Design, synthesis, and biological activity of novel 1,4-disubstituted piperidine/piperazine derivatives as CCR5 antagonist-based HIV-1 entry inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.019

A series of novel 1,4-disubstituted piperidine/piperazine derivatives were designed, synthesized and evaluated for their in vitro activities against HIV-1 Bal (R5) infection in CEMX174 5.25M7 cells. A majority of these compounds showed potent anti-HIV-1 activities with IC₅₀ at nanomolar levels. N-(4-Fluoro-benzyl)piperazine analog B07 hydrochloride exhibited potency against HIV-1 activity similar to that of TAK-220 hydrochloride, but it had much better water solubility (25 mg/ml in phosphate sodium buffer at 25 °C) and oral bioavailability (56%) than TAK-220 hydrochloride (a solubility of 2 mg/ml and oral bioavailability of 1.4%). These results suggest that B07 hydrochloride may serve as a better lead for the development of new anti-HIV-1 therapies or microbicides for treatment and prevent of HIV-1 infection.

Full text of DOI:10.1016/j.bmcl.2012.03.019

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3284–3286
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological activity of novel 1,4-disubstituted
piperidine/piperazine derivatives as CCR5 antagonist-based HIV-1
entry inhibitors
Ming-xin Dong a, Lu Lu b, Haitao Li a, Xiaohua Wang a, Hong Lu b, Shibo Jiang b,c, , Qiu-yun Dai a,
⇑
⇑
a Beijing Institute of Biotechnology, Beijing 100071, China
b MOE/MOH Key Laboratory of Medical Molecular Virology, Institute of Pathogenic Microbiology, Shanghai Medical College, Fudan University, Shanghai 200032, China
c Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,4-disubstituted piperidine/piperazine derivatives were designed, synthesized and
evaluated for their in vitro activities against HIV-1 Bal (R5) infection in CEMX174 5.25M7 cells. A majority
of these compounds showed potent anti-HIV-1 activities with IC50 at nanomolar levels. N-(4-Fluoro-
benzyl)piperazine analog B07 hydrochloride exhibited potency against HIV-1 activity similar to that of
TAK-220 hydrochloride, but it had much better water solubility (25 mg/ml in phosphate sodium buffer
at 25 °C) and oral bioavailability (56%) than TAK-220 hydrochloride (a solubility of 2 mg/ml and oral bio-
availability of 1.4%). These results suggest that B07 hydrochloride may serve as a better lead for the
development of new anti-HIV-1 therapies or microbicides for treatment and prevent of HIV-1 infection.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 October 2011
Revised 21 February 2012
Accepted 6 March 2012
Available online 11 March 2012
Keywords:
CCR5 receptor antagonist
Anti-HIV-1
SAR
CCR5, a member of the seven transmembrane G-protein cou-
pled receptor (GPCR) family, plays an integral role in the R5-tropic
HIV-1 entry process by serving as a critical co-receptor for the viral
envelope protein gp120.1 Over the last 10 years or so, researchers
in the pharmaceutical industry have increased their efforts to de-
velop CCR5 antagonist-based HIV-1 entry inhibitors. As a result,
many CCR5 antagonist chemotypes have been explored and several
of them have entered clinical development stage.2 For example,
TAK-220 (Fig. 1) developed by Takeda Pharmaceutical Co. Ltd
was shown to strongly inhibit the replication of CCR5-using HIV-
1 clinical isolates in human peripheral blood mononuclear cells
with a median effective concentration (EC50) about 1 nM.3 In
August 2007, Takeda licensed TAK-220 to Tobira Therapeutics un-
der the new name TBR-220, and this compound has entered Phase I
clinical trials.
(Fig. 1) and found that compound 1 showed potency against HIV-
1 activity (EC50 = 2.7 nM) similar to that of TAK-220.6 Long and
co-workers generated a novel series of 1,4-disubstituted piperidine
derivatives based on TAK-220 and compound 2, and reported that
compound 3 (Fig. 1) exhibited an EC50 value about 2.5 nM.7
Here, we designed and synthesized a new series of CCR5 antag-
onists by replacing 4-benzylpiperidine of TAK-220 using different
approaches. Applying the strategy of retroisosterism, we first cre-
ated compound A1 by inverting the amido bond of lead compound
3 (see Scheme 1). The commercially available N-Boc-piperidine-4-
carboxylic acid 4 was selected as the starting material. Using HBTU
as the coupling agent, the carboxylic acid was coupled with
ethylamine hydrochloride to yield compound 5. In the presence
Using a single-round infection assay with human CCR5 mole-
cule and its mutants, the binding mode for TAK-220 was proven.4,5
The observed loss in binding (647-fold) to the CCR5 E283A mutant
suggested a strong salt-bridge interaction between Glu283 and the
N atom of piperidine in TAK-220. Another strong interaction of
TAK-220 with CCR5 is predicted to be a hydrophobic interaction
with the acetyl group and Ile198. Roche Pharmaceutical Inc. cre-
ated an analogous compound 1 by replacing 4-benzylpiperidine
of TAK-220 with an octahydro-pyrrolo[3,4-c]pyrrole template
O
O
O
N
N
NH2
N
N
N
O
N
O
N
N
N
Cl
Cl
O
1
TAK-220
O
O
S
O
N
N
O
N
O
O
N
N
N
Cl
⇑
Corresponding authors.
E-mail addresses: sjiang@nybloodcenter.org (S. Jiang), qy_dai@yahoo.com
2
3
(Q.-y. Dai).
Figure 1. Structure of CCR5 antagonists.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.019

M. Dong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3284–3286
3285
O
N
N
O
O
O
N
O
N
d
a
b
c
boc N
boc N
O
N
N
boc N
HN
OH
NH
O
Cl
4
5
6
7
A1
Scheme 1. Preparation of representative compound A1. Reagents and conditions: (a) HBTU, DIEA, ethylamine hydrochloride, DMF, rt, overnight; (b) benzyl bromide, KOtBu,
DMF, 0 °C, 88% (steps a–b); (c) TFA, CH2Cl2, 100%; (d) 1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-chloropropyl)piperidine-4-carboxamide, KI, K2CO3, DMF, 52%.
Table 1
N
Anti-HIV activities and cytotoxicities of 1,4-disubstituted piperidine analogs
N
N
N
N
O
N
N
Figure 2. The design of 1,4-disubstituted piperazine fragment.
O
N
R
Cl
b
a
boc N
NH
boc N
N R
HN
N R
10
8
9
Compd
R
IC50 (nM)
CC50
(l
M)
SI (CC50/IC50
)
Scheme 2. Preparation of N-substituted piperazine. Reagents: (a) RX, K2CO3, DMF;
(b) TFA, DCM.
*
N
A1
59.89 11.25
83.85 2.09
1399
O
O
O
Table 2
N
A2
A3
A4
20.33 3.27
87.48 4.72
1833 294
>52.40
>2581
>593
10
N
H
*
*
Anti-HIV activities and cytotoxicities of 1,4-disubstituted piperazine analogs
N
>51.93
O
N
H
S
N
N
N
O
N
R
HN
17.91 0.67
N
*
*
*
Cl
N
HN
A5
A6
10.16 2.18
52.27 1.45
>55.10
>56.60
>5333
>1081
N
O
Compd
B01
R
IC50 (nM)
CC50
(
l
M)
SI (CC50/IC50
6797
)
14.88 2.90
101.17 5.34
97.76 10.94
*
of KOtBu, compound 5 reacted with benzyl bromide to give com-
pound 6. After removal of the Boc group using TFA/CH2Cl2, the
key intermediate 7 could be generated. 1-Acetyl-N-(3-chloro-4-
methylphenyl)-N-(3-chloropropyl) piperidine-4-carboxamide was
prepared as the literature3 and reacted with intermediate 7 in
the presence of potassium iodide and potassium carbonate to af-
ford the target compound A1.
1-Acetyl-N-(3-chloro-4-methylphenyl)-N-(3-chloropropyl)pipe
ridine-4-carboxamide reacted with another commercially avail-
able 4-substituted piperidine fragment to give target compounds
A2–6, as listed in Table 1. Compounds A1–6 were evaluated for
their in vitro antiviral activity against infection of HIV-1 Bal (R5)
strain in CEMX174 5.25M7 cells and for their cytotoxicities.8,9
Compound A1 exhibited potent antiviral activity (IC50 = 59.89 nM)
O
B02
147.37 4.72
663
*
N
B03
B04
B05
B06
B07
11.62 1.45
9.07 1.45
32.67 1.45
3.63 0.73
6.17 3.09
>52.36
>56.70
>56.70
>53.13
>100
>4507
>6154
*
S
Cl
N
*
*
*
*
>1739
N
N
Cl
F
>14,545
>16,207
O
S
B08
3.99 0.36
>49.28
>12,121
*
*
O
N
B09
B10
B11
56.26 6.53
6.56 3.82
9.01 2.96
>58.30
>1039
5726
and low cytotoxicity (CC50 = 83.85 lM) values. Compounds A2–3,
Cl
37.56 3.68
>100
which were generated by changing the position of the carbonyl
group in compound A1 retained antiviral activity. Replacement of
the carbonyl group in compound A1 with methylene resulted in
the generation of compounds A4–5. Compound A4 had a substan-
tial reduction of the anti-HIV-1 activity, while compound A5 exhib-
*
*
CN
>11,098
F
B12
1.85 0.17
30.47 7.99
16,470
*
F
F
TAK-220
3
2.48 0.66
5.23 1.64
>100
36.21 2.89
>40,322
6924
ited
a five fold improvement of viral inhibitory activity
(IC50 = 10.16 nM). We performed a docking analysis of A4 and A5
with CCR5 based on the binding model of TAK-220 with the
CCR5.4,5 The results showed that there is a H-bond interaction be-
tween pyrimidine moiety of A5 and I116, but found no interaction
of A4 with this residue in CCR5, perhaps the 4-methyl group of A4
affects the orientation of pyridine group.
Based on the structural characteristic of 1,4-disubstituted
piperidine fragment in TAK-220 and octahydro-pyrrolo[3,4-c]pyr-
role template in compound 1, we also designed an N-substituted
piperazine structure to replace 4-benzylpiperidine of TAK-220, as
shown in Figure 2.
The synthesis of N-substituted piperazine fragments was
presented in Scheme 2. In the presence of potassium carbonate,
commercially available 1-boc-piperazine 8 reacted with different
halogenated compounds to give the corresponding product 9. After

3286
M. Dong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3284–3286
Table 3
Pharmacokinetic property of B07 hydrochloride and TAK-220 hydrochloride in rata
Parameter
B07 hydrochloride
TAK-220 hydrochloride
IV (1 mg/kg)
PO (5 mg/kg)
IV (1 mg/kg)
2.25 0.60
PO (5 mg/kg)
T1/2 (h)
0.59 0.23
209.39 31.88
0.76 0.09
587.28 402.00
56%
4.25 0.65
22.67 9.20
1.4%
AUC0–12 (ng h/mL)b
328.03 101.50
%Fc
a
b
c
Drug was administered orally (PO) or intravenously (IV) to fasted SD rats (n = 3). Data are expressed as the means SD.
AUC0–12, AUC from time zero to 12 h.
F = AUCpo/(AUCiv  5)  100%.
removal of the Boc group using TFA/CH2Cl2, the intermediate 10
could be generated.
Replacement of the 4-benzylpiperidine fragment in TAK-220 with
N-(4-fluoro-benzyl)piperazine resulted in the creation of com-
pound B07, which exhibited attractive antiviral properties, selec-
tivity and bioavailability. This study suggests that B07 and other
1,4-disubstituted piperazine derivatives are promising for develop-
ment as new anti-HIV-1 therapeutics or microbicides for treatment
and prevention of HIV-1 infection.
1-Acetyl-N-(3-chloro-4-methylphenyl)-N-(3-chloropropyl)pipe
ridine-4-carboxamide reacted with N-substituted piperazine 10 to
afford target compounds B01–12. The antiviral activity and cyto-
toxicities of these compounds were summarized in Table 2. TAK-
220 and compound 3 were also tested as control compounds.
The majority of the test compounds inhibited HIV-1 replication
at the low nanomolar concentration range and exhibited low cyto-
toxicity. Replacement of the N-benzyl group in compound B01
(IC50 = 14.88 nM) with the N-benzoyl group resulted in compound
B02 (IC50 = 147.37 nM) with 10-fold reduction in antiviral activity.
An electron withdrawing substituent in the 4-benzylpiperidine
moiety in compound B01 could enhance anti-HIV-1 activity in
the following order: B12 (substituted with CF3), B08 (substituted
with CH3SO2), B07 (substituted with F), B10 (substituted with Cl)
and B12 (substituted with CN). In addition, some compounds
containing heteroaromatic ring instead of the N-benzyl group in
compound B01 also exhibited potent antiviral activity.
Acknowledgments
We thank Dr. Xinbo Jiang from GenScript USA Inc. for the tests
of inhibitory activity of human ERG channels, Prof. Zhengqing
Zhang from Beijing Institute of Toxicology and Pharmacology for
the determination of pharmacokinetics. This work was supported
by the key National Science and Technology Program—‘Major
New Drug Development During the 11th Five-Year Period of China’
(2009ZX09103-628) to Q.Y.D. and by Grants from the Natural Sci-
ence Foundation of China #81102373 to D.M.X. and #81173098 to
S.J.
Because compound B0710 was easily synthesized and exhibited
high anti-HIV-1 activity and lower cytotoxicity (IC50 = 6.17 nM,
References and notes
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The pharmacokinetic properties of B07 hydrochloride and TAK-
220 hydrochloride in rat were shown in Table 3.11 B07 hydrochlo-
ride exhibited much higher oral bioavailability (56%) than TAK-220
hydrochloride (1.4%) in exposure, but shorter terminal phase half-
life (0.76 h) than TAK-220 hydrochloride (4.25 h) in exposure. This
phenomenon may arise from the insufficient metabolic stability of
4-fluoro derivative in B07 hydrochloride.3 The solubility of B07
hydrochloride in phosphate sodium buffer (10 mM Na2HPO4,
150 mM NaCl, pH 7.4) was about 25 mg/mL at 25 °C, much higher
than TAK-220 hydrochloride (2 mg/ml) and compound 3 hydro-
chloride (<2 mg/ml) under the same condition, suggesting that it
may be a good candidate for development of anti-HIV-1 microbi-
cide formulation for preventing sexual transmission of HIV-1.
It has been reported that some CCR5 antagonists were with-
drawn from clinical trials because of their high binding to hu-
man-ERG channel, which leads to the side-effect of QT interval
prolongation.12,13 Here we tested the binding of B07 hydrochloride
to the human-ERG channel as previously described.14,15 The result
showed that B07 hydrochloride inhibited the hERG currents in a
10. Spectral data of B07: 1H NMR (400 MHz, CDCl3) d 7.28 (m, 4H), 7.18 (d,
J = 1.96 Hz, 1H), 6.99 (m, 3H), 4.52 (d, J = 13.44 Hz, 1H), 3.76 (d, J = 13.72 Hz,
1H), 3.65 (t, J = 7.84 Hz, 2H), 3.46 (s, 2H), 2.84 (m, 1H), 2.48 (m, 4H), 2.42 (s,
3H), 2.34 (m, 5H), 2.05 (s, 3H), 1.64 (m, 6H). 13C NMR (100 MHz, CDCl3) d
173.75, 167.94, 164.03, 161.57, 140.69, 135.71, 133.97, 132.22, 128.50, 127.47,
125.77, 115.88, 115.67, 57.54, 53.35, 47.84, 47.48, 45.89, 44.90, 39.93, 39.09,
28.67, 28.03, 21.89, 21.26, 19.37. MS (Turbo Spray): m/z (M+1)+ 528.8.
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concentration-dependent manner with an IC50 at 1.82 0.12
which was 295-fold of its antiviral IC50 value.
lM,
In summary, we have designed and synthesized a novel series of
1,4-disubstituted piperidine/piperazine embodied CCR5 antago-
nists with potent antiviral activity against R5 HIV-1 strains.
15. Yap, Y. G.; Camm, J. BMJ 2000, 320, 1158.