Discovery of ITX 4520: A highly potent orally bioavailable hepatitis C virus entry inhibitor

Article abstract of DOI:10.1016/j.bmcl.2012.06.038

The manuscript reports an identification of a highly potent, orally bioavailable hepatitis C virus entry inhibitor through optimization of a previously reported class of molecules (1) that were not stable in the rat plasma. Compound 39 (ITX 4520) exhibited an excellent PK profile in both rats and dogs with good oral exposure, half-life and oral bioavailability. The compound is also well-tolerated in the preliminary in vivo toxicity studies and has been selected as a pre-clinical candidate for our HCV clinical pipeline.

Full text of DOI:10.1016/j.bmcl.2012.06.038

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4955–4961
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of ITX 4520: A highly potent orally bioavailable hepatitis C virus
entry inhibitor
Gopi Kumar Mittapalli, Fang Zhao, Andrew Jackson, Hongfeng Gao, Haekyung Lee, Stephine Chow,
⇑
Maninder Pal Kaur, Natalie Nguyen, Robert Zamboni, Jeffrey McKelvy, Flossie Wong-Staal ,
James E. Macdonald
⇑
iTherX Pharmaceuticals, Inc., PO Box 910530, San Diego, CA 92191-0530, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The manuscript reports an identification of a highly potent, orally bioavailable hepatitis C virus entry
inhibitor through optimization of a previously reported class of molecules (1) that were not stable in
the rat plasma. Compound 39 (ITX 4520) exhibited an excellent PK profile in both rats and dogs with good
oral exposure, half-life and oral bioavailability. The compound is also well-tolerated in the preliminary
in vivo toxicity studies and has been selected as a pre-clinical candidate for our HCV clinical pipeline.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 May 2012
Revised 10 June 2012
Accepted 12 June 2012
Available online 19 June 2012
Keywords:
Hepatitis C virus
Entry inhibitors
Bioisostere
Oral bioavailability
Scavenger receptor B-1
Hepatitis C virus is a leading cause of chronic liver disease,
cirrhosis, liver failure and primary liver cancer that affects almost
3% of the world’s population or 170 million people worldwide.1,2
There is currently no vaccine available to prevent hepatitis C virus
infection due to its high degree of strain variation. The standard of
care (SOC), until recently, has been a combination therapy with
different stages of the HCV life cycle. We have recently reported13
a series of highly potent small molecule HCV entry inhibitors as
exemplified by compound 1 (Fig. 1) that showed an IC50 of
1.5 nM in the HCV2aCH-Rluc entry assay. Based on binding compe-
tition and resistance profiles,14 the class of compounds appears to
be mechanistically similar to ITX 506115 and targets the scavenger
receptor B-1 (SR-B1), one of the cellular co-factors required for
HCV entry. The series of compounds, though potent, is rapidly
metabolized in plasma due to the presence of the labile methyl
ester group. The corresponding amide derivatives were found to
be stable in plasma; however, the compounds are not only less
potent than compound 1 but also have poor pharmacokinetic prop-
erties such as low exposure, shorter half-life and no oral bioavail-
ability. Here, we report the identification of methyl ester
bioisosteric groups and an orally bioavailable HCV entry inhibitor
pegylated a-interferon and ribavirin and effective only in less than
50% of patients infected with most common genotype-1.3 Most of
the efforts are focused on the development of viral protease and
polymerase inhibitors that are crucial for viral replication. Very
recently, the FDA approved two protease inhibitors Incivek™ and
Victrelis™ to use in combination with pegylated a-interferon and
ribavirin. However, the occurrence of side effects and rapidly
emerging resistant mutants suggest the need for new, more effec-
tive and safer antivirals targeting multiple steps of the HCV life cy-
cle for the treatment of HCV.4–6
Virus entry into the host cell is a crucial step in the HCV life cy-
cle, requiring at least five cellular co-factors, CD-81,7 claudin,8,9
occludin,10 scavenger receptor-B111 and the more recently discov-
ered epidermal growth factor receptors 1 and 2,12 and represents
an alternative potential target for therapeutic intervention. The en-
try inhibitors can prevent the re-infection of cured cells and can be
most effective in combination with other antivirals targeting
O
6
O
N
NH
N
N
O
O
1
⇑
HCVcc IC50 = 1.5 nM
Corresponding authors. Tel.: +1 858 824 1114; fax: +1 858 824 1112.
E-mail addresses: fwongstaal@itherx.com (F. Wong-Staal), 2jemacd@gmail.com
(J.E. Macdonald).
Figure 1. Previously reported HCV entry inhibitor.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.038

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G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4955–4961
Table 1
39 (ITX 4520) with a good pharmacokinetic profile that has been
selected as a pre-clinical candidate.
Exploration of groups at the 6-position
We have investigated various groups at the 6-position to im-
prove the pharmacokinetics and to retain high antiviral potency.
We previously identified13 that the 4,6-disubstituted-2-oxo/ami-
no-pyrimidine or N,N-dimethyl sulfonamide groups with a three
carbon linker at the 9-position were optimal for the antiviral activ-
ity and utilized these best side chains to explore the groups at the
6-position. We have primarily explored a variety of hydrogen-bond
acceptor groups at the 6-position such as methyl sulfone, cyano,
nitro, as well as known bioisosteric groups of the methyl ester16a
such as oxadiazole,16b oxazole, triazole and tetrazoles since a
hydrogen-bond acceptor at this position seems to be essential for
the activity. The synthesis of target compounds 16–36 (Table 1)
is outlined in Schemes 1–3.
6
R
N
9
R1
Compound
R
R1
HCVcc IC50 (nM)
1.5
O
15
CO2CH3
N
N
NH
O
S
O
N
N
16
10
H3CO
OCH3
O
O
O
S
O
Fisher indole condensation of appropriate phenylhydrazines
with cyclohexanone provided the tetrahydrocarbazoles 2 and
4–6. The esterification of compound 2 gave intermediate 3. Com-
pounds 3–6 were then alkylated with different alkylating agents
to obtain the intermediates 7–14. The chloro displacement of
compounds 7–10 with 2-oxo pyrimidines afforded the target
compounds 15, 17, 18, 21, and 23. The intermediates 11 and
12, after tdeprotection of the Boc group, were reacted with 2-
chloro-4,6-dimethoxy pyrimidine to obtain the desired com-
pounds 16 and 22. The deprotection of the diethyl acetal of 13
and 14 followed by coupling with 1,2-diaminobenzene yielded
the compounds 20 and 25. Oxadiazole analogs 26–31 were pre-
pared by introducing different side-chains at the 9-position of
the intermediates 3a, 3b and 5a following the similar methods
used for the preparation of compounds 17–19 from 4. The 2-
methyl-1,3,4-oxadiazole intermediate 3b was prepared by the
reaction of intermediate 3 with hydrazine and subsequent cou-
pling with triethyl orthoacetate. The 5-methyl-1,2,4-oxadiazole
intermediate 5a was synthesized by the addition of hydroxyl-
amine to 5, followed by the condensation with methyl acetate.
The intermediate 3-methyl-1,2, 4-oxaxdiazole (3a) was prepared
by the condensation of methyl 2,3,4,9-tetrahydro-1H-carbazole-
6-carboxylate (3) with the acetamide oxime. The compound 15,
after ester hydrolysis, was coupled with ethanolamine to obtain
the corresponding amide analog 15a, which was subsequently
converted to oxazoline analog 32 with thionyl chloride and NaH-
CO3. The oxidation of oxazoline 32 using bromotrichloromethane
and DBU afforded the oxazole analog 33 (Scheme 2). The nitro
group of 21 was reduced with Zn/AcOH in MeOH and the result-
ing amine, after diazotization, was subsequently treated with
NaN3 to afford the intermediate 21a (Scheme 3). The azide
functionality of the intermediate 21a was then converted to tria-
zoles 34 and 35 via click chemistry using appropriate alkynes. The
tetrazole analogue 36 was prepared by reaction of the azide
group of 21a with triethyl orthoformate. All compounds were
characterized by 1H NMR, mass spectra17 and were tested in
the HCV2aCH-Rluc (HCVcc-2a) infectious assay.18–20 Since the
first tissue culture infectious clone is of genotype 2a,18 the origi-
nal, native HCV2a clone has long been the gold standard for
in vitro assays. Genotype 1 clones are chimeras, but are used in
some experiments to demonstrate genotype specificity, or lack
of. We did not run duplicate assays for both genotype 2a and 1
at all times, as the series continues to demonstrate the nearly
equal potency for both genotypes, as demonstrated in Table 6.
The antiviral activity data is presented in Table 1.
17
18
19
20
21
50
N
N
N
O
S
O
30
N
O
S
N
O
S
O
O
500
50
NH
O
S
O
N
O
O-
N
1.5
N
N
N
O
NH
N
N
N
22
23
15
15
H3CO
OCH3
O
N
N
O
N
N
S
N
24
25
50
50
O
NH
N
O
O
O
O
N
O
26
27
28
29
5
N
N
N
O
N
15
1.5
1.5
N
N
N
N
N
N
N
N
O
N
N
N
O
O
O
O
N
N
N
N
30
1.5
F3C
O
N
S
N
O
31
32
1.5
50
The replacement of the methyl ester group of 1 by the methyl
sulfone group as exemplified by 16–20 resulted in 6- to 300-fold
reduction of the potency depending on the type of side chain
present at the 9-position. While the nitrile analogs 22–25 were
10- to 30-fold less active, irrespective of the side chain present at
the 9-position, the nitro analog 21 was equally potent to the
N
O
O
N
N
N

G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4955–4961
4957
33 was equally potent as the 3-methyl-1,2,4-oxadiazole analog 29,
whereas the oxazoline analog 32 displayed 33-fold less activity
than the corresponding oxazole analog 29. We also explored tria-
zoles at 6-position as shown by 34 and 35, but observed inferior
activity compared to the corresponding 3-methyl-1,2,4-oxadiazole
analogs. The tetrazole analog 36 was found to be as potent as
3-methyl-1,2,4-oxadiazole analog 28.
With identification of the bioisosteric groups of methyl ester,
we have evaluated the in vivo pharmacokinetic properties of the
compounds 16, 29 and 31 in rats at 2 mg/kg via i.v. route (Table
2). The methylsulfone analog 16 showed better exposure and re-
duced clearance than the corresponding methyl ester analog 1,
but had shorter half-life. Surprisingly, the oxadiazole analogs 29
and 31 have higher clearance than the methyl ester analogue 1.
The MS analysis of microsomal samples of 29 revealed the oxida-
tive metabolism of the molecule (addition of 32 mass units to
the parent molecule), which probably occurs at the side chain
and on the tetrahydrocarbazole ring.
Table 1 (continued)
Compound
R
R1
HCVcc IC50 (nM)
1.5
O
O
O
O
O
33
34
35
36
N
N
N
N
N
N
F
N
N
N
50
15
1.5
N
N
N
N
N
N
nC3H7
N
N
N
N
Consequently, we focused efforts on modifying the side chain at
the 9-position and tetrahydro ring to improve the metabolic stabil-
ity and pharmacokinetics (Table 3). We have found that the
replacement of 2-oxopyrimidines with difluorosubstituted phenols
or thiophenols, as exemplified by 37–40, considerably improved
the metabolic stability and was also tolerable in terms of the
compound 1. Notably, 3-methyl-1,2,4-oxadiazole analogs 28–31
were as potent as the compound 1. Moreover, the 3-methyl-
1,2,4-oxadiazole group at 6-position was tolerable with different
side chains at 9-position. The regio-isomeric analogs of 3-
methyl-1,2,4-oxadiazole 26 and 27 were 3- and 10-fold less potent
compared to the corresponding compound 29. The oxazole analog
R
R
R
R
i
iii
iv
N
N
N
H
NHNH2
n
n
X
R1
ii
R = CO2H, SO2CH3,
CN, NO2
2
R = CO2H
3 R = CO2CH3
4 R = SO2CH3
15-18; 20-25
(for R1 see Table 1)
7 X = Cl; R = CO2CH3; n= 2
8
X = Cl; R = SO2CH3; n= 2
9 X = Cl; R = CN; n= 2
10
5
R = CN
6 R = NO2
X = Cl; R = NO2; n= 2
11 X = NHBoc; R = SO2CH3; n = 2
12
X = NHBoc; R = CN; n = 2
13 X = CH(OC2H5)2; R = SO2CH3; n = 1
14
X = CH(OC2H5)2; R = CN; n = 1
19 X = SO2N(CH3)2; R = SO2CH3; n =2
v
R
3
R
N
N
N
O
iii
iv
;
27 R =
26
R =
O
N
N
H
N
5
vi
O
N
N
N
N
3b R =
O
N
O
5a
26 - 27
R =
N
N
O
O
N
O
30 R1 =
31 R1 =
R1 =
N
N
N
N
N
N
N
28
29
N
iii
iv
O
vii
F3C
O
O
N
N
H
N
O
H
O
R1
R1
=
S
N
3
3a
O
28 - 31
Scheme 1. Reagents and conditions: (i) (a) cyclohexanone, AcOH, reflux, overnight; (ii) MeOH, H2SO4, reflux, overnight; (iii) for compounds 7–10: NaH, 1-bromo-3-chloro
propane, DMF, rt, 3 h; for compounds 11 and 12: NaH, DMF, BocNH-(CH2)3-OTs, rt, 18 h; for compounds 13 and 14: NaH, 3-chloropropionaldehyde diethylacetal, n-
Bu4NH4+Iꢀ, DMF, 50 °C, 24 h; for compound 19 and 31: NaH, 3-chloro-N,N-dimethylpropane-1-sulfonamide, n-Bu4NH4+Iꢀ, DMF, rt, 16 h; (iv) for compounds 16 and 22: (a)
TFA, CH2Cl2, 0 °C to rt, 3 h; (b) 2-chloro-4,6-dimethoxy pyrimidine, DMF, DIEA, 80 °C, overnight; for compound 20 and 25: (a) AcOH, aq HCl, 0 °C to rt, 3 h; (b) o-phenylene
diamine, DMF, 150 °C, overnight; for compounds 17, 18, 21, 23, 26–30: 2-oxo pyrimidines, K2CO3, 80 °C, overnight; (v) (a) NH2NH2, MeOH, reflux, 16 h; (b) triethyl
orthoacetate, MeOH, reflux, 16 h; (vi) (a) NaOMe, NH2OH.HCl, MeOH, reflux, 2 days; (b) MeOAc, K2CO3, reflux, 16 h; (vii) acetamide oxime, KOtBu, THF, reflux, 16 h.

4958
G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4955–4961
O
O
O
O
HO
N
N
N
H
O
i
ii
iii
N
N
N
N
O
O
O
O
N
N
N
N
N
N
N
N
33
15a
32
15
Scheme 2. Reagents and conditions: (i) (a) 2 N aq NaOH, MeOH, 60 °C, 3 h; (b) ethanolamine, HATU, Et3N, CH2Cl2, rt, 6 h; (ii) SOCl2, CH2Cl2, 10 min, then NaHCO3, 30 min, rt;
(iii) BrCCl3, DBU, CH2Cl2, 0 °C to rt, 16 h.
O2N
N3
R
N
N
N
N
i
;
F
N
35 R =
N
ii
34 R =
36 R =
N
N
N
2
O
O
O
N
N
N
N
N
N
N
N
N
N
21a
21
34 - 36
Scheme 3. Reagents and conditions: (i) (a) Zn, MeOH/AcOH, 3 h; (b) NaNO2, AcOH/H2O, 0 °C, 20 min, then aq.NaN3, rt, 16 h; (ii) for compounds 34 and 35: 1-alkynes, 1 M
sodium ascorbate, tBuOH/H2O, 0.3 M CuSO4, rt, 16 h; for compound 36: triethyl orthoformate, 2-methoxy ethanol, NaN3, Yb(OTf)3ꢁH2O, 100 °C, 16 h.
antiviral activity (Table 3). Compound 43 with a 5-fluoro benz-
imidazole side chain also displayed good microsomal stability in
rats, but was not stable in human microsomes. The difluorophenyl
sulfone and difluorophenyl sulfoxide side chain analogs 41 and 42
were found to have moderate stability in rats and poor stability in
human microsomes. We have introduced the fluorine atom into
the carbon-linker of the side chain as exemplified by 46, which
not only had good microsomal stability in both rat and human
Table 2
Intravenous pharmacokinetic properties of selected compounds in ratsa
Compd AUCall (
lG/L h) Cmax (lg/L) t1/2 (h) Vz (L/kg) Cl (L/h/kg)
1
521
902
497
520
182
2.16
0.63
3.22
0.71
11.28
3.51
18.94
3.96
3.75
2.21
4.09
3.97
16
29
31
1076
1051
1240
a
iv 2 mg/kg.
Table 3
Exploration of side chains at 6-position
N
R2
O
6
N
R3
N 9
X
R1
Compd
R1
R2 and R3
X
HCVcca IC50 (nM)
HLMb (t1/2
)
RLMb (t1/2
)
O
37
H
H
H
H
1.5
55
53
F
F
O
38
39
40
1.5
1.5
1.5
22
43
27
34
59
25
F
F
S
F
F
S
F
F
O
S
O
41
H
5
9
19
F
F

G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4955–4961
4959
Table 3 (continued)
Compd
R1
R2 and R3
X
HCVcca IC50 (nM)
HLMb (t1/2
)
RLMb (t1/2
)
O
S
42
43
H
15
4
18
F
F
NH
N
H
1.5
8
110
F
F
F
O
S
46
51
F
1.5
5
65
8
49
22
F
F
O
O
H
O
N
N
N
52
55
H
1.5
8
28
O
F
F
N
H
H
15
15
8
8
S
F
F
56
18
18
F
F
a
HCV2aCH-Rluc entry assay.
b
1 lM of compound was incubated with 0.5 mg/mL of human or rat hepatic microsomes; t1/2 in minutes; HLM: Human liver microsomes; RLM: Rat liver microsomes.
N
N
N
N
N
N
N
N
O
O
O
O
iii
i
ii
N
F
N
H
N
N
F
OH
O
F
O
O
3a
45
46
44
F
F
Scheme 4. Reagents and conditions: (i) NaH, ( )-epichlorohydrin, DMF, n-Bu4NH4+Iꢀ, 2 h; (ii) 3,5-difluorophenol, Cs2CO3, THF, 60 °C, 16 h; (iii) DAST, CH2Cl2, 0 °C to rt, 1 h.
S
N
N
N
N
O
N
O
O
O
O
O
O
O
51 R1 =
52 R1 =
I
I
I
iv
N
iii
O
i
ii
N
N
F
F
NH2
NH2
N
H
N
H
N
O
47
48
50
49
R1
N
N
51 and 52
Scheme 5. Reagents and conditions: (i) acetamide oxime, K2CO3, C2H5OH, reflux, 2 days; (ii) 1,3-cyclohexane dione, p-TsOH, toluene, reflux, 16 h; (iii) Pd(OAc)2, tri(o-
tolyl)phosphine, NaOAc, DMF, microwave, 160 °C, 30 min; (iv) for compound 52: a) NaH, 1-bromo-3-chloro propane, DMF, rt, 3 h; (b) 4,6-diethyl-2-oxopyrimidine, K2CO3,
80 °C, overnight; for compound 51: NaH, (3-chloropropyl)-(3,5-difluorophenyl)sulfane, n-Bu4NH4+Iꢀ, DMF, 80 °C, overnight.
microsomes but also exhibited high antiviral activity
(IC50 = 1.5 nM). Parallel to side chain modifications, we have also
modified the tetrahydro ring of tetrahydrocarbazole by introducing
electron-withdrawing groups to minimize the oxidative metabo-
lism. The 4-oxo analogs 51 and 52 were potent but had poor met-
abolic stability in human microsomes and moderate stability in
rats. The 3,30-difluoro analogs 55 and 56 were less potent and
had poor microsomal stability.
The synthesis of compound 46 is outlined in Scheme 4. The
alkylation of intermediate 3a with racemic-epichlorohydrin pro-
vided the intermediate 44, which was then reacted with 3,5-difluo-
rophenol to obtain the 45. The hydroxyl group of the 45 was

4960
G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4955–4961
S
N
N
N
O
O
O
O
F
F
F
HO
55 R1 =
56 R1 =
ii
N
HO
i
F
iii
iv, v
F
F
F
F
N
H
N
H
NH2NH2
N
O
53
54
R1
N
N
55 and 56
Scheme 6. Reagents and conditions: (i) 4,4-difluoro cyclohexanone, AcOH, reflux, overnight. (ii) a) MeOH, H2SO4, reflux, overnight; (b) acetamide oxime, K2CO3, C2H5OH,
reflux, 2–4 days; (iii) for compound 56: a) NaH, 1-bromo-3-chloro propane, DMF, rt, 3 h; (b) 4,6-diethyl-2-oxopyrimidine, K2CO3, 80 °C, overnight; for compound 55: (3-
chloropropyl)-(3,5-difluorophenyl)sulfane, n-Bu4NH4+Iꢀ, DMF, 80 °C, overnight.
Table 4
The synthesis of compounds 51 and 52 is shown in Scheme 5.
The commercially available compound 47 was reacted with acet-
Intravenous pharmacokinetic properties of selected compounds in rats and dogsa
amide oxime to obtain the intermediate 48, which was then con-
densed with cyclohexane-1,3-dione to yield the enaminone 49.
The cyclisation of enaminone 49 to tetrahydrocarbazolone 50
was achieved using intramolecular Heck reaction under micro-
wave irradiation. The alkylation of tetrahydrocarbazolone 50 with
1-bromo-3-chloro propane and subsequent chloro displacement
of the resulting intermediate furnished the target compound 52.
Compound 51 was obtained by the alkylation of tetra-
hydrocarbazolone 50 with 3-chloropropyl-3,5-difluorophenyl
sulfane. Compounds 55 and 56 were prepared similarly to
compound 51 and 52, by alkylation of the intermediate 54
(Scheme 6).
Compd
AUCall (
l
G/L h)
Cmax
(
l
g/L)
t1/2 (h)
Vz (L/kg)
Cl (L/h/kg)
37
46
39
39b
404
691
1184
1746
821
3.76
1.42
1.5
22.09
5.52
3.64
1.8
3.64
2.72
1.68
1.14
1708
2989
3876
1.1
a
iv 2 mg/kg.
PK data in dog.
b
Table 5
Oral pharmacokinetic properties of selected compounds in rat and dog
Compd
AUCall (
lG/L h)
Cmax
(l
g/L)
Tmax (h)
t1/2 (h)
%Fa
The in vivo pharmacokinetics were subsequently studied in rats
using both i.v. and p.o. routes for potent and metabolically stable
compounds 37, 39 and 46, and the PK parameters are shown in
Table 4 and 5. Of these, compound 39 (ITX 4520) displayed good
46b
39b
39d
3138
4226
2010
747
955
625
2.67
2.33
2.0
1.46
6.01
4.07
38
42c
34
Formulation: 5% dimethylacetamide + 10% preconcentrate21 + 85% saline.
PK data in rats; dose 20 mg/kg.
Oral bioavailability of the compound varies from 40% to 100% depending on the
a
b
c
plasma oral exposure (AUC = 9.61 lM h), moderate clearance and
good oral bioavailability. The PK of compound 39 was also studied
at higher doses orally (50, 200 and 500 mg/kg) in rats, which
showed dose-dependent plasma oral exposure up to 200 mg/kg.
Compound 39 displayed a similar PK profile when dosed orally at
10 mg/kg in dogs (Table 5). Compound 46 was also orally bioavail-
able, but had higher clearance and lower exposure compared to 39.
Since the sulfur atom in compound 39 is a potentially metaboli-
cally labile, we investigated formation of the sulfoxide metabolite
(42) in vivo but did not observe the metabolite when administered
via i.v. at 2 mg/kg. However, when administered orally at 20 mg/
kg, we noticed minor formation of the sulfoxide metabolite (AU-
formulation.
d
PK data in dogs; dose:10 mg/kg.
Table 6
Profile for compound 39 (ITX 4520)
N
O
N
N
S
Call = 143 lG/L h).
We have selected compound 39 for further evaluation based on
its potency (IC50 = 1.5 nM; IC90 = 50 nM) and pharmacokinetic
properties. The compound was equally potent against both HCV
genotype-1 and genotype-2 and did not inhibit the entry of the re-
lated virus bovine viral diarrhea (BVDV) and heterologous VSV-G
pseudo particles. Compound 39 also did not inhibit HCV RNA rep-
lication as confirmed by testing the compound in replicon assay.
We have screened compound 39 against 97 off-target assays (Cer-
ep Diversity Profile)22 and compound 39 displayed only 30–50% of
binding at A3-adenosine and cannabinoid-1 (CB-1) receptors in
F
F
Molecular weight
cLogP
tPSA
HCVcc 1a IC50
HCVcc 1a IC90
HCVcc 2a IC50
HCVcc 2a IC90
Solubility (formulation)
439.5
6.5
37.2
1.5 nM
150 nM
1.5 nM
50 nM
>1 mg/mL
in vitro toxicity (CC50-Huh7)
AMES
100
negative
>10
lM
radio-ligand binding assay at 10
was also tested in hERG and AMES assay at 10
l
M. Additionally, compound 39
M, which did not
hERG IC50
t1/2 (rat)
t1/2 (dog)
%F (rat)
%F (dog)
lM
l
6.01 h
4.07 h
40–100%a
34%
have activity in both the assays. The properties compound 39 are
shown in Table 6.
We have performed preliminary toxicological studies by repeat
dosing of compound 39 in rats orally at 20 mg/kg for 7 days and
have examined the levels of liver enzymes aspartate transaminase
(AST), alanine transaminase (ALT), alkaline phosphatase (ALK) at 0,
1, 4 and 7 days since the site of action is liver. No elevation of en-
zymes was observed. We also noticed neither mortality nor abnor-
mality in weight of the animals.
a
Oral bioavailability of the compound varies from 40–100% depending on the
formulation.
converted to flouro using the reagent (diethylamino)sulfur trifluor-
ide (DAST) to yield the target compound 46.

G. K. Mittapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4955–4961
4961
12. Lupberger, J.; Zeisel, M. B.; Xiao, F.; Thumann, C.; Fofana, I.; Zona, L.;
Davis, C.; Mee, C. J.; Turek, M.; Gorke, S.; Royer, C.; Fischer, B.; Zahid,
M. N.; Lavillette, D.; Fresquet, J.; Cosset, F. L.; Rothenberg, M. S.;
Pietschmann, T.; Patel, A. H.; Pessaux, P.; Doffoël, M.; Raffelsberger, W.;
Poch, O.; McKeating, J. A.; Brino, L.; Baumert, T. F. Nat. Med. 2011, 17,
589.
13. Mittapalli, G. K.; Jackson, A.; Zhao, F.; Lee, H.; Chow, S.; Mckelvy, J.; Wong-Staal,
F.; Macdonald, J. E. Bioorg. Med. Chem. Lett. 2011, 21, 6852.
14. The mechanism of action of the series was investigated with compound ITX
4520 Wong-Staal, F.; Syder, A. J.; McKelvy, J. Viruses 2010, 2, 1718.
15. Syder, A. J.; Lee, H.; Zeisel, M. B.; Grove, J.; Soulier, E.; Macdonald, J.; Chow, S.;
Chang, J.; Baumert, T. F.; McKeating, J. A.; McKelvy, J.; Wong-Staal, F. J. Hepatol.
2011, 54, 48.
In summary, we have identified a highly potent hepatitis C virus
entry inhibitor with excellent in vivo pharmacokinetics through
optimization of previously reported series of compounds that were
unstable in plasma. Compound 39 (ITX 4520) exhibited a good
drug-like profile and has been selected as a pre-clinical candidate.
Acknowledgments
We would like to thank Dr. Dennis Chapman, Vice President at
Pacific BioLabs for advice and discussion.
16. (a) Meanwell, N. A. J. Med. Chem. 2011, 54, 2529; (b) Böstrom, J.; Hogner, A.;
Llina`s, A.; Wellner, E.; Plowright, A. T. J. Med. Chem. 1817, 2012, 55.
17. Spectral data for compound 32: 1H NMR (500 MHz CDCl3) d: 8.28 (d, J = 1.15,
1H), 7.87 (dd, J = 1.5, 8.56, 1H), 7.33 (d, J = 8.57, 1H), 6.78–6.7 (m, 2H), 6.64–
6.57 (m, 1H), 4.19 (t, J = 6.93, 2H), 2.9 (t, J = 6.92, 2H), 2.75 (t, J = 6.12, 2H), 2.69
(t, J = 5.8, 2H), 2.47 (s, 3H), 2.11 (q, J = 6.9, 2H), 1.98–1.92 (m, 2H), 1.9–1.85 (m,
2H). ESI-MS (m/z): (M+H)+ 441.
18. Wakita, T.; Pietschmann, T.; Kato, T.; Date, T.; Miyamoto, M.; Zhao, Z.; Murthy,
K.; Habermann, A.; Krausslich, H. G.; Mizokami, M.; Bartenschlager, R.; Liang, T.
J. Nat. Med. 2005, 11, 791.
19. Lindenbach, B. D.; Evans, M. J.; Syder, A. J.; Wolk, B.; Tellinghuisen, T. L.; Liu, C.
C.; Maruyama, T.; Hynes, R. O.; Burton, D. R.; McKeating, J. A.; Rice, C. M. Science
2005, 309, 623.
20. Zhong, J.; Gastaminza, P.; Cheng, G.; Kapadia, S.; Kato, T.; Burton, D. R.;
Wieland, S. F.; Uprichard, S. L.; Wakita, T.; Chisari, F. V. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 9294.
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