Identification and profiling of 3,5-dimethyl-isoxazole-4-carboxylic acid [2-methyl-4-((2S,3′S)-2-methyl-[1,3′]bipyrrolidinyl-1′-yl) phenyl] amide as histamine H3 receptor antagonist for the treatment of depression

Article abstract of DOI:10.1016/j.bmcl.2013.09.081

Lead optimization guided by histamine H₃ receptor (H ₃R) affinity and calculated physico-chemical properties enabled simultaneous improvement in potency and PK properties leading to the identification of a potent, selective, devoid of hERG issues, orally bioavailable, and CNS penetrable H₃R antagonist/inverse agonist 3h. The compound was active in forced-swimming tests suggesting its potential therapeutic utility as an anti-depressive agent. This Letter further includes its cardiovascular and neuropsychological/behavioral safety assessments.

Full text of DOI:10.1016/j.bmcl.2013.09.081

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6269–6273
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and profiling of 3,5-dimethyl-isoxazole-4-carboxylic
acid [2-methyl-4-((2S,30S)-2-methyl-[1,30]bipyrrolidinyl-10-yl)phenyl]
amide as histamine H3 receptor antagonist for the treatment of
depression
b,
Zhongli Gao a, , William J. Hurst , Werngard Czechtizky d, Daniel Hall b, Nicolas Moindrot c,
⇑
Raisa Nagorny b, Philippe Pichat c, David Stefany b, James A. Hendrix b,à, Pascal G. George c
a LGCR SMRPD Chemical Research, Sanofi US, 153-1-122, 153 2nd Ave, Waltham, MA 02451, USA
b Sanofi US, Route 202-206, PO Box 6800, Bridgewater, NJ 08807, USA
c Sanofi R&D, 1 Avenue Pierre Brossolette, Chilly-Mazarin, France
d Sanofi R&D, Deutschland GmbH, Industriepark Hoechst, Frankfurt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Lead optimization guided by histamine H3 receptor (H3R) affinity and calculated physico-chemical
properties enabled simultaneous improvement in potency and PK properties leading to the identification
of a potent, selective, devoid of hERG issues, orally bioavailable, and CNS penetrable H3R antagonist/
inverse agonist 3h. The compound was active in forced-swimming tests suggesting its potential
therapeutic utility as an anti-depressive agent. This Letter further includes its cardiovascular and
neuropsychological/behavioral safety assessments.
Received 20 August 2013
Revised 24 September 2013
Accepted 25 September 2013
Available online 3 October 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Histamine H3 receptor antagonist/inverse
agonist
Forced swimming test
Experimental models of anxiety and
depression
Anxiety
Depression
The histamine H3 receptor (H3R),1 a G-protein coupled receptor,
is largely expressed in the anterior part of the cortex, in the
hippocampus, striatum, and to a lesser extent in the hypothalamus
and spinal cord. It is a pivotal presynaptic autoreceptor and hetero-
receptor modulating the synthesis and release of histamine and
other neurotransmitters, including acetylcholine (ACh), dopamine
(DA), and norepinephrine (NE). Cloning of H3R uncovered the
existence of isoforms, inter-species pharmacological differences
and a high constitutive (spontaneous) activity of the receptor.2–5
Many isoforms of human H3R have been identified.6–8 The full
length 445aa configuration appears to be the most functionally
dominant and abundantly expressed. In addition, at least another
19 isoforms have been reported for the human H3R. Several
additional isoforms have also been identified in rat, guinea pig,
and mouse. Not surprisingly, this level of complexity of expression
and isoforms of H3R have invoked more complexity in in vivo H3R
biology and interesting therapeutic applications,9–14 such as Alz-
heimer’s disease,15,16 attention deficit hyperactivity disorder,17
schizophrenia,18–20 sleep disorder,21 neuropathic pain,22 and
obesity.23 Many structurally diverse H3R antagonists have been
reported and a few have advanced into development (for review
articles, see Refs. 24,25). In this Letter, we will describe our
continued lead optimization effort which led to the identification
of a novel, potent, selective and orally efficacious H3R antagonist/
inverse agonist as one of several pre-development candidates.
Previous work26,27 from our group revealed
a series of
compounds, represented by 5-fluoro-2-methyl-N-[2-methyl-4-
(2-methyl[1,30]bipyrrolidinyl-10-yl)phenyl] benzamide (1), that
displayed oral activity in a mouse food intake inhibition model.
However, the compound exhibited unacceptably high affinity to-
⇑
Corresponding author. Tel.: +1 781 434 3635.
E-mail addresses: zhongli.gao@sanofi.com (Z. Gao), william.hurst@sanofi.com
(W.J. Hurst), jhendrix@oligomerix.com (J.A. Hendrix).
wards the hERG channel (IC50 = 0.48 lM). Furthermore, the four
Address: Sanofi US, 55C-420A, 55 Corporate Drive, Bridgewater, NJ 08807, USA.
Tel.: +1 908 981 3723.
stereoisomers of 1 were synthesized and evaluated28 hoping that
one specific stereoisomer retained high H3R affinity while devoid
of hERG channel inhibition. Interestingly, the S,S diastereomer (2)
à
Current address: Oligomerix Inc., 3960 Broadway, Suite 340D, New York, NY
10032, USA. Tel.: +1 212 568 0365.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.081

6270
Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6269–6273
H3C
showed superior in vitro H3R affinity. However, all four stereoiso-
F
h-H3R binding Ki = 8.6 nM
rh-H3R binding Ki = 1.2 nM
r-H3R binding Ki = 16.5 nM
h-H3R GTPγS: EC50 = 1.1 nM
hERG IC50 = 0.48 µM
mers displayed similar hERG channel affinities indicating that the
elevated hERG affinity might be due to the high lipophilicity of 1
(clogP = 3.2). Thus, we focused our attention towards finding a po-
tential drug candidate with similar or better H3R activity as 2 but
an acceptable hERG profile. Besides the hERG issue, we also set
up a criterion to discover an H3R antagonist/inverse agonist with
low risk of potential phospholipidosis induction, one of the com-
mon issues of H3R ligands reported in the literature.29,30
To address the hERG issue, we used clogP and PSA to guide the
design of new compounds. This strategy was echoed by Levoin’s31
QSAR in which he stated that the lipophilic character of the mole-
cules (the sum of atomic polarizabilities, clogP, clogD), as well as
aromatic tendency reflect most important aspects for hERG
affinity.
H
N
O
N
N
CH3
H3C
1
H3C
F
H
h-H3R binding Ki = 3.4 nM
rh-H3R binding Ki = 2.5 nM
r-H3R binding Ki = 3.0 nM
hERG IC50 = 1.1 µM
N
O
N
N
CH3
H3C
2
Phospholipidosis is a storage disorder resulting in excessive
accumulation of phospholipids in lysosomes of the tissues. The
cause is not well defined. However, an amphiphilic character of
molecules displays a high risk of inducing phospholipidosis. In or-
der to increase our odds to identify H3R ligands with low risk of
phospholipidosis induction potential, we screened compounds
using an in silico phospholipidosis model.32,33 Given the fact that
H3R is a biogenic amine receptor and our current lead compound
possessed a basic amine, it was hypothesized that lowering the
pKa and increasing the polarity (lowering clogP and increasing po-
lar surface area, PSA) should be the most direct way to bring the
H3C
H
N
O
N
Het-Ar
N
H3C
3
Figure 1. Lead structures of the H3R antagonists/inverse agonists and design
strategies.
Table 1
rh-H3R affinity and calculated physico-chemical properties
H3C
O
N
N
H
Het-Ar
N
H3C
.
3
No.
Het-Ar
rh-H3R bindinga Ki (nM)
pKa (basic center)
clogP
clogD7.4
MW
395
PSA (Å2)
36
2
2.5
10.1
3.2
0.7
3a
1.4
10.1
3.3
0.8
378
49
N
3b
3c
1.8
3.9
10.1
10.1
3.0
2.1
0.5
353
353
49
49
O
ꢀ0.4
O
3d
4.7
12.1
1.7
ꢀ0.8
367
64
N
N
3e
3f
10.8
1.1
10.1
10.1
1.6
2.0
ꢀ0.8
ꢀ0.5
381
368
53
62
N
N
O
N
O
3g
3h
4.6
0.4
10.1
10.1
2.8
2.8
0.4
0.4
368
382
62
62
N
N
O
a
Binding assay was performed as in Ref. 26; Ki data were presented as an average of multiple experiments (n P 3). Standard deviation <50%; ND = not determined.

Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6269–6273
6271
H3C
H3C
H
N
a
H2N
N
N
O
N
N
Ar
H3C
H3C
4
3
Scheme 1. Syntheses of analogs 3a–3h. Reagents and conditions: (a) ArCOCl, CH2Cl2, pyridine, rt, 16 h, 62–80% yield.
Table 2
rh-H3R affinity and calculated physico-chemical properties
H3C
N
N
z
N
H
Ar
N
H3C
pKa
5
No.
Ar
Z
rh-H3R binding Kia (nM)
clogP
clogD7.4
MW
PSA (Å2)
.
2
5a
5b
5d
5e
2.5
3.0
2.5
4.9
4.2
10.1
9.9
10.0
9.9
3.2
3.2
3.4
3.3
4.4
0.7
1.0
1.1
1.1
2.2
395
364
436
378
414
36
48
74
48
48
Ph
3,4-F2-Ph
Benzyl
–CO–
–SO2–
–CO–
–CO–
1-Naphthalene
9.9
a
Binding assay was performed as in Ref. 26; Ki data were presented as an average of multiple experiments (n P 3). Standard deviation <50%; ND = not determined.
O
O
O
O
b
HN
a
N+
Cl
N+
N
+
N
N
N
N
H3C
H3C
H3C
H3C
6
7
O
Ar
c
N
H
N
H2N
N
N
N
N
N
H3C
H3C
H3C
H3C
8
5
Scheme 2. Syntheses of analogs 5a–5e. Reagents and conditions: (a) powdered potassium carbonate, DMSO, 85 °C (external temperature), overnight, 87% yield; (b) 5% Pd/C,
MeOH, H2 (40 psi), rt, 4 h. 100% yield; (c) ArCOCl, CH2Cl2, pyridine, rt, 16 h, 62% yield; or ArCO2H, CH2Cl2, DMF, EDC, HOBt, N-methyl-morpholine, rt, overnight, 75–82% yield.
calculated values into a more desirable range. However, the previ-
ous SAR studies26,27 suggested that introduction of polar groups on
the terminal aromatic ring was detrimental to H3R affinity.
Rather than taking heteroatom-containing functional groups as
substituents at the terminal and the central aromatic rings to lower
clogP and increase PSA, we chose hetero-aryl, the ‘embedded’
heteroatoms, to replace the corresponding aromatic rings. In addi-
tion, our continued optimization started from the lead (2) with the
preferred S,S stereochemistry which was superior in H3R affinity.28
Our first attempt was to investigate whether or not we could
replace the terminal substituted aryl in lead (2) with a substituted
hetero-aryl without compromising H3R affinity while lowering
clogP and increasing PSA; therefore analogs of the general type 3
were designed (Fig. 1).
Analogs 3a–3h (Table 1) were synthesized according to
Scheme 1 by coupling commercially available hetero-aryl acid
chlorides with the chiral amine 428 in DCM catalyzed by pyridine
in good yield.
The analogs 3a–3h were evaluated in an H3R binding assay by
displacement of [3H]N-
a-methyl-histamine in membranes isolated
from a CHO cell line stably transfected with the recombinant
rhesus monkey H3 receptor (rh-H3R)26 (Table 1). Gratifyingly,
substituted hetero-aryl replacement of aryl in 2 did not cause a
substantial drop in H3R affinity for most of the compounds. As

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Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6269–6273
Table 3
In vitro profiling of 3h
Profiling assays
hERG
Results
Conditions
IC50 = 37
lM
Patch-clamp technique in the whole-cell
configuration on Chinese hamster ovary (CHO)
cells
Ames II test
MNT in vitro
Negative
Negative
Concentrations ranged from 3 to 1000
lg/mL
Concentration ranged from 5 to 950 g/mL in
l
the presence or absence of metabolic activation
by human liver microsomes
In silico prediction of phopholipidosis risk34
Panel screen (CEREP)
Low risk
% Inhibition in binding assay <50%, except
inhibition @ 10
<5% Metabolized
r
(1 and/or 2): 73%
78 receptors, 16 enzymes, 26 kinases, and 38
ion channels
Human, mouse, rat, rabbit, macaque and dog,
except in guinea pig in which 35% of 3h was
metabolized
lM
Metabolic lability in liver microsomes
Metabolic stability in plasma
<5% Metabolized (the compound was spiked to each of the blank
plasmas at a concentration of 100 ng/ml. The spiked plasma
samples were incubated in a water bath at 37 °C for up to 4 h)
Cl = 0.040 0.010 mL hꢀ1 (106 hepatocytes)ꢀ1 (n = 4)
No induction
Human, mouse, rat, rabbit, macaque and dog.
Intrinsic clearance in human hepatocytes
Cyp induction for CYP1A1, CYP1A2, and
CYP3A4
Drug concentration ranged from 1 to 60 lM.
MBI
No inhibition
Cyp inhibition (IC50) for 3A4, 2C9, 2C19
IC50 >100
lM
Incubated for 4 h at 37 °C
Table 4
300
250
200
150
100
Pharmacokinetics of 3h in male OF1 mice and male Sprague-Dawley rats
*
Male OF1 micea Male Sprague-Dawley ratsb
**
**
Plasma
Brain
Plasma
Brain
iv
AUC0ꢀ1 (ng h/mL)
t1/2 (h)
Cl (L h/kg)
Vd (L/kg)
670
0.65
3.0
1100
0.93
1100
1.2
1.8
1200
1.8
1.7
2.8
po
AUC0–1 (ng h/mL)
Cmax (ng/mL)
tmax (h)
1900
1680
0.17
1.8
3100
2620
0.17
4.7
3150
749
0.5
3400
599
1.0
50
0
t1/2 (h)
7.8
9.4
0
1
3
10
im i60
F (%)
57
56
B/P ratioc
1.6
1.1
3h Dose (mg/kg, po)
a
Administration at 2 mg/kg iv and 10 mg/kg po; iv formulation: 50% 1-methyl-2-
Figure 2. Forced-swimming test in rats. Dose–response relationship of 3h
(formulation: MC/0.6% + H2O) dosing at mg/kg, po; Positive control: imipramine
(60 mg/kg, po).
pyrrolidinone in saline; concentration = 1.0 mg/mL, dosing 2 mL; po formulation:
5% DMSO/0.5% MC/0.2% Tween80, concentration = 1.0 mg/mL; dosing 10.0 mL.
b
Administration at 2 mg/kg iv and 10 mg/kg po; iv formulation: saline, concen-
tration = 0.5 mg/mL; dosing 4.0 mL; po formulati.on: 0.5% MC/0.2% T80; concen-
using ACD/Labs methods). Compound 3h displayed an affinity for
human and rat H3R with Ki values of 0.1 and 0.9 nM, respectively.
In a human H3 (H445)-CHO-CRE-Luc assay, 3h showed an EC50 of
0.7 nM (n = 1). The crystalline free base of 3h34 showed a good
solubility (0.11 mg/mg in water, >2 mg/mL in GI track simulated
media). Compound 3h was further profiled (see Table 3). There
were no significant issues identified which prohibited the develop-
ment of 3h. Particularly, 3h showed a low risk of phospholipidosis
induction potential in our internal in silico screen.35
tration = 1.0 mg/mL, dosing 10 mL.
c
B/P ratio is brain to plasma ratio calculated with iv AUC0ꢀ1 exposure.
for the 5-membered hetero-aryl derivatives 3b–3h, a certain SAR
trend was obvious. Particularly, the clogP, clogD, and PSA were
substantially improved for all the compounds compared to lead 2.
Next, the tolerability of replacing the central core aromatic ring
by
a hetero-aromatic ring was explored and analogs 5a–5e
(Table 2) was designed. The syntheses of analogs 5a–5e (Scheme 2)
commenced from the condensation of the commercially available
6-chloro-2-methyl-3-nitro-pyridine with the amine 628 to yield
an adduct (7). Hydrogenation of the nitro compound 7 afforded
amine (8) which was coupled with aryl acid chlorides or sulfonyl
chlorides to obtain the designed analogs 5a–5e.
The H3R affinity data of analogs 5a–5e are listed in Table 2.
Introduction of heteroatoms in the central core had little effect
on H3R affinity. Interestingly, clogP of these compounds was not
substantially lowered as compared to the lead (2).
When dosed at 10 mg/kg, po, 3h displayed low plasma
clearance and elimination half-life (t1/2 = 1.8 h, mice; t1/2 = 7.8 h,
rat), high exposure (1900 ng h/mL, mice, 3150 ng h/mL, rat, respec-
tively) and good oral bioavailability (57% in mice, 56% in rat)
(Table 4). The brain exposures in mice and rats were 3100 ng h/
mL and 3400 ng h/mL, respectively. The corresponding brain to
plasma ratios were 1.6 and 1.1 for mice and rats, respectively.
These results indicated a good correlation between in vivo PK
and in vitro ADME data (Table 4).
The in vivo pharmacology of 3h was then studied for anti-
depression-like effects in rats. The compound dose-dependently
decreased the immobility time in the forced swimming test. The
reduction of immobility time was significant at 3 and 10 mg/kg
with imipramine (60 mg/kg, po) as a positive control (Fig. 2).
The effects of 3h on hERG current were investigated in vitro
using patch-clamp technique in the whole-cell configuration on
In consideration of the multiple parameters of H3R affinity and
physico-chemical properties in a balanced manner, 3h excelled.
The compound displayed a rh-H3R Ki of 0.3nM with the most
noticeable improvements being
a reduced clogP (3.2 ? 2.8),
clogD7.4 (0.7 ? 0.4), and an increased PSA (35.6 ? 61.6) as com-
pared to the lead (2) (physico-chemical properties were calculated

Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6269–6273
6273
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44
l
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mice). Compound 3h was well tolerated and showed no evidence
of any behavioral side effects (social interaction, motility, precon-
vulsant and convulsions) at doses of 30 mg/kg, po. Tremors were
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in an independent oral exploratory general behavior study (Irwin
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10, and 30 mg/kg. The MED for FST is 10 mg/kg; the tolerable dose
is 30 mg/kg. Therefore, TI for mouse was estimated to be 3ꢁ.
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negligible effect on the coronary pressure, suggesting no effect
on coronary smooth muscle cells; and a weak to moderate de-
crease on the ventricular contraction at higher concentrations
(23% decrease at 10
lM, 33% decrease at 30 lM), indicating an
inhibition of L-type calcium current.
In conclusion, lead optimization guided by histamine H3 recep-
tor (H3R) affinity and calculated physico-chemical properties en-
abled simultaneous improvement in potency and PK properties
leading to the identification of a potent, selective, devoid of hERG
issues, orally bioavailable, and CNS penetrable H3R antagonist/in-
verse agonist 3h. The compound was active in forced-swimming
tests suggesting its potential therapeutic utility as an anti-depres-
sive agent. After assessment of the cardiovascular and neuropsy-
chological/behavior safety of 3h, the compound was extensively
profiled as a pre-clinical candidate.
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Acknowledgments
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The authors greatly appreciate Sanofi R&D management for the
strong support, H3R project team members for their contributions,
the Sanofi analytical department for their support on analytical
studies in confirmation of the structures, solid state characteriza-
tions and formulation support, and the Sanofi DMPK and toxicol-
ogy department for PK and in vitro/in vivo safety assessments.
34. Analytical of 3h: LCMS: LC method: SYNERGI 2U HYDRO-RP 20 ꢁ 4.0 mm
column, 0.1% TFA in water/acetonitrile 5–40% acetonitrile in 2 min then, to 95%
acetonitrile at 5 min at flow rate of 1.0 mL/min; LCMS: tR = 1.54 min, MS: 383
(M+H+).
References and notes
1H NMR (CDCl3, 300 MHz), d (ppm): 7.44 (m, 1H), 6.92 (br s, 1H), 6.40 (br s, 1H),
6.39 (bs, 1H), 3.50 (m, 1H), 3.4–3.2 (m, 4H), 3.00 (m, 1H), 2.78 (m, 1H), 2.66 (br
s, 3H), 2.48 (br s, 3H), 2.5 (m, 1H), 2.26 (s, 3H), 2.18 (m, 1H), 2.00 (m, 2H), 1.79
(m, 2H), 1.48 (m, 1H), 1.14 (d, 6.3 Hz, 3H). Calcd for C22H30N4O2 382.4992; C
69.08, H 7.91, N 14.65, O 8.37; Found: C 69.05, H 8.17, N 14.69; Optical
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rotation: ½aꢂD +27.45° (c 0.532, MeOH); Chiral purity: 100% (chiral HPLC).
35. Proprietary internal in silico model prediction. The model is based on carefully
filtered internal experimental data. It was developed using relevant two-and
three dimensional descriptors to capture molecular properties like shape,
lipophilicity and electrostatic potential combined with powerful statistical
methods. The model was carefully validated using test sets of novel molecules.