Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR

Article abstract of DOI:10.1016/j.bmc.2011.11.031

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.

Full text of DOI:10.1016/j.bmc.2011.11.031

Bioorganic & Medicinal Chemistry 20 (2012) 58–68
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel pyrazolo[1,5-a]pyridines as p110
a
-selective PI3 kinase inhibitors:
Exploring the benzenesulfonohydrazide SAR
Jackie D. Kendall a,b, , Anna C. Giddens a, Kit Yee Tsang a, , Raphaël Frédérick a,à, Elaine S. Marshall a,
⇑
Ripudaman Singh 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,§, 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:
Structure–activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show
that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110
Received 31 August 2011
Revised 9 November 2011
Accepted 16 November 2011
Available online 25 November 2011
a
selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated
around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity.
The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phos-
phorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for
evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft
studies. This work has provided suggestions for the design of more soluble analogues.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
PI3 kinase
PI3K
PIK3CA
p110a
Pyrazolo[1,5-a]pyridine
Sulfonohydrazide
1. Introduction
(PIP3). PIP3 then recruits PH domain containing proteins such as
protein kinase B (PKB, also known as Akt) to the cell membrane.
Phosphoinositide-3-kinases (PI3Ks) are lipid kinases which
phosphorylate the 30-hydroxyl group of phosphatidylinositol 4,5-
diphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate
Once recruited, PKB is phosphorylated and activated, leading to a
cascade of cell signalling which control a range of cellular pro-
cesses like cell proliferation, growth and survival.1 PIK3CA, the gene
encoding for the p110
a isoform, is often over-expressed and mu-
tated in many cancer types.2,3
Inhibition of PI3 kinase, and in particular, selective inhibition of
Abbreviations: ATP, adenosine triphosphate; AUCinf, area under the curve to time
infinity; Cmax, maximum concentration; DMF, N,N-dimethylformamide; DMSO,
p110
ment. A recent report indicates that inhibiting p110
block tumour progression,4 however there are only a handful of re-
ports of p110
selective PI3 kinase inhibitors in the literature.4–8
a
could prove to be an important new strategy in cancer treat-
dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; i.p., intraperitoneal;
MEM, minimum essential medium; MTD, maximum tolerated dose; PDB, protein
a
alone can
data bank; PEG-400, polyethylene glycol with molecular weight 380–420 g molꢀ1
;
a
PH domain, pleckstrin homology domain; PIP2, phosphatidylinositol 4,5-diphos-
phate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PI3 kinase, phosphoinositide-
3-kinase; PKB, protein kinase B; q.d., daily; SAR, structure–activity relationships;
t1/2, biological half-life; TLC, thin layer chromatography; Tris, tris(hydroxy-
methyl)aminomethane.
Many groups are now trying to exploit PI3 kinase inhibitors as an
approach to cancer therapy, as exemplified by the number of small
molecule PI3 kinase inhibitors currently going through clinical tri-
als,9,10 however few of these are selective for p110
a.
⇑
Corresponding author.
In our earlier work, we found that pyrazolo[1,5-a]pyridine sul-
E-mail address: j.kendall@auckland.ac.nz (J.D. Kendall).
Present address: Department of Chemistry, University of Hong Kong, Pokfulam
Road, Hong Kong.
fonohydrazides provided a potent template for p110
a
selective PI3
kinase inhibitors.11 We investigated the structure–activity rela-
tionship (SAR) around the pyrazolo[1,5-a]pyridine ring and found
that only the 5-position was tolerant of substitution. Furthermore,
only small substituents were tolerated with 5-cyano (1, Table 1)
the most active of the analogues made. Although 1 showed good
Present address: Drug Design and Discovery Centre (D3C), University of Namur,
à
FUNDP, 61 Rue de Bruxelles, 5000 Namur, Belgium.
§
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.2011.11.031

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
59
in vitro activity, as demonstrated by its inhibition of phosphoryl-
ation of PKB, a downstream marker of PI3 kinase activity, it had
only modest in vivo activity in a human tumour xenograft model.
This was attributed to its poor aqueous solubility limiting the dose
level that could be administered. In this study, we investigate
further the SAR around 1 to understand better the mechanisms
which control potency and selectivity, and to identify potential
ways of preparing more soluble analogues.
Scheme 3. Reagents and conditions: (i) MeHNNH2, EtOH; (ii) 2, MeOH.
Table 1
Inhibition of PI3 kinase isoforms and cell proliferation for 1, 4a–f and 5
2. Results and discussion
2.1. Synthesis
N
N
O2N
NC
All the hydrazides were made starting from aldehyde 2.11 Con-
densation of 2 with 2-methyl-5-nitrobenzenesulfonohydrazide12
gave sulfonohydrazide 3 (Scheme 1). N-alkylation was then carried
out with either NaH and iodoethane or benzyl bromide to form
compounds 4a–b, or with an appropriate bromoalkyl amine and
Cs2CO3 to form 4c–f (Table 1).
N
RN
S
Me
O2
Cmpd
R
IC50 (nM)a
IC50 (l
M)a
p110
a p110b p110d NZB5 NZOV9
1
Me
Et
CH2Ph
(CH2)2NMe2
(CH2)2NEt2
(CH2)2N(morpholine) 4.5
(CH2)2N(piperidine)
(CH2)2OH
0.9
0.5
27
77
82
46
62
49
9.3
0.06
0.03
n.d.
0.36
3.5
0.50
1.1
0.06
1.4
0.09
2.5
0.39
1.7
0.54
0.49
0.14
Hydroxyethyl compound 5 was made from 2 by condensation
with 2-hydroxyethylhydrazine followed by sulfonylation
(Scheme 2). Sulfonohydrazides 6a–f, h–w with a range of substit-
uents around the phenyl ring (Table 2) were then made from 2
by condensation with methylhydrazine sulfate followed by sulf-
onylation with the appropriate sulfonyl chloride in a one-pot pro-
cedure. This reaction was carried out using either NaHCO3 or 2,6-
lutidine. The use of NaHCO3 necessitated an aqueous work-up to
remove inorganic impurities, whereas we later found that the pro-
cedure could be simplified by using 2,6-lutidine where the product
precipitated out of the reaction mixture and could be filtered off
without usually requiring further purification. Amino compound
6g was prepared by cleaving the trifluoroacetamide group of 6f
with Cs2CO3. Acyl hydrazides 6x,y (Table 2) were made similarly
but with using an acyl chloride instead of a sulfonyl chloride.
Finally, hydrazone 9 was made by reaction of benzylic chloride
7 with methylhydrazine using the method of Butler et al.,13 fol-
lowed by condensation with aldehyde 2 (Scheme 3).
4a
4b
4c
4d
4e
4f
5
>1000 32
490
>1000 110
230
>1000 89
51 54
180
10
140
1.3
a
All IC50 values are the mean of duplicate or triplicate measurements.
Table 2
Inhibition of PI3 kinase isoforms and cell proliferation for 6a–y and 9
N
N
NC
R
N
N
X
Me
Cmpd
X
R
IC50 (nM)a
IC50 (l
M)a
2.2. In vitro biology
p110
700
>1000 >1000 >1000 >20
a p110b p110d NZB5 NZOV9
6a
6b
6c
6d
6e
SO2 2-Me
>1000 2000
>20
12
In our previous paper, we found that 1 was a potent and selec-
SO2 2-Me, 3-NO2
SO2 2-Me, 4-NO2
SO2 2-Me, 6-NO2
SO2 2-Me, 5-
>20
>20
7.6
>20
tive inhibitor of the p110a
isoform of PI3 kinase.11 This encouraged
65
14
>1000 >1000 >20
340 100 3.7
>1000 >1000 >1000 >20
us to look further into the SAR around these compounds, in partic-
ular around the hydrazone and aryl sulfonyl moieties.
NHCOMe
6f
SO2 2-Me, 5-
>1000 >1000 >1000 2.3
9.1
NHCOCF3
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
9
SO2 2-Me, 5-NH2
SO2 2-Me, 5-CO2H
SO2 2-Me, 5-CO2Me
SO2 2-Me, 5-CN
SO2 2-Me, 5-SO2Me
SO2 2-Me, 5-CF3
SO2 2,5-Me2
>1000 >1000 >1000 n.d.
>1000 >1000 >1000 >20
n.d.
>20
1.9
0.21
0.62
1.3
990
2.4
350
21
>1000 400
2.4
130
830
60
59
200
35
0.28
0.50
0.98
100
55
13
13
36
>1000 >1000 2.1
0.88
14
Scheme 1. Reagents and conditions: (i) 2-methyl-5-nitrobenzenesulfonohydrazide,
MeOH; (ii) NaH, RX, DMF; (iii) Br(CH2)2NR2ꢁHBr, Cs2CO3, DMF.
SO2 2-Me, 5-F
SO2 2-Me, 5-Cl
SO2 2-Me, 5-Br
550
360
620
1200
280
300
44
310
230
180
410
13
39
120
24
>20
0.67
0.70
0.38
0.17
0.18
0.48
0.88
0.09
0.04
>20
1.8
0.54
0.57
0.69
0.08
0.26
0.40
0.35
0.05
0.10
>20
6.0
b
SO2 3-NO2
SO2 2-Et, 5-NO2
SO2 2-CHMe2, 5-NO2 1.8
SO2 2-F, 5-NO2
SO2 2-Cl, 5-NO2
SO2 2-OMe, 5-NO2
SO2 2-NMe2, 5-NO2
1.5
4.7
0.5
0.8
2.2
17
64
63
22
44
24
CO
CO
2-Me, 5-NO2
3-NO2
68
67
8.6
16
b
11
CH2 2-Me, 5-NO2
40
79
37
1.7
1.9
a
All IC50 values are the mean of duplicate or triplicate measurements.
3-NO2 is the equivalent of 5-NO2 when the phenyl ring does not bear another
Scheme 2. Reagents and conditions: (i) H2N(CH2)2OH, MeOH then 2,6-lutidine, 2-
methyl-5-nitrobenzenesulfonyl chloride; (ii) MeHNNH2ꢁH2SO4, NaHCO3 or 2,6-
lutidine, MeOH then sulfonyl chloride or acyl chloride; (iii) Cs2CO3, MeOH, H2O.
b
substituent.

60
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
Firstly we looked at the hydrazone nitrogen substituent (Ta-
potency and reduced selectivity between the p110
forms, a binding mode was predicted in the apo p110
where the pyrazolo[1,5-a]pyridine core was buried in the ATP-
binding site (Fig. 1). This identified potential interactions between
the heterocyclic core and the backbone amide NH group of Val851,
and its cyano group with the sidechain of Tyr836. The aryl nitro
a
and p110d iso-
structure
ble 1). We showed previously that a substituent on this nitrogen
a
was essential for good activity.11 Replacement of the methyl with
ethyl 4a gave similar activity for p110a, but the selectivity over
p110d dropped from 50-fold for 1 to only 20-fold. The larger benzyl
4b and aminoalkyl 4c–f substituents carried on this trend with a
decrease in p110d selectivity, and additionally they also gave a
group may also interact with either the p110
a specific Arg770 or
drop in p110
compounds with IC50 4.5 nM for p110
Section 2.3. Hydroxyethyl 5 however, retained excellent p110
potency and selectivity.
We next investigated the aryl nitro group (Table 2). Removal of
the nitro group (6a) gave a large loss of p110 activity compared
a
activity. Morpholine 4e was the most active of these
; this is discussed further in
Ser774 on the N-terminal lobe wall, or even the side chain
hydroxyl groups of Thr856 and Ser919 on the C-terminal lobe wall.
Maintaining an interaction between the aryl nitro group and the
active site is clearly important for potency as demonstrated by
the data for compound 6a. The absence of an interaction between
a
a
a
the sulfonyl moiety and the p110a specific Gln859, as observed in
with 1, as did moving it to the 3-position (6b). 4-Nitro compound
6c was 70-fold less potent than 1, and 6-nitro compound 6d was
only 15-fold less potent. Several nitro group replacements were
also investigated at the 5-position. Acetamide 6e, trifluoro-
acetamide 6f and amino 6g all gave a large loss of activity, as did
carboxylic acid 6h and methyl ester 6i. However, nitrile 6j only lost
compound 1,11 provides some support for the observed loss in
isoform selectivity for 4e and related compounds, and is consistent
with reduced selectivity in absence of the sulfonyl group (com-
pound 9). The predicted binding mode with the highest Chemscore
value after rescoring and minimisation had the hydrazone mor-
pholine side chain directed toward the basic residue Lys802 at
the catalytic centre (Fig. 1). In this orientation, the morpholine
oxygen may interact with this residue, while the more basic side
chains of 4c, 4d and 4f are less preferred. This interaction along
with the lower pKa of morpholine may provide some basis for
the difference in activity between 4e and 4f.
twofold activity for p110
p110b and p110d. In contrast, while methyl sulfone 6k and tri-
fluoromethyl 6l retained some (albeit less) p110 activity, they
a whilst retaining good selectivity over
a
lost virtually all selectivity against p110d. Methyl 6m and halo sub-
stituents 6n–p showed moderate potency and selectivity for
p110a.
The 2-position of the aryl ring was much more tolerant of sub-
2.4. Aqueous solubility
stituent size. A substituent here was required, as evidenced by its
removal in compound 6q with a 40-fold loss of p110
a
activity.
Compounds 4a, 5, 6v and 6w were selected for further investi-
Such a large loss of potency suggests that this methyl group con-
tributes to the relative orientation of the phenyl ring and hence
location of the nitro group. Other small substituents (6r–w) were
all well-tolerated with negligible loss of potency or selectivity.
Finally, replacement of the sulfonyl group (6x, 6y, 9) gave a
gation since they exhibited the best potency for PI3 kinase p110a,
selectivity over p110b and p110d, and inhibition of cell prolifera-
tion in the two cell lines tested. Measurement of aqueous solubility
showed that 5 was the most soluble and was 12-fold more soluble
than 1 (Table 3). Thus 5 was selected for further investigation.
slight decrease in p110a potency, but had negligible selectivity
over p110d. Interestingly when the sulfonyl group was replaced
2.5. Further in vitro screening of 5
by carbonyl (6x, y), the aryl methyl group was now not necessary
for retaining p110
ity against p110
potent for p110
tant for p110
a
potency since both 6x and 6y had similar activ-
Compound 5 was then screened against additional kinases
(Table 4). Compound 5 had similar potency against the two most com-
mon p110a mutations (E545K and H1047R) to that of wild-type
a
a
. Methylene-linked compound 9 was slightly less
. This suggests that the sulfonyl group is impor-
selectivity, but that the aryl nitro and the substitu-
a
ents on the pyrazolo[1,5-a]pyridine ring are more important for
overall PI3 kinase potency.
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
tumour (medulloblastoma), and has the wild-type gene for p110
a.
The NZOV9 cell line was derived from an ovarian tumour (poorly
differentiated endometrioid adenocarcinoma), and contains
mutant p110
a
a
enzyme with a single amino acid substitution in
the kinase domain (Y1021C).
In general, the most active compounds in the p110
a
assay were
also good inhibitors of cell proliferation, and in addition, inhibition
in the two cell lines tracked reasonably well with all values being
within fourfold of each other. Notably, two of the compounds
where the nitro group was moved around the aryl ring (6c,d),
and the two acylhydrazides (6x,y) were rather less active at inhib-
iting cell proliferation than they were for p110
they may not have good cell penetration.
a. This suggests that
2.3. Molecular modelling
Compounds with large hydrazone nitrogen substitutions such
as those in Table 1 were not expected to be potent PI3 kinase inhib-
Figure 1. Predicted binding mode for 4e (cyan ball and stick) in the p110
a ATP-
itors based on our model of compound 1 bound to p110a,
11 where
binding site. Amino acids likely to interact with the inhibitor are represented as
yellow ball and stick with possible interactions illustrated by black dashed lines.
(Image generated with PyMol).
the substituted hydrazone was adjacent to the side chains of
Gln859 and Met772. Using compound 4e as an example of good

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
61
Table 3
The plasma concentration peaked at 5 min with a Cmax of 7575 nM.
Despite a relatively short terminal t1/2 of 1.13 h, plasma AUCinf was
3163 nM h.
Aqueous solubility of selected compounds
Compound
Solubility (lg/mL)
1
4a
5
6v
6w
0.1
0.2
1.2
0.2
0.5
2.7. In vivo antitumour efficacy
Compound 5 was evaluated alongside compound 1 in an HCT-
116 human tumour xenograft model. Both compounds were dosed
at MTD (5: 40 mg/kg, 1: 20 mg/kg) daily by i.p. injection. Com-
pound 5 demonstrated antitumour activity, causing a tumour
growth delay on day 7 of 43.8 5.5% (P <0.001) relative to controls
and 37.0 6.2% (P <0.005) relative to 1 (Fig. 3A). However, its treat-
ment was associated with toxicity in 1/7 mice and moderate body-
weight loss in the remaining mice requiring treatment
discontinuation on day 7, when mean bodyweight loss exceeded
15% (Fig. 3B). Dosing was also suspended early due to bodyweight
loss for compound 1, prior to its final tumour measurement on day
10, when it had a tumour growth delay of 21.2 4.9% (P <0.01)
versus controls. The improved solubility of 5 over 1 was evident
by the presence of drug precipitation in the i.p. cavity on post-mor-
tem examination in mice treated with 1, but not in mice dosed
with 5.
Table 4
Inhibition of PI3 kinase p110
a
mutations, p110
c, and other selected kinases by 5
Kinase
% Activity remaining
1
lM
10
lM
p110
p110
p110
a
a
c
E545K
H1047R
(0.8 nM)a
(0.8 nM)a
(11 nM)a
(0.3 nM)a
(234 nM)a
DNA-PKb
mTORb
MKK1c
65
59
18
73
59
76
16
8
5
13
5
15
8
1
1
2
2
1
2
1
p38
c
MAPKc
6
2
2
9
2
6
RSK1c
TrkAc
PKAc
IRc
An alternative human tumour xenograft model, SK-OV-3, fea-
14
2
MINK1c
turing the same H1047R mutation in p110
a that is present in
HCT-116 cells, was used to test 5 at a reduced dose level (30 mg/
kg) i.p. daily for 14 days (Fig. 3C and D). At the completion of
dosing, 5 demonstrated tumour growth inhibition of 49.9 9.1%
(P <0.05) relative to controls. Compound 5 was tolerated through-
out the 14-day treatment duration, except in one mouse which
reached 20% bodyweight loss.
a
Values in parentheses represent IC50’s.
Assays were performed by Invitrogen Drug Discovery Services (Madison, WI,
b
USA).
c
Assays were performed by The National Centre for Protein Kinase Profiling
(Dundee, UK).
p110a, and eightfold selectivity over the Class Ib PI3 kinase p110c.
PI3 kinase inhibitors also commonly inhibit the closely related
kinases DNA-PK (DNA-dependent protein kinase) and mTOR
(mammalian target of rapamycin).14 Compound 5 is a potent inhib-
itor of DNA-PK but has 200-fold selectivity over mTOR. Previous
lead compound 1 showed activity against a selection of kinases
3. Conclusions
This investigation of the SAR around the pyrazolo[1,5-a]pyri-
dine PI3 kinase inhibitors shows that substitution off the hydra-
zone nitrogen and replacement of the sulfonyl both reduced the
at 1 and 10
at two concentrations (Table 4). Compound 5 showed weaker
activity for all seven kinases than 1 at 1 M, and furthermore
l
M,11 hence 5 was screened against these same kinases
p110a selectivity, with the exception of hydroxyethyl compound
5. Limited substitutions were tolerated around the phenyl ring,
and in particular the 2,5-substitution pattern was important for
PI3 kinase activity. Compound 5 also showed good inhibition of cell
proliferation and inhibition of phosphorylation of PKB, a down-
stream marker of PI3 kinase activity. It had suitable aqueous solu-
bility and pharmacokinetics for evaluation in vivo, and showed
tumour growth inhibition in two human xenograft models.
l
inhibited only two of these at >50% at this concentration: RSK1
(p90 ribosomal S6 kinase 1) and MINK1 (misshapen-like kinase
1). Compound 5 has less off-target kinase activity than 1.
The effect of 5 on a downstream marker of PI3 kinase, the phos-
phorylation of PKB, was also assessed in HCT-116 cells with the
H1047R mutation in p110a (Fig. 2). Compound 5 showed good
inhibition of phosphorylation at both Ser473 and Thr308 sites with
IC50s of 82 and 49 nM, respectively.
4. Experimental
4.1. Chemistry
2.6. Pharmacokinetics
The pharmacokinetics of compound 5 was then assessed in CD-1
NMR spectra were recorded on a Bruker Avance 400 spec-
mice.Itwasadministeredasasingledoseat10 mg/kgbyi.p.injection.
trometer; chemical shifts are reported in d using SiMe4 as the
pPKB
Total PKB
pPKB (Ser 473)
pPKB (Thr 308)
Figure 2. The effect of 5 on the phosphorylation of PKB in HCT-116 cells at Ser473 (IC50 82 nM) and Thr308 (IC50 49 nM).

62
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
Figure 3. In vivo antitumour efficacy (A, C) and bodyweight change (B, D) of 1 and 5 in mice with HCT-116 tumours (n = 6–7) (A, B), or 5 in mice with SK-OV-3 tumours
(n = 5–6) (C, D). All treatments were administered daily by i.p. injection.
internal 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 chromatog-
raphy was performed using 200–320 mesh silica gel obtained from
APS Finechem Ltd. Yields have not been optimised. Compounds 2
and 3 were prepared as described earlier.11
hexanes:EtOAc 2:1 to 1:1) gave 4a as a yellow solid (23 mg,
36%). 1H NMR d (400 MHz, CDCl3) 8.85 (d, J = 2.4 Hz, 1H), 8.55
(dd, J = 7.2, 0.9 Hz, 1H), 8.36 (dd, J = 8.4, 2.4 Hz, 1H), 8.23 (s, 1H),
8.21 (s, 1H), 8.13 (dd, J = 1.8, 0.9 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H),
7.01 (dd, J = 7.2, 1.8 Hz, 1H), 3.89 (q, J = 7.1 Hz, 2H), 2.76 (s, 3H),
1.34 (t, J = 7.1 Hz, 3H). LC–MS (APCI+) 413 (MH+, 100%). Anal. Calcd
for C18H16N6O4S: C, 52.42; H, 3.91; N, 20.38. Found C, 52.66; H,
4.08; N, 20.10.
4.1.1.2.
N-Benzyl-N0-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)-
methylene)-2-methyl-5-nitrobenzenesulfonohydrazide (4b).
NaH (6.9 mg, 60% in oil, 0.17 mmol) was added to a solution of 3
(60 mg, 0.16 mmol) in dry DMF (5 mL) at room temperature. After
1 h, benzyl bromide (27 mg, 0.16 mmol) in DMF (0.5 mL) was
added. After a further 1 h, the reaction mixture was diluted with
water and extracted twice with EtOAc. The combined extracts were
washed twice with water then with brine, dried (Na2SO4), and the
solvent removed in vacuo. Chromatography (eluting with hex-
anes:EtOAc 4:1 to 2:1) gave 4b as a yellow solid (36 mg, 49%). 1H
NMR d (400 MHz, CDCl3) 8.92 (d, J = 2.4 Hz, 1H), 8.49 (dd, J = 7.2,
0.9 Hz, 1H), 8.38 (dd, J = 8.4, 2.4 Hz, 1H), 8.07 (dd, J = 1.8, 0.9 Hz,
1H), 8.04 (s, 1H), 7.89 (s, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.38 (m,
4H), 7.31 (m, 1H), 6.97 (dd, J = 7.2, 1.8 Hz, 1H), 5.09 (s, 2H), 2.84
(s, 3H). LC–MS (APCI+) 475 (MH+, 100%). Anal. Calcd for
4.1.1. Synthesis of N-substituted sulfonohydrazides 4a–f
Unless otherwise stated, N-substituted sulfonohydrazides were
made by the following method. A suspension of N0-((5-cyanopyra-
zolo[1,5-a]pyridin-3-yl)methylene)-2-methyl-5-nitrobenzene-
sulfonohydrazide (3) (20 mg, 0.052 mmol), Br(CH2)2NR2ꢁHBr (2 equiv)
and Cs2CO3 (5 equiv) in DMF (3 mL) was stirred at room tempera-
ture for 2 h. The solution was diluted with water, extracted with
CH2Cl2, the extracts were dried (Na2SO4) and the solvents removed
in vacuo. Chromatography (eluting with a CH2Cl2:MeOH gradient)
gave the substituted hydrazide.
C23H18N6O4S: C, 58.22; H, 3.82; N, 17.71. Found C, 58.15; H, 3.97;
N, 17.45.
4.1.1.1. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-N-
ethyl-2-methyl-5-nitrobenzenesulfonohydrazide (4a).
NaH
4.1.1.3.
N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methy-
(6.9 mg, 60% in oil, 0.17 mmol) was added to a solution of 3
(60 mg, 0.16 mmol) in dry DMF (5 mL) at room temperature. After
1 h, iodoethane (25 mg, 0.16 mmol) in DMF (0.5 mL) was added.
After a further 2 h, the reaction mixture was diluted with water
and extracted twice with EtOAc. The combined extracts were
washed twice with water then with brine, dried (Na2SO4), and
the solvent removed in vacuo. Chromatography (eluting with
lene)-N-(2-(dimethylamino)ethyl)-2-methyl-5-nitrobenzene-
sulfonohydrazide (4c). Reaction of 3 (40 mg, 0.10 mmol) and
2-bromo-N,N-dimethylethylamine
hydrobromide
(36 mg,
0.15 mmol), after chromatography (eluting with CH2Cl2:MeOH
99:1 to 98:2 to 97:3) gave 4c as a yellow solid (22 mg, 47%). 1H
NMR d (400 MHz, CDCl3) 8.88 (d, J = 2.4 Hz, 1H), 8.54 (dd, J = 7.2,
1.0 Hz, 1H), 8.35 (dd, J = 8.4, 2.4 Hz, 1H), 8.33 (s, 1H), 8.23 (s, 1H),

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
63
8.12 (dd, J = 1.9, 1.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.00
(dd, J = 7.2, 1.9 Hz, 1H), 3.92 (t, J = 6.8 Hz, 2H), 2.78 (s, 3H), 2.61
(t, J = 6.8 Hz, 2H), 2.33 (s, 6H). LC–MS (APCI+) 456 (MH+, 100%).
Anal. Calcd for C20H21N7O4S.0.67 MeOH: C, 52.06; H, 5.00; N,
20.57. Found C, 51.99; H, 4.76; N, 20.45.
formylpyrazolo[1,5-a]pyridine-5-carbonitrile (2) (1 equiv) in
MeOH (5 mL). After all of the aldehyde was consumed, sulfonyl
chloride or acyl 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 or trituration then afforded
the hydrazides. Alternatively, methylhydrazine sulfate (1.2 equiv)
and 2,6-lutidine (5 equiv) were added to a solution of 2 (1 equiv)
in MeOH (5 mL). After all of the aldehyde was consumed, sulfonyl
chloride or acyl chloride (1.3 equiv) was added and the reaction
mixture stirred for a further 30 min. The hydrazide was then fil-
tered off, washed with MeOH and dried.
4.1.1.4. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-N-
(2-(diethylamino)ethyl)-2-methyl-5-nitrobenzenesulfono-
hydrazide (4d).
Reactionof 3 (20 mg, 0.052 mmol) and2-bromo-
N,N-diethylethylamine hydrobromide (27 mg, 0.10 mmol), after
chromatography (eluting with CH2Cl2:MeOH 99:1 to 98:2) gave 4d
as a yellow solid (10 mg, 40%). 1H NMR d (400 MHz, CDCl3) 8.88 (d,
J = 2.4 Hz, 1H), 8.53 (dd, J = 7.2, 1.0 Hz, 1H), 8.38 (s, 1H), 8.35 (dd,
J = 8.4, 2.4 Hz, 1H), 8.21 (s, 1H), 8.10 (s, 1H), 7.56 (d, J = 8.4 Hz, 1H),
6.99 (dd, J = 7.2, 1.8 Hz, 1H), 3.93 (m, 2H), 2.78 (m, 5H), 2.64 (m,
4H), 1.07 (m, 6H). LC–MS (APCI+) 484 (MH+, 100%). Anal. Calcd for
C22H25N7O4S.0.5 MeOH: C, 53.99; H, 5.49; N, 19.50. Found C, 54.27;
H, 5.51; N, 19.26.
4.1.3.1.
N,2-dimethylbenzenesulfonohydrazide (6a).
(30 mg, 0.18 mmol) and 2-methylbenzenesulfonyl chloride
(51 L, 0.35 mmol) using NaHCO3, after chromatography (eluting
N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
Reaction of 2
l
4.1.1.5. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
methyl-N-(2-morpholinoethyl)-5-nitrobenzenesulfono-
with hexanes:EtOAc 3:1 to 2:1 to 1:1) gave 6a as a yellow solid
(50 mg, 81%). 1H NMR d (400 MHz, CDCl3) 8.46 (dd, J = 7.2,
0.9 Hz, 1H), 8.24 (m, 1H), 8.12 (s, 1H), 7.86 (dd, J = 1.8, 0.9 Hz,
1H), 7.76 (s, 1H), 7.57–7.62 (m, 2H), 7.34 (m, 1H), 6.92 (dd,
J = 7.2, 1.8 Hz, 1H), 3.45 (s, 3H), 2.60 (s, 3H). LC–MS (APCI+) 354
(MH+, 100%). Anal. Calcd for C17H15N5O2S: C, 57.78; H, 4.28; N,
19.82. Found C, 57.89; H, 4.29; N, 20.02.
hydrazide (4e).
Reaction of 3 (40 mg, 0.10 mmol) and 4-(2-
bromoethyl)morpholine hydrobromide (43 mg, 0.16 mmol), after
chromatography (eluting with CH2Cl2:MeOH 99:1 to 98:2) gave
4e as a yellow solid (41 mg, 79%). 1H NMR d (400 MHz, CDCl3)
8.85 (d, J = 2.4 Hz, 1H), 8.55 (dd, J = 7.2, 1.0 Hz, 1H), 8.39–8.33 (m,
2H), 8.23 (s, 1H), 8.10 (dd, J = 1.8, 1.0 Hz, 1H), 7.57 (d, J = 8.5 Hz,
1H), 7.01 (dd, J = 7.2, 1.8 Hz, 1H), 3.93 (t, J = 6.6 Hz, 2H), 3.70 (m,
4H), 2.78 (s, 3H), 2.67 (t, J = 6.6 Hz, 2H), 2.55 (m, 4H). LC–MS
(APCI+) 498 (MH+, 100%). Anal. Calcd for C22H23N7O5S.0.25 H2O:
C, 52.63; H, 4.72; N, 19.53. Found C, 52.61; H, 4.71; N, 19.24.
4.1.3.2.
N,2-dimethyl-3-nitrobenzenesulfonohydrazide (6b).
N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
Reac-
tion of 2 (30 mg, 0.18 mmol) and 2-methyl-3-nitrobenzenesulfonyl
chloride (83 mg, 0.35 mmol) using NaHCO3, after chromatography
(eluting with hexanes:EtOAc 3:1 to 2:1 to 1:1) gave 6b as a yellow
solid (42 mg, 60%). 1H NMR d (400 MHz, d6-DMSO) 8.96 (dd, J = 7.2,
1.0 Hz, 1H), 8.41 (s, 1H), 8.36 (dd, J = 8.0, 1.1 Hz, 1H), 8.22 - 8.15 (m,
2H), 8.13 (dd, J = 1.9, 1.0 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.32 (dd,
J = 7.2, 1.9 Hz, 1H), 3.41 (s, 3H), 2.61 (s, 3H). LC–MS (APCI+) 399
(MH+, 100%). Anal. Calcd for C17H14N6O4S.0.1 hexanes: C, 51.82;
H, 3.78; N, 20.72. Found C, 52.08; H, 3.71; N, 21.05.
4.1.1.6. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
methyl-5-nitro-N-(2-(piperidin-1-yl)ethyl)benzenesulfono-
hydrazide (4f). Reaction of 3 (40 mg, 0.10 mmol) and 1-(2-bromo-
ethyl)piperidine hydrobromide (43 mg, 0.16 mmol), after chroma-
tography (eluting with CH2Cl2:MeOH 99:1 to 98:2) gave 4f as a
yellow solid (43 mg, 83%). 1H NMR d (400 MHz, CDCl3) 8.86 (d,
J = 2.4 Hz, 1H), 8.53 (dd, J = 7.2, 0.9 Hz, 1H), 8.39–8.33 (m, 2H),
8.21 (s, 1H), 8.08 (m, 1H), 7.56 (d, J = 8.4 Hz, 1H), 6.99 (dd, J = 7.2,
1.9 Hz, 1H), 3.96 (t, J = 6.5 Hz, 2H), 2.78 (s, 3H), 2.64 (t, J = 6.5 Hz,
2H), 2.48 (m, 4H), 1.59 (m, 4H), 1.46 (m, 2H). LC–MS (APCI+) 496
(MH+, 100%). Anal. Calcd for C23H25N7O4S: C, 55.74; H, 5.08; N,
19.79. Found C, 56.13; H, 5.39; N, 19.56.
4.1.3.3.
N,2-dimethyl-4-nitrobenzenesulfonohydrazide (6c).
N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
Reac-
tion of 2 (30 mg, 0.18 mmol) and 2-methyl-4-nitrobenzenesulfonyl
chloride15 (92 mg, 0.35 mmol) using NaHCO3, after chromatogra-
phy (eluting with CH2Cl2:MeOH 99.5:0.5) gave 6c as a yellow solid
(41 mg, 59%). 1H NMR d (400 MHz, d6-DMSO) 8.95 (dd, J = 7.2,
1.0 Hz, 1H), 8.40 (s, 1H), 8.32 (s, 1H), 8.27 (m, 2H), 8.17 (s, 1H),
8.13 (dd, J = 1.9, 1.0 Hz, 1H), 7.31 (dd, J = 7.2, 1.9 Hz, 1H), 3.41 (s,
3H), 2.71 (s, 3H). LC–MS (APCI+) 399 (MH+, 100%). Anal. Calcd for
4.1.2. Synthesis of N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)-
methylene)-N-(2-hydroxyethyl)-2-methyl-5-nitrobenzene-
sulfonohydrazide (5)
2-Hydroxyethylhydrazine (134 mg, 1.76 mmol) was added to a
solution of aldehyde 2 (200 mg, 1.17 mmol) in MeOH (30 mL).
After 1 h, 2,6-lutidine (0.27 mL, 2.3 mmol) and 2-methyl-5-nitro-
benzenesulfonyl chloride (413 mg, 1.75 mmol) were added and
the reaction mixture stirred for a further 1.5 h. The precipitated
product was filtered off, washed with a little MeOH and dried to
leave 5 as a yellow solid (302 mg, 60%). 1H NMR d (400 MHz, d6-
DMSO) 8.96 (dd, J = 7.2, 1.0 Hz, 1H), 8.73 (d, J = 2.5 Hz, 1H), 8.45–
8.38 (m, 3H), 8.08 (dd, J = 1.9, 1.0 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H),
7.32 (dd, J = 7.2, 1.9 Hz, 1H), 5.05 (t, J = 5.8 Hz, 1H), 3.99 (t,
J = 5.8 Hz, 2H), 3.68 (q, J = 5.8 Hz, 2H), 2.69 (s, 3H). LC–MS (APCI+)
429 (MH+, 100%). Anal. Calcd for C18H16N6O5S: C, 50.46; H, 3.76;
N, 19.62. Found C, 50.09; H, 3.86; N, 19.27.
C17H14N6O4S: C, 51.24; H, 3.54; N, 21.09. Found C, 50.95; H, 3.55;
N, 21.10.
4.1.3.4.
N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-6-nitrobenzenesulfonohydrazide (6d). 2-Methyl-
6-nitroaniline (1.01 g, 6.62 mmol) was suspended in concentrated
HCl (5 mL) at 0 °C. A solution of NaNO2 (690 mg, 10 mmol) in water
(2 mL) was added dropwise over 5 min, and the solution stirred for
45 min. Meanwhile, AcOH (6 mL) was saturated with SO2, then
CuCl2ꢁ2H2O (338 mg, 1.98 mmol) was added and SO2 bubbled
through for a further 10 min. The AcOH mixture was cooled to
5 °C, then the diazonium solution added over 5 min. The resulting
mixture was stirred for a further 2 h at 0 °C then 1 h at room tem-
perature. The solution was diluted with water and extracted four
times with CH2Cl2. The combined organic extracts were dried
(Na2SO4) and the solvent removed in vacuo. Chromatography
(hexanes:EtOAc 95:5 to 4:1) gave 2-methyl-6-nitrobenzenesulfonyl
chloride as a yellow powder (1.107 g, 71%). 1H NMR d (400 MHz,
4.1.3. Synthesis of hydrazides 6a–y
These were made using NaHCO3 or 2,6-lutidine as detailed
below, unless otherwise stated. Methylhydrazine sulfate
(1.2 equiv) and NaHCO3 (5 equiv) were added to a solution of 3-

64
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
CDCl3) 7.71 (t, J = 7.8 Hz, 1H), 7.60 (dm, J = 7.8 Hz, 1H), 7.48 (dm,
J = 7.8 Hz, 1H), 2.87 (s, 3H). LC–MS (APCI-) 216 (MꢀCl+O, 100%).
Reaction of 2 (201 mg, 1.18 mmol) and the above sulfonyl chloride
(587 mg, 2.49 mmol) using 2,6-lutidine, after recrystallisation from
CH2Cl2–MeOH, gave 6d as a yellow solid (213 mg, 45%). 1H NMR d
(400 MHz, d6-DMSO) 8.95 (dd, J = 7.2, 0.8 Hz, 1H), 8.41 (s, 1H), 8.21
(dd, J = 1.8, 0.8 Hz, 1H), 8.15 (s, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.76 (d,
J = 7.7 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.31 (dd, J = 7.2, 1.8 Hz, 1H),
3.44 (s, 3H), 2.63 (s, 3H). LC–MS (APCI+) 399 (MH+, 100%). Anal.
Calcd for C17H14N6O4S.0.55 H2O: C, 50.01; H, 3.73; N, 20.58. Found
C, 51.25; H, 3.54; N, 21.09.
J = 8.4 Hz, 1H), 6.89 (dd, J = 7.2, 1.9 Hz, 1H), 3.48 (s, 3H), 2.57 (s,
3H). LC–MS (APCI+) 465 (MH+, 100%). Anal. Calcd for
C19H15F3N6O3S: C, 49.14; H, 3.26; N, 18.10. Found C, 48.81; H,
3.26; N, 18.45.
4.1.3.7. 5-Amino-N0-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)-
methylene)-N,2-dimethylbenzenesulfonohydrazide (6g). Cs2CO3
(178 mg, 0.55 mmol) was added to a solution of 6f (39 mg,
84 lmol) in MeOH (5 mL) and H2O (2 mL), and at room tempera-
ture for 3 days. The MeOH was removed in vacuo, and the aqueous
residue extracted twice with CH2Cl2. The combined extracts were
dried (Na2SO4) and the solvent removed in vacuo. Chromatography
(eluting with CH2Cl2:MeOH 99.5:0.5) gave 6g as a yellow solid
(2.0 mg, 6%). HPLC purity 83%. 1H NMR d (400 MHz, CDCl3) 8.47
(dd, J = 7.2, 0.9 Hz, 1H), 8.12 (s, 1H), 7.92 (dd, J = 1.8, 0.9 Hz, 1H),
7.73 (s, 1H), 7.57 (d, J = 2.6 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.92
(dd, J = 7.2, 1.8 Hz, 1H), 6.88 (dd, J = 8.1, 2.6 Hz, 1H), 3.44 (s, 3H),
2.43 (s, 3H). LC–MS (APCI+) 369 (MH+, 100%). HRMS (ESI+) Calcd
for C17H17N6O2S: 369.1128; found (MH+) 369.1122.
4.1.3.5. N-(3-(2-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
1-methylhydrazinylsulfonyl)-4-methylphenyl)acetamide (6e). N-
p-Tolylacetamide (500 mg, 3.35 mmol) was added portionwise to
ClSO3H (1.3 mL, 20 mmol) at 0 °C. After 10 min, the reaction mix-
ture was heated to 100 °C for 2 h. The solution was dripped slowly
onto ice and stood until all of the ice had melted. Then the oily
product was extracted twice with CH2Cl2, the combined extracts
were dried (Na2SO4) and the solvent removed in vacuo to leave
5-acetamido-2-methylbenzenesulfonyl chloride as an off-white
solid (636 mg, 77%). 1H NMR d (400 MHz, d6-DMSO) 9.86 (br s,
1H), 7.79 (d, J = 2.3 Hz, 1H), 7.59 (dd, J = 8.1, 2.3 Hz, 1H), 7.02 (d,
J = 8.1 Hz, 1H), 2.44 (s, 3H), 2.00 (s, 3H). NOESY’s were observed
from the signal at 9.86 to 7.79, 9.86 to 7.59, and 2.44 to 7.02.
LC–MS (APCI+) 248 (MH+ with 35Cl, 100%), 250 (MH+ with 37Cl,
30%). Reaction of 2 (30 mg, 0.18 mmol) and the above sulfonyl
chloride (87 mg, 0.35 mmol) using NaHCO3, after chromatography
(eluting with CH2Cl2:MeOH 99:1 to 98.5:1.5) gave 6e as a yellow
solid (48 mg, 67%). 1H NMR d (400 MHz, CDCl3) 8.47 (dd, J = 7.2,
0.9 Hz, 1H), 8.18 (d, J = 2.2 Hz, 1H), 8.12 (s, 1H), 8.11 (br s, 1H),
7.96 (dd, J = 8.3, 2.2 Hz, 1H), 7.75 (s, 1H), 7.63 (br s, 1H), 7.28 (d,
J = 8.3 Hz, 1H), 6.92 (dd, J = 7.2, 1.9 Hz, 1H), 3.49 (s, 3H), 2.52 (s,
3H), 2.20 (s, 3H). LC–MS (APCI+) 411 (MH+, 100%). Anal. Calcd for
4.1.3.8. 3-(2-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
1-methylhydrazinylsulfonyl)-4-methylbenzoic acid (6h).
p-Toluic acid (500 mg, 3.67 mmol) was added portionwise to
ClSO3H (1.5 mL, 23 mmol) at 0 °C. After 10 min, the reaction mix-
ture was heated to 100 °C for 2 h. The solution was dripped slowly
onto ice and stood until all of the ice had melted. The precipitated
product was then filtered off, washed with water and dried to leave
3-(chlorosulfonyl)-4-methylbenzoic acid as a pale brown solid
(746 mg, 87%). 1H NMR d (400 MHz, d6-DMSO) 8.32 (d, J = 1.9 Hz,
1H), 7.76 (dd, J = 7.8, 1.9 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 5.55 (br
s, 1H), 2.58 (s, 3H). LC–MS (APCIꢀ) 233 (MꢀH+ with 35Cl, 100%),
235 (MꢀH+ with 37Cl, 40%). Reaction of 2 (30 mg, 0.18 mmol) and
the above sulfonyl chloride (82 mg, 0.35 mmol) using NaHCO3,
after chromatography (eluting with CH2Cl2:MeOH 98:2 to 19:1 to
9:1) gave 6h as a yellow solid (42 mg, 60%). 1H NMR d (400 MHz,
d6-DMSO) 13.30 (br s, 1H), 8.95 (dd, J = 7.2, 0.9 Hz, 1H), 8.49 (d,
J = 1.6 Hz, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H), 8.09 (dd,
J = 7.9, 1.6 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.30 (dd, J = 7.2,
1.9 Hz, 1H), 3.34 (s, 3H), 2.66 (s, 3H). LC–MS (APCI+) 398 (MH+,
100%). Anal. Calcd for C18H15N5O4S.0.66 MeOH: C, 53.55; H, 4.25;
N, 16.73. Found C, 53.37; H, 4.01; N, 16.76.
C19H18N6O3S.0.5 H2O: C, 54.41; H, 4.57; N, 20.04. Found C, 54.30;
H, 4.61; N, 20.02.
4.1.3.6. N-(3-(2-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
1-methylhydrazinylsulfonyl)-4-methylphenyl)-2,2,2-trifluoro-
acetamide (6f).
2,2,2-Trifluoro-N-p-tolylacetamide (316 mg,
1.56 mmol) was added portionwise to ClSO3H (0.62 mL, 9.3 mmol)
at 0 °C. After 10 min, the reaction mixture was heated to 100 °C for
2 h. The solution was dripped slowly onto ice and stood until all of
the ice had melted. Then the oily product was extracted twice with
CH2Cl2, the combined extracts were dried (Na2SO4) and the solvent
removed in vacuo. Chromatography (eluting with hexanes:EtOAc
98:2 to 9:1) gave firstly 5-methyl-2-(2,2,2-trifluoroacetamido)-
benzenesulfonyl chloride as a white solid (166 mg, 35%). 1H NMR
d (400 MHz, CDCl3) 9.82 (br s, 1H), 8.40 (d, J = 8.5 Hz, 1H), 7.88
(d, J = 1.9 Hz, 1H), 7.61 (dd, J = 8.5, 1.9 Hz, 1H), 2.47 (s, 3H).
NOESY’s were observed from the signal at 2.47 to 7.61, and 2.47
to 7.88. 13C NMR d 155.1 (q, J = 39 Hz), 137.8, 137.0, 132.8, 130.7,
129.0, 123.5, 115.4 (q, J = 289 Hz), 20.9. Followed by 2-methyl-5-
(2,2,2-trifluoroacetamido)benzenesulfonyl chloride as a white so-
lid (51 mg, 11%). 1H NMR d (400 MHz, CDCl3) 8.15 (d, J = 2.3 Hz,
1H), 8.06 (br s, 1H), 8.02 (dd, J = 8.4, 2.3 Hz, 1H), 7.48 (d,
J = 8.4 Hz, 1H), 2.78 (s, 3H). NOESY was observed from the signal
at 2.78 to 7.48. 13C NMR d 155.1 (q, J = 38 Hz), 143.4, 135.8,
134.5, 133.8, 126.8, 120.4, 115.5 (q, J = 289 Hz), 19.8. LC–MS
(APCI-) 300 (MꢀH+ with 35Cl, 100%), 302 (MꢀH+ with 37Cl, 30%).
Reaction of 2 (29 mg, 0.17 mmol) and 2-methyl-5-(2,2,2-trifluoro-
acetamido)benzenesulfonyl chloride (51 mg, 0.17 mmol) using
NaHCO3, after chromatography (eluting with CH2Cl2:MeOH
99.5:0.5) gave 6f as a yellow solid (54 mg, 69%). 1H NMR d
(400 MHz, CDCl3) 8.47 (dd, J = 7.2, 0.9 Hz, 1H), 8.42 (d, J = 2.4 Hz,
1H), 8.35 (br s, 1H), 8.13 (s, 1H), 7.82–7.77 (m, 3H), 7.40 (d,
4.1.3.9.
Methyl
3-(2-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)
methylene)-1-methylhydrazinylsulfonyl)-4-methylbenzoate (6i).
3-(Chlorosulfonyl)-4-methylbenzoic acid (200 mg, 0.85 mmol) was re-
fluxed in SOCl2 (1.0 mL) for 1 h. The solvent was removed in vacuo, then
MeOH (5 mL) added and the solution stirred for 1 h. The solvent was re-
moved in vacuo to leave methyl 3-(chlorosulfonyl)-4-methylbenzoate
as a pale brown solid (127 mg, 60%). 1H NMR d (400 MHz, CDCl3) 8.72
(d, J = 1.7 Hz, 1H), 8.25 (dd, J = 7.9, 1.7 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H),
3.97 (s, 3H), 2.86 (s, 3H). LC–MS (APCIꢀ) 229 (MꢀCl+O, 100%). Reaction
of 2 (30 mg, 0.18 mmol) and the above sulfonyl chloride (87 mg,
0.35 mmol) using NaHCO3, after chromatography (eluting with hex-
anes:EtOAc 2:1 to 1:1 to EtOAc) gave 6i as a yellow solid (53 mg,
74%). 1H NMR d (400 MHz, CDCl3) 8.78 (d, J = 1.5 Hz, 1H), 8.49 (d,
J = 7.2 Hz, 1H), 8.21 (dd, J = 7.9, 1.5 Hz, 1H), 8.15 (s, 1H), 7.98 (dd,
J = 1.8, 0.8 Hz, 1H), 7.84 (s, 1H), 7.45 (d, J = 7.9 Hz, 1H), 6.94 (dd,
J = 7.2, 1.8 Hz, 1H), 3.95 (s, 3H), 3.44 (s, 3H), 2.69 (s, 3H). LC–MS (APCI+)
412 (MH+, 100%). Anal. Calcd for C19H17N5O4S: C, 55.47; H, 4.16; N,
17.02. Found C, 55.72; H, 4.26; N, 17.18.
4.1.3.10.
methylene)-N,2-dimethylbenzenesulfonohydrazide (6j).
Amino-4-methylbenzonitrile (407 mg, 3.09 mmol) was suspended
in concentrated HCl (3 mL) at 0 °C. A solution of NaNO2 (320 mg,
4.64 mmol) in water (1 mL) was added dropwise over 5 min, and
5-Cyano-N0-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)-
3-

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
65
the solution stirred for 45 min. Meanwhile, AcOH (3 mL) was satu-
rated with SO2, then CuCl2ꢁ2H2O (158 mg, 0.93 mmol) was added
and SO2 bubbled through for a further 5 min. The AcOH mixture
was cooled to 5 °C, then the diazonium solution added over
5 min. The resulting mixture was stirred for a further 1.5 h, then
the precipitate filtered off, washed with a little water and dried
to leave 5-cyano-2-methylbenzenesulfonyl chloride as a yellow
2 (30 mg, 0.18 mmol) and 2,5-dimethylbenzenesulfonyl chloride
(43 mg, 0.21 mmol) using 2,6-lutidine, gave 6m as a yellow solid
(45 mg, 70%). 1H NMR d (400 MHz, CDCl3) 8.47 (dd, J = 7.2,
0.9 Hz, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.77 (s, 1H),
7.39 (dd, J = 7.7, 1.2 Hz, 1H), 7.24 (d, J = 7.7 Hz, 1H), 6.93 (dd,
J = 7.2, 1.9 Hz, 1H), 3.43 (s, 3H), 2.55 (s, 3H), 2.49 (s, 3H). LC–MS
(APCI+) 368 (MH+, 100%). Anal. Calcd for C18H17N5O2S: C, 58.84;
H, 4.66; N, 19.06. Found C, 58.73; H, 4.51; N, 19.03.
solid (167 mg, 25%). 1H NMR
d (400 MHz, CDCl3) 8.36 (d,
J = 1.7 Hz, 1H), 7.87 (dd, J = 7.9, 1.7 Hz, 1H), 7.58 (d, J = 7.9 Hz,
1H), 2.88 (s, 3H). LC–MS (APCIꢀ) 196 (MꢀCl+O, 100%). Reaction
of 2 (30 mg, 0.18 mmol) and the above sulfonyl chloride (45 mg,
0.21 mmol) using NaHCO3, after chromatography (eluting with
CH2Cl2:MeOH 99.75:0.25) gave 6j as a yellow solid (32 mg, 48%).
1H NMR d (400 MHz, d6-DMSO) 8.96 (dd, J = 7.2, 1.0 Hz, 1H), 8.41
(s, 1H), 8.32 (d, J = 1.8 Hz, 1H), 8.21 (dd, J = 1.9, 1.0 Hz, 1H), 8.16
(s, 1H), 8.05 (dd, J = 8.0, 1.8 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.32
(dd, J = 7.2, 1.9 Hz, 1H), 3.37 (s, 3H), 2.68 (s, 3H). LC–MS (APCI+)
379 (MH+, 100%). Anal. Calcd for C18H14N6O2S.0.2 H2O: C, 56.59;
H, 3.80; N, 22.00. Found C, 56.57; H, 3.86; N, 22.02.
4.1.3.14. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-5-
fluoro-N,2-dimethylbenzenesulfonohydrazide (6n).
Reac-
tion of 2 (30 mg, 0.18 mmol) and 5-fluoro-2-methylbenzene-
sulfonyl chloride (73 mg, 0.35 mmol) using NaHCO3, after chroma-
tography (eluting with hexanes:EtOAc 3:1 to 2:1 to 1:1) gave 6n as
a yellow solid (51 mg, 78%). 1H NMR d (400 MHz, CDCl3) 8.50 (dd,
J = 7.2, 0.9 Hz, 1H), 8.15 (s, 1H), 7.98 (dd, J = 1.8, 0.9 Hz, 1H), 7.85
(dd, J = 8.4, 2.7 Hz, 1H), 7.82 (s, 1H), 7.26–7.37 (m, 2H), 6.96 (dd,
J = 7.2, 1.8 Hz, 1H), 3.42 (s, 3H), 2.59 (s, 3H). LC–MS (APCI+) 372
(MH+, 100%). Anal. Calcd for C17H14FN5O2S: C, 54.98; H, 3.80; N,
18.86. Found C, 55.28; H, 3.80; N, 19.18.
4.1.3.11. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-(methylsulfonyl)benzenesulfonohydrazide
4.1.3.15.
methylene)-N,2-dimethylbenzenesulfonohydrazide
Reaction of (30 mg, 0.18 mmol) and 5-chloro-2-methyl-
5-Chloro-N0-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)-
(6o).
(6k).
4-(Methylsulfonyl)toluene (250 mg, 1.47 mmol) was
added portionwise to ClSO3H (0.6 mL, 9.0 mmol) at 0 °C. After
10 min, the reaction mixture was heated to 100 °C for 2 h. The solu-
tion was dripped slowly onto ice and stood until all of the ice had
melted. The precipitated product was then filtered off, washed with
water and dried, to leave 2-methyl-5-(methylsulfonyl)benzene-
sulfonyl chloride as a white solid (327 mg, 83%). 1H NMR d
(400 MHz, CDCl3) 8.62 (d, J = 1.9 Hz, 1H), 8.17 (dd, J = 8.0, 1.9 Hz,
1H), 7.66 (d, J = 8.0 Hz, 1H), 3.12 (s, 3H), 2.91 (s, 3H). LC–MS (APCIꢀ)
249 (MꢀCl+O, 100%). Reaction of 2 (30 mg, 0.18 mmol) and the
above sulfonyl chloride (94 mg, 0.35 mmol) using NaHCO3, after
chromatography (eluting with hexanes:EtOAc 2:1 to 1:1 to EtOAc)
gave 6k as a yellow solid (66 mg, 87%). 1H NMR d (400 MHz, CDCl3)
8.54 (d, J = 2.0 Hz, 1H), 8.52 (dd, J = 7.2, 1.0 Hz, 1H), 8.21 (dd, J = 1.8,
1.0 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J = 8.0, 2.0 Hz, 1H), 7.89 (s, 1H),
7.58 (d, J = 8.0 Hz, 1H), 6.99 (dd, J = 7.2, 1.8 Hz, 1H), 3.41 (s, 3H),
3.09 (s, 3H), 2.80 (s, 3H). LC–MS (APCI+) 432 (MH+, 100%). Anal. Calcd
for C18H17N5O4S2: C, 50.10; H, 3.97; N, 16.23. Found C, 50.35; H,
3.97; N, 16.26.
2
benzenesulfonyl chloride (47 mg, 0.21 mmol) using 2,6-lutidine,
after recrystallisation from CH2Cl2-hexanes gave 6o as a yellow
solid (31 mg, 46%). 1H NMR d (400 MHz, CDCl3) 8.51 (dd, J = 7.2,
0.9 Hz, 1H), 8.16 (s, 1H), 8.10 (d, J = 2.3 Hz, 1H), 8.01 (dd, J = 1.8,
0.9 Hz, 1H), 7.84 (s, 1H), 7.52 (dd, J = 8.2, 2.3 Hz, 1H), 7.30 (d,
J = 8.2 Hz, 1H), 6.97 (dd, J = 7.2, 1.8 Hz, 1H), 3.41 (s, 3H), 2.61 (s,
3H). LC–MS (APCI+) 388 (MH+ with 35Cl, 100%), 390 (MH+ with
37Cl, 40%). Anal. Calcd for C17H14ClN5O2S: C, 52.65; H, 3.64; N,
18.06. Found C, 52.86; H, 3.67; N, 18.15.
4.1.3.16.
methylene)-N,2-dimethylbenzenesulfonohydrazide
Reaction of (30 mg, 0.18 mmol) and 5-bromo-2-methyl-
benzenesulfonyl chloride16 (57 mg, 0.21 mmol) using 2,6-luti-
dine, gave 6p as
yellow solid (47 mg, 62%). 1H NMR
5-Bromo-N0-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)-
(6p).
2
a
d
(400 MHz, CDCl3) 8.51 (dd, J = 7.2, 1.0 Hz, 1H), 8.23 (d, J = 2.1 Hz,
1H), 8.16 (s, 1H), 8.03 (dd, J = 1.8, 1.0 Hz, 1H), 7.84 (s, 1H), 7.67
(dd, J = 8.2, 2.1 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 6.98 (dd, J = 7.2,
1.8 Hz, 1H), 3.40 (s, 3H), 2.59 (s, 3H). LC–MS (APCI+) 432 (MH+ with
79Br, 100%), 434 (MH+ with 81Br, 100%). Anal. Calcd for
4.1.3.12. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-N,2-
dimethyl-5-(trifluoromethyl)benzenesulfonohydrazide
(6l).
4-Methylbenzotrifluoride (250 mg, 1.56 mmol) was added portion-
wise to ClSO3H (0.6 mL, 9.0 mmol) at 0 °C. After 10 min, the reaction
mixture was heated to 100 °C for 2 h. The solution was dripped slowly
onto ice and stood until all of the ice had melted. The oily product was
extracted twice with CH2Cl2, the combined extracts were dried
(Na2SO4) and the solvent removed in vacuo. Chromatography (eluting
with hexanes:EtOAc 98:2) gave 2-methyl-5-(trifluoromethyl)benzene-
sulfonyl chloride as a colourless oil (150 mg, 37%). 1H NMR d (400 MHz,
CDCl3) 8.33 (m, 1H), 7.86 (dd, J = 8.0, 1.4 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H),
2.87 (s, 3H). LC–MS (APCIꢀ) 239 (MꢀCl+O, 100%). Reaction of 2 (30 mg,
0.18 mmol) and the above sulfonyl chloride (54 mg, 0.21 mmol)
using NaHCO3, after chromatography (eluting with CH2Cl2:MeOH
99.75:0.25) gave 6l as a yellow solid (36 mg, 49%). 1H NMR d
(400 MHz, CDCl3) 8.51 (dd, J = 7.2, 0.9 Hz, 1H), 8.33 (m, 1H), 8.16 (s,
1H), 8.02 (dd, J = 1.8, 0.9 Hz, 1H), 7.87 (s, 1H), 7.79 (dd, J = 8.0, 1.6 Hz,
1H), 7.51 (d, J = 8.0 Hz, 1H), 6.97 (dd, J = 7.2, 1.8 Hz, 1H), 3.41 (s, 3H),
2.73 (s, 3H). LC–MS (APCI+) 422 (MH+, 100%). Anal. Calcd for
C17H14BrN5O2S.0.05 hexanes: C, 47.59; H, 3.39; N, 16.04. Found C,
47.72; H, 3.42; N, 16.32.
4.1.3.17. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N-methyl-3-nitrobenzenesulfonohydrazide (6q).
Reaction
of 2 (30 mg, 0.18 mmol) and 3-nitrobenzenesulfonyl chloride
(78 mg, 0.35 mmol) using NaHCO3, after chromatography (eluting
with hexanes:EtOAc 3:1 to 2:1 to 1:1) gave 6q as a yellow solid
(53 mg, 79%). 1H NMR d (400 MHz, CDCl3) 8.71 (t, J = 1.9 Hz, 1H),
8.57 (d, J = 7.2 Hz, 1H), 8.48 (m, 1H), 8.40 (s, 1H), 8.29 (d,
J = 7.8 Hz, 1H), 8.21 (s, 1H), 8.02 (s, 1H), 7.83 (t, J = 8.0 Hz, 1H),
7.05 (dd, J = 7.2, 1.9 Hz, 1H), 3.32 (s, 3H). LC–MS (APCI+) 385
(MH+, 100%). Anal. Calcd for C16H12N6O4S.0.33 CH2Cl2: C, 47.56;
H, 3.09; N, 20.38. Found C, 47.72; H, 3.13; N, 20.40.
4.1.3.18. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
ethyl-N-methyl-5-nitrobenzenesulfonohydrazide
(6r).
(30 mg, 0.18 mmol) and 2-ethyl-5-nitro-
C18H14F3N5O2S: C, 51.30; H, 3.35; N, 16.62. Found C, 51.15; H, 3.34;
Reaction of
2
N, 16.48.
benzenesulfonyl chloride17 (88 mg, 0.35 mmol) using NaHCO3,
after chromatography (eluting with hexanes:EtOAc 4:1 to 1:1 to
EtOAc) gave 6r as a yellow solid (66 mg, 92%). 1H NMR d
(400 MHz, CDCl3) 8.89 (d, J = 2.4 Hz, 1H), 8.52 (dd, J = 7.2, 1.0 Hz,
4.1.3.13. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2,5-trimethylbenzenesulfonohydrazide (6m).
Reaction of

66
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
1H), 8.41 (dd, J = 8.5, 2.4 Hz, 1H), 8.18 (s, 1H), 8.07 (dd, J = 1.8,
1.0 Hz, 1H), 7.89 (s, 1H), 7.62 (d, J = 8.5 Hz, 1H), 6.98 (dd, J = 7.2,
1.8 Hz, 1H), 3.44 (s, 3H), 3.21 (q, J = 7.5 Hz, 2H), 1.33 (t, J = 7.5 Hz,
3H). LC–MS (APCI+) 413 (MH+, 100%). Anal. Calcd for C18H16N6O4S:
C, 52.42; H, 3.91; N, 20.38. Found C, 52.41; H, 3.93; N, 20.21.
C17H14N6O5S: C, 49.27; H, 3.41; N, 20.28. Found C, 49.31; H, 3.41;
N, 20.09.
4.1.3.23. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
(dimethylamino)-N-methyl-5-nitrobenzenesulfonohydrazide
(6w).
Reaction of 2 (30 mg, 0.18 mmol) and 2-(dimethyl-
4.1.3.19. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
amino)-5-nitrobenzenesulfonyl chloride19 (93 mg, 0.35 mmol)
using NaHCO3, after chromatography (eluting with hexanes:EtOAc
2:1 to 1:1 to EtOAc) gave 6w as a yellow solid (22 mg, 29%). 1H
NMR d (400 MHz, d6-DMSO) 8.95 (dd, J = 7.2, 1.0 Hz, 1H), 8.66 (d,
J = 2.8 Hz, 1H), 8.39 (s, 1H), 8.28 (dd, J = 9.3, 2.8 Hz, 1H), 8.15 (s,
1H), 8.05 (dd, J = 1.9, 1.0 Hz, 1H), 7.38 (d, J = 9.3 Hz, 1H), 7.30 (dd,
J = 7.2, 1.9 Hz, 1H), 3.39 (s, 3H), 2.99 (s, 6H). LC–MS (APCI+) 428
(MH+, 100%). Anal. Calcd for C18H17N7O4S.0.05 hexanes: C, 50.91;
H, 4.13; N, 22.71. Found C, 51.06; H, 4.03; N, 22.91.
isopropyl-N-methyl-5-nitrobenzenesulfonohydrazide
(6s).
1-Isopropyl-4-nitrobenzene (500 mg, 3.03 mmol) was added por-
tionwise to ClSO3H (1.2 mL, 18 mmol) at 0 °C. After 10 min, the
reaction mixture was heated to 100 °C for 2 h. The solution
was dripped slowly onto ice and stood until all of the ice had
melted. The oily product was extracted twice with CH2Cl2, the
combined extracts were dried (Na2SO4) and the solvent removed
in vacuo. Chromatography (eluting with hexanes:EtOAc 9:1) gave
2-isopropyl-5-nitrobenzenesulfonyl chloride as a pale yellow so-
lid (127 mg, 16%). 1H NMR d (400 MHz, CDCl3) 8.92 (d, J = 2.4 Hz,
1H), 8.50 (dd, J = 8.7, 2.4 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 4.15
(septet, J = 6.8 Hz, 1H), 1.40 (d, J = 6.8 Hz, 6H). LC–MS (APCIꢀ)
244 (MꢀCl+O, 100%). Reaction of 2 (30 mg, 0.18 mmol) and the
above sulfonyl chloride (55 mg, 0.21 mmol) using NaHCO3, after
chromatography (eluting with hexanes:EtOAc 3:1 to 2:1) gave
6s as a yellow solid (41 mg, 55%). 1H NMR d (400 MHz, CDCl3)
8.91 (d, J = 2.4 Hz, 1H), 8.51 (dd, J = 7.2, 1.0 Hz, 1H), 8.44 (dd,
J = 8.7, 2.4 Hz, 1H), 8.17 (s, 1H), 8.05 (dd, J = 1.8, 1.0 Hz, 1H),
7.87 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 6.97 (dd, J = 7.2, 1.8 Hz,
1H), 4.09 (septet, J = 6.8 Hz, 1H), 3.44 (s, 3H), 1.25 (d,
J = 6.8 Hz, 6H). LC–MS (APCI+) 427 (MH+, 100%). Anal. Calcd for
4.1.3.24. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzohydrazide (6x).
Reaction of 2
(30 mg, 0.18 mmol) and 2-methyl-5-nitrobenzoyl chloride
(70 mg, 0.35 mmol) using NaHCO3, after trituration with MeOH
gave 6x as a yellow solid (40 mg, 63%). 1H NMR d (400 MHz, d6-
DMSO) 8.91 (dd, J = 7.2, 0.9 Hz, 1H), 8.42 (s, 1H), 8.28 (s, 1H),
8.24 (dd, J = 8.4, 2.5 Hz, 1H), 8.19 (d, J = 2.5 Hz, 1H), 7.68 (d,
J = 8.4 Hz, 1H), 7.23 (dd, J = 7.2, 1.9 Hz, 1H), 7.09 (dd, J = 1.9,
0.9 Hz, 1H), 3.54 (s, 3H), 2.30 (s, 3H). LC–MS (APCI+) 363 (MH+,
100%). Anal. Calcd for C18H14N6O3: C, 59.67; H, 3.89; N, 23.19.
Found C, 59.46; H, 3.94; N, 23.11.
C19H18N6O4S: C, 53.51; H, 4.25; N, 19.71. Found C, 53.56; H,
4.1.3.25. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-
4.42; N, 19.84.
N-methyl-3-nitrobenzohydrazide (6y).
Reaction of
2
(30 mg, 0.18 mmol) and 3-nitrobenzoyl chloride (65 mg,
0.35 mmol) using NaHCO3, after trituration with MeOH gave 6y
as a yellow solid (55 mg, 90%). 1H NMR d (400 MHz, d6-DMSO)
8.93 (dd, J = 7.2, 0.9 Hz, 1H), 8.46–8.41 (m, 2H), 8.37 (ddd, J = 8.2,
2.4, 1.1 Hz, 1H), 8.30 (s, 1H), 8.06 (dt, J = 7.7, 1.3 Hz, 1H), 7.85 (m,
1H), 7.59 (s, 1H), 7.26 (dd, J = 7.2, 1.9 Hz, 1H), 3.54 (s, 3H). LC–MS
(APCI+) 349 (MH+, 100%). Anal. Calcd for C17H12N6O3: C, 58.62; H,
3.47; N, 24.13. Found C, 58.46; H, 3.54; N, 24.38.
4.1.3.20. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
fluoro-N-methyl-5-nitrobenzenesulfonohydrazide
Reaction of (250 mg, 1.46 mmol) and 2-fluoro-5-nitro-
benzenesulfonyl chloride18 (455 mg, 1.90 mmol) using 2,6-luti-
dine, gave 6t as
yellow solid (485 mg, 82%). 1H NMR
(6t).
2
a
d
(400 MHz, CDCl3) 8.93 (dd, J = 5.8, 2.9 Hz, 1H), 8.53 (dd, J = 7.2,
1.0 Hz, 1H), 8.49 (ddd, J = 9.0, 4.0, 2.9 Hz, 1H), 8.30 (dd, J = 1.8,
1.0 Hz, 1H), 8.19 (s, 1H), 7.98 (s, 1H), 7.40 (t, J = 8.9 Hz, 1H), 7.01
(dd, J = 7.2, 1.8 Hz, 1H), 3.46 (d, J = 1.6 Hz, 3H). LC–MS (APCI+)
403 (MH+, 100%). Anal. Calcd for C16H11FN6O4S: C, 47.76; H, 2.76;
N, 20.89. Found C, 48.04; H, 2.97; N, 20.69.
4.1.4. Synthesis of 3-((2-Methyl-2-(2-methyl-5-nitrobenzyl)
hydrazono)methyl)pyrazolo[1,5-a]pyridine-5-carbonitrile (9)
A solution of 2-(chloromethyl)-1-methyl-4-nitrobenzene20 (7)
(200 mg, 1.08 mmol) and methylhydrazine (0.28 mL, 5.3 mmol)
in EtOH (6 mL) was refluxed for 1 h. The solvent was removed in
vacuo, then taken up in CH2Cl2, washed with 1 M aqueous NaOH,
dried (Na2SO4) and the solvent removed in vacuo to leave 1-
methyl-1-(2-methyl-5-nitrobenzyl)hydrazine (8) as a yellow oil
(210 mg, 100%). 1H NMR d (400 MHz, CDCl3) 8.20 (d, J = 2.5 Hz,
1H), 8.04 (dd, J = 8.3, 2.5 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 3.65 (s,
2H), 3.00 (br s, 2H), 2.57 (s, 3H), 2.47 (s, 3H). LC–MS (APCI+) 196
4.1.3.21.
methylene)-N-methyl-5-nitrobenzenesulfonohydrazide (6u).
Reaction of (30 mg, 0.18 mmol) and 2-chloro-5-nitro-
2-Chloro-N0-((5-cyanopyrazolo[1,5-a]pyridin-3-yl)-
2
benzenesulfonyl chloride (90 mg, 0.35 mmol) using NaHCO3, after
chromatography (eluting with hexanes:EtOAc 3:1 to 1:1) gave 6u
as a yellow solid (20 mg, 27%). 1H NMR d (400 MHz, CDCl3) 9.16
(d, J = 2.7 Hz, 1H), 8.50 (dd, J = 7.2, 0.9 Hz, 1H), 8.40 (dd, J = 8.7,
2.7 Hz, 1H), 8.16 (s, 1H), 8.00 (dd, J = 1.8, 0.9 Hz, 1H), 7.86 (s, 1H),
7.72 (d, J = 8.7 Hz, 1H), 6.96 (dd, J = 7.2, 1.8 Hz, 1H), 3.57 (s, 3H).
LC–MS (APCI+) 419 (MH+ with 35Cl, 100%), 421 (MH+ with 37Cl,
30%). Anal. Calcd for C16H11ClN6O4S.0.33 H2O: C, 45.24; H, 2.77;
N, 19.79. Found C, 45.33; H, 2.79; N, 20.08.
(MH+, 100%).
A suspension of 2 (30 mg, 0.18 mmol) and 8
(35 mg, 0.18 mmol) was stirred in MeOH (10 mL) for 3 days. The
precipitate was filtered off and dried to leave 9 as a yellow solid
(40 mg, 66%). 1H NMR d (400 MHz, CDCl3) 8.45 (dd, J = 7.2,
0.9 Hz, 1H), 8.27 (dd, J = 1.8, 0.9 Hz, 1H), 8.18 (d, J = 2.4 Hz, 1H),
8.12 (dd, J = 8.3, 2.4 Hz, 1H), 8.07 (s, 1H), 7.49 (s, 1H), 7.41 (d,
J = 8.3 Hz, 1H), 6.86 (dd, J = 7.2, 1.8 Hz, 1H), 4.50 (s, 2H), 2.95 (s,
3H), 2.51 (s, 3H). LC–MS (APCI+) 349 (MH+, 100%). Anal. Calcd for
4.1.3.22. N0-((5-Cyanopyrazolo[1,5-a]pyridin-3-yl)methylene)-2-
methoxy-N-methyl-5-nitrobenzenesulfonohydrazide
(6v).
Reaction of (30 mg, 0.18 mmol) and 2-methoxy-5-nitro-
2
C18H16N6O2: C, 62.06; H, 4.63; N, 24.12. Found C, 61.76; H, 4.57;
benzenesulfonyl chloride (88 mg, 0.35 mmol) using NaHCO3, after
chromatography (eluting with CH2Cl2:MeOH 99.5:0.5) gave 6v as a
yellow solid (56 mg, 77%). 1H NMR d (400 MHz, d6-DMSO) 8.93
(dd, J = 7.2, 0.9 Hz, 1H), 8.72 (d, J = 2.9 Hz, 1H), 8.51 (dd, J = 9.2,
2.9 Hz, 1H), 8.37 (s, 1H), 8.12 (s, 1H), 8.07 (dd, J = 1.9, 0.9 Hz, 1H),
7.46 (d, J = 9.2 Hz, 1H), 7.29 (dd, J = 7.2, 1.9 Hz, 1H), 4.02 (s, 3H),
3.45 (s, 3H). LC–MS (APCI+) 415 (MH+, 100%). Anal. Calcd for
N, 24.06.
4.2. Enzyme assays
The Class I PI3 kinase assays were performed using a basic thin
layer chromatography technique, as described previously.21 The
PI3 kinase isoforms were prepared in-house, as described previ-

J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
67
ously.21 Reactions were made containing 0.1
enzyme, 10 -phosphatidylinositol, inhibitor (DMSO only or
DMSO + inhibitor to a final concentration of 1%), 2X Lipid Kinase
lg recombinant
vation in serum-free media. Cells were then exposed to varying con-
centrations 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 Signaling Technology (Ser473
catalogue# 9271, Thr308 catalogue# 9275).21
lg L-a
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
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
4.7. Pharmacokinetics
added, the biphasic reactions mixed and centrifuged briefly, and
the inorganic phase was removed and discarded. Following this
Age-matched specific pathogen-free male CD-1 mice were
administered a single 10 mg/kg dose by i.p. injection of 5 in 5%
DMSO, 45% PEG-400, 20% 2-hydroxypropyl-b-cyclodextrin, 30%
water. Blood samples were collected at multiple time-points after
dosing and the plasma supernatant was retained after centrifuga-
tion for 10 min at 6000 rpm at 20 °C. Quantitative analysis of
plasma was carried out by LC-MS/MS as previously described.4
Pharmacokinetic parameters were determined by non compart-
mental analysis using Phoenix WinNonlin 6.2 (Pharsight).
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
spotted onto TLC plates (Merck Ltd) and developed for 1 h in 1-pro-
panol: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.
4.8. Xenograft studies
4.3. Molecular modelling
Age-matched specific pathogen-free male Rag1ꢀ/ꢀ mice and
female NIH III nude mice were subcutaneously inoculated with
5 ꢂ 106 HCT-116 cells and 5 ꢂ 106 SK-OV-3 cells, respectively, in
phosphate buffered saline. Tumour volume (mm3) was calculated
The p110a apo structure (PDB code 2RD0) was prepared as pre-
viously described as were the ligands.11 Docking calculations were
also performed as previously described.11
using the formula (L ꢂ w2) ꢂ
p/6 (where; L = longest tumor diame-
ter and w = perpendicular diameter). Dosing began when tumours
reached a volume of approximately 150 mm3. Compounds 1 and 5
were administered q.d. in solution in 7.5% DMSO, 42.5% PEG-400,
20% 2-hydroxypropyl-b-cyclodextrin, 30% water by i.p. injection.
Control animals were treated with the dosing vehicle alone. Animal
bodyweight was measured daily, and tumour volume was mea-
sured three times per week. Treatment was discontinued if mean
bodyweight loss exceeded 15% of starting weight and 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 sig-
nificance of mean tumour volume at study completion was deter-
mined by 1-way ANOVA with Holm–Sidak multiple comparison
analysis using SigmaPlot 11.0.
4.4. Cellular assay
The early passage cell lines used in this study were developed in
this laboratory and cultured as previously described.22 Cell lines
were grown in a-modified minimal essential growth medium sup-
plemented insulin, transferrin, selenite and 5% foetal bovine serum.
Individual wells of 96-well tissue culture plates contained 1000
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
6 h. Cells were harvested and the incorporated radioactivity was
measured. Duplicate samples were analyzed 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%.
l
L. Drugs were added at 10-fold concen-
lM and plates were incubated
l
Acknowledgements
The authors would like to thank Sisira Kumara for the aqueous
solubility measurements, Maruta Boyd for the NMR spectra, and
Aaron Thompson for technical assistance. This work was funded
by the Health Research Council of New Zealand, Maurice Wilkins
Centre for Molecular Biodiscovery, and Pathway Therapeutics Inc.
4.5. Aqueous solubility
The solid compound sample was mixed with water (to make a
2 mM solution), and the suspension was sonicated for 15 mins
and then centrifuged at 13000 rpm for 6 min. An aliquot of the
clear supernatant was diluted 2-fold with water and then centri-
References and notes
1. Shuttleworth, S.; Silva, F.; Tomassi, C.; Cecil, A.; Hill, T.; Rogers, H.; Townsend,
P. Prog. Med. Chem. 2009, 48, 81.
fuged again at 13000 rpm for 6 min. A 100 lL aliquot of the clear
2. Cleary, J. M.; Shapiro, G. I. Curr. Oncol. Rep. 2010, 12, 87.
3. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Disc. 2009, 8, 627.
4. Jamieson, S.; Flanagan, J. U.; Kolekar, S.; Buchanan, C.; Kendall, J. D.; Lee, W.-J.;
Rewcastle, G. W.; Denny, W. A.; Singh, R.; Dickson, J.; Baguley, B. C.; Shepherd,
P. R. Biochem. J. 2011, 438, 53.
5. Hayakawa, M.; Kaizawa, H.; Kawaguchi, K.; Ishikawa, N.; Koizumi, T.; Ohishi,
T.; Yamano, M.; Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Waterfield,
M. D.; Parker, P.; Workman, P. Bioorg. Med. Chem. 2007, 15, 403.
6. Hayakawa, M.; Kawaguchi, K.; Kaizawa, H.; Koizumi, T.; Ohishi, T.; Yamano, M.;
Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Parker, P.; Workman, P.;
Waterfield, M. D. Bioorg. Med. Chem. 2007, 15, 5837.
supernatant was injected into the HPLC and the peak area mea-
sured. The solubility was calculated by comparing the peak area
obtained with that from a standard solution of the compound in
DMSO (after allowing for varying dilution factors and injection
volumes).
4.6. Phospho-PKB assay
7. Sun, M.; Hillmann, P.; Hofmann, B. T.; Hart, J. R.; Vogt, P. K. Proc. Natl. Acad. Sci.
U.S.A. 2010, 107, 15547.
8. Schmidt-Kittler, O.; Zhu, J.; Yang, J.; Liu, G.; Hendricks, W.; Lengauer, C.;
Gabelli, S. B.; Kinzler, K. W.; Vogelstein, B.; Huso, D. L.; Zhou, S. Oncotarget
2010, 1, 339.
HCT-116 cells were grown in MEM alpha supplemented with 10%
(v/v) foetal bovine serum, 100 units/ml penicillin and 100 lg/ml
streptomycin (all from Invitrogen). For inhibition studies, cells were
seeded in 12-well plates and grown for 1 day before overnight star-

68
J. D. Kendall et al. / Bioorg. Med. Chem. 20 (2012) 58–68
9. Ciraolo, E.; Morello, F.; Hirsch, E. Curr. Med. Chem. 2011, 18, 2674.
10. Dienstmann, R.; Rodon, J.; Markman, B.; Tabernero, J. Recent Pat. Anti-Cancer
Drug Disc. 2011, 6, 210.
16. Chen, Q.-H.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med. Chem. 2005, 13, 2459.
17. Hansch, C.; Schmidhalter, B.; Reiter, F.; Saltonstall, W. J. Org. Chem. 1956, 21,
265.
11. Kendall, J. D.; O’Connor, P. D.; Marshall, A. J.; Frédérick, R.; Marshall, E. S.; Lill, C.
L.; Lee, W.-J.; Kolekar, S.; Chao, M.; Malik, A.; Yu, S.; Chaussade, C.; Buchanan,
C.; Rewcastle, G. W.; Baguley, B. C.; Flanagan, J. U.; Jamieson, S. M. F.; Denny, W.
A.; Shepherd, P. R. Bioorg. Med. Chem. 2011, doi:10.1016/j.bmc.2011.11.029.
12. Fel’dman, I. K.; Mikhailova, V. N. Zh. Obshch. Khim. 1963, 33, 38.
13. Butler, D. E.; Alexander, S. M.; McLean, J. W.; Strand, L. B. J. Med. Chem. 1971, 14,
1052.
18. Courtin, A. Helv. Chim. Acta 1982, 65, 546.
19. Abramovitch, R. A.; Chellathurai, T.; Holcomb, W. D.; McMaster, I. T.;
Vanderpool, D. P. J. Org. Chem. 1977, 42, 2920.
20. Maulding, D. R.; Lotts, K. D.; Robinson, S. A. J. Org. Chem. 1983, 48, 2938.
21. Chaussade, C.; Rewcastle, G. W.; Kendall, J. D.; Denny, W. A.; Cho, K.; Gronning,
L. M.; Chong, M. L.; Anagnostou, S. H.; Jackson, S. P.; Daniele, N.; Shepherd, P. R.
Biochem. J. 2007, 404, 449.
14. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.; Goldenberg, D. D.;
Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.;
Williams, R. L.; Shokat, K. M. Cell 2006, 125, 733.
22. Marshall, E. S.; Baguley, B. C.; Matthews, J. H. L.; Jose, C. C.; Furneaux, C. E.;
Shaw, J. H. F.; Kirker, J. A.; Morton, R. P.; White, J. B.; Rice, M. L.; Isaacs, R. J.;
Coutts, R.; Whittaker, J. R. Oncol. Res. 2004, 14, 297.
15. Courtin, A. Helv. Chim. Acta 1976, 59, 379.