Discovery and structure-activity relationship of 1,3-cyclohexyl amide derivatives as novel mGluR5 negative allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2012.12.078

A novel series of trans-1,3-cyclohexyl diamides was discovered and characterized as mGluR5 negative allosteric modulators (NAMs) lacking an alkyne moiety. Conformational constraint of one of the amide bonds in the diamide template led to a spirooxazoline template. A representative compound (24d) showed good in vitro potency, high CNS penetration and, upon subcutaneous dosing, demonstrated efficacy in the mouse marble burying test, generally used as indicative of potential anxiolytic activity.

Full text of DOI:10.1016/j.bmcl.2012.12.078

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1398–1406
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and structure–activity relationship of 1,3-cyclohexyl amide
derivatives as novel mGluR5 negative allosteric modulators
Hao Zhou a, Sidney W. Topiol a, , Michel Grenon a, Hermogenes N. Jimenez a, Michelle A. Uberti b,
Daniel G. Smith b, Robbin M. Brodbeck b, Gamini Chandrasena c, Henrik Pedersen d,
Jens Christian Madsen d,à, Darío Doller a, Guiying Li a,
⇑
a Discovery Chemistry & DMPK, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
b Molecular Pharmacology & SAR, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
c Bioanalysis & Physiology, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
d Discovery Chemistry & DMPK, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of trans-1,3-cyclohexyl diamides was discovered and characterized as mGluR5 negative
allosteric modulators (NAMs) lacking an alkyne moiety. Conformational constraint of one of the amide
bonds in the diamide template led to a spirooxazoline template. A representative compound (24d)
showed good in vitro potency, high CNS penetration and, upon subcutaneous dosing, demonstrated effi-
cacy in the mouse marble burying test, generally used as indicative of potential anxiolytic activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 9 November 2012
Revised 17 December 2012
Accepted 21 December 2012
Available online 5 January 2013
Keywords:
mGluR5
Negative allosteric modulator
Glutamic acid is the major excitatory neurotransmitter in the
central nervous system (CNS) and exerts its signaling function by
activating ionotropic (iGluRs) and metabotropic glutamate recep-
tors (mGluRs). iGluRs are ligand-gated ion channels that mediate
fast excitatory transmission in many physiological functions, and
have been divided into three classes based on their selective
strategies have succeeded in delivering highly selective orthosteric
mGluR ligands.7 However, in general, these suffer from poor selec-
tivity within mGluR subtypes, which is generally believed to share
a relatively highly conserved binding region, although this hypoth-
esis has recently been challanged.8 Allosteric binding sites exist in
the receptor cell transmembrane domain, which is housed by what
is believed to be the less-conserved amino acids than at the orthos-
teric site of the receptors. Thus, the strategy of designing ligands
targeting this region has often produced selective receptor modu-
lation.9 mGluR5 negative allosteric modulators have been actively
pursued as a potential treatment for anxiety, depression, pain,
levodopa-induced dyskinesia in Parkinson’s disease (LID-PD), gas-
troesophageal reflux disease (GERD), cocaine addiction and Fragile
X Syndrome (FXS).10–12 Some of these indications already have
Phase II clinical proof of concept, including FXS,13 LID-PD,14
GERD,15 and anxiety.16 Currently, there are a number of mGluR5
NAMs in the clinic, including Novartis’ AFQ056 (1, Phase II/III for
FXS and LID-PD), Roche’s RO4917523 (2, Phase II for FXS and treat-
ment-resistant depression (TRD)), Addex’s ADX48621 (3, Phase II
for LID-PD) and Seaside’s STX107 (4, Phase I for FXS). Similar to
MPEP (5), these compounds contain an alkyne moiety as a key
structural component. Recent chemistry work has focused on
exploring non-alkyne chemotypes, in particular after termination
of the development of Addex’s first generation alkyne-containing
mGluR5 NAM, ADX10059 (6), due to observed liver function
abnormalities in the clinical studies.17 These efforts yielded a
number of mGluR5 NAMs free from alkyne functionalitites, such
interactions with different ligands:
methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA), and
N-methyl-
-aspartic acid (NMDA).1,2 The mGluRs belong to the
a-amino-3-hydroxy-5-
D
class C of G-protein coupled receptor (GPCR) superfamily and
mediate slower neurotransmission.3 Eight different mGluR sub-
types have been identified and divided into group I (mGluR1 and
5), group II (mGluR2 and 3), and group III (mGluR4, 6, 7 and 8)
based on sequence homology, second messenger coupling and
pharmacology.4 mGluR5s are mostly localized postsynaptically,
couple to Gq/11 proteins and activate phospholipase C, leading to
phosphoinositide hydrolysis and formation of two intracellular
second messengers: inositol triphosphate (IP3), which induces
intracellular Ca2+ release, and diacylglycerol (DAG), which can
activate protein kinase C.5,6 Orthosteric ligands of mGluRs bind in
the extracellular amino-terminal domain. Some recent design
⇑
Corresponding author. Tel.: +201 350 0140.
E-mail address: guli@lundbeck.com (G. Li).
Present address: 3D-2drug, PO Box 184, Fair Lawn, NJ 07410, USA.
Present address: Bruker Biospin Scandinavia AB, Vallgatan 5, SE-17067 Solna,
à
Sweden.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.078

H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
1399
as compounds 7–12 (see Fig. 1).18a–f Progress in non-alkyne chem-
ical space has been summarized in a recent review.18g Herein we
report the rational design, synthesis, and structure–activity rela-
tionships (SAR) of 1,3-cyclohexyl amide derivatives as a novel
series of mGluR5 NAMs lacking an alkyne moiety.
well, and project the outer aromatic groups in the right directions
resulting in good overlaying with compounds 13 and 14.
To test this binding hypothesis, and encouraged by the good
cLogP and CNS drug-likeness multi parameter optimization
(MPO22) values (2.7 and 5.0, respectively) of model compounds
15a and 15b, their preparation was undertaken using a synthetic
route that expeditiously provided both the cis- and trans-isomers
after preparative thin layer chromatography (TLC) separation
(Scheme 1). Cis or trans stereochemical assignments were made
using 1H NMR and HSQC spectroscopic analysis.23 The mGluR5
functional antagonistic activity of these compounds was evaluated
in vitro measuring their ability to inhibit the calcium mobilization
caused by the EC80 concentration of glutamate in HEK293 cells
expressing human mGluR5.24 We were delighted to find that in
this assay, the trans-isomer 15a showed an IC50 value of 531 nM.
The cis-isomer 15b was inactive. In addition, the allosteric nature
of 15a was supported by a displacement radioligand binding assay
using [3H]-ABP688,25 where 15a showed a Ki of 1538 nM, while
15b was also inactive (Table 1).
These positive results encouraged us to design a small library of
analogs, mostly with the active trans stereochemistry. To this end,
the synthetic route was modified to stereospecifically provide
either trans or cis analogs, as well as to further confirm the struc-
tural assignments. A number of compounds, including both iso-
mers 15a and 15b, were prepared from the corresponding trans-
or cis-3-((tert-butoxycarbonyl)amino)cyclohexane-carboxylic acid
(17a and 17b) as shown in Scheme 2. Stereospecific Curtius rear-
rangement with DPPA, followed by the treatment with tert-butyl
alcohol, afforded trans- or cis-1,3-cyclohexanedi(tert-butoxycar-
In search for novel allosteric mGluR5 chemical space, a compu-
tational overlay model was built reflecting a simple binding
hypothesis.19 It was observed that two literature compounds
1320a and 1420b (Fig. 2) with different multi-ring systems had sim-
ilar (hetero)aryl rings at both ends. We hypothesized that other
compounds which would present these terminal aromatic rings
in the same three-dimensional disposition, while traversing the
same bridging region, would also have activity. Thus, an in silico
core hopping strategy that preserved the geometric arrangement
of the outer aromatic groups in compounds 13 and 14 was exe-
cuted using the FlexAlign algorithm in MOE (Fig. 2).21 A replace-
ment was sought for the regions of 13 and 14 highlighted in
green, acting as a linker to optimally position the left- and right-
hand-side (hetero)aryls for binding to an mGluR5 allosteric site.
The model was constructed using structures 13 and 14, which were
first overlayed using FlexAlign. These overlayed structures were
then held in fixed geometries and used as the initial model. A num-
ber of possible linkers (green region in Fig. 2), such as different ring
systems and/or substituents were then selected ‘by hand’ to create
new hypothetical structures. These new structures were subjected
to an overlay with the initial model to see which ones fit well with
respect to the terminal aromatic rings. It was found that a cyclo-
hexyl core with an amide (e.g., –NHCO– or –CONH–) or alkyne lin-
ker on 1- and/or 3-positions (scaffold 15) would fit our hypothesis
N
O
F
N
N
O
N
Cl
H
H
N
N
OH
N
F
1
3
2
(AFQ056, mavoglurant)
(RO4917523 series)
Roche
(ADX48621, dipraglurant)
Novartis
Addex
CN
F
NH2
F
N
S
N
N
N
4
5
6
(STX107 series)
(MPEP)
(ADX10059, raseglurant)
Seaside
Addex
N
F
O
O
Cl
O
N
NC
N
N
Br
F
N
N
N
N
N
718a
H
CN
O
N
818b
918c
(AZD6538)
AstraZeneca
Merz
GSK
N
O
S
HN
O
N
N
N
N
Cl
S
N
NC
F
N
O
N
O
N
S
Cl
1018d
Gedeon Richter
1218f
1118e
Vanderbilt University
Lundbeck
Figure 1. Structures of select mGluR5 NAMs.

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H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
H
N
N
H
N
N
CN
CN
N
N
N
N
+
N
N
N
CN
Cl
1320a
1420b
FLIPR IC50 10 nM
FLIPR IC50 20 nM
Overlay of 13 and 14
X
Y
Ar
Ar'
X, Y: CONH, NHCO, alkyne
15
Overlay of 13 (purple), 14 (cyan) and 15 (gold)
Figure 2. Overlay model.
N
N
H2N
NH2
H
N
H
N
H
N
H
N
a, b
NC
NC
O
O
O
O
16
, cis/trans
~3:1 mixture
15a
15b
(trans)
(cis)
Scheme 1. Reagents and conditions: (a) (i) picolinic acid (1 equiv), BOP (1 equiv), NEt3 (4 equiv), CH2Cl2, room temp, overnight, (ii) m-CN-PhCO2H (1 equiv), BOP (1 equiv),
NEt3 (4 equiv), CH2Cl2, room temp, overnight; (b) separation by preparative TLC (ethyl acetate/hexane: 1/1).
bonyl)amine (18a and 18b),26 respectively. Removal of the Boc
group with TFA, followed by amidation with picolinic acid, and
then amidation with 3-cyanobenzoic acid under standard condi-
tions afforded 15a or 15b, respectively. Likewise, by appropriate
choice of the carboxylic acid or acyl chloride, analogs 15c–15ag
was prepared. Table 1 shows the in vitro screening results of these
compounds for mGluR5 functional activity and binding affinity, as
well as select in silico properties.
Early SAR observations pointed to the 3-chlorophenyl as a pre-
ferred fragment in this core, in agreement with previous work.18f,27
When R2 is pyridin-2-yl, replacement of 3-cyanophenyl (15c) with
3-chlorophenyl (15f) enhanced the functional potency by 4-fold.
Analogs where the 3-chlorophenyl (15f) is replaced with 3-anisyl
(15i) or 3-tolyl (15j) were significantly less active. The mGluR5
NAM activity of symmetrically substituted diamides was briefly
explored. Again, 3-chlorophenyl-bearing bis-amide 15k was more
potent than 3-fluorophenyl and pyridine-2-yl bis-amides 15l and
15m, respectively. At this point the decision was made to fix one
side of the amide as a 3-chlorophenyl (R1) and optimize the R2 sub-
stituent. Among pyridyl analogs, pyridin-2-yl (15f) was about 2- to
5-fold more active compared to pyridin-3-yl (15n) and pyridin-4-
yl (15o) in the functional assay. A methyl group on the 6-position
of the pyridyl ring (15p) was tolerated, but on 3- or 4-position it
significantly reduced the activity (15q and 15r). The 3-cyano-pyri-
din-2-yl fragment provided ca. 11-fold more functional potency
than 3-methyl-pyridin-2-yl (15s vs. 15r). Phenyl (15t) was well
tolerated. Walking the fluorine atom to positions 2, 3 or 4 in the
R2 phenyl ring did not have a significant impact on potency (15u,
15v and 15y). Electron donating methoxy group was tolerated both
at 3-or 4-position (15z and 15aa, respectively), while dimethyl-
amino group was tolerated at 4-, but not at 3-position (15ab and
15ac). Introduction of the second nitrogen into the pyridine ring
such as in pyrimidin-2-yl amide 15ad or pyrazin-2-yl amide
15ae reduced functional activity by 2- and 10-fold, respectively.
Aliphatic amides such as cyclohexyl (15af) or pyridin-2-ylmethyl
(15ag) drastically reduced the activity.
To explore the effect of a retro-amide linker, compounds 21a–
21c was prepared according to Scheme 2. Reversing one of the
amide bonds (i.e., –NH–CO– to –CO–NH–) has significantly nega-
tive impact on the activity (21a vs. 15a, 21b and 21c vs. 15f,
Scheme 3). A retro-amide tether on the 3-chlorophenyl side was
tolerated, but on the pyridin-2-yl side it abolished the activity.
To investigate the importance of the hydrogen donor of the -
NHCO- moiety, N-methyl and N,N0-dimethyl derivatives (22 and
23) of symmetrically substituted 15k were made (albeit in low
yields) according to Scheme 4. These analogs were inactive in the

H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
1401
Table 1
In vitro mGluR5 activity and in silico properties of select 1,3-cyclohexyl diamides
H
H
N
R1
N
R2
O
O
a
CMPD
R1
R2
cLogP
MPO
IC50 (nM)
Kia (nM)
LLEb
15a, trans
15b, cis
3-Cyanophenyl
3-Cyanophenyl
3-Cyanophenyl
3-Cyanophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Methoxylphenyl
3-Methylphenyl
3-Chlorophenyl
3-Fluorophenyl
Pyridin-2-yl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
—
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
3-Chlorophenyl
3-Fluorophenyl
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
6-Methylpyridin-2-yl
4-Methylpyridin-2-yl
3-Methylpyridin-2-yl
3-Cyanopyridin-2-yl
Phenyl
2-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
3-Methoxyphenyl
4-Methoxyphenyl
4-Dimethylaminophenyl
3-Dimethylaminophenyl
Pyrazin-2-yl
2.7
2.7
2.7
2.7
3.7
3.7
3.7
3.7
3.0
3.3
5.0
3.8
2.3
3.3
3.3
4.2
4.2
3.8
3.3
4.1
4.0
4.4
4.4
4.4
4.4
4.3
4.3
4.4
4.4
2.9
2.9
2.5
4.3
3.8
5.0
5.0
5.0
5.0
4.3
4.3
4.3
4.3
4.5
4.8
3.3
4.2
5.3
4.7
4.7
3.8
3.8
3.8
4.4
4.0
3.9
3.7
3.7
3.7
3.7
3.7
3.7
3.5
3.5
5.1
5.1
5.1
3.8
3.1
531 88
>10,000
231 52
>10,000
314 97
62 15
>10,000
1538 497
—
1275 480
—
3.6
—
4.0
—
15c, trans-(R,R)c
15d, trans (S,S)c
15e, cis/transd
15f, trans-(R,R)c
15g, trans-(S,S)c
15h, cis
—
—
205 46
—
—
—
—
—
—
—
3.5
1.3
2.2
—
1510 110
>10,000
15i, cis/transd
15j, trans
1000 69
139 27
593 57
1150 241
612 92
300 45
290 47
3400 410
7840 354
671 102
228 28
2.8
1.9
2.4
3.6
2.9
3.2
2.3
1.3
1.3
2.9
2.6
2.8
2.7
2.8
0.6
2.3
1.8
2.3
1.9
—
15k, cis/transd
15l, trans
15m, trans
15n, trans
15o, trans
15p, trans
15q, trans
—
445 65
582 95
—
15r, trans
—
15s, cis/transd
15t, trans
1246 125
672 107
760 119
320 64
211 62
—
492 99
—
674 178
—
15u, trans
15v, trans
175
86 20
59
6
15w, trans-(R,R)c
15x, trans-(S,S)c
15y, trans
2
9600 247
194 14
789 116
266 16
473 30
>10,000
1370 176
425 187
>10,000
15z, trans
15aa, trans
15ab, trans
15ac, trans
15ad, trans
15ae, trans
15af, trans
15ag, trans
MPEP
—
—
3.0
3.5
—
1.7
4.9
Pyrimidin-2-yl
Pyridin-2-ylmethyl
Cyclohexyl
3790 467
—
—
857 73
2.3 0.3
—
3.4 0.5
a
Human mGluR5 IC50 (FLIPR functional assay24) and Ki (binding assay25) values are expressed as mean S.E.M (nM) of three or four separate experiments except for 15n
(FLIPR IC50 is the average value of two experiments).
b
LLE = pIC50 ꢀ cLogP.
c
Absolute configurations arbitrarily assigned.
d
cis/trans refers to an achiral mixture of cis- and trans-isomers made from 16 (cis/trans ꢁ3:1).
H
N
H
N
BocHN
BocHN
NHBoc
H2N
NH2
R1
R2
CO2H
a
b
c
O
O
17a
18a
19a:
15a-ag
: 1,3-trans
: 1,3-trans
1,3-trans
17b: 1,3-cis
18b: 1,3-cis
19b: 1,3-cis
d
O
O
H
BocHN
e
R1
N
NHR2
NHR2
O
20 (from 17a)
21
Scheme 2. Synthesis of trans- or cis-diamides. Reagents and conditions: (a) (i) DPPA (1 equiv), NEt3 (1 equiv), toluene, reflux, 1 h, (ii) t-BuOH (2 equiv), reflux, 12 h, 40–60%;
(b) TFA, CH2Cl2, room temp, 1 h, 100% conversion, used without purification; (c) (i) NEt3 (4 equiv), R1CO2H (1 equiv), BOP (1 equiv), CH2Cl2, room temp, overnight; or NEt3
(3 equiv), R1COCl (1 equiv), CH2Cl2, 0 °C to room temp 20–45%, (ii) NEt3 (4 equiv), R2CO2H (1 equiv), BOP (1 equiv), CH2Cl2, room temp overnight; or NEt3 (3 equiv), R2COCl
(1 equiv), CH2Cl2, 0 °C to room temp; 60–90%; (d) R2NH2 (1 equiv), BOP (1 equiv), NEt3 (2 equiv), CH2Cl2, room temp, overnight; ꢁ70%; (e) (i) TFA, CH2Cl2, 30 min, (ii) NEt3
(4 equiv), R1CO2H (1 equiv), BOP (1 equiv), CH2Cl2, room temp, overnight; ꢁ25–60%.
functional assay (IC50 >10,000 nM). This lack of activity may result
from a need for a binding interaction with the amide N–H, or may
be due to energetically unfavorable conformational changes
caused by introduction of a methyl group on the amide moiety.
Select compounds were evaluated in a binding assay (Table 1).
The affinities (Ki) of these compounds shifted to the right by 1.5–9
fold compared to IC50 values. Racemic mixtures of key compounds
15a, 15e and 15u were resolved by chiral HPLC28 into the corre-
sponding trans homochiral analogs 15c and 15d; 15f and 15g;
and 15w and 15x, respectively. Absolute configurations were
assigned arbitrarily. Screening these compounds showed that
the mGluR5 NAM activity in this chemotype resides in one

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H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
H
N
H
N
H
N
H
N
NC
N
Cl
N
O
O
O
O
15a
15f
IC50 = 62 15 nM
IC50 = 531 88 nM
O
O
O
H
N
H
N
H
N
NC
N
H
N
Cl
N
H
N
Cl
N
H
N
O
O
O
21a
21c
21b
IC50 > 10,000 nM
IC50 = 468 178 nM
IC50 = 5,000 2070 nM
Scheme 3. mGluR5 NAM activity of a set of amides and retroamides.
Me
N
H
N
Cl
Cl
O
O
H
N
H
N
a
b
22
IC50 > 10,000 nM
Cl
Cl
O
O
15k
IC50 = 139 nM
Me
N
Me
N
Cl
Cl
O
O
23
IC50 > 10,000 nM
Scheme 4. Reagents and conditions: (a) NaH (2 equiv), MeI (2 equiv), DMF, room temp, 16 h, 3%; (b) NaH (5 equiv), MeI (10 equiv), DMF, room temp, 16 h, 14%.
enantiomer. The three active enantiomers 15c, 15f and 15w were
further profiled in a rat mGluR1 functional screen (to explore
mGluR Group 1 potential cross-reactivity), human, rat and mouse
liver microsomal stability, human plasma protein binding and
physicochemical property assays, and the results are summarized
in Table 2. Compounds 15w and 15f are equipotent in vitro, and
have about 4-fold more efficacy than 15c in the functional screen,
and about 6-fold higher affinity than 15c in the binding assay. All
three compounds showed good selectivity against rmGluR1, appro-
priate plasma free fraction, and their human liver microsomal
intrinsic clearances are below human liver hepatic blood flow.
Compound 15w has high LogD7.4 (>5) and low aqueous solubility
0.34 and unbound brain to unbound plasma concentrations ratio
(Kupp) of 0.67 1 h post-dosing. However, the free brain concentra-
tion in mouse at this dose and time point (170 nM ꢂ 0.22 = 37 nM)
only covered
a fraction of Ki due to suboptimal affinity
(Ki = 205 nM). We deemed further gains in affinity and functional
potency were required.
We set to explore whether the affinity at mGluR5 could be im-
proved by constraining the conformation of one of the amide
bonds in 15. Among a number of options conceived, a spirooxazo-
line template (Fig. 3) was designed and found to satisfy the binding
hypothesis using our computational overlay model. Compounds
24a–i were made according to Scheme 5. Their in silico properties
and in vitro mGluR5 activities are shown in Table 4. For these con-
strained analogs, the binding affinities (Ki) are comparable to their
IC50 values. Unlike the diamide template, where a methyl at 6-po-
sition of pyridyl has no significant impact on affinity and functional
potency (15p (racemate) vs. des-methyl analog 15f (chiral)), a
methyl at 6-position of the pyridyl in the spirooxazoline template
enhances the affinity and potency by about 5-fold (24d vs. 24g);
pyridin-3-yl and pyridin-4-yl analogs in the diamide template
are active (15n and 15o, IC50 <650 nM), while the corresponding
analogs in the spirooxazoline template are inactive at highest
at pH 7.4 (5 lM). Compound 15f has the best balance of properties
among these three compounds. We proceeded to explore brain
partition in mouse, as well as rat and mouse brain homogenate free
fraction and pharmacokinetic properties (Table 3). In rat, this com-
pound showed high oral bioavailability (F = 0.8), moderate volume
distribution (Vss = 3.4 L/kg), high plasma clearance (CLp = 4.1 L/h/
kg, 85% of rat hepatic blood flow) and short half life (T1/2 = 0.6 h).
Plasma and brain free fractions in rodent were high. Upon sc dos-
ing in mouse at a 10 mg/kg dose, CNS penetration was deemed
appropriate, with brain to plasma concentrations ratio (B/P) of
Table 2
Properties of active homochiral analogs
rmGluR129
Liver microsomal CLint
Human PPB%31
Solubility32
(lM)
LogD7.4
30,a
33
CMPD
EC50 (nM)
IC50 (nM)
Human (L/min)
Rat (mL/min)
Mouse (mL/min)
15c
15f
15w
>10,000
>10,000
>10,000
>10,000
>10,000
2100
0.5
1.1
0.8
8.3
41
9.4
1.8
9
nd
84.3
93.5
96.1
240
240
5.4
—
3.2
>5
a
Human hepatic blood flow = 1.5 L/min; rat hepatic blood flow = 20 mL/min; mouse hepatic blood flow = 1.8 mL/min.

H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
1403
Table 3
Brain homogenate free fraction and in vivo PK of compound 15f
Rat free
Mouse free
fraction%31
Exposure in mousea
In vivo rat PKb
fraction%31
B
P
B
P
B (nM) P (nM) B/P
Kupp Cmax (ng/mL) Tmax (h) CLp (L/h/kg) Vss (L/kg) T1/2 (h) AUC0-inf (ng.h/mL)
F
13
14
22
11
170
500
0.34 0.67
220
2
4.1
3.4
0.6
260 (IV)
530 (PO)
0.8
a
Brain (B) and plasma (P) exposures determined at 1 h following 10 mg/kg, SC in CD-1 mice (n = 2) using 20% hydroxypropyl-b-cyclodextrin in water as dosing vehicle.
Average values (n = 2) are given. Limit of quantification was 2 ng/g and 2 ng/mL for brain homogenate and plasma, respectively.
b
The study was performed in SD rats (n = 2) in a cross-over manner with 24 h washout period. IV: 1 mg/kg; PO: 2 mg/kg; vehicle: 20% hydroxypropyl-b-cyclodextrin in
water.
compounds are highly selective against mGluR1, have high liver
microsomal intrinsic clearance, good CNS penetration, and high
R2
H
N
H
N
H
N
O
plasma and brain exposure in mouse 1 h after sc dosing. Com-
pound 24d was also evaluated in a mouse marble burying test35
and demonstrated anxiolytic-like activity determined 1 h after sc
administration of a single 30 mg/kg dose (Fig. 4). In the absence
of exposure from this study, an extrapolated free brain concentra-
tion at 30 mg/kg based on an exposure study at 10 mg/kg, 1 h
would be 3 nM, which is lower than Ki (30 nM). However, actual
exposures may be higher due to combination of potentially non-
linear pharmacokinetics from 10 to 30 mg/kg and experimental
errors between the two studies. Since both compounds have
R1
R2
R1
N
O
O
O
Figure 3. Design of spirooxazoline.
testing concentration (10 lM), indicating differences in SAR be-
tween the two chemotypes. Compounds 24d and 24g were further
profiled for their selectivity against mGluR1, in vitro metabolic sta-
bility, exposure in rodent and free brain fraction (Table 5). Both
O
O
H
N
H
N
H
N
H
N
OH
O
O
R1
NH
NH
O
a
CO2H
b
c
Cbz
Cbz
Cbz
O
25
26
27
28
Cl
H
N
H
N
HO
O
R1
R1
NH2
N
d
e
O
O
24
29
f
Cl
Cl
H
O
H
R1
N
O
N
R1
N
+
N
O
O
24-trans
24-cis
g
Cl
Cl
H
N
H
N
O
O
+
R1
R1
N
N
O
O
24-(S,R)
24-(R,S)
Scheme 5. Reagents and conditions: (a) (i) LHMDS (1.1 equiv), THF, EtOAc (1.1 equiv) ꢀ78 °C; (ii) LiOHꢃH2O (10 equiv), THF/H2O (1:1), rt, overnight; two-step crude yield
75%; (b) DPPA (1 equiv), toluene, TEA (1 equiv), rt, 30 min, then reflux, 8 h, 74%; (c) (i) Pd/C, MeOH, H2, 50 psi, 4 h, crude yield 95%; (ii) R1CO2H (1 equiv), TEA (4 equiv), BOP
(1 equiv), DCM; ꢁ70%; (d) LiOHꢃH2O (10 equiv), dioxane/H2O (1:1), 70 °C, 5 h, crude yield 85%; (e) 3-chloro-benzimidic acid ethyl ester (1.5 equiv), EtOH, 1 drop 4 M HCl in
dioxane, 100 °C, microwave, 20–30 min, ꢁ60%; (f) silica gel chromatography (hexane/ethyl acetate); less polar fraction assigned as trans and more polar fraction assigned cis;
(g) resolution by chiral HPLC: Chiralpak AD (Diacel), 250 ꢂ 20 mm; mobile phase: 20% isopropanol, 79.9% hexane, 0.1% diethylamine; flow rate: 14 mL/min; UV at 254 nM;
front peak arbitrarily assigned as (S,R) and back peak arbitrarily assigned as (R,S). Cis assignment (as defined above) was based on the NMR spectroscopy analyses (COSY,
ROESY, edited HSQC overlaid with HMBC) of compound 24d.34

1404
H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
Table 4
In vitro mGluR5 activity and in silico properties of cyclohexyl spirooxazolines
a
CMPD
R1
cLogP
MPO
3.8
IC50 (nM)
Kia (nM)
LLEb
24a, trans
24b-, cis
24c, cis-(S,R)
24d, cis-(R,S)
24e, cis/trans mixture
24f, cis-(S,R)
24g, cis-(R,S)
24h, cis/trans mixture
24i, cis/trans mixture
6-Methylpyridin-2-yl
4.8
1505
72
5225
23
—
—
—
1.0
2.4
0.5
2.9
1.9
1.3
2.7
—
30
—
4
Pyridin-2-yl
4.3
4.1
647
2910
115
>10,000
>10,000
—
178
—
3
Pyridin-3-yl
Pyridin-4-yl
3.9
3.9
4.3
4.3
—
—
a,b
Same as described in Table 1.
Table 5
mGluR1 selectivity and DMPK profile of compounds 24d and 24g
CMPD rmGluR129
Liver microsomal CLint
Mouse free
fraction%31
Exposure in rodent at 10 mpk, 1 ha
Rat, PO Mouse
B (nM) P (nM) B/P B (nM) P (nM)
PO: 17
30
EC50 (IC50) (nM)
Human (L/min) Rat (mL/min) Mouse (mL/min)
B
P
B/P Kupp
24d
24g
>10,000
(>10,000)
>10,000
13
13
48
12.5
8.8
0.3
0.2
ND
5
ND
8
ND PO: 9
SC: 910
0.6 SC: 1165 SC: 1165 1.0 SC: 1.6
0.5 PO: 0.8
SC: 1175 0.8 SC: 1.1
160
0.5
0.3
(>10,000)
a
Same conditions as described in Table 3.
20
15
10
5
**
**
0
V
BUS
3
10
30
Compound 24d, SC, mg/kg
n = 12
V: 20% HPβCD in water, vehicle group
BUS: positive control group treated with buspirone (10 mg/kg, free base in distilled water, SC)
Treatment time: 30 min ** P< 0.01 vs. vehicle group (ANOVA with post-hoc Dunnett’s test)
Figure 4. Effect of compound 24d on marble burying in male CD-1 mice.
extremely low oral exposure, a chemical stability test of compound
24g under acidic conditions was performed.36 More than 50% of the
compound was missing after incubation in methanol at 37 °C, pH
1.5 for 1 h, forming a single major product with molecular weight
18 amu higher than 24g, presumably due to hydrolytic opening of
the oxazolinyl ring. Therefore, both first-pass metabolism and
chemical instability at acidic pH contributed to the low oral expo-
sure of compounds in this chemotype. At this point, the decision
was made to deprioritize further work related to compound 24d,
and to not establish its absolute configuration. Instead, resources
were directed towards solving the chemotype’s instability prob-
lems. This led to a novel spirocyclic lactam template, the absolute
stereochemistry of which was found to be R,R.38 This work will be
the focus of future communications.
was discovered as negative allosteric modulators of the mGlu5
receptor lacking an alkyne moiety. Compounds in this chemotype
are characterized by tractable SAR, reasonable in vitro potency
and good brain penetration in rodents. Conformational constraint
of one of the amide bonds in the diamide series led to a spirooxaz-
oline template that demonstrated good in vitro mGluR5 activity,
high selectivity against mGluR1, good CNS penetration and
in vivo efficacy in the mouse marble burying model (representative
compound 24d). The poor oral exposure of compounds in the
spirooxazoline template was established as arising from high
first-pass metabolism, as well as chemical instability under acidic
conditions. Further modifications of these two chemotypes were
explored seeking improvements in functional potency, binding
affinity, chemical stability and in vivo clearance, which led to a
number of non-alkyne chemotypes with enhanced proper-
ties.28,37,38 These will be the topic of future reports.
In summary, using a computational overlay model built based
on literature compounds, a series of trans-1,3-cyclohexyl-diamides

H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
1405
22. Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. ACS Chem. Neurosci. 2010, 1,
Acknowledgments
435.
23.
A mixture of cyclohexane-1,3-diamine (50 mg, 0.44 mmol), picolinic acid
We are grateful to Drs. Mark Hayward, Qing-ping Han, Chi
Zhang and Xu Zhang for solubility and LogD7.4 determinations,
compound purification, and racemic compound resolution, to Xiao-
sui Pu, Noel J. Boyle and Veena Menon for in vitro pharmacology
determinations, to Manuel Cajina, Megan E. Nattini and Kimloan
Nguyen for in vitro and in vivo DMPK data and bioanalytical sup-
port, to Christina Sveigaard for rodent plasma and brain free frac-
tions determination, to Dr. Jens-Jakob Karlsson for coordinating
in vitro DMPK studies at a CRO. We acknowledged Dr. Lena Tag-
mose for her constructive comments to this manuscript.
(54 mg, 0.44 mmol), TEA (0.24 mL, 1.76 mmol) and BOP (194 mg, 0.44 mmol)
in DCM (2 mL) was stirred at rt overnight. To the reaction mixture was added
3-cyanobenzoic acid (65 mg, 0.44 mmol), (0.24 mL, 1.76 mmol) and BOP
(194 mg, 0.44 mmol). After stirring at rt overnight, the reaction mixture was
diluted with DCM, washed with ½ saturated NaHCO3 and brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was purified on
a
preparative TLC plate (hexane/ethyl acetate: 1/1). 1H NMR and HSQC
indicated that the compound less polar on the TLC plate was trans (15a) and
the compound more polar on the TLC plate was cis (15b), where the 1H NMR
signals for the CH2-groups in the cyclohexane ring showed characteristic
patterns. For 15b, the protons were clearly axial/equatorial pairs with large
differences in chemical shifts in accordance with a chair conformation which is
preferred with the cis-configuration. This could be seen unambiguously for
CH2’s in position 2 and 5 from the standard HSQC spectrum and allowed the
assignment: H-2ax 1.37 ppm, quartet, J = 11.7 Hz; H-2eq 2.50 ppm, br d; H-5ax
1.60, multiplet; H-5 equiv 1.93 ppm, multiplet. For 15a, the chemical shift of
the protons within the same CH2-groups are almost identical in agreement
with a boat/twist boat conformation, which would be expected for the trans-
configuration. 15a: 1H NMR (500.13 MHz, CHCl3-d) d 8.57 (d, J = 4.5 Hz, 1H),
8.21 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.08 (s, 1H), 8.00 (d, J = 7.8 Hz,
1H), 7.85 (dt, J = 7.8 Hz, 1.9 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz,
1H), 7.44 (ddd, J = 7.8 Hz, 4.5 Hz, 1.9 Hz, 1H), 6.34 (d, J = 6.7 Hz, 1H), 4.33–4.45
(m, 2H), 1.86–2.05 (m, 4H), 1.60–1.86 (m, 4H). 15b: 1H NMR (500.13 MHz,
CHCl3-d) d 8.53 (d, J = 4.6 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.02 (s, 1H), 7.99 (d,
J = 7.8 Hz, 1H), 7.85 (dt, J = 7.8 Hz, 1.6 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.56 (t,
J = 7.8 Hz, 1H), 7.44 (dd, J = 7.8 Hz, 4.6 Hz, 1H), 6.00 (d, J = 7.3 Hz, 1H), 4.06–
4.19 (m, 2H), 2.50 (br d, 1H), 2.09 (m, 2H). 1.89–1.96 (m, 1H), 1.53–1.64 (m,
1H), 1.37 (q, J = 11.7 Hz, 1H), 1.19–1.33 (m, 2H).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.078.
Reference and notes
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M 20 Fluo-4/0.01% pluronic acid in assay
cells dye-loaded (25 L/well) with 3
l
l
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25 lL/well. Basal fluorescence is monitored in a fluorometric imaging plate
reader (FLIPR, Molecular Devices, Sunnyvale, CA) with an excitation
wavelength of 488 nM and an emission range of 500–560 nM. Laser
excitation energy was adjusted so that basal fluorescence readings were
approximately 10,000 relative fluorescent units. Cells were stimulated with an
EC20 or an EC80 concentration of glutamate in the presence of a compound to be
tested, both diluted in assay buffer, and relative fluorescent units were
measured at defined intervals (exposure = 0.6 s) over a 3 min period at room
temperature. Basal readings derived from negative controls were subtracted
from all samples. Maximum change in fluorescence was calculated for each
well. Concentration–response curves derived from the maximum change in
fluorescence were analyzed by nonlinear regression (Hill equation). An
antagonist can be identified from these concentration–response curves if a
compound produces
glutamate response.
a concentration dependent inhibition of the EC80
25. Binding assays were performed described in [O’Brien, J. A. et al. Mol. Pharmacol.
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at pH 7.4 to a final assay concentration of 40 l
g protein/well for [3H] 1,3-((6-
methylpyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime ([3H] ABP-688,
American Radiolabeled Chemicals, Inc., St. Louis, MO) filtration binding.
Incubations included 5 nM [3H]-ABP 688, membranes and either buffer or
varying concentrations of compound. Samples were incubated for 60 min at
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room temperature with shaking. Non-specific binding was defined with 10 lM
of RO4917523 (compound 2 in Fig. 1). After incubation, samples were filtered
over a GF/C filter (presoaked in 0.25% polyethyleneimine (PEI)) and then
washed four time using a TomtecÒ Harvester 96Ò Mach III cell harvester
(Tomtec, Hamden, CT) with 0.5 mL ice-cold 50 mM Tris–HCl (pH 7.4). IC50
values were derived from the inhibition curve and Ki values were calculated
according to the Cheng and Prusoff equation of Ki = IC50/(1 + [L]/Kd) described
in [Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099] where [L] is
the concentration of radioligand and Kd is its dissociation constant at the
receptor, derived from the saturation isotherm.
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28. Column: ChiralpakÒ AD(Daicel), 250 ꢂ 20 mm. Mobile phase: 20% isopropanol,
79.9% hexane, 0.1% diethylamine. Flow rate: 14 mL/min. UV detector: 254 nM.
29. Rat mGluR1 FLIPR assay: The cDNA for rat metabotropic glutamate receptor 1
was a generous gift from S. Nakanishi (Kyoto University, Kyoto, Japan). The
19. Topiol, S. W.; Li, G.; Zhou, H.; Jimenez, H.; Doller, D. in preparation.
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21. MOE; Chemical Computing Group: Montreal, Quebec, Canada, 2009.

1406
H. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1398–1406
1006, http://www.htdialysis.com). Test compounds (10
rmGluR1 was stably expressed in a HEK 293 cell line and grown in Dulbecco’s
l
M) were spiked into
Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) with supplements
(1.5% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin, 100l g/mL
streptomycin and 0.75 mM G1418) at 37 °C, 5% CO2. Twenty-four hours prior
to assay, cells were seeded into 384-well black wall microtiter plates coated
serum from human or rat, or rat brain homogenate, and dialyzed against
phosphate buffered saline (pH 7.4) via a >5000 molecular weight dialysis
membrane for 2.5 h in an incubator set to maintain 37 °C with 5% CO2. The
dialysis block was placed on an orbital shake. After 2.5 h incubation, buffer and
serum samples (or brain homogenate samples) were collected and transferred
into a 96-well matrix plate with tube inserts. Internal standards were added
and the samples were vortexed and mixed, and analyzed by LC–MS/MS.
32. Kinetic solubility was measured from 10 mM DMSO stock solution of the test
with poly-
loaded (25
D
-lysine. Just prior to assay, media was aspirated and cells dye-
l
L/well) with 3 M 20 Fluo-4/0.01% pluronic acid in assay buffer
l
(Hank’s Balanced Saline Solution (HBSS)): 150 mM NaCl, 5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, plus 20 mM N-2-hydroxyethylpiperazine-N0-2-
ethanesulfonic acid (HEPES), pH 7.4, 0.1% bovine serum albumin (BSA) and
2.5 mM probenecid) for 1 h in 5% CO2 at 37 °C. After excess dye was discarded,
cells were washed in assay buffer and layered with a final volume equal to
compound (300
lL) diluted with 2 ꢂ 200 lL of 25 mM potassium phosphate
buffer in a Titer plate shaker at a speed of 1.8 for 4 h. The supernatant was
analyzed by using a Waters Acquity UPLC system (Column:Acquity UPLC BEH
25
l
L/well. Basal fluorescence is monitored in a fluorometric imaging plate
C-18, 1.7
l
m
2.1 ꢂ 50 mm column and PDA detector at 254 nM for
reader (FLIPR, Molecular Devices, Sunnyvale, CA) with an excitation
wavelength of 488 nM and an emission range of 500–560 nM. Laser
excitation energy was adjusted so that basal fluorescence readings were
approximately 10,000 relative fluorescent units. Cells were stimulated with an
EC20 or an EC80 concentration of glutamate in the presence of a compound to be
tested, both diluted in assay buffer, and relative fluorescent units were
measured at defined intervals (exposure = 0.6 s) over a 3 min period at room
temperature. Basal readings derived from negative controls were subtracted
from all samples. Maximum change in fluorescence was calculated for each
well. Concentration–response curves derived from the maximum change in
fluorescence were analyzed by nonlinear regression (Hill equation).
quantitation) with a gradient (mobile phase A: 2% w/v ammonium formate
and 20% acetonitrile in water, mobile phase B: 100% acetonitrile) for 2 min.
33. LogD at pH 7.4 was determined with solid sample using classic shake flask
method described in Gulyaeva, N.; Zaslavsky, A.; Lechner, P.; Chlenov, M.;
Chait, A.; Zaslavsky, B. Eur. J. Pharm. Sci. 2002, 17, 81.
34. NMR analysis of compound 24d: From the standard HSQC and COSY spectra
24d proton 7 (CH, 4.10 ppm, br s) and protons 4 (CH2, AB-system, 3.77 ppm (d,
J = 14.9 Hz) and 3.81 ppm (d, J = 14.9 Hz) could be assigned. In the 2D-ROESY
spectrum the 4 and 7 protons show a distance correlation. This can only be true
in the case of cis-configuration with the spiro-6-ring in a chair conformation
with oxygen 1 and amide-N in an equatorial position. 1H NMR (600.13 MHz,
DMSO-d6) d 8.66 (d, J = 8.3 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.8–7.86 (m, 3H),
7.63 (ddd, J = 7.9 Hz, 2.2 Hz, 0.9 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.47 (d,
J = 7.5 Hz, 1H), 4.1 (br s, 1H), 3.81 (d, J = 14.9 Hz, 1H), 3.77 (d, J = 14.9 Hz, 1H),
2.53 (s, 3H), 2.03 (d, J = 6.0 Hz, 2H), 1.68–1.86 (m, 4H), 1.58–1.68 (m, 1H), 1.45–
1.54 (m, 1H). Spectra are in Supplemental.
30. Determination of metabolic stability in liver microsomes: This study was
conducted by incubating 1 lM concentration of test compounds at 37 °C in
pooled male rat or pooled human liver microsomes (0.5 mg/mL) in 0.1 M
potassium phosphate buffer (pH 7.4) supplemented with NADPH-
regenerating system (1.3 mM NADP+, 3 mM MgCl2, 3.5 mM glucose-6-
phosphate, and 4 units glucose-6-phosphate dehydrogenase). Aliquots were
taken at 0.25, 5, 15, 30 and 60-minute time points. The aliquots were added
to a 96-well plate containing an equal volume of acetonitrile in order to
terminate the reaction. The samples were vortexed and centrifuged at
3000 rpm for 15 min. A known volume of internal standard was added to the
supernatant. The samples were injected in an LC–MS/MS system to monitor
the disappearance of the parent compound. The half-life was measured by
plotting the log of the remaining compound versus time. The in vitro half-life
data were used to calculate the intrinsic clearance (CLint), according to the
equation shown below:
35. Mouse marble burying:
(1)Test subjects: Male CD1 mice were obtained from Charles River Laboratories
(Kingston, NY), and weighed 25–30 g. Animals were group-housed in
a
standard colony room with a 12:12 light/dark cycle (lights on at 6:00 am)
for at least one week prior to testing. Food and water were provided ad libitum.
Animals were weighed, tail marked, and randomly assigned to treatment
groups before testing.
(2) Apparatus: Standard mouse home cages were used as test cages. These
cages were individually filled with 1.5 in of Aspen bedding (PWI brand). The
bedding was flattened down and two parallel rows of 10 marbles (20 marbles
per test cage total) were placed on opposite sides of each cage. Filter tops were
used to cover each test cage.
(3) Treatment: Animals were treated with compound 24d (3, 10 and 30 mg/kg,
dissolved in 20% HPbCD), buspirone (positive control, free base, 10 mg/kg,
dissolved in distilled water) and vehicle (20% HPbCD). All animals were dosed
subcutaneously with an injection volume of 5 mL/kg.
(4) Testing procedure: Mice were individually placed in the test cages for a
session lasting 30 min after which they were removed and returned to their
home cages. The number of fully visible marbles (less than 2/3 covered with
bedding) were counted and subtracted from 20 to arrive at the number of
marbles buried. Twelve mice were tested per each group.
1
g liver weight
mL incubation
CLint ¼ 0:693 ꢂ
ꢂ
ꢂ
T1=2ðminÞ kg body weight mg microsomal protein
mg microsomal protein
ꢂ
ꢂ kg body weight
g liver weight
where, liver weight is 20 g/kg in human and 45 g/kg in rat, and
body weight is 70 kg for human and 250 g in rat. In this equa-
tion, it was assumed that 1 g of liver contains 45 mg of micro-
somal protein and the binding of the drug to microsomal
proteins was assumed to be zero, hence the unbound drug frac-
(5) Data Analysis: Data were analyzed using One-Way ANOVA with post-hoc
Dunnett’s test.
36. Chemical stability study: A solution of 0.5 mg of compound 24g in MeOH
(1 mL) and aq HCl (1 mL) at pH 1.5 was shacked at 37 °C for 1 h. The solution
was then analyzed by HPLC/MS.
tion, fu,mic = 1.
37. Jimenez, H. N.; Li, G.; Doller, D.; Grenon, M.; White, A. D.; Guo, M.; Ma, G. US
7947680.
38. Zhou, H.; Li, G.; Doller, D.; Ma, G. U.S. Pat. Appl. Publ. US 20110098299, 2011.
31. Human and rat protein binding, and rat brain homogenate free fraction were
determined by using a 96-well format equilibrium dialyzer (HTD96b, Catalog#