing
2
2
1
polarity of the mixture) to obtain the titled product as a colorless solid (6.94 g, 83%). H
NMR (CDCl ): d 12.69 (br s, D O exchangeable, 1H), 8.13 (s, 1H), 7.69 (s, 1H), 6.31 (d,
3
2
J = 5.4 Hz, 1H), 5.62 (t, J = 5.4 Hz, 1H), 5.47 (dd, J = 4.2, 5.4 Hz, 1H), 4.45 (m, 1H), 4.40
+
(
d, J = 4.2 Hz, 2H), 2.18 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H). ES-MS (m/z+1) : 419.
2.4. 4-Chloro-7-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-
carbonitrile (8)
3
,4-Dihydro-4-oxo-7-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-
carbonitrile, 7 (4.18 g, 10 mmol) was dissolved in phosphorous oxychloride (25 ml) and
refluxed for 1 h. The reaction mixture was cooled to room temperature and the volatiles
evaporated. The residue was suspended in ice cold water and stirred vigorously. The solids
separated were filtered and dried (P O ) under reduced pressure to obtain the titled crude
2
5
product in a quantitative yield. The hygroscopic solid was used in the next reactions with-
+
out further purification. ES-MS (m/z+1) : 437.
2
pyrrolo[2,3-d]pyrimidine-5-carbonitrile 10 exemplifies the general method for the synthesis
.5. The following procedure for the synthesis of 4-methylamino-7-(b-D-ribofuranosyl)-
4
of mono N -alkyl Toyocamycin derivatives 10–15. The rest of the compounds in the series
were characterized by LC-MS analysis
4
-Chloro-7-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitri-
le 8 (0.3 mmol) was treated with a solution of methylamine (3 ml, 2 M) in THF was added
and the reaction flask was closed tightly. The reaction mixture stirred at room temperature
for 16 h. The volatiles were evaporated and the residue was purified over flash silica gel

28
C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
chromatography using CH Cl and MeOH as eluants with increasing polarity to obtain 4-
2
2
1
methylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile 10 (63%).
H
NMR (CD OD): d 8.13 (s, 1H), 8.02 (s, 1H), 6.00 (d, J = 6.3 Hz, 1H), 4.62 (dd,
3
J = 5.4, 6.3 Hz, 1H), 4.27 (dd, J = 3.0, 5.4 Hz, 1H), 4.13 (dd, J = 3.0, 6.0 Hz, 1H), 3.80
+
(
ddd, J = 2.4, 12.3 and 33.2 Hz, 2H), 3.05 (s, 3H). ES-MS (m/z+1) : 306.
2
.6. 4-Chloro-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile (9)
-Chloro-7-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitri-
4
le 8 (1.9 g, 4.36 mmol) was treated with a cold (À20 ꢁC) solution of saturated methanolic
ammonia and the reaction mixture kept at 5 ꢁC for 2.5 h with occasional shaking. The vol-
atiles evaporated and the residue was purified over flash silica gel chromatography using
CH Cl and MeOH mixture with increasing polarity to obtain the title product (1.1 g,
2
2
1
8
1.5%). H NMR (CD OD): d 8.16 (s, 1H), 8.01 (s, 1H), 6.12 (d, J = 5.4 Hz, 1H), 4.40
3
(
(
t, J = 5.4 Hz, 1H), 4.27 (t, J = 3.9, 5.4 Hz, 1H), 4.09 (dd, J = 3.0, 6.6 Hz, 1H), 3.80
ddd, J = 2.7, 12.0 and 32 Hz, 2H). ES-MS (m/z+1) : 312.
+
2
.7. 4-Dimethylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile (16)
To a solution of 4-chloro-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile
9
(0.3 g, 0.96 mmol) in EtOH (10 ml) a solution of dimethylamine (10 ml, 2 M) in THF
was added and the reaction mixture was refluxed for 1 h. The volatiles evaporated and
the residue was purified by flash silica gel chromatography using CH Cl /MeOH mixture
2
2
1
with increasing polarity to obtain the titled product (0.2 g, 65.15%). H NMR (CD OD): d
3
8
.34 (s, 1H), 8.24 (s, 1H), 6.13 (d, J = 5.1 Hz, 1H), 4.52 (t, J = 5.1 Hz, 1H), 4.27 (dd,
J = 3.9, 5.1 Hz, 1H), 4.12 (dd, J = 2.7, 6.6 Hz, 1H), 3.82 (ddd, J = 2.4, 12.3 and
+
3
7.5 Hz, 2H), 3.39 (s, 6H). ES-MS (m/z+1) : 320.
2
.8. 4-O-Methyl-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile (17)
A solution of 4-chloro-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile 9
(
0.310 g, 1 mmol) and sodium methoxide (0.054 g, 5 mmol) in methyl alcohol (10 ml)
was refluxed for 16 h. The reaction mixture was cooled to room temperature and volatiles
evaporated. The residue was purified over flash silica gel chromatography (CH Cl /MeOH
2
2
1
mixture as eluant) to obtain the titled product (0.16 g, 52%) as a white solid. H NMR
CD OD): d 8.77 (s, 1H), 8.58 (s, 1H), 6.20 (d, J = 5.4 Hz, 1H), 4.53 (t, J = 5.4 Hz,
(
3
1
1
H), 4.22 (dd, J = 3.9, 4.8 Hz, 1H), 4.13 (dd, J = 3.0, 7.2 Hz, 1H), 3.80 (ddd, J = 2.7,
2.3 and 34.2 Hz, 2H), 4.02 (s, 3H). ES-MS (m/z+1) : 307.
+
2
.9. 4-Methylthio-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile (18)
A suspension of 4-chloro-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbonitrile
9
(0.310 g, 1 mmol) and sodium methanethiolate (0.105 g, 1.5 mmol) in isopropyl alcohol
(
10 ml) was refluxed for 1 h. The reaction mixture cooled to room temperature and vola-
tiles evaporated. The residue was purified over flash silica gel chromatography (CH Cl /
2
2
1
MeOH mixture as eluant) to obtain the titled product (0.23 g, 72%) as a white solid. H
NMR (CD OD): d 8.67 (s, 1H), 8.48 (s, 1H), 6.23 (d, J = 5.4 Hz, 1H), 4.51 (t,
3

C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
29
J = 5.4 Hz, 1H), 4.29 (dd, J = 3.9, 4.8 Hz, 1H), 4.12 (dd, J = 3.0, 7.2 Hz, 1H), 3.82 (ddd,
+
J = 2.7, 12.3 and 34.2 Hz, 2H), 2.72 (s, 3H). ES-MS (m/z+1) : 322.
2
(
.10. 4-Methylamino-7-(b-D-ribofuranosyl) pyrrolo[2,3-d]pyrimidine-5-carboxamidoxime
23)
To a solution of 4-methylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-car-
bonitrile 10 (0.305 g, 1 mmol) in EtOH (10 ml) an aqueous solution of hydroxylamine
0.5 ml, 50% w/w) was added and the reaction mixture refluxed for 16 h. The volatiles were
(
evaporated and the residue was recrystallized from minimum amount of methanol to
1
obtain the title product (0.21 g, 62%) as a white solid. H NMR (CD OD) d: 8.07 (s,
3
1
H), 7.68 (s, 1H), 5.97 (d, J = 6.6 Hz, 1H), 4.61 (dd, J = 5.4, 6.6 Hz, 1H), 4.26 (dd,
J = 2.7, 5.1 Hz, 1H), 4.10 (dd, J = 3.0, 5.7 Hz, 1H), 3.79 (ddd, J = 34.8, 12.3 and
+
2
.7 Hz, 2H), 3.02 (s, 3H). ES-MS (m/z+1) : 339.
2
.11. 4-Methylamino-7-(b-D-ribofuranosyl) pyrrolo[2,3-d]pyrimidine-5-carboxamidine (24)
To a solution of 4-methylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-car-
boxamidoxime 23 (0.14 g, 0.41 mmol) in a mixture of ethanol and water (10 ml, 1:1)
ammonium chloride (0.022 g, 0.41 mmol) and thoroughly washed Raney nickel (0.5 ml)
in water was successively added. The reaction mixture was shaken well under hydrogen
atmosphere (50 psi) at room temperature for 16 h, filtered over celite and the filtrate
1
was evaporated to obtain the titled product (0.1 g, 67%) as an hydrochloride salt. H
NMR (CD OD) d: 8.28 (s, 1H), 8.14 (s, 1H), 6.10 (d, J = 5.7 Hz, 1H), 4.59 (t,
3
J = 5.4 Hz, 1H), 4.29 (dd, J = 4.8, 3.3 Hz, 1H), 4.14 (dd, J = 6.3, 3.0 Hz, 1H), 3.79
+
(
ddd, J = 32.4, 12.3 and 2.4 Hz, 2H), 3.10 (s, 3H). ES-MS (m/z+1) : 323.
2
(
.12. 4-Dimethylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamidoxime
25)
To a solution of 4-dimethylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-
carbonitrile, 16 (0.1 g, 0.31 mmol) in EtOH (10 ml) an aqueous solution of hydroxylamine
0.5 ml, 50% w/w) was added and the reaction mixture refluxed for 16 h. The volatiles were
evaporated and the residue was purified over flash silica gel chromatography (CH Cl /
(
2
2
MeOH mixture as eluant) to obtain the title product (0.06 g, 54.5%) as a white solid.
1
H NMR (CD OD) d: 8.14 (s, 1H), 7.57 (s, 1H), 6.06 (d, J = 6.0 Hz, 1H), 4.58 (t,
3
J = 5.7 Hz, 1H), 4.27 (dd, J = 5.4, 3.0 Hz, 1H), 4.09 (dd, J = 6.0, 2.7 Hz, 1H), 3.77
+
(
ddd, J = 33.3, 12.3 and 2.7 Hz, 2H), 3.17 (s, 6H). ES-MS (m/z+1) : 353.
2
(
.13. 4-Dimethylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamidine
26)
To a solution of 4-dimethylamino-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-
carboxamidoxime 25 (0.1 g, 0.28 mmol) in a mixture of ethanol and water (5 ml, 1:1)
ammonium chloride (0.015 g, 0.28 mmol) and thoroughly washed Raney nickel (0.5 ml)
in water was successively added. The reaction mixture was shaken well under hydrogen
atmosphere (50 psi) at room temperature for 16 h. The reaction mixture was filtered over

30
C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
celite and the filtrate evaporated to obtain the titled product (0.05 g, 49.5%) as an hydro-
1
chloride salt. H NMR (CD OD) d: 8.29 (s, 1H), 8.23 (s, 1H), 6.16 (d, J = 6.0 Hz, 1H),
3
4
.58 (t, J = 5.4 Hz, 1H), 4.30 (dd, J = 4.5, 3.9 Hz, 1H), 4.13 (br d, J = 2.7 Hz, 1H), 3.82
+
(
ddd, J = 29.1, 12.6 and 2.7 Hz, 2H), 3.14 (s, 6H). ES-MS (m/z+1) : 337.
2
(
.14. 4-Methythio-7-(b-D-ribofuranosyl)pyrrolo[2,3- d]pyrimidine-5-carboxamidoxime,
27)
To a solution of 4-methylthio-7-(b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carbo-
nitrile, 18 (0.2 g, 0.62 mmol) in EtOH (10 ml) an aqueous solution of hydroxylamine
0.5 ml, 50% w/w) was added and the reaction mixture refluxed for 16 h. The volatiles were
evaporated and the residue was purified over flash silica gel chromatography (CH Cl /
(
2
2
1
MeOH mixture as eluant) to obtain the product (0.21 g, 72.7%) as a white solid. H
NMR (CD OD) d: 8.57 (s, 1H), 7.81(s, 1H), 6.21 (d, J = 5.4 Hz, 1H), 4.56 (t,
3
J = 6.0 Hz, 1H), 4.29 (br m, 1H), 4.10 (br m,1H), 3.79 (br m, 2H), 2.62 (s, 3H). ES-MS
+
(
m/z+1) : 356.
NH
2
OH
NC
NC
N
N
HO
HO
N
N
N
i
N
O
O
OH
OH
OH
OH
4
6
ii
Cl
N
NC
OH
NC
N
N
AcO
AcO
N
N
N
O
O
iii
OAc
OAc
OAc
OAc
8
7
2 2 3
Scheme 1. Reagents: (i) NaNO , water, glacial AcOH; (ii) Ac O, Py.; (iii) POCl .

C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
31
3
. Results and discussion
The present study involved modifications of exocyclic amino group and cyano group, at
C4 and C5 positions, respectively, of Toyocamycin (4). To explore the various substitu-
4
tions tolerated on exocyclic amino group several N -alkyl derivatives of 4 were synthesized
(
Scheme 2). The desired intermediate chloro derivative (8) was synthesized as shown in
Scheme 1. Thus, Toyocamycin 4 was diazotized by a modified procedure [8] to obtain
desaminotoyocamycin (6) which was protected as the corresponding triacetate (7) in high
yield. Hydroxy derivative 7 was then refluxed [9] in POCl for 16 h to obtain the crucial
3
intermediate 8 in quantitative yield.
The treatment of intermediate 8 with saturated methanolic ammonia at 5 ꢁC for 2.5 h
furnished 4-chloro-pyrrolo[2,3-d]pyrimidine nucleoside (9). The derivatives (10–18) modi-
fied at C4 position of Toyocamycin 4 were obtained from either of the intermediates 8 or
4
9
. Thus mono N -alkyl derivatives 10–15 (Table 1) were then generated in a small library
fashion by exposing 8 to respective amines. Although the larger size of alkyl group has
improved the activity more than two fold (10 vs. 13) it also increased the cytotoxicity mul-
4
tiple times. Apart from mono N -alkyl derivatives (10–15), the corresponding dialkyl
NC
Cl
NC
Cl
N
N
AcO
HO
N
N
N
N
O
O
i
OAc
OAc
OH
OH
8
9
ii
iii
X
N
NC
NHR
NC
N
N
HO
HO
N
N
N
O
O
OH
OH
OH
OH
1
0-15: R = Alkyl
16: X = NMe
2
1
1
7: X = OMe
8: X = SCH3
Scheme 2. Reagents: (i) Saturated methanolic ammonia; (ii) RNH
MeOH (or) NaSMe, isopropyl alcohol.
2 2
, THF; (iii) NHMe , THF (or) NaOMe,

32
C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
Table 1
SAR of the substitution at C4 position of Toyocamycin
Compound
R
EC50(lM)
CC50(lM)
300
1
1
0
1
NHMe
NH
11
8
100
1
1
1
2
3
4
NH
7
50
40
75
NH
NH
N
O
4.5
5
NH
N
15
5
100
1
1
1
6
7
8
NMe
OCH
SCH
2
3
0.4
0.4
250
40
250
3
3
4
4
(
N ,N -dimethyl) derivative [9] (16) was also synthesized to understand the influence of
substituting both protons of exocyclic amino group at C4 of Toyocamycin 4.
Thus, chloro derivative 9 upon exposure to dimethyl amine in refluxing THF afforded
4
4
the N ,N -dimethyl derivative 16. Derivative 16 was found to show better activity than
4
that of mono N -alkyl derivatives 10–15. This indicated that the two protons on the exo-
cyclic amino group at C4 in Toyocamycin 4 were not essential for the activity. This was
further validated by the altogether replacement of exocyclic amino group by different het-
ero-atom groups as in the case of methoxy (17) and methylthio (18) derivatives [9]. These
derivatives (17 and 18) were obtained from 9 by the reaction with NaOMe or NaSMe.
Between the derivatives 17 and 18, the latter was found to be less toxic than the former.
The excellent activity of derivatives 17 and 18 over that of 10–15 might suggest that the
hydrogen bond acceptors were better tolerated than the hydrogen bond donors at C4 posi-
tion in the absence of any modification of cyano group at C5 position of Toyocamycin 4.
After establishing the modifications allowed at C4 position of Toyocamycin 4, to
improve the activity of original lead adenosine derivative 1, we then focused our attention
on modifications involving both C4 and C5 positions (Table 2). Initially, the C5 cyano
group in desaminotoyocamycin 6 was modified. The target derivatives carboxamide
Table 2
SAR of the substitution at C4 and C5 positions of Toyocamycin
Compound
EC50(lM)
CC50(lM)
1
2
2
2
2
2
2
2
2
9
0
1
2
3
4
5
6
7
4
33
300
300
250
200
300
300
300
200
33
100
25
0.6
3.7
9
2.5
1

C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
33
(
(
19), carboxamido oxime (20), carboxamidine (21) and thioamide (22) were prepared
Scheme 3) as described previously in literature [8]. Of these 5-carboxamido derivative
1
9 had shown comparable activity as that of compounds 10–18 derived from single mod-
ification. Although carboxamide 19 exhibited good activity but it’s also found to be rela-
tively toxic (CC : 33 lM), whereas derivatives 20–22 were weakly active but they were not
5
0
toxic.
This prompted us to explore carboxamide oxime and carboxamidine modifications of
4
4
4
cyano group in N -methyl 10 and N ,N -dimethyl 16 and methylthio 18 derivatives.
Accordingly, derivatives 10, 16 and 18 were converted into the corresponding carboxam-
ide oximes (23, 25 and 27), respectively, upon reaction with aqueous hydroxylamine in
refluxing ethanol. The corresponding carboxamidines (24 and 26) of the carboxamide oxi-
mes 23 and 25 were obtained under catalytic hydrogenation conditions.
Among the carboxamide oximes 20, 23, 25 and 27 of this study only the carboxamide
oxime 23 derived from nitrile 10 was found to have highest activity. A closer look of oxime
2
3 revealed that it also has good selectivity [selectivity index (SI): 333] only next to that of
nitrile derivative 18 (SI: 625). Furthermore, the carboxamidines 24 and 26 were also found
to have good activity and selectivity over that of 21. This confirms to the fact that absence
of hydrogen bond donors, such as substituted amino groups, at C4 of Toyocamycin 4
enhances the activity even when cyano group at C5 is modified. The thioamides (not
shown) derived from compounds 10, 16 and 18 were also found to be inactive as that
of 22. Thus, simultaneous modification of C4 and C5 positions of Toyocamycin 4 did
not enhance the activity to a large extent over modifications at C4 alone.
In conclusion, we have synthesized and screened a variety of Toyocamycin 4 analogues
(
1
10–27) for their ability to inhibit HCV RNA replication in replicon cells. The derivatives
7 and 18 from single modifications at C4 and analogues 23 and 27 from combined mod-
ifications of C4 and C5 positions of Toyocamycin 4 showed good activity over the rest of
the compounds examined in this study. However derivatives 18, 23 and 27 were found to
have good selectivity in decreasing order.
X
X
NC
Y
N
N
HO
HO
N
N
(
i) or (ii) or (iii)
N
N
O
O
OH
OH
OH
OH
6
: X = OH
1
1
1
0: X = NHMe
X = OH
X = NHMe
6: X = NMe
8: X = SCH
2
3
19: Y = CONH2
20: Y = C(=NH)NHOH
iv
23: Y = C(=NH)NHOH
24: Y = C(=NH)NH2
iv
iv
2
2
1: Y = C(=NH)NH2
2: Y = CSNH2
X = NMe2
X = SMe
27: Y = C(=NH)NHOH
2
2
5: Y = C(=NH)NHOH
6: Y = C(=NH)NH2
Scheme 3. Reagents: (i) H
2
O
2
, NH
4
OH; (ii) aq NH
2
OH, EtOH; (iii) H
2
3 2
S, NEt , py.; (iv) Raney Ni, H .

34
C.V.N.S. Varaprasad et al. / Bioorganic Chemistry 35 (2007) 25–34
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