Discovery of the Aryl-phospho-indole IDX899, a Highly Potent Anti-HIV Non-nucleoside Reverse Transcriptase Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.5b01430

Here, we describe the design, synthesis, biological evaluation, and identification of a clinical candidate non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a novel aryl-phospho-indole (APhI) scaffold. NNRTIs are recommended components of highly active antiretroviral therapy (HAART) for the treatment of HIV-1. Since a major problem associated with NNRTI treatment is the emergence of drug resistant virus, this work focused on optimization of the APhI against clinically relevant HIV-1 Y181C and K103N mutants and the Y181C/K103N double mutant. Optimization of the phosphinate aryl substituent led to the discovery of the 3-Me,5-acrylonitrile-phenyl analogue R_P-13s (IDX899) having an EC₅₀ of 11 nM against the Y181C/K103N double mutant.

Full text of DOI:10.1021/acs.jmedchem.5b01430

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Article
Discovery of the Aryl-Phospho-Indole (IDX899), a highly
potent anti-HIV non-nucleoside reverse transcriptase inhibitor
Cyril Dousson, François-René Alexandre, Agnès Amador, Séverine Bonaric, Stéphanie Bot,
Catherine Caillet, Thierry Convard, Daniel da Costa, Marie-Pierre Lioure, Arlène Roland, Elodie
Rosinovsky, Sébastien Maldonado, Christophe Parsy, Christophe Trochet, Richard Storer, Alistair
Stewart, Jingyang Wang, BENJAMIN A MAYES, Chiara Musiu, Barbara Poddesu, Luana Vargiu,
Michel Liuzzi, Adel Moussa, Jocelyn Jakubik, Luke Hubbard, Maria Seifer, and David Standring
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01430 • Publication Date (Web): 23 Jan 2016
Downloaded from http://pubs.acs.org on January 26, 2016
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Journal of Medicinal Chemistry
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Discovery of the Aryl-Phospho-Indole (IDX899), a highly potent
anti-HIV non-nucleoside reverse transcriptase inhibitor
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Cyril Doussona‡, François-René Alexandrea*‡, Agnès Amadora,†, Séverine Bonarica,†, Stéphanie
Bota, Catherine Cailleta,†, Thierry Convarda, Daniel da Costaa, Marie-Pierre Liourea,† , Arlène
Rolanda,†, Elodie Rosinovskya,†, Sébastien Maldonadoa,†, Christophe Parsya, Christophe
Trocheta,†, Richard Storera,†, Alistair Stewartb,†, Jingyang Wangb,†, Benjamin A. Mayesb,†, Chi-
ara Musiub,†, Barbara Poddesub,†, Luana Vargiub,†, Michel Liuzzib,†, Adel Moussab,†, Jocelyn
Jakubikb,†, Luke Hubbardb,†, Maria Seiferb,†, David Standringb,†
aIDENIX, an MSD Company, 1682 rue de la Valsière, Cap Gamma, BP 50001, 34189 MONTPELLIER Cedex 4,
France and bIDENIX, 320 Bent Street - 4th Floor, Cambridge, MA 02139, USA
†Former Idenix employees
ABSTRACT: Here, we describe the design, synthesis, biological evaluation and identification of a clinical candidate
non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a novel Aryl-Phospho-Indole (APhI) scaffold. NNRTIs
are recommended components of highly active antiretroviral therapy (HAART) for the treatment of HIV-1. Since a
major problem associated with NNRTI treatment is the emergence of drug resistant virus, this work focussed on op-
timization of the APhI against clinically relevant HIV-1 Y181C and K103N mutants and the Y181C/K103N double mu-
tant. Optimization of the Phosphinate Aryl substituent led to the discovery of the 3-Me,5-Acrylonitrile-Phenyl ana-
logue RP-13s (IDX899) having an EC50 of 11 nM against the Y181C/K103N double mutant.
The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is a key enzyme in the replica-
tion cycle.1 Inhibition of the HIV-1 RT leads to viral load declines in patients as shown with currently approved NNR-
TIs: nevirapine (NVP) 1, efavirenz (EFV) 2, delavirdine (DLV) 3, etravirine (ETV) 4 and rilpivirine (RPV) 5. NNRTIs
bind to an allosteric hydrophobic pocket close to the polymerase active site of the HIV-RT, triggering a conforma-
,
tional change that inhibits the progression of viral DNA synthesis along the RNA template 2 3. However, a single ami-
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no acid mutation in the RT enzyme can lead to dramatic loss of efficacy of first generation NNRTIs such as NVP and
1
2
,
,
EFV.4 5 6 Clinically relevant mutants generated by first generation NNRTIs include Y181C, K103N and the Y181C/K103N
3
4
double mutant (DM). The second generation NNRTI, etravirine, has an improved coverage of RT-mutants7. However,
5
6
there is still a need for novel, chemically diverse, second generation NNRTIs with broader mutant coverage and an
7
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, ,
improved pharmaceutical profile8 9 10
.
9
Our goal in this work report was to develop a clinical candidate that retains good activity against the Y181C, K103N
and Y181C/K103N mutants compared to EFV. The potency optimization of this series of APhI was performed using a
cell-based HIV-1 assay to identify compounds that were potent enzyme inhibitors, and also had the ability to cross the
cell membrane and inhibit the HIV-1 virus replication cycle in a living cell.
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Figure 1. Marketed NNRTIs
In a previous communication11a, we described the discovery of the APhI scaffold11b-d. A widely accepted strategy to
improve the activity of an NNRTI against viruses containing the expected class resistant mutations is to build strong
,
,
,
interactions of the drug with conserved regions of the NNI Binding Pocket (NNIBP)12 13 14 15, such as highly conserved
side chain residues or protein backbone atoms. Ideally, the candidate NNRTI should also demonstrate decreased
interactions with mutable residues and exhibit some degree of conformational flexibility so that its binding can adapt
to accommodate possible mutated amino acids16. Furthermore, it is known that in order to get robust potency, the
ligand should have at least three points of interaction with the protein, and, ideally, these three points should not be
aligned but form a triangle.
Based on reported structures (1FK9, 1FKO, 1JKH) of EFV, and other inhibitors17 and the geometry requirements re-
vealed by these structures, we postulated that:
- A substituted bicyclic scaffold that generates hydrogen bond interactions with backbone of K101 and lipophilic in-
teractions or п/п interactions with F227.
- A branched tetrahedral linker could direct the substitution with the right angle towards the W229 conserved resi-
due.
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Phosphorus atom was selected as a key component of this linker because of its tetrahedral conformation, its novelty
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W229
W229
EFV from 1FK9
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Ar
R3
F227
F227
P
F3C
O
R2
O
Cl
Cl
O
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N
N
H
O
H
H
H
N
O
N
O
R
R
K101
K101
in this framework as well as for the opportunity of substitution allowed by its PV oxidation state (Figure 2).
(a) (b) (c)
Figure 2. (a) X-ray of EFV in 1FK9 (some residues have been omitted for clarity). (b) Schematic view of EFV in 1FK9. (c)
Selection of phospho-indole as novel scaffold.
In an earlier study we identified APhI 6 with the methyl phosphinate linker as lead series.11a The further optimiza-
tion of this lead is described in the present report.
Chemistry
Figure 3. Lead APhI 6
Two main synthesis strategies have been used to successfully give phospho-indole 13 with various substituents on
the phosphorus (scheme 1 and 2), both started from key intermediate 711b.
Scheme 1. Indole phosphinate synthesis – chlorophosphonate route
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(a) n-BuLi, THF, -90 °C, 5 min; (b) 8 in THF, 20-80%; (c) TMSBr, DMF, 55 °C; (d) P(OMe)3, 90 °C, 12-64%; (e) NH3, MeOH,
50 °C, 13-77%; (f) (Me)3SiCHN2, MeOH, 5-27%; (g) Pd(OAc)2, TlOAc, tri-o-tolylphosphine, acrylonitrile, DMF, ꢀW, 110 °C, 2-
56%.
Scheme 2. Indole phosphinate synthesis – indole H-phosphinate route
R2
16
R2
H
H
I
MeO
EtO
MeO
P
P
P
O
O
O
Cl
Cl
Cl
O
O
a, b, c
O
d
e
7
N
N
NH2
N
OEt
NH2
H
H
SO2Ph
13
14
15
(a) n-BuLi, THF, -90 °C, 5 min; (b) Cl-P(OEt)2; (c) HCl 0.5M/THF, 64% (3 steps); (d) NH3, MeOH 50 °C, 42%; (e) 16,
Pd(PPh3)4, Et3N, CH3CN, ꢀW, 85 °C, 8-10%.
The classical and linear chlorophosphonate route was first used as described previously11a-c for aromatic, heteroaro-
matic and alkyl R2 substituents (Scheme 1) before more convergent indole H-ethyl-phosphinate 14 was developed
(scheme 2). Attempts to directly give key intermediate indole H-Methyl phosphinate 15 succeeded by treatment with
ammonia in methanol. Intermediate 14 was obtained from 3-bromo indole analogue via lithium-bromine exchange
with n-BuLi at low temperature and nucleophilic substitution on chlorodiethylphosphite followed by indole-
phosphonite acid monohydrolysis in 64 % overall yield. This route proved to be also generally useful for the prepara-
tion of APhI with different substitutions on the phenyl ring (compounds 13c and 13t).
Results and Discussion
During the course of this program, an extensive structure activity relationship (SAR) of the indole scaffold was con-
ducted and more than 300 methyl phosphinate analogues were synthesized and evaluated in cellular assays using
standard procedure. In this article we wish to report a focused SAR of 2-carboxamide,5-chloro indole phosphinates
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and results are reported in Tables 1, 2, 3 and 4. The majority of the APhI synthesized were racemates at phosphorus
and only a few selected compounds were resolved to test the two enantiomers. Cytotoxicities were evaluated during
the course of the optimization of APhI and selectivity indexes proved to be over 1000 for most of the APhI (data not
shown11d). Mono substituted phenyl phosphino-indoles were generally very potent against wild type HIV-1, but only
those with long lipophilic groups tended to exhibit good activity against the mutant viruses as shown for compounds
13a and 13b (Table 1). The 3-E-acrylonitrile 13b displayed single nanomolar potency on WT and the single mutants
and, below 50 nM activity on the DM.
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Table 1. Structure and Activity of APhI-carboxamides 6, 13a-13b
R2
MeO
P
O
Cl
O
N
NH2
H
EC50 (µM)a
WT
Compd
EFV 2
611a
R2
K103N
0.037
Y181C
0.0018
0.0220
0.0084
0.0018
K103N/Y181C
0.0580
>1
0.0007
0.0003
0.0005
0.0034
H
0.0100
0.0016
0.0032
13a
3-iPr
0.0926
0.0495
13bb
3-acrylonitrile (E)
aPrimary MT-4 cell type assay. bSecondary MT-4 cell type assay.
Based on this result, other 3-substituted phenyl analogues with larger substituents were synthesized and tested.
However, as shown in Table 2, 3-acrylonitrile (13b) was not sufficient to retain good activity against the DM when
phenyl was substituted with 3-pyridine (13c). Furthermore, heterocycles such as 1-pyrazole or 2-thiophene (13d, 13e),
cycloalkyl (13f) or carboaromatics (13g) did not improve the DM activity.
Table 2. Structure and Activity of APhI-carboxamides 13c-13g
EC50 (µM)
Compd
R3
WT
K103N
Y181C
K103N/Y181C
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13c
13d
0.001
0.0025
0.0095
0.1025
0.192
>1
1
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3
4
0.0045
0.059
5
6
7
8
9
13e
13f
2-thiophene
Cyclohexyl
1-naphtyl
0.0003
0.0025
0.0175
0.0034
0.219
0.0086
0.1008
0.7272
>1
>1
>1
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13g
0.357
Phenyl substitutions on the phosphorus linker proved to have a significant influence on mutant activity. To further
evaluate the effect of substituted phenyl groups on DM activity, we synthesized and tested di-substituted phenyl
analogues as reported in Table 3. It appears that 3,5 disubstitution is better than 3,4 (13h vs. 13i), which may be ex-
plained by the close proximity of W229 in the NNIBP. This steric requirement can also be observed when both me-
thyls (13i) are replaced by fluorine (13j), chlorine (13k) or trifluoromethyl (13l), with the bulkier trifluoromethyl ana-
logue giving the weakest activity. Further, it appears that fluorine may be too small to fulfill alkyl hydrophobic inter-
actions with the P95 side chain of the protein, while methyl or chlorine substituents provided the best potency. The
electronic density of the phenyl ring also played a role in the DM activity in this binding region; keeping the me-
thyl/P95 interaction while modulating electronic density with small substituents led to the following ranking of activ-
ity against the DM: F>Cl>CN>Me>Br>OMe (13i, 13m to 13q). This ranking can be explained in part by π/π interac-
tions (Figure 5) with the electron rich aromatic ring of the Y188 residue that lies stacked above the phenyl ring of the
inhibitor; these π/π interactions become stronger with electron poor phenyl rings. This electronic requirement was
further confirmed when comparing electron donating groups of 13t, 13r and the electron withdrawing acrylonitrile
(13s), all having a similar length.
Compound 13t proved to be the best WT inhibitor of this series displaying an exceptional EC50 value of 80 pM;
however, it lost more than 1000-fold activity against the DM (EC50 = 111 nM). In contrast, compound 13s exhibited the
best DM activity and overall profile in the simple carboxamide series.
Table 3. Structure and Activity of APhI-carboxamides 13h-13t
R2
MeO
P
O
Cl
O
N
NH2
H
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EC50 (µM)
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2
3
4
5
6
7
8
9
Compd
13h
13i
R2
3-Me-4-Me
WT
K103N
0.0229
0.0040
0.0065
0.0017
0.5268
0.0005
0.0056
0.006
Y181C
0.0350
0.0150
0.0255
0.0199
0.9468
0.0034
0.0188
0.0250
0.0230
0.0184
0.0035
0.0030
0.0087
K103N/Y181C
0.7920
0.2380
0.6420
0.2480
>1
0.0002
0.0010
0.0007
0.0003
0.0246
0.0002
0.0011
3-Me-5-Me
13j
3-F-5-F
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13k
13l
3-Cl-5-Cl
3-CF3-5-CF3
3-F-5-Me
13m
13n
13o
13p
13q
13r
0.0690
0.1141
3-Cl-5-Me
3-Br-5-Me
0.0030
0.0004
0.0010
0.0007
0.0029
0.00008
0.3685
0.663
3-OMe-5-Me
3-CN-5-Me
0.009
0.0038
0.0007
0.0030
0.0018
0.1618
3-(CH2)2CN-5-Me
3-CH=CHCN-5-Me (E)
3-OCH2CN-5-Me
0.0285
0.0166
0.1110
13s
13t
With compound 13s exhibiting the best overall profile, its enantiomers were separated (RP-13s and SP-13s) by chiral
preparative HPLC. Consistent with the X-ray data, only the RP enantiomer was active with a DM activity difference
between the RP and SP enantiomers of over 110 fold. Compared to EFV, RP-13s demonstrated over 30- and 4-fold better
activity against the K103N and DM, respectively (Table 4 and Figure 4).
Table 4. Structure and Activity of enantiomers of 13s
EC50 (µM)
Compd
Structure
WT
K103N
0.0013
Y181C
K103N/Y181C
RP-13s
0.001
0.0028
0.011
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1
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SP-13s
0.19
0.377
0.824
>1.25
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Figure 4. X-ray co-crystal top view of APhI RP-13s (PDB 5FDL)
An X-ray structure of RP-13s (Figure 4) bound to the NNIBP of HIV-RT DM was obtained at a resolution of 3.3 Å
(PDB 5FDL). The corresponding ligand interaction diagram shows the residues interacting with the NNRTI (Figure 5).
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W229
1
2
N
3
4
5
Y188
F227
6
7
π
8
9
V106
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O
O
O
P
L234
Cl
NH2
O
L100
π
H235
N
H
Y318
P236
H
N103
O
N
K101
Style keys:Red large dashed line: hydrogen-bond interactions. Black dashed line: van der Waals, π and lipophilic interac-
tions. Grey residues: positioned behind RP-13s.
Figure 5. Ligand interaction diagram of RP-13s with HIV-RT DM.
To further evaluate the drug potential of the APhI, the aqueous solubility of some analogues were determined.
Compared to EFV (<0.5 ꢀM), this series proved to be more soluble (Table 5). The bioavailability and pharmacokinetic
profile were evaluated in three animal species (Table 6) and RP-13s proved to have a good exposure in monkey when
given orally as opposed to the low rat and dog AUC/dose values. RP-13s in vitro data in hepatocytes (Table 7) suggest a
better in vivo metabolic stability profile in human than the other species, with rat and dog giving the lowest values as
previously observed in vivo. Given its excellent in vitro and in vivo profile, RP-13s 5-chloro-3-[[3-[(E)-2-cyanovinyl]-5-
methyl-phenyl]-(R)-methoxy-phosphoryl]-1H-indole-2-carboxamide (IDX899) was selected as a clinical candidate for
human dosing18.
Table 5. Solubility profile of APhIs
pH7.0 Aqueous solubility
Compound
(ꢀM)
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EFV
13a
<0.5
0.8
0.9
1.7
1
2
3
4
5
6
7
8
13i
13m
RP-13s
20
9
10
11
12
13
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Table 6. PK profile of APhIs
Cmax
(ng/mL)
88.0
Tmax
(h)
-
t1/2
AUC
Cl
Vd
F
Compound
Species a Route b Dose
(h) /Dose (mL/kg/h) (mL/kg) (%)
Rat
Rat
IV
PO
IV
2.0
10.0
1.0
0.2
5.9
3.9
2.4
14.0
5.0
71.00
NC
NC
NC
NC
NC
NC
-
38.0
-
25.0
0.4
-
-
1.83
-
Dog
290.0
18.0
520.0
22.0
RP-13s
Dog
PO
IV
5.0
1.0
1.5
-
4.0
-
Monkey
Monkey
1465.0
945.0
2.0 3026.0
3.1 1267.0
0.33
-
PO
5.0
3.0
42.0
a Mean PK Parameters (n=3 or 2)
b Vehicle : PEG400
Table 7. Metabolic stability in hepatocytes
RP-13s
Species
Rat
Parent cpd depletion: t1/2 (min) a
17 ± 15
21 ± 10
Dog
Monkey
Human
52 ± 24
125 ± 55
a Independent experiments from 5 to 7 donors
Conclusion
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We have successfully developed and optimized a new valuable NNRTI series, namely APhI, which proved to have
substantial benefits in term of mutant activities, physicochemical properties and pharmacological profile. This work
led to the discovery of RP-13s (IDX899) as a new highly potent second generation NNRTI drug candidate. 19 Further to
this work, RP-13s progressed in phase IIb clinical trial where its development was suspended due to reports of seizures
as part of a clinical trial involving treatment-experienced patients.20
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Experimental Methods
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Synthesis of Aryl Phospho Indoles. The synthesis of (±)-13s only is described here, while the synthesis of all other
compounds is described in the Supporting Information.
All reactions were performed with reagent-grade materials under an atmosphere of nitrogen. Solvents were rea-
gent-grade or better. Evaporation of the solvents was carried out in a rotary evaporator under reduced pressure. Thin
layer chromatography (TLC) was performed on precoated aluminium sheets of silica gel 60 F254 (Merck, Art. 5554),
visualization of products being accomplished by UV absorbance at 254 nm; column chromatography on silica 60 (40-
63μm, Merck 11567). 1H (400 MHz), 13C (100 MHz) and 31P (162 MHz) NMR spectra were recorded on a Brucker AC300
spectrometer using DMSO-d6 or CDCl3 as solvents. NMR chemical shift (δ) are quoted in parts per million (ppm)
referenced to the residual solvent peak [DMSO-d6] set at 2.49 ppm or [CDCl3] set at 7.26 ppm. The accepted abbrevia-
tions are as followed: s, singulet; d, doublet; t, triplet; q, quartet; m, multiplet. Low-resolution LC mass spectra (LR
LCMS) were recorded on a WATERS unit [Alliance 2695, Photodiode Array detector 2996, ZQ 2000 (ESCI source),
Mass Lynx 4.1] using a reverse phase analytical column Synergy 4 μm Fusion 80A 50x2.0 mm (Phenomenex 100B-
4424-BO). The compound to be analyzed was eluted using a linear gradient of 5% -95% acetonitrile in water with 0.05
% formic acid programmed over a 8 or 20 minutes period with a flow rate of 0.4 mL/min. Purity of all compounds was
determined to be >95% by analytical HPLC using a WATERS unit [Alliance 2695, Photodiode Array detector 2996]
using a reverse phase analytical column Waters Novapack C18 4ꢀm 150x3.9 mm. The compound to be analyzed was
eluted using a linear gradient of 5–95% acetonitrile over a 20 min period with a flow rate of 1 mL/min and the chro-
matogram was recorded using an UV detection from 210 to 400 nm (PDA Max Plot). Microwave assisted chemistry
was performed with a CEM discover apparatus.
Ethyl 1-(benzenesulfonyl)-5-chloro-3-[(3-iodo-5-methyl-phenyl)-methoxy-phosphoryl] indole-2-carboxylate, (±)-
10s: n-BuLi (2.5M in hexane, 2.17 mL, 5.42 mmol) was added dropwise to a stirred and cooled (to about -90 °C) solu-
tion of bromoindole 7 (2.0 g, 4.52 mmol) in anhydrous THF (45 mL) under nitrogen. After keeping the solution at
about -90 °C for about 5 min, an appropriate chorophosphosphorus reagent 8s (R2=3-I,5-Me) (1.87 g, 5.42 mmol) was
added dropwise to the solution at the same temperature. The reaction was allowed to warm up slowly to about -40°C
(TLC monitoring, eluent CH2Cl2/EtOAc 9/1). Water (50 mL) was added and the reaction mixture was extracted with
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EtOAc (3x 50 mL), dried and evaporated. The crude oil was purified by chromatography on silica gel to give (±)-10s
(1.75 g, 49%) as a yellowish solid; 1H NMR (DMSO-d6, 300 MHz) δ (ppm) 1.40 (t, J = 7.12 Hz, 3H), 2.31 (s, 3H), 3.70 (d, J
= 11.59 Hz, 3H), 4.46 (q, J = 7.12 Hz, 2H), 7.55 (dd, J = 2.13 Hz and J = 9.01 Hz, 1H), 7.61-7.72 (m, 3H), 7.79-7.91 (m, 4H),
8.06-8.12 (m, 3H); 31P NMR (DMSO-d6, 121.49 MHz) δ (ppm) 22.09; MS (ESI) m/z = 658.4 (MH+).
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5-chloro-3-[[3-[(E)-2-cyanovinyl]-5-methyl-phenyl]-methoxy-phosphoryl]-1H-indole-2-carboxamide (±)-13s: In a
microwave tube, intermediate (±)-10s (0.467 g, 0.71 mmol) and acrylonitrile (17.75 mmol) in DMF (4.5 mL) were
stirred at room temperature. This mixture was flushed few minutes with nitrogen. Tl(OAc) (0.187 g, 0.71 mmol), tri-o-
tolylphosphine (0.086 g, 0.284 mmol) and Pd(OAc)2 (0.037 g, 0.142 mmol) were added. The reaction mixture was
heated under microwave irradiations under pressure at 110 °C (maximum power input 200 W) during 45 min. After
concentration, the reaction mixture was purified by preparative HPLC to give the expected N-deprotected
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intermediate. H NMR (DMSO-d6, 300 MHz) δ (ppm) 1.11 (t, J = 7.07 Hz, 3H), 2.34 (s, 3H), 3.65 (d, J = 11.48 Hz, 3H),
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4.14-4.25 (m, 2H), 6.5 (d, J = 16.69 Hz, 1H), 7.38-7.42 (m, 1H), 7.58-7.77 (m, 5H), 8.35-8.36 (m, 1H), 12.98 (brs, 1H); P
NMR (DMSO-d6, 121.49 MHz) δ (ppm) 28.69; MS (ESI) m/z = 443.4 (MH+). This compound was dissolved in methanol
(10 mL) in a pressure tube and was stirred and cooled to 0°C. The solution was saturated with NH3 gas for about 10
minutes. The mixture was stirred at about 50° C overnight, and after TLC monitoring, excess ammonia and methanol
were evaporated in vacuo. The crude residue was purified by chromatography on silica gel column to give the
expected carboxamide (±)-13s (0.164 g, 56%) as a white solid; 1H NMR (CDCl3, 300 MHz) δ (ppm) 2.36 (s, 3H), 3.85 (d,
J = 11.7 Hz, 3H), 5.87 (d, J = 16.72 Hz, 1H), 6.20 (brs, 1H), 7.29-7.36 (m, 2H), 7.39 (brs, 1H), 7.53-7.66 (m, 4H), 10.97 (brs,
1H), 11.13 (brs, 1H); 31P NMR (CDCl3, 121.49 MHz) δ (ppm) 31.73; MS (ESI) m/z = 414 (MH+).
HPLC chiral separation. Enantiomers of (±)-13s were separated by Supercritical Fluid Chromatography preparative
method at Chiral Technologies. Preparative column: Chiralpak AD-H (250x20 mm) with CO2/MeOH +1% diethyla-
mine 80/20 as the mobile phase, flow rate 60 mL/min. Analytical column: Chiralcel OD-H (250x4.6 mm) with n-
heptane/ethanol/diethylamine 70/30/0.1 as the mobile phase, flow rate 1.0mL/min. First eluting enantiomer RP-13s,
tR=4.9 min, second eluting enantiomer SP-13s, tR=16.9 min, orders of elution refer to those observed on the preparative
column.
HIV-1 infected cells inhibition experiments. The antiviral activity of compounds was measured by the inhibition of
virus-induced cytopathogenicity in MT-4 cells using the HIV strain BH10 wild-type or the resistant viruses Y181C,
K103N and Y181C/K103N as described in Hamann, M., Pierra, C., Sommadossi, J.P., Musiu, C., Vargiu, L., Liuzzi, M.,
Storer, R., and Gosselin, G. Bioorg. Med. Chem. 2009, 17, 2321-2326.
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Pharmacokinetic profile of RP-13s in rats, dogs and monkeys. Fed male Cynomolgus monkey, fed Beagle dogs and
fasted Sprague Dawley rats were used. Dogs and monkeys were dosed at 1mg/kg intraveinous (IV) and 5 mg/kg orally
(PO), rats at 5 mg/kg IV and 10 mg/kg PO, in a formulation of 10% dimethylacetamide/ 10% ethanol/ 80% PEG400.
After dose administration, blood samples were collected from each animal predose and at 0.083, 0.25, 0.5, 1, 2, 3, 4, 8,
12 and 24 hours postdose via a femoral vein and transferred into tubes containing lithium-heparin anticoagulant.
Plasma was spearated by centrifugation and stored at -70 °C until analysis. Concentration of unlabeled IDX899 within
plasma samples were identified through high performance liquid chromatographics (HPLC) methods that incorpo-
rated tandem mass spectrometric detection (LC-MS/MS).
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Supporting information. Synthetic procedure and characterization data of all compounds. This material is available free of
charge via the internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* To whom correspondence should be addressed. Phone: +33(0)499522252; fax: +33(0)499522250. E-mail: francois-
rene.alexandre@merck.com
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Sareum Ltd (Cambridge, UK) Dr. P.M. Leonard, Dr. M. Fisher, Dr. M.B.AC. Lamers and Dr. J. Reader for X-ray
structure determination of K103N/Y181C Mutant HIV-1 Reverse Transcriptase (RT) in Complex with the ligand (RP-13s).
ABBREVIATIONS
APhI, aryl-phospho-indole; DM, double mutant; EFV, efavirenz; ETV, etravirine; HAART, highly active antiretroviral thera-
py; HIV-1, human immunodeficiency virus type 1; NNIBP, nonnucleoside inhibitor-binding pocket; NNRTI, non-nucleoside
reverse transcriptase inhibitor; NVP, nevirapine; PK, pharmacokinetic; RPV rilpivirine, RT, reverse transcriptase; WT, wild
type.
Accession Codes
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Crystal structure of K103N/Y181C Mutant HIV-1 Reverse Transcriptase (RT) in Complex with RP-13s (IDX899) has been de-
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posited in the RCSB Protein Data Bank under the PDB ID code 5FDL.
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RP-13s (IDX899) was evaluated in phase 2 clinical trial and was licensed to GlaxoSmithKline as of February 2009 and its name
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