Synthesis and HIV-integrase strand transfer inhibition activity of 7-hydroxy[1,3]thiazolo[5,4-b]pyridin-5(4H)-ones

Article abstract of DOI:10.1016/j.bmcl.2006.08.007

An efficient synthesis of methyl 7-hydroxy[1,3]thiazolo[5,4-b]pyridin-5(4H)-one-6-carboxylates (8-10 and 16) and 6-carboxamides (17-20) is described. Sub-micromolar enzyme inhibition of HIV integrase was achieved with several carboxamide analogs which were superior to their carboxylic ester congeners.

Full text of DOI:10.1016/j.bmcl.2006.08.007

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5668–5672
Synthesis and HIV-integrase strand transfer inhibition activity
of 7-hydroxy[1,3]thiazolo[5,4-b]pyridin-5(4H)-ones
Eric E. Boros,* Brian A. Johns, Edward P. Garvey, Cecilia S. Koble and Wayne H. Miller
GlaxoSmithKline Research & Development, Five Moore Drive, Research Triangle Park, NC 27709, USA
Received 30 June 2006; revised 26 July 2006; accepted 1 August 2006
Abstract—An efficient synthesis of methyl 7-hydroxy[1,3]thiazolo[5,4-b]pyridin-5(4H)-one-6-carboxylates (8–10 and 16) and 6-carb-
oxamides (17–20) is described. Sub-micromolar enzyme inhibition of HIV integrase was achieved with several carboxamide analogs
which were superior to their carboxylic ester congeners.
Ó 2006 Elsevier Ltd. All rights reserved.
Acquired immunodeficiency syndrome (AIDS) in
humans results from infection by the human immunode-
ficiency virus type 1 (HIV-1). Highly active antiretrovi-
ral therapy (HAART) for HIV currently targets two
of the three virally encoded enzymes; namely, reverse
transcriptase and protease. This approach to therapy
usually involves treating the patient with an HIV-prote-
ase inhibitor in combination with both a nucleoside
reverse transcriptase inhibitor and a non-nucleoside re-
verse transcriptase inhibitor. Although HAART therapy
is highly successful in warding off the development of
AIDS, incidences of drug resistance and toxic side effects
emphasize the need for new targets in the treatment of
HIV.1
is needed for viral infectivity and there are no known
host cell counterparts.
The search for a clinically viable HIV-integrase inhibitor
for HIV therapy has spanned more than a decade and
recently led to clinical evaluations of heteroaryl diketone
S-1360 (1)5 and naphthyridine carboxamide L-870,810
(2).6 These molecules inhibit the strand transfer step of
HIV-1 integrase and possess potent antiviral activities.
More recently, other heteroaromatic scaffolds with
HIV-integrase inhibition activity have emerged, includ-
ing the naphthyridinone carboxamides 3.7 All of these
compounds (1–3) contain a diketoacid-like motif impli-
cated in binding of metal ions within the catalytic core
domain of the integrase enzyme.8 The substituted benzyl
group in 1–3 is also necessary for potent antiviral activ-
ity. In this report, we describe an expedient synthesis of
7-hydroxythiazolopyridinones 8–10 and 16–20, and
their HIV-integrase strand-transfer inhibition activity.
Significantly, the third and final HIV-encoded enzyme,
HIV-integrase (IN), has yet to yield to drug discovery
efforts.2 Integrase catalyzes the integration of reverse
transcribed viral DNA into host cell DNA through a
two-step, metal-dependent process. Step one, known as
30 processing,3 effects cleavage of two nucleotides from
the two 30 ends of double stranded viral DNA. The sec-
ond step, strand transfer,4 integrates the viral DNA into
the host cell DNA through a series of phosphodiester
transesterification reactions. A final non-integrase-de-
pendent step, involving cellular DNA repair enzymes,
fills in the remaining gaps. Integrase is an especially
attractive target for HIV therapy because the enzyme
Synthesis of the title compounds was achieved by the
chemistry depicted in Schemes 1, 2 and Eqs. 1–3. Struc-
tures of the title compound esters (8–10 and 16) and
carboxamides (17–20) are shown in Charts 1 and 2,
respectively. Enzyme inhibition data are provided in
Tables 1 and 2. The requisite intermediate 5-aminothiaz-
oles 6a–i were synthesized from commercially available
methyl isocyanoacetate 4 and the appropriate isothiocy-
anate 5a–i (Scheme 1).9 Treatment of 6a–i with methyl
malonyl chloride in 1,2-dichloroethane (DCE) at reflux
temperature afforded malonyl amides 7a–i which were
cyclized under basic conditions to thiazolopyridinones
8a, 8c–e, 9a–b, and 10a–c (see Chart 1).
Keywords: HIV-integrase; Strand-transfer inhibition.
*
Corresponding author. Tel.: +1 919 483 8116; fax: +1 919 315
0430; e-mail: eric.e.boros@gsk.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.007

E. E. Boros et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5668–5672
5669
O
OH
O
O
O
OH
N
HO
N
O
N
N
H
N
N
N
F
H
O
S
F
N
R
F
N
N
O
H
3
2
1
O
Bromine–magnesium exchange10 of 12a,b with isopropyl
magnesium bromide followed by reaction with 4-fluoro-
benzaldehyde afforded intermediate alcohols 13a,b
which were N-deprotected and reduced to 5-aminothiaz-
oles 14a,b in one step with TFA/triethylsilane.11 Reac-
tion of 14a,b with malonyl chloride and treatment of
the resulting malonyl amides 15a,b with NaOMe/MeOH
afforded the thiazolopyridinones 16a,b.
O
i
N
OMe
R
+
RNCS
NC
MeO
S
N
H
5a-i (R)
6a-i
5a (PMB); 5b (Me);
4
5c (Et); 5d (iPr); 5e (Ph);
5f (4-(MeO)Ph); 5g (3-(NO2)Ph);
5h (3-(CF3)Ph); 5i (3-(SMe)Ph)
ii
O
Equations 1-3. Synthesis of Compounds 17-20
O
O
OH
N
OMe
R
O
OH
O
O
OH
iii
N
OMe
RNH2
R
S
N
S
N
N
S
O
N
S
(1)
R
H
N
R
R
O
O
N
R
N
R
neat, 100-120˚C
MeO
O
8-10
7a-i
8-10 and 16
17-20
Scheme 1. Synthetic route to compounds 8a, 8c–e, 9a,b, and 10a–c.
Reagents and conditions: (i) t-BuOK, THF; (ii) methyl malonyl
chloride, DCE, reflux; (iii) NaOMe, MeOH.
TFA (8a) or TFA/TfOH (17a)
(2)
(3)
8a, 17a
8b, 17b
CH2Cl2
Treatment of aminothiazoles 6b and 6e with NBS in ace-
tonitrile provided 2-bromothiazoles 11a,b, respectively
(Scheme 2). Amino group protection of 11a,b with
(Boc)2O/DMAP produced N-Boc derivatives 12a,b.
MCPBA
CH2Cl2
oxone, H2O
19c
19e
19d
MeOH, CH2Cl2
O
O
O
HO
N
O
i
N
S
iii
ii
N
S
O
O
S
Br
6b, 6e
Br
N R
N R
BOC
N R
H
BOC
F
11a: R = Me
11b: R = Ph
13a,b
12a,b
iv
O
O
OH
O
N
O
N
S
N
S
O
O
vi
v
S
N R
O
O
N
R
N R
H
O
F
F
F
O
15a,b
16a,b
14a,b
Scheme 2. Synthetic route to compounds 16a,b. Reagents and conditions: (i) NBS, CH3CN; (ii) (Boc)2O, DMAP, THF, reflux; (iii) i-PrMgBr,
4-fluorobenzaldehyde, THF; (iv) Et3SiH, TFA, CH2Cl2; (v) methyl malonyl chloride, DCE, reflux; (vi) NaOMe, MeOH.

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E. E. Boros et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5668–5672
Methyl esters 8–10 and 16 were significantly less active
O
O
O
OH
OH
N
in the strand transfer assay12 (Table 1) compared with
S-1360 (1) (IC50 = 0.16 lM); esters containing substitut-
ed (8a), linear (8d) or branched (8e) N-4 alkyl groups
showed weak inhibition activity (IC50 = 4–11 lM); N-4
aryl derivatives (9a,b and 10a–c) were inactive. Interest-
ingly, incorporation of a p-fluorobenzyl moiety at C-2 in
the ester series (16a,b) enhanced inhibition activity com-
pared to 8c and 9a.
1
3
N
S
N
S
OMe
OMe
2
4
N
O
R
8a: R = PMB
8b: R = H
8c: R = Me
8d: R = Et
8e: R = i-Pr
X
9a: X = H
9b: X = OMe
Conversion of carboxylic esters 8–10 and 16 to carbox-
amides 17–20 improved inhibition activity with the
exception of 8a and 16a. In these latter cases, conversion
to the corresponding p-fluorobenzylcarboxamides pro-
duced compounds of similar (17a) or lesser (20a) poten-
cy. Carboxamides having N-4 ethyl (17d) and N-4
isopropyl (17e) substitution were the most potent ana-
logs (IC50 = 0.03–0.19 lM). Replacing the fluorine atom
of 17c with chlorine (17f) produced a 3-fold drop in
strand transfer inhibition activity.
O
OH
F
N
S
OMe
O
O
OH
N
O
N
OMe
S
N
R
X
16a: R = Me
16b: R = Ph
10a: X = NO2; R = H
10b: X = CF3; R = H
10c: X = SMe; R = H
In the N-4 aryl series (18–19), meta-phenyl substitution
(19a–e) reduced strand transfer inhibition activity
compared to the unsubstituted phenyl compound 18a.
Oxidation of thioanisole derivative 19c to the corre-
sponding sulfone (19d) and sulfoxide (19e) improved
potency by less than an order of magnitude. Interest-
ingly, none of the title compounds showed antiviral
activity separate from cellular toxicity; this observation
is unusual considering that many potent strand transfer
inhibitors have submicromolar cellular potency
values.13
Chart 1.
Methyl esters 8–10 and 16 were converted to carboxa-
mides 17–20 by reaction with the appropriate amines
under neat thermal conditions (Eq. 1). Reaction of the
p-methoxybenzyl (PMB) analogs 8a and 17a with TFA
or TFA/triflic acid afforded compounds 8b and 17b,
respectively (Eq. 2). Oxidation of m-thioanisole analog
19c with MCPBA or oxone gave the corresponding sul-
fone (19d) and sulfoxide (19e) products, respectively
(Eq. 3).
In summary, an efficient synthesis of thiazolo[5,4-b]pyri-
din-5(4H)-one HIV integrase strand transfer inhibitors
was presented. The methodology described herein
O
O
OH
OH
N
N
N
H
N
H
F
X
S
S
O
O
N
N
R
17a: R = PMB; X = F
17b: R = H; X = F
17c: R = Me; X = F
17d: R = Et; X = F
17e: R = i-Pr; X = F
17f: R = Me; X = Cl
X
18a: X = H
18b: X = OMe
O
OH
N
F
N
S
O
O
N
OH
H
N
S
F
N
O
H
F
N
R
X
20a: R = Me
20b: R = Ph
19a: X = NO2
19b: X = CF3
19c: X = SMe
19d: X = SO2Me
19e: X = S(O)Me
Chart 2.

E. E. Boros et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5668–5672
5671
Table 1. HIV integrase strand transfer inhibition activity of methyl
7-hydroxy[1,3]thiazolo[5,4-b]pyridin-5(4H)-one-6-carboxylates 8–10,
References and notes
and 16a
1. Richman, D. D. Nature 2001, 410, 995.
2. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev.
Drug Disc. 2005, 4, 236.
3. Benard, C.; Zouhiri, F.; Normand-Bayle, M.; Danet, M.;
Desmaele, D.; Leh, H.; Mouscadet, J.-F.; Mbemba, G.;
Thomas, C.-M.; Bonnenfant, S.; Bret, M. L.; d’Angelo, J.
Bioorg. Med. Chem. Lett. 2004, 14, 2473.
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Lett. 2004, 14, 4815.
5. Fujishita, T.; Yoshinaga, T.; Sato, A. WO00/39086 A1,
2000; Chem Abstr. 2000, 133, 89529.
6. Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.; Jolly, S. M.;
Wai, J. S.; Zhuang, L.; Fisher, T. E.; Embrey, M.; Guare,
J. P., Jr.; Egbertson, M. S.; Vacca, J. P.; Huff, J. R.;
Felock, P. J.; Witmer, M. V.; Stillmock, K. A.; Danovich,
R.; Grobler, J.; Miller, M. D.; Espeseth, A. S.; Jin, L.;
Chen, I.-W.; Lin, J. H.; Kassahun, K.; Ellis, J. D.; Wong,
B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.; Cortese, R.;
Emini, E.; Summa, V.; Holloway, M. K.; Young, S. D.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11233.
c,d,e
Compound
Nb
IC50
(lM)
8a
8b
2
1
1
1
1
1
1
1
1
1
1
1
4.2 3.6
>500
>500
1.23
8c
8d
8e
9a
11
>500
>500
>500
>500
>500
35
9b
10a
10b
10c
16a
16b
282
a See Chart 1 for molecular formulas.
b N = number of experiments.
c Data are expressed as means SE (N > 1).
d IC50 data for S-1360 (1) = 0.16 0.04 lM (N = 33).
e See Ref. 12 for assay conditions.
7. Egbertson, M.; Melamed, J. Y.; Langford, H. M.; Young,
S. D. WO03/062204 A1 2003; Chem. Abstr. 2003, 139,
133554.
8. Kiyama, R.; Kawasuji, T. WO01/95905 A1 2001; Chem.
Abstr. 2001, 136, 53570.
9. Suzuki, M.; Moriya, T.; Matsumoto, K.; Miyoshi, M.
Synthesis 1982, 874.
10. Abarbri, M.; Thibonnet, J.; Berillon, L.; Dehmel, F.;
Rottlander, M.; Knochel, P. J. Org. Chem. 2000, 65, 4618.
11. Carey, F. A.; Tremper, H. S. J. Am. Chem. Soc. 1969, 91,
2967.
Table 2. HIV integrase strand transfer inhibition activity of
7-hydroxy[1,3]thiazolo[5,4-b]pyridin-5(4H)-one-6-carboxamides 17–
20a
c,d,e
Compound
Nb
IC50
(lM)
17a
17b
17c
17d
17e
17f
2
2
2
1
2
2
2
1
2
1
2
2
2
1
2
4
2
1.5 0.1
0.7 0.3
0.03
0.19 0.03
2.1 0.1
0.4 0.1
1
12. Compounds were tested as inhibitors of recombinant
HIV integrase in the following in vitro strand transfer
assay. A complex of integrase and biotinylated donor
DNA–streptavidin-coated SPA beads was formed by
incubating 2 lM recombinant integrase with 0.66 lM
biotinylated donor DNA–4 mg/ml streptavidin-coated
SPA beads in 25 mM sodium MOPS, pH 7.2, 23 mM
NaCl, 10 mM MgCl2, and 10 mM dithiothreitol, and
10% DMSO for 5 min at 37 °C. Beads were pelleted by
centrifugation, supernatant removed, and then beads
resuspended in 25 mM sodium MOPS, pH 7.2, 23 mM
NaCl, and 10 mM MgCl2. Beads were again spun down,
supernatant removed, and then beads resuspended in a
volume of 25 mM sodium MOPS, pH 7.2, 23 mM NaCl,
10 mM MgCl2 that would give 570 nM integrase (assum-
ing all integrase bound the DNA-beads). Test com-
pounds dissolved and diluted in DMSO were added to
the integrase–DNA complex to give 6.7% DMSO (typ-
ically 1 ll of compound added to 14 ll of integrase
complex), and preincubated for 60 min at 37 °C. Then
[3H] target DNA substrate was added to give a final
concentration of 7 nM substrate, and the strand transfer
reaction mixture was incubated at 37 °C typically for 25–
45 min which allowed for a linear increase in covalent
attachment of the donor DNA to the radiolabeled target
DNA. A 20 ll reaction was quenched by adding 60 ll of
the following: 50 mM sodium EDTA pH 8, 25 mM
sodium MOPS, pH 7.2, 0.1 mg/ml salmon testes DNA,
and 500 mM NaCl. Streptavidin-coated SPA were from
GE Healthcare, oligos to make the donor DNA were
from Oligos Etc, and [3H] target DNA was a custom
synthesis from Perkin Elmer. Sequences of donor and
target DNA were previously described (Hazuda, D. J.;
Hastings, J. C.; Wolfe, A. L.; Emini, E. A. Nucleic Acid
Res. 1994, 22, 1121) with the addition of seven terminal
A’s on each end of the target DNA that allowed for the
18a
18b
19a
19b
19c
19d
19e
20a
20b
17
7
3
7
3
0.93 0.04
2.0 0.2
151
4
2
a See Chart 2 for molecular formulas.
b N = number of experiments.
c Data are expressed as means SE (N > 1).
d IC50 data for S-1360 (1) = 0.16 0.04 lM (N = 33).
e See Ref. 12 for assay conditions.
affords rapid access to diverse analogs in this series and
provides a general perspective on their structure–activity
relationships. Potent enzyme inhibition of HIV integrase
was achieved with several C-6 carboxamide-containing
derivatives which were generally more active than their
ester counterparts. Within the carboxamide series, sim-
ple N-4 alkyl substituents showed greater potency with
IC50 values as low as 0.03 lM. These findings contribute
to the growing understanding of SAR in the field of
2-metal-binding HIV integrase inhibitors.
Acknowledgment
This work was conducted as part of a research collabo-
ration between GlaxoSmithKline and Shionogi & Co.
Ltd.

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E. E. Boros et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5668–5672
incorporation of 14 tritiated T’s (specific activity of
no DMSO and no difference was observed (with poten-
cies in very good agreement with literature values if
applicable).
target DNA approximately 1300 Ci/mmol). The Km of
substrate target DNA and IC50 of standard inhibitors
were checked with this percentage of DMSO (6.7%) vs
13. Young, S. D. Annu. Rep. Med. Chem. 2003, 38, 173.