Synthesis and SAR of potent inhibitors of the Hepatitis C virus NS3/4A protease: Exploration of P2 quinazoline substituents

Article abstract of DOI:10.1016/j.bmcl.2010.05.029

Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.

Full text of DOI:10.1016/j.bmcl.2010.05.029

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4004–4011
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of potent inhibitors of the Hepatitis C virus NS3/4A protease:
Exploration of P2 quinazoline substituents
Magnus Nilsson a, Anna Karin Belfrage a,c, Stefan Lindström a, Horst Wähling a, Charlotta Lindquist a,
Susana Ayesa a, Pia Kahnberg a, Mikael Pelcman a, Kurt Benkestock a, Tatiana Agback a, Lotta Vrang a,
Ylva Terelius a, Kristina Wikström a, Elizabeth Hamelink a, Christina Rydergård a, Michael Edlund a,
Anders Eneroth a, Pierre Raboisson b, Tse-I Lin b, Herman de Kock b, Piet Wigerinck b, Kenneth Simmen b,
Bertil Samuelsson a, Åsa Rosenquist a,
*
a Medivir AB, PO Box 1086, SE-141 22 Huddinge, Sweden
b Tibotec – A Division of Janssen Pharmaceutical, Turnhoutseweg 30, 2340 Beerse, Belgium
c Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, BMC, PO Box 574, SE-751 23 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized.
High potency inhibitors displaying advantageous PK properties have been obtained through the optimi-
zation of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic
acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the
P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes.
By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopen-
tane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excel-
lent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered
quinazoline substituted P2 proline urea series.
Received 19 March 2010
Revised 10 May 2010
Accepted 11 May 2010
Available online 21 May 2010
Keywords:
HCV
NS3/4A protease
Inhibitors
P2 substituent
Quinazoline
Replicon assay
In vitro
Ó 2010 Elsevier Ltd. All rights reserved.
DMPK
In vivo PK
According to The World Health Organization about 170 million
people, 3% of the global population, are infected by Hepatitis C
virus (HCV), with 3–4 million new infections occurring each year.1
Individuals who become chronically infected usually progress to
end-stage liver diseases and approximately 2–4% develop hepato-
cellular carcinomas. Untreated HCV infection is the leading cause
of liver transplantation due to liver cirrhosis, hepatocellular carci-
noma, and ultimately liver failure.
response in patients co-infected with HIV is notably poor, that is,
30–40%, in genotype 1 patients.3
The limitations of today’s treatments are substantial,4 including
inconvenient dosing regimes and severe side effects such as hemo-
lytic anemia, flu-like symptoms, and neuropsychiatric events. The
severe side effects result in 10–20% of premature withdrawals
from therapy. The need for new efficacious drugs for HCV genotype
1 infected patients and for patients not responding to standard of
care is urgent. High SVR rates, shortened duration of treatment
and improved safety profiles together with more convenient once
daily dosing regimens are some of the goals for future treatments.
The virally encoded NS3/4A serine protease, a hetero dimeric
serine protease essential for viral replication, is an intensively
studied HCV drug target. Highly promising NS3/4A inhibitors have
been developed, and there are today several drug candidates in the
clinical pipeline. The most advanced candidates are VX-950 (tela-
previr)5 and SCH-503034 (boceprevir)6 which currently are in
phase III clinical trials. Both telaprevir (I) and boceprevir (II) are
The current standard of care treatment for chronic HCV is a
combination of pegylated interferon-
a and ribavirin. Approxi-
mately 42–46% of genotype 1 infected patients respond fully to
PEG-IFN/ribavirin combination therapy. Patients infected with
HCV genotype 2 and 3 show substantially higher response rates,
with sustained virological response (SVR) of around 90% and 80%,
respectively, after 24 weeks of therapy.2 The sustained virological
* Corresponding author.
E-mail address: asa.rosenquist@medivir.se (Å. Rosenquist).
linear peptidomimetic inhibitors incorporating an
a-ketoamide
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.029

M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
4005
R1
NO2
X
R1
NH2
NH2
iii
ii
O
O
3: R1 = OCH3
6: R1 = H
1: R1 = OCH3, X = OH
2: R1 = OCH3, X = NH2
i
O
R2
NH
NH2
R1
N
R2
R1
iv
N
OH
O
5a: R1 = OCH3, R2 = Phenyl
4a: R1 = OCH3, R2 = Phenyl
5b: R1 = OCH3, R2 = 4-isopropylthiazol-2-yl
5c: R1 = OCH3, R2 = 4-Fluorophenyl
8b: R1 = H, R2 = 4-isopropylthiazol-2-yl
4b: R1 = OCH3, R2 = 4-isopropylthiazol-2-yl
4c: R1 = OCH3, R2 = 4-Fluorophenyl
7b: R1 = H, R2 = 4-isopropylthiazol-2-yl
Scheme 1. Reagents and condition: (i) oxalyl chloride, DMF, DCM, then NH3 71%; (ii) Raney-Ni, EtOH, H2, 3.5 bar, 95%; (iii) R2-COOH, HOBt, EDAC, TEA, DMF, 55–90%; (iv)
Na2CO3, EtOH/H2O 1:1, reflux, 77–90%.
RO
NH2
CN
9: R = H
10: R = CH3
30 %
i
58 %
ii
O
NH2
NH2
O
i
NH2
OX
O
O
14: X = H
11
iii
15: X = CH3
v
R2
NH
NH2
O
R2
NH
O
O
O
OX
O
O
16a: R2 = Phenyl, X = CH3
12d: R2 = 3-Fluorophenyl
12e: R2 = 4-Methoxyphenyl
12h: R2 = Pyridin-3-yl
12i: R2 = Pyridin-4-yl
16c: R2 = 4-Fluorophenyl, X = CH3
16g: R2 = 6-Methylpyridin-2-yl, X = CH3
17a: R2 = Phenyl, X = CH3
vi
17c: R2 = 4-Fluorophenyl, X = CH3
17g: R2 = 6-Methylpyridin-2-yl, X = CH3
vii
iv
O
N
R2
N
OH
13a: R2 = Phenyl
13c: R2 = 4-Fluorophenyl
13d: R2 = 3-Fluorophenyl
13e: R2 = 4-Methoxyphenyl
13f: R2 = H
13g: R2 = 6-Methylpyridin-2-yl
13h: R2 = Pyridin-3-yl
13i: R2 = Pyridin-4-yl
Scheme 2. Reagents and conditions: (i) MeI, K2CO3, DMF; (ii) 2 M NaOH, EtOH; (iii) R2-COCl, pyridine, THF; (iv) Na2CO3, EtOH/H2O 1:1, reflux, 60–95% over two steps, note:
13f was formed as a biproduct when the procedure was performed in DMF rather than in EtOH; (v) R2-COOH, pyridine, DCM, 50–56%; (vi) 1 M LiOH, EtOH, 60 °C; (g)
formamide, 150 °C, 71–89% over two steps.

4006
M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
that reacts covalently with the catalytic Ser139 in the active site
via a reversible mechanism. Another class of NS3/4A inhibitors is
the non-covalent binding macrocyclic inhibitors chemically char-
acterized by a hydrocarbon linkage from the P1-side chain to the
P3-side chain, as in TMC4357 and ITMN-191/R-72278 or by a link-
age from the P2 substituent to the P4-side chain as in MK-7009.9
Key features of these three compounds are the P1 sulfone amides
interacting with the oxyanion pocket of the active site as well as
relatively large quinoline and indoline P2 substituents.
The syntheses of the corresponding 4-quinazolinols are out-
lined in Schemes 1 and 2. The carboxylic acid 112 was treated with
oxalyl chloride in dichloromethane/dimethylformamide followed
by the displacement of the so-formed acyl chloride with ammonia
leading to amide 2. Subsequent reduction of the nitro group fur-
nished amine 3. Amines 3 and 6 were coupled with carboxylic
acids (Table 1 for R2 groups) under standard conditions which gave
intermediates 4a–c and 7b. Subsequent condensation in refluxing
ethanol–water in the presence of sodium carbonate provided the
quinazolinols 5a–c and 8b.
We now report on quinazolines as novel P2 substituents in the
previously described cyclopentane7,10 and proline urea11 based
macrocyclic NS3/4A inhibitors.
The preparation of 8-methyl substituted quinazolinols is out-
lined in Scheme 2. Alkylation of 913 furnished the methyl ether
Table 1
SAR for the P2 cyclopentane series
Structure
Compound
R1
R2
R3
Ki
EC50
(nM)
HCV
Clint
L/min/mg)
Papp
(nM)
HCV
NS3 1a
(
l
(cm/s  10À6
)
NS3 1b
25
31
32
H
H
H
H
H
H
0.4
6.2
1.2
52
58
Nd
Nd
21
Nd
Nd
O
390
830
N
N
N
N
39b
45a
45b
H
H
H
H
0.8
0.4
1.2
120
14
Nd
88
76
Nd
13
6
S
S
MeO
MeO
R3
R1
N
O
R2
N
58
N
F
45c
51a
51c
MeO
MeO
MeO
H
0.2
0.1
0.4
11
5
32
<6
<6
20
13
3
O
O
O
H
N
S
O
N
H
Me
Me
O
N
F
F
10
51d
MeO
Me
0.4
20
10
5
O
51e
51f
MeO
MeO
Me
Me
0.6
8
11
9
H
>1000
>1000
Nd
Nd
Nd, not determined.
OH
O
O
O
i
ii
O
O
H
O
X
N
N
OH
O
O
O
H
18
19
O
O
O
20: X = OH
21: X = N-methylhex-5-enyl
iii
Scheme 3. Reagents and conditions: (i) (1R,2S)-ethyl 1-amino-2-vinylcyclopropanecarboxylate, HATU, DIPEA, 2:1 DCM/DMF, rt, 1 h; (ii) H2O, DIPEA, microwaves 100 °C,
40 min; (iii) N-methylhex-5-en-1-amine, HATU, DIPEA, DCM/DMF (4:1), 0 °C to rt, 73% over three steps.

M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
4007
10. Hydrolysis of the nitrile 10 under basic conditions delivered a
mixture of 11 and 14 (58% and 30% yield, respectively, over the
two steps) which was separated. Compound 11 was reacted with
the corresponding acid chlorides to obtain amides 12d, 12e, 12h,
and 12i, which were cyclized providing the corresponding quinaz-
olinols 13d–f and 13h–i in 60–95% yields, respectively. Starting
from 14, the carboxylic acid was esterified into 15 and subse-
quently coupled with the corresponding carboxylic acids under
standard conditions rendering the amides 16a, 16c, and 16g which
after ester hydrolysis and reaction with formamide at elevated
temperatures gave the corresponding quinazolinols 13a, 13c, and
13g in 71–89% yield.
with N-methylhex-5-en-1-amine gave the common intermediate
21 in 73% overall yield over three steps.
Two routes using the intermediate 21 were employed. Reacting
21 with 4-chloro-2-phenylquinazoline15 in the presence of sodium
hydride in DMF gave a mixture of ester 22 and acid 23, where 22
was hydrolyzed to 23 in 49% yield and coupled with cyclopropane-
sulfonamide under microwave irradiation16 giving 24 in 90% crude
yield. Finally ring-closing olefin metathesis using Hoveyda–Grubbs
2nd generation catalyst under microwave irradiation furnished the
target product 25 in 29% yield over the two steps. Alternatively, es-
ter hydrolysis of 21 afforded the carboxylic acid 26 which was cou-
pled with cyclopropanesulfonamide providing 27 in 77% yield over
the two steps. Reaction of 27 as described, vide supra, rendered the
chloro derivative 28 in 79% yield. Nucleophilic aromatic substitu-
tion of 28 using neat amines under microwave irradiation gave
derivatives 29 and 30 (52% and 82% yield, respectively), which
were subsequently subjected to ring-closing olefin metathesis,
providing target products 31 and 32 (56% and 22% yield,
respectively).
The synthetic routes to the target products are outlined in
Schemes 3–6. Initially, the route outlined in Schemes 3 and 4
was used for synthesis of the targets products in the cyclopentane
series.
Carboxylic acid 1810a (Scheme 3) was coupled with (1R,2S)-
ethyl-1-amino-2-vinylcyclopropanecarboxylate14 providing inter-
mediate 19. Subsequent lactone hydrolysis to acid 20 and coupling
OH
O
H
ii
21
N
N
X
O
O
26: X = OH
iii
O
O
S
27: X =
N
H
i
i
N
N
O
N
O
R2
N
O
H
O
O
O
H
N
N
N
N
S
X
N
H
O
O
O
O
28: R2 = Cl
22: X = OEt
23: X = OH
ii
v
29: R2 = 4-morpholinyl
iii
O
O
30: R2 = 4-methylpiperazinyl
S
24: X =
N
H
iv
iv
N
O
R2
N
N
O
N
O
O
O
O
O
O
H
N
H
S
N
S
O
O
N
H
N
H
O
O
N
N
25
31: R2 = 4-morpholinyl
32: R2 = 4-methylpiperazinyl
Scheme 4. Reagents and conditions: (i) 2,4-dichloroquinazoline or 4-chloro-2-phenylquinazoline, NaH, DMF, 0 °C to rt; (ii) aq 2 M LiOH DMF, microwaves 130 °C, 30 min; (iii)
cyclopropanesulfonamide, EDAC, DMAP, DBU, 1:1 DCM/DMF, microwaves 100 °C, 30 min; (iv) Hoveyda–Grubbs catalyst 2nd generation, DCE, microwaves 150 °C, 15 min; (v)
neat 4-morpholine or 4-methylpiperazine or 2-pyrazoline, microwaves 120 °C, 10 min.

4008
M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
The target products in the cyclopentane series were also pre-
cyclopropylsulfonamide gave target products 39b, 45a–c, 51a,
and 51c–f in moderate yields (10–40%) after preparative-HPLC
purification.
pared from the earlier described building block 337,10 as outlined
in Scheme 5.
Reacting 33 with quinazolinols 5a–c, 8b, 13a, and 13c–f under
Mitsunobu conditions gave 34b, 40a–c, 46a, and 46c–f in 68–99%
yield. Acidic hydrolysis of the tert-butyl esters of 34b, 40a–c,
46a, and 46c–f followed by HATU promoted coupling with
N-methylhex-5-enamine delivered 36b, 42a–c, 48a, and 48c–f in
good yields (60–85%). Ring-closing olefin metathesis of 36b, 42a–
c, 48a, and 48c–f using Hoveyda–Grubbs catalyst (1st or 2nd)
provided 37b, 43a–c, 49a, and 49c–f in moderate to good yields
(30–80%). Ester hydrolysis of 37b, 43a–c, 49a, and 49c–f under
basic conditions followed by EDAC promoted coupling to
The synthesis of the P2 proline urea target products from the
key intermediate 5211 is outlined in Scheme 6.
N-deprotection11 of 52 resulted in 53 (95% crude yield), fol-
lowed by treatment with phosgene yielding 54 and subsequent
coupling with N-methylhex-5-enamine delivering the proline urea
55 (90% yield over the two steps). Ring-closing olefin metathesis of
55 using Hoveyda–Grubbs 2nd generation catalyst provided 56 in
74% yield. Selective hydrolysis of the 4-nitrobenzyl ester of 56
afforded the alcohol 57 in 80% yield. Reacting 57 with quinazoli-
nols 13a, 13c, 13e, and 13g–i under Mitsunobu conditions
R3
R1
N
O
R2
N
OH
O
O
i
iii
H
H
N
O
N
X
O
O
O
O
O
O
33
34b: R1 = R3 = H, X = O-t-Bu
35b: R1 = R3 = H, X = OH
ii
ii
ii
40a-c: R1 = OCH3, R3 = H, X = O-t-Bu
41a-c: R1 = OCH3, R3 = H, X = OH
46a, c-f: R1 = OCH3, R3 = CH3, X = O-t-Bu
47a, c-f: R1 = OCH3, R3 = CH3, X = OH
R3
R3
R1
N
O
R2
R1
N
R2
N
N
O
O
H
N
vi
O
iv
H
N
O
N
Y
O
O
N
O
O
36b: R1 = R3 = H
37b: R1 = R3 = H, Y = OEt
38b: R1 = R3 = H, Y = OH
43a-c: R1 = OCH3, R3 = H, Y = OEt
44a-c: R1 = OCH3, R3 = H, Y = OH
49a, c-f = R1 = OCH3, R3 = CH3, Y = OEt
50a, c-f = R1 = OCH3, R3 = CH3, Y = OH
42a-c: R1 = OCH3, R3 = H
v
48a, c-f = R1 = OCH3, R3 = CH3
v
v
R3
R1
N
O
R2
N
O
O
O
H
N
S
O
N
H
O
N
39b: R1 = R3 = H
45a-c: R1 = OCH3, R3 = H
51a, c-f: R1 = OCH3, R3 = CH3
Scheme 5. Reagents and conditions: (i) quinazolinol, PPh3, DIAD, THF, 0 °C to rt overnight; (ii) 2:1 DCM/TFA, Et3SiH, 0 °C to rt, 1 h; (iii) N-methylhex-5-enamine, HATU,
DIPEA, DMF, 0 °C to rt, 1–3 h; (iv) Hoveyda–Grubbs catalyst (1st or 2nd), 1,2-dichloroethane, reflux, 4–12 h; (v) 2:1:1 THF/MeOH/H2O, aq 1 M LiOH; (vi) EDAC, DCM, rt, 3–
12 h, then cyclopropylsulfonamide, DBU 6–12 h. R2 substituents are defined in Schemes 1 and 2 (a–f).

M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
4009
delivered 58a, 58c, 58e, and 58g–i in moderate to good yields (30–
85%). Ester hydrolysis of 58a, 58c, 58e, and 58g–i followed by cou-
pling to cyclopropylsulfonamide gave target products 60a, 60c,
60e, and 60g–i in 40–85% yields over the two steps. Similarly the
target compounds 66a and 66c were obtained from quinazolinols
13a and 13c in 20% and 12% yield, respectively, over the six steps.
N-deprotection of 66a and 66c under acidic conditions gave target
compounds 67a and 67c (62% and 38% yield, respectively).
The novel quinazoline containing macrocyclic inhibitors were
evaluated in a biochemical assay for their inhibitory capacities on
the full-length HCV NS3/4A protease incorporating the central part
of the cofactor NS4A as described earlier.17 The inhibition con-
stants (Ki values)18,19 were determined, and are listed in Tables 1
and 2. The cell-based activities (EC50) were measured in the
Huh7-replicon cell line containing subgenomic bicistronic replicon
clone ET with a luciferase readout.20 For initial in vitro PK profiling,
the stability in human liver microsomes (HLM), measured as the
intrinsic clearance (Clint), and the A–B apparent permeability coef-
ficient in Caco-2 cells (Papp), were evaluated for most of the final
compounds in Tables 1 and 2.
The initial lead 25 in this series, having a 7-phenylquinazoline
substituent, displayed high potency on enzyme (Ki = 0.4 nM), mod-
erate potency in the cell-based HCV replicon assay (EC50 = 52 nM),
moderate stability in human liver microsomes (Clint = 58 lL/min/
mg), and excellent permeability in the Caco-2 cell-based assay
(Papp = 21 Â 10À6 cm/s). These data encouraged us to continue
exploration of the quinazoline P2 substituent in both the cyclopen-
tane series as well as the earlier described macrocyclic proline urea
series. Quinazoline modifications were evaluated by introducing
substituents in positions 2, 7, and 8. While introduction of a meth-
oxy substituent in position 7 of the quinazoline (45a) resulted in
improved cell-based potency (EC50 = 14 nM) other key in vitro
DMPK properties (Clint = 88
The introduction of a 4-isopropylthiazolyl moiety as in derivatives
39b (Ki = 0.8 nM, EC50 = 120 nM) and 45b (Ki = 1.2 nM, EC50
lL/min/mg) were not improved upon.
=
58 nM) resulted in loss of potency at both the enzyme level and
in the replicon cell-based assay. This is in contrast to our earlier
findings of quinazoline substitutions and could possibly be ex-
plained by repulsion between the nitrogen’s in the quinazoline
and the hetero atoms in the thiazolyl substituent, inducing the
NO2
NO2
O
O
O
O
O
O
i
H
N
H
iii
N
O
N
N
O
R
O
O
O
O
52
53: R = H
ii
54: R = COCl
NO2
O
O
OR
R4
N
O
O
vi
iv
H
N
H
N
N
N
O
O
O
n
O
O
O
N
R4
55: R4 = CH3, n = 1
61: R4 = p-MeOBn, n = 2
n
56: R = 4-NO2-benzoyl, R4 = CH3, n = 1
57: R = H, R4 = CH3, n = 1
v
62: R = 4-NO2-benzoyl, R4 = p-MeOBn, n = 2
63: R = H, R4 = p-MeOBn, n = 2
v
R3
R3
O
N
O
R2
O
N
O
R2
N
N
O
viii
O
O
O
H
N
H
N
N
N
S
O
O
OR
N
H
O
O
N
N
R4
R4
n
n
60a, c, e, g-i: R3 = CH3, n = 1
66a, c: R3 = CH3, R4 = p-MeOBn, n = 2
67a, c: R3 = CH3, R4 = H, n = 2
58a, c, e, g-i: R = Et, R3 = CH3, n = 1
vii
vii
59a, c, e, g-i: R = H, R3 = CH3, n = 1
ix
64a, c: R = Et, R3 = CH3, R4 = p-MeOBn, n = 2
65a, c: R = H, R3 = CH3, R4 = p-MeOBn, n = 2
Scheme 6. Reagents and conditions; (i) 2:1 DCM/TFA, 0 °C to rt, 1.5 h; (ii) phosgene in toluene, THF, NaHCO3, rt, 1 h; (iii) DCM, NaHCO3, N-methylhex-5-enamine or hept-6-
enyl-(p-methoxybenzyl)-amine, rt overnight, 85–90% over three steps; (iv) Hoveyda–Grubbs catalyst 2nd generation, 1,2-dichloroethane, reflux, 4–12 h; (v) aq 1 M LiOH,
H2O, THF, MeOH, 0 °C, 4 h, 55–65% over two steps; (vi) quinazolinol, PPh3, DIAD, THF, 0 °C to rt overnight, 30–85%; (vii) 2:1:1 THF/MeOH/H2O, aq 1 M LiOH; (viii) EDAC, DCM,
rt 5 h, then cyclopropylsulfonamide, DBU, rt overnight, 45–80%; (ix) 2:1 DCM/TFA, rt 30 min, 38–62%.

4010
M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
Table 2
SAR for the P2 proline urea series
Structure
Compound
R2
R3
R4
n
Ki
EC50
(nM)
HCV
Clint
L/min/mg)
Papp
(nM)
HCV
NS3 1a
(
l
(cm/s  10À6
)
NS3 1b
60a
Me
Me
1
0.2
11
17
26
F
60c
60e
60g
Me
Me
Me
Me
Me
Me
1
1
1
0.3
0.6
0.9
16
3
<6
8
32
33
Nd
R3
O
O
N
O
R2
N
130
Nd
N
60h
60i
Me
Me
Me
Me
1
1
2.8
0.2
13
7
Nd
14
Nd
0.4
O
N
N
O
O
H
N
N
S
O
N
H
O
N
R4
67a
67c
Me
Me
H
H
2
2
0.3
0.2
23
16
26
20
38
32
n
F
Nd, not determined.
Table 3
Mean plasma levels (n = 2) together with pharmacokinetic parameters of the four macrocyclic compounds 51a, 60a, 60c, and 67c after a single intravenous (2 mg base-equiv/kg)
and oral administration (20 mg base-equiv/kg) in the male Sprague–Dawley rats
Compound
51a
60a
60c
67c
Iv (2 mg/kg, n = 2)
Cl (L/h/kg)
Vdss
1
0.69
1.1
8.4
38
0.56
2
Nd
Nd
1.6
1.2
Nd
2.5
0.56
Nd
Nd
AUC (
lM h)
Liver/plasma ratio (6 h)
Oral (10 mg/kg, n = 2)
AUC ( M h)
l
3.3
0.66
2
15
3.4
5
11
2.6
5
1.7
0.39
2
Cmax M)
(l
Tmax (h)
T1/2 (h)
F (%)
Liver/plasma ratio (6 h)
3.8
40
Nd
Nd
73
32
Nd
Nd
42
Nd
38
100
Nd, not determined.
thiazolyl substituent to adopt a non-coplanar and for interacting
with the NS3 enzyme less optimal conformation. The metabolic
stability was improved by modifications of the phenyl substituent
in position 2 of the quinazoline as well as the introduction of a
methyl group in position 8 of the quinazoline. This is exemplified
a 10-fold potency drop in both the enzymatic- and in the cell-
based assays. Possibly this could be due to the stringent criteria
for the conformation of the P2 substituent.
The quinazoline P2 substituents were also explored in the pro-
line urea series. In the 14-membered N-methyl proline urea series,
the enzymatic and cellular potency as well as stability in human li-
ver microsomes were comparable to the data seen for the cyclo-
pentane series. For this series further improved Caco-2
permeability was obtained with Papp values of 26 Â 10À6 and
32 Â 10À6 cm/s for 60a and 60c, respectively. The two compounds
prepared in the 15-membered NH proline urea series 67a
(EC50 = 23 nM, Papp = 38 Â 10À6 cm/s) and 67c (EC50 = 16 nM,
Papp = 32 Â 10À6 cm/s) had similar properties compared with the
14-membered analogs (60a and 60c), but displaying decreased
by compounds 51a (Clint < 6
lL/min/mg), 51c (Clint < 6
lL/min/
mg), 51d (Clint = 10 L/min/mg), and 51e (Clint = 11
l
lL/min/mg)
demonstrating excellent stability in human liver microsomes with
retained potency. Surprisingly, for compound 51c the introduction
of the 8-methyl substituent led to an unexpected reduction of
Caco-2 permeability (Papp = 3 Â 10À6 cm/s), possibly due to lower
solubility resulting from increased lipophilicity.
Complete removal of the substituent in the position 2, as for
compound 51f, resulted in almost complete loss in activity indicat-
ing that an aromatic- or a heteroaromatic substituent in this
position plays a critical role for the binding interactions of these
metabolic stability (Clint = 26 and 20 lL/min/mg). Further im-
proved potency in the cell-based replicon assay was obtained with
the introduction of a 4-methoxyphenyl substituent, that is, 60e,
where the overall favorable in vitro DMPK properties were retained
inhibitors. Interestingly, compounds 31 (Ki = 6.2 nM, EC50
=
390 nM) and 32 (Ki = 12 nM, EC50 = 830 nM) having non-aromatic
substituents in position 2 of the quinazoline gave approximately
(Clint = 8
l
L/min/mg, Papp = 33 Â 10À6 cm/s). Similarly 4-pyridyl

M. Nilsson et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4004–4011
4011
6. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu,
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7. Raboisson, P.; de Kock, H.; Rosenquist, Å.; Nilsson, M.; Salvador-Oden, L.; Lin,
T.-I.; Roue, N.; Ivanov, V.; Wähling, H.; Wikström, K.; Edlund, M.; Vrang, L.;
Venderville, S.; Van De Vreken, W.; McGowan, D.; Tahri, A.; Hu, Li.; Lenz, O.;
Delouvroy, F.; Pille, G.; Surleaux, D.; Wigerinck, P.; Samuelsson, B.; Simmen, K.
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substitution (60i) resulted in improved potency (EC50 = 7 nM),
however with loss of permeability in the Caco-2 assay
(Papp = 0.4 Â 10À6 cm/s). The corresponding pyridyl substitutions
of 60h, and in particular of 60g however resulted in decreased po-
tency (EC50 = 13 and 130 nM, respectively).
Based on the excellent potency and in vitro DMPK properties for
these quinazoline containing inhibitors four compounds were se-
lected for evaluation of their in vivo pharmacokinetic properties
in rats. This comprised examples from all three series, that is, the
cyclopentane series (51a), the 14-membered N-methyl proline
urea series (60a and 60c), as well as the 15-membered NH proline
urea series (67c) and the results are shown in Table 3.
The mean plasma levels, liver-to-plasma ratios together with
pharmacokinetic parameters were determined after a single intra-
venous and oral administration in male Sprague–Dawley rats. The
proline urea derivatives 60a, 60c, and 67c were all found to have
favorable liver-to-plasma ratios (32–100) after 6 h and were hence
well distributed to the liver. The cyclopentane derivative 51a did
however not reach detectable concentrations and the liver-to-plas-
ma ratio could thus not be determined. When comparing the two
compounds 51a (Cmax = 0.66 lM) and 60a (Cmax = 3.4 lM), the
mean maximum plasma concentrations (Cmax) were achieved after
2 h for the cyclopentane derivative and after 5 h for the proline
compound indicating a slower absorption for 60a but also a higher
Cmax and hence a larger area under the curve (AUC).
The 15-membered NH proline urea derivative 67c exhibited
three times higher clearance (1.6 L/h/kg), associated with a signif-
icantly lower Cmax (0.39 lM) and AUC (1.7 lM h) compared with
the two 14-membered N-methyl proline urea derivatives 60a and
60c.
Taken together, these in vivo PK results support further SAR
studies on quinazoline substituted P2 proline ureas of the 14-
membered macrocyclic ring series to access potent HCV protease
inhibitors displaying high oral bioavailability in combination with
high liver-to-plasma ratios.
11. Vendeville, S.; Nilsson, M.; de Kock, H.; Lin, T.-I.; Antonov, D.; Classon, B.;
Ayesa, S.; Ivanov, V.; Johansson, P.-O.; Kahnberg, P.; Eneroth, A.; Wikström, K.;
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A.; Hu, L.; Lenz, O.; Delouvroy, R.; Van Dooren, M.; Kindermans, N.; Surleraux,
D.; Wigerinck, P.; Rosenquist, Å.; Samuelsson, B.; Simmen, K.; Raboisson, P.
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