Synthesis of novel substituted 2-phenylpyrazolopyridines with potent activity against herpesviruses

Article abstract of DOI:10.1016/j.bmc.2005.05.043

Herpesviruses are a significant source of human disease; amongst these herpes simplex virus 1 (HSV-1) and HSV-2 are very prevalent and cause recurrent infections. We recently identified a pyrazolo[1,5-a]pyridine scaffold that showed promising activity aga

Full text of DOI:10.1016/j.bmc.2005.05.043

Bioorganic & Medicinal Chemistry 13 (2005) 5346–5361
Synthesis of novel substituted 2-phenylpyrazolopyridines
with potent activity against herpesviruses
Kristjan S. Gudmundsson,a,* Brian A. Johns,a Zhicheng Wang,a Elizabeth M. Turner,a
Scott H. Allen,a George A. Freeman,a F. Leslie Boyd, Jr.,a Connie J. Sexton,b
Dean W. Selleseth,b Kelly R. Monirib and Katrina L. Creechb
aDepartment of Medicinal Chemistry, GlaxoSmithKline Research and Development, Five Moore Drive,
Research Triangle Park, NC 27709-3398, USA
bDepartment of Virology, GlaxoSmithKline Research and Development, Five Moore Drive,
Research Triangle Park, NC 27709-3398, USA
Received 28 February 2005; revised 20 May 2005; accepted 23 May 2005
Available online 21 July 2005
Abstract—Herpesviruses are a significant source of human disease; amongst these herpes simplex virus 1 (HSV-1) and HSV-2 are
very prevalent and cause recurrent infections. We recently identified a pyrazolo[1,5-a]pyridine scaffold that showed promising activ-
ity against HSV-1 and HSV-2 in Vero cell antiviral assays. Here, we describe the synthesis and anti-herpetic activity of several
3-pyrimidinyl-2-phenylpyrazolo[1,5-a]pyridines with differing 2-phenyl substitution patterns. Approaches to rapidly access a num-
ber of analogs with different 2-phenyl substitution patterns are outlined. Several of the compounds described have comparable activ-
ity to acyclovir against HSV-1 and HSV-2.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
viruses is their ability to cause latent infection of neu-
rons, which upon reactivation leads to recurrent lesions
at the innervated dermatome, the mucocutaneous area
of initial infection. Frequency of recurrence is higher
for HSV-2 than HSV-1, occurring about 2–3 times every
100 days. Thus, once latency is established, the primary
infection of HSV is not completely cleared but persists
chronically throughout life.3
The herpesvirus family (Herpesviridae) is highly dissem-
inated in nature and most animal species host at least
one herpesvirus. This family contains eight known
human viruses, herpes simplex virus 1 (HSV-1), HSV-
2, varicella zoster virus (VZV), human cytomegalovirus
(HCMV), Epstein–Barr virus (EBV), and human herpe-
sviruses 6–8 (HHV-6, HHV-7, and HHV-8).1 These her-
pesviruses are associated with a diverse set of diseases,
ranging in severity from asymptomatic to life-threaten-
ing illness.
Previous antiviral research on HSVs has primarily
focused on the development of nucleoside analogs that
target the viral polymerase.4 Early nucleosides included
idoxuridine, vidarabine, and trifluridine. These are still
used topically, but are too toxic for use as first-line treat-
ment.5 Development of acyclovir (1, Zovirax),6 a potent,
specific, and well-tolerated nucleoside inhibitor of her-
pes DNA polymerase, was a milestone in the develop-
ment of antiviral drugs in the late 1970s and spurred
development of a number of other nucleoside analogs.
Currently, only the nucleosides acyclovir, valacyclovir
(2),7 famciclovir (3),8 and penciclovir (4) are recom-
mended for the treatment of herpes simplex disease
(Fig. 1).
Much research has been focused on HSV-1 and HSV-2
as these viruses have a high incidence rate (ꢀ1.6 million
new cases of HSV-2 predicted per year in the United
States) and a high prevalence of 50–95% (HSV-1) and
6–50% (HSV-2) depending on other factors such as
sex, age, and marital status.2 HSV-1 and HSV-2 cause
mucocutaneous infections, such as cold sores (HSV-1)
and genital infection (HSV-2). A key feature of these
Keywords: Pyrazolopyridines; Herpesviruses; Anti-viral compounds.
*
Though numerous strategies and considerable effort
have been expended in the search for the next generation
Corresponding author. Tel.: +1 9194835862; fax: +1 9194836053;
e-mail: Kristjan.S.Gudmundsson@gsk.com
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.043

K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5347
2. Chemistry
For this SAR study, we chose to keep the C-7 position
substituted with either a cyclopentylamino or a n-
butylamino group.17 The C-3 pyrimidine moiety was
also kept unchanged so we could focus our attention
on identifying optimal substitution on the 2-phenyl
moiety.
Key intermediates in the synthesis of the desired pyrazol-
opyridines were 7-chloro-2-phenylpyrazolopyridines (F).
These were synthesized from 6-chloropicoline (B) and
appropriately substituted benzoate esters (C) as outlined
in Scheme 1 using previously described methodology.16
The 3-pyrimidinyl-2-phenylpyrazolopyridines A were
subsequently synthesized from F using either of two meth-
ods for construction of the 3-pyrimidinyl moiety as
outlined in Schemes 1 and 2, respectively.
Briefly, treatment of F with Ac2O and BF3OEt2 in tolu-
ene gave the 3-acetyl derivatives G. The desired C-7
amine was installed using the Pd(O)-mediated Buch-
wald–Hartwig18 amination, using Pd(OAc)2, BINAP,
Cs2CO3 in toluene to give H. Refluxing in dimethyl-
formamide dimethylacetal (DMF-DMA) or dimethyl-
formamide di-tert-butyl acetal (DMF-DTBA) gave the
vinylogous amide I. Treatment of this vinylogous amide
with guanidines (J) gave the desired compound A. Alter-
natively, F could be formylated under Vilsmeier Haack
conditions as outlined in Scheme 2. The resulting 3-for-
myl derivatives (K) were treated with an ethynyl Grig-
nard reagent to give alcohols (L). Oxidation of the
alcohols (L) by MnO2 gave the alkynyl ketones (M) that
were treated with guanidines (J) to give the 7-chloro
derivatives (N). Buchwald–Hartwig methodology or
thermal displacement was then used to install the C-7
amine to give the desired product A.
Figure 1. Drugs approved for treatment of herpes simplex infections
(1–4) and recently described pyrazolo[1,5-a]pyridine (5).
anti-herpetic therapy, it has been proved difficult to
outperform acyclovir.9 Vaccines,10 interleukins,11 inter-
ferons,12 therapeutic proteins, and antibodies with spe-
cific or non-specific mode of action have lacked either
the specificity or the safety to replace the currently avail-
able nucleosides as first-line treatment. Small molecule
drugs have recently received considerable attention as
potential next generation anti-herpetics. Immunomodu-
lators (imiquimod and resiquimod),13 non-nucleoside
viral polymerase inhibitors (4-hydroxyquinoline-3-carb-
oxamides),14 and viral helicase inhibitors (thiazolylphe-
nyl and thiazolylamide)15 appear to be among the
most promising investigational drugs.
We recently identified pyrazolo[1,5-a]pyridine scaffold 5
that showed activity similar to acyclovir against HSV-1
and HSV-2 in our cellular assay.16 Interestingly, few re-
ports exist in the literature regarding the chemistry of
pyrazolopyridines. We have previously described the
methodology developed for the synthesis of 5: here, we
report structure–activity trends (SAR) observed when
the substituents on the 2-phenyl portion of the pyrazolo-
pyridine are varied (Fig. 2).
Figure 2. Pyrazolopyridines, SAR focused on varying substituents on
the 2-phenyl moiety (R1 and R2 = cyclopentyl or n-butyl).
Scheme 1. Synthetic route for general procedure I.

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K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
Scheme 2. Synthetic route for general procedure II.
Yield, scaleability, and ease of purification dictated
whether general procedure I or II was used for the syn-
thesis of individual derivatives. Initially, general proce-
dure I was used to synthesize a set of para-F analogs,
where R1 and R2 were either cyclopentyl or n-butyl
(compounds 5–7; Table 1). Then, a set of halogenated
derivatives was synthesized to investigate how para vs.
meta or ortho halogen (F, Cl, Br) substituent would af-
fect activity (compounds 8–13, synthesized according to
procedure I from appropriate halogenated benzoic acid
derivative). The unsubstituted 2-phenyl derivative 14
(X = H), the m-CH3 derivative (15), and the m-CF3
derivative (16) were also synthesized using the method-
ologies outlined in Schemes 1 and 2.
Scheme 4. Synthesis of various amine derivatives.
Because of the promising activity of the p- and m-chlo-
ro-substituted compounds, we chose to synthesize the
isosteric methoxy derivatives. These methoxy derivatives
17 and 18 showed very promising anti-HSV activity and
we became interested in synthesizing additional alkoxy
analogs (Scheme 3). For the synthesis of these, the meth-
oxy derivatives 17 and 18 were demethylated using BBr3
in dichloromethane to give the corresponding phenols
19 and 20. The phenols were alkylated with a variety
of alkyl bromides in the presence of Cs2CO3 or K2CO3
to give alkoxy derivatives 21–27. The bipenyl ether 28
was synthesized by condensing the p-phenol derivative
19 with phenylboronic acid in the presence of Cu(OAc)2
and Et3N using conditions similar to those described by
Evans et al.19
To further explore electron-donating substituents on the
2-phenyl portion of the pyrazolopyridines, the bromo
derivatives 11 and 12 were used to access amine deriva-
tives 29 and 30 (Scheme 4). These were synthesized via
Buchwald amination, where the bromo derivatives 11
and 12, respectively, were treated with benzophenone
imine, Pd2dba3, and BINAP in the presence of base to
give imines that were subsequently hydrolyzed in aque-
ous acid to give the desired amines 29 and 30. To further
investigate how changes in the basicity and electron-do-
nating effect of the amine would affect anti-viral activity,
the acetylated and sulfonylated derivatives 31 and 32
were synthesized. In addition, prepared from 29 were
alkylamine derivatives 33 and 34.
To explore the steric tolerance around the 2-phenyl moi-
ety, biphenyl derivatives 35 and 36 were prepared from
bromo derivatives 11 and 12 and phenyl boronic acids
using Suzuki coupling methodology (Scheme 5).20
Scheme 3. Synthesis of alkoxy derivatives 17–28.

K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5349
Finally, we were interested in the synthesis of a carbox-
ylic acid derivative, to investigate how a negative charge
at physiological pH would affect the anti-viral activity.
We opted to synthesize the carboxylic acid via nitrile
derivative 37 (Scheme 6). The nitrile derivative 37 was
synthesized by palladium (Pd2dba3)-mediated coupling
of 12 and Zn(CN)2 using conditions similar to those pre-
viously described in the literature.21 Treatment of 37
with KOH at elevated temperature gave the desired acid
38. Additionally, treatment of 37 with ammonium
hydroxide and hydrogen peroxide gave the amide 39.22
The pyrazolopyridines showed similar activity against
HSV-1 and HSV-2 in Vero cells. Several of the com-
pounds showed activity similar to acyclovir against
HSV-1 and HSV-2. Several representative compounds
have also been tested for the activity against other virus-
es (e.g., HIV and HBV) and have not shown significant
activity. We are continuing to investigate the SAR of
these novel pyrazolopyridines and these studies will be
the subject of future publications.
4. Conclusions
In this study, we have described the synthesis of a
diverse set of substituted 2-phenylpyrazolopyridines.
Halogen- and alkoxy-substituted derivatives were identi-
fied as the most promising leads.
3. Results and discussion
The new pyrazolopyridine derivatives were evaluated for
activity against HSV-1 (SC-16) and for cytotoxicity in
Vero cells (Table 1). Several of the pyrazolopyridine
analogs in Table 1 show similar or better anti-HSV
activity than acyclovir.
Several of these novel compounds show similar activity
against HSV-1 and HSV-2 to that of the gold standard
acyclovir. Furthermore, this new series of inhibitors
are not DNA synthesis inhibitors, like the currently
marketed nucleoside analogs.23 This new class of inhib-
itors shows potent antiviral activity separated from
cytotoxicity that suggest its potential use as a therapeu-
tic intervention in HSV-infected patients.
The choice of R1 and R2 as cyclopentyl or n-butyl does
not significantly affect the antiviral activity (com-
pounds 5, 6, and 7 in Table 1). On the other hand,
the choice of substituent on the 2-phenyl can signifi-
cantly change the antiviral activity as well as the toxic-
ity profile of the pyrazolopyridines. In general, small
substituents appear to be preferred over large substitu-
ents, thus halogens, alkyl, alkoxy, and cyano substitu-
ents gave compounds with better anti-viral activity
than bulky phenyl (35 and 36) or amine (31 and 32)
substituents. Hydrophobic substituents were also gen-
erally preferred over hydrophilic substituents. Thus,
halogen (5–13) and alkoxy (17–26) substituted deriva-
tives showed better activity than amino (29–34) or car-
boxyl (38–39) substituted derivatives. Electronic nature
of the substituent did not affect the antiviral activity to
a significant extent. Strongly donating groups such as
methoxy did not give compounds with better antiviral
activity than strongly withdrawing substituents such
as fluorine. In addition, for smaller substituents
whether the substituent was at the para or meta posi-
tion did not make significant difference (e.g., methoxy
derivatives 17 and 18 or fluoro derivatives 5 and 8
showed similar activity) (Table 1).
5. Experimental
5.1. General
1H and 13C NMR spectra were obtained on Varian
Unity Plus NMR spectrometers at 300 or 400 MHz
and 75 or 100 MHz, respectively. 19F NMR spectra were
recorded at 282 MHz. Mass spectra were obtained on
Micromass Platform or ZMD mass spectrometers from
Micromass (Altrincham, UK), using either atmospheric
A selected set of halogenated and alkoxy derivatives that
showed good activity against HSV-1 and good separa-
tion between anti-HSV activity and toxicity along with
promising developability profile were tested for activity
against HSV-2 (Table 2).
Scheme 5. Synthesis of biphenyl derivatives.
Scheme 6. Synthesis of nitriles, acids, and amides.

5350
K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
Table 1. Activity against HSV-1 (SC-16 strain) and cytotoxicity in Vero cells
Compound
X
R1
R2
IC50 (lM)
CC50 (lM)
5
6
p-F
p-F
c-Pent
n-Bu
n-Bu
c-Pent
n-Bu
0.26 (0.01)
0.51 (0.05)
0.41 (0.06)
0.23 (0.01)
0.86 (0.25)
0.17 (0.03)
2.82 (1.14)
0.31 (0.06)
0.85 (0.06)
0.62 (0.11)
0.22 (0.03)
0.27 (0. 05)
0.22 (0.02)
0.20 (0.02)
10.1 (4.6)
0.66 (0.02)
0.20 (0.03)
3.13 (0.40)
0.58 (0.05)
0.17 (0.02)
0.17 (0.04)
0.35
>160
>250
>40
7
p-F
m-F
c-Pent
c-Pent
c-Pent
c-Pent
n-Bu
8
c-Pent
c-Pent
c-Pent
n-Bu
>40
>40
9
p-Cl
m-Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
1
>40
>40
p-Br
m-Br
o-Br
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
n-Bu
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
n-Bu
>100
>40
>40
H
m-CH3
m-CF3
>40
>40
p-OCH3
m-OCH3
p-OH
>100
>40
>50
m-OH
>40
p-OCH2CH@CH2
p-O-n-Bu
p-O-sec-Bu
p-OCH2c-Pr
m-OCH2c-Pr
p-OCH2c-Bu
p-OCH2CO2Et
p-OPh
>100
>100
>100
>100
>40
>100
>100
>40
3.29 (0.21)
0.90 (0.39)
1.07
p-NH2
m-NH2
19
c-Pent
c-Pent
c-Pent
n-Bu
c-Pent
c-Pent
c-Pent
n-Bu
0.64 (0.02)
2.26 (0.13)
2.09 (0.45)
3.24 (0.81)
1.70 (0.46)
2.79 (0.47)
2.18 (0.64)
0.28 (0.04)
3.08 (0.37)
1.67 (0.32)
0.39 (0.01)
m-NHCOCH3
m-NHSO2CH3
p-NH-c-Hexyl
p-N(CH3)2
p-Ph
>40
>40
>40
n-Bu
n-Bu
n-Bu
n-Bu
m-Ph
m-CN
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
c-Pent
>40
>20
>40
>30
>200
m-COOH
m-CONH2
ACV
Vero cells, SC-16 strain. IC50 is the concentration at which 50% efficacy in the anti-viral assay is observed, standard error is shown in brackets. CC50
is the concentration at which 50% cytotoxicity is observed. c-Pent is cyclopentyl; n-Bu is n-butyl.
chemical ionization (APCI) or electrospray ionization
(ESI). Solvents were purchased as anhydrous grade
and used without further purification. Unless otherwise
stated, column chromatography for the purification of
some compounds, used Merck Silica gel 60 (230–400
mesh), and the stated solvent system under pressure.
All compounds were characterized as their free-base
form unless otherwise stated. On occasion, the corre-
sponding hydrochloride salts were formed to generate
solids as noted. Combustion analyses were performed
by Atlantic Microlabs (Norcross, GA, USA). In general,
Table 2. Activity of selected compounds against HSV-1 (SC-16 strain
in vero cells) and HSV-2
Compound
IC50 (HSV-1)
(lM)
IC50 (HSV-2)
(lM)
CC50
(lM)
5
12
0.26
0.31
0.22
0.20
0.58
0.17
0.35
0.39
0.2
>160
>100
>100
>40
0.25
0.20
0.12
0.50
0.1
17
18
23
24
>100
>100
>100
>200
26
1 (ACV)
0.21
0.23
1
compound purity was assessed by high field H NMR
and HPLC. Biological results were obtained with com-
pounds of >98% purity as determined by the above
methods.
IC50 is the concentration at which 50% efficacy in the anti-viral assay is
observed. CC50 is the concentration at which 50% cytotoxicity is
observed.

K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5351
5.2. General procedure I (Scheme 1)
5.2.5.
1-[7-Cyclopentylamino-2-phenylpyrazolo[1,5-a]-
pyridin-3-yl]ethanone (H). To a solution of ketone G
(1 equiv.) in toluene (10 mL/g) was added successive-
ly racemic-BINAP (0.06 equiv.), cesium carbonate
(1.5 equiv.), cyclopentylamine (5 equiv.), and palladi-
um (II) acetate (0.04 equiv.). The resultant mixture
was heated at 100 °C until the reaction was as com-
plete judged by TLC. The solution was cooled to
room temperature and ether was added. The organic
layer was washed with water and brine. The aqueous
layer was extracted with ether, and the combined
organics dried over magnesium sulfate. Filtration
and concentration followed by silica gel chromatogra-
phy gave the desired compound H (where R1 is
cyclopentyl).
5.2.1. Synthesis of 2-(6-chloro-2-pyridinyl)-1-phenyletha-
none derivatives (D). To a cold (0 °C) solution of
6-chloro-2-picoline (B, 1 equiv.) and ethyl benzoate
(C, 2 equiv.) in THF, lithium bis(trimethylsilyl)amide
(2 equiv. in THF) was added dropwise via a pressure
equalizing funnel over 1 h. Upon complete addition, the
cold bath was removed and the resultant solution was
heated at 45 °C until the reaction was complete as judged
by TLC. The mixture was cooled to room temperature
and quenched by the addition of water. Ether was added
and the organic layer was washed with brine. The aqueous
layer was extracted with ether, and the combined organics
were dried over magnesium sulfate. Filtration and
concentration gave a product D that was purified by
recrystallization or silica gel chromatography.
5.2.6. 1-[7-Cyclopentylamino-2-phenylpyrazolo[1,5-a]-
pyridin-3-yl]-3-(dimethylamino)-2-propen-1-one (I). A
5.2.2. Synthesis of 2-(6-chloro-2-pyridinyl)-1-phenyletha-
none oximes (E). To a solution of ketone D (1 equiv.) in
MeOH (ꢀ10 mL/g), hydroxylamine hydrochloride (5
equiv.) was added followed by NaOH (5 equiv., 10%
aqueous). The resultant suspension was heated to reflux
until complete by TLC and then cooled to room temper-
ature. The mixture was concentrated in vacuo and the
residue taken up in ether and water. The organic layer
was washed with brine. The aqueous layer was extracted
with ether, and the combined organics were dried over
magnesium sulfate. Filtration and concentration gave
a residue that was purified by recrystallization or silica
gel chromatography to give E.
solution of H (1 equiv.) in N,N-dimethylformamide
dimethyl acetal (8 mL/g) was heated to reflux for 6 days.
The mixture was cooled to room temperature, and
EtOAc was added followed by water. The organic layer
was washed with brine. The aqueous layer was extracted
with EtOAc, and the combined organics were dried over
magnesium sulfate. Filtration and concentration fol-
lowed by silica gel chromatography (EtOAc) provided
the desired vinylogous amide I.
5.2.7. Synthesis of guanidines (J). N-cyclopentylguani-
dine hydrochloride16 and N-butylguanidine sulfate24
were synthesized as described in the literature.
5.2.3. Formation of 7-chloro-2-(phenyl)pyrazolo[1,5-a]-
pyridines (F). To a solution of E (1 equiv.) in 1,2-
dimethoxyethane (1 mL/g) at 0 °C was added trifluoro-
acetic anhydride (1 equiv.), keeping the temperature
below 10 °C. After the addition was complete, the reac-
tion was allowed to warm to room temperature. The
solution was then cooled to 4 °C and a solution of tri-
ethylamine (2 equiv.) in 1,2-dimethoxyethane was add-
ed over 0.5 h. After warming to room temperature, the
mixture was stirred until the starting material was con-
sumed. To this, iron(II)chloride (0.1 equiv.) was added,
and the reaction was heated to reflux for 8 h. The reac-
tion was concentrated, and the resulting solid was puri-
fied by recrystallization or silica gel chromatography to
give F.
5.2.8. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-phenylpyrazolo[1,5-a]pyridin-7-amine (A). To a
solution of I (1 equiv.) in EtOH (10 mL/g) the desired
guanidine (J) (1.2 equiv.) was added, followed by anhy-
drous K2CO3 (2.5 equiv.). The resultant solution was
heated at reflux until reaction was complete. Upon cool-
ing to room temperature, ether was added followed by
water. The organics were washed with brine, and the
aqueous layer was extracted with ether. The combined
organics were dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue was purified
by silica gel chromatography to give the desired
product A.
5.3. General procedure II (Scheme 2)
5.2.4. 1-[7-Chloro-2-phenylpyrazolo[1,5-a]pyridin-3-yl]-
ethanone (G). To a solution of F (1 equiv.) in toluene
(22 mL/g) at room temperature, acetic anhydride
(1.25 equiv.) was added. Boron trifluoride diethyleth-
erate (1.1 equiv.) was then added dropwise, and the
resultant solution was heated to reflux until complete
as determined by TLC. The reaction mixture was
cooled to room temperature and quenched by the
dropwise addition of aqueous sodium bicarbonate.
Ether was added and the organic layer was washed
with brine. The aqueous layer was extracted with
ether, and the combined organics were dried over
magnesium sulfate, filtered, and concentrated. The
residue was purified by recrystallization or silica gel
chromatography to give G.
5.3.1. 7-Chloro-2-phenylpyrazolo[1,5-a]pyridine-3-carbal-
dehydes (K). DMF (5–10 mL/g) was cooled to 0 °C and
treated with phosphorous oxychloride (1.5–2 equiv.).
After the addition was complete, the mixture was
warmed to room temperature and stirred for 1 h. To
this, the desired F (1 equiv.) was added and the resultant
solution was stirred overnight. Water was added, fol-
lowed by dichloromethane. The aqueous layer was
extracted with dichloromethane. The combined organics
were washed with brine, dried over magnesium sulfate,
filtered, and concentrated. The residue was recrystallized
to give the desired aldehyde K.
5.3.2. 1-[7-Chloro-2-phenylpyrazolo[1,5-a]pyridin-3-yl]-2-
propyn-1-ol (L). To a cold (ꢁ78 °C) solution of aldehyde

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K (1 equiv.) in THF (0.15 M) was added alkynyl magne-
sium bromide (2.5 equiv.) in THF (0.5–1.0 M) dropwise.
The resulting mixture was allowed to warm to 0 °C and
stirred at that temperature until the reaction was com-
plete as judged by TLC or LC/MS methods. The resul-
tant solution was poured into water and extracted
with ether. The organic layer was washed with water
and brine, and the combined organics were dried over
sodium sulfate. Filtration and concentration followed
by flash chromatography or recrystallization provide
the desired alcohol L.
5.4. N-Butyl-3-[2-(butylamino)-4-pyrimidinyl]-2-(4-fluoro-
phenyl)pyrazolo[1,5-a]pyridin-7-amine (6)
5.4.1. 1-[7-(Butylamino)-2-(4-fluorophenyl)pyrazolo-[1,5-
a]pyridin-3-yl]ethanone (40). 1-[7-Chloro-2-(4-fluoro-
phenyl)pyrazolo[1,5-a]pyridin-3-yl]ethanone (969 mg,
3.4 mmol) was treated according to the procedure I to
give after flash chromatography (2:1, hexanes/ethyl ace-
1
tate) 40 (921 mg, 84%) as an oil. H NMR (CDCl3): d
7.71 (d, 1H), 7.63 (dd, 2H), 7.49 (t, 1H), 7.23 (t, 2H),
6.16 (d, 1H), 6.10 (m, 1H), 3.40 (m, 2H), 2.18 (s, 3H),
1.78 (m, 2H), 1.52 (m, 2H ), 1.02 (t, 3H); MS m/z 326
(M + 1); Rf 0.6 (1:1 hexanes/ethyl acetate).
5.3.3. 1-[7-Chloro-2-phenylpyrazolo[1,5-a]pyridin-3-yl]-2-
propyn-1-one (M). To a solution of alcohol L (1 equiv.)
in CH2Cl2 (0.05 M), manganese dioxide (40 equiv.) was
added. The reaction mixture was stirred at room tem-
perature until complete as judged by TLC or LC/MS
methods. The suspension was filtered through a pad of
Celite, and the filtrate was concentrated in vacuo to give
ketone M. This material is used without further
purification.
5.4.2. (2E)-1-[7-(Butylamino)-2-(4-fluorophenyl)-pyrazolo-
[1,5-a]pyridin-3-yl]-3-(dimethylamino)-2-propen-1-one
(41). 1-[7-(Butylamino)-2-(4-fluorophenyl)-pyrazolo[1,5-
a]pyridin-3-yl]ethanone (40, 880 mg, 2.7 mmol) was
treated as described in the general procedure I to give
1
41 (971 mg, 95%) as yellow foam. H NMR (CDCl3):
d 7.67 (dd, 2H), 7.60 (d, 1H), 7.53 (d, 1H), 7.27 (t,
1H), 7.09 (t, 2H), 5.96–5.93 (m 2H), 5.01 (d, 1H), 3.30
(m, 2H), 3.10–2.30 (br, 6H), 1.69 (m, 2H), 1.43 (m,
2H), 0.93 (t, 3H); MS m/z 381 (M + 1).
5.3.4. 4-[7-Chloro-2-phenylpyrazolo[1,5-a]pyridin-3-yl]-
N-cyclopentyl-2- pyridinamine (N). To a dry round bot-
tom flask added sodium metal (1.3 equiv.). Ethanol
(0.15 M) was added and allowed to react with sodium
at room temperature until completely dissolved. Guan-
idinium salt J (1.3 equiv.) was added, and the mixture
was allowed to stir at room temperature for 10 min.
To the resultant mixture, ketone M (1 equiv.) was add-
ed, and the reaction mixture was stirred at room temper-
ature or heated to 70 °C until complete as judged by
TLC or LC/MS. The reaction mixture was cooled to
room temperature and diluted with water. The mixture
was extracted with ethyl acetate, and the combined ex-
tracts were washed with water and brine. The organic
layer was dried over sodium sulfate. Filtration and
concentration followed by flash chromatography or
recrystallization provided pyrimidine N. An alternative
5.4.3. N-Butyl-3-[2-(butylamino)-4-pyrimidinyl]-2-(4-flu-
orophenyl)pyrazolo[1,5-a]pyridin-7-amine (6). In a similar
manner as described in the general procedure I, from 41
(34 mg, 0.09 mmol) and N-butylguanidine sulfate, 6
1
(33 mg, 85%) was obtained as a yellow solid. H NMR
(CDCl3): d 7.98 (d, 1H), 7.68 (d, 1H), 7.59 (m, 2H),
7.27 (t, 1H), 7.10 (t, 2H), 6.25 (d, 1H), 6.00–5.96 (m,
2H), 5.05 (m, 1H), 3.41 (m, 2H), 3.33 (m, 2H), 1.71
(m, 2H), 1.60 (m, 2H), 1.50–1.36 (m, 4H), 0.97–0.91
(m, 6H); MS m/z 433 (M + 1); Rf 0.67 (1:1, hexanes/ethyl
acetate).
5.5. N-Butyl-3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-
fluorophenyl)pyrazolo[1,5-a]pyridin-7-amine (7)
method involved heating
a solution of alkynyl
ketone M (1 equiv.) guanidine J (1.3 equiv.), and
K2CO3 (1.3 equiv.) in NMP or DMF (0.15 M) at
120 °C to give N.
In a similar manner as described in the general procedure
I, from 41 (258 mg, 0.68 mmol) and N-cyclopentylguani-
dine, 7 (260 mg, 87%) was obtained as a yellow solid. 1H
NMR (CDCl3): d 8.01 (d, 1H), 7.73 (d, 1H), 7.62 (m,
2H), 7.29 (t, 1H), 7.12 (t, 2H), 6.28 (d, 1H), 6.05 (m,
1H), 5.98 (d, 1H), 5.27 (br, 1H), 4.32 (m, 1H), 3.34 (m,
2H), 2.10–2.00 (m, 2H), 1.77–1.41 (m, 10H), 0.97 (t,
3H); 13C NMR (CDCl3) d 163.03 (d, JCF = 244.5 Hz),
162.14, 161.41, 156.74, 152.10, 142.99, 141.02, 131.36 (d,
JCF = 8.0 Hz) 129.92 (d, JCF = 3.0 Hz), 128.31, 115.36
(d, JCF = 21.0 Hz), 108.58, 107.9, 105.17, 89.21, 52.79,
42.27, 33.40, 31.06, 23.65, 20.13, 13.70; 19F NMR
(CDCl3) d ꢁ113.42; MS m/z 445 (M + 1).
5.3.5. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-phenylpyrazolo[1,5-a]pyridin-7-amine (A). To a
solution of chloride N (1 equiv.) in toluene (0.2 M), suc-
cessively racemic-BINAP (6 mol%), cesium carbonate
(1.5 equiv.), cyclopentylamine (5 equiv.), and palladium
(II) acetate (4 mol%) were added. The resultant mixture
was heated to 95 °C until the reaction was complete as
judged by TLC or LC/MS. The solution was cooled to
room temperature and ether was added. The organic layer
was washed with water and brine. The aqueous layer was
extracted with ether and the combined organics dried over
sodium sulfate. Purification by flash chromatography or
recrystallization provided 7-amino derivative A (where
R1 is cyclopentyl).
5.6. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(3-fluorophenyl)pyrazolo[1,5-a]pyridin-7-amine (8)
Compound 8 was obtained according to the general pro-
cedure I. Rf 0.43 (99:1, dichloromethane/methanol); H
1
Alternatively, chloride N was heated in either neat
amine at 130–150 °C or a solution of excess amine in
ethanol at reflux followed by similar workup to provide
the desired 7-amino derivative A.
NMR (CDCl3): d 8.06 (d, 1H), 7.76 (d, 1H), 7.47–7.30
(m, 4H), 7.17 (m, 1H), 6.35 (d, 1H), 6.10–6.04 (m,
2H), 5.15 (d, 1H), 4.36 (m, 1H), 4.05 (m, 1H), 2.21–
2.07 (m, 4H), 1.88–1.54 (m, 12H); MS m/z 457 (M + 1).

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5353
5.7. 2-(4-Chlorophenyl)-N-cyclopentyl-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (9)
5.9.6. (2E)-1-[2-(4-Bromophenyl)-7-(butylamino)-pyrazol-
o[1,5-a]pyridin-3-yl]-3-(dimethylamino)-2-propen-1-one (47).
In a similar manner as described in the general proce-
dure I, from 46 (5.5 g, 14.2 mmol), 47 (5.78 g,
Compound 9 was obtained according to procedure I. 1H
NMR (CDCl3): d 8.02 (d, 1H), 7.72 (m, 1H), 7.60 (d,
2H), 7.41 (d, 2H), 7.35 (m, 1H), 6.31 (d, 1H), 6.03 (m,
2H), 5.17 (br, 1H), 4.32 (m, 1H), 4.00 (m, 1H), 2.18–
2.05 (m, 4H), 1.82–1.50 (m, 12H). MS m/z 474 (M + 1).
1
92%) was obtained as a brown oil. H NMR (CDCl3):
d 7.57 (m, 6H), 7.28 (t, 1H), 5.95 (m, 2H), 5.03
(d, 1H), 3.32 (q, 2H), 2.92 (bs, 3H), 2.52 (bs, 3H),
1.71 (m, 2H), 1.44 (m, 2H), 0.94 (t, 3H); MS m/z 441
(M + 1).
5.8. 2-(3-Chlorophenyl)-N-cyclopentyl-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (10)
5.9.7. 2-(4-Bromophenyl)-N-butyl-3-[2-(butylamino)-4-
pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (11). In a sim-
ilar manner as described in the general procedure I, from
47 (5.78 g, 13.1 mmol) and N-butylguanidine sulfate, 11
Compound 10 was prepared according to procedure I as a
yellow solid. Rf 0.48 (49:1 dichloromethane/methanol);
1H NMR (DMSO-d6): d 8.02 (d, 1H), 7.73–7.67 (br,
2H), 7.56–7.44 (m, 2H), 7.36 (t, 1H), 7.01 (d, 1H), 6.61
(d, 1H), 6.22–6.17 (m, 2H), 4.09 (br, 1H), 3.98 (m, 1H),
2.04 (m, 2H), 1.84 (m, 2H), 1.72–1.48 (m, 12H); MS m/z
473 (M + 1). Anal. Calcd for C27H29ClN6: C, 68.56; H,
6.18; N, 17.77. Found: C, 68.55; H, 6.20; N, 17.64.
1
(5.18 g, 80%) was obtained as a pale yellow solid. H
NMR (CDCl3): d 7.98 (d, 1H), 7.71 (d, 1H), 7.56 (m,
4H), 7.33 (t, 1H), 6.35 (d, 1H), 6.03 (m, 2H), 3.46 (q,
2H), 3.38 (q, 2H), 1.81–1.40 (m, 8H), 0.98 (m, 6H);
MS m/z 493 (M + 1).
5.10. 2-(3-Bromophenyl)-N-cyclopentyl-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (12)
5.9. 2-(4-Bromophenyl)-N-butyl-3-[2-(butylamino)-4-pyri-
midinyl]pyrazolo-[1,5-a]pyridin-7-amine (11)
5.10.1.
1-(3-Bromophenyl)-2-(6-chloro-2-pyridinyl)etha-
5.9.1.
1-(4-Bromophenyl)-2-(6-chloro-2-pyridinyl)etha-
none (48). In a similar manner as described in the proce-
dure I, from ethyl 3-bromobenzoate (50.6 g, 220 mmol)
and 6-chloro-2-picoline (24 mL, 220 mmol), 48 (59.4 g,
87%) was obtained as a yellow solid. 1H NMR (CDCl3):
d 7.96 (br s, 1H), 7.71 (d, 1H), 7.35–7.25 (m, 3H), 6.98 (t,
1H), 6.81 (d, 1H), 6.58 (d, 1H), 5.84 (s, 1H); MS m/z 310
(M + 1).
none (42). In a similar manner as described in the general
procedure I, from ethyl 4-bromobenzoate (12.8 mL,
78.3 mmol) and 6-chloro-2-picoline (4.3 mL, 39.2 mmol),
42 (9.6 g, 82%) was obtained as a crystalline solid existing
as a keto–enol tautomeric mixture. 1H NMR (CDCl3): for
the keto tautomer d 7.95 (d, 2H), 7.74–7.56 (m, 3H), 7.27
(m, 2H), 4.47 (s, 2H); MS m/z 310 (M + 1).
5.10.2. 1-(3-Bromophenyl)-2-(6-chloro-2-pyridinyl)ethanone
oxime (49). In a similar manner as described in the pro-
cedure I, from 48 (59.1 g, 190 mmol), 49 (58.3 g, 94%)
was obtained as a white solid. 1H NMR (CDCl3): d
7.96 (s, 1H), 7.67–7.50 (m, 3H), 7.28–7.18 (m, 3H),
4.80 (br, 1H), 4.39 (s, 2H); MS m/z 325 (M + 1).
5.9.2. 1-(4-Bromophenyl)-2-(6-chloro-2-pyridinyl)-ethanone
oxime (43). In a similar manner as described in the pro-
cedure I, from 42 (9.5 g, 30.6 mmol), 43 (10.0 g, 99%)
was obtained as a white solid. 1H NMR (CDCl3): d
9.78 (br, 1H), 7.74–7.47 (m, 5H ), 7.21–7.17 (m, 2H),
4.39 (s, 2H); MS m/z 325 (M + 1).
5.10.3. 2-(3-Bromophenyl)-7-chloropyrazolo[1,5-a]pyri-
dine (50). In a similar manner as described in the pro-
cedure I, from 49 (59.1 g, 181.5 mmol), 50 (24.5 g,
44%) was obtained as a yellow solid. 1H NMR
(CDCl3): d 8.13 (dd, 1H), 7.90 (d, 1H), 7.47
(m, 2H), 7.29 (t, 1H), 7.05 (t, 1H), 6.88 (m, 2H);
5.9.3. 2-(4-Bromophenyl)-7-chloropyrazolo[1,5-a]pyridine
(44). In a similar manner as described in the procedure I,
from 43 (45.2 g, 139 mmol), 44 (30.5 g, 72%) was ob-
tained as a pale yellow crystalline solid. 1H NMR
(CDCl3): d 7.85 (dd, 2H), 7.54 (dd, 2H), 7.46 (d, 1H),
7.04 (m, 1H), 6.87 (m, 2H); MS m/z 307 (M + 1).
MS m/z 307 (M + 1).
5.9.4.
1-[2-(4-Bromophenyl)-7-chloropyrazolo[1,5-a]-
5.10.4.
1-[2-(3-Bromophenyl)-7-chloropyrazolo[1,5-a]-
pyridin-3-yl]ethanone (45). In a similar manner as de-
scribed in the general procedure I, from 44 (10.0 g,
32.5 mmol), 45 (7.63 g, 67%) was obtained as pink nee-
dles. H NMR (CDCl3): d 8.37 (d, 1H), 7.62 (d, 2H),
7.43 (m, 3H), 7.14 (d, 1H), 2.13 (s, 3H); MS m/z 349
(M + 1).
pyridin-3-yl]ethanone (51). In a similar manner as de-
scribed in the procedure I, from 50 (16.5 g, 53.6 mmol),
51 (8.6 g, 46%) was obtained as pinkish needles. 1H
NMR (CDCl3): d 8.46 (d, 1H), 7.83 (s, 1H), 7.69 (d,
1H), 7.59–7.40 (m, 3H), 7.22 (d, 1H), 2.21 (s, 3H); MS
m/z 349 (M + 1).
1
5.9.5. 1-[2-(4-Bromophenyl)-7-(butylamino)pyrazolo[1,5-
a]pyridin-3-yl]ethanone (46). In a similar manner as de-
scribed in the general procedure I, from 45 (2.55 g,
7.3 mmol), 46 (2.15 g, 76%) was obtained as a yellow
5.10.5. 1-[2-(3-Bromophenyl)-7-(cyclopentylamino)-pyr-
azolo[1,5-a]pyridin-3-yl]ethanone (52). In a similar man-
ner as described in the procedure I, from 51 (3.00 g,
8.6 mmol), 52 (1.90 g, 56%) was obtained as a yellow
1
1
oil. H NMR (CDCl3): d 7.61 (m, 3H), 7.43 (m, 3H),
syrup. H NMR (CDCl3): d 7.74 (dd, 1H), 7.61–7.57
6.08 (d, 1H), 6.02 (bs, 1H ), 3.33 (q, 2H), 2.12 (s, 3H),
1.70 (m, 2H), 1.44 (m, 2H), 0.94 (t, 3H); MS m/z 386
(M + 1).
(m, 2H), 7.50 (d, 1H), 7.41 (t, 1H), 7.32 (t, 1H), 6.10
(dd, 1H), 5.99 (d, 1H), 3.95 (m, 1H), 2.12–2.05 (m,
5H), 1.78–1.63 (m, 6H); MS m/z 398 (M + 1).

5354
K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5.10.6. (2E)-1-[2-(3-Bromophenyl)-7-(cyclopentylamino)-
pyrazolo[1,5-a]pyridin-3-yl]-3-(dimethylamino)-2-propen-
1-one (53). In a similar manner as described in the pro-
cedure I, from 52 (1.90 g, 4.8 mmol), 53 (1.87 g, 86%)
5.11.6.
(2E)-1-[2-(2-Bromophenyl)-7-(cyclopentylami-
no)pyrazolo[1,5-a]pyridin-3-yl]-3-(dimethylamino)-2-pro-
pen-1-one (59). In a similar manner as described in the
procedure I, from 58 (0.93 g, 2.3 mmol), 59 (0.57 g,
54%) was obtained as a brown syrup. 1H NMR
(CDCl3): d 7.78 (d, 1H), 7.66 (d, 1H), 7.53 (d, 1H),
7.46 (m, 1H), 7.39 (t, 1H), 7.31–7.22 (m, 2H), 6.02 (d,
1H), 5.85 (d, 1H), 4.80 (d, 1H), 3.95 (m, 1H), 2.90 (br
s, 3H), 2.30 (br s, 3H), 2.08 (m, 2H), 1.77–1.63 (m,
6H); MS m/z 455 (M + 1).
1
was obtained as a brown syrup. H NMR (CDCl3): d
7.88 (s, 1H) 7.64–7.49 (m, 4H), 7.30–7.24 (m, 2H),
6.00 (d, 1H), 5.93 (d, 1H), 5.03 (d, 1H), 3.95 (m, 1H),
3.10–2.35 (br, 6H), 2.10–2.06 (m, 2H), 1.77–1.62 (m,
6H); MS m/z 455 (M + 1).
5.10.7. 2-(3-Bromophenyl)-N-cyclopentyl-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (12).
In a similar manner as described in the procedure I,
from 53 (500 mg, 1.1 mmol) and N-cyclopentylguani-
dine, the title compound 12 (500 mg, 88%) was obtained
as a yellow solid. 1H NMR (CDCl3): d 8.00 (d, 1H), 7.83
(s, 1H), 7.65 (d, 1H), 7.51 (m, 2H), 7.29–7.22 (m, 2H),
6.28 (d, 1H), 6.00 (d, 1H), 5.96 (d, 1H), 5.00 (d, 1H),
4.27 (m, 1H), 3.97 (m, 1H), 2.11–2.00 (m, 4H), 1.79–
1.46 (m, 12H); MS m/z 517 (M + 1).
5.11.7. 2-(2-Bromophenyl)-N-cyclopentyl-3-[2-(cyclopen-
tylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine
(13). In a similar manner as described in the procedure I,
from 59 (200 mg, 0.46 mmol), 13 (110 mg, 48%) was ob-
1
tained as a yellow foam. H NMR (CDCl3): d 7.90 (m,
2H), 7.66 (d, 1H), 7.44 (m, 1H), 7.39 (t, 1H), 7.32–7.27
(m, 2H), 6.04–5.96 (m, 3H), 5.02 (m, 1H), 4.21 (m,
1H), 3.95 (m, 1H), 2.10–1.96 (m, 4H), 1.75–1.43 (m,
12H); MS m/z 517 (M + 1).
5.11. 2-(2-Bromophenyl)-N-cyclopentyl-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (13)
5.12. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-phenylpyrazolo[1,5-a]pyridin-7-amine (14)
5.11.1. 1-(2-Bromophenyl)-2-(6-chloro-2-pyridinyl)-etha-
none (54). In a similar manner as described in the proce-
dure I, from ethyl 2-bromobenzoate (50.0 g, 218 mmol)
and 6-chloro-2-picoline (24 mL, 218 mmol), 54 (52.4 g,
77%) was obtained as a yellow solid. 1H NMR (CDCl3):
d 7.53 (d, 1H), 7.45 (d, 1H), 7.25 (d, 1H), 7.05 (d, 1H),
6.97 (d, 1H), 6.67 (d, 1H), 6.53 (d, 1H), 5.28 (s, 1H);
MS m/z 310 (M + 1).
To a solution of 13 (100 mg, 0.19 mmol) in toluene
(10 mL) tributyltin hydride (112 mg, 0.39 mmol) and
2,20-azobisisobutyronitrile (9.4 mg, 0.057 mmol) were
added. After heating at reflux for 4 h, the reaction
mixture was allowed to cool to room temperature.
Concentration followed by purification with flash
chromatography (40:60, ethyl acetate/hexanes) gave 14
1
as a yellow foam (39 mg, 46%). H NMR (CDCl3): d
7.98 (d, 1H), 7.78 (d, 1H), 7.64 (m, 2H), 7.45 (m, 2H),
7.33–7.26 (m, 2H), 6.27 (d, 1H), 6.04 (m, 2H), 5.12 (d,
1H), 4.35 (m, 1H), 4.00 (m, 1H), 2.09–2.03 (m, 4H),
1.81–1.59 (m, 12H); MS m/z 439 (M + 1).
5.11.2. 1-(2-Bromophenyl)-2-(6-chloro-2-pyridinyl)-etha-
none oxime (55). In a similar manner as described in
the procedure I, from 54 (52.4 g, 169 mmol) 55 (36.5 g,
66%) was obtained as a pale yellow solid. 1H NMR
(CDCl3): d 7.50–7.45 (m, 2H), 7.23–7.07 (m, 6H), 4.29
(s, 2H); MS m/z 325 (M + 1).
Alternatively, 14 could be prepared from ethyl benzoate
using general procedure I.
5.11.3. 2-(2-Bromophenyl)-7-chloropyrazolo[1,5-a]pyri-
dine (56). In a similar manner as described in the proce-
dure I, from 55 (36.5 g, 112 mmol), 56 (21.0 g, 61%) was
5.13. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(3-methylphenyl)-pyrazolo[1,5-a]pyridin-7-amine
(15)
1
obtained as a yellow solid. H NMR (CDCl3): d 7.83
(dd, 1H), 7.65 (d, 1H), 7.51 (d, 1H), 7.37 (t, 1H), 7.21
(m, 1H), 7.07 (m, 2H), 6.89 (d, 1H); MS m/z 307
(M + 1).
5.13.1. 2-(6-Chloro-2-pyridinyl)-1-(3-methylphenyl)-etha-
none (60). In a similar manner as described in the proce-
dure I, from ethyl 3-methylbenzoate (30 g, 183 mmol),
60 (33.6 g, 75% yield) was obtained as a mixture of ke-
tone and enol tautomers. This mixture was used directly
in the next step.
5.11.4.
1-[2-(2-Bromophenyl)-7-chloropyrazolo[1,5-a]-
pyridin-3-yl]ethanone (57). In a similar manner as de-
scribed in the procedure I, from 56 (21.0 g, 68.3 mmol),
57 (15.7 g, 66%) was obtained as orange needles. 1H
NMR (CDCl3): d 8.48 (dd, 1H), 7.72 (d, 1H), 7.49–7.36
(m, 4H), 7.18 (dd, 1H), 2.06 (s, 3H); MS m/z 349 (M + 1).
5.13.2. 2-(6-Chloro-2-pyridinyl)-1-(3-methylphenyl)-etha-
none oxime (61). In a similar manner as described in
the procedure I, from 60 (33.6 g, 137 mmol), 61
(26.1 g, 73% yield) was obtained as a white solid.
1H NMR (CDCl3): d 7.54–7.47 (m, 3H), 7.26–7.15
(m, 4H), 4.40 (s, 2H), 2.34 (s, 3H). MS m/z 243
(M + 1).
5.11.5. 1-[2-(2-Bromophenyl)-7-(cyclopentylamino)pyraz-
olo[1,5-a]pyridin-3-yl]ethanone (58). In a similar manner
as described in the procedure I, from 57 (3.00 g,
8.6 mmol), 58 (0.93 g, 27%) was obtained as a yellow
syrup. 1H NMR (CDCl3): d 7.73–7.68 (m, 2H), 7.43
(m, 3H), 7.35–7.31 (m, 1H), 6.12 (d, 1H), 5.98 (d, 1H),
3.95 (m, 1H), 2.08 (m, 2H), 1.98 (s, 3H), 1.76–1.53 (m,
6H); MS m/z 398 (M + 1).
5.13.3. 7-Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]-pyri-
din (62). In a similar manner as described in the proce-
dure I, from 61 (13 g, 50 mmol), 62 (11.5 g, 99% yield)
was obtained as a yellow crystalline solid. 1H NMR

K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5355
(CDCl3): d 7.86 (s, 1H), 7.79 (d, 1H), 7.48 (d, 1H), 7.34
(t, 1H), 7.20 (d, 1H), 7.05 (dd, 1H), 6.91 (s, 1H), 6.88 (d,
1H), 2.44 (s, 3H). MS m/z 243 (M + 1).
5.14. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-[3- (trifluoromethyl)phenyl]pyrazolo[1,5-a]pyridin-
7-amine (16)
5.13.4. 7-Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]pyri-
dine-3-carbaldehyde (63). 7-Chloro-2-(3-methylphe-
nyl)pyrazolo[1,5-a]pyridine (62, 16.3 g, 67 mmol) was
treated according to procedure II to give after recrystal-
lization from diethyl ether and hexanes 63 (14.3 g, 79%)
The title compound was prepared using procedure II in
a similar fashion as outlined for 15. Rf 0.22 (4:1, hex-
anes/ethyl acetate); 1H NMR (300 MHz, CDCl3): d
8.06–8.02 (m, 2H), 7.84 (d, 1H), 7.71–7.67 (m, 2H),
7.55 (t, 1H), 7.34 (t, 1H), 6.32 (d, 1H), 6.07 (d, 1H),
6.03 (d, 1H), 5.08 (d, 1H), 4.28 (m, 1H), 4.03 (m, 1H),
2.17 (m, 2H), 2.04 (m, 2H), 1.86–1.51 (m, 12H); MS
m/z 507 (M + 1).
1
as a pale yellow solid. H NMR (CDCl3): d 10.11 (s,
1H), 8.41 (dd, 1H), 7.63 (s, 1H), 7.58 (d, 1H), 7.49
(dd, 1H), 7.42 (t, 1H), 7.33 (d, 1H), 7.20 (dd, 1H),
2.45 (s, 3H); MS m/z 271 (M + 1).
5.15. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimid-
inyl]-2-(4-methoxyphenyl)pyrazolo[1,5-a]pyridin-7-amine
(17)
5.13.5.
pyridin-3-yl]-2-propyn-1-ol (64). 7-Chloro-2-(3-methyl-
phenyl)pyrazolo[1,5-a]pyridine-3-carbaldehyde (63,
1-[7-Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]-
10.38 g, 36.2 mmol) was treated according to procedure
II to give after flash chromatography (4:1, hexanes/ethyl
acetate to 7:3, hexanes/ethyl acetate) 64 (11.3 g, 77%) as
a yellow foam. 1H NMR (CDCl3) d 8.03 (d, 1H), 7.60 (s,
1H), 7.54 (d, 1H), 7.37 (t, 1H), 7.26 (d, 1H), 7.17 (dd,
1H), 6.98 (d, 1H) , 2.67 (s, 1H), 2.43 (s, 3H), 2.38 (s,
1H); MS m/z 297 (M + 1).
5.15.1. 2-(6-Chloro-2-pyridinyl)-1-(4-methoxyphenyl)-eth-
anone (67). 6-Chloro-2-picoline (18.3 mL, 166.5 mmol)
and ethyl 4-methoxybenzoate (30.0 g, 166.5 mmol) were
treated as outlined in the general procedure I to give 67
(37.4 g, 86%) as a yellow solid. H NMR (CDCl3): d
7.99 (d, 2H), 7.57 (t, 1H), 7.22–7.19 (m, 2H), 6.90 (d,
2H), 4.39 (s, 2H), 3.83 (s, 3H); MS m/z 262 (M + 1).
1
5.13.6. 1-[7-Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]pyri-
din-3-yl]-2-propyn-1-one (65). 1-[7-Chloro-2-(3-methyl-
phenyl)pyrazolo[1,5-a]pyridin-3-yl]-2-propyn-1-ol (64,
11.3 g, 36.2 mmol) in chloroform (300 mL) was treated
according to procedure II to give after purification by
flash chromatography (7:3, hexanes/ethyl acetate) 65
(4.57 g, 41%) as a pale yellow crystalline. 1H NMR
(CDCl3): d 8.48 (d, 1H), 7.51 (m, 3H), 7.33 (t, 1H), 7.29
(d, 1H), 8.21 (dd, 1H), 2.89 (s, 1H), 2.42 (s, 3H); MS m/z
295 (M + 1).
5.15.2. 2-(6-Chloro-2-pyridinyl)-1-(4-methoxyphenyl)-eth-
anone oxime (68). 2-(6-Chloro-2-pyridinyl)-1-(4-meth-
oxyphenyl)ethanone (67, 37.4 g, 142.9 mmol) was
treated as outlined in the general procedure I to give
1
68 (38.7 g, 97%) as a white solid. H NMR (CDCl3): d
8.23 (br, 1H), 7.63 (d, 2H), 7.48 (d, 1H), 7.12 (m, 2H),
6.83 (dd, 2H), 4.33 (s, 2H), 3.76 (s, 3H); MS m/z 277
(M + 1).
5.15.3. 7-Chloro-2-(4-methoxyphenyl)pyrazolo[1,5-a]pyr-
idine (69). 2-(6-Chloro-2-pyridinyl)-1-(4-methoxyphe-
nyl)ethanone oxime (68, 38.7 g, 140 mmol) was treated
as outlined in the general procedure I to give 69
(18.7 g, 52%) as pale yellow needles. 1H NMR (CDCl3):
d 7.91 (d, 2H), 7.43 (d, 1H), 7.01 (t, 1H), 6.95 (d, 2H),
6.81 (d, 1H), 6.80 (s, 1H), 3.83 (s, 3H); MS m/z 259
(M + 1).
5.13.7.
4-[7-Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]-
pyridin-3-yl]-N-cyclopentyl- 2-pyrimidinamine (66). 1-[7-
Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]pyridin-3-yl]-
2-propyn-1-one (65, 4.04 g, 13.0 mmol) and cyclopentyl
guanidine hydrochloride (6.36 g, 39 mmol) were treated
according to procedure II to give after purification by
silica gel flash chromatography (3:10, ethyl acetate/
1
hexanes) 66 (2.39 g, 43%) as a yellow foam. H NMR
5.15.4. 1-[7-(Chloro)-2-(4-methoxyphenyl)pyrazolo[1,5-
a]pyridin-3-yl]ethanone (70). 7-Chloro-2-(4-methoxyphe-
nyl)pyrazolo[1,5-a]pyridine (69, 18.7 g, 72.4 mmol) was
treated as outlined in the general procedure I to give
after recrystallization from ethyl acetate hexanes 70
(14.2 g, 65%) as reddish needles.1H NMR (CDCl3): d
8.37 (dd, 1H), 7.49 (dd, 2H), 7.39 (dd, 1H), 7.10 (dd,
1H), 6.98 (dd, 2H), 3.84 (s, 3H), 2.13 (s, 3H); MS m/z
301 (M + 1).
(CDCl3): d 8.47 (d, 1H), 7.99 (d, 1H), 7.48 (s, 1H), 7.39
(d, 1H), 7.33–7.24 (m, 3H), 7.05 (d, 1H), 6.33 (d, 1H),
5.38 (br s, 1H), 4.36 (m, 1H), 2.40 (s, 3H), 2.10 (m, 2H),
1.78 (m, 2H), 1.67 (m, 2H), 1.58 (m, 2H); MS m/z 404
(M + 1).
5.13.8.
N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(3-methylphenyl)pyrazolo[1,5-a]pyridin-7-amine
(15). 4-[7-Chloro-2-(3-methylphenyl)pyrazolo[1,5-a]pyri-
din-3-yl]-N-cyclopentyl-2-pyrimidinamine (66, 500 mg,
1.24 mmol) in cyclopentylamine (50 mL) was treated
according to procedure II to give after purification by
flash chromatography (4:1, hexanes/ethyl acetate) 15
5.15.5.
1-[7-(Cyclopentylamino)-2-(4-methoxyphenyl)-
pyrazolo[1,5-a]pyridin-3-yl]ethanone (71). 1-[7-(Chloro)-
2-(4-methoxyphenyl)pyrazolo[1,5-a]pyridin-3-yl]ethanone
(70, 5.0 g, 16.6 mmol) was treated as outlined in the gen-
eral procedure I to give after purification by flash chro-
matography (4:1, hexanes/ethyl acetate) 71 (5.66 g, 97%)
as a yellow foam. 1H NMR (CDCl3): d 7.65 (d, 1H), 7.48
(d, 2H), 7.39 (t, 1H), 6.99 (d, 2H), 6.09 (d, 1H), 6.01 (d,
1H), 3.95 (m, 1H), 3.84 (s, 3H), 2.09 (s, 3H), 2.09–2.00
(m, 2H) 1.76–1.22 (m, 6H); MS m/z 350 (M + 1).
1
(360 mg, 64%) as a yellow foam. H NMR (CDCl3): d
7.94 (d, 1H), 7.79 (d, 1H), 7.46 (s, 1H), 7.40 (d, 1H),
7.34–7.29 (m, 2H), 7.24 (d, 1H), 6.27 (d, 1H), 6.03 (m,
2H), 5.12 (br s, 1H), 4.35 (m, 1H), 3.99 (m, 1H), 2.40
(s, 3H), 2.14–2.04 (m, 4H), 1.81–1.52 (m, 12H); MS m/z
453 (M + 1).

5356
K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5.15.6. (2E)-1-[7-(Cyclopentylamino)-2-(4-methoxyphe-
nyl)pyrazolo[1,5-a]pyridin-3-yl]-3-(dimethylamino)-2-
propen-1-one (72). 1-[7-(Cyclopentylamino)-2-(4-meth-
5.16.5. 1-[7-Chloro-2-(3-methoxyphenyl)pyrazolo[1,5-a]-
pyridin-3-yl]-2-propyn-1-ol (77). In a similar manner as de-
scribed in the procedure II, from 76 (17.25 g, 60.1 mmol)
and ethynylmagnesium bromide, 77 (19.0 g, 100%) was
obtained. H NMR (CDCl3): d 8.02 (d, 1H), 7.38–7.32
(m, 3H), 7.17 (t, 1H), 6.98–6.97 (d, 2H), 5.82 (m, 1H),
3.86 (s, 3H), 2.67 (s, 1H), 2.53 (d, 1H); MS m/z 313.
oxyphenyl)-pyrazolo[1,5-a]pyridin-3-yl]ethanone
(71,
1
5.56 g, 15.9 mmol) in N,N-dimethylformamide dimethyl
acetal (25 mL) was heated at reflux for 5 days as out-
lined in the general procedure I to give after flash chro-
matography (7:3, ethyl acetate/acetone) 72 (5.97 g, 93%)
1
as a colored syrup. H NMR (CDCl3): d 7.96–7.59 (m,
5.16.6. 1-[7-Chloro-2-(3-methoxyphenyl)pyrazolo[1,5-a]-
pyridin-3-yl]-2-propyn-1-one (78). In a similar manner as
described in the procedure II, from 77 (19 g, 60.8 mmol)
78 (14.4 g, yield 76%) was obtained as an orange colored
solid. 1H NMR (CDCl3): d 8.47 (d, 1H), 7.52 (t, 1H), 7.36
(t, 1H), 7.29–7.21 (m, 3H), 7.02 (dd, 1H), 3.86 (s, 3H), 2.92
(s, 1H); MS m/z 311.
3H), 7.53 (d, 1H), 7.23 (dd, 1H), 6.93 (d, 2H), 5.97–
5.94 (m, 2H), 5.07 (d, 1H), 3.95 (m, 1H), 3.81 (s, 3H),
3.0–2.3 (br, 6H), 2.07 (m, 2H), 1.76–1.60 (m, 6H); MS
m/z 405 (M + 1).
5.15.7. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-methoxyphenyl)pyrazolo[1,5-a]pyridin-7-amine
(17).
(2E)-1-[7-(Cyclopentylamino)-2-(4-methoxyphenyl)-
5.16.7. 4-[7-Chloro-2-(3-methoxyphenyl)pyrazolo[1,5-a]-
pyridin-3-yl]-N-cyclopentyl-2-pyrimidinamine (79). In a
similar manner as described in the procedure II, from
78 (1.0 g, 3.2 mmol) and cyclopentyl guanidine hydro-
chloride 79 (0.34 g, yield 25%) was obtained as a pale
pyrazolo[1,5-a]pyridin-3-yl]-3-(dimethylamino)-2-pro-
pen-1-one (72, 5.97 g, 14.7 mmol) was treated as
outlined in the general procedure I to give after purifica-
tion by flash chromatography (4:6, ethyl acetate/hexane)
1
1
17 (5.02 g, 73%) as a yellow solid. H NMR (CDCl3): d
yellow foam. H NMR (CDCl3): d 8.52 (d, 1H), 8.05
7.95 (d, 1H), 7.72 (d, 1H), 7.54 (dd, 2H), 7.25 (t, 1H),
6.94 (dd, 2H), 6.28 (d, 1H), 6.00–5.97 (m, 2H), 5.03 (d,
1H), 4.33–4.31 (m, 1H), 3.96 (m, 1H), 3.83 (s, 3H), 2.10–
2.01 (m, 4H), 1.77–1.50 (m, 12H); MS m/z 469 (M + 1);
Anal. Calcd for C28H32 N6O: C, 71.77; H, 6.88; N,
17.93. Found: C, 71.41; H, 7.02; N, 17.89.
(d, 1H), 7.43–7.23 (m, 4H), 7.11 (d, 1H), 7.03 (dd,
1H), 6.40 (d, 1H), 5.45 (br s, 1H), 4.40 (m, 1H), 3.86
(s, 3H), 2.10 (m, 2H), 1.85–1.59 (m, 6H); MS m/z 420.
5.16.8. N-Cyclopentyl-3-[2-(cyclopenylamino)-4-pyrimidi-
nyl]-2-(3-methoxyphenyl)pyrazolo[1,5-a]pyridin-7-amine (18).
In a similar manner as described in the procedure
II, from 79 (500 mg, 1.19 mmol) and cyclopentylamine,
18 (389 mg, yield 70%) was obtained as a yellow foam.
1H NMR (CDCl3): d 7.92 (d, 1H), 7.79 (d, 1H),
7.38–7.30 (m, 2H), 7.21–7.18 (m, 2H), 6.99 (d, 1H), 6.30
(d, 1H), 6.04 (m, 2H), 5.38 (br s, 1H), 4.36 (m, 1H), 4.00
(m, 1H), 3.82 (s, 3H), 2.14–2.06 (m, 4H), 1.82–1.57 (m,
12H); MS m/z 469.
5.16. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimid-
inyl]-2-(3-methoxyphenyl)pyrazolo[1,5-a]pyridin-7-amine
(18)
5.16.1. 2-(6-Chloro-2-pyridinyl)-1-(3-methoxyphenyl)-eth-
anone (73). In a similar manner as described in the pro-
cedure I, from ethyl 3-methoxybenzoate (30 g, 166
mmol) and 6-chloropicoline (21.2 g, 166 mmol), 73
was obtained as a mixture of ketone and enol tauto-
mers. This product was used directly in the next step.
5.17. 4-{7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]pyrazolo[1,5-a]pyridin-2-yl}phenol (19)
5.16.2. 2-(6-Chloro-2-pyridinyl)-1-(3-methoxyphenyl)-eth-
anone oxime (74). In a similar manner as described in the
procedure I, from 73 74 (33.1 g, yield for the two steps
To a solution of 17 (1.16 g, 2.48 mmol) in dichlorometh-
ane (50 mL) at –78 °C was added boron tribromide
(9.92 mL, 1.0 M in dichloromethane, 9.92 mmol) drop-
wise. The resulting solution was allowed to warm to
room temperature. After stirring for 15 h at room tem-
perature, the mixture was cooled to 0 °C and quenched
by the addition of water. The mixture was extracted with
ethyl acetate. The organic phase was dried (magnesium
sulfate), filtered, and concentrated to give a solid residue
which was purified by flash chromatography (95:5, chlo-
roform/methanol). 19 was obtained as a yellow solid
1
72%) was obtained as a white solid. H NMR (CDCl3):
d 8.22 (br s, 1H), 7.52 (t, 1H), 7.34 (m, 1H), 7.26–7.25
(m, 2H), 7.17–7.05 (d, 2H), 6.90 (m, 1H), 4.36 (s, 2H),
3.81 (s, 3H); MS m/z 277.
5.16.3. 7-Chloro-2-(3-methoxyphenyl)pyrazolo[1,5-a]pyr-
idine (75). In a similar manner as described in the proce-
dure I, from 74 (33.0 g, 119 mmol) 75 (23.1 g, 75% yield)
1
1
was obtained as a pale yellow solid. H NMR (CDCl3):
(0.80 g, 71%). H NMR (CDCl3): d 7.91 (d, 1H), 7.70
d 7.59 (m, 2H), 7.49 (d, 1H), 7.37 (t, 1H), 7.07 (t, 1H),
6.95–6.94 (dd, 1H), 6.91 (s, 1H), 6.88 (d, 1H), 3.90 (s,
3H); MS m/z 259.
(d, 1H), 7.46 (d, 2H), 7.26 (t, 1H), 6.86 (d, 2H), 6.27
(d, 1H), 6.00–5.97 (m, 2H), 5.06 (d, 1H), 4.33 (m, 1H),
3.96 (m, 1H), 2.10–2.03 (m, 4H), 1.78–1.47 (m, 12H);
MS m/z 455 (M + 1).
5.16.4. 7-Chloro-2-(3-methoxyphenyl)pyrazolo[1,5-a]pyr-
idine-3-carbaldehyde (76). In a similar manner as de-
scribed in the procedure II, from 75 (23 g, 88.9 mmol)
76 (21.6 g, 84%) was obtained as a white solid. 1H
NMR (CDCl3): d 10.13 (s, 1H), 8.40 (d, 1H), 7.52–
7.42 (m, 2H), 7.35 (m, 2H), 7.21 (d, 1H), 7.06 (d, 1H),
3.89 (s, 3H); MS m/z 287.
5.18. 3-{7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]pyrazolo[1,5-a]pyridin-2-yl}phenol (20)
In a similar manner as described above, from 18 (150 mg,
0.32 mmol) 20 (126 mg, yield 87%) was obtained as a yel-
low foam. 1H NMR (CDCl3): d 7.83 (d, 1H), 7.77 (d, 1H),

K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5357
7.35–7.28 (m, 2H), 7.21 (d, 1H), 7.06 (s, 1H), 6.95 (dd,
1H), 6.39 (d, 1H), 6.05–6.02 (m, 2H), 5.29 (s, 1H), 5.22
(m, 1H), 4.31 (m, 1H), 3.99 (m, 1H), 2.12–1.98 (m, 4H),
1.80–1.46 (m, 12H); MS m/z 455.
5.23. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-[3- (cyclopropylmethoxy)phenyl]pyrazolo[1,5-a]pyri-
din-7-amine (25)
To a solution of 20 (400 mg, 0.88 mmol) in acetonitrile
(80 mL) cesium carbonate (315 mg, 0.97 mmol) and
(bromomethyl)cyclopropane (0.26 mL, 2.64 mmol) were
added. The reaction mixture was heated at reflux for 6 h.
After the reaction was cooled to room temperature, eth-
yl acetate was added and the organic phase was washed
with water, brine and dried over magnesium sulfate. Fil-
tration and concentration followed by purification with
silica gel chromatography (3:2, hexanes/ethyl acetate)
gave 25 (320 mg, 71%) as yellow solid. 1H NMR
(CDCl3): d 7.97 (d, 1H), 7.78 (d, 1H), 7.33 (m, 2H),
7.18 (m, 2H), 6.98 (m, 1H), 6.30 (d, 1H), 6.02 (m, 2H),
5.08 (d, 1H), 4.35 (m, 1H), 3.99 (m, 1H), 3.80 (d, 2H),
2.014 (m, 4H), 1.83–1.55 (m, 12H), 1.22 (m, 1H), 0.61
(m, 2H), 0.35 (m, 2H). MS m/z 509 (M + 1).
5.19. 2-[4-(Allyloxy)phenyl]-N-cyclopentyl-3-[2-(cyclopen-
tylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine
(21)
To a solution of 19 (100 mg, 0.22 mmol) in N,N-di-
methylformamide (5 mL), allyl bromide (21 lL,
0.24 mmol) and potassium carbonate (122 mg,
0.88 mmol) were added. The mixture was heated at re-
flux for 3 h. The mixture was allowed to cool to room
temperature and water was added. The mixture was
extracted with ethyl acetate. The ethyl acetate phase
was dried (magnesium sulfate), filtered, and concentrat-
ed to a solid. The residue was purified by flash chroma-
tography (1:1, ethyl acetate/hexanes) to give 21 (67 mg,
1
61%) as a yellow solid. H NMR (CDCl3): d 7.94 (d,
1H), 7.73 (d, 1H), 7.53 (d, 2H), 7.24 (t, 1H), 6.95 (d,
2H), 6.28 (d, 1H), 6.08–5.96 (m, 3H), 5.41 (dd, 1H),
5.27 (dd, 1H), 5.10 (d, 1H), 4.56 (d, 2H), 4.32 (m,
1H), 3.95 (m, 1H), 2.10–2.00 (m, 4H), 1.76–1.45 (m,
12H); MS m/z 495 (M + 1); Anal. Calcd for
C30H34N6O: C, 72.86; H, 6.93; N,16.99. Found: C,
72.47; H, 7.05; N, 16.75.
5.24. 2-[4-(Cyclobutylmethoxy)phenyl]-N-cyclopentyl-3-
[2-(cyclopentylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyri-
din-7-amine (26)
In a similar manner as described above, from 19 (67 mg,
0.15 mmol) and (bromomethyl)cyclobutane, 26 (50 mg,
65%) was formed as a yellow foam. H NMR (CDCl3):
1
d 7.99 (d, 1H), 7.78 (d, 1H), 7.57 (d, 2H), 7.30 (t, 1H),
6.98 (d, 2H), 6.34 (d, 1H), 6.05–6.00 (m, 2H), 5.13 (d,
1H), 4.37 (m, 1H), 3.99 (m, 3H), 2.82 (m, 1H), 2.20–1.50
(m, 22H); MS m/z 523 (M + 1).
5.20. 2-(4-Butoxyphenyl)-N-cyclopentyl-3-[2-(cyclopen-
tylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (22)
In a similar manner as described above, from 19 (100 mg,
0.22 mmol) and butyl bromide, 22 (80 mg, 71%) was
formed as a yellow solid. H NMR (CDCl3): d 8.00 (d,
1H), 7.78 (d, 1H), 7.57 (d, 2H), 7.28 (t, 1H), 6.97 (d,
2H), 6.34 (d, 1H), 6.05 (d, 1H), 6.00 (d, 1H), 5.22 (d,
1H), 4.37 (m, 1H), 4.02 (m, 3H), 2.12–2.05 (m, 4H),
1.82–1.49 (m, 16H), 1.00 (t, 3H); MS m/z 511 (M + 1).
5.25. Ethyl (4-{7-(cyclopentylamino)-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]-pyrazolo[1,5-a]pyridin-2-yl}phen-
oxy)acetate (27)
1
To a solution of 19 (100 mg, 0.22 mmol) in acetone
(10 mL) ethyl a-bromoacetate (49 lL, 0.44 mmol) and
potassium carbonate (304 mg, 2.2 mmol) were added.
The mixture was heated to reflux for 3 h. The reaction
mixture was cooled to room temperature and water
was added. The solution was extracted with ethyl ace-
tate. The organics were dried (magnesium sulfate), fil-
tered, and concentrated, followed by purification with
flash chromatography (1:1, ethyl acetate/hexanes) to
give 27 (85 mg, 71%) as a brown oil. 1H NMR (CDCl3):
d 7.94 (d, 1H), 7.70 (d, 1H), 7.55 (d, 2H), 7.23 (t, 1H),
6.95 (d, 2H), 6.25 (d, 1H), 5.98–5.95 (m, 2H), 5.13 (d,
1H), 4.62 (s, 2H), 4.31–4.21 (m, 3H), 3.94 (m, 1H),
2.07–1.99 (m, 4H), 1.75–1.46 (m, 12H), 1.28 (t, 3H);
MS m/z 541 (M + 1).
5.21. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidinyl]-
2-(4-isobutoxyphenyl)pyrazolo[1,5-a]pyridin-7-amine (23)
In a similar manner as described above, from 19
(100 mg, 0.22 mmol) and isobutyl bromide, 23 (76 mg,
68%) was formed as a yellow foam. H NMR (CDCl3):
1
d 8.00 (d, 1H), 7.78 (d, 1H), 7.57 (d, 2H), 7.29 (t, 1H),
6.98 (d, 2H), 6.35 (d, 1H), 6.05 (d, 1H), 6.01 (d, 1H),
5.16 (d, 1H), 4.37 (m, 1H), 4.00 (m, 1H), 3.79 (d, 2H),
2.16–2.05 (m, 5H), 1.81–1.52 (m, 12H), 1.06 (d, 6H);
MS m/z 511 (M + 1).
5.22. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-[4-(cyclopropyl- methoxy)phenyl]pyrazolo[1,5-a]-
pyridin-7-amine (24)
5.26. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidinyl]-
2-(4- phenoxyphenyl)pyrazolo[1,5-a]pyridin-7-amine (28)
In a similar manner as described above, from 19 (100 mg,
0.22 mmol), Cs2CO3, (bromomethyl)cyclopropane, 24
(48 mg, 44%) was formed as a yellow foam. H NMR
(CDCl3): d 7.99 (d, 1H), 7.77 (d, 1H), 7.57 (dd, 2H),
7.30 (t, 1H), 6.97 (d, 2H), 6.33 (d, 1H), 6.04–6.00 (m,
2H), 5.08 (d, 1H), 4.37 (m, 1H), 4.00 (m, 1H), 3.87 (d,
2H), 2.14–2.07 (m, 4H), 1.82–1.52 (m, 12H), 1.32 (m,
1H), 0.65 (m, 2H), 0.38 (m, 2H); MS m/z 509 (M + 1).
To a solution of 19 (100 mg, 0.22 mmol) in dichloro-
methane (5 mL), copper (II) acetate (40 mg, 0.22 mmol),
phenylboronic acid (80 mg, 0.66 mmol), triethylamine
(92 lL, 0.66 mmol) and molecular sieves were added.
The mixture was stirred at room temperature for 24 h.
Solids were removed by filtration and the filtrate was
concentrated, followed by purification by flash chroma-
tography (2:3, ethyl acetate/hexanes) to give 28 (14 mg,
1

5358
K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
1
12%) as a yellow foam. H NMR (CDCl3): d 8.05–6.85
(m, 12H), 6.40 (d, 1H), 6.06 (m, 2H), 5.12 (d, 1H),
4.40 (m, 1H) 4.01 (m, 1H), 2.20–2.09 (m, 4H), 1.82–
1.59 (m, 12H); MS m/z 531 (M + 1).
1H), 7.80–7.75 (m, 3H), 7.48 (m, 1H), 7.41 (m, 2H),
7.27 (m, 5H), 7.23–7.12 (m, 4H), 7.04 (s, 1H), 6.80 (d,
1H), 6.03-5.97 (m, 3H), 4.39 (m, 1H), 4.00 (m, 1H),
2.15–2.05 (m, 4H), 1.83–1.56 (m, 12H); MS m/z 618
(M + 1). The N-cyclopentyl-3-[2-(cyclopentylamino)-4-
pyrimidinyl]-2-{3-[(diphenylmethylene)amino]phenyl}-
pyrazolo[1,5-a]pyridin-7-amine (2.61 g, 4.22 mmol) in
tetrahydrofuran (30 mL) at 0 °C was treated with 4 N
hydrochloric acid (20 mL) as described above to give
after recrystallization from ethyl acetate–hexanes, 30
5.27. N-{4-[2-(4-Aminophenyl)-7-(butylamino)pyrazolo-
[1,5-a]pyridin-3-yl]-2-pyrimidinyl}-N-butylamine (29)
To a solution of 11 (1.0 g, 2.0 mmol) in toluene (20 mL),
racemic-2,20-bis(diphenylphosphino)-1,10-binaphthyl (200
mg, 0.30 mmol), tris(dibenzylideneacetone)dipalladi-
um(0) (92 mg, 0.10 mmol), diphenylimine (1.02 mL,
6.1 mmol), and sodium tert-butoxide (582 mg, 6.1 mmol)
were added. The mixture was heated to 100 °C for
50 min. The resultant solution was cooled to room tem-
perature and diluted with ether and water was added.
The organic layer was washed with brine. The aqueous
layer was extracted with ether, and the combined organ-
ics dried over magnesium sulfate. Filtration and concen-
tration followed by flash chromatography (4:1, hexanes/
ethyl acetate with 1% triethylamine) provided N-butyl-3-
[2-(butylamino)-4-pyrimidinyl]-2-{4-[(diphenylmethylene)-
amino]phenyl}-pyrazolo[1,5-a]pyridin-7-amine (1.0 g, 84%)
as a yellow solid. 1H NMR (CDCl3): d 7.90 (d, 1H), 7.80–
7.28 (m, 12H), 7.19–7.16 (m, 2H), 6.80 (d, 2H), 6.07 (d,
1H), 6.02 (t, 1H), 5.97 (d, 1H), 4.99 (t, 1H), 3.48 (m,
2H), 3.36 (m, 2H), 1.80–1.43 (m, 8H), 1.00–0.96 (m,
6H); MS m/z 594 (M + 1).
1
(1.78 g, 93%) as a pale yellow solid. H NMR (CDCl3):
d 7.92 (d, 1H), 7.82 (d, 1H), 7.32 (t, 1H), 7.22 (m, 2H),
7.00-6.94 (m, 2H), 6.78 (m, 1H), 6.34 (d, 1H), 6.04 (m,
2H), 4.38 (m, 1H), 4.00 (m, 1H), 3.75 (br, 2H),
2.14-2.05 (m, 4H), 1.83–1.54 (m, 12H); MS m/z 454
(M + 1).
5.29. N-(3-{7-(cyclopentylamino)-3-[2-(cyclopentylamino)-
4-pyrimidinyl]pyrazolo[1,5-a ]pyridin-2-yl}phenyl)acetam-
ide (31)
To a suspension of 2-(3-aminophenyl)-N-cyclopentyl-3-
[2-(cyclopentylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyri-
din-7-amine (30, 150 mg, 0.33 mmol) in dimethylform-
amide (10 mL) was added triethylamine (51 lL,
0.36 mmol). The reaction mixture was cooled to 0 °C
and flushed with nitrogen, then acetyl chloride
(26 lL, 0.36 mmol) was added dropwise. The reaction
mixture was stirred at room temperature overnight.
Water was added and the resulting mixture was
extracted with ethyl acetate. The ethyl acetate phase
was dried (magnesium sulfate), filtered and concentrat-
ed to a solid. This solid was purified by flash chroma-
tography (95:5 chloroform/methanol) to give 31
(151 mg, 92%) as a tan solid. 1H NMR (CDCl3): d
8.19 (s, 1H), 7.95 (d, 1H), 7.82 (d, 1H), 7.75 (d, 1H),
7.63 (s, 1H), 7.36–7.25 (m, 3H), 6.28 (d, 1H), 6.00
(m, 2H), 5.17 (d, 1H), 4.32 (m, 1H), 3.96 (m, 1H),
2.11 (s, 3H), 2.11–2.02 (m, 4H), 1.76–1.48 (m, 12H);
MS m/z 496 (M + 1).
To the solution of N-butyl-3-[2-(butylamino)-4-pyrimidi-
nyl]-2-{4-[(diphenylmethylene)- amino]phenyl}pyrazol-
o[1,5-a]pyridin-7-amine (1.0 g, 1.7 mmol) in tetrahydro-
furan (50 mL) was added hydrochloric acid (10 mL, 4 N
aqueous). The resultant solution was stirred at room
temperature for 30 min. Ether was added and the
solution was made basic by the slow addition of saturated
aqueous sodium bicarbonate. The organic layer was
washed with brine. The aqueous layer was extracted with
ether and the combined organics dried over magnesium
sulfate. Filtration and concentration followed by flash
chromatography (1:1 hexanes/ethyl acetate) provided
1
29 as an orange oil. H NMR (CDCl3): d 8.02 (d, 1H),
5.30. N-(3-{7-(cyclopentylamino)-3-[2-(cyclopentylamino)-
4-pyrimidinyl]pyrazolo[1,5-a]pyridin-2-yl}phenyl)meth-
anesulfonamide (32)
7.81 (d, 1H), 7.47 (d, 2H), 7.31 (m, 1H), 6.77 (d, 2H),
6.42 (d, 1H), 6.08 (m, 1H), 6.00 (d, 1H), 5.18 (br, 1H),
3.86 (br, 2H), 3.53 (m, 2H), 3.39 (m, 2H), 1.82-1.64 (m,
4H), 1.58-1.46 (m, 4H), 1.04-0.99 (m, 6H); MS m/z 430
(M + 1).
To a suspension of 2-(3-aminophenyl)-N-cyclopentyl-3-
[2-(cyclopentylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyri-
din-7-amine (30, 150 mg, 0.33 mmol) in N,N-dimethyl-
formamide (5 mL) was added pyridine (40 lL,
0.49 mmol). The reaction mixture was cooled to 0 °C
under nitrogen, then methanesulfonyl chloride (28 lL,
0.36 mmol) was added dropwise. After stirring at room
temperature for 18 h, the reaction mixture turned clear.
Ethyl acetate and water were added and the phases sep-
arated. The organic phase was washed with water and
brine, then dried over magnesium sulfate. Filtration
and concentration, followed by flash chromatography
(95:5 chloroform/methanol), gave 32 (170 mg, 96%) as
a yellow syrup. 1H NMR (CDCl3): d 7.93 (d, 1H),
7.66 (d, 1H), 7.42 (m, 1H), 7.35–7.25 (m, 5H), 6.25 (m,
1H), 5.99 (m, 2H), 5.62 (br, 1H), 4.29 (m, 1H), 3.96
(m, 1H), 2.94 (s, 3H), 2.10–1.99 (m, 4H), 1.77–1.49 (m,
12H). MS m/z 532 (M + 1).
5.28. 2-(3-Aminophenyl)-N-cyclopentyl-3-[2-(cyclopentyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (30)
2-(3-Bromophenyl)-N-cyclopentyl-3-[2-(cyclopentylami-
no)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (12,
3.00 g, 5.8 mmol) in toluene (60 mL) was treated with
benzophenone imine (3.15 g, 17.4 mmol), tris(dibenzy-
lideneacetone)dipalladium (0.26 g, 0.3 mmol), rac-2,20-
bis(diphenylphosphino)-1,10-binaphthyl (0.54 g, 0.15
mmol) and sodium tert-butoxide (1.67 g, 17.4 mmol) as
described above to give after flash chromatography
(4:6 ethyl acetate/hexanes) N-cyclopentyl-3-[2-(cyclo-
pentylamino)-4-pyrimidinyl]-2-{3-[(diphenyl-methylene)
amino]phenyl}-pyrazolo[1,5-a]pyridin-7-amine (2.61 g,
1
73%) as a yellow solid. H NMR (CDCl3): d 7.89 (d,

K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5359
5.31. N-Butyl-3-[2-(butylamino)-4-pyrimidinyl]-2-[4-(cyclo-
hexylamino)phenyl]pyrazolo[1,5-a]pyridin-7-amine (33)
5.34. 2-[1,10-Biphenyl]-3-yl-N-cyclopentyl-3-[2-(cyclopen-
tylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine
(36)
A
solution of N-{4-[2-(4-aminophenyl)-7-(butylami-
no)pyrazolo[1,5-a]pyridin-3-yl]-2-pyrimidinyl}-N-butyl-
amine (29, 62 mg, 0.15 mmol) in 1,2-dichloroethane
(2 mL) was treated with cyclohexanone (0.02 mL,
0.22 mmol), acetic acid (0.04 mL, 0.72 mmol), and sodi-
um triacetoxyborohydride (61 mg, 0.29 mmol). The
resultant solution was stirred at room temperature for
18 h. Saturated aqueous sodium bicarbonate was added
dropwise followed by ether. The organic layer was
washed with brine. The aqueous layer was extracted
with ether and the combined organics dried over magne-
sium sulfate. Filtration and concentration followed
by flash chromatography (4:1 to 2:1 hexanes–ethyl
acetate) provided 33 (54 mg, 73%) as an orange oil.
1H NMR (CDCl3): d 7.94 (d, 1H), 7.73 (d, 1H), 7.39
(d, 2H), 7.23 (m, 1H), 6.59 (d, 2H), 6.41 (d, 1H), 6.03
(m, 1H), 5.91 (d, 1H), 5.11 (br, 1H), 3.66 (br, 1H),
3.44 (m, 2H), 3.30 (m, 3H), 2.05 (m, 2H), 1.75–1.12
(m, 16H), 0.95–0.92 (m, 6H); MS m/z 512 (M + 1).
This material was treated with anhydrous hydrochloric
acid in ether to provide the corresponding hydrochloride
salt.
To a solution of 2-(3-bromophenyl)-N-cyclopentyl-3-[2-
(cyclopentylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-
7-amine (12, 100 mg, 0.19 mmol) in dimethylformamide
(6 mL) was added phenylboronic acid (47 mg,
0.39 mmol), palladium (II) acetate (4.3 mg, 0.02 mmol),
potassium carbonate (54 mg, 0.39 mmol) and triphenyl
phosphine (30 mg, 0.08 mmol). The reaction was heated
at 100 °C for 24 h. After allowing the reaction mixture
to cool to room temperature, ethyl acetate and water
were added. The organic layer was separated and
washed with water, then brine and dried (magnesium
sulfate). Filtration and concentration, followed by puri-
fication with flash chromatography (4:6 ethyl acetate/
1
hexanes) gave 36 as a yellow foam (87 mg, 89%). H
NMR (CDCl3): d 8.01 (d, 1H), 7.92 (s, 1H), 7.81 (d,
1H), 7.69–7.62 (m, 4H), 7.53 (t, 1H), 7.47–7.43 (m,
2H), 7.38–7.32 (m, 2H), 6.40 (d, 1H), 6.09–6.05 (m,
2H), 5.16 (m, 1H), 4.37 (m, 1H), 4.03 (m, 1H), 2.15–
2.06 (m, 4H), 1.82–1.64 (m, 12H); MS m/z 515 (M + 1).
5.35. 3-{7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]pyrazolo[1,5-a]pyridin-2-yl}benzonitrile (37)
5.32. N-Butyl-3-[2-(butylamino)-4-pyrimidinyl]-2-[4-(di-
methylamino)phenyl]pyrazolo[1,5-a]pyridin-7-amine (34)
To a solution of 2-(3-bromophenyl)-N-cyclopentyl-3-[2-
(cyclopentylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-
7-amine (12, 500 mg, 0.97 mmol) in N,N-dimethylform-
amide (25 mL) was added zinc cyanide (68 mg,
In a similar manner as described above from N-{4-[2-(4-
aminophenyl)-7-(butylamino)pyrazolo[1,5-a]pyridin-3-
yl]-2-pyrimidinyl}-N-butylamine (50 mg, 0.12 mmol),
formaldehyde (13 lL, 37% aqueous solution,
0.17 mmol) and sodium triacetoxyborohydride (99 mg,
0.47 mmol) was prepared 34 (40.5 mg, 76%) as a pale
0.58 mmol),
tris(dibenzylidineacetone)bipalladium(0)
(888 mg, 0.97 mmol) and 1,10-bis(diphenylphosphino)
ferrocene (1.29 g, 2.3 mmol). The resultant mixture
was heated at 120 °C for 20 h. After cooling to room
temperature, ethyl acetate was added to the reaction
mixture. The organic phase was washed with water,
brine and dried over magnesium sulfate. Filtration and
concentration, followed by purification with flash chro-
matography (40:60 ethyl acetate/hexanes) gave 37
(0.19 g, yield 42%) as a yellow foam. 1H NMR (CDCl3):
d 8.07 (m, 2H), 7.89 (d, 1H), 7.70 (d, 1H), 7.62 (d, 1H),
7.52 (t, 1H), 7.33 (t, 1H), 6.33 (d, 1H), 6.06 (d, 1H), 6.00
(d, 1H), 5.16 (d, 1H), 4.25 (m, 1H), 4.02 (m, 1H), 2.15
(m, 2H), 2.03 (m, 2H), 1.85–1.51 (m, 12H). MS m/z
464 (M + 1).
1
yellow solid. H NMR (CDCl3): d 7.80 (m, 2H), 7.50
(d, 2H), 7.33 (t, 1H), 6.79 (d, 2H), 6.44 (d, 1H), 6.10
(t, 1H), 6.03 (d, 1H), 3.54 (q, 2H), 3.38 (q, 2H), 3.03
(s, 6H), 1.80–1.43 (m, 8H), 0.99 (t, 6H); MS m/z 458
(M + 1).
5.33. 2-[1,10-Biphenyl]-4-yl-N-butyl-3-[2-(butylamino)-4-
pyrimidinyl]pyrazolo[1,5-a ]pyridin-7-amine (35)
To a solution of 2-(4-Bromophenyl)-N-butyl-3-[2-(butyl-
amino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-7-amine (11,
52 mg, 0.11 mol) in tetrahydrofuran was added phenylbo-
ronic acid (26 mg, 0.21 mmol), sodium carbonate
(0.21 mL, 2 M aqueous, 0.42 mmol), and dichlorobistri-
phenylphosphine palladium (II) (7.5 mg, 0.01 mmol).
The mixture was heated at reflux for 3.5 h. The resultant
solution was cooled to room temperature and diluted
with ether and water was added. The organic layer was
washed with brine. The aqueous layer was extracted
with ether and the combined organics dried over
magnesium sulfate. Filtration and concentration
followed by flash chromatography provided 35 (37 mg,
5.36. 3-{7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]pyrazolo-[1,5-a]pyridin-2-yl}benzoic acid (38)
To a solution of 3-{7-(cyclopentylamino)-3-[2-(cyclo-
pentylamino)-4-pyrimidinyl]pyrazolo[1,5-a]pyridin-2-yl}-
benzonitrile (37, 50 mg, 0.1 mmol) in methanol was
added 4 N potassium hydroxide. After heating at
85 °C for 2 days, the reaction mixture was cooled to
room temperature and acidified with 2 N hydrochloric
acid. The solution was extracted with ethyl acetate.
The organic phases were combined and dried over mag-
nesium sulfate. Filtration and concentration, followed
by purification by flash chromatography (90:10 ethyl
acetate/methanol) gave 38 (10 mg, 19%) as a brown
1
73%) as a yellow solid. H NMR (CDCl3): d 8.05 (d,
1H), 7.83–7.69 (m, 7H), 7.51 (m, 2H), 7.43–7.33 (m,
2H), 6.44 (d, 1H), 6.12 (t, 1H), 6.05 (d, 1H), 5.16 (br,
1H), 3.52 (m, 2H), 3.42 (m, 2H), 1.84–1.43 (m, 8H),
1.05–0.99 (m, 6H); MS m/z 491 (M + 1).
1
foam. H NMR (CDCl3): d 8.11 (s, 1H), 8.00 (d, 1H),

5360
K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
7.96 (d, 1H), 7.80 (br s, 1H), 7.64 (d, 1H), 7.46 (t, 1H),
7.37 (t, 1H), 6.98 (d, 1H), 6.57 (d, 1H), 6.21 (d, 1H), 6.11
(br, 1H), 4.12 (m, 1H), 4.01 (m, 1H), 2.06 (m, 2H), 1.88
(m, 2H), 1.69–1.49 (m, 12H). MS m/z 483 (M + 1); 481
(M ꢁ 1).
subsequent addition of SuperSignalÒ substrate (Pierce,
Rockford, MD). The resulting chemiluminescent signal
from compound-treated cells was compared to that of
compound-free cells to obtain percent inhibitions, which
were used to construct dose response curves to derive
50% inhibitory concentrations (IC50).
5.37. 3-{7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]pyrazolo[1,5-a]pyridin-2-yl }benzamide (39)
5.39. Cytotoxicity assay
3-{7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]pyrazolo[1,5-a]pyridin-2-yl}benzonitrile (37,
45 mg, 0.097 mmol) was dissolved in hot methanol
(2 mL). Subsequently, the solution was cooled down to
room temperature and 30% ammonium hydroxide
(2 mL) was added. The reaction mixture was then cooled
to 0 °C, and 30% hydrogen peroxide was added. After
stirring at room temperature for 8 h, water was added
and the resulting mixture extracted with ethyl acetate.
The organics were dried over magnesium sulfate. Filtra-
tion and concentration, followed by purification with
flash chromatography (95:5 dichloromethane/metha-
Compounds were dissolved in DMSO at a stock con-
centration of 10 mM and serially diluted twofold in
DMSO. Dilutions were carried out in columns 1–9 of
a 96-well, v-bottom polypropylene plate. Columns 10,
11, and 12 were control columns that did not contain
compound. Plates were then seeded at 10,000 cells/well,
columns 1–11, in minimum essential medium contain-
ing EarleÕs salt and L-glutamine and supplemented
with 10% heat-inactivated fetal bovine serum, penicil-
lin–streptomycin, and 1% L-glutamine resulting in a fi-
nal 250-fold drug dilution. Thus, the maximum
compound concentration used in the assay is 40 lM
while the final DMSO concentration is 0.4%. Plates
were incubated at 37 °C in a humidified, 5% CO2
atmosphere and growth inhibition was measured after
3 days.
1
nol), gave 39 (18 mg, 39% yield) as a yellow solid. H
NMR (CDCl3): d 8.12 (s, 1H), 7.99 (d, 1H), 7.92 (d,
1H), 7.79 (d, 1H), 7.70 (d, 1H), 7.51 (t, 1H), 7.32 (t,
1H), 6.28 (d, 1H), 6.18 (br, 1H), 6.05 (d, 1H), 6.02 (d,
1H), 5.82 (br, 1H), 5.25 (d, 1H), 4.30 (m, 1H), 4.00
(m, 1H), 2.15 (m, 2H), 2.03 (m, 2H), 1.81–1.50 (m,
12H). MS m/z 482 (M + 1).
Cytotoxicity was determined in vero cells using the
CellTiter 96Ò Aqueous Non-Radioactive Cell Prolifera-
tion Assay (Promega Corporation, G1111). Metaboli-
cally active cells will convert methylthiazol tetrazolium
inner salt (MTS), through the actions of cellular dehy-
drogenases, into a colorized formazan end product.
On day 3, MTS solution was added to the assay plates,
incubated for 1.5 to 2 h at 37 °C in a humidified, 5%
CO2 atmosphere, and absorbance was measured at
490 nm using the Wallac Victor2 1420 multilabel count-
er (Perkin-Elmer, Wellesley, MA). RoboFit 2000 curve-
fitting software was then used to obtain a CC50 (50%
cytotoxicity concentration) value from the curves
generated.
5.38. HSV anti-viral assay
5.38.1. Cell culture and HSV infection. Vero 76 cells
(American Type Culture Collection, Manassas, VA)
were grown and passed in MEM with EarleÕs salts,
L-glutamine, penicillin, and streptomycin (Invitrogen,
Carlsbad, CA) supplemented with 8% fetal bovine ser-
um (FBS) (Hyclone Laboratories, Logan, UT). The
amount of FBS was reduced to 2% for assays.
Vero cells were infected in suspension with either HSV-1
or HSV-2 for 45 min at 37 °C at a multiplicity of infec-
tion of 0.001. Infected cells were plated at a density of
50,000 cells/well into 96-well tissue culture plates con-
taining anti-viral test compounds and incubated at
37 °C for 40–48 h.
References and notes
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USA 1977, 74, 5716; (b) Elion, G. B. J. Med. Virol. Suppl.
1993, 1, 2.
5.38.2. HSV DNA hybridization. The effect of com-
pounds on HSV replication was assessed by convention-
al DNA hybridization. Cell lysates were prepared for
hybridization by removing growth medium from HSV-
infected Vero cells 2 days post-infection and adding
150 lL lysis buffer (0.2 N NaOH with 1% NP-40) to
each well. The lysates were then incubated at room tem-
perature for 5 days in a humidified chamber to ensure
complete DNA hydrolysis. Samples of the lysates were
neutralized in a phosphate-buffered guanidine isothiocy-
anate (GuSCN) solution and combined with a digoxi-
genin-labelled 710 bp DNA fragment of the HSV
UL15 open reading frame. The hybridization solution
was heated to 90 °C for 6 min and incubated at 42 °C
overnight. Immobilized hybrids were detected by incu-
bation with anti-digoxigenin HRP-conjugated antibody
(Boehringer Mannheim, Indianapolis, IN) and
7. (a) Beutner, K. R. Antiviral Res. 1995, 28, 281; (b) Perry,
C. M.; Faulds, D. Drugs 1996, 52, 754.
8. Jarvest, R. L.; Sutton, D.; Vere Hodge, R. A. Pharm.
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K. S. Gudmundsson et al. / Bioorg. Med. Chem. 13 (2005) 5346–5361
5361
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