Design and synthesis of selenazole-substituted ritonavir analogs

Article abstract of DOI:10.1016/j.bmcl.2018.06.027

With the help of Surflex-Dock calculation, two ritonavir analogs in which one thioazole unit was replaced by selenazole have been designed and synthesized. The key selenazole structure was constructed from β-azido diselenide through a cascade diselenide cleavage/selenocarbonylation/Staudinger reduction/aza-Wittig reaction and a following MnO₂ oxidation. The accordingly prepared compounds exhibited good anti-HIV-1 (IIIB) activities comparable to that of the original ritonavir, as well as the positive SI values.

Full text of DOI:10.1016/j.bmcl.2018.06.027

Accepted Manuscript
Design and Synthesis of Selenazole-Substituted Ritonavir Analogs
Junfei Qiao, Chuanfang Zhao, Jun Liu, Yuguo Du
PII:
S0960-894X(18)30516-X
https://doi.org/10.1016/j.bmcl.2018.06.027
BMCL 25908
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 April 2018
1 June 2018
14 June 2018
Please cite this article as: Qiao, J., Zhao, C., Liu, J., Du, Y., Design and Synthesis of Selenazole-Substituted Ritonavir
Analogs, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design and Synthesis of Selenazole-Substituted Ritonavir Analogs
Junfei Qiaoa,b, Chuanfang Zhaob, Jun Liua, Yuguo Dua,b*
aState Key Laboratory of Environmental Chemistry and Eco-toxicology, Research
Center for Eco-Environmental Sciences, Chinese Academy of Science, Beijing
100085, China.
bSchool of Chemistry and Chemical Engineering, University of Chinese Academy of
Sciences, Beijing 100049, China.
Human immunodeficiency virus (HIV-1) is a unique invader of the immune
system that eventually leads to acquired immune deficiency syndrome (AIDS) if not
treated.1 HIV has affected over 71 million people around the world since its
emergence according to UNAIDS reports.2 The main action mode of HIV is believed
to invade CD4 T-lymphocytes, resulting in proliferation of the virus and an increased
apoptosis rate of host cells.3,4 As the immune system is compromised, patients with
CD4 cell count levels below 200 cells/
L (< 14%) are classified as living with AIDS.5-7
ꢀ
The worst is that no protective vaccine yet available and the disease is not curable by
the current therapeutic strategies.8,9
Ritonavir, as HIV-1 protease inhibitor (PI), is one of the key components of
current antiretroviral therapies.10 Ritonavir-boosted regimens combine ritonavir with a
second PI, as well as the other nucleoside reverse transcriptase inhibitors (NRTIs), to
achieve higher sustained levels of the second PI to overcome the emerging drug
resistance and allow less frequent dosing.11,12 In another aspect, selenium is of critical
roles in viral infection as several members of the selenoprotein family regulate or are
regulated by cellular redox tone, which is an important modulator of immune cell
signaling and function.5,13 It is reported in a clinical trial that 24-month selenium
supplementation significantly reduced the rate of CD4 cell count decline among
HIV-positive patients.5 In addition, some selenium-containing compounds have been
prepared and their bioactivities against HIV-1 in vitro seemed to be promising.14-20
Taking advantages from the above information, we here would like to report our
results for design, synthesis and anti-HIV activity of selenazole substituted ritonavir.
We were expecting that replacement of sulfur with selenium on ritonavir (to form
Se-ritonavir) might generate a new class of protease inhibitors (PIs). Thus,
Surflex-Dock in SYBYL software was applied firstly to mimic the potential

interaction between Se-ritonavir and its bio-target HIV-1 protease.21 TotalScore
analysis indicated that Se-ritonavir (1a) showed a comparable binding ability with
respect to ritonavir itself, as well as the similar binding sites with HIV-1 protease in
docking model. Further calculation suggested that the replacement of isopropyl group
on selenazole 1a with phenyl could generate another PI candidate 1b. According to
the overlay, the phenyl of ligand 1b laid closer to the PRO-81 and VAL-82, and the
thioazole unit of 1b formed H-bond with ASP-29 and ASP30 as well. This is
somehow different from ritonavir and 1a (Figure 1 and Table 1).
R
Ph
N
N
O
OH
H
N
H
N
X
N
O
S
N
H
O
O
Ph
Ritonavir X = S, R = isopropyl
1a X = Se, R = isopropyl
1b X = Se, R = Ph
Fig. 1. Structures of ritonavir, compounds 1a and 1b, and the complex of ritonavir, 1a
and 1b (labeled in magenta) with HIV-1 protease (A chain labeled in green and B
chain labeled in cyan), respectively. In overlay, ritonavir labeled in magenta, while 1a
in yellow and 1b in orange. The hydrogen bond interaction labeled in red dot line.

Table 1. TotalScore of ritonavir, 1a and 1b binds with HIV-1 Protease
Ligand
Ritonavir
14.91
1a
1b
TotalScore
15.7
16.1
It is obvious that the target compound 1 (1a, 1b) could be assembled from the
corresponding selenazole fragment 2 (2a, 2b) and thiazole segment 3 (Scheme 1). We
envisaged that the core structure of selenazole could be constructed with our newly
developed one-pot cascade protocol.22
Compounds 1a, 1b
R
Ph
O
OH
S
N
H
H
N
N
+
N
N
O
Se
OH
H2N
O
O
Ph
2a
R = isopropyl
2b R = Ph
3
Scheme 1. Retrosynthetic analysis of the target compounds
N
S
N
a
p-NO2PhO
O
HO
S
O
4
5
Ph
BocHN
Ph
RHN
OH
N
OH
b
H
N
NH2
Ph
O
S
O
Ph
6
7 R = Boc
R = H
c
3
Scheme 2. Reagents and conditions: (a) 4-nitrophenyl chloroformate/Et3N, DCM, rt,
4 h, 95%; (b) DIPEA/THF, 50 °C, 5 h, 90%; (c) TFA, DCM, rt, 2 h.
Compound 3 was prepared in a modified route23 as shown in Scheme 2.
5-Hydroxymethylthiazole 4 was activated with 4-nitrophenyl chloroformate ( 5),
then coupled with commercially available 6 in the presence of diisopropylethylamine
o
(DIPEA) at 50 C to yield carbamate 7, which was subjected to trifluoroacetic acid
(TFA) promoted hydrolysis to afford the key building block 3 (Scheme 2).
To prepare the core selenazole building block 2, N-Boc-L-serine methyl ester (8)
was treated with Ph3P, Br2, and imidazole in dry dichloromethane (DCM) to convert

hydroxyl group into the corresponding bromide in 86% yield (Scheme 3). The
bromide was added into a DMF solution containing a mixture of elemental selenium,
hydrazine monohydrate, and NaOH. The reaction between selenium and hydrazine in
the presence of NaOH gave Na2Se2, which condensed with the bromide to give the
N-Boc-protected homoselenocystine 9. It is worth to note that the molar ratio between
selenium and NaOH had better be kept at 1:1 to ensure the predominant product
diselenide 9. Treatment of 9 with 10% TFA in DCM removed Boc group, followed by
ZnSO4 promoted azide transfer using freshly prepared TfN3,24 obtained the expected
β-azido diselenide 10 in 44% overall yield. Condensation of 10 with benzoic acid or
2-propionic acid in the presence of pentafluorophenyl diphenylphosphate (FDP), Ph3P,
o
and Et3N in CH3CN at 70 C for 5h afforded selenazoline which was subjected to
MnO2 catalyzed oxidation generating selenazoles in good yield (65% for 11a, 79% for
11b). Transformation of 11 into the corresponding aldehydes 12 with DIBAL-H was
straightforward, and the subsequent reduction of 12 using NaBH4 in MeNH2 gave
selenazole substituted secondary amines 13 efficiently. In the following urea
formation, triphosgene promoted condensation was found to be promising in the
presence of secondary amine. Thus, compound 14 was prepared via the active
intermediate isocyanate, which was generated in situ from L-Valine methyl ester upon
treatment with triphosgene.25 It is worth to note that traditional urea formation
conditions, such as carbamoyl chlorides,26 carbonylimidazolide,27,28 and
carbamoylimidazolium salts29 were insufficient in this condensation, possibly because
of the weak electron withdraw ability of selenazole comparing to its thioazole
counterpart. Finally, saponification of ester 14 with LiOH in THF/H2O30 to the
corresponding acid 2 and subsequent coupling with amine 7 in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 1-hydroxy-benzotrizole
(HOBt)31-33 furnished the target compound 1.

O
N3
NHBoc
Se
a, b
c, d
HO
OMe
MeO
MeO
Se
NHBoc
2
2
O
O
9
10
8
Se
Se
R
N
Se
N
e, f
h
g
R
R
OMe
H
NHMe
N
O
O
11a R = isopropyl
11b R = Ph
12a
12b
13a
13b
R = isopropyl
R = Ph
R = isopropyl
R = Ph
R
R
N
Se
N
O
H
N
O
i
j
H
Se
N
N
N
OH
OMe
O
O
14a
14b
R = isopropyl
R = Ph
2a
R = isopropyl
2b R = Ph
R
Ph
+ 3
O
N
OH
N
H
H
N
N
O
Se
N
k
N
S
H
O
O
Ph
1a
R = isopropyl
1b R = Ph
Scheme 3. Synthesis of Se-Ritonavir analogs. Reagents and conditions: (a) compound
8 (1.0 equiv), Br2 (2.0 equiv), Ph3P (2.0 equiv), imidazole (2.0 equiv) in DCM, rt, 2 h,
86%; (b) N2H4•H2O/Se/NaOH/DMF, rt, 3 h, 70%; (c) TFA/DCM, rt, 3 h; (d)
NaN3/Tf2O, ZnSO4, Et3N, CH3OH/H2O/DCM, 0 ºC, overnight, 44% over two steps;
(e) 2-propionic acid acid or benzoic acid, FDP, Et3N, Ph3P, CH3CN, 70℃, 5 h; (f)
MnO2, DCE, reflux, 8 h, over two steps 65% for 11a, 79% for 11b; (g) DIBAL-H,
DCM, -78 ºC, 2 h, 86% for 12a, 90% for 12b; (h) 2 N CH3NH2, NaBH4, THF, rt, 8 h,
80% for 13a; 83% for 13b; (i) triphosgene, L-Valine methyl ester hydrochloride,
DIPEA, toluene, rt, 5 h, 86% for 14a, for 81% 14b; (j) LiOH, THF/H2O, rt, 2 h; (k)
EDCI, HOBt, DIPEA, THF, rt, N2, 5 h, 86% for 1a, 79% for 1b.
The anti-HIV-1 (IIIB) activities for the synthesized compounds 1a and 1b were
further evaluated with acutely infected MT-4 cells for three times in parallel.34,35 The
results were summarized in Table 2. The selenazole-derived compounds 1a and 1b
showed good antiviral activity against HIV-1 with EC50 at 550 and 170 nM,
respectively, possessing the similar HIV-1 inhibitory activity compared to the
marketed ritonavir (EC50 at 150 nM from this report, and 90 nM from literature20).
Moreover, the selenazole-derivatives 1a and 1b exhibited low cytotoxicity with
remarkable selectivity index values (SI at 76 and 124 for 1a and 1b respectively),

with a behavior similar to that observed for ritonavir. Interestingly, compound 1b
showed a better inhibition result in acutely infected MT-4 cells testing in consistence
with the computational prediction.
Table 2. Anti-HIV-1 Activities Determined by MTT in MT-4 Cellsa
Compound
1a
EC50 (nM)a,c
SId
CC50 (M)b,c
550
170
150
90
42
21
76
124
> 7
1b
Ritonavir
Ritonavir34
> 1
> 5.5
> 61
aEC50: concentration of compound required to achieve 50% protection of MT-4 cells
b
from HIV induced cytopathogenicity, as determined by the MTT method. CC50:
concentration of compound that reduces the viability of mock-infected cells by 50%,
c
as determined by the MTT method. All data represent mean values for at least three
separate experiments. dSI: selectivity index, ratio of CC50/EC50.
In conclusion, we have designed two new ritonavir derivatives with the help of
Surflex-Dock calculation. The target compounds 1a and 1b (Se-ritonavir), possessing
a selenazole instead of thioazole unit in ritonavir, have been successfully synthesized
and their antiviral activities against HIV-1 were evaluated with MT-4 cells culture.
Compounds 1a and 1b exhibited promising HIV-1 inhibitory activities comparable to
that of the original ritonavir, as well as the positive SI values. This result provides a
constructive idea to design and synthesize new anti-HIV agents through
selenium-sulfur exchanging.
Supplementary data
Supplementary data associated with this article, including the detailed experimental
procedures and the characteristics of new compounds, can be found in the online
version at https://
Acknowledgments
This work was supported in part by the Strategic Priority Research Program of CAS
(XDB14040201), the MOST (project 2015CB931903), and NNSFC (projects
21672255 and 21621064). We thank Xuefeng Sun for high resolution mass
determination.

References
1) Levy JA, Trends Immunol. 2001;22:312.
2) UNAIDS, World AIDS Day 2014 Report - Fact Sheet 2014, http://www.unaids.
org/en/resources/campaigns/World-AIDS-Day-Report-2014/factsheet.
3) Danner SA, Carr A, Leonard JM, Lehman LM, Gudiol F., Gonzales J, Raventos A,
Rubio R, Bouza E, Pintado V, Aguado AG, de Lomas JG, Delgado R, Borleffs JC,
Hsu A, Valdes J, Boucher CA, Cooper DA, New. Engl. J. Med. 1995;333:1528.
4) Kempf DJ, Marsh KC, Kumar G, Rodrigues AD, Denissen JF, Mcdonald E,
Kukulka MJ, Hsu A, Granneman GR, Baroldi PA, Sun E, Pizzuti D, Plattner J.J,
Norbeck DW, Leonard JM, Antimicrob. Agents Chemother. 1997;41:654.
5) Kamwesiga J, Mutabazi V, Kayumba J, Tayari JCK, Uwimbabazi JC, Batanage G,
Uwera G, Baziruwiha M, Ntizimira C, Murebwayire A, Haguma JP,
Nyiransabimana J, Nzabandora JB, Nzamwita P, Mukazayire E, AIDS
2015;29:1045.
6) Alqudah MAY, Yaseen MMM, Yaseen MMS, HIV & AIDS Rev 2016;15:1.
7) Xu H, Wang X, Veazey RS, Immunol. Rev. 2013;254:10.
8) Ensoli B, Cafaro A, Monini P, Marcotullio S, Ensoli F, Front. Immunol.
2014;5:417.
9) Ellegard R, Shankar EM, Larsson M, Curr. Opin. HIV AIDS 2011;6:435.
10) Ghosh AK, Parham GL, Martyr CD, Nyalapatla PR, Osswald HL, Agniswamy J,
Wang Y F, Amano M, Weber IT, Mitsuya H, J. Med. Chem. 2013; 56:6792.
11) Zeldin RK, Petruschke RA, J. Antimicrob. Chemother. 2004;53:4.
12) Youle M, J. Antimicrob. Chemother. 2007;60:1195.
13) Huang Z, Rose AH, Hoffmann PR, Antioxid Redox Sign 2012;16:705.
14) Koketsu M, Ishihara H, Curr Org Chem. 2003;7:175.
15) Baba M, Antiviral Res. 1997;33:141.
16) Chu CK, Ma L, Olgen S, Pierra C, Du J, Gumina G, Gullen E, Cheng YC,
Schinazi RF, J. Med. Chem. 2000;43:3906.
17) Jeong LS, Choi Y N, Tosh DK, Choi WJ, Kim HO, Choi J, Bioorg. Med. Chem.
2008;16:9891.
18) Zhan P, Liu X, Fang Z, Pannecouque C, Clercq ED, Bioorg. Med. Chem.
2009;17:6374.
19) Thenin-Houssier S, S. de Vera IM, Pedro-Rosa L, Brady A, Richard A, Konnick
B, Opp S, Buffone C, Fuhrmann J, Kota S, Billack B, Pietka-Ottlik M,
Tellinghuisen T, Choe H, Spicer T, Scampavia L, Diaz-Griffero F, Kojetin DJ,
Valente ST, Antimicrob. Agents Chemother. 2016;60:2195.
20) Sancineto L, Mariotti A, Bagnoli L, Marini F, Desantis J, Iraci N, Santi C,
Pannecouque C, Tabarrini O, J. Med. Chem. 2015;58:9601.
21) Shityakov S, Dandekar T, Bioinformation 2010;4:295.

22) Liu Y, Liu J, Qi XB, Du Y, J. Org. Chem. 2012;77:7108.
23) Kempf DJ, Sham HL, Marsh KC, Flentge CA, Betebenner D, Green BE,
McDonald E, Vasavanonda S, Saldivar A, Wideburg NE, Kati WM, Ruiz L, Zhao
C, Fino L, Patterson J, Molla A, Plattner JJ, Norbeck DW, J. Med. Chem.
1998;41:602.
24) Loos P, Ronco C, Riedrich M, Arndt HD, Eur. J. Org. Chem. 2013;3290.
25) Eder M, Schafer M, Bauder-Wust U, Hull WE, Wangler C, Mier W, Haberkorn U,
Eisenhut M, Bioconjugate Chem. 2012;23:688.
26) D'Souza MJ, Kevill DN, Int. J. Mol. Sci. 2016;17.
27) Padiya KJ, Gavade S, Kardile B, Tiwari M, Bajare S, Mane M, Gaware V,
Varghese S, Harel D, Kurhade S, Org. Lett. 2012;14:2814.
28) Liu H, Xu L, Hui H, Vivian R, Callebaut C, Murray BP, Hong A, Lee MS, Tsai
LK, Chau JK, Stray KM, Cannizzaro C, Choi YC, Rhodes GR, Desai MC, Bioorg
& Med. Chem. Lett. 2014;24:989.
29) Grzyb JA, Shen M, Yoshina-Ishii C, Chi W, Brown RS, Batey RA, Tetrahedron
2005;61:7153.
30) Kankanamalage ACG, Kim Y, Weerawarna PM, Uy RAZ, Damalanka VC,
Mandadapu SR, Alliston KR, Mehzabeen N, Battaile KP, Lovell S, Chang KO,
Groutas WC, J. Med. Chem. 2015;58:3144.
31) Sevrioukova IF, Poulos TL, J. Med. Chem. 2013;56:3733.
32) Xu L, Liu H, Hong A, Vivian R, Murray BP, Callebaut C, Choi YC, Lee MS,
Chau J, Tsai LK, Stray KM, Strickley RG, Wang J, Tong L, Swaminathan S,
Rhodes GR, Desai MC, Bioorg & Med. Chem. Lett. 2014;24:995.
33) Xu L, Liu H, Murray BP, Callebaut C, Lee MS, Hong A, Strickley RG, Tsai LK,
Stray KM, Wang Y, Rhodes GR, Desai MC, ACS Med. Chem. Lett. 2010;1:209.
34) Pannecouque C, Daelemans D, de Clercq E, Nat. Protoc. 2008;3:427.
35) Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P,
Desmyter J, de Clercq EJ, Virol. Methods 1988;20:309.

Highlights

New method to make selenazole from β-azido diselenide and alkyl acids in
one-pot


Two new selenazole-containing ritonavir analogs were designed and synthesized
The designed compounds exhibited promising anti-HIV-1 activities and SI values

Design and Synthesis of Selenazole-Substituted Ritonavir Analogs
Junfei Qiao, Chuanfang Zhao, Jun Liu and Yuguo Du*
Two new ritonavir analogs were designed and synthesized taking advantages of one-pot cascade
condensation of β-azido diselenide and alkyl acids. The prepared compounds exhibited good
anti-HIV-1 activities as well as the positive SI values.
Ph
R
S
O
OH
N
H
N
H
N
N
O
Se
N
N
H
O
O
Ph
R = Isopropyl or Phenyl