Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.029

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC₅₀ 0.9 nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.

Full text of DOI:10.1016/j.bmc.2011.11.029

Bioorganic & Medicinal Chemistry 20 (2012) 69–85
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of pyrazolo[1,5-a]pyridines as p110a-selective PI3 kinase inhibitors
Jackie D. Kendall a,b, , Patrick D. O’Connor a, Andrew J. Marshall a, Raphaël Frédérick a, , Elaine S. Marshall a,
⇑
Claire L. Lill c, Woo-Jeong Lee c, Sharada Kolekar c, Mindy Chao c, Alisha Malik c, Shuqiao Yu c,
Claire Chaussade b,c,à, Christina Buchanan b,c, Gordon W. Rewcastle a,b, Bruce C. Baguley a,b
Jack U. Flanagan a,b, Stephen M.F. Jamieson a,b, William A. Denny a,b, Peter R. Shepherd b,c
,
a Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
c Department of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their
Received 30 August 2011
Revised 9 November 2011
Accepted 16 November 2011
Available online 25 November 2011
selectivity for the p110
a isoform over the other Class Ia PI3 kinases. We investigated the SAR around the
pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110
a
IC50 0.9 nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream
marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
PI3 kinase
PI3K
PIK3CA
p110a
Pyrazolo[1,5-a]pyridine
Sulfonohydrazide
1. Introduction
proteins such as protein kinase B (PKB, also known as Akt) to the
cell membrane. Once recruited, PKB is phosphorylated and acti-
Phosphoinositide-3-kinases (PI3 kinases) are lipid kinases
which phosphorylate the 30-hydroxyl group of phosphatidyl-
inositol 4,5-diphosphate (PIP2) to phosphatidylinositol 3,4,5-tri-
phosphate (PIP3). PIP3 then recruits PH domain containing
vated, leading to a cascade of cell signalling which control a range
of cellular processes like cell proliferation, growth and survival.1
Furthermore, lipid phosphatase PTEN which dephosphorylates
PIP3, is often deleted or inactivated in many cancer types, leading
to increased levels of PIP3 and increased tumour survival.2
The PI3 kinases are split into three sub-families (class I, II and
III), and class I is further split into class Ia and Ib based upon their
mechanism of activation. The class Ia PI3 kinases are heterodimer-
Abbreviations: ATP, adenosine triphosphate; Boc, tert-butoxycarbonyl; CDI, 1,10-
carbonyldiimidazole; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
DNPH, O-(2,4-dinitrophenyl)hydroxylamine; EDTA, ethylenediaminetetraacetic
acid; ip, intraperitoneal; MEM, minimum essential medium; MSH, O-(mesitylsulfo-
nyl)hydroxylamine; PDB, protein data bank; PEG-400, polyethylene glycol with
molecular weight 380–420 g molꢀ1; PH domain, pleckstrin homology domain; PIK-
ic, consisting of a catalytic subunit (p110
complex with a regulatory subunit.1 PIK3CA, the gene encoding
for p110 , is often over-expressed and mutated in many cancer
a, p110b or p110d) in
a
75,
N0-((6-bromoimidazo[1,2-a]pyridin-3-yl)methylene)-N,2-dimethyl-5-nitro-
types. Two of the most common of these mutations (E545K and
H1047R) have been confirmed as activating mutations and hence
increase levels of PIP3. Mutations in p110b and p110d have not
yet been reported.3
benzenesulfonohydrazide; PIP2, phosphatidylinositol 4,5-diphosphate; PIP3, phos-
phatidylinositol 3,4,5-triphosphate; PI3 kinase, phosphoinositide-3-kinase; PKB,
protein kinase B; PTEN, phosphatase and tensin homologue; p-TsOH, p-toluene-
sulfonic acid monohydrate; q.d., daily; SAR, structure–activity relationship; THF,
tetrahydrofuran; TFA, trifluoroacetic acid; tlc, thin layer chromatography; Tris,
tris(hydroxymethyl)aminomethane.
Inhibitors of PI3 kinase, and in particular selective inhibitors of
⇑
p110
treatment. A recent report indicates that inhibiting p110
can block tumour progression4 however there are few reports of
p110
selective PI3 kinase inhibitors in the literature.4–8 Many
groups are now trying to exploit PI3 kinase inhibitors as an
approach to cancer therapy, as exemplified by the number of small
a
, could prove to be an important new strategy in cancer
Corresponding author.
E-mail address: j.kendall@auckland.ac.nz (J.D. Kendall).
Present address: Drug Design and Discovery Centre (D3C), University of Namur,
FUNDP, 61 rue de Bruxelles, 5000 Namur, Belgium.
a
alone
a
à
Present address: Centre for Cell Signalling, Barts Cancer Institute, Queen Mary
University of London, John Vane Science Centre, Charterhouse Square, London EC1M
6BQ, UK.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.029

70
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
molecule PI3 kinase inhibitors currently going through clinical
trials,3,9 however few of these are selective for p110
PIK-75 (Fig. 1) is an experimental compound that shows good
selectivity for p110 over the other class Ia PI3 kinases and has
Compounds 5t–x were made from methyl isonicotinate (8) by
N-amination with MSH and cyclisation as before (Scheme 3).
Diester 3t was converted to mono-carboxylic acid 7s by refluxing
in 40% aqueous H2SO4, and was then re-esterified to ester 7t with
HCl in MeOH. A Vilsmeier reaction followed by ester hydrolysis
afforded carboxylic acid 4s, and then further elaboration was car-
ried out by activation with thionyl chloride, followed by reaction
with amines to form carboxamides 4u–w. Alternatively, carboxylic
acid 7s was converted to carboxamide 7u by activation with
iBuOCOCl followed by reaction with ammonia gas. Vilsmeier
conditions then introduced the formyl group and concomitantly
dehydrated the carboxamide to form nitrile 4x. Condensation and
sulfonylation afforded sulfonohydrazides 5t–x.
Disubstituted compound 5z was made starting from 3-fluoro-
isonicotinaldehyde (9) by protecting the aldehyde as an acetal fol-
lowed by N-amination and cyclisation to 3y as before (Scheme 4).
The aldehyde was then unmasked and oxidised to carboxylic acid
3z. The ethyl ester was then decarboxylated with 40% aqueous
H2SO4, and the remaining carboxylic acid converted to primary
carboxamide 7z. Vilsmeier conditions then firstly dehydrated the
amide and then installed the required aldehyde group. Condensa-
tion and sulfonylation afforded sulfonohydrazide 5z.
a
.
a
shown activity in a human cancer xenograft model.6 However,
there has been minimal literature around the structure–activity
relationships (SARs) of PIK-75 beyond the original papers from
Hayakawa et al.5,6 Schmidt-Kittler et al.8 have published analogues
of PIK-75 with extra substituents on the benzene ring and showed
that two of these compounds inhibit the formation of tumour
metastases in mice, although they only had a minor growth delay
effect in an HCT-116 cancer xenograft model. We reported on
replacing the imidazo[1,2-a]pyridine ring with other heterocycles,
and found that a suitably positioned nitrogen available for hydro-
gen bonding was essential for activity.10 In this paper, we continue
with this work and investigate the SAR around the novel pyra-
zolo[1,5-a]pyridine ring system.
2. Results and discussion
2.1. Synthesis
Compounds 5aa–ac were made from 4-methoxypyridine (10) by
N-amination and cyclisation as before (Scheme 5). Decarboxylation
of ester 3aa with 40% aqueous H2SO4 also cleaved the methyl ether;
this was then reintroduced with iodomethane to afford ether 7aa. A
Vilsmeier reaction of pyrazolo[1,5-a]pyridines 7aa and 7ab intro-
duced the formyl groups, and then subsequent hydroxyl acetylation
of 4ab afforded ester 4ac. Aldehydes 4aa and 4ac did not react satis-
factorily under the usual methylhydrazine sulfate reaction condi-
tions. Reaction was slow for these aldehydes and decomposition
was observed. However, 4aa and 4ac condensed in good yield with
2-methyl-5-nitrobenzenesulfonohydrazide,14 and then subsequent
N-methylation without isolation using diazomethane afforded 5aa
and 5ac. Acetate 5ac was then cleaved to phenol 5ab with NaHCO3.
Sulfonohydrazides 13b–d and 14b–d were made by N-amination
of isonicotinonitrile (11) with MSH, followed by cyclisation with an
alkynyl ketone (Scheme 6). 3-Butyn-2-one gave methyl ketone 12b
in reasonable yield. We chose however, to make ethyl and propyl ke-
tones 12c and 12d using the trimethylsilyl alkynes due to ease of
handling of the reagents. The addition of potassium fluoride was
found to significantly improve the yields in these instances. Conden-
sation of 4x and 12b–d with 2-methyl-5-nitrobenzenesulfonohyd-
razide gave compounds 13a–d, and then N-methylation proceeded
smoothly with diazomethane to afford 14b–d.
The sulfonohydrazides were assembled (Scheme 1) by conver-
sion of an appropriately substituted pyridine (2) through to
pyrazolo[1,5-a]pyridines containing a 3-ester group (3b–j) by
initial N-amination with either O-(mesitylsulfonyl)hydroxylamine
(MSH)11 or O-(2,4-dinitrophenyl)hydroxylamine (DNPH),12 fol-
lowed by a 1,3-dipolar cycloaddition with ethyl propiolate.13
Although DNPH was more stable and easier to handle than MSH,
it was also less reactive and did not react well with pyridines
containing an electron-withdrawing group. MSH proved far
superior for these pyridines. In the case of 3-substituted pyridines
(3-bromopyridine and 3-methylpyridine) both possible isomeric
products were formed. These were easily separated by silica gel
chromatography.
The ester functionality was then converted to an aldehyde (4b–
j) by one of two methods. A reduction of the ester to the primary
alcohol followed by reoxidation with MnO2 worked in many cases,
but we found that a one-pot ester hydrolysis and decarboxylation
by refluxing in 40% aqueous H2SO4, followed by a Vilsmeier reac-
tion to install the aldehyde was more efficient. Finally, conversion
to sulfonohydrazides 5a–j was achieved by condensation with
methylhydrazine sulfate under weakly basic conditions followed
by sulfonylation without isolation of the intermediate N-
methylhydrazone.
Finally, compounds 13e and 14e were made starting from tert-
butyl pyridin-4-ylcarbamate (6) (Scheme 7). N-Amination and cyc-
lisation with 3-butyn-2-one gave ketone 12e, which on cleavage of
the tert-butoxycarbonyl group and diazotisation, gave bromo com-
pound 12f. As before, condensation gave compound 13e which was
N-methylated with diazomethane to afford 14e.
The sulfonohydrazides containing functional groups which
would not tolerate these reaction conditions were made by
alternative methods. Compounds 5m–r were made starting from
tert-butyl pyridin-4-ylcarbamate (6) (Scheme 2). N-Amination
and cyclisation to ester 3k was carried out as before. Removal of
the Boc protecting group with trifluoroacetic acid followed by
diazotisation introduced a 5-halo substituent. Decarboxylation
and Vilsmeier reaction afforded aldehydes 4m–o. Additionally,
bromo compound 4m underwent Stille reactions to make
cyclopropane 4p and alkene 4q, and a Sonogashira reaction to form
alkyne 4r. Finally, condensation and sulfonylation as before
afforded sulfonohydrazides 5m–r.
2.2. In vitro biology
In a previous paper, we described modifications to the hetero-
cyclic core of PIK-75.10 Subsequently, we found that the pyra-
zolo[1,5-a]pyridine ring system (5a) gave a similar potency to-
wards p110a in comparison with the corresponding imidazo[1,2-
a]pyridine (1) which did not contain the critical bromo substituent,
whilst retaining good selectivity over p110b and p110d (Table 1).
This encouraging result led us to the present investigation of the
SAR around this novel class of PI3 kinase inhibitors.
Firstly we looked at the effect of substituents around the 6-
membered ring of the pyrazolo[1,5-a]pyridine ring system. Of the
bromo isomers 5b, 5c and 5m, only the 5-bromo compound 5m
Figure 1. Structures of PIK-75 and 1.
showed any significant activity against p110a, with a 250-fold

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
71
Scheme 1. Reagents: (i) MSH, CH2Cl2 or DNPH, MeCN then ethyl propiolate, K2CO3, DMF; (ii) 40% H2SO4, reflux; (iii) POCl3, DMF; (iv) NaOH, H2O, EtOH; (v) CDI, THF then
NaBH4; (vi) MnO2, CH2Cl2; (vii) LiAlH4, THF; (viii) MeHNNH2.H2SO4, NaHCO3, MeOH then 2-methyl-5-nitrobenzenesulfonyl chloride.
Scheme 2. Reagents: (i) DNPH, CH2Cl2 then ethyl propiolate, K2CO3, DMF; (ii) TFA, CH2Cl2; (iii) NaNO2, H2O, HBr then CuBr, HBr; (iv) NaNO2, HCl, H2SO4, H2O then urea then
KI, H2O; (v) 40% H2SO4, reflux; (vi) NaNO2, CuCl, H2O, HCl; (vii) POCl3, DMF; (viii) RSnBu3, Pd(PPh3)4, PhMe, reflux; (ix) Me3SiCCH, (Ph3P)2PdCl2, CuI, NEt3, DMF then K2CO3,
MeOH; (x) MeHNNH2.H2SO4, NaHCO3, MeOH then 2-methyl-5-nitrobenzenesulfonyl chloride.
Scheme 3. Reagents: (i) MSH, CH2Cl2 then ethyl propiolate, K2CO3, DMF; (ii) 40% H2SO4, reflux; (iii) MeOH, HCl; (iv) iBuOCOCl, NEt3, CH2Cl2 then NH3(g); (v) POCl3, DMF; (vi)
NaOH, H2O, EtOH; (vii) SOCl2, reflux then amine, CH2Cl2; (viii) MeHNNH2.H2SO4, NaHCO3, MeOH then 2-methyl-5-nitrobenzenesulfonyl chloride.
Scheme 4. Reagents: (i) ethylene glycol, p-TsOH, benzene, Dean–Stark, reflux; (ii) DNPH, MeCN then ethyl propiolate, K2CO3, DMF; (iii) HClO4, dioxane, acetone; (iv) NaClO2,
H2O2, NaH2PO4, H2O, MeCN; (v) 40% H2SO4, reflux; (vi) CDI, CH2Cl2, then NH3, MeOH; (vii) POCl3, DMF; (viii) MeHNNH2.H2SO4, NaHCO3, MeOH then 2-methyl-5-
nitrobenzenesulfonyl chloride.
improvement in potency over 5a, and good selectivity over p110b
and p110d (60-fold and 40-fold, respectively). A similar pattern
was observed for the methyl isomers 5d–g, although 5g was less
active than 5m.
We further investigated 5-substitution with a range of large and
small, and electron-donating and electron-withdrawing substit-
uents. Ethyl 5h and phenyl 5j substituents gave a decrease in
still retained some p110
active at 2.4 nM for p110
potent than bromo compound 5m, whereas chloro compound 5o
had similar potency. The methyl ester 5t and primary carboxamide
5u retained weak activity, but adding further substitution to the
a
activity, and ethynyl 5r was particularly
. Iodo compound 5n was slightly less
a
amide (5v and 5w) further decreased the p110
5x was the most potent of the 5-substituents (IC50 0.9 nM for
p110 ) with 50-fold selectivity over both p110b and p110d. Of
a activity. Nitrile
p110a
activity, however trifluoromethyl 5i and vinyl 5q analogues
a

72
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
Scheme 5. Reagents: (i) DNPH, MeCN then ethyl propiolate, K2CO3, DMF; (ii) 40% H2SO4, reflux; (iii) MeI, K2CO3, DMF; (iv) POCl3, DMF; (v) Ac2O, NEt3, CH2Cl2; (vi) 2-methyl-
5-nitrobenzenesulfonohydrazide, MeOH then CH2N2, Et2O, THF; (vii) NaHCO3, H2O, MeOH.
Table 1
Inhibition of PI3 kinase isoforms and cell proliferation for PIK-75, 1 and compounds 5
a
a
Compd
R
IC50 (nM)
IC50
NZB5
(lM)
p110
a
p110b
p110d
NZOV9
Scheme 6. Reagents: (i) MSH, CH2Cl2, then 3-butyn-2-one, K2CO3, DMF; (ii) MSH,
CH2Cl2, then R1COC„CSiMe3, KF, K2CO3, DMF; (iii) 2-methyl-5-nitrobenzene-
sulfonohydrazide, MeOH; (iv) CH2N2, Et2O, THF.
PIK-75b
1b
5a
5b
5c
5m
5d
5e
5f
5g
5h
5i
5j
5n
5o
5p
5q
5r
5t
5u
5v
5w
5x
5z
5aa
5ab
5ac
7.8
860
960
>1000
>1000
3.8
1600
>1000
>1000
110
>1000
150
>1000
16
2.9
1100
130
2.4
360
530
>1000
>1000
0.9
45
190
340
910
0.069
0.35
0.47
>20
>20
0.31
6.0
0.066
0.68
0.66
4.5
6.5
0.05
1.2
>10000
>10000
>1000
>1000
230
>1000
>1000
>1000
>1000
>1000
>1000
>1000
370
5400
3400
>1000
>1000
150
>1000
>1000
>1000
700
>1000
>1000
>1000
170
H
6-Br
4-Br
5-Br
7-Me
6-Me
4-Me
5-Me
5-Et
5-CF3
5-Ph
5-I
10
18
8.0
>20
0.07
0.56
0.09
10
0.05
0.55
0.67
0.58
0.11
1.5
10
10
10
1.4
n.d.
0.52
>20
8.9
0.05
0.41
0.07
10
0.04
n.d.
0.74
0.99
0.16
1.8
0.60
10
10
0.06
n.d.
0.94
>20
8.2
Scheme 7. Reagents: (i) DNPH, MeCN then 3-butyn-2-one, K2CO3, DMF; (ii) TFA,
CH2Cl2; (iii) NaNO2, 48% HBr, H2O, CuBr; (iv) 2-methyl-5-nitrobenzenesulfonohyd-
razide, MeOH, reflux; (v) CH2N2, Et2O, THF.
5-Cl
390
90
5-cyclopropyl
5-CH@CH2
5-C„CH
5-CO2Me
5-CONH2
5-CONHMe
5-CONMe2
5-CN
>1000
>1000
30
>1000
>1000
>1000
>1000
46
>1000
>1000
41
690
the O-linked compounds 5aa–ac, the methoxy 5aa was the most
potent at 190 nM for p110a. Overall, it appears that small groups
are tolerated best at the 5-position, and we selected the 5-bromo
and 5-cyano substituents to carry out further SAR analysis on com-
pounds carrying modifications elsewhere on the molecule.
We also investigated substitution on the hydrazone linker (Ta-
ble 2). Removal of the R3 methyl group (13a) gave a 14-fold de-
>1000
>1000
>1000
49
2500
>1000
>1000
>1000
5-CN, 6-F
5-OMe
5-OH
1800
>1000
>1000
>1000
2600
620
5-OCOMe
crease in p110
a potency compared to 5x. In comparison, ketone-
a
derived hydrazones 13b–e where R3 was hydrogen were at least
10-fold more potent than their methylated counterparts 14b–e,
suggesting that the presence of substituents at both R2 and R3 were
causing a change in conformation and having a detrimental effect
on PI3 kinase activity. The size of the R2 substituent was also crit-
All IC50 values are the mean of duplicate or triplicate measurements.
Data from Ref. 10.
b
compounds bearing either no substituent at the 5-position of the
pyrazolo[1,5-a]pyridine ring (5a), or an alkyl substituent (5g–i
and 5p) demonstrated greater potency in the NZB5 cells than for
ical; the p110
a
potency decreased as R2 increased in size from
methyl to propyl (13b–d, 14b–d). Finally, 5-bromo 13e and 14e
were less active than their 5-cyano analogues 13b and 14b.
The compounds were also assayed for their effect on cell prolif-
eration in a thymidine depletion assay in two early passage cell
lines (Tables 1 and 2). The NZB5 cell line was derived from a brain
p110a. This may be driven by off-target activity, and additionally
the divergences in IC50 values for the two cell lines may also point
to the existence of off-target effects that reflect individual differ-
ences in the cell lines.
tumour (medulloblastoma) and has the wild-type gene for p110
a
.
2.3. Molecular modelling
The NZOV9 cell line was derived from an ovarian tumour (poorly
differentiated endometrioid adenocarcinoma), and contains
mutant p110
the kinase domain (Y1021C).
a
The PI3 kinase ATP binding site can be divided into three consti-
tutive sites important in ligand binding, the adenine binding
pocket (containing the hinge region backbone amide acceptor site
that interacts with the nucleotide), an affinity pocket (extending
from the adenine binding pocket), and a hydrophobic site on the
C-terminal lobe of the catalytic domain. To identify potential
interactions between the pyrazolo[1,5-a]pyridine based inhibitors
a
enzyme with a single amino acid substitution in
In general, the most active compounds in the p110
a
assay were
also effective inhibitors of cell proliferation, particularly for the
NZB5 cell line. Compounds 13a and 13b with no hydrazone N-sub-
stituents, were notable by their large drop in activity between
p110a
and NZB5, possibly due to a lack of cell penetration. The
and the p110a active site, docking studies were performed using

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
73
Table 2
Inhibition of PI3 kinase isoforms and cell proliferation for compounds 13a–e and
14b–e
R1
R2
R3
IC50 (nM)
IC50
NZB5
(lM)
a
a
Compd
p110
a
p110b
p110d
NZOV9
13a
13b
13c
13d
13e
14b
14c
14d
14e
CN
CN
CN
CN
Br
CN
CN
CN
Br
H
Me
Et
H
H
H
H
13
7
10
39
30
620
410
>1000
>1000
480
580
900
440
310
220
2.6
1.1
4.9
2.1
0.36
0.64
0.35
7.5
3.7
2.3
>20
0.088
0.37
0.42
4.4
0.41
0.68
>20
Pr
Me
Me
Et
Pr
Me
H
Me
Me
Me
Me
80
600
510
390
610
1200
>1000
>1000
>1000
>1000
>1000
>1000
a
All IC50 values are the mean of duplicate or triplicate measurements.
a model based on the apo p110
a X-ray crystal structure (PDB code
2RD0).15
Most binding modes predicted for compounds 5a, 5m, 5g and
5x buried the pyrazolo[1,5-a]pyridine core in the adenine binding
pocket, with a potential hydrogen bond interaction between the
pyrazolo N atom and the backbone amide NH group of Val851. This
is consistent with the characteristic hydrogen bond found in other
PI3 kinase inhibitor complexes.16 In this mode, the pyridyl ring of
5x with its substituent cyano group extends into the affinity pocket
where it forms a T-shape interaction with Tyr836 (Fig. 2a), an
interaction also predicted for the related imidazo[1,2-a]pyridine
core of PIK75.10,17–19 The occupation of this pocket by the 5 posi-
tion substituent is consistent with the substitution data reported
in Table 1 that suggest smaller groups at the 5 position are better
tolerated, and furthermore the cyano group may interact with the
side chain hydroxyl of Tyr836 improving affinity over the bromine
substituted compounds (13b vs 13e and 14b vs 14e).
Figure 2. (a) Predicted binding mode for 5m (cyan ball and stick) and 5x (magenta
stick) in the p110a ATP-binding site. Amino acids within 5 Å of 5m are illustrated,
and those likely to interact with the pyrazolo[1,5-a]pyridine core and sulfono-
hydrazide moiety are represented as yellow ball and stick with the interaction
illustrated by black dashed lines. (b) Possible interactions predicted for the aryl
nitro group of 5m and 5x. Compound 5m is represented as cyan ball and stick, with
alternative aryl nitro orientations including 5x represented as magenta sticks.
A range of possible interaction sites were predicted between the
rest of the inhibitor and the p110a active site. Where the substi-
tuted pyrazolo[1,5-a]pyridine core accessed the affinity pocket
these included a possible interaction between the sulfonyl moiety
Ligand atoms potentially involved in p110
a specific interaction are shown as balls.
and the side chain carboxamide NH2 group of the p110
a specific
p110 ATP-binding site residues within 5 Å of 5m are illustrated and those
a
residue Gln859 (Fig. 2a), while aryl nitro group interactions in-
cluded the Lys802 side chain donor group in the catalytic centre,
the backbone amide NH groups of Asn853 and Ser854 at the C-ter-
minal end of the hinge region, and the side chains of Ser774 or the
predicted to interact with the ligand are represented as yellow ball and stick with
the interactions illustrated by black dashed lines. (Images generated using PyMol.)
p110a
specific Arg770 on the N-terminal wall of the catalytic cleft.
2.4. Further in vitro screening of 5x
Other modes also contacted the side chains of Gln859, Thr856 and
Ser919 on the C-terminal wall of the active site. Those interactions
As the most active compound against PI3 kinase p110a that also
which identified regions with p110
were preferred. In these models, Gln859 is potentially involved in
ligand binding and the aryl nitro group may interact with the
a
specific sequence differences
showed good inhibition of cell proliferation, 5x was selected for
other investigation by screening against other kinases (Table 3).
Compound 5x showed similar potency against the two most
p110
tions illustrated in Figure 2b. Although Ser774 is found in other
PI3 kinase isoforms, the surrounding residues are variable.10
a
specific Arg770 or Ser774, with the alternative conforma-
common p110
type p110 , and 15-fold selectivity over the class Ib PI3 kinase
p110
a mutations (E545K and H1047R) to that of wild-
a
A
c.
binding site in this region is consistent with GDC0941 binding to
both p110c20 and p110d,16 as well as AS15 binding to p110d.16
The central hydrazone moiety transitions from the pyrazolo[1,5-
a]pyridine core buried at the base of the active site through to
the aryl nitro binding site higher on the N-terminal lobe wall,
and is adjacent to residues Thr856, Met922 and Gln859 on the C-
terminal lobe, and N-terminal lobe residues Trp780 and Met772.
Some flexibility around the sulfonohydrazide may be required to
for optimal binding through this region as indicated by the data
presented in Table 2.
PI3 kinase inhibitors also commonly inhibit the closely related
kinases DNA-PK (DNA-dependent protein kinase) and mTOR
(mammalian target of rapamycin).21 Compound 5x is a potent
inhibitor of DNA-PK but has 300-fold selectivity over mTOR
(Table 3). PIK-75 has activity against a range of other kinases
(<10% activity remaining at 10
l
M),4 and a representative seven
M (Table 3).
of these were chosen for screening at 1 and 10
l
Compound 5x has a similar level of potency to PIK-75 against five
of these kinases, but weaker activity for TrkA (neurotrophic tyro-
sine kinase receptor, type 1) and IR (insulin receptor).

74
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
Table 3
Inhibition of PI3 kinase p110
the poor solubility of 5x prevented all the drug from being absorbed
a
mutations, p110
c
, and other selected kinases by 5x
and may have limited its in vivo tolerance, indicating that further
investigation of the SAR around these compounds is required with
a view to designing more soluble analogues.
Kinase
% Activity remaining
1
lM
10
l
M
p110
p110
p110
a
a
c
E545K
H1047R
(0.7 nM)a
(0.5 nM)a
(14 nM)a
(1.1 nM)a
(300 nM)a
4. Experimental
4.1. Chemistry
DNA-PKb
mTORb
MKK1c
10
18
4
2
4
1
1
0
2
1
8
9
2
13
1
42
2
2
1
1
1
0
3
1
p38
c
MAPKc
NMR spectra were recorded on a Bruker Avance 400 spec-
trometer; chemical shifts are reported in d using SiMe4 as the inter-
nal standard when measured in CDCl3, and the residual DMSO as
internal standard when measured in d6-DMSO. Low resolution
mass spectra were recorded on a Thermo Finnigan MSQ single
quadrupole mass spectrometer. High resolution mass spectra were
obtained on a Bruker micrOTOF-QII mass spectrometer using elec-
trospray ionisation (ESI). Analyses were carried out in The Camp-
bell Microanalytical Laboratory, University of Otago, Dunedin,
New Zealand. Tested compounds were assessed as P95% purity,
as determined by combustion analysis, or the purity was measured
by HPLC conducted on an Agilent 1100 system using a reverse-
phase C8 column with diode array detection. Silica gel chroma-
tography was performed using 200–320 mesh silica gel obtained
from APS Finechem Ltd. Yields have not been optimised.
RSK1c
TrkAc
PKAc
IRc
19
1
55
5
MINK1c
a
Values in parentheses represent IC50’s.
Assays were performed by Invitrogen Drug Discovery Services (Madison, WI,
USA).
Assays were performed by The National Centre for Protein Kinase Profiling
(Dundee, UK).
b
c
Since benzoyl hydrazones have previously been reported as
irreversible inhibitors of Cathepsin S,22 5x was tested for its ability
to be washed off p110
in an in vitro assay using wortmannin1 as a
a
control for irreversible binding and LY2940021 as a control for
reversible ATP competitive binding. In this assay 70% of the inhib-
itory activity of 5x at a concentration of 100 nM was washed off.
Compound 5x was assayed for influence on cell signalling by
measuring its effect on the phosphorylation of PKB, a downstream
marker of PI3 kinase activity. This was carried out in HCT-116 cells
4.1.1. Synthesis of ethyl pyrazolo[1,5-a]pyridine-3-carboxylates
3
These were made using MSH or DNPH as detailed below, unless
otherwise stated. A fresh solution of MSH23 in CH2Cl2 (1 equiv) was
added to the substituted pyridine (1 equiv) in CH2Cl2 (10 mL) at
0 °C. After 2 h, the solvent was removed in vacuo. Alternatively, a
solution of DNPH12 (1 equiv) and the substituted pyridine (1 equiv)
in MeCN (40 mL) was heated at 40 °C for 18 h. The solvent was re-
moved in vacuo. The method continues by taking the residue from
either method in dry DMF (8 mL), and then ethyl propiolate
(1 equiv) and K2CO3 (2 equiv) were added, and the suspension stir-
red at room temperature for 18 h. The reaction mixture was diluted
with water and extracted twice with EtOAc. The combined organic
phases were washed three times with water then with brine, dried
(Na2SO4) and the solvent removed in vacuo. Chromatography
(eluting with a hexanes: EtOAc gradient, unless otherwise stated)
gave the pyrazolo[1,5-a]pyridine.
which contain the p110a mutation H1047R (Fig. 3). Compound 5x
inhibited the phosphorylation at both Ser473 and Thr308 with
IC50s of 23 and 26 nM, respectively.
2.5. In vivo antitumour efficacy
Compound 5x was then evaluated in an HCT-116 human xeno-
graft model in female Rag1ꢀ/ꢀ mice. The control vehicle, PIK-75
and 5x were dosed ip at MTD (20 mg/kg) daily for 14 days (Fig. 4).
Compound 5x reduced tumour growth by 39.1 4.3% (P <0.005)
compared to controls by the completion of the study. PIK-75 also re-
duced tumour growth compared to controls, but this difference
(23.2 3.7%) did not reach statistical significance. Both drugs were
similarly tolerated throughout the duration of the study. After an
initial reduction following the first dose, animal bodyweight was
stable until day 10, then steadily declined so that significant body-
weight loss (15–20% loss of initial weight) was observed in 3/7 mice
treated with 5x and 1/7 mice with PIK-75. Although the toxicity of 5x
4.1.1.1. Ethyl 6-bromopyrazolo[1,5-a]pyridine-3-carboxylate
(3b) and ethyl 4-bromopyrazolo[1,5-a]pyridine-3-carboxylate
(3c).
Reaction of 3-bromopyridine (843 mg, 5.34 mmol) with
MSH, after chromatography (eluting with hexanes: EtOAc 98:2 to
97:3 to 19:1 to 9:1) gave firstly 3b as a pale brown solid
(255 mg, 29%). 1H NMR d (400 MHz, CDCl3) 8.67 (dd, J = 1.6,
0.8 Hz, 1H), 8.36 (s, 1H), 8.07 (dd, J = 9.4, 0.8 Hz, 1H), 7.47 (dd,
J = 9.4, 1.6 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H).
LC–MS (APCI+) 269 (MH+ with 79Br, 100%), 271 (MH+ with 81Br,
80%). Followed by 3c as a pale yellow solid (255 mg, 29%). 1H
NMR d (400 MHz, CDCl3) 8.52 (dd, J = 6.9, 1.0 Hz, 1H), 8.43 (s,
1H), 7.64 (dd, J = 7.4, 1.0 Hz, 1H), 6.80 (t, J = 7.2 Hz, 1H), 4.38 (q,
J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 269 (MH+
with 79Br, 80%), 271 (MH+ with 81Br, 100%).
may have resulted from its improved potency against p110
a or
through off-target activity against other protein or lipid kinases,
the late decline in bodyweight loss and presence of drug precipitate
in the peritoneal cavity of all treated mice on post-mortem examina-
tion suggests that poor solubility may at least be partially responsi-
ble for drug toxicity. Therefore, the design of soluble analogues of 5x
may help improve drug tolerance to provide a substantial improve-
ment in therapeutic index over PIK-75.
3. Conclusions
4.1.1.2. Ethyl 7-methylpyrazolo[1,5-a]pyridine-3-carboxylate
We have found that the pyrazolo[1,5-a]pyridines are a new series
(3d).
Reaction of 2-methylpyridine (251 mg, 2.7 mmol) with
of p110
around the ring system. Compound 5x showed good selectivity for
PI3 kinase p110 over p110b and p110d, substantial inhibition of
cell proliferation, and inhibition of phosphorylation of PKB, a down-
stream marker of PI3 kinase activity. It also showed modest tumour
growth inhibition in an HCT-116 human xenograft study. However,
a selective PI3 kinase inhibitors and have explored the SAR
MSH, after chromatography (eluting with hexanes: EtOAc 97:3 to
19:1) gave 3d as a yellow solid (205 mg, 37%). 1H NMR d
(400 MHz, CDCl3) 8.44 (s, 1H), 8.08 (d, J = 8.9 Hz, 1H), 7.35 (dd,
J = 8.9, 7.0 Hz, 1H), 6.81 (d, J = 7.0 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H),
2.80 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 205 (MH+, 100%).
a

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
75
pPKB
Total PKB
pPKB (Ser 473)
pPKB (Thr 308)
Figure 3. The effect of 5x on the phosphorylation of PKB in HCT-116 cells at Ser473 (IC50 23 nM) and Thr308 (IC50 26 nM).
Figure 4. Average tumour volume (A) and bodyweight change (B) in Rag1ꢀ/ꢀ mice with HCT-116 tumours treated with 5x, PIK-75 and the control vehicle (n = 7 per group). All
treatments were administered by ip injection daily for 14 days.
4.1.1.3. Ethyl 6-methylpyrazolo[1,5-a]pyridine-3-carboxylate
(3e) and ethyl 4-methylpyrazolo[1,5-a]pyridine-3-carboxylate
(441 mg, 3.00 mmol) with MSH, after chromatography (eluting
with hexanes: EtOAc 19:1) gave 3i as an off-white solid (276 mg,
36%). 1H NMR d (400 MHz, CDCl3) 8.62 (d, J = 7.3 Hz, 1H), 8.47–
8.50 (m, 2H), 7.11 (dd, J = 7.3, 2.0 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H),
1.43 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 259 (MH+, 100%).
(3f).
Reaction of 3-methylpyridine (497 mg, 5.34 mmol) with
MSH, after chromatography (eluting with hexanes: EtOAc 98:2 to
97:3 to 19:1 to 9:1) gave firstly 3f as a pale yellow solid
(152 mg, 21%). 1H NMR d (400 MHz, CDCl3) 8.41 (s, 1H), 8.38 (d,
J = 6.9 Hz, 1H), 7.12 (d, J = 6.9 Hz, 1H), 6.83 (t, J = 6.9 Hz, 1H), 4.34
(q, J = 7.1 Hz, 2H), 2.84 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). LC–MS
(APCI+) 205 (MH+, 100%). Followed by 3e as a yellow solid
(160 mg, 23%). 1H NMR d (400 MHz, CDCl3) 8.33 (s, 1H), 8.32 (dd,
J = 2.3, 1.1 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 7.26 (dd, J = 9.0, 1.5 Hz,
1H), 4.37 (q, J = 7.1 Hz, 2H), 2.39 (s, 1H), 1.41 (t, J = 7.1 Hz, 3H).
LC–MS (APCI+) 205 (MH+, 100%).
4.1.1.7. Ethyl 5-phenylpyrazolo[1,5-a]pyridine-3-carboxylate
(3j).
Reaction of 4-phenylpyridine (388 mg, 2.50 mmol) with
MSH, after chromatography (eluting with hexanes: EtOAc 97:3 to
19:1 to 9:1) gave 3j as a yellow solid (192 mg, 29%). 1H NMR d
(400 MHz, CDCl3) 8.56 (dd, J = 7.2, 0.8 Hz, 1H), 8.41 (s, 1H), 8.37
(dd, J = 2.0, 0.8 Hz, 1H), 7.73–7.68 (m, 2H), 7.54–7.48 (m, 2H),
7.47–7.42 (m, 1H), 7.22 (dd, J = 7.2, 2.0 Hz, 1H), 4.41 (q, J = 7.1 Hz,
2H), 1.43 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 267 (MH+, 100%).
4.1.1.4. Ethyl 5-methylpyrazolo[1,5-a]pyridine-3-carboxylate
(3g).
Reaction of 4-methylpyridine (248 mg, 2.66 mmol) with
4.1.1.8. Ethyl 5-(tert-butoxycarbonylamino)pyrazolo[1,5-a]pyr-
MSH, after chromatography (eluting with hexanes: EtOAc 97:3 to
95:5 to 9:1) gave 3g as a pale brown solid (175 mg, 53%). 1H
NMR d (400 MHz, CDCl3) 8.39 (d, J = 7.1 Hz, 1H), 8.34 (s, 1H), 7.93
(s, 1H), 6.76 (dd, J = 7.1, 1.9 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 2.47
(s, 3H), 1.41 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 205 (MH+, 100%).
idine-3-carboxylate (3k).
Reaction of tert-butyl pyridin-4-
ylcarbamate (6) (1.77 g, 8.89 mmol) with DNPH, after chroma-
tography (eluting with CH2Cl2: MeOH 99.5:0.5) gave 3k as a yellow
solid (1.25 g, 46%). 1H NMR d (400 MHz, CDCl3) 8.38 (d, J = 7.6 Hz,
1H), 8.32 (s, 1H), 7.95 (d, J = 1.9 Hz, 1H), 7.28 (m, 1H), 6.71 (s,
1H), 4.37 (q, J = 7.1 Hz, 2H), 1.55 (s, 9H), 1.41 (t, J = 7.1 Hz, 3H).
LC–MS (APCI+) 306 (MH+, 100%).
4.1.1.5. Ethyl 5-ethylpyrazolo[1,5-a]pyridine-3-carboxylate
(3h).
Reaction of 4-ethylpyridine (215 mg, 2.01 mmol) with
DNPH, after chromatography (eluting with hexanes: EtOAc 19:1
to 9:1) gave 3h as a pale yellow solid (310 mg, 71%). 1H NMR d
(400 MHz, CDCl3) 8.41 (d, J = 7.1 Hz, 1H), 8.34 (s, 1H), 7.95 (m,
1H), 6.80 (dd, J = 7.1, 1.9 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 2.76 (q,
J = 7.6 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H), 1.32 (t, J = 7.6 Hz, 3H). LC–
MS (APCI+) 219 (MH+, 100%).
4.1.1.9. Ethyl 5-aminopyrazolo[1,5-a]pyridine-3-carboxylate tri-
fluoroacetate (3l).
A solution of 3k (1.25 g, 4.10 mmol) and
trifluoroacetic acid (3.2 mL, 41.5 mmol) in CH2Cl2 (50 mL) was
stirred at room temperature for 18 h. The solvents were removed
in vacuo to leave 3l as a pale brown solid (1.72 g, 97%). 1H NMR
d (400 MHz, CDCl3) 8.37 (dd, J = 7.4, 0.6 Hz, 1H), 8.25 (s, 1H), 7.22
(dd, J = 2.6, 0.6 Hz, 1H), 6.40 (dd, J = 7.4, 2.6 Hz, 1H), 4.35 (q,
J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 206 (MH+,
100%).
4.1.1.6. Ethyl 5-(trifluoromethyl)pyrazolo[1,5-a]pyridine-3-car-
boxylate (3i).
Reaction of 4-(trifluoromethyl)pyridine

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J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
4.1.1.10. Ethyl 5-bromopyrazolo[1,5-a]pyridine-3-carboxylate
(3m). A solution of NaNO2 (411 mg, 5.96 mmol) in water
(MgSO4) and the solvent removed in vacuo. The solid residue
was taken up in CH3CN (70 mL) and 0.16 M NaH2PO4 (10 mL), then
cooled to 0 °C and 30% v/v H2O2 added in a single portion. A solu-
tion of NaClO2 (750 mg, 8.29 mmol) in water (5 mL) was added
dropwise over 5 min and the reaction was stirred at room temper-
ature for 3 h. The reaction was quenched with saturated aqueous
Na2S2O3, and then the CH3CN removed in vacuo. The remaining
aqueous layer was diluted with 0.5 M NaOH and extracted with
CH2Cl2. The aqueous layer was acidified to pH 4 with 1 M HCl
and extracted with CH2Cl2. This last extract was dried (MgSO4)
and the solvent removed in vacuo to afford 3z as a colourless white
solid (320 mg, 77%). 1H NMR d (400 MHz, CDCl3) 8.89 (d, J = 7.2 Hz,
1H), 8.57 (d, J = 5.2 Hz, 1H), 8.48 (s, 1H), 4.43 (q, J = 7.1 Hz, 2H),
1.44 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 253 (MH+, 100%).
(6 mL) was added dropwise to a solution of 3l (1.72 g, 3.97 mmol)
in concentrated HBr (4 mL) at 0 °C over 5 min. After 10 min, a solu-
tion of CuBr (1.14 g, 7.95 mmol) in concentrated HBr (4 mL) was
added, then the reaction mixture heated to 50 °C for 15 min until
gas evolution ceased. Then the reaction mixture was basified to
pH 5 with 6 M NaOH and extracted twice with CH2Cl2. The com-
bined extracts were dried (Na2SO4) and the solvent removed in
vacuo. Chromatography (eluting with hexanes: EtOAc 19:1 to
9:1) gave 3m as a pale yellow solid (695 mg, 65%). 1H NMR d
(400 MHz, CDCl3) 8.53–8.20 (m, 3H), 7.03 (dd, J = 7.3, 2.0 Hz, 1H),
4.39 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 269
(MH+ with 79Br, 100%), 271 (MH+ with 81Br, 90%).
4.1.1.11. Ethyl 5-iodopyrazolo[1,5-a]pyridine-3-carboxylate
4.1.1.15. Ethyl 5-methoxypyrazolo[1,5-a]pyridine-3-carboxylate
(3n).
NaNO2 (36 mg, 0.52 mmol) was added to a solution of
(3aa).
Reaction of 4-methoxypyridine (10) (244 mg,
3l (150 mg, 0.40 mmol) in concentrated HCl (3 mL), H2SO4 (1 mL)
and water (3 mL) at 0 °C. After 1 h, urea (2.4 mg, 0.04 mmol) was
added, then after a further 15 min, a solution of KI (132 mg,
0.80 mmol) in water (3 mL) was added. After 1 h at room temper-
ature, the reaction mixture was basified to pH 3 with 1 M NaOH
and extracted twice with CH2Cl2. The combined extracts were
dried (Na2SO4) and the solvent removed in vacuo. Chromatography
(eluting with hexanes: EtOAc 5:1) gave 3n as a pale yellow solid
(57 mg, 45%). 1H NMR d (400 MHz, CDCl3) 8.59 (dd, J = 1.9,
0.7 Hz, 1H), 8.33 (s, 1H), 8.23 (dd, J = 7.2, 0.7 Hz, 1H), 7.18 (dd,
J = 7.2, 1.9 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H).
LC–MS (APCI+) 317 (MH+, 100%).
2.24 mmol) with DNPH, after chromatography (eluting with
hexanes: EtOAc 9:1 to 4:1) gave 3aa as a yellow solid (114 mg,
23%). 1H NMR d (400 MHz, CDCl3) 8.31 (d, J = 7.5 Hz, 1H), 8.28 (s,
1H), 7.43 (d, J = 2.8 Hz, 1H), 6.61 (dd, J = 7.5, 2.8 Hz, 1H), 4.37 (q,
J = 7.1 Hz, 2H), 3.93 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). LC–MS (APCI+)
221 (MH+, 100%).
4.1.2. Synthesis of pyrazolo[1,5-a]pyridine-3-carbaldehydes 4
These were made by decarboxylation/Vilsmeier or hydrolysis/
reduction/reoxidation as detailed below, unless otherwise stated.
Decarboxylation was carried out by refluxing a solution of the
ester (1 equiv) in 40% aqueous H2SO4 (3 mL) for 18 h. The solution
was then cooled in ice and neutralised to pH 7 with 6 M NaOH,
then extracted twice with CH2Cl2. The combined extracts were
dried (Na2SO4) and the solvent removed in vacuo to leave the
decarboxylated material. The pyrazolo[1,5-a]pyridine was then
reacted under Vilsmeier conditions in dry DMF (2 mL) with POCl3
(3 equiv) at 0 °C under an atmosphere of N2. The reaction mixture
was then warmed to room temperature and stirred for 2 h. The
solution was poured onto ice, basified to pH 10 with 1 M NaOH,
stirred for 1 h then extracted twice with CH2Cl2. The combined
extracts were washed twice with water, dried (Na2SO4) and the
solvent removed in vacuo to leave the aldehyde.
4.1.1.12. 3-Ethyl 5-methyl pyrazolo[1,5-a]pyridine-3,5-dicar-
boxylate (3t).
Reaction of methyl isonicotinate (8) (2.45 g,
17.9 mmol) with MSH, after chromatography (eluting with
hexanes: EtOAc 19:1 to 9:1) gave 3t as a pale brown solid
(1.88 g, 42%). 1H NMR d (400 MHz, CDCl3) 8.86 (dd, J = 1.8, 0.9 Hz,
1H), 8.55 (dd, J = 7.2, 0.9 Hz, 1H), 8.47 (s, 1H), 7.52 (dd, J = 7.2,
1.8 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 3.99 (s, 3H), 1.44 (t, J = 7.1 Hz,
3H). LC–MS (APCI+) 249 (MH+, 100%).
4.1.1.13. Ethyl 5-(1,3-dioxolan-2-yl)-6-fluoropyrazolo[1,5-a]pyr-
idine-3-carboxylate (3y).
To a solution of 3-fluoroisonicotin-
Alternatively, the ester was hydrolysed by refluxing a solution of
the ester (1 equiv) in 1 M NaOH (3 equiv) and EtOH (5 mL) for 6 h.
The EtOH was removed in vacuo, and then the aqueous residue acid-
ified to pH 1 with 1 M HCl. The precipitated carboxylic acid was fil-
tered off, washed with water and dried. The carboxylic acid was
reduced by adding CDI (1.5 equiv) to a suspension of carboxylic acid
(1 equiv) in dry THF (10 mL) under an atmosphere of N2. After stir-
ring for 18 h, the resulting solution was added dropwise to a solution
of NaBH4 (5 equiv) in H2O (10 mL) and stirred for 30 min. The reac-
tion was then quenched by the addition of 1 M HCl and stirred for a
further 30 min. The solution was neutralised with saturated aque-
ous NaHCO3 and extracted twice with CH2Cl2. The combined organic
layers were dried (Na2SO4) and the solvent removed in vacuo. Chro-
matography (eluting with a hexanes: EtOAc gradient) gave the alco-
hol. Reoxidation was carried out by stirring a suspension of the
pyrazolo[1,5-a]pyridine-3-methanol (1 equiv) and MnO2 (10 equiv)
in CH2Cl2 (2 mL) at room temperature for 4 days. The reaction mix-
ture was then filtered through celite, washed with CH2Cl2, and the
solvent removed from the filtrate in vacuo to leave the aldehyde.
aldehyde (9) (9.83 g, 78.5 mmol) and ethylene glycol (9.74 g,
157 mmol) in benzene (100 mL) was added p-TsOHꢁH2O (14.2 g,
82.5 mmol) and the mixture was refluxed under a Dean–Stark
apparatus. After 12 h, the reaction was cooled and quenched with
20% w/w NaOH. The benzene layer was removed and the remain-
ing aqueous layer was extracted with EtOAc. The combined organic
fractions were washed with brine, dried (MgSO4) and the solvents
removed in vacuo. Chromatography (eluting with hexanes: EtOAc
5:1) gave 4-(1,3-dioxolan-2-yl)-3-fluoropyridine as a colourless
oil (11.4 g, 86%). 1H NMR d (400 MHz, CDCl3) 8.48 (d, J = 1.8 Hz,
1H), 8.45 (dd, J = 4.8, 0.8 Hz, 1H), 7.45 (dd, J = 6.0, 4.8 Hz, 1H),
6.09 (s, 1H), 4.16–4.04 (m, 4H). LC–MS (APCI+) 170 (MH+, 100%).
Reaction of the above pyridine (6.87 g, 40.7 mmol) with DNPH,
after chromatography (eluting with CH2Cl2: MeOH 98:2) gave a
mixture of pyridine starting material and isomerically pure prod-
uct (3y) (462 mg, 4.7%). 1H NMR d (400 MHz, CDCl3) 8.47 (dd,
J = 4.8, 0.4 Hz, 1H), 8.40 (s, 1H), 8.31 (d, J = 7.1 Hz, 1H), 6.14 (s,
1H), 4.39 (q, J = 7.1 Hz, 2H), 4.20–4.08 (m, 4H), 1.42 (t, J = 7.1 Hz,
3H). LC–MS (APCI+) 281 (MH+, 100%).
4.1.2.1.
6-Bromopyrazolo[1,5-a]pyridine-3-carbaldehyde
(4b).
4.1.1.14. 3-(Ethoxycarbonyl)-6-fluoropyrazolo[1,5-a]pyridine-5-
Decarboxylation of ester 3b (253 mg, 0.94 mmol) gave 6-bromo-
pyrazolo[1,5-a]pyridine as a pale yellow oil (93 mg, 50%). 1H NMR d
(400 MHz, CDCl3) 8.63 (m, 1H), 7.92 (d, J = 2.3 Hz, 1H), 7.44 (d,
J = 9.4 Hz, 1H), 7.17 (dd, J = 9.4, 1.6 Hz, 1H), 6.54 (d, J = 2.3 Hz, 1H).
LC–MS (APCI+) 196 (MH+ with 79Br, 90%), 198 (MH+ with 81Br, 100%).
carboxylic acid (3z).
70% w/w Perchloric acid (30 mL) was
added to a stirred solution of 3y (400 mg, 1.42 mmol) in dioxane
(30 mL) and acetone (2 mL), and stirred for 3 h. The reaction mix-
ture was then extracted with EtOAc, washed with brine, dried

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
77
Vilsmeier reaction of the above compound (92 mg, 0.47 mmol) gave 4b
as a pale pink solid (87 mg, 83%). 1H NMR d (400 MHz, CDCl3) 10.05 (s,
1H), 8.73 (dd, J = 1.6, 0.8 Hz, 1H), 8.36 (s, 1H), 8.21 (dd, J = 9.3, 0.8 Hz,
1H), 7.60 (dd, J = 9.3, 1.6 Hz, 1H). LC–MS (APCI+) 224 (MH+ with 79Br,
80%), 226 (MH+ with 81Br, 100%).
1H), 8.45 (d, J = 6.9 Hz, 1H), 7.27 (m, 1H), 6.94 (t, J = 6.9 Hz, 1H),
2.79 (s, 3H). LC–MS (APCI+) 161 (MH+, 100%).
4.1.2.6. 5-Methylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4g).
Hydrolysis of ester 3g (173 mg, 0.85 mmol) gave 5-methylpyra-
zolo[1,5-a]pyridine-3-carboxylic acid as a white solid (136 mg,
91%). 1H NMR d (400 MHz, d6-DMSO) 12.3 (br s, 1H), 8.70 (d,
J = 7.0 Hz, 1H), 8.30 (s, 1H), 7.86 (s, 1H), 6.96 (dd, J = 7.0, 1.7 Hz,
1H), 2.44 (s, 3H). Reduction of the above acid (134 mg, 0.76 mmol),
after chromatography (eluting with hexanes: EtOAc 2:1 to 1:1 to
EtOAc) gave (5-methylpyrazolo[1,5-a]pyridin-3-yl)methanol as a
colourless oil (18 mg, 15%). 1H NMR d (400 MHz, CDCl3) 8.33 (d,
J = 7.1 Hz, 1H), 7.89 (s, 1H), 7.38 (s, 1H), 6.60 (dd, J = 7.1, 1.8 Hz,
1H), 4.83 (d, J = 5.4 Hz, 2H), 2.36 (s, 3H), 1.41 (t, J = 5.4 Hz, 1H).
LC–MS (APCI+) 163 (MH+, 100%). Reoxidation of the above alcohol
(18 mg, 0.11 mmol) gave 4g as a white solid (14 mg, 78%). 1H
NMR d (400 MHz, CDCl3) 10.00 (s, 1H), 8.44 (d, J = 7.0 Hz, 1H),
8.32 (s, 1H), 8.09 (s, 1H), 6.89 (dd, J = 7.0, 1.7 Hz, 1H), 2.50 (s,
3H). LC–MS (APCI+) 161 (MH+, 100%).
4.1.2.2. 4-Bromopyrazolo[1,5-a]pyridine-3-carbaldehyde (4c).
Hydrolysis of ester 3c (253 mg, 0.94 mmol) gave 4-bromopyra-
zolo[1,5-a]pyridine-3-carboxylic acid as a white solid (212 mg,
93%). 1H NMR d (400 MHz, d6-DMSO) 12.50 (br s, 1H), 8.88 (d,
J = 6.9 Hz, 1H), 8.44 (s, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.02 (t, J = 7.2 Hz,
1H). Reduction of the above acid (210 mg, 0.87 mmol) after chroma-
tography (eluting with hexanes: EtOAc 3:1 to 2:1 to 1:1), gave (4-
bromopyrazolo[1,5-a]pyridin-3-yl)methanol as
a yellow solid
(165 mg, 83%). 1H NMR d (400 MHz, CDCl3) 8.44 (d, J = 7.0 Hz, 1H),
8.02 (s, 1H), 7.38 (d, J = 7.2 Hz, 1H), 6.65 (t, J = 7.2 Hz, 1H), 5.03 (d,
J = 6.2 Hz, 2H), 1.77 (t, J = 6.2 Hz, 1H). LC–MS (APCI+) 226 (MH+ with
79Br, 80%), 228 (MH+ with 81Br, 100%). Reoxidation of the above alco-
hol (165 mg, 0.73 mmol) gave 4c as a white solid (146 mg, 89%). 1H
NMR d (400 MHz, CDCl3) 10.77 (s, 1H), 8.61–8.58 (m, 2H), 7.74 (dd,
J = 7.5, 0.8 Hz, 1H), 6.89 (t, J = 7.3 Hz, 1H). LC–MS (APCI+) 224 (MH+
with 79Br, 100%), 226 (MH+ with 81Br, 80%).
4.1.2.7. 5-Ethylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4h).
Decarboxylation of ester 3h (310 mg, 1.42 mmol) gave 5-ethyl-
pyrazolo[1,5-a]pyridine as a pale brown oil (171 mg, 82%). 1H NMR
d (400 MHz, CDCl3) 8.37 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 2.1 Hz, 1H),
7.29 (m, 1H), 6.60 (dd, J = 7.2, 1.7 Hz, 1H), 6.39 (d, J = 2.1 Hz, 1H),
2.67 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). LC–MS (APCI+) 147
(MH+, 100%). Vilsmeier reaction of the above compound (170 mg,
1.16 mmol) gave 4h as a brown oil (203 mg, 100%). 1H NMR d
(400 MHz, CDCl3) 10.01 (s, 1H), 8.46 (d, J = 7.1 Hz, 1H), 8.33 (s, 1H),
8.10 (m, 1H), 6.93 (dd, J = 7.1, 1.9 Hz, 1H), 2.79 (q, J = 7.5 Hz, 2H),
1.33 (t, J = 7.5 Hz, 3H). LC–MS (APCI+) 175 (MH+, 100%).
4.1.2.3. 7-Methylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4d).
Decarboxylation of ester 3d (97 mg, 0.55 mmol) gave 7-methyl-
pyrazolo[1,5-a]pyridine as a pale brown oil (62 mg, 85%). 1H
NMR
d (400 MHz, CDCl3) 8.00 (d, J = 2.2 Hz, 1H), 7.47 (d,
J = 8.8 Hz, 1H), 7.06 (dd, J = 8.8, 6.8 Hz, 1H), 6.62 (d, J = 6.8 Hz,
1H), 6.56 (d, J = 2.2 Hz, 1H), 2.76 (s, 3H). LC–MS (APCI+) 133
(MH+, 100%). Vilsmeier reaction of the above compound (62 mg,
0.47 mmol) gave 4d as a white solid (48 mg, 64%). 1H NMR d
(400 MHz, CDCl3) 10.06 (s, 1H), 8.42 (s, 1H), 8.21 (d, J = 8.7 Hz,
1H), 7.47 (dd, J = 8.7, 7.0 Hz, 1H), 6.93 (d, J = 7.0 Hz, 1H), 2.83 (s,
3H). LC–MS (APCI+) 161 (MH+, 100%).
4.1.2.8. 5-(Trifluoromethyl)pyrazolo[1,5-a]pyridine-3-carbalde-
hyde (4i).
LiAlH4 (4.3 mL, 1.0 mol Lꢀ1 in THF, 4.3 mmol) was
added to a solution of ester 3i (276 mg, 1.07 mmol) in dry THF
(10 mL) at 0 °C under an atmosphere of N2. The reaction mixture
was then warmed to room temperature and stirred for 1 h. The
reaction was quenched by the dropwise addition of water, then fil-
tered through a plug of celite and washed with CH2Cl2. The solvent
was removed from the filtrate in vacuo. Chromatography (eluting
with hexanes: EtOAc 2:1 to 1:1) gave (5-(trifluoromethyl)pyra-
zolo[1,5-a]pyridin-3-yl)methanol as a white solid (28 mg, 12%).
1H NMR d (400 MHz, CDCl3) 8.54 (d, J = 7.3 Hz, 1H), 8.05 (s, 1H),
8.00 (d, J = 1.9 Hz, 1H), 6.94 (dd, J = 7.3, 1.9 Hz, 1H), 4.93 (d,
J = 4.2 Hz, 2H), 3.48 (br s, 1H). LC–MS (APCI+) 217 (MH+, 100%).
Reoxidation of the above alcohol (28 mg, 0.13 mmol) gave 4i as
an off-white solid (25 mg, 89%). 1H NMR d (400 MHz, CDCl3)
10.12 (s, 1H), 8.69 (d, J = 7.2 Hz, 1H), 8.63 (m, 1H), 8.50 (s, 1H),
7.24 (dd, J = 7.2, 2.0 Hz, 1H). LC–MS (APCI+) 215 (MH+, 100%).
4.1.2.4. 6-Methylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4e).
Hydrolysis of ester 3e (158 mg, 0.77 mmol) gave 6-methylpyra-
zolo[1,5-a]pyridine-3-carboxylic acid as
a pale yellow solid
(109 mg, 80%). 1H NMR d (400 MHz, d6-DMSO) 12.30 (br s, 1H),
8.68 (s, 1H), 8.30 (m, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.42 (dd, J = 9.0,
0.9 Hz, 1H), 2.35 (s, 3H). Reduction of the above acid (107 mg,
0.61 mmol), after chromatography (eluting with hexanes: EtOAc
2:1 to 1:1 to EtOAc) gave (6-methylpyrazolo[1,5-a]pyridin-3-
yl)methanol as a colourless oil (55 mg, 56%). 1H NMR d (400 MHz,
CDCl3) 8.25 (m, 1H), 7.87 (s, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.01 (dd,
J = 9.0, 1.4 Hz, 1H), 4.84 (s, 2H), 2.34 (s, 3H), 1.43 (br s, 1H). LC–MS
(APCI+) 163 (MH+, 100%). Reoxidation of the above alcohol (55 mg,
0.34 mmol) gave 4e as a white solid (48 mg, 89%). 1H NMR d
(400 MHz, CDCl3) 10.01 (s, 1H), 8.38 (m, 1H), 8.32 (s, 1H), 8.19 (d,
J = 8.9 Hz, 1H), 7.39 (dd, J = 8.9, 1.4 Hz, 1H), 2.43 (s, 3H). LC–MS
(APCI+) 161 (MH+, 100%).
4.1.2.9. 5-Phenylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4j).
Decarboxylation of ester 3j (192 mg, 0.72 mmol) gave 5-phenyl-
pyrazolo[1,5-a]pyridine as a pale brown oil (80 mg, 57%). 1H NMR
d (400 MHz, CDCl3) 8.52 (d, J = 7.3 Hz, 1H), 7.97 (d, J = 2.2 Hz, 1H),
7.72 (dd, J = 2.0, 0.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.51–7.45 (m, 2H),
7.42–7.37 (m, 1H), 7.03 (dd, J = 7.3, 2.0 Hz, 1H), 6.56 (dd, J = 2.2,
0.7 Hz, 1H). LC–MS (APCI+) 195 (MH+, 100%). Vilsmeier reaction of
the above compound (80 mg, 0.41 mmol) gave 4j as a pale brown
oil (91 mg, 99%). 1H NMR d (400 MHz, CDCl3) 10.07 (s, 1H), 8.61
(dd, J = 7.2, 0.8 Hz, 1H), 8.51 (dd, J = 2.0, 0.8 Hz, 1H), 8.39 (s, 1H),
7.75–7.70 (m, 2H), 7.55–7.43 (m, 3H), 7.34 (dd, J = 7.2, 2.0 Hz, 1H).
LC–MS (APCI+) 223 (MH+, 100%).
4.1.2.5. 4-Methylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4f).
Hydrolysis of ester 3f (158 mg, 0.77 mmol) gave 4-methylpyra-
zolo[1,5-a]pyridine-3-carboxylic acid as a white solid (118 mg,
91%). 1H NMR d (400 MHz, d6-DMSO) 12.20 (br s, 1H), 8.64 (d,
J = 6.9 Hz, 1H), 8.36 (s, 1H), 7.27 (d, J = 6.9 Hz, 1H), 7.01 (t,
J = 6.9 Hz, 1H), 2.75 (s, 3H). Reduction of the above acid (116 mg,
0.66 mmol), after chromatography (eluting with hexanes: EtOAc
2:1 to 1:1 to EtOAc) gave (4-methylpyrazolo[1,5-a]pyridin-3-
yl)methanol as a white solid (58 mg, 54%). 1H NMR d (400 MHz,
CDCl3) 8.32 (d, J = 6.9 Hz, 1H), 7.90 (s, 1H), 6.89 (d, J = 6.9 Hz, 1H),
6.68 (t, J = 6.9 Hz, 1H), 4.90 (d, J = 4.6 Hz, 2H), 2.70 (s, 3H), 1.46
(t, J = 4.6 Hz, 1H). LC–MS (APCI+) 163 (MH+, 100%). Reoxidation of
the above alcohol (58 mg, 0.36 mmol) gave 4f as a white solid
(56 mg, 98%). 1H NMR d (400 MHz, CDCl3) 10.17 (s, 1H), 8.49 (s,
4.1.2.10. 5-Bromopyrazolo[1,5-a]pyridine-3-carbaldehyde (4m).
Decarboxylation of ester 3m (695 mg, 2.58 mmol) gave 5-bromo-
pyrazolo[1,5-a]pyridine (7m) as a pale brown solid (489 mg,

78
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
96%). 1H NMR d (400 MHz, CDCl3) 8.34 (d, J = 7.4 Hz, 1H), 7.95
(d, J = 2.1 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H), 6.84 (dd, J = 7.4, 2.1 Hz,
1H), 6.48 (d, J = 1.8 Hz, 1H). LC–MS (APCI+) 197 (MH+ with 79Br,
100%), 199 (MH+ with 81Br, 90%). Vilsmeier reaction of 7m (438 mg,
2.22 mmol) gave 4m as a pale brown solid (482 mg, 96%). 1H NMR d
(400 MHz, CDCl3) 10.02 (s, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.42 (d,
J = 7.3 Hz, 1H), 8.37 (s, 1H), 7.16 (dd, J = 7.3, 2.1 Hz, 1H). LC–MS
(APCI+) 225 (MH+ with 79Br, 100%), 227 (MH+ with 81Br, 95%).
(400 MHz, CDCl3) 10.03 (s, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.34 (s,
1H), 8.19 (d, J = 1.9 Hz, 1H), 7.18 (dd, J = 7.2, 1.9 Hz, 1H), 6.80 (dd,
J = 17.5, 10.9 Hz, 1H), 5.97 (d, J = 17.5 Hz, 1H), 5.55 (d, J = 10.9 Hz,
1H). LC–MS (APCI+) 173 (MH+, 100%).
4.1.2.15. 5-Ethynylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4r).
Ethynyltrimethylsilane (75
of 4m (60 mg, 0.27 mmol), CuI (5.1 mg, 27
(9.4 mg, 13 mol) in DMF (3 mL) and NEt3 (3 mL) which had been
lL, 0.53 mmol) was added to a solution
lmol) and (Ph3P)2PdCl2
l
4.1.2.11. 5-Iodopyrazolo[1,5-a]pyridine-3-carbaldehyde (4n).
Decarboxylation of ester 3n (57 mg, 0.18 mmol) gave 5-iodopyra-
zolo[1,5-a]pyridine (7n) as a pale brown solid (42 mg, 95%). 1H
deoxygenated by bubbling N2 through it. After heating to 60 °C for
2 h the solvents were removed in vacuo, then K2CO3 (111 mg,
0.80 mmol) and MeOH (10 mL) were added and the reaction stirred
for a further 2 h. The solvent was removed in vacuo, and the residue
taken up in water and extracted twice with CH2Cl2. The combined
extracts were dried (Na2SO4) and the solvent removed in vacuo.
Chromatography (eluting with hexanes: EtOAc 4:1) gave 4r as a
white solid (14 mg, 31%). 1H NMR d (400 MHz, CDCl3) 10.05 (s,
1H), 8.50 (dd, J = 7.1, 0.9 Hz, 1H), 8.43 (m, 1H), 8.40 (s, 1H), 7.07
(dd, J = 7.1, 1.8 Hz, 1H), 3.36 (s, 1H). LC–MS (APCI+) 171 (MH+, 100%).
NMR
d (400 MHz, CDCl3) 8.21 (d, J = 7.3 Hz, 1H), 7.95 (d,
J = 1.7 Hz, 1H), 7.90 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 7.3, 1.7 Hz,
1H), 6.44 (d, J = 2.2 Hz, 1H). LC–MS (APCI+) 245 (MH+, 100%). Vils-
meier reaction of 7n (42 mg, 0.17 mmol) gave 4n as a pale brown
solid (45 mg, 96%). 1H NMR d (400 MHz, CDCl3) 10.02 (s, 1H), 8.74
(d, J = 1.7 Hz, 1H), 8.33 (s, 1H), 8.28 (d, J = 7.2 Hz, 1H), 7.31 (dd,
J = 7.2, 1.7 Hz, 1H). LC–MS (APCI+) 273 (MH+, 100%).
4.1.2.12. 5-Chloropyrazolo[1,5-a]pyridine-3-carbaldehyde (4o).
Decarboxylation of ester 3l (1.29 g, 2.98 mmol), except with carry-
ing out the aqueous extraction from pH 12, gave pyrazolo[1,5-
a]pyridin-5-amine (7l) as a pale brown solid (310 mg, 78%). 1H
4.1.2.16. Methyl 3-formylpyrazolo[1,5-a]pyridine-5-carboxylate
(4t).
Decarboxylation of 3t (2.65 g, 10.7 mmol), except for
basifying the aqueous reaction mixture to pH 2 with 6 M NaOH,
then the precipitated solid was filtered off, washed with water
and dried to leave pyrazolo[1,5-a]pyridine-5-carboxylic acid (7s)
as an off-white solid (1.60 g, 92%). 1H NMR d (400 MHz, d6-DMSO)
12.5 (br s, 1H), 8.74 (d, J = 7.3 Hz, 1H), 8.34 (m, 1H), 8.11 (d,
J = 2.3 Hz, 1H), 7.25 (dd, J = 7.3, 1.9 Hz, 1H), 6.89 (dd, J = 2.3,
0.8 Hz, 1H). LC–MS (APCIꢀ) 161 (MꢀH, 100%). A solution of 7s
(31 mg, 0.19 mmol) and concentrated HCl (three drops) in MeOH
(10 mL) was refluxed for 4 h. The solvent was removed in vacuo,
saturated aqueous NaHCO3 was added to the residue, and then it
was extracted twice with CH2Cl2. The combined extracts were
dried (Na2SO4) and the solvent removed in vacuo to leave methyl
pyrazolo[1,5-a]pyridine-5-carboxylate (7t) as an off-white solid
(25 mg, 74%). 1H NMR d (400 MHz, CDCl3) 8.52 (dt, J = 7.3, 0.9 Hz,
1H), 8.33 (dd, J = 1.8, 0.9 Hz, 1H), 8.04 (d, J = 2.3 Hz, 1H), 7.34 (dd,
J = 7.3, 1.8 Hz, 1H), 6.75 (dd, J = 2.3, 0.9 Hz, 1H), 3.96 (s, 3H). LC–
MS (APCI+) 177 (MH+, 100%). Vilsmeier reaction of 7t (25 mg,
0.14 mmol) gave 4t as a yellow solid (22 mg, 76%). 1H NMR d
(400 MHz, CDCl3) 10.12 (s, 1H), 8.95 (dd, J = 1.8, 0.9 Hz, 1H), 8.61
(dd, J = 7.2, 0.9 Hz, 1H), 8.47 (s, 1H), 7.64 (dd, J = 7.2, 1.8 Hz, 1H),
4.01 (s, 3H). LC–MS (APCI+) 205 (MH+, 100%).
NMR
d (400 MHz, CDCl3) 8.23 (d, J = 7.4 Hz, 1H), 7.79 (d,
J = 2.0 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.22 (dd, J = 7.4, 2.4 Hz,
1H), 6.13 (d, J = 2.0 Hz, 1H), 3.81 (s, 2H). LC–MS (APCI+) 134
(MH+, 100%). A solution of NaNO2 (27 mg, 0.39 mmol) in water
(1 mL) was added dropwise to a solution of 7l (40 mg, 0.30 mmol)
and CuCl (74 mg, 0.75 mmol) in concentrated HCl (1 mL) at 0 °C
over 2 min. After 30 min, the reaction mixture was heated to
80 °C for 15 min, and then cooled to room temperature, basified
to pH 10 with 1 M NaOH, filtered through a plug of celite and
washed with CH2Cl2. The layers of the filtrate were separated
and the aqueous layer extracted with CH2Cl2. The combined ex-
tracts were dried (Na2SO4) and the solvent removed in vacuo.
Chromatography (eluting with hexanes: EtOAc 3:1) gave 5-chloro-
pyrazolo[1,5-a]pyridine (7o) as a white solid (6 mg, 13%). 1H NMR
d (400 MHz, CDCl3) 8.38 (d, J = 7.4 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H),
7.53 (d, J = 1.8 Hz, 1H), 6.71 (dd, J = 7.4, 2.2 Hz, 1H), 6.47 (d,
J = 1.8 Hz, 1H). LC–MS (APCI+) 153 (MH+ with 35Cl, 100%), 155
(MH+ with 37Cl, 30%). Vilsmeier reaction of 7o (6 mg, 0.039 mmol)
gave 4o as a white solid (7 mg, 100%). 1H NMR d (400 MHz, CDCl3)
10.02 (s, 1H), 8.48 (d, J = 7.3 Hz, 1H), 8.38 (s, 1H), 8.33 (d, J = 2.3 Hz,
1H), 7.04 (dd, J = 7.3, 2.3 Hz, 1H). LC–MS (APCI+) 181 (MH+ with
35Cl, 100%), 183 (MH+ with 37Cl, 30%).
4.1.2.17. 3-Formylpyrazolo[1,5-a]pyridine-5-carboxylic acid (4s).
Hydrolysis of 4t (1.23 g, 6.0 mmol) gave 4s as a white solid (0.98 g,
85%). 1H NMR d (400 MHz, d6-DMSO) 13.8 (br s, 1H), 10.10 (s, 1H),
9.01 (dd, J = 7.1, 0.8 Hz, 1H), 8.76 (s, 1H), 8.74 (m, 1H), 7.58 (dd,
J = 7.1, 1.9 Hz, 1H). LC–MS (APCI+) 191 (MH+, 100%).
4.1.2.13. 5-Cyclopropylpyrazolo[1,5-a]pyridine-3-carbaldehyde
(4p).
Pd(PPh3)4 (31 mg, 0.027 mmol) was added to a solution
of 4m (60 mg, 0.27 mmol) and tributyl(cyclopropyl)stannane
(115 mg, 0.35 mmol) in toluene (10 mL) which had been deoxy-
genated by bubbling N2 through it. After refluxing for 18 h, the sol-
vent was removed in vacuo. Chromatography (eluting with
hexanes: EtOAc 9:1 to 4:1) gave 4p as a pale yellow solid (37 mg,
74%). 1H NMR d (400 MHz, CDCl3) 9.98 (s, 1H), 8.41 (dd, J = 7.2,
0.7 Hz, 1H), 8.30 (s, 1H), 7.97 (d, J = 1.9 Hz, 1H), 6.74 (dd, J = 7.2,
1.9 Hz, 1H), 2.02 (m, 1H), 1.15 (m, 2H), 0.89 (m, 2H). LC–MS (APCI+)
187 (MH+, 100%).
4.1.2.18. 3-Formylpyrazolo[1,5-a]pyridine-5-carboxamide (4u).
A solution of 4s (60 mg, 0.32 mmol) in SOCl2 (1 mL) was refluxed
for 1 h. The solvent was removed in vacuo, and then the residue
was taken up in CH2Cl2 (5 mL) and added to concentrated aqueous
NH3 (5 mL). After 30 min the reaction mixture was acidified to pH
1 with 1 M HCl, saturated with NaCl and extracted four times with
CH2Cl2. The combined extracts were dried (Na2SO4) and the solvent
removed in vacuo to leave 4u as a white solid (29 mg, 48%). 1H
NMR d (400 MHz, d6-DMSO) 10.09 (s, 1H), 9.04 (dd, J = 7.2,
0.9 Hz, 1H), 8.75 (s, 1H), 8.73 (dd, J = 1.9, 0.9 Hz, 1H), 8.41 (br s,
1H), 7.77 (br s, 1H), 7.62 (dd, J = 7.2, 1.9 Hz, 1H). LC–MS (APCIꢀ)
188 (MꢀH, 100%).
4.1.2.14. 5-Vinylpyrazolo[1,5-a]pyridine-3-carbaldehyde (4q).
Pd(PPh3)4 (26 mg, 0.022 mmol) was added to a solution of 4m
(50 mg, 0.22 mmol) and tributyl(vinyl)tin (84 lL, 0.29 mmol) in
toluene (10 mL) which had been deoxygenated by bubbling N2
through it. After refluxing for 2 h, the solvent was removed in
vacuo. Chromatography (eluting with hexanes: EtOAc 9:1 to
85:15) gave 4q as a pale yellow solid (35 mg, 92%). 1H NMR d
4.1.2.19. 3-Formyl-N-methylpyrazolo[1,5-a]pyridine-5-carbox-
amide (4v).
A solution of 4s (60 mg, 0.32 mmol) in SOCl2
(1 mL) was refluxed for 1 h. The solvent was removed in vacuo,

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
79
then the residue taken up in CH2Cl2 (5 mL) and added to a suspen-
sion of MeNH2ꢁHCl (64 mg, 0.95 mmol) and NEt3 (0.22 mL,
1.6 mmol) in CH2Cl2 (5 mL). After 3 h, water was added, the reac-
tion mixture was acidified to pH 1 with 1 M HCl and stirred for
30 min. The reaction mixture was then basified to pH 10 with
1 M NaOH, the layers were separated, and the aqueous layer was
extracted with CH2Cl2. The combined organic extracts were dried
(Na2SO4) and the solvent removed in vacuo. Chromatography
(eluting with EtOAc) gave 4v as an off-white solid (51 mg, 80%).
1H NMR d (400 MHz, CDCl3) 10.08 (s, 1H), 8.62 (dd, J = 7.1, 0.9 Hz,
1H), 8.56 (dd, J = 2.0, 0.9 Hz, 1H), 8.45 (s, 1H), 7.60 (dd, J = 7.1,
2.0 Hz, 1H), 6.39 (br s, 1H), 3.08 (d, J = 4.9 Hz, 3H). LC–MS (APCI+)
204 (MH+, 100%).
(dd, J = 2.4, 1.4 Hz, 1H), 6.60 (br s, 1H), 5.90 (br s, 1H). LC–MS (APCI+)
180 (MH+, 100%). Vilsmeier reaction of 7z (113 mg, 179 mmol), after
chromatography (eluting with hexanes: EtOAc 7:1) gave 6-fluoro-
pyrazolo[1,5-a]pyridine-5-carbonitrile as
a
colourless solid
(101 mg, 88%). 1H NMR d (400 MHz, CDCl3) 8.55 (d, J = 4.4 Hz, 1H),
8.07 (d, J = 2.4 Hz, 1H), 7.98 (d, J = 6.3 Hz, 1H), 6.81 (dd, J = 2.4,
0.7 Hz, 1H). LC–MS (APCI+) 162 (MH+, 100%). Vilsmeier reaction of
the above compound (89 mg, 0.55 mmol), after chromatography
(eluting with hexanes: EtOAc 3:1) gave 4z as a colourless solid
(55 mg, 52%). 1H NMR d (400 MHz, CDCl3) 10.10 (s, 1H), 8.73 (d,
J = 6.4 Hz, 1H), 8.67 (d, J = 3.8 Hz, 1H), 8.52 (s, 1H).
4.1.2.23.
(4aa)24
5-Methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde
Decarboxylation of 3aa (114 mg, 0.52 mmol) gave pyr-
.
4.1.2.20. 3-Formyl-N,N-dimethylpyrazolo[1,5-a]pyridine-5-car-
azolo[1,5-a]pyridin-5-ol(7ab) as a pale brownsolid (61 mg, 88%). 1H
NMR d (400 MHz, d6-DMSO) 10.06 (s, 1H), 8.45 (d, J = 7.5 Hz, 1H),
7.79 (d, J = 2.2 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.46 (dd, J = 7.5,
2.4 Hz, 1H), 6.23 (d, J = 2.2 Hz, 1H). LC–MS (APCI+) 135 (MH+,
boxamide (4w).
A solution of 4s (100 mg, 0.53 mmol) in
SOCl2 (1.5 mL) was refluxed for 1 h. The solvent was removed in
vacuo, then the residue taken up in CH2Cl2 (5 mL) and added to a
suspension of Me2NHꢁHCl (86 mg, 1.05 mmol) and NEt3 (0.37 mL,
2.7 mmol) in CH2Cl2 (5 mL). After 3 h, water was added, the reac-
tion mixture was acidified to pH 1 with 1 M HCl and stirred for
30 min. The reaction mixture was then basified to pH 10 with
1 M NaOH, the layers were separated, and the aqueous layer was
extracted with CH2Cl2. The combined organic extracts were dried
(Na2SO4) and the solvent removed in vacuo. Chromatography
(eluting with EtOAc) gave 4w as a pale yellow solid (86 mg, 75%).
1H NMR d (400 MHz, CDCl3) 10.06 (s, 1H), 8.61 (d, J = 7.1 Hz, 1H),
8.43 (s, 1H), 8.32 (s, 1H), 7.15 (dd, J = 7.1, 1.8 Hz, 1H), 3.16 (s,
3H), 3.08 (s, 3H). LC–MS (APCI+) 218 (MH+, 100%).
100%). Iodomethane (23 lL, 0.37 mmol) was added to a suspension
of 7ab (25 mg, 0.19 mmol) and K2CO3 (52 mg, 0.38 mmol) in DMF
(2 mL). After 3 h, the reaction mixture was diluted with water and
extracted twice with CH2Cl2. The combined extracts were dried
(Na2SO4) and the solvent removed in vacuo to leave 5-methoxy-
pyrazolo[1,5-a]pyridine (7aa) as a pale brown oil (24 mg, 86%). 1H
NMR d (400 MHz, CDCl3) 8.28 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 2.2 Hz,
1H), 6.74 (d, J = 2.7 Hz, 1H), 6.44 (dd, J = 7.6, 2.7 Hz, 1H), 6.32 (d,
J = 2.2 Hz, 1H), 3.84 (s, 3H). LC–MS (APCI+) 149 (MH+, 100%). Vilsmeier
reaction of 7aa (24 mg, 0.16 mmol) gave 4aa as a pale yellow solid
(27 mg, 93%). 1H NMR d (400 MHz, CDCl3) 9.96 (s, 1H), 8.37 (d,
J = 7.5 Hz, 1H), 8.27 (s, 1H), 7.60 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 7.5,
2.7 Hz, 1H), 3.96 (s, 3H). LC–MS (APCI+) 177 (MH+, 100%)
4.1.2.21. 3-Formylpyrazolo[1,5-a]pyridine-5-carbonitrile (4x).
NEt3 (2.08 mL, 14.9 mmol) and isobutyl chloroformate (1.94 mL,
15.0 mmol) were added to a solution of 7s (2.20 g, 13.6 mmol) in
dry THF (100 mL) at 0 °C. After 1 h, NH3 was bubbled through the
solution for ca. 2 min. The reaction mixture was warmed to room
temperature and stirred for a further 1 h before diluting with CH2Cl2
and water. It was then basified to pH 12 with 1 M NaOH, saturated
with NaCl, and extracted four times with CH2Cl2. The combined ex-
tracts were dried (Na2SO4) and the solvents removed. The resulting
solid was triturated with hexanes-CH2Cl2 to leave pyrazolo[1,5-
a]pyridine-5-carboxamide (7u) as a pale brown solid (1.78 g, 81%).
1H NMR d (400 MHz, d6-DMSO) 8.72 (d, J = 7.3 Hz, 1H), 8.25 (dd,
J = 1.9, 0.8 Hz, 1H), 8.11 (br s, 1H), 8.07 (d, J = 2.3 Hz, 1H), 7.52 (br
s, 1H), 7.28 (dd, J = 7.3, 1.9 Hz, 1H), 6.80 (dd, J = 2.3, 0.8 Hz, 1H).
LC–MS (APCI+) 162 (MH+, 100%). Vilsmeier reaction of 7u (2.36 g,
14.7 mmol), except for using 5 equiv of POCl3, and filtration of the
product from the basified aqueous reaction mixture, gave 4x as a
pale brown solid (1.94 g, 77%). 1H NMR d (400 MHz, CDCl3) 10.12
(s, 1H), 8.70 (m, 1H), 8.66 (d, J = 7.1 Hz, 1H), 8.52 (s, 1H), 7.19 (dd,
J = 7.1, 1.7 Hz, 1H). LC–MS (APCI+) 172 (MH+, 100%).
4.1.2.24.
(4ab).
5-Hydroxypyrazolo[1,5-a]pyridine-3-carbaldehyde
Vilsmeier reaction of 7ab (36 mg, 0.27 mmol), except
for reacidifying the aqueous mixture to pH 3 before extracting,
gave 4ab as a red-brown solid (42 mg, 95%). 1H NMR d (400 MHz,
d6-DMSO) 11.10 (s, 1H), 9.83 (s, 1H), 8.71 (d, J = 7.4 Hz, 1H), 8.44
(s, 1H), 7.42 (d, J = 2.6 Hz, 1H), 6.77 (dd, J = 7.4, 2.6 Hz, 1H). LC–
MS (APCI+) 163 (MH+, 100%).
4.1.2.25. 3-Formylpyrazolo[1,5-a]pyridin-5-yl acetate (4ac).
A solution of 4ab (42 mg, 0.26 mmol), Ac2O (37
and NEt3 (54 L, 0.39 mmol) in CH2Cl2 (10 mL) was stirred at room
temperature for 3 days. The reaction mixture was diluted with
CH2Cl2 and washed with water, dried (Na2SO4) and the solvent
removed in vacuo. Chromatography (eluting with hexanes: EtOAc
3:1 to 2:1) gave 4ac as a pale brown solid (48 mg, 91%). 1H NMR
d (400 MHz, CDCl3) 10.02 (s, 1H), 8.54 (dd, J = 7.4 Hz, 1H), 8.38 (s,
1H), 8.05 (d, J = 2.5 Hz, 1H), 6.91 (dd, J = 7.4, 2.5 Hz, 1H), 2.37 (s,
3H). LC–MS (APCI+) 205 (MH+, 100%).
lL, 0.39 mmol)
l
4.1.2.22.
nitrile (4z).
6-Fluoro-3-formylpyrazolo[1,5-a]pyridine-5-carbo-
Decarboxylation of 3z (320 mg, 1.27 mmol) gave
4.1.3. Synthesis of sulfonohydrazides 5
Unless otherwise stated, sulfonohydrazides 5 were made by the
following method. Methylhydrazine sulfate (1.2 equiv) and
NaHCO3 (5 equiv) were added to a solution of aldehyde (1 equiv)
in MeOH (5 mL). After all of the aldehyde was consumed, 2-
methyl-5-nitrobenzenesulfonyl chloride (1.3 equiv) was added
and the reaction mixture stirred for a further 30 min. The solvent
was removed in vacuo and the residue taken up in CH2Cl2 and
water. The layers were separated and the aqueous phase extracted
with CH2Cl2, then the combined organic layers were dried (Na2SO4)
and the solvent removed in vacuo. Chromatography (eluting with a
hexanes: EtOAc gradient, unless otherwise stated) afforded 5.
6-fluoropyrazolo[1,5-a]pyridine-5-carboxylic acid as a white solid
(208 mg, 91%). 1H NMR d (400 MHz, CDCl3) 8.52 (d, J = 5.8 Hz, 1H),
8.36 (d, J = 7.2 Hz, 1H), 8.04 (d, J = 2.4 Hz, 1H), 6.81 (d, J = 2.4 Hz,
1H). LC–MS (APCI+) 181 (MH+, 100%). CDI (561 mg, 3.46 mmol)
was added to a stirred suspension of the above acid (208 mg,
1.15 mmol) in CH2Cl2 (25 mL). After 30 min the starting material
had dissolved and a solution of NH3 (2 mL of a 6 M solution in MeOH,
12 mmol) was added in a single portion. The reaction mixture was
stirred for an additional 30 min before the solvent was removed in
vacuo. Chromatography (eluting with hexanes: EtOAc 1:1) gave 6-
fluoropyrazolo[1,5-a]pyridine-5-carboxamide (7z) as a colourless
white solid (113 mg, 55%). 1H NMR d (400 MHz, CDCl3) 8.51 (d,
J = 6.8 Hz, 1H), 8.45 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 6.78
4.1.3.1. N,2-Dimethyl-5-nitro-N0-(pyrazolo[1,5-a]pyridin-3-yl-
methylene)benzenesulfonohydrazide (5a).
Reaction of pyra-

80
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
zolo[1,5-a]pyridine-3-carbaldehyde25 (4a) (43 mg, 0.29 mmol),
after chromatography (eluting with hexanes: EtOAc 3:1 to 2:1 to
1:1) gave 5a as a yellow solid (63 mg, 57%). 1H NMR d (400 MHz,
CDCl3) 9.01 (d, J = 2.4 Hz, 1H), 8.46 (d, J = 7.0 Hz, 1H), 8.28 (dd,
J = 8.4, 2.4 Hz, 1H), 8.04 (s, 1H), 7.95 (s, 1H), 7.87 (d, J = 8.9 Hz, 1H),
7.48 (d, J = 8.4 Hz, 1H), 7.28 (m, 1H), 6.90 (td, J = 7.0, 1.3 Hz, 1H),
3.41 (s, 3H), 2.76 (s, 3H). LC–MS (APCI+) 374 (MH+, 100%). Anal. Calcd
for C16H15N5O4S: C, 51.47; H, 4.05; N, 18.76. Found: C, 51.72; H, 4.13;
N, 18.98.
4.1.3.7. N,2-Dimethyl-N0-((5-methylpyrazolo[1,5-a]pyridin-3-yl)-
methylene)-5-nitrobenzenesulfonohydrazide (5g). Reaction
of 4g (14 mg, 0.09 mmol) after chromatography (eluting with
CH2Cl2 to CH2Cl2: MeOH 99.5:0.5) gave 5g as a yellow solid
(14 mg, 41%). 1H NMR d (400 MHz, CDCl3) 9.00 (d, J = 2.4 Hz, 1H),
8.34 (d, J = 7.1 Hz, 1H), 8.30 (dd, J = 8.4, 2.4 Hz, 1H), 8.00 (s, 1H),
7.99 (s, 1H), 7.61 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.72 (dd, J = 7.1,
1.9 Hz, 1H), 3.39 (s, 3H), 2.76 (s, 3H), 2.38 (s, 3H). LC–MS (APCI+)
388 (MH+, 100%). Anal. Calcd for C17H17N5O4S.0.1 EtOAc: C, 52.75;
H, 4.53; N, 17.67. Found: C, 52.66; H, 4.48; N, 17.49.
4.1.3.2. N0-((6-Bromopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5b).
Reac-
4.1.3.8. N0-((5-Ethylpyrazolo[1,5-a]pyridin-3-yl)methylene)-N,2-
tion of 4b (85 mg, 0.38 mmol) after chromatography (eluting with
hexanes: EtOAc 2:1 to 1:1 to 1:2) gave 5b as a yellow solid
(116 mg, 68%). 1H NMR d (400 MHz, CDCl3) 8.99 (d, J = 2.4 Hz,
1H), 8.61 (dd, J = 1.7, 0.8 Hz, 1H), 8.29 (dd, J = 8.4, 2.4 Hz, 1H),
8.01 (s, 1H), 7.88 (s, 1H), 7.80 (dd, J = 9.4, 0.8 Hz, 1H), 7.49 (d,
J = 8.4 Hz, 1H), 7.34 (dd, J = 9.4, 1.7 Hz, 1H), 3.41 (s, 3H), 2.75 (s,
3H). LC–MS (APCI+) 452 (MH+ with 79Br, 95%), 454 (MH+ with
81Br, 100%). Anal. Calcd for C16H14BrN5O4S: C, 42.49; H, 3.12; N,
15.48. Found: C, 42.71; H, 3.07; N, 15.45.
dimethyl-5-nitrobenzenesulfonohydrazide (5h).
Reaction
of 4h (50 mg, 0.29 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 2:1) gave 5h as a yellow solid (65 mg,
57%). 1H NMR d (400 MHz, CDCl3) 8.98 (d, J = 2.4 Hz, 1H), 8.36 (d,
J = 7.1 Hz, 1H), 8.29 (dd, J = 8.4, 2.4 Hz, 1H), 8.01 (s, 1H), 8.00 (s,
1H), 7.66 (m, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 7.1, 1.9 Hz,
1H), 3.39 (s, 3H), 2.76 (s, 3H), 2.66 (q, J = 7.6 Hz, 2H), 1.25 (t,
J = 7.6 Hz, 3H). LC–MS (APCI+) 402 (MH+, 100%). Anal. Calcd for
C18H19N5O4S: C, 53.85; H, 4.77; N, 17.45. Found: C, 54.11; H,
4.83; N, 17.24.
4.1.3.3. N0-((4-Bromopyrazolo[1,5-a]pyridin-3-yl)methylene)-
4.1.3.9.
zolo[1,5-a]pyridin-3-yl)methylene)benzenesulfonohydrazide
(5i). Reaction of 4i (25 mg, 0.12 mmol) after chromatography
(eluting with hexanes: EtOAc 3:1 to 2:1) gave 5i as a yellow solid
(45 mg, 87%). 1H NMR d (400 MHz, CDCl3) 8.92 (d, J = 2.4 Hz, 1H),
8.55 (d, J = 7.3 Hz, 1H), 8.32 (dd, J = 8.4, 2.4 Hz, 1H), 8.17 (s, 1H),
8.10 (m, 1H), 7.94 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.03 (dd, J = 7.3,
2.0 Hz, 1H), 3.45 (s, 3H), 2.76 (s, 3H). LC–MS (APCI+) 442 (MH+,
100%). Anal. Calcd for C17H14F3N5O4S.0.1 hexanes: C, 46.98; H,
3.45; N, 15.56. Found: C, 46.99; H, 3.35; N, 15.84.
N,2-Dimethyl-5-nitro-N0-((5-(trifluoromethyl)pyra-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5c).
Reac-
tion of 4c (90 mg, 0.40 mmol) after chromatography (eluting with
hexanes: EtOAc 5:1 to 3:1 to 2:1) gave 5c as a yellow solid (27 mg,
15%). 1H NMR d (400 MHz, CDCl3) 8.86 (d, J = 2.4 Hz, 1H), 8.65 (s,
1H), 8.45 (d, J = 6.9 Hz, 1H), 8.30 (s, 1H), 8.29 (dd, J = 8.5, 2.4 Hz,
1H), 7.45–7.52 (m, 2H), 6.69 (t, J = 7.1 Hz, 1H), 3.39 (s, 3H), 2.86
(s, 3H). LC–MS (APCI+) 452 (MH+ with 79Br, 100%), 454 (MH+ with
81Br, 90%). Anal. Calcd for C16H14BrN5O4S. 0.1 hexanes: C, 43.26;
H, 3.37; N, 15.19. Found: C, 43.53; H, 3.37; N, 15.10.
4.1.3.4.
yl)methylene)-5-nitrobenzenesulfonohydrazide (5d).
N,2-Dimethyl-N0-((7-methylpyrazolo[1,5-a]pyridin-3-
Reac-
4.1.3.10.
a]pyridin-3-yl)methylene)benzenesulfonohydrazide
N,2-Dimethyl-5-nitro-N0-((5-phenylpyrazolo[1,5-
(5j).
tion of 4d (48 mg, 0.30 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 2:1) gave 5d as a yellow solid (78 mg, 67%).
1H NMR d (400 MHz, CDCl3) 9.01 (d, J = 2.4 Hz, 1H), 8.28 (dd,
J = 8.4, 2.4 Hz, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.79 (d, J = 8.9 Hz, 1H),
7.48 (d, J = 8.4 Hz, 1H), 7.23 (dd, J = 8.9, 7.0 Hz, 1H), 6.77 (d,
J = 7.0 Hz, 1H), 3.40 (s, 3H), 2.75 (s, 6H). LC–MS (APCI+) 388 (MH+,
100%). Anal. Calcd for C17H17N5O4S: C, 52.70; H, 4.42; N, 18.08.
Found: C, 52.90; H, 4.61; N, 17.93.
Reaction of 4j (45 mg, 0.20 mmol) after chromatography (eluting
with hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5j as a yellow solid
(83 mg, 91%). 1H NMR d (400 MHz, CDCl3) 8.86 (d, J = 2.4 Hz, 1H),
8.51 (dd, J = 7.2, 0.9 Hz, 1H), 8.20 (dd, J = 8.4, 2.4 Hz, 1H), 8.12
(dd, J = 2.0, 0.9 Hz, 1H), 8.09 (s, 1H), 8.07 (s, 1H), 7.57 (m, 2H),
7.54–7.44 (m, 3H), 7.42 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 7.2, 2.0 Hz,
1H), 3.39 (s, 3H), 2.76 (s, 3H). LC–MS (APCI+) 450 (MH+, 100%).
Anal. Calcd for C22H19N5O4S.0.1 EtOAc: C, 58.71; H, 4.35; N,
15.28. Found: C, 58.98; H, 4.49; N, 15.15.
4.1.3.5.
yl)methylene)-5-nitrobenzenesulfonohydrazide (5e).
N,2-Dimethyl-N0-((6-methylpyrazolo[1,5-a]pyridin-3-
Reac-
4.1.3.11. N0-((5-Bromopyrazolo[1,5-a]pyridin-3-yl)methylene)-
tion of 4e (30 mg, 0.19 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5e as a yellow solid (44 mg,
60%). 1H NMR d (400 MHz, CDCl3) 9.01 (d, J = 2.4 Hz, 1H), 8.28 (dd,
J = 8.4, 2.4 Hz, 1H), 8.26 (m, 1H), 7.97 (s, 1H), 7.94 (s, 1H), 7.77 (d,
J = 9.0 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 9.0, 1.4 Hz, 1H),
3.39 (s, 3H), 2.75 (s, 3H), 2.36 (s, 3H). LC–MS (APCI+) 388 (MH+,
100%). Anal. Calcd for C17H17N5O4S: C, 52.70; H, 4.42; N, 18.08.
Found: C, 52.85; H, 4.55; N, 17.78.
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5m).
Reac-
tion of 4m (22 mg, 0.10 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5m as a yellow solid (43 mg,
98%). 1H NMR d (400 MHz, CDCl3) 9.01 (d, J = 2.4 Hz, 1H), 8.36 (dd,
J = 8.4, 2.4 Hz, 1H), 8.28 (d, J = 7.3 Hz, 1H), 8.03 (s, 1H), 7.89 (s, 1H),
7.76 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 7.3,
2.2 Hz, 1H), 3.43 (s, 3H), 2.74 (s, 3H). LC–MS (APCI+) 452 (MH+ with
79Br, 80%), 454 (MH+ with 81Br, 100%). Anal. Calcd for
C16H14BrN5O4S: C, 42.49; H, 3.12; N, 15.48. Found: C, 42.77; H,
3.07; N, 15.47.
4.1.3.6.
yl)methylene)-5-nitrobenzenesulfonohydrazide (5f).
N,2-Dimethyl-N0-((4-methylpyrazolo[1,5-a]pyridin-3-
Reac-
4.1.3.12.
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5n).
N0-((5-Iodopyrazolo[1,5-a]pyridin-3-yl)methylene)-
Reac-
tion of 4f (54 mg, 0.34 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1) gave 5f as a yellow solid (80 mg, 61%). 1H
NMR d (400 MHz, CDCl3) 8.83 (d, J = 2.4 Hz, 1H), 8.36 (s, 1H), 8.34
(d, J = 6.9 Hz, 1H), 8.29 (dd, J = 8.4, 2.4 Hz, 1H), 8.25 (s, 1H), 7.50
(d, J = 8.4 Hz, 1H), 7.02 (m, 1H), 6.75 (t, J = 6.9 Hz, 1H), 3.32 (s,
3H), 2.83 (s, 3H), 2.60 (s, 3H). LC–MS (APCI+) 388 (MH+, 100%).
Anal. Calcd for C17H17N5O4S: C, 52.70; H, 4.42; N, 18.08. Found:
C, 52.95; H, 4.51; N, 17.96.
tion of 4n (45 mg, 0.17 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5n as a yellow solid (60 mg,
72%). 1H NMR d (400 MHz, CDCl3) 8.99 (d, J = 2.4 Hz, 1H), 8.36 (dd,
J = 8.4, 2.4 Hz, 1H), 8.16 (dd, J = 7.2, 0.7 Hz, 1H), 8.05 (m, 1H), 8.00
(s, 1H), 7.90 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.09 (dd, J = 7.2, 1.9 Hz,
1H), 3.43 (s, 3H), 2.75 (s, 3H). LC–MS (APCI+) 500 (MH+, 100%). Anal.

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
81
Calcd for C16H14IN5O4S.0.15 hexanes: C, 39.63; H, 3.17; N, 13.67.
Found: C, 39.63; H, 2.95; N, 13.69.
d6-DMSO) 8.84 (dd, J = 7.3, 0.8 Hz, 1H), 8.68 (d, J = 2.5 Hz, 1H), 8.40
(dd, J = 8.4, 2.5 Hz, 1H), 8.34 (dd, J = 1.9, 0.8 Hz, 1H), 8.31 (s, 1H),
8 = 24 (s, 1H), 8.07 (br s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.58 (br s, 1H),
7.40 (dd, J = 7.3, 1.9 Hz, 1H), 3.39 (s, 3H), 2.74 (s, 3H). LC–MS (APCI+)
417 (MH+, 100%). Anal. Calcd for C17H16N6O5S: C, 49.03; H, 3.87; N,
20.18. Found: C, 49.29; H, 3.92; N, 20.30.
4.1.3.13. N0-((5-Chloropyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5o).
Reac-
tion of 4o (7 mg, 0.039 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 1:1) gave 5o as a yellow solid (13 mg, 81%).
1H NMR d (400 MHz, CDCl3) 9.01 (d, J = 2.4 Hz, 1H), 8.32–8.38 (m,
2H), 8.04 (s, 1H), 7.89 (s, 1H), 7.56 (d, J = 1.9 Hz, 1H), 7.53 (d,
J = 8.4 Hz, 1H), 6.83 (dd, J = 7.3, 2.3 Hz, 1H), 3.42 (s, 3H), 2.75 (s,
3H). LC–MS (APCI+) 408 (MH+ with 35Cl, 100%), 410 (MH+ with
37Cl, 25%). Anal. Calcd for C16H14ClN5O4S: C, 47.12; H, 3.46; N,
17.17. Found: C, 47.06; H, 3.53; N, 16.97.
4.1.3.19. N-Methyl-3-((2-methyl-2-(2-methyl-5-nitrophenylsul-
fonyl)hydrazono)methyl)pyrazolo[1,5-a]pyridine-5-carboxam-
ide (5v).
Reaction of 4v (51 mg, 0.25 mmol) after
chromatography (eluting with hexanes: EtOAc 1:1 to EtOAc) gave
5v as a yellow solid (80 mg, 74%). 1H NMR d (400 MHz, CDCl3)
8.91 (d, J = 2.4 Hz, 1H), 8.49 (dd, J = 7.2, 0.9 Hz, 1H), 8.35 (dd,
J = 1.9, 0.9 Hz, 1H), 8.31 (dd, J = 8.4, 2.4 Hz, 1H), 8.11 (s, 1H), 7.97
(s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.33 (dd, J = 7.2, 1.9 Hz, 1H), 6.36
(br s, 1H), 3.42 (s, 3H), 3.09 (d, J = 4.9 Hz, 3H), 2.77 (s, 3H). LC–
MS (APCI+) 431 (MH+, 100%). Anal. Calcd for C18H18N6O5S.0.33
H2O: C, 49.54; H, 4.31; N, 19.26. Found: C, 49.58; H, 4.25; N, 18.97.
4.1.3.14. N0-((5-Cyclopropylpyrazolo[1,5-a]pyridin-3-yl)methyl-
ene)-N,2-dimethyl-5-nitrobenzenesulfonohydrazide
(5p).
Reaction of 4p (37 mg, 0.20 mmol) after chromatography (eluting
with hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5p as a yellow solid
(37 mg, 45%). 1H NMR d (400 MHz, CDCl3) 8.98 (d, J = 2.4 Hz, 1H),
8.32-8.24 (m, 2H), 8.00-7.96 (m, 2H), 7.62 (d, J = 1.9 Hz, 1H), 7.49
(d, J = 8.4 Hz, 1H), 6.53 (dd, J = 7.2, 2.0 Hz, 1H), 3.39 (s, 3H), 2.75
(s, 3H), 1.90 (m, 1H), 1.10 (m, 2H), 0.73 (m, 2H). LC–MS (APCI+)
414 (MH+, 100%). Anal. Calcd for C19H19N5O4S: C, 55.20; H, 4.63;
N, 16.94. Found: C, 54.80; H, 4.57; N, 16.65.
4.1.3.20. N,N-Dimethyl-3-((2-methyl-2-(2-methyl-5-nitrophen-
ylsulfonyl)hydrazono)methyl)pyrazolo[1,5-a]pyridine-5-carbox-
amide (5w).
Reaction of 4w (43 mg, 0.20 mmol) after chro-
matography (eluting with hexanes: EtOAc 1:1 to EtOAc) gave 5w
as a yellow solid (83 mg, 94%). 1H NMR d (400 MHz, d6-DMSO)
8.93 (d, J = 2.4 Hz, 1H), 8.45 (dd, J = 7.1, 0.9 Hz, 1H), 8.34 (dd,
J = 8.4, 2.4 Hz, 1H), 8.09 (s, 1H), 7.93 (s, 1H), 7.75 (dd, J = 1.8,
0.9 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 6.89 (dd, J = 7.1, 1.8 Hz, 1H),
3.42 (s, 3H), 3.15 (s, 3H), 3.04 (s, 3H), 2.73 (s, 3H). LC–MS (APCI+)
445 (MH+, 100%). Anal. Calcd for C19H20N6O5S: C, 51.34; H, 4.54;
N, 18.91. Found: C, 51.20; H, 4.66; N, 19.09.
4.1.3.15. N,2-Dimethyl-5-nitro-N0-((5-vinylpyrazolo[1,5-a]pyri-
din-3-yl)methylene)benzenesulfonohydrazide (5q).
Reac-
tion of 4q (35 mg, 0.20 mmol) after chromatography (eluting
with hexanes: EtOAc 4:1 to 2:1 to 1:1) gave 5q as a yellow solid
(36 mg, 44%). 1H NMR d (400 MHz, CDCl3) 8.98 (d, J = 2.4 Hz, 1H),
8.37 (d, J = 7.3 Hz, 1H), 8.29 (dd, J = 8.4, 2.4 Hz, 1H), 8.02 (s, 1H),
7.97 (s, 1H), 7.74 (d, J = 1.9 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.03
(dd, J = 7.3, 1.9 Hz, 1H), 6.67 (dd, J = 17.5, 10.9 Hz, 1H), 5.82 (d,
J = 17.5 Hz, 1H), 5.48 (d, J = 10.9 Hz, 1H), 3.41 (s, 3H), 2.75 (s, 3H).
LC–MS (APCI+) 400 (MH+, 100%). Anal. Calcd for C18H17N5O4S: C,
54.13; H, 4.29; N, 17.53. Found: C, 54.42; H, 4.29; N, 17.38.
4.1.3.21. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5x).
Reac-
tion of 4x (49 mg, 0.29 mmol) after chromatography (eluting with
CH2Cl2: MeOH 99.5:0.5) gave 5x as a yellow solid (59 mg, 52%). 1H
NMR d (400 MHz, CDCl3) 8.91 (d, J = 2.4 Hz, 1H), 8.52 (d, J = 7.2 Hz,
1H), 8.37 (dd, J = 8.4, 2.4 Hz, 1H), 8.18 (s, 1H), 8.04 (m, 1H), 7.90 (s,
1H), 7.57 (d, J = 8.4 Hz, 1H), 6.98 (dd, J = 7.2, 1.7 Hz, 1H), 3.45 (s,
3H), 2.79 (s, 3H). LC–MS (APCI+) 399 (MH+, 100%). Anal. Calcd for
C17H14N6O4S: C, 51.25; H, 3.54; N, 21.09. Found: C, 50.95; H,
3.54; N, 20.90.
4.1.3.16. N0-((5-Ethynylpyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5r).
Reac-
tion of 4r (14 mg, 0.08 mmol) after chromatography (eluting with
hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5r as a yellow solid
(24 mg, 73%). 1H NMR d (400 MHz, CDCl3) 9.04 (d, J = 2.4 Hz, 1H),
8.39-8.29 (m, 2H), 8.05 (s, 1H), 7.90 (s, 1H), 7.64 (m, 1H), 7.52 (d,
J = 8.4 Hz, 1H), 6.86 (dd, J = 7.2, 1.8 Hz, 1H), 3.44 (s, 3H), 3.29 (s,
1H), 2.73 (s, 3H). LC–MS (APCI+) 398 (MH+, 100%). Anal. Calcd for
C18H15N5O4S: C, 54.40; H, 3.80; N, 17.62. Found: C, 54.37; H,
3.87; N, 17.26.
4.1.3.22.
N0-((5-Cyano-6-fluoropyrazolo[1,5-a]pyridin-3-yl)-
methylene)-N,2-dimethyl-5-nitrobenzenesulfonohydrazide (5z).
Reaction of 4z (55 mg, 0.29 mmol) after chromatography (eluting
with CH2Cl2: iPrOH 99:1) and trituration with Et2O, gave 5z as a yel-
low solid (107 mg, 85%). HPLC purity 97%. 1H NMR d (400 MHz,
CDCl3) 8.88 (d, J = 2.4 Hz, 1H), 8.52 (dd, J = 4.1, 0.7 Hz, 1H), 8.37
(dd, J = 8.4, 2.4 Hz, 1H), 8.16 (s, 1H), 8.11 (d, J = 6.4 Hz, 1H), 7.85
(s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 3.45 (s, 3H), 2.78 (s, 3H). LC–MS
(APCI+) 417 (MH+, 100%). Anal. Calcd for C17H13FN6O4S: C, 49.04;
H, 3.15; N, 20.18. Found: C, 48.81; H, 2.89; N, 20.04. HRMS (ESI+)
Calcd for C17H13FN6O4SNa: 439.0595; found (M+Na+) 439.0604.
4.1.3.17. Methyl 3-((2-methyl-2-(2-methyl-5-nitrophenylsulfo-
nyl)-hydrazono)methyl)pyrazolo[1,5-a]pyridine-5-carboxylate
(5t).
Reaction of 4t (22 mg, 0.11 mmol) after chromatography
(eluting with hexanes: EtOAc 3:1 to 2:1 to 1:1) gave 5t as a yellow
solid (43 mg, 93%). 1H NMR d (400 MHz, CDCl3) 8.95 (d, J = 2.4 Hz,
1H), 8.51 (dd, J = 1.9, 0.9 Hz, 1H), 8.47 (dd, J = 7.2, 0.9 Hz, 1H),
8.32 (dd, J = 8.4, 2.4 Hz, 1H), 8.13 (s, 1H), 7.96 (s, 1H), 7.50 (d,
J = 8.4 Hz, 1H), 7.42 (dd, J = 7.2, 1.9 Hz, 1H), 3.98 (s, 3H), 3.44 (s,
3H), 2.78 (s, 3H). LC–MS (APCI+) 432 (MH+, 100%). Anal. Calcd for
C18H17N5O6S.0.2 H2O: C, 49.70; H, 4.03; N, 16.10. Found: C, 49.65;
H, 4.08; N, 15.79.
4.1.3.23. N0-((5-Methoxypyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide
(5aa).
A
solution of 4aa (27 mg, 0.15 mmol) and 2-methyl-5-nitro-
benzenesulfonohydrazide14 (39 mg, 0.17 mmol) in MeOH (5 mL)
was stirred at room temperature for 4 h. THF (2 mL) was then
added, followed by the dropwise addition of an ethereal solution
of CH2N2 solution until reaction was complete by tlc analysis. The
solvents were removed in vacuo. Chromatography (eluting with
CH2Cl2: MeOH 99.5:0.5) gave 5aa as a yellow solid (36 mg, 58%).
1H NMR d (400 MHz, d6-DMSO) 8.67 (d, J = 2.5 Hz, 1H), 8.62 (d,
J = 7.6 Hz, 1H), 8.39 (dd, J = 8.4, 2.5 Hz, 1H), 8.15 (s, 1H), 8.12 (s,
1H), 7.74 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 2.8 Hz, 1H), 6.74 (dd,
4.1.3.18.
3-((2-Methyl-2-(2-methyl-5-nitrophenylsulfonyl)-
hydrazono)methyl)pyrazolo[1,5-a]pyridine-5-carboxamide (5u).
Reaction of 4u (28 mg, 0.15 mmol) except that after the reaction sol-
vent was removed in vacuo, the residue was taken up in CH2Cl2 and
water. The solid was filtered off, washed with water and CH2Cl2 then
dried to leave 5u as a yellow solid (30 mg, 48%). 1H NMR d (400 MHz,

82
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
J = 7.6, 2.8 Hz, 1H), 3.81 (s, 3H), 3.29 (s, 3H), 2.71 (s, 3H). LC–MS
(APCI+) 404 (MH+, 100%). Anal. Calcd for C17H17N5O5S: C, 50.61; H,
4.25; N, 17.36. Found: C, 50.85; H, 4.29; N, 17.51.
J = 7.4 Hz, 2H), 1.14 (t, J = 7.4 Hz, 3H), 0.24 (s, 9H). LC–MS (APCI+)
155 (MH+, 100%). Reaction of 11 (529 mg, 5.08 mmol) and the above
ketone (520 mg, 3.38 mmol) with the addition of KF (589 mg,
10.1 mmol), after chromatography (eluting with CH2Cl2) gave 12c
as an off-white solid (479 mg, 71%). 1H NMR d (400 MHz, CDCl3)
8.81 (dd, J = 1.8, 1.0 Hz, 1H), 8.61 (dd, J = 7.2, 1.0 Hz, 1H), 8.46 (s,
1H), 7.11 (dd, J = 7.2, 1.8 Hz, 1H), 2.96 (q, J = 7.4 Hz, 2H), 1.27 (t,
J = 7.4 Hz, 3H). LC–MS (APCI+) 200 (MH+, 100%).
4.1.3.24.
3-((2-Methyl-2-(2-methyl-5-nitrophenylsulfonyl)-
hydrazono)methyl)pyrazolo[1,5-a]pyridin-5-yl acetate (5ac).
A solution of 4ac (48 mg, 0.24 mmol) and 2-methyl-5-nitro-
benzenesulfonohydrazide14 (60 mg, 0.26 mmol) in MeOH (5 mL)
was stirred at room temperature for 4 h. THF (2 mL) was then
added, followed by the dropwise addition of an ethereal solution
of CH2N2 solution until reaction was complete by tlc analysis.
The solvents were removed in vacuo. Chromatography (eluting
with CH2Cl2: MeOH 99.5:0.5) gave 5ac as a yellow solid (72 mg,
71%). 1H NMR d (400 MHz, CDCl3) 9.02 (d, J = 2.4 Hz, 1H), 8.41
(dd, J = 7.5, 0.7 Hz, 1H), 8.30 (dd, J = 8.4, 2.4 Hz, 1H), 8.03 (s, 1H),
7.92 (s, 1H), 7.51 (d, J = 2.5 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 6.71
(dd, J = 7.5, 2.5 Hz, 1H), 3.42 (s, 3H), 2.72 (s, 3H), 2.39 (s, 3H). LC–
MS (APCI+) 432 (MH+, 100%). Anal. Calcd for C18H17N5O6S.0.5
H2O: C, 49.09; H, 4.12; N, 15.90. Found: C, 49.31; H, 4.00; N, 15.91.
4.1.4.3. 3-Butyrylpyrazolo[1,5-a]pyridine-5-carbonitrile (12d).
AlCl3 (3.08 g, 23.1 mmol) was added to a solution of butyryl chlo-
ride (2.00 mL, 19.3 mmol) and bis(trimethylsilyl)acetylene
(4.80 mL, 21.2 mmol) in dry CH2Cl2 (50 mL) at 0 °C. After 30 min,
the reaction mixture was warmed to room temperature, stirred
for
a further 2 h, and then poured onto ice-1 M HCl (1:1,
200 mL). The layers were separated, and the aqueous layer was ex-
tracted twice with CH2Cl2. The combined organic layers were dried
(Na2SO4) and the solvent removed in vacuo. Chromatography
(eluting with hexanes: Et2O 98:2) gave 1-(trimethylsilyl)hex-1-
yn-3-one as a brown oil (2.98 g, 92%). 1H NMR d (400 MHz, CDCl3)
2.54 (t, J = 7.3 Hz, 2H), 1.70 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H), 0.24 (s,
9H). LC–MS (APCI+) 169 (MH+, 100%). Reaction of 11 (137 mg,
1.32 mmol) and the above ketone (147 mg, 0.88 mmol) with the
addition of KF (153 mg, 2.63 mmol), after chromatography (eluting
with hexanes: EtOAc 85:15 to 4:1) gave 12d as an off-white solid
(153 mg, 82%). 1H NMR d (400 MHz, CDCl3) 8.82 (dd, J = 1.9,
1.0 Hz, 1H), 8.61 (dd, J = 7.2, 1.0 Hz, 1H), 8.46 (s, 1H), 7.12 (dd,
J = 7.2, 1.9 Hz, 1H), 2.89 (t, J = 7.3 Hz, 2H), 1.82 (m, 2H), 1.04 (t,
J = 7.4 Hz, 3H). LC–MS (APCI+) 214 (MH+, 100%).
4.1.3.25.
ene)-N,2-dimethyl-5-nitrobenzenesulfonohydrazide
N0-((5-Hydroxypyrazolo[1,5-a]pyridin-3-yl)methyl-
(5ab).
Saturated aqueous NaHCO3 (5 mL) was added to a suspension of
5ac (52 mg, 0.12 mmol) in MeOH (10 mL) and stirred for 2 h. The
MeOH was removed in vacuo, and the resulting solid filtered off
and washed with water. Chromatography (eluting with CH2Cl2:
MeOH 99:1 to 19:1) gave 5ab as a yellow solid (26 mg, 55%). 1H
NMR d (400 MHz, d6-DMSO) 10.58 (br s, 1H), 8.69 (d, J = 2.5 Hz,
1H), 8.54 (d, J = 7.5 Hz, 1H), 8.41 (dd, J = 8.4, 2.5 Hz, 1H), 8.12 (s,
1H), 8.06 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 2.6 Hz, 1H),
6.60 (dd, J = 7.5, 2.6 Hz, 1H), 3.27 (s, 3H), 2.69 (s, 3H). LC–MS
(APCI+) 390 (MH+, 100%). Anal. Calcd for C16H15N5O5S: C, 49.35;
H, 3.88; N, 17.99. Found: C, 49.51; H, 3.94; N, 17.72.
4.1.4.4. tert-Butyl 3-acetylpyrazolo[1,5-a]pyridin-5-ylcarbamate
(12e).
A solution of 6 (4.94 g, 25.4 mmol) and DNPH (5.05 g,
25.4 mmol) in MeCN (60 mL) was heated at 40 °C for 18 h. The sol-
vent was removed in vacuo. The residue taken up in DMSO
(150 mL), then K2CO3 (7.02 g, 50.8 mmol) followed by 3-butyn-2-
one (0.87 mL, 37.9 mmol) were then added. The suspension stirred
at room temperature for 18 h, then diluted with cold water. The
aqueous layer was extracted three times with ethyl acetate, and
the combined organic extracts were washed with 1 M HCl, fol-
lowed by water then brine. The organic layer was dried (Na2SO4)
and the solvent removed in vacuo. Filtration through a plug of neu-
tral Al2O3 (eluting with EtOAc) gave 12e as a pale yellow solid
(4.20 g, 60%). 1H NMR d (400 MHz, CDCl3) 8.41 (d, J = 7.6 Hz, 1H),
8.26 (s, 1H), 8.08 (d, J = 2.3 Hz, 1H), 7.58 (m, 1H), 6.96 (s, 1H),
2.71 (s, 3H), 1.55 (s, 9H). LC–MS (APCI+) 276 (MH+, 100%).
4.1.4. Synthesis of pyrazolo[1,5-a]pyridine-3-ketones 12b–f
Unless otherwise stated, the following method was used. A fresh
solution of MSH23 in CH2Cl2 (1 equiv) was added to the pyridine
(1 equiv) in CH2Cl2 (10 mL) at 0 °C. After 2 h, the solvent was re-
moved in vacuo. The residue was taken up in dry DMF (8 mL), then
the alkynyl ketone (1 equiv) and K2CO3 (2 equiv) were added, and
the suspension stirred at room temperature for 18 h. The reaction
mixture was diluted with water and extracted twice with EtOAc.
The combined organic phases were washed three times with water
then with brine, dried (Na2SO4) and the solvent removed in vacuo.
Chromatography (eluting with a hexanes: EtOAc gradient, unless
otherwise stated) gave the pyrazolo[1,5-a]pyridine.
4.1.4.5. 1-(5-Bromopyrazolo[1,5-a]pyridin-3-yl)ethanone (12f).
A solution of 12e (81 mg, 0.29 mmol) and TFA (0.43 mL, 5.8 mmol)
in CH2Cl2 (5 mL) was stirred at room temperature for 18 h. The sol-
vents were removed in vacuo to leave 1-(5-aminopyrazolo[1,5-
a]pyridin-3-yl)ethanone trifluoroacetate as a brown solid (86 mg,
100%). 1H NMR d (400 MHz, d6-DMSO) 8.33 (d, J = 7.4 Hz, 1H),
8.22 (s, 1H), 7.51 (d, J = 2.6 Hz, 1H), 6.46 (dd, J = 7.4, 2.6 Hz, 1H),
2.52 (s, 3H). LC–MS (APCI+) 176 (MH+, 100%). A solution of NaNO2
(0.378 g, 5.5 mmol) in H2O (4 mL) was added dropwise to a sus-
pension of the above amine (1.7 g, 4.2 mmol) in 48% HBr (5 mL)
in an ice-salt bath at a rate so that the internal temperature of
the reaction was maintained below ꢀ5 °C. The reaction was stirred
for 10 min, then a solution of CuBr (1.51 g, 10.5 mmol) in 48% HBr
(5 mL) was added dropwise to maintain the internal temperature
of the reaction between ꢀ5 and 0 °C. After complete addition of
the CuBr, the reaction was stirred at 0 °C for a further 30 min,
and then at room temperature for 1 h. The reaction was cooled
and carefully neutralised to pH 8 with 2 M NaOH. A solution of
EDTA (3 g) in water (100 mL), followed by CH2Cl2 (100 mL) were
added and the reaction was stirred vigorously for 15 min. The
4.1.4.1. 3-Acetylpyrazolo[1,5-a]pyridine-5-carbonitrile (12b).
Reaction of 11 (1.19 g, 11.4 mmol) and 3-butyn-2-one (0.89 mL,
11.4 mmol), after chromatography (eluting with hexanes: EtOAc
4:1 to 3:1 to 2:1 to 1:1) gave 12b as a pale yellow solid (746 mg,
35%). 1H NMR d (400 MHz, CDCl3) 8.80 (dd, J = 1.8, 1.0 Hz, 1H),
8.61 (dd, J = 7.2, 1.0 Hz, 1H), 8.45 (s, 1H), 7.13 (dd, J = 7.2, 1.9 Hz,
1H), 2.60 (s, 3H). LC–MS (APCI+) 186 (MH+, 100%).
4.1.4.2. 3-Propionylpyrazolo[1,5-a]pyridine-5-carbonitrile (12c).
AlCl3 (921 mg, 6.91 mmol) was added to a solution of propionic
anhydride (0.74 mL, 5.76 mmol) and bis(trimethylsilyl)acetylene
(1.43 mL, 6.31 mmol) in dry CH2Cl2 (20 mL) at 0 °C. After 30 min,
the reaction mixture was warmed to room temperature, stirred
for a further 1 h, and then poured onto ice-1 M HCl (1:1, 80 mL).
The layers were separated, and the aqueous layer was extracted
with CH2Cl2. The combined organic layers were dried (Na2SO4)
and the solvent removed in vacuo. Chromatography (eluting with
hexanes: Et2O 98:2) gave 1-(trimethylsilyl)pent-1-yn-3-one as a
brown oil (739 mg, 83%). 1H NMR d (400 MHz, CDCl3) 2.58 (q,

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
83
solution was filtered and the layers separated. The aqueous layer
was extracted twice with EtOAc, washed with water and brine.
The combined organic extracts were dried (Na2SO4) and the sol-
vent removed in vacuo. Chromatography (eluting with hexanes
J = 2.5 Hz, 1H), 8.67 (d, J = 7.3 Hz, 1H), 8.44 (dd, J = 8.4, 2.5 Hz,
1H), 8.39 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H),
7.11 (dd, J = 7.3, 2.2 Hz, 1H), 2.77 (s, 3H), 2.32 (s, 3H). LC–MS
(APCI+) 452 (MH+ with 79Br, 100%), 454 (MH+ with 81Br, 100%).
Anal. Calcd for C16H15BrN5O4S.0.04 hexanes: C, 43.16; H, 3.30; N,
15.74. Found: C, 42.80; H, 3.22; N, 15.37.
to hexanes: EtOAc 85:15) gave 12f as
a pale yellow solid
(677 mg, 51%). 1H NMR d (400 MHz, CDCl3) 8.61 (dd, J = 2.2,
0.8 Hz, 1H), 8.37 (dd, J = 7.3, 0.8 Hz, 1H), 8.32 (s, 1H), 7.10 (dd,
J = 7.3, 2.2 Hz, 1H), 2.55 (s, 3H). LC–MS (APCI+) 239 (MH+ with
79Br, 100%), 241 (MH+ with 81Br, 80%).
4.1.6. Synthesis of sulfonohydrazides (14b–e) by methylation
An ethereal solution of CH2N2 was added dropwise to a solution
of the sulfonohydrazide (1 equiv) in THF (5 mL) until reaction was
complete by tlc analysis. The solvents were removed in vacuo.
Chromatography (eluting with a hexanes: EtOAc gradient, unless
otherwise stated) gave the methylated hydrazide.
4.1.5. Synthesis of sulfonohydrazides 13a–e
A solution of the aldehyde or ketone (1 equiv) and 2-methyl-5-
nitrobenzenesulfonohydrazide (1.1 equiv) in MeOH (5 mL) was re-
fluxed for 18 h unless otherwise stated. After cooling to room tem-
perature, the precipitated solid was filtered off, washed with a
little MeOH and dried.
4.1.6.1. N0-(1-(5-Cyanopyrazolo[1,5-a]pyridin-3-yl)ethylidene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide
(14b).
Reaction of hydrazide 13b (50 mg, 0.13 mmol) after chromatogra-
phy (eluting with CH2Cl2: MeOH 99.75:0.25 to 99.5:0.5) gave 14b
as a yellow solid (35 mg, 67%). 1H NMR d (400 MHz, CDCl3) 8.76
(d, J = 2.4 Hz, 1H), 8.59 (dd, J = 7.2, 1.0 Hz, 1H), 8.42 (dd, J = 8.4,
2.5 Hz, 1H), 8.38 (s, 1H), 8.30 (dd, J = 1.9, 1.0 Hz, 1H), 7.63 (d,
J = 8.4 Hz, 1H), 7.04 (dd, J = 7.2, 1.9 Hz, 1H), 3.02 (s, 3H), 2.72 (s,
3H), 2.67 (s, 3H). LC–MS (APCI+) 413 (MH+, 100%). Anal. Calcd for
C18H16N6O4S.0.33 H2O: C, 51.68; H, 4.01; N, 20.09. Found: C,
51.69; H, 4.07; N, 19.81.
4.1.5.1. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
methyl-5-nitrobenzenesulfonohydrazide (13a).
Reaction of
4x (150 mg, 0.88 mmol) at room temperature for 18 h gave 13a
as a yellow solid (305 mg, 91%). 1H NMR d (400 MHz, d6-DMSO)
11.99 (s, 1H), 8.94 (dd, J = 7.2, 0.9 Hz, 1H), 8.72 (d, J = 2.5 Hz, 1H),
8.42 (s, 1H), 8.38 (dd, J = 8.4, 2.5 Hz, 1H), 8.20 (s, 1H), 8.10 (dd,
J = 1.9, 0.9 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.31 (dd, J = 7.2,
1.9 Hz, 1H), 2.77 (s, 3H). LC–MS (APCI+) 385 (MH+, 100%). Anal.
Calcd for C16H12N6O4S: C, 50.00; H, 3.15; N, 21.86. Found: C,
50.21; H, 3.15; N, 21.80.
4.1.6.2.
lidene)-N,2-dimethyl-5-nitrobenzenesulfonohydrazide
N0-(1-(5-Cyanopyrazolo[1,5-a]pyridin-3-yl)propy-
(14c).
4.1.5.2. N0-(1-(5-Cyanopyrazolo[1,5-a]pyridin-3-yl)ethylidene)-
Reaction of hydrazide 13c (70 mg, 0.17 mmol) after chromatogra-
phy (eluting with CH2Cl2: MeOH 99.75:0.25) gave 14c as a yellow
solid (40 mg, 56%). 1H NMR d (400 MHz, CDCl3) 8.77 (d, J = 2.4 Hz,
1H), 8.59 (dd, J = 7.2, 1.0 Hz, 1H), 8.44 (dd, J = 8.4, 2.4 Hz, 1H), 8.37
(s, 1H), 8.26 (dd, J = 1.9, 1.0 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.04
(dd, J = 7.2, 1.9 Hz, 1H), 3.11 (q, J = 7.7 Hz, 2H), 3.01 (s, 3H), 2.71
(s, 3H), 1.37 (t, J = 7.7 Hz, 3H). LC–MS (APCI+) 427 (MH+, 100%).
Anal. Calcd for C19H18N6O4S: C, 53.51; H, 4.25; N, 19.71. Found:
C, 53.43; H, 4.35; N, 19.36.
2-methyl-5-nitrobenzenesulfonohydrazide (13b).
Reaction
of 12b (50 mg, 0.27 mmol) gave 13b as a yellow solid (82 mg,
76%). HPLC purity 93%. 1H NMR d (400 MHz, d6-DMSO) 11.15 (s,
1H), 8.91 (dd, J = 7.2, 0.6 Hz, 1H), 8.77 (d, J = 2.5 Hz, 1H), 8.55 (s,
1H), 8.40 (dd, J = 8.4, 2.5 Hz, 1H), 7.93 (s, 1H), 7.76 (d, J = 8.4 Hz,
1H), 7.28 (dd, J = 7.2, 1.9 Hz, 1H), 2.78 (s, 3H), 2.35 (s, 3H). LC–MS
(APCI+) 399 (MH+, 100%). HRMS (ESI+) Calcd for C17H15N6O4S:
399.0870; found (MH+) 399.0879.
4.1.5.3. N0-(1-(5-Cyanopyrazolo[1,5-a]pyridin-3-yl)propylidene)-
4.1.6.3. N0-(1-(5-Cyanopyrazolo[1,5-a]pyridin-3-yl)butylidene)-
2-methyl-5-nitrobenzenesulfonohydrazide (13c).
Reaction
N,2-dimethyl-5-nitrobenzenesulfonohydrazide
(14d).
of 12c (71 mg, 0.36 mmol), after chromatography (eluting with
CH2Cl2: MeOH 99.5:0.5) followed by trituration with MeOH gave
13c as a yellow solid (105 mg, 71%). HPLC purity 96%. 1H NMR d
(400 MHz, d6-DMSO) 11.37 (s, 1H), 8.93 (dd, J = 7.2, 0.9 Hz, 1H),
8.76 (d, J = 2.4 Hz, 1H), 8.56 (s, 1H), 8.40 (dd, J = 8.4, 2.4 Hz, 1H),
7.92 (dd, J = 1.8, 0.9 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.29 (dd,
J = 7.2, 1.8 Hz, 1H), 2.82 (q, J = 7.6 Hz, 2H), 2.76 (s, 3H), 1.10 (t,
J = 7.6 Hz, 3H). LC–MS (APCI+) 413 (MH+, 100%). HRMS (ESI+) Calcd
for C18H17N6O4S: 413.1027; found (MH+) 413.1021.
Reaction of hydrazide 13d (44 mg, 0.10 mmol) after chromatogra-
phy (eluting with hexanes: EtOAc 4:1 to 3:1) gave 14d as a yellow
solid (42 mg, 93%). 1H NMR d (400 MHz, CDCl3) 8.75 (d, J = 2.4 Hz,
1H), 8.58 (dd, J = 7.2, 1.0 Hz, 1H), 8.43 (dd, J = 8.4, 2.4 Hz, 1H), 8.35
(s, 1H), 8.26 (dd, J = 1.9, 1.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.04
(dd, J = 7.2, 1.9 Hz, 1H), 3.06 (m, 2H), 3.00 (s, 3H), 2.70 (s, 3H),
1.78 (m, 2H), 1.11 (t, J = 7.4 Hz, 3H). LC–MS (APCI+) 441 (MH+,
100%). Anal. Calcd for C20H20N6O4S.0.25 EtOAc: C, 54.54; H, 4.79;
N, 18.17. Found: C, 54.34; H, 4.73; N, 18.19.
4.1.5.4. N0-(1-(5-Cyanopyrazolo[1,5-a]pyridin-3-yl)butylidene)-
4.1.6.4. N0-(1-(5-Bromopyrazolo[1,5-a]pyridin-3-yl)ethylidene)-
2-methyl-5-nitrobenzenesulfonohydrazide (13d).
Reaction
N,2-dimethyl-5-nitrobenzenesulfonohydrazide
(14e).
of 12d (57 mg, 0.27 mmol), after chromatography (eluting with
CH2Cl2: MeOH 99.75:0.25) followed by trituration with MeOH gave
13d as a yellow solid (68 mg, 60%). HPLC purity 97%. 1H NMR d
(400 MHz, d6-DMSO) 11.36 (s, 1H), 8.91 (dd, J = 7.2, 0.9 Hz, 1H),
8.76 (d, J = 2.5 Hz, 1H), 8.55 (s, 1H), 8.39 (dd, J = 8.4, 2.5 Hz, 1H),
7.96 (dd, J = 1.9, 0.9 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.27 (dd,
J = 7.2, 1.9 Hz, 1H), 2.81–2.75 (m, 5H), 1.54 (m, 2H), 0.96 (t,
J = 7.3 Hz, 3H). LC–MS (APCI+) 427 (MH+, 100%). HRMS (ESI+) Calcd
for C19H19N6O4S: 427.1183; found (MH+) 427.1184.
Reaction of hydrazide 13e (49 mg, 0.11 mmol) after chromatogra-
phy (eluting with hexanes: EtOAc 4:1 to 3:1) gave 14e as a
cream-coloured solid (27 mg, 53%). 1H NMR d (400 MHz, CDCl3)
8.78 (d, J = 2.4 Hz, 1H), 8.38 (dd, J = 8.4, 2.4 Hz, 1H), 8.36 (dd,
J = 7.3, 0.7 Hz, 1H), 8.24 (s, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.60 (d,
J = 8.4 Hz, 1H), 7.02 (dd, J = 7.3, 2.2 Hz, 1H), 3.00 (s, 3H), 2.71 (s,
3H), 2.63 (s, 3H). LC–MS (APCI+) 466 (MH+ with 79Br, 90%), 468
(MH+ with 81Br, 100%). Anal. Calcd for C17H16BrN5O4S. 0.33 EtOAc:
C, 44.42; H, 3.79; N, 14.14. Found: C, 44.36; H, 3.77; N, 14.14.
4.1.5.5. N0-(1-(5-Bromopyrazolo[1,5-a]pyridin-3-yl)ethylidene)-
4.2. Enzyme assays
2-methyl-5-nitrobenzenesulfonohydrazide (13e).
Reaction
of 12f (60 mg, 0.25 mmol) gave 13e as an orange solid (88 mg,
The Class I PI3 kinase assays were performed using a basic thin
layer chromatography technique, as described previously.26 The
78%). 1H NMR d (400 MHz, d6-DMSO) 10.97 (br s, 1H), 8.79 (d,

84
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 69–85
PI3 kinase isoforms were prepared in-house, as described previ-
ously.26 Reactions were made containing 0.1
g recombinant
enzyme, 10 -phosphatidylinositol, inhibitor (DMSO only or
4.4. Cellular assay
l
lg
L
-a
The early passage cell lines used in this study were developed in
this laboratory and cultured as previously described.31 Cell lines
were grown in a-modified minimal essential growth medium sup-
DMSO + inhibitor to a final concentration of 1%), 2ꢂ Lipid Kinase
Buffer (40 mM Tris–HCl pH 7.4, 200 mM NaCl, 1 mM EDTA), and
activated upon the addition of an ATP mix (5 mM MgCl2, 100
lM
plemented insulin, transferrin, selenite and 5% foetal bovine serum.
Individual wells of 96-well tissue culture plates contained 1000
ATP, 0.1 L [
l
c
33P]ATP). Reactions were incubated at room temper-
ature for 1 h following which the reactions were stopped by the
addition of 1 M HCl. The lipids were then extracted using a two
step procedure. Firstly, 200 lL of chloroform:methanol (1:1) was
added, the biphasic reactions mixed and centrifuged briefly, and
cells in a volume of 150
tration steps to a maximum of 20
under an atmosphere of 5% O2, 5% CO2 and 90% N2 for five days,
with 3H-thymidine (0.04
Ci per well) being added over the last
l
L. Drugs were added at 10-fold concen-
l
M and plates were incubated
l
the inorganic phase was removed and discarded. Following this
6 h. Cells were harvested and the incorporated radioactivity was
measured. Duplicate samples were analysed for each drug dose
with multiple control samples and data were fitted to a least-
squares regression of the form y = y0 + aeꢀbx, where y is the incor-
porated radioactivity, x is the drug concentration and y0, a and b
are variables. The IC50 value was defined as the drug concentration
reducing 3H-thymidine incorporation by 50%.
80
lL of methanol:hydrochloric acid (1:1) was added and the same
procedure followed. Next, 70
lL of the organic phase was trans-
ferred to a clean 1.6 mL tube and the reactions were dried using
a speed vac, with no heating, for 30 min. The reactions were spot-
ted onto TLC plates (Merck Ltd) and developed for 1 h in 1-propa-
nol:2 M acetic acid (13:7). The TLC plates were then dried at room
temperature and quantified using a phosphorimager (Storm-
Imager, Amersham). Nine inhibitor concentrations were used to
determine the IC50. Each experiment was performed twice and
the average IC50 value used.
4.5. Phospho-PKB assay
HCT-116 cells were grown in MEM alpha supplemented with
10% (v/v) foetal bovine serum, 100 units/mL penicillin and
4.3. Molecular modelling
100 lg/mL streptomycin (all from Invitrogen). For inhibition
studies, cells were seeded in 12-well plates and grown for 1 day
before overnight starvation in serum-free media. Cells were then
exposed to varying concentrations of inhibitor dissolved in DMSO
(final concentration of DMSO in media 0.1%) for 15 min before
stimulation with 500 nM insulin for 5 min. Protein isolation and
immunoblotting for phospho-PKB was carried out according to
the methods previously described, with antibodies from Cell Sig-
naling Technology (Ser473 catalogue# 9271, Thr308 catalogue#
9275).26
The p110
a apo structure (PDB code 2RD0) was prepared by
stripping all waters and assigning protons using SYBYL8.0.3
(Tripos, St. Louis). Prior to this, sidechain orientations were
checked using Molprobity27 and only those within the docking
cavity were adjusted if there was clear evidence, and included
residues His855 and His917. The side chain of Met772 was reposi-
tioned between the side chains of Trp780 and Pro778 as it was
thought that its orientation in the native structure may be influ-
enced by the Ras binding domain loop from a neighbouring
p110
tation is more consistent with that observed in the wortmannin
bound p110 structure (PDB code 3HHM). The side chain of
a
molecule located in the ATP-binding site.15 The new orien-
4.6. Xenograft studies
a
Age-matched specific pathogen-free female Rag1ꢀ/ꢀ mice
were subcutaneously inoculated with 5 ꢂ 106 HCT-116 cells in
phosphate buffered saline. Tumour volume (mm3) was calculated
Ser863 was also redirected toward the affinity pocket. All sidechain
adjustments used the Lovell rotamer library as implemented in
SYBYL8.0.3. As the neighbouring molecule may also affect the
using the formula (L ꢂ w2) ꢂ
p/6 (where; L = longest tumour diam-
p110
MODELLER29 (9v3) was used to incorporate a C-terminal helix into
the 2RD0 structure after K12 based on that present in the p110
structure 1EU7. The p110 residues Lys1065 to Asp1070 were
aligned to the p110 residues Leu1036 to Lys1041, with the
p110 residues Gln1042 to His1065 modelled based on corre-
a
C-terminal tail, predicted by PSI-PRED28 to be helical,
eter and w = perpendicular diameter). Dosing began when tumours
reached a volume of approximately 150 mm3. Compound 5x
and PIK-75 were administered in solution in 7.5% DMSO, 42.5%
PEG-400, 20% 2-hydroxypropyl-b-cyclodextrin, 30% water by ip
injection at 20 mg/kg q.d. for 14 days. Control animals were admin-
istered the control vehicle alone. Animal bodyweight was mea-
sured daily, and tumour volume measured three times per week.
Mice were culled if bodyweight loss exceeded 20% of starting
weight. All animal experiments followed protocols approved by
the Animal Ethics Committee of The University of Auckland. The
statistical significance of mean tumour volume at study comple-
tion was determined by 1-way ANOVA with Holm-Sidak multiple
comparison analysis using SigmaPlot 11.0.
a
c
c
a
a
sponding positions in 1EU7. All other 2RD0 amino acids were kept
in their original position. A loop was also generated by Modeller for
the activation residues Lys941 to Glu950 while the remaining
structure was kept fixed. These additional structures were not
included in the docking cavity.
Low energy ligand conformations were built by OMEGA2.2.1
(Openeyes, Santa Fe) using default conditions with the MMFF94s
forcefield option and dielectric set to 80. Subsequent modifications
were performed with the SKETCHER module of SYBYL8.0.3 with
optimisation by MAXIMIN2 using the MMFF94s forcefield with
MMFF94 charges and a distance dependent dielectric function with
a dielectric constant of 80, and converged to 0.05 Kcal/mol ꢃ Å.
Ligands were docked into an 18 Å cavity centred on Ile800 using
GOLD30 (v5.0.1) with search efficiency set at 200%. The Chemscore
scoring function with kinase specific modification was used, and 20
dockings were performed with all poses kept. All ligand flexibility
options were turned on with the exception of internal hydrogen
bonds, the flip option was selected when available, and the solvate
all option was also turned off as well. Poses were then mimimised
and rescored in GOLD using the same scoring function.
Acknowledgments
The authors would like to thank Maruta Boyd for the NMR
spectra, and Ripudaman Singh and Ping Shang for technical assis-
tance. This work was funded by the Health Research Council of
New Zealand and Maurice Wilkins Centre for Molecular
Biodiscovery.
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