Design of a series of bicyclic HIV-1 integrase inhibitors. Part 2: Azoles: Effective metal chelators

Article abstract of DOI:10.1016/j.bmcl.2010.07.081

Synthesis of a diverse set of azoles and their utilizations as an amide isostere in the design of HIV integrase inhibitors is described. The Letter identified thiazole, oxazole, and imidazole as the most promising heterocycles. Initial SAR studies indicat

Full text of DOI:10.1016/j.bmcl.2010.07.081

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5909–5912
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of a series of bicyclic HIV-1 integrase inhibitors. Part 2: Azoles:
Effective metal chelators
Giang Le a, Nick Vandegraaff a, David I. Rhodes a, Eric D. Jones a, Jonathan A. V. Coates a,
Neeranat Thienthong a, Lisa J. Winfield a, Long Lu b, Xinming Li b, Changjiang Yu b, Xiao Feng b,
John J. Deadman a,
*
a Avexa Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia
b Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Lu, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a diverse set of azoles and their utilizations as an amide isostere in the design of HIV integr-
ase inhibitors is described. The Letter identified thiazole, oxazole, and imidazole as the most promising
heterocycles. Initial SAR studies indicated that these novel series of integrase inhibitors are amenable
to lead optimization. Several compounds with low nanomolar inhibitory potency are reported.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 June 2010
Revised 18 July 2010
Accepted 20 July 2010
Available online 27 July 2010
Keywords:
Integrase
Inhibitor
Metal chelator
Synthesis
Human immuno-deficiency virus type 1 (HIV-1) is the etiologi-
cal agent that causes acquired immuno-deficiency syndrome
(AIDS). The treatment for HIV-1 infected patients is known as
highly active antiretroviral therapy (HAART) and typically consists
of a combination of two nucleoside reverse transcriptase inhibitors
(NRTIs) and either a non-nucleoside reverse transcriptase inhibitor
(NNRTI) or a protease inhibitor (PI). Recently, however, a new drug
(Raltegravir, Isentress™) targeting the third viral enzyme integrase
(IN) has been approved by the FDA.1 This drug interrupts the crit-
ical process of integration, whereby newly made viral DNA is in-
serted into the host cellular chromosome. IN achieves this via a
two-step enzymic process in which the 30 terminal two nucleotides
on each newly made viral DNA strand are (30-processing reaction).
IN then facilitates the concerted integration of both trimmed ends
of the viral DNA into cellular chromosomal DNA in a process called
strand transfer. Within the active site of IN lies a highly conserved
trio of basic amino acids (Asp-64, Asp-116, and Glu-152) that to-
tors of HIV integrase.3 It was demonstrated that varying the substi-
tutions of the bicyclic rings provided an effective approach to
optimize the interaction of the metal binding element with the
protein. This strategy has resulted in the discovery of potent, nano-
molar inhibitors of HIV integrase in both enzymatic and cell-based
assays. We sought to further improve the activity and pharmacoki-
netic properties of these compounds in whole cell assays by replac-
ing the amide group. The chelating scaffolds of these inhibitors
consist of one phenolic hydroxyl group and two amide functional-
ities serving as co-ordinating components for divalent Mg2+ ions in
the active site of the integrase protein. Since azoles are well known
amide isosteres having been successfully utilized in design of po-
tent inhibitors for HIV protease and apoptosis proteins,4 we em-
barked upon studies to examine the possibility of using diverse
azoles in place of the exo amide group to provide the essential che-
lation of the Mg2+ ions. We reasoned that changing the nature of
the heterocycles from azoles to imidazoles or oxazoles, would offer
additional means to fine-tune electronic properties, and thus metal
binding capabilities of the scaffolds (Fig. 1). In addition, the hetero-
cylic azoles would also allow modification of the flexible linker
that connects the metal chelator and the p-fluoro substituted phe-
nyl ring. Herein, we report our findings of successful utilization of
azoles as effective metal chelators in designing a new class of po-
tent HIV integrase inhibitors.
gether co-ordinate two divalent metal ions (physiologically Mg2+
)
that are critical for the architecture of the active site and enzyme
catalysis. Raltegravir and related integration strand transfer inhib-
itors (INSTIs) are proposed to interrupt viral DNA binding of integr-
ase by co-ordinating these metal ions via an hydrogen bond
acceptor–donor–acceptor motif.2 Recently we have disclosed the
discovery of new bicyclic pyrimidine derivatives as potent inhibi-
The key starting material for azoles is the methyl ester 3 of
which the synthesis was described previously.3 The ester group re-
acted readily with hydrazine hydrate to give the mono-hydrazide,
* Corresponding author. Tel.: +61 408538488.
E-mail address: jjdeadman@bigpond.com (J.J. Deadman).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.081

5910
G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5909–5912
-DD(35)E-
O
O
N
O
O
O
O
N
-DD(35)E-
N
OBn
O
N
N
N
O
N
a
N
O
O
O
O
O
O
O
O
O
O
OH
Mg
O
O
11
O
O
Mg
O
Mg
Mg
3
F
F
O
O
N
b
O
N
O
X
Het
N
O
N
N
H
N
O
O
OBn
N
OBn
H
N
N
N
Flexible linker
X = O or N
c
N
Y
Flexible linker
N
O
O
13
12
R
R
Figure 1. Design of azoles for amide replacement.
d
O
N
which was further acylated with an acid chloride to give interme-
diate 4 (Scheme 1).
OH
N
14 15 16
NH
N
Y
O
S
Cyclization of the hydrazide group followed by Bn deprotection
with TMSI as Lewis acid furnished the final 1,3,4-oxadiazole 5.
Alternatively, using Lawesson’s reagent as cyclizing agents led to
1,3,4-thiadiazole 6. Triazole 7 was also prepared from ester 3
which readily underwent amidation with aqueous ammonium
hydroxide. The resulting amide was reacted with Lawesson’s re-
agent, and subsequently with MeI to provide an S-Me thioamide
intermediate that cyclized with 4-F-phenyl acetohydrazide to form
triazole 7. Preparation of the asymmetrical 1,2,4-oxadiazole 10 re-
quired a different strategy. In the first step, ester 3 was converted
to nitrile 8 via amidation with ammonia and dehydration with tri-
chlorotriazine. Next, the nitrile group was reacted with hydroxyl-
amine to give N-hydroxyamidine, which was acylated with an
appropriate phenylacetyl chloride to afford precursor 9. Formation
of the 1,2,4-oxadiazole ring was achieved readily by refluxing
intermediate 9 in toluene.
Y
R
Scheme 2. Reagents and conditions: (a) LiOH, H2O, THF, 80%; (b) EDCIꢀHCl, HOBt,
Et3N, NH2–CH2–C(O)–R, DMF; 74%; (c) Ph3P, CCl4, Et3N for Y = O, 50%; Lawesson’s
reagent, toluene, reflux for Y = S, 35%; ammonium acetate, AcOH, xylene, reflux for
Y = NH, 38%; (d) TMSI/CH3CN, 54% for 14, 30% for 15, 76% for 16.
tuted 2-oxo-ethylamine to give the key intermediate 12. Following
the cyclization steps, removal of the Bn group was affected by TMSI
in acetonitrile to complete the synthetic sequence.
Oxazole 14 and thiazole 15 rings can also be prepared from acid
11 and 2-bromo aldehyde 18 (Scheme 3). The latter was conve-
niently synthesized in two steps via Heck coupling of commercially
available iodo-benzene derivatives 17, followed by bromination.
The acid group of 11 was alkylated with 2-bromo aldehyde 18 in
the presence of a proton sponge to act as acid scavenger to afford
ester 19. Under similar conditions, thioacid 20, which was prepared
from 11 via reaction with sodium hydrosulfide and subsequent
Similar cyclization approaches were also employed successfully
for the preparation of oxazole 14, thiazole 15, or imidazole 16
(Scheme 2). The ester group of compound 3 was hydrolyzed with
LiOH to give acid 11 which underwent amidation with a substi-
O
N
O
O
O
N
O
O
N
R1
OBn
Br
N
OBn
O
N
O
I
N
N
a
N
a
O
O
R
O
+
R
HN
4
O
N
H
R
OH
3
11
17
18
f
b
b
c
d
O
O
O
O
N
N
N
OBn
CN
OH
Y
N
N
O
O
N
N
R1
R1
N
OBn
N
OBn
SH
N
N
8
R
O
5
6 7
O
N
O
O
Y
O S NH
d
O
Ar
19
20
c
O
O
O
O
N
OBn
b
e
N
OH
N
N
N
NH 2
N
N
R1
O
N
OBn
Y
O
N
O
N
N
O
R1
10
9
OBn
c
R
N
N
R
N
S
O
Scheme 1. Reagents and conditions: (a) (i) hydrazine hydrate, MeOH, 81%; (ii)
RCOCl, 94%; (b) Y = O,S, (i) Ph3P, CCl4, Et3N for Y = O, 43%; Lawesson’s reagent,
toluene, reflux for Y = S, 35%; (ii) TMSI/CH3CN, 41% for 5, 35% for 6; (c) (i) NH3, H2O,
50 °C, 59%; (ii) 2,4,6-trichloro-1,3,5-triazine, 88%; (d) (i) NH2OHꢀHCl, NaHCO3, 92%;
EtOH, reflux; (ii) RCOCl, 97%; (e) (i) toluene, reflux, 75%; (ii) FeCl3, CH2Cl2, 30%; (f)
Y = N, (i) NH3, H2O, 50 °C, 59%; (ii) Lawesson’s reagent, toluene, reflux, 45%; (iii) MeI,
50%; 4-F-phenyl acetohydrazide, AcOH, reflux, 55%; (iv) TMSI/CH3CN, 50%.
13
O
Ar
R
21
Scheme 3. Reagents and conditions: (a) (i) All-OH, Heck coupling, 50%; (ii) TMSBr,
Br2, CH2Cl2, 75%; (b) proton sponge, CH2Cl2, reflux; (c) ammonium acetate, AcOH,
toluene, reflux, 30–40% yield over steps (b) and (c); (d) CDI, NaSH, 60–8%.

G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5909–5912
5911
acylation with 18 was converted to thioester 21. Both 20 and 21
were cyclized readily under heating with ammonium acetate to
the corresponding oxazole or thiazole 13.
It is worth noting that it has been previously reported that the
nitrogen atom of the naphthyridine ring is able to co-ordinate
Mg2+, an observation that formed the basis of a scaffold for a series
of potent HIV integrase inhibitors.7 Replacement of the heteroatom
neighbor to the chelating nitrogen proved most beneficial for the
inhibitory activity. In particular, thiazole 28, oxazole 29, and imid-
azole 30 were all significantly more potent compared to the corre-
sponding thiadiazole 26, oxadiazole 23, and triazole 25, as well as
the parent amide 22.
We next examined the SAR of other substituents of the bicyclic
ring systems using the identified thiazole, oxazole, or imidazole as
metal binding motifs. Introduction of halogen substitutions in
position 9 improved the potency several fold resulting in low
nanomolar inhibitors 31 and 32 (Table 2). Similarly the electron-
donating cyclic sulfonamide group on the thiazole (34) was bene-
ficial. Surprisingly the same group caused a significant reduction in
activity when applied to oxazole and imidazole scaffolds (35 and
36) demonstrating the existence of a subtle difference between
these heterocycles and the azole rings. In contrast, a range of nitro-
gen containing substitutions was well tolerated when combined
with the imidazole as the amide isostere.
The inhibitory potency of the azole derivatives were assessed
initially in an enzymatic assay using recombinant HIV WT integr-
ase,5 and compounds exhibiting IC50 values <1
lM were subjected
to single-round HIV-1-based infectivity assays to determine the
more therapeutically relevant EC50 values. The amide 22, selected
as the parent compound for our investigations displayed an IC50
of 775 nM and a reasonable EC50 of 215 nM in these assays. Grati-
fyingly, the easily accessible oxadiazoles 23 and 24, triazole 25, and
thiadiazole 26 all displayed nanomolar potency akin to that of the
parent amide. These results suggested that five-membered ring
azoles are capable of functionally replacing an amide at this posi-
tion. We postulated that in these azoles the nitrogen atom (high-
lighted in red, Table 1) furthest from the 4-F benzyl substituent
would be positioned in a favorable geometry to chelate with the
divalent Mg2+ ion. Changing the electronic properties of the azoles
by inclusion of oxygen, nitrogen, or sulfur in the ring influenced the
activity, although only with marginal effects. Similarly, the posi-
tion of the non-chelating nitrogen and oxygen atoms were not cru-
cial for potency (23 vs 24). The nature of the co-ordinating atom
was critical as evidenced by compound 27, which was inactive de-
spite containing a similar asymmetrical oxadiazole as 24. The data
from these two compounds suggest that a nitrogen atom, and not
an oxygen atom is the preferred metal chelator within the hetero-
cyclic setting. This result is intriguing given the ability of the amide
carbonyl oxygen to co-ordinate Mg2+ as demonstrated in the recent
X-ray crystal structure of Raltegravir within the active site of the
Foamy Virus integrase.6
Sultam 37, which is a close analog of sulfonamide 36, exhibited
a surprisingly good EC50 of 11 nM while cyclic carbamate 38, cyclic
lactam 39, or amide 40 all showed a reasonable level of potency
ranging from 39 nM to 86 nM in the cell-based assays. Among
Table 2
SAR of azoles as amide isotere
O
O
N
N
OH
N
N
R9
X
Table 1
F
Enzymatic and in vivo activities of compounds 22–30
28-41
O
O
N
O
O
N
N
OH
N
N
OH
N
X
O
F
Het
HN
23-30
F
22
X
Het
Heterocycle
—
IC50 (nM) EC50 spread (nM) EC50 Luc (nM)
22
775
310
28
215
na
N
N
23
24
25
26
27
28
29
30
215
O
N
O
450
na
99
na
14
na
16.5
6
na
N
N
N
197.5
na
N
N
N
N
225
>10,000
20
S
O
>1000
32
N
N
S
N
59
21.5
62
O
N
45
na
N

5912
G. Le et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5909–5912
37, 3100; (b) Thompson, S. K.; Eppley, A. M.; Frazee, J. S.; Darcy, M. G.; Lum, R. T.;
Tomaszek, T. A., Jr.; Ivanoff, L. A.; Morris, J. F.; Sternberg, E. Bioorg. Med. Chem.
Lett. 1994, 4, 2441; (c) Cohen, F.; Koehler, M. F. T.; Bergeron, P.; Elliott, L. O.;
Flygare, J. A.; Franklin, M. C.; Gazzard, L.; Keteltas, S. F.; Lau, K.; Ly, C. Q.; Tsui, V.;
Fairbrother, W. Bioorg. Med. Chem. Lett. 2010, 20, 2229.
the studied substitutions, cyclic urea 41 appeared the most prom-
ising with an EC50 value of 6 nM. Importantly, the inhibitory
activity of this compound displayed a small shift (<threefold) in
cell-based assays conducted in a high (50%) serum environment
(data not shown).
5. (a) Ovenden, S. P.; Yu, J.; Wan, S. S.; Sberna, G.; Tait, R. M.; Rhodes, D.; Cox, S.;
Coates, J.; Walsh, N. G.; Meurer-Grimes, B. M. Phytochemistry 2004, 65, 3255; (b)
In summary we have presented data that supports the utility of
azoles as a component of a metal binding motif in the acceptor–do-
nor–acceptor pharmacophore of HIV-1 integrase inhibitors. We
have examined eight different five-member ring azoles and estab-
lished that they can efficiently serve as an amide isostere when
placed adjacent to a 6,6 bicyclic pyrimidine core. Of the heterocy-
cles tested, introduction of the thiazole, oxazole and imidazole into
the scaffold resulted in the best anti-integrase and antiviral poten-
cies. We further demonstrated that in contrast to that of the amide
setting, within the heterocyclic environment a nitrogen atom
rather than an oxygen atom appears to be the preferred hetero-
atom for metal co-ordination. Finally, the initial SAR presented
here demonstrated that these compounds are highly potent leads
amenable to further optimization to generate inhibitors of HIV
integrase. A comprehensive lead optimization of these promising
leads is underway and the results will be reported in due course.8
Combined strand transfer assay: briefly, for
a 96-well format, 400 ng IN is
incubated with 30 nM pre-processed substrate DNA, consisting of annealed U5
LTR sequence oligonucleotides tagged with Digoxigenin (DIG; 50-ACTG
CTAGAGATTTTCCACACTGACTAA-AAGGGTC-DIG-30) or biotin (50-Bio-GACCC
TTTTAGTCAGTGT-GGAAAATCTCTAGCA-30) so that each substrate has either a
DIG or Bio tag on opposite strands. Reactions are carried out for 2 h at 37 °C,
products generated as a result of 30 processing and strand transfer activity are
bound to streptavidin plates and detected using anti-DIG-alkaline phosphatase
conjugate and p-nitro phenyl phosphate substrate.; (c) Inhibition of HIV
replication: cells are seeded into 96-well microtitre plates at 50,000 cells per
50
prepared to 4ꢁ final concentration in RF-10/2, and 30
(40 l in RF-10/2 containing 1600 pfu) is added to each well or 40
negative controls and for assaying compound cytotoxicity. After 24 h, an
additional 90
l of media or media containing 1ꢁ compound is added to each
well. At 4 days post infection, 100 l of media is removed from each well and
replaced with 100 l of fresh media with or without compound. Forty eight
hours later supernatants are harvested and levels of extracellular p24
determined. Supernatants are diluted in 10,000 and p24 levels assayed
l
l per well in RF-10 containing 2
l
g/ml polybrene (RF-10/2). Compounds are
l added to cells. Virus
l RF-10/2 for
l
l
l
l
l
l
1
using the Vironostika p24 assay kit. EC50 is calculated as the concentration
required to inhibit HIV p24 production to 50%.; (d) Chang, T. L.-Y.; Francois, F.;
Mosiam, M.; Klotman, M. E. J. Virol. 2003, 77, 6777.
6. Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Nature 2010, 464,
232.
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