Tricyclic HIV integrase inhibitors V. SAR studies on the benzyl moiety

Article abstract of DOI:10.1016/j.bmcl.2009.02.092

SAR studies on the para-fluorobenzyl moiety of tricyclic HIV integrase inhibitors are discussed and lead compounds with potency and PK properties comparable to raltegravir were identified.

Full text of DOI:10.1016/j.bmcl.2009.02.092

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2263–2265
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tricyclic HIV integrase inhibitors V. SAR studies on the benzyl moiety
*
Haolun Jin , Sammy Metobo, Salman Jabri, Michael Mish, Rachael Lansdown, Xiaowu Chen,
Manuel Tsiang, Matthew Wright, Choung U. Kim
Gilead Sciences, Inc. 333 Lakeside Drive, Foster City, CA 94404, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR studies on the para-fluorobenzyl moiety of tricyclic HIV integrase inhibitors are discussed and lead
compounds with potency and PK properties comparable to raltegravir were identified.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 20 November 2008
Revised 22 February 2009
Accepted 24 February 2009
Available online 27 February 2009
Keywords:
AIDS
HIV
Integrase inhibitor
Tricyclic
Benzyl moiety
N
N
The integrase (IN) of Human immunodeficiency virus (HIV), the
causative pathogen of AIDS, is an essential enzyme encoded in the
HIV pol gene, together with reverse transcriptase (RT) and protease
(PR). The recent progress in researches directed towards identify-
ing clinically useful IN inhibitors has lead to the market approval
of raltegravir (MK-0518, 1),1 a powerful addition to HIV therapeu-
tics aimed at overcoming drug resistance problems and improving
the life of AIDS patients (Fig. 1).
In our previous publications we described the design and SAR
studies of a class of novel and highly organized tricyclic HIV IN
inhibitors.2 The lead compound from the series, compound 2,
exhibited not only excellent potency against both IN strand trans-
fer activity in the enzymatic assay and HIV replication in the cell
culture, but also good pharmacokinetics in both rat and dog.2d
In the systematic efforts to optimize both potency and pharma-
cokinetic profile of the tricyclic integrase inhibitors, we started
examining the roles of hydrophobic p-fluorobenzyl moiety high-
lighted in 2, in addition to other portions of the scaffold that have
been evaluated previously.2 A modeling system developed in-
house3 indicated that besides the p-fluorine, many other hydro-
phobic/hydrophilic substituents could be accommodated at site 1
(Fig 2). We noticed that SAR studies on the hydrophobic p-fluorob-
enzyl moiety on a raltegravir-based pyrimidinone scaffold were
recently published by Merck scientists.4 In this manuscript, we
report the results of our investigations on the roles of substitutions
on the phenyl portion of p-fluorobenzyl moiety and a preferred
substitution pattern emerged from these studies.
O
O
O
O
F
O
S
N
HN
F
N
N
N
H
N
N
O
O
O
H
O
H
1 (raltegravir, MK-0518)
Figure 1. The structures of 1 and 2.
2
* Corresponding author. Tel.: +1 650 522 5460; fax: +1 650 522 5899.
E-mail address: hjin@gilead.com (H. Jin).
Figure 2. Modeling indicates that the hydrophobic pocket (site 1) may accommo-
date additional substituents on the p-F-benzyl moiety.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.092

2264
H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2263–2265
Table 1
OH
O
O
Integration strand transfer inhibition and anti-HIV proliferation assay results for
a.
compounds 2, 7–13a
MeO
MeO
N
MeO
a.
N
EC50 (10%FBS)c
EC50 (HSP)d
Shift fold (HSP/FBS)
b
N
Compd
IC50
O
O
OH
2
7
8
9
10
11
12
13
28
120
52
61
39
135
74
5
1.7
35
3.5
6
6
5
32
52
4
11.4
nd
12
18
17
18
90
nd
20
6.7
nd
3.4
3
2.8
3.6
2.8
nd
5
MeO
3
4
O
O
O
O
S
S
N
N
b; c
d; e
1e (raltegravir)
40
HN
MeO
MeO
N
N
N
Ph
a
Values are means of at least two experiments, given in nM, nd: not determined.
Ref. 5a.
Ref. 5b.
O
O
O
b
c
O
PMB
Ph
O
d
HSP: human serum proteins adjusted EC50, obtained by assaying compounds in
6
5
the presence of physiological concentrations of human serum albumin and AAG;
see Ref. 5c for details.
R
e
In-house data.
7
H
O
S
8
9
3-Cl-4-F
2-F-3-Cl
5-Cl-2,4-F
N
Table 2
Pharmacokinetics of 2, 8 and 10 in rat and doga
10
N
Species
Rat
Dog
R
N
11 3-Cl-2,4-F
O
F (%) T1/2 (h) CL (L/h/kg) F (%) T1/2 (h)
CL (L/h/kg)
OH
7-13
12 2-CONHMe-4-F
13 2-NHCOMe-4-F
2
8
15
7
9
1.1
1.36
1.38
0.28
0.23
0.29
45
41
79
7
7.13
16
0.4
0.15
0.02
10
Scheme 1. PMB: p-methoxybenzyl. Reagents and conditions: (a) For conditions
see: Ref. 2a and 2d; (b) TFA, triethylsilane, rt 1 h then 78 °C, 2 h, 78%; (c) Cs2CO3, p-
methoxybenzyl chloride, tetrabutylammonium iodide, 65°C, 2 h, 80%; (d) substi-
tuted benzyl bromides, sodium hydride, DMF, 0 °C (see Ref. 6 for for the preparation
of the substituted benzyl bromides); (e) TFA, triethylsilane, rt, 50–93%.
1 (raltegravir)b 37
2
2.34
45
11 (b T1/2
)
0.36
F (%): fraction absorbed upon oral dosing testing compounds as compared to iv
dosing, calculated based on AUC from iv and po groups, expressed as %; CL: total
body clearance obtained from ivdosing groups.
a
All compounds were dosed as free parent in a solution form (EtOH, PG, PEG400;
and citric acid; pH 3.3 for iv and pH 2.2 for p.o.); and T1/2 was generated from the iv
dosing group. The values were means of data obtained from samples of three ani-
mals in each study.
To access a variety of substituted benzyl moieties in 2, we
envisaged a convergent synthetic plan utilizing key intermediate
6 shown in Scheme 1. Compound 3 was prepared from 2,4-dime-
thoxybenzylamine and succinic anhydride under heating
condition. Dieckmann condensation between 3 and dimethyl 2,3-
pyridinedicarboxylate yielded 4 which was converted to C5 aza
intermediate 5 according to the procedures described in our previ-
ous publications.2a,d Upon treating 5 with acid in the presence of
triethylsilane, free phenol was generated which was re-protected
as p-methoxybenzyl ether 6. Alkylation of 6 with benzyl bromide
or 3-Cl-4-F-benzyl bromide yielded 7 and 8. Other substituted ben-
zylating agents were prepared with the standard procedures and
used to convert 6 to 9–12.6
b
Data adapted from Ref. 1.
moiety of tricyclic scaffold were hydrophobic functional groups.
This trend is in contrast to the raltegravir based scaffold, which tol-
erated carboxamides in anti-HIV assay.4 One of the possible expla-
nations is the reduced capability of these compounds to penetrate
the cell membrane since their enzymatic activity is comparable to
the other analogs.
The results of pharmacokinetic studies performed on both 8 and
10inratanddogaresummarizedinTable2. Similarto 2,2d both8and
10 were found to be orally bioavailable in these two species. A
remarkable feature of 8 and 10 is their excellent PK profiles in dog.
All analogs prepared were tested for their activity in both HIV
integrase strand transfer assay and anti-HIV assay in cell culture.
The data are summarized in Table 1.
Both compounds exhibited low clearance, long half-life time (T1/2
)
and high oral bioavailability. The major PK parameters compare very
favourably to those of raltegravir (1) reported by the Merck group.1
The noticeably low systemic clearance of both 8 and 10 in dog PK
studies can be a combination of a few factors including their intrinsic
metabolic stabilities and plasma protein binding in dog.
In conclusion, analogs of the C5 aza tricyclic quinoline derived
by modifying the p-fluorobenzyl moiety were prepared. Examina-
tion of SAR revealed a preferred substitution pattern that favors
the incorporation of two or three halogens with flexible substituted
positions. More notably, addition of halogens greatly improved dog
PK properties. These lead compounds were selected for the clinical
investigation and the results will be reported in due course.
All the analogs tested showed enzymatic activity in the IN
strand transfer assay at submicromolar concentrations. An analysis
of the potency revealed a unique substitution pattern that is pre-
ferred in the series. First, the role of the fluorine for the potency
is essential since removal of the fluorine (7) led to a 20-fold loss
in potency by comparing 7 to 2. All the multi-halogenated analogs
(8–11) were highly potent in anti-HIV assay in cell culture. Further
more, the cell-based potency of these compounds was shifted by
human serum proteins (HSP) to small degrees (2.8–3.6 folds of
shifting) and they were equipotent to raltegravir (1) when tested
in parallel. A noteworthy feature is that these halogens can be
incorporated in different combinations of substituted positions to
maintain good activities.
Acknowledgments
On the other hand, installing relatively polar functional groups
such as carboxamide at the ortho position (12 and 13) compro-
mised the cell culture activity in anti-HIV assay. These observa-
tions suggest that the preferred substituents on the benzyl
The authors are grateful to Xubin Zheng and Bing Lu for analyz-
ing samples in PK studies; Vahid Zia and co-workers in the formu-

H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2263–2265
2265
with anti-DIG antibody-conjugated horse radish peroxidase detection was
used.; (b) For antiviral assay, 50
diluted drug in culture medium were added to each well of a 96-well plate (9
concentrations) in triplicate. MT-2 cells were infected with HIV-1 IIIB at an m.o.i.
of 0.01 for 3 h. Fifty microlitres of infected cell suspension in culture medium
(ꢀ1.5 Â 104 cells) were then added to each well containing the drug dilutions.
lation group for supporting the PK studies; Gregg Jones for carrying
out biological assays.
ll of 2Â test concentration of fivefold serially
References and notes
The plates are incubated at 37 °C for
5 days. One hundred microlitres of
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5. (a) Strand transfer assay modified from a previous report (Hazuda et al., Nucleic
Acid Res. 1994, 22, 1121). Biotinylated donor DNA was bound to Reacti-Bind
High Binding Capacity Streptavidin coated white plates. DIG-tagged target DNA
CellTiter-GloTM Reagent (catalog # G7571, Promega Biosciences, Inc., Madison,
WI) were then added to each well. Cell lysis was allowed to complete by
incubating at room temperature for 10 min. Chemiluminescence was then read.
For the cytotoxicity assay, the protocol is identical to that of the antiviral assay,
except that uninfected cells and a threefold serial dilution of drugs were used.;
(c) The effect of compounds binding to serum protein components was
evaluated by determining the antiviral EC50 in MT-2 cells in 10% FBS in the
presence or absence of serum concentrations of HSA (35 mg/ml) or
a1-AGP
(1.5 mg/ml). From the EC50 data in the presence of each individual protein, the
EC50 resulting from the combined effect of both proteins (as in serum) can be
calculated. The derivation of the appropriate equation for this calculation can be
made through competitive binding assumptions.
6. For 3-Cl-4-F-benzyl bromide (8): DeGraw, J. I.; Cory, M.; Skinner, W. A.; Theisen,
M. C.; Mitoma, C. J. Med Chem. 1967, 10, 64; For 5-Cl-2,4-F-benzyl bromide (9 and
10), 2-CONHMe-4-F benzyl analog () and 2-NHAc-4-F-benzyl analog (13):
US2007/72831; For 3-Cl-2,4-F-benzyl bromide (11): WO2007/61670 example
68.