Parallel synthesis of 5-cyano-6-aryl-2-thiouracil derivatives as inhibitors for hepatitis C viral NS5B RNA-dependent RNA polymerase

Article abstract of DOI:10.1016/j.bioorg.2005.10.001

From random screening of our compound libraries, we identified a hit compound with an IC₅₀ of 27 μM against hepatitis C viral NS5B RNA-dependent RNA polymerase. By using a parallel synthetic strategy, a series of its derivatives were synthesize

Full text of DOI:10.1016/j.bioorg.2005.10.001

Bioorganic Chemistry 34 (2006) 26–38
www.elsevier.com/locate/bioorg
Parallel synthesis of 5-cyano-6-aryl-2-thiouracil
derivatives as inhibitors for hepatitis C viral NS5B
RNA-dependent RNA polymerase
*
Yili Ding , Jean-Luc Girardet, Kenneth L. Smith, Gary Larson,
Brett Prigaro, Jim Z. Wu, Nanhua Yao
Valeant Pharmaceuticals International, 3300 Hyland Avenue Costa Mesa, CA 92626, USA
Received 17 August 2005
Available online 19 December 2005
Abstract
From random screening of our compound libraries, we identified a hit compound with an IC50 of
27 lM against hepatitis C viral NS5B RNA-dependent RNA polymerase. By using a parallel syn-
thetic strategy, a series of its derivatives were synthesized. From their anti-HCV activity screening,
compounds with single digital 3.8 micromolar activity were obtained.
Ó 2005 Elsevier Inc. All rights reserved.
Keywords: HCV; NS5B; 5-Cyano-6-aryl-2-thiouracil derivatives; Inhibitors
1. Introduction
Chronic viral infection caused by hepatitis C virus (HCV) has been recognized as one of
the leading causes of liver impairment such as cirrhosis and hepatocellular carcinoma. It is
estimated that 3% of the world population or about 170 million people are infected with
hepatitis C virus [1]. The recommended standard of care treatment, the pegylated interfer-
on a in the combination with ribavirin, provides a sustained response in about 50% of the
treated patients, but side effects could be severe [2]. The need of a more efficacious and
*
Corresponding author.
E-mail address: yding@valeant.com (Y. Ding).
0045-2068/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2005.10.001

Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
27
better tolerated treatment has spurred intense research efforts in pharmaceutical industry
to develop novel anti-HCV agents.
HCV is a 9.6 kb positive strand RNA virus of the flaviviridae, genus Hepacivirus. It con-
tains a single open reading frame coding for a ꢀ3000 amino acid polyprotein, which is further
processed into various structural and non-structural viral proteins by host and viral proteas-
es. The NS5B RNA-dependent RNA polymerase (RdRp) is the central enzyme that is
responsible for replication of the viral genome, and has since become a target of choice for
the screening and design of small molecular inhibitors, which in principle, should interfere
with viral replication [3]. Various institutions and pharmaceutical companies have reported
structurally diverse non-nucleoside small molecular inhibitors of NS5B polymerase [4].
S
N
NH
IC50 = 27µM
O
CN
1
Our efforts towards identifying an HCV NS5B RdRp inhibitor started with a high
throughput screening of compound libraries using an NS5B RdRp directed RNA synthe-
sis assay [5]. The effort culminated in identification of a 5-cyano-6-aryl-2-thiouracil com-
pound 1 that had an IC50 (inhibitor concentration for 50% inhibition) of 27 lM. Although
its potency was not impressive, compound 1 had no apparent structural liability and
seemed to possess a pharmacophore of uracil, the nucleobase of uridine that is utilized
by an RNA polymerase. In addition, this class of molecule has been previously reported
to possess antiviral activity against polio, coxsackie, and semliki forest viruses [6]. A recent
patent application also revealed that 5-cyano-6-aryl-2-thiouracils are active against HCV
NS3 helicase [7]. In this communication, we report the synthesis of various thiouracil
derivatives with different substituents across the ring skeleton and their inhibitory activity
against HCV NS5B RdRp.
2. Results and discussion
Based on the structure of compound 1, we made the chemical modifications on aryl
group at C-6 of the ring, alkylation on the S atom, and substitutions on the N of the ring
through the parallel synthesis strategy. We like to find out the structure–activity relation of
these modifications. The efficient synthetic strategy is summarized in Scheme 1. The thi-
oxopyrimidine heterocyclic cores were constructed by a cyclo-condensation of equivalent
molar quantities of 10 substituted aromatic aldehydes 1a–1j, thiourea, and ethyl cyanoac-
etate in the parallel fashion. After neutralization with acetic acid, the desired 5-cyano-4-
oxo-6-aryl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine derivatives 2a–2j were obtained as
solids.
Benzylation of compounds 2a–2j with different substituted benzyl bromides in ethanol
in the presence of K2CO3 gave 6-aryl-5-cyano-2-benzylthio-4-oxopyrimidines 3aa–3je in
high yields. During these reactions, in some cases, the S and N disubstituted pyrimidine
derivatives such as compound 3a were also isolated from the reaction mixture as minor
products.

28
Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
A
S
a R=2, 4-difluoro
b R=2,4-dichloro
c R=4-bromo
f R=2-methyl
OEt
O
O
HN
NH
a
g R=3,5-difluoro
h R=2-trifluoromethyl
i R=3,5-dichloro
j R=4-isopropyl
R
R
+
H
O
d R=3-methyl
CN
e R=3-trifluoromethyl
CN
b
1a-1j
2a-2j
3aa R=2, 4-difluoro, R1=3,5-dimethoxyl 3ec R=3-CF3, R1=3-NO2
3ab R=2,4-difluoro, R1=3-Me
3ac R=2,4-difluoro, R1=2-Br
3ad R=2,4-difluoro, R1=3-NO2
3ba R = 2,4-dichloro, R1=3,5-dimethoxyl
3bb R = 2,4-dichloro, R1=3-Me
3bc R = 2,4-dichloro, R1=4-OCF3
3ca R=4-Br, R1=3,5-dimethoxyl
3cb R=4-Br, R1=3-Me
3cc R=4-Br, R1=2-Br
3cd R=4-Br, R1=3-NO2
3ce R=4-Br, R1=2-CF3
3cf R=4-Br, R1=4-OCF3
3fa R=2-Me, R1=3,5-dimethoxyl
3fb R=2-Me, R1=3-Me
3fc R=2-Me, R1=3-Br
S
R1
N
NH
3fd R=2-Me, R1=3-NO2
3ga R=3,5-difluoro, R1=3,5-dimethoxyl
3gb R=3,5-difluoro, R1=3-Me
3ha R=2-CF3, R1=3,5-dimethoxyl
3hb R=2-CF3, R1=3-Me
3hc R=2-CF3, R1=2-Br
3hd R=2-CF3, R1=4-OCF3
3ia R=3,5-dichloro, R1=3,5-dimethoxyl
3ib R=3,5-dichloro, R1=3-Me
3ja R=4-isopropyl, R1=3,5-dimethoxyl
3jb R=4-isopropyl, R1=3-Me
3jc R=4-isopropyl, R1=2-Br
3jd R=4-isopropyl, R1=3-NO2
3je R=4-isopropyl, R1=4-OCF3
R
O
CN
3aa-3je
NO2
3da R=3-Me, R1=3,5-dimethoxyl
3db R=3-Me, R1=3-Me
S
NO2 3dc R=3-Me, R1=3-NO2
3dd R=3-Me, R1=4-OCF3
N
N
F
3ea R=3-CF3, R1=3,5-dimethoxyl
3eb R=3-CF3, R1=3-Me
O
CN
F
R1
3a
S
N
NH
c
R
4aa-4ha, 4ja-4jc
2a-2h, 2j
O
CN
4aa R=2, 4-difluoro, R1=(CH2)4CH3
4ba R = 2,4-dichloro, R1=(CH2)4CH3
4bb R = 2,4-dichloro, R1=(CH2)3Ph
4ca R=4-Br, R1=(CH2)4CH3
4eb R=3-CF3, R1=(CH2)3Ph
4fa R=2-Me, R1=(CH2)4CH3
4ga R=3,5-difluoro, R1=(CH2)3Ph
4gb R=3,5-difluoro, R1=(tetrahydro-2H-pyran)methene
4ha R=2-CF3, R1=(CH2)4CH3
4cb R=4-Br, R1=CH2CH2OCOPh
4cc R=4-Br, R1=(tetrahydro-2H-pyran)methene
4da R=3-Me, R1=(CH2)4CH3
4db R=3-Me, R1=(tetrahydro-2H-pyran)methene
4ea R=3-CF3, R1=(CH2)4CH3
4ja R=4-isopropyl, R1=(CH2)4CH3
4jb R=4-isopropyl, R1=(CH2)3Ph
4jc R=R=4-isopropyl, R1=(tetrahydro-2H-pyran)methene
B
S
d
2a, 2b, 2e, 2f, 2g, 2i
N
N
a R1=2,4-difluoro
b R1=2,4-dichloro
e R1=3-methyl
f R1=3-trifluoromethyl
g R1=2-methyl
R1
O
CN
e
5b, 5e, 5f, 5g, 5i
S
i R1=4-bromo
N
N
R1
O
CN
F3C
N
7a R=2-(3-methoxylphenyl)-ethyl, R1=H
7b R=cyclbutyl, R1=H
7c R=2-(phenyl)-ethyl, R1=H
7d R=2-(ethyl)-heptanyl, R1=H
7e R=4-(methylamino) piperidine, R1=Me
7f R=2-(3,4-dimethoxylphenyl)-ethyl, R1=H
7g R=(2-pyrimidy)-piperazine, R1=H
7h R=2-(1-methylpyrrolidine)-ethyl
7i R=CH2CF3, R1=H
6a, 6b, 6e, 6f, 6g, 6i
F3C
S
S
f
g
N
3ce
N
N
Cl
NRR1
CN
Br
CN
Br
7j R=2-(3, 4-benzodioxole)-ethyl, R1=H
7
7a-7j

Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
29
Alkylation of compounds 2a–2h, and 2j with various alkyl bromides in DMF in the
presence of triethylamine provided the 6-aryl-5-cyano-2-alkylthio-4-oxopyrimidines 4aa–
4ha, 4ja–4jc. Treatment of compounds 2a, 2b, 2e, 2f, 2g, and 2i with 1,2-dibromoethylene
or 1,3-dibromopropane in DMF in the presence of triethylamine afforded the five-mem-
bered ring bicyclic thiazolo[3,2-a]pyrimidines 5b, 5e, 5f, 5g, 5i, and the six-membered ring
bicyclic pyrimido[2,1-b]thiazines 6a, 6b, 6e, 6f, 6g, and 6i. We tried to get the seven-mem-
bered ring bicyclic derivatives from the reactions with 1,4-dibromobutane, however, no
desired compound was isolated from the reaction mixture even under strong reaction con-
ditions (higher temperature and longer reaction time in DMF or DMSO).
Compound 3ce was halogenated from the reaction with phosphoryl chloride to yield 6-(4-
bromophenyl)-4-chloro-2-(2-trifluoromethyl-benzylthio)-pyrimidine-5-carbonitrile
(7).
This highly activated intermediate was then reacted with different amines to yield 6-(4-brom-
ophenyl)-4-alkylamino-2-(2-trifluoromethyl-benzylthio)-pyridine- 5-carbonitriles 7a–7j.
Each compound was purified on preparative TLC in a parallel fashion. The identity of
the compounds described above was confirmed by ESMS (data not shown) and 1H NMR
spectra. They normally had more than 90% purity judged by HPLC and LCMS.
All the compounds were evaluated for inhibitory activity against HCV NS5B RdRp
using a publishing procedure [5]. In the assay, a compound was first dissolved in DMSO,
and then transferred into the assay buffer. Every compound was tested in duplicate. IC50
determined for each compound generally carried with a standard deviation <10%.
A systematic study of importance of the substitution on the aryl ring and benzyl ring
for compounds 3aa–3je is listed in Table 1. The inhibitory activities of 2-alkylthio-6-ar-
yl-3,4-dihydro-6-oxopyrimidine-5-carbonitrile derivatives 4aa–4jc are listed in Table 2.
Some of these compounds showed single digital micromolar activity including compounds
3ce (7.1 lM), 3cf (8.7 lM), 3dd (8.6 lM), 3hd (9.2 lM), 3jc (3.8 lM), and 3je (9.9 lM)
against hepatitis C viral NS5B RNA-dependent RNA polymerase. The best compound
3jc was further assayed for its ability to inhibit hepatitis C viral subgenome replication
assay in Huh-7 cells. Its activity was weak with an EC50 of 32 lM.
The para-substitution on both C-6 aryl group and the S-benzyl group improved the
inhibitory activity two-fold over the lead compound. However, a combination of para-
substitution on C-6 aryl group and a ortho-substitution on S-benzyl group led to a five-
fold increase over the lead compound activity. Non-benzyl alkyl substitution on sulfur
did not improve the activity very much.
Five-membered ring bicyclic heterocycles 5b, 5e, 5f, 5g, and 5f, six-membered ring bicy-
clic heterocycles 6a, 6b, 6e, 6f, 6g, and 6i, 4-alkylamino-6-(4-bromophenyl)-2-(2-trifluo-
romethyl-benzylthio)-pyrimidine-5-carbonitrile analogues 7a–7j, and the S and N
disubstituted pyrimidine derivative 3a did not show any inhibitory activity
(IC50 > 100 lM). All of these compounds have the substitution at N of the ring, indicating
free NH of the ring is necessary for the activity.
b
Scheme 1. Reagents and conditions: (a) 1a–1j, thiourea, K2CO3, ethanol, refluxing; (b) 2a–2j, substituted benzyl
bromides, K2CO3, DMF, room temperature, 10 h; (c) 2a–2h, 2j, alkyl bromides, triethylamine, DMF, 80 °C, 10 h;
(d) 2b, 2e, 2f, 2g, and 2i, BrCH2CH2Br, triethylamine, DMF, 80 °C, 10 h; (e) 2a, 2b, 2e, 2f, 2g, and 2i,
BrCH2CH2CH2Br, triethylamine, DMF, 80 °C, 10 h; (f) 3ce, POCl3, 70 °C, 100%; (g) 7, alkyl amines,
triethylamine, CH3CN, room temperature, 10 h.

30
Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
Table 1
SAR of the substitution on the aryl and benzyl groups of compound 1
R2
S
N
NH
O
R1
CN
Compound
R1
R2
IC50 (lM)
MeO
F
3aa
19
F
F
F
F
MeO
Me
F
3ab
3ac
3ad
3ba
3bb
3bc
3ca
3cb
3cc
3cd
3ce
3cf
57
13
32
34
33
11
13
11
10
14
7.1
8.7
30
Br
F
O2N
F
MeO
Cl
Cl
Cl
Cl
MeO
Me
Cl
Cl
F3CO
MeO
Br
Br
Br
Br
Br
MeO
Me
Br
O2N
CF3
Br
F3CO
MeO
Me
3da
MeO

Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
31
Table 1 (continued)
Compound
R1
R2
IC50 (lM)
Me
Me
3db
3dc
3dd
3ea
3eb
3ec
3fa
3fb
3fc
22
O2N
Me
20
8.6
11
14
13
25
14
32
37
15
Me
F3CO
MeO
F3C
F3C
F3C
MeO
Me
O2N
MeO
Me
Me
Me
Me
MeO
Me
Br
O2N
3fd
3ga
F
MeO
F
F
MeO
Me
3gb
3ha
3hb
3hc
3hd
27
F
MeO
12
CF3
CF3
CF3
CF3
MeO
Me
14
Br
21
9.2
F3CO
(continued on next page)

32
Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
Table 1 (continued)
Compound
R1
R2
IC50 (lM)
Cl
MeO
3ia
3ib
34
Cl
Cl
MeO
Me
29
Cl
MeO
3ja
3jb
3jc
3jd
3je
25
MeO
Me
12
Br
3.8
10
O2N
9.9
F3CO
All the compounds described here were tested for cellular activity using a cell-based
HCV subgenomic replicon assay. They did not show any toxicity up to 250 lM.
3. Conclusions
From the screening of our compound libraries using an NS5B-directly RNA synthesis
assay, we identified a hit compound 1 with an IC50 of 27 lM. By using a parallel synthetic
strategy, we were able to synthesize a series of 5-cyano-6-aryl-2-thiouracil derivatives for
anti-HCV agent screening. Modification of the hit has resulted in a series of inhibitor with
improved potency. The SAR studies suggest that the free NH of the uracil ring seems to
be necessary for the inhibitory activity, the para-substitution on both C-6 aryl group and
the S-benzyl group slightly improved the activity, and a combination of the para-substitution
on the aryl group and an ortho-substitution on the sulfur atom increase the activity. Based on
these results, further optimization, mechanism of action, and structural studies are in pro-
gress to elucidate their mode of action and improve their potency against HCV NS5B RdRp.
4. Experimental
4.1. General
1H spectra were obtained using a Varian Gemini 300 NMR. The proton NMR spectra
were recorded at 300 MHz. All reagents and chemicals were obtained from Aldrich Chem-

Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
33
Table 2
SAR at the substitution of aryl group and the non-beznyl alkyl group on sulfur atom of the core structure
SR2
N
NH
O
R1
CN
Compound
R1
R2
IC50 (lM)
F
4aa
CH3(CH2)4
12
F
Cl
Cl
4ba
CH3(CH2)4
17
Cl
4bb
4ca
4cb
4cc
4da
Ph(CH2)3
12
11
30
50
18
Cl
CH3(CH2)4
PhCO2(CH2)2
Br
Br
Br
CH2
O
Me
CH3(CH2)4
Me
4db
4ea
4eb
>100
12
CH2
O
F3C
F3C
CH3(CH2)4
PhCO2(CH2)2
13
Me
4fa
CH3(CH2)4
Ph(CH2)3
18
F
4ga
4gb
>100
>100
F
F
CH2
O
F
CF3
4ha
4ja
4jb
4jc
CH3(CH2)4
CH3(CH2)4
Ph(CH2)3
20
12
>100
17
CH2
O

34
Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
ical (USA) and were used as received unless otherwise noted. All the reactions were per-
formed in a Reacto-Station RS 6000 synthesizer. Each compound was purified on a pre-
parative TLC. The purity of the compounds was determined by LCMS (waters Micromass
ZQ).
1
4.2. Spectral data: H NMR data
3aa (CD3OD): d 7.72 (m, 1H), 7.20 (m, 2H), 6.54 (s, 2H), 6.37 (s, 1H), 4.23 (s, 2H), 3.68
(s, 6H).
1
3ab H NMR (CD3OD): d 7.53 (q, 1H), 7.21 (s, 1H), 7.17–7.01 (m, 5H), 4.33 (s, 2H),
2.29 (s, 3H).
3ac 1H NMR (CD3OD): d7.56 (m, 2H), 7.23 (t, 1H, J = 7.2 Hz), 7.13 (m, 3H), 4.51 (s, 2H).
3ad 1H NMR (CD3OD): d 8.31 (s, 1H), 8.09 (d, 1H, J = 8.4 Hz), 7.88 (d, 1H,
J = 8.4 Hz), 7.52 (m, 2H), 7.10 (m, 2H), 4.53 (s, 2H).
3a 1H NMR (CD3OD): d 8.27 (s, 1H), 8.19 (m, 1H), 8.12 (s, 1H), 8.06 (d, 1H,
J = 8.1 Hz), 7.80 (d, 1H, J = 8.1 Hz), 7.8 (m, 2H), 7.48 (t, 1H, J = 8.1 Hz), 7.37 (m,
1H), 7.01 (m, 2H), 4.46 (d, 2H, J = 8.4 Hz), 4.28 (d, 2H, J = 8.4 Hz).
1
3ba H NMR (CD3OD): d 7.69 (s, 1H), 7.53 (s, 1H), 7.52 (s, 1H), 6.51 (s, 2H), 6.36 (s,
1H), 4.39 (s, 2H), 3.68 (s, 6H).
3bb 1H NMR (CD3OD): d 7.59 (d, 1H, J = 2.1 Hz), 7.45 (dd, 1H, J = 8.7, 2.1 Hz), 7.37
(d, 1H, J = 8.7 Hz), 7.20 (s, 1H), 7.16 (s, 1H), 7.15 (d, 1H, J = 6.6 Hz), 7.02 (d, 1H,
J = 6.6 Hz), 4.36 (s, 2H), 2.27 (s, 3H).
3bc 1H NMR (CD3OD): d 7.59 (s, 1H), 7.52 (d, 2H, J = 8.7 Hz), 7.38 (q, 2H,
J = 8.4 Hz), 7.16 (d, 2H, J = 8.7 Hz); 4.38 (s, 2H).
3ca 1H NMR (DMSO-d6): d 7.74 (d, 2H, J = 8.7 Hz), 7.68 (d, 2H, J = 8.7 Hz), 6.56 (s,
2H), 6.32 (s, 1H), 4.24 (s, 2H), 3.64 (s, 6H).
3cb 1H NMR (DMSO-d6): d 7.79 (d, 2H, J = 8.4 Hz), 7.73 (d, 2H, J = 8.4 Hz), 7.18 (m,
2H), 7.16, (s, 1H), 7.04 (m, 1H), 4.35 (s, 2H), 2.22 (s, 3H).
3cc 1H NMR (DMSO-d6): d 7.86 (d, 2H, J = 6.9 Hz), 7.79 (d, 2H, J = 6.9 Hz), 7.64 (d,
1H, J = 6.9 Hz), 7.53 (d, 1H, J = 6.9 Hz), 7.32 (t, 1H, J = 6.9 Hz), 7.23 (t, 1H,
J = 6.9 Hz), 4.60 (s, 2H).
3cd 1H NMR (CD3OD): d 8.33 (s, 1H), 8.09 (d, 2H, J = 8.1 Hz), 7.86 (d, 2H,
J = 8.1 Hz), 7.73 (d, 2H, J = 8.7 Hz), 7.64 (d, 2H, J = 8.7 Hz), 7.52 (t, 1H, J = 8.1 Hz),
4.51 (s, 2H).
3ce 1H NMR (DMSO-d6): d 7.78 (m, 6H), 7.64 (t, 1H, J = 7.5 Hz), 7.52 (t, 1H,
J = 7.5 Hz), 4.68 (s, 2H).
1
3cf H NMR (CD3OD): d 7.71 (d, 2H, J = 8.4 Hz), 7.63 (d, 2H, J = 8.4 Hz), 7.53 (d,
1H, J = 8.2 Hz), 7.17 (d, 2H, J = 8.2 Hz), 4.41 (s, 2H).
3da 1H NMR (CD3OD): d 7.78 (s, 1H), 7.76 (d, 1H), 7.41 (m, 2H), 6.59 (s, 2H), 6.36 (s,
1H), 4.47 (s, 2H), 3.64 (s, 6H), 2.43 (s, 3H).
1
3db H NMR (CD3OD): d 7.61 (s, 2H), 7.33 (m, 2H), 7.17 (m, 3H), 7.01 (d, 1H,
J = 6.0 Hz), 4.40 (s, 2H), 2.39 (s, 3H), 2.27 (s, 3H).
3dc 1H NMR (DMSO-d6): d 8.27 (s, 1H), 8.06 (d, 1H, J = 8.1 Hz), 7.87 (d, 1H,
J = 8.1 Hz), 7.58 (t, 1H, J = 8.1 Hz), 7.49 (d, 1H), 7.48 (s, 1H), 7.31 (t, 1H, J = 8.1 Hz),
7.24 (t, 1H, J = 8.1 Hz), 4.38 (s, 2H), 2.33 (s, 3H).
3dd 1H NMR (CDCl3): d 7.47 (d, 1H), 7.44 (s, 1H), 7.11 (m, 4H), 6.89 (d, 2H,
J = 8.1 Hz), 4.12 (s, 2H), 2.15 (s, 3H).

Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
35
3ea 1H NMR (CD3OD): d 8.30 (s, 1H), 8.25 (d, 1H, J = 6.9 Hz), 7.90 (d, 1H,
J = 6.9 Hz), 7.77 (t, 1H, J = 6.9 Hz), 6.57 (s, 2H), 6.36 (s, 1H), 4.49 (s, 2H), 3.65 (s, 6H).
3eb 1H NMR (CD3OD): d 8.10 (s, 1H), 8.07 (d, 1H, J = 8.8 Hz), 7.80 (d, 1H,
J = 7.8 Hz), 7.68 (t, 1H, J = 8.8 Hz), 7.23 (s, 1H), 7.19 (d, 1H, J = 8.8 Hz), 7.14 (t, 1H,
J = 8.8 Hz), 7.02 (d, 1H, J = 8.8 Hz), 4.35 (s, 2H), 2.28 (s, 3H).
3ec 1H NMR (DMSO-d6): d 8.27 (s, 1H), 8.05 (t, 1H, J = 9.0 Hz), 7.99 (s, 1H), 7.86 (d,
1H, J = 7.5 Hz), 7.83 (d, 1H, J = 7.5 Hz), 7.69 (t, 1H, J = 9.0 Hz), 7.57 (t, 1H,
J = 9.0 Hz), 4.39 (s, 2H).
3fa 1H NMR (CD3OD): d 7.20 (m, 4H), 6.56 (s, 2H), 6.34 (s, 1H), 4.40 (s, 2H), 3.66 (s,
6H), 2.32 (s, 3H).
3fb 1H NMR (CD3OD): d 7.38–7.11 (m, 7H), 7.02 (d, 1H, J = 6.6 Hz), 4.33 (s, 2H), 2.27
(s, 3H), 2.28 (s, 3H).
3fc 1H NMR (CD3OD): d 7.40–7.11 (m, 7H), 7.02 (d, 1H, J = 6.6 Hz), 4.38 (s, 2H), 2.27
(s, 3H).
3fd 1H NMR (CDCl3): d 8.20 (s, 1H), 8.02 (t, 1H), 7.82 (d, 1H, J = 7.5 Hz), 7.60 (t, 1H),
7.20 (m, 4H), 4.20 (s, 2H), 2.20 (s, 3H).
3ga 1H NMR (CD3OD): d 7.59 (d, 1H), 7.57 (d, 1H), 7.22 (m, 1H), 6.58 (s, 2H), 6.37 (s,
1H), 4.24 (s, 2H), 3.69 (s, 6H).
3gb 1H NMR (CD3OD): d 7.41 (m, 2H), 7.23 (s, 1H), 7.17 (d, 1H, J = 7.5 Hz), 7.15 (t,
1H, J = 7.5 Hz), 7.06 (m, 1H), 7.02 (d, 1H, J = 7.5 Hz), 4.34 (s, 2H), 2.29 (s, 3H).
1
3ha H NMR (CD3OD): d 7.91 (d, 1H, J = 7.2 Hz), 7.77 (t, 2H, J = 7.2 Hz), 7.62 (d,
1H, J = 7.2 Hz), 6.47 (s, 2H) 6.34 (s, 1H), 4.37 (s, 2H), 3.64 (s, 6H).
3hb 1H NMR (CD3OD): d 7.81 (d, 1H, J = 7.5 Hz), 7.68 (q, 2H, J = 7.5 Hz), 7.47 (d, 1H,
J = 7.5 Hz), 7.19 (s, 1H), 7.14 (m, 2H), 7.01 (d, 1H, J = 6.0 Hz), 4.31 (s, 2H), 2.28 (s, 3H).
1
3hc H NMR (CD3OD): d 7.82 (d, 1H, J = 8.4 Hz), 7.72 (t, 2H, J = 8.4 Hz), 7.66 (d,
1H, J = 8.4 Hz), 7.55 (d, 1H, J = 8.4 Hz), 7.53 (d, 1H, J = 8.4 Hz), 7.47 (d, 1H,
J = 8.4 Hz), 7.21 (t, 1H, J = 8.4 Hz), 7.12 (t, 1H, J = 8.4 Hz), 4.49 (s, 2H).
3hd 1H NMR (CDCl3): d 7.67 (d, 1H, J = 7.8 Hz), 7.45 (t, 1H, J = 7.8 Hz), 7.33 (t, 1H,
J = 7.8 Hz), 7.16 (d, 2H, J = 8.4 Hz), 7.09 (d, 2H, J = 7.8 Hz), 6.93 (d, 2H, J = 8.4 Hz),
4.15 (s, 2H).
1
3ia H NMR (CD3OD): d 7.77 (s, 2H), 7.58 (s, 1H), 6.60 (s, 2H), 6.33 (s, 1H), 3.77 (s,
2H), 3.72 (s, 6H).
1
3ib H NMR (CD3OD): d 7.75 (d, 2H, J = 2.1 Hz), 7.57 (t, 1H, J = 2.1 Hz), 7.24 (s,
1H), 7.18 (s, 1H), 7.04 (s, 1H), 7.02 (s, 1H), 4.33 (s, 2H), 2.29 (s, 3H).
3ja 1H NMR (DMSO-d6): d 7.90 (d, 2H, J = 9.1 Hz), 7.43, (d, 2H, J = 9.1 Hz), 6.57 (s,
2H), 6.34 (s, 1H), 4.43 (s, 2H), 4.36 (s, 1H, NH), 3.55 (s, 6H), 2.96 (m, 1H), 1.27 (d, 6H,
J = 6.9 Hz).
1
3jb H NMR (CD3OD): d 7.75 (s, 1H), 7.74 (s, 1H), 7.57 (t, 1H, J = 2.1 Hz), 7.24 (s,
1H), 7.18 (s, 1H), 7.14 (d, 1H), 7.02 (d, 1H), 4.33 (s, 2H), 2.29 (s, 3H).
3jc 1H NMR (DMSO-d6): d 7.84 (d, 2H, J = 7.8 Hz), 7.38 (d, 1H, J = 7.8 Hz), 7.30 (d,
1H, J = 7.8 Hz), 7.13 (d, 2H, J = 7.8 Hz), 6.96 (t, 2H, J = 7.8 Hz), 4.36 (s, 2H), 2.85 (m,
1H), 1.29 (d, 6H, J = 6.6 Hz).
3jd 1H NMR (CD3OD): d 8.34 (s, 1H), 8.12 (d, 1H, J = 9.3 Hz), 7.88 (d, 2H,
J = 8.4 Hz), 7.84 (d, 1H, J = 9.3 Hz), 7.54 (t, 1H, J = 9.3 Hz), 7.40 (d, 2H, J = 8.4 Hz),
4.65 (s, 2H), 3.02 (m, 1H), 1.30 (d, 6H, J = 6.6 Hz).
3je 1H NMR (CDCl3): d 7.61 (d, 2H, J = 8.1 Hz), 7.04 (m, 4H), 6.83 (d, 4H,
J = 8.1 Hz), 4.03 (s, 2H), 2.80 (m, 1H), 1.13 (d, 6H, J = 6.6 Hz).

36
Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
1
4aa H NMR (CD3OD): d 7.58 (q, 1H, J = 8.4 Hz), 7.08 (t, 2H, J = 8.4 Hz), 3.09 (t,
2H, J = 7.6 Hz), 1.70 (m, 2H), 1.36 (m, 4H), 0.90 (t, 3H, J = 7.6 Hz).
4ba 1H NMR (CD3OD): d 7.59 (s, 1H), 7.43 (m, 2H), 3.09 (t, 2H, J = 7.6 Hz), 1.70 (m,
2H), 1.36 (m, 4H), 0.90 (t, 3H, J = 7.6 Hz).
4bb 1H NMR (CD3OD): d 7.59 (s, 1H), 7.43 (q, 2H, J = 7.6 Hz), 7.21 (m, 5H), 3.09 (t,
2H, J = 7.6 Hz), 2.70 (t, 2H, J = 7.6 Hz), 2.01 (m, 2H).
4ca 1H NMR (CD3OD): d 7.79 (d, 1H, J = 7.5 Hz), 7.63 (t, 2H, J = 7.5 Hz), 3.09 (t, 2H,
J = 7.2 Hz), 1.75 (m, 2H), 1.40 (m, 4H), 0.90 (t, 3H, J = 7.2 Hz).
1
4cb H NMR (CD3OD): d 7.74 (d, 2H, J = 6.6 Hz), 7.65 (d, 2H, J = 6.6 Hz), 7.34 (m,
3H), 7.12 (m, 2H), 4.10 (t, 2H, J = 7.4 Hz), 3.40 (d, 1H, J = 7.4 Hz).
1
4cc H NMR (CD3OD): d 7.74 (d, 1H, J = 6.6 Hz), 7.65 (d, 1H, J = 6.6 Hz), 3.95 (dd,
1H, J = 11.1, 2.7 Hz), 3.58 (m, 1H), 3.44 (m, 1H), 3.35 (dd, 1H), 3.08 (q, 1H, J = 13.5 Hz),
1.82 (m, 2H), 1.52 (m, 3H), 1.31 (m, 1H).
4da 1H NMR (CD3OD): d 7.62 (s, 1H), 7.59 (d, 1H, J = 7.2 Hz), 7.31 (m, 2H), 3.09 (t,
2H, J = 7.2 Hz), 1.75 (m, 2H), 1.40 (m, 4H), 0.90 (t, 3H, J = 7.2 Hz).
4db 1H NMR (DMSO-d6): d 7.70 (d, 1H, J = 5.7 Hz), 7.67 (s, 1H), 7.42 (t, 1H,
J = 5.7 Hz), 7.40 (d, 1H, J = 5.7 Hz), 4.42 (s, 1H, NH), 3.93 (dd, 1H), 3.56 (m, 1H),
3.44 (m, 1H), 3.35 (dd, 1H, J = 14.1, 4.8 Hz), 3.17 (q, 1H, 13.5), 2.19 (s, 3H), 1.82 (m,
2H), 1.52 (m, 3H), 1.35 (m, 1H).
1
4ea H NMR (CD3OD): d 8.11 (s, 1H), 8.10 (d, 1H), 7.80 (d, 1H, J = 7.2 Hz), 7.68 (t,
1H, J = 7.2 Hz), (m, 2H), 3.13 (t, 2H, J = 7.5 Hz), 1.75 (m, 2H), 1.40 (m, 4H), 0.91 (t, 3H,
J = 7.2 Hz).
4eb 1H NMR (CDCl3): d 8.15 (s, 1H), 7.95 (m, 2H), 7.89 (m, 2H), 7.60 (m, 3H), 7.38 (m,
2H), 4.61 (t, 2H, J = 6.0 Hz), 3.65 (t, 2H, J = 6.0 Hz).
4fa 1H NMR (CD3OD): d 7.26 (m, 4H), 3.07 (t, 2H, J = 7.5 Hz), 2.27 (s, 3H), 1.75 (m,
2H), 1.40 (m, 4H), 0.91 (t, 3H, J = 7.2 Hz).
4ga 1H NMR (CD3OD): d 7.21 (m, 8H), 4.04 (t, 2H, J = 6.3 Hz), 2.72 (t, 2H,
J = 6.3 Hz), 1.92 (m, 2H).
4gb 1H NMR (CD3OD): d 7.50 (m, 2H), 7.10 (m, 1H), 3.95 (dd, 1H, J = 11.1, 2.7 Hz),
3.56 (m, 1H), 3.45 (m, 1H), 3.35 (dd, 1H), 3.12 (q, 1H, J = 13.5 Hz), 1.82 (m, 2H), 1.52 (m,
3H), 1.35 (m, 1H).
1
4ha H NMR (CD3OD): d 7.80 (d, 1H, J = 7.5 Hz), 7.69 (t, 1H, J = 7.5 Hz), 7.64 (t,
1H, J = 7.5 Hz), 7.46 (d, 1H, J = 7.5 Hz), 3.09 (t, 2H, J = 7.2 Hz), 1.70 (m, 2H), 1.36
(m, 4H), 0.90 (t, 3H, J = 7.2 Hz).
1
4ja H NMR (CD3OD): d 7.76 (d, 2H, J = 8.1 Hz), 7.33 (d, 2H, J = 8.1 Hz), 3.11 (t,
2H, J = 7.2 Hz), 2.98 (m, 1H), 1.71 (m, 2H), 1.40 (m, 4H), 1.30 (d, 6H, J = 6.9 Hz),
0.91 (t, 3H, J = 7.2 Hz).
1
4jb H NMR (CD3OD): d 7.77 (d, 2H, J = 8.4 Hz), 7.34 (d, 2H, J = 8.4 Hz), 7.18 (m,
5H), 3.14 (d, 1H, J = 7.2 Hz), 2.98 (m, 1H), 2.75 (t, 2H, J = 7.2 Hz), 2.03 (m, 2H).
1
4jc H NMR (CD3OD): d 7.76 (d, 2H, J = 8.4 Hz), 7.34 (d, 2H, J = 8.4 Hz), 3.94 (dd,
1H, J = 10.5, 2.7 Hz), 3.67 (m, 1H), 3.44 (m, 1H), 3.33 (dd, 1H, J = 13.8, 4.5 Hz), 3.13 (q,
1H, J = 7.5 Hz), 2.99 (m, 1H), 1.83 (m, 2H), 1.58 (m, 3H), 1.35 (m, 1H), 1.30 (d, 6H,
J = 6.9 Hz).
5b 1H NMR (CD3OD): d 7.67 (s, 1H), 7.51 (s, 1H), 7.50 (s, 1H), 4.61 (t, 2H,
J = 7.8 Hz), 3.68 (t, 2H, J = 7.8 Hz).
5e 1H NMR (CDCl3): d 7.75 (s, 1H), 7.73 (t, 1H, J = 4.8 Hz), 7.40 (d, 2H, J = 4.8 Hz),
4.57 (t, 2H, J = 7.8 Hz), 3.65 (t, 2H, J = 7.8 Hz).

Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
37
5f 1H NMR (CD3OD): d 8.23 (s, 1H), 8.22 (d, 1H, J = 6.6 Hz), 7.89 (d, 1H, J = 6.6 Hz),
7.75 (t, 1H, J = 6.6 Hz), 4.59 (t, 2H, J = 7.8 Hz), 3.67 (t, 2H, J = 7.8 Hz).
5g 1H NMR (CD3OD): d 7.25 (m, 4H), 4.60 (t, 2H, J = 7.8 Hz), 3.60 (t, 2H,
J = 7.8 Hz), 2.30 (s, 3H).
1
5i H NMR (CDCl3): d 7.96 (d, 2H, J = 8.1 Hz), 7.61 (d, 2H, J = 8.1 Hz), 4.60 (t, 2H,
J = 7.8 Hz), 3.60 (t, 2H, J = 7.8 Hz).
1
6a H NMR (CD3OD): d 7.67 (m, 1H), 7.18 (m, 2H), 4.15 (m, 2H), 3.30 (m, 2H), 2.34
(m, 2H).
6b 1H NMR (CD3OD): d 7.65 (s, 1H), 7.48 (s, 1H), 7.47 (s, 1H), 4.17 (t, 2H,
J = 7.8 Hz), 3.30 (t, 2H), 2.35 (m, 2H).
6e 1H NMR (CDCl3): d 7.88 (s, 1H), 7.86 (t, 1H, J = 8.1 Hz), 7.69 (d, 2H, J = 8.1 Hz),
4.13 (t, 2H, J = 7.8 Hz), 3.30 (t, 2H), 2.33 (m, 2H).
6f 1H NMR (CD3OD): d 8.213 (s, 1H), 8.22 (d, 1H, J = 7.8 Hz), 7.89 (d, 1H,
J = 7.8 Hz), 7.73 (t, 1H, J = 7.8 Hz), 4.16 (t, 2H), 3.30 (t, 2H), 2.34 (m, 2H).
1
6g H NMR (CDCl3): d 7.25 (m, 4H), 4.60 (d, 2H, J = 7.8 Hz), 3.30 (m, 2H), 2.31 (m,
2H), 2.30 (s, 3H).
1
6i H NMR (CD3OD): d 7.65 (s, 1H), 7.49 (m, 2H, J = 6.6 Hz), 4.18 (m, 4H), 2.35 (d,
2H).
1
7a H NMR (DMSO-d6): d 8.31 (t, 1H, NH), 7.74 (m, 6H), 7.63 (t, 1H, J = 7.8 Hz),
7.51 (d, 1H, J = 7.8 Hz), 7.15 (t, 1H, J = 7.8 Hz), 6.74 (d, 1H, J = 8.1 Hz), 6.72 (s, 1H),
6.70 (d, 1H, J = 8.1 Hz), 4.62 (s, 2H), 3.64 (s, 3H), 3.63 (m, 2H), 2.79 (t, 2H, J = 7.5 Hz).
7b 1H NMR (CDCl3): d 7.81 (d, 2H, J = 8.4 Hz), 7.69 (d, 1H, J = 7.5 Hz), 7.67 (d, 1H,
J = 7.5 Hz), 7.61 (d, 2H, J = 8.4 Hz), 7.48 (t, 1H, J = 7.5 Hz), 7.37 (t, 1H, J = 7.2 Hz),
5.75 (t, 1H, NH), 4.65 (s, 2H), 4.58 (m, 1H), 2.35 (m, 2H), 1.95 (m, 2H), 1.78 (m, 2H).
7c 1H NMR (CDCl3): d 7.81 (d, 2H, J = 8.4 Hz), 7.67 (m, 4H), 7.49 (t, 1H, J = 7.2 Hz),
7.36 (t, 1H, J = 7.2 Hz), 7.28 (m, 3H), 7.16 (d, 2H, J = 7.8 Hz), 5.72 (t, 1H, NH), 4.67 (s,
2H), 3.75 (q, 2H), 2.87 (t, 2H, J = 8.2 Hz).
7d 1H NMR (CDCl3): d 7.84 (d, 2H, J = 8.7 Hz), 7.69 (d, 1H, J = 7.2 Hz), 7.66 (d, 1H,
J = 7.2 Hz), 7.62 (d, 2H, J = 8.7 Hz), 7.48 (t, 1H, J = 7.2 Hz), 7.37 (t, 1H, J = 7.2 Hz),
5.64 (t, 1H, NH), 4.65 (s, 2H), 3.46 (t, 2H, J = 6.3 Hz), 1.34 (m, 1H), 1.27 (m, 8H),
0.88 (t, 6H, J = 6.0 Hz).
7e 1H NMR (CDCl3): d 7.73 (d, 2H, J = 8.2 Hz), 7.67 (d, 1H, J = 8.2 Hz), 7.64 (d, 1H,
J = 8.15 Hz), 7.60 (d, 2H, J = 8.2 Hz), 7.49 (t, 1H, J = 8.2 Hz), 7.39 (t, 1H, J = 8.2 Hz),
4.61 (s, 2H), 4.58 (m, 1H), 3.18 (s, 3H), 2.95 (m, 2H), 2.31 (s, 3H), 2.00 (m, 3H), 1.80
(m, 3H), 1.10 (m, 1H).
1
7f H NMR (CDCl3): d 7.82 (d, 2H, J = 8.4 Hz), 7.68 (t, 2H, J = 9.0 Hz), 7.65 (d, 2H,
J = 8.4 Hz), 7.49 (t, 1H, J = 9.0 Hz), 7.37 (t, 1H, J = 9.0 Hz), 6.82 (d, 1H, J = 8.1 Hz),
6.71 (d, 1H, J = 8.1 Hz), 6.70 (s, 1H), 5.73 (t, 1H, NH), 4.67 (s, 2H), 3.86 (s, 3H), 3.84
(s, 3H), 3.77 (m, 2H), 2.83 (t, 2H, J = 6.9 Hz).
1
7g H NMR (CDCl3): d 8.21 (m, 1H), 7.74 (d, 2H, J = 8.7 Hz), 7.68 (m, 1H), 7.65 (d,
2H, J = 8.7 Hz), 7.55 (d, 1H), J = 8.4 Hz), 7.53 (t, 1H, J = 8.4 Hz), 7.37 (t, 1H,
J = 8.4 Hz), 6.69 (m, 1H), 6.63 (d, 1H, J = 8.4 Hz), 4.64 (s, 2H), 4.06 (t, 4H,
J = 4.5 Hz), 3.68 (t, 4H, J = 4.5 Hz).
7h 1H NMR (CDCl3): d 9.5 (s, 1H, NH), 7.81 (d, 2H, J = 8.7 Hz), 7.67 (d, 1H,
J = 7.4 Hz), 7.65 (d, 1H, J = 7.4 Hz), 7.58 (d, 2H, J = 8.7 Hz), 7.48 (t, 1H, J = 7.4 Hz),
7.46 (t, 1H, J = 7.4 Hz); 4.65 (s, 2H), 3.80 (m, 1H), 3.50 (td, 1H), 3.20 (m, 1H), 2.80
(m, 1H), 2.60 (m, 1H), 2.38 (s, 3H), 2.02 (dt, 2H), 1.99 (m, 2H), 1.82 (m, 2H).

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Y. Ding et al. / Bioorganic Chemistry 34 (2006) 26–38
7i 1H NMR (CDCl3): d 7.85 (d, 2H, J = 8.7 Hz), 7.70 (d, 1H, J = 7.5 Hz), 7.69 (d, 1H,
J = 7.5 Hz), 7.65 (d, 2H, J = 8.7 Hz), 7.50 (t, 1H, J = 7.5 Hz), 7.38 (t, 1H, J = 7.5 Hz),
5.99 (t, 1H, NH), 4.65 (s, 2H), 4.20 (m, 2H).
7j 1H NMR (CDCl3): d 7.84 (d, 2H, J = 8.4 Hz), 7.63 (d, 1H, J = 7.5 Hz), 7.61 (d, 2H,
J = 7.5 Hz), 7.60 (t, 2H, J = 8.4 Hz), 7.50 (t, 1H, J = 7.5 Hz); 7.39 (t, 1H, J = 7.5 Hz), 6.74
(d, 1H, J = 8.1 Hz), 6.62 (d, 1H, J = 8.1 Hz), 6.61 (s, 1H), 5.94 (s, 2H), 5.72 (t, 1H, NH),
4.66 (s, 2H), 3.70 (m, 2H), 2.78 (t, 2H, J = 6.6 Hz).
References
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