Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.11.078

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.

Full text of DOI:10.1016/j.bmcl.2016.11.078

Accepted Manuscript
Novel Pyrazolo[1,5-a]pyridines with Improved Aqueous Solubility as p110α-
Selective PI3 Kinase Inhibitors
Jackie D. Kendall, Anna C. Giddens, Kit Yee Tsang, Elaine S. Marshall, Claire
L. Lill, Woo-Jeong Lee, Sharada Kolekar, Mindy Chao, Alisha Malik, Shuqiao
Yu, Claire Chaussade, Christina Buchanan, Stephen M.F. Jamieson, Gordon W.
Rewcastle, Bruce C. Baguley, William A. Denny, Peter R. Shepherd
PII:
S0960-894X(16)31243-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.078
BMCL 24473
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 November 2016
23 November 2016
24 November 2016
Please cite this article as: Kendall, J.D., Giddens, A.C., Yee Tsang, K., Marshall, E.S., Lill, C.L., Lee, W-J., Kolekar,
S., Chao, M., Malik, A., Yu, S., Chaussade, C., Buchanan, C., Jamieson, S.M.F., Rewcastle, G.W., Baguley, B.C.,
Denny, W.A., Shepherd, P.R., Novel Pyrazolo[1,5-a]pyridines with Improved Aqueous Solubility as p110α-
Selective PI3 Kinase Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.11.078
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel Pyrazolo[1,5-a]pyridines with Improved Aqueous Solubility as p110α-Selective PI3
Kinase Inhibitors
Jackie D. Kendall,*,a,b Anna C. Giddens,a Kit Yee Tsang,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,d
Christina Buchanan,b,c Stephen M.F. Jamieson,a,b Gordon W. Rewcastle,a,b Bruce C. Baguley,a,b
William A. Denny,a,b Peter R. Shepherd.b,c
aAuckland Cancer Society Research Centre, School of Medical and Health Sciences, The University
of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
bMaurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag
92019, Auckland 1142, New Zealand.
cDepartment of Molecular Medicine and Pathology, School of Medical and Health Sciences, The
University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
dPresent address: Galderma R&D, Nestle Skin Health, Sophia Antipolis, France.
*To whom correspondence should be addressed. E-mail: j.kendall@auckland.ac.nz.
Abstract
As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase
inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic
amine. The compounds demonstrated comparable p110α potency and selectivity to earlier
compounds but with up to 1000x greater aqueous solubility, as the hydrochloride salts. The
compounds also displayed good activity in a cellular assay of PI3 kinase activity.
Keywords
PI3 kinase; PI3K; p110α; pyrazolo[1,5-a]pyridine; sulfonohydrazide; aqueous solubility.
Phosphoinositide-3-kinases (PI3 kinases) are lipid kinases which phosphorylate the 3’-hydroxyl group
of phosphatidylinositol 4,5-diphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3).
PIP3 subsequently recruits pleckstrin homology (PH) domain containing proteins, primarily protein
kinase B (PKB, also known as Akt) to the cell membrane. Once recruited, PKB is phosphorylated
and activated, leading to a cascade of cell signalling which control a range of cellular processes like
cell proliferation, growth and survival.

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 and sequence homology. The class Ia PI3
kinases are heterodimeric, consisting of a catalytic subunit (p110α, p110β or p110δ) in complex with
a regulatory subunit.1 PIK3CA, the gene encoding for p110α, is often over-expressed and mutated in
many cancer types. Two of the most common of these mutations (E545K and H1047R) have been
confirmed as activating mutations and hence increase levels of PIP3.2 Mutations in p110β and p110δ
are much less common.1
Inhibitors of PI3 kinase are widely regarded as an important new strategy in cancer treatment,3, 4
where they cover a wide range of structural types and isoform selectivity profiles.5 The different
isoforms each have a different purpose: p110α in solid tumours, p110β in thrombosis and p110δ in
haematological cancers.5 Indeed the p110δ-selective inhibitor idelalisib (CAL-101), has now been
approved for use in the clinic for relapsed chronic lymphocytic leukaemia (CLL), relapsed follicular
B-cell non-Hodgkin lymphoma (NHL) and relapsed small lymphocytic leukaemia (SLL).6
However, few highly p110α-selective PI3 kinase inhibitors have been reported, notably A667 and
closely related alpelisib, also known as BYL719 (Figure 1)8 which is currently in phase II clinical
trials. Alpelisib is showing promise in combination with aromatase inhibitor letrozole, where 9 out of
26 patients achieved a lack of disease progression at 6 months in a phase Ib study in breast cancer.9
The majority of these patients had ER+/PIK3CA mutant breast cancer. These results suggest that the
development of isoform selective PI3 kinase inhibitors is still of benefit in the clinic encouraged us to
continue in our study of p110α-selective PI3 kinase inhibitors.
Figure 1. Structures of some p110α-selective PI3 kinase inhibitors.
In our previous papers we had made a series of pyrazolo[1,5-a]pyridine compounds and shown that
they have good PI3 kinase potency and selectivity for the p110α isoform.10, 11 In particular, compound
1 had a p110α IC50 of only 1.3 nM and selectivity of around 40-fold over p110β and p110δ. This
compound also showed tumour growth inhibition in a HCT-116 human tumour xenograft model in
mice.11 However, this compound was deemed not sufficiently water-soluble for further development.

In the current work we aimed to improve the aqueous solubility of these compounds and investigate
whether this improvement would result in better efficacy in vivo.
The previous work showed us that the benzene ring was tolerant to substitution, and molecular
modelling indicated that such substitutions would be pointing toward solvent,11 hence we chose to
incorporate a range of basic amine groups off that ring to improve aqueous solubility.
The aldehyde derived hydrazones were all prepared starting from either aldehyde 2a or 2b10 according
to Scheme 1. They were converted to fluorophenyl compounds 3a-c, 7a-b by our previously
published method of condensation with methylhydrazine sulfate in the presence of 2,6-lutidine
followed by addition of either a sulfonyl chloride or an acyl chloride. Substitution of the fluoride with
alcohols was achieved by initial deprotonation with sodium hydride to make 4a-c, 5a, 6a, and with
amines in THF or DMSO, to afford 4d-v, 5b-d, 6b-d, 8a-b.
Scheme 1. Reagents: i. MeHNNH2.H2SO4, 2,6-lutidine, MeOH then arylsulfonyl chloride; ii. alcohol,
NaH, THF; iii. amine, THF or DMSO; iv. MeHNNH2.H2SO4, 2,6-lutidine, MeOH then acyl chloride.

The ketone derived hydrazones were made by a slightly different method, where methyl ketones 9a-b
were reacted firstly with hydrazine hydrate followed by sulfonylation to afford 10a-b (Scheme 2).
Next, fluoro substitution with amines gave 11a-f, and finally N-methylation was achieved by reaction
with diazomethane solution to give 12a-f.
Scheme 2. Reagents: i. N2H4.H2O, MeOH, reflux; ii. 2-fluoro-5-nitrobenzenesulfonyl chloride,
pyridine, CH2Cl2; iii. amine, THF; iv. CH2N2 solution in Et2O, THF.
The compounds were all assayed against the three class I PI3 kinase isoforms: p110α, p110β and
p110δ, as well as their effect on the cell proliferation in two early passage cell lines: NZB5 and
NZOV9. The NZB5 cell line was derived from a brain tumour (medulloblastoma) and has the wild-
type gene for p110α, and the NZOV9 cell line was derived from an ovarian tumour (poorly
differentiated endometrioid adenocarcinoma) and contains a mutant p110α with a single amino acid
substitution in the kinase domain (Y1021C).
The compounds with the amine side chain linked through either oxygen (4a-c) or a secondary amine
(4d-f) all had moderate to good p110α potency and selectivity over the other isoforms, however the
cell potency was markedly less. In contrast, the compounds with an NMe linker between the aryl ring
and the amine side chain (4g-v) gave much better results against both p110α and in the cell
proliferation assay. In general, the more basic alkyl amines (4g-4l) had slightly higher IC50s for
p110α than the less basic morpholines (4m, n), pyridines (4o-t) and imidazoles (4u, v), although
dimethylaminopropyl compound 4j is an exception to this with IC50 16 nM. All of these compounds
demonstrated selectivity over the p110β and p110δ isoforms. The potency in the cell proliferation

assay gave more variable results however. Six compounds had IC50 <10 nM in the cell line NZB5 (4j,
4r-v) although there were large differences observed between the two cell lines.
We had previously found that replacement of the aryl nitro group with cyano gave only slightly
reduced potency against p110α,11 however in this instance, the comparison of 5a-d with their nitro
analogues 4b, 4i, 4n and 4o, respectively, gave higher IC50s against p110α by between 2-fold and 20-
fold. Similarly, replacement of the cyano pyrazolo[1,5-a]pyridine substituent with bromo (6a-d)
which we had previously shown was well tolerated10 gave higher p110α IC50s, although 6b was
unexpectedly potent in the cell proliferation assay against both cell lines suggesting the possibility of
off-target activity. Acyl hydrazides 8a and 8b were both much less potent than their corresponding
sulfonyl hydrazide analogues 4i and 5b against p110α and both cell lines although 8a did demonstrate
modest p110δ selectivity.
Finally, ketone hydrazones 11a-f and 12a-f all had poor activity in the cell proliferation assay even
though the isolated enzyme potencies varied widely. Pyridine compounds 11c, 11f, 12c and 12f were
the most potent of these compounds, and in particular 11f and 12c showed excellent selectivity for
p110α over p110β (800-fold and 40-fold, respectively) and p110δ (2000-fold and 100-fold,
respectively). The poor cell potency however suggests that these compounds may suffer from a lack
of cell penetration.
Table 1. Inhibition of PI3 kinase isoforms and cell proliferation.
IC50a (nM)
p110α p110β p110δ NZB5 NZOV9
Cpd Str R1
R2
R3
1b
1.3
75
21
6.0
430
9
51
54
60
140
170
410
660
640
>20000
680
57
A
A
A
A
A
A
A
A
CN NO2 O(CH2)2NMe2
>1000 >1000 100
4a
4b
4c
4d
4e
4f
CN NO2 O(CH2)2(4-morph)
CN NO2 OCH2(2-pyridyl)
CN NO2 NH(CH2)2NMe2
CN NO2 NH(CH2)2(4-morph)
CN NO2 NHCH2(2-pyridyl)
CN NO2 NMe(CH2)2NH2
CN NO2 NMe(CH2)2NHMe
220
94
440
5600
950
1300
>1000 >1000 750
>1000 27
>1000 350
>1000 470
9400
25
50
65
1100
29
4g
4h
1000
280
24
11

A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
C
C
C
C
C
C
C
C
C
C
C
C
CN NO2 NMe(CH2)2NMe2
CN NO2 NMe(CH2)3NMe2
CN NO2 NMe(CH2)2NEt2
46
16
75
79
19
26
10
5.8
15
9
670
76
230
100
66
49
55
4i
4
25
4j
330
4200
1000
510
450
530
390
410
180
160
150
740
500
92
340
49
4k
4l
CN NO2 NMe(CH2)2(1-piperid)
CN NO2 NMe(CH2)2(4-morph)
CN NO2 NMe(CH2)3(4-morph)
CN NO2 NMeCH2(2-pyridyl)
CN NO2 NMeCH2(3-pyridyl)
CN NO2 NMeCH2(4-pyridyl)
CN NO2 NMe(CH2)2(2-pyridyl)
CN NO2 NMe(CH2)2(3-pyridyl)
450
74
260
71
210
560
330
630
480
410
740
250
45
4m
4n
4o
170
130
160
150
60
90
540
44
4p
4q
4r
5
9
55
<2
4s
CN NO2 NMe(CH2)2(4-pyridyl) 17
61
<2
4t
CN NO2 NMe(CH2)2(1-imid)
CN NO2 NMe(CH2)2(4-imid)
31
100
88
5
4u
4v
18
3
57
CN CN
CN CN
CN CN
CN CN
O(CH2)2(4-morph)
NMe(CH2)2NMe2
360
81
>1000 680
2000 380
>1000 370
400
55
390
47
5a
5b
5c
NMe(CH2)3(4-morph)
NMeCH2(2-pyridyl)
240
40
21
360
960
550
<2
130
270
<2
5d
6a
Br
Br
Br
Br
NO2 O(CH2)2(4-morph)
NO2 NMe(CH2)2NMe2
NO2 NMe(CH2)3(4-morph)
NO2 NMeCH2(2-pyridyl)
51
2900
350
510
370
180
580
630
51
6b
6c
94
670
580
3100
6200
2000
9500
3200
420
3200
2800
860
2400
2300
930
3800
3000
>1000
230
93
6d
8a
CN NO2 NMe(CH2)2NMe2
68
5600
2900
460
2400
97
CN CN
NMe(CH2)2NMe2
8b
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
CN
CN
CN
Br
H
H
H
H
H
H
NMe(CH2)2NMe2
900
676
NMe(CH2)3(4-morph)
NMeCH2(2-pyridyl)
NMe(CH2)2NMe2
333
4.8
207
11
208
448
7.3
>1000 >1000 420
900
Br
NMe(CH2)3(4-morph)
NMeCH2(2-pyridyl)
NMe(CH2)2NMe2
Br
5600
292
96
CN Me
CN Me
CN Me
328
>1000
5.2
>1000 658
1800
3300
2200
NMe(CH2)3(4-morph)
NMeCH2(2-pyridyl)
NMe(CH2)2NMe2
9400
575
Br
Br
Br
Me
Me
Me
>1000
>1000
1040
>1000 3500
5100
NMe(CH2)3(4-morph)
NMeCH2(2-pyridyl)
2600
a All IC50 values are the mean of duplicate or triplicate measurements.

b Data from ref.11
The most potent compounds against both p110α and at least one of the two cell lines were assessed
for their aqueous solubility (Table 2). As hydrochloride salts, the three pyridine compounds 4r, 4s
and 4t had the lowest solubility of those tested at <50 µg/mL. It should be noted however that these
still show an improvement over compound 1 at only 1.2 µg/mL.11 Much better solubility was seen
with dimethylamino compound 4j (1200 µg/mL) and imidazoles 4u and 4v (1000 µg/mL and 900
µg/mL, respectively).
Table 2. Aqueous solubility of selected compounds as their hydrochloride salts.
Compound Aqueous solubility (µg/mL)
1a
1.2
1200
5.7
4j
4r
4s
4t
24
45
1000
900
4u
4v
a Compound 1 is neutral and hence not a salt. Data from ref.11
The three most soluble compounds were then tested in an assay of their cellular potency by measuring
the inhibition of phosphorylation of PKB at Ser473, a down-stream marker of PI3 kinase activity, in
HCT-116 cells containing the H1047R mutation of p110α (Figure 1). The IC50s for 4j, 4u and 4v
were 72 nM, 111 nM and 67 nM, respectively. This compares favourably with compound 1 with IC50
82 nM in the same assay.11

Figure 2. Inhibition of phosphorylation of PKB at Ser473 in HCT-116 cells.
Finally, compounds 4j and 4v were assessed for their tolerability in mice, and disappointingly both
compounds were found to be more toxic than 1, and so were not evaluated further.
This toxicity may indeed be related to the disparity between the potencies in the p110α enzyme assay
and the cell proliferation assay. A comparison of the IC50s against p110α and the NZB5 and NZOV9
cell lines for 4j (0.25 and 1.6 for NZB5/p110α and NZOV9/p110α, respectively), 4v (0.17 and 3.2,
respectively) and 1 (46 and 108, respectively) would suggest that 4j and 4v are behaving differently in
cells to 1, and the lower IC50s for 4j and 4v in cells may be an indicator of off-target activity resulting
in the observed toxicity. Clearly, there are a number of other factors operating in cells which are not
fully understood.
In conclusion, we made a set of novel analogues of compound 1 and found that we could greatly
improve the aqueous solubility by the addition of a basic amine group while retaining good PI3 kinase
activity and p110α selectivity. Our earlier work had directed us to investigate substitution off the 2-
position of the benzene ring. We found this position to be tolerant of quite large basic substituents,
which is consistent with the molecular modelling indicating that substituents at this position would
point out towards solvent. Unfortunately, these more soluble compounds were not suitable for further
evaluation in an in vivo efficacy model due to their toxicity.

Acknowledgements
The authors would like to thank Sisira Kumara for the aqueous solubility measurements, and Maruta
Boyd for the NMR spectra. This work was funded by the Health Research Council of New Zealand
(grant number 06/062), Maurice Wilkins Centre for Molecular Biodiscovery, and Pathway
Therapeutics Inc.
Supplementary Data
Experimental details for the synthesis and characterisation of target compounds and their
intermediates. Experimental details for all assays.
References
1.
2.
3.
4.
5.
6.
7.
Cui, W.; Cai, Y.; Zhou, X. Pathology 2014, 46, 169.
Ciraolo, E.; Morello, F.; Hirsch, E. Curr. Med. Chem. 2011, 18, 2674.
Thorpe, L. M.; Yuzugullu, H.; Zhao, J. J. Nat. Rev. Cancer 2015, 15, 7.
Wang, X.; Ding, J.; Meng, L. Acta Pharmacol. Sin. 2015, 36, 1170.
Sabbah, D. H.; Hu, J.; Zhong, H. A. Curr. Top. Med. Chem. 2016, 16, 1413.
Markham, A. Drugs 2014, 74, 1701.
Sun, M.; Hillmann, P.; Hofmann, B. T.; Hart, J. R.; Vogt, P. K. Proc. Natl. Acad. Sci. U. S. A.
2010, 107, 15547.
8.
Furet, P.; Guagnano, V.; Fairhurst, R. A.; Imbach-Weese, P.; Bruce, I.; Knapp, M.; Fritsch,
C.; Blasco, F.; Blanz, J.; Aichholz, R.; Hamon, J.; Fabbro, D.; Caravatti, G. Bioorg. Med. Chem. Lett.
2013, 23, 3741.
9.
Mayer, I. A.; Abramson, V.; Formisano, L.; Balko, J. M.; Estrada, M. V.; Sanders, M.; Juric,
D.; Solit, D.; Berger, M. F.; Won, H.; Li, Y.; Cantley, L. C.; Winer, E. P.; Arteaga, C. L. Clin. Cancer
Res. 2016, http://dx.doi.org/10.1158/1078-0432.CCR-16-0134.
10.
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. 2012, 20, 69.
11.
Kendall, J. D.; Giddens, A. C.; Tsang, K. Y.; Frédérick, R.; Marshall, E. S.; Singh, R.; 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. 2012, 20, 58.

Graphical abstract