Synthesis and bioactivity of novel bis(heteroaryl)piperazine (BHAP) reverse transcriptase inhibitors: Structure-activity relationships and increased metabolic stability of novel substituted pyridine analogs

Article abstract of DOI:10.1021/jm960269m

The major route of metabolism of the bis(heteroaryl)piperazine (BHAP) class of reverse transcriptase inhibitors (RTIs), atevirdine and delavirdine, is via oxidative N-dealkylation of the 3-ethyl- or 3-isopropylamino substituent on the pyridine ring. This

Full text of DOI:10.1021/jm960269m

J . Med. Chem. 1996, 39, 5267-5275
5267
Syn th esis a n d Bioa ctivity of Novel Bis(h eter oa r yl)p ip er a zin e (BHAP ) Rever se
Tr a n scr ip ta se In h ibitor s: Str u ctu r e-Activity Rela tion sh ip s a n d In cr ea sed
Meta bolic Sta bility of Novel Su bstitu ted P yr id in e An a logs
Michael J . Genin,† Toni J . Poel,† Yoshihiko Yagi,‡ Carolyn Biles,† Irene Althaus,‡ Barbara J . Keiser,‡
Laurice A. Kopta,§ J an M. Friis,| Fritz Reusser,‡ Wade J . Adams,| Robert A. Olmsted,§ Richard L. Voorman,|
Richard C. Thomas,† and Donna L. Romero*,†
Pharmacia & Upjohn, Kalamazoo, Michigan 49001
Received April 5, 1996X
The major route of metabolism of the bis(heteroaryl)piperazine (BHAP) class of reverse
transcriptase inhibitors (RTIs), atevirdine and delavirdine, is via oxidative N-dealkylation of
the 3-ethyl- or 3-isopropylamino substituent on the pyridine ring. This metabolic pathway is
also the predominant mode of metabolism of (alkylamino)piperidine BHAP analogs (AAP-
BHAPs), compounds wherein a 4-(alkylamino)piperidine replaces the piperazine ring of the
BHAPs. The novel AAP-BHAPs possess the ability to inhibit non-nucleoside reverse
transcriptase inhibitor (NNRTI) resistant recombinant HIV-1 RT and NNRTI resistant variants
of HIV-1. This report describes an approach to preventing this degradation which involves
the replacement of the 3-ethyl- or 3-isopropylamino substituent with either a 3-tert-butylamino
substituent or a 3-alkoxy substituent. The synthesis, bioactivity and metabolic stability of
these analogs is described. The majority of analogs retain inhibitory activities in enzyme and
cell culture assays. In general, a 3-ethoxy or 3-isopropoxy substituent on the pyridine ring, as
in compounds 10, 20, or 21, resulted in enhanced stabilities. The 3-tert-butylamino substituent
was somewhat beneficial in the AAP-BHAP series of analogs, but did not exert a significant
effect in the BHAP series. Lastly, the nature of the indole substitution sometimes plays a
significant role in metabolic stability, particularly in the BHAP series of analogs.
In tr od u ction
panel of recombinant RTs containing mutations known
to confer resistance to other NNRTIs.14,15 Unfortu-
nately, N-dealkylation of these compounds occurs even
more rapidly than in their piperazine forerunners.
Thus, developing metabolically stable 3-pyridine sub-
stituents became critically important to the discovery
of pharmaceutically acceptable AAP-BHAP clinical
candidates.
The development of non-nucleoside inhibitors of HIV-1
reverse transcriptase (NNRTIs) as potential candidates
for the treatment of HIV infection has been the focus of
extensive research efforts in many laboratories.1-6 Such
research at Upjohn led to the discovery of the bis-
(heteroaryl)piperazine (BHAP) class of HIV-1 RT inhibi-
tors.7,8 This discovery served as the impetus for an
extensive structure-activity relationship (SAR) pro-
gram9 which led to the development of the clinical
candidates atevirdine mesylate (1)10 and delavirdine
mesylate (2).11,12 Both of these compounds are potent
inhibitors of HIV-1 RT in vitro and viral replication in
cell culture.
Since cytochrome P450 mediated N-dealkylation pro-
ceeds through decomposition of a carbinolamine which
is formed via either a one-electron oxidation on nitrogen
or direct proton abstraction R to the nitrogen,16,17
replacing the ethyl or isopropyl group with a group
containing a quaternary carbon such as tert-butyl should
preclude metabolism via the carbinolamine. Consider-
ation of other potential substituents that would fit the
structural criteria required for bioactivity led to the
possibility of employing ethers. Even though metabo-
lism of ethers is reported to occur via a pathway similar
to N-dealkylation (i.e. via abstraction of an R-hydrogen
and formation of an unstable hemiacetal which decom-
poses to the alcohol),18 the relative rate of such a process
compared to N-dealkylation was unknown for the BHAP
chemical class. Literature reports indicating that the
rate of O-dealkylation is decreased as the n-alkyl chain
of an aromatic ether is lengthened18,19 supported our
decision to pursue the synthesis of the targeted ether
analogs. Consequently, the piperazine-containing BHAP
analogs 6-13 (Table 1) and the piperidine-containing
AAP-BHAP analogs 17-23 (Table 2) were synthesized
and tested for HIV-1 RT inhibitory activity. The active
analogs which resulted from this effort were further
evaluated to determine their metabolic stability in vitro.
The major route of metabolism in vivo for both 1 and
2 is via oxidative N-dealkylation of the ethylamino or
isopropylamino substituent on the pyridine ring (Scheme
1). The ratio of drug to metabolite circulating in vivo
has been determined to be approximately 1:1.13 Unfor-
tunately the amino metabolites, 3 and 4, are both
inactive in the in vitro assay for RT inhibition (Table
1). Therefore, we have been interested in replacing the
ethylamino and isopropylamino groups of 1 and 2 with
appropriate surrogates in order to slow metabolic deg-
radation while at the same time retaining potency.
More recently, we have shown that BHAP analogs
containing an (alkylamino)piperidine (AAP-BHAPs) in
place of the piperazine, as in compound 14 (Table 2),
possess remarkable broad spectrum activity against a
† Medicinal Chemistry Research.
| Drug Metabolism Research.
‡ Chemical and Biological Screening.
§ Infectious Diseases Research.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
S0022-2623(96)00269-5 CCC: $12.00 © 1996 American Chemical Society

5268 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Genin et al.
Sch em e 1
Syn th esis
The ethylamine analog of delavirdine (5) was obtained
by treating the precursor 5-aminoindole BHAP with
methanesulfonyl chloride in a manner similar to the
methods described by us previously.8,9 Coupling of 1-[3-
[(1,1-dimethylethyl)amino]-2-pyridyl]piperazine20 with
5-methanesulfonamidoindole-2-carboxylic acid afforded
tert-butylamine congener 6. The synthesis of the alkoxy-
BHAP analogs 7-13 began with the preparation of the
2-bromo-3-alkoxypyridines 25-28 (Scheme 2). The
isopropyl, ethyl, and benzyl ethers were prepared in
high yields by heating the commercially available
2-bromo-3-pyridinol (24) with the appropriate alkyl
iodide in DMF. Standard methods for tert-butylation
with isobutylene and acid failed under various condi-
tions due to very poor solubility of the starting 2-bromo-
3-pyridinol. Thus, the tert-butyl ether of 2-bromo-3-
pyridinol was synthesized via a modification of the
procedure developed by J ackson et al.21 Alcohol 24 was
treated with an excess of tert-butyl trichloroacetimidate
and catalytic boron trifluoride etherate in a THF/
cyclohexane mixture to afford the tert-butyl ether.
These ethers were heated with excess piperazine in a
stainless steel autoclave to afford the (pyridyl)pipera-
zines 29-32 as oils in yields of 59-81%. Subsequent
coupling with an indole carboxylic acid derivative
provided the desired products 7-13.
Sch em e 2
The synthesis of the AAP-BHAPs 14-16 was straight-
forward and began with the condensation of 1-benzyl-
4-(N-methylamino)piperidine with 2-chloro-3-nitro-
pyridine.15 The remaining steps were conducted as
described for the piperazine containing BHAPs: nitro
group reduction, reductive alkylation, debenzylation,
and coupling to indole-2-carboxylic acids. tert-Butyla-
tion of 1-benzyl-4-[N-methyl-N-(3-amino-2-pyridyl)ami-
no]piperidine20,22 followed by deprotection and coupling
to 5-methanesulfonamidoindole-2-carboxylic acid af-
forded analog 17. Alkoxypiperidine analogs 18-21 were
prepared in a manner similar to that of the correspond-
ing piperazine congeners (Scheme 3). Condensation of
the piperidine moiety with 2-bromo-3-isopropoxypyri-
dine was more difficult than experienced in the pipera-
zine series. Although the same conditions were used,
lower yields were usually obtained. Subsequent debenz-
ylation and coupling with the appropriate indole-2-
carboxylic acid proceeded uneventfully. The deuterated
analogs 22 and 23 were prepared as depicted in Scheme
4 via reductive alkylation of (3-aminopyridyl)piperidine
37 with acetone and sodium borodeuteride for 38 or
acetone-d6 and sodium cyanoborohydride for 39. De-
benzylation and coupling of 38 and 39 with 5-methane-
sulfonamidoindole-2-carboxylic acid led to the deuter-
ated AAP-BHAPs 22 and 23.
Sch em e 3
piperazine and piperidine types are potent inhibitors
of HIV-1 RT in vitro and HIV-1 replication in cell
culture.23
Resu lts a n d Discu ssion
Specifically, the isopropyl and ethyl ethers (7-10)
retain activity comparable to the parent drugs 1 and 2,
demonstrating that the alkoxy group is a good surrogate
for the alkylamine moiety. The tert-butylamine and
tert-butyl ether analogs 6 and 13 were also quite active.
In fact the tert-butylamino compound 6 possessed
potency similar to the parent drug, delavirdine (2), in
the tissue culture assay measuring inhibition of viral
replication. Finally, both benzyl ethers 11 and 12
showed no activity in the initial assay. This result was
The analogs 5-23 were initially evaluated for their
ability to inhibit recombinant HIV-1 RT in an in vitro
assay.8,9 The compounds were tested at 100 µM, and
IC50 values were determined for most analogs. These
analogs were further tested in a cell culture assay to
determine their ability to inhibit HIV1 replication in
human lymphocytes (PBMC) as previously described.8
These results are summarized in Tables 1 and 2. From
these data, it is clear that several analogs of both the

Novel BHAP RT Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5269
Sch em e 4
tert-butylamines can still be dealkylated, any diminu-
tion in the efficiency of the initial hydroxylation reaction
relative to N-dealkylation of the isopropylamine would
result in increased metabolic stability for tert-butyl-
amine 6. Preliminary results with a closely related
compound that lacked indole substitution indicated that
the analog containing the tert-butylamino substituent
reduced the amount of N-desalkyl metabolite observed
relative to compound containing the isopropylamino
substituent.8 Similarly, the dealkylated material was
not observed as the major metabolite in the case of 6.
The fact that the presence of the tert-butylamine did
not increase the half-life in this assay, which measured
disappearance of starting material, suggests that other
equally efficient metabolic processes such as the side-
chain hydroxylation or pyridine ring hydroxylation were
competitive.
not surprising since the corresponding benzylamines
were previously shown to be inactive.8
Alkoxy-BHAPS containing 5-unsubstituted indoles
such as 7 and 9 were much less metabolically stable in
comparison to their 5-methanesulfonamido-substituted
counterparts 8 and 10. This was also observed in the
case of the (ethylamino)- and (isopropylamino)-BHAPs
with unsubstituted versus substituted indoles and is
likely due to hydroxylative metabolism at the 5-position
of the indole.26 A dramatic increase in metabolic
stability was observed in the case of compound 10 which
contains the requisite 5-substituted indole and a 3-
ethoxypyridine. This compound possessed a half-life
3-4 times longer than that of either atevirdine or
delavirdine, and approximately 4 times longer than that
of the corresponding isopropyl ether 8. Moreover, direct
comparison of the half-lives of ethyl ether 10 to its
ethylamino counterpart 5 demonstrated that the ether
is superior in its metabolic stability. Unfortunately, the
isopropyl ether 8 and tert-butyl ether 13 do not possess
similar enhanced stabilities compared to the isopropyl-
amine 2 or tert-butylamine 6. In summary, the alkoxy
BHAPs follow the same general trends in metabolic
stability as the (alkylamino)-BHAPs, i.e. isopropyl
ethers and tert-butyl ethers have similar half-lives, both
of which are shorter than the ethyl ethers. The longer
half-life of ether 10 suggests that dealkylation was
slowed or that metabolism was shunted to a less
efficient pathway such as pyridine ring hydroxylation
and/or hydroxylation of the ether side chain.18
The AAP-BHAPs containing a 3-alkylamino sub-
stituent such as compounds 14-17 exhibited very good
potency in the in vitro antiviral assay. Replacing the
3-ethylamino group with a 3-ethoxy group resulted in
a loss of anti-HIV-1 potency in the cell-based assay (e.g.
18 vs 14). However within the alkoxy series, the
piperazine-linked compounds appeared to possess ac-
tivities comparable to the piperidine-linked compounds
(9 vs 18, 10 vs 20). Even though preliminary results
indicated that substitution with the alkoxy group di-
minished antiviral potency in the AAP-BHAP series,
several compounds containing the 3-alkoxypyridine
possessed sufficient antiviral potency to warrant further
evaluation. Therefore, selected potent analogs were
evaluated for metabolic stability (t1/2) in the presence
of hepatic microsomal cytochrome P450 in vitro. The
results of these experiments are also summarized in
Tables 1 and 2.
In the piperazine-linked series reference compound
3-(ethylamino)pyridine 5 was more metabolically stable
than either of the closely related parent drugs 1 or 2
with a half-life 3 times that of the corresponding
3-(isopropylamino)pyridine, delavirdine (2). This result
parallels the trend in metabolic stabilities previously
observed in other 5-substituted indole series such as
atevirdine (1) and its 3-(isopropylamino)pyridine con-
gener.24 Compound 5 differs from atevirdine (1) solely
in the substitution at the 5-position of the indole.
Whereas atevirdine contains a 5-methoxy-substituted
indole, 5 contains a 5-methanesulfonamido-substituted
indole. This particular substitution resulted in a more
than 2-fold increase in half-life.
Unfortunately, no enhancement in metabolic stability
was obtained for the (tert-butylamino)pyridine 6 relative
to delavirdine. As detailed above, it was anticipated
that the absence of an abstractable hydrogen R to the
heteroatom would significantly decrease the dealkyla-
tive metabolism of 6, since carbinolamine formation is
precluded. Literature precedent indicates that N-dealky-
lation of tert-butylamines can occur via a cytochrome
P450 mediated oxidation of one of the methyl groups.25
The resulting alcohol is converted to the carboxylic acid
via two consecutive oxidations by alcohol dehydrogenase
and aldehyde dehydrogenase. Finally, decarboxylation
of the acid to the isopropylamine, subsequent oxidation
by cytochrome P450 to the carbinolamine, and decom-
position provide the dealkylated amine.16 Even though
The exciting discovery that certain AAP-BHAPs had
the ability to inhibit various mutant RT enzymes,
particularly the RT containing the P236L mutation
linked to BHAP resistance,14 led to the investigation of
their pharmacokinetic properties. This in turn led to
the disappointing discovery that these compounds were
unsuitable from a pharmacokinetic standpoint due to
their rapid metabolism.15 The piperidine AAP-BHAPs
possessed half-lives much shorter than the correspond-
ing piperazine BHAPs (e.g. 16 vs 2, and 17 vs 6). Once
again the 3-tert-butylamino substituent as found in
compound 17 provided only modest protection from
metabolic degradation, although, relatively, it contrib-
uted more to metabolic stability in the piperidine series
than in the piperazine series. In any case, we were
anxious to apply the alkoxy-substituted pyridine solu-
tion to this series of compounds to see if benefits similar
to those obtained in the piperazine series (Table 1) could
be realized.
We were gratified to find that comparison of the

5270 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Genin et al.
Ta ble 1. Biological Activities and Metabolic Stability (t1/2) of BHAP Analogs
RT inhibitiona in vitro
% at 100 µM IC50 (µM)
92 5.2
PBMCb D34
ED50 (µM)
compound
X
Y
t1/2c (min)
atevirdine (1)
delavirdine (2)
OMe
NHSO2Me
OMe
NHSO2Me
NHSO2Me
NHSO2Me
H
NHSO2Me
H
NHSO2Me
H
NHSO2Me
NHSO2Me
NHEt
NH-i-Pr
0.001
0.0001
ND
14.7
10.8
ND
98
0
1.1
ND
ND
2.7
4.8
1.8
1.1
2.1
1.9
ND
ND
ND
3
4
5
6
7
8
9
NH2
NH2
0
ND
ND
NHEt
NH-tert-Bu
O-i-Pr
O-i-Pr
OEt
96
97
98
99
98
98
0
0.01-0.1
0.0001
0.001-0.01
0.001-0.01
0.1
0.01
ND
ND
0.1
36.5
10.1
3.1
11.7
6.1
46.8
ND
ND
10
11
12
13
OEt
OBn
OBn
0
92
O-tert-Bu
10.6
a
The HIV-1 RT in vitro assay was carried out with recombinant enzyme32 using the template:primer poly(rA):(dT)10 and dTTP as the
mononucleotide substrate as described.8,33 IC50 values were determined by assaying at four drug concentrations. See footnote 34 for a
b
description of the assay. c Half-life of parent compound upon microsomal incubation. ND ) not determined.
Ta ble 2. Biological Activities and Metabolic Stability (t1/2) of AAP-BHAP Analogs
RT inhibitiona in vitro
PBMCb D34
compound
X
Y
% at 100 µM
IC50 (µM)
ED50 (µM)
t1/2 (min)
14
15c
16
17
18
19
20
21
22
23
H
H
NHEt
96
96
98
97
94
96
96
91
98
98
2.2
2.0
2.2
3.1
3.2
2.0
3.4
1.4
ND
ND
0.0001
0.0001
<0.0001
<0.0001
0.01-0.1
ND
0.01-0.1
ND
ND
ND
0.9
1.03
1.4
3.6
2.8
ND
22.0
10.0
1.88
1.25
NH-i-Pr
NH-i-Pr
NH-tert-Bu
OEt
O-i-Pr
OEt
O-i-Pr
NHCD(CH3)2
NHCH(CD3)2
NHSO2Me
NHSO2Me
H
H
NHSO2Me
NHSO2Me
NHSO2Me
NHSO2Me
a
The HIV-1 RT in vitro assay was carried out with recombinant enzyme32 using the template:primer poly(rA):(dT)10 and dTTP as the
mononucleotide substrate as described.8,33 IC50 values were determined by assaying at four drug concentrations. See footnote 34 for a
description of the assay. A less than (<) symbol indicates that the ED50 was below the lowest concentration tested. c Compound 15 was
tested as the mesylate salt. ND ) not determined.
b
3-ethoxy-substituted AAP-BHAP, 18, with the corre-
sponding 3-ethylamino congener, 14, demonstrated a
3-fold improvement in the half-life. Moreover, an even
greater benefit was derived from the analog which
possessed an indole 5-methanesulfonamido group in
concert with a 3-isopropoxy substituent (21), relative to
its 3-isopropylamino-substituted counterpart (16) wherein
a 7-fold enhancement in half-life was achieved. The
trend wherein a 3-ethoxy substituent, as in compound
20, proved to be more robust than the 3-isopropoxy
substituent (21) continued. In short, a half-life similar
to that of clinical candidate delavirdine was realized
with AAP-BHAP 21 and a half-life 2-fold longer was
realized with AAP-BHAP 20. The enhanced stability
of the alkoxypyridines carries over into the AAP-BHAP
series of analogs.
anism of metabolic degradation involves a direct proton
abstraction, then a significant primary deuterium iso-
tope effect (kH/kD ) 10) should be observed. Since
production of deuterated 22 involves a simple modifica-
tion of the chemical procedure utilized to produce 16,
such an effect might easily be used to our advantage to
afford a compound with increased metabolic stability.
The hexadeuterated compound 23 was also synthesized
for comparison. Only a small increase in the half-life
of 22 was observed, indicative of a secondary isotope
effect (kH/kD ) 2). Thus it appears likely that cyto-
chrome P450 is acting via a one-electron oxidation of
the nitrogen.
The effect of alkoxy versus (alkylamino)pyridine sub-
stituents on the broad spectrum activity of the AAP-
BHAPs 15-21 was assessed in enzyme assays employ-
ing recombinant RTs containing a mutation at amino
acid 236 (proline to leucine)27 or 181 (tyrosine to
cysteine)28 (Table 3). The alkoxy-AAP-BHAPs 18-21
demonstrated slightly lower IC50 values against WT RT
Another approach to enhancing the metabolic stability
of the AAP-BHAP template involved the synthesis of
compound 22 wherein the R-hydrogen of the isopropyl-
amine is replaced by a deuterium atom. If the mech-

Novel BHAP RT Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5271
Ta ble 3. Activities of Selected AAP-BHAP Analogs against
IC90 value for the alkoxy analog 21 versus L-drug
resistant virus was 5-fold higher than the IC90 for the
alkylamino analog 16. The relative susceptibilities of
these two viruses to 16 and 21 are in accordance with
the in vitro inhibitory activities observed for these two
compounds against the mutant RTs (Table 3). In short,
the 3-alkoxy substitution preserved the in vitro inhibi-
tory activities against both the wild-type and P236L RT
enzymes. However, the alkoxy substituents conferred
a reduction in potency against the Y181C RT in vitro
and L-drug resistant virus expressing the Y181C RT.
Nevertheless, we evaluated the pharmacokinetic pa-
rameters of isopropoxy AAP-BHAP 21 upon intrave-
nous and oral administration to rats at 15 mg/kg. The
enhanced metabolic stability observed in liver mi-
crosomes correlated with the in vivo results obtained
in rats. For example, alkoxy analog 21 had an iv
clearance rate 0.8-fold that of its isopropylamino coun-
terpart 16, and an absolute oral bioavailability 3-fold
greater than that of 16.15 These results indicate sub-
stantial improvement in the metabolic stability and/or
absorption efficiency of the AAP-BHAP template as a
result of the alkoxy substitution. The oral bioavailabil-
ity of 21 was 40% that of delavirdine15 at comparable
doses. Thus even with the improvements offered by the
alkoxy substituent, compounds containing the AAP-
BHAP template possess lower oral bioavailabilities than
the corresponding compounds containing the piperazine
template.
Wild-Type and Mutant RT Enzymes
IC50 (µM)a
fold resistantb
compound
WT
P236L Y181C P236L/WT Y181C/WT
15c
16
17
18
19
20
21
0.23
0.50
0.39
0.18
0.09
0.06
0.10
0.74
1.5
1.5
1.5
1.27
1.5
0.80
1.1
0.51
4.8
3.7
2.0
3.2
3.0
3.8
3.5
2.2
1.3
26.7
41.1
33.3
20.0
5.6
14.1
16.7
12.0
1.2
2.0
a
RNA-dependent DNA polymerase activity of mutant RTs was
assayed as described in the Experimental Section. IC50 values
were determined by nonlinear least-squares fit of data from
duplicate points at six drug concentrations. Differences in the IC50
values against WT RT in Table 3 and those in Table 2 are
presumed to reflect the fact that two sets of experiments were
performed in different laboratories using somewhat different assay
b
conditions (cf. Experimental Section). Expressed as the ratio of
IC50 mutant RT/IC50 WT RT. c Compound 15 was tested as the
mesylate salt.
Ta ble 4. Comparison of the Antiviral Activity of an
Alkoxy-Substituted AAP-BHAP to an Alkylamino AAP-BHAP
against NNRTI Resistant Viruses
IC90 (µM)
U-90152R HIV-1MF
(P236L)
L-697,661R HIV-1IIIB
(Y181C)
compound
16
21
5.3
4.0
1.0
5.1
than those of the (alkylamino)-AAP-BHAPs 15-17,
while both types of analogs possessed comparable
activities against the P236L enzyme. However, when
activities against the Y181C mutant enzyme were
compared, the alkoxy analogs (18-21) were less potent
(i.e. higher IC50 values) than their alkylamino counter-
parts (15-17). Moreover, comparison of the P236L RT
or Y181C RT IC50 values to WT RT IC50 values to
generate fold resistance ratios for each compound
revealed that the mutant RTs were more resistant to
the alkoxy-AAP-BHAPs (Table 3). For example, the
fold resistance of the Y181C RT to alkoxy compounds
18-21 ranged from 20 to 41.1, whereas the fold resis-
tance to alkylamino compounds 15-17 ranged from 1.3
to 3.5. This effect is due to the increased activity of the
alkoxy compounds against WT RT in concert with their
decreased activity against Y181C RT. Differences in
P236L RT fold resistance ratios were observed between
the two sets of analogs but were of lesser magnitude.
The ratios also indicated that the Y181C mutation
conferred a higher level of resistance to the alkoxy
compounds 18-21 than did the substitution at residue
236. This trend was not observed for the alkylamino
compounds 15-17 as indicated by the fold resistance
ratios for both mutant enzymes which were comparable.
An assessment of the effects of the alkylamino and
alkoxy substituents on antiviral activity is shown in
Table 4. The antiviral activities of 16 and 21 were
compared in cell culture assays that employed drug
resistant virus stocks derived by serial passage of virus
in the presence of the NNRTIs U-90152 (BHAP) or
Merck’s L-697,661 (pyridinone). From genotypic analy-
ses of these two HIV-1 variants, the BHAP and L-drug
resistant viruses contained the P236L and Y181C RT
substitutions, respectively. Comparison of the IC90
values revealed 16 and 21 to be comparable in inhibiting
the replication of BHAP resistant virus; however, the
Con clu sion
The synthesis of tert-butylamine and various alkyl
ether BHAP and AAP-BHAP analogs designed to
possess enhanced metabolic stability relative to the
parent drugs 1 and 2 was accomplished. The majority
of the analogs synthesized retained potent RT inhibitory
activities. Compounds which contained indoles unsub-
stituted in the 5-position in either the alkylamine or
alkyl ether series possessed metabolic stabilities less
than the parent compounds, as evidenced by their
relative half-lives in the in vitro microsomal assay. In
addition, the nature of the 5-indole substituent was
found to be an important contributor to the metabolic
stability of the BHAPs. The 5-methanesulfonamido
substituent found in delavirdine decreased the metabo-
lism of the piperazine BHAP analogs relative to the
5-methoxy group found in atevirdine. The relative order
of metabolic stability among the 3-pyridine substituents
in both the alkylamine or alkyl ether series in the BHAP
compounds which contained the 5-methanesulfonami-
doindole was ethyl > isopropyl ) tert-butyl. As a result,
the piperazine-linked analogs which incorporated a
5-substituted indole in concert with an 3-(ethylamino)-
pyridine or 3-ethoxypyridine, such as compounds 5 and
10, possessed the longest half-lives, 3-4 times those of
the parent drugs.
In general, the AAP-BHAPs were metabolically
degraded in this assay more rapidly than their pipera-
zine counterparts. The presence or absence of a 5-indole
substituent did not appreciably alter the metabolic
stability. On the other hand, a 3-(tert-butylamino)-
pyridine substituent resulted in relatively greater sta-
bility in this series than it did in the piperazine series.
Incorporation of a 3-alkoxy substituent exerted a very
beneficial effect on metabolic stability providing analogs
with half-lives similar to or greater than delavirdine.

5272 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Genin et al.
1.12 g (81%), was isolated as a yellow oil: 1H NMR (CDCl3) δ
7.69 (dd, J ) 1.5, 4.9 Hz, 1 H), 6.86 (dd, J ) 1.4, 7.8 Hz, 1 H),
6.60 (dd, J ) 4.8, 7.8 Hz, 1 H), 4.32-4.40 (m, 1 H), 3.20-3.24
(m, 4 H), 2.85-2.88 (m, 4 H), 2.50 (s, 1 H), 1.16 (d, J ) 6.1 Hz,
6 H); 13C NMR (CDCl3) δ 153.32, 144.61, 139.24, 121.80,
116.58, 70.44, 49.25, 45.94, 22.02; HRMS calcd for C12H19N3O1
221.1521, found 221.1528. Anal. (C12H19N3O‚0.25H2O) C, H,
N.
Once again the ethoxy-substituted analog was more
stable than the isopropoxy analog. The compounds
embodying these changes, 20 and 21, possessed good
activities in cell culture and against the WT and P236L
RT enzymes. Activities against the Y181C RT in vitro
and virus harboring a Y181C containing a Y181C
mutation were diminished. Therefore we are continuing
to investigate alternate solutions to this problem.
1-(3-Eth oxy-2-p yr id yl)p ip er a zin e (30): yield 59%; 1H
NMR (CDCl3) δ 7.86 (dd, J ) 1.5, 4.9 Hz, 1 H), 7.01 (dd, J )
1.4, 7.9 Hz, 1 H), 6.80 (dd, J ) 4.9, 7.9 Hz, 1 H), 4.03 (q, J )
7.0 Hz, 2 H), 3.38-3.41 (m, 4 H), 3.04-3.07 (m, 4 H), 2.75 (s,
1 H), 1.46 (t, J ) 7.0 Hz, 6 H); 13C NMR (CDCl3) δ 151.88,
145.71, 138.41, 118.15, 116.44, 63.29, 49.04, 45.58, 14.38;
HRMS calcd for C11H17N3O1 207.1372, found 207.1528. Anal.
(C11H17N3O‚H2O) C, N; H: calcd, 8.50; found, 7.78.
1-[3-(Ben zyloxy)-2-p yr id yl]p ip er a zin e (31): yield 49%;
1H NMR (CDCl3) δ 7.89 (dd, J ) 1.4, 4.9 Hz, 1 H), 7.28-7.41
(m, 5 H), 7.10 (dd, J ) 1.4, 7.9 Hz, 1 H), 6.82 (dd, J ) 4.9, 7.8
Hz, 1 H), 5.09 (s, 2 H), 4.52 (br s, 1 H), 3.53 (br s, 4 H), 3.11
(br s, 4 H); 13C NMR (CDCl3) δ 151.86, 145.70, 139.29, 136.23,
128.53, 128.00, 127.00, 119.65, 116.92, 70.30, 48.13, 45.08;
HRMS calcd for C16H19N3O 269.1528, found 269.1522.
1-(3-ter t-Bu toxy-2-p yr id yl)p ip er a zin e (32): yield 78%;
1H NMR (CDCl3) δ 7.95 (dd, J ) 4.77, 1.54 Hz, 1 H), 7.18 (dd,
J ) 7.78, 1.71 Hz, 1 H), 6.77 (dd, J ) 7.84, 4.79 Hz, 1 H),
3.61-3.58 (m, 4 H), 1.33 (s, 9 H). Anal. (C13H21N3O‚0.6H2O)
C, H, N.
1-Ben zyl-4-[N-m et h yl-N-(3-et h oxy-2-p yr id yl)a m in o]-
p ip er id in e (33). A neat mixture of 1-benzyl-4-(methylamino)-
piperidine15 (2.92 g, 14.3 mmol) and 26 (1.44 g, 7.15 mmol)
was treated as described above for 29: yield 63%; 1H NMR
(CDCl3) δ 7.82 (dd, J ) 1.5, 4.8 Hz, 1H), 7.32-7.22 (m, 5H),
6.97 (dd, J ) 1.5, 7.8 Hz, 1H), 6.70 (dd, J ) 4.8, 7.8 Hz, 1H),
4.01 (q, J ) 7.0 Hz, 2H), 3.78 (m, 1H), 3.50 (s, 2H), 2.97 (m,
2H), 2.88 (s, 3H), 2.05-1.85 (m, 4H), 1.69 (m, 2H), 1.45 (t, J )
7.0 Hz, 3H). Anal. (C20H27N3O) C, H, N.
Exp er im en ta l Section
Flash chromatography utilized E. Merck silica gel (230-
400 mesh). Melting points were taken on a Thomas-Hoover
capillary melting point apparatus and are uncorrected. High-
resolution mass spectra and combustion analysis were ob-
tained from the Structural, Analytical and Medicinal Chem-
istry Department at Pharmacia & Upjohn. Proton NMR
spectra were recorded on a Bruker Aspect 3000 300-MHz
spectrometer. Carbon NMR spectra were performed on the
same instrument at 75 MHz.
Gen er a l P r oced u r e for P r ep a r a t ion of 3-Alk oxy-
p yr id in es 25-27. 2-Br om o-3-isop r op oxyp yr id in e (25). A
mixture of 2-bromo-3-pyridinol (1.74 g, 10 mmol), 2-iodopro-
pane (2 mL, 20 mmol), and K2CO3 (2.5 g, 18 mmol) in dry DMF
(20 mL) was heated to 80 °C for 2 h. The solvent was removed
in vacuo, and the residue was partitioned between EtOAc and
H2O. The aqueous layer was extracted with EtOAc and the
combined organic layers were washed with H2O and brine and
dried (Na2SO4). Removal of solvent in vacuo gave a tan liquid
which was homogeneous by TLC and used without further
1
purification: yield 2.15 g (99%); H NMR (CDCl3) δ 7.80 (dd,
J ) 2.4, 3.8 Hz, 1 H), 7.03-7.10 (m, 2 H), 4.39-4.51 (m, 1 H),
1.24 (d, J ) 6.1 Hz, 6 H); 13C NMR (CDCl3) δ 151.24, 140.87,
133.96, 123.08, 121.48, 72.15, 21.56; HRMS calcd for C8H10N10
OBr 214.9946, found 214.9945.
-
2-Br om o-3-eth oxyp yr id in e (26): yield 92%; 1H NMR
(CDCl3) δ 7.31 (dd, J ) 1.8, 4.4 Hz, 1 H), 6.53-6.62 (m, 2 H),
3.48 (q, J ) 7.0 Hz, 2 H), 0.84 (t, J ) 7.0 Hz, 3 H); 13C NMR
(CDCl3) δ 152.30, 140.95, 132.89, 123.43, 119.56, 64.94, 14.52;
HRMS calcd for C7H8N10OBr 200.9790, found 200.9801.
2-Br om o-3-(ben zyloxy)pyr idin e (27): yield 66%; 1H NMR
(CDCl3) δ 7.86-7.91 (m, 1 H), 7.37-7.41 (m, 2 H), 7.22-7.34
(m, 3 H), 7.05-7.13 (m, 2 H), 5.03 (s, 2 H); 13C NMR (CDCl3)
δ 151.69, 141.19, 135.33, 132.83, 128.47, 128.01, 126.83,
123.35, 120.17, 70.48; HRMS calcd for C12H10NOBr 262.9946,
found 262.9944. Anal. (C12H10NOBr) C, H, N.
1-Ben zyl-4-[N-m eth yl-N-[3-(1-m eth yleth oxy)-2-pyr idyl]-
1
a m in o]p ip er id in e (34): yield 51%; H NMR (CDCl3) δ 7.82
(dd, J ) 1.5, 4.8 Hz, 1H), 7.32-7.23 (m, 5H), 6.98 (dd, J ) 1.4,
9.3 Hz, 1H), 6.70 (dd, J ) 4.9, 7.9 Hz, 1H), 4.53 (sept, J ) 6.0
Hz, 2H), 3.82 (m, 1H), 3.49 (s, 2H), 2.97 (m, 2H), 2.86 (s, 3H),
1.97 (m, 4H), 1.69 (m, 2H), 1.34 (d, J ) 6.1 Hz, 6H). Anal.
(C21H29N3O) C, H, N.
Gen er a l P r oced u r e for t h e Deb en zyla t ion of Ben z-
ylp ip er id in es 33 a n d 34 To Affor d 35 a n d 36. 4-[N-
Meth yl-N-(3-eth oxy-2-p yr id yl)a m in o]p ip er id in e (35). A
mixture of 33 (1.40 g, 4.30 mmol), 10% Pd/C (1.4 g), and
ammonium formate (813 mg, 12.9 mmol) in methanol (25 mL)
was degassed, stirred at 65 °C for 45 min, cooled to RT, and
filtered through Celite to remove catalyst. The filtrate was
concentrated in vacuo to provide 832 mg (83%) of the title
compound as a colorless film: 1H NMR (CDCl3) δ 7.83 (d, J )
4.8 Hz, 1H), 6.97 (d, J ) 7.7 Hz, 1H), 6.71 (dd, J ) 4.9, 7.5 Hz,
1H), 4.02 (q, J ) 6.9 Hz, 2H), 3.88 (m, 1H), 3.15 (bd, J ) 11.9
Hz, 2H), 2.88 (s, 3H), 2.63 (m, 2H), 1.75 (m, 4H), 1.46 (t, J )
6.9 Hz, 3H); HRMS (EI) calcd for C13H21N3O: 235.1685, found
235.1688.
4-[N-Meth yl-N-[3-(1-m eth yleth oxy)-2-p yr id yl]a m in o]-
p ip er id in e (36): yield 80%; 1H NMR (CDCl3) δ 7.82 (m, 1H),
6.98 (d, J ) 7.5 Hz, 1H), 6.71 (m, 1H), 4.54 (m, 1H), 3.92 (m,
1H), 3.15 (bd, J ) 11.5 Hz, 2H), 2.87 (s, 3H), 2.62 (m, 2H),
1.74 (m, 5H), 1.35 (d, J ) 5.8 Hz, 6H); HRMS (EI) calcd for
C14H23N3O 249.1841, found 249.1843.
1-[(1,1-Dim et h ylet h oxy)ca r b on yl]-4-[N-m et h yl-N-[3-
[(1-d e u t e r io-1-m e t h yle t h yl)a m in o]-2-p yr id yl]a m in o]-
piper idin e (38). 1-[(1,1-Dimethylethoxy)carbonyl]-4-[N-methyl-
N-(3-amino-2-pyridyl)amino]piperidine (0.61 g, 2.0 mmol) was
dissolved in 2.5 mL of acetone and 0.72 mL of acetic acid-d.
The solution was cooled to 0 °C, and sodium borodeuteride
(0.146 g, 3.5 mmol) was added. The ice bath was removed,
and the reaction mixture was stirred at room temperature for
30 min. Then another 0.07 g of sodium borodeuteride was
added, and the reaction mixture was stirred another 30 min.
The reaction mixture was quenched with D2O, and the mixture
was partitioned between 1 N NaOH and CHCl3. The organic
2-Br om o-3-ter t-bu toxyp yr id in e (28). 2-Bromo-3-pyridi-
nol (0.5 g, 2.9 mmol) was dissolved in a minimum amount of
dry THF, and cyclohexane was added until the solution became
turbid. Then tert-butyl trichloroacetimidate (2.1 mL) was
added followed quickly by BF3‚Et2O (0.06 mL). The reaction
mixture became homogeneous and was stirred at room tem-
perature for 1 h after which time additional tert-butyl trichlo-
roacetimidate (1 mL) was added. The reaction mixture was
stirred overnight, and solid NaHCO3 was added. The mixture
was filtered through a plug of silica gel, and the solvent was
removed in vacuo. The white solid thus obtained was tritu-
rated in hexane, filtered, and washed with copious amounts
of hexane. The filtrate was concentrated in vacuo to a colorless
liquid which was chromatographed on a 2 × 40 cm column
with EtOAc/hexane (1:3). The product was isolated as a
colorless liquid in a yield of 0.42 g (64%): 1H NMR (CDCl3) δ
7.96 (dd, J ) 4.6, 1.6 Hz, 1 H), 7.29 (dd, J ) 8.01, 1.68 Hz, 1
H), 7.09 (dd, J ) 8.05, 4.48 Hz, 1 H), 1.36 (s, 9 H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 29-34 via
Con d en sa tion of P ip er a zin e or 1-Ben zyl-4-(m eth yla m i-
n o)p ip er id in e w ith 3-Alk oxyp yr id in es 25-28. 1-(3-Iso-
p r op oxy-2-p yr id yl)p ip er a zin e (29). A neat mixture of
piperazine (2.69 g, 31.25 mmol) and 25 (1.35 g, 6.25 mmol)
was heated in a sealed stainless steel autoclave at 165 °C for
24 h. The resulting mixture was partitioned between CHCl3
and 1 M NaHCO3 solution. The organic layer was washed with
H2O and dried (Na2SO4). Removal of solvent in vacuo gave a
brown oil which was chromatographed on a 2 × 30 cm column
with MeOH/CHCl3 (1:9) as the eluting solvent. The product,

Novel BHAP RT Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5273
layer was dried over Na2SO4 and concentrated in vacuo. Flash
column chromatography (20% ethyl acetate/hexane) afforded
0.51 g of the title compound (73%) as an oil: 1H NMR (CDCl3)
δ 7.58 (dd, J ) 1.6, 4.7 Hz, 1H), 6.80 (dd, J ) 4.7, 8.0 Hz, 1H),
6.71 (dd, J ) 1.6, 8.0 Hz, 1H), 4.36 (br, 1H), 3.98 (br, 2H),
3.11 (tt, J ) 3.7, 10.8 Hz, 1H), 2.65 (br t, 2H), 2.51 (s, 3H),
1.63 (m, 2H), 1.42 (m, 2H), 1.35 (s, 9H), 1.11 (s, 6H); MS (EI)
m/e (rel int): 350 (26), 349 (41), 166 (41), 165 (59), 164 (40),
57 (100); HRMS calcd for C19H31DN4O2 349.2588, found
349.2588.
isop r op oxy-2-p yr id yl)p ip er a zin e (8): yield 83%; mp 210-
211 °C; 1H NMR (CDCl3) δ 9.65 (s, 1 H), 7.88 (dd, J ) 1.5, 4.9
Hz, 1 H), 7.60 (s, 1 H), 7.42, (d, J ) 8.7 Hz, 1 H), 7.69 (dd, J
) 2.1, 8.7 Hz, 1 H), 7.09 (d, J ) 6.6 Hz, 1 H), 6.86, (dd, J )
4.9, 7.9 Hz, 1 H), 6.83 (s, 1 H), 6.78 (s, 1 H), 4.52-4.64 (m, 1
H), 4.07 (br s, 4 H), 3.55-3.58 (m, 4 H), 2.97 (s, 3 H), 1.40 (d,
J ) 6.1 Hz, 6 H); 13C NMR (CDCl3) δ 162.02, 144.72, 138.84,
134.13, 130.74, 129.36, 127.87, 121.33, 120.99, 117.26, 116.42,
112.84, 105.30, 70.71, 48.29, 38.79, 22.09; HRMS calcd for
C22H27N5O4S: 457.1784, found 457.1809. Anal. (C22H27N5O4S)
C, H, N.
1-[(1,1-Dim et h ylet h oxy)ca r b on yl]-4-[N-m et h yl-N-[3-
[[1-(1,1,1-tr ideu ter iom eth yl)-2,2,2-tr ideu ter ioeth yl]am in o]-
2-p yr id yl]a m in o]p ip er id in e (39). 1-[(1,1-Dimethylethoxy)-
ca r bon yl]-4-[N -m et h yl-N -(3-a m in o-2-p yr id yl)a m in o]-
piperidine (0.5 g, 1.63 mmol) was dissolved in 3.2 mL of CH3-
OH and cooled to 0 °C. Then acetone-d6 (0.18 mL, 2.4 mmol)
and acetic acid (0.92 mL, 16 mmol) were added. NaCNBH3
(0.15 g, 2.4 mmol) was added in one portion, and the reaction
was warmed to room temperature. Since the reaction pro-
ceeded only modestly, additional acetone-d6 (0.5 mL), acetic
acid (0.5 mL), and NaCNBH3 (0.1 g) were added in batches
four times, at which point the reaction mixture was complete
as detected by TLC. Then the reaction mixture was parti-
tioned between 1 N NaOH and CHCl3. The organic layer was
washed with brine, dried over Na2SO4, and concentrated in
vacuo. Flash column chromatography (20% ethyl acetate/
hexane) afforded 0.57 g (99%) of the title compound: 1H NMR
(CDCl3) δ 7.54 (dd, J ) 1.6, 4.7 Hz, 1H), 6.77 (dd, J ) 4.7, 8.0
Hz, 1H), 6.67 (dd, J ) 1.6, 8.0 Hz, 1H), 3.84 (br, 2H), 3.33 (s,
1H), 3.02 (tt, J ) 3.7, 10.8 Hz, 1H), 2.50 (br t, 2H), 2.44 (s,
3H), 1.51 (m, 2H), 1.33 (m, 2H), 1.26 (s, 9H); MS (EI) m/e (rel
int): 355 (22), 354 (100), 211 (21), 171 (29), 170 (89); HRMS
calcd for C19H26D6N4O2 354.2902, found 354.2897.
1-(In d ol-2-ylca r b on yl)-4-(3-et h oxy-2-p yr id yl)p ip er a -
zin e (9): yield 78%; mp 160-162 °C; 1H NMR (CDCl3) δ 9.50
(s, 1 H), 7.88 (dd, J ) 1.4, 4.9 Hz, 1 H), 7.66 (d, J ) 8.0 Hz, 1
H), 7.44 (d, J ) 8.2 Hz, 1 H), 7.26-7.31 (m, 1 H), 7.06-7.16
(m, 2 H), 6.84-6.89 (m, 2 H), 4.10 (t, J ) 7.0 Hz, 2 H), 4.05-
4.12 (m, 4 H), 3.55-3.58 (m, 4 H), 1.50 (t, J ) 7.0 Hz, 3 H);
13C NMR (CDCl3) δ 162.42, 151.06, 146.01, 138.33, 135.58,
129.19, 127.36, 124.27, 121.76, 120.43, 118.68, 117.21, 111.67,
105.23, 63.82, 48.34, 14.69; HRMS calcd for C20H22N4O2
350.1743, found 350.1740. Anal. (C20H22N4O2) C, H, N.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-(3-
eth oxy-2-p yr id yl)p ip er a zin e (10): yield 79%; mp 195-197
°C; 1H NMR (CDCl3) δ 9.43 (s, 1 H), 7.88 (dd, J ) 1.4, 4.9 Hz,
1 H), 7.60 (s, 1 H), 7.43, (d, J ) 8.7 Hz, 1 H), 7.17 (dd, J ) 2.1,
8.7 Hz, 1 H), 7.08 (d, J ) 7.9 Hz, 1 H), 6.87 (dd, J ) 4.9, 7.9
Hz, 1 H), 6.80 (s, 1 H), 6.55 (s, 1 H), 4.09 (q, J ) 7.0 Hz, 2 H),
4.10 (br s, 4 H), 3.55-3.58 (m, 4 H), 2.97 (s, 3 H), 1.50 (t, J )
7.0 Hz, 3 H); 13C NMR (DMSO-d6) δ 161.78, 145.36, 138.13,
133.60, 130.77, 130.37, 126.89, 119.43, 119.11, 117.02, 114.28,
112.45, 104.01, 63.38, 47.87, 38.22, 14.43; HRMS calcd for
C21H25N5O4S 443.1627, found 443.1627. Anal. (C21H25N5O4S)
C, H, N, S.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-[3-
(eth yla m in o)-2-p yr id yl]p ip er a zin e (5). 1-[(5-Aminoindol-
2-yl)carbonyl]-4-[3-(ethylamino)-2-pyridyl]piperazine15 (0.16 g,
0.43 mmol) was dissolved in 4 mL of CH2Cl2 and cooled to 0
°C. Then pyridine (0.037 mL, 0.45 mmol) was added followed
by methanesulfonyl chloride (0.035 mL, 0.45 mmol), and the
reaction mixture was slowly warmed to room temperature and
stirred for 24 h. The reaction mixture was diluted with CHCl3,
washed with aqueous NaHCO3, and dried over Na2SO4.
Purification by flash column chromatography (90% EtOAc/
hexane) provided 0.11 g (58%) of the title compound: mp 215-
216 °C (EtOAc/hexane); 1H NMR (CD3OD) δ 7.48 (m, 2H), 7.34
(d, J ) 8.8 Hz, 1H), 7.07 (dd, J ) 2.1, 8.8 Hz, 1H), 6.90 (m,
2H), 6.75 (s, 1H), 3.95 (br, 4H), 3.11 (q, J ) 7.0 Hz, 2H), 3.02
(m, 4H), 2.80 (s, 3H), 1.19 (t, J ) 7.0 Hz, 3H). Anal.
(C21H26N6O3S) C, H, N, S.
1-(In d ol-2-ylca r bon yl]-4-[3-(ben zyloxy)-2-p yr id yl]p ip -
er a zin e (11): yield 46%; mp 247-249 °C; 1H NMR (CDCl3) δ
11.59 (s, 1 H), 7.82-7.85 (m, 1 H), 7.61 (d, J ) 8.0 Hz, 1 H),
7.30-7.51 (m, 7 H), 7.16-7.20 (m, 1 H), 7.01-7.07 (m, 1 H),
6.92 (dd, J ) 4.8, 7.9 Hz, 1 H), 6.83 (s, 1 H), 5.17 (s, 2 H), 3.88
(br s, 4 H), 3.40-3.50 (m, 4 H); 13C NMR (CDCl3) δ 162.15,
151.26, 145.41, 138.72, 136.80, 135.97, 128.61, 127.96, 127.45,
126.87, 123.26, 121.41, 120.05, 119.78, 117.25, 112.11, 104.18,
69.74, 48.16; HRMS calcd for C25H24N4O2 412.1899, found
412.1892. Anal. (C25H24N4O2‚H2O) C, H, N.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-[3-
(ben zyloxy)-2-pyr idyl]piper azin e (12): yield 79%; mp 260-
262 °C; 1H NMR (DMSO-d6) δ 11.65 (s, 1 H), 9.37 (s, 1 H),
7.85 (d, J ) 3.5 Hz, 1 H), 7.30-7.52 (m, 8 H), 7.12, (d, J ) 8.7
Hz, 1 H), 6.94 (dd, J ) 4.8, 7.8 Hz, 1 H), 6.85, (s, 1 H), 5.19 (s,
2 H), 3.88 (br s, 4 H), 3.44 (br s, 4 H), 2.88 (s, 3 H); 13C NMR
(DMSO-d6) δ 162.06, 151.34, 145.48, 138.83, 136.88, 133.85,
130.99, 130.67, 128.69, 128.05, 127.53, 127.15, 120.12, 119.71,
117.35, 114.53, 112.70, 104.27, 69.82, 48.25, 38.49; HRMS
calcd for C26H27N5O4S 505.1784, found 505.1786. Anal.
(C26H27N5O4S‚0.75H2O) C, H, N, S.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-(3-
ter t-bu toxy-2-p yr id yl)p ip er a zin e (13): yield 86%; mp 238-
239 °C; 1H NMR (DMSO-d6) δ 11.45 (s, 1 H), 9.18 (s, 1 H),
7.76 (dd, J ) 4.7, 1.7 Hz, 1 H), 7.31 (d, J ) 1.88 Hz, 1 H), 7.21
(d, J ) 8.72 Hz, 1 H), 7.14 (dd, J ) 7.77, 1.67 Hz, 1 H), 6.93
(dd, J ) 8.75, 2.10 Hz, 1 H), 6.67-6.71 (m, 2 H), 3.71 (br s, 4
H), 3.26 (br s, 4 H), 2.70 (s, 3 H), 1.16 (s, 9 H); 13C NMR
(DMSO-d6) δ 161.86. 155.94, 141.74, 141.38, 133.66, 130.94,
130.81, 130.46, 126.96, 119.49, 116.62, 114.32, 112.49, 104.09,
80.46, 47.94, 38.30, 28.37; HRMS calcd for C23H29N5O4S
471.1940, found 471.1933. Anal. (C23H29N5O4S) C, H, N.
Gen er a l P r oced u r e for Syn th esis of 18-19 via CDI
Cou p lin g of In d ole-2-ca r boxylic Acid s w ith P yr id ylp ip -
er a zin es or 4-[N-m eth yl-N-(3-a lk oxy-2-p yr id yl)a m in o]-
p ip er id in es. 1-(In d ol-2-ylca r b on yl)-4-[N-m et h yl-N-(3-
eth oxy-2-pyr idyl)am in o]piper idin e (18). Indole-2-carboxylic
acid (86 mg, 0.53 mmol) and CDI (0.085 g, 0.533 mmol) were
dissolved in 2.7 mL of THF and stirred at room temperature
for 1.5 h. A solution of 35 (0.114 g, 0.484 mmol) in 7 mL of
THF was added, and the mixture was stirred for 40 h and then
concentrated in vacuo. The residue was dissolved in CH2Cl2,
washed with saturated NaHCO3, water, and brine, dried over
Gen er a l P r oced u r e for Syn th esis of 6-13 via EDC
Cou p lin g of In d ole-2-ca r boxylic Acid s w ith P yr id ylp ip -
er a zin es or 4-(N-Meth yl-N-(3-a lk oxy-2-p yr id yl)a m in o]-
p ip er id in es. 1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r -
b o n y l]-4-[3-[(1,1-d im e t h y le t h y l)a m in o ]-2-p y r id y l]-
p ip er a zin e (6). 5-Methanesulfonamidoindole-2-carboxylic
acid29 (0.46 g, 1.80 mmol), 1-[3-[(1,1-dimethylethyl)amino]-2-
pyridyl]piperazine8 (0.42 g, 1.80 mmol), and EDC (0.35 g, 2.2
mmol) were coupled in 3 mL of THF and 3 mL of DMF as
described for 7. Workup, chromatography (60% EtOAc/hexane
to 10% CH3OH/EtOAc), and crystallization (DMF) afforded
0.41 g (48%) of the title compound: mp 259-260 °C; 1H NMR
(DMSO) δ 7.53 (m, 1H), 7.44 (m, 1H), 7.34 (d, J ) 8.7 Hz, 1H),
7.07 (m, 2H), 6.88 (dd, J ) 4.7, 7.9 Hz, 1H), 6.80 (s, 1H), 4.56
(s, 1H), 3.87 (br, 4H), 2.93 (br, 4H), 2.83 (s, 3H), 1.32 (s, 9H).
Anal. (C23H30N6O3S‚0.5H2O) C, H, N, S.
1-(In d ol-2-ylca r b on yl)-4-(3-isop r op oxy-2-p yr id yl)p ip -
1
er a zin e (7): yield 71%; mp 169-170 °C; H NMR (CDCl3) δ
9.43 (s, 1 H), 7.88 (dd, J ) 1.5, 4.9 Hz, 1 H), 7.66 (d, J ) 7.9
Hz, 1 H), 7.44 (d, J ) 8.2 Hz, 1 H), 7.26-7.31 (m, 1 H), 7.07-
7.17 (m, 2 H), 6.83-6.87 (m, 2 H), 4.54-4.62 (m, 1 H), 4.09
(br s, 4 H), 3.55-3.58 (m, 4 H), 1.39 (d, J ) 6.1 Hz, 6 H); 13C
NMR (CDCl3) δ 162.27, 152.0, 144.49, 138.50, 135.42, 129.11,
127.26, 124.15, 121.64, 121.19, 120.32, 116.90, 111.52, 105.11,
70.52, 48.13, 21.86; HRMS calcd for C21H24N4O2 364.1899,
found 364.1903. Anal. (C21H24N4O2) C, H, N.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-(3-

5274 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Genin et al.
Na2SO4, and concentrated in vacuo. Purification by flash
column chromatography (10-50% ethyl acetate/hexane) pro-
vided the product as a white solid (0.123 g, 67%): 1H NMR
(CDCl3) δ 9.47 (bs, 1H), 7.85 (dd, J ) 1.5, 4.8 Hz, 1H), 7.66 (d,
J ) 8.0 Hz, 1H), 7.01 (dd, J ) 1.4, 7.8 Hz, 1H), 6.81 (d, J )
1.3 Hz, 1H), 6.76 (dd, J ) 5.0, 7.8 Hz, 1H), 4.86 (bd, J ) 12.8
Hz, 2H), 4.15 (m, 1H), 4.05 (q, J ) 7.0 Hz, 2H), 3.05 (bs, 2H),
2.88 (s, 3H), 1.94 (m, 4H), 1.48 (t, J ) 7.0 Hz, 3H). Anal.
(C22H26N4O2) C, H, N.
1-(In d ol-2-ylca r b on yl)-4-[N -m e t h yl-N -(3-(1-m e t h yl-
eth oxy)-2-p yr id yl)a m in o]p ip er id in e (19): yield 94%; 1H
NMR (CDCl3) δ 9.68 (bs, 1H), 7.85 (dd, J ) 1.5, 4.8 Hz, 1H),
7.65 (d, J ) 7.9 Hz, 1H), 7.44 (d, J ) 8.2 Hz, 1H), 7.27 (m,
1H), 7.13 (td, J ) 0.9, 8.0 Hz, 1H), 7.03 (dd, J ) 1.4, 7.9 Hz,
1H), 6.81 (d, J ) 1.3 Hz, 1H), 6.75 (dd, J ) 4.8, 7.9 Hz, 1H),
4.86 (bd, J ) 12.8 Hz, 2H), 4.56 (sept, J ) 6.1 Hz, 1H), 4.18
(m, 1H), 2.87 (s, 3H), 1.92 (m, 4H), 1.37 (d, J ) 6.1 Hz, 6H).
Anal. (C23H28N4O2) C, H, N.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-[N-
m eth yl-N-(3-eth oxy-2-pyr idyl)am in o]piper idin e (20). 5-Ni-
troindole-2-carboxylic acid and 35 were treated as described
above for 18: yield 79%; 1H NMR (CDCl3) δ 10.84 (bs, 1H),
8.63 (d, J ) 2.2 Hz, 1H), 8.15 (dd, J ) 2.2, 9.0 Hz, 1H), 7.86
(dd, J ) 1.4, 4.8 Hz, 1H), 7.49 (d, J ) 9.1 Hz, 1H), 7.04 (dd, J
) 1.4, 7.9 Hz, 1H), 6.97 (d, J ) 1.5 Hz, 1H), 6.78 (dd, J ) 4.9,
7.8 Hz, 1H), 4.86 (bs, 2H), 4.20 (m, 1H), 4.06 (q, J ) 7.0 Hz,
2H), 3.4-2.8 (bm, 2H), 2.90 (s, 3H), 1.96 (m, 4H), 1.49 (t, J )
6.9 Hz, 3H); HRMS (EI) calcd for C22H25N5O4 423.1906, found
423.1906.
1-[(5-Nitroindol-2-yl)carbonyl]-4-[N-methyl-N-(3-ethoxy-2-
pyridyl)amino]piperidine (from above, 350 mg, 0.83 mmol) was
dissolved in 20 mL of DMF, and 20 mL of methanol and 10%
Pd/C (88 mg) were added. The mixture was degassed, put
under a hydrogen atmosphere for 2.5 h, filtered through Celite,
and concentrated in vacuo. The residue obtained was chro-
matographed (5% methanol/CH2Cl2) to provide 103 mg of the
amine (28%). The amine product was dissolved in CH2Cl2 (5
mL), and pyridine (41 µL, 0.51 mmol) was added. Then the
reaction mixture was treated with methanesulfonyl chloride
(21 µL, 0.267 mmol) and stirred for 16 h. The reaction mixture
was diluted with CH2Cl2, washed with water, and brine, dried
over Na2SO4, and concentrated in vacuo. Purification on three
silica gel plates, eluting with 5% CH3OH/CHCl3 (Rf ) 0.27-
0.40) afforded 96 mg (80%) of the product as a light brown,
amorphous solid: 1H NMR (CDCl3) δ 9.81 (bs, 1H), 7.85 (m,
1H), 7.61 (s, 1H), 7.42 (d, J ) 8.7 Hz, 1H), 7.16 (dd, J ) 2.0,
8.7 Hz, 1H), 7.02 (d, J ) 7.8 Hz, 1H), 6.98 (s, 1H), 6.75 (m,
2H), 4.83 (bd, J ) 12.6 Hz, 2H), 4.16 (m, 1H), 4.05 (q, J ) 7.0
Hz, 2H), 3.20-2.85 (bs, 2H), 2.96 (s, 3H), 1.95 (m, 4H), 1.48
(t, J ) 6.9 Hz, 3H); HRMS (EI) calcd for C23H29N5O4S 471.1940,
found 471.1931.
ethyl acetate/hexane) afforded the product which was recrys-
tallized from CH3OH/CHCl3/ether to provide 0.6 g of the title
compound (88%): 1H NMR (DMSO-d6): δ 7.58 (dd, J ) 1.6,
4.7 Hz, 1H), 7.44 (m, 1H), 7.35 (d, J ) 8.7 Hz, 1H), 7.04 (dd,
J ) 2.0, 8.7 Hz, 1H), 6.89 (m, 2H), 6.74 (br, 1H), 4.97 (br s,
1H), 4.38 (m, 2H), 3.32 (m, 1H), 2.85 (s, 3H), 2.53 (s, 3H), 1.78
(m, 2H), 1.56 (m, 2H), 1.14 (s, 6H); MS (EI) m/e (rel int): 486
(42), 485 (59), 166 (52), 165 (81), 164 (63), 130 (100); HRMS
calcd for C24H31DN6O3S 485.2319, found 485.2314. Anal.
(C24H31DN6O3S‚0.6H2O) C, H, N.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-[N-
m eth yl-N-[3-[[1-(1,1,1-tr id eu ter iom eth yl)-2,2,2-tr id eu te-
r ioeth yl]a m in o]-2-p yr id yl]a m in o]p ip er id in e (23). Pre-
pared as described for 22 from 39: yield 81%; 1H NMR (CDCl3)
δ 9.89 (s, 1H), 7.53 (dd, J ) 1.6, 4.7 Hz, 1H), 7.38 (d, J ) 2.0
Hz, 1H), 7.20 (m, 2H), 6.95 (dd, J ) 2.0, 8.7 Hz, 1H), 6.75 (dd,
J ) 4.7, 8.0 Hz, 1H), 6.65 (dd, J ) 1.6, 8.0 Hz, 1H), 6.51 (d, J
) 1.5 Hz, 1H), 4.44 (br, 2H), 3.46 (br s, 1H), 3.32 (m, 1H), 3.40-
2.82 (br, 2H), 2.76 (s, 3H), 2.44 (s, 3H), 1.75 (m, 2H), 1.46 (m,
2H). MS (EI) m/e (rel int) 491 (8), 490 (28), 237 (43), 222 (36),
130 (100); HRMS calcd for C24H26D6N6O3S 490.2633, found
490.2641.
HIV-RT Assa y P r otocol. The RT assays described in
Table 3 were carried out at 28 °C for 10 min in a 100 µL
reaction mixture consisting of 2 mM dithiothreitol, 60 mM
NaCl, 10 mM MgCl2, 50 mM Tris-HCl (pH 8.3), 0.05% Nonidet
P-40, 50 µM thymidine triphosphate with 12 µCi/mL [methyl-
3H]thymidine 5′-triphosphate (Amersham), 200 nM template-
primer (poly rA600:oligo dT10 from Pharmacia), and 4 µg/mL of
purified recombinant HIV RT. The RT enzymes (WT, P236L,
and Y181C) were used as the homodimers and were obtained
as previously described.30,31 The reaction mixture was stopped
with 100 µL of 10% (v/v) trichloroacetic acid. The acid
precipitated materials, recovered on glass fiber filters, were
analyzed for radioactivity. Non-nucleoside drugs were added
from DMSO stocks. In all cases the final DMSO concentration
was 1% (v/v).
Assa y for Meta bolic Sta bility. Analogs were screened
for metabolic stability in the presence of hepatic microsomal
cytochrome P450 by measuring loss of the parent drug in the
following manner. Hepatic microsomes (0.4 mg), obtained
from untreated rats, were diluted into 1 mL (final volume) of
50 mM potassium phosphate buffer, pH 7.4, 0.1 mM EDTA.
Drug was added from 1 mM stock in MeOH to a final
concentration of 5 µM. Following a preincubation at 37 °C,
reaction mixture was started by addition of an NADPH
generating system consisting of (final concentration) 0.5 mM
NADPH, 5 mM isocitrate, 5 mM MgCl2, and 0.4 unit of
isocitrate dehydrogenase. At 2 min intervals between 0 and
12 min from start of reaction, 100 µL aliquots of incubation
were removed and quenched with 100 µL acetonitrile followed
by addition of an appropriate internal standard contained in
10 µL of acetonitrile. Samples were vortex mixed and centri-
fuged to pellet protein. The supernatant was diluted 1:1 with
HPLC buffer, and a 100 µL sample was assayed by reverse
phase HPLC using a Zorbax Rx-C8 column (15 × 0.49 cm).
The mobile phase consisted of a 12 min gradient of 20-60%
acetonitrile balanced by 100 mM ammonium acetate, pH 4.0,
flowing at 1 mL/min. Detection was by UV absorbance at 295
nm. Drug concentration was estimated by peak height ratio
to the internal standard. Concentration data were fitted to a
monoexponential equation, yielding the rate constant (k) for
loss of drug, which was converted to half-life by t1/2 ) ln 2/k.
For purposes of comparison, half-lives of comparator com-
pounds were normalized to delavirdine mesylate or atevirdine
mesylate which were run as controls in each assay.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-[N-
m et h yl-N-[3-(1-m et h ylet h oxy)-2-p yr id yl]a m in o]p ip er i-
d in e (21). Prepared as described for 20 using 5-nitroindole-
1
2-carboxylic acid (600 mg, 2.91 mmol) and 36: yield 74%; H
NMR (CDCl3) δ 9.98 (bs, 1H), 7.84 (dd, J ) 1.5, 4.8 Hz, 1H),
7.60 (d, J ) 1.9 Hz, 1H), 7.41 (d, J ) 8.7 Hz, 1H), 7.20 (s, 1H),
7.16 (dd, J ) 2.1, 8.7 Hz, 1H), 7.03 (dd, J ) 1.4, 8.0 Hz, 1H),
6.75 (m, 2H), 4.83 (bd, J ) 12.9 Hz, 2H), 4.56 (sept, J ) 6.1
Hz, 1H), 4.18 (m, 1H), 3.10 (bm, 2H), 2.96 (s, 3H), 2.86 (s, 3H),
1.90 (m, 4H), 1.37 (d, J ) 6.0 Hz, 6H); HRMS (EI) calcd for
C24H31N5O4S 485.2097, found 485.2092.
1-[(5-Met h a n esu lfon a m id oin d ol-2-yl)ca r b on yl]-4-[N-
m e t h y l-N -[3-[(1-d e u t e r i o -1-m e t h y le t h y l)a m i n o ]-2-
p yr id yl]a m in o]p ip er id in e (22). 38 (0.5 g, 1.43 mmol) was
dissolved in 15 mL of 4 N HCl in dioxane for 15 min at room
temperature. The reaction mixture was concentrated in vacuo,
dissolved in CHCl3, and concentrated again. The residue was
dried on a vacuum pump for 1 h, dissolved in 3 mL of THF,
and 5-methanesulfonylindole-2-carboxylic acid (0.43 g, 1.7
mmol), triethylamine (0.237 mL, 1.7 mmol), and EDC (0.33 g,
1.7 mmol) were added. The reaction mixture was stirred for
18 h at room temperature, concentrated in vacuo, and parti-
tioned between CHCl3 and 1 N NaOH. The organic layer was
separated, diluted with methanol, dried over Na2SO4, and
concentrated in vacuo. Flash column chromatography (75%
Ack n ow led gm en t. We thank Satish K. Sharma and
David B. Evans for developing the procedure to prepare
the mutant enzymes used for screening. In addition,
we thank J ean L. Sarcich and Alfredo G. Tomesselli for
aiding in the large-scale isolation of the same mutant
enzymes.

Novel BHAP RT Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5275
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1
× 106 cells were infected with an inoculum
containing 1 ng of p24 of the HIV-1 D34 isolate. The level of
HIV-1 replication was determined 4 days after infection by
measuring the levels of supernatant p24. Drugs were tested at
four concentrations via a series of 10-fold dilutions. When the
apparent ED50 fell between two such dilutions, the range is
reported. Cell viability was determined by measuring the levels
of mitogen-stimulated cell proliferation.
(16) Miwa, G. T.; Walsh, J . S.; Kedderis, G. L.; Hollenberg, P. F. The
Use of Intramolecular Isotope Effects to Distinguish between
Deprotonation and Hydrogen Atom Abstraction Mechanisms in
Cytochrome P-450- and Peroxidase-catalyzed N-Demethylation
Reactions. J . Biol. Chem. 1983, 258, 14445-14449.
J M960269M