Difluoromethylbenzoxazole pyrimidine thioether derivatives: A novel class of potent non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm200766b

This paper reports the synthesis and antiviral properties of new difluoromethylbenzoxazole (DFMB) pyrimidine thioether derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors. By use of a combination of structural biology study and traditional medicinal chemistry, several members of this novel class were synthesized using a single electron transfer chain process (radical nucleophilic substitution, S_RN1) and were found to be potent against wild-type HIV-1 reverse transcriptase, with low cytotoxicity but with moderate activity against drug-resistant strains. The most promising compound 24 showed a significant EC₅₀ value close to 6.4 nM against HIV-1 IIIB, a moderate EC₅₀ value close to 54 μM against an NNRTI resistant double mutant (K103N + Y181C), but an excellent selectivity index >15477 (CC₅₀ > 100 μM).

Full text of DOI:10.1021/jm200766b

Article
pubs.acs.org/jmc
Difluoromethylbenzoxazole Pyrimidine Thioether Derivatives: A
Novel Class of Potent Non-Nucleoside HIV-1 Reverse Transcriptase
Inhibitors†
Jeremie Boyer,‡ Eric Arnoult,§ Maurice Medebielle,*,‡ Jerome Guillemont,*,§ Johan Unge,*,∥
́
́
́
́
̂
and Dirk Jochmans⊥,#
‡Universite
UMR CNRS-UCBL-INSA, Equipe “Synthes
11 Novembre 1918, F-69622 Villeurbanne, France
́
de Lyon, Universite
́
Claude Bernard Lyon 1, Institut de Chimie et Biochimie Molec
́
ulaires et Supramolec
́
ulaires (ICBMS),
̀
e de Molecules d’Interet Therapeutique (SMITH)”, Bat
́
́
̂
́
̂
iment Curien, 43 Boulevard du
§Chemistry Lead Antimicrobial Research, Janssen-Cilag/Tibotec, Campus de Maigremont BP615, F-27106 Val de Reuil Cedex,
France
∥MAX-lab, Lund University, P.O. Box 118, SE-221 00 Lund, Sweden
⊥Tibotec-Virco BVBA, A Division of Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, BE-2340 Beerse,
Belgium
S
* Supporting Information
ABSTRACT: This paper reports the synthesis and antiviral
properties of new difluoromethylbenzoxazole (DFMB) pyrimidine
thioether derivatives as non-nucleoside HIV-1 reverse transcriptase
inhibitors. By use of a combination of structural biology study and
traditional medicinal chemistry, several members of this novel class
were synthesized using a single electron transfer chain process
(radical nucleophilic substitution, SRN1) and were found to be
potent against wild-type HIV-1 reverse transcriptase, with low
cytotoxicity but with moderate activity against drug-resistant strains. The most promising compound 24 showed a significant
EC50 value close to 6.4 nM against HIV-1 IIIB, a moderate EC50 value close to 54 μM against an NNRTI resistant double mutant
(K103N + Y181C), but an excellent selectivity index >15477 (CC50 > 100 μM).
dines 4 (RDEA-640) and 5 (RDEA-427, preclinical),4 have
been recently disclosed and displayed similar or better potency
than etravirine (Figure SI-1 of Supporting Information).
Additional structures have been disclosed from major
INTRODUCTION
■
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
have been shown to be a key component of highly active
antiretroviral therapy (HAART). The use of NNRTIs has
become part of standard combination antiviral therapies
producing very effective drugs with clinical efficacy comparable
to that of other antiviral regimens. There is, however, a critical
issue with the emergence of drug-resistant HIV-1 mutants, and
a need has arisen for novel NNRTIs with improved activity
pharmaceutical companies, and some of them are already in
clinical trials such as trisubstituted thioacetanilinide triazole 6
(RDEA-806, phase II),4 arylphosphoindole derivative 7 (IDX-
899/GSK-2248761, phase II),5 diaryl ether/pyrazolo[3,4-b]-
pyridine 8 (MK-4965, phase I) (Figure SI-1 of Supporting
Information).6 Compound 9 (L-696,229)7 and some analogues
profiles. There are currently three commercially available first
generation NNRTIs: efavirenz, nevirapine, and delavirdine
(Figure 1).1
(10 and 11) are potent NNRTIs (Figure SI-2 of Supporting
Information) and were discovered in a screening program at
Merck. Although their clinical development was suspended
because of the emergence of resistance, the pyridine-2(1H)-
ones remained an attractive pharmacophore and various
synthetic programs were initiated in order to discover new
molecules that could circumvent the resistance of the Merck
pyridinones.8,9 Despite that a number of molecules have been
shown to be very potent at nanomolar concentrations against
clinically relevant mutant strains, none of them have been
pushed forward to clinical trials.
Recently, the second generation NNRTI etravirine (TMC-
125, Figure 1), a diarylpyrimidine (DAPY) derivative, has been
approved by the FDA and has demonstrated activity toward a
number of clinically observed mutations.2 Further research to
develop an even better DAPY derivative has led to the
discovery of an advanced NNRTI, rilpivirine (TMC-278, Figure
1), which has been approved by the FDA in February 2011 and
showed better potency and pharmacological profiles than
etravirine.2
Quite close analogues of the DAPY family, such as
Received: June 14, 2011
Published: October 21, 2011
diarylpyridine 1,3 diarylanilines 2 and 3,3 and pyrrolopyrimi-
© 2011 American Chemical Society
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reductive cleavage of 2-(bromodifluoromethyl)benzoxazole
and bromodifluoromethyloxadiazole derivatives with subse-
quent coupling reactions of the corresponding stable
difluoromethyl anions with commercially available heteroalde-
hyde and ketones. Unfortunately the corresponding carbinols
thus obtained were devoid of any activity when tested against
HIV-1 virus (IC50 > 50 μM; Figure 2). At that time, it was
postulated that the hydroxyl group may block efficient binding
to the enzyme; therefore, in a subsequent work the hydroxyl of
such carbinols was transformed into other functionalities via
straightforward synthetic sequences.12 However these new
molecules bearing −CF2CH(OR)− (R = Me, COCH3, COAr),
−CF2CHF−, −CF2CO−, −CF2CF2−, and −CF2CH2− linkers
were again devoid of significant activity (IC50 > 50 μM; Figure
2).12 However, with a heteroatom (Y = S, O) replacing one of
the −CH2, a number of molecules that were obtained using the
18
S
1
RN
coupling methodology of 2-(bromodifluoromethyl)-
benzoxazole with commercially available heterocyclic thiols and
phenolic compounds were found to be moderately to quite
active (Figure 3).12 In this last series, the two most active
compounds 12 and 13 tested at the NIH were found to be
quite potent with 12 being the most active (EC50 = 64.6 nM)
against HIV-1 IIIB. It was also found that the pyrimidine
thioether 14 was 10 times more active than its nonfluorinated
analogue 15 (Figure 3). Although 12 was quite active, it was
inactive against a number of mutant strains of HIV. Despite the
fact that these molecules were only active against HIV-1 wild-
type, a new class of potent anti-HIV-1 agents were thus
obtained in only two steps using commercially available
reagents (aldehydes, thiols, and phenols). However, there was
still some unclear reasons why the carbinols and their
chemically derived compounds were totally inactive compare
to the thioethers and also to a lesser extent the ether
derivatives. Worth mentioning was that at the time we started
the project in 1997−1998, there were two relevant papers from
Pharmacia & Upjohn Company19,20 including two patents21,22
describing promising results with pyrimidine thioether
derivatives; however, for unknown reasons these series were
not pursued.
Figure 1. Currently marketed NNRTIs and the most advanced
NNRTI TMC-278.
Some years ago, a research program directed to the
development of efficient and mild approaches to prepare
difluoromethylene substituted aromatics and heterocycles of
biological interest was launched. As part of these investigations,
we envisaged selectively incorporating fluorine substituents into
structural analogues of 9 in order to determine the impact of
such substitution on biological activity.10−12 Dramatic enhance-
ments of activity have been reported in many cases for partially
fluorinated analogues of biologically active compounds;13
fluorinated substituted aromatics and heterocycles have found
broad applications in agrochemicals and anticancer and antiviral
agents. The similarity in size makes the fluorine an obvious
candidate to replace hydrogen, often without significant
disturbance of the molecular geometry and shape. In addition
it is now recognized that fluorine substitution can reduce or
block metabolic pathways selectively, affect substantially the
pKa of a molecule to modify the binding and the
pharmacokinetic properties of pharmaceutical agents and
change also the lipophilicity of a drug candidate, to cite a
few.14−16 In 1998 the first series of simpler analogues of 9
where the −CH2CH2− spacer was replaced with a
−CF2CHOH−10 were prepared using a novel methodology
based on the tetrakis(dimethylamino)ethylene (TDAE)17
In a collaborative project with Tibotec, it was then decided to
re-evaluate the biological activity of 12 and 13, to test them
against clinically relevant double mutant strain (K103N +
Y181C), and to improve the overall antiviral profile of the hit
12 using structural biology and medicinal chemistry. The
Figure 2. Benzoxazole derivatives with a CF2 group.
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Figure 3. Biologically active benzoxazoles with a −CF2S− spacer.
medicinal chemistry effort concentrated on structural mod-
ifications on the western wing (benzoxazole) and eastern wing
(pyrimidine), keeping the −CF2S− linker intact.
RESULTS
■
By use of standard antiviral assays, it was found that 12 and 13
were indeed potent HIV-1 inhibitors with similar activities as
previously reported by NIH (Figure 4). Compound 12, with an
Figure 4. Biological data and physicochemical properties of the hit 12
and its analogue 13.
EC50 equal to 111 nM against HIV-1 IIIB and with low
cytotoxicity (CC50 > 100 μM, SI > 879), remained the most
active compound but with still a poor anti-HIV activity (EC50
≈
67 μM) against the double mutant (K103N + Y181C).
Moreover, compound 12 displayed very good physicochemical
properties (Figure 4) but also an excellent permeability in the
Caco-2 cell-based assay (Papp = 34.6 × 10−6 cm s−1), a good
stability in human liver microsomes (90% recovery after 15
min), and no issue for cytochrome P450 inhibition (<30%
inhibition for CYP3A4, CYP2C9, CYP2D6). These encourag-
ing data incited us to continue exploration of the aryl moieties.
Structural Biology. Cocrystal structures could provide
information that could help in the design of inhibitors. To
support this process, two HIV-1 reverse transcriptase mutants,
Phe227Cys and Phe227Leu, were cocrystallized with com-
pound 12 and the structures were solved at a resolution of 2.75
and 2.1 Å, respectively, and with R/Rfree of 26/33 and 25/29,
respectively (see Table 1 in Supporting Information). F227C/
E478Q and F227L/E7478Q double mutants were chosen, since
better (big) crystals were usually produced, and in addition the
E478Q mutation induces reduction of flexibility by local
reduction of negative charges in the active site of RNasH.
The key interactions of compound 12 with the non-
nucleoside inhibitor binding pocket (NNIBP) of HIV-1 RT
mutants Phe227Cys and Phe227Leu are shown in Figure 5.
The distances between the compound and the mutated residue
are 5.2 Å (Leu) and 5 Å (Cys). In both structures, it is obvious
that the mutated residues Phe227Leu and Phe227Cys are too
Figure 5. NNRTI binding pocket of (a) F227L (PDB access number
2ykn) and (b) F227C (PDB access number 2ykm) HIV reverse
transcriptase bound with compound 12. The inhibitor is shown in
capped sticks. The mutated amino acids F227L and F227C are
highlighted in magenta. The crystallized water molecule, located in this
area, is shown as a red sphere. For clarity, only the first layer amino
acids surrounding the inhibitor are shown (at a distance of 5 Å). The
hydrogen atoms are not shown. The structures were created with
Benchware 3D Explorer on a Windows workstation (Tripos
Associates, St. Louis, MO, U.S.).
far from the ligand for any direct interaction. It is unlikely that
the binding of the 12 is affected by any of these mutations.
The compound, which adopts a typical butterfly-like
conformation frequently found in most of the NNRTIs,23
was easily interpreted in the structures. The position of the
benzoxazole group of compound 12 is stabilized by π-stacking
interactions with aromatic side chains of Tyr181 at the shortest
distance of 3.6 Å and of Tyr188 at a distance of 3.8 Å. The
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continuous electron density between the benzoxazole and this
Tyr188 side chain reinforces this assumption. We also found an
edge−face π-systems interaction to the benzene ring of Trp229,
with a closest distance of 3.8 Å between the closest atoms
(Figure SI-3 of Supporting Information). The pyrimidine ring
makes another edge−face interaction to Tyr318 in the eastern
wing, 3.6 Å apart.
These ring interactions are further stabilized by the snug fit
into the binding groove of the protein. Concerning the linker,
the fluorine atoms rest on the interface of two NNIBP β
strands. The sulfur atom is situated in a well-formed
hydrophobic pocket, constituted by the hydrophobic parts of
residues Leu100, Val179, Gly190, and Lys103 at typical van Der
Waals distances. The shape of the fluorine and the sulfur
pockets determine the orientation of the compound, and larger
structures in the linker region would not fit into these
hydrophobic pockets. In the case of other NNRTIs, rilpivirine,
for instance,24 whose linker region contains a ring structure, the
ligand is rotated, leaving the linker region outside these pockets
in order to accommodate the extra atoms (Figure 6).
The role of the fluorine atom for the enhanced activity
compared to hydrogen atoms in the linker merits some
comments. Fluorine is known to have a larger van der Waals
radius, and it may be speculated that the fluorine atoms prevent
the ligand from moving too deep into the pocket, thereby
maintaining a better position for the π-stacking bonds of the
benzoxazole group. One can also think that the fluorine atom
may interact favorably with backbone and side chain amide
groups in the enzyme pocket as previously observed in some
other studies;16 however, it is not obvious from our
crystallographic data that such interactions do occur really
with 12. In addition to the fluorine effect, a critical key to
observe any inhibitory activity is the presence of a sulfur atom,
since the molecules having a −CF2− adjacent to a carbon are
totally inactive. A cocrystallized water molecule was found in
both structures, at the same place, between one of the nitrogen
of the pyrimidine and the sulfur atom (compound 12), the
backbone of Lys101, and the side chain of Lys101/103, offering
to this water molecule plenty of H-bonds network. A
comparison of rilpivirine25 and 12 cocrystal structures (Figure
6) shows a pretty good superimposition of this water molecule
and the nitrogen of the pyrimidine in rilpivirine, showing the
same H-bonds interactions. The H-bonds network is believed
to be an important additional contributing factor for RT
inhibitors binding affinity.
Medicinal Chemistry. On the basis of structural biology
data, a first series of compounds 17−28 were designed (Figure
7). These new molecules modified on either the benzoxazole or
pyrimidine rings were developed with the aim to improve in the
first instance the activity against the wild type and then on the
double mutant. Basically we wanted to explore the impact on
the antiviral activity when replacing the methyl moiety on the
pyrimidine ring of 12 with larger substitutions such as
cyclopropyl, ethyl, and isopropyl and see if sterically more
demanding substitutions will be tolerated. Structural mod-
ifications (methyl, chlorine) on the benzoxazole ring were also
envisaged to determine if such moieties will induce better π-
stacking with both Tyr188 and Tyr181, as well as more
favorable interactions with Tyr318 and Trp229. From the
cocrystallized structures of 12, there appear to be enough space
to introduce such modifications that maybe well tolerated. On
the pyrimidine ring additional substitutions (NH2, OH,
CH2OMe) were also thought in order to find out if they will
Figure 6. Comparison of binding mode of (a) compound 12 and (b)
TMC278 (PDB accession number 3MEE). (b) The TMC278 X-ray
structure was superimposed onto the NNRTI binding site of
compound 12. The water molecule that crystallizes in the presence
of compound 12 is in close contact with the aminopyrimidine of
TMC278, probably displaced by the nitrogen atom in the TMC278-
RT binding site.
create more proper hydrogen bonds with key amino acids
Lys101 and Lys103 that are missing to some extent with hit 12.
Such typical variations have been already well studied with
other NNRTIs and particularly TMC278 and have been proven
to be beneficial for improving the activity against wild type and
mutants. In addition introducing flexibility in the binding mode
of the NNRTI to accommodate the “wiggle and jiggle”
hypothesis will be desirable, however, these latter modifications
are far more difficult to introduce by design and perhaps only
feasible by a trial and error approach.25
All the compounds are obtained from the coupling of a
suitable thiopyrimidine anion (generated in situ with sodium
hydride in anhydrous N,N-dimethylformamide) in the presence
of the corresponding 2-(bromodifluoromethyl)benzoxazole,
following the SRN1 mechanism.11 The starting 2-
(bromodifluoromethyl)benzoxazoles 29−33 are prepared
from the cyclization of the corresponding amides 34−37 in
the presence of polyphosphoric acid under heating following
our published procedure (Scheme 1).26
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Figure 7. New designed compounds based on structural biology.
a
Scheme 1
a
(a) Claycop = Cu(NO3)2 + montmorillonite K10 clay for 42, 48%. (b) Zn + AcOH/MeOH: 38, 64%; 39, 81%. (c) BrCF2CO2Et + Et3N/EtOAc
under reflux: 34, 76%; 35, 72%; 36, 22%; 37, 78%. (d) PPA at 150 °C, then aqueous NH3,:29, 45%; 30, 43%; 31, 37%; 32, 80%. (e) Pivaloyl chloride
+ Et3N/CH2Cl2: 46, >95%. (f) n-BuLi (2.4 equiv) + C2Cl6/THF: 47, 72%. (g) HBr at 120 °C for 40, 92%.
a
Scheme 2
a
(a) Neat, reflux or neat, 110 °C (sealed tube): 52, 66%; 53; 39%; 54, 50%; 55, 45%. (b) MeONa/EtOH, 24 h: 48, 47%; 49; 10%; 50, 38%; 51,
38%.
The amides 34−37 are prepared from key amino phenols
38−41 (41 is commercially available) that are obtained (a) in
one step for 39 from the zinc reduction in acetic acid and
MeOH of commercially available 43, (b) in two steps for 38
through nitration of 44 using copper(II) nitrate supported on
Montmorillonite clay K1027 and further reduction of 42 with
zinc in acetic acid and MeOH, and (c) in three steps for 40
from known 4628 using an ortho-lithiation strategy and final
one-pot deprotection of the methoxy group and carbamate of
47 in HBr at 120 °C. Compound 47 is known from a patent,29
but we have improved its yield (72% vs 41% in the patent)
using 2.4 equiv of n-BuLi in THF without TMEDA (instead of
1 equiv of n-BuLi in Et2O in the presence of TMEDA). The
amides thus obtained by reaction with ethyl bromodifluoro-
acetate in refluxing ethyl acetate in the presence of triethyl-
amine, are further cyclized into the corresponding benzoxazoles
using polyphosphoric acid (PPA) at 150 °C with careful
workup with aqueous NH3, which prevents the conversion of
the benzoxazoles back to the amides, by acid-catalyzed
hydrolysis. The benzoxazoles are usually isolated by silica gel
chromatography or can be distilled (larger scale).
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Thiopyrimidines 48,30 49,31 50, and 51 are prepared from
the condensation of thiourea and suitable enamino ketones
52,32 53,33 54,34 and 5535 that can be prepared from N,N-
dimethylformamidedimethyl acetal 56 and appropriate com-
mercially available ketones 57−60 (Scheme 2). Most of the
enaminoketones were obtained in moderate yields after silica
gel chromatography, since the conversion of the starting
ketones is not complete, with also the formation of
regioisomeric enaminoketones. Classical thiopyrimidine ring
formation was done using thiourea under basic conditions using
MeONa as the base (excess) in refluxing absolute ethanol as
solvent. The final products were usually obtained after silica gel
chromatography (to remove unreacted thiourea) and purifica-
tion of the resulting solid from precipitation with MeOH or
CH2Cl2/pentane mixture (to remove unreacted enaminoke-
tone). Surprisingly the thiopyrimidine 49 was only obtained in
a 10% isolated yield. 4-Amino-2-thiopyrimidine 61 that is
required to prepare targets 21, 22, and 26 is commercially
available as well as 2-thiouracil 62 to prepare target 27.
a
Scheme 4
a
(a) mCPBA (8 equiv), CH2Cl2, 0 °C to room temp, 15 h: 63, 46%.
(b) Ac2O, pyridine, DMAP cat., 55 °C, 7 h: 64, 35%.
Table 2. Data of Compounds 12, 13, 17−28, 63, and 64
K103N + Y181C
a
b
c
compd IIIB EC50 (μM)
CC50 (μM)
SI
EC50 (μM)
12
13
17
18
19
20
21
22
23
24
25
26
28
63
64
0.11
0.95
0.20
0.14
0.17
0.098
0.83
>28
>100.0
>98.4
>98.4
>98.4
>98.4
>98.4
20.04
28.83
77.48
>98.0
>98.36
33.45
>98.4
61.0
>800
>100
>490
>700
>570
67.0
77.2
55.4
60.0
>98.4
79.0
With all the reactants in hand, the SRN1 reactions of 29−33
with the appropriate thiopyrimidines 48−51 and 61−62 were
run in anhydrous DMF at 60−100 °C. The reactions were
usually monitored by TLC and 19F NMR until almost complete
conversion of the starting 2-(bromodifuoromethyl)benzoxazole
(Scheme 3). Despite many attempts, the coupling product 27
>1000
24
>20
<1.0
>28
0.16
0.0064
0.13
0.26
0.66
7.9
484.2
>15000
>774.0
129.1
>141.9
8
67.93
57.0
>98.4
>33
a
Scheme 3
>98.4
>61
43.34
>98.4
>2.3
>98.36
a
Values are the mean determined from at least two experiments.
b
Effective concentration for 50% inhibition. Concentration for 50%
cytotoxicity. Selective index SI = CC50/EC50.
c
The antiviral activity of most compounds is similar to the
activity of compound 12. A modification at position 4 or 7 of
the benzoxazole ring, keeping the methyl substitution on the
pyrimidine ring, does not influence antiviral activity (17, 18, 19,
and 20). More variations in antiviral activity are observed when
the Me is replaced by another substituent. Replacing this by an
NH2 moiety slightly decreases activity (26) with concomitant
increase of the cytotoxicity. And combining this substitution
(NH2) with a modification on the benzoxazole ring further
reduces activity (21 and 22). Substituting the methyl for a
CH2OMe or NHCOCH3 also results in a reduced antiviral
activity (28, 64). Introduction of a cyclopropyl (23) or
isopropyl (25) moiety were well tolerated indicating that steric
effects may not be an issue. Sulfone derivative (63) did also
reduce the activity. One modification that results in a very
considerable increase of activity is the replacement of the
methyl with an ethyl moiety (24). This replacement increases
the potency by a factor of 17, reaching an EC50 in the low
nanomolar range. Compared to the cocrystallized compound
12, compound 24 is decorated with an ethyl group (same
position) that points in the direction of a hydrophobic pocket
(F227 in the WT, V106, L234, P236). This hydrophobic pocket
seems to be large enough to accept more than a methyl group.
The ethyl group may then create more favorable van der Waals
interaction than the methyl.
a
(a) NaH (2.5−3.5 equiv), DMF, 60−100 °C, 15−18 h: 17, 43%; 18;
56%; 19, 80%; 20, 47%; 21, 24%; 22; 54%; 23, 75%; 24, 10%; 25,
35%; 26; 80%; 27, 0%; 28, 50%.
from the reaction of 33 with 2-thiouracil 62 was unsuccessful
even at temperature close to 100 °C. Surprisingly the SRN1
reaction product 24 was only obtained in 10%, since the
starting thiol 49 was contaminated with some thiourea and
therefore required careful purification.
Two additional compounds 63 and 64 were also synthesized
from hit 12 and coupling product 26 (Scheme 4). The sulfone
63 was obtained in a moderate 46% yield after silica gel
chromatography from the mCPBA oxidation (8 equiv) of
sulfanyl compound 12 in dichloromethane, since a mixture of
sulfoxide and unidentified fluorinated impurities was always
obtained. Amide compound 64 was obtained in a nonoptimized
35% yield from the acetic anhydride reaction in pyridine in the
presence of a catalytic amount of N,N-dimethylaminopyridine
from coupling product 26.
None of the modifications prepared seem to have an
influence on the reduced sensitivity of the double mutant
(K103N + Y181C). The presence of these mutations reduces
the antiviral activity of all compounds at least by a factor of 30.
Antiviral Activity. All the compounds thus synthesized
were evaluated against HIV-1 IIIB and the double mutant
(K103N + Y181C) NNRTI resistant strains (Table 2).
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delay of 0.1 s. Samples were supplied as 0.5−1 mg/mL in methanol
and/or acetonitrile with 5 μL injected on a partial loop fill. The purity
of the final compounds was determined by HPLC as described above
and is 95% or higher unless specified otherwise. Thin-layer
chromatography (TLC) was performed on silica gel GF254 plates.
Macherey-Nagel silica gel 60M (0.04−0.063 mm) was used for silica
gel chromatography. Solvents for chromatography, workup, and
recrystallization are acetone (AC), ethyl acetate (EA), diethyl ether
(DE), dichloromethane (DCM), petroleum ether (PE), pentane (PT),
hexane (HX), cyclohexane (CY), methanol (ME), absolute ethanol
(ET), and chloroform (CL). Melting points (uncorrected) were
CONCLUSION
■
Dramatic enhancements of activity have been reported in many
cases for partially fluorinated analogues of biologically active
compounds.13 This study is another successful example of the
potential of this concept by applying it to HIV-1 NNRTI
syntheses.
We re-evaluated the antiviral activity of 12 and 13 and tested
them against clinically relevant double mutant strains (K103N
+ Y181C). Our objective was to improve the antiviral activity of
the hit 12 using structural biology and medicinal chemistry.
The medicinal chemistry effort concentrated on structural
modifications on the western wing (benzoxazole) and eastern
wing (pyrimidine), keeping the −CF2S− linker intact.
Our efforts in structural biology resulted in a detailed analysis
of the binding mode of 12 in the NNRTI pocket of HIV-1,
based on two crystal structures obtained by cocrystallization. In
comparison with previously published NNRTI-RT crystal
structures, the binding of this NNRTI in the pocket involves
a H2O molecule. On the basis of these crystal structures, we
synthesized different compounds with the objective of
improving the interactions with the enzyme. Our results were
successful in the sense that the potency on wild-type HIV-1
(IIIB) could be improved from EC50 = 110 nM for 12 to EC50
= 6.4 nM for compound 24. However, none of the compounds
showed significant activity on a relevant double mutant NNRTI
resistant strain.
determined in capillary tubes on a Buchi apparatus.
̈
Typical Procedure for the Preparation of Amino Phenols (38
and 39). 2-Methyl-6-aminophenol (38). To a solution of 307.5
mg (2.01 mmol) of 6-methyl-2-nitrophenol 42 in 40 mL of methanol
and 10 mL of acetic acid is added 799.6 mg (12 mmol) of zinc. This
mixture is stirred for 10 min at room temperature and then filtered.
The filtrate is diluted with 60 mL of water, and the mixture is extracted
by 5 × 80 mL of diethyl ether. The combined organic layers are then
washed with 3 × 100 mL of a saturated NaHCO3 solution, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product is purified on a silica gel column (CY/EA = 60:40). 157.3 mg
(64%) of pure 38 is obtained as a brown viscous oil. 1H NMR
(CDCl3) δ 6.68 (m, 3H), 3.96 (br, 3H), 2.23 (s, 3H).
6-Chloro-2-aminophenol (39). Yield is 81% from 1.02 g (5.76
mmol) of 6-chloro-2-and 2.29 g (35 mmol) of zinc (not activated) to
afford 666.3 mg after purification on a silica gel column (PT/EA =
70:30), brown solid, mp 70 °C. 1H NMR (CDCl3) δ 6.6−6.7 (m, 3H),
5.58 (s, 1H), 3.86 (s, 2H).
2-Amino-3-chlorophenol (40). A suspension of 7.98 g (33.1
mmol) of N-(2-chloro-6-methoxyphenyl)-2,2-dimethylpropionamide
47 in 190 mL of HBr is heated to 120 °C overnight. After cooling to
room temperature, the mixture is diluted with 300 mL of water. Then
3000 mL of a 25% aqueous ammonia solution is added to the solution
(pH > 9). This mixture is extracted with 4 × 250 mL of ethyl acetate.
The combined organic layers are dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product is purified by
precipitation from pentane. An amount of 4.37 g (92%) of pure 39 is
Further efforts are necessary to optimize these molecules
toward activity on NNRTI-resistant HIV; cocrystallization
studies of 24 will be performed in due course in order to
propose a model for its better ability to bind to the wild type.
Additional derivatives are being investigated with other
modifications on the benzoxazole and pyrimidine rings based
on our structural biology and current antiviral data. Molecular
docking studies combined with our current and new structural
biology data will also be planned in order to provide better
rationale and support for our hypotheses. The results when
available will be presented in a future publication.
1
obtained as a gray solid, mp 113 °C. H NMR (CDCl3) δ 6.82 (m,
1H), 6.64 (m, 1H), 6.55 (m, 1H), 4.60 (br s, 3H).
2-Methyl-6-nitrophenol (42). To a suspension of 2.30 g of
claycop in 14 mL of carbon tetrachloride and 7 mL of acetic anhydride
is added 1 mL (9.52 mmol) of 2-methylphenol. This mixture is stirred
5 min at room temperature and then filtered. The filtrate is diluted
with 50 mL of dichloromethane, and the mixture is washed with 3 ×
100 mL of water. The organic layers are dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude product is
purified on a silica gel column (CY/EA = 95:5). An amount of 701.2
EXPERIMENTAL SECTION
Chemistry. Solvents were of the highest purity and anhydrous and
■
were purchased from Acros Organics and Sigma Aldrich. Reagents
were used without further purification. H, 19F, and 13C NMR spectra
1
were recorded with a Bruker Avance 300 or a DRX300 spectrometer
(in acetone-d6, CDCl3, or DMSO-d6) at 300, 282, and 75 MHz,
respectively. Chemical shifts are given in parts per million (ppm)
1
mg (48%) of pure 42 is obtained as a yellowish viscous oil. H NMR
(CDCl3) δ 7.94 (dd, J = 8.4 and 0.9 Hz, 1H), 7.43 (dd, J = 7.2 and 0.9
Hz, 1H), 6.87 (m, 1H), 2.33 (s, 3H).
relative to the residual peak of solvent (δ H = 7.26 for CHCl3, δH
=
2.50 for DMSO, δc = 77.0 for CDCl3 and δc = 39.52 for DMSO-d6) or
CFCl3 (19F). The following abbreviations are used to describe peak
patterns: s (singlet), d (doublet), dd (double doublet), t (triplet), m
(multiplet), q (quadruplet), and br (broad). Coupling constants are
given in hertz. Mass spectra were recorded using a Finnigan MAT 95
(electrospray ionization, chemical ionization mode, or electron impact
mode). The purities of target compounds were ≥95%, measured by
LC/MS, which was performed on a Waters Acquity UPLC system
connected to a Waters Quattro-micro mass spectrometer. Separations
were carried out on Waters Acquity BEH (bridged ethylsiloxane/silica
hybrid) C18 column (1.7 μm, 2.1 mm × 100 mm) with a flow rate of
0.343 mL/min at 40 °C. Two mobile phases (mobile phase A of 95% 7
mM ammonium acetate/5% acetonitrile; mobile phase B of 100%
acetonitrile) were employed to run a gradient condition from 84.2% A
and 15.8% B (hold for 0.49 min) to 10.5% A and 89.5% B in 2.18 min,
hold for 1.94 min, and back to the initial conditions in 0.73 min, hold
for 0.73 min. An injection volume of 2 μL was used. Cone voltage was
20 V for positive and negative ionization mode. Mass spectra were
acquired by scanning from 100 to 1000 in 0.2 s using an interscan
N-(2-Chloro-6-methoxyphenyl)-2,2-dimethylpropionamide
(47). To a solution of 1.016 g (4.91 mmol) of 46 in 10 mL of dry
tetrahydrofuran is slowly added at −10 °C, under nitrogen, 4.7 mL
(11.75 mmol) of a 2.5 M solution of n-BuLi in hexane. This mixture is
allowed to warm to room temperature and stirred for 1 h and 30 min.
This solution is then added dropwise under nitrogen to a cooled (−10
°C) solution of 1.79 g (7.49 mmol) of hexachloroethane in 3.5 mL of
dry tetrahydrofuran. This mixture is stirred 2 h at −10 °C and then
overnight at room temperature. An amount of 50 mL of water is then
added, and that mixture was extracted with 3 × 50 mL of
dichloromethane. The combined organic layers are dried over
Na2SO4, filtered, and concentrated under reduced pressure The
crude product is purified on a silica gel column (HX/EA = 90:10 to
HX/EA = 80:20). An amount of 770.4 mg (65%) of pure 47 is
1
obtained as a pale yellowish viscous oil. H NMR (CDCl3) δ 7.06 (m,
1H), 7.05 (br s, 1H), 6.93 (m, 1H), 6.73 (m, 1H), 3.72 (s, 1H), 1.27
(s, 9H).
Typical Procedure for the Preparation of 2-Bromo-2,2-
difluoro-N-(2-hydroxy-3-aryl)acetamides (34−37). 2-Bromo-
7980
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Journal of Medicinal Chemistry
Article
(s), 108.3 (t, J = 300.7 Hz). HRMS (CI) calculated for [M + H]+
C8H4BrClF2NO 281.9133, found 281.9133.
2,2-difluoro-N-(2-hydroxy-3-methylphenyl)acetamide (34). To
a solution of 117.5 mg (0.96 mmol) of 38 in 6 mL of ethyl acetate are
added 0.13 mL (0.99 mmol) of ethyl bromodifluoroacetate and 0.15
mL (1.07 mmol) of triethylamine. The reaction mixture is heated at
reflux for 5 h. After the mixture is cooled to room temperature, 10 mL
of ethyl acetate is added and the solution is washed with 20 mL of
dilute HCl (one part concentrated HCl to nine parts of water). The
aqueous layer is extracted with 4 × 20 mL of ethyl acetate. The
combined organic layers are dried over Na2SO4 and concentrated
under reduced pressure. The crude product is purified on a silica gel
column (CY/EA = 80:20). An amount of 203.1 mg (76%) of pure 34
2-(Bromodifluoromethyl)-4-chlorobenzoxazole (31). Yield is
37% from 386.6 mg (1.29 mmol) of 36 and 1.24 g of polyphosphoric
acid to afford 134.2 mg of pure 31 after purification on a silica gel
1
column (HX/EA = 90:10), pale orange solid, mp 35 °C. H NMR
(CDCl3) δ 7.56 (m, 1H), 7.45 (m, 2H). 19F NMR (CDCl3) δ −52.4
(s). 13C NMR (CDCl3) δ 156.0 (t, J = 33.0 Hz), 151.0 (s), 137.6 (s),
128.2 (s), 126.7 (s), 126.1 (s), 110.2 (s), 108.3 (t, J = 300.9 Hz).
HRMS (EI) calculated for [M]+ C8H3BrClF2NO 280.9055, found
280.9055.
1
is obtained as a brown oil. H NMR (CDCl3) δ 8.45 (br s, 1H), 7.70
2-(Bromodifluoromethyl)-4-methylbenzoxazole (32). Yield
is 80%, starting from 1.84 g (6.57 mmol) of 37 and 6.3 g of
polyphosphoric acid to afford 698 mg of pure 32 after purification on a
silica gel column (PE/EA = 90:10), colorless liquid. 1H NMR
(CDCl3) δ 7.43 (m, 2H), 7.27 (m, 1H), 2.67 (s, 3H). 19F NMR
(CDCl3) δ −51.6 (s). 13C NMR (CDCl3) δ 154.9 (t, J = 32.3 Hz),
150.3 (s), 139.0 (s), 132.6 (s), 127.4 (s), 126.2 (s), 108.9 (t, J = 300.3
Hz), 108.7 (s), 16.3 (s). HRMS (EI) calculated for [M]+
C9H6BrF2NO 260.9601, found 260.9601.
Typical Procedure for the Preparation of Thiopyrimidines
(50, 51). (4-Isopropyl-2-mercaptopyrimidine (50). To a mixture
of 358.0 mg (2.54 mmol) of (E)-1-(dimethylamino)-4-methylpent-1-
en-3-one 54 and 218.4 mg (2.84 mmol) of thiourea in 10 mL of
absolute ethanol is added 290 mg (5.20 mmol) of sodium methoxide.
The reaction mixture is then refluxed in a sealed tube for 24 h. After
the mixture is cooled to room temperature, the volatiles are removed
under reduced pressure and the crude product is purified on a silica gel
column (DCM/ME = 95:5). The yellow solid thus obtained is
dissolved in a minimum of dichloromethane and precipitated with
pentane. An amount of 149.7 mg (38%) of pure 50 is obtained, yellow
solid, mp 86 °C. 1H NMR (DMSO-d6) δ 13.66 (br s, 1H), 7.98 (d, J =
6.0 Hz, 1H), 6.79 (d, J = 6.0 Hz, 1H), 2.86 (m, 1H), 1.16 (d, J = 6.9
Hz, 6H). 13C NMR (DMSO-d6) δ 181.0 (s), 176.1 (s), 158.2 (s),
107.1 (s), 34.8 (s), 20.7 (s). HRMS (CI) calculated for C7H11N2S [M
+ H]+ 153.0486 found 153.0484.
(d, J = 7.5 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.89 (m, 1H), 5.90 (br s,
1H), 2.30 (s, 3H). 19F NMR (CDCl3) δ −60.6 (s). 13C NMR (CDCl3)
δ 157.9 (t, J = 54.8 Hz), 144.6 (s), 128.4 (s), 125.3 (s), 123.6 (s),
121.1 (s), 119.3 (s), 111.3 (t, J = 314.4 Hz), 15.9 (s). HRMS (CI)
calculated for [M + H]+ C9H9BrF2NO2 279.9785, found 279.9785.
2-Bromo-2,2-difluoro-N-(2-hydroxy-3-chlorophenyl)-
acetamide (35). Yield is 64%, starting from 139.6 mg (0.97 mmol)
of 6-chloro-2-aminophenol 39 and 0.14 mL (1.07 mmol) of ethyl
bromodifluoroacetate to afford 187.4 mg of pure 35 after purification
on a silica gel column (PE/EA = 90:10), red crystals, mp 54 °C
1
(DCM/HX). H NMR (CDCl3) δ 8.39 (s, 1H), 8.17 (m, 1H), 7.20
(m, 1H), 6.95 (m, 1H), 5.97 (s, 1H). 19F NMR (CDCl3) δ −61.1 (s).
13C NMR (CDCl3) δ 157.3 (t, J = 27.8 Hz), 141.2 (s), 125.5 (s), 124.8
(s), 121.6 (s), 119.8 (s), 119.3 (s), 111.2 (t, J = 314.7 Hz). HRMS
(CI) calculated for [M + H]+ C8H6BrClF2NO2 299.9239, found
299.9239.
2-Bromo-2,2-difluoro-N-(2-hydroxy-6-chlorophenyl)-
acetamide (36). Yield is 22% from 4.36 g (30.4 mmol) of 2-amino-
2-chlorophenol 40 and 4.4 mL (33.6 mmol) of ethyl bromodi-
fluoroacetate to afford 2.01 g of pure 36 after purification on a silica
gel column (HX/EA = 80:20), brown crystals, mp 83 °C (EA/PT). 1H
NMR (CDCl3) δ 8.40 (br s, 1H), 7.18 (m, 1H), 7.04 (m, 2H), 5.90
(br s, 1H). 19F NMR (CDCl3) δ −60.7 (s). 13C NMR (CDCl3) δ
159.2 (t, J = 28.2 Hz), 150.9 (s), 129.2 (s), 127.7 (s), 121.8 (s), 120.3
(s), 119.3 (s), 110.6 (t, J = 313.5 Hz). HRMS (CI) calculated for [M
+ H]+ C8H6BrClF2NO2 299.9239, found 299.9239.
2-Bromo-2,2-difluoro-N-(2-hydroxyphenyl)acetamide (37).
Yield is 72% from 2.30 g (179 mmol) of 41 and 2.60 mL (199 mmol)
of ethyl bromodifluoroacetate to afford a brown powder that is
recrystallized (CL/HX) to give 3.89 g of pure 37, brown crystals, mp
90 °C. 1H NMR (CDCl3) δ 7.76 (s, 1H), 7.15 (m, 1H), 6.86 (m, 2H),
6.46 (s, 1H). 19F NMR (CDCl3) δ −60.7 (s). 13C NMR (CDCl3) δ
159.2 (t, J = 27.8 Hz), 150.6 (s), 134.4 (s), 129.0 (s), 122.9 (s), 120.6
(s), 116.1 (s), 111.1 (t, J = 316.1 Hz), 17.7 (s). HRMS (CI) calculated
for [M + H]+ C9H8BrF2NO2 278.9706, found 278.9706.
4-(Methoxymethyl)-2-mercaptopyrimidine (51). Yield is 38%
under reflux for 19 h from 450.5 mg (3.15 mmol) of (E)-4-
(dimethylamino)-1-methoxybut-3-en-2-one 55 and 267.8 mg (3.48
mmol) of thiourea to afford 128.9 mg of pure 51 after purification on a
silica gel column (DCM/ME = 95:5) and precipitation from a
MeOH/pentane mixture, yellow solid, mp 152 °C. 1H NMR (DMSO-
d6) δ 13.74 (br s, 1H), 8.05 (d, J = 6.0 Hz, 1H), 6.81 (d, J = 6.0 Hz,
1H), 4.33 (s, 2H), 3.36 (s, 3H). 13C NMR (DMSO-d6) δ 180.5 (s),
175.4 (s), 134.3 (s), 106.0 (s), 58.5 (s), 30.6 (s). HRMS (CI)
calculated for C6H9N2OS [M + H]+ 157.0436 found 157.0436.
Typical Procedure for the Preparation of 2-[Difluoro-
[(substituted-pyrimidinyl)thio]methyl]-Substituted Benzoxa-
zoles. 2-[Difluoro-[(4-methylpyrimidinyl)thio]methyl]-7-meth-
ylbenzoxazole (17). To a suspension of 57 mg (1.43 mmol) of NaH
(60% in mineral oil) in 2.5 mL of dry DMF is added under nitrogen
117 mg (0.71 mmol) of 4-methyl-2-mercaptopyrimidine. After 10 min
of stirring a solution of 88 mg (0.34 mmol) of 2-(bromodifluor-
omethyl)-7-chlorobenzoxazole 29 in 2.5 mL of dry DMF is added. The
mixture is then heated at 60 °C (oil bath temperature) for 17 h. After
the mixture is cooled to room temperature, 15 mL of water is added
and the mixture is extracted with 5 × 20 mL of CH2Cl2. The combined
organic layers are then washed with 3 × 100 mL of water, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product is purified on a silica gel column (CY/EA = 80:20). An
amount of 44.8 mg (43%) of pure 17 is obtained, brown solid, mp 67
Typical Procedure for the Preparation of 2-(Bromodifluor-
omethyl)-Substituted Benzoxazoles. 2-(Bromodifluorometh-
yl)-7-methylbenzoxazole (29). A mixture of 210.9 mg (0.75
mmol) of 34 and 0.74 g of polyphosphoric acid is heated at 150 °C
for 30 min. Then 9 mL of crushed ice and 2 mL of a 25% aqueous
ammoniac solution are added to the flask. After dissolution of the
solids, the resulting solution is extracted with 5 × 40 mL of CHCl3.
The combined organic layers are dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product is purified on
a silica gel column (CY/EA = 90:10). An amount of 87.7 mg (45%) of
pure 29 is obtained as a colorless liquid. 1H NMR (CDCl3) δ 7.67 (m,
1H), 7.34 (m, 2H), 2.58 (s, 3H). 19F NMR (CDCl3) δ −51.6 (s). 13
C
NMR (CDCl3) δ 155.4 (t, J = 32.1 Hz), 149.9 (s), 139.2 (s), 128.5 (s),
125.8 (s), 122.3 (s), 119.0 (s), 109.0 (t, J = 300.3 Hz), 15.0 (s).
HRMS (CI) calculated for [M + H]+ C9H7BrF2NO 261.9680, found
261.9680.
1
°C (CY/PT/DCM). H NMR (CDCl3) δ 8.23 (d, J = 5.1 Hz, 1H),
7.62 (m, 1H), 7.27 (m, 2H), 6.82 (d, J = 5.1 Hz, 1H), 2.57 (s, 3H),
2.20 (s, 3H). 19F NMR (CDCl3) δ −77.2 (s). 13C NMR (CDCl3) δ
168.2 (s), 166.4 (t, J = 5.7 Hz), 157.5 (t, J = 31.8 Hz), 157.1 (s), 149.8
(s), 140.0 (s), 127.6 (s), 125.2 (s), 122.0 (s), 120.7 (t, J = 271.7 Hz),
118.6 (s), 118.0 (s), 23.7 (s), 15.1 (s). HRMS (CI) calculated for [M
+ H]+ C14H12ClF2N3OS 308.0669, found 308.0669. HPLC purity:
97%
2-(Bromodifluoromethyl)-7-chlorobenzoxazole (30). Yield is
43%, starting from 1.18 g (3.94 mmol) of 35 and 3.77 g of
polyphosphoric acid to afford 479.1 mg of pure 30 after purification on
a silica gel column (PT/EA = 95:5), colorless liquid. 1H NMR
(CDCl3) δ 7.75 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.40 (m,
1H). 19F NMR (CDCl3) δ −52.0 (s). 13C NMR (CDCl3) δ 155.9 (t, J
= 32.9 Hz), 147.2 (s), 140.7 (s), 128.0 (s), 126.6 (s), 120.2 (s), 117.0
7981
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Journal of Medicinal Chemistry
Article
2-[Difluoro-[(4-methylpyrimidinyl)thio]methyl]-7-chloro-
benzoxazole (18). Yield is 56% at 60 °C for 17 h from 210.0 mg
(0.74 mmol) of 2-(bromodifluoromethyl)-7-chlorobenzoxazole 30 and
245 mg (1.53 mmol) of 4-methyl-2-mercaptopyrimidine to afford
136.9 mg of pure 18 after purification on a silica gel column (CY/EA =
80:20), brown solid, mp 66 °C (CY/PT/DCM). 1H NMR (CDCl3) δ
8.21 (d, J = 5.1 Hz, 1H), 7.69 (m, 1H), 7.45 (m, 1H), 7.35 (m, 1H),
6.82 (d, J = 5.1 Hz, 1H), 2.18 (s, 3H). 19F NMR (CDCl3) δ −77.8 (s).
13C NMR (CDCl3) δ 168.2 (s), 166.2 (t, J = 5.9 Hz), 158.4 (t, J = 32.5
(s), 126.9 (s), 125.3 (s), 121.4 (s), 120.3 (t, J = 271 Hz), 116.7 (s),
111.4 (s), 16.7 (s), 11.7 (s). HRMS (CI) calculated for C15H12F2N3OS
[M + H]+ 320.0669, found 320.0668. HPLC purity: 96.4%
2-[Difluoro-[(4-ethylpyrimidinyl)thio]methyl]benzoxazole
(24). Yield is 10% at 70 °C for 15 h from 313 mg (1.27 mmol) of 2-
(bromodifluoromethyl)benzoxazole and 350 mg of crude 4-ethyl-2-
mercaptopyrimidine 48 to afford 39 mg of pure 24 after purification
on a silica gel column (HX/EA = 80:20) and two recrystallizations
1
from CY/PT/DCM, yellowish solid, mp 76 °C. H NMR (CDCl3) δ
Hz), 157.1 (s), 147.1 (s), 141.6 (s), 127.1 (s), 126.0 (s), 120.4 (t, J =
272.0 Hz), 119.8 (s), 118.1 (s), 116.6 (s), 23.6 (s). HRMS (CI)
calculated for [M + H]+ C13H9ClF2N3OS 328.0123, found 328.0123.
HPLC purity: 98.2%
8.25 (d, J = 5.4 Hz, 1H), 7.80 (m, 1H), 7.62 (m, 1H), 7.43 (m, 2H),
6.81 (d, J = 5.1 Hz, 1H), 2.41 (q, J = 7.6 Hz, 2H), 0.94 (t, J = 7.6 Hz,
3H). 19F NMR (CDCl3) δ −77.5 (s). 13C NMR (CDCl3) δ 173.0 (s),
166.4 (t, J = 6 Hz), 157.8 (t, J = 32 Hz), 157.2 (s), 150.5 (s), 140.4 (s),
126.8 (s), 125.3 (s), 124.3 (s), 121.4 (s), 120.6 (t, J = 271 Hz), 116.9
(s), 111.4 (s), 30.5 (s), 12.0 (s). HRMS (CI) calculated for
C14H12F2N3OS [M + H]+ 308.0669, found 308.0669. HPLC purity:
96.8%
2-[Difluoro-[(4-isopropylpyrimidinyl)thio]methyl]-
benzoxazole (25). Yield is 35% at 70 °C for 16 h from 154.0 mg
(0.62 mmol) of 2-(bromodifluoromethyl)benzoxazole and 185.7 mg
(1.21 mmol) of 4-isopropyl-2-mercaptopyrimidine 50 to afford 69.7
mg of pure 25 after purification on a silica gel column (HX/EA =
80:20), white crystals (CY/PT/DCM), mp 77 °C. 1H NMR (CDCl3)
δ 8.29 (d, J = 5.1 Hz, 1H), 7.80 (m, 1H), 7.62 (m, 1H), 7.43 (m, 2H),
6.81 (d, J = 5.1 Hz, 1H), 2.61 (m, 1H), 0.91 (d, J = 6.9 Hz, 6H). 19F
NMR (CDCl3) δ −77.3 (s). 13C NMR (CDCl3) δ 176.8 (s), 166.4 (t, J
= 6 Hz), 157.8 (t, J = 32 Hz), 157.5 (s), 150.6 (s), 140.5 (s), 126.8 (s),
125.3 (s), 121.4 (s), 120.6 (t, J = 271 Hz), 115.6 (s), 111.4 (s), 35.6
(s), 21.0 (s). HRMS (CI) calculated for C15H14F2N3OS [M + H]+
322.0826, found 322.0826. HPLC purity: 96.7%
2-[Difluoro-[(4-aminopyrimidinyl)thio]methyl]benzoxazole
(26). Yield is 80% at 60 °C for 18 h from 157 mg (0.64 mmol) of 2-
(bromodifluoromethyl)benzoxazole and 168 mg (1.28 mmol) of 4-
methyl-2-mercaptopyrimidine to afford 149 mg of pure 26 after
purification on a silica gel column (PE/EA = 60:40), white solid, mp
115 °C (CY/CL). 1H NMR (CDCl3) δ 7.86 (m, 2H), 7.64 (d, J = 7.5
Hz, 1H), 7.44 (m, 2H), 6.06 (d, J = 5.7 Hz, 1H), 4.74 (s, 2H). 19F
NMR (CDCl3) δ −77.0 (s). 13C NMR (DMSO-d6) δ 164.2 (t, J = 5.4
Hz), 163.2 (s), 157.0 (t, J = 31.8 Hz), 155.0 (s), 150.0 (s), 139.7 (s),
127.5 (s), 125.8 (s), 121.2 (s), 120.8 (t, J = 268.3 Hz), 111.9 (s), 103.3
(s). HRMS (EI) calculated for C12H8F2N4OS 295.0465, found
295.0466. HPLC purity: 96.4%
2-[Difluoro-[(4-methylpyrimidinyl)thio]methyl]-4-methyl-
benzoxazole (19). Yield is 80% at 75 °C for 18 h from 197.1 mg
(0.75 mmol) of 2-(bromodifluoromethyl)-4-methylbenzoxazole 32
and 251 mg (1.53 mmol) of 4-methyl-2-mercaptopyrimidine to afford
184.6 mg of pure 19 after purification on a silica gel column (PE/EA =
80:20), white solid, mp 80 °C (CY/DCM). 1H NMR (CDCl3) δ 8.23
(d, J = 5.1 Hz, 1H), 7.42 (m, 1H), 7.33 (m, 1H), 7.19 (m, 1H), 6.82
(d, J = 5.1 Hz, 1H), 2.63 (s, 3H), 2.20 (s, 3H). 19F NMR (CDCl3) δ
−76.8 (s). 13C NMR (CDCl3) δ 168.2 (s), 166.4 (t, J = 5.7 Hz), 157.1
(s), 156.9 (t, J = 32.1 Hz), 150.3 (s), 139.9 (s), 132.1 (s), 126.5 (s),
125.7 (s), 120.7 (t, J = 271.5 Hz), 118.0 (s), 108.5 (s), 23.7 (s), 16.5
(s). HRMS (EI) calculated for C14H12F2N3OS 308.0669, found
308.0670. HPLC purity: 97.5%.
2-[Difluoro-[(4-methylpyrimidinyl)thio]methyl]-4-chloro-
benzoxazole (20). Yield is 47% at 70 °C for 16 h from 2-
(bromodifluoromethyl)-4-chlorobenzoxazole 31 and 177 mg (1.08
mmol) of 4-methyl-2-mercaptopyrimidine to afford 82.0 mg of pure
20 after purification on a silica gel column (HX/EA = 80:20), white
solid, mp 83 °C (CY/PT/DCM). 1H NMR (CDCl3) δ 8.20 (d, J = 5.1
Hz, 1H), 7.53 (dd, J = 1.8 and 7.5 Hz, 1H), 7.39 (m, 2H), 6.82 (d, J =
5.1 Hz, 1H), 2.17 (s, 3H). 19F NMR (CDCl3) δ −77.5 (s). 13C NMR
(CDCl3) δ 168.2 (s), 166.1 (t, J = 6.0 Hz), 158.4 (t, J = 32.6 Hz),
157.1 (s), 151.0 (s), 138.5 (s), 127.2 (s), 126.1 (s), 125.6 (s), 120.4 (t,
J = 271.8 Hz), 118.1 (s), 110.0 (s), 23.7 (s). HRMS (CI) calculated
for [M + H]+ C13H9ClF2N3OS 328.0123, found 328.0123. HPLC
purity: 97.5%
2-[Difluoro-[(4-aminopyrimidinyl)thio]methyl]-4-methyl-
benzoxazole (21). Yield is 24% at 75 °C for 15 h and 30 min from
178.1 mg (0.68 mmol) of 2-(bromodifluoromethyl)-4-methylbenzox-
azole 32 and 180 mg (1.37 mmol) of 4-amino-2-mercaptopyrimidine
to afford 50.2 mg of pure 21 after purification on a silica gel column
2-[Difluoro-[(4-(methoxymethyl)pyrimidinyl)thio]methyl]-
benzoxazole (28). Yield is 50% at 70 °C for 18 h from 84.0 mg
(0.34 mmol) of 2-(bromodifluoromethyl)benzoxazole and 101.8 mg
(1.21 mmol) of 4-(methoxymethyl)-2-mercaptopyrimidine 51 to
afford 55.0 mg of pure 28 after purification on a silica gel column
1
(PE/EA = 60:40), yellow solid, mp 135 °C (CY/DCM). H NMR
(CDCl3) δ 7.84 (d, J = 5.7 Hz, 1H), 7.42 (m, 1H), 7.34 (m, 1H), 7.19
(m, 1H), 6.07 (d, J = 5.7 Hz, 1H), 2.63 (s, 3H), 2.20 (s, 3H). 19F
NMR (CDCl3) δ −76.4 (s). HRMS (ESI) calculated for [M + H]+
C13H11F2N4OS 309.0622, found 309.0622. HPLC purity: 97.4%
2-[Difluoro-[(4-aminopyrimidinyl)thio]methyl]-4-chloroben-
zoxazole (22). Yield is 54% at 70 °C for 17 h from 222.0 mg (0.79
mmol) of 2-(bromodifluoromethyl)-4-chlorobenzoxazole 31 and 206.5
mg (1.57 mmol) of 4-amino-2-mercaptopyrimidine to afford 138.6 mg
of pure 22 after washing the crude product with pentane, methanol,
1
(HX/EA = 80:20), yellowish thick oil. H NMR (CDCl3) δ 8.36 (d, J
= 5.1 Hz, 1H), 7.80 (m, 1H), 7.62 (m, 1H), 7.43 (m, 2H), 7.11 (d, J =
5.1 Hz, 1H), 4.11 (s, 2H), 3.27 (s, 3H). 19F NMR (CDCl3) δ −77.2
(s). 13C NMR (CDCl3) δ 168.8 (s), 166.1 (t, J = 6 Hz), 158.0 (s),
157.7 (t, J = 32 Hz), 150.5 (s), 140.4 (s), 126.9 (s), 125.3 (s), 121.4
(s), 120.6 (t, J = 272 Hz), 115.0 (s), 111.4 (s), 73.4 (s), 58.9 (s).
HRMS (CI) calculated for C14H12F2N3O2S [M + H]+ 324.0618, found
324.0618. HPLC purity: 97.1%
1
and diethyl ether, yellow solid, mp (dec) 243 °C (AC). H NMR
(acetone-d6) δ 7.80 (d, J = 7.5 Hz, 1H), 7.40 (dd, J = 1.5 and 8.1 Hz,
1H), 7.31 (m, 1H), 7.20 (dd, J = 1.5 and 8.1 Hz, 1H), 6.45 (d, J = 7.5
Hz, 1H). 19F NMR (acetone-d6) δ −66.9 (s). HRMS (CI) calculated
for [M + H]+ C12H8ClF2N4OS 329.0075, found 329.0074. HPLC
purity: 97.8%
2-[Difluoro-[(4-cyclopropylpyrimidinyl)thio]methyl]-
benzoxazole (23). Yield is 75% at 70 °C for 15 h from 230.4 mg
(0.93 mmol) of 2-(bromodifluoromethyl)benzoxazole and 283.4 mg
(1.08 mmol) of 4-cyclopropyl-2-mercaptopyrimidine 48 to afford
224.4 mg of pure 23 after purification on a silica gel column (HX/EA
= 80:20), white crystals (CY/PT/DCM), mp 80 °C. 1H NMR
(CDCl3) δ 8.15 (d, J = 5.1 Hz, 1H), 7.81 (m, 1H), 7.62 (m, 1H), 7.43
(m, 2H), 6.84 (d, J = 5.1 Hz, 1H), 1.69 (m, 1H), 0.81 (m, 2H), 0.64
(2H, m). 19F NMR (CDCl3) δ −77.1 (s). 13C NMR (CDCl3) δ 173.4
(s), 166.4 (t, J = 6 Hz), 157.7 (t, J = 32 Hz), 156.1 (s), 150.6 (s), 140.4
2-[Difluoro-(4-methylpyrimidin-2-ylsulfonyl)methyl]-
benzoxazole (63). An amount of 234 mg (0.8 mmol) of 12 was
dissolved in dichloromethane (1 mL), and the resulting pale yellow
solution, cooled to 0 °C, is treated dropwise by a dichloromethane
solution (8 mL) of 1.1 g (64 mmol) of mCPBA (70%). The resulting
yellowish-orange solution is then warmed to room temperature and
stirred for 15 h. The solution is then filtered and the solid washed with
dichloromethane (4 × 2 mL). The combined solutions were
evaporated to dryness. The crude product is then purified on a silica
gel column (PE/EA = 30:70). An amount of 0.12 g (46%) of pure 63
1
is obtained, red crystals, mp 110−111 °C. H NMR (CDCl3) δ 8.70
(d, J = 5.1 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H),
7.50 (m, 3H), 2.60 (s, 3H). 19F NMR (CDCl3) δ −103.7 (s). MS m/z
(%) 674 (29), 673 (2M + 2Na, 100), 348 (M + Na, 58), 326 (M + 1,
7982
dx.doi.org/10.1021/jm200766b|J. Med. Chem. 2011, 54, 7974−7985

Journal of Medicinal Chemistry
Article
11), 168 (12). HRMS (EI) calculated for C12H8F2N4OS 295.0465,
found 295.0466. HPLC purity: 97.3%
(NH4)2SO4, 50 mM Hepes, pH 7.2, 5 mM MgCl2, and 300 mM KCl)
was combined with 5 μL of the protein solution and equilibrated
against the crystallization buffer. The droplets were seeded with
microcrystals. Crystals appeared after 3 days and grew to a size of 0.3
mm × 0.2 mm × 0.2 mm within 1 month. Data were collected at
beamline I911-2 at MAX-lab to 2.9 and 2.1 Å for the F227C, E478Q
and the F227L, E478Q crystals, respectively (Table 1 in Supporting
Information). The two crystal structures were solved with molecular
replacement using AMORE and rigid body refinement using Refmac
with PDB entries as 1IKX template. The final structures including 276
water molecules and one Ca ion were refined with CNS and Refmac to
final Rfree values (Table 1 in Supporting Information). Amino acids
B219-231 and B358-361 could not be traced confidently and were
excluded in both structures. The electron densities at the active site
including compound 12 are very clear and were unambiguously
interpreted early in refinement. The structures were deposited with the
PDB with identity codes 2ykm (F227C, E478Q*12) and 2ykn
(F227L, E478Q*12).
2-[Difluoro-[(4-(acetamidyl)pyrimidinyl)thio]methyl]-
benzoxazole (64). To a solution of 52.8 mg (0.18 mmol) of 2-
[difluoro-[(4-aminopyrimidinyl)thio]methyl]benzoxazole 26 in 0.45
mL of pyridine containing 0.43 mL of acetic anhydride is added a
catalytic amount of DMAP. The reaction mixture is heated to 55 °C
for 7 h. After cooling to room temperature, the mixture is concentrated
and the crude is purified on a silica gel column (HX/EA = 60:40). The
solid thus obtained is washed with pentane and diethyl ether, and after
filtration, 21.2 mg (35%) of pure 64 is obtained, brown solid, mp 183
°C. 1H NMR (CDCl3) δ 8.26 (d, J = 5.7 Hz, 1H), 7.83 (m, 1H), 7.77
(br s, 1H), 7.64 (m, 1H), 7.47 (m, 2H), 2.11 (s, 3H). 19F NMR
(CDCl3) δ −77.1 (s). 13C NMR (DMSO-d6) δ 170.7 (s), 164.1 (t, J =
5 Hz), 159.0 (s), 158.2(s), 156.2 (t, J = 32 Hz), 150.0 (s), 139.5 (s),
127.6 (s), 125.8 (s), 124.2 (s), 120.6 (s), 116.9 (t, J = 271 Hz), 116.9
(s), 107.4 (s), 24.1 (s). HRMS calculated for [M + H]+
C14H11F2N4O2S 337.0572, found 337.0572. HPLC purity: 96.2%
Biology. Antiviral assays and toxicity measurements were
performed as described previously.36
The human T lymphotropic virus type 1 transformed human T
lymphoblastoid cell line MT4 was kindly provided by Naoki
Yamamoto (National Institute of Infectious Diseases, AIDS Research
Center, Tokyo, Japan), maintained in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-
glutamine, 0.1% NaHCO3, and antibiotics (0.02% gentamicin, 0.8%
G418), and incubated in a humidified incubator with a 5% CO2
atmosphere at 37 °C. MT4-LTR-EGFP cells were obtained by
transfecting MT4 cells with a selectable construct encompassing the
sequences coding for the HIV long terminal repeat (LTR) as a
promoter for the expression of enhanced green fluorescent protein
(EGFP) and subsequent selection of permanently transfected cells.
HIV-1 IIIB was provided by Guido van der Groen (Institute of
Tropical Medicine, Antwerp, Belgium).
The double mutant (K103N + Y181C) was generated by site-
directed mutagenesis. Site-directed mutant RT-coding sequence was
generated from a pGEM vector containing the HIV-1 clone HXB2
protease- and RT-coding sequence by using a QuikChange site-
directed mutagenesis kit (Stratagene) and high-performance liquid
chromatography purified primers (Genset Oligos). Plasmid was
sequenced to confirm that it contained the desired mutations. Mutant
virus was created by recombination of the mutant protease-RT
sequence with a protease-RT-deleted HIV-1 HXB2 proviral clone.37
The antiviral activity on HIV-1 (IIIB and the double mutant) was
determined in a cell-based virus replication assay. Here MT4-LTR-
EGFP cells (150 000 cells/mL) are infected (multiplicity of infection
(MOI) of 0.01) in the presence or absence of different inhibitor
concentrations. After 3 days of incubation, the amount of virus
replication is quantified by measuring the EGFP fluorescence and
expressed as the 50% effective concentration (EC50). The toxicity of
inhibitors is determined in parallel on mock-infected MT4 cells (150
000 cells/mL) stably transformed with a CMV-EGFP reporter gene
and cultured in the presence or absence of test compound
concentrations. After 3 days of incubation, cell proliferation is
quantified by measuring the EGFP fluorescence and expressed as
CC50 values (cytotoxic concentration of drug that reduced the viable
cell number by 50%).
Structural Biology. The RT gene (HIV-1, BH10 isolate 19+0-
3587) was isolated by PCR. Through the construct the peptide
sequence was provided with a C-terminal histidine affinity tag.
Mutants were made by PCR by amplification using PfuUltra
(Stratagene) polymerase of the entire expression vector with help of
two complementary oligonucleotides containing the mutation and
subsequent digestion with DpnI (Stratagene) of the parental vector,
prior to transformation to the cell line TOP10 (Invitrogen) for
cloning. Expression was done with help of the vector pCRT7 and as
described previously.38 The inhibitor complexes were formed by
addition of the inhibitor in DMSO solution to a 2-fold molar excess to
the RT solution (20 mg/mL). Crystals were produced by vapor
diffusion in which an amount of 5 μL of crystallization buffer (1.4 M
ASSOCIATED CONTENT
* Supporting Information
■
S
Figures showing new leads, NNRTIs, and Merck pyridinone
NNRTIs; schematic of electronic density surrounding Tyr181,
Tyr188, and compound 12; Table 1 containing crystallographic
information. This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
†Protein Data Bank entries are the following: 2ykm (F227C,
E478Q*12), 2ykn (F227L, E478Q*12), 3MEE (TMC278).
AUTHOR INFORMATION
Corresponding Author
■
*For M.M: phone: phone, +33 4 7243 1989; e-mail, maurice.
medebielle@univ-lyon1.fr. For J.G: phone, +33 2 3261 7458; e-
mail, jguillem@its.jnj.com. For J.U: phone, +46 7 0143 6965; e-
mail, johan.unge@maxlab.lu.se.
Present Address
#Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
ACKNOWLEDGMENTS
This investigation was supported by Tibotec Pharmaceuticals
and in part by the Centre National de la Recherche Scientifique
■
́
(CNRS) and Universite Claude Bernard Lyon 1. We thank L.
Dehuyser for making some additional 12 and 13 and C. Perez
for the preparation of 63. We also thank Torsten Unge for
initial crystallographic work and enzyme production. We are
grateful to the Centre Commun de Spectromet
́
rie de Masse
(CCSM) of Universite Claude Bernard Lyon 1 (F. Albrieux, C.
́
Duchet, N. Enriques) for the MS analyses. M.M. thanks Dr.
Luc Van Hijfte (Johnson and Johnson Research and Develop-
ment, Beerse, Belgium) for support at the early stage of this
work, Kenny Simmen (Johnson & Johnson Research and
Development, Mechelen, Belgium), and Pr. William R. Dolbier,
Jr and Dr. Conrad Burkholder (University of Florida,
Gainesville, FL, U.S.) for their early contributions to this
project.
ABBREVIATIONS USED
■
CC50, concentration for 50% cytotoxicity; DAPY, diary-
lpyrimidines; DFMB, difluoromethylbenzoxazoles; EC50, effec-
tive concentration for 50% inhibition; HAART, highly active
antiretroviral therapy; HIV, human immunodeficiency virus;
NIH, National Institutes of Health; NNRTI, non-nucleoside
7983
dx.doi.org/10.1021/jm200766b|J. Med. Chem. 2011, 54, 7974−7985

Journal of Medicinal Chemistry
Article
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