Discovery of the CCR1 antagonist, BMS-817399, for the treatment of rheumatoid arthritis

Article abstract of DOI:10.1021/jm5003167

High-affinity, functionally potent, urea-based antagonists of CCR1 have been discovered. Modulation of PXR transactivation has revealed the selective and orally bioavailable CCR1 antagonist BMS-817399 (29), which entered clinical trials for the treatment of rheumatoid arthritis.

Full text of DOI:10.1021/jm5003167

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Article
The Discovery of CCR1 Antagonist, BMS-817399,
for the Treatment of Rheumatoid Arthritis
Joseph Santella, John V. Duncia, Daniel Gardner, Hong Wu, Murali T. G. Dhar, Cullen L.
Cavallaro, Andrew Joseph Tebben, Percy H Carter, Joel C. Barrish, Mellissa Yarde, Stephanie
Briceno, Mary Ellen Cvijic, Robert Grafstrom, Richard Liu, Sima Patel, Andrew Watson,
Guchen Yang, Anne Rose, Rodney Vickery, Janet Caceres Cortes, Christian Caporuscio,
Daniel Camac, Javed Khan, Yongmi An, William Foster, Paul Davies, and John Hynes
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5003167 • Publication Date (Web): 07 Aug 2014
Downloaded from http://pubs.acs.org on August 16, 2014
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The Discovery of CCR1 Antagonist, BMS-817399, for the
Treatment of Rheumatoid Arthritis
Joseph B. Santella, III*, Daniel S. Gardner, John V. Duncia*, Hong Wu, Murali Dhar,
Cullen Cavallaro, Andrew J. Tebben, Percy H. Carter, Joel C. Barrish, Melissa Yarde,
Stephanie W. Briceno, Mary Ellen Cvijic, R. Robert Grafstrom, Richard Liu, Sima R.
Patel, Andrew J.Watson, Guchen Yang, Anne V. Rose, Rodney D. Vickery, Janet Caceres-
Cortes, Christian Caporuscio, Daniel M. Camac, Javed A. Khan, Yongmi An, William R.
Foster, Paul Davies, and John Hynes, Jr.
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Bristol Myers Squibb Company, P.O. Box 4000, Princeton, New Jersey, 08543-4000
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Graphical Abstract
ABSTRACT: High affinity, functionally potent, urea-based antagonists of CCR1 have been discovered.
Modulation of PXR transactivation has revealed the selective and orally bioavailable CCR1 antagonist
BMS-817399 (29), which entered clinical trials for the treatment of rheumatoid arthritis.
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The Discovery of CCR1 Antagonist, BMS-817399, for the
Treatment of Rheumatoid Arthritis
Joseph B. Santella, III*, Daniel S. Gardner, John V. Duncia*, Hong Wu, Murali Dhar,
Cullen Cavallaro, Andrew J. Tebben, Percy H. Carter, Joel C. Barrish, Melissa Yarde,
Stephanie W. Briceno, Mary Ellen Cvijic, R. Robert Grafstrom, Richard Liu, Sima R.
Patel, Andrew J.Watson, Guchen Yang, Anne V. Rose, Rodney D. Vickery, Janet Caceres-
Cortes, Christian Caporuscio, Daniel M. Camac, Javed A. Khan, Yongmi An, William R.
Foster, Paul Davies, and John Hynes, Jr.
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Bristol Myers Squibb Company, P.O. Box 4000, Princeton, New Jersey, 08543-4000
ABSTRACT: High affinity, functionally potent, urea-based antagonists of CCR1 have been discovered.
Modulation of PXR transactivation has revealed the selective and orally bioavailable CCR1 antagonist
BMS-817399 (29), which entered clinical trials for the treatment of rheumatoid arthritis.
INTRODUCTION.
CCR1 (CC Chemokine Receptor-1) is a G-protein coupled receptor (GPCR) that is expressed on
1
the surface of monocytes/macrophages, osteoclasts, T-cells, and neutrophils. Eleven different
chemokines, including MIP-1α (CCL3), RANTES (CCL5), and Leukotactin-1 (CCL15), interact with this
receptor to induce leukocyte activation and migration, processes that are critical for the progression of
2
inflammatory diseases.
Pre-clinical studies in rodents suggested that CCR1 blockade represents a viable strategy for the
3
development of therapies for inflammatory disorders. Several small molecule antagonists of CCR1 have
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appeared in the literature and a number have advanced to human clinical trials (Figure 1). These
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6,10,11
include BX-471 (1) for multiple sclerosis , CP-481715 (2) for rheumatoid arthritis (RA)
, AZD-
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13
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818 (3) for chronic obstructive pulmonary disease , and MLN-3897 (4) for RA. Unfortunately,
these molecules failed to achieve their targeted clinical endpoints in Phase 2 studies.
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Figure 1. Structures of CCR1 antagonists with reported data from Phase 2 clinical trials.
In spite of earlier Phase 2 clinical failures, CCR1 has continued to be implicated in maintaining
the population of inflammatory cells within the synovial tissues and fluid of joints in human RA
1
0,16
patients.
A number of authors have suggested that the Phase 2 clinical failures were due to inadequate
2b,16
blockade of the CCR1 receptor over the course of the trial.
One molecule countering that trend is the
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7
CCR1 antagonist CCX354 (structure unknown), which recently showed clinical efficacy in a three
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8,19,20
month Phase 2 RA study.
We have also been exploring CCR1 antagonists and have previously
21,22
reported on our efforts leading to BMS-457 (8a).
We now report the discovery of BMS-817399 (29),
which entered Phase 2 human clinal trials for the treatment of rheumatoid arthritis.
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CHEMISTRY
The syntheses of the CCR1 antagonists in this report are shown in Scheme 1. (S)-4-(4-
2
3
Chlorophenyl)-3,3-dimethylpiperidin-4-ol (5) was coupled with BOC-D-valine followed by acid
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a
(
a) BOC-D-Val, HOBT, EDC, TEA, DCM, rt, 24h. (b) 4N HCl in dioxane, dioxane, 2h, rt, 44%. (c)
o
o
PhO(CO)Cl, TEA, DCM, 1.5h, 0 C, 53%. (d) RNH , TEA, MeCN, 20-30 min. microwave, 100 C. (e)
2
RCOOH, EDC, HOBT, DIEA, DCM and/or DMF, rt, 1-24h. (f) free base of 6 (obtained via chiral
o
normal-phase HPLC), α,α-dimethyl-γ-butyrolactone, neat, 120 C, 6h, 30%. (g) RNH-(CO)-OPh, TEA,
o
o
MeCN, microwave, 100 C, 1h then 150 C, 1h. (h) 1-pyrrolidinecarbonyl chloride, TEA, DCM, rt, 20h,
8
6%. (i) RNCO, TEA, THF, rt.
deprotection to yield valine amide 6. This could be either coupled with carboxylic acids to yield amides
a, 10, 12, 14, 16, 18, 24, 26, 28, and 30 or converted to carbamate 7 then reacted with amines to yield
8
ureas 9, 11, 13, 15, 17, 19, 22, 25, 27, and 29. The coupling partners can be reversed so that phenyl
carbamates of amines, such as R- or S-sec-butylamine, were reacted with valine amide 6 to yield ureas 20
and 21. Reaction of 6 with an aminecarbonylchloride such as pyrrolidinylcarbonylchloride yielded urea
2
3. Amide 30 was obtained by reacting the free base of 6 (obtained via chiral normal-phase HPLC) with
o
α,α-dimethyl-γ-butyrolactone neat at 120 C.
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RESULTS AND DISCUSSION
We have previously disclosed the discovery of 8a (Figure 2), a potent CCR1 antagonist nominated
2
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for advanced pre-clinical development. While this compound fulfilled the advancement criteria
excellent target affinity, functional potency, selectivity, in vitro safety, and pharmacokinetics), mild QT
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(
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prolongation was observed in a rabbit cardiovascular safety model. This was later confirmed in a
conscious telemetrized dog study, thereby halting the development of this compound. Additionally, in
vivo conversion to ketone (8b, Figure 2) was observed in rats. This ketone displays increased pregnane X
receptor (PXR) transactivation. Increased PXR transactivation leads to upregulation of CYP3A4 in an
attempt by the body to clear foreign substances, including drugs, via their metabolism. Thus increased
25
PXR transactivation can potentially lead to undesirable drug-drug interactions. Although ketone 8b was
not initially observed in significant concentrations in human, rat, and mouse liver microsomes, incubation
of 8a at higher concentrations revealed more significant conversion to 8b (data not shown).
Figure 2. Replacement of the rightmost amide of preclinical candidate 8a with a urea.
To seek out new SAR in an effort to circumvent these cardiovascular and metabolism issues, we
replaced the rightmost amide of 8a with a variety of differently substituted ureas yielding compounds such
as 9. We hypothesized that the more polar urea could modulate the liabilities associated with 8a while
also providing an additional conformational restriction of the terminal substituent. Fortunately, the ureas
were often equipotent to their amide counterparts in both the binding and chemotaxis assays. For
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example, phenylurea 11 was equipotent with phenylamide 10 with respect to CCR1 binding affinity
2
2
(
Table 1, IC column). Likewise, cyclopentylurea 13 was equipotent with cyclopentylamide 12 with
50
respect to both binding affinity and chemotaxis inhibition. The metabolic stability of compounds in
mouse, rat, and human liver microsomes was determined in order to see whether there is the potential to a)
form undesired potentially toxic or pharmacologically inactive metabolites due to phase I metabolism or
b) to accumulate in the body due to lack of metabolism. Liver microsome stability is therefore a tool
which can to some extent predict the metabolic fate of compounds and is expressed as % remaining intact
after incubation with the microsomes. Both cyclopentylamide 12 and cyclopentylurea 13 exhibited poor
liver microsome stability (see Table 1, column entitled “% remaining in liver microsomes”). From
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experience, we knew that a single hydroxyl group could have a powerful effect on metabolic stability.
The addition of a 3-hydroxyl group to amide 12 yielded four diastereomeric cyclopentanol amides (8a, 14,
16, and 18) of which two showed a dramatic improvement in liver microsome stability (8a and 14) while
2
2
the other two only moderate (16 and 18). Unfortunately, 8a and 14 were converted to the ketone in vitro
when assayed at higher concentrations in liver microsomes (data not shown). We synthesized the four
diastereomers of 3-hydroxycyclopentylurea, namely 9, 15, 17, and 19. The ureas were once again
essentially equipotent with their corresponding amides with regards to binding affinity as well as in the
inhibition of THP-1 cell chemotaxis, with the only marginal outlier being urea 9.
Three
hydroxycyclopentylureas showed a dramatic improvement in liver microsome stability (9, 15, and 19)
over cyclopentylurea 13, while 17 showed only a moderate improvement. Although metabolically more
stable in liver microsomes than 13, cyclopentylurea 9 was less potent in inhibiting chemotaxis while 15
and 19 displayed PXR transactivation activity (15: EC = 5.7 ꢀM; 19: EC = 19 ꢀM; these micromolar
50
50
EC values, even though weak, are considered to show some transactivation activity). Thus, none of the
50
3-hydroxycyclopentylureas were progressed further.
We synthesized a wide variety of urea analogs at R , only some of which are listed in Table 1
1
(
compounds 20-23). Many of these urea analogs at R yielded good CCR1 binding affinity.
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Unfortunately, many analogs, including 20 – 23, displayed PXR transactivation activity and/or human
liver microsomal instability.
The published X-ray crystal structure of PXR revealed that it has a large binding pocket that
28,29
allows it to interact with a wide range of hydrophobic substrates.
The addition of a polar hydroxyl
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group to cyclopentane yielded mixed results, inhibiting PXR transactivation activity in only four (8a, 9,
4, and 17) out of eight compounds (8a, 9, 14-19) (Table 1). We continued to apply this strategy to
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acyclic alkyl amides and ureas and the results are shown in Table 2. Surprisingly, S-isopropanol urea 25
had a more potent binding IC than its corresponding amide 24. In addition, urea 25 did not induce PXR
5
0
transactivation and displayed respectable liver microsome stability. Compound 25, however, could
potentially have its secondary hydroxyl group metabolized to the ketone. For the R-isopropanol series,
both amide 26 and urea 27 displayed equipotent CCR1 binding affinities. However, urea 27, did not show
any PXR transactivation activity, reminiscent of the lack of observed PXR transactivation activity with
urea 25. The strategy apparently worked with the additional polar hydroxyl group, eliminating the PXR
transactivation activity for the ureas. To alleviate the risk for secondary alcohol metabolism, we
converted urea-isopropanols 25 and 27 to the achiral tertiary alcohol 29 via the substitution of a single
methyl group. Compound 29 exhibited CCR1 binding affinity and chemotaxis inhibition potencies of 1
and 6 nM, respectively, while displaying no PXR transactivation up to 25 ꢀM. Furthermore, tertiary
alcohol 29 did not exhibit the metabolic instability issues in liver microsomes seen with secondary
alcohols 8a and 14 (Table 1). Compound 29 also demonstrated generally favorable pharmacokinetic (PK)
profiles in all species tested. The calculated oral bioavailabilities (F%) of 29 when dosed as a solution in
the mouse, rat, dog and cynomolgus monkey were 79%, 46%, 100% and 40%, respectively (Figure 3).
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Table 1. Binding and Chemotaxis Data for Ureas versus Amides.
Cl
OH
O
N
R1
amide: n=0
urea: n=1
N
N
H
H
O
n
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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43
44
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47
48
49
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55
56
57
58
59
60
Inhibition of Inhibition of
CCR1 binding
MIP-1α-
induced
chemotaxis
PXR trans-
activation
% remaining
in liver micro-
somes (h,r,m)
a
a,d
Compound
n
affinity
Issues
a
e
IC (nM)
A: EC (ꢀM)
50
20
IC (nM)
B: EC (ꢀM)
5
0
50
2
2
2
f
1
1
1
0
1
2
0
1
0
1
0
1
0
1
0
1
0
1
1
1
1
1
18
20±18 (2)
1±0.5 (177)
2±0.5 (2)
10±5 (2)
N.D.
A, 1.1
N.D.
N.A.
Potency
Potency
PXR, metstab
PXR, metstab
ketone formation
chemotaxis
ketone formation
PXR
PXR, metstab
metstab
PXR, metstab
PXR
PXR, metstab
PXR, metstab
PXR, metstab
PXR, metstab
b
N.D.
47,50,26
77,57,68
98,87,88
100,84,100
95,73,90
99,96,100
43,75,60
76,75,86
67,67,65
92,83,100
59,85,72
65,89,87
3,1,2
2,g
c
3±2 (72)
3±0 (2)
2±0.1 (2)
19±8 (4)
12±3 (2)
10±3 (2)
3±2 (2)
4±1 (2)
1±0.1 (2)
6±3 (2)
6±0.3 (2)
2±0.3 (2)
N.D.
A, 0.5
A, 7.1
A, 41
B, >50
A, >50
B, 5.7
A, 5.3
B, >50
A, 1.8
B, 19
1
8a
9
3
2
2
2
2
2±0.6 (4)
4
6
3
14
1
1
1
1
1
2
2
2
2
5
6
7
8
9
0
1
2
3
22
22
1±0.2 (2)
2
2
3
3
2
1
9
A, 5.2
A, 8.7
A, 2.2
A, 0.8
N.D.
2,11,19
a
Values without standard deviations represent a single determination. Values in parentheses represent number of
determinations. Assay variability was measured using compound 12 as an internal standard with <50% variability for
b
c
the CCR1 binding assay (n = 177) and <66% for the chemotaxis inhibition assay (n = 72). Not done. metstab =
d
metabolic instability in human, rat, and/or mouse liver microsomes. Listing of both EC and EC values denotes a
2
0
50
e
o
change in the assay reporting format during the course of the work. Compounds were incubated at 37 C for 10
f
minutes in the presence of human (h), rat (r), and mouse (m) liver microsomes. Substrate concentration = 3 ꢀM. Not
available.
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Table 2. Hydroxyl-substituted aliphatic amides and ureas.
Cl
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
0
1
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3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
R1
amide: n=0
urea: n=1
N
N
H
H
O
n
Inhibition of
MIP-1α-
Inhibition of
PXR trans-
activation
CCR1 binding
% remaining
in liver micro-
1
a
Compound
n
R
affinity
induced
IC (nM) (n) chemotaxis
a,
Issues
a
d
somes (h,r,m)
50
EC (ꢀM)
50
IC (nM) (n)
50
b
2
2
2
2
2
2
3
4
5
6
7
8
9
0
0
1
0
1
0
1
0
S-CH
S-CH
R-CH
R-CH
2
2
CH(OH)CH
CH(OH)CH
CH(OH)CH
CH(OH)CH
3
3
11
2
2
N.D.
N.D.
>25
7.8
>25
2.3
N.D.
potency
c
7±2 (2)
N.D.
6±2 (3)
N.D.
6±2 (8)
22±12 (2)
88,79,95 (0.5 ꢀM)
metstab
PXR, metstab
metstab
87,79,85
2
3
3
2±0.4 (6)
3
1±0.4 (6)
2±0.3 (3)
88,78,86 (0.5 ꢀM)
100,84,100
2
CH
CH
CH
C(OH)(CH
)
)
PXR
NONE
chemotaxis, PXR
2
2
2
3
2
C(OH)(CH
CH
>25
25
100,74,86 (0.5 ꢀM)
91,35,74 (0.5 ꢀM)
3
2
C(OH)(CH
)
2
3
2
a
Values without standard deviations represent a single determination. Values in parentheses represent number of
determinations. Assay variability was measured using compound 12 as an internal standard with <50% variability for
b
c
the CCR1 binding assay (n = 177) and <66% for the chemotaxis inhibition assay (n = 72). Not done. metstab =
d
o
metabolic instability in human, rat, and/or mouse liver microsomes. Compounds were incubated at 37 C for 10
minutes in the presence of human (h), rat (r), and mouse (m) liver microsomes. Substrate concentration = 3 ꢀM unless
specified as 0.5 ꢀM
.
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Figure 3. Intravenous (i.v.) and oral (p.o.) PK curves together with bioavailabilities (%F) for compound
29 in the (a) mouse, (b) rat, (c) dog, and (d) cyno.
Compound 29 binds to human peripheral blood mononuclear cells (PBMCs) with an IC = 4.2 ±
5
0
125
125
0
.9 nM using I-hMIP-1α as the ligand. Again, using I-hMIP-1α as the ligand, compound 29 did not
bind to mouse, rat (both non-PBMC), and monkey (PBMC) CCR1 (IC s > 50,000 nM each) and only
5
0
bound marginally to dog CCR1 (PBMC) with an IC = 635 ± 785 nM (n=4). Most compounds in the
5
0
program displayed selectivity only for human CCR1 (data not shown) due most likely to reduced CCR1
3
0
homology amongst different species.
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1
1
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5
5
6
In addition to MIP-1α (CCL3), compound 29 potently inhibited chemotaxis induced by other
CCR1 ligands. These data are summarized in Table 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
Table 3. Inhibition (IC , nM) of different chemokine-induced chemotaxis by compound 29.
50
c
b
MIP-1α
RANTES
CCL5
MCP-3
CCL7
HCC-1
CCL14a
LKN-1
HCC-4
CCL16
MPIF-1
CCL23
CCL3
CCL15
a
b
5
.5± 2.4 (8)
3.3± 1.5 (2)
9.3± 2.8 (2)
7.5± 0.1 (2)
16.2± 6.0 (6)
3.2± 2.3 (2)
14.3± 4.6 (2)
a
b
c
Number in parentheses = n. Based on 50% of a Ymax of 60%. ꢁ-24 N-truncated forms.
CCR1 activation stimulates a variety of responses that include chemotaxis and cellular adhesion
3
1
mediated partly via the up-regulation of the β integrin receptor, CD11b. Compound 29 blocked MIP-
2
1
α- and LKN-1-mediated up-regulation of CD11b in human whole blood with IC values of 25 ± 10 nM
5
0
and 42 ± 8 nM, respectively. This measurement in whole blood defines an early critical event in cellular
arrest at the endothelial surface. We infer from these data that compound 29 is not highly protein bound
50
in human blood since the MIP-1α CD11b IC value is only a few fold different or the same as the IC50
values for binding and chemotaxis in the presence of MIP-1α (1 and 6 nM, respectively, Table 2). Actual
protein binding measurements showed that it is 19 % free in human serum.
As mentioned earlier, several authors believe failure of CCR1 antagonists in the clinic was
possibly due to inadequate blockade of the CCR1 receptor over time. The oral bioavailabilities of
compound 29 in the species tested should enable coverage of multiples of the human whole blood
functional CD11b IC (200 nM) over 24 hours in the clinic and results will be reported in the future.
9
0
To test the effect of CCR1 antagonism on chemotaxis driven by synovial fluid (SF) from patients
with RA, we obtained human SF samples and confirmed that they were chemotactic for THP-1 cells,
giving a window similar to that elicited by commercial CCR1 ligands. Compound 29 completely inhibited
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Journal of Medicinal Chemistry
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1
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1
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2
2
2
2
2
2
2
2
3
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6
chemotaxis in 8/14 samples. The mean IC for the eight samples was 12.9 ± 7.1 nM. In the remaining 6
50
samples, inhibition was either absent (1/14) or incomplete (5/14) at a maximum compound concentration
of 100 nM. This may be due to the presence of chemotactic agents that act independently of CCR1.
Compound 29 did not bind to other CC family receptors, including CCR2, CCR3, CCR4, CCR5,
CXCR2, CXCR3, and CXCR5. Nor did compound 29 bind to a broad panel of other G-protein coupled
receptors and biogenic amine transporters. Advanced counter screen profiling did not reveal any
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
2+
significant safety (hERG, Na , Ca channel), or drug-drug interaction (cytochrome P450 enzyme) issues.
Because of its potency in the various assays described heretofore and its clean counterscreen profile,
compound 29 was advanced into extensive pre-clinical safety evaluation prior to human clinical trials.
This compound entered Phase II testing in rheumatoid arthritis patients.
Rationale for PXR Selectivity
32
A 2.8Å resolution X-ray crystal structure of amide 28 in PXR (Figure 4A) showed that the
piperidine amide carbonyl and hydroxyl are forming hydrogen bonds with Q285 and H407, respectively.
In addition to these direct interactions, the piperidine amide carbonyl is H-bonded to S247 through a
bridging water. An intramolecular hydrogen bond is also observed between the isopropanol OH and the
isopropyl amide NH. This stabilizes a compact ligand conformation that is compatible with the PXR
ligand binding site. A similar scenario can be invoked for amide 30 when overlapped onto amide 28 in the
observed binding site of PXR. A compact conformation can be achieved when the isopropanol OH is
hydrogen bonded to the bridging water with only the terminal isopropanol groups being slightly shifted
from one another to enable accomodation (Figure 4B). However, the rigidity of the urea linker in 25, 27
and 29 forces these compounds into a more extended conformation that is predicted to cause steric clashes
within the PXR binding site. An alignment of the most similar conformation of urea 29 onto amide 28
yields a steric interaction between one of the isopropanol gem-dimethyls and PXR’s binding pocket, in
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1
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1
1
2
2
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2
2
2
2
2
2
2
3
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3
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3
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5
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6
particular with F288 (Figure 4C). Furthermore, all of the lowest energy rotational conformers for both
the cis- and trans-urea isomers of compound 29 displayed steric interactions with PXR’s binding pocket
(Figure 5). Thus there is no low energy urea conformation available to 29 that avoids a steric clash with
the PXR binding site while maintaining the polar interactions with H407 and Q285. The urea moieties of
0
1
2
3
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9
0
1
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3
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1
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9
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8
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0
1
2
3
4
5
6
7
8
9
0
compounds 25 and 27 would likely mimic the steric clashes within the PXR binding site of compound 29.
Figure 4. (A) Crystal structure of compound 28 in PXR. Compound 28 is represented in yellow tubes, and
PXR residues within 4Å are green lines. The binding site is shown as a surface view with the surface of
the front most residues turned off for clarity. Hydrogen bonds to H407 and Q285 are shown as green
dashed lines, hydrogen bonds to the bridging water (red sphere) are shown as red dashed lines and the
intramolecular hydrogen bond as a yellow dashed line. (B) Predicted binding mode of compound 30
(cyan) in PXR. Hydrogen bonds are colored as above. (C) Superposition of the best fitting conformation
of compound 29 onto compound 28 from the PXR crystal structure. Steric clashes with F288 that block
binding of compound 29 can be seen by the penetration of the gem-dimethyl moiety through the surface.
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49
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56
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58
59
60
Figure 5. Surface view of the PXR ligand binding site with low energy conformations of compound 29
superimposed. The low energy cis-urea conformations are shown in white and salmon sticks. The low
energy trans-ureas are shown as magenta and purple. No conformation is completely enclosed within the
surface, indicating the presumed steric clashes between the urea and the PXR binding site.
Conclusion.
The entire sequence of events leading to the discovery of compound 29 is shown in Scheme 2.
Beginning with lead 31, which was obtained from screening our corporate compound collection, it was
21
discovered that the D-valine linker is optimal as shown in benzamide 32. Replacement of the piperidine
2
2
with a gem-dimethylpiperidinol yielded benzamide 10 with improved chemotaxis inhibition potency.
Replacement of the benzamide with an alkyl/cycloalkyl amide (cyclopentane amide 12) led to yet another
2
2
increase in binding and chemotaxis inhibition potencies. However, these alkyl/cycloalkyl ureas suffered
from metabolic instability in human liver microsomes and exhibited increased PXR transactivation
activity. We hypothesized that the addition of a hydroxyl group should increase metabolic stability and
decrease PXR transactivation activity. Indeed this proved to be the case with hyroxycyclopentane 8a
compared to its des-hydroxy parent 12. We were fortunate that replacement of the amide linker with a
urea maintained binding potency (9) as well as metabolic stability and low PXR transactivation activity.
Unfortunately, the hydroxycyclopentylureas could not be progressed. We then turned out attention to the
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acyclic alkyl ureas such as 25 which were more potent than their amide counterparts (24) with regards to
binding affinity. Both acyclic alkyl ureas 25 and 27 displayed a complete lack of PXR transactivation
activity unlike their amide counterpart (26). Conversion of the secondary alcohols of 25 and 27 to a
metabolically more stable and achiral tertiary alcohol via the substitution of a single methyl group resulted
in the discovery of clinical candidate BMS-817399 (29). The polar hydroxyl group in 29 initially was
thought to eliminate PXR activation activity by providing a repulsive interaction with PXR’s hydrophobic
pocket. We now believe it is the rigidity of the urea linker that induces a more extended conformation in
which there is no conformation devoid of steric clashes within the PXR binding site, resulting in
selectivity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In conclusion, SAR development of lead 8a led us to compound 29, a potent, selective, and orally
bioavailable CCR1 antagonist suitable for clinical development. Additional studies on the structure-
activity relationships of compound 8a led to a different structural solution to the selectivity problems, and
these will be the subject of a future report.
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Journal of Medicinal Chemistry
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Experimental Section.
1
Proton magnetic resonance ( H NMR) spectra were recorded on either a Bruker Avance 400 or a
JEOL Eclipse 500 spectrometer and are reported in ppm relative to the reference solvent of the sample in
which they were run. HPLC and LCMS analyses were conducted using a Shimadzu SCL-10A liquid
chromatograph and a SPD UV-vis detector at 220 nm with the MS detection performed with either a
Micromass Platform LC spectrometer or a Waters Micromass ZQ spectrometer. All flash column
chromatography was performed on EM Science silica gel 60 (particle size of 40-60 µm). All reagents were
purchased from commercial sources and used without further purification unless otherwise noted. All
reactions were performed under an inert atmosphere. HPLC analyses were performed using the following
conditions. All final compounds had an HPLC purity of ≥ 95% unless specifically mentioned.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HPLC Methods. HPLC analyses and preparatory reverse-phase scale purifications were performed using
the following conditions.
Method A. A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (solvent A) and 95%
acetonitrile, 5% water, and 0.05% TFA (solvent B); t = 0 min, 10% B; t= 15 min, 100% B was employed
on a SunFire C18 3.5 µ 3.5 mm x 150 mm column. Flow rate was 0.5 mL/min and UV detection was set
to 220 nm. The LC column was maintained at ambient temperature.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Method B. A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (solvent A) and 95%
acetonitrile, 5% water, and 0.05% TFA (solvent B); t = 0 min, 10%B; t = 15 min, 100%B (20 min.) was
employed on a XBridge Ph 3.5 µ 3.0 mm x 150 mm column. Flow rate was 0.5 mL/min and UV
detection was set to 220 nm. The LC column was maintained at ambient temperature.
Method C. A linear gradient using 10% methanol, 90% water, 0.1% TFA (solvent A) and 90% methanol,
10% water, and 0.1% TFA (solvent B); t = 0 min, 20%B; t = 14 min, 100%B was employed on a Waters
Sunfire C-18 19 mm x 50 mm column. Flow rate was 20 mL/min and UV detection was set to 220 nm.
The LC column was maintained at ambient temperature. Fractions containing the product were
concentrated in vacuo to yield product.
Method D. An isocratic run using a polar organic phase consisting of a 4:1 mixture of
methanol/isopropanol with 0.1% diethylamine was employed on a Chiralpak AD-H 5 x 25 cm, 5 ꢀM
column. Flow rate was 60 mL/min and UV detection was set to 220 nm. The HPLC column was
o
maintained at 35 C. Fractions containing the product were concentrated in vacuo.
Method E. A linear gradient using 100% water, 0.05% TFA (solvent A) and 100% acetonitrile and
0
.05% TFA (solvent B); t = 0 min, 0%B; t = 30 min, 100%B was employed on a Phenomenex Luna 5ꢀ
C-18(2) 21.2 mm x 250 mm column. Flow rate was 15 mL/min and UV detection was set to 220 nm. The
LC column was maintained at ambient temperature. Fractions containing the product were lyophilized to
yield product.
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Journal of Medicinal Chemistry
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(R)-2-Amino-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methylbutan-1-one,
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HCl (6). Part A. Synthesis of tert-butyl (R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-
1-yl)-3-methyl-1-oxobutan-2-ylcarbamate. (R)-2-(tert-Butoxycarbonylamino)-3-methylbutanoic acid
0
1
2
3
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0
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9
0
1
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9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
(BOC-D-Val) (13.16 g, 60.6 mmol, 1.1 eq), EDC (11.61 g, 60.6 mmol, 1.1 eq), and HOBT (9.27 g, 60.6
mmol, 1.1 eq) were mixed in methylene chloride (200 mL) at room temperature with stirring until a clear
solution was obtained. This was followed by the addition of (S)-4-(4-chlorophenyl)-3,3-
24
dimethylpiperidin-4-ol (13.2 g, 55.1 mmol, 1.0 eq), followed by triethylamine (9.21 mL, 66.1 mmol, 1.2
eq) via an addition funnel over 5 minutes at room temperature. A slight exotherm was observed. The
reaction was stirred overnight. Workup entailed rinsing the organic layer with water (1 X 50 mL), 1N
HCl (1 X 50 mL), saturated sodium bicarbonate (1 X 50 mL), and brine (1 X 50 mL). The organic layer
was dried over anhydrous sodium sulfate. Concentration under reduced pressure provided 28 grams of a
light tan glass. The crude product was purified over silica gel in 9:1 to 3:1 to 1:1 hexanes/EtOAc to 100%
EtOAc. tert-Butyl (R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-
1
oxobutan-2-ylcarbamate (24 g, 54.7 mmol) was obtained as a white glass. H NMR (400 MHz, CDCl ) δ
3
7
.42 - 7.29 (m, 4H), 5.39 (dd, J=18.0, 9.2 Hz, 1H), 4.71 - 4.63 (m, 0.5H), 4.57 (td, J=9.8, 5.7 Hz, 1H),
4.17 - 4.10 (m, 0.5H), 3.89 (d, J=13.1 Hz, 0.5H), 3.68 - 3.55 (m, 1H), 3.39 (d, J=13.1 Hz, 0.5H), 3.15 (td,
J=13.0, 3.0 Hz, 0.5H), 3.05 (d, J=13.1 Hz, 0.5H), 2.73 - 2.65 (m, 0.5H), 2.65 - 2.57 (m, 0.5H), 2.03 - 1.87
(m, 1H), 1.69 - 1.60 (m, 2H), 1.58 - 1.51 (m, 0.5H), 1.47 - 1.40 (m, 9H), 1.06 (d, J=6.8 Hz, 1H), 0.94 (d,
J=6.8 Hz, 3H), 0.90 (d, J=6.8 Hz, 2H), 0.85 (d, J=2.5 Hz, 3H), 0.78 (d, J=11.3 Hz, 3H). LCMS (ESI) m/z
+
Calcd for C H ClN O [M + H] 439.24. Found, 439.28.
1
8
28
2
2
Part B. tert-Butyl (R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-
oxobutan-2-ylcarbamate (from part A) (24 g, 54.7 mmol, 1 eq) was dissolved in dioxane (50 mL) at room
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6
temperature with stirring. To this solution at rt was added 4N HCl in dioxane (27.3 mL, 109 mmol, 2 eq)
dropwise via an addition funnel over 5 minutes. After 4 hours, LCMS detects about 80% completion.
Another one equivalent of 4N HCl in dioxane was added thereto. After 2 additional hours, the reaction
was evaporated 5 times from methylene chloride to yield crude product as a white glass. The glass was
stirred in 200 mL of acetone overnight. The precipitate was filtered and redissoved in methanol, followed
by the addition of ethyl ether and the mixture was evaporated to yield a white amorphous glass which was
subsequently dried under high vacuum for 8 hours. Product (R)-2-amino-1-((S)-4-(4-chlorophenyl)-4-
0
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9
0
hydroxy-3,3-dimethylpiperidin-1-yl)-3-methylbutan-1-one, HCl (10.0 g, 26.6 mmol, 44% yield over two
1
steps) was obtained as an amorphous white solid. H NMR (400 MHz, CD OD) δ 7.51 (d, J=8.8 Hz,
3
1
H), 7.49 (d, J=8.8 Hz, 1H), 7.33 (d, J=8.8 Hz, 2H), 4.62 - 4.53 (m, 0.5H), 4.39 (dd, J=12.3, 4.4 Hz, 1H),
.04 (dd, J=13.0, 0.5 Hz, 1H), 3.88 (d, J=13.2 Hz, 0.5H), 3.78 - 3.72 (m, 0.5H), 3.71 - 3.62 (m, 2H), 3.61 -
4
3.55 (m, 0.5H), 3.28 – 3.21 (m, 0.5H), 3.21 – 3.15 (m, 0.5H), 2.82 - 2.60 (m, 1H), 2.35 (dq, J=11.4, 6.9
Hz, 0.5H), 2.27 - 2.13 (m, 0.5H), 1.63 (d, J=14.1 Hz, 0.5H), 1.56 (d, J=13.6 Hz, 0.5H), 1.22 (d, J=7.0 Hz,
1
.5H), 1.10 (t, J=7.7 Hz, 3H), 1.01 (d, J=7.0 Hz, 1.5H), 0.89 - 0.80 (m, 4.5H), 0.77 (s, 1.5H). LCMS
+
(
ESI) m/z Calcd for C H ClN O [M + H] 339.18. Found, 339.24.
1
2
2
2
Phenyl (R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-
-ylcarbamate (7). (R)-2-Amino-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
2
methylbutan-1-one, HCl (6) (1.0 g, 2.66 mmol, 1 eq) was added to methylene chloride (15 mL) with
stirring at rt under nitrogen. Triethylamine (0.743 mL, 5.33 mmol, 2 eq) was added and the colorless
solution was cooled to 0 °C. To this was added a methylene chloride solution of phenyl chloroformate
(0.417 g, 2.66 mmol, 1 eq) dropwise via an addition funnel over 5 minutes. After 1 hour, LCMS showed
the reaction to be about 90% complete. An additional 0.2 equivilents of TEA were added followed by 0.2
equivalents of phenyl chloroformate at 0 °C. The organic mixture was rinsed with 1N HCl (1 x 15 mL),
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followed by saturated aqueous sodium carbonate (1 x 15 mL) and brine (1 x 15 mL). The organic layer
was dried over anhydrous sodium sulfate. The mixture was filtered and the solvent removed in vacuo to
provide a colorless oil. The oil was stirred at rt with 20 mL of 1:1 acetonitrile/heptane mixture and the
precipitate then filtered and dried yielding phenyl (R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-
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dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-ylcarbamate (650 mg, 1.42 mmol, 53% yield) as a white
1
solid. H NMR (400 MHz, CD OD) δ 7.51 - 7.45 (m, 2H), 7.40 - 7.28 (m, 4H), 7.25 - 7.17 (m, 1H), 7.12
3
(
d, J=8.6, Hz, 1H), 7.08 (d, J=8.6, Hz, 1H), 4.60 (m, 1H), 4.07 (d, J=13.0 Hz, 1H), 3.70 - 3.59 (m, 1H),
.48 (d, J=13.1, Hz, 1H), 3.25 - 3.18 (m, 0.5H), 3.15 (d, J=12.3 Hz, 0.5H), 2.78 - 2.59 (m, 1H), 2.21 (dq,
J=13.5, 6.8, Hz, 0.5H), 2.09 (dq, J=13.6, 6.7 Hz, 0.5H), 1.61 (d, J=14.1 Hz, 0.5H), 1.53 (d, J=13.6 Hz,
3
1
4
H), 1.11 (d, J=6.8 Hz, 1.5H), 1.07 (d, J=6.8 Hz, 1.5H), 1.01 (dd, J=6.8, 4.2 Hz, 3H), 0.84 - 0.79 (m,
+
.5H), 0.77 (s, 1.5H). LCMS (ESI) m/z Calcd for C H ClN O [M + H] 459.21. Found, 459.13.
2
5
32
2
4
1
3
-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-
-(2-hydroxy-2-methylpropyl)urea (29). Phenyl (R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-
dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-ylcarbamate (7) (920 mg, 2.004 mmol, 1 eq) was added to
acetonitrile (10 mL). To this mixture was then added triethylamine (0.279 mL, 2.004 mmol, 1 eq) and 1-
amino-2-methylpropan-2-ol (179 mg, 2.004 mmol, 1 eq). The reaction was heated at 100 °C for 30
minutes in the microwave. Upon cooling, a precipitate formed. Ethyl ether (10 mL) was added and the
mixture was stirred for 10 minutes. The precipitate was filtered and the product obtained was dried under
high vacuum to yield (638 mg, 1.54 mmol, 70% yield) of a white solid.
1
H NMR (500 MHz, CD OD) (approx. 1:1 mixture of rotamers) δ 7.47 (dd, J=15.4, 8.8 Hz, 2H), 7.31
3
(dd, J=8.5, 5.2 Hz, 2H), 4.71 (dd, J=12.1, 6.1 Hz, 1H), 4.54 (ddd, J=12.9, 2.5, 2.2 Hz, 0.5H), 4.08 – 3.98
(m, 1H), 3.68 – 3.58 (m,1H), 3.48 (dd, J=12.9, 1.4 Hz, 0.5H), 3.21 – 3.13 (m, 1H), 3.14 – 3.06 (m, 2H),
2
.70 (td, J=13.6, 4.97 Hz, 0.5H), 2.61 (td, J=13.5, 5.0 Hz, 0.5H), 2.09 (dq, J=13.2, 6.6 Hz, 0.5H), 1.95
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(dq, J=13.3, 6.7 Hz, 0.5H), 1.60 (ddd, J=13.9, 2.5, 2.3 Hz, 0.5H), 1.51 (ddd, J=14.2, 2.6, 2.5 Hz, 0.5H),
1
.16 (s, 3H), 1.14 (d, J=1.7 Hz, 3H), 1.05 (d, J=7.2 Hz, 1.5H), 0.98, (d, J=7.2 Hz, 1.5H), 0.94 (d, J=6.6
1
3
Hz, 1.5H), 0.91 (d, J=6.6 Hz, 1.5H), 0.82 (s, 1.5H), 0.81 (s, 1.5H), 0.79 (s, 1.5H), 0.75 (s, 1.5H).
C
NMR (126 MHz, CD OD) (mixture of rotamers) δ 173.62, 173.30, 161.14, 160.78, 144.78, 144.62,
3
0
1
2
3
4
5
6
7
8
9
0
1
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33.82 (2 C, s), 130.20 (4 C, s), 128.28 (4 C, s), 76.01, 75.95, 71.70, 71.66, 55.87, 55.22, 55.08, 51.77 (2
C, s), 51.08, 43.01, 40.38, 39.86, 39.29, 34.78, 33.67, 33.12, 32.43, 27.16 (2 C, s), 27.06 (2 C, s), 23.13,
+
2
2.79, 21.35, 21.11, 20.32, 19.82, 17.94, 17.71. (LCMS (ESI) m/z Calcd for C H ClN O [M + H]
23 36 3 4
4
54.25. Found, 454.32. HPLC Purity: 96.8%, t = 7.66 min (method A); 99.1%, t = 8.23 min (method
r r
B).
N-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-
yl)benzamide (10). (R)-2-Amino-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
methylbutan-1-one, HCl salt (6) (30 mg, 0.089 mmol, 1.0 eq), benzoic acid (10.81 mg, 0.089 mmol, 1.0
eq), EDC (20.37 mg, 0.106 mmol, 1.2 eq), HOBT (16.27 mg, 0.106 mmol, 1.2 eq) and N,N-
diisopropylethylamine (0.031 mL, 0.177 mmol, 2.0 eq) were mixed in methylene chloride (3 mL) and
DMF (0.5 mL) at rt with stirring for 1 h. The product was purified by reverse-phase preparative HPLC
(
Method C) (t = 12.54 min). Product fractions were collected and the solvent removed in vacuo to yield
r
N-((R)-1-((S)-4-(4- chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-
1
yl)benzamide (30 mg, 0.064 mmol, 73% yield) was obtained as a white solid. H NMR (400 MHz,
CD OD) (mixture of rotamers) δ 7.87 - 7.80 (m, 2H), 7.59 - 7.44 (m, 5H), 7.32 (d, J=8.6 Hz, 1H), 7.29 (d,
3
J=8.6 Hz, 1H), 5.11 (d, J=7.7 Hz, 0.5H), 5.04 (d, J=7.5 Hz, 0.5H), 4.64 - 4.55 (m, 0.5H), 4.21 (d, J=11.2
Hz, 0.5H), 4.30 – 4.10 (m, 0.5H), 3.71 - 3.59 (m, 2H), 3.22 (td, J=12.9, 3.0 Hz, 0.5H), 3.15 (d, J=12.8 Hz,
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0.5H), 2.75 (td, J=13.5, 4.6 Hz, 0.5H), 2.64 (td, J=13.6, 4.8 Hz, 0.5H), 2.35 - 2.25 (m, 0.5H), 2.18 (dq,
J=14.0, 6.9 Hz, 0.5H), 1.64 (d, J=14.3 Hz, 0.5H), 1.53 (d, J=13.6 Hz, 0.5H), 1.12 (d, J=6.6 Hz, 1.5H),
1
.07 (d, J=6.6 Hz, 1.5H), 1.00 (d, J=6.8 Hz, 3H), 0.84 (d, J=10.1 Hz, 3H), 0.79 (d, J=5.9 Hz, 3H). LCMS
+
(ESI) m/z Calcd for C H ClN O [M + H] 443.21. Found, 443.50. HPLC Purity: 98.0%, t = 9.81 min
2
5
31
2
3
r
0
1
2
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4
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8
9
0
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(
method A); 99.1%, t = 11.24 min (method B).
r
(R)-1-sec-Butyl-3-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-
oxobutan-2-yl)urea (20).
Part A. Phenyl (R)-sec-butylcarbamate. (R)-Butan-2-amine (1.00 g, 13.7 mmol, 1 eq) was dissolved in
acetonitrile (10 mL) at rt under nitrogen with stirring. To this solution was added triethylamine (3.81 mL,
o
2
7.3 mmol, 2 eq) and the reaction cooled to 0 C with an ice bath. An acetonitrile solution of phenyl
chloroformate (1.72 mL, 13.7 mmol, 1 eq) was then added dropwise via an addition funnel over 5
minutes. A precipitate formed. After 3 hours, the solvent was evaporated followed by the addition of
ethyl acetate (10 mL) and water (10 mL). The layers were separated and the ethyl acetate layer was rinsed
with 1N HCl (2 x 15 mL), and brine (1 x 15 mL). The organic layer was dried over sodium sulfate and the
solvent removed in vacuo to yield a colorless oil. The oil was purified over silica gel in 9:1 n-
heptane/ethyl acetate. The product (R)-phenyl sec-butylcarbamate (1.20 g, 6.21 mmol) was obtained as a
1
colorless oil which solidified upon standing. H NMR (400 MHz, CD OD) δ 7.38 - 7.33 (m, 2H), 7.21 -
3
7
.16 (m, 1H), 7.08 (d, J=7.9 Hz, 2H), 3.57 (sxt, J=6.7 Hz, 1H), 1.53 (quin, J=7.1 Hz, 2H), 1.18 (d, J=7.0
+
Hz, 3H), 0.97 (t, J=7.5 Hz, 3H). LCMS (ESI) m/z Calcd for C H NO [M + H] 194.12. Found,
1
1
16
2
194.22.
Part B. (R)-2-Amino-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methylbutan-1-
one, HCl (6) (40 mg, 0.107 mmol, 1 eq), (R)-phenyl sec-butylcarbamate (from part A) (20.59 mg, 0.107
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mmol, 1 eq), and triethylamine (0.030 ml, 0.215 mmol, 2 eq) were mixed in acetonitrile (3 mL) at rt. The
o
mixture was heated in the microwave at 100 C for 1 hour. LCMS showed product and both starting
materials to be present. The reaction was transferred to a new microwave tube. Additional triethylamine
o
(0.030 ml, 0.215 mmol, 2 eq) was added and the contents heated once more in the microwave at 150 C
0
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9
0
for 1 hour. The solvent was removed and the residue was purified over silica gel in 3:1 hexanes/EtOAc to
1
00% EtOAc. The product 1-sec-butyl-3-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-
dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)urea (37 mg, 0.084 mmol, 79% yield) was obtained as
1
a white amorphous solid. H NMR (400 MHz, CD OD) (mixture of rotamers) δ 7.48 (d, J=8.6 Hz, 2H),
3
7.31 (d, J=8.6 Hz, 2H), 6.10 (dd, J=15.6, 8.6 Hz, 0.5H), 5.96 (dd, J=16.5, 9.4 Hz, 0.5H), 4.77 - 4.66 (m,
1
H), 4.59 - 4.49 (m, 0.5H), 4.02 (d, J=11.4 Hz, 1H), 3.70 - 3.53 (m, 2H), 3.53 - 3.45 (m, 0.5H), 3.22 - 3.15
m, 0.5H), 3.12 (d, J=13.2 Hz, 0.5H), 2.76 - 2.56 (m, 1H), 2.15 - 2.02 (m, 0.5H), 1.94 (dq, J=13.2, 6.7 Hz,
0.5H), 1.61 (d, J=14.1 Hz, 0.5H), 1.51 (d, J=14.1 Hz, 0.5H), 1.48 - 1.36 (m, 2H), 1.29 (br. s., 0.5H), 1.12 -
(
1
.03 (m, 4.5H), 1.00 - 0.84 (m, 8.5H), 0.84 - 0.78 (m, 4.5H), 0.75 (s, 1.5H). LCMS (ESI) m/z Calcd for
+
C H ClN O [M + H] 438.25. Found, 438.33. HPLC Purity: 93.4%, t = 9.06 min (method A);
23 37
3
3
r
9
5.0%, t = 10.19 min (method B).
r
N-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-
yl)pyrrolidine-1-carboxamide (23). (R)-2-Amino-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-
dimethylpiperidin-1-yl)-3-methylbutan-1-one, HCl (6) (30 mg, 0.080 mmol, 1 eq), TEA (0.022 mL, 0.160
mmol, 2 eq) and pyrrolidine-1-carbonyl chloride (10.68 mg, 0.080 mmol, 1 eq) were mixed and stirred in
methylene chloride (1 ml) at rt for 20 hours. The solvent was removed and the residue was purified over
silica gel in 3:1 hexanes /EtOAc to 1:1 hexanes /EtOAc to 100% EtOAc. The product N-((R)-1-((S)-4-
(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)pyrrolidine-1-
1
carboxamide (30 mg, 0.069 mmol, 86 % yield) was obtained as a white solid. H NMR (400 MHz,
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CD OD) (mixture of rotamers) δ 7.45 (d, J=8.8 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.28 (dd, J=8.3, 4.8 Hz,
3
2
H), 5.62 (d, J=8.8 Hz, 1H), 5.54 (d, J=8.8 Hz, 1H), 4.78 - 4.72 (m, 0.5H), 4.72 - 4.66 (m, 0.5H), 4.52 (d,
J=11.0 Hz, 0.5H), 4.10 - 3.97 (m, 1H), 3.65 - 3.55 (m, 1H), 3.49 - 3.43 (m, 0.5H), 3.20 - 3.13 (m, 0.5H),
3
0
.13 - 3.05 (m, 0.5H), 2.73 - 2.54 (m, 1H), 2.14 - 2.02 (m, 0.5H), 2.01 - 1.85 (m, 4.5H), 1.84 - 1.78 (m,
0
1
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0
.5H), 1.58 (d, J=14.1 Hz, 0.5H), 1.48 (d, J=14.1 Hz, 0.5H), 1.28 – 1.12 (m, 1H), 1.03 (d, J=6.6 Hz,
1.5H), 0.96 (d, J=6.6 Hz, 1.5H), 0.91 (d, J=7.0 Hz, 1.5H), 0.88 (d, J=6.2 Hz, 1.5H), 0.78 (d, J=6.6 Hz,
+
3
H), 0.74 (d, J=7.9 Hz, 3H). LCMS (ESI) m/z Calcd for C H ClN O [M + H] 436.24. Found, 436.28.
2
3
35
3
3
HPLC Purity: 96.9%, t = 8.74 min (method A); 96.3%, t = 9.77 min (method B).
r
r
N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-
-hydroxy-4-methylpentanamide (30). The free base of (R)-2-amino-1-((S)-4-(4-chlorophenyl)-4-
4
hydroxy-3,3-dimethylpiperidin-1-yl)-3-methylbutan-1-one, HCl salt, (6) was obtained from preparative
chiral normal-phase HPLC (t = 10.27 min) (Method D). Thus (R)-2-amino-1-((S)-4-(4-chlorophenyl)-4-
r
hydroxy-3,3-dimethylpiperidin-1-yl)-3-methylbutan-1-one (40 mg, 0.118 mmol, 1 eq) and 5,5-
dimethyldihydrofuran-2(3H)-one (40.4 mg, 0.354 mmol, 3 eq) were mixed together neat and heated in a
closed vial behind a safety shield at 120 °C with stirring. The reaction became a dark amber solution
during heating. After 6 hours, the heating was stopped. The reaction was evaporated and the residue
purified by reverse-phase preparative HPLC (Method C) (t = 11.39 min). The product N-((R)-1-((S)-4-
r
(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-4-hydroxy-4-
1
methylpentanamide (16 mg, 0.035 mmol, 30%) was obtained as an off-white solid. H NMR (400 MHz,
CD OD) (mixture of rotamers) δ 7.49 (d, J=8.8 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.32 (d, J=8.8 Hz, 1H),
3
7
.31 (d, J=8.8 Hz, 1H), 4.81 (d, J=7.3 Hz, 0.5H), 4.60 - 4.50 (m, 0.5H), 4.10 (dd, J=13.5, 2.1 Hz, 0.5H),
4.03 (dd, J=12.7, 1.8 Hz, 0.5H), 3.66 - 3.58 (m, 1H), 3.58 - 3.48 (m, 0.5H), 3.23 - 3.13 (m, 0.5H), 3.10 (d,
J=12.7 Hz, 0.5H), 2.71 (td, J=13.5, 4.8 Hz, 0.5H), 2.61 (td, J=13.6, 4.7 Hz, 0.5H), 2.40 - 2.29 (m, 2H),
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2
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2
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3
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2.16 (dq, J=13.6, 6.8 Hz, 0.5H), 2.09 - 1.95 (dq, J = 13.6, 6.8 Hz, 0.5H), 1.80 – 1.70 (m, 2H), 1.60 (dt,
J=13.8, 2.4 Hz, 0.5H), 1.52 (dt, J=13.8, 2.2 Hz, 0.5H), 1.20 (s, 3H), 1.19 (s, 3H), 1.05 (d, J=6.6 Hz, 1.5H),
1
.00 (d, J=6.8 Hz, 1.5H), 0.93 (d, J=6.8 Hz, 3H), 0.83 (s, 1.5H), 0.81 (s, 1.5H), 0.76 (s, 3H). LCMS (ESI)
+
m/z Calcd for C H ClN O [M + H] 453.25. Found, 453.28. HPLC Purity: 96.4%, t = 7.89 min
24
38
2
4
r
0
1
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8
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0
1
2
3
4
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6
7
8
9
0
(
method A); 95.0%, t = 8.57 min (method B).
r
1
-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-
3
-phenylurea (11). (R)-2-Amino-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
methylbutan-1-one, HCl (6) (25 mg, 0.067 mmol, 1 eq) was mixed with THF (2 mL) and methylene
chloride (2 ml) at rt. To this mixture was added triethylamine (0.019 ml, 0.133 mmol, 2 eq) followed by
phenyl isocyanate (0.015 ml, 0.133 mmol, 2 eq). After 1 hour, the solvent was removed in vacuo and the
residue taken up in a minimum of THF to solubilize and the mixture purified over silica gel in 3:1 to 1:1
hexanes/EtOAc to 100% THF. The product 1-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-
dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3-phenylurea (9.0 mg, 0.020 mmol, 30 %) was
1
obtained as a white solid. H NMR (500 MHz, CD OD) (mixture of rotamers) δ 7.50 (d, J=8.8 Hz, 1H),
3
7
.47 (d, J=8.5 Hz, 1H), 7.38 - 7.28 (m, 4H), 7.27 - 7.21 (m, 2H), 6.97 (q, J=7.5 Hz, 1H), 4.60 (s, 1H), 4.04
d, J=11.0 Hz, 1H), 3.73 (t, J=6.3 Hz, 1H), 3.66 (d, J=13.2 Hz, 1H), 3.51 - 3.43 (m, 1H), 3.19 - 3.15 (m,
1H), 2.04 - 1.98 (m, 0.5H), 1.87 (dt, J=6.5, 3.2 Hz, 1H), 1.63 (d, J=14.0 Hz, 0.5H), 1.52 (d, J=14.8 Hz,
(
0
1
.5H), 1.25 (t, J=7.1 Hz, 3H), 1.11 (d, J=6.6 Hz, 1.5H), 1.01 (d, J=6.9 Hz, 1.5H), 0.99 (d, J=6.6 Hz,
.5H), 0.94 (d, J=6.9 Hz, 1.5H), 0.84 (d, J=4.9 Hz, 3H), 0.82 (s, 1.5H), 0.77 (s, 1.5H). LCMS (ESI) m/z
+
Calcd for C H ClN O [M + H] 458.22. Found, 458.28. HPLC Purity: 95.2%, t = 9.85 min (method
2
5
33
3
3
r
A); 95.8%, t = 10.93 min (method B).
r
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Page 27 of 52
Journal of Medicinal Chemistry
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N-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-
yl)cyclopentanecarboxamide (12). The title compound was synthesized by the procedure used to make
N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-
yl)benzamide (10). The crude product was purified via flash chromatography over silica gel in 9:1 to 1:1
0
1
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4
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8
9
0
1
2
3
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9
0
1
Hexanes/EtOAc. H NMR (400 MHz, CDCl ) (mixture of rotamers) δ 7.42 - 7.38 (m, 1H), 7.37 - 7.29
3
(m, 3H), 6.33 (d, J=8.8 Hz, 0.5H), 6.27 (d, J=8.8 Hz, 0.5H), 4.97 (dt, J=8.9, 5.4 Hz, 1H), 4.71 - 4.63 (m,
0
.5H), 4.13 (dd, J=13.2, 1.8 Hz, 0.5H), 3.95 (dt, J=13.3, 2.4 Hz, 0.5H), 3.60 (d, J=13.6 Hz, 0.5H), 3.42
dd, J=13.4, 1.5 Hz, 0.5H), 3.17 (td, J=13.0, 3.1 Hz, 0.5H), 3.06 (d, J=13.2 Hz, 0.5H), 2.74 - 2.66 (m,
0.5H), 2.64 (d, J=5.7 Hz, 0.5H), 2.62 - 2.53 (m, 1H), 2.12 - 1.66 (m, 7H), 1.45 (d, J=13.6 Hz, 0.5H), 1.27
(
(br. s., 1H), 1.05 (d, J=6.6 Hz, 1.5H), 0.94 (dd, J=6.6, 3.1 Hz, 3H), 0.90 (d, J=7.0 Hz, 2.5H), 0.86 (d,
+
J=3.5 Hz, 3H), 0.77 (d, J=1.3 Hz, 3H). LCMS (ESI) m/z Calcd for C H ClN O [M + H] 435.24.
2
4
36
2
3
Found, 435.20. HPLC Purity: 92.9%, t = 9.66 min (method A); 91.5%, t = 11.06 min (method B).
r
r
1
-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-
3-cyclopentylurea (13). The title compound was synthesized by the procedure used to make 1-((R)-1-
((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3-phenylurea
(11). The crude product was purified via flash chromatography over silica gel in 3:1 to 1:1
1
hexanes/EtOAc to 100% EtOAc. H NMR (400 MHz, CD OD) (mixture of rotamers) δ 7.49 (d, J=8.8 Hz,
3
1
H), 7.46 (d, J=8.8 Hz, 1H), 7.16 (d, J=8.8 Hz, 1H), 7.31 (d, J=8.8 Hz, 1H), 6.26 (dd, J = 15.2, 7.5 Hz,
0.5H), 5.91 (dd, J = 14.6, 9.4 Hz, 0.5H), 4.75 - 4.65 (m, 1H), 4.54 (d, J=8.4 Hz, 1H), 4.02 (d, J=10.8 Hz,
1
0
H), 3.99 - 3.89 (m, 1H), 3.69 - 3.57 (m, 1H), 3.51 - 3.43 (m, 0.5H), 3.22 - 3.15 (m, 0.5H), 3.15 - 3.08 (m,
.5H), 2.77 - 2.54 (m, 1H), 2.07 (dd, J=12.9, 6.7 Hz, 0.5H), 1.90 (ddd, J=18.5, 12.1, 6.6 Hz, 2.5H), 1.76 -
1.47 (m, 5H), 1.38 (dt, J=12.2, 6.0 Hz, 2H), 1.05 (d, J=6.8 Hz, 1.5H), 0.96 (d, J=6.8 Hz, 1.5H), 0.93 (d,
J=6.8 Hz, 1.5H), 0.89 (d, J=6.8 Hz, 1.5H), 0.83 (s, 1.5H), 0.81 (d, J=2.6 Hz, 3H), 0.75 (s, 1.5H). LCMS
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Journal of Medicinal Chemistry
Page 28 of 52
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+
(ESI) m/z Calcd for C H ClN O [M + H] 450.25. Found, 450.36. HPLC Purity: 98.7%, t = 9.30 min
2
4
37
3
3
r
(
method A); 98.9%, t = 10.42 min (method B).
r
(
1R,3R)-N-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-
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0
1-oxobutan-2-yl)-3-hydroxycyclopentanecarboxamide (8a). The title compound was synthesized by the
procedure used to make N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
methyl-1-oxobutan-2-yl)benzamide (10). The crude product was purified via flash chromatography over
silica gel in 9:1 methylene chloride/ethyl acetate to 4:1 to 1:1 methylene chloride/ethyl acetate to 100%
1
EtOAc to 9:1 ethyl acetate/methanol. H NMR (500 MHz, CD OD) (mixture of rotamers) δ 7.49 (d,
3
J=8.8 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.31 (dd, J=8.8 Hz, 2H), 4.55 (d, J=12.6 Hz, 0.5H), 4.38 - 4.31 (m,
1
-
H), 4.09 (d, J=13.2 Hz, 0.5H), 4.03 (d, J=12.6 Hz, 0.5H), 3.65 - 3.55 (m, 1H), 3.55 - 3.50 (m, 0.5H), 3.21
3.14 (m, 0.5H), 3.11 (d, J=12.6 Hz, 0.5H), 3.06 - 2.96 (m, 1H), 2.74 - 2.66 (m, 0.5H), 2.66 - 2.57 (m,
0.5H), 2.21 - 2.13 (m, 0.5H), 2.12 - 2.01 (m, 1.5H), 2.00 - 1.67 (m, 4H), 1.67 - 1.56 (m, 1.5H), 1.51 (d,
J=13.7 Hz, 0.5H), 1.04 (d, J=6.6 Hz, 1.5H), 1.00 (d, J=6.6 Hz, 1.5H), 0.93 (m, 3H), 0.82 (d, J=4.9 Hz,
+
3
H), 0.75 (d, J=7.7 Hz, 3H). LCMS (ESI) m/z Calcd for C H ClN O [M + H] 451.24. Found, 451.33.
2
4
36
2
4
HPLC Purity: 99.5%, t = 7.68 min (method A); 99.4%, t = 8.24 min (method B).
r
r
1
-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-
oxobutan-2-yl)-3-((1R,3R)-3-hydroxycyclopentyl)urea (9). The title compound was synthesized by the
procedure used to make 1-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
methyl-1-oxobutan-2-yl)-3-(2-hydroxy-2-methylpropyl)urea (29). The crude product was purified via
flash chromatography over silica gel in 1:1 hexanes/EtOAc to 100% EtOAc to 9:1 methylene
1
chloride/MeOH. H NMR (400 MHz, CD OD) (mixture of rotamers) δ 7.48 (d, J=8.8 Hz, 2H), 7.46 (d,
3
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Page 29 of 52
Journal of Medicinal Chemistry
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J=8.8 Hz, 2H), 7.32 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 4.70 (t, J=5.3 Hz, 1H), 4.53 (d, J=10.8 Hz,
0
-
.5H), 4.30 (d, J=4.0 Hz, 1H), 4.23 - 4.11 (m, 1H), 4.02 (d, J=12.5 Hz, 1H), 3.62 (d, J=13.2 Hz, 1H), 3.50
3.43 (m, 0.5H), 3.22 - 3.15 (m, 0.5H), 3.15 - 3.08 (m, 0.5H), 2.76 - 2.56 (m, 1H), 2.18 - 1.88 (m, 4H),
1.69 - 1.46 (m, 3H), 1.36 (qd, J=13.9, 6.6 Hz, 1H), 1.05 (d, J=6.6 Hz, 1.5H), 0.95 (dd, J=8.7, 6.9 Hz, 3H),
0
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9
0
0
.89 (d, J=6.8 Hz, 1.5H), 0.83 (s, 1.5H), 0.80 (d, J=3.1 Hz, 3H), 0.75 (s, 1.5H). LCMS (ESI) m/z Calcd
+
for C H ClN O [M + H] 466.25. Found, 466.25. HPLC Purity: 95.3%, t = 7.51 min (method A);
24 37
3
4
r
95.3%, t = 7.91 min (method B).
r
(1S,3S)-N-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-
oxobutan-2-yl)-3-hydroxycyclopentanecarboxamide (14). The title compound was synthesized by the
procedure used to make N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
methyl-1-oxobutan-2-yl)benzamide (10). The crude product was purified via preparative reverse-phase
1
HPLC (Method E). H NMR (500 MHz, CD OD) (mixture of rotamers) δ 7.51 - 7.44 (m, 2H), 7.34 - 7.28
3
(m, 2H), 4.55 (d, J=12.1 Hz, 0.5H), 4.36 (dt, J=5.5, 2.7 Hz, 1H), 4.13 - 4.01 (m, 1H), 3.66 - 3.55 (m, 1H),
3
.53 – 3.50 (m, 1H), 3.28 - 3.07 (m, 2H), 3.02 (dq, J=16.9, 8.7 Hz, 1H), 2.75 - 2.66 (m, 0.5H), 2.62 (td,
J=13.5, 4.9 Hz, 0.5H), 2.22 - 2.12 (m, 0.5H), 2.09 - 1.80 (m, 4.5H), 1.79 - 1.57 (m, 2.5H), 1.52 (d, J=13.7
Hz, 0.5H), 1.04 (d, J=6.6 Hz, 1.5H), 0.99 (d, J=7.1 Hz, 1.5H), 0.92 (d, J=6.6 Hz, 3H), 0.82 (d, J=5.5 Hz,
+
3
4
H), 0.76 (d, J=5.5 Hz, 3H). LCMS (ESI) m/z Calcd for C H ClN O [M + H] 451.24. Found,
24 36
2
4
51.50. HPLC Purity: 95.5%, t = 7.61 min (method A); 95.2%, t = 8.24 min (method B).
r
r
1
-((R)-1-((S)-4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-
oxobutan-2-yl)-3-((1S,3S)-3-hydroxycyclopentyl)urea (15). The title compound was synthesized by the
procedure used to make 1-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-
methyl-1-oxobutan-2-yl)-3-(2-hydroxy-2-methylpropyl)urea (29). The crude product was purified via
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