Urea-PETT compounds as a new class of HIV-1 reverse transcriptase inhibitors. 3. Synthesis and further structure-activity relationship studies of PETT analogues

Article abstract of DOI:10.1021/jm990095j

The further development of allosteric HIV-1 RT inhibitors in the urea analogue series of PETT (phenylethylthiazolylthiourea) derivatives is described here. The series includes derivatives with an ethyl linker (1-5) and racemic (6-16) and enantiomeric (17-

Full text of DOI:10.1021/jm990095j

4150
J . Med. Chem. 1999, 42, 4150-4160
Ur ea -P ETT Com p ou n d s a s a New Cla ss of HIV-1 Rever se Tr a n scr ip ta se
In h ibitor s. 3. Syn th esis a n d F u r th er Str u ctu r e-Activity Rela tion sh ip Stu d ies of
P ETT An a logu es
Marita Ho¨gberg,*,† Christer Sahlberg,† Per Engelhardt,† Rolf Nore´en,† J ussi Kangasmetsa¨,†
Nils Gunnar J ohansson,† Bo O¨ berg,†,‡ Lotta Vrang,† Hong Zhang,† Britt-Louise Sahlberg,† Torsten Unge,*,§
Seved Lo¨vgren,§ Kerstin Fridborg,§ and Kristina Ba¨ckbro§
Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden, MTC, Center for Microbiology and Tumorbiology, The Karolinska
Institute, Stockholm, Sweden, and Department of Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden
Received March 3, 1999
The further development of allosteric HIV-1 RT inhibitors in the urea analogue series of PETT
(phenylethylthiazolylthiourea) derivatives is described here. The series includes derivatives
with an ethyl linker (1-5) and racemic (6-16) and enantiomeric (17-20) cis-cyclopropane
compounds. The antiviral activity was determined both at the RT level and in cell culture on
both wild-type and mutant forms of HIV-1. Most compounds have anti-HIV-1 activity on the
wt in the nanomolar range. Resistant HIV-1 was selected in vitro for some of the compounds,
and the time for resistant HIV-1 to develop was longer for urea-PETT compounds than it was
for reference compounds. Preliminary pharmacokinetics in rats showed that compound 18 is
orally bioavailable and penetrates well into the brain. The three-dimensional structure of
complexes between HIV-1 RT and two enantiomeric compounds (17 and 18) have been
determined. The structures show similar binding in the NNI binding pocket. The propionylphe-
nyl moieties of both inhibitors show perfect stacking to tyrosine residues 181 and 188. The
cyclopropyl moiety of the (+)-enantiomer 18 exhibits optimal packing distances for the
interactions with leucine residue 100 and valine residue 179.
In tr od u ction
in combination with nucleoside analogues. A number
of such agents are or have been in clinical trials
including 3,4-dihydroquinoxaline-2(1H)-thione deriva-
tive (HBY 097),5 R-anilinophenylacetamide (R-APA,
loviridine),6 tetrahydroimidazobenzodiazepinthione (tivi-
rapine),7 and isopropyluracil derivatives (HEPT, MKC-
442),8 and some of the other more recent additions to
the group include alkenyldiarylmethane derivative
(ADAM),9 1H-imidazol-2-ylmethyl carbamate derivative
(S-1153),10 and thiocarboxanilide derivatives (UC 84,
UC 38).11
Because NNRTIs interact with a specific binding site
on the enzyme, any slight variation brought about by a
single point mutation can have a significant impact on
the sensitivity of virus to members of this group, and
high-level resistance can develop quickly.1 The rapid
development of resistance to NNRTIs is an important
factor influencing the failure of today’s treatment ap-
proaches to HIV infection.12a,b As part of the preclinical
evaluation of new treatments for HIV, the activity of
all new agents are investigated in combination with
other antiretroviral compounds against a number of
both laboratory and clinical HIV isolates, to evaluate
potential synergistic or antagonistic activity.13,14 Com-
bination therapy can slow the selection of drug-resistant
strains and is associated with significant clinical ben-
efit.15 Several combinations are used, and several are
currently undergoing clinical evaluation, including com-
binations of two or more RT inhibitors and combinations
of RT inhibitors with protease inhibitors.2d,16 There is
a further need for the discovery and development of
improved NNRTIs.
The life cycle of HIV has been extensively studied,
and a number of stages identified for possible interven-
tion to prevent viral replication.1 These include forma-
tion of proviral DNA by the reverse transcriptase
enzyme (RT), integration of proviral DNA into the host
DNA by the integrase enzyme, and cleavage of the
precursor viral proteins by the protease enzyme. Clini-
cally relevant agents which have been successfully
developed are RT inhibitors and protease inhibitors. RT
inhibitors are divided into two categories: nucleoside
(NRTIs) and nonnucleoside (NNRTIs) reverse tran-
scriptase inhibitors. NRTIs are phosphorylated by cel-
lular enzymes to an active metabolite, analogous to a
natural nucleotide, for incorporation into the growing
viral DNA chain by HIV RT in a competitive reaction.
Once inserted into the chain, no further nucleosides can
be added, and therefore viral replication ceases. NNRTIs
are a diverse group of compounds which inhibit HIV RT
by an allosteric mechanism of action. NNRTIs are not
incorporated into the growing strand of HIV DNA but
directly inhibit the viral RT by binding to an allosteric
site.
Thus far, three NNRTIs, nevirapine (Viramune),2
delavirdine (Rescriptor),3 and DMP-266 (Sustiva),4 have
been approved for treatment of HIV-1-infected adults
* Author for correspondence: M. Ho¨gberg; e-mail, marita.hogberg@
medivir.se. Author for crystallography: T. Unge, Department of
Molecular Biology, BMC, Box 590, S-751 24 Uppsala, Sweden; e-mail,
torsten@alpha2.bmc.uu.se.
† Medivir AB.
‡ The Karolinska Institute.
§ Uppsala University.
We have recently described a series of thiourea
10.1021/jm990095j CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/14/1999

Urea-PETT Compounds as New HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4151
Sch em e 1
of this reaction was 9%. The corresponding coupling
reactions with 2-amino-5-bromopyridine and 2-amino-
5-chloropyridine gave higher yields.
The synthesis of compound 17 described in Scheme 3
is representative for the enantiomeric cyclopropane
compounds 17-20. Acylation of 3-fluorophenol (34) with
propionyl chloride followed by Fries rearrangement with
AlCl3 afforded intermediate 36 in very high yield
without formation of any regioisomers. Compound 36
was alkylated with methyl iodide to give 4′-fluoro-2′-
methoxypropiophenone (37). This compound was acetal-
protected by reaction with ethylene glycol, and further
reaction of the resulting intermediate 38 with n-BuLi
and DMF afforded aldehyde 39. A Wittig reaction on
compound 39 gave styrene 40. The next step, asym-
metric cyclopropanation reaction with ethyl diazoac-
etate, catalyzed by copper(I) triflate and enantiomeric
ligand22 gave ester 41 (cis/ trans ratio of 3.0, cis purified
by column chromatography, the yield of pure cis-ester
was 45%). It was notable that the cis/ trans ratio of the
product in this asymmetric cyclopropanation reaction
was reversed compared to the above-mentioned. We
have no explanation for this high cis-selectivity, but one
speculation is that substituents of the phenyl ring
stabilize a transition state that leads to the cis-
compound. The corresponding reaction using styrene
gives a cis/ trans ratio of 0.3.22 Cyclopropanecarboxylic
acid 43 was obtained by hydrolysis of cis-ester 41 first
with HCl/dioxane/water to give deketalized compound
42, followed by hydrolysis with lithium hydroxide in
methanol/water. Curtius reaction on the acid 43 fol-
lowed by reaction with 2-amino-5-bromopyridine as
outlined also in Scheme 2 yielded compound 44, which
was demethylated with boron trichloride in methylene
chloride to afford the desired compound 17. The enan-
tiomeric purity of this compound was determined by
high-performance liquid chromatography (HPLC) on a
Chiral-AGP column to be 98.6% ee.
derivatives, the PETT (phenylethylthiazolylthiourea)
compounds, which belong to the NNRTIs.17-20 The lead
compound in this series was LY73497 which inhibited
wild-type HIV-1 with ED50 of 1.3 µM and HIV-1 RT with
IC50 of 0.9 µM. The first generation of PETT compounds
resulted in trovirdine with ED50 of 20 nM and IC50 of
15 nM. The further optimization of the PETT series has
led to urea analogues briefly described in our prelimi-
nary paper.21 We can now more comprehensively con-
firm that urea analogues have better toxicological and
pharmacokinetic properties than the thiourea com-
pounds and their antiviral activity in cell culture in the
presence of human serum is better than that of the
thiourea compounds.
In this study we report the synthesis and anti-HIV-1
activity of 20 urea-PETT compounds including both
phenethyl and phenylcyclopropyl derivatives, develop-
ment of resistant HIV-1 in vitro for some of the
compounds, and also pharmacokinetic studies for one
of the compounds. The three-dimensional structure of
the complexes between HIV-1 RT and an enantiomeric
pair of a PETT cyclopropane compound has been
determined.
Ch em istr y
Compounds 1-5 (Table 1) with an ethyl linker
described in the present study were synthesized accord-
ing to Scheme 1. Conversion of the previously reported
thiourea compounds20 to the corresponding urea ana-
logues was achieved either with N-bromosuccinimide
(NBS) or with silver nitrate (AgNO3). The yields of these
reactions were only about 30%, but both agents were
efficient and rapid to afford these urea compounds. It
was important that the products of these reactions were
purified carefully, because it was known that the
starting materials were highly potent HIV-1 RT inhibi-
tors.
A representative synthesis for racemic cyclopropane
compounds 6-16 is described in Scheme 2. Ethylation
of 2-chloro-4-fluorophenol (28) with ethyl iodide in the
presence of potassium carbonate in refluxing acetone
afforded intermediate 29 which was converted to alde-
hyde 30 using n-BuLi and DMF. 2-Chloro-3-ethoxy-6-
fluorostyrene (31) was prepared from 2-chloro-3-ethoxy-
6-fluorobenzaldehyde (30) by reaction with an appropriate
Wittig reagent. The yields of the three first steps were
high: 89-98%. Cyclopropanation of the styrene 31 with
ethyl diazoacetate in dichloroethane using copper(I)
iodide and palladium(II) acetate as catalysts afforded
the ester 32 (cis/ trans ratio 0.45). cis-Ester was purified
by column chromatography (the yield of pure cis-ester
was 12%) and then hydrolyzed with potassium hydrox-
ide in ethanol/water to give cyclopropanecarboxylic acid
33. Curtius reaction on the acid using diphenyl phos-
phorazidate followed by reaction with 2-amino-5-cyano-
pyridine afforded compound 10. The unoptimized yield
Biologica l Resu lts a n d Discu ssion
An tivir a l Activity. All compounds 1-20 in this
study were tested in HIV-1 RT enzyme assays with wild-
type and three constructed mutants, Ile100, Cys181,23
and Asn103, and in cell culture using MT-4 cells24 and
both wild-type virus and virus containing a mutation
in residue 100, 181, or 103. The amino acid substitutions
Ile100 and Cys181 were observed at an early stage when
studying PETT compounds in cell culture,20 and virus
carrying Cys181 or Asn103 has been identified in
clinical isolates after administration of nevirapine,
delavirdine, and DMP-266.1,12a,b The most active com-
pounds were tested in a cell culture assay containing
50% human serum to simulate protein binding in the
physiological environment. DMP-266, HBY-097, nevi-
rapine, and delavirdine were chosen as reference com-
pounds. The IC50 and ED50 values from these assays are
presented in Table 1.
All the present compounds can be divided into four
groups: (i) phenethyl derivatives, compounds 1-5, (ii)
racemic cyclopropane derivatives with 2,6-dihalogen-3-
alkoxy or 2,6-dihalogen-3-hydroxy substitution of the
phenyl ring, compounds 6-12, (iii) racemic cyclopropane
derivatives with 2-hydroxy-3-acetyl or propionyl-6-fluoro

4152 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ho¨gberg et al.
Sch em e 2a
a
The compounds are racemic; only one enantiomer of the cis-compound is shown.
Sch em e 3
As we have found earlier the cis-cyclopropane deriva-
tives are more potent than the ethyl-linked analogues
especially on mutants.21 cis-Cyclopropane compounds
have a more restricted conformation compared with
ethyl analogues which appears to lead to better interac-
tion with the mutant enzymes. The most favorable
substitution of the phenyl ring in the second group,
which contained cis-cyclopropane derivatives, was the
2-fluoro-3-ethoxy-6-fluoro substitution of compounds 7
and 8. Replacement of the pyridyl ring on the right-hand
side of the molecule with a pyridazine ring, compounds
9 and 11, and replacement of the 3-ethoxy substituent
with a 3-hydroxy substituent on the phenyl ring,
compound 12, led to decreased activity.
Racemic cis-cyclopropane derivatives described in this
paper, compounds 13-16 of the third group, represent
a new type of 2-hydroxy-3-acetyl- or propionyl-6-fluo-
rophenyl ring substitution. They represent a very potent
group of PETT-urea compounds on both wild-type and
mutant HIV-1 RT and virus. All of them were more
active on mutant HIV-1 RT (Asn103) than any com-
pound hitherto. Hydrogen bonding between the 2-hy-
droxy and 3-acetyl or 3-propionyl substituent of the
phenyl ring contributes to the stability of the compounds
and consequently is beneficial for activity.
The last group, compounds 17-20, included pure (-)-
and (+)-enantiomers of 2-hydroxy-3-propionyl-6-fluo-
rophenyl cis-cyclopropane derivatives with bromo- and
chloropyridine rings on the opposite side of the urea
function, herein referred to as the right-hand side of the
molecule. Both (-)- and (+)-enantiomers were very
active. (+)-Enantiomers, compounds 18 and 20, were
about 10-fold more potent on RT mutant (Ile100) and
2-3-fold more potent on mutant HIV-1 (Ile100) than
(-)-enantiomers, compounds 17 and 19, while (-)-
enantiomers were 2-4-fold more potent on mutant
HIV-1 (Asn103) than (+)-enantiomers.
Enantiomeric cis-cyclopropane compounds 17-20 were
also tested in a wild-type HIV-1 assay which contained
50% human serum. The same assay in our earlier work
contained 15% human serum.21 The presence of human
serum resulted in a slight decrease in activity, which
was less than that observed with delavirdine and DMP-
266.
3-D Str u ctu r e of HIV-1 RT In h ibitor Com p lexes.
The three-dimensional structures of the complexes
between HIV-1 RT and inhibitors 17 and 18 have been
determined with 2.9 and 2.8 Å diffraction data, respec-
tively. The structures show similar binding of the two
inhibitors in the NNI binding pocket (Figure 1). The
substitution of the phenyl ring, compounds 13-16, and
(iv) enantiomeric cyclopropane derivatives, compounds
17-20.
Compound 2 with a 2-fluoro-3,6-dimethoxy-substi-
tuted phenyl ring was the most active of the phenethyl
derivatives in the first group but not as active as its
thiourea analogue as we have reported earlier.20 3-Acetyl
substitution of the phenyl ring in compounds 4 and 5
decreased activity significantly on both wild-type and
mutant HIV-1 RT and virus. Compound 1 with a
dimethylamino group in the 3-position of the phenyl ring
was synthesized to enhance water solubility, but this
substituent was not favorable for activity.

Urea-PETT Compounds as New HIV-1 RT Inhibitors
Ta ble 1. Inhibition of HIV-1 RT and HIV-1 (IIIB) Replication
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4153
HIV-1 RT (rCdG), IC50 (µM)b
wt Ile100 Cys181 Asn103 wt
0.0250 >2.5
HIV-1 MT-4 cells, ED50 (µM)c
wtd
Ile100 Cys181 Asn103
0.500 nde
0.224 0.028 nd
optical
activity
compd
R2
R3
R6
Ar
1
2
3
4
5
6
7
8
F
F
NMe2
OMe
OEt
F
5-bromopyrid-2-yl
1.5
0.140
>2.5
>20
3.8
>20
>5
>20
1.13
OMe 5-chloropyrid-2-yl
0.0030
0.0040
0.224
0.140
1.68
26.2
11.2
0.081
0.052
0.027
1.0
Cl
F
F
5-bromopyrid-2-yl
5-chloropyrid-2-yl
0.240 >2.4
0.070 nd
0.819 nd
0.273 nd
>10
>5
>5
OMe COMe
12.7
10.1
0.014
0.021
0.020
1.0
>2.7
>2.7
0.473 0.031 nd
0.237 0.009 nd
0.192 0.004 nd
>5
>5
>5
F
F
F
F
COMe OMe 5-chloropyrid-2-yl
0.109
>20
>20
3.28
OEt
OEt
OEt
OEt
OEt
OEt
OH
F
5-chloropyrid-2-yl
5-chloropyrid-2-yl
5-cyanopyrid-2-yl
(+,-) 0.0016
(+,-) 0.0013
(+,-) 0.0027
0.816
0.139
0.053
0.163
0.104
0.200
0.544
0.780
0.400
Cl
Cl
Cl
F
9
F
5-chloropyridazin-2-yl (+,-) 0.0052
5-cyanopyrid-2-yl (+,-) 0.0043
5-chloropyridazin-2-yl (+,-) 0.0160
>2.6
0.183 0.008 nd
0.018 nd
>5
0.234
4.4
>5
>5
10
11
12
13
14
15
16
17
18
19
20
Cl
Cl
Cl
0.200
0.140
4.1
0.130
0.270 >5
F
F
F
F
F
F
F
F
0.260 >2.6
0.078 nd
0.110 nd
2.3
>5
>5
>5
5-chloropyrid-2-yl
5-chloropyrid-2-yl
5-cyanopyrid-2-yl
5-cyanopyrid-2-yl
5-bromopyrid-2-yl
5-bromopyrid-2-yl
5-bromopyrid-2-yl
5-chloropyrid-2-yl
5-chloropyrid-2-yl
(+,-) 0.135
(+,-) 0.0090
(+,-) 0.0068
(+,-) 0.0053
(+,-) 0.0035
(-) 0.0038
(+) 0.0048
(-) 0.0032
(+) 0.0027
0.0040
4.3
>2.7
>5
OH COEt
OH COEt
OH COMe
OH COMe
OH COEt
OH COEt
OH COEt
OH COEt
0.076
0.032
0.013
0.018
0.171
0.014
0.115
0.014
0.050
0.009
0.130
0.051
0.016
0.016
0.008
0.016
0.010
0.019
0.004
0.006
0.009
0.114
0.057 0.027 nd
0.097 0.003 nd
0.098 0.008 nd
0.070 0.021 nd
0.083 0.008 0.058
0.041 0.012 0.056
0.082 0.009 0.051
0.077 0.013 0.080
0.061 0.005 0.050
0.056 0.009 0.320
0.625 0.176 >5
0.220
0.069
0.040
0.050
0.100
0.053
0.100
0.075
0.060
0.072
0.340
0.046
0.230
0.113
0.069
0.095
0.100
0.100
0.016
0.073
0.350
0.137
0.104
0.561
0.080
0.358
0.200
0.470
0.140
0.500
F
F
DMP-266
HBY-097
delavirdine
nevirapine
0.008
0.0013
>5
1.6
>5
>20
>5
>20
0.170
17.1
>50
3.8
0.230 0.850
a
b
All compounds have the cis-configuration, + and - reflecting the 1S,2S and 1R,2R configurations respectively. The HIV-1 RT assay
which used (poly)rC‚(oligo)dG as the template/primer is described in ref 23. c Anti-HIV activity assay: MT4 cells (human T cell line)
grown in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin were seeded into 96-well microplates
(2 × 104 cells/well) and infected with 10-20 TCID50 of HIV-1 (IIIb) per well. Test compounds in different concentrations were added. The
cultures were incubated at 37 °C in CO2 atmosphere, and the viability of cells was determined at day 5 or 6 with XTT vital dye.24 The
anti-HIV-1 activity was measured as the reduction in cytopathic effect caused by the virus. Mutant HIV-1 was achieved by passaging the
virus in MT4 cells in stepwise increasing concentrations of different NNRTIs. When virus was growing in highest possible, nontoxic
concentration of the compound, it was passaged once without compound. Cellular DNA was analyzed with respect to nucleotide sequence
d
in the HIV-1 RT gene, and the supernatant was frozen in aliquots at -70 °C for cross-resistance studies. The assay contains 50%
human AB serum. e Not determined.
conformation of both inhibitors is stabilized by a hy-
drogen bond network involving one amide hydrogen of
the urea moiety, the nitrogen in the pyridine ring, the
hydrogen of the phenolic hydroxyl, and the carbonyl
oxygen of the propionyl moiety. The hydrogen bond
interactions to the protein differ between compounds 17
and 18. Compound 18 has two hydrogen bonds to the
protein. One of the urea nitrogen atoms and the carbo-
nyl oxygen atom bind to the main chain carbonyl oxygen
(2.6 Å) and amide nitrogen of K101 (3.0 Å), respectively.
Compound 17 has only one hydrogen bond to the
protein, namely between one of the urea nitrogens and
the carbonyl oxygen of K101 (2.8 Å).
The propionylphenyl moiety of the inhibitor stacks to
the tyrosine residues 181 and 188 and shows also van
der Waals interaction perpendicular to W229. The
pyridine moiety close packs to P236 and the oxygen of
H235 on the side and to K103 side and main chain
atoms. The packing to the H235 oxygen and to the
phenolic oxygen of Y318 forces the plane of the pyridine
ring to deviate from planarity with the plane of the urea
moiety by an angle of 10°. The inhibitor is covered from
above by the side chains of L100, L234, and Y318. V106
interacts with the inhibitor from below. The bromo
moiety of the pyridyl ring and the propionylphenyl
moiety of the phenyl ring pack against F227 and the
backbone of P235. The fluoro substituent of the phenyl
ring packs in both complexes against the carbon chain
of E1138 (residue 138 in the p51 subunit) (Figure 2).
The orientation of the cyclopropyl ring differs between
17 and 18. It points downward relative to the plane of
the inhibitor in compound 17, and in the enantiomeric
compound 18 it points up. The cyclopropyl ring close
packs to L100, V179, and Y181 in compound 17. In
addition to these interactions this ring in compound 18
close packs extensively to E1138 (Figure 2). This causes
a small alteration in the positioning of the propio-
nylphenyl moiety.
Stu d y of Resista n ce Develop m en t. Selection of
resistant HIV-1 in vitro was performed for pheneth-
ylurea compound 2 and racemic cyclopropane com-
pounds 8 and 13. Delavirdine and nevirapine were used
as reference compounds. A curve showing maximal
concentration of test compound permitting virus growth
in each passage is presented in Figure 3. The time
before virus appeared in 0.1 µg/mL delavirdine, nevi-
rapine, and compounds 2, 8, and 13 was 2, 5, 6, 13, and
17 weeks, respectively. There is a correlation between
the activity on mutant forms of HIV-1 and the length
of time for resistance development for urea-PETT
compounds. The results depicted in Figure 3 clearly
demonstrate the superiority of compounds 8 and 13
compared to the reference compounds in terms of
resistance development. This makes the cyclopropyl-

4154 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ho¨gberg et al.

Urea-PETT Compounds as New HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4155
F igu r e 3. Selection of resistant HIV-1 in vitro. MT4 cells were
seeded into microplates (2 × 104 cells/well) and infected with
10-20 TCID50/well of HIV-1(IIIb) in the presence of 5-fold
serial dilutions of a test compound. RT activity was determined
in the supernatant after 6 days. Virus replicating in wells
containing subinhibitory concentrations of test compound was
passaged into (a) cells without test compound, (b) cells with
test compound at the same concentration as used in the
preceding selection, and (c) cells with test compound at 5-fold
higher concentration. Virus was passaged once a week until
replicating virus was detected in cells cultured in the presence
of the highest nontoxic concentration of the test compound.
Finally, virus was grown to give a virus stock, and sequence
analysis of the RT gene was performed.
were analyzed. High levels of 18 were detected in both
brain and plasma: i.e., at 18.5 min the concentration of
18 was 1.2 µg/mL in plasma and 5 µg/g in brain tissue,
at 62 min 1.2 µg/mL and 6 µg/g, respectively, and at
120 min 0.7 µg/mL and 2 µg/g, respectively. These
results clearly indicate that compound 18 penetrates
well into the brain, and levels comparable with the
concentration in plasma are detected. This compound
was also dosed orally at 11.4 mg/kg in 10% ethanol, 10%
Capmul, and 80% oleic acid. These studies show that
this compound is orally available, and a rather flat AUC
curve was observed, thus making it hard to calculate
the absolute oral availability. Cmax values of 0.07 µg/
mL (0.17 µM) at 4 and 6 h were detected indicating that
the compound is slowly absorbed which is most likely
due to its expected poor water solubility.
F igu r e 2. Cyclopropyl moiety, which differs in conformation
between the two enantiomeric inhibitors 17 (a) and 18 (b), has
close-packing interactions (dotted lines) with L100 and V179
and Y181 in both cases, but in addition to these interactions
the cyclopropyl moiety of 18 has extensive hydrophobic close-
packing interactions with E1138. The program Molscript was
used for drawing the figures.33
urea-PETT compounds very interesting potential drugs
against HIV/AIDS. Resistant virus from four cultures
exposed to compound 13 were isolated after 23 passages.
Sequence analysis of a portion of the RT gene coding
for aa 90-250 showed the following aa changes: (L100I,
Y181N), (L100I, Y181N), (K101E, I132L, Y181N), or
(Y181C, Y188H).
P h a r m a cok in etic Stu d ies. Compound 18 was se-
lected for preliminary pharmacokinetic studies in male
rats. These studies also included determination of
concentration in the brain since it is of great importance
that drugs against HIV/AIDS can penetrate into the
brain. A DMSO solution of compound 18 was intrave-
nously injected at a dose of 13 mg/kg into three rats.
The rats were sacrificed after 18.5, 62, and 120 min,
respectively, and concentrations in brain and plasma
Con clu sion s
In conclusion, cyclopropane urea analogues were more
potent than the ethyl-linked urea compounds. A study
of conformationally restricted cyclopropane thiourea
compounds has been communicated.25 In that study all
compounds with significant activity were cis-isomers
and they were more potent than phenethyl compounds
with the same substitution pattern on the phenyl ring
and the same right-hand side of the molecule. The same
pattern is found for urea cyclopropane compounds.
Compounds bearing 2-hydroxy-3-acetyl or propionyl-6-
F igu r e 1. Three-dimensional structure of the complex between HIV-1 RT and (a) 17 and (b) 18. Positioning of HIV-1 RT inhibitors
17 and 18 in the NNI binding site of the palm subdomain. In order to make the figure less complex, the residues L100, E1138
(residue 138 in the p51 subunit), H235, and P236 have been excluded, though they are important ligands. The two inhibitors
bind similarly. The striking features of the binding mode of these inhibitors are the internal stabilizing hydrogen bond network,
the hydrogen bonds between the inhibitor urea moiety and the main chain atoms of K101, and the efficient utilization of the van
der Waals interactions with the propionylphenyl moiety of the inhibitor and the aromatic amino acid residues Y181, Y188, and
W229. Moreover, there is an extensive utilization of close-packing interactions of predominantly hydrophobic nature between
these inhibitors and the protein. The program Molscript was used for drawing the figures.33

4156 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ho¨gberg et al.
from 2-fluoro-4-methoxyacetophenone.The thiourea compound
was converted to corresponding urea compound as described
for compound 4: mp 189 °C; 1H NMR (250 MHz, CDCl3) δ 9.18
(br s, 1H), 8.98 (br s, 1H), 7.96 (d, 1H), 7.80 (t, 1H), 7.52 (dd,
1H), 6.90 (d, 1H), 6.70 (d, 1H), 3.82 (s, 3H), 2.98 (m, 4H), 2.50
(d, 3H). Anal. (C17H17ClFN3O3) C, H, N.
Gen er a l P r oced u r e for Syn th esis of Cyclop r op a n e
Com p ou n d s 6-12: N-[cis-2-(2-Ch lor o-3-et h oxy-6-flu o-
r op h en yl)cyclop r op yl]-N′-[2-(5-cya n op yr id yl)]u r ea (10).
2-Chloro-4-fluorophenol (28) (25 g, 170.6 mmol), ethyl iodide
(27.3 mL, 341.2 mmol), and K2CO3 (47.2 g, 341.2 mmol) in
acetone (350 mL) were stirred at 55 °C overnight. The reaction
mixture was filtered and evaporated. n-Hexane was added and
the mixture was filtered again. The evaporation gave 29.26 g
(98%) of crude 1-chloro-2-ethoxy-5-fluorobenzene (29): 1H
NMR (250 MHz, CDCl3) δ 7.15-7.11 (m, 1H), 6.93-6.86 (m,
2H), 4.07 (q, 2H), 1.46 (t, 3H).
Compound 29 (29.26 g, 167.7 mmol) was dissolved in dry
THF (350 mL) and the mixture was cooled to -65 °C; 2.5 M
n-BuLi in hexanes (73.8 mL, 184.5 mmol) was added under
nitrogen and the mixture was stirred at the same temperature
for 0.5 h. DMF (14.3 mL, 184.5 mmol) was added to the
mixture and it was allowed to warm to room temperature. The
reaction mixture was poured onto ice and extracted with
diethyl ether. The organic layer was washed with 0.01 N HCl,
H2O, and brine, dried over Na2SO4, and evaporated. Crystal-
lization from n-hexane gave 30.8 g (91%) of 2-chloro-3-ethoxy-
6-fluorobenzaldehyde (30): 1H NMR (250 MHz, CDCl3) δ 10.43
(s, 1H), 7.12-7.01 (m, 2H), 4.09 (q, 2H), 1.46 (t, 3H).
Dry methyltriphenylphosphonium bromide (54.6 g, 151.6
mmol) was mixed with THF (750 mL) under nitrogen at room
temperature; 2.5 M n-BuLi in hexanes (65.6 mL, 164.0 mmol)
was added dropwise and the temperature of the reaction
mixture rose to 36 °C. The temperature was then allowed to
fall to 30 °C. Compound 30 (30.7 g, 151.6 mmol) was dissolved
in THF (100 mL) and added dropwise to the reaction mixture.
The temperature of the mixture rose to 45 °C. The mixture
was stirred for 20 min. It was evaporated to the volume of
about 200 mL, diethyl ether (700 mL) was added, and the
mixture was washed with H2O and brine, filtered twice
through an Al2O3 column, and evaporated to give 27.18 g (89%)
of 2-chloro-3-ethoxy-6-fluorostyrene (31): 1H NMR (250 MHz,
CDCl3) δ 6.99-6.75 (m, 3H), 6.01-5.92 (m, 1H), 5.69-5.63 (m,
1H), 4.08 (q, 2H), 1.46 (t, 3H).
fluoro substituents on the phenyl ring on the left-hand
side of the molecule and 5-bromo-, 5-chloro-, or 5-cyano-
pyridine on the right-hand side of the molecule were
most potent inhibitors of HIV-1. The time for resistance
development was longer for our compounds than for
reference compounds, and the pharmacokinetic proper-
ties were promising. Further development aimed at a
drug against HIV/AIDS is ongoing in our laboratories.
Exp er im en ta l Section
Ch em istr y. All melting points were determined on an
Electrothermal 9100 capillary melting point apparatus and are
uncorrected. Analytical results are indicated by atom symbols
and are within 0.4% of theoretical values except where
indicated. 1H NMR spectra were recorded on a Bruker AC-
250 spectrometer (250 MHz) using TMS as the internal
standard. Hewlett-Packard 5890 series II gas chromatograph
equipped with fused silica capillary column and thin-layer
chromatography with precoated silica gel 60 F256 from Merck
were used for monitoring reactions. The high-performance
liquid chromatography (HPLC) analyses of enantiomeric com-
pounds were performed on a Chiral-AGP column. Optical
rotations were determined on Perkin-Elmer polarimeter 241.
Merck silica gel, 230-400 mesh, was used for chromatography.
Yields were not optimized.
Gen er a l P r oced u r e for Syn th esis of 1-3 fr om Th io-
u r ea An a logu es. The corresponding thiourea compound (1
equiv) was dissolved in dioxane (7 mL/mmol) and H2O (1.4 mL/
mmol), and N-bromosuccinimide (4.7 equiv) dissolved in H2O
(2 mL/mmol) was added. The reaction mixture was stirred at
room temperature for 20 min. The mixture was washed with
0.01 N NaOH and brine, dried over Na2SO4, and evaporated.
The crude material was crystallized from acetone.
N-[2-(2,6-Diflu or o-3-(d im eth yla m in o)p h en eth yl)]-N′-[2-
(5-br om op yr id yl)]u r ea (1). The title compound was prepared
from the corresponding thiourea analogue:20 mp 135 °C; 1H
NMR (250 MHz, CDCl3) δ 9.65 (s, 1H), 9.21 (br s, 1H), 8.13 (d,
1H), 7.65 (dd, 1H), 6.86-6.75 (m, 3H), 3.63 (q, 2H), 3.01 (t,
2H), 2.75 (s, 6H). Anal. (C16H17BrF2N4O) H, N; C: calcd, 48.1;
found, 47.6.
N-[2-(3,6-Dim eth oxy-2-flu or op h en eth yl)]-N′-[2-(5-ch lo-
r op yr id yl)]u r ea (2). The title compound was prepared from
1
the corresponding thiourea analogue:20 mp 185 °C; H NMR
Compound 31 (27.0 g, 134.6 mmol) was dissolved in 1,2-
dichloroethane (400 mL), and Cu(I)I (40 mg) and Pd(OAc)2 (40
mg) were added. The mixture was heated to reflux. Ethyl
diazoacetate (28.3 mL, 269.2 mmol) in 1,2-dichloroethane (100
mL) was added dropwise under 2 h and the mixture was
refluxed for 1 h. The cooled mixture was evaporated, and the
cis-ester was purified by column chromatography (silica gel,
5-50% EtOAc in n-hexane) to give 4.0 g (12%) of the ethyl
ester of cis-2-(2-chloro-3-ethoxy-6-fluorophenyl)cyclopropan-
ecarboxylic acid (32): 1H NMR (250 MHz, CDCl3) δ 6.88 (t,
1H), 6.76 (dd, 1H), 4.01 (qq, 4H), 2.26-2.19 (m, 2H), 1.79-
1.68 (m, 1H), 1.62-1.53 (m, 1H), 1.43 (t, 3H), 1.10 (t, 3H).
Compound 32 (3.0 g, 11.6 mmol) was added to the mixture
of KOH (1.95 g, 34.8 mmol) in EtOH (30 mL) and H2O (10
mL). The mixture was refluxed overnight. The cooled mixture
was washed twice with n-hexane, acidified with concentrated
HCl, extracted with diethyl ether, dried over Na2SO4, and
evaporated to give 2.4 g (80%) of cis-2-(2-chloro-3-ethoxy-6-
fluorophenyl)cyclopropanecarboxylic acid (33): 1H NMR (250
MHz, CDCl3) δ 6.88 (t, 1H), 6.77 (dd, 1H), 4.05 (q, 2H), 2.32-
2.17 (m, 2H), 1.72-1.61 (m, 2H), 1.45 (t, 3H).
Compound 33 (0.3 g, 1.16 mmol), diphenyl phosphorazidate
(0.28 mL, 1.28 mmol), and triethylamine (0.18 mL, 1.28 mmol)
in toluene (7 mL) were refluxed for 40 min. 2-Amino-5-
cyanopyridine20 (152 mg, 1.28 mmol) in 2 mL of DMF was
added, and reflux was continued for 4 h. Toluene was evapo-
rated and diethyl ether was added to the mixture which was
washed with 0.01 N HCl, H2O, and brine, dried over Na2SO4,
and evaporated. Crude material was crystallized from acetone
and recrystallized from acetonitrile to give 40 mg (9%) of the
(250 MHz, CDCl3) δ 9.51 (br s, 1H), 9.07 (br s, 1H), 8.02 (d,
1H), 7.52 (dd, 1H), 6.88 (d, 1H), 6.79 (t, 1H), 6.53 (dd, 1H),
3.83 (s, 3H), 3.74 (s, 3H), 3.60 (q, 2H), 3.00 (dt, 2H). Anal.
(C16H17ClFN3O3) C, H, N.
N-[2-(2-Ch lor o-3-et h oxy-6-flu or op h en et h yl)]-N′-[2-(5-
br om op yr id yl)]u r ea (3). The title compound was prepared
from the corresponding thiourea analogue:20 mp 202 °C; 1H
NMR (250 MHz, DMSO-d6) δ 9.26 (br s, 1H), 8.29 (d, 1H), 7.92
(dd, 1H), 7.70 (t, 1H), 7.57 (d, 1H), 7.28-7.08 (m, 2H), 4.20 (q,
2H), 3.53 (q, 2H), 3.08 (t, 2H), 1.47 (t, 3H). Anal. (C16H16
BrClFN3O2) C, H, N.
-
N-[2-(3-Acetyl-6-flu or o-2-m eth oxyp h en eth yl)]-N′-[2-(5-
ch lor op yr id yl)]u r ea (4). The title compound was prepared
from the corresponding thiourea analogue20 which was syn-
thesized from 4-fluoro-2-methoxyacetophenone. The thiourea
analogue (1.0 g, 2.6 mmol) was dissolved in H2O/dioxane (1:
10, 20 mL). AgNO3 (2.0 g, 11.8 mmol) dissolved in H2O (5 mL)
was added. The reaction mixture was stirred at room temper-
ature for 2 h. EtOAc was added to the mixture and it was
filtered through Celite, washed with H2O and brine, dried over
Na2SO4, and evaporated. The crude material was purified by
column chromatography (silica gel, 50% EtOAc in n-hexane
followed by EtOAc) to give 0.3 g (32%) of the title compound:
1
mp 180 °C; H NMR (250 MHz, DMSO-d6) δ 9.74 (br s, 1H),
8.81 (br s, 1H), 8.06 (s, 1H), 7.62 (d, 1H), 7.54 (t, 1H), 7.09 (d,
1H), 6.88 (t, 1H), 3.80 (s, 3H), 3.58 (q, 2H), 3.01 (t, 2H), 2.56
(t, 3H). Anal. (C17H17ClFN3O3) C, H, N.
N-[2-(3-Acetyl-2-flu or o-6-m eth oxyp h en eth yl)]-N′-[2-(5-
ch lor op yr id yl)]u r ea (5). The title compound was prepared
from corresponding thiourea analogue20 which was synthesized

Urea-PETT Compounds as New HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4157
title compound: mp 201 °C; 1H NMR (250 MHz, CDCl3) δ 9.83
(br s, 1H), 9.36 (br s, 1H), 8.14 (d, 1H), 7.71 (dd, 1H), 6.98-
6.78 (m, 3H), 4.11 (q, 2H), 3.29 (m, 1H), 2.11 (q, 1H), 1.64 (q,
1H), 1.53 (t, 3H), 1.33 (m, 1H). Anal. (C18H16ClFN4O2) C, H,
N.
N-[cis-2-(2,6-Diflu or o-3-et h oxy)cyclop r op yl]-N′-[2-(5-
ch lor op yr id yl)]u r ea (6). The title compound was prepared
from 2,4-difluorophenol as described for compound 10. 2-Amino-
5-chloropyridine was used instead of 2-amino-5-cyanopyri-
dine: mp 158 °C; 1H NMR (250 MHz, CDCl3) δ 9.24 (br s, 1H),
8.89 (br s, 1H), 7.87 (d, 1H), 7.48 (dd, 1H), 6.86-6.73 (m, 3H),
4.04 (q, 2H), 3.30 (m, 1H), 2.10 (q, 1H), 1.54 (m, 1H), 1.45 (t,
3H), 1.35 (m, 1H). Anal. (C17H16ClF2N3O2) C, H, N.
13.2 (d, 1H), 9.81 (br s, 1H), 8.30 (d, 1H), 8.06-7.94 (m, 2H),
7.83 (br s, 1H), 7.41 (d, 1H), 6.84 (dd, 1H), 3.18-3.01 (m, 3H),
1.94 (q, 1H), 1.43 (m, 1H), 1.16-1.03 (m, 4H). Anal. (C19H17
-
FN4O3) C, H, N.
N-[cis-2-(3-Acetyl-6-flu or o-2-h yd r oxyp h en yl)cyclop r o-
p yl]-N′-[2-(5-cya n op yr id yl)]u r ea (15). The title compound
was prepared from 3-fluorophenol as described for compound
17. Acetyl chloride instead of propionyl chloride was used in
the first step and the cyclopropanation step was carried out
as described for compound 10: mp 236.4 °C; 1H NMR (250
MHz, DMSO-d6) δ 13.14 (s, 1H), 9.79 (br s, 1H), 8.34 (d, 1H),
8.04 (dd, 1H), 7.96 (dd, 1H), 7.71 (br s, 1H), 7.46 (d, 1H), 6.85
(dd, 1H), 3.07 (m, 1H), 2.65 (s, 3H), 1.94 (q, 1H), 1.42 (m, 1H),
1.09 (m, 1H). Anal. (C18H15FN4O3) H, N; C: calcd, 61.0; found,
60.5.
N-[cis-2-(3-Acetyl-6-flu or o-2-h yd r oxyp h en yl)cyclop r o-
p yl]-N′-[2-(5-br om op yr id yl)]u r ea (16). The title compound
was prepared as described for compound 15. 2-Amino-5-
bromopyridine was used instead of 2-amino-5-cyanopyridine:
mp 226 °C; 1H NMR (250 MHz, DMSO-d6) δ 13.13 (s, 1H), 9.51
(br s, 1H), 7.98-7.92 (m, 2H), 7.84-7.79 (m, 2H), 7.24 (d, 1H),
6.83 (dd, 1H), 3.04 (m, 1H), 2.65 (s, 3H), 1.92 (q, 1H), 1.41 (m,
1H), 1.07 (m, 1H). Anal. (C17H15BrFN3O3) C, H; N: calcd, 10.3;
found, 9.7.
Gen er a l P r oced u r e for Syn th esis of En a n tiom er ic
Cyclop r op a n e Com p ou n d s 17-20: 3-F lu or o-1-p r op ion yl-
oxyben zen e (35). Propionyl chloride (20 mL, 0.255 mol) was
added to a solution of 3-fluorophenol (34) (22.4 g, 0.2 mol) and
pyridine (24 mL, 0.3 mol) in dichloromethane (200 mL) at room
temperature over a period of 5 min. The reaction was exother-
mic. The solution was stirred for 30 min at room temperature,
dichloromethane was added, and the solution was washed with
saturated NaHCO3 and H2O, dried over MgSO4, and concen-
trated in vacuo to give 33.8 g (100%) of the title compound:
1H NMR (250 MHz, CDCl3) δ 7.34 (m, 1H), 6.98-6.83 (m, 3H),
2.60 (q, 2H), 1.27 (t, 3H).
4′-F lu or o-2′-h yd r oxyp r op iop h en on e (36). Compound 35
(33.8 g of crude material from the previous step) was reacted
with AlCl3 (33.3 g, 0.25 mol) at 150 °C for 10 min. After careful
quenching with water, the reaction mixture was extracted with
diethyl ether; the organic layer was dried over MgSO4 and
evaporated to give 29.5 g (88%) of the title compound: 1H NMR
(250 MHz, CDCl3) δ 12.68 (d,1H), 7.77 (dd, 1H), 6.67-6.56 (m,
2H), 2.99 (q, 2H), 1.23 (t, 3H).
N-[cis-2-(6-Ch lor o-3-et h oxy-2-flu or op h en yl)cyclop r o-
p yl]-N′-[2-(5-ch lor op yr id yl)]u r ea (7). The title compound
was prepared from 4-chloro-2-fluorophenol as described for
compound 10. 2-Amino-5-chloropyridine was used instead of
1
2-amino-5-cyanopyridine: mp 176.9 °C; H NMR (250 MHz,
CDCl3) δ 9.47 (br s, 1H), 9.27 (br s, 1H), 7.81 (d, 1H), 7.46 (dd,
1H), 7.14 (dd, 1H), 6.81 (t, 2H), 4.06 (q, 2H), 3.33 (m, 1H), 2.09
(q, 1H), 1.62 (m, 1H), 1.45 (t, 3H), 1.33 (m, 1H). Anal. (C17H16
Cl2FN3O2) C, H, N.
-
N-[cis-2-(6-Ch lor o-3-et h oxy-2-flu or op h en yl)cyclop r o-
p yl]-N′-[2-(5-cya n op yr id yl)]u r ea (8). The title compound
was prepared from 4-chloro-2-fluorophenol as described for
compound 10: mp 211-212 °C; 1H NMR (250 MHz, CDCl3) δ
9.77 (br s, 1H), 9.32 (br s, 1H), 8.16 (d, 1H), 7.71 (dd, 1H),
7.15 (dd, 1H), 6.98-6.78 (m, 2H), 4.08 (q, 2H), 3.34 (m, 1H),
2.12 (q, 1H), 1.65 (q, 1H), 1.47 (t, 3H), 1.31 (m, 1H). Anal.
(C18H16ClFN4O2) H, N; C: calcd 57.7; found, 57.2.
N-[cis-2-(6-Ch lor o-3-et h oxy-2-flu or op h en yl)cyclop r o-
p yl]-N′-[2-(5-ch lor op yr id a zin yl)]u r ea (9). The title com-
pound was prepared from 4-chloro-2-fluorophenol as described
for compound 10. 2-Amino-5-chloropyridazine was used instead
of 2-amino-5-cyanopyridine: mp 218.9 °C; 1H NMR (250 MHz,
DMSO-d6) δ 9.87 (s, 1H), 8.10 (d, 1H), 7.81 (d, 1H), 7.33 (d,
1H), 7.17 (t, 1H), 7.01 (s, 1H), 4.16 (q, 2H), 3.33 (m, 1H), 2.15
(q, 1H), 1.60 (q, 1H), 1.41 (t, 3H), 1.22 (m, 1H). Anal. (C16H15
Cl2FN4O2) C, H, N.
-
N-[cis-2-(2-Ch lor o-3-et h oxy-6-flu or op h en yl)cyclop r o-
p yl]-N′-[2-(5-ch lor op yr id a zin yl)]u r ea (11). The title com-
pound was prepared from 2-chloro-4-fluorophenol as described
for compound 10. 2-Amino-5-chloropyridazine was used instead
1
of 2-amino-5-cyanopyridine: mp 192 °C; H NMR (250 MHz,
CDCl3) δ 10.88-10.54 (m, 2H), 8.08-7.90 (m, 1H), 7.37 (d, 1H),
4′-F lu or o-2′-m eth oxyp r op iop h en on e (37). Compound 36
(29.5 g, 0.176 mol) was dissolved in 200 mL of acetone, and
K2CO3 (42.0 g, 0.3 mol) and methyl iodide (25.0 mL, 0.4 mol)
were added. The reaction mixture was heated at 40 °C for 12
h and filtered, and acetone was evaporated. The residue was
dissolved in diethyl ether, washed with 0.01 N NaOH and H2O,
dried over MgSO4, and evaporated to give 31.2 g (97%) of the
title compound: 1H NMR (250 MHz, CDCl3) δ 7.82-7.73 (m,
1H), 6.72-6.63 (m, 2H), 3.40 (s, 3H), 2.97 (q, 2H), 1.17 (t, 3H).
1-[1,1-(Eth ylen ed ioxy)p r op yl]-4-flu or o-2-m eth oxyben -
zen e (38). A solution of compound 37 (31.2 g, 0.171 mol),
ethylene glycol (10.5 mL, 0.188 mol), and p-toluenesulfonic acid
(1 g) in benzene (300 mL) was refluxed in a Dean-Stark
apparatus for 12 h. After cooling the reaction mixture was
washed several times with 1 M NaOH and dried over Na2SO4
and K2CO3. Evaporation gave 38 g (86%) of the title com-
pound: 1H NMR (250 MHz, CDCl3) δ 7.46-7.40 (m, 1H), 6.68-
6.56 (m, 2H), 4.02 (t, 2H), 3.85 (s, 3H), 3.84 (t, 2H), 2.12 (q,
2H), 0.82 (t, 3H).
3-[1,1-(Eth ylen ed ioxy)p r op yl]-6-flu or o-2-m eth oxyben -
za ld eh yd e (39). 2.5 M n-BuLi in hexanes (128 mL, 0.32 mol)
was added dropwise to a solution of compound 38 (38 g, 0.168
mol) in THF (450 mL) at -65 °C under nitrogen. The
temperature was allowed to reach -40 °C and then recooled
to -65 °C. While keeping the temperature at about -65 °C, a
solution of DMF (25 mL, 0.32 mol) in THF (50 mL) was added.
The reaction mixture was allowed to reach room temperature
slowly and no starting material was left after 30 min as
monitored by GC. After an additional 1 h the reaction mixture
was quenched with saturated NH4Cl solution, extracted with
6.86 (t, 1H), 6.76-6.67 (m, 1H), 4.04 (q, 2H), 3.44 (m, 1H),
2.16 (q, 1H), 1.64 (q, 1H), 1.52-1.40 (m, 4H). Anal. (C16H15
Cl2FN4O2) C, H, N.
-
N-[cis-2-(2-Ch lor o-6-flu or o-3-h yd r oxyp h en yl)cyclop r o-
p yl]-N′-[2-(5-ch lor op yr id yl)]u r ea (12). The title compound
was prepared from 2-chloro-4-fluorophenol as described for
compound 10. The last step, dealkoxylation, was carried out
as in the last step for the compound 17 and 2-amino-5-
chloropyridine was used instead of 2-amino-5-cyanopyridine:
mp 206.5 °C; 1H NMR (250 MHz, DMSO-d6) δ 9.44 (br s, 1H),
8.03 (br s, 1H), 7.88 (d, 1H), 7.72 (dd, 1H), 7.20 (d, 1H), 7.01
(t, 1H), 6.90 (dd, 1H), 3.13 (m, 1H), 2.03 (q, 1H), 1.46 (m, 1H),
1.02 (m, 1H). Anal. (C15H12Cl2FN3O2) C, H; N: calcd, 11.8;
found, 11.3.
N-[cis-2-(6-F lu or o-2-h yd r oxy-3-p r op ion ylp h en yl)cyclo-
p r op yl]-N′-[2-(5-ch lor op yr id yl)]u r ea (13). The title com-
pound was prepared from 3-fluorophenol as described for
compound 17. The cyclopropanation step was carried out as
described for compound 10. 2-Amino-5-chloropyridine was used
instead of 2-amino-5-cyanopyridine: mp 223 °C; 1H NMR (250
MHz, DMSO-d6) δ 13.21 (d, 1H), 9.42 (br s, 1H), 8.01-7.88
(m, 2H), 7.83 (d, 1H), 7.71 (dd, 1H), 7.24 (d, 1H), 6.82 (dd, 1H),
3.17-2.98 (m, 3H), 1.91 (q, 1H), 1.42 (m, 1H), 1.15-1.01 (m,
4H). Anal. (C18H17ClFN3O3) C, H; N: calcd, 11.1; found, 10.6.
N-[cis-2-(6-F lu or o-2-h yd r oxy-3-p r op ion ylp h en yl)cyclo-
p r op yl]-N′-[2-(5-cya n op yr id yl)]u r ea (14). The title com-
pound was prepared from 3-fluorophenol as described for
compound 17. The cyclopropanation step was carried out as
described for compound 10: 1H NMR (250 MHz, DMSO-d6) δ

4158 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ho¨gberg et al.
diethyl ether, and dried over Na2SO4. Evaporation gave the
residue which was purified on a silica gel column eluting with
EtOAc/n-hexane (1:9) to give 10 g (25%) of the title com-
pound: 1H NMR (250 MHz, CDCl3) δ 10.4 (s, 1H), 7.8-7.7 (m,
1H), 6.9 (t, 1H), 4.15-4.0 (m, 2H), 3.97 (s, 3H), 3.95-3.8 (m,
2H), 2.1 (q, 2H), 0.85 (t, 3H).
ethylamine (0.41 mL, 2.94 mmol) and diphenyl phosphorazi-
date (0.635 mL, 2.94 mmol) were added to a solution of
compound 43 (0.782 g, 2.94 mmol) in dry toluene (2.5 mL).
The solution was stirred at room temperature under argon for
30 min and then heated to 120 °C. After 15 min a solution of
2-amino-5-bromopyridine (0.56 g, 3.23 mmol) in DMF (1 mL)
was added and heating was continued for 3 h. Toluene was
evaporated; the residue was dissolved in benzene, washed with
1 N HCl, H2O, and brine, dried over Na2SO4, and evaporated.
The residue was purified by column chromatography on silica
gel by eluting with EtOAc/n-hexane (1:2 to 1:1) to give 0.49 g
(39%) of the title compound: 1H NMR (250 MHz, CDCl3) δ
9.72 (br s, 1H), 9.08 (br s, 1H), 7.80 (d, 1H), 7.62-7.47 (m,
2H), 6.92-6.78 (m, 2H), 3.91 (s, 3H), 3.42 (m, 1H),2.90 (q, 2H),
2.12 (q, 1H), 1.56 (q, 1H), 1.38 (q, 1H), 1.13 (t, 3H).
3-[1,1-(Eth ylen ed ioxy)p r op yl]-6-flu or o-2-m eth oxysty-
r en e (40). 2.5 M n-BuLi in hexanes (16 mL, 40 mmol) was
added to a suspension of methyltriphenylphosphonium bro-
mide (14.3 g, 40 mmol) in THF (250 mL) at room temperature
under nitrogen followed by the addition of compound 39 (10
g, 39.5 mmol) in THF (30 mL). The reaction mixture was
stirred at room temperature for 2 h. n-Hexane and brine were
added to the mixture and the organic phase was washed twice
with brine and once with water. After evaporation of the
solvent the residue was filtered through a funnel filled with
alumina (aluminum oxide 90 acc. Brockmann from Merck) and
eluting with EtOAc/n-hexane (1:9) to remove the generated
triphenylphosphine oxide. Evaporation of the organic solvent
gave a residue which was finally purified on silica gel eluting
with EtOAc/n-hexane (1:9) to give 6.9 g (70%) of the title
compound: 1H NMR (250 MHz, CDCl3) δ 7.4-7.3 (m, 1H),
6.85-6.7 (m, 2H), 6.05-5.95 (m, 1H), 5.65-5.55 (m, 1H), 4.1-
4.0 (m, 2H), 3.95-3.8 (m, 2H), 3.8 (s, 3H), 2.1 (q, 2H), 0.85 (t,
3H).
(1R,2R)-N-[cis-2-(6-F lu or o-2-h yd r oxy-3-p r op ion ylp h e-
n yl)cyclop r op yl]-N′-[2-(5-br om op yr id yl)]u r ea (17). A 1 M
solution of BCl3 in CH2Cl2 (3.3 mL, 3.3 mmol) was added to a
solution of compound 44 (0.49 g, 1.1 mmol) in CH2Cl2 (10 mL)
at -60 °C under argon. Stirring was continued overnight and
the temperature was allowed to rise slowly to room temper-
ature. The solution was diluted with CH2Cl2 and washed with
an aqueous solution of NaHCO3, H2O, and brine. The organic
layer was dried over Na2SO4 and concentrated. The crude
material was purified by chromatography on silica gel by
eluting with EtOH/CH2Cl2 (1:10) and by crystallization from
acetonitrile to give 0.24 g (52%) of the titled compound: ee
98.6% as determined by HPLC on a chiral-AGP column, eluent
11% acetonitrile in sodium phosphate buffer, flow 0.9 mL/min,
Eth yl Ester of (1R,2S)-cis-2-[3-(1,1-(Eth ylen ed ioxy)-
p r op yl)-6-flu or o-2-m e t h oxyp h e n yl]cyclop r op a n e ca r -
boxylic Acid (41). Cu(I)OTf (100 mg, 0.2 mmol) (very
sensitive to air)26 was added to 40 mL of chloroform (chloroform
used in this reaction was ethanol-free, degassed, and purified
through a basic alumina column), and the mixture was stirred
at room temperature under argon for 15 min. The chiral ligand
2,2′-isopropylidenebis[(4S)-4-tert-butyl-2-oxazoline] (commer-
cially available from Aldrich) (295 mg, 1.0 mmol) was added
and the mixture, which became green, was stirred for 30 min
at room temperature under argon. Compound 40 (5.0 g, 19.8
mmol) in 20 mL of chloroform was added and the solution was
cooled to 0 °C on ice. Ethyl diazoacetate (8.0 mL, 80 mmol) in
40 mL of chloroform was added dropwise under a period of 4
h. The color of the reaction mixture became yellow and then
dark brown. The mixture was stirred at room temperature for
2 h (GC showed complete reaction and cis/trans ratio of 3.0)
and then evaporated. The residue was dissolved in 200 mL of
EtOAc, washed with saturated NaHCO3, water, and brine,
dried over Na2SO4, and evaporated. The residue was purified
by column chromatography on silica gel by eluting with 5-10%
EtOAc in n-hexane to give 3.0 g (45%) of the title compound:
1H NMR (250 MHz, CDCl3) δ 7.34 (m, 1H), 6.80 (m, 1H), 4.12-
3.68 (m, 7H), 2.31-1.98 (m, 4H), 1.69-1.45 (m, 3H), 1.33 (m,
1H), 1.10 (t, 3H), 0.81 (t, 3H).
1
UV detection at 254 nm, tR 14.26 min; mp 198.9 °C; H NMR
(250 MHz, DMSO-d6) δ 13.22 (s, 1H), 9.42 (br s, 1H), 8.04-
7-77 (m, 4H), 7.16 (d, 1H), 6.78 (t, 1H), 3.11 (q, 2H), 3.02 (m,
1H), 1.93 (q, 1H), 1.42 (q, 1H), 1.11 (t, 3H), 1.00 (m, 1H). Anal.
22
(C18H17BrFN3O3) C, H, N. [R]D ) -153.8° (c ) 0.005 g/mL,
CH2Cl2).
(1S,2S)-N-[cis-2-(6-F lu or o-2-h yd r oxy-3-p r op ion ylp h e-
n yl)cyclop r op yl]-N′-[2-(5-br om op yr id yl)]u r ea (18). The
title compound was prepared as described for compound 17.
The chiral ligand that was used in the cyclopropanation step
was 2,2′-isopropylidenebis[(4R)-4-tert-butyl-2-oxazoline] (for
preparation see the Supporting Information in ref 22): ee
98.6% as determined by HPLC on a chiral-AGP column, eluent
11% acetonitrile in sodium phosphate buffer, flow 0.9 mL/min,
UV detection at 254 nm, tR 20.56 min; mp 198-199 °C; 1H
NMR (250 MHz, CDCl3) δ 13.32 (d, 1H), 9.10 (br s, 1 H), 8.53
(br s, 1H), 7.83 (br s, 1H), 7.72-7.67 (m, 1H), 7.57 (dd, 1H),
6.76 (br s, 1 H), 6.60 (t, 1H), 3.20-3.17 (m, 1H), 3.06-2.97
(m, 2H), 2.05-1.94 (m, 1H), 1.62-1.52 (m, 2H), 1.28 (t, 3H).
22
Anal. (C18H17BrFN3O3) C, H, N. [R]D ) +149.8° (c ) 0.005
g/mL, CH2Cl2).
E t h yl E st er of (1R,2S)-cis-2-(6-F lu or o-2-m et h oxy-3-
pr opion ylph en yl)cyclopr opan ecar boxylic Acid (42). Com-
pound 41 (2.2 g, 6.5 mmol) was dissolved in 1,4-dioxane (13
mL). 6M HCl (2.6 mL) was added and the mixture was stirred
for 1.5 h at room temperature. The mixture was extracted with
diethyl ether, the organic layer was washed with brine, dried
over Na2SO4 and evaporated to give 1.85 g (97%) of the title
compound: 1H NMR (250 MHz, CDCl3) δ 7.54-7.48 (m, 1H),
6.85 (t, 1H), 4.03-3.95 (m, 2H), 3.86 (t, 3H), 2.94 (q, 2H), 2.26-
2.18 (m, 2H), 1.71-1.52 (m, 2H), 1.20-1.08 (m, 6H).
(1R,2S)-cis-2-(6-F lu or o-2-m eth oxy-3-p r op ion ylp h en yl)-
cyclop r op a n eca r boxylic Acid (43). Compound 42 (1.8 g,
6.12 mmol) was dissolved in solution of LiOH (0.59 g, 24.5
mmol) in MeOH (25 mL)/H2O (10 mL) and stirred at 80 °C for
2.5 h. The reaction solution was concentrated and acidified
with 4 N HCl. The mixture was extracted with CH2Cl2; the
organic layer was washed with brine, dried over Na2SO4, and
evaporated to give 1.5 g (92%) of the title compound: 1H NMR
(250 MHz, CDCl3) δ 7.51-7.45 (m, 1H), 6.84 (t, 1H), 3.82 (s,
3H), 2.93 (q, 2H), 2.29 (q, 1H), 2.15 (q, 1H), 1.63-1.57 (m, 2H),
1.16 (t, 3H).
(1R,2R)-N-[cis-2-(6-F lu or o-2-h yd r oxy-3-p r op ion ylp h e-
n yl)cyclop r op yl]-N′-[2-(5-ch lor op yr id yl)]u r ea (19). The
title compound was prepared as described for compound 17.
2-Amino-5-chloropyridine was used instead of 2-amino-5-
bromopyridine: ee of the corresponding ester 98.4% as deter-
mined by HPLC on a chiral column, eluent 11% acetonitrile
in sodium phosphate buffer, flow 0.9 mL/min, UV detection
1
at 220 nm, tR 33.42 min; mp 196.5-198.5 °C; H NMR (250
MHz, CDCl3) δ 13.32 (d, 1H), 9.30-8.66 (m, 2H), 7.78-7.67
(m, 2H), 7.46 (dd, 1H), 6.81 (m, 1H), 6.58 (t, 1H), 3.20 (m, 1H),
3.00 (q, 2H), 2.01 (q. 1H), 1.57 (q, 1H), 1.33-1.19 (m, 4H). Anal.
22
(C18H17ClFN3O3) C, H, N. [R]D ) -176.8°(c ) 0.005 g/mL,
CH2Cl2).
(1S,2S)-N-[cis-2-(6-F lu or o-2-h yd r oxy-3-p r op ion ylp h e-
n yl)cyclop r op yl]-N′-[2-(5-ch lor op yr id yl)]u r ea (20). The
title compound was prepared as described for compound 18.
2-Amino-5-chloropyridine was used instead of 2-amino-5-
bromopyridine: ee of the corresponding ester 96.0% as deter-
mined by HPLC on a chiral column, eluent 11% acetonitrile
in sodium phosphate buffer, flow 0.9 mL/min, UV detection
at 220 nm, tR 20.61 min; mp 196-197 °C; 1H NMR (250 MHz,
CDCl3) δ 13.34 (d, 1H), 9.14 (br s, 2H), 7.79.7.62 (m, 2H), 7.42
(dd, 1H), 6.79 (m, 1H), 6.59 (t, 1H), 3.21 (m, 1H), 3.04 (q, 2H),
(1R,2R)-N-[cis-2-(6-F lu or o-2-m eth oxy-3-p r op ion ylp h e-
n yl)cyclop r op yl]-N′-[2-(5-b r om op yr id yl)]u r ea (44). Tri-

Urea-PETT Compounds as New HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4159
Discovery, Synthesis, and Bioactivity of Bis(heteroaryl)-pipera-
zines. 1. A Novel Class of Non-Nucleoside HIV-1 Reverse
Transcriptase Inhibitors. J . Med. Chem. 1994, 37, 999-1014.
(b) Davey J r., R. T.; Chaitt, D. G.; Reed, G. F.; Freimuth, W.
W.; Herpin, B. R.; Metcalf, J . A.; Eastman, P. S.; Falloon, J .;
Kovacs, J . A.; Polis, M. A.; Walker, R. E.; Masur, H.; Boyle, J .;
Coleman, S.; Cox, S. R.; Wathen, L.; Daenzer, C. L.; Lane, H. C.
Randomized, Controlled Phase I/II Trial of Combination Therapy
with Delavirdine (U-90152S) and Conventional Nucleosides in
Human Immunodeficiency Virus Type 1-Infected Patients. An-
timicrob. Agents Chemother. 1996, 40, 1657-1664.
2.00 (q, 1H), 1.58 (q, 1H), 1.36-1.19 (m, 4H). Anal. (C18H17
-
22
ClFN3O3) C, H, N. [R]D ) +176.8° (c ) 0.005 g/mL, CH2Cl2).
P h a r m a cok in etic Eva lu a tion . Br a in sa m p le p r ep a r a -
tion : Brains were weighed and homogenized in approximately
30 mL of methanol/chloroform (1/1). Internal standard (com-
pound 13) was added, and the homogenate was filtered. The
tissue precipitate was extracted three times with 5 mL of
methanol/chloroform (1/1) and the combined extracts were
added to the filtrate. One part of water was added to 5 parts
of the filtrate and the mixture was shaken in 5 min. An aliquot
(0.5 mL) of the organic phase was evaporated to dryness and
the residue was dissolved in 20 µL of DMSO and 0.5 mL of
the mobile phase. The sample was centrifuged (1 min, 14 000
rpm) and 50 µL of the supernatant was injected into a
reversed-phase HPLC system using UV detection at 250 nm.
P la sm a sa m p le p r ep a r a tion : 40-100 µL of plasma was
mixed with an equal volume of acetonitrile (10 s; Vibrofix).
The sample was centrifuged (3 min, 14 000 rpm) and 30-50
µL of the supernatant was injected into a reversed-phase
HPLC system using UV detection at 250 nm.
(4) Young, S. D.; Britcher, S. F.; Tran, L. O.; Payne, L. S.; Lumma,
W. C.; Lyle, T. A.; Huff, J . R.; Anderson, P. S.; Olsen, D. B.;
Carroll, S. S.; Pettibone, D. J .; O’Brien, J . A.; Ball, R. G.; Balani,
S. K.; Lin, J . H.; Chen, I.-W.; Schleif, W. A.; Sardana, V. V.; Long,
W. J .; Byrnes, V. W.; Emini, E. A. L-743,726 (DMP-266):
a
Novel, Highly Potent Nonnucleoside Inhibitor of the Human
Immunodeficiency Virus Type 1 Reverse Transcriptase. Anti-
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M.; Schneweis, K. E.; Winkler, I.; Riess, G. Preclinical Evaluation
of HBY 097,
a New Nonnucleoside Reverse Transcriptase
Inhibitor of Human Immunodeficiency Virus Type 1 Replication.
Antimicrob. Agents Chemother. 1995, 39, 2253-2257.
Cr ysta llogr a p h y. The details of the crystallization and the
structure determination will be published elsewhere. Briefly,
complexes of HIV-1 RT with the compounds 17 and 18 were
crystallized in the space group C2221. The cell parameters
were a ) 119.5 Å, b ) 156.6 Å, and c ) 156.4 Å. X-ray
diffraction data to 2.95 and 2.7 Å resolution, respectively, were
collected at the synchrotron stations D41 at LURE Paris,
France (compound 18), and 1711 at MAX-lab Lund, Sweden
(compound 17). Data were processed with DENZO and scaled
with Scalepack.27,28 The completeness of data was 99% with
an Rsym value29 of 16.1% for the RT/17 complex, and for the
RT/18 complex the corresponding values were 94% and 10.2%.
The protein model coordinates from 1hni were used for rotation
and translation functions and refinements using the program
(6) (a) Pauwels, R.; Andries, K.; Debyser, Z.; Van Daele, P.; Schols,
D.; Stoffels, P.; De Vreese, K.; Woestenborghs, R.; Vandamme,
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A. J . Potent and Highly Selective Human Immunodeficiency
Virus Type 1 (HIV-1) Inhibition by a Series of Alpha-anilinophe-
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(b) Staszewski, S.; Miller, V.; Rehmet, S.; Stark, T.; De Cre´e, J .;
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Breslin, H. J .; Woestenborghs, R.; Desmyter, J .; J anssen, M. A.
C.; De Clercq, E.; J anssen, P. A. J . New Tetrahydroimidazo[4,5,1-
jk][1,4]-Benzodiazepin-2(1H)-One and -Thione Derivatives Are
Potent Inhibitors of Human Immunodeficiency Virus Type 1
Replication and Are Synergistic with 2′,3′-Dideoxynucleoside
Analogues. Antimicrob. Agents Chemother. 1994, 38, 2863-2870.
(8) Okamoto, M.; Makino, M.; Yamada, K.; Nakade, K.; Yuasa, S.;
Baba, M. Complete inhibition of viral breakthrough by combina-
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package XPLOR.30 The structures were refined to Rcryst
-
values31 of 21.4% (Rfree ) 29.1%) using 2.95 Å data for the RT/
17 complex and 24% (Rfree ) 28.3%) using 2.7 Å data for the
RT/18 complex. Model construction employed the program O.32
Ack n ow led gm en t. We thank C. Rydega˚rd, C. A° h-
gren, and B. Golas for technical assistance with the
biological assays, L. Pettersson for excellent secretarial
help, and the late K. Frykman for performing the rat
studies. Philip Dumas Strasbourg, France, is thanked
for sharing with us his unpublished protocol for crystal-
lization of HIV-1 RT with nevirapine. This work was
supported by the Swedish Medical Research Council
(MFR, K79-16X-09505-07A).
(9) Cushman, M.; Golebiewski, W. M.; Graham, L.; Turpin, J . A.;
Rice, W. G.; Fliakas-Boltz, V.; Buckheit, J r., R. W. Synthesis
and Biological Evaluation of Certain Alkenyldiarylmethanes as
Anti-HIV-1 Agents Which Act as Non-Nucleoside Reverse Tran-
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Y.; Kodama, M.; Wu, Y.; Chen, L. B.; Harada, H.; Sugimoto, H.;
Hatanaka, M.; Hinuma, Y. S-1153 Inhibits Replication of Known
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