Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment. 12. Structure-activity relationships associated with 4-fluoro-6-azaindole derivatives leading to the identification of 1-(4-benzoylpiperazin-1-yl)-2-(4- fluoro-7-[1,2,3]triazol-1-yl-1H-

Article abstract of DOI:10.1021/jm3016377

A series of highly potent HIV-1 attachment inhibitors with 4-fluoro-6-azaindole core heterocycles that target the viral envelope protein gp120 has been prepared. Substitution in the 7-position of the azaindole core with amides (12a,b), C-linked heterocycl

Full text of DOI:10.1021/jm3016377

Article
pubs.acs.org/jmc
Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-1)
Attachment. 12. Structure−Activity Relationships Associated with
4‑Fluoro-6-azaindole Derivatives Leading to the Identification of
1‑(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-
yl‑1H‑pyrrolo[2,3‑c]pyridin-3-yl)ethane-1,2-dione (BMS-585248)
Alicia Regueiro-Ren,*,† Qiufen M. Xue,† Jacob J. Swidorski,† Yi-Fei Gong,‡ Marina Mathew,§
Dawn D. Parker,§ Zheng Yang,§ Betsy Eggers,‡ Celia D’Arienzo,§ Yongnian Sun,‡ Jacek Malinowski,§
Qi Gao,∥ Dedong Wu,∥ David R. Langley,⊥ Richard J. Colonno,‡ Caly Chien,# Dennis M. Grasela,#
Ming Zheng,§ Pin-Fang Lin,‡ Nicholas A. Meanwell,† and John F. Kadow†
‡
§
∥
†Department of Medicinal Chemistry, Virology, Pharmaceutical Candidate Optimization, Analytical Research and Development,
⊥
and Computer-Assisted Drug Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford,
Connecticut 06492, United States
#Department of Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb, Pennington, New Jersey, United States
S
* Supporting Information
ABSTRACT: A series of highly potent HIV-1 attachment inhibitors with 4-fluoro-6-azaindole core
heterocycles that target the viral envelope protein gp120 has been prepared. Substitution in the 7-
position of the azaindole core with amides (12a,b), C-linked heterocycles (12c−l), and N-linked
heterocycles (12m−u) provided compounds with subnanomolar potency in a pseudotype infectivity
assay and good pharmacokinetic profiles in vivo. A predictive model was developed from the initial
SAR in which the potency of the analogues correlated with the ability of the substituent in the 7-
position of the azaindole to adopt a coplanar conformation by either forming internal hydrogen
bonds or avoiding repulsive substitution patterns. 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-
[1,2,3]triazol-1-yl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-585248, 12m) exhibited much improved in vitro
potency and pharmacokinetic properties than the previous clinical candidate BMS-488043 (1). The predicted low clearance in
humans, modest protein binding, and good potency in the presence of 40% human serum for 12m led to its selection for human
clinical studies.
interaction of gp120 with CD4 utilizing small molecules
presents an alternative approach for inhibiting HIV-1 infection
which these laboratories have pursued.20−30
Proof-of-concept for this approach was validated by clinical
studies with BMS-488043 (1, Figure 1), a small molecule that
binds to a relatively conserved region of the viral envelope in
gp120.12−19,31 However, in order to attain the exposure
INTRODUCTION
■
Current therapies to treat HIV-1 have transformed this
infection into a manageable chronic disease for many patients.
These treatments rely on long-term administration of triple
drug combinations called highly active combination antire-
troviral therapy (cART).1,2 However, long-term toxicities,
including persistent inflammation3 and immunological activa-
tion4 due to residual viral replication as well as the appearance
of other non-AIDS related events such as cardiovascular,5 liver,6
and kidney disease,7 cancers,8 premature aging, and cognitive
decline,9 have been associated with the persistent use of these
therapeutic agents. More importantly, more than 70% of
patients develop resistance and about 15% of new infections
carry key resistance mutations.10,11 It is desirable to have
additional therapies that offer improved safety profiles and that
target events in HIV-1 replication other than protease,
integrase, or reverse transcriptase (RT). HIV-1 entry inhibitors
have the advantage of preventing the establishment of infection
in host cells. The first stage of viral entry involves the
interaction of the viral envelope gp120 with the primary cell
receptor CD4, the attachment step.12−19 Interfering with the
Figure 1. BMS-488043 (1) and BMS-585428 (12m).
Received: November 6, 2012
Published: January 29, 2013
© 2013 American Chemical Society
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Table 1. Antiviral in Vitro Potency, Cytotoxicity, Dihedral Angle at Minimun Energy,56 and in Vitro Pharmacokinetic Profiling
Data for Compounds 12a−u
a
b
The data reported are the mean of two or more experiments. The minimum were identified using Jaguar56 B3LYP/6-31G**. The dihedral angle is
c
depicted by the dark bonds on the model system (above right). Y is the heavier atom relative to X. The Caco-2 Pc for metroprolol, a highly
d
permeable marker, was 132 nm/s, whereas the Caco-2 Pc for mannitol, a poorly permeable marker, was 15 nm/s. Low-clearance category: CL < 21
mL min−1 kg−1. Intermediate-clearance category: 21 mL min−1 kg−1 ≤ CL ≤ 29 mL min−1 kg−1. High clearance category: CL > 29 mL min−1 kg−1.
required for efficacious antiviral activity, it was necessary to
administer 1 in high dose (800 or 1800 mg b.i.d.) in
combination with a high fat meal. Both requirements were
considered liabilities that needed to be avoided. One plausible
approach to solving these issues involved the preparation of
prodrugs designed to increase the exposure of the parent
compound,32 which is characterized as BCS class 2, by
overcoming dissolution-limited absorption.33,34 Herein, we
describe a portion of the work from an alternative approach
that focuses on identifying compounds with improved
preclinical properties that ultimately translated into an
enhanced clinical profile.
in 1 provided a boost in potency.41,42 Therefore, we focused
our attention on the synthesis of 4-fluoro-6-azaindole analogues
with carboxamides and heterocycles incorporated at the 7-
position.
CHEMISTRY
■
The synthetic pathways utilized in the preparation of the 7-
substituted-4-fluoro-6-azaindole inhibitors presented in Table 1
are outlined in Schemes 1−4. All compounds were prepared
through the common intermediate 7-bromo-4-fluoro-1H-
pyrrolo[2,3-c]pyridine (7), which was initially synthesized as
described in Scheme 1.
We previously reported the in vitro and in vivo evaluation of
indole35−37 and 6-azaindole HIV-1 attachment inhibitors.38−40
In the indole series, the introduction of a fluorine in the 4
position rendered compounds with excellent potency. The 6-
azaindole series provided compounds such as 1 with improved
pharmaceutical and pharmacokinetic profiles. In an effort to
identify further improved HIV-1 attachment inhibitors, we
explored the introduction of substituents to the 4-fluoro-6-
azaindole template. Initial structure−activity relationship (SAR)
studies indicated that introducing an sp2 center at the 7-
position of the azaindole instead of the methoxy moiety found
5-Fluoro-2-methoxypyridine (3) was prepared via a slightly
modified literature procedure.43 Thus, 5-amino-2-methoxypyr-
idine was converted into the diazonium salt 2 in 99% yield. Salt
2 was slowly added in portions to a preheated (100 °C) toluene
solution to afford compound 3. Demethylation of 3 with HCl
(35%) generated the hydroxypyridine 4 which was nitrated to
produce 5-fluoro-3-nitro-2-hydroxypyridine (5) in 30% overall
yield from the diazonium salt 3. Compound 5 was converted to
the corresponding bromide 6 in 97% yield by treatment with
POBr3 and catalytic DMF. No chromatography was utilized for
purification throughout the sequence leading to 6. A Bartoli
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a
chain at the 3-position was achieved by acylation with
ethyloxalyl chloride in a mixture of aluminum chloride and
ethylmethylimidazolium chloride47,48 to provide acids 11a and
11b. Coupling of these acids with 1-benzoylpiperazine
generated amides 12a and 12b.
Scheme 1. Synthesis of 4-Fluoro-7-bromo,6-azaindole (7)
Oxazole 12c was prepared by treating bromide 7 with n-BuLi
at −78 °C followed by the addition of DMF to the resulting
carbanion to render aldehyde 13. Reaction with toluenesulfo-
nylmethyl isocyanide (TOSMIC reagent)49 afforded oxazole 14
which was acylated at C-3 followed by coupling with 1-
benzoylpiperazine as described above to provide oxazole 12c
(Scheme 3).
a
Reagents and conditions: (a) HBF4, NaNO2, 99%; (b) toluene, 100
°C; (c) concentrated HCl, sealed tube 140 °C; (d) fuming HNO3,
H2SO4, 30% from 2; (e) POBr3, DMF (cat.), 110 °C, 97%; (f)
vinylmagnesium bromide, THF, −20 to 0 °C, 23−33%.
Alternatively, 7-C-linked heterocyclic compounds 12i−l and
7-N-linked heterocyclic compounds 12m−u, as shown in
Scheme 4, were prepared in a more convergent fashion from
intermediate 7 by first installing the side chain in the C-3
position as described above to afford 1-(4-benzoylpiperazin-1-
yl)-2-(7-bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-
1,2-dione (16) in which the C-7 bromide was utilized as a
convenient functionality to introduce heterocycles. The
syntheses of the 7-C-linked heterocyclic analogues 12d−g
were readily accomplished via a Stille coupling between
bromide 16 and commercially available stannylated hetero-
cycles. Pyrazole 12h was also prepared by a Stille coupling of
16 with ethyl 3-(tributylstannyl)-1H-pyrazole-5-carboxylate.50
Subsequent treatment of 12h with an amine generated the
series of pyrazoleamides 12i−l. Copper-assisted coupling
between N-containing heterocycles and bromide 9 in the
presence of potassium carbonate was employed for the
preparation of the N-linked heterocyclic analogues 12m−u.
reaction between 5-fluoro-3-nitro-2-bromopyridine and vinyl-
magnesium bromide afforded the desired 4-fluoro-7-bromo,6-
azaindole 7 in 23−33% yield.44,45
Amides 12a and 12b and oxazole 12c were prepared from
intermediate 7 by first modifying the 7-position of the azaindole
followed by attachment of the side chain at the 3-position, as
described in Schemes 2 and 3, respectively. Amides 12a and
a
Scheme 2. Synthesis of Amides 12a and 12b
RESULTS AND DISCUSSION
■
The in vitro antiviral activity and cytotoxicity associated with
the targeted compounds are compiled in Table 1 along with in
vitro pharmacokinetic profiling for select compounds. The 4-
fluoro-6-azaindole analogues were tested for inhibitory activity
toward HIV-1 infection in a single cycle pseudotype assay in
which the viral Nef gene was replaced with a luciferase
reporter.20,35,37 A proviral clone of LAI virus in which the env
gene is replaced by the firefly luciferase gene was co-transfected
into HEK-293 cells with a plasmid expressing a JRFL virus
envelope. After 48 h the supernatant containing the
recombinant pseudovirus was harvested and the titer
determined by performing serial dilutions in HeLa67 cells,
which expresses the primary receptor CD4 and the HIV-1 co-
receptors CXCR4 and CCR5. Virus growth was quantified by
measuring lucirerase activity (luciferase gene reporter assay kit
by Roche) 3 days after infection. For the analysis of the antiviral
activity of text compounds, 4-fold serial dilutions of compounds
were added to pseudovirus-infected HeLa67 cells at the time of
infections. After 3 days, the extent of luciferase activity was
compared to controls where no compound was added and the
data were used to calculate the EC50 values for individual
compounds. The cytotoxicity of the compounds was
determined in parallel on HeLa cells.51 Human liver micro-
somal stability (HLM)52 and Caco-2 permeability53 were used
as predictors of systemic clearance and permeability,
respectively.
a
(a) CuCN, DMF, 150 °C; (b) (1) NaOH, EtOH, 100 °C; (2) HCl;
(c) R1R2N, DEPBT, DMF; (d) ClCOCOOMe, AlCl3, ethyl-
methylimidazolium chloride; (e) 1-benzoylpiperazine, NMM, EDAC,
DMF.
a
Scheme 3. Synthesis of Oxazole 12c
a
Reagents and conditions: (a) n-BuLi, THF, −78 °C, then DMF; (b)
TOSMIC reagent, K2CO3, MeOH; (c) ClCOCOOEt, AlCl3, ethyl-
methylimidazolium chloride; (d) 1-benzoylpiperazine, DIPEA,
DEPBT, DMF.
12b were prepared by treating bromide 7 with copper cyanide
to give nitrile 8, which was converted to the acid 9 under
standard conditions. DEPBT-mediated amide bond formation
between 8 and methylamine or ammonium chloride provided
amides 10a and 10b, respectively.46 Installation of the side
We first focused attention on the C-7 amides 12a and 12b
and the unsubstituted C-linked heterocycle 12c−g derivatives
because they exhibited excellent potency and a good
therapeutic index, as shown in Table 1. Interestingly, some of
the most potent compounds in this series, the methylamide 12a
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a
Scheme 4. Synthesis of 7-C-Linked Heterocyclic Analogues 12d−l and 7-N-Linked Analogues 12m−u
a
Reagents and conditions: (a) ClCOCOOEt, AlCl3, ethylmethylimidazolium chloride; (b) 1-benzoylpiperazine, DIPEA, DEPBT, DMF; (c) Bu3SnR,
Pd(Ph3P)4, dioxane, 145 °C; (d) ethyl 3-(tributylstannyl)-1H-pyrazole-5-carboxylate, Pd(Ph3P)4, dioxane, 145 °C; (e) R1R2N, rt to 70 °C; (f) N-
containing heterocycle, Cu(0), K2CO3.
and unsubstituted amide 12b (EC50 of 0.04 and 0.09 nM,
respectively), pyrazine 12f (EC50 = 0.06 nM), and pyrazole 12g
(EC50 = 0.07 nM) can potentially form internal hydrogen
bonds with the indole NH moiety.54 Moreover, the amides 12a
and 12b can also establish additional hydrogen bonding
interactions between the NH and the pyridine nitrogen atom
of the core heterocycle.55 These interactions would be expected
to favor a coplanar arrangement between the azaindole core
and the moiety at C-7. A single crystal X-ray structure of
pyrazole 12g shows the presence of internal hydrogen bonding
between N-1 of the pyrazole and NH group of the indole in the
solid state which confers coplanarity between the azaindole and
C-7 pyrazole rings, clearly noticeable in Figure 2.
at the minimun energy conformation for different C7
substituents (data captured in Table 1).56 In the case of the
oxazole 12c, although the internal hydrogen bonding is absent,
the coplanarity between this C-7 substituent and the core
heterocycle can still be preserved by means of a positive
electrostatic interaction between the O atom of the oxazole and
the indole NH. This conformation also avoids a negative
interaction between the lone pairs of electrons associated with
the oxazole O and pyridine N atoms.57,58 Only a slight
reduction in potency occurred with this compound, EC50
=
0.15 nM, compared to the above-mentioned compounds.
However, a further loss of potency is observed when
coplanarity cannot be readily achieved, as exemplified by
pyrimidine 12e which exhibits EC50 = 0.38 nM.
However, a significant loss of potency is observed with the
oxazole 12d despite the potential for establishment of an
internal H-bond between the oxazole N atom and azaindole
NH that would favor a coplanar topographical arrangement.
The oxazole oxygen atom in this case appears to establish a
negative dipole interaction with the pyridine N atom. Hence, a
complementary CH or hydrogen-bond donor, such as in 12c,
appears to be preferable.59,60
Figure 2. Single crystal X-ray structure of pyrazole 12g.
In vitro pharmacokinetic profiling of methylamide 12a and
oxazole 12c predicted that both should have good intestinal
membrane permeability with Caco-2 values of 124 and 135
To further evaluate the coplanarity hypothesis, quantum
mechanics calculations were used to estimate the dihedral angle
a
Table 2. In Vivo Pharmacokinetic Properties of Selected Compounds in Rats
b
b
compd
F (%)
AUC, 24 h (μM·h)
Cmax (nM)
CL, iv ( mL min−1 kg−1
)
Vss, iv (L/kg)
1
90
63
68
20
33
64
95
77
61
6.3 2.7
1.5 0.28
2.6 0.12
12.0 3.8
12.5 2.3
86 33
1889 647
326 121
13 4.0
1.1 0.22
0.74 0.09
1.4 0.21
0.14 0.01
0.25 0.19
0.49 0.26
0.26 0.04
0.18 0.01
0.6 0.07
12a
12b
12c
12g
12m
12o
12p
12t
31 5.9
603 29
19.3 0.65
1.4 0.29
2.2 0.3
1.6 0.2
0.7 0.12
0.39 0.07
16.5 3.3
1289 187
1400 680
9484 2796
8764 810
13777 217
498.1 193
83.7 9.8
160 11
2.1 0.4
a
b
Vehicle: poly(ethylene glycol) 400 (PEG 400) and ethanol (90:10 v/v). Dose, 5 mg/kg po. Oral Cmax and AUC adjusted to 5 mg/kg.
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nm/s, respectively, and low potential for clearance in man and
rat based on their stability upon incubation with human and rat
liver microsomes. Both the pyrazole 12g and methylamide 12a
also showed good stability in HLM and RLM assays. However,
the Caco-2 value for pyrazole 12g was low, only 7 nm/s, which
is predictive of poor absorption, while the permeability of 12b
could not be determined, probably because of poor solubility.
Nevertheless, these four compounds were chosen for in vivo
pharmacokinetic evaluation in rats, with the results shown in
Table 2. Pyrazine 12f showed only intermediate stability in
HLMs and a Caco-2 value less than 15 nm/s; hence, it was not
evaluated in vivo.
Oral bioavailability was acceptable for amides 12a and 12b.
However, both compounds displayed a much higher clearance
when compared with the benchmark compound 1. This may
contribute to the reduced exposure (AUC) which is only about
half of that of 1 for amide 12b and about one-quarter of 1 for
amide 12a. Oxazole 12c exhibited approximately twice the
exposure (AUC) demonstrated by 1, although the oral
bioavailability was poor. For this particular compound, the
clearance was also improved over 1. Despite a low Caco-2
value, pyrazole 12g provided a better-than-predicted oral
bioavailability, low clearance, and an AUC that was twice that
of 1.
These structure−activity relationship observations offered a
new direction for further effort. On the basis of the belief that
coplanarity between the C-7 substituent and the core
heterocycle provided a beneficial effect on potency, we
investigated the incorporation of N-linked heterocycles,
primarily pyrazoles and triazoles, at the 7-position of the
azaindole ring. In comparison with imidazoles, both pyrazoles
and triazoles should, in principle, be capable of establishing a
hydrogen-bonding interaction with the indole NH group. It was
anticipated that those regioisomers carrying a C−H H-bond
donor could display maximum potency. Moreover, such
compounds were expected to show improved permeability
and overall exposure when compared with pyrazole 12g
because of the absence of a NH group in the newly
incorporated heterocycle.64,65
The antiviral activity of the compounds comprising this new
series, 12m−u, are shown in Table 1. The structure−activity
relationships are consistent with the predictive model that relies
on hydrogen-bonding between the C-7 substituent and the core
heterocycle to favor an overall planar arrangement. Consistent
with this model, the 1,2,3-triazole 12m, the 1,2,4-triazoles
12o,p, and the pyrazoles 12s,t are more potent than the
imidazole 12r, which cannot engage in intramolecular hydro-
gen-bonding. Substitution at position 3 of the heterocycle is
well-tolerated, as demonstrated by the potency of the 3-methyl
heteroaryl derivatives triazole 12p and pyrazole 12t, while
substitution at the 5-position causes considerable loss of
potency, as observed with the triazole 12q and pyrazole 12u.
The 2-substituted 1,2,3-triazole 12n is 80 times less potent than
its 1-substituted regioisomer 12m, presumably because 12n has
an N atom that is proximal to the N of the pyridine ring when
adopting a coplanar arrangement and that may establish an
unfavorable dipole interaction.
The in vitro and in vivo pharmacokinetic properties of four
compounds, 12m, 12o, 12p, and 12t, are summarized in Tables
1 and 2, respectively. All four compounds were found to have
good stability in human and rat liver microsomal assays that
predicted low clearance in both species. None of the human
metabolizing enzymes assayed were inhibited by these
compounds, suggesting low potential for drug−drug inter-
actions (low CYP liability) with other medications. This may
prove to be important, as these compounds would be used in
drug combination therapy that is critical in the treatment of
HIV-1 infection. All four of the compounds displayed good to
excellent oral bioavailability, and only pyrazole 20h showed
lower exposure and higher clearance than compound 1. All
three triazoles 12m, 12o, and 12p showed good permeability
and superior overall pharmacokinetic profiles in rats when
compared to 1. Compounds 12m and 12o produced AUCs 13-
fold higher than 1, while exposure over the time of study for
12p was 25-fold of that of 1. Cmax and clearance were also
greatly improved in all three cases. Next, the stability of these
three triazoles was evaluated in an in vitro assay using human
hepatocytes in an effort to estimate clearance in humans. 1,2,4-
Triazoles 12o and 12p showed values of 12.3 and 10.8 mL
min−1 kg−1, respectively, which indicated a potential for higher
clearance than the 1,2,3-triazole 12m for which disappearance
was not measurable. Thus, the hepatocyte incubation assay
predicted that 12m would display a very low clearance in
humans.21 The total body clearance was also low in dogs and
monkeys. Moreover, the potency of 12m in the whole virus
assay (EC50 = 0.8 nM) was superior to that of the 1,2,4-triazoles
12o and 12p (EC50= 4.3 nM and EC50= 2.3 nM, respectively).
Oxazole 12c and pyrazole 12g were further evaluated in
whole virus assays,61 in either the presence or absence of
human serum (Table 3). The potency of oxazole 12c was
Table 3. Whole Virus Data and PB for Selected
Compounds61
EC50 against HIV Bru (nM)
compd
−HS
+HS
FC
HuPB (%)
12b
12c
12g
12m
12o
12p
0.36
0.34
0.64
0.8
0.99
7.80
2.8
2.8
23
4
96
99.2
2.3
2.8
6.2
8
95.3
97
4.31
3.01
26.8
24.2
97
greatly reduced in the presence of 40% human serum (EC50
=
0.34 nM vs EC50 = 7.8 nM, >20× shift). For pyrazole 12g, the
potency was minimally affected by the presence of human
serum (EC50 shifted 4-fold from 0.64 to 2.80 nM). Consistent
with this result, the oxazole 12c was found to be 99% protein-
bound to human plasma as assessed by an ultrafiltration
method at a plasma concentration of 1 μg/mL. These
compounds were not advanced further because only com-
pounds with superior exposure multiples were desired.62,63
The high potency associated with pyrazole 12g and its
promising in vivo pharmacokinetic properties prompted us to
evaluate additional pyrazoles with substituents adjacent to the
nitrogen with the goal of improving permeability. A series of
pyrazole amides were prepared, and their biological activity and
in vitro pharmacokinetic properties are shown in Table 1. All
compounds, including the ethyl ester intermediate 12h, showed
excellent potency with the primary amide 12i and mono-
methylamide 12j being more potent than amide 12k, which
incorportes a single, larger substituent, and the disubstituted
amide 12l. Although the Caco-2 permeability is improved for
most of these molecules in comparison to pyrazole 12g, their
metabolic stability in both human and rat liver microsomal
preparations was variable and further studies were not pursued.
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Furthermore, when measured in the presence of 40% human
serum, the potency of the 1,2,4-triazoles 12o and 12p was
reduced by a modest 6-fold while the potency of 12m was
reduced by only 2- to 3-fold. This correlated with the relatively
modest protein binding for the latter, which was determined to
be 95% in human plasma by dialysis.
Since HIV-1 heterogeneity is largely dependent on the
envelope glycoprotein, it is critical that any aniviral agent must
have the potency and spectrum coverage to treat this diverse
group of viruses. Therefore, the ability of 12m to block
infection of five different laboratory strains of B subtype was
examined next. The potency displayed by 12m against both T-
(SI) and M-(NSI) tropic HIV-1 strains was superior to that of
1, with an overall increase in spectrum coverage (Table 4). The
Figure 3. Mean plasma concentration−time profiles of 12m after
single doses of 100−1200 mg in healthy subjects (n = 6 per dose
group).
Table 4. Anti-HIV-1 Activity of 1 and 12m against Subtype B
a
Laboratory Strains
Figure 4 shows the mean plasma concentration−time profiles
for 12m (time 0−24) after single-dose administrations of 100−
EC50 (nM)
HIV-1 strain co-receptor usage NSI/SI
host
1
12m
LAI
X4
X4
X4
R5
R5
SI
MT-2
MT-2
MT-2
PM1
3.44
5.58
8.32
28.5
3.0
0.87
1.32
5.17
0.6
NL4-3
MN
SI
SI
BAL
JRFL
NSI
NSI
PM1
<0.23
a
Viral replication of T-tropic strains was determined by measuring RT
activity. Replication of M-tropic strains was detected using a P24 assay.
anti-HIV-1 specificity of 12m was examined in a VSV-G-
pseudotyped HIV-1 assay using efavirenz (NNRTI) as a
possitive control (EC50 = 0.43 nM). Compound 12m showed
no activity at 100 μM, suggesting that its activity against HIV-1
env is not due to host cellular target and is specific against HIV-
1 env.
Figure 4. Mean plasma concentration−time profiles of 12m (time 0−
24 h) after single-dose administrations of 100−1200 mg in healthy
subjects.
Safety profiling of 12m showed no sign of mutagenicity in an
Ames reverse mutation assay at 1600 μg/mL plate or any
evidence for cardiac liability in a cloned hERG channel patch
clamp assay and a rabbit purkinjie fiber assay. On the basis of its
overall potency, pharmacokinetic, and safety profiles, 12m was
advanced into pre-IND toxicology studies as a prelude to
clinical evaluation. In the initial clinical trial, six sequential dose
groups were scheduled in healthy human subjects at doses of
100, 200, 400, 800, 1200, and 1800 mg. Eight subjects were
enrolled in each group and were randomized in a 3:1 drug-to-
placebo ratio. The first five groups received drug doses of 100,
200, 400, 800, and 1200 mg, administered orally as a spray-
dried capsule formulation in a fasted state. The 1800 mg cohort
was not dosed because of early termination of the study. Dose
escalation did not occur until day 5 safety data (including
reported adverse events, clinical laboratory results, vital signs,
and ECG data) from the prior dose group was available,
reviewed, and deemed safe and tolerable. An additional eight
subjects were randomized in a 3:1 drug/placebo ratio to 20 mg
of 12m administered as an oral solution in a fasted state. Figure
3 shows the mean plasma concentration−time profiles of 12m
after single-dose administrations of 100−1200 mg in healthy
subjects over time (0−96 h). For doses (mg) of
100:200:400:800:1200 (ratio 1:2:4:8:12), the geometric mean
of Cmax was in the ratio 1.0:1.3:1.5:2.5:2.7 and the geometric
mean of AUC(0−T) was in the ratio 1.0:1.7:1.6:2.7:3.3. These
data indicate much less than dose-proportional increases in
exposure with increasing doses.
1200 mg in healthy subjects and includes a comparator for the
protein-binding-adjusted EC90 vs B-clade virus (48 ng/mL). As
can be seen from the figure, at 12 h, the levels of drug at even
the highest dose of 1200 mg do not quite equal 2× the protein-
binding-adjusted EC90 of the drug.
Figure 5 shows the comparative PK profiles (time 0−96 h) of
12m following 200 mg of spray-dried capsule vs 20 mg of oral
solution. The mean T1/2 of 12m measured between 2 and 12 h
postdose was about 1.4 h after dosing with the solution
Figure 5. Comparative PK profiles (time 0−96 h) of 12m following
200 mg of spray-dried capsule vs 20 mg of oral solution.
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TFA; 4 min gradient; 4 mL/min flow rate. Method 2: column,
Phenomenex Luna 10 μm C18, 4.6 mm × 50 mm; 5% ACN−95%
H2O−10 mM NH4OAc to 95% ACN−5% H2O−10 mM NH4OAc; 4
min gradient; 4 mL/min flow rate. Method 3: column, Phenomenex
Luna 10 μm C18, 4.6 mm × 50 mm; 5% ACN−95% H2O−10 mm
NH4OAc to 95% ACN−5% H2O−10 mm NH4OAc; 3 min gradient; 4
mL/min flow rate. Method 4: column, Phenomenex Luna 10 μm C18,
3.0 mm × 50 mm; 10% MeOH−90% H2O−0.1% TFA to 90%
MeOH−10% H2O−0.1% TFA; 3 min gradient; 4 mL/min flow rate.
Method 5: column, YMC ODS-A S7, 3.0 mm × 50 mm; 10% MeOH−
90% H2O−0.1% TFA to 90% MeOH−10% H2O−0.1% TFA; 2 min
gradient; 5 mL/min flow rate. Method 6: column, XTERRA S7, 3.0
mm × 50 mm; 10% MeOH−90% H2O−0.1% TFA to 90% MeOH−
10% H2O−0.1% TFA; 2 min gradient; 5 mL/min flow rate. Method 7:
column, Phenomenex Luna C18, 3 μm, 2.0 mm × 30 mm; 5%
MeOH−95% H2O−10 mM NH4OAc to 5% H2O−95% MeOH−10
mM NH4OAc; 4 min gradient; 1 mL/min flow rate. Analytical HPLC
analyses were performed using one of the following conditions.
Method A: column, YMC Pro-C18, 4.6 mm × 50 mm, 5 μm; 10%
ACN−90% H2O−0.1% TFA to 90% MeOH−10% ACN−0.1% TFA;
4 min gradient; 4 mL/min flow rate. Method B: column, Xterra, 5 μm,
4.6 mm × 50 mm; 10% MeOH−90% H2O−0.2% H3PO4 to 80% (90%
MeOH−10% H2O−0.2% H3PO4); 4 min gradient; 4 mL/min flow
rate. Method C: column, Xterra, 5 μm, 4.6 mm × 50 mm; 10%
MeOH−90% H2O−0.1% TFA to 80% (90% MeOH−10% H2O−0.1%
TFA); 4 min gradient; 4 mL/min flow rate. Method D: column,
Gemini C18, 4.6 mm × 150 mm; 5% MeOH−95% H2O−10 mM
NH4HCO3 to 95% MeOH−5% H2O−10 mM NH4HCO3; 20 min
gradient; 1 mL/min flow rate. Preparative reverse phase (rp) HPLC
was performed using two Shimadzu LC-8A pumps, a SPD-10AV UV−
vis detector set at 220 nm, a YMC C18 S5 20 mm × 100 mm column
with an initial gradient (40% B, 60% A) ramp to final gradient (100%
B, 0% A) over 20 min and a hold for 3 min (100% B, 0%A), where
solvent A = 10% MeOH−90% H2O−0.1% TFA and solvent B = 90%
MeOH−10% H2O−0.1% TFA. NMR (1H, 13C, and 19F) spectra were
recorded on one of the following instruments: Bruker DRX-500f at
500 MHz or Bruker DPX-300B or Varian Gemini 300 at 300 MHz as
stated. Shifts are provided in ppm. High-resolution mass spectra were
recorded on one of the following mass spectrometers: a LCT, TOF, or
a LTQ-Orbitrap instrument.
formulation. The levels of 12m in samples collected beyond 16
h postdose were all below assay LOQ. Both the Cmax and AUC
from the solution formulation tended to be less variable
compared to the capsule formulation. The bioavailability of
12m from the capsule formulation was about 30% relative to
the solution formulation based on dose-normalized AUC(INF)
.
The prolonged terminal phase (12−96 h) observed from the
PK profile of the capsule formulation suggests that absorption
of 12m continued in the lower GI tract (i.e., colon). 12m was
concluded to be safe and well tolerated at all doses of the
human study. However, the study was terminated early and
clinical development of 12m was discontinued because of
inadequate pharmacokinetic properties (as reflected in C12
values) and exposure multiples targeted for an optimal
antiretroviral response.
The aqueous crystalline solubility of 12m was determined to
be low at only 7 μg/mL, believed to be responsible for the poor
oral bioavailability at high doses. Previously disclosed
phosphate prodrug methodology32 was successfully applied to
12m to overcome dissolution-limited absorption. Similar to the
published data from a similar prodrug for 1, overall exposure
was significantly increased but Cmin concentrations were still
too low to be viable from a standard formulation.32
Concomitantly, a compound with a more promising overall
profile was discovered in the 4-methoxy, 6-azaindole core series,
and that molecule, BMS-626529, was also advanced to pre-IND
studies along with a phosphonooxymethyl prodrug BMS-
663068. This compound has progressed into phase II clinical
studies.66−70
CONCLUSION
■
A series of HIV-1 attachment inhibitors based on a 4-fluoro,6-
azaindole pharmacophore was prepared. 7-Carbon-linked
amide and heterocycle derivatives showed excellent potency,
and the potency correlated with an ability of the azaindole and
C-7 substituent to adopt a coplanar arrangement. By use of this
model, 7-N-linked heteroaromatic analogues were designed and
synthesized that conserved the excellent potency and also
showed improved in vitro and in vivo pharmacokinetic
properties.
7-Bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridine (7). 5-Amino-2-
methoxypyridine (200 g, 1.6 mol) was added to a stirring mixture of
absolute ethanol (1.1 L) and HBF4 (48% in H2O, 688 mL) and cooled
to 0 °C. NaNO2 (516 g, 7.6 mmol) was dissolved in H2O (210 mL)
and added portionwise over 1 h. The stirring was continued at 0 °C for
2 h. The reaction mixture was diluted with Et2O (4 L). The solid
product formed was collected by filtration and washed with EtOH/
Et2O (50:50, 2 L) and subsequently several times with ether until the
product was slightly pinkish in color. The pale pink solid containing
diazonium salt 2 (360 g, 100% yield) was kept in a desiccator over
P2O5 for 48 h. Anhydrous toluene (1.3 L) was preheated under
nitrogen to 100 °C (internal temperature) in a 2 L, three-neck round-
bottom flask provided with a mechanical stirrer. Diazonium salt 2 (206
g, 0.92 mol) was added portionwise over a period of 1 h via a scoop
through a powder funnel that was attached to an adapter with slight
outward positive nitrogen flow. During the addition (approximately 1
h), the internal temperature was maintained between 99 and 102 °C
(set at 100 °C) and the mixture was stirred vigorously. After the
addition was finished, the reaction mixture was heated at 110 °C for 1
h. The heating mantle was removed, and stirring was stopped. The
reaction mixture was allowed to stand for 2 h (until ambient
temperature achieved). Safety Note: The reaction mixture contains
BF3, and working with the hot reaction mixture exposed vapors that
caused skin irritation on some occasions. No incidents were noted at
ambient temperature. The toluene was decanted into a 4 L Erlenmeyer
flask, and the residual dark brown oil was washed with toluene (50
mL) and combined with the toluene extract. NaOH (1 N, 1.5 L) was
added to the toluene solution. The organic layer was separated and
washed with brine (100 mL). The toluene solution containing 5-
The in vitro potency and the in vitro and in vivo
pharmacokinetic profile of 12m were superior to those of 1
and resulted in the selection of this compound for advanced
preclinical studies in higher species. The data from these studies
prompted the nomination of 7-(1,2,3-triazole)-4-fluoro,6-
azaindole 12m as a candidate for clinical development. Findings
on 12m from the clinic resulted in further work in another
series, resulting in the selection of the current clinical candidate
BMS-663068 now in phase II trials.
EXPERIMENTAL SECTION
■
Chemistry. All nonaqueous reactions were carried out under a
nitrogen atmosphere at room temperature unless otherwise indicated.
All reagents and anhydrous solvents were purchased from commercial
sources and were used without further purification or distillation unless
otherwise noted. Flash column chromatography was performed on EM
Science silica gel 60 (particle size of 40−63 μm). Analytical high
pressure liquid chromatography (HPLC) and LCMS analyses were
conducted using Shimadzu LC-10AS pumps and a SPD-10AV UV−vis
detector set at 220 nm with the MS detection performed with a
Micromass Platform LC spectrometer for LC in electrospray mode.
LCMS analyses were performed using one of the following conditions:
Method 1: column, Phenomenex Luna 10 μm C18, 3.0 mm × 50 mm;
10% MeOH−90% H2O−0.1% TFA to 90% MeOH−10% H2O−0.1%
1
fluoro-2-methoxypyridine 3 was used as is in the next step. H NMR
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(500 MHz, CDCl3) δ 7.92 (d, J = 3.0 Hz, 1H), 7.27 (m, 1H), 6.64 (m,
1H), 3.84 (s, 3H).
19F NMR (471 MHz, CDCl3) δ ppm −138.97 (s, 1 F); 13C NMR (126
MHz, CDCl3) δ 100.66, 118.93, 123.48 (d, J = 20.2 Hz, 1C), 125.23
(d, J = 25.2 Hz, 1C), 128.24, 133.93, 153.97 (d, J = 257.0 Hz, 1C).
Anal. LCMS: (ES+) m/z (M + H)+ = 215.02, tR = 2.25 min (method
1). Anal. rp-HPLC: tR = 1.718 min (method A, purity = 100 %).
HRMS for C7H4BrFN2, (M + H)+ calcd 214.9620, found 214.9616.
3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-N-
methyl-1H-pyrrolo[2,3-c]pyridine-7-carboxamide (12a). Repre-
sentative Procedure for the Synthesis of Carboxamides 12a,b
from 7. A mixture of CuCN (430 mg, 4.8 mmol) and 7-bromo-4-
fluoro-1H-pyrrolo[2,3-c]pyridine (7) (510 mg, 2.4 mmol) in
anhydrous DMF (5 mL) was heated in a sealed tube at 150 °C for
1 h. The reaction mixture was cooled to room temperature. NH4OH
(28% aqueous, 10 mL) and Et2O (50 mL) were added. The organic
layer was separated, dried over Na2SO4, filtered, concentrated in vacuo,
and chromatographed over silica gel to afford 4-fluoro-1H-pyrrolo[2,3-
The toluene solution containing 3 was placed in several sealed tubes
and treated with HCl (35% aq, 300 mL) at 145 °C for 2 h. The
toluene solution was decanted and discarded. The aqueous phase was
washed with EtOAc and concentrated to remove volatiles to afford a
brown solid containing 4-fluoro-2-hydroxypyridine (4) (76 g). 1H
NMR (500 MHz, DMSO-d6) δ 11.33 (s, 1 H), 7.66 (t, J = 3.05 Hz, 1
H), 7.49−7.56 (m, 1 H), 6.45 (dd, J = 9.61, 4.43 Hz, 1 H); 13C NMR
(126 MHz, DMSO-d6) δ ppm 116.95 (d, J = 6.3 Hz, 1C), 125.01 (d, J
= 31.5 Hz, 1C), 130.41 (d, J = 21.4 Hz, 1C), 149.34 (d, J = 230.6 Hz,
1C), 160.60. LCMS: method 2, tR = 0.423 min, mass not apparent.
Anal. rp-HPLC: tR = 0.292 min (method A, purity = 100 %). HRMS
for C5H5OFN2, (M + H)+ calcd 114.0350, found 114.0349. This solid
was taken to next step without further purification. (Note: We have
subsequently improved the method by decanting the toluene layer
prior to heating to reduce volumes.)
1
c]pyridine-7-carbonitrile (8) (255 mg, 66%) as a brownish solid. H
NMR (300 MHz, CD3OD) δ 6.56 (s, 1H), 6.14 (d, J = 3.0 Hz, 1H),
The solid containing 4 (76 g) was dissolved in H2SO4 (170 mL) at
room temperature and then heated to 65 °C. A preformed solution of
fuming HNO3 (63 mL) and H2SO4 (17 mL) was added dropwise. The
temperature was kept between 65 and 80 °C (the reaction is
exothermic, and although the bath was set at 65 °C, the temperature
rises, usually to 75−80 °C). After the addition was complete, the
reaction mixture was heated at 65 °C for an additional hour. The
reaction mixture was cooled to room temperature and poured into a
flask containing ice (1.5 kg, 20 g of ice/g compound). Evolution of gas
occurred. A solid precipitated and was collected by filtration and
discarded. The aqueous layer was extracted with AcOEt (3 × 100 mL)
and the combined extracts were concentrated on a rotary evaporator
under vacuum to afford a solid that was triturated with Et2O to afford
5-fluoro-3-nitropyridin-2-ol (5) (39.5 g, 27% yield from 2) as a bright
yellow solid. 1H NMR (500 MHz, CD3OD) δ 8.57 (m, 1H), 8.03 (m,
1H). MS (ESI) m/z 159 (M + H)+; MS (ESI) m/z 157 (M − H)−. 5-
Fluoro-3-nitropyridin-2-ol (5) (39.5 g, 0.25 mol) was treated with
POBr3 (315 g, 0.75 mol) and a catalytic amount of DMF (15 mL) at
room temperature (DMF was added carefully, gas evolution was
observed). After 5 min at room temperature, the solution was heated
at 110 °C for 3 h and then allowed to cool to room temperature. The
reaction flask was placed in an ice bath, and ice was added portionwise
into the flask (gas evolved because of HBr formation). The liquid and
black solid that formed were poured into a beaker containing ice.
EtOAc (100 mL) was added, and the mixture was extracted with
EtOAc (3 × 75 mL). The organic layer was washed with saturated
aqueous NaHCO3 (75 mL), H2O (75 mL), and brine (75 mL); dried
over Na2SO4; filtered; and concentrated under reduced pressure. The
product was dried under high vacuum overnight to provide 2-bromo-
5-fluoro-3-nitropyridine (6) as a brown solid (42.5 g, 77% yield) which
was taken to the next step without further purification. 1H NMR (500
MHz, CDCl3) δ 8.53 (m, 1H), 7.94 (m, 1H); 13C NMR (500 MHz,
CDCl3) δ 158.9, 156.8, 141.5, 128.1, 121.6. Vinylmagnesium bromide
(800 mL, 1 M in THF) was cooled below −60 °C with vigorous
stirring under N2. A solution of 2-bromo-5-fluoro-3-nitropyridine (6)
(43.3g, 0.192 mol) in THF (200 mL) was added dropwise via an
addition funnel at such a rate that the temperature was kept below −60
°C (over a period of approximately 75 min). The reaction mixture was
warmed to −40 to −50 °C and stirred for an additional 1 h. Saturated
aqueous NH4Cl (1 L) was added slowly and cautiously. At first,
foaming occurred and considerable solid was present, but this
dissolved as the addition was completed and the material warmed to
room temperature. The layers were separated, and the aqueous layer
was extracted with EtOAc (3 × 75 mL). The organic extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated to
afford ∼50 g of a black gummy solid which contained ∼57−58%
product as determined by HPLC. CH2Cl2 was added and the solid
formed was collected by filtration and washed with CH2Cl2 to afford 7-
bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridine (7) (11.1 g) as a brown
solid. The CH2Cl2 solution was concentrated in vacuo and the residue
purified using silica gel to afford 7 (3.3 g). Total amount of 7 was 14.4
1
5.23 (d, J = 3.0 Hz, 1H); H NMR (500 MHz, DMSO-d6) δ ppm
12.95 (br s, 1 H), 8.30 (d, J = 1.83 Hz, 1 H), 7.89 (d, J = 3.05 Hz, 1
H), 6.85 (d, J = 3.05 Hz, 1 H); 19F NMR (471 MHz, DMSO-d6) δ
−128.56 (s, 1 F); 13C NMR (126 MHz, DMSO-d6) δ 98.18, 112.70,
115.54, 122.61 (d, J = 20.1 Hz, 1C), 126.10 (d, J = 23.9 Hz, 1C),
132.67, 137.60, 154.38 (d, J = 262.08 Hz, 1C). Anal. LCMS: (ES+) m/
z (M + H)+ = 162.1110, tR = 1.922 min (method 4). Anal. rp-HPLC:
tR = 1.718 min (method A, purity = 100 %). HRMS for C8H5FN3, (M
+ H)+ calcd 162.046 20, found 162.046 52. A mixture of NaOH (50%
aqueous, 2 mL) and 4-fluoro-1H-pyrrolo[2,3-c]pyridine-7-carbonitrile
(8) (100 mg, 0.62 mmol) in EtOH (5 mL) was heated in a sealed tube
at 110 °C for 18 h and then cooled to room temperature. HCl (6 N)
was added until the pH was adjusted to 2 and a brown precipitate
appeared. The solid was filtered off and the solution was concentrated
to dryness to afford 4-fluoro-1H-pyrrolo[2,3-c]pyridine-7-carboxylic
1
acid (9) as a pale yellow solid. H NMR (300 MHz, CD3OD) δ 8.34
(d, J = 1.5 Hz, 1H), 8.20 (s, 1H), 7.12 (d, J = 1.5 Hz, 1H). This
compound was taken to the next step without further purification.
A mixture of 4-fluoro-1H-pyrrolo[2,3-c]pyridine-7-carboxylic acid
(9) (218 mg, 1.21 mmol), MeNH2 (2 M in THF, 2.42 mL, 4.84
mmol), NMO (0.27 mL, 2.42 mmol), and EDAC (0.48 g, 2.42 mmol)
was stirred in THF (4 mL) at room temperature overnight. H2O (10
mL) and EtOAc (25 mL) were added to the reaction mixture. The
organic layer was separated, dried over Na2SO4, filtered, concentrated
and the residue purified over silica gel using EtOAc/hexanes (10−
50%) as eluent to afford 4-fluoro-N-methyl-1H-pyrrolo[2,3-c]pyridine-
7-carboxamide (10a) (194 mg, 83%) as a brown solid. 1H NMR (300
MHz, CDCl3) δ 8.01 (d, J = 2.0 Hz, 1H), 7.48 (m, 1H), 6.68 (m, 1H),
3.07 (d, J = 5.1 Hz, 3H). 4-Fluoro-N-methyl-1H-pyrrolo[2,3-
c]pyridine-7-carboxamide (10a) (194 mg, 1.0 mmol) was added to a
mixture of AlCl3 (800 mg, 6.0 mmol) and ethylmethylimidazolium
chloride (293 mg, 2.0 mmol). Ethyl oxalylchloride (0.22 mL, 2.0
mmol) was added slowly to the solution and the mixture stirred at
room temperature for 48 h (the mixture became a brown sticky goo).
The reaction flask was placed in an ice bath, and H2O was slowly
added until a suspension formed. The solid was collected by filtration
and dried in the open air to afford 2-(4-fluoro-7-(methylcarbamoyl)-
1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetic acid (11b) as a white solid.
Anal. LCMS: (ES+) m/z (M + H)+ = 266.01, tR = 0.7 min (method 6).
This compound was taken to the next step without further purification.
A mixture of 2-(4-fluoro-7-(methylcarbamoyl)-1H-pyrrolo[2,3-c]-
pyridin-3-yl)-2-oxoacetic (266 mg, 1.0 mmol) (11a), 1-benzoylpiper-
azine hydrochloride (227 mg, 1.0 mmol), NMM (0.52 mL, 6.0 mmol),
and EDAC (380 mg, 2.0 mmol) was stirred in DMF (6 mL) at room
temperature for 8 h. The solvent was removed in vacuo, and H2O (20
mL) and EtOAc (50 mL) were added. The organic layer was
separated, dried over Na2SO4, filtered, concentrated, and purified over
silica gel using CH2Cl2/MeOH (0−10%) as eluent to afford 3-(2-(4-
benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-N-methyl-1H-pyrrolo-
[2,3-c]pyridine-7-carboxamide (12a) (45 mg, 10% for two steps) as a
white solid. 1H NMR (500 MHz, DMSO-d6) δ 12.85 (br s, 1 H), 8.94
(d, J = 4.58 Hz, 1 H), 8.36 (s, 1 H), 8.29 (d, J = 3.36 Hz, 1 H), 7.44
1
g (35%). H NMR (500 MHz, CDCl3) δ ppm 8.65 (br s, 1 H), 7.92
(d, J = 1.83 Hz, 1 H), 7.42 (t, J = 2.90 Hz, 1 H), 6.71−6.84 (m, 1 H);
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(br s, 5 H), 3.28−3.87 (m, 8 H), 2.89 (d, J = 4.5 Hz, 3 H); 19F NMR
(471 MHz, DMSO-d6) δ ppm −120.70 (s, 1 F); 13C NMR (126 MHz,
DMSO-d6) δ ppm 25.66, 40.65, 45.25, 112.07 (d, J = 6.3 Hz, 1C),
119.54 (d, J = 17.6 Hz, 1C), 126.94, 127.19 (d, J = 26.5 Hz, 1C),
128.36, 129.63, 131.92 (d, J = 3.8 Hz, 1C), 134.62 (d, J = 10.1 Hz,
1C), 135.44, 141.44, 153.80 (d, J = 264.6 Hz, 1C), 164.17, 165.55,
Hz, 1C), 125.29, 126.95, 128.19, 128.38, 129.51, 129.64, 135.41,
141.06, 148.14, 152.05 (d, J = 263.3 Hz, 1C), 152.55, 165.61, 169.20,
184.07. Anal. LCMS: (ES+) m/z (M + H)+ = 448.13, tR = 1.370 min
(method 3). Anal. rp-HPLC: tR = 1.693 min (method A, purity = 100
%). HRMS for C23H19FN5O4, (M + H)+ calcd 448.141 56, found
448.142 43.
2-(7-Bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxo-
acetic Acid (15). 7-Bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridine (7)
(15.0 g, 69.7 mmol) was divided into three portions (5 g, 23.2 mmol).
Each portion was added to a mixture of AlCl3 (18.5 g, 139 mmol) and
ethylmethylimidazolium chloride (6.8 g, 46.4 mmol). Ethyl oxalyl-
chloride (5.18 mL, 46.4 mmol) was added slowly to each of the above
solutions, and the mixtures were stirred at room temperature for 8 h
(reaction became a brown sticky goo). The reaction flasks were placed
in ice baths, and H2O was added slowly until a suspension formed.
The solids from all portions were collected by filtration and air-dried
to afford ketoacid 15 (17.0 g, 85%). Anal. LCMS: (ES+) m/z (M +
H)+ = 288.97, tR = 0.69 min (method 7). The solid was taken to the
next step without further purification.
1-(4-Benzoylpiperazin-1-yl)-2-(7-bromo-4-fluoro-1H-
pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (16). A mixture of 2-
(7-bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetic acid
(15) (17 g, 59.2 mmol), 1-benzoylpiperazine hydrochloride (13.4 g,
59.2 mmol), DIPEA (20.6 mL, 118.4 mmol), and DEPBT (35.5 g,
118.4 mmol) was stirred in DMF (300 mL) at room temperature
overnight. The reaction mixture was diluted with EtOAc (1 L) and
washed with water (3 × 200 mL) and brine (200 mL). The organic
layer was dried over MgSO4, concentrated under reduced pressure and
the residue recrystallized from EtOAc to afford 16 (17.7 g, 65%) as a
brown solid. This material was used without further purification in the
next step. A portion of this material was purified using rp-HPLC for
full characterization. 1H NMR (500 MHz, DMSO-d6) δ 13.43 (br s, 1
H), 8.49 (s, 1 H), 8.17 (s, 1 H), 7.44 (br s, 5 H), 3.22−3.87 (m, 8 H);
19F NMR (471 MHz, DMSO-d6) δ −128.26 (s, 1 F); 13C NMR (126
MHz, DMSO-d6) δ 40.60, 45.20, 113.63 (d, J = 3.8 Hz, 1C), 119.65
(d, J = 21.4 Hz, 1C), 120.14, 126.94, 128.15 (d, J = 27.7 Hz, 1C),
128.37, 129.64, 134.75 (d, J = 8.8 Hz, 1C), 135.41, 140.62, 152.77 (d, J
= 259.6 Hz, 1C), 165.43, 169.21, 184.20. Anal. LCMS: (ES+) m/z (M
+ H)+ = 459, tR = 2.723 min (method 1). Anal. rp-HPLC: tR = 1.790
min (method A, purity =100%). HRMS for C20H16BrFN4O3, (M +
H)+ calcd 459.046 81, found 459.0466.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(oxazol-2-yl)-1H-
pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12d). Representa-
tive Procedure for the Stille Coupling of 9 with a Tin
Heterocycle for the Preparation of 7-C-Linked Heterocyles
12d−g. A mixture of 1-(4-benzoylpiperazin-1-yl)-2-(7-bromo-4-
fluoro-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (16) (200 mg,
0.43 mmol), 2-(tri-n-butylstannyl)oxazole (0.14 mL, 0.65 mmol),
(Ph3P)4Pd(0) (50 mg, 0.043 mmol) in 1,4-dioxane (2 mL) was heated
in a sealed tube for 18 h at 110 °C. The reaction mixture was cooled
and filtered through filter paper. The filtrate was diluted with MeOH
and purified using rp-HPLC to give a pale yellow solid which was
recrystallized from MeOH to afford 12d (37 mg, 19%). 1H NMR (500
MHz, CDCl3) δ 11.32 (br s, 1 H), 8.54−8.29 (m, 2 H), 7.92 (s, 1 H),
7.56−7.33 (m, 6 H), 4.02−3.50 (m, 8 H); 13C NMR (126 MHz,
DMSO-d6) δ 40.75, 45.26, 112.76, 119.27, 126.93, 127.64, 128.49 (d, J
= 34.0 Hz, 1C), 129.61, 133.14, 135.46, 140.92, 141.06, 152.97 (d, J =
264.6 Hz, 1C), 158.14, 165.5, 169.24, 183.96. Anal. LCMS: (ES+) m/z
(M + H)+ = 448.53, tR = 2.017 min (method 1). Anal. rp-HPLC: tR =
1.777 min (method A, purity = 100 %). HRMS for C23H19FN5O4, (M
+ H)+ calcd 448.1416, found 448.1417.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(pyrimidin-5-yl)-
1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12e). 12e was
prepared according to the procedure described for 12d using 5-
(tributylstannyl)pyrimidine instead of 2-(tri-n-butylstannyl)oxazole.
1H NMR (500 MHz, DMSO-d6) δ 13.32 (br s, 1 H), 9.35 (s, 1 H),
9.24 (s, 2 H), 8.46−8.54 (m, 2 H), 7.41−7.50 (m, 5 H), 3.40−3.78
(m, 8 H); 19F NMR (471 MHz, DMSO-d6) δ −126.43 (s, 1 F); 13C
NMR (126 MHz, DMSO-d6) δ 40.62, 45.24, 112.70 (d, J = 6.3 Hz,
1C), 119.16 (d, J = 20.16 Hz, 1C), 126.95, 128.23, 128.38, 128.61 (d, J
169.20, 183.95. Anal. LCMS: (ES+) m/z (M + H)+ = 438.46, tR
=
2.080 min (method 4). Anal. rp-HPLC: tR = 1.703 min (method A,
purity =100%). HRMS for C22H21FN5O4, (M + H)+ calcd 438.1572,
found 438.1575.
3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-1H-
pyrrolo[2,3-c]pyridine-7-carboxamide (12b). 12b was prepared
according to the procedure described for 19a using NH4Cl instead of
MeNH2. 1H NMR (500 MHz, CDCl3) δ 8.30 (bs, 1H), 8.20 (bs, 1H),
7.41 (bs, 5H), 3.96−3.47 (m, 8 H); 1H NMR (500 MHz, DMSO-d6) δ
12.79 (br s, 1 H), 8.35 (s, 1 H), 8.27−8.31 (m, 2 H), 7.87 (br s, 1 H),
7.44 (br s, 5 H), 3.40−3.80 (m, 8 H); 19F NMR (471 MHz, DMSO-
d6) δ −120.34 (s, 1 F). Anal. LCMS: (ES+) m/z (M + H)+ = 424.28, tR
= 2.123 min (method 4). Anal. rp-HPLC: tR = 1.600 min (method 1,
purity = 92.6 %). HRMS for C21H19FN5O4, (M + H)+ calcd 424.141
56, found 424.142 16.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(oxazol-5-yl)-1H-
pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12c). n-BuLi (2.5 M
in hexanes, 2.1 mL, 5.25 mmol) was added dropwise to a solution of 7-
bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridine (7) (500 mg, 1.74 mmol)
in THF (5 mL) at −78 °C. The mixture was stirred at this temperature
for 15 min and then at 0 °C for 30 min. The reaction mixture was
cooled to −78 °C, and DMF (0.7 mL, 8.7 mmol) was added. After the
mixture was stirred at this temperature for 30 min, water (5 mL) was
added to quench the reaction and the organic layer was extracted with
EtOAc (2 × 10 mL), dried over MgSO4, filtered, concentrated, and
chromatographed over silica gel to afford 4-fluoro-1H-pyrrolo[2,3-
1
c]pyridine-7-carbaldehyde (13) as a yellow solid (208 mg, 73%). H
NMR (500 MHz, CDCl3) δ 8.28 (s, 1H), 7.50 (s, 1H), 6.75 (s, 1H).
Anal. LCMS: (ES+) m/z (M + H)+ = 164.98: tR = 0.440 min (method
5). A mixture of 4-fluoro-1H-pyrrolo[2,3-c]pyridine-7-carbaldehyde
(13) (50 mg, 0.3 mmol), TOSMIC reagent (60 mg, 0.3 mmol), and
K2CO3 (42 mg, 0.3 mmol) in MeOH (3 mL) was refluxed for 2 h. The
reaction mixture was cooled to room temperature, quenched with ice−
water (10 mL), and extracted with Et2O (2 × 25 mL). The organic
layer was washed with HCl (0.1 N, 10 mL), H2O (10 mL), and brine
(10 mL); dried over Na2SO4; filtered; concentrated; and chromato-
graphed over silica gel to afford 5-(4-fluoro-1H-pyrrolo[2,3-c]pyridin-
1
7-yl)oxazole (14) (61 mg, 100%). H NMR (500 MHz, CD3OD) δ
8.43 (s, 1H), 8.04 (s, 1H), 7.81 (s, 1H), 7.67 (s, 1H), 6.73 (s, 1H). 1-
Ethyl-3-methylimidazolium chloride (82 mg, 0.56 mmol) was added to
a flask containing 5-(4-fluoro-1H-pyrrolo[2,3-c]pyridin-7-yl)oxazole
(14) (56 mg, 0.28 mmol), and the mixture was cooled to 0 °C. AlCl3
(336 mg, 2.52 mmol) was added in one portion, followed by
ethyloxalyl chloride (0.06 mL, 0.56 mmol), and the mixture was stirred
at room temperature for 48 h. Ice−water (10 mL) was added to
quench the reaction. The solid was collected by filtration and washed
with H2O and Et2O and dried in air to afford 2-(4-fluoro-7-(oxazol-5-
yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetic acid (58 mg, 38%).
Anal. LCMS: (ES+) m/z (M + H)+ = 276.15, tR = 0.85 min (method
5). This compound was used in the next step without further
purification. A mixture of 2-(4-fluoro-7-(oxazol-5-yl)-1H-pyrrolo[2,3-
c]pyridin-3-yl)-2-oxoacetic acid (50 mg, 0.18 mmol), 1-benzoylpiper-
azine hydrochloride (61 mg, 0.27 mmol), DIPEA (0.08 mL, 0.45
mmol), and DEPBT (54 mg, 0.18 mmol) was stirred in DMF (1 mL)
at room temperature for 48 h. The reaction mixture was diluted with
EtOAc (10 mL) and washed with H2O (2 × 10 mL). The organic layer
was dried over MgSO4, concentrated in vacuo, chromatographed over
silica gel using EtOAc/hexanes (80−100%) followed by EtOAc/
MeOH (0−10%) as eluent to afford 12c (25 mg, 28%) as a white solid
after recrystallization from MeOH. 1H NMR (500 MHz, DMSO-d6) δ
12.95 (br s, 1 H), 8.68 (s, 1 H), 8.44 (s, 1 H), 8.39 (s, 1 H), 7.92 (s, 1
H), 7.44 (br s, 5 H), 3.42−3.77 (m, 8 H); 19F NMR (471 MHz,
DMSO-d6) δ −125.43 (s, 1 F); 13C NMR (126 MHz, DMSO-d6) δ
40.47, 45.25, 110.31, 112.61 (d, J = 6.3 Hz, 1C), 119.46 (d, J = 21.4
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= 25.2 Hz, 1C), 129.64, 130.45, 134.09 (d, J = 8.8 Hz, 1C), 135.41,
135.85 (d, J = 3.8 Hz, 1C), 141.04, 152.49 (d, J = 262.1 Hz, 1C),
156.44, 158.24, 165.68, 169.22, 184.18. Anal. LCMS: (ES+) m/z (M +
H)+ = 459.3, tR = 2.192 min (method 1). Anal. rp-HPLC: tR = 1.548
min (method 1, purity = 93.0 %). HRMS for C24H19FN6O3, (M + H)+
calcd 459.1581, found 459.1578.
MHz, DMSO-d6) δ 25.58, 40.69, 45.26, 103.60, 112.38 (d, J = 4.8 Hz,
1 C), 118.91 (d, J = 20.2 Hz, 1 C), 126.93, 127.78 (d, J = 25.2 Hz, 1
C), 128.34, 129.60, 131.79 (d, J = 8.6 Hz, 1 C), 133.62, 135.45, 139.84,
140.57, 149.07, 151.84 (d, J = 260.8 Hz, 1 C), 159.34, 165.69, 169.25,
184.15. Anal. LCMS: (ES+) m/z (M + H)+ = 504.41, tR = 1.670 min
(method 4). Anal. rp-HPLC: tR = 1.588 min (method A, purity =
100%). HRMS for C25H23FN7O4, (M + H)+ calcd 504.1796, found
504.1791.
3-(3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-1H-
pyrrolo[2,3-c]pyridin-7-yl)-N-(2-morpholinoethyl)-1H-pyra-
zole-5-carboxamide (12k). 12k was prepared according to the
procedure described for 12i using 2-morpholinoethanamine instead of
MeNH2 and isolated as a white solid. 1H NMR (500 MHz, DMSO-d6)
δ 14.28 (br s, 1 H), 12.25 (br s, 1 H), 9.67 (br s, 1 H), 8.90 (br s, 1 H),
8.28−8.42 (m, 2 H), 7.65 (s, 1 H), 7.45 (br s, 5 H), 4.03 (d, J = 12.82
Hz, 2 H), 3.32−3.78 (m, 16 H), 3.17 (br s, 2 H). 19F NMR (471 MHz,
DMSO-d6) δ −126.92 (s, 1 F); 13C NMR (126 MHz, DMSO-d6) δ
33.22, 40.25, 45.24, 51.23, 55.28, 63.21,104.31, 112.36, 119.20, 126.94,
127.88 (d, J = 23.9 Hz, 1 C), 128.37, 129.64, 131.71 (d, J = 8.8 Hz, 1
C), 135.39, 140.73, 151.91 (d, J = 260.8 Hz, 1 C), 165.63, 169.22,
184.16. Anal. LCMS: (ES+) m/z (M + H)+ = 603.26, tR = 1.485 min
(method 3). Anal. rp-HPLC: tR = 1.417 min (method A, purity =
100%). HRMS for C30H32FN8O5, (M + H)+ calcd 603.2474, found
603.2478.
3-(3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-1H-
pyrrolo[2,3-c]pyridin-7-yl)-N,N-dimethyl-1H-pyrazole-5-car-
boxamide (12l). 12l was prepared according to the procedure
described for 12i using Me2NH (40% in H2O) instead of MeNH2 and
isolated as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 12.35 (br
s, 1 H), 8.36 (s, 1 H), 8.32 (d, J = 2.44 Hz, 1 H), 7.45 (br s, 5 H), 7.30
(br s, 1 H), 3.39−3.84 (m, 8 H), 3.23−3.31 (m, 3 H), 3.06 (s, 3 H);
19F NMR (471 MHz, DMSO-d6) δ −127.05 (s, 1 F); 13C NMR (126
MHz, DMSO-d6) δ 35.47, 38.34, 40.24, 45.24, 105.03, 112.33 (d, J =
5.04 Hz, 1 C), 118.91 (d, J = 21.42 Hz, 1 C), 126.95, 127.74 (d, J =
25.2 Hz, 1 C), 128.37, 129.64, 131.80 (d, J = 7.6 Hz, 1 C), 133.52,
135.40, 139.36, 140.66, 149.52, 151.83 (d, J = 260.8 Hz, 1 C), 160.46,
165.65, 169.22, 184.15. Anal. LCMS: (ES+) m/z (M + H)+ = 518.38,
tR = 2.370 min (method 4). Anal. rp-HPLC: tR = 1.637 min (method
A, purity = 97.5%). HRMS for C26H25FN7O4, (M + H)+ calcd
518.1947, found 518.1949.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(pyrazin-2-yl)-1H-
pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12f). 12f was pre-
pared according to the procedure described for 12d using 2-
1
(tributylstannyl)pyrazine instead of 2-(tri-n-butylstannyl)oxazole. H
NMR (500 MHz, DMSO-d6) δ 12.93 (br s, 1 H), 9.66 (s, 1 H), 8.83−
8.85 (m, 1 H), 8.78 (d, J = 2.44 Hz, 1 H), 8.50 (s, 1 H), 8.38 (s, 1 H),
7.40−7.51 (m, 5 H), 3.42−3.80 (m, 8 H); 19F NMR (471 MHz,
DMSO-d6) δ −123.23 (s, 1 F); 13C NMR (126 MHz, CDCl3) δ 41.89,
46.22, 114.13 (d, J = 5.0 Hz, 1C), 120.18 (d, J = 18.9 Hz, 1C), 127.20,
128.82, 129.25 (d, J = 25.2 Hz, 1C), 130.35, 134.53 (d, J = 3.8 Hz,
1C), 134.91 (d, J = 8.8 Hz, 1C), 135.05, 138.30, 142.42, 144.23,
144.37, 150.71, 153.86 (d, J = 265.9 Hz, 1C), 165.85, 170.83, 183.75.
Anal. LCMS: (ES+) m/z (M + H)+ = 459.16, tR = 1.585 min (method
3). Anal. rp-HPLC: tR = 1.918 min (method A, purity = 96.6 %).
HRMS for C24H19FN6O3, (M + H)+ calcd 459.1581, found 459.1576.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(1H-pyrazol-3-yl)-
1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12g). 12g was
prepared according to the procedure described for 12d using 3-
(tributylstannyl)-1H-pyrazole instead of 2-(tri-n-butylstannyl)oxazole.
1H NMR (500 MHz, DMSO-d6) δ 13.36 (s, 1 H), 12.34 (br s, 1 H),
8.33 (s, 1 H), 8.27 (d, J = 3.36 Hz, 1 H), 7.97 (s, 1 H), 7.45 (br s, 5
H), 6.98 (s, 1 H), 3.40−3.81 (m, 8 H); 19F NMR (471 MHz, DMSO-
d6) δ −127.79 (s, 1 F); 13C NMR (101 MHz, DMSO-d6) δ 40.05,
44.61, 102.74, 111.63 (d, J = 5.4 Hz, 1 C), 118.07 (d, J = 23.9 Hz, 1
C), 126.29, 126.95 (d, J = 24.2 Hz, 1 C), 127.71, 128.97, 129.58,
131.07 (d, J = 7.7 Hz, 1 C), 131.84, 133.94, 134.78, 139.71, 150.94 (d,
J = 259.6 Hz, 1 C), 165.08, 168.57, 183.52. Anal. LCMS: (ES+) m/z
(M + H)+ = 447.15, tR = 1.45 min (method 1). Anal. rp-HPLC: tR =
1.562 min (method A, purity = 95.1%). HRMS for C23H19FN6O3, (M
+ H)+ calcd 447.1581, found 447.1573.
Ethyl 3-(3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluo-
ro-1H-pyrrolo[2,3-c]pyridin-7-yl)-1H-pyrazole-5-carboxylate
(12h). 12h was prepared according to the procedure described for 12d
using pyrazol-3-carboxylic ethyl ester-5-tributyltin instead of 2-(tri-n-
butylstannyl)oxazole. 1H NMR (500 MHz, DMSO-d6) δ 12.41 (br s, 1
H), 8.38 (br s, 1 H), 8.34 (br s, 1 H), 7.45 (br s, 6 H), 4.38 (q, J = 7.0
Hz, 2 H), 3.48 (br s, 8 H), 1.36 (t, J = 7.0 Hz, 3 H); 19F NMR (471
MHz, DMSO-d6) δ −126.501 (s, 1 F). Anal. LCMS: (ES+) m/z (M +
H)+ = 519.15, tR = 1.705 min (method 3). Anal. rp-HPLC: tR = 2.010
min (method A, purity = 100%). HRMS for C26H24FN6O5, (M + H)+
calcd 519.1792, found 519.1785.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(1H-1,2,3-triazol-
1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12m)
and 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(2H-1,2,3-tria-
zol-2-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12n).
Representative Procedure for the Copper Catalyzed Coupling
of 9 with N-Containing Heterocyles for Preparation of 7-N-
Linked Heterocycles 12m−u. A mixture of 1-(4-benzoylpiperazin-
1-yl)-2-(7-bromo-4-fluoro-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-
dione (16) (300 mg, 0.64 mmol), 2H-1,2,3-triazole (0.8 mL, 12.8
mmol), K2CO3 (166 mg, 1.28 mmol), and Cu (0) (43 mg, 0.64 mmol)
was heated at 140 °C in a sealed tube for 6 h. The reaction mixture
was cooled to room temperature and filtered through filter paper to
remove the solids. The filtrate was diluted with MeOH and purified
using rp-HPLC to afford compounds 12m (24 mg, 5.4%) and 12n (10
mg, 2.2%), both as white solids.
3-(3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-1H-
pyrrolo[2,3-c]pyridin-7-yl)-1H-pyrazole-5-carboxamide (12i).
Representative Procedure for the Reaction between Pyrazole
Ester 12h and an Amine for the Preparation of Pyrazole
Amides 12i−l. A mixture of ethyl 3-(3-(2-(4-benzoylpiperazin-1-yl)-
2-oxoacetyl)-4-fluoro-1H-pyrrolo[2,3-c]pyridin-7-yl)-1H-pyrazole-5-
carboxylate (12h) (80 mg, 0.154 mmol) and NH3 in MeOH (5 mL)
was heated at 70 °C in a sealed tube for 48 h. The solvent was
removed in vacuo and the residue was purified using rp-HPLC to
1
12m: H NMR (500 MHz, DMSO-d6) δ 13.09 (s, 1 H), 9.02 (s, 1
1
H), 8.38 (s, 1 H), 8.33 (s, 1 H), 8.13 (d, J = 1.22 Hz, 1 H), 7.45 (br s,
5 H), 3.41−3.83 (m, 8 H); 19F NMR (471 MHz, DMSO-d6) δ
−125.86 (s, 1 F); 13C NMR (126 MHz, DMSO-d6) δ 40.66, 45.25,
112.98 (d, J = 5.76 Hz, 1 C), 121.81 (d, J = 21.4 Hz, 1 H), 122.85,
125.90 (d, J = 27.7 Hz, 1 H), 126.95, 128.37, 129.64, 131.45, 134.02,
135.42, 141.56, 152.15 (d, J = 259.56, 1 H), 165.45, 169.20, 183.94.
Anal. LCMS: (ES+) m/z (M + H)+ = 447.90, tR = 2.245 min (method
1). Anal. rp-HPLC: tR = 1.890 min (method A, purity = 100 %).
HRMS for C22H19FN7O3, (M + H)+ calcd 448.1528, found 448.1519.
Anal. Calcd for C22H18FN7O3·0.76H2O: C, 59.06; H, 4.06; N, 21.91.
Found: C, 56.91; H, 4.05; N, 21.35.
afford 12i as a pale yellow solid (24 mg, 32%) . H NMR (500 MHz,
DMSO-d6) δ 14.12 (br s, 1 H), 12.30 (br s, 1 H), 8.26−8.40 (m, 2 H),
8.10 (br s, 1 H), 7.56−7.69 (m, 2 H), 7.44 (br s, 5 H), 3.30−3.82 (m,
8 H); 19F NMR (471 MHz, DMSO-d6) δ −127.18 (s, 1 F). Anal.
LCMS: (ES+) m/z (M + H)+ = 490.28, tR = 2.510 min (method 1).
Anal. rp-HPLC: tR = 1.515 min (method A, purity = 94.1%). HRMS
for C24H21FN7O4, (M + H)+ calcd 490.1639, found 490.1641.
3-(3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-fluoro-1H-
pyrrolo[2,3-c]pyridin-7-yl)-N-methyl-1H-pyrazole-5-carboxa-
mide (12j). 12j was prepared according to the procedure described
for 12i using MeNH2 (40% in H2O) instead of NH3 and isolated as a
pale yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 12.34 (br s, 1 H),
8.61 (d, J = 2.75 Hz, 1 H), 8.36 (s, 1 H), 8.32 (s, 1 H), 7.57 (s, 1 H),
7.39−7.51 (m, 5 H), 3.36−3.84 (m, 8 H), 2.83 (d, J = 4.88 Hz, 3 H);
19F NMR (471 MHz, DMSO-d6) δ −127.02 (s, 1 F); 13C NMR (126
12n: 1H NMR (500 MHz, DMSO-d6) δ 12.90 (br s, 1 H), 8.37 (s, 1
H), 8.34 (s, 2 H), 8.29 (br s, 1 H), 7.45 (br s, 5 H), 3.40−3.79 (m, 8
H); 19F NMR (471 MHz, DMSO-d6) δ −126.55 (s, 1 F); 13C NMR
(126 MHz, DMSO-d6) δ 40.75, 45.22, 113.03 (d, J = 6.3 Hz, 1 C),
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121.53 (d, J = 20.2 Hz, 1 C), 125.49 (d, J = 7.6 Hz, 1 C), 125.9 (d, J =
27.7 Hz, 1 C), 126.94, 128.37, 129.64, 133.13, 135.41, 137.10, 140.95,
152.09 (d, J = 259.6 Hz, 1 C), 165.48, 169.21, 184.01. Anal. LCMS:
(ES+) m/z (M + H)+ = 448.56, tR = 1.942 min (method 1). Anal. rp-
HPLC: tR = 1.728 min (method A, purity = 100 %). HRMS for
C22H19FN7O3, (M + H)+ calcd 448.1528, found 448.1519.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(3-methyl-1H-pyr-
azol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12t)
and 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(5-methyl-1H-
pyrazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione
(12u). 12u was prepared according to the procedure described for
12m using 3-methylpyrazole instead of 1,2,3-triazole.
12t: 1H NMR (500 MHz, DMSO-d6) δ 12.52 (br s, 1 H), 8.59 (d, J
= 2.44 Hz, 1 H), 8.30 (s, 1 H), 8.14 (s, 1 H), 7.45 (br s, 5 H), 6.48 (d,
J = 2.44 Hz, 1 H), 3.41−3.78 (m, 8 H), 2.43 (s, 3 H); 19F NMR (471
MHz, DMSO-d6) δ −130.535 (s, 1 F); 13C NMR (126 MHz, DMSO-
d6) δ 13.53, 40.61, 45.21, 108.09, 112.69 (d, J = 5.0 Hz, 1 C), 121.35
(d, J = 20.2 Hz, 1 C), 124.18 (d, J = 8.8 Hz, 1 C), 125.33 (d, J = 26.5
Hz, 1 C), 126.95, 128.25, 128.37, 129.64, 133.91, 135.40, 140.20,
150.85 (d, J = 254.5 Hz, 1 C), 151.32, 165.57, 169.22, 184.13. Anal.
LCMS: (ES+) m/z (M + H)+ = 461.42, tR = 2.112 min (method 4).
Anal. rp-HPLC: tR = 2.372 min (method A, purity = 97.4%). HRMS
for C24H22FN6O3, (M + H)+ calcd 461.173 19, found 461.174 06.
12u: 1H NMR (500 MHz, DMF) δ 13.10 (br s, 1 H), 8.69 (s, 1 H),
8.61 (s, 1 H), 8.23 (s, 1 H), 7.87 (br s, 5 H), 6.85 (s, 1 H), 3.83−4.19
(m, 8 H), 3.04 (s, 3 H); 19F NMR (471 MHz, DMSO-d6) δ −128.47
(s, 1 F). Anal. LCMS: (ES+) m/z (M + H)+ = 461.17, tR = 1.847 min
(method 3). Anal. rp-HPLC: tR = 2.260 min (method A, purity = 100
%). HRMS for C24H22FN6O3, (M + H)+ calcd 461.173 19, found
461.173 94.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(1H-1,2,4-triazol-
1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12o). 12o
was prepared according to the procedure described for 12m using
1,2,4-triazole instead of 1,2,3-triazole. 1H NMR (500 MHz, DMSO-d6)
δ 12.89 (br s, 1 H), 9.50 (s, 1 H), 8.52 (s, 1 H), 8.35 (s, 1 H), 8.26 (s,
1 H), 7.44 (br s, 5 H), 3.39−3.80 (m, 8 H); 19F NMR (471 MHz,
DMSO-d6) δ −126.76 (s, 1 F); 13C NMR (126 MHz, DMSO-d6) δ
40.70, 45.26, 112.88 (d, J = 5.0 Hz, 1 C), 121.78 (d, J = 21.4 Hz, 1 C),
124.80 (d, J = 8.8 Hz, 1 C), 125.76 (d, J = 27.7 Hz, 1 C), 126.94,
128.37, 129.64, 131.72, 135.41, 141.08, 142.72, 151.98 (d, J = 258.3
Hz, 1 C), 153.00, 165.43, 169.20, 183.97. Anal. LCMS: (ES+) m/z (M
+ H)+ = 448.17, tR = 1.422 min (method 3). Anal. rp-HPLC: tR
=
1.730 min (method A, purity = 100%). HRMS for C22H19FN7O3, (M
+ H)+ calcd 448.152 79, found 448.153 56.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(3-methyl-1H-
1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-
dione (12p) and 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(5-
methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-
ethane-1,2-dione (12q). 12q was prepared according to the
procedure described for 12m using 3-methyl-1,2,4-triazole instead of
1,2,3-triazole.
Virus Single Cycle HIV-1 Infection Assay. Virus-single-round
infectious reporter virus was produced by co-transfecting human
embryonic kidney 293 cells with an HIV-1 envelope DNA expression
vector and a proviral cDNA containing an envelope deletion mutation
and the luciferase reporter gene inserted in place of the HIV-1 nef
sequences. Transfections were performed using lipoafectAMINE
PLUS reagent as described by the manufacturer (Life Technologies,
Gaithersburg, MD).
Whole Virus Assay. MT-2 cells were infected with laboratory
strains at a multiplicity of infection (MOI) of 0.005, followed by
incubation in the presence of serially diluted inhibitors for 4−5 days in
the presence or absence of 40% heat activated human serum. Virus
yields were quantitated by using a reverse-transcriptase assay.34
12p: 1H NMR (500 MHz, DMSO-d6) δ 12.66 (br s, 1 H), 9.34 (s, 1
H), 8.36 (s, 1 H), 8.22 (br s, 1 H), 7.45 (br s, 5 H), 3.42−3.77 (m, 8
H), 2.48−2.51 (m, 3 H); 19F NMR (471 MHz, DMSO-d6) δ −127.67
(s, 1 F). Anal. LCMS: (ES+) m/z (M + H)+ = 462.17, tR = 1.808 min
(method 2). Anal. rp-HPLC: tR = 1.808 min (method A, purity =
100%). HRMS for C23H21FN7O3, (M + H)+ calcd 462.168 44, found
462.169 40;.
12q: 1H NMR (500 MHz, DMSO-d6) δ 12.85 (br s, 1 H), 8.35 (s, 1
H), 8.25 (s, 2 H), 7.44 (br s, 5 H), 3.40−3.78 (m, 8 H), 3.17 (d, J =
5.19 Hz, 3 H); 19F NMR (471 MHz, DMSO-d6) δ −126.50 (s, 1 F).
Anal. LCMS: (ES+) m/z (M + H)+ = 462.17, tR = 1.447 min (method
2). Anal. rp-HPLC: tR = 1.757 min (method A, purity = 100 %).
HRMS for C23H21FN7O3, (M + H)+ calcd 462.168 44, found 462.169
55.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional HPLC purity methods for key compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org. Chrystallographic data have been deposited at the
Cambridge Crystallographic Data Center (CCDC reference
number 913996), and data can be obtained free of charge via
the internet at http://www.ccdc.cam.ac.uk.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(1H-imidazol-1-yl)-
1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12r). 12r was
prepared according to the procedure described for 12m using
1
imidazole instead of 1,2,3-triazole. H NMR (500 MHz, DMSO-d6)
δ 13.67 (br s, 1 H), 9.67 (s, 1 H), 8.71 (s, 1 H), 8.38 (s, 1 H), 8.23 (s,
1 H), 7.96 (s, 1 H), 7.45 (br s, 5 H), 3.31−3.88 (m, 8 H); 19F NMR
(471 MHz, DMSO-d6) δ −123.899 (s, 1 F); 13C NMR (126 MHz,
DMSO-d6) δ 40.78, 45.29, 113.32 (d, J = 5.76 Hz, 1 C), 121.20, 121.38
(d, J = 20.16, 1 C), 121.90, 126.68 (d, J = 27.72, 1 C), 126.94, 128.39,
129.32 (d, J = 9.60 Hz, 1 C), 129.67, 130.09, 135.38, 136.54, 141.85,
153.11 (d, J = 262.08, 1 C), 165.40, 169.25, 184.07. Anal. LCMS:
(ES+) m/z (M + H)+ = 447.47, tR = 1.577 min (method 4). Anal. rp-
HPLC: tR = 1.330 min (method A, purity = 100 %). HRMS for
C23H20FN6O3, (M + H)+ calcd 447.1575, found 447.1580.
AUTHOR INFORMATION
Corresponding Author
*Phone: 203-677-7416. E-mail: alicia.regueiroren@bms.com.
■
Notes
The authors declare no competing financial interest.
1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-(1H-pyrazol-1-yl)-
1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (12s). 12s was
prepared according to the procedure described for 12m using pyrazole
ACKNOWLEDGMENTS
■
The authors are grateful to Dr. Gregrory Yamanaka (deceased)
for his contributions in the preparation of the virology data
presented and Dr. Xiaohua Stella Huang for assistance in
obtaining NMR data.
1
instead of 1,2,3-triazole. H NMR (500 MHz, DMSO-d6) δ 12.74 (br
s, 1 H), 8.74 (d, J = 2.44 Hz, 1 H), 8.29 (s, 1 H), 8.19 (s, 1 H), 8.02 (d,
J = 1.22 Hz, 1 H), 7.45 (br s, 5 H), 6.68−6.72 (m, 1 H), 3.41−3.81
(m, 8 H); 19F NMR (471 MHz, DMSO-d6) δ −129.41 (s, 1 F); 13C
NMR (126 MHz, DMSO-d6) δ 40.63, 45.27, 108.02, 112.71 (d, J =
5.04 Hz, 1 C), 121.51 (d, J = 20.16 Hz, 1 C), 124.36 (d, J = 7.6 Hz, 1
C), 125.41 (d, J = 26.5 Hz, 1 C), 126.95, 127.82, 128.37, 129.64,
133.86, 135.41, 140.55, 142.38, 151.17 (d, J = 257.0 Hz, 1 C), 165.55,
ABBREVIATIONS USED
■
HIV-1, human immunodeficiency virus type 1; cART, highly
active combination antiretroviral therapy; AIDS, acquired
immunodeficiency syndrome; RT, reverse transcriptase;
gp120, glycoprotein 120; CD4, cluster of differentiation 4;
b.i.d., twice a day (“bis in die”); BCS, biopharmaceutics
classification system; SAR, structure−activity relationship;
169.20, 184.04. Anal. LCMS: (ES+) m/z (M + H)+ = 447.40, tR
=
1.993 min (method 4). Anal. rp-HPLC: tR = 2.153 min (method A,
purity = 95.3 %). HRMS for C23H20FN6O3, (M + H)+ calcd 447.157
54, found 447.158 43.
1666
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