Discovery and characterization of N-(1,3-dialkyl-1H-indazol-6-yl)-1H-pyrazolo[4,3-b]pyridin-3-amine scaffold as mGlu4 positive allosteric modulators that mitigate CYP1A2 induction liability

Article abstract of DOI:10.1016/j.bmcl.2018.06.034

Previous reports from our laboratory disclosed the structure and activity of a novel 1H-pyrazolo[4,3-b]pyridine-3-amine scaffold (VU8506) which showed excellent potency, selectivity and in vivo efficacy in preclinical rodent models of Parkinson's disease.

Full text of DOI:10.1016/j.bmcl.2018.06.034

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and characterization of N-(1,3-dialkyl-1H-indazol-6-yl)-1H-
pyrazolo[4,3-b]pyridin-3-amine scaffold as mGlu positive allosteric
4
modulators that mitigate CYP1A2 induction liability
Darren W. Engersa,b, Sean R. Bollingera,b, Julie L. Engers , Joseph D. Panaresea,b,
a,b
a,b
a,b
a,b
c
c
Megan M. Breiner , Alison Gregro , Anna L. Blobaum , Joanne J. Bronson , Yong-Jin Wu ,
c
a,b
a,b
a,b
John E. Macor , Alice L. Rodriguez , Rocio Zamorano , P. Jeffrey Conn
,
a,b,d
a,b,e
a,b,d,⁎
Craig W. Lindsley
Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232, USA
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA
Bristol-Myers Squibb Co., Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA
Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
Vanderbilt Kennedy Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA
, Colleen M. Niswender
, Corey R. Hopkins
a
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
mGlu4 PAM
Positive allosteric modulator
CYP induction
Parkinson’s disease
Structure-activity relationship
Previous reports from our laboratory disclosed the structure and activity of a novel 1H-pyrazolo[4,3-b]pyridine-
3-amine scaffold (VU8506) which showed excellent potency, selectivity and in vivo efficacy in preclinical rodent
models of Parkinson’s disease. Unfortunately, this compound suffered from significant CYP1A2 induction as
measured through upstream AhR activation (125-fold) and thus was precluded from further advancement in
chronic studies. Herein, we report a new scaffold developed recently which was systematically studied in order
to mitigate the CYP1A2 liabilities presented in the earlier scaffolds. We have identified a novel structure that
maintains the potency and selectivity of other mGlu
vation = 2.3-fold).
4 4
PAMs, leading to 9i (hmGlu EC50 = 43 nM; AhR acti-
Parkinson’s disease (PD) is a debilitating neurodegenerative disease
that affects > 1 million US adults. PD is a chronic movement disorder
stemming from the loss of dopaminergic neurons in the basal ganglia
4 6 7 8
of mGlu , mGu , mGlu , and mGlu . The extracellular binding domain
1
,2
contains the orthosteric binding site and the 7TM domain is the binding
site for most of the known allosteric modulators. Due to the conserved
nature of the orthosteric binding site, the development of selective
(
BG) and is characterized by tremor, rigidity, bradykinesia and posture
3
12
instability as its core disturbances. There are no cures for PD, nor are
there any disease-modifying therapies available, only medications for
subclass-specific agonists has proven difficult. Due to these chal-
lenges, our efforts have been concentrated in developing positive al-
losteric modulators (PAMs) in order to capitalize on potential ad-
vantages perceived in discovering selective agents.9
4
,5
temporary symptomatic relief. Over the past several years, one pro-
mising receptor class has emerged that has the potential to be disease-
,13,14
modifying, the metabotropic glutamate receptor 4 (mGlu
cation in the indirect pathway of the BG.
4
), due its lo-
To this end, our laboratory has discovered multiple scaffolds as
6–8
mGlu
group (e.g., VU4770, 1, Fig. 1).
very successful in early stage validation of mGlu
4
PAMs, mostly centered around a privileged picolinamide core
15,16
The metabotropic glutamate receptors (mGlu receptors) are family
C members of the GPCR family and are characterized by a large ex-
tracellular amino-terminal binding domain (venus fly trap) in addition
This scaffold moiety proved to be
PAMs as selective and
However, many of these com-
4
brain penetrant tool compounds.1
7,18
9
–11
to a seven-transmembrane spanning (7TM) domain.
There are eight
pounds suffered from significant metabolic stability issues which pre-
cluded them from any further advancement. Utilizing the picolinamide
as a template, we recently disclosed the ring closure of the picolinamide
cloned mGlu receptors and these have been further differentiated into
three sub-classes due to their ligand specificity and preferred coupling
1
1
mechanisms. The group I subfamily consists of mGlu
1
and mGlu
5
,
into the 1H-pyrazolo[4,3-b]pyridine-3-amine core scaffold (e.g.,
VU8506, 2).1
9,20
These analogs exhibited equal or better potency,
group II include mGlu
2
and mGlu
3
and the group III family is composed
⁎
Corresponding author at: Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, USA.
E-mail address: corey.hopkins@unmc.edu (C.R. Hopkins).
https://doi.org/10.1016/j.bmcl.2018.06.034
Received 3 May 2018; Received in revised form 15 June 2018; Accepted 16 June 2018
0960-894X/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Engers, D.W., Bioorganic & Medicinal Chemistry Letters (2018), https://doi.org/10.1016/j.bmcl.2018.06.034

D.W. Engers et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 1
Initial SAR around the 6,5-aryl portion.
Cmpd
R
hmGlu
4
EC50 (nM)a
%Glu Maxa,b
110.4
5a
870
n = 2
5
5
5
b
257
105.1
54.6
83.8
Fig. 1. Evolution of initial picolinamide, 1 to the final scaffold, 3.
selectivity against other mGlu receptors, and brain penetration that led
to further validation and efficacy in advanced preclinical models of PD.
Unfortunately, after further study of this scaffold, it was discovered that
c
> 10,000
3590
1
9
2
suffered from significant CYP1A2 induction. Since the major me-
d
tabolizing CYP enzyme of 2 was also CYP1A2, which led to a confirmed
risk of auto-induction in vivo, this facet of the compound proved im-
possible to overcome and thus relegated 2 to a tool compound only for
use in acute dosing paradigms. Next, we set out to explore and better
understand the SAR surrounding CYP1A2 induction and either a) sig-
nificantly reduce the overall liability of the CYP1A2 induction, or b)
lessen the overall extent of a single CYP metabolizing enzyme. Herein,
we report the results of this study in the identification of a series of
compounds that have overcome the CYP1A2 induction liability.
From previously disclosed work, we knew that further expansion of
the meta-position on the right-hand aryl moiety was not well tolerated
5e
1430
72.4
5f
Inactive
5g
Inactive
(
3
e.g., Cl position in 1 and 2); thus, we took the approach to cyclize the
,4-positions into an additional ring system and then attempt to build
1
5
off the para-position (e.g., F position in 2). First, we needed to confirm
that cyclization of the right-hand aryl group could be a productive
option. The synthesis of the first analogs starts with the commercially
available, 4, and the corresponding coupling partners (Scheme 1). Thus,
under palladium catalyzed reaction conditions, the initial targets
a
b
4
Calcium mobilization mGlu assay, n = 1 (unless otherwise noted).
Amplitude of response in the presence of 30 μM test compound, normalized
to a standard compound, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-
a-carboxamide, PHCCC (100% Glu Max).
1
(
5a–g) were obtained using the appropriately substituted 6,5-biaryl
2
1
halides in modest to good overall yields.
A number of indazole, indole and other polyaza-systems were in-
itially investigated to determine a baseline SAR around this change
(
Table 1). The initial 5-substituted-1-methyl-1H-indazole, 5a, was ac-
tive (EC50 = 870 nM; %Glu Max = 110.4) and was followed by the 6-
substituted-1-methyl-1H-indazole, 5b, which was also an active analog
(
EC50 = 257 nM; %Glu Max = 105.1). Interestingly, replacing the 6-
indazole, 5b, with the 6-benzimidazole, 5c, led to significant loss of
activity. In addition, the indole and benzotriazole exhibited modest
activity (5d, EC50 = 3,590 nM; 5e, EC50 = 1,430 nM). Lastly, the two
[
1,2,4]triazolo[4,3-a]triazines were inactive.
Based on the initial success of the replacement of the phenyl group
with the indazole moiety, we next looked to further elaborate around
the N-1 and C-3 positions of this group. A variety of synthetic proce-
dures were needed in order to obtain these products and these are
outlined in Schemes 2–4. As shown in Scheme 2, the bisalkylated in-
dazole compounds were synthesized starting with 6 followed by N-al-
2 3
Scheme 2. Reagents and conditions: (a) Cs CO , R–X (80–87%, ∼3:1 ratio,
t
t
t
t
7a:7b); (b) NaO Bu, BuXphos, Buxphos palladacycle, BuOH, 60 °C; (c) TFA,
150 °C, μW (15–55%, 2 steps).
2 3
kylation (Cs CO , R-X) which gave both the regioisomeric products
which were carried on to the next step. As seen above, the final targets
were realized after palladium-catalyzed cross-coupling with 8 followed
by separation of the regioisomers. The NH analog, 9h, could be directly
synthesized by the cross-coupling protocol.
For 3-substituted analogs that were not commercially available, we
were able to obtain these by building the indazole ring system as shown
in Scheme 3. First, 4-bromo-2-fluoropicolinamide, 11, was reacted with
an appropriate alkyl Grignard reagent followed by TPAP, NMO
t
t
t
Scheme 1. Reagents and conditions: (a) NaO Bu, BuXphos, BuXphos pallada-
t
cycle, BuOH, 60 °C, R-Br; (b) TFA, 150 °C, μW (60–86%, 2 steps).
2

D.W. Engers et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 2
Further elaboration of the right-hand indazole moiety.
Cmpd
X
Y
R
hmGlu
78.1
4
EC50 (nM)a
%Glu Maxa,c
98.0
9a
N
CH
Scheme 3. Reagents and conditions: (a) cyPr-MgBr, THF; (b) TPAP, NMO,
CH Cl , MeCN (24–33%, 2steps); (c) Methylhydrazine, n-BuOH, 150 °C μW; (d)
2 2
t
t
t
t
NaO Bu, Buxphos, BuXphos palladacycle, BuOH, 60 °C; (e) TFA, 150 °C, μW
45–54%, 3 steps).
9b
N
N
CH
CH
345
815
104.7
67.0
(
9c
9
9
9
d
e
f
N
N
N
CH
CH
CH
37.9
95.1
9890
94.5
78.3
62.0
Scheme 4. Reagents and conditions: (a) oxalyl chloride, MeOH; (b)
t
Methylhydrazine, n-BuOH, 170 °C, μW (60–70%,
2
steps); (c) NaO Bu,
CO
t
t
t
BuXphos, BuXphos palladacycle, BuOH, 60 °C; (d) TFA, 150 °C, μW; (e) K
2
3
,
R-X(5–30%, 3 steps).
9
9
g
N
N
CH
CH
119
452
77.5
76.5
h
9
9j
i
N
CH
N
CH
N
N
43b
43.2
19.8
88.7
94.8
100.8
b
Scheme 5. Reagents and conditions: (a) Pd
2
(dba)
3
, Xantphos, Cs
2
Co
3
, 1,4-di-
b
9k
oxane, μW, 140 °C, 1 h (46–50%); (b) 1 N LiOH, MeOH.1,4-dioxane, 45 °C
(
(
86–90%); (c) Amine, HATU, DIEA, DMF, (d) TFA, μW, 150 °C, 30–45 min
26–83%, 2 steps).
9
l
N
N
CH
CH
468
75.0
oxidation to the ketone, 12. Next, reaction with methylhydrazine fol-
lowed by cyclization formed the azaindazole scaffold which was then
coupled to the left-hand coupling partner under palladium-catalyzed
conditions in good overall yield.
An additional methyl group on the 6- or 5-substituted indazole led
to an increase in potency (9a, EC50 = 78.1 nM vs. 5b, EC50 = 257 nM
and 9b, EC50 = 345 nM vs. 5a, EC50 = 870 nM). Further elaboration of
the N- or C-position corresponding to a para-relationship with the
pyrazolo[4,3-b]pyridine head group led to slight increases in potency
9
m
Inactive
(
9d, EC50 = 37.9 nM; 9e, EC50 = 95.1 nM); however, replacement of a
methyl with a chloro did not prove productive (9c vs. 9e). As has been
seen in previous work, extending sterically in the presumed meta-po-
sition led to significantly reduced potency (9f, 9m, 9n). Electron-
(
continued on next page)
3
withdrawing groups were also tolerated (CF , 9g, EC50 = 119 nM).
3

D.W. Engers et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 3
Ether and amide substitutions on right-hand indazole.
Table 2 (continued)
Cmpd
X
Y
R
hmGlu
4
EC50 (nM)a
%Glu Maxa,c
36.2
9n
N
CH
1210
Cmpd
R
hmGlu
413
4
EC50 (nM)a
%Glu Maxa,b
70.9
9
9
o
p
N
CH
CH
N
36.2
160
97.4
75.7
17a
1
7b
231
104
86.7
9
9
9
q
r
s
N
CH
N
CH
N
N
67.9b
129
24.9
83.7
84.0
94.8
17c
107.3
9
t
N
N
CH
CH
95.5
265
62.7
93.8
17d
85.6
100.5
9
u
1
1
7e
7f
45.4
85.1
88.3
1880
9v
N
CH
46.8
93.2
2
2
1a
1b
827
764
41.6
55.5
a
Calcium mobilization mGlu
n = 2.
Amplitude of response in the presence of 30 μM test compound, normalized
4
assay, n = 1.
b
c
to a standard compound, PHCCC (100% Glu Max).
2
1c
907
289
41.9
57.8
Increasing the size of the alkyl substituent was also a particularly pro-
ductive change – the cyclopropyl substituent proved to yield com-
pounds that were very potent mGlu
EC50 = 43 nM; 9k, EC50 = 19.8 nM). In addition, the left-hand head
group can contain the pyrazolo[4,3-b]pyridine (9i), pyrazolo[3,4-b]
pyridine (9j) or the pyrazolo[3,4-b]pyrazine (9k) group without any
loss of activity. Removal of the N-methyl group from 9i did result in a
PAMs (9i, EC50 = 43 nM;22 9j,
21d
21e
4
653
794
70.3
74.0
1
0-fold loss of potency (9h, EC50 = 452 nM). Concerned that the cy-
clopropyl group may be metabolically unstable, we were able to obtain
the quaternary methyl cyclopropyl analogs and these were also shown
to be active (9q–s). Lastly, larger ring systems were less active (cyclo-
pentyl, 9u, EC50 = 265 nM); however, potency could be returned by
inserting a methylene spacer group (9v, EC50 = 47 nM). The corre-
sponding regiosiomer compounds, e.g., 10, generally led to ∼15-fold
loss of potency.
Lastly, we wanted to explore more fully the steric tolerability of the
right-side of this scaffold. To do this, we next introduced synthetic
handles to elaborate the functional groups. Our first attempts were to
incorporate ether substituents as shown in Scheme 4. To this end, 4-
bromo-2-fluoropicolinic acid, 14, was converted to the acid chloride
2
2
1f
1g
116
52.2
(
oxalyl chloride, MeOH) followed by ring closure to the 6-bromo-1-
methyl-1,2-dihydro-3H-indazol-3-one, 15 in good yield. Cross-coupling
with 4 led to the penultimate indazol-3-one which could be alkylated
under basic conditions with a variety of agents (17a–f).
(
continued on next page)
The amide analogs were synthesized as outlined in Scheme 5.
Starting from the commercially available, 4, and 18, cross coupling led
4

D.W. Engers et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
substituent analogs (Table 2). Both heteroaryl and cycloheteroalkyl
Table 3 (continued)
substituents are tolerated (17c, EC50 = 104 nM; 17d, EC50 = 86 nM;
Cmpd
R
hmGlu
498
4
EC50 (nM)a
%Glu Maxa,b
89.5
17e, EC50 = 45 nM). These larger substituents were slightly more active
than the substituted alkyl ethers (17a and 17b). However, addition of a
basic amine led to a significant loss of potency (17f, EC50 = 1,880 nM).
A variety of secondary and tertiary amides were also investigated;
however, all of the amides tested were much less active than the other
analogs that were synthesized (21a–h).Table 3.
On the basis of potency and efficacy of these compounds, we next
determined if these compounds displayed improved pharmacokinetic
properties compared to the previously identified, 2. As can be seen in
Table 4, the selected indazole compounds are somewhat less stable than
2 in rat liver microsomes; however, they displayed moderate-to-good
free fraction in rat plasma. Interestingly, many of the compounds have a
significantly improved CYP1A2 induction profile over 2, as measured
by upstream AhR activation studies. Compound 9i was identified as a
representative lead molecule based on its overall product profile and
this was then investigated in an in vivo brain distribution study to assess
brain levels. Satisfyingly, 9i was shown to be highly brain penetrant
21h
a
b
4
Calcium mobilization mGlu assay, n = 1.
Amplitude of response in the presence of 30 μM test compound, normalized
to a standard compound, PHCCC (100% Glu Max).
Table 4
In vitro DMPK properties of selected compounds (Predicted hepatic clearance,
PPB, CYP inhibition, AhR activation).
Cmpd Rat CLHEP
Plasma protein binding
CYP1A2
AhR fold
mL/min/kg)a
b
c
activationd
(
(%f
u
)
(μM)
Human
Rat
2
5
5
9
9
9
9
9
12.1
40.1
10.7
18.6
30.8
21.5
16.1
22.4
1.7
11.8
9.3
5.8
3.1
2.2
3.0
3.0
2.8
12.7
6.1
8.9
1.4
2.4
3.1
3.5
< 0.1
0.53
0.11
< 0.1
0.28
0.35
2.6
125
28.0
154
25.0
13.5
2.3
(
K
Table 4) at 1 h after PO dosing (rat brain:plasma
p,uu = 0.3). Lastly, 9i, was investigated for potential efflux and none
was noted (MDCK-MDR1 ER: 1.2).
p
K = 1.04,
a
b
a
h
i
A representation of the SAR around the AhR activation is shown in
Fig. 2. Cyclization around the right-hand side of compound 2 (AhR
activation = 125-fold) led to the two indazole scaffolds (5b, AhR acti-
vation = 154; 5a, AhR activation = 28), which was the first indication
that we could mitigate this potential CYP1A2 induction liability. An
additional methyl to 5b led to reduction in the AhR activation (9a, 25-
fold). Further steric bulk (9h) helped in reducing the upstream acti-
vation of AhR. Combining the 3-cyclopropyl with the N-Me substitution
pattern led to the identification of a set of molecules that no longer
suffer from the AhR activation and CYP1A2 induction liability that
hindered the advancement of 2.
j
k
2.6
2.3
0.48
In vivo brain distribution (PO, 10 mg/kg)
Time
1h
Plasma (ng/mL)
787
Brain (ng/g)
816
K
p
K
p,uu
9i
1.04
0.30
a
Predicted hepatic clearance based on intrinsic clearance in rat liver mi-
crosomes.
b
%
f
u
= % fraction unbound.
c
In summary, we report the discovery and characterization of a novel
N-(1,3-dialkyl-1H-indazol-6-yl)-1H-pyrazolo[4,3-b]pyridin-3-amine
IC50 determination of CYP1A2 enzyme inhibition in human liver micro-
somes using specific probe substrate-metabolite pairs.
d
scaffold as potent mGlu
right-hand aryl group into fused indazole systems is well tolerated. We
have shown that 9i, is potent (hmGlu EC50 = 43 nM and
rmGlu = 89 nM) mGlu PAM in both rat and human and has favorable
4
PAMs. We have shown the elaboration of the
Human AhR activation measured as Emax fold-activation over baseline
compared to known positive controls.
a
4
to the elaborated ester, 19. Saponification to the acid, 20, proceeded in
high yield followed by amide formation and finally PMB-deprotection
led to the final compounds, 21a–h. Both of the ether and amide syn-
thetic strategies provided a method for rapid elucidation of SAR via a
convergent pathway.
4
4
in vitro PK properties. In addition, 9i is highly brain penetrant. Further,
we have shown that the CYP1A2 induction activity can be mitigated via
systematic SAR evaluation around the indazole core. Further work
around these and related scaffolds is on-going and will be reported in
due course.
The alkylated ether substituents were active as mGlu
4
PAMs; how-
ever, there was a loss of potency compared to the simple alkyl
Fig. 2. Representation of the SAR around the AhR activation in the indazole scaffold.
5

D.W. Engers et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Acknowledgments
13. Robichaud AJ, Engers DW, Lindsley CW, Hopkins CR. ACS Chem Neurosci.
2
011;2:433–449.
4. Lindsley CW, Niswender CM, Engers DW, Hopkins CR. Curr Top Med Chem.
009;9:949–963.
15. Engers DW, Niswender CM, Weaver CD, et al. J Med Chem. 2009;52:4115–4118.
6. Jones CK, Bubser M, Thompson AD, et al. J Pharmacol Exp Ther. 2012;340:404–421.
7. Engers DW, Field JR, Le U, et al. J Med Chem. 2011;54:1106–1110.
8. Jones CK, Engers DW, Thompson AD, et al. J Med Chem. 2011;54:7639–7647.
19. Engers DW, Blobaum AL, Gogliotti RD, et al. ACS Chem Neurosci. 2016;7:1192–1200.
20. Niswender CM, Jones CK, Lin X, et al. ACS Chem Neurosci. 2016;7:1201–1211.
1. Moss TA, Addie MS, Nowak T, Waring MJ. Synlett. 2012;23:285–289.
1
The authors would like to acknowledge the technical assistance of
Daryl Venable and Shan Liang in performing in vitro pharmacology
studies. The authors would also like to thank the Michael J. Fox
Foundation for Parkinson’s Research and Bristol-Myers Squibb for their
support of the program to develop mGlu
K. Warren, Jr. and the William K. Warren Foundation who funded the
William K. Warren, Jr. Chair in Medicine (to C.W.L.).
2
1
1
1
4
PAMs. We also thank William
2
2
2. Synthesis of 9i: In a microwave vial, was added 1-(4-methoxybenzyl-1H-pyrazolo
[
4,3-b]pyridine-3-amine (1 eq.), 6-bromo-3-cyclopropyl-1-methyl-1H-indazole
t
t
t
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indazol-6-yl)-1H-pyrazolo[4,3-b]pyridine-3-amine, 9i. LCMS: R
T
= 0.795 min, >
8% @ 215 and 254 min, m/z = 305.2 [M+H]+
1
9
. H NMR (400 MHz, d-DMSO): δ
9
.33 (s, 1H), 8.45 (dd, J = 4.0, 0.8 Hz, 1H), 8.14 (d, J = 1.2 Hz, 1H), 7.89 (dd,
J = 8.4, 1.2 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.41 (dd, J = 8.4, 4.4 Hz, 1H), 7.35
dd, J = 8.8, 1.6 Hz, 1H), 3.84 (s, 3H), 2.23–2.16 (m, 1H), 0.99–0.90 (m, 4H).
10. Conn PJ, Pin J-P. Annu Rev Pharmacol Toxicol. 1997;37:205–237.
11. Niswender CM, Conn PJ. Annu Rev Pharmacol Toxicol. 2010;50:295–322.
12. Selvam C, Lemasson IA, Brabet I, et al. J Med Chem. 2018;61:1969–1989.
(
[M+H+], 305.15; found 305.15.
HRMS, calc’d for C17
17 6
H N
6