Design of a series of bicyclic HIV-1 integrase inhibitors. Part 1: Selection of the scaffold

Article abstract of DOI:10.1016/j.bmcl.2010.07.079

HIV integrase inhibitors based on a novel bicyclic pyrimidinone core is presented. Nine variations of the core scaffold are evaluated leading to optimization of the 6:6 core giving compound 48 with an EC₅₀ of 3 nM against wild type HIV infected T-cells.

Full text of DOI:10.1016/j.bmcl.2010.07.079

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5913–5917
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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 1: Selection of
the scaffold
Eric D. Jones a, Nick Vandegraaff a, Giang Le a, Neil Choi a, William Issa a, Katherine Macfarlane a,
Neeranat Thienthong a, Lisa J. Winfield a, Jonathan A. V. Coates a, Long Lu b, Xinming Li b, Xiao Feng b,
Changjiang Yu b, David I. Rhodes a, 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:
HIV integrase inhibitors based on a novel bicyclic pyrimidinone core is presented. Nine variations of the
core scaffold are evaluated leading to optimization of the 6:6 core giving compound 48 with an EC50 of
3 nM against wild type HIV infected T-cells.
Received 10 June 2010
Revised 18 July 2010
Accepted 20 July 2010
Available online 27 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Integrase
Inhibitor
Synthesis
3-Hydroxy-pyrido[12-a]pyrimidin-4-one
The Human Immunodeficiency Virus (HIV) is the causative
agent of acquired immunodeficiency syndrome (AIDS), a disease
for which there is presently no cure. Therapy currently consists
of a combination of three drugs targeting two or more critical tar-
gets within the viral life cycle in what has proved to be an extre-
mely successful treatment regimen known as highly active
antiretroviral therapy (HAART). Raltegravir (Isentress™),1 a drug
approved by the FDA in October 2007 for the treatment of HIV
infection, targets the viral integrase (IN) protein that is responsible
for recombining newly synthesized HIV-1 DNA into the host cellu-
lar 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 trimmed (30-processing [30] reaction) prior to inser-
tion into the host cellular DNA (strand transfer [ST] reaction). As-
says that recapitulate these two reactions in vitro using
recombinant IN rely on the presence of divalent metal ions (such
as Mg2+ or Mn2+) that are known to be coordinated by a trio of con-
served basic residues (D64, D116, and E152) within the active site.
Raltegravir and related integration strand transfer inhibitors (INS-
TIs Fig. 1) are proposed2 to interrupt viral DNA binding of integrase
by coordinating these metal ions via a hydrogen bond acceptor–
donor–acceptor motif.
structure with viral DNA bound confirms this interaction.3 Since
6-carboxamido-5,4-hydroxypyrimidines were known as a metal
coordinating motif,4 we chose the para-fluorobenzyl 6-carboxam-
K+
OH
O
F
O
N
Cl
O
O
N
N
N
H
N
O
F
N
O
O
O
HN
OH
(a) raltegravir
F
(b)
GS9137
O
O
N
N
O
OH
O
N
OH
N
O
O
HN
HN
F
While no crystal structure of HIV integrase with viral or host
DNA and its inhibitors is yet available, the foamy virus integrase
OH
GSK364735
Figure 1. Reported INSTIs.
GSK1349572
* 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.2010.07.079

5914
E. D. Jones et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5913–5917
O
N
O
O
O
O
R1
R2
OH
F
O
O
HO
N
O
HO
a
b
O
H
OH
O
N
O
HO
OH
OH
O
O
O
Scaffold A
O
N
O
c
R2
F
OH
OH
F
N
N
R2
H
H
N
N
N
N
O
d
N
O
R1
R1
O
O
HO
CO Me
2
OAc
CO Me
2
Scaffold C
O
Scaffold B
F
O
R1
N
MeO C
2
OH
MeO C
2
OAc
R1
N
OH
OH
F
N
N
H
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 0 °C, 1 h then reflux 3 h, 85%;
(b) NBS, CCl4, reflux, 4 h; (c) Na2S2O4, H2O, rt, 2 h, 30% overall b and c; (d) pTsOH,
reflux, 24 h, 72%.
NH
N
N
N
R2
O
O
Scaffold E
Scaffold D
F
O
O
R1
OH
NH
R1
N
OH
F
Table 1
N
H
IC50 achieved with scaffold selection
S
N
N
S
N
Compound
Scaffold
A
R1
R2
H
IC50 (lM)
O
O
4
7-Br
0.445
0.080
Scaffold G
O
Scaffold F
O
F
7-
5
A
H
O
N
OH
F
OH
O
O
6
7
8
A
B
C
C
D
D
D
E
E
F
F
F
G
G
H
I
H
CH3
H
CH3
H
H
CH3
H
CH3
H
H
H
H
H
0.587
0.0768
>100
20.25
>100
13.65
>30
N
N
H
R1
N
NH
N
N
R1
9
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
7-Ph
8-CN
H
Scaffold H
Scaffold I
Figure 2. Target scaffolds A–I.
H
H
H
H
H
H
H
H
>10
0.626
33.25
4.25
1.13
>50
>30
5.1
>10
>10
7-CH3
7-Morph
9-OCH3
H
7-CH3
H
ide side chain on a 4-keto-5-hydroxy pyrimidine to mimic the
acceptor–donor–acceptor motif. We reasoned that incorporation
of a bicyclic system could firstly modulate the electronics of the
coordinating motif and optimize the binding to the catalytic metal
and secondly provide a scaffold to introduce functionality to en-
hance the interaction with the integrase enzyme. Accordingly a
series of bi- and tricyclic novel compounds were prepared (Fig. 2).
Previously a single report5 had described a route to unsubstitut-
ed 5:6 and 6:6 bicyclic pyrimidinones 3, from the reaction of
diacetoxy fumerate (DAF) 2 with the appropriate 2-amino hetero-
cycle 1 (Scheme 1). From 3 introduction of the required benzyl side
chain was achieved by reaction with excess amine.
In our hands the procedure of Tisler was found to be low yield-
ing (5–40%) and attempts to optimize the yield by varying reaction
time, temperature, and catalyst used were not successful. In order
to access larger quantities of core materials, it was necessary to
prepare kilogram quantities of DAF as described in Scheme 2.
With the synthetic route on hand the examples of the target
scaffolds in Figure 2 were prepared and assayed and the results
are presented in Table 1.
H
H
I
7-Morph
Activity of the compounds were assessed in an isolated protein
assay using full length integrase whereby a model 50-biotin labeled
DNA substrate already in the pre-processed form with a 30-CA se-
quence is inserted into an acceptor 30-digoxin DNA strand as previ-
ously described.6 This strand transfer assay format was selected
over the combined 30-ST assay to allow optimization for the second
strand transfer step of integrase function. It was encouraging to
find that a range of scaffolds were active against the enzyme with
sub micromolar IC50s as shown by 4–7 and 14, and that some pre-
liminary SAR could be established. For scaffold E, unsubstituted
nitrogen in the ring was not tolerated (13 vs 14) and extension
O
H
O
N
H
O
O
N
H
O
N
H
O
R1
H+
N
O
O
R1
R1
R2
N
H
N
A
O
N
F
A
H
A
+
N
H
O
O
R2
O
R2
O
O
O
1
2
3
F
Scheme 1.

E. D. Jones et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5913–5917
5915
O
N
O
O
N
OH
F
N
N
OH
F
H
N
N
H
N
O
N
N
O
O
J
R
O
K
O
(IC50 25nM; EC50in 10% NHS 31 nM)7
Figure 3. Bi-functionalized pyrimido-pyrimidine.
O
O
Br
Br
N
N
N
a
N
b
c
N
N
NO
2
NO
2
NO
2
NH2
NH2
NH2
X
24
23
25
d,e
O
O
N
O
f
N
OH
F
N
N
H
N
N
NH2
R
O
R
26
27
Scheme 3. Reagents and conditions: (a) (i) ClCOOEt, Py 93%; (ii) HNO3, H2SO4, 30 °C, 20 h, 68%; (iii) KOH/EtOH/H2O, 75%; (b) morpholine, 140 °C, 10 h, 85%; (c) NaNO2, H2SO4,
H2O, 0 °C, 2 h then 80 °C 2 h, 75% for X = OH or CuBr, X = Br 80%; (d) X = OH then DIAD, Ph3P, THF, ROH, 0 °C, 2 h, 75% or from X = Br with isothiazolidine-1,1-dioxide [entry 43
and ROH, 44], DMEDA, CuI/K2CO3, toluene, 80 °C, 5 h, 43.1%; (e) SnCl2, EtOH/H2O, 80 °C, 5 h, 70–90%; (f) (i) HOAc, DAF, sealed tube 90o, 20 h C; (ii) 4-F-benzyl amine, MeOH,
reflux, 3 h for 38 15.2% overall.
to the tricyclic systems was not tolerated for scaffolds C and I (7 vs
9).
Table 2
Effect on activity by introduction of an EDG at C9 of 5
Compounds that showed IC50s of ꢀ1
lM were assessed for anti-
HIV-1 activity in Hut78 cells infected with HIV (NL4-3 strain). Of
note were examples of scaffolds A, 4 and 5, B compound 7, and F
compound 17 showing sub micromolar inhibition in (EC50’s 0.65,
O
O
OH
F
N
N
H
N
0.028, 0.113, and 0.48
lM, respectively) while showing no cytotox-
N
icity up to 10 M. These three classes represent potentially novel
l
R
O
inhibitors of HIV and herein we report the optimization of Scaffold
A. Optimization of scaffolds B and F will be the subject of future
publications.
Compound
R
IC50 (nM)
EC50 (nM)
10% FBS
215
9.5
50% NHS
In the course of this work, researchers at Merck reported7 their
discovery of the pyrimido-pyrimidine core as a new class of HIV-1
integrase inhibitors. This program focused on derivatives at posi-
tion 9, (structure J in Fig. 3) achieving low nM potencies against
HIV. We decided to exploit our different route of synthesis by
directing our efforts into investigating the effect of a substitution
at the C9 position of 5 (K in Fig. 3).
In order to assess the effect of substitution at the C9 of 5, the
route as in Scheme 3 was devised. Starting from 3-amino-5-bro-
mopyridine, regioselective nitration allowed SnAr conditions to
introduce the required morpholine 24. Diazotization allowed the
introduction of bromine (25) or Mitsunobu reaction to give the aryl
ether and subsequent reduction afforded the amino pyridine 26.
Core formation by reaction with DAF and aminolysis provided
the desired targets 27.
5
28
29
H
OMe
OiPr
80
170
245
ND
1400
ND
5
O
30
31
32
429
275
755
12
590
ND
O
80
F
N
O
730
1550
Initially we investigated the effects of introducing an electron
withdrawing substituent at C9 of 5. Compounds were again as-
sayed in the isolated enzyme system and subjected to single-round

5916
E. D. Jones et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5913–5917
O
O
N
O
b,c
a
N
O
OH
F
O
O
N
N
N
N
H
N
O
O
N
N
NH2
O
N
O
Br
26
S
Br
O
34
35
Scheme 4. Reagents and conditions: (a) (i) DAF, HOAc, MeOH, reflux, 24 h; (ii) BnBr, K2CO3, DMF, 80 °C, 6 h, 25% two-steps; (b) Pd2(dba)3, xanthphos, Cs2CO3, dioxane, 100 °C,
4 h, 67%; (c) (i) TFA, 70 °C, 5 h; (ii) pF-benzylamine, MeOH, reflux, 15 h, 50% overall two-steps.
O
O
N
Br
O
O
N
c
b
Br
O
O
Br
a
Br
N
N
O
N
N
O
O
NH 2
NH 2
N
S
I
I
O
38
37
36
d
X
O
N
X
O
N
O
O
N
OH
H
N
F
e, f
N
N
O
N
O
O
N
O
N
S
S
O
O
35a
39
Scheme 5. Reagents and conditions: (a) I2, HIO4ꢁH2O, AcOH, H2SO4ꢁH2O, 75 °C, 7 h, 70%; (b) (i) DAF, pTSOH, 100 °C, 30 h; (ii) BnBr, K2CO3, DMF, 70 °C, 16 h, 16% two-steps;
(c) Pd2(dba)3, xanthphos, Cs2CO3, dioxane, 2-methyl-[1,2,5]thiadiazolidine-1,1-dioxide, 80 °C, 6 h, 61% or MeSO2NHMe, 50 °C, 16 h, 85%; (d) Pd2(dba)3, xanthphos, Cs2CO3,
dioxane, 90 °C, 15 h, 40%; (e) TFA, 70 °C, 5 h, 90%; (f) pF-benzylamine, MeOH, reflux, 15 h, 39%.
HIV-1-based infectivity assays, which allowed the determination
of EC50 values in low serum (10% FBS) and the more therapeutically
relevant high serum level (50% NHS). Table 2 summarizes the
results.
OH gave the intermediate 37. The increased reactivity of the C9-
iodo to Buchwald conditions was exploited to introduce polar sub-
stituents as C9 followed by a second amination to functionalize at
C7.
Improvements in potency against the enzyme by incorporation
of an electron donating group, EDG, into the base compound 5 as
shown by 28–32 was not obtained. In contrast, potency in the
anti-HIV assay was greatly enhanced by the inclusion of alkoxy
groups (28–31). The anti-HIV result for 29 compared to the C9-
smaller 28 and larger 30 and 31 suggests a finite aliphatic binding
pocket in the region of C9, and the discordance between the iso-
lated integrase enzyme and cell-based data indicates that the en-
zyme may adopt a slightly different conformation in the whole
cell system. Although low nanomolar potency has been achieved
in 28 the >100-fold shift of inhibitory activity in cell-based assays
conducted in a high serum environment indicate the detrimental
effect of plasma protein binding. In order to explore this, a series
of analogues containing EDG at C9 were prepared by modification
of the chemistry described in Scheme 3. As Scheme 4 shows, direct
core formation of the intermediate 26 from Scheme 3, followed by
benzyl protection of the 2-OH afforded the C9-halogenated scaffold
33 which was primed for further extension at C9 by a series of me-
tal mediated aminations.
The introduction of an electron withdrawing group, EWG, at
R1 as in Scheme 4 continued to provide low nanomolar potency
against HIV (40–42). Increasing the polarity by introduction of a
sultam, made by the literature method,8 at C9 in 43 was also tol-
erated, and a reduced shift of less than twofold in 50% NHS
(Table 3). Solubility needed to be balanced, and although the
dichlorobenzyl amine compound 44 was potent, it showed a dras-
tic shift in the presence of serum compared to 43. Introduction of
a piperazine functionality R1 at C7 to improve solubility was also
tolerated in 45, although the N-des-methyl version 46 was not.
Scheme 5 allowed the introduction of a basic piperazine at R1
with
a linear sulfonamide in 47, which was potent (EC50
19.8 nM) and showed a threefold shift in serum. However, the
more basic N-methyl thiadiazolidine, made following the litera-
ture method9, when introduced into position C9 in 48 with an
EC50 3 nM gave a minimal fold change in the presence of 50%
serum.
In summary, the work presented here has defined a range of no-
vel scaffolds that form the basis of HIV integrase inhibitors. Opti-
mization of the 6:6 bicyclic scaffold has lead to significant
improvements in antiviral activity superior to that of the launched
drug raltegravir. Further optimization of this series will be pre-
sented in due course.
In order to explore the R1 group at position C7 in structures
based on 35 beyond morpholine, the route as in Scheme 5 was de-
vised where the di-halo intermediate 36 was formed under stan-
dard core formation conditions followed by protection of the C3-

E. D. Jones et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5913–5917
5917
Table 3
References and notes
Effect on activity EWG at C9 of scaffold A
1. Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.; Fiore,
F.; Gardelli, C.; Paz, O. G.; Hazuda, D. J.; Jones, P.; Kinzel, O.; Laufer, R.;
Monteagudo, E.; Muraglia, E.; Nizi, E.; Orvieto, F.; Pace, P.; Pescatore, G.;
Scarpelli, R.; Stillmock, K.; Witmer, M. V.; Rowley, M. J. Med. Chem. 2008, 51,
5843.
O
R1
OH
F
N
H
N
N
2. Kawasuji, Y. T.; Fuji, M.; Yoshinga, T.; Sato, A.; Fujiwara, T.; Kiyama, R. Bioorg.
Med. Chem. 2006, 14, 8420.
R2
O
3. Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Nature 2010, 464,
23.
4. Sunderland, J.; Botta, M.; Aime, S.; Raymond, K. N. Inorg. Chem. 2001, 40, 6746.
5. Tisler, M.; Zupet, R. Org. Prep. Proced. Int. 1990, 224, 532.
Compound R1
R2
IC50
(nM)
EC50 (nM)
10%
FBS
50%
NHS
6. (a) Ovenden, S. P. B.; Yu, J.; Wan, S. S.; Sberna, G.; Tait, M. R.; Rhodes, D.; Cox, S.;
Coates, J.; Walsh, N. G.; Meurer-Grimes, B. Phytochemistry, 2004, 65, 3255.
(b) 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-
ACTGCTAGAGATTTTCCACACTGACTAA-AAGGGTC-DIG-30) or biotin (50-Bio-
GACCCTTTTAGTCAGTGT-GGAAAATCTCTAGCA-30) so that each substrate has
either a DIG or Bio tag on opposite strands. Reactions are carried out for 2 h at
37ꢀC, 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
O
O
O
O
N
N
N
N
40
41
42
I
85
6
50
70
Br
Cl
130
135
7
11
158
O
N
N
43
O
195
10.3
18
S
50,000 cells per 50
Compounds are prepared to 4ꢂ final concentration in RF-10/2, and 30
to cells. Virus (40 l in RF-10/2 containing 1600 pfu) is added to each well or
40 l RF-10/2 for negative controls and for assaying compound cytotoxicity.
After 24 h, an additional 90
l of media or media containing 1ꢂ 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.
ll per well in RF-10 containing 2
l
g/ml polybrene (RF-10/2).
ll added
O
O
N
l
44a
45
130
110
ND
0.2–1.2
8
43
40
43
O
Br
O
S
l
l
l
N
N
l
O
Forty eight hours later supernatants are harvested and levels of extracellular
p24 determined. Supernatants are diluted 1 in 10,000 and p24 levels assayed
using the Vironostika p24 assay kit. EC50 is calculated as the concentration
required to inhibit HIV p24 production to 50%.
O
HN
N
N
N
46
280
S
7. (a) Kinzel, O. D.; Ball, R. G.; Donghi, M.; Maguire, C. K.; Muraglia, E.; Pesci, S.;
Rowley, M.; Summa, V. Tetrahedron Lett. 2008, 49, 6556; (b) Donghi, M.; Kinzel,
O. D.; Summa, V. Bioorg. Med. Chem. Lett. 2009, 19, 1930.
8. White, E. H.; Lim, H. M. J. Org. Chem. 1987, 52, 2162.
9. Kim, S. J.; Park, H. B.; Lee, J. S.; Joa, N. H.; Yoo, K. H.; Baek, D.; Kang, B.-W.; Choa,
J.-H.; Oha, C. H. Eur. J. Med. Chem. 2007, 42, 1176.
O
N
N
N
47
ND
19.8
61.5
O
O
S
O
N
N
S
48
ND
3
4
N
Raltegravir
130
9.4
7.1
a
3,5-Dichlorobenzylamine analogue of 43.