Studies on the structure-activity relationship of 1,3,3,4-tetra-substituted pyrrolidine embodied CCR5 receptor antagonists. Part 1: Tuning the N-substituents

Article abstract of DOI:10.1016/j.bmcl.2010.05.102

A novel series of CCR5 antagonists has been identified, utilizing the lead, nifeviroc, which were further modified based on bioisosteric principles. Lead optimization was pursued by balancing potential toxicity and potency. Potent analogues with low toxic properties were successfully developed by formation of urea and amide bonds at the nitrogen at position 4- of the pyrrolidine ring.

Full text of DOI:10.1016/j.bmcl.2010.05.102

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4012–4014
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies on the structure–activity relationship of 1,3,3,4-tetra-substituted
pyrrolidine embodied CCR5 receptor antagonists. Part 1: Tuning
the N-substituents
Li Ben a, , Eric Dale Jones b, , Enkun Zhou c, , Chen Li d, Dean Cameron Baylis b, Shanghai Yu e, Miao Wang e,
Xing He c, Jonathan Alan Victor Coates b, David Ian Rhodes b, Gang Pei a, John Joseph Deadman b, , Xin Xie c,
,
*
Dawei Ma e,
*
a Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 319 Yueyang Lu, Shanghai 200031, China
b Avexa Limited, 576 Swan Street, Richmond Victoria 3121, Australia
c State Key Laboratory of Drug Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road,
Shanghai 201203, China
d Shanghai Targetdrug Co. Ltd, 500 Caobao Lu, Shanghai 20023, China
e State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of CCR5 antagonists has been identified, utilizing the lead, nifeviroc, which were further
modified based on bioisosteric principles. Lead optimization was pursued by balancing potential toxicity
and potency. Potent analogues with low toxic properties were successfully developed by formation of
urea and amide bonds at the nitrogen at position 4- of the pyrrolidine ring.
Received 12 April 2010
Revised 11 May 2010
Accepted 27 May 2010
Available online 1 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CCR5 antagonists
Anti-HIV
Human immunodeficiency virus (HIV) infects millions of people
worldwide and causes the fatal disease Acquired Immune Defi-
ciency Syndrome (AIDS). Although highly active antiretroviral
therapy (HAART), a combination of drugs targeting HIV-1 protease,
reverse transcriptase and integrase, has been successful in sup-
pressing viral replication and in reducing HIV-1-associated mortal-
ity, issues relating to the development of viral resistance and the
long term toxicity of the component drugs remain prominent.1
Accordingly it seems urgent to discover new classes of drugs for
therapeutic intervention with better efficacy and safety profiles.2
The discovery that chemokine receptors function as HIV-1 co-
receptors has provided us novel targets for controlling HIV-1 infec-
tion.3,4 Although several chemokine receptors have been impli-
cated in HIV entry in various experimental systems, CCR5, a
member of the G-protein-coupled receptor (GPCR) superfamily, is
the principal co-receptor for the R5 HIV-1 strains that is most com-
monly transmitted between individuals and predominates during
the early years of infection.5,6 With the combination of high-
throughput screening and medicinal chemistry-based structure
optimization, a considerable number of potent CCR5 antagonists
with excellent in vitro anti-viral activities have been discov-
ered.7–11
Our efforts have led to the discovery of a potent CCR5 antago-
nist, nifeviroc (1), featuring a 1,3,3,4-tetra-substituted pyrrolidine
template (Fig. 1). This compound Antagonizes RANTES-induced cal-
cium elevation and GTPcS binding in CCR5-expressing CHO cells
with IC50 values of 3.5 nM and 2.9 nM, respectively. It also showed
excellent in vitro anti-HIV activity in PBMC cells (EC50 <0.4 nM).12
However, further development of 1 could be limited the undesir-
HO
N
O
N
O
N
O
NO2
nifeviroc (1)
* Corresponding authors.
E-mail addresses: jdeadman@avexa.com.au (J.J. Deadman), xiexin-2004@163.
com (X. Xie), madw@mail.sioc.ac.cn (D. Ma).
These authors contributed equally to this work.
Figure 1. Structure of nifeviroc.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.102

L. Ben et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4012–4014
4013
then extended this series by replacing the carbamate linkage with
an amide linker by coupling 2 with substituted or unsubstituted
phenylacetic acids to afford the amides 5.
HO
N
HO
N
N
O
N
HN
N
1. ArCH2OH
O
triphosgene
DIPEA
With all the designed compounds in hand, we evaluated their
ability to inhibit RANTES-stimulated
[ cS binding to
35S]-GTP
2. 2, DIPEA
62-85%
CCR5-expressing CHO cell membranes, and antiviral activity in
R5-tropic HIV-1 Ba-L infected peripheral blood mononuclear cells
(PBMCs) with 10% FBS. The biological data for the carbamates 3
is summarized in Table 1. We have previously reported that dele-
tion of the nitro group or introduction of electron-donating groups
resulted in a dramatic loss of antiviral activity, with different ef-
fects on excellent binding inhibition (compare 1, 3a with 3b and
3c).12 In contrast, in this study we found that the addition of elec-
tron-withdrawing groups in the 4-position of the terminal phenyl
group (3d, 3e, 3f, 3g) restored excellent antiviral potency
(EC50 <3 nM), although incorporation of these groups was detri-
mental to CCR5 binding inhibition. Substitution with halogens
was accompanied by a slight reduction in potency (3h, 3i, 3j).
Unfortunately, all these new analogues displayed similar hERG
channel affinity potency, except for methylsulfonyl analogue 3f
O
O
3
R
2
1. ArCH2NH2, triphosgene,DIPEA
2. 2, DIPEA
ArCH2COOH, EDCI
HOBt, DIPEA, 73-95%
65-78%
HO
N
HO
N
N
N
N
N
O
O
NH
O
O
R
5
R
4
Scheme 1.
which showed a moderate drop of hERG affinity at 1 lM.
It can be seen from Table 1 that IC50’s measured in the assay
that uses RANTES as the ligand for the CCR5 receptor do not follow
the same trends as the EC50 measured in the whole cell assay
where the GP120 protein on the virus itself is the ligand.
In order to study the effects of the carbamate linker on the
potencyinmoredepth, andto decrease thepotentialtoxicityfurther,
we developed a series of urea analogues. From the results of Table 2,
we found that the urea compounds (except 4a) showed slightly less
hERG affinity than the corresponding carbamates, while showing
excellent binding inhibition and antiviral activity. Among this series
of compounds, 4e matched the potency as the lead, nifeviroc. How-
ever, it maintained high hERG affinity. Of note, 4i and 4j showed
weak hERG binding activity, but also had excellent potency.
able aromatic nitro-moiety, a possible mutagenic and carcinogenic
functionality.13–15 Secondly, the high hERG affinity (IC50 <1.0
M)
of 1 suggested a narrow safety window. In a continuation of our
work, a series of analogues of 1 were prepared aimed at eliminat-
ing any potential toxicity liability of the aromatic nitro group and
decreasing hERG channel affinity.
As depicted in Scheme 1, we accessed further analogues of 1 via
the key intermediate 2.12 A round of carbamates 3 was prepared by
reacting substituted benzyl alcohols with triphosgene and then
trapping the resulting chloro-formates with 2. Urea analogues 4
were assembled by condensation of 2 with carbamic chlorides gen-
erated in situ from benzyl amines with triphosgene. Finally we
l
Next we turned our attention to the amide series 5. As indicated
in Table 3, a wide range of para-substituents on the terminal phe-
nyl group featuring an amide linker were examined and most led
to a drop in antiviral potency, although good CCR5 binding inhibi-
tion was retained (5a–g). However, similar to 3f the methylsulfo-
nyl analogue (5h) displayed encouraging binding and antiviral
Table 1
Biological data for carbamates 3
HO
N
N
O
N
O
Table 2
Biological data for ureas 4
O
3
R
HO
N
N
N
a
b
R
IC50 (nM) EC50 (nM) hERG bindingc hERG bindingd
O
NH
4-NO2 (1)
H (3a)
4-OMe (3b)
2.9
<0.4
78
>100
62.0
—
—
98.0
—
—
8.75
6.22
O
O
O
R
4
122
>100
—
—
a
b
R
IC50 (nM) EC50 (nM) hERG bindingc hERG bindingd
(3c)
H (4a)
4-F (4b)
4-Cl (4c)
4-NO2 (4d)
4-CF3 (4e)
4-OCF3 (4f)
2,4-F2 (4g)
3,4-F2 (4h)
4-SO2NHCH3 (4i)
4-SO2N(CH3)2(4j)
37.7
1.71
2.66
3.2
1.09
6.15
1.62
1.69
2.93
5
>100
30
1.54
3
<0.4
2.5
16
34.7
40.6
50.3
37.2
51.1
87.9
—
—
7.3
13.1
84.1
82.4
97.8
83.1
104
100.8
—
—
19.9
73.9
4-CF3 (3d)
>30
6.15
37.3
2
2.5
<0.4
80.8
87.9
44.1
104.6
100.8
96.3
4-OCF3 (3e)
4-SO2Me (3f)
N
O
113.6
<0.4
72.6
102.2
N
(3g)
8
4-F (3h)
4-Cl (3i)
4-Br (3j)
220.6
1.09
20.2
18
2.8
65
70.2
76.2
—
101.9
97.9
—
2.8
0.9
a
b
c
a
b
c
SPA GTP
Antiviral activity in PBMC.
% Inhibition at 1 M.
M.
cS assay.
SPA GTPcS assay.
Antiviral activity in PBMC.
% Inhibition at 1 M.
M.
l
l
d
d
% Inhibition at 10
l
% Inhibition at 10
l

4014
L. Ben et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4012–4014
Table 3
Biological data for amides 5
possibly by interaction with Thr623 and Ser624 of the selectivity
filter region of the hERG protein.
The pharmacokinetics and the ability of 5j to inhibit HIV repli-
cation in various strains was examined (Table 4). Consistent with
5j being an antagonist of CCR5, potent activity against R5 tropic
strains (HIV-1 006) was observed (EC50 <0.8 nM). However with
X4 tropic (HIV-1 599) or X4/R5 dual tropic (HIV-1 593) strains anti-
viral activity was lacking (EC50 >1000 nM).
HO
N
N
N
O
In rats, 5j showed rapid plasma clearance, possibly by phase 1
metabolism as predicted by exposure of 5j to isolated human liver
microsomes. Additionally systemic exposure appears limited with
PAMPA assays reveling low permeability consistent with a low PO
O
R
5
a
b
R
IC50
EC50
hERG
hERG
(nM)
(nM)
bindingc
bindingd
Cmax (0.031 lM) and CACO-2 data suggesting 5j maybe a substrate
for efflux transporters.
H (5a)
3.5
14.6
5.3
11
3.6
17.8
149.2
4
2.1
1.53
3.32
>100
>100
10
>100
>100
>100
70
34
8
0.8
<0.4
44.6
40.7
68.9
43.9
40.1
—
92
4-OMe (5b)
4-NO2 (5c)
4-F (5d)
4-Cl (5e)
4-Br (5f)
4-CF3 (5g)
4-SO2Me (5h)
4-SO2NH2 (5i)
4-SO2NHCH3 (5j)
4-SO2N(CH3)2
(5k)
88.7
96.1
91.5
94.8
—
In summary, SAR studies around the carbamate linker and sub-
stituents on the left phenyl portions of nifeviroc have led to the
identification of 4-sulfamoyl analogues, 4i, 4j, 5j and 5k as highly
potent CCR5 antagonists with significantly excellent antiviral
activity, very weak hERG affinity and lacking the undesirable aro-
matic nitro functionality. Further SAR and pharmacokinetic opti-
mization on this series will be the subject of future publications.
—
—
23.9
14.4
18.2
20.3
71.1
16.6
34.1
98.6
Acknowledgements
a
b
c
SPA GTP
Antiviral activity in PBMC.
% Inhibition at 1 M.
M.
cS assay.
The authors are grateful to the National Natural Science
Foundation of China (Grant 90713047), the Ministry of Science
and Technology (Grant 2008DFB30150, 2009ZX09302-001 and
2009ZX09501-009), and the Chinese Academy of Sciences for their
financial support.
l
d
% Inhibition at 10
l
Table 4
Anti-HIV activity and pharmacokinetics for 5j
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HIV-1 599 (X4)
HIV-1 593 (X4/R5)
Rat pharmacokineticsa
Clp (L/h/kg)
Vdss (L/kg)
Cmax (lg/L)
t1/2 (h)
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activity with attenuated hERG affinity. These findings prompted us
to prepare the sulfonamides analogues 5i–k and interestingly, 5i
hardly inhibited the hERG channel at 1 and 10 lM and kept excel-
lent potency (EC50 = 8 nM). Introduction of a mono-methyl sulfon-
amide (5j), and dimethyl-sulfonamide (5k) increased slightly hERG
channel affinity, but also improved the antiviral activity. The key
finding from this survey was that a hydrophilic group in the termi-
nal phenyl, such as SO2Me, SO2NH2, SO2NHCH3 or SO2N(CH3)2 de-
tuned hERG activity.
A recent review16 has noted that polar or electronegative
groups in the para position of phenyl rings may increase binding,
16. Mitcheson, J. S. Chem. Res. Toxicol. 2008, 21, 1005.