Novel Hepatitis C virus replicon inhibitors: Synthesis and structure-activity relationships of fused pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.01.096

The synthesis of several pyrido[2,3-d]pyrimidine and pyrimido[4,5-d] pyrimidine analogs is described with one such analog possessing subnanomolar potency in both genotype 1a and 1b cell culture HCV replicon assays.

Full text of DOI:10.1016/j.bmcl.2012.01.096

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2212–2215
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel Hepatitis C virus replicon inhibitors: Synthesis and structure–activity
relationships of fused pyrimidine derivatives
⇑
A. Chris Krueger , Darold L. Madigan, David W. Beno, David A. Betebenner, Robert Carrick, Brian E. Green,
Wenping He, Dachun Liu, Clarence J. Maring, Keith F. McDaniel, Hongmei Mo, Akhteruzzaman Molla,
Christopher E. Motter, Tami J. Pilot-Matias, Michael D. Tufano, Dale J. Kempf
Abbott Labs, Global Pharmaceutical Research and Development, Department R4DH, Building AP52-N, 200 Abbott Park Road, Abbott Park, IL 60064, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of several pyrido[2,3-d]pyrimidine and pyrimido[4,5-d]pyrimidine analogs is described
with one such analog possessing subnanomolar potency in both genotype 1a and 1b cell culture HCV
replicon assays.
Received 6 December 2011
Revised 24 January 2012
Accepted 25 January 2012
Available online 2 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C
Replicon inhibitors
Pyrido[2,3-d]pyrimidines
Pyrimido[4,5-d]pyrimidines
Hepatitis C virus (HCV) is a (+)-strand RNA virus of the Flaviviridae
family that was first identified in 1989.1 HCV is a common pathogen
that can lead to cirrhosis, hepatocellular carcinoma (HCC) and liver
failure. It is estimated that 170 million people were infected world-
wide in the year 2000, and that the virus is responsible for at least
10,000 deaths annually in the United States alone.2 HCV has six
major genotype classes, with genotypes 1 and 2 being the most
prevalent in the United States, Europe, and Japan.3 Within genotype
1, the majority of patients are infected with subgenotypes 1a or 1b.
Recently two new protease inhibitor drugs, boceprevir and telapre-
vir, were approved by the FDA. However these drugs still must be
used in combination with the standard of care which imparts
several drug-related toxicities. Thus there is still an urgent need
for new HCV drugs with diverse modes of action.4
Our research team has been pursuing inhibition of the Hepatitis
C virus by targeting several non-structural viral enzymes.5 We
have also initiated a broad screening program towards identifying
novel leads that have antiviral potency in a HCV subgenomic
replicon assay.6 One such lead was a substituted naphthyridine
derivative (1), shown in Table 1. We were pleased with the geno-
type 1b replicon potency of (EC50 18 nM) as a starting point and
hoped to improve upon the EC50 for genotype 1a, which was
100-fold higher than in 1b. Thus we initially explored the effect
of replacing each of the nitrogen atoms independently in the
naphthyridine ring system with carbon atoms to generate quino-
line derivatives 2 and 3 (Scheme 1). The data indicate that both
nitrogens are required for obtaining sub micromolar potency
against the 1b replicon strain in this series. In an effort to obtain
sub micromolar potency against both 1a and 1b virus strains and
to decrease the cytotoxicity associated with the lead structures
(MTT values), we embarked upon an effort to explore further mod-
ifications to the fused ring system.
We were able to construct a series of substituted pyrido
[2,3-d]pyrimidine analogs by employing a short synthetic se-
quence (Scheme 2).7 A variety of methyl ketones 14 were treated
with sodium metal and ethyl formate to generate the sodium salt
of alkyl substituted hydroxyl enal compounds 15 which were
cyclized with 2-cyanoacetamide to form the corresponding substi-
tuted oxopyridines 16. Conversion to the corresponding bromo-
pyridines 17 and subsequent reaction with ammonia under high
pressure generated the aminopyridines 18, of which the amino
group could be converted to the dimethylamidine functionality.
Reaction of these substituted pyrido dimethylamidines 19 with
the substituted phenylsulfanyl anilines 5–10 from Scheme 1 in
the presence of hot acetic acid generated a wide variety of the
desired pyrido[2,3-d]pyrimidines 20–47.8 The data from Table 2
show that when the methyl substituted pyrido[2,3-d]pyrimidine
20 was compared to napthyridine analog 1, analog 20 was less
potent but displayed somewhat lower toxicity in the assay. Within
the pyrido[2,3-d]pyrimidine series we also generated several
analogs that had substituents on the para position of the phenyl
thio ring (R2 group in Scheme 2). In several cases, a hydroxyl group
⇑
Corresponding author. Tel.: +1 847 938 2944; fax: +1 847 938 2756.
E-mail address: a.chris.krueger@abbott.com (A. Chris Krueger).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.096

A. Chris Krueger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2212–2215
2213
Table 1
Cell culture replicon potency of analogs 1–3
HN
S
A
B
MTTc
(lM) TD50
a,b
a,b
Compd
A
B
Replicon 1a EC50
(
lM)
Replicon 1b EC50
(
lM)
1
2
3
N
N
C
N
C
N
1.8
15.7
—
0.018
9.9
1.8
8.6
15.9
2.6
a
b
c
EC50 values in all tables are means of at least two independent determinations, with a maximum of threefold value variations.
Assay run with 5% fetal calf serum.
Cytotoxicity measured in Huh-7 cells.
Cl
a,b,c
d
HN
+
H2N
S
S
N
O2N
OH
11
N
R
2
4
5 : R= H
9 : R= F
6 : R= OH 10 : R= OMe
7 : R= NHAc
8 : R= NHSO2Me
Cl
O
O
e, f
g
HN
N
OEt
S
N
N
H
12
13
3
Scheme 1. Reagents and conditions: (a) trifluoroacetic anhydride, Et3N, CH2Cl2, 24 h, 90–100%; (b) Na2CO3, DMF, 80 °C, 18 h, 90–98%; (c) 10 equiv Fe, 10 equiv NH4Cl, MeOH,
water, reflux, 2 h, 79–100%; (d) compound 5, NaOtBu, Pd2(dba)3, 2,8,9-tri-iso-butyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3] undecane, toluene, 100 °C, 18 h, 25%; (e) NaOH,
water, 150 psi, 180 °C, 16 h, 86%; (f) excess POCl3, 60 °C, 2.5 hrs, 88%; (g) compound 5, EtOH, 80 °C, 24 h, 11%.
CN
O
CN
b
ONa
c
O
a
R1
R1
R1
N
H
16
O
R1
N
Br
14
15
17
d
HN
N
CN
N
f
e
S
CN
N
R1
N
N
R1
N
R2
R1
N
NH2
19
20 - 47
18
Scheme 2. Reagents and conditions: (a) ethyl formate, Na°, Et2O, 0 °C to 25 °C, 18 h, 45–63%; (b) 2-cyanoacetamide, water, piperidine acetate, reflux, 2 h, 55–78%; (c)
tetrabutylammonium bromide, P2O5, toluene, reflux, 5 h, 90–98%; (d) ammonia, EtOH, sealed tube, 130 °C, 20 h, 82–93%; (e) N,N-dimethylforamide dimethyl acetal, toluene,
reflux, 3 h, 98–100%; (f) compound 5–10, acetic acid solvent, reflux, 1.5 h, 10–54%.
at this position provided the greatest potency boost when all other
parts of the analogs were the same.
This was especially true in the cases of analogs 22 and 33.
Analog 33 was the most potent of the pyrido[2,3-d]pyrimidines
prepared, having submicromolar potency against both subtype 1a
and 1b replicons. Seemingly small modifications were made to this
isopropyl analog 33, such as changing the isopropyl group to a
cyclopropyl (compound 39) or isobutyl (compound 45), which
produced a potency loss of at least a 100-fold in the 1b replicon
and roughly 50-fold in the 1a replicon. In the absence of knowledge

2214
A. Chris Krueger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2212–2215
Table 2
Cell culture replicon potency of analogs 20–47
HN
S
N
R1
N
N
R2
Compd
R1
R2
Replicon 1a EC50
(l
M)
Replicon 1b EC50
(lM)
MTT (lM)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
Ethyl
Ethyl
n-Propyl
n-Propyl
n-Propyl
i-Propyl
i-Propyl
i-Propyl
i-Propyl
i-Propyl
i-Propyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclobutyl
Cyclobutyl
Cyclobutyl
i-Butyl
H
NHAc
OH
OMe
F
H
NHAc
OH
H
NHAc
OH
H
NHAc
OH
OMe
F
NHSO2Me
H
NHAc
OH
H
NHAc
OH
H
NHAc
OH
13.3
>50
23.4
44.5
10.5
18.9
>12.5
62.2
9.5
13.8
7.1
10.3
2.6
0.17
11.4
3.2
18.9
10.4
15.2
10.5
6.1
15.8
11.3
13.3
27.4
14.3
4.6
3.84
>50
0.05
13.3
3.0
22.2
>50
>100
28.6
6.1
22.7
>12.5
78.0
11.8
>25
31.1
8.0
28.8
22.9
8.8
4.8
>50
9.9
29.8
17.4
3.6
49.2
31.8
7.5
38.5
11.2
7.1
3.4
6.9
10.0
0.32
0.63
0.25
3.4
13.8
0.016
1.7
1.8
11.2
3.7
1.7
1.6
1.6
3.7
3.1
1.0
3.3
2.4
0.47
0.88
i-Butyl
i-Butyl
tert-Butyl
Cyclohexyl
OH
OH
2.7
>3.12
of the mechanism of inhibition and structural information on
inhibitor binding, it is difficult to rationalize these results. Other
difficult to interpret SAR data include the good potency of analogs
22 and 30 against replicon 1b, verses the lack of potency for analog
27. Another large 1b potency difference is seen between analogs 21
and 29. Some calculated properties of the analogs from Table 2
were obtained in order to help find some correlation between
the observed SAR. The cLogP of these analogs varied between 5
and 8, and the PSA was in the 50–80 range. Unfortunately these
values did not help us to find any reasonable correlations within
the SAR. We also generated other analogs that contained a pyrim-
ido[4,5-d]pyrimidine bicyclic ring system (Scheme 3).7 The reac-
tion of 2-alkyl-2-thiopseudourea hydrobromide 48 or 49 with
(ethoxymethylene)malononitrile (50) generated an amino-(alkyl-
sulfanyl)pyrimidine 51 or 52. The amine in 51 was converted to
an amidine functionality and cyclized with aniline 6 in hot acetic
CN
CN
N
b
NC
CN
HBr
a
NH
N
N
+
R
S
NH2
R
S
N
NH2
S
N
N
OEt
51, R=Me
52, R=Ph
48, R=Me
49, R=Ph
53
50
c
cpd 52
f,g,b,c
d or e
HN
HN
N
or
HN
N
S
S
S
N
N
N
N
N
N
R1
N
R2
N
N
OH
RO
N
OH
S
N
OH
58 - 61
54
55 - 57
Scheme 3. Reagents and conditions: (a) Hunig’s base, ethanol, 4 h, 39–45%; (b) N,N-dimethylforamide dimethyl acetal, toluene, reflux, 3 h, 100%; (c) 6, acetic acid solvent,
reflux, 1.5 h, 15–45%; (d) excess sodium alkoxide, EtOH, heating, 32%; (e) excess morpholine or piperidine, microwave irradiation, 180 °C, 2 h, 38–65%; (f) mCPBA, CH2Cl2,
95%; (g) excess ethyl amine or methyl amine, EtOH, heating, 37–44%.

A. Chris Krueger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2212–2215
2215
Table 3
Cell culture replicon potency of analogs 54–61
HN
HN
and
S
S
N
N
N
N
R1
N
N
N
OH
RO
N
N
OH
R2
55 - 57
58 - 61
Compd
R
R1
R2
Replicon 1a EC50
(lM)
Replicon 1b EC50
(lM)
MTT (lM)
54
55
56
57
58
59
60
61
see Scheme 3
4.8
>8
7.3
5.3
14.1
22.0
47.6
>50
0.01
>8
12.0
3.5
7.3
2.8
46.9
>8
Methyl
Ethyl
Phenyl
—
—
—
—
—
—
—
—
—
29.7
17.1
>50
>50
>50
>50
Morpholine
Piperidine
Ethyl
H
30.0
>50
—
Methyl
Methyl
acid to provide the ethylsulfanyl analog 54. The ethylsulfanyl
functionality could be displaced with other nucleophiles to provide
the corresponding ether 55–57 and amine 58, 59 analogs directly.
Alternately, the acyclic amine analogs 60 and 61 were generated by
first oxidizing the benzyl mercapto group in 52 to the correspond-
ing benzyl sulfone followed by displacement of this group with the
corresponding amines. Amidine formation and cyclization pro-
vided the desired amine analogs 60 and 61. The ethylsulfanyl-
pyrimidopyrimidine analog 54 displayed excellent 1b replicon
potency (EC50 10 nM), however it was much weaker against the
assessed for inhibitory potency in genotypes 1a and 1b HCV
replicon assays. Overall we observed that the SAR was very sensi-
tive to seemingly small structural modifications and that the most
potent analog displayed submicromolar potency in one cell culture
assay containing 40% human serum.
References and notes
1. Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
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Moyer, L. A.; Kaslow, R. A.; Margolis, H. S. N. Engl. J. Med. 1999, 341, 556; (b)
Hepatology, 1997, 26, pp. 2S–10S.
1a replicon (EC50 4.8 lM, Table 3). Unfortunately the EC50s of all
of the ether 55–57 and amine 58–61 analogs were greater than 2
micromolar in both assays.
3. Simmonds, P. J. Hepatol. 1999, 31, 54.
4. Rice, C. Nature 2011, 474, S8.
The most potent analog, iso-propyl pyrido[2,3-d]pyrimidine 33,
was retested in the replicon assay in the presence of 40% human
serum to access how much potency loss would occur due to unpro-
ductive protein binding. The potency loss was 10-fold in the 1b
replicon assay (16 to 170 nm). We also evaluated the pharmacoki-
netic properties of analog 33 after i.v. and oral administration in
rats (2.5 mg/kg as a solution). Compound 33 displayed high
clearance (1.58 L/h/kg) and a relatively short half-life (3.1 h). Cmax
5. (a) Randolph, J. T.; Zhang, X.; Huang, P. P.; Klein, L. L.; Kurtz, K. A.;
Konstantinidis, A. K.; He, W.; Kati, W. M.; Kempf, D. J. Bioorg. Med. Chem. Lett.
2008, 18, 2745; (b) Krueger, A. C.; Madigan, D. L.; Green, B. E.; Hutchinson, D.
K.; Jiang, W. W.; Kati, W. M.; Liu, Y.; Maring, C. J.; Masse, S. V.; McDaniel, K. F.;
Middleton, T. R.; Mo, H.; Molla, A.; Montgomery, D. A.; Ng, T. I.; Kempf, D. J.
Bioorg. Med. Chem. Lett. 2007, 17, 2289.
6. Ikeda, M.; Yi, M.; Li, K.; Lemon, S. M. J. Virol. 2002, 76, 2997; (b) Blight, K. J.;
Kolykhalov, A.; Rice, C. M. Science 2000, 290, 1972.
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Rockway, T. W.; Cooper, C. S.; Anderson, D. D.; Donner, P. L.; Green, B. E.;
Kempf, D. J.; Liu, D.; McDaniel, K. F.; Madigan, D. L.; Motter, C. E.; Pratt, J. K.;
Shanley, J. P.; Tufano, M. D.; Wagner, R.; Zhang, R.; Molla, A.; Mo, H.; Pilot-
Matias, T. J.; Masse, S. VL.; Carrick, R. J.; He, W.; Lu, L.; Grampovnik, D. J. Patent
Application WO 2007/081517, 19 July 2007.
8. Tsou, H-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
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9. Krishnan, P.; Pilot-Matias, T. J. unpublished results.
(0.07 lM), plasma AUC (1.58 lg h/mL) and bioavailability (10%)
after oral administration were low. In a future publication we will
disclose our efforts related to improving upon this pharmacoki-
netic profile. Finally, based on resistance studies which will be
published in the future,9 we believe that the target of these HCV
replicon inhibitors is the HCV NS5A protein which has been shown
to be critical for viral replication.10
10. Nettles, R. E.; Gao, M.; Bifano, M.; Chung, E.; Persson, A.; Marbury, T. C.;
Goldwater, R.; DeMicco, M. P.; Rodriquez-Torres, M.; Vutikullird, A.; Fuentes,
E.; Lawitz, E.; Lopez-Talavera, J. C.; Grasela, D. M. Hepatology 2011, 54, 1956.
In summary, a series of substituted pyrido[2,3-d]pyrimidine
and pyrimido[4,5-d]pyrimidine analogs were synthesized and