Development of a Novel Human Parathyroid Hormone Receptor 1 (hPTHR1) Agonist (CH5447240), a Potent and Orally Available Small Molecule for Treatment of Hypoparathyroidism

Article abstract of DOI:10.1021/acs.jmedchem.8b00182

During the course of derivatization of HTS hit 4a, we have identified a novel small-molecule hPTHR1 agonist, 1-(3,5-dimethyl-4-(2-((2-((1R,4R)-4-methylcyclohexyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1-methylurea (CH5447240, 14l). Compound 14l exhibited a potent in vitro hPTHR1 agonist effect with EC₂₀ of 3.0 μM and EC₅₀ of 12 μM and showed excellent physicochemical properties, such as high solubility in fasted state simulated intestinal fluid and good metabolic stability in human liver microsomes. Importantly, 14l showed 55% oral bioavailability and a significantly elevated serum calcium level in hypocalcemic model rats.

Full text of DOI:10.1021/acs.jmedchem.8b00182

Subscriber access provided by NORTHERN KENTUCKY UNIV STEELY
Article
Development of a Novel Human Parathyroid Hormone
Receptor 1 (hPTHR1) Agonist (CH5447240), a Potent and Orally
Available Small-Molecule for Treatment of Hypoparathyroidism
YOSHIKAZU NISHIMURA, TORU ESAKI, YOSHIAKI ISSHIKI, NAOKI OKAMOTO, Yoshiyuki
Furuta, TOMOYA KOTAKE, YOSHIAKI WATANABE, MASATERU OHTA, Toshito Nakagawa,
HIROSHI NODA, MASARU SHIMIZU, Hitoshi Saito, Tatsuya Tamura, and Haruhiko Sato
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00182 • Publication Date (Web): 22 Jun 2018
Downloaded from http://pubs.acs.org on June 23, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Development of a Novel Human Parathyroid Hormone
Receptor 1 (hPTHR1) Agonist (CH5447240), a Potent
and Orally Available SmallꢀMolecule for Treatment of
Hypoparathyroidism
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Yoshikazu Nishimura*, Toru Esaki, Yoshiaki Isshiki, Naoki Okamoto, Yoshiyuki Furuta, Tomoya Kotake, Yoshiaki
Watanabe, Masateru Ohta, Toshito Nakagawa, Hiroshi Noda, Masaru Shimizu, Hitoshi Saito, Tatsuya Tamura,
Haruhiko Sato
Chugai Pharmaceutical Co., Ltd., Research Division, Komakado 1ꢀ135, Gotemba, Shizuoka, Japan
ABSTRACT
During the course of derivatization of HTS hit 4a, we have identified a novel smallꢀmolecule hPTHR1 agonist,
1ꢀ(3,5ꢀdimethylꢀ4ꢀ(2ꢀ((2ꢀ((1R,4R)ꢀ4ꢀmethylcyclohexyl)ꢀ4ꢀoxoꢀ1,3,8ꢀtriazaspiro[4.5]decꢀ1ꢀenꢀ8ꢀyl)sulfonyl)ethyl)p
henyl)ꢀ1ꢀmethylurea (CH5447240, 14l). Compound 14l exhibited a potent in vitro hPTHR1 agonist effect with
EC20 of 3.0 nM and EC50 of 12 nM and showed excellent physicochemical properties, such as high solubility in
fasted state simulated intestinal fluid and good metabolic stability in human liver microsomes. Importantly, 14l
showed 55% oral bioavailability, and a significantly elevated serum calcium level in hypocalcemic model rats.
INTRODUCTION
Hypoparathyroidism is an endocrine disorder most commonly seen after surgical removal of a parathyroid gland
in patients with thyroid cancer, and is also associated with hereditary or acquired abnormalities, such as
autoimmunity, DiGeorge syndrome, mitochondrial dysfunction, and activating mutations of the calciumꢀsensing
receptor. It is caused by insufficient production of parathyroid hormone (PTH) from the parathyroid glands or by
tissue insensitivity to PTH, a polypeptide containing 84 amino acids that is secreted by the parathyroid glands to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 49
1
2
3
4
5
6
7
8
9
maintain serum calcium and phosphate levels through the PTH type 1 receptor (PTHR1) in bone and kidney1,2.
Hypoparathyroidism is characterized by hypocalcemia and hyperphosphatemia and the clinical signs of
hypocalcemiaꢀassociated hypoparathyroidism are tetany, seizures, mental disturbances, congestive heart failure, or
stridor, while hyperphosphatemia can contribute to major longꢀterm complications, such as ectopic mineralization
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
in the soft tissues (kidney, brain, eye and vascular)3ꢀ5
.
To maintain the serum calcium level of hypoparathyroidism patients, highꢀdose oral calcium and active vitamin
D have conventionally been used. However, longꢀterm vitamin D therapy causes not only marked swings in blood
calcium (hypercalcemia and hypocalcemia) but also hypercalciuria, resulting in nephrolithiasis and impaired renal
function6,7. In contrast, PTH can maintain serum calcium level without hypercalciuria8,9, and recombinant human
PTH(1ꢀ84) was recently launched as an alternative option for patients with hypoparathyroidism whose calcium
level cannot be controlled on calcium and vitamin D10.
However, because of its peptidic nature, recombinant PTH is subcutaneously injected. Although some oral
deliveries of PTH have been investigated, no successful formulation has been reported so far. Therefore, the advent
of orally available smallꢀmolecule hPTHR1 agonists is still eagerly anticipated. At the time of writing, two
smallꢀmolecule hPTHR1 antagonists, compound 1 (SW106)11,12 and compound 213, have been reported, and only
one smallꢀmolecule agonist, compound 3 (AH3960)12,14, although its clinical development has not been reported.
Antagonist
Agonist
(AH3960)
3
(SW106)
1
2
Figure 1. Smallꢀmolecule PTHR1 Ligands.
ACS Paragon Plus Environment

Page 3 of 49
Journal of Medicinal Chemistry
1
2
3
4
Recently we have reported an orallyꢀactive small molecule PTHR1 agonist, 1ꢀ{3,5ꢀdimethylꢀ4ꢀ[2ꢀ({4ꢀoxoꢀ2ꢀ
5
6
7
[4ꢀ(trifluoromethoxy)phenyl]ꢀ1,3,8ꢀtriazaspiro[4.5]decꢀ1ꢀenꢀ8ꢀyl}
8
9
sulfonyl)ethyl]phenyl}ꢀ5,5ꢀdimethylimidazolidineꢀ2,4ꢀdione (PCO371), and its potential application for
hypoparathyroidism15. In this report, we explain the optimization of an HTS hit 4a into compound 14l
(CH5447240), which became the lead compound in the discovery of PCO371, and outline its characterization as a
highly orally active PTHR1 agonist.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HTS Hit 4a
± 120
μ
M
hPTHR1 cAMP EC20 530
μ
M
EC50 >1000
Figure 2. Structure and hPTHR1 agonistic activity of HTS hit 4a.
CHEMISTRY
Novel hPTHR1 smallꢀmolecule agonists reported herein were synthesized as shown in Schemes 1ꢀ4. The
synthesis of 3ꢀchlorophenyl sulfonamides 4a-h started from the reaction of piperidinꢀ4ꢀone hydrochloride (5) and
3ꢀchlorobenzenesulfonyl chloride (6), as shown in Scheme 1. The resulting ketone 7 was converted to aminonitrile
8 by Strecker synthesis. Compound 8 was then reacted with various acyl chlorides or carboxylic acids to give 9a-h.
Finally, compounds 9a-h were treated with hydrogen peroxide under basic conditions to afford spiroꢀimidazolone
derivatives 4a-h.
Scheme 1. Synthesis of 3ꢀchlorobenzenesulfonamide derivatives
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 49
1
2
3
4
HCl
a
b
5
6
7
6
5
7
8
8
9
c or d
e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9a-h
4a-h
: R = 2,4ꢀdiꢀClꢀPh
: R = 4ꢀClꢀPh
: R = 2ꢀClꢀPh
: R = Ph
: R = 4ꢀCO HꢀPh
: R = 4ꢀPy
: R = cHex
: R = transꢀ4ꢀMeꢀcHex
4a
4b
4c
4d
4e
4f
4g
4h
: R = 2,4ꢀdiꢀClꢀPh
: R = 4ꢀClꢀPh
: R = 2ꢀClꢀPh
9a
9b
9c
9d
9e
9f
9g
9h
: R = Ph
: R = 4ꢀCO MeꢀPh
2
2
: R = 4ꢀPy
: R = cHex
: R = transꢀ4ꢀMeꢀcHex
Reagents and conditions: (a) K2CO3/CHCl3ꢀH2O, room temperature, 72%. (b) KCN, NH4OAc, MeOH, room
temperature, 86%. (c) RꢀCOCl, Na2CO3, CH2Cl2, room temperature, 44%–90%. (d)
(1R,4R)ꢀ4ꢀmethylcyclohexaneꢀ1ꢀcarboxylic acid, HATU, DIPEA, DMF, room temperature, 90%. (e) 30% H2O2 aq.,
6M NaOH aq., EtOH, 80ºC, 45%–76%.
Compounds 14a and 14d-f were prepared as shown in Scheme 2. Initially, aminonitrile 10 was reacted with acid
chlorides to afford amide nitriles 11a-c. Cyclization of 11a-c to 12a-c, followed by the deprotection of BOC group
under acidic conditions, gave piperidines 13a-d. Acyl piperidines 14a and 14d-f were finally obtained by coupling
of sulfonyl chlorides with amine 13a-d.
Scheme 2. Synthesis of sulfonylpiperidines
ACS Paragon Plus Environment

Page 5 of 49
Journal of Medicinal Chemistry
1
2
3
4
a
b
5
6
7
8
9
10
12a: R = 2,4ꢀdiꢀClꢀPh
12b: R= cHexyl
12c: R= 4ꢀMeꢀcHexyl
11a: R = 2,4ꢀdiꢀClꢀPh
11b: R = cHexyl
11c: R= 4ꢀMeꢀcHexyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
e
14a, 14d, 14e
Ph
2
14a: R = CH
c or d
2 2
14d: R = (CH ) ꢀNaphtyl
14e: R = Naphtyl
e
13a: R = 2,4ꢀdiꢀClꢀPh (2HCl salt)
13b: R = cHexyl (2TFA salt)
13c: R = cHexyl (2HCl salt)
13d: R = transꢀ4ꢀMeꢀcHexyl (2HCl salt)
14f
Reagents and conditions: (a) RꢀCOCl, Et3N, DMAP, CH2Cl2, room temperature, 30%–71%. (b) 30% H2O2 aq., 6M
NaOH aq., EtOH, 80ºC, 43%–86%. (c) 4M HClꢀdioxane, room temperature, 99%–100%. (d) TFA, CH2Cl2, room
temperature, 92%. (e) RꢀSO2Cl, Et3N, CH2Cl2, room temperature, 39%–90%.
Arylethylsulfonamides 14b-c, 14g-i, and 14k were prepared by following the synthetic route depicted in Scheme
3. Piperidines 13c-d were reacted with 2ꢀchloroethaneꢀ1ꢀsulfonyl chloride to give vinylsulfonamides 15a-b. Heck
reaction of 15a with aryl bromides afforded transꢀethenesulfonamides 17b-c, 17g-i, and 17k, followed by
hydrogenation of olefin to give arylethylsulfonamides 14b-c, 14g-i, and 14k.
Scheme 3. Synthesis of arylethylsulfonamides via Heck reaction
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 49
1
2
3
4
2HCl
b
a
5
6
7
8
ArꢀBr
16b, c, g, h, i, k
15a: R = H
15b: R = Me
13c: R = H
13d: R = Me
17b, c, g, h, i, k
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16b, 17b, 14b: Ar = Ph
16c, 17c, 14c: Ar = 4ꢀPy
16g, 17g, 14g: Ar = 2ꢀMeꢀPh
16h, 17h, 14h: Ar = 2ꢀMeꢀ4ꢀNHAcꢀPh
16i, 17i, 14i: Ar = 2,6ꢀdiꢀMeꢀ4ꢀNHAcꢀPh
c or d or e
14b, c, g, h, i, k
16k, 17k, 14k: Ar = 2,6ꢀdiꢀMeꢀ4ꢀNꢀimidazolineꢀ2ꢀoneꢀPh
f, g
18
16k
Reagents and conditions: (a) 2ꢀchloroethaneꢀ1ꢀsulfonyl chloride, Et3N, CH2Cl2, room temperature, 66%–80%. (b)
ArBr, Pd(dba)2, (tꢀBu)3PꢀHBF4, methyl dicyclohexylamine, NMP, 110ºC, 51%–87%. (c) 10% Pd/C, H2,
EtOHꢀAcOEtꢀAcOH, room temperature, 68%–75%. (d) 10% Pd/C, H2, EtOHꢀDMF, 30 bar, 40ºC (HꢀCube®), 63%–
66%. (e) 20% Pd(OH)2/C, H2, MeOH, room temperature, 41%–78%. (f) 2ꢀchloroethyl isocyanate, DMF, room
temperature. (g) NaH, DMF, room temperature, 81% from 18.
The synthetic route of Nꢀmethylurea derivatives 14j and 14l is illustrated in Scheme 4. Heck reaction of
vinylsulfonamides 15a-b with aryl bromide 19 gave transꢀolefins 20a-b. NꢀMethylurea derivatives 21a-b were
prepared by the reaction of 20a-b with sodium cyanate under acidic conditions. Finally hydrogenation of the olefin
moiety of 21a-b afforded 14j and 14l. Aryl bromide reagent 19 was prepared by methylation and deprotection from
22.
Scheme 4. Synthesis of Nꢀmethylurea derivatives
ACS Paragon Plus Environment

Page 7 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
a
b
7
8
9
15a: R = H
15b: R = Me
20a: R = H
20b: R = Me
21a: R = H
21b: R = Me
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
c
14j: R = H
14l: R = Me
d
e
19
23
22
Reagents and conditions: (a) 4ꢀbromoꢀN,3,5ꢀtrimethylaniline (19), Pd(dba)2, (tꢀBu)3PꢀHBF4, methyl
dicyclohexylamine, NMP, 110ºC, 77%–79%. (b) NaOCN, AcOH, CH2Cl2, room temperature, 82%–97%. (c) 20%
Pd(OH)2/C, H2, MeOH, room temperature, 62%–80%. (d) MeI, NaH, DMF, room temperature, 93%. (e) TFA,
CH2Cl2, room temperature, 99%.
RESULTS AND DISCUSSION
With the aim of identifying novel orally available smallꢀmolecule hPTHR1 agonists, we conducted an HTS
campaign on more than one million compounds in the ChugaiꢀRoche library using a sensitive cellꢀbased assay16
and have identified spiroꢀimidazolone derivative 4a as a hit. hPTHR1 agonistic activity was evaluated in a cAMP
production assay in HKRKꢀB7 cells that stably express hPTHR1 LLCꢀPK1 cells. Parent LLCꢀPK1 cells without
hPTHR1 were used as a counter assay. Hit compound 4a shows the cAMP activity in HKRKꢀB7 cells expressing
hPTHR1, whereas no cAMP activity was observed in LLCꢀPK1 cells without hPTHR1. To enhance the activity of
agonist 4a, of which the EC20 value was in the subꢀmillimolar range, we conducted structureꢀactivity relationship
(SAR) studies focusing on the two phenyl rings and the sulfonyl linker. The first SAR studies, focusing on the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 49
1
2
3
4
5
6
7
8
9
phenyl ring at the 2ꢀposition of spiroꢀimidazolone, are shown in Table 1. Compounds 4a-h were tested in vitro on
hPTHR1ꢀexpressing HKRKꢀB7 cells, and 1 mM of each of those compounds produced various extents of cAMP
activity. In comparison with 4a, mono chlorophenyl derivatives 4b and 4c produced similar agonistic activity with
values of 22% and 18%, respectively. Surprisingly, the agonistic activity of nonꢀsubstituted phenyl derivative 4d
was almost completely undetected, and moreover, the hydrophilic derivatives 4e-f were also inactive. On the other
hand, cyclohexyl derivatives 4g-h showed more potent agonistic activity than 4a. From these results, hydrophobic
substituents on the imidazolone moiety were suggested to be important for the PTHR1 agonistic activity. These
findings focused our next efforts on derivatization at the phenylsulfonyl moiety of 4g. Because the derivatized
compounds had weak PTHR1 agonistic activity at the beginning of the hit to lead optimization, the agonistic
activity at 1 mM concentration and the EC20 value were used as indicators for structural modifications.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. SAR of 2ꢀsubstituted imidazolone derivatives
cAMP Production at 1
No.
4a
4b
4c
R
mM (%)*1
23 ± 2.6
22*2
18*2
1.9*2
1.3*2
4d
4e
ACS Paragon Plus Environment

Page 9 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4f
4g
4h
1.0*2
45 ± 3.6
35 ± 5.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*1 Values for cAMP production at 1 mM are the ratio of cAMP production stimulated by a concentration of 1 mM
of compound to the maximal cAMP production in HKRKꢀB7 cells6, which was based on cAMP production
stimulated by 100 nM hPTH(1ꢀ34). Values represent the mean ± SEM of two experiments (n=2), with each
experiment performed in duplicate, except for values marked *2, which are from one experiment (n=1) performed
in duplicate. All compounds showed no response in LLCꢀPK1 cells without hPTHR1.
For SAR analysis of the phenylsulfonyl moiety, compounds 14a-f were evaluated as hPTHR1 agonists by the
cAMP assay. Although benzyl derivative 14a did not show any agonistic activity, phenethyl derivative 14b showed
similar agonistic activity to 4g. Importantly, naphthylethyl derivative 14d showed 35 times more potent agonistic
activity (EC20=15 µM) than 4g; however, pyridylethyl derivative 14c was inactive. When compared to 14d, the
activity levels of 14e and 14f were 10 times less effective, with EC20 values of 220 and 150 µM, respectively.
Solubility and human liver microsome (hLM) stability of 14d were measured and revealed 14d to be poorly
waterꢀsoluble and metabolically unstable (Table 3). These drawbacks were suggested to be due to the existence of a
hydrophobic naphthyl ring.
Table 2. SAR of piperidine substituent R2
R2
R1
hPTHR1 cAMP
No.
R1
R2
EC20 ( M)*1
µ
140 ± 18
4g
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 49
1
2
3
4
5
6
7
8
9
14a
14b
14c
>1000*2
240 ± 87
>1000*2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15 ± 0.8
220 ± 110
150 ± 69
14d
14e
14f
*1 Values for EC20 represent the concentration of a compound that produces 20% of the maximal cAMP production
in HKRKꢀB7 cells6, and represent the mean ± SEM of two experiments (n=2), with each experiment performed in
duplicate, except for values marked *2, which are from one experiment (n=1) performed in duplicate. The EC20
value of PTH(1ꢀ34) was 0.2 ± 0.05 nM. All compounds showed no response in LLCꢀPK1 cells without hPTHR1.
Therefore, we concentrated on modifying the naphthyl moiety in the next SAR analysis. Our specific concept in
the subsequent derivatization was to replace the naphthalene ring with a phenyl ring, having a hydrophilic
substituent, to improve the waterꢀsolubility and metabolic stability.
Table 3. Profile of compound 14d
hPTHR1 EC20 ( M)
15 ± 0.8
>1000
µ
hPTHR1 EC50 ( M)
µ
ACS Paragon Plus Environment

Page 11 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
Solubility (PBS) ( g/ml)
BLQ*
3.1
µ
Solubility (FaSSIF) ( g/ml)
µ
6
7
Human LM CL ( L/min/mg)
300
µ
8
9
*BLQ means belowꢀtheꢀlimitꢀofꢀquantitation. Intrinsic clearance (CL) was identified using hLM with NADPH in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
vitro.
For this purpose, derivatives 14g-l were tested in a solubility assay and an in vitro metabolic stability assay, in
addition to the hPTHR agonist assay, as shown in Table 4. Although insertion of acetamide at 4ꢀposition on the
phenyl ring improved hPTHR1 agonistic activity, solubility, and hLM metabolic stability in the absence of NADPH
compared to a nonꢀsubstituted phenyl ring, the hLM clearance of acetanilides 14h and 14i was higher than that of
14g. We considered that hydrolysis of acetanilide is one of the major metabolic pathways in hLM, so to improve
metabolic stability, acetanilide was changed to urea. 14j and 14l showed good hLM stability with or without
NADPH condition and potent agonist activity with EC20 values of 8.1 and 3.0 µM, respectively. These results
suggested the importance of carbonyl oxygen for agonistic activity (14h-j, and 14l) and the inadequate size of the
cyclic urea in 14k. Except for 14k, most derivatives showed the expected improved solubility in sodium phosphate
buffer (PBS) and fasted state simulated intestinal fluid (FaSSIF) assays. Compared with 14d, all tested compounds
showed enhanced stability in metabolic stability assays.
Table 4. SAR of arylethylsulfonamide derivatives
hPTHR1
Solubility
hLM CL
(µM)
(µg/ml)
(µL/min/mg)
No.
R
H
Ar
EC20*1
EC50*1
>1000
PBS
FaSSIF
NADPH(+)
NADPH(ꢀ)
30 ± 11
11
15
100
0
14g
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 49
1
2
3
4
5
6
7
8
9
H
H
H
H
11 ± 1.8
6.4 ± 0.6
8.1 ± 1.0
240*2
50 ± 20
26 ± 3.9
67 ± 5.8
>1000
280
8.0
400
38
46
39
23
62
45
33
18
14h
14i
14j
14k
14l
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
290
450
0
BLQ*3
99
BLQ*3
250
3.4
0.14
Me
3.0 ± 0.2
12 ± 1.0
(CH5447240)
*1 Values for EC20 and EC50 represent the concentration of a compound that produces, respectively, 20% and 50%
of the maximal cAMP production in HKRKꢀB7 cells6 and are the mean of two experiments (n=2), which were each
performed in duplicate, except for values marked *2, which are from one experiment (n=1) performed in duplicate.
EC20 and EC50 values of PTH(1ꢀ34) were 0.6 ± 0.1 nM and 2.5 ± 0.4 nM, respectively. All compounds showed no
response in LLCꢀPK1 cells without hPTHR1. *3 BLQ means belowꢀtheꢀlimitꢀofꢀquantitation.
Based on the wellꢀbalanced in vitro profiles of 14l, its pharmacokinetics (PK) was studied in female rats. In this
study 14l exhibited good exposure with bioavailability of 55% (Table 5). The in vitro hPTHR1 agonistic activity
and PK profile of 14l encouraged us to perform an in vivo pharmacological assay, which tested the compound’s
ability to restore the serum calcium level in a thyroparathyroidectomized (TPTX) rat model (Figure 3). The results
showed that 14l exhibits PTHꢀlike calcemic and hypophosphatemic activity by oral administration. Furthermore,
14l at 19 and 60 mg/kg doseꢀdependently increased serum calcium levels and decreased serum phosphorus levels
with more prolonged profiles than those of subcutaneous (sc) hPTH(1ꢀ34). It is likely that the prolonged duration of
calcemic activity of 14l in rats can be attributed to better bioavailability, lower clearance and longer halfꢀlife than
hPTH(1ꢀ34). A previous study showed that sc administration of PTH(1ꢀ34) in rats gave Tmax of 15 min and T1/2 of
ACS Paragon Plus Environment

Page 13 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
30 min17, and these are shorter than those of 14l. Bone anabolic activity of 14l has not been tested in vivo; however,
14l has a longer PK profile than PTH(1ꢀ34), so it is unlikely to exert anabolic activity on bone, which requires a
sharp and transient PK profile 18, 19, 20, 21. In a competition assay, membranes prepared from COSꢀ7 cells expressing
human PTHR1 were used with a radiolabelled tracer of 125Iꢀ[Aib1,3,Nle8,Gln10,Har11,Ala12,Trp14,Tyr15]–PTH(1–15),
which interacts mainly with the transmembrane domain of PTHR115. 14l at 300 µM inhibited 75% of the binding
by the radioꢀlabeled tracer, but the IC50 value of 14l was 93.7 µM (compared to 0.08 nM for hPTH(1ꢀ34)), which
was almost eight times weaker than its cAMP agonistic activity (EC50=12.0 µM). The binding affinity of 14l was
assessed from the displacement of a PTH peptide tracer, whereas the cAMP agonistic activity of 14l was assessed
from the accumulation of intracellular cAMP when a phosphodiesterase inhibitor, IBMX, was added. These
different assay methods may partly explain the discrepancy between the values for binding affinity and cAMP
agonistic activity of 14l in vitro. For the preliminary safety assessment, an inhibition assay of the human
etherꢀaꢀgoꢀgo–related gene channel was conducted, using an automated patch clamp platform22, and in this assay
14l showed low inhibitory activity with 10% inhibition at 10 µM.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. PK parameters of 14l in female rats at the 3 mg/kg dose
Cmax
AUCinf
CL
Vss
F
Route
T1/2 (h) Tmax (h)
(ng/mL)
(ng*h/mL)
(mL/h/kg)
(L/kg)
(%)
i.v.
1.1
1.0
̶
̶
4820
2650
624
0.395
̶
̶
p.o.
1.0
1360
1140
55.0
Plasma concentrations were measured by LCꢀMS/MS. PK parameters were calculated by noncompartmental
analysis using Watson7.1. Data are expressed as mean (n=2).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 49
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. (a) Calcemic and (b) hypophosphatemic effects of 14l (CH5447240) in TPTX rat model. Treatment
began 6 days after TPTX surgery. N = 5, sixꢀweekꢀold rats, Data are given as the mean ± SEM; *p < 0.05, **p <
0.01, ***p < 0.001 for 14l versus TPTX with vehicle, or ##p < 0.01 for PTH(1ꢀ34) versus PC buffer.
CONCLUSION
We identified a novel smallꢀmolecule hPTHR1 agonist 4a as a hit from an HTS campaign and have successfully
optimized 4a to 14l, which had agonist activity 100 times more potent than 4a. 14l exhibited, orally and
doseꢀdependently, a potent calcium effect in TPTX rats. From these findings, 14l was considered to be a candidate
drug for patients with hypoparathyroidism, and being orally available, was thought to have great benefit for patients
who have to take drugs over a long period. However, studies of 14l in hLM detected a GSHꢀtrapped reactive
metabolite, and a program of further optimization study was needed before the clinical candidate PCO371 could be
obtained.
EXPERIMENTAL SECTION
ACS Paragon Plus Environment

Page 15 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used without
further purification. Silica gel chromatography purification was performed using prepacked silica gel cartridges
(Biotage, Shoko Scientific). Reverseꢀphase column chromatography purification was performed using Wakosil
25C18 (Wako Pure Chemical Industries). Nuclear magnetic resonance (NMR) spectra were determined with a
Varian MRꢀ400 spectrometer (400 MHz, Agilent). Chemical shifts are reported in parts per million (ppm, δ units).
The following NMR abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs =
broad singlet. High resolution mass spectrometric analysis was performed on a Xevo G2ꢀS Tof instrument (Waters).
All biologically tested compounds were determined as ≥95% pure by UHPLC analysis measured at 230 nm. The
UHPLC analysis was carried out on a Waters ACQUITY UPLC IꢀClass system including a ACQUITY UPLC PDA.
Method: The gradient was 5% B to 100% B for 0–1.0 min; 100% B (1.0 mL/min flow rate) for 1.0–1.4 min. Mobile
Phase A: 10 mM AcONH4 in water. Mobile Phase B: methanol. Column: Ascentis Express C18 column (2.1
mmI.D. × 50 mm). Column temperature: 35 °C.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8-((3-Chlorophenyl)sulfonyl)-2-(2,4-dichlorophenyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4a). To a stirred
mixture of 2,4ꢀdichloroꢀNꢀ(1ꢀ((3ꢀchlorophenyl)sulfonyl)ꢀ4ꢀcyanopiperidinꢀ4ꢀyl)benzamide (40.5 mg, 0.086 mmol)
(9a) in EtOH (430 µl), 6 mol/L NaOH (143 µl, 0.857 mmol) and 30% aq. H2O2 (61 µl, 0.60 mmol) were added at
0ºC. The mixture was stirred at 80ºC for 2 h. This mixture was evaporated and EtOAc and H2O were added. The
organic layer was washed with H2O and sat. aq. NaCl, and dried over anhyd. Na2SO4, and evaporated in vacuo. The
mixture was purified by column chromatography (silica gel, EtOAcꢀHexane) to afford 4a (20.9 mg, 51%) as a
white solid. 1HꢀNMR (CDCl3) δ: 8.55 (1H, brs), 7.82 (1H, d, J = 8.4 Hz), 7.79 (1H, dd, J = 1.7, 1.7 Hz), 7.70ꢀ7.67
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 49
1
2
3
4
5
6
7
8
9
(1H, m), 7.60ꢀ7.57 (1H, m), 7.49 (1H, dd, J = 8.4, 8.4 Hz), 7.48 (1H, d, J = 2.0 Hz), 7.36 (1H, dd, J = 8.4, 2.0 Hz),
3.78ꢀ3.73 (2H, m), 3.12ꢀ3.05 (2H, m), 2.17ꢀ2.10 (2H, m), 1.72ꢀ1.66 (2H, m). 13CꢀNMR (CDCl3) δ: 184.6, 156.4,
138.7, 138.6, 135.5, 133.0, 132.8, 132.1, 130.5, 130.5, 128.1, 127.6, 126.2, 125.7, 67.7, 42.1, 32.3. HRMS
(ESIꢀTOF) m/z calcd for C19H17Cl3N3O3S (M+H), 472.0056; Found 472.0050. Purity 99%, RT 1.00 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-(4-Chlorophenyl),-8-((3-chlorophenyl)sulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4b). 4b was prepared
from 9b (39.1 mg, 0.089 mmol) using a procedure similar to that described for the preparation of 4a. (White solid,
17.7 mg, 45%) 1HꢀNMR (DMSOꢀd6) δ: 11.67 (1H, brs), 7.90 (2H, d, J = 7.8 Hz), 7.88ꢀ7.85 (1H, m), 7.84 (1H, dd,
J = 1.7, 1.7 Hz), 7.81ꢀ7.78 (1H, m), 7.74 (1H, dd, J = 7.8, 7.8 Hz), 7.58 (2H, d, J = 7.8 Hz), 3.66ꢀ3.60 (2H, m),
2.94ꢀ2.88 (2H, m), 1.87ꢀ1.79 (2H, m), 1.63ꢀ1.57 (2H, m). 13CꢀNMR (DMSOꢀd6) δ: 186.1, 157.9, 137.9, 136.5,
134.1, 133.2, 131.6, 128.8, 128.7, 127.2, 126.8, 126.1, 67.4, 42.1, 31.8. HRMS (ESIꢀTOF) m/z calcd for
C19H18Cl2N3O3S (M+H), 438.0446; Found 438.0436. Purity 99%, RT 1.00 min.
2-(2-Chlorophenyl)-8-((3-chlorophenyl)sulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4c). 4c was prepared
from 9c (80 mg, 0.183 mmol) using a procedure similar to that described for the preparation of 4a. (White solid, 29
mg, 36%) 1HꢀNMR (CDCl3) δ: 8.48 (1H, brs), 7.85ꢀ7.82 (1H, m), 7.80ꢀ7.78 (1H, m), 7.70ꢀ7.67 (1H, m), 7.59ꢀ7.56
(1H, m), 7.49 (1H, dd, J = 7.9, 7.9 Hz), 7.46ꢀ7.44 (2H, m), 7.39ꢀ7.35 (1H, m), 3.79ꢀ3.73 (2H, m), 3.13ꢀ3.06 (2H,
m), 2.18ꢀ2.11 (2H, m), 1.73ꢀ1.68 (2H, m). 13CꢀNMR (CDCl3) δ: 184.6, 157.3, 138.7, 135.5, 133.0, 132.8, 132.1,
131.1, 130.7, 130.5, 127.8, 127.6, 127.6, 125.7, 67.6, 42.1, 32.3. HRMS (ESIꢀTOF) m/z calcd for C19H18Cl2N3O3S
(M+H), 438.0446; Found 438.0449. Purity 99%, RT 0.93 min.
ACS Paragon Plus Environment

Page 17 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
8-((3-Chlorophenyl)sulfonyl)-2-phenyl-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4d). 4d was prepared from 9d
(41.9 mg, 0.104 mmol) using a procedure similar to that described for the preparation of 4a. (White solid, 22.1 mg,
53%) 1HꢀNMR (DMSOꢀd6) δ: 11.62 (1H, brs), 7.91ꢀ7.85 (3H, m), 7.84 (1H, dd, J = 1.7, 1.7 Hz), 7.82ꢀ7.79 (1H, m),
7.74 (1H, dd, J = 7.8, 7.8 Hz), 7.58ꢀ7.53 (1H, m), 7.51ꢀ7.46 (2H, m), 3.66ꢀ3.60 (2H, m), 2.97ꢀ2.89 (2H, m),
1.87ꢀ1.80 (2H, m), 1.62ꢀ1.55 (2H, m). 13CꢀNMR (DMSOꢀd6) δ: 186.2, 158.7, 137.9, 134.1, 133.2, 131.7, 131.6,
128.6, 128.3, 126.8, 126.8, 126.1, 67.3, 42.1, 31.8. HRMS (ESIꢀTOF) m/z calcd for C19H19ClN3O3S (M+H),
404.0836; Found 404.0832. Purity 100%, RT 0.93 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-(8-((3-Chlorophenyl)sulfonyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-2-yl)benzoic acid (4e). 4e was prepared
from 9e (29.9 mg, 0.065 mmol) using a procedure similar to that described for the preparation of 4a. (White solid,
14.1 mg, 49%) 1HꢀNMR (DMSOꢀd6) δ: 13.3 (1H, brs), 11.73 (1H, brs), 8.05ꢀ7.98 (4H, m), 7.89ꢀ7.85 (1H, m), 7.84
(1H, dd, J = 1.7, 1.7 Hz), 7.82ꢀ7.79 (1H, m), 7.74 (1H, dd, J = 7.8, 7.8 Hz), 3.67ꢀ3.61 (2H, m), 2.96ꢀ2.89 (2H, m),
1.88ꢀ1.81 (2H, m), 1.65ꢀ1.59 (2H, m). 13CꢀNMR (DMSOꢀd6) δ: 186.0, 166.6, 158.3, 137.9, 134.1, 133.6, 133.2,
131.9, 131.6, 129.5, 127.1, 126.8, 126.1, 67.6, 42.1, 31.7. HRMS (ESIꢀTOF) m/z calcd for C20H19ClN3O5S (M+H),
448.0734; Found 448.0734. Purity 99%, RT 0.75 min.
8-((3-Chlorophenyl)sulfonyl)-2-(pyridin-4-yl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4f). 4f was prepared
from 9f (60 mg, 0.148 mmol) using a procedure similar to that described for the preparation of 4a. (light yellow
solid, 41 mg, 68%) 1HꢀNMR (CDCl3) δ: 10.30 (1H, brs), 8.81 (2H, dd, J = 4.5, 1.7 Hz), 7.82 (1H, dd, J = 1.7, 1.7
Hz), 7.73ꢀ7.70 (3H, m), 7.65ꢀ7.62 (1H, m), 7.53 (1H, dd, J = 7.9, 7.9 Hz), 3.84ꢀ3.79 (2H, m), 3.11ꢀ3.03 (2H, m),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 49
1
2
3
4
5
6
7
8
9
2.24ꢀ2.17 (2H, m), 1.69ꢀ1.64 (2H, m). 13CꢀNMR (CDCl3) δ: 187.5, 156.6, 150.9, 138.7, 135.5, 135.3, 133.1, 130.5,
127.6, 125.7, 120.5, 69.6, 42.2, 32.3. HRMS (ESIꢀTOF) m/z calcd for C18H18ClN4O3S (M+H), 405.0788; Found
405.0787. Purity 95%, RT 0.86 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8-((3-Chlorophenyl)sulfonyl)-2-cyclohexyl-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4g). 4g was prepared from
9g (46.1 mg, 0.112 mmol) using a procedure similar to that described for the preparation of 4a. (White solid, 35.1
mg, 76%) 1HꢀNMR (CDCl3) δ: 8.60 (1H, brs), 7.78 (1H, dd, J = 1.8, 1.8 Hz), 7.69ꢀ7.65 (1H, m), 7.59ꢀ7.55 (1H, m),
7.48 (1H, dd, J = 7.9, 7.9 Hz), 3.73ꢀ3.68 (2H, m), 3.05ꢀ2.97 (2H, m), 2.42ꢀ2.35 (1H, m), 2.07ꢀ1.98 (2H, m),
1.89ꢀ1.78 (4H, m), 1.74ꢀ1.68 (1H, m), 1.57ꢀ1.50 (2H, m), 1.40ꢀ1.20 (5H, m). 13CꢀNMR (CDCl3) δ: 186.5, 165.0,
138.7, 135.4, 132.9, 130.4, 127.6, 125.6, 67.4, 42.1, 39.4, 32.3, 29.7, 25.6, 25.4. HRMS (ESIꢀTOF) m/z calcd for
C19H25ClN3O3S (M+H), 410.1305; Found 410.1301. Purity 99%, RT 0.97 min.
8-((3-Chlorophenyl)sulfonyl)-2-((1R,4R)-4-methylcyclohexyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4h). 4h
was prepared from 9h (46.4 mg, 0.109 mmol) using a procedure similar to that described for the preparation of 4a.
(White solid, 33.2 mg, 72%) 1HꢀNMR (CDCl3) δ: 8.63 (1H, brs), 7.72 (1H, dd, J = 1.7, 1.7 Hz), 7.63ꢀ7.60 (1H, m),
7.53ꢀ7.50 (1H, m), 7.42 (1H, dd, J = 7.8, 7.8 Hz), 3.68ꢀ3.63 (2H, m), 2.99ꢀ2.91 (2H, m), 2.30ꢀ2.22 (1H, m),
2.01ꢀ1.93 (2H, m), 1.86ꢀ1.80 (2H, m), 1.77ꢀ1.71 (2H, m), 1.50ꢀ1.44 (2H, m), 1.37ꢀ1.27 (3H, m), 0.98ꢀ0.87 (2H, m),
0.86 (3H, d, J = 6.6 Hz). 13CꢀNMR (CDCl3) δ: 186.6, 165.1, 138.8, 135.4, 132.9, 130.5, 127.6, 125.6, 67.4, 42.1,
39.3, 34.1, 32.3, 32.0, 29.7, 22.4. HRMS (ESIꢀTOF) m/z calcd for C20H27ClN3O3S (M+H), 424.1461; Found
424.1456. Purity 98%, RT 1.01 min.
ACS Paragon Plus Environment

Page 19 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1-((3-Chlorophenyl)sulfonyl)piperidin-4-one (7). To a stirred mixture of piperidinꢀ4ꢀone hydrochloride (5) (0.5
g, 3.69 mmol) and K2CO3 (1.27 g, 9.22 mmol) in CHCl3 (9 ml) and H2O (9 ml) was added 3ꢀchlorobenzenesulfonyl
chloride (6) (0.57 ml, 4.06 mmol) at room temperature. The mixture was stirred at room temperature for 12 h under
nitrogen atmosphere. The reaction mixture was diluted with CH2Cl2 and separated. The organic layer was washed
with H2O and sat. aq. NaCl, and dried over anhyd. Na2SO4, and evaporated in vacuo. The residue was triturated
with EtOAcꢀHexane to afford 7 (728 mg, 72%) as a white solid. 1HꢀNMR (CDCl3) δ: 7.80ꢀ7.78 (1H, m), 7.71ꢀ7.67
(1H, m), 7.62ꢀ7.59 (1H, m), 7.51 (1H, dd, J = 7.9, 7.9 Hz), 3.44 (4H, t, J = 6.2 Hz), 2.57 (4H, t, J = 6.2 Hz).
13CꢀNMR (CDCl3) δ: 205.2, 138.5, 135.8, 133.5, 130.8, 127.6, 125.7, 46.0, 40.8.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-amino-1-((3-chlorophenyl)sulfonyl)piperidine-4-carbonitrile (8). To a stirred mixture of
1ꢀ((3ꢀchlorophenyl)sulfonyl)piperidinꢀ4ꢀone (7) (700 mg, 2.56 mmol) and ammonium acetate (217 mg, 2.81 mmol)
in MeOH (7 ml) was added KCN (167 mg, 2.56 mmol) at room temperature. The mixture was stirred at room
temperature for 5 h under nitrogen atmosphere. NaHCO3 (107 mg, 1.279 mmol) was added at 0ºC and the mixture
was evaporated. The residue was diluted with CH2Cl2 and H2O and separated. The organic layer was washed with
H2O and sat. aq. NaCl, and dried over anhyd. Na2SO4, and evaporated in vacuo. The mixture was purified by
column chromatography (silica gel, EtOAcꢀHexane) to afford 8 (657 mg, 86%) as a white solid. 1HꢀNMR (CDCl3)
δ: 7.76ꢀ7.75 (1H, m), 7.66ꢀ7.63 (1H, m), 7.62ꢀ7.58 (1H, m), 7.50 (1H, dd, J = 7.9, 7.9 Hz), 3.62ꢀ3.55 (2H, m),
2.96ꢀ2.88 (2H, m), 2.13ꢀ2.06 (2H, m), 1.88ꢀ1.81 (2H, m), 1.75 (2H, brs). 13CꢀNMR (CDCl3) δ: 138.2, 135.8, 133.4,
130.7, 127.7, 125.6, 122.7, 48.9, 42.4, 36.5.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 49
1
2
3
4
5
6
7
8
9
2,4-dichloro-N-(1-((3-chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)benzamide (9a). To a stirred mixture of
4ꢀaminoꢀ1ꢀ((3ꢀchlorophenyl)sulfonyl)piperidineꢀ4ꢀcarbonitrile (8) (50 mg, 0.167 mmol) and DIPEA (35 µl, 0.20
mmol) in CH2Cl2 (834 µl) was added 2,4ꢀdichlorobenzoyl chloride (26 µl, 0.18 mmol) at 0ºC. The mixture was
stirred at room temperature for 12 h. The reaction mixture was diluted with CH2Cl2, quenched by H2O and
separated. The organic layer was washed with H2O and sat. aq. NaCl, and dried over anhyd. Na2SO4, and
evaporated in vacuo. The mixture was purified by column chromatography (silica gel, EtOAcꢀHexane) to afford 9a
(67.7 mg, 86%) as a white solid. 1HꢀNMR (DMSOꢀd6) δ: 9.13 (1H, s), 7.92ꢀ7.89 (1H, m), 7.86 (1H, dd, J = 1.8, 1.8
Hz), 7.83ꢀ7.80 (1H, m), 7.77 (1H, dd, J = 7.8, 7.8 Hz), 7.73 (1H, d, J = 1.8 Hz), 7.54 (1H, dd, J = 8.3, 1.8 Hz), 7.47
(1H, d, J = 8.3 Hz), 3.21ꢀ3.10 (4H, m), 2.40ꢀ2.32 (2H, m), 2.30ꢀ2.22 (2H, m). 13CꢀNMR (DMSOꢀd6) δ: 165.6,
136.8, 135.2, 134.3, 134.3, 133.5, 131.6, 131.0, 130.5, 129.2, 127.5, 127.0, 126.3, 119.1, 48.4, 41.7, 32.3. HRMS
(ESIꢀTOF) m/z calcd for C19H17Cl3N3O3S (M+H), 472.0056; Found 472.0050.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-Chloro-N-(1-((3-chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)benzamide (9b). 9b was prepared from 8
(50 mg, 0.167 mmol) and 4ꢀchlorobenzoyl chloride using a procedure similar to that described for the preparation
of 9a. (White solid, 54.8 mg, 75% yield) 1HꢀNMR (DMSOꢀd6) δ: 8.89 (1H, s), 7.92ꢀ7.89 (1H, m), 7.87ꢀ7.80 (4H,
m), 7.77 (1H, dd, J = 7.8, 7.8 Hz), 7.62ꢀ7.58 (2H, m), 3.58ꢀ3.51 (2H, m), 2.87ꢀ2.80 (2H, m), 2.50ꢀ2.44 (2H, m),
2.17ꢀ2.09 (2H, m). 13CꢀNMR (DMSOꢀd6) δ: 165.9, 137.0, 136.9, 134.3, 133.5, 131.9, 131.6, 129.6, 128.5, 126.9,
126.2, 118.9, 49.2, 42.3, 32.8. HRMS (ESIꢀTOF) m/z calcd for C19H18Cl2N3O3S (M+H), 438.0446; Found
438.0446.
ACS Paragon Plus Environment

Page 21 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2-Chloro-N-(1-((3-chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)benzamide (9c). 9c was prepared from 8 (50
mg, 0.167 mmol) and 2ꢀchlorobenzoyl chloride using a procedure similar to that described for the preparation of 9a.
(White solid, 58.6 mg, 80% yield) 1HꢀNMR (DMSOꢀd6) δ: 9.03 (1H, s), 7.89ꢀ7.86 (1H, m), 7.82 (1H, dd, J = 1.7,
1.7 Hz), 7.79ꢀ7.76 (1H, m), 7.73 (1H, dd, J = 7.7, 7.7 Hz), 7.48ꢀ7.46 (2H, m), 7.40ꢀ7.37 (2H, m), 3.19ꢀ3.06 (4H, m),
2.34ꢀ2.23 (4H, m). 13CꢀNMR (DMSOꢀd6) δ: 166.4, 136.6, 135.4, 134.2, 133.4, 131.5, 131.3, 129.6, 129.5, 129.0,
127.2, 126.9, 126.2, 119.2, 48.1, 41.5, 32.1. HRMS (ESIꢀTOF) m/z calcd for C19H16Cl2N3O3S (MꢀH), 436.0290;
Found 436.0285.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(1-((3-chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)benzamide (9d). 9d was prepared from 8 (50 mg,
0.167 mmol) and benzoyl chloride using a procedure similar to that described for the preparation of 9a. (White
solid, 59.8 mg, 89%). 1HꢀNMR (DMSOꢀd6) δ: 8.81 (1H, s), 7.92ꢀ7.89 (1H, m), 7.88ꢀ7.86 (1H, m), 7.84ꢀ7.74 (4H,
m), 7.64ꢀ7.59 (1H, m), 7.54ꢀ7.49 (2H, m), 3.54ꢀ3.48 (2H, m), 2.90ꢀ2.83 (2H, m), 2.50ꢀ2.45 (2H, m), 2.20ꢀ2.12 (2H,
m). 13CꢀNMR (DMSOꢀd6) δ: 167.0, 136.9, 134.3, 133.5, 133.3, 132.0, 131.6, 128.3, 127.7, 127.0, 126.2, 119.1,
49.0, 42.3, 32.7. HRMS (ESIꢀTOF) m/z calcd for C19H19ClN3O3S (M+H), 404.0836; Found 404.0827.
Methyl 4-((1-((3-chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)carbamoyl)benzoate (9e). 9e was prepared
from 8 (50 mg, 0.167 mmol) and methyl 4ꢀ(chlorocarbonyl)benzoate using a procedure similar to that described for
the preparation of 9a. (White solid, 38.5 mg, 50% yield) 1HꢀNMR (DMSOꢀd6) δ: 9.00 (1H, s), 8.06ꢀ8.03 (2H, m),
7.92ꢀ7.89 (2H, m), 7.88ꢀ7.85 (1H, m), 7.83 (1H, dd, J = 1.7, 1.7 Hz), 7.80ꢀ7.77 (1H, m), 7.73 (1H, dd, J = 7.8, 7.8
Hz), 3.89 (3H, s), 3.55ꢀ3.50 (2H, m), 2.84ꢀ2.76 (2H, m), 2.50ꢀ2.42 (2H, m), 2.14ꢀ2.07 (2H, m). 13CꢀNMR
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 49
1
2
3
4
(DMSOꢀd6) δ: 166.2, 165.6, 137.3, 137.0, 134.3, 133.5, 132.4, 131.7, 129.1, 128.1, 126.9, 126.2, 118.8, 52.5, 49.3,
5
6
7
42.4, 32.7. HRMS (ESIꢀTOF) m/z calcd for C21H21ClN3O5S (M+H), 462.0890; Found 462.0884.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(1-((3-Chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)isonicotinamide (9f). 9f was prepared from 8 (50 mg,
0.167 mmol) and 4ꢀpyridinecarbonyl chloride using a procedure similar to that described for the preparation of 9a.
(White solid, 29.4 mg, 44%) 1HꢀNMR (DMSOꢀd6) δ: 9.13 (1H, s), 8.78 (2H, dd, J = 4.4, 1.6 Hz), 7.92ꢀ7.89 (1H, m),
7.87 (1H, dd, J = 1.7, 1.7 Hz), 7.84ꢀ7.80 (1H, m), 7.77 (1H, dd, J = 7.8, 7.8 Hz), 7.73 (2H, dd, J = 4.4, 1.6 Hz),
3.59ꢀ3.54 (2H, m), 2.88ꢀ2.80 (2H, m), 2.52ꢀ2.45 (2H, m), 2.18ꢀ2.10 (2H, m). 13CꢀNMR (DMSOꢀd6) δ: 165.5, 150.3,
140.2, 137.0, 134.3, 133.5, 131.7, 126.9, 126.2, 121.5, 118.7, 49.3, 42.3, 32.7. HRMS (ESIꢀTOF) m/z calcd for
C18H18ClN4O3S (M+H), 405.0788; Found 405.0785.
N-(1-((3-Chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)cyclohexanecarboxamide (9g). 9g was prepared
from 8 (50 mg, 0.167 mmol) and cyclohexylcarbonyl chloride using a procedure similar to that described for the
preparation of 9a. (White solid, 61.7 mg, 90%) 1HꢀNMR (DMSOꢀd6) δ: 8.19 (1H, s), 7.87ꢀ7.84 (1H, m), 7.80 (1H,
dd, J = 1.7, 1.7 Hz), 7.77ꢀ7.74 (1H, m), 7.71 (1H, dd, J = 7.6, 7.6 Hz), 3.19ꢀ3.13 (2H, m), 2.95ꢀ2.87 (2H, m),
2.27ꢀ2.20 (2H, m), 2.12ꢀ1.99 (3H, m), 1.69ꢀ1.52 (5H, m), 1.28ꢀ1.10 (5H, m). 13CꢀNMR (DMSOꢀd6) δ: 175.6, 136.7,
134.3, 133.5, 131.6, 126.9, 126.3, 119.5, 47.7, 43.3, 41.8, 32.5, 28.8, 25.3, 25.0. HRMS (ESIꢀTOF) m/z calcd for
C19H25ClN3O3S (M+H), 410.1305; Found 410.1295.
(1R, 4R)-N-(1-((3-Chlorophenyl)sulfonyl)-4-cyanopiperidin-4-yl)-4-methylcyclohexane-1-carboxamide (9h).
To a stirred mixture of 4ꢀaminoꢀ1ꢀ((3ꢀchlorophenyl)sulfonyl)piperidineꢀ4ꢀcarbonitrile (8) (50 mg, 0.167 mmol),
ACS Paragon Plus Environment

Page 23 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(1R, 4R)ꢀ4ꢀmethylcyclohexaneꢀ1ꢀcarboxylic acid (26 mg, 0.183 mmol) and DIPEA (35 µl, 0.200 mmol) in DMF
(834 µl) at 0ºC was added HATU (76 mg, 0.200 mmol). The mixture was stirred at room temperature for 12 h. The
reaction mixture was quenched by H2O. The mixture was extracted with EtOAc. The organic layer was washed
with H2O and sat. aq. NaCl, and dried over anhyd. Na2SO4, and evaporated in vacuo. The mixture was purified by
column chromatography (silica gel, EtOAcꢀHexane) to afford 9h (63.4 mg, 90%) as a white solid. 1HꢀNMR
(DMSOꢀd6) δ: 8.26 (1H, s), 7.91ꢀ7.88 (1H, m), 7.83 (1H, dd, J = 1.7, 1.7 Hz), 7.81ꢀ7.77 (1H, m), 7.75 (1H, dd, J =
7.6, 7.6 Hz), 3.24ꢀ3.18 (2H, m), 2.97ꢀ2.90 (2H, m), 2.31ꢀ2.23 (2H, m), 2.10ꢀ2.01 (3H, m), 1.72ꢀ1.58 (4H, m),
1.35ꢀ1.23 (3H, m), 0.92ꢀ0.82 (2H, m), 0.88 (3H, d, J = 6.4 Hz). 13CꢀNMR (DMSOꢀd6) δ: 175.7, 136.7, 134.3, 133.5,
131.6, 126.9, 126.3, 119.4, 47.7, 43.2, 41.8, 33.8, 32.5, 31.5, 28.8, 22.5. HRMS (ESIꢀTOF) m/z calcd for
C20H27ClN3O3S (M+H), 424.1461; Found 424.1454.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tert-Butyl 4-Cyano-4-(2,4-dichlorobenzamido)piperidine-1-carboxylate (11a). To a stirred mixture of
tertꢀbutyl 4ꢀaminoꢀ4ꢀcyanopiperidineꢀ1ꢀcarboxylate (10) (12.2 g, 54.2 mmol) and triethylamine (15.1 ml, 108
mmol) in CH2Cl2 (200 ml) at room temperature was added 2,4ꢀdichlorobenzoyl chloride (8.4 ml, 59.9 mmol). Then
dimethylaminopyridine (331 mg, 2.71 mmol) was added. The mixture was stirred at room temperature for 1 h. This
mixture was quenched by H2O. The mixture was extracted with CH2Cl2. The organic layer was washed with H2O
and sat. aq. NaCl, and dried over anhyd. Na2SO4, and evaporated in vacuo. The mixture was triturated with
EtOAcꢀHexane to afford 11a (6.54 g, 30%) as a white solid. 1HꢀNMR (CDCl3) δ: 7.74 (1H, d, J = 8.4 Hz), 7.44 (1H,
d, J = 2.0 Hz), 7.36 (1H, dd, J = 8.4, 2.0 Hz), 6.55 (1H, brs), 4.01ꢀ3.90 (2H, m), 3.40ꢀ3.31 (2H, m), 2.53ꢀ2.44 (2H,
m), 1.97ꢀ1.88 (2H, m), 1.47 (9H, s). 13CꢀNMR (CDCl3) δ: 164.6, 154.4, 138.1, 132.2, 131.5, 131.5, 130.3, 128.1,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 49
1
2
3
4
118.3, 80.7, 51.0, 39.9, 34.7, 28.5. HRMS (ESIꢀTOF) m/z calcd for C18H20Cl2N3O3 (MꢀH), 396.0882; Found
5
6
7
396.0881.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tert-Butyl 4-Cyano-4-(cyclohexanecarboxamido)piperidine-1-carboxylate (11b). 11b was prepared from 10
(8.42 g, 25.1 mmol) and cyclohexylcarbonyl chloride using a procedure similar to that described for the preparation
of 11a. (White solid, 5.33 g, 63%) 1HꢀNMR (CDCl3) δ: 5.59 (1H, brs), 4.01ꢀ3.87 (2H, m), 3.30ꢀ3.20 (2H, m),
2.47ꢀ2.32 (2H, m), 2.14ꢀ2.07 (1H, m), 1.87ꢀ1.64 (7H, m), 1.49ꢀ1.41 (2H, m), 1.46 (9H, s), 1.30ꢀ1.20 (3H, m).
13CꢀNMR (CDCl3) δ: 175.7, 154.4, 118.8, 80.5, 50.2, 45.3, 40.0, 34.8, 29.5, 28.5, 25.7, 25.6. HRMS (ESIꢀTOF) m/z
calcd for C18H28N3O3 (MꢀH), 334.2131; Found 334.2136.
tert-Butyl 4-Cyano-4-(4-methylcyclohexane-1-carboxamido)piperidine-1-carboxylate (11c). 11c was
prepared from 10 (4.07 g, 18.1 mmol) and 4ꢀmethylcyclohexane carbonyl chloride (cisꢀtrans mixture) using a
procedure similar to that described for the preparation of 11a. (White solid, 4.46 g, 71%, cis : trans = 4 : 6)
1HꢀNMR (CDCl3) δ: 5.54 (0.4H, brs, cis), 5.50 (0.6H, brs, trans), 4.00ꢀ3.90 (2H, m), 3.30ꢀ3.22 (2H, m), 2.44ꢀ2.36
(2H, m), 2.30ꢀ2.25 (0.4H, m, cis), 2.06ꢀ1.98 (0.6H, m, trans), 1.88ꢀ1.30 (11H, m), 1.46 (9H, s), 0.94 (1.2H, d, J =
6.7 Hz, cis), 0.89 (1.8H, d, J = 6.5 Hz, trans).
tert-Butyl 2-(2,4-Dichlorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-ene-8-carboxylate (12a). To a stirred
mixture of tertꢀbutyl 4ꢀcyanoꢀ4ꢀ(2,4ꢀdichlorobenzamido)piperidineꢀ1ꢀcarboxylate (11a) (6.22 g, 18.1 mmol) in
EtOH (100 ml), 6 mol/L aq. NaOH (36 ml) and 30% aq. H2O2 (16 ml) were added at room temperature. The
mixture was stirred at 80ºC for 4 h. This mixture was evaporated, and EtOAc and H2O were added. The organic
ACS Paragon Plus Environment

Page 25 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
layer was washed with H2O and sat.aq.NaCl, and dried over anhyd. Na2SO4, and evaporated in vacuo. The mixture
was purified by column chromatography (silica gel, EtOAcꢀHexane) to afford 12a (3.11 g, 43%) as a white solid.
1HꢀNMR (CDCl3) δ: 8.84 (1H, brs), 7.93 (1H, d, J = 8.4 Hz), 7.50 (1H, d, J = 2.0 Hz), 7.40 (1H, dd, J = 8.4, 2.0
Hz), 4.12ꢀ3.95 (2H, m), 3.48ꢀ3.37 (2H, m), 1.98ꢀ1.88 (2H, m), 1.59ꢀ1.52 (2H, m), 1.48 (9H, s). 13CꢀNMR (CDCl3)
δ: 185.7, 156.1, 154.9, 138.5, 133.0, 132.3, 130.6, 128.2, 126.7, 79.9, 69.1, 39.8, 32.5, 28.6. HRMS (ESIꢀTOF) m/z
calcd for C18H20Cl2N3O3 (MꢀH), 396.0882; Found 396.0876.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tert-Butyl 2-Cyclohexyl-4-oxo-1,3,8-triazaspiro[4.5]dec-1-ene-8-carboxylate (12b). 12b was prepared from
11b (5.33 g, 15.9 mmol) using a procedure similar to that described for the preparation of 12a. (White solid, 4.43 g,
83%) 1HꢀNMR (CDCl3) δ: 8.74 (1H, brs), 4.01ꢀ3.89 (2H, m), 3.45ꢀ3.36 (2H, m), 2.48ꢀ2.41 (1H, m), 1.96ꢀ1.91 (2H,
m), 1.86ꢀ1.69 (5H, m), 1.49ꢀ1.24 (7H, m), 1.47 (9H, s). 13CꢀNMR (CDCl3) δ: 187.4, 164.4, 154.8, 79.6, 68.6, 40.1,
39.2, 32.4, 29.7, 28.5, 25.7, 25.5. HRMS (ESIꢀTOF) m/z calcd for C18H28N3O3 (MꢀH), 334.2131; Found 334.2129.
tert-Butyl 2-((1R,4R)-4-Methylcyclohexyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-ene-8-carboxylate (12c). 12c
was prepared from 11c (4.46 g, 12.8 mmol) using a procedure similar to that described for the preparation of 12a.
(White solid, 3.83 g, 86%) 1HꢀNMR (CDCl3) δ: 8.78 (1H, brs), 4.03ꢀ3.87 (2H, m), 3.46ꢀ3.34 (2H, m), 2.42ꢀ2.33
(1H, m), 1.99ꢀ1.92 (2H, m), 1.86ꢀ1.76 (5H, m), 1.49ꢀ1.41 (4H, m), 1.47 (9H, s), 1.08ꢀ0.96 (2H, m), 0.93 (3H, d, J =
6.5 Hz). 13CꢀNMR (CDCl3) δ: 187.4, 164.5, 154.8, 79.6, 68.6, 40.0, 39.2, 34.2, 32.4, 32.0, 29.7, 28.5, 22.4. HRMS
(ESIꢀTOF) m/z calcd for C19H30N3O3 (MꢀH), 348.2287; Found 348.2290.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 49
1
2
3
4
5
6
7
8
9
2-(2,4-Dichlorophenyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one dihydrochloride (13a). The mixture of tertꢀbutyl
2ꢀ(2,4ꢀdichlorophenyl)ꢀ4ꢀoxoꢀ1,3,8ꢀtriazaspiro[4.5]decꢀ1ꢀeneꢀ8ꢀcarboxylate (12a) (30 mg, 0.075 mmol) and 4
mol/L HClꢀdioxane (377 µl, 1.51 mmol) was stirred at room temperature for 2 h. This mixture was evaporated to
afford 13a (28 mg, 100%) as a white solid. 1HꢀNMR (CD3OD) δ: 7.85 (1H, d, J = 8.4 Hz), 7.84 (1H, d, J = 2.0 Hz),
7.67 (1H, dd, J = 8.4, 2.0 Hz), 3.68ꢀ3.61 (2H, m), 3.54ꢀ3.47 (2H, m), 2.40ꢀ2.33 (2H, m), 2.24ꢀ2.17 (2H, m).
13CꢀNMR (CD3OD) δ: 179.6, 167.5, 142.3, 135.3, 134.0, 132.2, 129.6, 123.5, 65.2, 40.5, 29.7. HRMS (ESIꢀTOF)
m/z calcd for C13H14Cl2N3O (M+H), 298.0514; Found 298.0516.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Cyclohexyl-1,3,8-triazaspiro[4.5]dec-1-en-4-one bis(2,2,2-trifluoroacetate) (13b). To a mixture of tertꢀbutyl
2ꢀcyclohexylꢀ4ꢀoxoꢀ1,3,8ꢀtriazaspiro[4.5]decꢀ1ꢀeneꢀ8ꢀcarboxylate (12b) (4.43 g, 13.2 mmol) in CH2Cl2 (60 ml)
was added trifluoroacetic acid (20 ml) at room temperature. The mixture was stirred at room temperature for 5 h
and evaporated. The residue was triturated with CH2Cl2 to afford 13b (5.66 g, 92%) as a white solid. 1HꢀNMR
(DMSOꢀd6) δ: 8.82 (1H, brs), 8.62 (1H, brs), 3.40ꢀ3.33 (2H, m), 3.22ꢀ3.11 (2H, m), 2.54ꢀ2.47 (1H, m), 1.99ꢀ1.86
(4H, m), 1.79ꢀ1.62 (5H, m), 1.50ꢀ1.39 (2H, m), 1.35ꢀ1.13 (3H, m). 13CꢀNMR (DMSOꢀd6) δ: 182.4, 172.4, 158.3 (q,
J = 35.1 Hz), 115.9 (q, J = 293.2 Hz), 63.7, 37.9, 28.5, 28.4, 25.0, 24.7. HRMS (ESIꢀTOF) m/z calcd for
C13H22N3O (M+H), 236.1763; Found 236.1766.
2-Cyclohexyl-1,3,8-triazaspiro[4.5]dec-1-en-4-one dihydrochloride (13c). 13c was prepared from 12b (43.1
mg, 0.128 mmol) using a procedure similar to that described for the preparation of 13a. (White solid, 39.8 mg,
100%) 1HꢀNMR (CD3OD) δ: 3.66ꢀ3.60 (2H, m), 3.58ꢀ3.51 (2H, m), 2.97ꢀ2.88 (1H, m), 2.41ꢀ2.28 (4H, m),
ACS Paragon Plus Environment

Page 27 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2.16ꢀ2.09 (2H, m), 1.99ꢀ1.93 (2H, m), 1.87ꢀ1.80 (1H, m), 1.68ꢀ1.58 (2H, m), 1.55ꢀ1.30 (3H, m). 13CꢀNMR
(CD3OD) δ: 180.4, 178.0, 63.0, 40.2, 39.7, 29.9, 29.8, 29.4, 26.2. HRMS (ESIꢀTOF) m/z calcd for C13H22N3O
(M+H), 236.1763; Found 236.1772.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-((1R,4R)-4-Methylcyclohexyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one dihydrochloride (13d). 13d was
prepared from 12c (100 mg, 0.286 mmol) using a procedure similar to that described for the preparation of 13a.
(White solid, 91.1 mg, 99%) 1HꢀNMR (DMSOꢀd6) δ: 9.46 (2H, brs), 3.56ꢀ3.48 (2H, m), 3.41ꢀ3.35 (2H, m),
2.82ꢀ2.75 (1H, m), 2.19ꢀ2.00 (6H, m), 1.85ꢀ1.79 (2H, m), 1.75ꢀ1.65 (2H, m), 1.47ꢀ1.36 (1H, m), 1.06ꢀ0.96 (2H, m),
0.93 (3H, d, J = 6.5 Hz). 13CꢀNMR (DMSOꢀd6) δ: 177.9, 177.2, 62.8, 38.3, 37.3, 33.1, 31.0, 28.1, 27.8, 22.1.
HRMS (ESIꢀTOF) m/z calcd for C14H24N3O (M+H), 250.1919; Found 250.1942.
8-(Benzylsulfonyl)-2-cyclohexyl-1,3,8-triazaspiro[4.5]dec-1-en-4-one (14a). To a stirred mixture of
2ꢀcyclohexylꢀ1,3,8ꢀtriazaspiro[4.5]decꢀ1ꢀenꢀ4ꢀone bis(2,2,2ꢀtrifluoroacetate) 13b (60 mg, 0.129 mmol) and
triethylamine (63 µl, 0.452 mmol) in CH2Cl2 (1.5 ml) was added benzylsulfonyl chloride (30 mg, 0.157 mmol) at
0ºC. The mixture was stirred at room temperature for overnight. This mixture was evaporated and the residue was
purified by column chromatography (silica gel, EtOAcꢀhexane) to afford 14a (19.5 mg, 39%) as a white solid.
1HꢀNMR (CDCl3) δ: 8.02 (1H, brs), 7.45ꢀ7.35 (5H, m), 4.22 (2H, s), 3.60ꢀ3.53 (2H, m), 3.30ꢀ3.23 (2H, m),
2.45ꢀ2.37 (1H, m), 1.95ꢀ1.81 (6H, m), 1.76ꢀ1.70 (1H, m), 1.50ꢀ1.25 (7H, m). 13CꢀNMR (CDCl3) δ: 186.7, 165.0,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 49
1
2
3
4
131.5, 129.6, 129.4, 129.3, 68.2, 57.8, 42.6, 39.8, 33.3, 30.3, 26.3, 26.1. HRMS (ESIꢀTOF) m/z calcd for
5
6
7
C20H28N3O3S (M+H), 390.1851; Found 390.1852. Purity 100%, RT = 0.90 min.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Cyclohexyl-8-(phenethylsulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (14b). To a stirred solution of
(E)ꢀ2ꢀcyclohexylꢀ8ꢀ(styrylsulfonyl)ꢀ1,3,8ꢀtriazaspiro[4.5]decꢀ1ꢀenꢀ4ꢀone (17b) (28.2 mg, 0.070 mmol) in EtOH
(1.0 ml), AcOEt (0.5 ml) and AcOH (0.15 ml) were added 10% Pd/C (10 mg) and this mixture was stirred at room
temperature for 2 days under hydrogen atmosphere. The mixture was filtered and evaporated. The residue was
purified by column chromatography (silica gel, hexaneꢀEtOAc) to afford 14b (19.3 mg, 68%) as a white solid.
1HꢀNMR (CDCl3) δ: 7.87 (1H, brs), 7.35ꢀ7.30 (2H, m), 7.28ꢀ7.24 (1H, m), 7.24ꢀ7.20 (2H, m), 3.77ꢀ3.71 (2H, m),
3.43ꢀ3.36 (2H, m), 3.22ꢀ3.12 (4H, m), 2.45ꢀ2.38 (1H, m), 2.00ꢀ1.90 (4H, m), 1.86ꢀ1.80 (2H, m), 1.75ꢀ1.70 (1H, m),
1.62ꢀ1.54 (2H, m), 1.45ꢀ1.20 (5H, m). 13CꢀNMR (CDCl3) δ: 186.1, 164.8, 138.5, 129.2, 128.7, 127.3, 67.7, 51.3,
42.0, 39.5, 33.0, 30.1, 29.7, 26.0, 25.8. HRMS (ESIꢀTOF) m/z calcd for C21H30N3O3S (M+H), 404.2008; Found
404.2021. Purity 98%, RT 0.95 min.
2-Cyclohexyl-8-((2-(pyridin-4-yl)ethyl)sulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (14c). 14c was
prepared from 17c (20 mg, 0.050 mmol) using a procedure similar to that described for the preparation of 14b.
(White solid, 15 mg, 75%) 1HꢀNMR (CDCl3) δ: 8.57ꢀ8.55 (2H, m), 7.82 (1H, brs), 7.18ꢀ7.15 (2H, m), 3.78ꢀ3.72
(2H, m), 3.44ꢀ3.38 (2H, m), 3.22ꢀ3.12 (4H, m), 2.46ꢀ2.38 (1H, m), 2.05ꢀ1.90 (4H, m), 1.86ꢀ1.80 (2H, m), 1.76ꢀ1.70
(1H, m), 1.65ꢀ1.55 (2H, m), 1.46ꢀ1.20 (5H, m). 13CꢀNMR (CDCl3) δ: 185.6, 164.5, 150.2, 147.1, 123.7, 67.3, 49.7,
ACS Paragon Plus Environment

Page 29 of 49
Journal of Medicinal Chemistry
1
2
3
4
41.7, 39.2, 32.6, 29.7, 28.7, 25.6, 25.4. HRMS C20H29N4O3S (ESIꢀTOF) m/z calcd for 405.1960 (M+H),; Found
5
6
7
405.1963. Purity 95%, RT 0.78 min.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Cyclohexyl-8-((2-(naphthalen-1-yl)ethyl)sulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (14d). 14d was
prepared from 13b (1.00 g, 2.16 mmol) and 1ꢀnaphthylethylsulfonyl chloride using a procedure similar to that
described for the preparation of 14a. (White solid, 877 mg, 90%) 1HꢀNMR (CDCl3) δ: 8.97 (1H, brs), 8.03 (1H, d, J
= 8.4 Hz), 7.88 (1H, d, J = 8.4 Hz), 7.78 (1H, d, J = 7.8 Hz), 7.60ꢀ7.55 (1H, m), 7.54ꢀ7.49 (1H, m), 7.42 (1H, t, J =
7.8 Hz), 7.39ꢀ7.37 (1H, m), 3.82ꢀ3.75 (2H, m), 3.65ꢀ3.60 (2H, m), 3.43ꢀ3.37 (2H, m), 3.32ꢀ3.28 (2H, m), 2.44ꢀ2.36
(1H, m), 2.03ꢀ1.95 (2H, m), 1.93ꢀ1.87 (2H, m), 1.83ꢀ1.77 (2H, m), 1.72ꢀ1.65 (1H, m), 1.60ꢀ1.54 (2H, m), 1.45ꢀ1.18
(5H, m). 13CꢀNMR (CDCl3) δ: 187.3, 165.4, 134.5, 134.3, 131.7, 129.4, 128.2, 127.0, 126.9, 126.2, 125.9, 123.3,
68.0, 50.4, 42.1, 39.6, 32.9, 29.9, 26.9, 25.9, 25.7. HRMS (ESIꢀTOF) m/z calcd for C25H32N3O3S (M+H), 454.2164;
Found 454.2169. Purity 100%, RT 1.04 min.
2-Cyclohexyl-8-(naphthalen-1-ylsulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (14e). 14e was prepared from
13c (15 mg, 0.049 mmol) and naphthaleneꢀ1ꢀsulphonyl chloride using a procedure similar to that described for the
preparation of 14a. (White solid, 13.8 mg, 67%) 1HꢀNMR (CDCl3) δ: 8.75 (1H, d, J = 8.6 Hz), 8.39 (1H, brs), 8.23
(1H, d, J = 6.4 Hz), 8.07 (1H, d, J = 8.1 Hz), 7.93 (1H, d, J = 8.1 Hz), 7.68ꢀ7.51 (3H, m), 3.84ꢀ3.76 (2H, m),
3.28ꢀ3.19 (2H, m), 2.40ꢀ2.31 (1H, m), 2.00ꢀ1.91 (2H, m), 1.89ꢀ1.75 (4H, m), 1.73ꢀ1.65 (1H, m), 1.54ꢀ1.46 (2H, m),
1.40ꢀ1.16 (5H, m). 13CꢀNMR (CDCl3) δ: 186.4, 164.7, 134.5, 134.4, 133.6, 130.2, 129.0, 128.9, 128.1, 126.9, 125.2,
ACS Paragon Plus Environment