Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.09.068

Herein, we report the synthesis and structure-activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the disco

Full text of DOI:10.1016/j.bmcl.2013.09.068

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6325–6330
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Azetidines and spiro azetidines as novel P2 units in hepatitis C virus
NS3 protease inhibitors
Lavanya Bondada a, Ramu Rondla a, Ugo Pradere a, Peng Liu a, Chengwei Li a, Drew Bobeck b,
Tamara McBrayer b, Philip Tharnish b, Jerome Courcambeck c, Philippe Halfon c, Tony Whitaker b,
Franck Amblard a, Steven J. Coats b, Raymond F. Schinazi a,
⇑
a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Veterans Affairs Medical Center, 1670
Haygood Drive, NE, Atlanta, GA 30322, USA
b RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
c Genoscience-Pharma, 10 rue d’Iena, 13006 Marseille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the synthesis and structure–activity relationship studies of new analogs of boceprevir 1
and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyr-
rolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c
Received 12 August 2013
Revised 19 September 2013
Accepted 23 September 2013
Available online 30 September 2013
(EC50 = 0.8 lM).
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
HCV
Protease inhibitor
Antiviral
Hepatitis C virus (HCV) is a major health hazard affecting an
estimated 170–200 million individuals worldwide and it is a lead-
ing cause of chronic liver disease in the United States.1 First gener-
126 was prepared from cyclohexyl cyanide 3 by reaction with
2-(benzyloxy)acetaldehyde in presence of LDA to give the hydroxyl
derivative 4 as a mixture of enantiomers (Scheme 1). The cyano
group was then reduced with LiAlH4 and the resulting amine 5
ation treatment involving
a-interferon (IFN) in combination with
ribavirin (RBV) was only partially effective with approximately
50% of genotype 1 HCV patients demonstrating sustained viral re-
sponse. Immense drug discovery efforts towards improved HCV
therapy in recent years has led to the 2011 FDA approval of two
HCV NS3 protease inhibitors (PI): boceprevir 1 (Victrelis) and tela-
previr 2 (Incivek).2,3 However, despite the existence of treatments
involving pegylated IFN and RBV, with these two PIs,4 (Fig. 1) the
limited efficacy and side effects emphasize the need for additional
improved therapeutic agents. Following the discovery of telaprevir
and boceprevir, numerous modifications have been investigated at
various positions of these peptidomimetics in order to improve
their overall therapeutic profile.5 Herein, we detail our studies in
the P2 area, specifically the introduction of various new azetidine
moieties, which resulted in inhibitors with good potency.
P2
P1'
O
H
N
NH2
N
H
N
H
N
O
O
O
P3-cap
O
1
boceprevir
P1
P3
P2
N
P4
O
H
H
N
P1-cap
N
In order to prepare telaprevir and boceprevir analogs 37a–i,
41a–f and 42a–b, we synthesized key 2-azaspiro[3.5]nonane inter-
mediate 12 (Scheme 1), 10,30-dihydrospiro[azetidine-3,20-indene]
intermediates 22 and 24 (Scheme 2) along with various P1 precur-
sors 29a–f (Scheme 3). Thus, 2-azaspiro[3.5]nonane intermediate
O
H
O
O
N
N
N
O
H
N
O
P1
P4-cap
P3
2
telaprevir
⇑
Corresponding author.
E-mail address: rschina@emory.edu (R.F. Schinazi).
Figure 1. Structures of FDA approved HCV protease inhibitors boceprevir 1 and
telaprevir 2.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.068

6326
L. Bondada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6325–6330
OH
CN
OH
OH
H
N
BocHN
BocHN
OH
, R4
R4
O2N
OH
d
a
b
c
a
b, c
O2N R3
R3
O
CN
R3
O
R3
O
26
OBn
OBn
OBn
H2N OH
5
BocNH OH
OH
25
27
28
3
4
6
3
=
f
29a
OH
R
=
e
d
H
OH
OBn
e
ClH.H2N
N
R4
, R4
, R4
=
=
=
3
3
3
29b
29c
R
R
=
=
OBn
R3
O
N
N
BocNH OMs
Boc
Boc
9
7
8
, R4
29d
R
=
h
g
i
Oi Pr
F
OMe
, R4
, R4
=
=
29e R3
29f R3
=
=
OiPr
OH
N
H
O
N
O
N
O
HCl
Boc
Boc
10
12
11
Scheme 3. Reagents and conditions: (a) glyoxalic acid, Et3N, MeOH, rt; (b) H2, Pd/C,
AcOH, rt; (c) Boc2O, NaOH, dioxane/H2O, 80–90% over three steps; (d) appropriate
amine, HOBt, EDCI, DIPEA, DMF, 0 °C to rt, 10 h, 60–80%; (e) 4 N HCl in dioxane, rt,
5–6 h, 90–95%.
Scheme 1. Reagents and Conditions: (a) 2-(benzyloxy)acetaldehyde, LDA, THF,
ꢀ78 °C to rt, 7 h, 65%; (b) AlCl3, LiAlH4, Et2O, ꢀ78 °C to rt, 14 h, 60%; (c) Boc2O,
CH2Cl2, rt, 24 h, 92%; (d) MeSO2Cl, Et3N, ꢀ10 °C to rt, CH2Cl2, 36 h, 67%; (e) NaH,
DMF, 45 °C, 1 h, 56%; (f) ammonium formate, 10% Pd/C, MeOH, 60 °C, 2 h, 76%; (g)
NaIO4, RuCl3, CCl4, rt, 2 h, 74%; (h) iPrOH, EDC, DMAP, 0–55 °C, 15 h, 63%; (i) 3 N HCl
in dioxane, 6 h, rt, 90%.
enol ether 16. Reaction with N-p-methoxyphenyl (PMP)-a-imino
ethyl glyoxylate, 17,7 in the presence of TMSOTf gave compound
18 as a mixture of enantiomers. Cyclisation to form 19 was
achieved by treatment with MeMgBr in 70% yield. Removal of
the PMP group with CAN, reduction of both the ester and the amide
groups with LAH and subsequent reprotection using Boc2O affor-
ded the hydroxyl derivative 21. Oxidation to the acid was per-
formed using NaIO4 and RuCl3 in 63% yield. Treatment of the acid
22 with iPrOH in presence of EDC and DMAP gave the Boc’ed ester
23. Finally, Boc removal with HCl in dioxane gave the desired spiro
azetidine ester 24.
The P1 portion (Fig. 1) of the targeted molecules were prepared
from various commercially available nitro alkyl derivatives by
reaction with glyoxalic acid in presence of triethylamine to give
intermediate 26 (Scheme 3). Nitro groups were then reduced by
catalytic hydrogenation and the resulting amines were protected
by reaction with Boc2O in presence of NaOH.8 Coupling of 27 with
various amines in presence of EDCI and HOBt was followed by
treatment with 4 N HCl in dioxane to afford the desired intermedi-
ates 29a–f in good overall yields.
O
O
Br
Br
a
b
COOMe
+
EtO
OEt
14
15
13
PMP
+
O
c
OEt
N
d
MeOOC
PMP
O
OEt
18
N
PMP
O
COOEt
17
NH
MeO
OTMS
19
16
OH
OH
OEt
g
f
e
O
N
N
N
H
O
O
Boc
Boc
21
22
20
With these intermediates in hand, synthesis of inhibitors 37a–i,
Oi Pr
OiPr
i
h
analogs of telaprevir, with either an azetidine,
a 2-azaspi-
N
H
N
O
O
ro[3.5]nonane or a 10,30-dihydrospiro[azetidine-3,20-indene] at P2
was achieved according to Scheme 4. Commercially available
cyclohexyl glycine 30 was Boc protected then coupled with tert-
butyl glycine ester 32 via a standard EDCI/HOBt protocol. Follow-
ing deprotection under acidic conditions, the pyrazine P4 cap
was introduced and the methyl ester saponified using LiOH to give
intermediate 34. Commercially available azetidine ester 35, 2-aza-
spiro[3.5]nonane, 12 or 10,30-dihydrospiro[azetidine-3,20-indene],
24 were then coupled (EDCI/HOBt) with 34 to generate, after
saponification of the isopropyl ester with LiOH, compound 36. Fi-
nally, coupling of intermediates 36 with 29a–f, followed by oxida-
tion under Dess–Martin conditions, allowed introduction of the
various relevant P1 and P1 caps, and lead to targeted molecules
37a–i.
Boc
24
23
Scheme 2. Reagents and conditions: (a) (i) Na, KOH, EtOH, Et2O, H2O, reflux, 5 h,
73%; (ii) 200 °C, 20 min to rt, MeOH, H2SO4, reflux, 1 h, 26% over two steps; (b) LDA,
TMSCl, THF, ꢀ78 °C, 30 min, 56%; (c) TMSOTf, CH2Cl2, 0 °C to rt, 12 h, 79%; (d)
MeMgBr, CH2Cl2, 0 °C to rt, 18 h, 70%; (e) CAN, CH3CN, H2O, 0 °C, 45 min, 56%; (f) (i)
LAH, THF, 0 °C to rt, 20 h; (ii) Boc2O, CH2Cl2, rt, 10 h, 46% over two steps; (g) NaIO4,
RuCl3, H2O, CH3CN, CCl4, rt, 2 h, 65%; (h) iPrOH, EDC, DMAP, CH2Cl2, 50 °C, 18 h, 39%;
(i) 3 N HCl in dioxane, 6 h, rt, 56%.
was Boc protected. Subsequently, formation of mesylate 7 and
cyclisation using NaH gave the 2-azaspiro[3.5]nonane derivative
8 in 56% yield. Palladium catalyzed hydrogenation in presence of
ammonium formate to remove the benzyl group followed by oxi-
dation of the formed alcohol with NaIO4 and RuCl3 lead to acid
10. Finally, esterification with iPrOH in presence of EDC and acidic
deprotection of the amine gave the desired 2-azaspiro[3.5]nonane
ester 12.
Synthesis of inhibitors 41a–f and 42a–b, analogs of boceprevir,
with a 10,30-dihydrospiro[azetidine-3,20-indene] at P2 was achieved
according to the general Scheme 5. 10,30-Dihydrospiro[azetidine-
3,20-indene], 22 was coupled to amino acid derivatives 29a–b
(EDCI/HOBt) to give, after Boc removal under acidic conditions,
compounds 38. Introduction of the P3 moiety was realized by cou-
pling 18 with commercially available NHBoc–tBu-glycine (EDCI/
HOBt). Desired compounds 41a–f and 42a–b were obtained after
oxidation, using Dess–Martin reagent, followed by Boc removal
and reaction with either substituted an isocyanate or alkyl carbox-
ylic acid, allowing the introduction a large variety of P3 caps.
10,30-Dihydrospiro[azetidine-3,20-indene] intermediates 22 and
24 were prepared by a sequence that started with condensation
of 1,2-bis(bromomethyl)benzene 13 with diethyl malonate 14
(Scheme 2). The resulting crude diethyl malonate derivative was
reacted with sulfuric acid in the presence of methanol at 200 °C
which resulted in a hydrolysis, decarboxylation and esterification
sequence to produce methyl ester 15. Treatment of 15 with LDA
produced the enolate which was trapped with TMSCl to give silyl

L. Bondada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6325–6330
6327
by extraction and amplification of both HCV RNA and ribosomal
RNA.10 First of all, replacement of the telaprevir P2 moiety with a
simple azetidine moiety proved to be counterproductive as 37d
provided inactive along with the related inactive compounds
37e–g. More interestingly, introduction of a 2-azaspiro[3.5]nonane
moiety at P2 as in compounds 37h and 37i displayed EC50values of
OMe
O
OMe
O
a
b
H2N
H
N
+
OH
OH
BocHN
H2N
H2N
O
O
O
30
33
31
32
2.3 and 5.4
Huh7 cells up to 10 l
l
M, respectively, with no apparent cytotoxicity in
12 R1/R1'
=
R1 R1'
*
*
O
OH
M. Replacement of the pyrole moiety of tela-
H
N
OiPr
O
previr with a 10,30-dihydrospiro[azetidine-3,20-indene] P2 lead to a
complete loss of potency (compound 37b). However, slight tuning
at P1 by replacing the propyl chain with a cyclobutyl or a cyclopro-
pyl chain gave compounds 37a and 37c that displayed potency
c, d
+
N
N
H
O
1
1'
24
R /R
=
N
H
N
O
*
*
35 R1/R1' = H/H
34
R1 R1'
similar to telaprevir with EC50 values of 0.8 and 0.7
lM, respec-
OH
tively, compared to 0.4 M for telaprevir. Unfortunately, com-
l
OH
H
O
N
O
O
H
e, f
ClH.H2N
N
pound 37a was found to be toxic toward Huh7 cells at a similar
level to the observed anti-HCV activity and thus the anti-HCV read-
out was most likely due to toxicity. Introduction of the same 10,30-
dihydrospiro[azetidine-3,28-indene] P2 moiety into the boceprevir
scaffold lead first to inactive compounds 41a–d and 42a–b but
introduction of a cyclohexyl amine as the P4-cap lead to low
micromolar derivatives 41e and 41f displaying EC50 values of 8.9
and 5.8 lM, respectively. However, these two compounds also
proved to be toxic to Huh7 cells and thus their anti-HCV activity
is most likely due to their toxic effects.
+
R4
N
N
N
H
R3
O
N
O
29a-f
36
O
NHR4
O
R1 R2
HN
O
R3
g
O
N
O
H
N
N
N
H
N
O
37a-i
Compound 37h was one of the first prepared that displayed sin-
gle digit micromolar activity and we chose to profile it further to
determine if the series might differentiate itself from the clinical
PIs such as telaprevir, boceprevir and MK-700911. First using a
panel of genotype 1b mutant NS3 protease enzymes conferring
resistance to other PIs, we found the profile to be similar to boce-
previr but less potent versus R155K, V36M and A156S relative to
boceprevir. In consideration of fold increase, the resistance profile
based on the mutants tested was better than telaprevir (on which
the 37h structure is based) and MK-7009 (Table 2), which encour-
aged us to profile 37h further.
We next looked at compound 37h side-by-side with telaprevir,
boceprevir and MK-7009 versus several human proteases to deter-
mine if it may have toxicities associated with inhibition of these
proteases. Elastase (Neutrophil), Plasmin, Thrombin, and Cathepsin
S were chosen as potentially contributing to the clinical observa-
tion of rashes with telaprevir. Elastase (or leukocyte elastase) also
known as ELA2 (elastase 2) is a serine protease in the same family
as chymotrypsin and has broad substrate specificity. Secreted by
neutrophils during inflammation, one of its primary roles is to de-
stroy bacteria in host tissue.12 Plasmin is a serine protease derived
from the conversion of plasminogen in blood by plasminogen acti-
vators.13 Plasmin degrades many plasma proteins, most notably,
fibrin clots. Plasmin is also involved in several pathological and
physiological processes such as inflammation, neoplasia, metasta-
sis, wound healing, angiogenesis, embryogenesis and ovulation.14
Thrombin is a protein in the blood that has many effects in the
coagulation cascade. It is a serine protease that converts soluble
fibrinogen into insoluble strands of fibrin, as well as catalyzing
many other coagulation-related reactions.15 Cathepsin S is a lyso-
somal cysteine protease that participates in the degradation of
antigenic proteins and therefore is key to immune response.16
Inspection of the human protease data presented in Table 3
reveals a profile for 37h that is quite similar to telaprevir. Interest-
ingly MK-7009 did not inhibit any of the four human proteases up
Scheme 4. Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2, rt, 18 h, 90-95%; (b)
(i) HOBt, EDCI, DIPEA, DMF, rt, 12 h, 55–65%; (ii) 4N HCl in dioxane, rt, 3 h, 60–70%;
(c) 2-pyrazine carboxylic acid, HOBt, EDCI, DMF, rt, 18 h, 76–80%; (d) LiOH, THF/
MeOH/H2O, rt, 6 h, 90–95%; (e) HOBt, EDCI, DMF, rt, 18 h, 63–70%; (f) LiOH, THF/
MeOH/H2O, rt, 6 h, 77–90%; (g) (i) HOBt, EDCI, DMF, rt, 18 h, 70–75%; (ii) Dess–
Martin reagent, CH2Cl2, rt, 18 h, 75–85%.
HO HN
OH
H
ClH.H2N
N
+
HN
O
O
OH
O
a, b
R3
R3
29a-b
O
N
H
N
Boc
22
38
HN
O
HO
O
HN
HN
O
c
HN
O
R3
R3
d
N
N
O
BocHN
BocHN
O
O
39
40
g, h
e, f
O
HN
O
O
HN
O
HN
O
HN
R3
R3
N
N
O
H
N
H
N
H
N
R
O
R
O
O
O
42a-b
41a-f
Scheme 5. Reagents and conditions: (a) HOBt, EDCI, DMF, 0 °C to rt, 12–16 h, 59–
75%; (b) 4 N HCl in dioxane, rt, 5–6 h, 90–95%; (c) BocNH–tBu-glycine, HOBt, EDCI,
DMF, 0 °C to rt, 12–16 h, 73–80%; (d) Dess–Martin reagent, CH2Cl2, rt, 6 h, 70–85%;
(e) 4 N HCl in dioxane, rt, 6 h, 90–95%; (f) R–N@C@O, NMM, CH2Cl2, 0 °C to rt, 12 h,
56–70%; (g) 4 N HCl in dioxane, rt, 5–6 h, 85–90%; (h) R–COOH, HOBt, EDCI, DIPEA,
DMF, 0 °C to rt, 65–76%.
to 100 lM, while boceprevir had a similar profile except for its sub-
micromolar inhibition of thrombin (see fig. 2).
Based on these results, our most promising compounds 37c and
37h along with a selection of the remaining compounds, were fur-
ther profiled by assessing their cytotoxicity in primary human PBM
(peripheral blood mononuclear) cells, CEM cells (a human-T-cell-
derived cell line) and Vero cells (African green monkey kidney
Synthesized compounds 37a–i, 41a–f and 42a–b were evalu-
ated for inhibition of HCV RNA replication in Huh7 cells using a
subgenomic HCV replicon system (Table 1).9 Cytotoxicity in
Huh7 cells was determined simultaneously with anti-HCV activity

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L. Bondada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6325–6330
Table 1
Chemical structure, anti-HCV activity and cytotoxicity of compounds 37a–i, 41a–f and 42a–b
O
HN R4
O
R2 R2'
HN
R3
O
H
N
R1
O
O
Compound
R1
R2/R2’
R3
R4
Anti-HCVa
EC50
(l
M)
Cytotoxicity, CC50
(
l
M) in
EC90
PBM
CEM
Vero
Huh7
1
2
N/A
N/A
N
N/A
N/A
N/A
N/A
N/A
N/A
0.01
0.4
0.03
0.9
>100
ND
>100
ND
>100
ND
>10
>10
O
*
*
*
*
37a
37b
37c
37d
37e
37f
37g
37h
37i
0.7
1.0
29
43
>100
>100
>100
>100
>100
>100
>100
27
1.4
N
HN
*
*
*
*
*
*
*
*
*
*
*
*
*
N
N
N
N
N
N
N
N
O
>10
0.8
>10
1.5
>100
12
72
>10
>10
>10
>10
>10
>10
>10
>10
N
N
N
N
N
N
N
N
HN
*
*
*
O
14
HN
*
*
*
*
*
*
*
O
*
*
H/H
>10
>10
>10
>10
2.3
>10
>10
>10
>10
6.4
>100
27
>100
30
HN
O
H/H
H/H
HN
F
*
*
O
80
52
HN
OMe
O
H/H
>100
32
38
HN
O
*
*
*
*
*
16
HN
F
O
*
5.4
9.7
32
5.4
21
HN
NH
*
*
*
*
*
*
*
*
41a
41b
41c
41d
41e
41f
13
31
>100
>100
15
ND
ND
ND
ND
ND
15
>100
95
27
*
*
*
*
*
*
*
*
*
*
*
*
*
*
NH
NH
>10
>10
>10
8.9
5.8
>10
>10
>10
>10
30
>10
>10
>10
8.2
5.5
>10
46
*
*
*
*
MeO
NH
22
43
NH
NH
30
44
*
9.8
>10
15
26
*
*
42a
44
35
46
(continued on next page)

L. Bondada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6325–6330
6329
Table 1 (continued)
Compound
R1
R2/R2’
R3
R4
Anti-HCVa
EC50
(l
M)
Cytotoxicity, CC50
(
l
M) in
EC90
>10
PBM
CEM
Vero
Huh7
*
*
*
42b
>10
31
22
43
10
*
*
a
HCV Replicon in Huh7 cells. All concentrations were run in triplicate and reported as mean values.
Table 2
HCV NS3/4A Protease Assay, The EC50/EC90
(lM) values are shown for genotype 1b wild type and mutants
HCV
Boceprevir
EC90
Telaprevir
37 h
MK-7009
Protease
EC50
EC50
EC90
EC50
0.78
>100 (>130)
5.5 (7.1)
0.4 (0.5)
6.9 (8.8)
43 (55)
EC90
9.81
>100 (>10)
64 (6.6)
3.3 (0.3)
75 (7.6)
>100 (>10)
8.7 (0.9)
8.2 (0.8)
EC50
EC90
Wild type
A156T
R155K
D168V
V36M
A156S
V170A
D168A
0.52
ꢁ1
0.06
0.78
0.0062
0.013
61 (120)
1.4 (2.7)
0.22 (0.4)
0.35 (0.7)
3.7 (7.1)
0.77 (1.5)
0.52 (1)
>100 (>100)
4.8 (ꢁ4.8)
0.9 (ꢁ0.9)
5.6 (ꢁ5.6)
P100 (P100)
7.7 (ꢁ7.7)
4.7 (ꢁ4.7)
46 (780)
0.8 (13)
0.08 (1.3)
0.51 (8.5)
2.2 (37)
0.2 (3.3)
0.58 (1)
>100 (>130)
8.3 (11)
0.74 (0.9)
5.1 (6.5)
52 (66)
0.91 (150)
>1 (>160)
>1 (>160)
0.045 (7.2)
>1 (>77)
>1 (>77)
>1 (>77)
0.10 (7.8)
0.017 (1.3)
0.010 (0.8)
11 (840)
<0.001 (<0.16)
0.0046 (0.7)
2.4 (390)
1.5 (1.9)
0.69 (0.9)
>1 (>1.2)
0.72 (0.9)
Fold Increase (FI) data are in parenthesis.
Table 3
Effect of HCV Protease Inhibitors on selected human proteases (l
M)a
Compound
Elastase
Plasmin
IC90
Thrombin
IC90
Cathepsin S
IC50
IC90
IC50
IC50
<1
>100
<1
IC50
IC90
37h
7.8 0.3
>100
8.0 1.6
P100
85 2.4
>100
>100
>100
>100
8.7 2.5
>100
>100
>100
110 32
>100
<1.7 0.4
>100
<1
<1.4 0.9
>100
>100
>100
>100
>100
MK-7009
Telaprevir
Boceprevir
78 14
>100
>100
<1
> 100
a
Results are average IC50 and IC90 values standard error from 2 independent assays.
the founder and a major shareholder of RFS Pharma, LLC. Emory re-
ceived no funding from RFS Pharma, LLC to perform this work and
vice versa.
N
O
N
O
O
O
H
N
S
O
N
References and notes
H
H
O
O
N
O
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O
MK-7009 (vaniprevir) 43
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