Synthesis of C-6 substituted pyrazolo[1,5-a]pyridines with potent activity against herpesviruses

Article abstract of DOI:10.1016/j.bmc.2005.09.015

A novel series of potent C-6 substituted pyrazolo[1,5-a]pyridine inhibitors of herpes simplex viruses has been identified. A synthetic methodology was developed involving functionalization of a C-6 trifluoromethyl pyrazolo[1,5-a]pyridine to allow facile a

Full text of DOI:10.1016/j.bmc.2005.09.015

Bioorganic & Medicinal Chemistry 14 (2006) 944–954
Synthesis of C-6 substituted pyrazolo[1,5-a]pyridines with
potent activity against herpesviruses
Scott H. Allen,a,* Brian A. Johns,a Kristjan S. Gudmundsson,a
George A. Freeman,a F. Leslie Boyd, Jr.,a Connie H. Sexton,b Dean W. Selleseth,b
Katrina L. Creechb and Kelly R. Monirib
aDepartment of Medicinal Chemistry, GlaxoSmithKline Research and Development, Five Moore Drive, PO Box 13398,
Research Triangle Park, NC 27709, USA
bDepartment of Virology, GlaxoSmithKline Research and Development, Five Moore Drive, PO Box 13398,
Research Triangle Park, NC 27709, USA
Received 28 July 2005; revised 6 September 2005; accepted 6 September 2005
Available online 4 October 2005
Abstract—A novel series of potent C-6 substituted pyrazolo[1,5-a]pyridine inhibitors of herpes simplex viruses has been iden-
tified. A synthetic methodology was developed involving functionalization of a C-6 trifluoromethyl pyrazolo[1,5-a]pyridine to
allow facile access to a diverse set of analogues from common late stage intermediates. The expansion of the SAR of this
series at the 6 position allows for modifications to developability parameters such as clogP, while maintaining potency com-
parable to acyclovir.
Ó 2005 Elsevier Ltd. All rights reserved.
F
1. Introduction
O
Herpesviruses are a large family of viruses that infect
N
most mammalian species.1 Of particular concern to hu-
mans are herpes simplex virus 1 (HSV-1) which causes
cold sores and herpes simplex virus 2 (HSV-2) which
causes genital infections. The current gold standard
therapy based on acyclovir (1) and valacyclovir is both
safe and efficacious; however, there is significant interest
in optimizing treatment for HSV in regard to lesion pain
relief, time to healing, viral shedding, and reduction of
episodes of reactivation.2
HN
N
3
H
N
N
7
N
H2N
HO
N
N
N
4
O
HN
Acyclovir (ACV) (1)
GW3733 (2)
Figure 1.
polymerase inhibitors. This prompted the directed
chemistry effort around this core to identify a potent
and safe agent with drug-like properties.
Our group recently reported a pyrazolo[1,5-a]pyridine
scaffold, as exemplified by GW 3733 (2), that showed
promising antiherpetic activity (Fig. 1).3,4
Although original antiviral activity indicated potency
for compounds containing non-polar amines at the
C-7 position,4 it remained unclear whether this was a
regiospecific requirement. It was therefore desired to
explore the SAR attributes of a series of C-6 substituted
pyrazolo[1,5-a]pyridines. To accomplish this, a strategy
was developed using a previously reported C-6 trifluo-
romethyl intermediate5 which would allow synthetic ac-
cess to both C-6 monosubstituted and C-6/C-7
disubstituted products (Fig. 2).
The pyrazolo[1,5-a]pyridines of this class were shown
not to be DNA synthesis inhibitors and thus have a
mechanism of action different from currently marketed
Keywords: Pyrazolo[1,5-a]pyridines; Alkynyl ketone; Pyrimidine
synthesis; HSV.
*
Corresponding author. Tel.: +1 9194836214; fax: +1 9194836053;
e-mail: scott.h.allen@gsk.com
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.015

S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
945
F
converted to the corresponding bromomethyl com-
pound 17. Tin hydride reduction provided the 6-methyl
pyrazolo[1,5-a]pyridine 18. Bromide 17 also provided a
route to the N-cyclopentylaminomethyl derivative 19
(Scheme 2).
F
N
N
N
R
N
N
N
F
R'
HN
The C-6 orthoester 9 provided a precursor for C-6/C-7
disubstituted products. Lithiation of the 7-position of
the pyrazolo[1,5-a]pyridine ring system,7 followed by
F
F
3
A
4
quenching with CCl4 , provided the 7-chloro species 20
Figure 2. Retrosynthetic scheme.
(Scheme 3). Thermal displacement of the chloride in
neat cyclopentylamine provided 21 in excellent yield.
Hydrolysis of the orthoester, followed by saponification,
provided carboxylic acid 23. The C-6 acid allowed for
facile access to a series of amido derivatives 24–29 via
an in situ generated acid chloride intermediate. The ester
22 could also be reduced with DIBAL-H to provide the
7-amino-6-hydroxymethyl derivative 30. Additionally,
the orthoester group in 20 could be hydrolyzed to pro-
vide ester 31, containing the useful 7-chloro substituent.
Interestingly, very few examples of Negishi-type dialkyl-
zinc couplings exist in the literature.8 However, treat-
ment of 31 with Me2Zn and Pd(Ph3P)4 provided the
desired 7-methyl pyrazolo[1,5-a]pyridine 32 in modest
yield. Saponification and standard amide formation pro-
vided 34 (Scheme 3).
2. Chemistry
Compounds 4–22 were synthesized as is outlined in
Schemes 1–3. The previously described pyrazolo[1,5-
a]pyridine 35 was chosen as a starting material as the
6-trifluoromethyl moiety was known to be labile to
alkanolysis and provided a synthetic handle at the 6-po-
sition. Vilsmeier–Haack formylation provided the C-3
aldehyde 4 in excellent yield. Grignard addition fol-
lowed by MnO2 oxidation of the resulting propargyl
alcohol gave the alkynyl ketone 6. Cyclization with the
cyclopentyl guanidine 76 under basic conditions provid-
ed clean conversion to the pyrimidine 8. The choice of
the alkynyl ketone as the substrate for cyclization was
based on a synthetic methodology developed for similar
molecules.3 Ethanolysis of the triflouromethyl group
provided the ortho ester 9 (Scheme 1).5
3. Results and discussion
The ortho ester 9 provided a straightforward route to a
diverse set of C-6 substituted pyrazolo[1,5-a]pyridines.
Acid catalyzed hydrolysis provided ethyl ester 10, which
was cleanly saponified to carboxylic acid 11. Curtius
rearrangement followed by deprotection of the resulting
tert-butylcarbamate provided the amine 13. Standard
reductive amination conditions provided the secondary
amines 14 and 15. The ethyl ester 10 proved to be a ver-
satile intermediate. DIBAL-H reduction of 10 provided
6-hydroxymethyl pyrazolo[1,5-a]pyridine 16, which was
Our initial interest was to gauge the effects of moving the
cyclopentylamino group from the 7- to the 6-position.
As can be seen in Table 1, the 6-cyclopentylamino ana-
logue 14 exhibited a activity and cytotoxicity profile sim-
ilar to those of GW 3733 (2). Evaluation of additional
small polar and non-polar groups indicated that substi-
tution at this position was well tolerated. Interestingly,
the methylene spaced cyclopentylamine (entry 6) showed
greater cytotoxicity and somewhat less antiviral activity
than related compounds in this series. The two possible
F
F
F
c
b
a
N
N
N
N
HO
O
N
N
H
CF3
CF3
CF3
5
4
3
F
F
F
HN NH2
.
NH
HCl
N
N
N
N
7
e
N
N
O
N
N
N
N
CF3
C(OEt)3
d
HN
HN
CF3
6
8
9
Scheme 1. Reagents and conditions: (a) POCl3, DMF, 3 days, 94%; (b) ethynylmagnesium bromide, THF, ꢀ78 °C, 96%; (c) MnO2, CHCl3, 0 °C,
69%; (d) NaOEt, EtOH, 98%; (e) NaOEt, EtOH, 60 °C, 92%.

946
S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
F
F
F
F
N
N
a
N
N
b
N
N
N
N
c
N
N
N
N
N
N
R
N
N
N
HN
CO2Et
CO2H
C(OEt)3
HN
H
HN
HN
12
13
R = Boc
R = H
11
10
d
9
f
e
F
F
F
F
N
N
h
i
N
N
N
N
N
N
N
N
N
N
N
H
N
N
N
CH3
N
HN
R
X
N
HN
HN
HN
H
18
16 X = OH
17
14 R = cyclopentyl
15 R = isopropyl
g
X = Br
Scheme 2. Reagents and conditions: (a) pTsOH, H2O/acetone, 85%; (b) aq LiOH, dioxane, 92%; (c) diphenylphosphoryl azide, Et3N, t-BuOH, 54%;
(d) 4 N HCl in dioxane, 27%; (e) alkylamine, HOAc, NaBH(OAc)3, 1,2 DCE; (f) DIBAL-H, CH2Cl2, ꢀ78 °C, 40%; (g) PBr3, CHCl3, 43%; (h)
Bu3SnH, AIBN, toluene, 95 °C, 12%; (i) cyclopentylamine, THF, 20%.
F
F
F
F
N
N
N
N
H
H
N
N
N
N
N
a
d
N
b
Cl
N
N
N
N
N
N
N
N
X
R
HN
HN
C(OEt)3
C(OEt)3
HN
O
HN
21 R = C(OEt)3
22 R = CO2Et
23 X = OH
20
9
e
c
24-29 X = NHR
c
F
F
F
F
f
N
N
g
N
N
N
N
N
H
d
Cl
N
N
N
N
N
N
N
N
N
N
X
CO2Et
CO2Et
HN
HN
HN
HN
O
OH
31
32
30
33 X = OH
e
34 X = NHCyclopropyl
Scheme 3. Reagents and conditions: (a) LDA, CCl4, THF, ꢀ78 °C, 46%; (b) cyclopentylamine, 80 °C, 96%; (c) p-TsOH, H2O/acetone; (d) aq LiOH,
dioxane; (e) SOCl2, alkylamine, CH2Cl2; (f) DIBAL-H, CH2Cl2, ꢀ78 °C, 59%; (g) Me2Zn, (PPh3)4Pd, THF, 60 °C, 45%.
factors for this cytotoxicity, the increased flexibility and
increased basicity of the amine, were not further investi-
gated. As this was the only methylene spaced alkylami-
no compound synthesized, it remains unclear whether
this is a significant SAR trend.
tain polar groups were tolerated at the 6 position (en-
tries and 3). Clearly, there was significant
2
cytotoxicity imparted by the introduction of a 6-amido
group (entries 5–10) and in these cases the antiviral
activity was not well established from the overlying tox-
icity. As stated earlier, one of the investigational goals of
C-6 substitution was to identify potent compounds with
lower clogPÕs than GW3733 (clogP = 6.7), thus impart-
ing a more desirable developability profile. Compounds
Having established that activity could be retained in 6-
substituted analogues, we wished to evaluate C-6/C-7
disubstituted analogues. As can be seen in Table 2, cer-

S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
Table 1. HSV-1 antiviral activity (Vero cells, SC-16 strain) and cytotoxicity
947
F
N
N
R1
R2
N
N
N
H
Entry
1
Compound
ACV
R1
—
R2
—
EC50 (lM)
CC50 (lM)
clogP
0.39
>100
—
H
N
2
3
4
5
6
7
8
9
2
H
0.26
>160
>40
>40
>40
5.8
6.7
14
15
13
19
18
11
16
H
0.74 (0.21)
2.09 (0.19)
0.71 (0.08)
4.8
6.7
5.9
4.3
6.0
5.6
5.1
4.6
N
H
H
H
H
H
H
H
N
H
NH2
H
N
2.0 (0.51)
0.55 (0.07)
1.8 (0.51)
>40
>40
>40
CH3
OH
O
OH
Vero cells, SC-16 strain. IC50 is the concentration at which 50% efficacy in the antiviral assay is observed.
CC50 is the concentration at which 50% cytotoxicity is observed.
Compounds showing overt cytotoxicity were not retested, but all others were retested with n P 2. Standard deviation is shown in brackets. clogP
values calculated by DaylightÓ Pomona College and BioByte Inc.
of note include 30 and 34, which have lower clogP val-
ues relative to GW3733, while maintaining potency and
a large therapeutic index.
substituents, provides a mechanism for improving the
drug-like properties of this class of molecules.
5. Experimental
5.1. General
4. Conclusions
In summary, a series of C-6 substituted and C-6/C-7
disubstituted pyrazolo[1,5-a]pyridines with potent anti-
viral activity has been identified. Synthetic accessibility
to the C-6 substituted compounds was provided through
a chemically labile C-6 trifluoromethyl group, providing
a C-6 ortho ester as a key synthetic intermediate. This
intermediate allowed rapid access to a variety of C-6
substituted and C-6/C-7 disubstituted pyrazolo[1,
5-a]pyridines of varying degrees of size and polarity.
Substitution at the 6-position of the monosubstituted
compounds was well tolerated with a variety of small
functional groups with varying polarity. Disubstitution
at the C-6/C-7 positions was also well tolerated, which
coupled with the clogP lowering effects of the polar
1H and 13C NMR spectra were obtained on Varian Uni-
ty Plus NMR spectrometers at 300 or 400 MHz, and 75
or 100 MHz, respectively. Mass spectra were obtained
on Micromass Platform or ZMD mass spectrometers
from Micromass Ltd. Altrincham, UK, using either
atmospheric chemical ionization (APCI) or electrospray
ionization (ESI). Solvents were purchased as anhydrous
grade and used without further purification. For TLC,
Merck precoated plates (silica gel 60 F254) were used.
Unless otherwise stated, column chromatography for
the purification of some compounds used Merck Silica
gel 60 (230–400 mesh), and the stated solvent system un-
der pressure. All compounds were characterized as their

948
S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
Table 2. HSV-1 antiviral activity (Vero cells, SC-16 strain) and cytotoxicity
F
N
N
R1
R2
N
N
N
H
Entry
1
Compound
R1
CH3
R2
EC50(lM)
CC50 (lM)
clogP
H
N
34
1.77 (0.30)
>40
4.9
O
H
N
OH
2
23
30
22
24
25
26
27
28
29
0.55 (0.07)
0.75 (0.21)
6.27 (0.21)
1.6
38
>40
>40
11
7.0
6.0
7.8
5.9
6.0
5.1
6.1
5.3
5.1
O
H
N
3
OH
H
N
OEt
4
O
O
H
N
N
5
H
N
H
N
N
N
6
0.43
0.63
22.4
22.1
7.2
O
O
H
N
H
N
OH
OH
7
4.26
H
N
H
N
8
5.05
N
O
O
O
O
H
N
N
9
2.07
H
N
H
N
10
1.12
1.1
OH
Vero cells, SC-16 strain. IC50 is the concentration at which 50% efficacy in the antiviral assay is observed.
CC50 is the concentration at which 50% cytotoxicity is observed.
Compounds showing overt cytotoxicity were not retested, but all others were retested with n P 2. Standard deviation is shown in brackets. clogP
values calculated by Daylight Ó Pomona College and BioByte Inc.
free-base form unless otherwise stated. The correspond-
ing hydrochloride salts were formed to generate solids
where noted. In lieu of elemental analysis, compound
nyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridine (3) (11.0 g,
39.3 mmol). The reaction mixture was stirred at room
temperature for 72 h, and then quenched with ice water.
The solid precipitate was collected on a filter to provide
1
purity was assessed by high field H NMR and HPLC.
1
HPLC was performed using Synergi Max-RP 3.5 l col-
umn, running 15–100% MeOH gradient (0.1% formic
acid). Biological results were obtained with compounds
of >98% purity as determined by the above methods.
4 (11.4 g, 94%). Rf 0.45 (4:1 hexanes/ethyl acetate); H
NMR (400 MHz, CDCl3): d 10.15 (s, 1H), 8.92 (s, 1H),
8.53 (d, J = 9.2 Hz, 1H), 7.80 (m, 2H), 7.70 (d, J = 9.3
Hz, 1H), 7.27 (t, J = 8.5 Hz, 2H).
5.2. Synthesis of N-cyclopentyl-4-[2-(4-fluorophenyl)-6-
(triethoxymethyl)pyrazolo[1,5-a]pyridin-3-yl]-2-pyrimi-
dinamine (9)
5.2.2. 1-[2-(4-Fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,
5-a]pyridin-3-yl]-2-propyn-1-ol (5). To a cold (ꢀ78 °C) sus-
pension of 2-(4-fluorophenyl)-6-(trifluoromethyl)pyrazol-
o[1,5-a]pyridine-3-carbaldehyde (4) (11.4 g, 37.0 mmol)
in tetrahydrofuran (100 mL) was added ethynylmagne-
sium bromide (111 mL, 0.5 M in tetrahydrofuran,
56 mmol). The reaction mixture was warmed to room tem-
perature and stirred for 14 h. The reaction mixture was
5.2.1. 2-(4-Fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-
a]pyridine-3-carbaldehyde (4). To a cold (0 °C) solution of
phosphorus oxychloride (8.0 mL, 86 mmol) in N,N-di-
methylformamide (160 mL) was added 2-(4-fluorophe-

S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
949
poured into water and adjusted to neutral pH with 1 N
aqueous hydrochloric acid. The aqueous mixture was
extracted with ethyl acetate. The combined extracts were
washed with water and brine. The organic layer was dried
over magnesium sulfate. Filtration and concentration pro-
vided5(11.9 g,96%)asatansolid. Rf 0.18(4:1hexanes/eth-
2H), 7.46 (dd, J = 9.3, 1.1 Hz, 1H), 7.14 (app t,
J = 8.7 Hz, 2H), 6.32 (d, J = 5.4 Hz, 1H), 5.14 (d,
J = 7.3 Hz, 1H), 4.35 (m, 1H), 3.43 (m, 6H), 2.09 (m,
2H), 1.81–1.51 (m, 6H), 1.21 (m, 9H); MS m/z 520
(M+1).
1
yl acetate); H NMR (300 MHz, CDCl3): d 8.81 (s, 1H),
5.3. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluorophe-
nyl)pyrazolo[1,5-a]pyridine-6-carboxylic acid hydrochloride
(11)
8.14 (d, J = 9.3 Hz, 1H), 7.76 (m, 2H), 7.34 (dd, J = 9.3,
1.2 Hz, 1 H), 7.19 (app t, J = 8.7 Hz, 2H), 5.76 (m, 1H),
2.70 (d, J = 2.2 Hz, 1H), 2.60 (d, J = 4.0 Hz, 1H); MS
m/z 335 (M+1).
5.3.1. Ethyl 3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-
fluorophenyl)pyrazolo[1,5-a]pyridine-6-carboxylate (10).
To a solution of N-cyclopentyl-4-[2-(4-fluorophenyl)-6-
(triethoxymethyl)pyrazolo[1,5-a]pyridin-3-yl]-2-pyrim-
idinamine (9) (1.0 g, 1.9 mmol) in acetone (40 mL) and
water (10 mL) was added p-toluenesulfonic acid mono-
hydrate (915 mg, 4.81 mmol). The reaction mixture
was stirred at room temperature for 2 h. The pH of
the reaction mixture was adjusted to slightly basic using
saturated aqueous sodium bicarbonate solution. The
reaction mixture was concentrated in vacuo to one-third
of the original volume and then diluted with water. The
precipitate was collected on a filter to provide 10
(722 mg, 85%) as an orange solid. Rf 0.15 (4:1 hex-
anes/ethyl acetate); 1H NMR (300 MHz, CDCl3): d
9.22 (s, 1H), 8.39 (d, J = 9.4 Hz, 1H), 8.08 (br, 1H),
7.85 (d, J = 9.4 Hz, 1H), 7.64 (m, 2H), 7.16 (app t,
J = 8.6 Hz, 2H), 6.34 (br, 1H), 5.27 (br, 1H), 4.44 (q,
J = 7.1 Hz, 2H), 4.35 (br, 1H), 2.10 (m, 2H), 1.82–1.52
(m, 6H), 1.44 (t, J = 7.1 Hz, 3H); MS m/z 446 (M+1).
5.2.3. 1-[2-(4-Fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-
a]pyridin-3-yl]-2-propyn-1-one (6). To a cold (0 °C) solution
of 1-[2-(4-fluorophenyl)-6-(trifluoromethyl)pyrazolo[1,5-a]
pyridin-3-yl]-2-propyn-1-ol (5) (5.00 g, 15.0 mmol) in chlo-
roform (400 mL) was added manganese dioxide (130 g,
1.50 mol). The reaction mixture was stirred at 0 °C for
1.5 h. The reaction mixture was filtered through a pad of
Celite. The filtrate was concentrated in vacuo to provide 6
(3.44 g, 69%) as a clear oil. Rf 0.39 (4:1 hexanes/ethyl ace-
tate); 1H NMR (400 MHz, CDCl3): d 8.90 (s, 1H), 8.61 (d,
1H), 7.72–7.69 (m, 3H), 7.17 (m, 2H), 3.06 (s, 1H); MS
m/z 333 (M+1).
5.2.4. N-Cyclopentyl-4-[2-(4-fluorophenyl)-6-(trifluorometh-
yl)pyrazolo[1,5-a]pyridin-3-yl]-2-pyrimidinamine (8). To a
suspension of N-cyclopentylguanidine hydrochloride (7)9
(2.20 g, 13.5 mmol) in ethanol (70 mL) was added sodium
ethoxide (4.5 mL, 3 M in ethanol, 14 mmol). The mixture
was stirred at room temperature for 30 min, and then cooled
to 0 °C. To this mixture was added 1-[2-(4-fluorophenyl)-6-
(trifluoromethyl)pyrazolo[1,5-a]pyridin-3-yl]-2-propyn-
1-one (6) (3.44 g, 10.4 mmol) portionwise. The reaction mix-
ture was stirred at 0 °C for 30 min, followed by room tem-
perature for 15 h. The reaction mixture was diluted with
water (400 mL). The solid precipitate was collected on a fil-
ter to provide 8 (4.48 g, 98%) as an orange solid. 1H NMR
(400 MHz, CDCl3): d 8.83 (s, 1H), 8.50 (d, J = 9.4 Hz,
1H), 8.10 (d, J = 5.1 Hz, 1H), 7.63 (m, 2H), 7.43 (d,
J = 9.3 Hz, 1H), 7.16 (app t, J = 8.7 Hz, 2H), 6.33 (d,
J = 5.1 Hz, 1H), 5.17 (d, J = 7.2 Hz, 1H), 4.34 (m, 1H),
2.10 (m, 2H), 1.81–1.53 (m, 6H); MS m/z 442 (M+1); mp
155–156 °C.
5.3.2. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluoro-
phenyl)pyrazolo[1,5-a]pyridine-6-carboxylic acid hydrochlo-
ride (11). To a solution of ethyl 3-[2-(cyclopentylamino)-4-
pyrimidinyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-
carboxylate (10) (385 mg, 0.864 mmol) in dioxane (9 mL)
and water (1 mL) was added lithium hydroxide monohy-
drate (109 mg, 2.60 mmol). The reaction mixture was heated
at 95 °C for 5 h. The reaction mixture was concentrated in
vacuo. A suspension of the concentrated residue in water
was acidified with 1 N aqueous hydrochloric acid. The solid
precipitate was collected on a filter to provide 11 (359 mg,
92%) as an orange solid. 1H NMR (400 MHz, DMSO-d6):
d 9.27 (s, 1H), 8.72 (br, 1H), 8.51 (br, 1H), 8.12 (br, 1H),
7.96 (br, 1H), 7.66 (m, 2H), 7.36 (app t, J = 8.7 Hz, 2H),
6.40 (br, 1H), 4.18 (br, 1H), 1.95 (br, 2H), 1.71 (br, 2H),
1.56 (br, 4H); MS m/z 418 (M+1).
5.2.5. N-Cyclopentyl-4-[2-(4-fluorophenyl)-6-(triethoxy-
methyl)pyrazolo[1,5-a]pyridin-3-yl]-2-pyrimidinamine (9).
To a dry round-bottomed flask was added sodium metal
(1.9 g, 83 mmol). Ethanol (110 mL) was added and al-
lowed to react with sodium at room temperature until
completely dissolved. N-Cyclopentyl-4-[2-(4-fluorophe-
nyl)-6-(trifluoromethyl)pyrazolo[1,5-a]pyridin-3-yl]-2-pyri-
midinamine (8) (4.48 g, 10.1 mmol) was added and the
reaction mixture was stirred at 60 °C for 18 h. The reac-
tion mixture was cooled and concentrated in vacuo to
approximately one-fourth of the original volume. The
resulting mixture was diluted with water and extracted
with ethyl acetate. The organic layer was washed with
water and brine, and then dried over magnesium sulfate.
Filtration and concentration provided 9 (4.86 g, 92%) as
an off-white solid. Rf 0.15 (4:1 hexanes/ethyl acetate); 1H
NMR (300 MHz, CDCl3): d 8.81 (s, 1H), 8.38 (d,
J = 9.3 Hz, 1H), 8.06 (d, J = 5.3 Hz, 1H), 7.62 (m,
5.4. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluorophe-
nyl)pyrazolo[1,5-a]pyridin-6-amine dihydrochloride (13)
5.4.1. tert-Butyl 3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-
(4-fluorophenyl)pyrazolo[1,5-a]pyridin-6-ylcarbamate (12).
To a suspension of 3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carbox-
ylic acid hydrochloride (11) (60 mg, 0.13 mmol) in tert-
butanol were added triethylamine (39 lL, 0.28 mmol)
and diphenylphosphoryl azide (34 lL, 0.16 mmol). The
reaction mixture was refluxed for 2.5 h. The reaction
mixture was cooled to room temperature and diluted
with ethyl acetate. The organic layer was washed with
5% aqueous citric acid solution, water, saturated aque-
ous sodium bicarbonate solution, and brine. The organic

950
S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
layer was dried over sodium sulfate. Filtration and con-
centration followed by flash chromatography (39:1
dichloromethane/methanol) provided 12 (35 mg, 54%)
as a light green oil. Rf 0.32 (29:1 dichloromethane/meth-
anol); 1H NMR (400 MHz, CDCl3): d 8.94 (br, 1H), 8.34
(d, J = 9.4 Hz, 1H), 8.00 (br, 1H), 7.59 (m, 2H), 7.18 (d,
J = 9.4 Hz, 1H), 7.12 (app t, J = 8.7 Hz, 2H), 6.51 (s,
1H), 6.30 (d, J = 5.3 Hz, 1H), 4.33 (m, 1H), 2.07 (m,
2H), 1.79–1.49 (m, 6H), 1.53 (s, 9H); MS m/z 489 (M+1).
(d, J = 5.6 Hz, 1H), 7.72 (d, J = 1.8 Hz, 1H), 7.55 (m,
2H), 7.19 (app t, J = 8.8 Hz, 2H), 7.03 (dd, J = 9.6,
2.0 Hz, 1H), 6.24 (d, J = 5.4 Hz, 1H), 4.22 (m, 1H),
3.52 (m, 1H), 2.02 (m, 2 H), 1.76 (m, 2H), 1.67–1.51
(m, 4H), 1.24 (d, J = 6.2 Hz, 6H); MS m/z 431 (M+1).
5.7. [3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluoro-
phenyl)pyrazolo[1,5-a]pyridin-6-yl]methanol (16)
To a cold (ꢀ78 °C) solution of ethyl 3-[2-(cyclopentyla-
mino)-4-pyrimidinyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]-
pyridine-6-carboxylate (10) (722 mg, 1.62 mmol) in
dichloromethane (14 mL) was added diisobutylalumi-
num hydride (6.5 mL, 1.0 M in hexanes, 6.5 mmol).
The reaction mixture was stirred at ꢀ78 °C for 1.5 h.
The reaction mixture was poured into saturated aqueous
Rochelle salt solution and stirred at room temperature
for 2 h. The resultant mixture was extracted with ethyl
acetate. The organic layer was washed with water and
brine, and then dried over magnesium sulfate. Filtration
and concentration followed by flash chromatography
(4:1 dichloromethane/acetone) provided 16 (261 mg,
40%) as a white solid. Rf 0.41 (4:1 dichloromethane/ac-
etone); 1H NMR (300 MHz, CDCl3): d 8.36 (s, 1H),
8.27 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 5.2 Hz, 1H), 7.56
(m, 2H), 7.23 (d, J = 9.1 Hz, 1H), 7.10 (app t,
J = 8.6 Hz, 2H), 6.26 (d, J = 5.3 Hz, 1H), 5.23 (d,
J = 7.2 Hz, 1H), 4.64 (s, 2H), 4.29 (m, 1H), 2.03 (m,
2H), 1.77–1.45 (m, 6H); MS m/z 404 (M+1).
5.4.2. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluorophe-
nyl)pyrazolo[1,5-a]pyridin-6-amine dihydrochloride (13). To
a solution of tert-butyl 3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridin-6-ylcarbamate
(12) (35 mg, 0.072 mmol) in dichloromethane was added
hydrogen chloride (144 lL, 4 N in dioxane, 0.58 mmol).
The reaction mixture was stirred at room temperature for
18 h. The reaction mixture was diluted with ether and the
precipitated solids were collected on a filter to provide 13
(9 mg, 27%) as a brownish yellow solid. 1H NMR
(300 MHz, DMSO-d6): d 8.23 (br, 1H), 7.96–7.92 (m,
2H), 7.53 (m, 2H), 7.26 (app t, J = 8.9 Hz, 2H), 7.06 (d,
J = 9.5 Hz, 1H), 6.15 (br, 1H), 3.97 (br, 1H), 1.85 (m,
2H), 1.66 (m, 2H), 1.48 (m, 4H); MS m/z 389 (M+1).
5.5. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidinyl]-
2-(4-fluorophenyl)pyrazolo[1,5-a]pyridin-6-amine (14)
To a suspension of 3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridin-6-amine
dihydrochloride (13) (90 mg, 0.20 mmol) in 1,2 dichloro-
ethane were added cyclopentanone (26 lL, 0.29 mmol),
acetic acid (56 lL, 0.98), and sodium triacetoxyborohy-
dride (82 mg, 0.39 mmol). The reaction mixture was stir-
red at room temperature 16 h and then quenched with
water. The resultant mixture was diluted with ethyl ace-
tate and saturated aqueous sodium bicarbonate solu-
tion. The organic layer was separated and washed with
water and brine. The organic layer was dried over mag-
nesium sulfate. Filtration and concentration followed by
flash chromatography (4:1 hexanes/ethyl acetate to 7:3
hexanes/ethyl acetate) provided 14 (40 mg, 45%) as a
5.8. N-Cyclopentyl-4-[2-(4-fluorophenyl)-6-methylpyraz-
olo[1,5-a]pyridin-3-yl]-2-pyrimidinamine (18)
5.8.1. 4-[6-(Bromomethyl)-2-(4-fluorophenyl)pyrazolo[1,5-a]-
pyridin-3-yl]-N-cyclopentyl-2-pyrimidinamine (17). To a
solution of [3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-
fluorophenyl)pyrazolo[1,5-a]pyridin-6-yl]methanol
(16)
(65 mg, 0.16 mmol) in chloroform (1 mL) was added phos-
phorus tribromide (6 lL, 0.06 mmol). The reaction mixture
was stirred at room temperature for 2 h, and then quenched
with saturated aqueous sodium bicarbonate solution. The
resultant mixture was extracted with dichloromethane.
The organic layer was washed with water and brine, and
then dried over sodium sulfate. Filtration and concentration
followed by flash chromatography (19:1 dichloromethane/
acetone) provided 17 (32 mg, 43%) as a yellow oil. Rf 0.68
(9:1 dichloromethane/acetone); 1H NMR (400 MHz,
CDCl3): d 8.49 (s, 1H), 8.39 (d, J = 9.3 Hz, 1H), 8.05 (d,
J = 5.3 Hz, 1H), 7.59 (m, 2H), 7.34 (dd, J = 9.2, 1.5 Hz,
1H), 7.13 (app t, J = 8.7 Hz, 2H), 6.30 (d, J = 5.3 Hz, 1H),
5.21 (br d, J = 7.2 Hz, 1H), 4.52 (s, 2H), 4.33 (m, 1H), 2.07
(m, 2H), 1.78–1.50 (m, 6H); MS m/z 467 (M+1).
1
green oil. Rf 0.25 (2:1 hexanes/ethyl acetate); H NMR
(300 MHz, CDCl3): d 8.24 (d, J = 9.4 Hz, 1H), 8.00 (d,
J = 5.4 Hz, 1H), 7.79 (d, J = 1.7 Hz, 1H), 7.61 (m,
2H), 7.13 (app t, J = 8.7 Hz, 2H), 6.85 (dd, J = 9.6,
2.1 Hz, 1H), 6.32 (d, J = 5.4 Hz, 1H), 5.23 (br d,
J = 6.5 Hz, 1H), 4.35 (m, 1H), 3.71 (m, 1H), 3.54 (d,
J = 5.8 Hz, 1H), 2.14–2.03 (m, 4H), 1.80–1.51 (m,
12H); MS m/z 457 (M+1). To a solution of the product
in ether was added 1 MHCl in ether. The precipitated
solid was isolated to give the corresponding HCl salt.
5.6. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluoro-
phenyl)-N-isopropylpyrazolo[1,5-a]pyridin-6-amine (15)
5.8.2.
N-Cyclopentyl-4-[2-(4-fluorophenyl)-6-methyl-
pyrazolo[1,5-a]pyridin-3-yl]-2-pyrimidinamine (18). To a
solution of 4-[6-(bromomethyl)-2-(4-fluorophenyl)pyr-
azolo[1,5-a]pyridin-3-yl]-N-cyclopentyl-2-pyrimidinamine
(17) (40 mg, 0.086 mmol) in toluene (5 mL) were added
tributyltin hydride (35 lL, 0.13 mmol) and 2,20-azobisi-
sobutyronitrile (2 mg, 0.009 mmol). The reaction mix-
ture was heated at 95 °C for 3 h. After cooling the
reaction mixture to room temperature, Celite was added
and the resultant mixture was concentrated in vacuo.
In a similar manner as described in for compound 14
from 3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-fluor-
ophenyl)pyrazolo[1,5-a]pyridin-6-amine dihydrochlo-
ride (13) (40 mg, 0.087 mmol) and acetone (10 lL,
0.13 mmol) was obtained 15 (16 mg, 43%) as a pale
green solid. Rf 0.21 (2:1 hexanes/EtOAc); 1H NMR
(400 MHz, CD3OD): d 8.25 (d, J = 9.5 Hz, 1H), 7.89

S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
951
Flash chromatography (19:1 dichloromethane/acetone)
provided a crude material which was triturated with
ether to provide 18 (4 mg, 12%) as a pale yellow solid.
Rf 0.63 (9:1 dichloromethane/acetone); 1H NMR
(300 MHz, CDCl3): d 8.36–8.29 (m, 2H), 8.00 (br, 1H),
7.60 (m, 2H), 7.23–7.11 (m, 3H), 6.31 (d, J = 5.2 Hz,
1H), 5.37 (br, 1H), 4.35 (m, 1H), 2.39 (s, 3H), 2.08 (m,
2H), 1.81–1.53 (m, 6H); MS m/z 388 (M+1).
5.10.2. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimid-
inyl]-2-(4-fluorophenyl)-6-(triethoxymethyl)pyrazolo[1,5-
a]pyridin-7-amine (21). A mixture of 4-[7-chloro-2-(4-flu-
orophenyl)-6-(triethoxymethyl)pyrazolo[1,5-a]pyridin-3-
yl]-N-cyclopentyl-2-pyrimidinamine (20) (2.37 g, 4.28 mmol)
and cyclopentylamine (10 mL, 100 mmol) was heated in a
sealed tube at 80 °C for 16 h. The reaction mixture was
cooled to room temperature and excess cyclopentylamine
was removed in vacuo. The crude solid was triturated in
water. The solids were collected on filter to provide 21
(2.48 g, 96%). Rf 0.42 (4:1 hexanes/ethyl acetate); 1H
NMR (300 MHz, CDCl3): d 8.03 (d, J = 5.3 Hz, 1H),
7.74 (d, J = 9.2 Hz, 1H), 7.68–7.60 (m, 3H), 7.12 (app t,
J = 8.6 Hz, 2H), 6.36–6.31 (m, 2H), 5.32 (m, 1H), 5.06
(d, J = 7.5 Hz, 1H), 4.35 (m, 1H), 3.45 (q, J = 7.2 Hz,
6H), 2.13–1.95 (m, 4H), 1.83–1.47 (m, 12H), 1.25 (t,
J = 7.2 Hz, 9H); MS m/z 603 (M+1).
5.9. N-Cyclopentyl-4-[6-[(cyclopentylamino)methyl]-2-(4-fluoro-
phenyl)pyrazolo[1,5-a]pyridin-3-yl]-2-pyrimidinamine (19)
To a suspension of 4-[6-(bromomethyl)-2-(4-fluorophe-
nyl)pyrazolo[1,5-a]pyridin-3-yl]-N-cyclopentyl-2-pyrimi-
dinamine (17) (30 mg, 0.064 mmol) in tetrahydrofuran
(2 mL) was added cyclopentylamine (730 lL, 7.4 mmol).
The reaction mixture was stirred at room temperature
for 3 h, and then diluted with ethyl acetate. The organic
layer was washed with saturated aqueous sodium bicar-
bonate solution, water, and brine. The organic layer was
dried over magnesium sulfate. Filtration and concentra-
tion followed by flash chromatography (39:1 dichloro-
methane/methanol to 35:5 dichloromethane/methanol)
provided 19 (6 mg, 20%) as a light yellow solid. Rf
0.56 (35:5 dichloromethane/methanol); 1H NMR
(400 MHz, CDCl3): d 8.46 (s, 1H), 8.36 (d, J = 9.3 Hz,
1H), 8.03 (d, J = 5.2 Hz, 1H), 7.60 (m, 2H), 7.36 (d,
J = 9.4 Hz, 1H), 7.12 (app t, J = 8.7 Hz, 2H), 6.30 (d,
J = 5.3 Hz, 1H), 5.10 (d, J = 7.2 Hz, 1H), 4.33 (m,
1H), 3.84 (s, 2H), 3.14 (quint, J = 6.7 Hz, 1H), 2.07
(m, 2H), 1.85 (m, 2H), 1.78–1.38 (m, 12H); MS m/z
471 (M+1).
5.10.3. Ethyl 7-(cyclopentylamino)-3-[2-(cyclopentylamino)-
4-pyrimidinyl]-2-(4-fluorophenyl) [1,5-a]pyridine-6-carboxyl-
ate (22). To a suspension of N-cyclopentyl-3-[2-(cyclopen-
tylamino)-4-pyrimidinyl]-2-(4-fluorophenyl)-6-(triethoxym-
ethyl)pyrazolo[1,5-a]pyridin-7-amine (21) (2.48 g, 4.11 mmol)
in acetone (80 mL) and water (20 mL) was added p-tolu-
enesulfonic acid monohydrate (1.95 g, 10.2 mmol). The
reaction mixture was stirred at room temperature for
3 h, then poured onto ice and neutralized with saturated
aqueous sodium bicarbonate solution. The aqueous mix-
ture was concentrated in vacuo to remove acetone. The
resulting aqueous mixture was extracted with ethyl acetate.
The organic layer was washed with water and brine, and
then dried over magnesium sulfate. Filtration and concen-
tration followed by flash chromatography (4:1 hexanes/
ethyl acetate to 100% ethyl acetate to 4:1 ethyl acetate/
methanol) provided 22 (1.9 g, 88%) as a light yellow solid.
5.10. Ethyl 7-(cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]-2-(4-fluorophenyl) [1,5-a]pyridine-6-carboxyl-
ate (22)
1
Rf 0.20 (4:1 hexanes/ethyl acetate); H NMR (300 MHz,
CDCl3): d 9.26 (d, J = 7.5 Hz, 1H), 8.08 (d, J = 5.3 Hz,
1H), 7.77 (d, J = 9.4 Hz, 1H), 7.64 (m, 2H), 7.47 (d,
J = 9.5 Hz, 1H), 7.12 (app t, J = 8.7 Hz, 2H), 6.32 (d,
J = 5.3 Hz, 1H), 5.51 (m, 1H), 5.10 (d, J = 7.2 Hz, 1H),
4.39–4.28 (m, 3H), 2.12–2.03 (m, 4H), 1.82–1.49 (m,
12H), 1.41 (t, J = 7.2 Hz, 3H); MS m/z 529 (M+1).
5.10.1. 4-[7-Chloro-2-(4-fluorophenyl)-6-(triethoxymethyl)
pyrazolo[1,5-a]pyridin-3-yl]-N-cyclopentyl-2-pyrimidinamine
(20). To a cold (0 °C) solution of diisopropylamine (4.1 mL,
29 mmol) in tetrahydrofuran (25 mL) was added butyllithi-
um(17 mL,1.6 Minhexanes, 28 mmol) dropwise. The reac-
tion mixture was stirred at 0 °C for 10 min and then cooled
to ꢀ78 °C. The reaction mixture was transferred via syringe
to a cold (ꢀ78 °C) solution of N-cyclopentyl-4-[2-(4-fluor-
ophenyl)-6-(triethoxymethyl)pyrazolo[1,5-a]pyridin-3-yl]-
2-pyrimidinamine (9) (4.86 g, 9.35 mmol) in tetrahydrofu-
ran (25 mL). The reaction mixture was stirred at ꢀ78 °C
for 30 min. Carbon tetrachloride (3.6 mL, 37 mmol) was
added and the resulting mixture was warmed to room tem-
perature and stirred 2 h. The reaction mixture was poured
onto ice. After the ice had melted, the aqueous mixture
was extracted with ethyl acetate. The organic layer was
washed with water and brine, and then dried over sodium
sulfate. Filtration and concentration followed by flash
chromatography (4:1 hexanes/ethyl acetate) provided 20
(2.37 g, 46%) as a yellow solid. Rf 0.36 (4:1 hexanes/ethyl
acetate); 1H NMR (400 MHz, CDCl3): d 8.36 (d,
J = 9.4 Hz, 1H), 8.08 (d, J = 5.2 Hz, 1H), 7.85 (d,
J = 9.5 Hz, 1H), 7.66 (m, 2H), 7.15 (app t, J = 8.6 Hz,
2H), 6.32 (d, J = 5.2 Hz, 1H), 5.15 (d, J = 7.2 Hz, 1H),
4.36 (m, 1H), 3.45 (m, 6H), 2.10 (m, 2H), 1.81–1.54 (m,
6H), 1.26 (m, 9H); MS m/z 554 (M+1).
5.11. 7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carbox-
ylic acid (23)
To a solution of ethyl 7-(cyclopentylamino)-3-[2-(cyclo-
pentylamino)-4-pyrimidinyl]-2-(4-fluorophenyl)pyraz-
olo[1,5-a]pyridine-6-carboxylate (22) (200 mg, 0.378 mmol)
in dioxane (5 mL) and water (700 lL) was added lithium
hydroxide monohydrate (48 mg, 1.1 mmol). The reaction
mixture was heated for 40 h at 100 °C. The reaction mix-
ture was cooled and concentrated in vacuo, and then dilut-
ed with water. The aqueous mixture was acidified with
1 N aqueous hydrochloric acid. The solid precipitate
was collected on a filter to provide 23 (125 mg, 58%) as
an orange solid. 1H NMR (400 MHz, DMSO-d6): d
13.06 (br, 1H), 9.46 (d, J = 7.4 Hz, 1H), 8.07 (d,
J = 5.6 Hz, 1H), 7.78 (d, J = 9.2 Hz, 1H), 7.65 (m, 2H),
7.34 (app t, J = 8.8 Hz, 2H), 6.28 (br, 1H), 5.37 (m, 1H),
4.10 (m, 1H), 1.99 (m, 2H), 1.87 (m, 2H), 1.74-1.47 (m,
12H); MS m/z 501 (M+1).

952
S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
5.12. N-Cyclopentyl-3-[2-(cyclopentylamino)-4-pyrimidinyl]-
2-(4-fluorophenyl)-6-(1-pyrrolidinylcarbonyl)pyrazolo[1,5-a]-
pyridin-7-amine (24)
added to a dry flask and cooled to ꢀ78 °C. Thionyl chlo-
ride (38 lL, 0.52 mmol) was added and the reaction
mixture was stirred at room temperature for 30 min.
The reaction mixture was concentrated under
a
7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carboxyl-
ic acid dihydrochloride (23) (19 mg, 0.033 mmol) was added
to a dry flask and cooled to ꢀ78 °C. Thionyl chloride
(14 lL, 0.19 mmol) was added and the reaction mixture
was stirred at room temperature for 30 min. The reaction
mixture was concentrated under a stream of nitrogen and
then placed under high vacuum (using base trap in vacu-
um line). To a solution of the crude residue in dichlo-
romethane (600 lL) was added pyrrolidine (150 lL,
1.80 mmol). The reaction mixture was stirred at room
temperature for 10 min and then partitioned between
ethyl acetate and saturated aqueous sodium bicarbon-
ate solution. The aqueous layer was extracted with eth-
yl acetate. The combined extracts were washed with
water and brine, and then dried over magnesium sul-
fate. Filtration and concentration followed by flash
chromatography (1:1 hexanes/ethyl acetate to 100%
ethyl acetate) provided 24 (9 mg, 49%) as a clear oil.
Rf 0.13 (1:1 hexanes/ethyl acetate); 1H NMR
(300 MHz, CDCl3): d 8.04 (d, J = 5.4 Hz, 1H), 7.72
(d, J = 9.1 Hz, 1H), 7.63 (m, 2H), 7.24 (m, 1H), 7.14
(app t, J = 8.6 Hz, 2H), 6.54 (d, J = 8.7 Hz, 1H), 6.29
(d, J = 5.3 Hz, 1H), 5.11 (d, J = 6.7 Hz, 1H), 4.31 (m,
1H), 4.20 (m, 1H), 3.66 (br, 2H), 3.38 (br, 2H), 2.11–
1.90 (m, 8H), 1.80–1.49 (m, 12H); MS m/z 554
(M+1). To a solution of the product in ether was add-
ed 1 M HCl in ether. The precipitated solid was isolat-
ed to give the corresponding HCl salt.
stream of nitrogen and then placed under high vacuum
(using base trap in vacuum line). In a separate flask,
a suspension of hydroxylamine hydrochloride (29 mg,
0.42 mmol) and potassium carbonate (29 mg,
0.21 mmol) in tetrahydrofuran (600 lL) was stirred at
room temperature for 30 min. To this suspension was
added a solution of the crude acid chloride in dichloro-
methane (600 lL). The resulting mixture was stirred at
room temperature for 16 h. The reaction mixture was
diluted with ethyl acetate and washed with saturated
aqueous sodium bicarbonate solution, water, and brine.
The organic layer was dried over magnesium sulfate.
Filtration and concentration followed by chromatogra-
phy (100% ethyl acetate to 9:1 ethyl acetate/methanol)
provided 29 (12 mg, 26%) as a brownish-yellow solid.
Rf 0.42 (19:1 ethyl acetate/methanol); 1H NMR
(400 MHz, CDCl3): d 8.05 (br, 2H), 7.60 (m, 2H), 7.49
(br, 1H), 7.21 (br, 1H), 7.13 (app t, J = 8.6 Hz, 2H),
6.29 (d, J = 5.2 Hz, 1H), 5.90 (br, 1H), 4.92 (br, 1H),
4.30 (m, 1H), 2.08–1.98 (m, 4H), 1.82–1.54 (m, 12H);
MS m/z 516 (M+1).
5.15. 7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)-N-[2-(4-morpholinyl)ethyl]pyraz-
olo[1,5-a]pyridine-6-carboxamide (27)
In a similar manner as described in for compound 24 from
7-(cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyrimid-
inyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carbox-
ylic acid dihydrochloride (23) (40 mg, 0.070 mmol) and 4-
(2-aminoethyl)morpholine (400 lL, 3.0 mmol) was
formed 27 (20 mg, 47%) as an orange solid. Rf 0.27 (27:3
ethyl acetate/methanol); 1H NMR (400 MHz, CDCl3): d
8.52 (br, 1H), 8.06 (d, J = 5.3 Hz, 1H), 7.65–7.61 (m,
3H), 7.40 (d, J = 9.3 Hz, 1H), 7.12 (app t, J = 8.7 Hz,
2H), 6.92 (br, 1H), 6.31 (d, J = 5.2 Hz, 1H), 5.11 (d,
J = 7.5 Hz, 1H), 5.00 (br, 1H), 4.30 (m, 1H), 3.74 (t,
J = 4.4 Hz, 4H), 3.55 (m, 2H), 2.63 (t, J = 5.9 Hz, 2H),
2.53 (br, 4H), 2.10–1.95 (m, 4H), 1.82–1.48 (m, 12H);
MS m/z 613 (M+1).
5.13. 7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)-N,N-dimethylpyrazolo[1,5-a]pyr-
idine-6-carboxamide (28)
In a similar manner as described in for compound 24 from
7-(cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyrimid-
inyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carbox-
ylic acid dihydrochloride (23) (41 mg, 0.071 mmol) and
dimethylamine (500 lL, 2.0 M in tetrahydrofuran,
0.25 mmol) was formed 28 (14 mg, 38%) as a clear oil.
Rf 0.12 (1:1 hexanes/ethyl acetate); 1H NMR (400 MHz,
CDCl3): d 8.04 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz,
1H), 7.63 (m, 2H), 7.19 (d, J = 8.8 Hz, 1H), 7.15 (app t,
J = 8.7 Hz, 2H), 6.51 (d, J = 8.4 Hz, 1H), 6.30 (d,
J = 5.3 Hz, 1H), 5.17 (d, J = 6.8 Hz, 1H), 4.32 (m, 1H),
4.13 (m, 1H), 3.09 (br, 6H), 2.11–1.95 (m, 4H), 1.80–
1.50 (m, 12H); MS m/z 528 (M+1). To a solution of the
product in ether was added 1 M HCl in ether. The precip-
itated solid was isolated to give the corresponding HCl
salt.
5.16. 7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)-N-[3-(1H-imidazol-1-yl)pro-
pyl]pyrazolo[1,5-a]pyridine-6-carboxamide (25)
In a similar manner as described in for compound 24
from 7-(cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-
carboxylic acid dihydrochloride (23) (40 mg, 0.070 mmol)
and 1-(3-aminopropyl)imidazole (400 lL, 3.4 mmol)
was formed 25 (19 mg, 43%) as a yellow solid. Rf 0.09
(27:3 ethyl acetate/methanol); 1H NMR (400 MHz,
CDCl3): d 8.44 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 5.2 Hz,
1H), 7.64–7.60 (m, 4H), 7.37 (d, J = 9.4 Hz, 1H), 7.14–
7.08 (m, 4H), 6.98 (s, 1H), 6.65 (t, J = 5.8 Hz, 1H),
6.30 (d, J = 5.2 Hz, 1H), 4.94 (m, 1H), 4.29 (m, 1H),
4.08 (m, 2H), 3.46 (m, 2H), 2.14 (m, 2H), 2.07–1.94
(m, 4H), 1.80–1.47 (m, 12H); MS m/z 608 (M+1).
5.14. 7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)-N-hydroxypyrazolo[1,5-a]pyr-
idine-6-carboxamide (29)
7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carboxyl-
ic acid dihydrochloride (23) (52 mg, 0.091 mmol) was

S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
953
5.17. 7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)-N-[2-hydroxy-1-(hydroxymeth-
yl)ethyl]pyrazolo[1,5-a]pyridine-6-carboxamide (26)
(400 MHz, CDCl3): d 8.36 (d, J = 9.5 Hz, 1H), 8.08 (d,
J = 5.1 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.65 (m, 2H),
7.14 (app t, J = 8.7 Hz, 2H), 6.30 (d, J = 5.4 Hz, 1H), 5.19
(d, J = 6.8 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 4.32 (m, 1H),
2.08 (m, 2H), 1.78–1.22 (m, 9H); MS m/z 480 (M+1).
In a similar manner as described in for compound 24
from 7-(cyclopentylamino)-3-[2-(cyclopentylamino)-4-
pyrimidinyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-
carboxylic acid dihydrochloride (23) (40 mg, 0.070 mmol)
and serinol (200 mg, 2.2 mmol) was formed 26 (8 mg,
5.19.2. Ethyl 3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-
fluorophenyl)-7-methylpyrazolo[1,5-a]pyridine-6-carboxyl-
ate (32). To a solution of ethyl 7-chloro-3-[2-(cyclopen-
tylamino)-4-pyrimidinyl]-2-(4-fluorophenyl)pyrazolo[1,5-
a]pyridine-6-carboxylate (31) (90 mg, 0.19 mmol) in tet-
rahydrofuran (1 mL) were added dimethylzinc (281 lL,
2.0 M in toluene, 0.56 mmol) and tetrakis(triphenylphos-
phine)palladium (21 mg, 0.018 mmol). The reaction mix-
ture was stirred at 60 °C for 16 h. The reaction mixture
was quenched with ice water and then extracted with eth-
yl acetate. The organic layer was washed with water and
brine, and then dried over magnesium sulfate. Filtration
and concentration followed by flash chromatography
(49:1 dichloromethane/methanol) provided 32 (40 mg,
1
20%) as an orange solid. H NMR (300 MHz, CDCl3):
d 8.47 (br d, J = 7.5 Hz, 1H), 7.96 (br, 1H), 7.67–7.55
(m, 4H), 7.43 (d, J = 9.2 Hz, 1H), 7.20–7.09 (m, 3H),
6.24 (d, J = 5.3 Hz, 1H), 4.99 (m, 1H), 4.32 (m, 1H),
4.17 (m, 1H), 3.97 (m, 4H), 2.10–1.94 (m, 4H), 1.83–
1.52 (m, 12H); MS m/z 572 (Mꢀ1).
5.18. [7-(Cyclopentylamino)-3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridin-6-yl]meth-
anol (30)
1
To a cold (ꢀ78 °C) solution of ethyl 7-(cyclopentylamino)-
3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-fluorophe-
nyl)pyrazolo[1,5-a]pyridine-6-carboxylate (22) (50 mg,
0.095 mmol) in dichloromethane (1 mL) was added dii-
sobutylaluminum hydride (490 lL, 1.0 M in hexanes,
0.49 mmol). The reaction mixture was stirred at
ꢀ78 °C for 2 h then poured into a stirring mixture of
ether and aqueous Rochelle salt solution. The resultant
mixture was stirred at room temperature for 16 h. The
organic layer was separated and the aqueous layer was
extracted with ethyl acetate. The combined organic lay-
ers were washed with water and brine. Concentration
and flash chromatography (29:1 dichloromethane/meth-
anol) provided 30 (27 mg, 59%) as a green solid. Rf 0.26
45%). H NMR (300 MHz, CDCl3): d 8.32 (d, J = 9.3
Hz, 1H), 8.10 (d, J = 5.0 Hz, 1H), 7.89 (d, J = 9.4 Hz,
1H), 7.70 (m, 2H), 7.19 (app t, J = 8.7 Hz, 2H), 6.36
(d, J = 5.5 Hz, 1H), 5.32 (br, 1H), 4.47 (q, J = 7.1 Hz,
2H), 4.38 (m, 1H), 3.26 (s, 3H), 2.12 (m, 2H), 1.85–
1.45 (m, 9H); MS m/z 460 (M+1).
5.19.3. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-2-(4-fluoro-
phenyl)-7-methylpyrazolo[1,5-a]pyridine-6-carboxylic acid
(33). To a solution of ethyl 3-[2-(cyclopentylamino)-4-pyri-
midinyl]-2-(4-fluorophenyl)-7-methylpyrazolo[1,5-a]pyri-
dine-6-carboxylate 32 (40 mg, 0.087 mmol) in dioxane
(600 lL) was added lithium hydroxide (300 lL, 1 M aque-
ous, 0.30 mmol). The reaction mixture was stirred at room
temperature 16 h. The reaction mixture was concentrated
in vacuo to remove dioxane and then diluted with water.
The aqueous mixture was acidified with 1 N aqueous
hydrochloric acid. Upon standing for 72 h, a solid precip-
itate had formed which was collected by filtration to pro-
vide (33) (31 mg, 82%). Rf 0.10 (19:1 dichloromethane/
methanol); MS m/z 432 (M+1).
1
(29:1 dichloromethane/methanol); H NMR (300 MHz,
CDCl3): d 7.99 (d, J = 5.2 Hz, 1H), 7.69 (d, J = 8.9 Hz,
1H), 7.61 (m, 2H), 7.22 (d, J = 8.9 Hz, 1H), 7.13 (app
t, J = 8.7 Hz, 2H), 6.28–6.23 (m, 2H), 5.08 (d,
J = 7.4 Hz, 1H), 4.79 (s, 2H), 4.46 (br, 1H), 4.29 (m,
1H), 2.10–2.00 (m, 4H), 1.85–1.47 (m, 12H); MS m/z
487 (M+1).
5.19. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-N-cyclopro-
pyl-2-(4-fluorophenyl)-7-methylpyrazolo[1,5-a]pyridine-6-
carboxamide (34)
5.19.4. 3-[2-(Cyclopentylamino)-4-pyrimidinyl]-N-cyclopro-
pyl-2-(4-fluorophenyl)-methylpyrazolo[1,5-a]pyridine-6-car-
boxamide (34). Thionyl chloride (200 lL, 2.7 mmol) was
added to 3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-(4-flu-
orophenyl)-7-methylpyrazolo[1,5-a]pyridine-6-carboxylic
acid 33 (31 mg, 0.072 mmol), pre-cooled to 0 °C. The
reaction mixture was stirred at room temperature for
1 h. The excess thionyl chloride was removed in vacuo
(using base trap). To a solution of the residue in dichloro-
methane (300 lL) was added cyclopropylamine (50 lL,
0.72 mmol). The reaction mixture was stirred at room
temperature for 30 min. The resultant mixture was
quenched with water and diluted with ethyl acetate. Satu-
rated aqueous sodium bicarbonate solution was added to
the bi-phasic mixture. The organic layer was washed with
water and brine, and then dried over magnesium sulfate.
Filtration and concentration, followed by flash chroma-
tography (3:2 hexanes/ethyl acetate to 2:3 hexanes/ethyl
acetate) provided (34) (15 mg, 44%) as a pale yellow
5.19.1. Ethyl 7-chloro-3-[2-(cyclopentylamino)-4-pyrimidi-
nyl]-2-(4-fluorophenyl)pyrazolo[1,5-a]pyridine-6-carboxylate
(31). To a solution of 4-[7-chloro-2-(4-fluorophenyl)-6-(tri-
ethoxymethyl)pyrazolo[1,5-a]pyridin-3-yl]-N-cyclopen-
tyl-2-pyrimidinamine (20) (375 mg, 0.677 mmol) in acetone
(8 mL) and water (2 mL) was added p-toluenesulfonic acid
monohydrate (321 mg, 1.69 mmol). The reaction mixture
was stirred at room temperature for 2 h and then quenched
with ice water. The reaction mixture was neutralized with
saturated aqueous sodium bicarbonate solution and then
concentrated invacuoto remove the majorityof the acetone.
The resultant mixture was extracted with ethyl acetate. The
organic layer was washed with water and brine, then
dried over magnesium sulfate. Filtration and concentration
and followed by flash chromatography (29:1 dichlorometh-
ane/methanol) provided 31 (175 mg, 54%) as a brown solid.
Rf 0.08 (29:1 dichloromethane/methanol); 1H NMR
solid. 1H NMR (400 MHz, CDCl3):
J = 9.2 Hz, 1H), 8.05 (d, J = 5.1 Hz, 1H), 7.63 (m, 2H),
d 8.28 (d,

954
S. H. Allen et al. / Bioorg. Med. Chem. 14 (2006) 944–954
7.30 (d, J = 9.2 Hz, 1H), 7.13 (app t, J = 8.6 Hz, 2H), 6.29
(d, J = 5.3 Hz, 1H), 5.10 (d, J = 7.3 Hz, 1H), 4.31 (m, 1H),
2.96 (s, 3H), 2.93 (m, 1H), 2.06 (m, 2H), 1.78–1.49 (m,
6H), 0.91 (m, 2H), 0.66 (m, 2H); MS m/z 471 (M+1).
1–11, in minimum essential medium containing EarleÕs
salt and L-glutamine and supplemented with 10% heat-
inactivated fetal bovine serum, 1% penicillin–streptomy-
cin, and 1% L-glutamine (200 mM) resulting in a final
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 are incubated at
37 °C in a humidified, 5% CO2 atmosphere and growth
inhibition is measured after 3 days.
5.20. HSV antiviral assay
5.20.1. 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 Life
Technologies, Carlsbad, CA) supplemented with 8% fe-
tal bovine serum (FBS) (Hyclone Laboratories, Logan,
UT). The amount of FBS was reduced to 2% for assays.
Cytotoxicity is determined in Vero cells using the CellT-
iter 96Ò Aqueous Non-Radioactive Cell Proliferation
Assay (Promega Corporation, G1111). Metabolically
active cells will convert methylthiazol tetrazolium inner
salt (MTS), through the actions of cellular dehydrogen-
ases, into a colorized formazan end product. On day 3,
MTS solution is added to the assay plates, incubated for
1.5–2 h at 37 °C in a humidified, 5% CO2 atmosphere,
and absorbance is measured at 490 nm using the Wallac
Victor2 1420 multilabel counter (Perkin-Elmer, Welles-
ley, NA). RoboFit 2000 curve-fitting software is then
used to obtain a CC50 (50% cytotoxicity concentration)
value from the curves generated.
Vero cells were infected in suspension with either HSV-1
for 45 min at 37 °C at a multiplicity of infection of
0.001. Infected cells were plated at a density of 50,000
cells/well into 96-well tissue culture plates containing anti-
viral test compounds and incubated at 37 °C for 40–48 h.
5.20.2. 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 two 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 five 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-labeled 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 over-
night. Immobilized hybrids were detected by incubation
with anti-digoxigenin HRP-conjugated antibody (Boeh-
ringer–Mannheim, Indianapolis, IN) and subsequent
addition of SuperSignal? substrate (Pierce, Rockford,
MD). The resulting chemiluminescent signal from com-
pound-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% inhibi-
tory concentrations (IC50).
References and notes
1. Roizman, B.; Pellett, P. E.. In Knipe, D. M., Howley, P.
M., Eds.; FieldsÕ Virology; Lippincott Williams and Wil-
kins: Philadelphia, PA, 2001; Vol. 2, p 2381.
2. Corey, L.; Tyring, S.; Sacks, S.; Warren, T.; Beutner, K.;
Patel, R.; Wald, A.; Mertz, G.; Paavonen, J. In Abstract of
papers, 42nd Interscience Conference on Antimicrobial
Agents and Chemotherapy, San Diego, CA; American
Society for Microbiology: Washington, DC. 2002; Abstract
LB3.
3. Johns, B. A.; Gudmundsson, K. S.; Turner, E. M.; Allen, S.
H.; Jung, D. K.; Sexton, C. J.; Boyd, F. L., Jr.; Peel, M. R.
Tetrahedron 2003, 59, 9001.
4. Johns, B. A.; Gudmundsson, K. S.; Turner, E. M.; Allen, S.
H.; Samano, V. A.; Ray, J. A.; Freeman, G. A.; Boyd, F.
L., Jr.; Sexton, C. J.; Selleseth, D. A.; Creech, K. L.;
Moniri, K. R. Bioorg. Med. Chem. 2005, 2397.
5. Alberti, M. J.; Baldwin, I. R.; Cheung, M.; Cockerill, S.;
Flack, S.; Harris, P. A.; Jung, D. K.; Peckham, G.; Peel, M.
R.; Stanford, J. B.; Stevens, K.; Veal, J. M. WO 02/16359,
2002; Chem Abstr. 2002, 136, 200184.
5.21. Cytotoxicity assay
6. Cyclopentyl guanidine 61 (NR3R4 = c-C5H9) was made via
a modification of methods described in Bannard, R. A. B.;
Casselman, A. A.; Cockburn, W. F.; Brown, G. M. Can. J.
Chem. 1958, 36.
Compounds were dissolved in DMSO at a stock concen-
tration of 10 mM and serially diluted 2-fold in DMSO.
Dilutions were carried out in columns 1–9 of a 96-well,
v-bottomed polypropylene plate. Columns 10, 11, and
12 are control columns that do not contain compound.
Plates are then seeded at 10,000 Vero cells/well, columns
7. (a) Finkelstein, B. L. J. Org. Chem. 1992, 5538; (b) Aboul-
Fadl, T.; Lober, S.; Gmeiner, P. Synthesis 2000, 1727.
8. (a) Herbert, J. M. Tetrahedron Lett. 2004, 87; (b) Erdik, E.
Tetrahedron 1992, 44, 9577.