Discovery of 2-aminothiazole-4-carboxamides, a novel class of muscarinic M3 selective antagonists, through solution-phase parallel synthesis

Article abstract of DOI:10.1248/cpb.53.437

Synthesis and structure-activity relationship of a new class of muscarinic M₃ selective antagonists were described. In the course of searching for a muscarinic M₃ antagonist with a structure distinct from those of the 2-(4,4-difluorocyclopentyl)-2-phenylacetamide derivatives, we identified a thiazole-4-carboxamide derivative (1) as a lead compound in our in-house chemical collection. Since this compound (1) showed relatively low binding affinity (K₁ = 140 nM) for M₃ receptors in the human binding assays, we tried to improve its potency and selectivity for M ₃ over M₁ and M₂ receptors by derivatization of 1 through a combinatorial approach. A solution-phase parallel synthesis effectively contributed to the optimization of each segment of 1. Thus, we have identified a cyclooctenylmethyl derivative (3e) and a cyclononenylmethyl derivative (3f) as representative M₃ selective antagonists in this class.

Full text of DOI:10.1248/cpb.53.437

April 2005
Notes
Chem. Pharm. Bull. 53(4) 437—440 (2005)
437
Discovery of 2-Aminothiazole-4-carboxamides, a Novel Class of
Muscarinic M3 Selective Antagonists, through Solution-Phase Parallel
Synthesis
Yufu SAGARA, Morihiro MITSUYA, Minaho UCHIYAMA, Yoshio OGINO, Toshifumi KIMURA,
Norikazu OHTAKE,* and Toshiaki MASE
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd.; Okubo-3, Tsukuba, Ibaraki 300-2611, Japan.
Received October 14, 2004; accepted January 7, 2005; published online January 19, 2005
Synthesis and structure–activity relationship of a new class of muscarinic M3 selective antagonists were
described. In the course of searching for a muscarinic M3 antagonist with a structure distinct from those of the
2-(4,4-difluorocyclopentyl)-2-phenylacetamide derivatives, we identified a thiazole-4-carboxamide derivative (1)
as a lead compound in our in-house chemical collection. Since this compound (1) showed relatively low binding
affinity (Kiꢀ140 nM) for M3 receptors in the human binding assays, we tried to improve its potency and selectivity
for M3 over M1 and M2 receptors by derivatization of 1 through a combinatorial approach. A solution-phase par-
allel synthesis effectively contributed to the optimization of each segment of 1. Thus, we have identified a
cyclooctenylmethyl derivative (3e) and a cyclononenylmethyl derivative (3f) as representative M3 selective antago-
nists in this class.
Key words muscarine M3 receptor; antagonist; 2-aminothiazole-4-carboxamide; parallel synthesis
To date, five distinct but homologous gene sequences cod- ing the structural complexity of 1 due to the five chiral cen-
ing for muscarinic receptors (m1, m2, m3, m4, m5) have ters, we first replaced the perhydronaphtylmethyl moiety
been identified and cloned.1—5) Pharmacologically, four sub- with a naphtylmethyl group, without regard for the binding
types (M1, M2, M3, M4) have been defined.6—8) Among these affinity of the compound. Optimization of the compound (2)
muscarinic receptor subtypes, M3 receptors are localized in by using a solution-phase parallel synthesis method led us to
smooth muscle and mucosal glands and mediate contraction the identification of M3 selective antagonists (3e, f) showing
and mucus secretion. M1 receptors, localized to the post-gan- high potency (Kiꢀca. 1 nM) for M3 receptors and greater than
glionic cholinergic nerve terminals and glands, facilitate neu- 100-fold selectivity for M3 over M1 and M2 receptors.
rotransmission and gastric secretion. Neuronal M2 receptors
In this paper, we describe the synthesis of aminothiazole
provide a functional negative feedback modulation of acetyl- derivatives, their binding affinities for M1—M3 receptors in
choline (ACh) release.9,10) Extensive efforts have been the binding assay, and their selectivity for M3 over M1 and
directed to the identification of potent and subtype-selective M2 receptors, and discuss their structure–activity and struc-
M3 antagonists to complete the classification of the receptor ture–selectivity relationships.
subtypes and to provide more ideal therapeutic agents,11—13)
however, few structure classes with sufficient M3 selectivity Chemistry
have been discovered to date.14)
Preparation of 3f was outlined in Chart 1 as a representa-
As a part of our program for developing a muscarinic M3 tive procedure for the series of 2-aminothiazole-4-carboxam-
receptor antagonist for the treatment of pulmonary or urinary ide derivatives. The 2-aminothiazole-4-carboxylic acid (5)
diseases, we pursued M3 antagonists that are structurally dis- was derived from a thiourea (4) and ethyl 2-bromopyruvate
tinct from a series of 2-(4,4-difluorocyclopentyl)-2-phenylac- by a conventional method in 86% yield. The (3S)-3-
etamide derivatives such as Compound A13) and have selec- aminomethylpiperidine (10), a component of 3f, was pre-
tivity for M3 receptors two orders of magnitude greater than pared from a racemic ethyl nipecotate (7).15) Optical resolu-
those for M1 and M2 receptors. As a result of screening of tion of ethyl nipecotate was performed using a standard
our in-house chemical collection, a thiazole-4-carboxamide method using L-tartaric acid to give a (3R)-ethyl nipecotate
derivative (1) was identified as a new lead structure. Avoid- (8). Following the reduction of 8 with LAH, the piperidine
Chart 1
∗ To whom correspondence should be addressed. e-mail: norikazu_ohtake@merck.com
© 2005 Pharmaceutical Society of Japan

438
Vol. 53, No. 4
Table 1. The Binding Affinity of the Compounds (14—16) for M3 Recep-
tors and the Selectivity for M3 over M1 and M2 Receptors
nitrogen was protected as a tert-butylcarbamate to afford the
alcohol, which was converted to an azide (9) via the mesylate
in 69% yield from 8. 9 was hydrogenated to produce the
amine (10) in quantitative yield. Coupling of the acid (5)
with 10 was achieved using a standard protocol (EDCI and
HOBT) to give an amide (6). Deprotection of the Boc group
in 6 under acidic conditions, followed by reductive alkylation
by treatment with an aldehyde (13)16) in the presence of
NaBH(OAc)3 afforded 3f in 76% yield.
M3
Selectivity
R
(Ki, nM)
M1/M3 M2/M3
14
15
16
250
1100
1600
4.6
1.3
1.7
55
25
27
Results and Discussion
Compounds prepared by a solution-phase parallel synthe-
sis were tested in an initial screen to assess the percentage of
inhibition at 1 mM in the binding assay for the muscarinic M3
receptor subtype in transfected CHO cells.17) Selected com- Table 2. Percent Inhibition of Compounds at 1mM to M3 Receptors
pounds showing greater than 50% inhibition at 1 mM were
subsequently purified or re-synthesized and tested in the
binding assay for muscarinic receptor subtypes (M1, M2, M3)
to determine the Ki values18) and subtype selectivity (M1/M3,
M2/M3).
R1
% inhibition at 1mM
Before applying the solution-phase parallel synthesis for
optimization of 2, we prepared the three compounds (14—
16) and tested their binding affinity to examine the necessity
of the asymmetric carbon on the piperidine ring of 2. Of
them, compound (14) bearing a (3S)-piperidine moiety
clearly showed the best binding and selectivity profiles
(Table 1).
Thus, the (3S)-piperidine part being fixed, we tried to
replace the 1,4-benzodioxane moiety of 14 with various
functional groups using parallel synthesis (Table 2). Among
26 kinds of substituents introduced into the 2 position (R1)
on the aminothiazole ring, only an N-methylphenylamino
group (14a) showed inhibitory activity comparable to 14.
The Ki values of 14a for the M1, M2 and M3 receptor sub-
types were confirmed to be 1200, 54000 and 230 nM, respec-
tively, and the selectivity for M3 over the M1 and M2 recep-
tors was 5- and 230-fold, respectively.
Table 3. Percent Inhibition of Compounds at 0.1mM to M3 Receptors
Comparison of these binding data with those of 14 indi-
cated that an N-methylphenylamino group played an impor-
tant role in improving the selectivity for M3 over M2 recep-
tors, while this moiety did not contribute to enhancement of
the M3 binding affinity. Thus, we selected an N-methyl-N-
phenylamino group as the optimized R1 segment.
R2
% inhibition at 0.1mM
Next, we tried to optimize the naphtylmethyl moiety of
14a, which was tentatively introduced into the piperidine
nitrogen to avoid the complexity of the stereocenters of the
perhydronaphthyl group in 1, by substituting this moiety with
various aromatic or cycloalkyl groups (Table 3). In this case,
compounds were screened by the percentage of inhibition for
M3 receptors at 0.1 mM. Two substituents, a cyclohexylethyl
and a cyclooctylmethyl group, seemed to be most effective in
enhancing the binding affinity among 23 kinds of functional
groups. Evaluation of the Ki values of the two compounds
(3a, b) for the three receptor subtypes indicated that 3b with
a cyclooctylmethyl moiety displayed more potent activity
(Kiꢀ20 nM) for M3 receptors than 3a. Also, 3b had better
selectivity for M3 over M2 receptors (M2/M3ꢀ74). Therefore,
further optimization of the R2 segment in 3b was conducted
by replacing the cyclooctylmethyl moiety with larger ring-
sized cycloalkylmethyl groups such as a cyclononyl- and
cyclodecylmethyl groups (Table 4).
Replacement with a cyclononylmethyl group (3c) resulted
in enhancement of the M3 binding affinity to some extent,
while the selectivity for M3 over M1 and M2 receptors was
maintained. Introduction of a cyclodecylmethyl group (3d)
dramatically improved the binding affinity and selectivity for
M3 over M2 receptors. In the process of identification of the
2-cyclopentyl-2-phenylacetamide derivatives,17) we found
that installment of a double bond into the piperidinyl side
chain was effective in enhancing M3 binding affinity. There-
fore, we prepared cycloalkenylmethyl derivatives (3e, f)

April 2005
439
Table 4. The Binding Affinity of the Compounds (3a—f, 14a) for M3 Re-
ceptors and the Selectivity for M3 over M1 and M2 Receptors
M3
Selectivity
M3
Selectivity
Compd. R2
Compd. R2
(Ki, nM) M1/M3 M2/M3
(Ki, nM) M1/M3 M2/M3
Fig. 1. Structures of Representative Compounds
3a
3b
3c
3d
52
20
10
33
34
42
32
74
3e
3f
1.1 110
0.92 110
560
310
enchnced selectivity for M3 over M1 receptors.
In conclusion, we have succeeded in identifying a new
class of M3 selective antagonists by derivatization of the lead
compound (1) through the combinatorial approach. In this
class, the cyclooctenylmethyl (3e) and cyclononenylmethyl
(3f) derivatives were found to show potent binding affinities
for M3 receptors, together with greater than 100-fold selec-
tivity for M3 over M1 and M2 receptors. These aminothiazole
derivatives are the alternative examples showing greater than
100-fold selectivity for M3 over M1 and M2 receptors,14) and
these might be useful tools for complete characterization of
the roles of M3 receptor subtype and better understanding for
the binding mode of the M3 selective antagonist.
93 14a
200
230
5.3 230
1.0 64
Table 5. The Binding Affinity of the Compounds (3g—m) for M3 Recep-
tors and the Selectivity for M3 over M1 and M2 Receptors
M3
Selectivity
M3
Selectivity
Compd. R3
Compd. R3
(Ki, nM) M1/M3 M2/M3
(Ki, nM) M1/M3 M2/M3
Experimental
Materials and Methods All reagents and solvents were of commercial
quality and used without further purification unless otherwise noted. Melting
points were determined with a Yanaco MP micromelting point apparatus and
3g
3h
3i
0.92 110 310
3k
1200
19
1.0
3.4
28
420
210
33
2.6
5.0 11
8.6 3l
31 110
1
were not corrected. H-NMR spectra were obtained on a JEOL AL400 with
tetramethylsilane as an internal standard. Mass spectrometry was performed
with a JEOL JMS-SX 102A. Elemental analysis was performed on an
EA-1108 FISONS Instruments CHNOS analyzer. TLC was done using
Merck Kieselgel F254 pre-coated plates. Silica gel column chromatography
was carried out on Wako gel C-200.
9.5 3m
8.3 110 290
3j
2-(N-Methyl-N-phenylamino)thiazole-4-carboxylic Acid (5) 1) To a
solution of 4 (3.0 g, 20 mmol) in EtOH (70 ml) was added ethyl bromopyru-
vate (3.0 ml, 1.24 mmol), and the mixture was heated at 90 °C for 3 h. After
cooling to room temperature, the solvent was removed under reduced pres-
sure, and the residue was diluted with EtOAc. The organic layer was washed
with aqueous NaHCO3 solution and brine, dried over MgSO4, and evapo-
rated. The suspension of the residue in toluene–hexane was heated, and the
insoluble material was removed by filtration. The filtrate was concentrated
under reduced pressure, and the resulting crystalline solid was collected by
filtration, washed with toluene and dried to produce ethyl 2-(N-phenyl-
amino)thiazole-4-carboxylate (3.4 g, 14 mmol, 70%).
and evaluated their binding affinities. As expected, the
cyclooctenylmethyl derivative (3e) displayed more potent
binding activity and much better selectivity for M3 over M1
and M2 receptors than the corresponding cyclooctylmethyl
derivative (3b). Similarly, the cyclononenylmethyl derivative
(3f) was 10-fold more active and approximately 3-fold more
selective for M3 over M2 receptors than the cyclononylmethyl
derivative (3c).
Further derivatization of 3f was performed for the opti-
mization of the R3 segment (Table 5). These derivatizations
indicated that the distance between the amide carbonyl and
cationic amine parts, and a suitable structural rigidity as the
R3 segment were important for the M3 binding affinity. Based
on these modifications, the (3S)-3-aminomethylpiperidine
moiety was an optimal R3 segment. It is interesting to note
that although both 3f and Compound B (Fig. 1)14) have excel-
lent M3 potency and selectivity for M3 over M1 and M2
receptors, each of them has a different stereochemical struc-
ture in terms of the 3-aminomethylpiperidine portion. One of
the reasons we assume is that the spatial arragangement of
the whole molecule plays a key role in the binding interac-
tion between the M3 receptors and the antagonists. On the
other hand, Compound A (Fig. 1) with a conservative struc-
ture had a good potency for M3 recptors, but was only selec-
tive for M3 over M2 receptors. Comparison of the structure of
Compound A with those of the M3 selective antagonists
(3f and Compound B) suggests that a larger size of the acid
2) To a solution of ethyl 2-(N-phenylamino)thiazole-4-carboxylate (2.5 g,
10 mmol) in DMF (25 ml) was added NaH (60% in mineral oil, 600 mg,
15 mmol) at 0 °C. After the mixture was stirred for 15 min at 0 °C, MeI
(3.1 ml, 50 mmol) was added and the mixture was stirred for 2 h. The reac-
tion was quenched by adding aqueous NH4Cl solution, and the mixture was
extracted with Et2O. The organic phase was dried (MgSO4), and evaporated.
The residue was purified by silica gel column chromatography (hexane–
EtOAc, 10 : 1 elution) to give ethyl 2-(N-methyl-N-phenylamino)thiazole-4-
carboxylate (2.2 g, 2.4 mmol, 84%) as a colorless oil.
3) To a solution of ethyl 2-(N-methyl-N-phenylamino)thiazole-4-carboxy-
late (1.9 g, 7.2 mmol) in MeOH (40 ml) was added 3 N NaOH (15 ml), and
the mixture was stirred for 15 h at room temperature. After completion of
the reaction, the mixture was washed with Et2O. The aqueous phase was
acidified with 3 N HCl, and extracted with CHCl3. The organic phase was
dried (MgSO4), and evaporated to give 5 (1.7 g, 7.3 mmol, quant.) as a white
1
solid: mp 159—162 °C; H-NMR (400 MHz, CDCl3) d: 3.56 (3H, s), 7.34
(1H, t, Jꢀ7.8 Hz), 7.37 (2H, d, Jꢀ7.8 Hz), 7.44 (1H, s), 7.46 (2H, t,
Jꢀ7.8 Hz); FAB-MS m/z 233 (C11H10N2O2S ꢁH)ꢁ.
N-[(3S)-1-(Cyclononen-1-ylmethyl)piperidin-3-ylmethyl]-2-(N-methyl-
N-phenylamino)thiazole-4-carboxamide (3f) 1) To
a mixture of 5
(551 mg, 2.4 mmol) and 10 (630 mg, 2.9 mmol) in CHCl3 (15 ml) were
added EDCI–HCl (500 mg, 2.6 mmol) and HOBt (540 mg, 3.5 mmol), and
the mixture was stirred at room temperature for 16 h. The reaction was
part or the cationic amine side chain would contribute to the quenched by adding aqueous NaHCO3 solution, and the mixture was

440
Vol. 53, No. 4
extracted with Et2O. The organic phase was washed with H2O and brine,
dried (MgSO4), and evaporated. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc, 1 : 1 elution) to give 6 (1.04 g,
M., Haga T., Haga K., Ichiyama A., Kanagawa K., Kojima M., Matsuo
H., Hirose T., Numa S., Nature (London), 323, 411—416 (1986).
2) Kubo T., Maeda A., Sugimoto K., Akiba I., Mikami A., Takahashi H.,
Haga T., Haga K., Ichiyama A., Kanagawa K., Matsuo H., Hirose T.,
Numa S., FEBS Lett., 209, 367—372 (1986).
3) Peralta E. G., Ashkenazi A., Winslow J. W., Smith D. H., Ramachan-
dran J., Capon D. J., EMBO J., 6, 3923—3929 (1986).
4) Bonner T. I., Buckley N. J., Young A. C., Brann M. R., Science, 237,
527—532 (1987).
5) Bonner T. I., Young A. C., Brann M. R., Buckley N. J., Neuron, 1,
403—410 (1988).
6) Hulme E. C., Birdsall N. J. M., Buckley N. J., Ann. Rev. Pharmacol.
Toxicol., 30, 633—673 (1990).
1
2.4 mmol, quant.) as a white foam: H-NMR (400 MHz, CDCl3) d: 1.20—
1.33 (1H, m), 1.45 (9H, s), 1.61—1.92 (4H, m), 2.72 (1H, t, Jꢀ11.0 Hz),
2.90 (1H, ddd, Jꢀ13.0, 11.0, 2.9 Hz), 3.18—3.48 (2H, m), 3.54 (3H, s),
3.75—4.06 (2H, m), 7.26—7.33 (3H, m), 7.38 (2H, d, Jꢀ7.8 Hz), 7.45 (2H,
t, Jꢀ7.8 Hz); FAB-MS m/z 431 (C22H30N4O3S + H)ꢂ.
2) A mixture of 6 (1.04 g) in 10% HCl–MeOH (10 ml) was stirred at room
temperature for 13 h. The reaction mixture was basified (pH 9) with aqueous
NaHCO3 solution and extracted with CHCl3. The organic phase was dried
(MgSO4) and evaporated to give the crude product, N-[(3S)-piperidin-
3-ylmethyl]-2-(N-methyl-N-phenylamino)thiazole-4-carboxamide (790 mg,
1
2.4 mmol, quant.) as a colorless oil: H-NMR (400 MHz, CDCl3) d: 1.13—
7) Caulfield M. P., Pharmacol. Ther., 58, 319—379 (1993).
8) Barnes P. J., Life Sci., 52, 521—527 (1993).
1.27 (1H, m), 1.40—1.53 (1H, m), 1.65—1.81 (3H, m), 1.83—1.91 (1H, m),
2.40 (1H, t, Jꢀ11.3 Hz), 2.58 (1H, td, Jꢀ11.3, 2.9 Hz), 3.00 (1H, d,
9) Minette V. A., Barnes P. J., J. Appl. Physiol., 64, 2532—2537 (1988).
Jꢀ12.2 Hz), 3.12 (1H, d, Jꢀ12.2 Hz), 3.22—3.39 (2H, m), 3.53 (3H, d, 10) Fryer A. D., Maclagan J., Eur. J. Pharmacol., 139, 147—151 (1991).
Jꢀ1.5 Hz), 7.26—7.34 (3H, m), 7.38 (2H, d, Jꢀ7.8 Hz), 7.44 (2H, t,
11) Alabaster V. A., Life Sci., 60, 1053—1060 (1997).
12) Miyachi H., Kiyota H., Segawa M., Biorg. Med. Chem. Lett., 8,
2163—2168 (1998).
Jꢀ7.8 Hz); FAB-MS m/z 331 (C17H22N4O3S + H)ꢂ.
3) To a solution of N-[(3S)-piperidin-3-ylmethyl]-2-(N-methyl-N-phenyl-
amino)thiazole-4-carboxamide (25 mg, 0.076 mmol) in THF (1 ml) was 13) Mitsuya M., Kobayashi K., Kawakami K., Satoh A., Ogino Y.,
added 13 (30 mg, 0.20 mmol), AcOH (6 mg, 0.10 mmol), and NaBH(OAc)3
(50 mg, 0.23 mmol), and the mixture was stirred at room temperature for
16 h. The reaction was quenched by adding saturated aqueous NaHCO3 solu-
tion and extracted with CHCl3. The organic phase was dried (Na2SO4), and
evaporated. The residue was purified by preparative TLC (CHCl3–
MeOHꢀ9 : 1) to give 3f (27 mg, 0.058 mmol, 76%) as a colorless oil: 1H-
Kakikawa T., Ohtake N., Kimura T., Hirose H., Sato A., Numazawa T.,
Hasegawa T., Noguchi K., Mase T., J. Med. Chem., 43, 5017—5029
(2000).
14) Recently, we succeeded in identifying M3 selective antagonists; Sagara
Y., Sagara T., Mase T., Kimura T., Numazawa T., Fujikawa T., Noguchi
K., Ohtake N., J. Med. Chem., 45, 984—987 (2002).
NMR (400 MHz, CDCl3) d: 1.01—2.01 (17H, m), 2.07—2.25 (4H, m), 15) Hilpert K., Ackermann J., Banner D. W., Gast A., Gubernator K.,
2.69—2.93 (4H, m), 3.28—3.38 (2H, m), 3.54 (3H, s), 5.44 (1H, t, Jꢀ
8.3 Hz), 7.25—7.35 (3H, m), 7.38 (2H, d, Jꢀ7.8 Hz), 7.44 (2H, t, Jꢀ
Hadváry P., Labler L., Müller K., Schmid G., Tschopp T. B., Water-
beemd H., J. Med. Chem., 37, 3889—3901 (1994).
7.8 Hz); HR-MS Calcd for C27H39N4SO (MꢂH)ꢂ: 467.2845, Found 16) Naya A., Sagara Y., Ohwaki K., Saeki T., Ichikawa D., Iwasawa Y.,
467.2820; Anal. Calcd for C27H39N4O·0.5H2O·0.3CHCl3: C, 64.10; H,
7.74; N, 10.95. Found: C, 64.40; H, 7.93; N, 10.70.
Noguchi K., Ohtake N., J. Med. Chem., 44, 1429—1435 (2001).
17) Mitsuya M., Mase T., Tsuchiya Y., Kawakami K., Hattori H.,
Kobayashi K., Ogino Y., Fujikawa T., Satoh A., Kimura T., Noguchi
K., Ohtake N., Tomimoto K., Bioorg. Med. Chem., 7, 2555—2567
(1999).
Binding Assay According to the reported method,17) the binding affini-
ties were determined by inhibition of specific binding of [3H]-NMS using
membranes from CHO cells expressing cloned human M1—M3 receptors.
The Ki values of compounds were expressed the means of two or more inde-
pendent assays.
18) Cheng Y. C., Prusoff W. H., Biochem. Pharmacol., 22, 3099—3108
(1973).
References and Notes
1) Kubo T., Fukuda K., Mikami A., Maeda A., Takahashi H., Mishina