Synthesis of pyrimidine and quinolone conjugates as a scaffold for dual inhibitors of HIV reverse transcriptase and integrase

Article abstract of DOI:10.1016/j.bmcl.2008.01.025

A series of conjugates combining a pyrimidine and a quinolone moiety were designed and synthesized. The assay results show that these compounds demonstrate both anti-RT activity and anti-IN activity and therefore provide a useful scaffold for identifying inhibitors with balanced dual activities.

Full text of DOI:10.1016/j.bmcl.2008.01.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1293–1296
Synthesis of pyrimidine and quinolone conjugates as a scaffold
for dual inhibitors of HIV reverse transcriptase and integrase
Zhengqiang Wang* and Robert Vince*
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St SE, Minneapolis, MN 55455, USA
Received 11 December 2007; revised 4 January 2008; accepted 8 January 2008
Available online 11 January 2008
Abstract—A series of conjugates combining a pyrimidine and a quinolone moiety were designed and synthesized. The assay results
show that these compounds demonstrate both anti-RT activity and anti-IN activity and therefore provide a useful scaffold for iden-
tifying inhibitors with balanced dual activities.
Published by Elsevier Ltd.
Human immunodeficiency virus (HIV), the etiological
agent of acquired immunodeficiency syndrome
(AIDS),1,2 continues to be a major health threat world-
wide despite FDA approval of around 30 anti-HIV
drugs.3 With the exception of two entry inhibitors, enfu-
virtide4 and maraviroc,5 and an integrase (IN) inhibitor,
raltegravir (MK-0518, 25),6 all FDA-approved anti-HIV
drugs inhibit either reverse transcriptase (RT) or prote-
ase (PR). Singly dosed antivirals are rarely used in clinic
due to the rapid emergence of resistant viral strains. This
problem is countered by combining multiple mechanisti-
cally distinct drugs to form highly active antiretroviral
therapy (HAART),7 which can successfully suppress vir-
al replication. However, the benefits of HAART as the
standard AIDS chemotherapy are compromised by high
cost and severe toxicity,8 both resulting from using mul-
tiple drugs. Toxicity in particular imposes a huge barrier
to excellent patient adherence, without which patients
experience viral rebound, and even worse, multi-drug
resistance (MDR).
limited by toxicity and the lack of a general design strat-
egy. Recently we disclosed the first rationally designed
RT/IN dual ligands (3, Fig. 1).13 Significantly, TNK-
651 (1) of the 1-[(2-hydroxyethoxy)methyl]-6-(phenyl-
thio)thymine (HEPT) family is a potent non-nucleoside
RT inhibitor of which the N-1 substituent extends from
the NNRTI binding pocket to the protein/solvent inter-
face.14 This would tolerate structural modifications at
the N-1 site of 1 and allow the incorporation of a second
pharmacophore to generate activity against IN.13
As an example of our design, compound 3 contains a
diketoacid (DKA) pharmacophore derived from the ste-
reotypical DKA IN inhibitor 2. Dual activities against
RT and IN were observed13 (3, Table 1), though the dis-
parity between these two activities prompts us to con-
tinue searching for dual inhibitors with better balanced
activities against these two enzymes. We were particu-
larly interested in a scaffold where the DKA pharmaco-
phore in compound 3 is replaced with an isosteric
quinolone carboxylic acid moiety (5–8, Fig. 1). Quino-
lone compounds constitute one of the largest families
of antimicrobial agents;15,16 and GS-913717,18 (4,
Fig. 1), which has a quinolone carboxylic acid core, is
a very potent IN inhibitor currently under clinical devel-
opment. Therefore, if dually active, conjugates combin-
ing quinolone pharmacophore with pyrimidine would
provide a useful scaffold in our search for RT/IN dual
inhibitors.
Designed multiple ligands (DMLs)9–11 are single struc-
tures engaging multiple biological targets, and therefore
could incur lower cost and toxicity than HAART drug
cocktails. Identification of multiple ligands through
rational design has been a subject of growing interest in
medicinal chemistry.10 Compounds active against both
RT and IN were reported,12 though their usability is
Keywords: HIV; Reverse transcriptase; Integrase; Dual inhibitor;
Rational design.
Inhibitors 6–8 were synthetically accessed through a
convergent strategy featuring a classic Gould–Jacobs
synthesis19 of quinolone carboxylate as illustrated by
the preparation of 8 (Scheme 1). In this event, a conju-
*
Corresponding authors. Tel.: +1 612 625 8253; fax: +1 612 625 8252
(Z.W.); tel.: +1 612 624 9911; fax: +1 612 625 2633 (R.V.); e-mail
addresses: wangx472@umn.edu; vince001@umn.edu
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.01.025

1294
Z. Wang, R. Vince / Bioorg. Med. Chem. Lett. 18 (2008) 1293–1296
O
N
O
N
O
N
HN
HN
HN
O
O
O
O
O
O
O
O
OH
O
OH
N
R
O
OH
5
, R = H
1
3
6, R = Me
7, R = Et
8
, R = CH2CH2OH
F
O
O
O
Cl
OH
OH
O
N
O
OH
OH
2
4
Figure 1. Design of dual inhibitors (5–8) based on RT inhibitor 1 and IN inhibitor 4.
O
OEt
OEt
O
OEt
OEt
H
N
NH2
a
c
EtO
+
HO
RO
O
O
11
12
O
, R = H
10
9
b
, R = MOM
O
O
O
Cl
O
OEt
e
MOMO
OEt
N
N
R
OAc
g
16
14, R = H
Br
OAc
O
O
N
d
15, R = CH2CH2OAc
13
HN
O
O
O
OTMS
N
O
OR1
f
TMSO
HN
19, R1 = Et, R2 = AC
8, R1 = R2 =H
N
h
O
N
H
17
N
OR2
18
Scheme 1. Synthesis of inhibitor 8. Reagents and conditions: (a) toluene, reflux, 3 h, 98%; (b) MOMCl, DIEA, rt, 15 h, 100%; (c) Ph2O, 230 °C,
20 min, 65%; (d) 13, K2CO3, DMF, 80 °C, 93%; (e) BCl3, CH2Cl2; (f) BSA, CH2Cl2, rt, 20 min; (g) 18, tetrabutylamonium iodide (TBAI), rt, 15 h; (h)
NaOH, CH2Cl2/EtOH, rt, 29% over two steps.
O
OEt
OEt
O
OEt
OEt
H
N
H
N
a
b
17
HO
O
Cl
O
O
20
11
O
N
O
N
HN
HN
c
O
O
O
O
O
OR
O
OEt
N
O
N
H
22
5
, R = Et
H
d
21
, R = H
EtO
O
Scheme 2. Synthesis of inhibitor 5. Reagents and conditions: (a) (HCHO)n, TMSCl; (b) 18, TBAI, rt, 15 h, 82%; (c) Ph2O, 230 °C, 20 min, 50%; (d)
NaOH, CH2Cl2/EtOH, rt, 89%.
gate addition of the commercially available aniline 9
onto malonate 10 furnished enamine intermediate 11.
The protection of the free hydroxyl group with MOM
then allowed a smooth thermal cyclization of intermedi-
ate 12 to produce quinolone ester 14, which was N-
alkylated with bromide 13 to give compound 15. The di-
rect generation of chloromethyl ether 16 from 15 was ef-
fected under the action of BCl3.20 The initial MOM

Z. Wang, R. Vince / Bioorg. Med. Chem. Lett. 18 (2008) 1293–1296
1295
Table 1. Anti-RT, anti-IN and anti-HIV assay results for compounds
5–8
anti-RT and anti-IN activities are considerably more
balanced in compound 8 than the original DKA dual
inhibitor 3. This is not trivial since achieving balanced
activities against distinct biological targets remains the
biggest challenge in designing dual inhibitors.10
Compound RT IC50 (lM) IN IC50 (lM) HIV EC50 (lM)
313
5
0.057
0.19
2.6
2.4
35
0.033
0.22
0.21
1.5
6
41
51
19
We expect that the anti-IN activity can be further im-
proved by structure–activity relationship (SAR) studies,
as the quinolone carboxylic acid in these conjugates rep-
resents only the minimal structural requirement for
binding to IN: the metal chelator and the hydrophobic
aromatic ring (Fig. 2).23,24 Interestingly, all IN inhibi-
tors (Fig. 2) that have entered clinical trial bear a benzyl
group that has a particular spatial relationship with the
chelating functionality.25 Such a benzyl group could be
crucial to achieving optimal binding to IN. Studies are
currently underway to determine the benzyl effect on
RT/IN dual inhibitor scaffolds.
7
1.1
3.7
8
2.2
protection was designed based on this transformation.
Compound 16 was then coupled with bissilylated pyrim-
idine 18 which was freshly prepared from pyrimidine
1721 to successfully produce key intermediate 19. Final-
ly, 19 was hydrolyzed to deliver the desired quinolone
carboxylic acid 8.22
Unfortunately, the direct generation of chloromethyl
ether from MOM group failed with intermediate 14
(Scheme 1), presumably due to the influence of the free
NH group. Alternatively, intermediate 11 was chlorom-
ethylated with paraformaldehyde in TMSCl,13 and the
resulting chloromethyl ether 20 was coupled with 18 to
yield enamine 21 (Scheme 2). Gratifyingly, upon heat-
ing, 21 was smoothly cyclized to deliver the desired quin-
olone ester 22, which after saponification produced
inhibitor 5. It is noteworthy that the strategy described
in Scheme 2 is not suitable for the synthesis of inhibitors
6–8 as the N-alkylated analogues of enamine 21 failed to
cyclize under thermal condition.
Acknowledgments
This research was supported by the Center for Drug De-
sign at the University of Minnesota. We thank Dr. Eric
Bennett for discussion, Dr. Christine Salomon for IN as-
say, and Christine Dreis for RT assay. We also thank
Roger Ptak at Southern Research Institute for cell-
based assay and Dr. Robert Craigie of NIH for the
integrase plasmid.
References and notes
The assay results for compounds 5–8 are summarized in
Table 1. Notably, these inhibitors demonstrate activity
against RT at low to sub-micromolar range, which con-
firms that introducing a second pharmacophore at the
N-1 pedant of HEPT compounds does not seriously
diminish their binding affinity to RT. In addition, mod-
erate activity against IN is also observed, which vali-
dates quinolone carboxylic acid as a pharmacophore
choice in designing RT/IN dual inhibitors. The anti-
HIV activity from cell-based assay falls into the same
range as the anti-RT activity, implying that the contri-
bution of IN activity to the overall activity might not
be significant. Nevertheless, it was also found that the
´
1. Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M.
T.; Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.;
Vezinet-Brun, F.; Rouzioux, C.; Rozenbaum, W.; Mon-
tagnier, L. Science 1983, 220, 868.
2. Gallo, R. C.; Salahuddin, S. Z.; Popovic, M.; Shearer, G.
M.; Kaplan, M.; Haynes, B. F.; Palker, T. J.; Redfield, R.;
Oleske, J.; Safai, B.; White, G.; Foster, P.; Markham, P.
D. Science 1984, 224, 500.
3. FDA, http://www.fda.gov/oashi/aids/virals.html, Drugs
used in the Treatment of HIV Infection.
4. Manfredi, R.; Sabbatani, S. Curr. Med. Chem. 2006, 13,
2369.
5. Girotto, J. E. Formulary 2007, 42, 601.
6. Markowitz, M.; Nguyen, B.-Y.; Gotuzzo, E.; Mendo, F.;
Ratanasuwan, W.; Kovacs, C.; Prada, G.; Morales-
Ramirez, J. O.; Crumpacker, C. S.; Isaacs, R. D.; Gilde,
L. R.; Wan, H.; Miller, M. D.; Wenning, L. A.; Teppler,
H. JAIDS 2007, 46, 125.
7. Yeni, P. J. Hepatol. 2006, 44, S100.
8. Haugaard, S. B. Expert Opin. Drug Metab. Toxicol. 2006,
2, 429.
OH
O
O
N
OH
N
N
H
O
N
N
NH
F
O
O
N
F
S
9. Morphy, R.; Kay, C.; Rankovic, Z. Drug Discov. Today
2004, 9, 641.
23
, S-1360
24, L-870810
10. Morphy, R.; Rankovic, Z. J. Med. Chem. 2005, 48,
6523.
F
O
O
F
O
Cl
OH
OH
N
N
N
11. Morphy, R.; Rankovic, Z. J. Med. Chem. 2006, 49, 4961.
12. Didierjean, J.; Isel, C.; Querre, F.; Mouscadet, J.-F.;
Aubertin, A.-M.; Valnot, J.-Y.; Piettre, S. R.; Marquet, R.
Antimicrob. Agents Chemother. 2005, 49, 4884.
13. Wang, Z.; Bennett, E. M.; Wilson, D. J.; Salomon, C.;
Vince, R. J. Med. Chem. 2007, 50, 3416.
H
H
N
N
O
N
O
N
O
O
OH
25,
4, GS-9137
MK-0518
Figure 2. Structure of important IN inhibitors. Red, metal chelator;
Green, hydrophobic aromatic ring; Blue, benzyl group.
14. Hopkins, A. L.; Ren, J.; Esnouf, R. M.; Willcox, B. E.;
Jones, E. Y.; Ross, C.; Miyasaka, T.; Walker, R. T.;

1296
Z. Wang, R. Vince / Bioorg. Med. Chem. Lett. 18 (2008) 1293–1296
Tanaka, H.; Stammers, D. K.; Stuart, D. I. J. Med. Chem.
1996, 39, 1589.
15. Andriole, V. T. Clin. Infect. Dis. 2005, 41, S113.
16. Van Bambeke, F.; Michot, J. M.; Van Eldere, J.; Tulkens,
P. M. Clin. Microbiol. Infect. 2005, 11, 256.
17. Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.;
Yamashita, M.; Ito, Y.; Kawakami, H.; Matsuzaki, Y.;
Watanabe, W.; Yamataka, K.; Ikeda, S.; Kodama, E.;
Matsuoka, M.; Shinkai, H. J. Med. Chem. 2006, 49, 1506.
18. Satoh, M.; Matsuda, T.; Okuda, S.; Kawakami, H.;
Aramaki, H.; Shinkai, H.; Matsuzaki, Y.; Watanabe, W.;
Yamataka, K.; Kiyonari, S.; Wamaki, S.; Takahashi, M.;
Yamada, N.; Nagao, A. WO Patent 2005113509, 2005.
19. Gould, R. G.; Jacobs, W. A. J. Am. Chem. Soc. 1939, 61,
2890.
as catalyst. The resulting mixture was stirred for 15 h and
quenched by adding 2 mL of a saturated aqueous solution
of NaHCO3. After standard workup, the crude residue
was saponified with NaOH (H2O/EtOH/CH2Cl2) and the
final product was purified with HPLC to give inhibitor 8.
1H NMR (600 MHz, CD3OD/CDCl3) d 8.87 (s, 1H), 8.48
(s, 1H), 7.83 (s, 2H), 7.59 (s, 1H), 7.34 (t, J = 7.8 Hz, 2H),
7.27(t,J = 7.8 Hz, 1H), 7.13 (d,J = 7.2 Hz, 2H), 5.30 (s,
2H), 4.85 (s, 2H), 4.57 (m, 2H), 4.24 (s, 2H), 4.00 (m, 2H),
3.39 (s, 1H), 2.86 (m, 1H), 1.22 (d, J = 6.6 Hz, 6H); 13C
NMR (150 MHz, CD3OD/CDCl3) d 182.6, 172.3, 167.1,
156.2, 154.0, 152.7, 143.2, 140.4, 139.3, 137.6, 133.3, 131.4,
131.3, 130.4, 129.6, 123.9, 121.3, 116.1, 77.3, 74.7, 63.1,
60.6, 50.6, 37.6, 24.1; HRMS (ESI-) calcd for C28H28N3O7
[M-H]À 518.1927, found 518.1937.
20. Goff, D. A.; Harris, R. N.; Bottaro, J. C.; Bedford, C. D.
J. Org. Chem. 1986, 51, 4711.
23. Dayam, R.; Sanchez, T.; Clement, O.; Shoemaker, R.; Sei,
S.; Neamati, N. J. Med. Chem. 2005, 48, 111.
21. Adrian, G.; Mignonac, S.; Lecoutteux, F. WO Patent
2000003999, PCT Int. Appl. 2000.
24. Dayam, R.; Sanchez, T.; Neamati, N. J. Med. Chem. 2005,
48, 8009.
22. Synthesis of inhibitor 8: to a solution of MOM ether 15
(97 mg, 0.26 mmol) in dry CH2Cl2 (0.5 mL) at 0 °C was
25. Wai, J. S.; Egbertson, M. S.; Payne, L. S.; Fisher, T. E.;
Embrey, M. W.; Tran, L. O.; Melamed, J. Y.; Langford,
H. M.; Guare, J. P., Jr.; Zhuang, L.; Grey, V. E.; Vacca, J.
P.; Holloway, M. K.; Naylor-Olsen, A. M.; Hazuda, D. J.;
Felock, P. J.; Wolfe, A. L.; Stillmock, K. A.; Schleif, W.
A.; Gabryelski, L. J.; Young, S. D. J. Med. Chem. 2000,
43, 4923.
added
a solution of BCl3 in CH2Cl2 (0.086 mL,
0.086 mmol). The resulting orange solution was stirred
at rt for 1 h and then added to freshly prepared 18
(0.17 mmol) in CH2Cl2 solution followed by the addition
of a small amount of tetrabutylammonium iodide (TBAI)