Discovery of Pyrazolopyrimidine Derivatives as Novel Dual Inhibitors of BTK and PI3Kδ

Article abstract of DOI:10.1021/acsmedchemlett.6b00356

The aberrant activation of B-cells has been implicated in several types of cancers and hematological disorders. BTK and PI3Kδ are kinases responsible for B-cell signal transduction, and inhibitors of these enzymes have demonstrated clinical benefit in certain types of lymphoma. Simultaneous inhibition of these pathways could result in more robust responses or overcome resistance as observed in single agent use. We report a series of novel compounds that have low nanomolar potency against both BTK and PI3Kδ as well as acceptable PK properties that could be useful in the development of treatments against B-cell related diseases.

Full text of DOI:10.1021/acsmedchemlett.6b00356

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Letter
Discovery of Pyrazolopyrimidine Derivatives
as Novel Dual Inhibitors of BTK and PI3K#
Brahmam Pujala, Anil K. Agarwal, Sandip MIddya, Monali Banerjee, Arjun Surya, Anjan K.
Nayak, Ashu Gupta, Sweta Khare, Rambabu Guguloth, Nitin A. Randive, Bharat U. Shinde,
Anamika Thakur, Dhananjay I. Patel, Mohd Raja, Michael J. Green, Jennifer Alfaro, Patricio
Avila, Felipe Pérez de Arce, Ramona G. Almirez, Stacy Kanno, Sebastián Bernales, David T.
Hung, Sarvajit Chakravarty, Emma McCullagh, Kevin P. Quinn, Roopa Rai, and Son M. Pham
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00356 • Publication Date (Web): 28 Oct 2016
Downloaded from http://pubs.acs.org on October 29, 2016
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ACS Medicinal Chemistry Letters
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Discovery of Pyrazolopyrimidine Derivatives as Novel Dual Inhibitors
of BTK and PI3Kδ
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Brahmam Pujala,‡ Anil K. Agarwal,‡ Sandip Middya,§ Monali Banerjee,§ Arjun Surya,§ Anjan K.
Nayak,‡ Ashu Gupta,‡ Sweta Khare,‡ Rambabu Guguloth,‡ Nitin A. Randive,‡ Bharat U. Shinde,‡
Anamika Thakur,‡ Dhananjay I. Patel,‡ Mohd. Raja,‡ Michael J. Green,† Jennifer Alfaro,∥ Patricio
Avila,∥ Felipe Pérez de Arce,∥,⊥ Ramona G. Almirez,† Stacy Kanno,† Sebastián Bernales,† David T.
Hung,† Sarvajit Chakravarty,† Emma McCullagh,† Kevin P. Quinn,† Roopa Rai† and Son M.
Pham*,†
†Medivation, Inc., 525 Market Street, 36th Floor, San Francisco, California 94105, United States
‡Integral BioSciences, Pvt. Ltd., C-64, Hosiery Complex Phase II Extension, Noida, Uttar Pradesh, 201306, India
§Curadev, Pvt. Ltd., B-87, Sector 83, Noida, Uttar Pradesh, 201305, India
∥Fundación Ciencia y Vida, Avenida Zañartu 1482, Ñuñoa, Santiago 7780272, Chile
⊥Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago
8370146, Chile
Keywords: BTK, PI3K, p110δ, inhibitor, B-cell, BCR, pyrazolopyrimidine
ABSTRACT: The aberrant activation of B-cells has been implicated in several types of cancers and hematological
disorders. BTK and PI3Kδ are kinases responsible for B-cell signal transduction and inhibitors of these enzymes have
demonstrated clinical benefit in certain types of lymphoma. Simultaneous inhibition of these pathways could result in
more robust responses or overcome resistance as observed in single agent use. We report a series of novel compounds
which have low nanomolar potency against both BTK and PI3Kδ as well as acceptable PK properties that could be useful
in
the
development
of
treatments
against
B-cell
related
diseases.
B-cell receptor (BCR) signaling is essential for normal
biphosphate
(PIP2)
to
phosphatidylinositol-3,4,5-
B-cell development, proliferation and survival.1
Constitutive activation of the signaling pathway
downstream of BCR is a hallmark of B-cell malignancies
such as chronic lymphocytic leukemia (CLL), mantle cell
lymphoma (MCL), follicular B-cell non-Hodgkin
lymphoma (FL), small lymphocytic lymphoma (SLL) and
diffuse large B-cell lymphoma (DLBCL).2,3 Inhibition of
BCR signaling is used clinically to treat B-cell
malignancies and there are several intervention points in
the downstream signaling pathway that are clinically
relevant including LYN, SYK, BTK and PI3Kδ.3,4
triphosphate (PIP3),
impacts cellular growth, proliferation, chemotaxis,
differentiation and survival.
a
key signaling molecule that
The approval of ibrutinib5 (formerly PCI-32765) and
idelalisib6 (formerly GS-1101) by the US FDA between 2013
and 2014 demonstrated the therapeutic benefit of
inhibiting BTK (ibrutinib) and PI3Kδ (idelalisib) for the
treatment of certain B-cell malignancies such as CLL and
MCL. However, de Rooij et al. reported that roughly one-
third of patients receiving ibrutinib for CLL and MCL
show primary resistance.7–9 Moreover, it is expected that a
percentage of responsive patients who receive prolonged
treatment with ibrutinib will develop resistance due to
mutations of BTK and phospholipase Cγ2 (PLCγ2), the
kinase immediately downstream of BTK.10,11 In contrast to
ibrutinib, mechanisms for resistance to idelalisib are not
as well understood.12 Although both kinases regulate BCR
signaling, they do so via parallel pathways,13 making
combination therapy, either by co-dosing or with a dual
agent, an attractive strategy for potentially achieving
Bruton’s tyrosine kinase (BTK) is a member of the TEC
family of non-receptor tyrosine kinases and is an
upstream activator of multiple antiapoptotic signaling
molecules and pathways which include phosphoinositide
3-kinase (PI3K). PI3K constitutes a family of lipid kinases
which, like BTK, are involved in cellular signaling and
control a broad number of cellular processes. The PI3K
family is comprised of four isoforms, α, β, γ and δ which
catalyze the phosphorylation of phosphatidylinositol-4,5-
1
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deeper durable responses as well as preventing resistance. the propeller-like bend and the bicyclic-group attached to
1
2
3
4
5
6
7
8
Herein we report the discovery of reversible dual
inhibitors of BTK and PI3Kδ for the potential treatment of
B-cell malignancies.
N-1 of the pyrazolopyrimidine was simplified.
The analogs were prepared following the route in
Scheme 1. Starting from iodide 3,18 a Suzuki coupling19
with boronic acid 4 afforded pyrazolopyrimidine 5 which
was subsequently modified with several mesylates.
Treatment of compounds 6-8 with BBr3 in CH2Cl2 cleaved
the methyl-groups to afford the desired compounds 9-11.
F
OH
O
NH2
N
NH2
N
N
Scheme 1a
N
N
N
N
9
N
Me
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
N
F
F
F
Me
O
OCH3
c
OH
OCH3
b
NH2
4
a
I
NH2
NH2
NH2
1, PCI-29732
2, SW13
N
N
N
N
N
N
N
N
N
N
H
N
H
N
N
N
N
N
Figure 1. Structural similarities between PCI-29732 and SW13.
R
R
3
5
6-8
9-11
In 2010, the Shokat group at UCSF reported a series of
PI3Kδ/γ dual inhibitors, the most potent being SW13
(PI3Kδ IC50 = 0.7 nM; PI3Kγ IC50 = 33 nM).14 Recognizing
the structural similarities between PCI-29732,15 a non-
covalent analog of ibrutinib, (1, BTK IC50 = 8.2 nM) and
SW13 (2), Figure 1, we speculated that appropriately
designed molecules could maintain reasonable affinity for
PI3Kδ while also demonstrating affinity for BTK.
aReagents
and
conditions:
(a)
3-Fluoro-5-
methoxyphenylboronic acid (4), Na2CO3, Pd(PPh3)4, DMF,
H2O, 75 °C, 2 h, 32%; (b) i. NaH, DMF, 0 °C-RT, ii. R-OMs, 80
°C, 2 h, 5-50%; (c) 1M BBr3 in CH2Cl2, 0 °C, 18 h, 3-50%.
Table 1. N-1 Pyrazolopyrimidine Modifications to
Give Compounds 6-11a
BTK
PI3Kδ
No.
2
R
(IC50, nM)
(IC50, nM)
NA
>10,000
1,970
584
891
<0.5
2,790
1,830
1,340
8
6
2-indanyl
1-indanyl
2-tetralinyl
2-indanyl
1-indanyl
2-tetralinyl
7
8
9
67
10
11
217
13
Figure 2: Docking of PCI-29732 (A) and an overlay of SW13
(B) to BTK (3GEN) using Glide. The bound ligand is removed
for ease of viewing.
86
9
aCompounds 7, 8, 10 and 11 were tested as racemates.
Towards that end, PCI-29732 was docked (Figure 2A)
and SW13 was overlaid (Figure 2B) onto the BTK binding
To our satisfaction, 9-11 not only retained PI3Kδ activity
but also gained activity against BTK, Table 1. In our assay,
SW13 (2) demonstrated sub-nanomolar inhibition of
PI3Kδ, which was in agreement with the value reported by
Shokat, and no inhibition of BTK. Replacing the
methylene-linked quinazolinone group of SW13 with a 2-
indanyl in 9, increased BTK activity to 67 nM while
maintaining single-digit nanomolar activity against
PI3Kδ. Although not as dramatic, similar results were
observed for 10 and 11. Interestingly, the corresponding
methoxy-containing analogs 6-8 had significantly
attenuated activity against both kinases compared to 9-11,
signifying that either the phenolic group was making
important interactions with both targets or the methoxy
group is sterically encumbered.
pocket
of
structure
3GEN16
which
has
a
pyrrolopyrimidine-containing ligand that is otherwise
identical to PCI-29732. As expected, the docked pose of
PCI-29732 makes the same important interactions as the
co-crystallized ligand, namely two hydrogen bonds with
each hinge residue, Glu475 and Met477, as well as a pi-pi
interaction with Phe540. The pose for SW13 was achieved
by superimposing the pyrazolopyrimidine of SW13 on to
the pyrrolopyrimidine of the 3GEN ligand The co-
crystallized ligand was then removed for ease of viewing.
This perspective view allowed us to quickly assess that the
large tolyl-quinazolinone of SW13 would need to be
modified to something smaller that would fit in this part
of the BTK pocket. Although the propeller-like
conformation is favorable for selective binding to PI3Kδ,17
we decided to address selectivity within the PI3K family at
a later time. With this in mind, our initial analogs were
designed with the methylene group removed to obviate
Having identified a scaffold which provided dual
activity against both BTK and PI3Kδ, we next turned our
attention towards evaluating the C-3 aryl group. As
outlined in Scheme 2, C-3 modifications were prepared by
alkylating pyrazolopyrimidine
3
with indan-2-yl
2
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methanesulfonate (12). Suzuki coupling of the resulting 3-
iodopyrazolopyrimidine, 13, with various boronic acids
provided compounds 14-21.
Table 2. C-3 Pyrazolopyrimidine Modifications to
Give Compounds 14-21
1
2
3
4
5
6
7
8
BTK
PI3Kδ
Scheme 2a
No.
14
Ar
(IC50, nM) (IC50, nM)
3-OH-Ph
NDa
142
7
NDb
4
15
3-Cl-5-OH-Ph
4-F-5-OH-Ph
4-Cl-5-OH-Ph
4-Br-5-OH-Ph
3-OH-4-Me-Ph
3-OH-4-OMe-Ph
3,4-diF-5-OH-Ph
9
16
17
61
33
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14
aReagents and conditions: (a) i. NaH, DMF, 0 °C-RT, ii. 12,
80 °C, 2 h, 51%; (b) Ar-B(OR)2, Na2CO3, Pd(PPh3)4, DMF,
H2O, 75 °C, 2-3 h, 30-70%.
18
19
20
21
12
43
84
27
172
32
3
Interestingly, removal of the fluoro-substituent had
little effect on BTK affinity but attenuated PI3Kδ activity
as demonstrated by comparing 9 and 14, Table 2.
Replacing the fluoro-substituent with
a
chloro-
aIC50 not determined, inhibition of BTK was 66% at 100 nM
b
substituent (15), maintained moderate BTK activity while
low nanomolar activity against PI3Kδ was restored,
suggesting that substitution at the meta-position may be
important for proper binding to PI3Kδ. This was further
demonstrated when the halo-substituent was moved to
the para-position as in 16-17. In these examples, the PI3Kδ
IC50 increased roughly 8-fold, compared to their
corresponding meta-substituted variants. To our surprise,
the BTK IC50 decreased nearly 10-fold in the same
examples. These were our first analogs where the potency
against BTK was greater than PI3Kδ. Increasing the size of
the para-substituent to bromo (18) maintained BTK
activity while enhancing PI3Kδ activity.
14; IC50 not determined, inhibition of PI3Kδ was 41% at 100
nM 14.
Table 3. In Vivo Mouse Pharmacokinetic Propertiesa
of Initial Hits
CL
Vz
AUCPO
(μM•h)
PO t
(h)
F
½
No.
9b
(L/h/kg) (L/kg)
(%)
5.10
9.36
7.29
3.82
1.87
9.25
6.01
11.5
0.177
0.033
0.211
0.213
2.37
3.1
2.2
0.8
3.7
6.9
11
15b
16b
18c
20b
21b
0.60
0.80
2.3
5.45
5.90
6.56
12.1
At this point, compound 18 was the most potent dual
BTK, PI3Kδ inhibitor we had made thus far. Replacing the
para-bromo group with a methyl- or methoxy-group
resulted in attenuated activity against both targets as
observed in 19 and 20, respectively. This was somewhat
surprising for BTK as we predicted that a pocket which
could accommodate the phenyl ether of PCI-29732 and
the bromo-group of 18 would be more than large enough
to accept moderately sized substituents such as methyl or
methoxy. This suggests that electronic effects on the aryl-
ring may also contribute given that electron donating
groups such as the methoxy-group in 20 appear to be
weaker BTK binders than the corresponding electron
withdrawing groups. Furthermore in the case of 20, the
disruption of an internal hydrogen bond between the
hydroxy and methoxy groups would also decrease binding
afffinity. Substituting both the meta- and para-positions
as in 21 affords a compound with good BTK activity and
excellent activity against PI3Kδ. Inspired by the in vitro
potency, we quickly sought to evaluate our compounds in
vivo. Selected compounds from our initial hits were
administered to mice and their pharmacokinetic (PK)
properties are summarized in Table 3.
6.3
0.073
0.95
1.7
b
aWinNonlin non-compartmental analysis; Dosed 5 mg/kg
IV and 15 mg/kg PO; cDosed 1 mg/kg IV and 5 mg/kg PO.
Unfortunately, all of our initial compounds performed
poorly in mouse PK studies. The results suggested that
the compounds generally suffered from high clearance
and poor bioavailability. Interestingly, 20 had markedly
lower clearance and a longer oral half-life compared to
the other examples. Appreciating that phenolic groups
could have the propensity to form O-linked conjugates in
vivo, we speculated whether the hydroxyl was a primary
contributor to the high clearance. The results for 18 and
20 would appear to corroborate this as the relative size of
the adjacent group could decrease the relative rate of
bioconjugation. In an effort to elucidate the factors which
contributed to our in vivo PK results, we tested the
compounds in a series of in vitro metabolism studies for
oxidation and glucuronidation stability, Table 4.
Table 4. In Vitro Metabolism Results of Initial Hitsa
mLM
(% R)
mUGT
(% R)
hLM
hUGT
(% R)
No.
(% R)
3
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Table 5. C-3 Pyrazolopyrimidine Optimizations
9
25
53
14
<1
13
67
66
52
75
1.3
5.2
7.0
3.7
1
2
3
4
5
6
7
8
BTKa
PI3Kδa
mUGTb
(% R)
18
20
21
No.
21
Ar
(% Inh) (% Inh)
12
3,4-diF-5-OH-Ph
3,4,5-triF-Ph
74
21
87
21
7.1
85
92
97
89
78
5.1
22
23
aPercent parent remaining (% R) after 30 min.
3-NH2-4-F-Ph
3-NH2-4,5-diF-Ph
3-NH2-4-OMe-Ph
79
63
72
8.2
26
14
The in vitro metabolism assays were performed with
either mouse or human liver microsmes which were
supplemented with either NADPH to assess oxidative
stability (mLM or hLM) or UDP-glucuronic acid to assess
glucuronidation transferase stability (mUGT or hUGT).
The results demonstrate high turnover of substrate and
identify glucuronidation as a clearance pathway in vitro
for compounds 9, 18, 20 and 21. Moreover in mice,
glucuronidation appeared to be the primary clearance
pathway for compounds 9, 18 and 21 given that the levels
of parent remaining were significantly higher under
NADPH conditions compared to when UDP-glucuronic
acid was added. In human liver microsomes, phase II
metabolism appeared to be more significant than
oxidative metabolism for all compounds tested.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
24
25
26
27
57
52
40
53
98
91
28
29
6-indolyl
46
24
78
NDc
90
5-benzimidazolyl
9.6
Armed with this information we set out to improve on
our initial hits with a directed effort to replace the
problematic phenolic group. Following Scheme 2, we
prepared a series of analogs in which the hydroxy-group
was replaced with either a fluoro- or amino-group, Table
5. In comparison to 21, trifluorophenyl-containing analog,
22, had significantly decreased inhibition of both
enzymes, however, stability towards bioconjugation was
significantly increased. This reaffirms the importance of
the phenol with respect to BTK and PI3Kδ affinity, while
demonstrating its liability to PK properties. Compounds
23-25 had substituted anilines in place of the phenol.
These compounds showed improvement in BTK
inhibition but continued to lack significant activity
against PI3Kδ. When reviewing our initial SAR, we
noticed that 20 had over a 5-fold improvement in PI3Kδ
IC50 compared to 19. This suggested to us that the
aKinase percent inhibition at 100 nM test article; bPercent
c
(% R) parent remaining after 30 min; Percent (% R) not
determined.
Table 6. N-1 Pyrazolopyrimidine Optimizations
BTK
PI3Kδ
No.
27
R
(IC50, nM) (IC50, nM)
2-indanyl
104
NDa
NDc
24
35
NDb
NDd
47
methoxy-group was able to adopt
a
favorable
(±)-30
(±)-31
(+)-32
(−)-32
(+)-33
(−)-33
2-tetralinyl
conformation when binding to PI3Kδ. We prepared cyclic
amino-ether 26 in an effort tie-back the methylene group
and preclude bond rotation. Gratifyingly, we observed
nearly a 3-fold improvement in PI3Kδ inhibition with a
moderate decrease in BTK inhibition and excellent
stability towards UGT activity. Adding a meta-fluoro-
substituent afforded 27, which further enhanced the
PI3Kδ inhibition while maintaining BTK inhibition and
UGT stability. Compound 27 was our first non-phenol
containing analog which achieved dual-inhibition at or
below IC50 = 100 nM but more importantly demonstrated
low levels of glucuronidation in our in vitro mouse UGT
assay. The indole-containing analog 28 had reduced
PI3Kδ affinity while the benzimidazole containing analog
29 lost nearly all activity against BTK compared to 27.
2-(5-F-indanyl)
2-(5-NH2-indanyl)
2-(5-NH2-indanyl)
2-(7-NH2-tetralinyl)
2-(7-NH2-tetralinyl)
32
16
176
55
19
72
aIC50 not determined, inhibition of BTK was 34% at 100 nM
b
(±)-30; IC50 not determined, inhibition of PI3Kδ was 52% at
100 nM (±)-30; cIC50 not determined, inhibition of BTK was
d
6.6% at 100 nM (±)-31; IC50 not determined, inhibition of
PI3Kδ was 26% at 100 nM (±)-31.
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Table
ACS Medicinal Chemistry Letters
7.
Biochemical
and
In
Vitro
ADME
Results
for
(+)-32
and
(−)-32
1
2
3
4
5
6
7
8
PPBf
(%)
Kinase Selectivity (Ratio)
PI3Kαa PI3Kβa PI3Kγa BMXb TECc hLM
In Vitro Metabolismd (% R)
No.
mLM
28
rLM dLM hUGT mUGT
Caco-2e
1.3
(+)-32
(−)-32
6
34
44
2
7
4
1
20
7
71
45
46
65
32
91
91
93
99
98
98
17
63
11
1.7
b
c
d
aRatio of PI3K IC50/PI3Kδ IC50; Ratio of BMX IC50/BTK IC50; Ratio of TEC IC50/BTK IC50; Percent parent remaining (% R)
after 30 min; eEfflux ratio, Papp B→A/ Papp A→B; fPercent mouse plasma protein binding (PPB)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Having identified a potential substitute for the phenol,
we next turned our attention towards improving the
biochemical affinity against both BTK and PI3Kδ. In a
similar manner to Scheme 1, we prepared optimized
compounds 30-33, Table 6. Substituting the indane of 27
with a tetralin affords (±)-30. Compared to 27, the larger
ring had little effect on PI3Kδ binding but diminished
affinity for BTK. Adding a fluoro-substituent on the aryl
portion of the indane reduced affinity for both enzymes
even further as observed in compound (±)-31. Amino-
substitutions such as in compounds 32 and 33 also had
little effect on PI3Kδ binding but now provided improved
BTK results compared to the unsubstituted analogs 27
and 30, respectively. Comparing docked poses of 27 and
32 suggested that the improvements in BTK binding were
the result of an additional H-bond interaction with
Asn484, Figure 3. Interestingly both enantiomers of 32
exhibited excellent dual-inhibition of BTK and PI3Kδ,
whereas (+)-33 had a 9-fold increase in BTK IC50
compared to (−)-33.
than (−)-32 to oxidative metabolism in mouse and dog
liver microsomes. More importantly, these analogs
showed good stability in the low affinity albeit high
capacity glucuronidation assay. Lastly, both enantiomers
were equal in regards to P-gp efflux potential on Caco-2
cells and mouse plasma protein binding.
Table 8. In Vivo Mouse Pharmacokinetic Propertiesa
of 32b
CL
Vz
AUCPO
(μM•h)
PO t
(h)
F
½
No.
(L/h/kg) (L/kg)
(%)
(+)-32
(−)-32
1.46
0.58
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9.28
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b
aWinNonlin non-compartmental analysis; Dosed 2 mg/kg
IV and 10 mg/kg PO.
Although both compounds appear to be more stable in
rats, as well as dogs, we chose to continue our studies in
mice. Firstly, we wanted to determine the extent of our
optimizations in comparison to our previous analogs
tested for mouse PK. Secondly, we were anticipating that
mice would be the primary species for our preclinical in
vivo efficacy models. Table 8 summarizes the mouse PK
properties for (+)-32 and (−)-32. Compared to our initial
leads, both compounds exhibited lower systemic
clearance which may largely be due their improved
stability against glucuronidation. Moreover, oral
exposures increased over 125-fold for both compounds
compared to 21, resulting in oral bioavailabilities of 57%
and 40% for (+)-32 and (−)-32, respectively.
We were pleased to observe that the systematic
optimizations starting from SW13 and our initial hits
resulted in a set of compounds which are: (1) potent dual-
inhibitors of BTK and PI3Kδ and (2) have improved
mouse PK properties. These compounds will serve as the
starting point for a subsequent round of optimizations
which will include cellular and in vivo proof-of-concept
(PoC) studies.
Figure 3. Docked poses of 27 (A) and 32 (B) with BTK
indicate that 32 makes an additional H-bond interaction with
Asn484.
We selected both enantiomers of 32 for additional
biochemical profiling in an effort to distinguish between
the two molecules, Table 7. In a lipid kinase panel
consisting of the various PI3K isoforms, (−)-32 was more
selective than (+)-32 when compared against PI3Kδ. The
opposite trend was observed for the two non-receptor
tyrosine kinases. When comparing against BTK, (+)-32
was slightly more selective against BMX, non-receptor
tyrosine kinase which is closely related to BTK, and TEC
versus (−)-32. In general, both compounds performed
similarly in our full metabolism panel which included rat
(rLM) and dog (dLM) liver microsomes. The only clear
distinction was that (+)-32 appeared to be more stable
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental procedures and analytical data for 3, 5-33;
descriptions for in vitro biochemical and metabolic stability
assays; descriptions for in vivo PK experiments; description
for molecular modeling. (PDF)
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Stilgenbauer, S.; Dreyling, M.; Jedrzejczak, W. W.; Johnson, P.;
AUTHOR INFORMATION
Corresponding Author
*E-mail: son.pham@medivation.com.
Notes
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2
3
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Spurgeon, S. E.; Li, L.; Zhang, L.; Newberry, K.; Ou, Z.; Cheng, N.;
Fang, B.; McGreivy, J.; Clow, F.; Buggy, J. J.; Chang, B. Y.;
Beaupre, D. M.; Kunkel, L. A.; Blum, K. A. Targeting BTK with
Ibrutinib in Relapsed or Refractory Mantle-Cell Lymphoma. N.
Engl. J. Med. 2013, 369, 507–516.
5
(9)
Byrd, J. C.; Furman, R. R.; Coutre, S. E.; Flinn, I. W.;
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This research was funded solely by Medivation, Inc. The
authors declare no competing financial interest.
Burger, J. A.; Blum, K. A.; Grant, B.; Sharman, J. P.; Coleman, M.;
Wierda, W. G.; Jones, J. A.; Zhao, W.; Heerema, N. A.; Johnson,
A. J.; Sukbuntherng, J.; Chang, B. Y.; Clow, F.; Hedrick, E.; Buggy,
J. J.; James, D. F.; O’Brien, S. Targeting BTK with Ibrutinib in
Relapsed Chronic Lymphocytic Leukemia. N. Engl. J. Med. 2013,
369, 32–42.
(10) Woyach, J. A.; Furman, R. R.; Liu, T.-M.; Ozer, H. G.;
Zapatka, M.; Ruppert, A. S.; Xue, L.; Li, D. H.-H.; Steggerda, S.
M.; Versele, M.; Dave, S. S.; Zhang, J.; Yilmaz, A. S.; Jaglowski, S.
M.; Blum, K. A.; Lozanski, A.; Lozanski, G.; James, D. F.;
Barrientos, J. C.; Lichter, P.; Stilgenbauer, S.; Buggy, J. J.; Chang,
B. Y.; Johnson, A. J.; Byrd, J. C. Resistance Mechanisms for the
Bruton’s Tyrosine Kinase Inhibitor Ibrutinib. N. Engl. J. Med.
2014, 370, 2286-2294.
ACKNOWLEDGMENT
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The authors would like to thank Anup Barde, Vijay Sharma,
Shailender Chauhan, Abhinandan Danodia, Kailas Thube and
Ankesh Sharma for their help with the scale-up of key
intermediates; Diksha Sharma, Vishal Budha and Rahul
Takalkar for their assistance in the purification of
intermediates and final compounds; and Shivam Dixit and
Meena Bisht for their assistance in acquiring NMR data. We
also thank Christopher Higgs for his analysis and comments
regarding the preparation of this manuscript.
(11)
Komarova, N. L.; Burger, J. A.; Wodarz, D. Evolution of
ABBREVIATIONS
Ibrutinib Resistance in Chronic Lymphocytic Leukemia (CLL).
Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13906–13911.
AUCpo, Area under the concentration-time curve from time
zero to the last quantifiable concentration after oral (PO)
administration; BCR, B-cell receptor; BMX, Bone marrow
kinase on chromosome X; CL, Total body clearance after
intravenous (IV) administration; F, Absolute oral
(12)
Wiestner, A. The Role of B-Cell Receptor Inhibitors in
the Treatment of Patients with Chronic Lymphocytic Leukemia.
Haematologica 2015, 100, 1495–1507.
(13)
Suzuki, H.; Matsuda, S.; Terauchi, Y.; Fujiwara, M.;
Ohteki, T.; Asano, T.; Behrens, T. W.; Kouro, T.; Takatsu, K.;
Kadowaki, T.; Koyasu, S. PI3K and Btk Differentially Regulate B
Cell Antigen Receptor-Mediated Signal Transduction. Nat.
Immunol. 2003, 4, 280–286.
(14) Williams, O.; Houseman, B. T.; Kunkel, E. J.;
Aizenstein, B.; Hoffman, R.; Knight, Z. A.; Shokat, K. M.
Discovery of Dual Inhibitors of the Immune Cell PI3Ks p110delta
and p110gamma: A Prototype for New Anti-Inflammatory Drugs.
Chem. Biol. 2010, 17, 123–134.
bioavailability
DoseIV/DosePO
determined
HPβCD, 2-Hydroxypropyl-β-cyclodextrin;
from
AUCpo/AUCiv
×
;
LM, Liver microsomes; LYN, Lck/Yes novel tyrosine kinase;
ND, Not determined; PEG, Polyethylene glycol; PO t1/2,
Terminal half-life after oral administration; SYK, Spleen
tyrosine kinase; TEC, Tyrosine-protein kinase Tec; UGT,
Uridine 5ʹ-diphospho-glucuronosyltransferase, Vz, Total
volume of distribution after IV administration.
(15)
Pan, Z.; Scheerens, H.; Li, S.-J.; Schultz, B. E.;
Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.;
Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.;
Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T.
Discovery of Selective Irreversible Inhibitors for Bruton’s
Tyrosine Kinase. ChemMedChem 2007, 2, 58–61.
(16) Marcotte, D. J.; Liu, Y.-T.; Arduini, R. M.; Hession, C.
A.; Miatkowski, K.; Wildes, C. P.; Cullen, P. F.; Hong, V.;
Hopkins, B. T.; Mertsching, E.; Jenkins, T. J.; Romanowski, M. J.;
Baker, D. P.; Silvian, L. F. Structures of Human Bruton’s Tyrosine
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and PI3Kδ for Therapy of Cancer and Autoimmune Disorders. P.
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Cameron, F.; Sanford, M. Ibrutinib: First Global
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FDA Approval: Idelalisib Monotherapy for the Treatment of
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De Rooij, M. F. M.; Kuil, A.; Kater, A. P.; Kersten, M. J.;
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Figure 1. Structural similarities between PCI-29732 and SW13.
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Figure 2: Docking of PCI-29732 (A) and an overlay of SW13 (B) to BTK (3GEN) using Glide. The bound
ligand is removed for ease of viewing.
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Scheme 1
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Scheme 2
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Table 5. C-3 Pyrazolopyrimidine Optimizations
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Table 5. C-3 Pyrazolopyrimidine Optimizations
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Table 6. N-1 Pyrazolopyrimidine Optimizations
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Figure 3. Docked poses of 27 (A) and 32 (B) and 2D-interaction diagrams of 27 (C) and 32 (D) with BTK
indicate that 32 makes an additional H-bond interaction with Asn484.
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Graphical Abstract
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