Design, synthesis and biological evaluation of HIV-1 protease inhibitors with morpholine derivatives as P2 ligands in combination with cyclopropyl as P1′ ligand

Article abstract of DOI:10.1016/j.bmcl.2020.127019

A series of novel HIV-1 protease inhibitors has been designed and synthesized, which contained morpholine derivatives as the P2 ligands and hydrophobic cyclopropyl as the P1′ ligand at the meantime in this study, with the aim of improving the interactions between the active sites of HIV-1 protease and the inhibitors. Twenty-eight compounds were synthesized and assessed, among which inhibitors m18 and m1 exhibited excellent inhibitory effect on the activity of HIV-1 protease with IC₅₀ value of 47 nM and 53 nM, respectively. The molecular modeling of m1 revealed possible hydrogen bondings or van der Waals between the inhibitor and the protease, worthy of in-depth study.

Full text of DOI:10.1016/j.bmcl.2020.127019

Bioorganic&MedicinalChemistryLetters30(2020)127019
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of HIV-1 protease inhibitors with
morpholine derivatives as P2 ligands in combination with cyclopropyl as P1′
ligand
⁎
Yue Doua, Mei Zhub, Biao Dongb, Ju-Xian Wangb, Guo-Ning Zhangb, Fan Zhanga,
,
⁎
Yu-Cheng Wangb,
a School of Pharmacy, Jinzhou Medical University, Jinzhou 121001, PR China
b Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel HIV-1 protease inhibitors has been designed and synthesized, which contained morpholine
derivatives as the P2 ligands and hydrophobic cyclopropyl as the P1′ ligand at the meantime in this study, with
the aim of improving the interactions between the active sites of HIV-1 protease and the inhibitors. Twenty-eight
compounds were synthesized and assessed, among which inhibitors m18 and m1 exhibited excellent inhibitory
effect on the activity of HIV-1 protease with IC50 value of 47 nM and 53 nM, respectively. The molecular
modeling of m1 revealed possible hydrogen bondings or van der Waals between the inhibitor and the protease,
worthy of in-depth study.
HIV-1 protease inhibitors
Morpholine
Cyclopropyl
Synthesis
Biological evaluation
Molecular modeling
As
a
degenerative disease of immune system, acquired im-
above, morpholine, with flexible ring including oxygen and nitrogen
groups, was introduced as the P2 ligands instead of the bis-THF struc-
tural template in the lead compound DRV (see in Fig. 1), for the sake of
improving hydrogen bonding interactions with amino acid residues of
Asp29 or Asp30.7,8 Furthermore, hydrophobic cyclopropyl group was
introduced into P1′ ligand in order to improve backbone bindings or
favorable van der Waals in the S1′ subsite,9,10 as well as different
phenylsulfonimide groups as the P2′ ligands.11
munodeficiency syndrome (AIDS), caused by human immunodeficiency
virus (HIV), is a social and medical problem of staggering dimensions.1
It is reported that there are approximately 1.7 million new infections
and 770 thousand people died of AIDS-related illnesses in 2018. So
effective chemotherapy drugs are urgently needed to combat the dis-
ease.
The highly active antiretroviral therapy treatment (HAART), con-
sisting of HIV-1 protease inhibitors (PIs) and two or more reverse
transcriptase inhibitors (RTIs), is certainly a blessing for patients with
AIDS.2 Ten HIV-1 PIs have been approved in clinical practice nowadays,
which could prevent the cleavage of viral precursor peptides Gag and
Gag-pol being enzymatic proteins and mature virions, so as to produce
morphologically immature and noninfectious viral particles.3 However,
there still exists serious problems, such as genetic diversity, side effects,
especially drug resistance.4 Hence, there is an urgent need for novel
HIV-1 PIs.
In Scheme 1, Intermediates 6–9 were prepared from the commer-
cially available material (2S, 3S)-1,2-epoxy-3-(boc-amino)-4-phe-
nylbutane (1), which was reacted with cyclopropanamine to provide
the chiral secondary amine 2 in 91.4% yield, followed by nucleophilic
substitution with 4-substituted-benzenesulfonyl chlorides to afford
sulfonamide derivatives 3–5 in good yields (95.1–99.3%). Boc-group
was effectively removed by trifluoroacetic acid to obtain compounds
6–8 in 74.1–80.6% yields.12 Corresponding aminosulfonamide 9 was
obtained by catalytic hydrogenation of 8 in MeOH with 5% Pd/C in
yield of 97.2%.13
One of the most effective design strategies for eliminating the re-
sistance is to increase the hydrogen bonds between inhibitor and the
protease. The bis-THF of P2 ligand in Darunavir (DRV) forms strong
hydrogen bonding interactions through cyclic ether oxygens with the
backbone amide NH of the protease residues in the S2 subsite, which
may explain the high resistance profile of DRV.5,6 Inspired by the
In Scheme 2, Commercially available 2-morpholinoethan-1-ol (A1)
was reacted with 4-nitrophenyl carbonochloridate in ether to provide
activated carbonate B1 in 91.2% yield. Alkoxycarbonylation of amines
6–9 with 2-morpholinoethyl (4-nitrophenyl) carbonate (B1) provided
inhibitors m1-4 in yields of 67.6–89.3%.14 N-Alkylation of A2-A4 with
⁎ Corresponding authors.
E-mail addresses: zhangfan830720@163.com (F. Zhang), wangyucheng@imb.pumc.edu.cn (Y.-C. Wang).
https://doi.org/10.1016/j.bmcl.2020.127019
Received 9 December 2019; Received in revised form 15 January 2020; Accepted 5 February 2020
Availableonline05February2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

Y. Dou, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127019
Fig. 1. Chemical structure of target molecules.
Scheme 1. Syntheses of Amines 6–9. Reagents and conditions: (a) cyclopropanamine, CH3CN, 80 ℃, 10 h, 91.4%; (b) Aryl sulfonyl chloride, DIEA, DMAP, THF,
0 ℃ ~ r.t, 7.5 h, 95.1–99.3%; (c) CH2Cl2-CF3COOH (1:1), r.t, 5 h, 74.1–80.6%; (d) H2 (gas), 50 psi, 5% Pd/C, MeOH-EA (2:1), r.t, 4 h, 97.2%.
bromoacetic acid proceeded with K2CO3 as the acid binding agent in
anhydrous DMF and then added 4 M HCl (aqueous) in dropwise to give
corresponding acids B2–B4 with yields of 85.3–88.2%. Treatment
compounds B2–B5 with amines 6–9 generated inhibitors m5-20 under
the condition of EDCI, HOBt and DMAP mediated coupling method in
yields of 51.1–73.4%.15 Inhibitors m21-24 were synthesized by alky-
lation of amines 6–9 with compound B6 under the catalytic condition of
DIEA and DMAP in yields of 50.9–69.3%. Inhibitors m25-28 were
obtained by the reaction of compound B7 and amines 6–9 with K2CO3
as the acid binder in yields of 60.3–72.8%.
In this study, fluorescence resonance energy transfer (FRET) was
adopted to assess the activity of the new HIV-1 PIs in vitro.16 DRV, the
most effective HIV-1 PI, was chosen as the positive control. The results
were reported in Tables 1 and 2. Although not very potent, there still
existed regularity. As a whole, inhibitors with amide chains (carbamate
or carbamido) showed enhancement of HIV-1 protease inhibition over
2

Y. Dou, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127019
Scheme 2. Syntheses of Inhibitors m1-28. Reagents and conditions: (e) ether, 0 ℃ ~ r.t, overnight, 91.2%. (f) DIEA, anhydrous DMF, Argon, 0 ℃ ~ r.t, 10 h,
67.6–89.3%. (g) (i) 2-Bromoacetic acid, K2CO3, anhydrous DMF, Argon, r.t, overnight; (ii) 4 M HCl, 0 ℃, 0.5 h, 85.3–88.2%. (h) EDCI, HOBt, DMAP, anhydrous DMF,
Argon, 0 ℃ ~ r.t, 5 h, 51.1–65.8%. (i) EDCI, HOBt, DMAP, anhydrous DMF, Argon, 0 ℃ ~ r.t, 5 h, 55.8%–73.4%. (j) DIEA, DMAP, anhydrous EtOH, Argon, reflux,
7 h, 50.9–69.3%. (k) K2CO3, CH3CN, 60 ℃, 1 h, 60.3–72.8%.
3

Y. Dou, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127019
Table 1
Table 2
Enzyme inhibitory activity of inhibitors with amide linker.
Enzyme inhibitory activity of inhibitors with aliphatic chain linkers.
linker
linker
R1
R1
O
O
O
R2
S
S
R2 nX
N
N
N
N
O
O
H
H
R4
OH
OH
b
R1
R2
Linkera
IC50 (μM)
Compd.
R1
R2
Linkera
IC50 (μM)
b
Compd.
m1
4-OCH3 Morpholine
0.054
0.004
O
O
O
O
m21
4-OCH3
Morpholine
Morpholine
Morpholine
Morpholine
0.18
1.26
> 10
1.15
0.099
O
O
O
O
O
O
O
O
m2
m3
m4
m5
4-CF3
4-NO2
4-NH2
Morpholine
Morpholine
Morpholine
0.12
0.011
0.006
0.005
0.084
m22
m23
m24
4-CF3
0.43
0.080
0.061
0.19
4-NO2
4-NH2
0.76
4-OCH3 (R)-2-
Methylmorpholine
O
O
O
O
O
O
O
O
m25
m26
m27
m28
4-OCH3
4-CF3
4-NO2
4-NH2
–
Morpholine
Morpholine
Morpholine
Morpholine
–
0.17
1.69
0.61
0.94
0.37
0.090
0.75
0.087
0.095
0.070
m6
4-CF3
4-NO2
4-NH2
(R)-2 Methylmorpholine
2.99
0.93
1.53
1.27
0.40
0.58
1.34
1.04
0.63
m7
(R)-2-
0.075
0.72
Methylmorpholine
DRV (nM)
–
m8
(R)-2-
Methylmorpholine
a
Linker was defined as chains connected backbone amino group to mor-
pholine of the P2 ligand.
m9
4-OCH3 (S)-2-Methylmorpholine
0.39
b
All assays were conducted in triplicate, and the data shown represent mean
values ( 1 standard deviation) derived from the results of three independent
experiments
m10
m11
m12
m13
4-CF3
4-NO2
4-NH2
(S)-2-Methylmorpholine
(S)-2-Methylmorpholine
(S)-2-Methylmorpholine
0.032
0.053
0.95
the backbone of the protease.15,17–20 Furthermore, carbamate inhibitors
exhibited the most potent activity with IC50 value in nanomolar order of
magnitude, which might account for the degree of freedom and flex-
ibility that can be better bound to protease. In addition, the morpholine
ring located in S2 subsite and the chain forced the benzenesulfonyl
group to extent inside S2′ subsite deeply. For acetamide compounds,
the activity of compounds with (R)-2-methylmorpholine in R3 fragment
was better than that with (S)-2-methylmorpholine in general, which
may be due to the difference of steric hindrance and conformation;
besides, the van der Waals of (R)-2-methylmorpholine made it better
matching the binding pocket of enzyme so as to improve the activity.
Compounds with the linker of non-substitution straight aliphatic
chains exhibited better activity than the corresponding compounds
with carbonyl substituted aliphatic chains such as m27 vs m23, which
may be due to the larger degree of freedom and the reduction of steric
hindrance.
4-OCH3 Morpholine
0.21
O
O
O
O
m14
m15
m16
m17
4-CF3
4-NO2
4-NH2
Morpholine
Morpholine
Morpholine
0.40
5.43
> 10
0.26
0.048
1.77
4-OCH3 Morpholine
0.083
0.004
O
O
O
O
m18
4-CF3
Morpholine
0.047
The functional groups in P2′ ligand also played important roles.
Compounds with 4-methoxyl, 4-amino and 4-trifluoromethyl sub-
stituents showed higher potency than the corresponding 4-nitro sub-
stituent compounds, except for m7, m11 and m27. The oxygen atom in
the 4-methoxyl group might form hydrogen bonds (O⋯H–N) with
Asp30′ or Asp29′ in the protease S2′ subsite, which increased the
binding ability of inhibitor and enzyme.21 The 4-amino group could
make weak hydrogen bonds involved directly interactions (N–H⋯N)
with the main chain amides and water-mediated interactions
(NH⋯H2O⋯HOOC) with the side chain oxygen of Asp30′ or Asp29′ in
S2′ active subsite.19,20 The 4-trifluoromethyl group is a weak electron
acceptor when connected with aromatic rings, and going forward, the
introduction of trifluoromethyl group may enhance cell membrance
permeability, which may be in favor of anti-HIV activity.22 The 4-nitro
group is a strong electron-withdrawing group, which would not only
reduce the electron density of oxygen atom itself by inductive effects,
m19
m20
4-NO2
4-NH2
–
Morpholine
Morpholine
–
0.79
0.64
0.37
0.037
0.077
0.070
DRV (nM)
a
–
Linker was defined as chains connected backbone amino group to mor-
pholine of the P2 ligand.
b
All assays were conducted in triplicate, and the data shown represent mean
values ( 1 standard deviation) derived from the results of three independent
experiments.
that with aliphatic chains (for instance, m14 vs m26). The reason may
be that amide chains mimic the peptide characteristics of HIV-1 pro-
tease substrate on the one hand. On the other hand, it may be that the
carbonyl group in amide compounds could form hydrogen bonds with
4

Y. Dou, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127019
Fig. 2. The molecular modeling for compound m1. (A) Docked pose of m1. (B) Ligplot interaction of m1. Ligand exposures are described as purple spheres. Hydrogen
bonding interactions between residues and ligands are represented as dashed arrows.
but also reduce the electron density of sulfonyl group by conjugative
effects. That could reduce the ability of hydrogen to bond with amides
of Asp30′ or Asp29′ in protease S2′ subsite.23
Acknowledgements
This work is supported by CAMS Innovation Fund for Medical
Sciences (CIFMS 2016-I2M-1-011) and Natural Science Foundation of
Liaoning Province (2019-ZD-0834).
In order to explored molecular insight into the interactions of in-
hibitors and HIV-1 protease, we have measured the computational
docking of m1 with active sites using a HIV-1 protease crystal structure
(PDB-ID: 4mc9) (Fig. 2).24 The inhibitor m1 fitted into the active site
cavity and the oxygen atom of hydroxyethyl amine could form hy-
drogen bonds with amide of IIe50/IIe50′ in flap, as well as van der
Waals were generated with the outer enzyme atoms. These interactions
might be responsible for the HIV-1 protease inhibitory activity. Also as
can be seen, oxygen atom of morpholine in P2 ligand could generate
interactions with the active site while there was no clean evidence for
interactions between the wrapped nitrogen atom and the active site,
and this could account for why the inhibition of morpholine analogues
were inferior to that of DRV containing two exposed oxygen atoms.
Furthermore, modeling studies indicated decreased van der Waals in-
teractions, although hydrophobic cyclopropyl of the P1′ ligand could fit
into the S1′ subsite of HIV-1 protease.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127019.
References
[1]. Gao BL, Zhang CM, Yin YZ, Tang LQ, Liu ZP. Bioorg Med Chem Lett.
2011;21:3730–3733.
[2]. Sun Y, Xu W, Fan N, et al. Bioorg Med Chem. 2017;25:1076–1084.
[3]. Lu XY, Wang LF, Jiang ZJ. IEEE. 2018 5th International Conference on Systems and
Informatics. 2018; 2018:1299–1304.
[4]. Bonini C, Chiummiento L, Di Blasio N, et al. Bioorg Med Chem. 2014;22:4792–4802.
[5]. Johnson ECB, Malito E, Shen Y, et al. J Mol Biol. 2007;373:573–586.
[6]. Pawar S, Wang YF, Wong-Sam A, et al. Biochem Biophys Res Commun.
2019;514:974–978.
In summary, a series of new HIV-1 PIs with morpholine derivatives as
P2 ligands as well as cyclopropyl as P1′ ligand was reported in this study.
The inhibitors were designed with the purpose of enhancing extensive
hydrogen bonding interactions or favorable van der Waals with the
protein skeleton of the active site of HIV-1 protease. Though not very
satisfactory, there still exhibited potent inhibitors such as m1, m3, m4
and m18 containing carbamate or carbamido as linkers along with 4-
methoxybenzenesulfonamide or 4-tifluoromethylbenzenesulfonamide as
P2′ ligands, which displayed inhibitory effect on the activity of HIV-1
protease with IC50 value of 54 nM, 80 nM, 61 nM and 47 nM in vitro,
respectively, and thus provided basis for further study. The rigid cyclo-
propyl system might decrease van der Waals interactions in the vicinity
of the S1′ subsite, which is worthy of ulteriorly optimization. Further
design and investigation of this kind of inhibitors, especially groups in
P1′ ligands are in progress.
[7]. Yang ZH, Bai XG, Zhou L, Wang JX, Liu HT, Wang YC. Bioorg Med Chem Lett.
2015;25:1880–1883.
[8]. Aruksankunwong O, Hannongbua S, Wolschann P. J Mol Struct. 2006;790:174–182.
[9]. Vondrasek AWAJ. Annu Rev Biophys Biomol Struct. 1998;27:249–284.
[10]. Midde NM, Patters BJ, Rao P, Cory TJ, Kumar S. Expert Opin Invest Drugs.
2016;25:1189–1200.
[11]. Mitchell ML, Xu L, Newby ZE, Desai MC. Tetrahedron Lett. 2017;58:1123–1126.
[12]. Zhu M, Du XN, Li YG, Zhang GN, Wang JX, Wang YC. Bioorg Med Chem Lett.
2019;29:357–361.
[13]. Yan J, Huang N, Li S, et al. Bioorg Med Chem Lett. 2012;22:1976–1979.
[14]. Ghosh AK, Gemma S, Simoni E, et al. Bioorg Med Chem Lett. 2010;20:1241–1246.
[15]. Bai X, Yang Z, Zhu M, et al. Eur J Med Chem. 2017;137:30–44.
[16]. Hendrix J, Debyser Z, Hofkens J, Engelborghs Y. Eur Biophys J Biophys Lett.
2011;40:48.
[17]. Qiu X, Zhao GD, Tang LQ, Liu ZP. Bioorg Med Chem Lett. 2014;24:2465–2468.
[18]. Ghosh AK, Jadhav RD, Simpson H, et al. Eur J Med Chem. 2018;160:171–182.
[19]. Yeung CM, Klein LL, Flentge CA, et al. Bioorg Med Chem Lett. 2005;15:2275–2278.
[20]. Perez MA, Fernandes PA, Ramos MJ. J Mol Graph Model. 2007;26:634–642.
[21]. Ghosh AK, Brindisi M, Nyalapatla PR, et al. Bioorg Med Chem. 2017;25:5114–5127.
[22]. Adrian Meredith J, Wallberg H, Vrang L, et al. Eur J Med Chem. 2010;45:160–170.
[23]. Zhu M, Dong B, Zhang GN, Wang JX, Cen S, Wang YC. Bioorg Med Chem Lett.
2019;29:1541–1545.
Declaration of Competing Interest
[24]. Ganguly AK, Alluri SS, Wang CH, et al. Tetrahedron. 2014;70:2894–2904.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
5