Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance properties. 1

Article abstract of DOI:10.1021/jm040071z

We have used a structure-based approach to design a novel series of non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Detailed analysis of a wide range of crystal structures of HIV-1 RT-NNRTI complexes together with data on drug resistance mutations has ide

Full text of DOI:10.1021/jm040071z

5912
J. Med. Chem. 2004, 47, 5912-5922
Design of Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase with
Improved Drug Resistance Properties. 1.
Andrew L. Hopkins,§,∞ Jingshan Ren,§ John Milton,∆ Richard J. Hazen,O Joseph H. Chan,O
David I. Stuart,§,‡ and David K. Stammers*,§,‡
Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive,
Oxford, OX3 7BN, UK, Oxford Centre for Molecular Sciences, New Chemistry Building, University of Oxford,
South Parks Road, Oxford, OX1 3QT, UK, GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, U.K. and
GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, North Carolina 27709
Received March 25, 2004
We have used a structure-based approach to design a novel series of non-nucleoside inhibitors
of HIV-1 RT (NNRTIs). Detailed analysis of a wide range of crystal structures of HIV-1 RT-
NNRTI complexes together with data on drug resistance mutations has identified factors
important for tight binding of inhibitors and resilience to mutations. Using this approach we
have designed and synthesized a novel series of quinolone NNRTIs. Crystal structure analysis
of four of these compounds in complexes with HIV-1 RT confirms the predicted binding modes.
Members of this quinolone series retain high activity against the important resistance mutations
in RT at Tyr181Cys and Leu100Ile.
Introduction
challenge the medicinal chemist faces in designing
improved NNRTIs is that every single-point NNRTI
resistance mutations can be assumed to be present in
the drug-naive HIV-1 population.15,16 In this paper we
attempt to synthesize the knowledge gained from crys-
tallographic and mutational studies of HIV-1 reverse
transcriptase into a set of medicinal chemistry strate-
gies to improve the drug resistance profile of NNRTIs.
Spatial Distribution of Resistant Mutations. In
the clinic the most commonly observed NNRTI resis-
tance mutations are Tyr181Cys and Lys103Asn. How-
ever, the majority of residues lining the drug binding
site have been observed to evolve drug resistance
mutations when selection pressure is exerted on HIV-1
in vitro with a wider range of NNRTIs.17-20
Mapping the wealth of drug resistance sequence data
onto the high-resolution protein crystal structures of the
inhibited HIV-1 reverse transcriptase complex enables
the medicinal chemist to observe trends that may be
exploited in drug design. The observed drug resistance
mutations selected by HIV-1 within the NNRTI pocket
can be classified into three distinct categories (Figure
1). The mutations Val106Ala, Tyr181Cys, Tyr188Cys
and Phe227Leu appear to confer resistance by reducing
the contact surface area compared to the wild-type
residues, thus presumably weakening the van der Waals
interactions between the protein and the bound
NNRTI.21-23 There are some biochemical data support-
ing this interpretation at least for two of the residues.
Studies of the pre-steady-state kinetics of the inhibition
of Tyr181Cys RT by nevirapine indicate the mutation
increases the Kd of inhibitor binding over 500-fold.24 The
decreased affinity of nevirapine is a consequence of the
faster dissociation rate between the inhibitor and the
Tyr181Cys RT enzyme, indicating a lower energy of
interaction between the inhibitor and the mutant
NNRTI pocket. Furthermore, steady-state kinetic ex-
periments demonstrate the Val106Ala mutation in-
creases the rate constant for nevirapine dissociation
Despite the advances in the available regimen of
drugs to treat HIV-1 infection, there remains vital need
to improve patient compliance, cost-of-goods and resis-
tance profiles of each of the classes of anti-retroviral
drugs. One of the four main classes of licensed anti-
retroviral drugs are the non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs), the other three classes
being nucleoside reverse transcriptase inhibitors, pro-
tease inhibitors and cell fusion inhibitors. The discovery
of the first NNRTIs, HEPT1,2 and TIBO,3,4 opened a
fruitful stream of research which eventually lead to
three licensed drugs: nevirapine, delavirdine and
efavirenz.5
The NNRTIs selectively inhibit HIV-1 reverse tran-
scriptase by binding an allosteric site in a noncompeti-
tive manner with respect to the substrate6 and displac-
ing the polymerase active site catalytic residues.7
Unusually, the allosteric site appears to be formed only
on binding of the NNRTIs to the viral enzyme. The
particular ‘plasticity’6,8,9 of the NNRTI binding site has
enabled the discovery of many structurally diverse
classes of allosteric enzyme inhibitors, providing the
medicinal chemist with a broad pallet from which to
design improved anti-retrovirals.10-12 However, NNRTIs
have proven to be susceptibility to the rapid selection
of drug resistance when evolutionary pressure is applied
to the nonconserved binding site.
The dynamics of HIV replication and massive viral
population constitute huge reservoirs of genetic variants
in every single HIV infected patient.13-15 Thus the
* To whom correspondence should be addressed. E-mail: daves@
strubi.ox.ac.uk.
§ Division of Structural Biology.
‡ Oxford Centre for Molecular Sciences.
∆ GSK Stevenage.
O GSK RTP.
∞ Current address: Molecular Informatics, Structure and Design,
Pfizer Global Research and Development, Sandwich, Kent, CT13 9NJ,
U.K.
10.1021/jm040071z CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/23/2004

Inhibitors of HIV-1 Reverse Transcriptase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5913
derivatives HBY 097 and S-2720, which dramatically
reduces viral replication competency by decreasing the
enzymatic activity of RT to only 5% that of wild type.27,28
The large increase in bulk on mutation from Gly to Glu
at position 190 could occupy the NNRTI binding site
and distort the polymerase catalytic site in an analogous
way to the NNRTIs, in addition to the unfavorable
positioning of a charged residue in the hydrophobic
pocket. Such a mechanism would explain the preference
for the virus to select for NNRTI mutations, which
generally decrease the size of the residue side chain.
The substitution of amino acid with larger residues may
provide sufficient steric hindrance to provide substantial
drug resistance but large amino acid substitutents may
also, by analogy with the binding of an NNRTI, prove
lethal by inducing conformational changes in the poly-
merase active site.
Structural Constraints on Drug Resistance. The
number and variety of NNRTI resistance mutations
may lead one to conclude the entire NNRTI pocket is
mutable. Ultimately this is so, however, in terms of
initial single and double drug resistance mutations
there appear to be important structural constraints on
protein stability and catalytic efficiency, which restrict
the initial options for HIV-1 to mutate. The spatial
relationship between the RT structure and the observed
drug resistance mutations is visualized in Figure 1. The
majority of the mutated residues cluster in a region near
Tyr181 and in the solvent channel between Lys101 and
Glu138(p51). Mapping of the mutational data demon-
strates the presence of a ‘conserved′ region in the
NNRTI pocket, comprising residues, which have not
been reported to mutate as primary sources of resis-
tance. The residues, which have not been primarily
selected as drug resistance mutations are Pro225,
Phe227, Trp229, Leu234 and Tyr318, all of which are
highly conserved between other lentiviral RTs. The
amino acids at positions 227 to 235 belong to the ‘primer
grip’ region, responsible for maintaining the 3′-primer
terminus in an orientation susceptible to nucleophilic
attack by the incoming dNTP.29-34
The single point mutations Pro225His35 and
Phe227Leu36 in recombinant RT increase resistance
against several NNRTIs; however, mutations at posi-
tions 225 and 227 have only been observed to evolve
as secondary mutations, appearing if the RT genetic
background already contains the primary Val106Ala
mutation.37 Depending on the specific inhibitor, under
dose-escalating passage experiments the selection of
Val106Ala mutation may lead to the subsequent addi-
tion of either Pro225His (S-272038 or as a triple mu-
tant against a Leu100Ile/Val106Ala background for
HBY09739) or Phe227Leu (UC-78137 and S-115336). Site-
directed mutation of Phe227 to Ala resulted in an RT
which lacked 5′-RNA-directed RnaseH catalysis,34 il-
lustrating the cost of mutations in the ‘primer grip’
region. Sequential selection of Pro225His against the
Val106Ala background has been proposed to arise due
the additional stability of a potential hydrogen bond
between the Nꢀ2 atom of the mutated Pro225His to
the main chain of residue 106.38 Pro227His has also
been observed as a double resistance mutation with
Lys103Asn from patients on combination therapy that
included efavirenz.40 Interestingly Pro225His35,38 and
Figure 1. Physicochemical mechanisms of drug resistance.
HIV-1 NNRTI resistance mutations are mapped onto the
binding site of the mapped onto the NNRTI binding pocket
from the crystal structure of the RT-2a complex. The inhibitor
is shown as a ball-and-stick representation, and the surround-
ing residues are shown as thin sticks. The atoms are colored
by type (grey - carbon; red - oxygen; blue - nitrogen; green
- chlorine) and the water molecule as a red sphere. Mutations
which confer resistance to NNRTIs by (a) loss of van der Waals
contacts are colored yellow; (b) change of electrostatic charge
or hydrogen bonding properties are colored orange; (c) steric
hindrance with the inhibitor are colored red. Conserved
residues, not been observed as drug resistant mutants are
colored green. Partially conserved residues, which have only
been observed as secondary or tertiary mutations against an
existing drug resistant background are colored in a corre-
sponding paler hue (residue 227 in pale yellow; residue 318
in pale orange; and residues 225 and 234 in pale red).
(koff) by 8.5-fold,25 supporting the assumption that this
mutation confers resistance by reducing stabilizing
interactions.
A second category of resistance mutations com-
prisesLys101Glu,Lys103Asn/Thr,Glu138Lys,Val179Asp,
Glu233Val and Lys238Thr. These residues are posi-
tioned around the solvent channel in the NNRTI binding
pocket, accessible to the bulk solvent. The mutations
belonging to this second category either change the
electrostatic charge or the hydrogen-bonding character
compared to wild type. Steady-state kinetic experiments
demonstrate the Lys103Asn mutation reduces the rate
constant (kon) for nevirapine binding25 which together
with structural studies6,26 indicate that this particular
mutation may influence the hydrogen bonding in the
unliganded enzyme.
A third category of mutations can involve steric
interference, either directly with the NNRTI or indi-
rectly via the subtle repositioning of neighboring resi-
dues which appears to account for the action of the
mutations Ala98Gly, Leu100Ile, Val108Ile, Gly190Ala/
Glu, Pro225His, Pro236Leu and Leu234Ile. An example
of this latter class would be the Val108Ile mutation,
which is likely to act by altering the conformation of
Tyr188. These residues, situated in the NNRTI pocket,
undergo mutations that create steric clashes by subtle
repositioning of the component atoms but generally do
not have large changes in side chain bulk, possibly
indicating that large residues would impair the function
of the enzyme. Loss of enzyme function is exemplified
by the Gly190Glu mutation, reported for quinoxaline

5914 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Hopkins et al.
Phe227Leu36 sensitize RT to delavirdine (BHAP). Po-
tentiation of delavirdine inhibition by the Pro225His
mutation can be explained by the conformation of this
residue, as observed in the RT-BHAP crystal struc-
ture.35
The discrepancy between the large variety of drug
resistant mutations reported in vitro and the far more
limited set of clinically observed resistance mutants can
be explained if one considers the tradeoff relationship
between drug resistance and enzymatic, and thus viral,
fitness. The widespread occurrence of Tyr181Cys and
Lys103Asn mutations in clinical use of NNRTIs is most
likely due to the ability of these specific mutations to
provide resistance without severely affecting enzyme
efficiency and hence viral replication. The limitations
on the repertoire of resistance mutations used by the
virus resulting from structural and fitness constraints
lead to a balance between resistance/fitness which can
provide an opportunity to devise design strategies which
takes these factors into account. In the next section we
attempt to apply the knowledge of structural constraints
to the design a series of NNRTIs with improved activity
against common drug resistant strains.
Structure-Based Design. A plausible strategy, to
improve the performance of an NNRTI against the
known drug resistant mutations, would be to target the
inhibitor to make strong interactions with the conserved
regions in the pocket, such as main chain atoms and
the conserved residues while decrease the dependence
on interactions with the mutable residues. In addition
to the rapid selection of drug resistance, a further
difficulty facing the rational design of an NNRTI is the
observed plasticity of the binding site inherent in the
structure, since this is an allosteric binding pocket
formed upon binding the inhibitor. The size and volume
of the NNRTI pocket, is greatly affected by the move-
ment of the Pro236 hairpin loop and the repositioning
of â10 strand (residues 232-234) and â11 strand
(residues 239-241). Although the Pro236 loop occupies
a fan of positions that track the size of the various
NNRTIs, each can be classified into two discrete binding
modes. The first mode is a nevirapine-type binding
pocket observed for the binding of small NNRTIs to RT
(such as nevirapine,6 9-Cl-TIBO,47 R-APA6 and
1051U916). The nevirapine-type NNRTI pocket is de-
fined by a hydrogen bond between the main chain
nitrogen of Lys103 and the main chain carbonyl oxygen
of Pro236. This hydrogen bond is formed when the
flexible Pro236 hairpin loop moves about 5 Å, to
encapsulate a small NNRTI. In a second binding mode
the NNRTI pocket appears able to adopt is a BHAP-
type pocket. On binding of larger NNRTIs, such as
BHAP9 or HEPT analogues8,43 the Pro236 hairpin loop
is closer to the apo conformation, forming a more open
pocket in which there is no hydrogen bond between
Lys103 and Pro236.
Trp229 is highly conserved among lentiviruses and
has never arisen as a site for primary or secondary drug
resistance mutation under selection pressure. The es-
sential role of residue Trp229 has been investigated by
mutagenesis, in which, the replacement of Trp229 with
Ala exhibits a reduction in RNA and DNA-primed
synthesis.31,33,34,41,42 The Trp229Ala results in a mutant
enzyme which shows a dramatic reduction in specific
RNaseH cleavage function33 and is incapable of forming
a stable RT-template-primer replication complex.32
Trp229 has been observed in two different conforma-
tions, where the ring system has been effectively flipped
180° by the Câ-Cγ bond, the differing conformations can
be observed even in structures of related inhibitor
analogues.8,43 Despite this structural flexibility, sys-
tematic site directed mutation of residue 229 reveals a
steric preference for trytophan.44 All other amino acids
lead to a dramatic reduction in enzyme function to less
than 1% of wild-type activity. Even aromatic substi-
tutents, Phe and Tyr exhibit only 0.7% and 1.1% wild-
type activity. Additionally recombinant virus strains
with mutation at residue 229 were found to be unvi-
able.32,44
A further ‘conserved’ residue in the region of the
‘primer grip’ part of the NNRTI pocket is Leu234. The
importance of conserving Leu234 is demonstrated by the
mutation of Leu234 to Asp resulting in noninfectious
virus and a reduction in reverse transcriptase activity,30
while the alteration of Leu234 to Ala produces a p66
subunit incapable of associating with the p51 subunit
to form a stable heterodimer.31 Recombinant Leu234Ile
RT enzyme is 22-fold less sensitive to S-1153 than wild
type, the most resistant single mutation against this
drug. However, the Leu234Ile resistance mutation has
only been observed as a part of a triple mutation with
Lys103Thr and Val106Ala after prolonged selection
pressure from S-1153.36 These observations can be
interpreted as evidence of the lower propensity of
Leu234 to mutate under drug selection pressure than
many other residues surrounding the NNRTI pocket.
In addition to the lower propensity of residues associ-
ated with the ‘primer grip’ to mutate under selection
pressure, residue Tyr318 appears to be generally highly
conserved, there is only one report of Tyr318 being
mutated (Tyr318Phe) in patients treated with NNRTIs,
where resistance to delavirdine was shown.45 Site-
directed mutatagenesis of residue 318 to either Cys,
Leu, Lys or His drastically reduced RT activity to only
5% of wild type.46 Only conservative mutations of
Tyr318 to Phe and Trp significantly retained enzyme
activity (95% and 54% of wild-type activity, respec-
tively). These mutagenesis experiments demonstrate the
importance of Tyr318 in HIV-1 RT. In the both unli-
ganded and NNRTI bound structures of RT the hydroxyl
group of the Tyr318 phenol side chain forms a hydrogen
bond to the main chain nitrogen of His235.6,7 This bond
links the connection domain and the palm domain and
may be important in forming the correct conformation
of the enzyme in this region.
The open conformation of the BHAP-type pockets
exposes the peptide backbone of Lys103 enabling the
possibility of forming a series of hydrogen bonds to the
main chain atoms of residues Lys101 to Lys103. Previ-
ously we have discussed the possible advantages of an
inhibitor mimicking a peptide â-sheet binding to the
conserved main chain residues. However, the difficulties
of accurately exploiting main chain interactions with
hydrophilic groups by design lead us to avoid this
approach.48 However, after our undertaking this current
work, our group has elucidated the crystal structure of
RT complexed with S-1153, which fortuitously fulfils our
earlier prediction that, providing there is little depen-

Inhibitors of HIV-1 Reverse Transcriptase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5915
Figure 2. Exploiting contacts to the conserved regions. The p-chloroaniline moiety (grey - carbon atoms; blue - amine nitrogen;
green - chlorine atom) makes good contacts to the conserved region of the NNRTI pocket. The benzyl ring is positioned to exploit
aromatic interactions with Tyr318. The amine nitrogen can form a hydrogen bond to the carbonyl oxygen of Lys101 and the
chloro group fits snugly into the Leu234 subpocket. The surrounding protein is shown as thin sticks (yellow - carbon atoms; red
- oxygen; blue - nitrogen). The solvent-accessible surface is colored blue (calculated using the program VOLUMES calculated by
rolling a 1.4 Å probe over the surface of the protein structure (R. Esnouf, unpublished)). The energy contour levels for the
hydrophobic probe (calculated using the program GRID53) are contoured at: -0.5 kcal mol-1 (yellow); -1.0 kcal mol-1 (orange);
-1.5 kcal mol-1 (red); -2.0 kcal mol-1 (brown).
dency on interactions with the side chain of Pro236,
forming a large number of van der Waals and hydrogen
bonding interactions of the Lys101-Lys103 backbone
may significantly improve the resistance profile of
NNRTIs.49
synthetically accessible. Hydrophobicity is a key feature
driving the potency of inhibitor binding to the NNRTI
site, and thus identification of hydrophobic binding sites
is an important consideration in the design of NNRTIs.
Analysis of the NNRTI site (see Experimental Section)
reveals the deepest hydrophobic minimum occurs in the
upper part of the pocket near to residue Trp229 and the
second deepest in the Leu234 subpocket (Figure 2).
These two minima are found in a general hydrophobic
space connecting the Leu234 subpocket with the upper
hydrophobic pocket along residues His235, Leu234,
Phe227, Tyr188, Trp229, Tyr181 and Val106. In con-
trast, the GRID water and NH amine hydrophilic probes
suggest there are relatively few opportunities for hy-
drogen bonds inside the NNRTI binding pocket. The
hydrophilic region of the pocket is situated along the
peptide backbone of residues Lys103 and Lys101 and
into the solvent channel past residue Glu138 (p51). Both
the water and the NH amine probes indicate that strong
hydrogen bonds can be formed to the carbonyl oxygen
of Lys101. Crystallographic observations and GRID
calculations confirm the strong potential to form a
hydrogen bond with the main chain carbonyl oxygen of
Lys101. A nitrogen atom was therefore placed at a
distance of 3.0 Å from the main chain carbonyl oxygen
of Lys101, in a similar position to the 3-NH of the
pyrimidine rings of the HEPT analogues to exploit this
The smaller, encapsulated nevirapine-type pocket also
provides opportunity for interactions with conserved
residues. The movement of Pro236, enclosing the pocket,
causes the side chains of residues Pro236, Pro225,
Phe227, Leu234 and the main chains of His235 and
Pro236 to partially collapse and form a compact sub-
pocket, henceforth known as the ‘Leu234 subpocket’.
The Leu234 subpocket appears hydrophobic in nature
and is surrounded by a cluster of residues, which have
not been observed as primary resistance mutations and
is flanked by the conserved residue Tyr318. We judged
for the purpose of a structure-based design exercise that
the exploitation of contacts to the more conserved
residues, Trp229, Leu234 and Tyr318, could be best
accomplished by designing inhibitors to fit into the small
nevirapine-type pockets and interact with the compact
Leu234 subpocket.
The inhibitor design strategy in this current work in-
volved determining which moieties and groups would
provide the best complementarity to each of the individ-
ual subpockets in the NNRTI binding site and connect-
ing the groups together in a scaffold which would prove

5916 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Hopkins et al.
anchor point. Exploitation of van der Waals contacts to
the hydrophobic and conserved Leu234 subpocket and
the aromatic character of the Tyr318 could provide
valuable interactions between the inhibitor and the
more conserved region of the NNRTI pocket. The
putative inhibitor was based on a benzene ring exploit-
ing aromatic π-π interactions with residue Tyr318. A
benzene ring was placed in an off-center stacking
position relative to the ring plane of Tyr318, with a
centroid-centroid distance of 4.5 Å between the two
aromatic rings. A small hydrophobic subpocket exists
in the NNRTI binding site next to Leu234. Exploitation
of the pocket by a tight fitting group, such as a chloro,
bromo, methoxy, methyl or nitrile, could make numer-
ous van der Waals interactions with the side chains of
Phe227 and Leu234 and the main chain atoms of His235
and Pro236. These three putative interactions with the
Lys101 main chain, the Tyr318 aromatic ring and the
Leu234 subpocket can be connected to form a p-
chloroaniline moiety (Figure 2). Interestingly many
chemically diverse second generation NNRTIs, such as
efavirenz, HBY-097, UC-781 and PETT-2 contain this
para-chloroaniline motif.
The upper part of the NNRTI binding site is a large
cavity, predominately of aromatic character, formed by
Tyr181, Tyr188, Phe227 and roofed by Trp229. Previ-
ously we have argued that the π-π aromatic interac-
tions which may be beneficial in the wild-type pocket
may be unfavorable in the Tyr181Cys mutant pocket.8
Ideally substitutents in the upper part of the NNRTI
pocket should maximize contacts with the conserved
residue Trp229 while avoiding the energetic complica-
tion of differing aromatic stacking environments be-
tween wild type and reducing the overall percentage of
contacts with the mutable residues Tyr181 and Tyr188.
Our previous studies on HEPT analogues has already
demonstrated that by positioning a cyclohexyl group in
the upper part of the NNRTI pocket an inhibitor can
utilize this region of the pocket for both the wild type
and Tyr181Cys mutant.43
A final key aspect of the compound design is the
provision of a substituent to occupy the subpocket at
the base of the Tyr181 side chain. Analysis of the
structures of RT complexed with HEPT analogues lead
to the hypothesis that a potent inhibitor should have a
substituent that fills the volume of the small Tyr181
subpocket, situated between the side chains of Val179
and Tyr181 and walled by the main chain atoms of
residues 180 and 181. Small hydrophobic groups such
as ethyl, propyl or isopropyl provide substitutents of the
correct size.
After several iterative design rounds (following the
procedures outlined in the Experimental Section) in-
volving building and evaluating a variety of distinct
chemical scaffolds, the final selection of quinolone
analogues was determined as a compromise between the
design principles outlined above and synthetic acces-
sibility (Figure 3). Given the promiscuity of the NNRTI
binding site a wide variety of other compounds could
be designed followed the criteria outlines above. To test
the hypothesis that knowledge gained from the struc-
tural analysis of the mechanisms of RT inhibition and
drug resistance can guide the design of improved ‘second
Figure 3. Proposed quinolone-based non-nucleoside inhibitors
of HIV-1 RT.
Scheme 1. Synthesis of 6-Chloro-4-cyclohexyloxy-3-
propylquinol-2-one (2a, R ) Pr)). Replacement of the
Diethyl Propylmalonate with Diethyl Isopropylmalonate
in the First Step Led to the Synthesis of 2b (R ) i-Pr)
a Reagent and conditions: (a) Ph2O, N2, 250 °C reflux, 20 h; (b)
bromocyclohexane, DMF, Et3N, K2CO3, N2, 21 h reflux.
generation’ inhibitors the quinolone analogues were
synthesized and evaluated.
Chemistry. The synthesis of the 4-alkyloxyquino-
lones and 4-alkylthioquinolones compounds was ac-
complished separately using two distinct synthetic
schemes from 4-hydroxyquinol-2-ones intermediates.
The general procedure to form 4-hydroxyquinol-2-ones
is by the reaction of the diethyl malonate ester with
aniline to form the malonanilide with the liberation of
ethanol. The ring closure of the malonanilide and
tautomerization leads to the formation of the common
intermediate 4-hydroxyquinol-2-one (1).
The complete syntheses of the alkyloxy quinolone
ethers, compounds 2a and 2b, are two-step processes
(Scheme 1). Scheme 1 enables the simple substitution
of groups at positions 3 and 6 on the quinolone, but is
limited to the synthesis of alkyloxy quinolone ethers
only. 4-Hydroxyquinol-2-ones (compounds 1a and 1b)
were heated with an alkyl bromide (cyclohexyl bromide)
under basic conditions (potassium carbonate and tri-
ethylamine) to form 4-cyclohexyloxyquinol-2-ones via
the Williamson synthesis of an unsymmetrical ether.
Scheme 2 provides a general method for synthesising
unsymmetrical ethers or thioethers, with any alkyloxy,
alkylthio, and aryloxy or arylthio substituents to the
4-position of the quinolone. The 4-hydoxy-quinol-2-one
(1) is brominated50 with heated phosphorus tribromide
to produce the 2,4-dibromoquinoline compound (3).
Hydrolysis of compound 3 to the 4-bromo-1H-quinol-2-
one (4) was achieved by heating in glacial acetic acid
containing a small amount of water. The displacement
of the bromide in compound 4 by the sodium mercaptan
salt (prepared in situ) leads to the formation of the
4-cyclohexylthioquinol-2-one (5). Oxidation of the sulfur,
in compound 5, with m-chloroperbenzoic acid (MCPBA)
led to the formation of the sulfinyl derivative (6). Details
of the all the reactions are given in the Experimental
Section.

Inhibitors of HIV-1 Reverse Transcriptase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5917
Table 2. Antiviral Activity of Quinolone Inhibitors in MT4
Scheme 2. Synthesis of 6-Chloro-4-cyclohexylthio-3-
propylquinol-2-one (5) (a, R ) Pr; b, i-Pr)
Cells
a
HIV-1IIIB
IC50
nevirapine-resistant HIV-1b
compd
IC50 (fold)c
2a
2b
5
0.035
0.051
0.100
0.242
0.607 (17)
0.710 (14)
0.368 (4)
2.210 (9)
6
a HIV-1IIIB virus b HIV-1 virus containing Tyr181Cys mutation
in RT. c Multiple increase in IC50 between wild-type (IIIB) and
nevirapine-resistant HIV-1 strains.
inhibition concentrations for both wild-type and nevi-
rapine-resistant HIV-1, relative to the other three.
To confirm if the quinolones bound to RT as predicted,
the actual binding mode of the compounds were ob-
served by determination of the X-ray crystallographic
structures of the four quinolone analogues, 2a, 2b, 5
and 6 complexed with HIV-1 reverse transcriptase.
Details of the crystallization and structure determina-
tions of the four RT-inhibitor complexes are outlined
in the Experimental Section. The overall structures of
the RT-quinolone complexes have the same general
description of the RT p66/p51 heterodimer as reported
for the other RT-NNRTI complexes and show no
significant global rearrangements. The electron-density
maps clearly and unambiguously show the positions,
orientations and conformations of the inhibitors bound
at the NNRTI site (Figure 4). The |Fobsd| - |Fcalcd| omit
map for RT-6 (Figure 4d) indicates the chirality of the
sulfinyl compound is modeled as the R enantiomer. The
protein-inhibitor interactions for the structures of the
quinolones compounds complexed to RT are very similar
for all four complexes. Comparison of the binding of the
quinolone inhibitors to RT between the crystal struc-
tures and the modeled structures reveal an extremely
close overlap (mean rms deviation between the crystal
structures and the models is 0.5 Å).
A strong hydrogen bond between the N1H of the
heterocylic ring of the quinolone moiety and the main
chain carbonyl oxygen of Lys101 is observed, as pre-
dicted. Water has only been built into the 2.6 Å
resolution map of the RT-2a complex. In this complex
the inhibitor is orientated into position by an additional
hydrogen bond between the carbonyl oxygen O2 of the
quinolone and a water molecule, which in turn forms a
hydrogen bond to the side chain of Lys103 (Figure 1).
This water molecule is situated in the channel that leads
to the bulk solvent but makes different hydrogen bonds
to the equivalent water molecule in the RT-MKC-442
and related structures, which bond to the side chains
of Glu138(p51) and Lys101. Although no water mol-
ecules have been modeled into the electron density maps
of the other three structures, the conformation of the
Lys103 side chains and the position of the O2 carbonyl
oxygen of the quinolones are such that water could
hydrogen bond in the same position as in the RT-2a
model.
a Reagents and conditions: (a) PBr3, 140 °C, reflux, 4.5 h; (b)
acetic acid, water, 125 °C reflux, 8 h; (c) dry DMF, N2, 20 h, 140
°C, sodium cyclohexane mercaptan; (d) MCBPA, room tempera-
ture, 1 h.
Table 1. Inhibition of Wild-Type and Mutant HIV-1 RT by
Quinolone Compounds. Data Are the Mean Value of Two
Independent Experiments
wild-type RT
IC50, µM
Cys181 RT
Ile100 RT
compd
IC50, µM (fold)a IC50, µM (fold)a
2a
0.71
0.63
0.24
0.38
0.30
1.13 (1.5)
2.50 (3.9)
1.36 (1.9)
1.96 (3.1)
2b
5
6
0.86 (3.6)
1.61 (6.7)
1.26 (3.3)
>100 (>100)
2.38 (6.2)
>100 (>100)
nevirapine
a Multiple fold increase in IC50 between wild-type and mutant
HIV-1 RT.
Results
All four of the lead quinolone compounds tested (2a,
2b, 5 and 6) proved to be potent and selective inhibitors
of HIV-1 RT (Table 1). The comparison with nevirapine
starkly shows the success of the designed quinolone
compounds in inhibiting the common drug resistant RT
mutants, Tyr181Cys and Leu100Ile. Whereas the sen-
sitivity of the mutant enzyme to nevirapine is reduced
more that 100 fold, the quinolone compounds, as a
series, show decreases between 1.5- and 7-fold. The
average increase in the IC50 values for the four inhibi-
tors against the Cys181 and Ile100 mutant RTs was
only a 3.1-fold and 4.5-fold, respectively. The most
potent inhibitor of the Cys181 mutant RT is, again,
compound 5, with an IC50 ) 0.86 µM, while the most
potent inhibitor of the Leu100Ile mutant is compound
2a (IC50 ) 1.36 µM). The 4-cyclohexyloxyquinolone
analogue, 2a, is the least affected by the resistance
mutations as indicated by only a 1.5-fold increase in IC50
against the Tyr181Cys mutant and only 1.9-fold in-
crease for Leu100Ile, when compared to the IC50 for
wild-type RT. The quinolone compounds (2a, 2b, 5 and
6) are demonstrated to be potent inhibitors of wild-type
(HIV-1IIIB) and nevirapine-resistant strains of HIV-1,
in the MT4 cell assay (Table 2). The 4-cyclohexyloxy
quinolone derivatives, 2a and 2b, are extremely potent
inhibitors of HIV-1, with IC50 ) 35 nM and IC50 ) 51
nM, respectively. The 4-cyclohexylthioquinolone deriva-
tive, 5, appears to be least affected by the Tyr181Cys
mutation in the nevirapine-resistant HIV-1 strain, with
an IC50 ) 0.368 µM which is only an increase of 3.6-
fold over the inhibition value for wild type. The sulfinyl
analogue, 6, displays the least impressive performance
of the four compounds, with significantly higher 50%
The position of the Pro236 loop is the same in all four
of the RT-inhibitor complexes and closely resembles the
position of Pro236 in the RT-TIBO complex, the refer-
ence structure used for the initial inhibitor design. The
quinolone moiety of the inhibitors sits between the
hydrophobic residues Val106 and Leu100 and is flanked
by Tyr318. The quinolone group of all four inhibitors is
in the same plane, with the benzene ring moiety

5918 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Hopkins et al.
Figure 4. Diagrams of the electron density omit |Fobsd| - |Fcalcd| maps contoured at 3σ of the quinolone inhibitors bound at the
NNRTI binding site of HIV-1 RT for (a) RT-2a, (b) RT-2b, (c) RT-5 and (d) RT-6 complexes. The inhibitors are shown as
ball-and-stick representations where the atoms are colored by type (grey - carbon; red - oxygen; blue - nitrogen; green - chlorine;
yellow - sulfur) and the surrounding protein is displayed as gray sticks. The omit maps are shown as green nets.
appearing to interact with the conserved residue Tyr318
by aromatic-aromatic, off-center π-stacking,51 there is
an average centroid to centroid distance of 4.5 Å
between the ring planes. The 7-chloro substituents of
the quinolones fit snugly into a subpocket created by
the encapsulating movement of the Pro236 hairpin loop.
The 7-chloro substituents form close van der Waals
(e3.7 Å Cl-C van der Waals contacts) to the surround-
ing residues Phe227, Leu234, His235, Pro236, side chain
and main chain atoms. The 3-propyl groups of 2a, 5 and
6 extend to fill the subpocket at the main chain Tyr181
and side chain of Val179. The terminal methyl of the
3-propyl group makes close contacts to main chain
atoms of Tyr181 and Tyr188 and the side chain of
Val179. The shorter 3-isopropyl group of 2b would still
clash with the apo conformation of Tyr181, but makes
less of a intrusion into this subpocket, making contacts
to Tyr188, since both the 3-isopropyl and 3-propyl
quinolone analogues are in the same plane. 2b does not
rearrange its orientation in the pocket to place the
3-isopropyl deeper into the Tyr181 subpocket. The main
differences between the four RT-quinolone complexes
are the subtle changes in the position and interactions
of the 4-cyclohexyloxy and 4-cyclohexylthio substituents
with the protein. The cyclohexyl ring of all the inhibitors
sits in the upper part of the NNRTI binding site, which
is mainly aromatic in character, surrounded by residues
Leu100, Tyr181, Tyr188, Phe227 and Trp229, the latter
lying at the top of the pocket, close to normal to the
plane of the tyrosine side-chains and cyclohexyl group.
The cyclohexyl ring in all four inhibitors adopts the
same chair conformation. Due to the longer carbon-
sulfur bond length and sharper bond angle the 4-cyclo-
hexylthio substituent fits slightly deeper into the pocket,
enabling closer contacts to the conserved residue Trp229.
The increased hydrophobic character of the thioether
linker in 5 as compared with the oxy ether linker of 2a
and 2b and the deeper penetration into the hydrophobic
pocket may account for the lower IC50 of 5 against RT
enzyme than 2a and 2b.
Conclusion
A detailed analysis of both the structure of the NNRTI
pocket and the available data on drug resistance for this
compound class led to a structure-based design ap-
proach to the synthesis of novel inhibitors of HIV-1 RT.
For reasons of synthetic accessibility, we decide to focus
our chemistry efforts on the quinolone scaffold, yet we
believe the general strategies, outline here, for tackling
drug resistance can be applied as design principles to
the search for other NNRTI chemotypes. These initial
results indicate the quinolone series was much more
favorable than nevirapine when comparing inhibition
of the clinically important Tyr181Cys mutation in HIV-1
RT. These initial results provided us with enough
confidence to extend this study to further explore the
potential of this approach in the design of further
analogues in the quinolone series of NNRTIs. In Part 2
of this work we provide further evidence from the
comprehensive screening of quinolone analogues against
a broad panel of HIV-1 RT drug resistance mutants for
the potential of this series and this approach in the
design of NNRTIs with improved drug resistance pro-
files.52
Experimental Section
Computational Chemistry. Iterative rounds of compound
design and retrosynthetic analysis were conducted in the
following manner. The NNRTI binding pocket was defined by

Inhibitors of HIV-1 Reverse Transcriptase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5919
calculating a solvent accessible surface using the program
VOLUMES (Dr. R. Esnouf, unpublished). The energy contours
for the hydrophobic and hydrophilic probes were calculated
using the program GRID (version 14).53 Molecules expected
to fulfill the design criteria were constructed and minimized
using the program SYBYL (Tripos Associates) on an R10000
Silicon Graphics workstation. The molecules were initially
manually docked into the NNRTI binding site of the RT model
guided by superposition models of the all the available RT-
inhibitors structures, using the protein model of the RT-9-
Cl-TIBO as the main reference structure (2.6 Å resolution;
PDB accession number 1REV47). The position of the docked
molecules were refined in the NNRTI pocket were subject to
energy minimization using the conjugate gradient method54
as implemented in X-PLOR55 with the protein fully restrained.
Protein-inhibitor interactions were calculated using X-PLOR.
The synthetic accessibilities of the molecular designs were
evaluated by searching the synthetic reaction database at
Daresbury Laboratory.56
with light petroleum ether and dried to give pure product
(12.16 g; 0.051mol). Yield 63.8%. Rf product ) 0.1 (10% EtOAc/
1
90% hexane). Melting point of product, 255-256°C. H NMR
200 MHz (DMSO-d6) 11.35 (s, 1H, NH), 10.10 (s, 1H, OH), 7.9
(s, 1H, 5-H), 7.45 (d, J ) 5 Hz, 1H, 7-H), 7.24 (d, J ) 6.5 Hz,
1H, 8-H), 3.4 (m, 1H, i-propyl 1′-H), 1.28 (d, J ) 5 Hz, 6H,
i-propyl 2′,3′-H). Anal. (C12H12NO2Cl) C, H, N.
6-Chloro-4-cyclohexyloxy-3-propyl-1H-quinol-2-one
(compound 2a). 6-Chloro-4-hydroxy-3-propyl-1H-quinol-2-one
(0.5 g; 2.1 mmol) was dissolved in DMF (50 mL). To this
solution was added 2 equiv of cyclohexyl bromide (0.52 mL;
4.2 mmol), 1.1 equiv of potassium carbonate (0.319 g; 2.31
mmol) and triethylamine (0.21 mL). The reaction mixture was
heated to 140 °C, under a nitrogen atmosphere, overnight. LC-
MS after this time indicated that about 30% of the starting
material had been converted to product. The mixture was
allowed to cool and filtered to remove insoluble potassium
carbonate. Volatiles were removed in vacuo. The remaining
dark red solid was dissolved in DCM (100 mL) and washed
with water and then brine. The organic layer was dried by
adding solid anhydrous sodium sulfate. The crude product was
concentrated to a solid by removing the organic solvent under
reduced pressure. The crude dark red solid was dissolved in a
minimum amount of ethyl acetate with a small addition of
acetone to aid solubility. The final product was isolated and
purified by silica column chromatography (solvent system
initially 100% hexane, slowly increasing the concentration of
ethyl acetate). The fractions containing the product were
pooled and volatiles removed in vacuo to recover 53 mg
amorphous solid of pure product: Yield 8%. Melting point of
solid product 214-215 °C. 1H NMR 200 MHz (CDCl3) 12.0 (s,
1H, NH), 7.75 (s, 1H, 5-H), 7.4 (d, J ) 5 Hz, 1H, 7-H), 7.3 (d,
J ) 10 Hz, 1H, 8-H), 4.1 (m, 1H cyclohexyloxy OCH), 2.7 (t, J
) 5 Hz, 2H, propyl 1′-H), 2.08 (m, 2H, C2ax, C6ax cyclohexyl-
H), 1.85 (m, 2H, C2eq, C6eq cyclohexyl-H), 1.67 (m, 2H, propyl
2′-H), 1.28 (m, 6H, C4, C5, C6 cyclohexyl-H), 1.03 (t, J ) 5 Hz,
3H, propyl 3′-H). Anal. (C18H22NO2Cl) C, H, N.
6-Chloro-4-cyclohexyloxy-3-isopropylquinol-2-one (com-
pound 2b). 6-Chloro-4-cyclohexyloxy-3-isopropyl-quinol-2-one
(0.5 g) was dissolved in dry DMF (50 mL). To this solution
was added triethylamine (0.45 mL), potassium carbonate (1
g) and 5 equiv of cyclohexyl bromide. The reaction mixture
was stirred and heated to 150 °C for 48 h. On cooling the
reaction mixture was filtered and volatiles were removed in
vacuo. The remaining residue was dissolved into 5 mL of ethyl
acetate. The product was obtained by silica column chroma-
tography (initial column 100% hexane, increasing the concen-
tration of ethyl acetate, Rfproduct ) 0.1 (10% ethyl acetate/90%
hexane)) to give 50 mg of product. Yield 8%. Melting point of
solid product 190-191 °C. 1H NMR 200 MHz (CDCl3) 12.4 (s,
1H, NH), 7.75 (s, 1H, 5-H), 7.4 (d, J ) 7.5 Hz, 1H, 7-H), 7.3 (d,
J ) 6 Hz, 1H, 8-H), 4.03 (m, 1H, cylcohexyloxy OCH), 3.45
(m, 1H, i-propyl C1H1), 2.08 (m, 2H, cyclohexyl-H C2ax, C6ax),
1.85 (m, 2H, cyclohexyl C2eq, C6eq), 1.52-1.75 (m, 6H, cyclo-
hexyl-H, C3, C4, C5), 1.48 (d, J ) 5 Hz, 6H, i-propyl C2-H3, C3-
H3). Anal. (C18H22NO2Cl) C, H, N.
Synthetic Chemistry. 1H NMR spectra were obtained on
a Bru¨cker AC250 (200 MHz) spectrometer, DMSO-d6 and
CDCl3 were used as solvents, with tetramethylsilane (TMS)
added as an internal standard in all cases. Data are reported
as follows: chemical shifts reported in ppm units relative to
the TMS internal reference, multiplicity (b ) broad, s )
singlet, d ) doublet, t ) triplet, m ) multiplet, q ) quartet),
coupling constant (Hz), integration and chemical group. LC-
MS (liquid chromatography-mass spectrometry) data were
collected on a Hewlett-Packard spectrometer. All compounds
1
were routinely checked by H NMR, LC-MS and TLC. NMR
spectral data were consistent with the indicated structures and
LC-MS data were consistent with expected masses. Reactions
were monitored by thin-layer chromatography, on glass plates
coated with silica gel and visualized by UV light. Elemental
analyses were determined by Butterworth Laboratories Ltd.
IR spectra were recorded on a Biorad FT-IR FTS155 spectro-
photomer with compounds pressed into KBr pellets. Melting
points were measured in open capillary tubes using a Bu¨chi
melting point apparatus and are uncorrected. Separations by
flash chromatography were performed with Merck silica gel
(Merck No. 60, 230-400 Mesh). All reagents and solvents were
of commercial quality. Solvents were dried using standard
procedures. Concentrations of solutions after reactions and
extractions involved the use of a rotary evaporator operating
at a reduced pressure of approximately 15 Torr. Organic
solutions were dried over sodium sulfate. Samples prepared
for physical data and biological testing were dried overnight
under high vacuum.
6-Chloro-4-hydroxy-3-propyl-1H-quinol-2-one (com-
pound 1a). 4-Chloroaniline (10 g: 0.08mol) and diethyl
propylmalonate (16.31 mL; 0.08mol) were dissolved in diphenyl
ether (100 mL). The reaction mixture was heated to reflux at
250 °C in a liquid metal bath and stirred. The reaction was
conducted under a nitrogen atmosphere with the nitrogen
blowing onto the surface of the reaction solution. After 20 h
the reaction was cooled. The reaction flask was placed in a 50
°C water bath to prevent the diphenyl ether from solidifying.
Colorless crystals of the product crystallized out of the warm
solution. These crystals were filtered off, washed with light
petroleum ether (bp 40-60 °C) and dried in a vacuum over
for several hours to give 11.63 g of pure product: Yield 61.3%.
Melting point of white crystals, 245 °C. 1H NMR 200 MHz
(DMSO-d6) 11.44 (s, 1H, NH), 10.25 (s, 1H, OH), 7.85 (s, 1H,
5-H), 7.49 (d, J ) 5 Hz, 1H, 7-H), 7.25 (d, J ) 5 Hz, 1H, 8-H),
2.52 (t, J ) 5 Hz, 2H, propyl 2′-H2), 1.45 (m, 2H, J ) 5 Hz,
propyl 1′-CH2), 0.9 (t, J ) 5 Hz, 3H, propyl 3′-H3). Anal. (C12H12-
NO2Cl) C, H, N.
2,4-Dibromo-6-chloro-3-propylquinoline (compound
3). 6-Chloro-4-hydroxy-2-quinolone (3.0363 g; 0.013 mol) was
mixed with phosphorus tribromide (50 mL). The reaction
mixture was stirred and heated to 140 °C. The crust that
formed on top of the reaction mixture was broken up at
intervals. After 4.5 h the mixture was cooled, poured into iced
water (500 mL) and left to stand overnight. The crude product
(3.57 g) was collected and crystallized from 95% ethanol to give
white, slender, needle crystals of pure product (800 mg). Yield
17%. Melting point of crystals 109-110 °C. 1H NMR (DMSO-
d6) 8.15 (s, 1H, 5-H), 8.05 (d, J ) 7.5 Hz, 1H, 7-H), 7.88 (d, J
) 7.5 Hz, 1H, 8-H), 3.1 (t, J ) 5 Hz, 2H, propyl 1′-CH2), 1.65
(m, 2H, propyl 2′-CH2), 1.05 (t, J ) 5 Hz, 3H, propyl 3′-CH3).
Anal. (C12H10NClBr2) C, H, N.
6-Chloro-4-hydroxy-3-isopropyl-1H-quinol-2-one (com-
pound 1b). 4-Chloroaniline (10 g; 0.08mol) and diethyl
isopropylmalonate (0.08mol; 16.1 g) were dissolved, by stirring,
into diphenyl ether (70 mL). The reaction mixture was heated
to 250 °C and refluxed, under a stream of nitrogen, for 24 h.
On cooling to 50 °C a white solid precipitated out of the
reaction solution. The solid product was filtered off, washed
4-Bromo-6-chloro-3-propyl-1H-quinol-2-one (compound
4). The 2,4-dibromoquinoline analogue (compound 3, 100 mg;
0.275 mmol) was mixed with a solution of glacial acetic acid

5920 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Hopkins et al.
(10 mL) and water (5 mL). The reaction mixture was heated
to 125 °C and refluxed for 8 h. On cooling a white solid
precipitated from the solution, which was collected and dried
to give pure product (80 mg; 97.5% yield). Melting point of
HPO4) at pH 5, using the sitting drop method at 4 °C. Each
drop containing 4 µL of enzyme-inhibitor solution and 4 µL
of PEG/buffer mix and were microseeded using a light brush
from a suspension of crushed RT-nevirapine crystals. The
crystals were subject to partial dehydration by equilibrating
with 50% PEG 3350 by gradually increasing the concentration
of PEG over a period of 3 days.
X-ray Data Collection. Diffraction data for the RT-2a,
RT-2b and RT-5 complexes were collected on Station 7.2 at
the SRS, Daresbury Laboratory, UK, using the oscillation
method with X-rays of 1.488 Å wavelength collimated to a
diameter of 200 µm. Data images of 1.5° oscillations were
recorded using 120 s exposures on a 18 cm MAR Research
imaging plate system which was positioned 130 mm from the
crystal. Diffraction data from the RT-6 complex were collected
on beamline BM-14 at the ESRF, Grenoble, France, with
X-rays of 0.918 Å wavelength using a 34.5 cm MAR Research
imaging plate. All crystals were flash-frozen in liquid propane
and maintained at 100 K in a stream of nitrogen gas, using
an Oxford Cryosystems Cryostream (Long Hanborough, Oxon.,
UK). All datasets were collected from single crystals for each
complex with negligible radiation damage.
Data images were auto-indexed and spot intensities were
integrated using the program DENZO.64 The observed data
was corrected for Lorentz and polarization effects and reduced
to a list of unique, indexed reflections with averaged intensities
using the program SCALEPACK.64 The orthorhombic crystals
were in space group P212121. From the cell parameters, all four
of the complexes were classified as belonging to RT cell form
F. Data collection and reduction statistics can be found in
Table 3.
Structure Solution and Refinement. The RT-inhibitor
complexes were solved by molecular replacement using X-
PLOR.55 All four of the structures were phased using the 2.6
Å resolution structure of the RT-9-Cl-TIBO complex47 (exclud-
ing water and inhibitor molecules), which possesses a similar
unit cell parameters (a ) 138.8, b ) 115.8, c ) 66.2) to each of
the complexes. Each of the data sets was separately refined
by similar refinement protocols with CNS65 using the Engh
and Huber protein force-field parameters.66 Initially, data from
10 to 6 Å resolution were used with the model as a single rigid
body; then the p66 and p51 subunits were used as two rigid
groups, and the nine domains in the heterodimer were each
treated as separate rigid groups and refined initially against
data from 10 to 4 Å resolution and finally against data from
10 Å to the high-resolution shell (2.6 Å for RT-2a, 2.8 Å for
RT-2b and RT-5 and 2.9 Å for RT-6).
Due to the low ratio of reflections to parameters, strong
restraints on atom positions greater than 25 Å from the CR
atom of Tyr188 in the NNRTI binding site were employed in
all subsequent refinements after the nine domain rigid body
refinements. Each of the models from the rigid body refinement
were subject to restrained positional refinement using data
from 30-2.6 Å resolution, for the RT-2a complex, 30-2.8 Å
for both the RT-2b and RT-5 complexes and 25-2.9 Å for
RT-6. To compensate for the weakness of the high-resolution
reflections and the anisotropy in the data, each of the RT-
quinolone complex datasets were sharpened using anisotropic
B factor scaling against the structure factors calculated from
a positional refined model where the atomic B factors were
reduced. The sharpened datasets were used in further posi-
tional and restrained individual B factor refinement, incorpo-
rating a bulk solvent correction.
The |Fobsd| - |Fcalcd| difference maps for each of the RT-
quinolone complexes clearly showed each of the inhibitors
bound at their respective NNRTI binding site. Models of the
complexes were built into the 2|Fobsd| - |Fcalcd| and |Fobsd| -
|Fcalcd| electron density maps using the program FRODO67 on
an Evans and Sutherland ESV workstation. The models were
improved with iterative rounds of manual model building and
positional refinement and restrained individual temperature
factor refinements. All four of the structures have been
assigned the secondary structure nomenclature as defined for
1
product 216-217 °C. H NMR (CDCl3) 11.45 (s, 1H, NH), 8.0
(s, 1H, 5-H), 7.45 (d, J ) 5 Hz, 1H, 7-H), 7.23 (d, J ) 5 Hz,
1H, 8-H), 2.92 (t, J ) 5 Hz, 2H, propyl 1′-CH2), 1.66 (m, 2H,
propyl 2′-CH2), 1.05 (t, J ) 4 Hz, 3H, propyl, 3′-CH3). Anal.
(C12H11NOClBr) C, H, N.
6-Chloro-4-Cyclohexylthio-3-propyl-1H-quinol-2-one
(compound 5). Sodium hydride (60% oil dispersion, 4 mmol;
0.16 g; 4 equiv) was mixed with cyclohexyl mercaptan (8 mmol;
0.98 mL; 8 equiv) in dry DMF (5 mL) at 0 °C for 45 min. Solid
4-bromo-quinol-2-one compound (300 mg; 0.001mol) was dis-
solved into dry DMF (5 mL) and added to the sodium
cyclohexyl mecaptan salt. The reaction mixture was heated
to 140 °C, under a nitrogen atmosphere, for 20 h. On cooling,
a solid white precipitate was filtered off and volatiles removed
in vacuo. The remaining solid was dissolved in ethyl acetate
(40 mL). On addition of a small amount of water (10 mL) to
wash the organic layer, a white precipitate was produced. The
solution was gently heated to redissolve the white precipitate.
The water layer was removed by use of a separating funnel
and the remaining organic layer dried with solid anhydrous
sodium sulfate. Volatiles were removed in vacuo, and the
product was recrystallized from ethyl acetate to give white
crystals of pure product (209 mg; yield 62%). Melting point of
white crystals 246-247 °C. 1H NMR 200 MHz (CDCl3) δ 10.89
(s, 1H, NH), δ 8.21 (s, 1H, 5-H), δ 7.41 (d, J ) 5 Hz, 1H, 7-H),
δ 7.20 (d, J ) 5 Hz, 1H, 8-H), δ 3.05 (t, J ) 5 Hz, 2H, propyl
1′-CH2), δ 3.0 (m, 1H, cyclohexylthio SCH), δ 1.9 (m, 2H,
cyclohexyl C2ax, C6ax), δ 1.78 (m, 2H, cyclohexyl C2eq, C6eq), δ
1.57 (m, 2H, propyl 2′-CH2), δ 1.25 (m, 6H, cyclohexyl C3, C4,
C5), δ 1.05 (t, J ) 4 Hz, 3H, propyl 3′-CH3). Structure confirmed
by NOE of protons on C5 quinolone and C2 and C6 cyclohexyl.
Anal. (C18H22NOSCl) C, H, N.
6-Chloro-4-(cyclohexylsulfinyl)-3-propyl-1H-quinol-2-
one (compound 6). 4-Cyclohexylthioquinolone (5) (80 mg;
0.239 mmol) was dissolved in 10 mL of 2-propanol. To this
solution was added 3-chloroperbenzoic acid (55% dispersion;
0.165 g; 0.526 mmol) mixed with 5 mL of 2-propanol. The
reaction mixture was stirred for 1 h at room temperature,
washed with saturated sodium hydrogencarbonate solution
and dried over solid anhydrous sodium sulfate. The volume of
2-propanol was reduced in vacuo, gently heated and left to cool.
Pure product was obtained from the solution in the form of
white crystal (50 mg; yield 59.6%). Melting point of crystals
215-216 °C. 1H NMR 200 MHz (CDCl3) 11.3 (s, 1H, NH), 8.7
(s, 1H, 5-H), 7.5 (d, J ) 5 Hz, 1H, 7-H), 7.28 (d, J ) 5 Hz, 1H,
8-H), 3.45 (m, 1H, cyclohexyl SCH), 3.0 (m, 2H, propyl 1′-CH2),
2.78 (m, 1H, cyclohexyl C2ax, C6ax), 2.45 (m, 1H, cyclohexyl C2eq
or C6eq), 2.40 (m, 1H, cyclohexyl C2eq or C6eq), 2.03 (m, 1H,
cyclohexyl C2eq or C6eq), 1.98 (m, 1H, cyclohexyl C2eq or C6eq),
1.58 (m, 2H, propyl 2′-CH2), 1.45-1.20 (m, 6H, cyclohexyl-H
C3, C4, C5), 1.08 (t, J ) 1m, 3H, propyl 3′-CH3). IR ν 1049.16
(sulfoxide SO). Anal. (C18H22NO2SCl) C, H, N.
Reverse Transcriptase Assay. Inhibition of recombinant
HIV-1 reverse transcriptase was performed in a manner
similar to previously published protocols57,58 by determining
the polymerase activity by measuring the incorporation of
radiolabeled nucleotide into the primer DNA strand.
Antiviral Assay. The compounds were tested for their
ability to inhibit wild-type HIV-1 (strain IIIB) and nevirapine-
resistant HIV-1 (containing the Tyr181Cys mutation in the
RT sequence). The activity of the compounds against HIV-1
in whole cell culture were measured using a propidium iodide-
based colorimeteric assay, to estimate DNA content, in human
T-cell lymphotropic virus-transformed cell line MT4 in a
following previous reported protocols.59-62
Crystallization. Crystals of RT complexed with the qui-
nolone compounds 2a, 2b, 5 and 6 were grown following the
reported procedure.63 Briefly, the crystals were grown by
equilibration against reservoirs of 7-10% PEG 3350 and
citrate-phosphate buffer (24.25 mM citric acid, 51.5 mM Na2-

Inhibitors of HIV-1 Reverse Transcriptase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5921
Table 3. Statistics for Crystallographic Structure Determinations
RT-2a
RT-2b
RT-5
RT-6
Data Collection Details
SRS PX7.2
data collection site
detector
SRS PX7.2
MAR 18 cm
1.488
SRS PX7.2
MAR 18 cm
1.488
ESRF BM-14
MAR 345 mm
0.918
MAR 18 cm
1.488
wavelength (Å)
unit cell dimensions (a, b, c in Å)
resolution range (Å)
observations
138.3, 115.1, 65.2
30.0-2.60
73067
138.0, 115.2, 65.3
30.0-2.80
70383
137.8, 115.2, 65.0
30.0-2.80
65563
137.5, 115.3, 65.4
25.0-2.90
88607
unique reflections
completeness (%)
average I/σ(I)
28424
24466
24383
23603
86.1
92.1
93.8
99.2
9.5
9.8
9.2
19.8
a
Rmerge
0.087
0.103
0.104
0.052
Outer Resolution Shell
resolution range (Å)
unique reflections
completeness (%)
average I/σ(I)
2.69-2.60
2405
74.6
1.2
2.90-2.80
2186
83.3
1.6
2.90-2.80
2127
84.7
1.4
2.97-2.90
1460
92.3
2.5
Refinement Statistics
resolution range (Å)
30.0-2.60
27006/1380
0.209/0.282
0.200
30.0-2.80
23198/1171
0.205/0.286
0.197
30.0-2.80
23194/1169
0.206/0.281
0.200
25.0-2.90
22415/1135
0.221/0.282
0.218
no. of reflections(working/test)
R-factorb (Rwork/Rfree
R-factorb (all data)
)
no. atoms (protein/inhibitor/water)
rms bond length deviation (Å)
rms bond angle deviation (deg)
mean B-factor (Å2)c
7550/22/37
0.0114
1.63
7576/22/-
0.0101
7576/22/-
0.0101
7567/23/-
0.0105
1.53
1.55
1.60
63/69/41/40
3.8
58/65/41/-
3.5
64/70/48/-
3.6
67/70/65/-
3.6
rms backbone B-factor deviation (Å2)
PDB code
1TKT
1TL3
1TKZ
1TL1
a Rmerge ) ∑|I - I |/∑ I . b R-factor ) ∑|Fo - Fc|/∑Fo. c Mean B-factor for main-chain, side-chain, inhibitor and water molecules.
the RT-nevirapine structure.6 Overall the electron density for
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C.; et al. non-nucleoside HIV-1 reverse transcriptase (RT)
each of the complexes was well defined, but a few small
inhibitors: past, present and future perspectives. Curr. Pharm.
stretches of sequence were omitted from the models where the
electron density maps showed disorder. Sections omitted from
Des. 2002, 8, 615-657.
(11) De Clercq, E. Perspectives for non-nucleoside reverse tran-
the models were as follows: RT-2a complex, residues 1-3,
64-70, 444-454, 538-560 from p66 and residues 1-6, 89-
94, 214-224, 357-361, 428-440 from p51; RT-2b complex,
residues 1-3, 64-69, 444-454, 538-560 from p66 and resi-
dues 1-6, 89-94, 215-224, 357-361, 428-440 from p51;
RT-5 complex, residues 1-3, 64-69, 444-454, 538-560 from
p66 and residues 1-5, 89-94, 215-224, 357-361, 428-440
from p51; RT-6 complex, residues 1-3, 64-69, 444-454,
538-560 from p66 and residues 1-6, 65-67, 89-94, 215-224,
357-361, 431-440 from p51. Water molecules were only
incorporated into the high resolution, RT-2a model, in posi-
tions where there was well-defined electron density and
hydrogen-bonding possibilities. Refinement and model quality
statistics are listed in Table 3.
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