Synthesis and biological evaluation of new HIV-1 protease inhibitors with purine bases as P2-ligands

Article abstract of DOI:10.1016/j.bmcl.2019.03.049

Introducing purine bases to P2-ligands might enhance the potency of Human Immunodeficiency Virus-1 (HIV-1) protease inhibitory because of the carbonyl and NH groups promoting the formation of extensive H-bonding interactions. In this work, thirty-three compounds are synthesized and evaluated, among which inhibitors 16a, 16f and 16j containing N-2-(6-substituted-9H-purin-9-yl)acetamide as the P2-ligands along with 4-methoxylphenylsulfonamide as the P2′-ligand, display potent inhibitory effect on the activity of HIV-1 protease with IC₅₀ 43 nM, 42 nM and 68 nM in vitro, respectively.

Full text of DOI:10.1016/j.bmcl.2019.03.049

Accepted Manuscript
Synthesis and biological evaluation of new HIV-1 protease inhibitors with pu-
rine bases as P2-ligands
Mei Zhu, Biao Dong, Guo-Ning Zhang, Ju-Xian Wang, Shan Cen, Yu-Cheng
Wang
PII:
S0960-894X(19)30204-5
https://doi.org/10.1016/j.bmcl.2019.03.049
BMCL 26363
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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20 March 2019
30 March 2019
Please cite this article as: Zhu, M., Dong, B., Zhang, G-N., Wang, J-X., Cen, S., Wang, Y-C., Synthesis and biological
evaluation of new HIV-1 protease inhibitors with purine bases as P2-ligands, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.03.049
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Synthesis and biological evaluation of new HIV-1 protease inhibitors
with purine bases as P2-ligands
Mei Zhu a, Biao Dong a, Guo-Ning Zhang a, Ju-Xian Wang a, Shan Cen a, *, Yu-Cheng
Wang a, *
a
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing 100050, China
Abstract: Introducing purine bases to P2-ligands might enhance the potency of
Human Immunodeficiency Virus-1 (HIV-1) protease inhibitory because of the
carbonyl and NH groups promoting the formation of extensive H-bonding interactions.
In this work, thirty-three compounds are synthesized and evaluated, among which
inhibitors 16a, 16f and 16j containing N-2-(6-substituted-9H-purin-9-yl)acetamide as
the P2-ligands along with 4-methoxylphenylsulfonamide as the P2'-ligand, display
potent inhibitory effect on the activity of HIV-1 protease with IC50 43 nM, 42 nM and
68 nM in vitro, respectively.
Keywords: purine bases; HIV-1 protease inhibitors; biological evaluation
1. Introduction
AIDS is one of the most challenging problems in medicine and causes severe
perniciousness situations for human health. More than thirty kinds of drugs have thus
far been approved for the treatment of HIV/AIDS for targeting different steps of the
1
HIV viral life cycle. It has become evident that combination chemotherapy is
2
significantly more effective than dosing drugs sequentially. The appearance of
HIV-1 protease inhibitors (PIs) in the mid-1990’s and their combination with reverse
transcriptase inhibitors marked the beginning of highly active antiretroviral therapy
3,4
(HAART).
However, there still exist severe problems, such as the emergence of
5-7
extensively cross-resistant strains of HIV-1, in addition to adverse effects.
Thus,
there is an urgent need for new anti-HIV drug candidates with increased potency,
improved pharmacokinetic properties, and reduced side effects.
HIV-1 protease inhibitors (HIV-1 PIs) play an important role in the treatment of
HIV/AIDS. 8,9 Although approximate ten HIV-1 PIs have been approved by the FDA,
the rapid emergence of multi-drug-resistant (MDR) strains of HIV-1 protease (PR)
10-13
has severely limited long-term treatment options.
HIV-1 PIs is urgent.
Thus, the design of novel
As is already known, nucleoside drugs are important antitumor, antifungal and
especially antiviral agents. 14-19 We introduced purine bases into P2 moieties of HIV-1
protease inhibitors in this work, aiming at increasing inhibitory activity, according to
the strategy to overcome drug resistance through increasing interactions between
inhibitors and protease, carbonyl and/or NH groups involved in newly introduced P2
moieties can promote extensive H-bonding interactions involved directly or
* Corresponding author. Tel. /Fax: +86-10-63165263 (Y.-C. Wang),
E-mail address: wangyucheng@imb.pumc.edu.cn (Y.-C. Wang); shancen@hotmail.com.(S. Cen).

water-mediated with the backbone NH groups of residues Asp29 and Asp30 of PR in
the corresponding S2 subsite (Fig. 1); these interactions may be responsible for the
impressive activities against MDR PR mutants and antiviral activities in cellular
20-25
assays.
According to the literatures and our previous studies, 4-substitutied
phenylsulfonamide on P2'-ligands showed better inhibitory effect on the activity of
HIV-1 protease than other substitutied phenylsulfonamide. 26-29 Both modifications of
the substitution pattern on the phenylsufonamide P2'-ligands of all series, and the
configuration of purine bases on P2-ligands, were synthesized for the
structure-activity relationship (SAR) study in this work.
Fig. 1. Design and general structure of target molecules
2. Results and Discussion
The syntheses of intermediates of substituted 2-(9H-purin-9-yl) acetic acids 2a-k
are outlined in Scheme 1. All starting materials are commercially available. The acids
2a and 2b were readily accomplished by substitution with bromoacetic acid using
potassium carbonate as base from corresponding 6-chloro-9H-purines (1a and 1b) in
30
moderate yields.
N-Alkylation of 1c and 1d with ethyl bromoacetate proceeded
using sodium hydride as the base in anhydrous DMF to give corresponding esters 3a
and 3b in yields of 52% and 59%, respectively. Subsequent saponification was
finished by sodium hydroxide to give acetic acids 2c and 2d in 55% and 45% yields,
respectively. However, acids 2e-g were synthesized directly by saponification using a
“one-pot” method after the N-alkylation of 1e-g in 43-62% yields. The
2-(6-methoxy-9H-purin-9-yl) acetic acids 2h-i were accomplished firstly by refluxing
1a-b with sodium methylate in methanol as 6-methoxy purines 4a-b, followed by
N-alkylation and then hydrolysis using the same method as described in 2c-e. For
synthesis of 6-hydroxy purines acetic acids 2j-k, 6-chloro-9H-purines (1a-b) were
heated at 130 ℃ with sodium benzyloxide in benzyl alcohol to give
6-benzyloxy-9H-purines (5a-b), followed by treatment of ethyl bromoacetate to
afford purine acetates (6a-b). The benzyl protecting group was removed by
hydrogenolysis under 25 bar of hydrogen using lead(Ⅱ) acetate as a catalyst in
methanol for 12 hours in a moderate yield of 6-hydroxy purine esters 7a-b.

Saponification of the ester moiety permitted conversion to acids 2j-k.

Scheme 1. Syntheses of substituted 2-(9H-purin-9-yl) acetic acids 2a-k. Reagents and conditions:
(a) Bromoacetic acid, K2CO3, anhydrous DMF, Ar, r.t, overnight; (b) Ethyl bromoacetate, NaH,
anhydrous DMF, Ar, 0 ℃~ r.t, 3 h; (c) (i) NaOH, H2O, r.t, 1 h; (ii) 4 M HCl, 0 ℃, 0.5 h; (d)
CH3ONa, CH3OH, reflux, 12 h; (e) (i) Ethyl bromoacetate, NaH, anhydrous DMF, Ar, 0 ℃~ r.t, 3
h; (ii) NaOH, H2O, r.t, 1 h; (iii) 4 M HCl, 0 ℃, 0.5 h; (f) (i) PhCH2OH, Na, 130 ℃, 2 h; (ii)
PhCH2OH, 130 ℃, 4 h; (g) H2 (gas), 25 bar, Pb(OAc)2, CH3OH, r.t, 12 h.
Compounds 13-15 were prepared from the commercially available material (2S,
3S)-1,2-epoxy-3-(boc-amino)-4-phenylbutane (9), as reported in the literature and
26-28
shown in Scheme 2.
Catalytic hydrogenation of 14 over 10% Pd/C in methanol
afforded the corresponding aminosulfonamide derivatives 15 29.
Scheme 2. Syntheses of amines 13-15. Reagents and conditions: (a) i-BuNH2, CH3CN, 80 ℃, 6 h;
(b) Aryl sulfonyl chloride, DIEA, DMAP(Cat.), THF, 0 ℃ ~ r.t, 3- 5 h; (c) CH2Cl2-CF3COOH

(1:1), 0 ℃~ r.t, 3 h; (d) H2 (gas), 50 psi, 10% Pd/C, CH3OH, r.t, 2 h.
The syntheses of inhibitors 16-18 shown in Scheme 3 were carried out by
coupling acids 2a-k with amines 13-15 under an EDCI/HOBt/DMAP-mediated
coupling method. 26-29 Catalytic hydrogenation of 17c-k over 10% Pd/C in methanol
afforded the corresponding aminosulfonamide derivatives18c-k.
Scheme 3. Syntheses of inhibitors 16-18. Reagents and conditions: (a) EDCI, HOBt, DMAP,
anhydrous DMF, Ar, 0 ℃~ r.t, 3 h; (b) H2 (gas), 50 psi, 10% Pd/C, CH3OH, r.t, 2 h.
The inhibitory activities of the synthetic compounds against HIV-1 wild-type
protease were evaluated in vitro using a fluorescence resonance energy transfer
(FRET) method. 31,32 As a reference of the best marketed compound, darunavir (DRV)
has also been included. Purine bases as P2-ligands were investigated in combination
with other phenylsulfonamide substituents as P2 '-ligands in the protease S2 ' subsite.
The results are presented in Table 1. Most of the inhibitors with purine base
amine-acetamide P2-ligands displayed micromolar potency. Inhibitors 16a, 16f and
16j containing N-2-(6-substituted-9H-purin-9-yl)acetamide as the P2-ligands along

with 4-methoxylphenylsulfonamide as the P2'-ligand, displayed potent inhibitory
activity of HIV-1 protease with IC50 43 nM, 42 nM and 68 nM in vitro, respectively.
As shown in Table 1, most of the series of purine derivatives exhibited micromolar
inhibitory potency. However, functional 2'-phenylsulfonamide ligand still plays a vital
role in this series of inhibitors, as reported in previously results. For example,
phenylsulfonamide derivatives with a 4-methoxy substituent displayed generally
higher potency in sub-micromolar potency than that with 4-amino and 4-nitro
substituents. The P2' oxygen atom of OMe is likely to form hydrogen bonds (O…H-N)
with the main-chain amide of Asp30' in the protease S2 ' subsite, similar to inhibitors
6,23
GRL-0489A, TMC-126, GRL-0467 and GRL-0519A.
Moreover, the strong
electron-withdrawing group 4-nitro would reduce the electron density of not only the
oxygen atom itself via inductive effects but also the oxygen atom on the sulfonyl
group via conjugative effects, reducing the ability of hydrogen to bond with the amide
of Asp30' (phenyl-Oinh…N-H) or water-mediated interactions with the amide of Ile50'
20-22
(SO2inh…H2O…N-H) in the generally conserved protease S2' subsite.
Among
these compounds, inhibitors with chloride atom at Ra and methoxy group at Rd
showed better potency than those with electron-donating groups at Ra and methoxy
group at Rd, i.e., 16a vs 16b, 16d, 16e, 16f, 16g, 16h, 16i, 16j and 16k. It also appears
that more substituted groups at the purine ring such 16a vs 16b, 16h and 16i were
detrimental to the inhibitory potency. This observation suggests that higher electron
density or bigger space volume on the purine ring might reduce the ability of nitrogen
atoms to act as hydrogen bond-accepting groups to form hydrogen bonds with the
amide of possible residues in the generally conserved protease S2 subsite.
Furthermore, methyl substituent in hexa-heterocycle of purine with a 4-methoxy
substituent in P2'-ligand shows better potency than that of methyl substituent in
penta-heterocycle of purine such 16f vs 16g, which might give a hint that the location
of the substituent might affect the combination of compounds with the protease S2
subsite. In addition, the purine ring might be improper to adapt to the cavity in the
protease S2 subsite very well, the inhibitory potency of which was inferior to that
with the pyrimidine ring in our later work. The reason might be that massive and rigid
structures were not matched with the residues of the protease S2 subsite very well,
just as previously reported that inhibitors with flexible heterocyclic moieties at P2
were considerably more potent than their inflexible and acyclic counterparts in both
enzyme inhibitory and antiviral assays. 25
In summary, we have reported the structure-based design of novel HIV-1
protease inhibitors incorporating purine bases as P2-ligands and phenylsulfonamides
as P2'-ligands. The inhibitors were designed with the purpose of making extensive
hydrogen binding interactions with the protein backbone of HIV-1 protease active
sites by introducing heterocyclic moieties, carbonyl groups, and amino groups on
purine bases. Both P2- and P2'-ligands were involved in hydrogen bonding
interactions with the backbone of both S2 and S2' subsites. It was found that inhibitors
16a, 16f and 16j containing N-2-(6-substituted-9H-purin-9-yl)acetamide as the
P2-ligands along with 4-methoxylphenylsulfonamide as the P2'-ligand, display potent
inhibitory effect on the activity of HIV-1 protease with IC50 43 nM, 42 nM and 68 nM
in vitro, respectively. Further study on the evaluation of another nucleobases,

pyrimidine bases are currently underway.
Table 1 Enzymatic inhibitory activities of inhibitors with purine base
amine-acetamide P2-ligands
Compd. Ra
Rb
H
Rc Rd
IC50a(μM)
Compd.
18f
Ra
Rb
H
H
H
H
H
H
H
Rc
H
Rd
IC50a(μM)
16a
17a
18a
16b
17b
18b
16c
17c
18c
16d
17d
18d
16e
17e
18e
16f
17f
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe 0.043±0.007
NO2 0.31±0.11
NH2 7.02±5.96
OMe 0.57±0.19
NO2 0.96±0.42
NH2 11.7±8.57
OMe 0.24±0.16
NO2 2.58±1.72
NH2 0.57±0.52
OMe 1.98±0.55
NO2 3.58±1.27
NH2 1.24±0.62
OMe 0.36±0.17
NO2 3.68±1.12
NH2 6.79±3.25
OMe 0.042±0.027
NO2 2.53±1.99
Me
H
NH2
3.76±0.90
Cl
H
16g
Me OMe 0.15±0.091
Cl
H
17g
H
Me NO2
Me NH2
0.57±0.18
0.64±0.26
Cl
NH2
NH2
NH2
H
18g
H
Cl
16h
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
-
OMe 0.18±0.08
Cl
17h
NO2
NH2
2.60±0.25
1.51±0.24
H
18h
H
H
16i
OMe NH2
OMe NH2
OMe NH2
OMe 0.46±0.21
H
H
17i
NO2
NH2
1.73±0.80
NH2
NH2
NH2
H
H
18i
0.19±0.069
H
16j
OH
OH
OH
OH
OH
OH
-
H
OMe 0.068±0.029
H
17j
H
NO2
NH2
2.43±0.51
1.81±0.34
NH2
NH2
NH2
H
18j
H
H
16k
NH2
NH2
NH2
-
OMe 0.080±0.020
H
17k
NO2
NH2
-
0.79±0.43
4.73±2.80
4.01±1.35
Me
Me
18k
H
DRV(nM)
a Values were means of three independent experiments.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Nos.
81473099, 81703366 and 81473098) and CAMS Innovation Fund for Medical
Sciences (CIFMS 2016-I2M-1-011, CIFMS 2016-I2M-3-014 and CIFMS
2017-I2M-3-019).
Conflicts of Interest
The authors declare no competing financial interests.
Supplementary data
Supplementary data associated with this article can be found in the online version.
The data include MOL files of the compounds described in this article.

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IC50 = 43 nM, 42 nM and 68 nM (in vitro)