Design, synthesis, and evaluation of conformationally restricted acetanilides as potent and selective β3 adrenergic receptor agonists for the treatment of overactive bladder

Article abstract of DOI:10.1021/jm4017224

A series of conformationally restricted acetanilides were synthesized and evaluated as β₃-adrenergic receptor agonists (β₃-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine β₃-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent β₃-AR mediated responses in a rat bladder hyperactivity model.

Full text of DOI:10.1021/jm4017224

Article
pubs.acs.org/jmc
Design, Synthesis, and Evaluation of Conformationally Restricted
Acetanilides as Potent and Selective β3 Adrenergic Receptor Agonists
for the Treatment of Overactive Bladder
Christopher R. Moyes,*,† Richard Berger,‡ Stephen D. Goble,§ Bart Harper,§ Dong-Ming Shen,§
Liping Wang,§ Alka Bansal,§,∥ Patricia N. Brown,† Airu S. Chen,§ Karen H. Dingley,† Jerry Di Salvo,†
Aileen Fitzmaurice,† Loise N. Gichuru,† Amanda L. Hurley,§ Nina Jochnowitz,† Randall R. Miller,†
Shruty Mistry,§ Hiroshi Nagabukuro,§,# Gino M. Salituro,§ Anthony Sanfiz,§,⊥ Andra S. Stevenson,†
Katherine Villa,§,∇ Beata Zamlynny,† Mary Struthers,† Ann E. Weber,† and Scott D. Edmondson†
†Early Development and Discovery Sciences, Merck and Co., Inc., 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United
States
‡Early Development and Discovery Sciences, Merck and Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United
States
§Early Development and Discovery Sciences, Merck and Co., Inc., 126 East Lincoln Avenue, Rahway, New Jersey 07065, United
States
S
* Supporting Information
ABSTRACT: A series of conformationally restricted acetanilides were synthesized and
evaluated as β3-adrenergic receptor agonists (β3-AR) for the treatment of overactive
bladder (OAB). Optimization studies identified a five-membered ring as the preferred
conformational lock of the acetanilide. Further optimization of both the aromatic and
thiazole regions led to compounds such as 19 and 29, which have a good balance of
potency and selectivity. These compounds have significantly reduced intrinsic
clearance compared to our initial series of pyridylethanolamine β3-AR agonists and thus have improved unbound drug
exposures. Both analogues demonstrated dose dependent β3-AR mediated responses in a rat bladder hyperactivity model.
muscarinic receptor selectivities,7 there remains a need for
therapies with an improved side effect profile. Within the last 15
INTRODUCTION
■
The discovery of the β3-adrenergic receptor (β3-AR) in the early
years, evidence for the presence of the β3-AR on human and rat
1980s1 and the demonstration that activation of this receptor by
detrusor muscle has emerged.8 In vitro studies have demon-
small molecule agonists induced thermogenic2 and antihyper-
strated that selective β3-AR agonists cause relaxation of isolated
glycaemic activity3 in rodents led to a period of intense activity
rat and human detrusor muscle strips, and compelling in vivo
within the pharmaceutical industry aimed at novel treatments for
type 2 diabeties and obesity.4 A number of compounds entered
clinical studies, however, ultimately they were unsuccessful,
either because of a lack of efficacy, an unfavorable cardiovascular
evidence to support a role for β3-AR in increasing bladder
capacity in anesthetized rats has emerged.9 This has led to
renewed activity within the pharmaceutical industry in an
attempt to discover potent and selective β3-AR agonists as a
side effect profile, or poor pharmacokinetics, which led to a
potential new treatment for OAB, with the possibility for fewer
downturn in interest in this receptor as a target.
Overactive bladder (OAB) is a chronic and sometimes
debilitating condition of the lower urinary tract that negatively
impacts the quality of life of millions of people. A large
side effect liabilities compared to antimuscarinics.10 Several
compounds such as 1 (YM-178, mirabegron)11 and 2
(GW427353, solabegron),12 as shown in Figure 1, have entered
the clinic and, in the case of 1, have been approved for the
treatment of overactive bladder with symptoms of urge urinary
incontinence, urgency, and urinary frequency.
multicountry survey recently estimated the prevalence of OAB at
11.8%, increasing with age.5 Given that the elderly are the fastest-
growing segment of the population, it is likely that this number
Previous work in our laboratories has described the discovery
will only increase. Muscarinic antagonists are the mainstay of
pharmacotherapy for OAB,6 and these drugs have been
demonstrated to improve urgency, frequency of micturition,
and urge incontinence, all of which are primary symptoms of
OAB. However, through their anticholinergic mechanism, these
of a series of pyridylethanolamine β3-AR agonists with excellent
potency and selectivity and possessing moderate oral bioavail-
ability, culminating in the discovery of our first-generation
clinical candidate 3.13 In an effort to address some of the liabilities
agents have also demonstrated adverse effects, most notably dry
mouth and constipation. While tolerability may be improved by
the use of extended release formulations and agents with differing
Received: November 8, 2013
Published: January 17, 2014
© 2014 American Chemical Society
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Figure 1. Structures of some of the β3-AR agonists to have entered the clinic.
Figure 2. Structures of second-generation pyrrolidine based β3-AR agonists.
a
Scheme 1. Preparation of RHS-Thiazole Intermediates
a
Reagents: (a) Br2, CHCl3, 0 °C; (b) thiourea, EtOH, reflux; (c) (Boc)2O, Et3N, DMAP, CH2Cl2; (d) LiOH, THF, MeOH, H2O; (e) tert-
butylnitrite, DMF, 50 °C; (f) R3-thioamide 36a−e, EtOH, reflux.
associated with this series,14 we proceeded to report the design
and synthesis of second generation β3-AR agonists containing a
novel pyrrolidine scaffold, exemplified by acetanilide derivative
4.15 Compound 4 demonstrated potent human β3-AR agonist
activity and was also very selective over the β1 and β2 receptors.
We have also disclosed the structure−activity relationships
(SAR) surrounding some acetanilide derivatives in a linear
(nonpyrrolidine) series and demonstrated the beneficial effects
on potency and selectivity of substitution at the position α to the
amide carbonyl group.16 We now wish to report the
incorporation and extension of this SAR into the novel
pyrrolidine based series.
We initially targeted simple substitution with a methyl group
to establish if the previously observed SAR would translate to this
series and then further elaborated with higher alkyl homologues
(5) (Figure 2). We have previously demonstrated in the linear
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a
Scheme 2. Preparation of LHS-Aromatic Intermediates
a
(a) Trimethylacetyl chloride, Et3N, THF; (b) (S)-4-benzyl-2-oxazolidinone, LiCl, THF; (c) ArCHO, MgCl2, TMS-Cl, Et3N, EtOAc; (d) TFA,
MeOH; (e) TBS-triflate, 2,6-lutidine, CH2Cl2; (f) H2O2, LiOH, THF, H2O; (g) DPPA, Et3N, 4-methoxybenzyl alcohol, toluene, 80 °C; (h) 4-
iodonitrobenzene, PdCl2(dppf), CuI, Et3N, DMF; (i) pyrrolidine, DMF, 85 °C; (j) AcOH, H2O; (k) TFA, CH2Cl2; (l) NaCNBH3, MeOH; (m)
(Boc)2O, i-Pr2NEt, CH2Cl2; (n) H2, 10% Pd/C, MeOH or Zinc, AcOH, EtOH 80 °C; (o) TBAF, THF; (p) H2 50 psi, 10% Pd/C, KOAc MeOH.
series that substitution with a methyl group on the thiazole-5-
position also improved potency. We therefore realized that with
an open C−H bond at the thiazole-5-position, there was the
opportunity to form a tether between the thiazole and the α
position (6). In principle, the introduction of this conformational
constraint could have a positive effect on potency through a
reduction in the entropic penalty required to achieve the optimal
compound−receptor interaction.
In this article, we will describe the synthesis and SAR of various
ring size tethers as well as compounds with modifications to both
the left-hand side (LHS) aromatic and to the thiazole-2-position.
Finally, we explored an alternative to the thiazole by replacement
with a pyrazole. These studies have led to the discovery of a novel
series of conformationally restricted acetanilide derivatives. In
this paper, we describe in vitro SAR, pharmacokinetic properties,
and evaluation of certain compounds from this new series in an in
vivo rat bladder hyperactivity model.
rearranged to the desired γ-brominated product by blowing wet
air through the reaction mixture,17 and the resultant crude
material is treated with thiourea in refluxing ethanol to afford
amino thiazoles 31a−g. N-Boc protection under standard
conditions was performed in order to facilitate purification of
later intermediates. Saponification of the esters 32a−g afforded
the acids 33a−g. A similar sequence was performed on ethyl 2-
oxocyclopentane carboxylate (30e), replacing thiourea with
thioamides 36a−d to yield the desired substituted thiazoles
37a−d. Hydrolysis of the ester under standard conditions
afforded acids 38a−d. The unsubstituted thiazole 35 was
prepared by direct deamination of amino-thiazole 31e by heating
with tert-butyl nitrite in DMF to give ester 34, followed by
hydrolysis to the acid 35.
Intermediates with modifications to the LHS aromatic ring
were prepared according to the procedure shown in Scheme 2. 5-
Hexynoic acid (39) was coupled with (S)-4-benzyl-2-oxazolidi-
nione to afford the N-acyloxazolidinone 40, which underwent
magnesium chloride catalyzed anti-Aldol reaction18 with
benzaldehydes, followed by desilylation with TFA in methanol
to give the adduct 41a−e in high diastereoselectivity. Protection
of the alcohol using TBS-triflate and 2,6-lutidine afforded
products 42a−e. Removal of the oxazolidinone auxiliary with
CHEMISTRY
■
Thiazole acid intermediates 33a−g, 35, and 38a−d were
synthesized following the routes illustrated in Scheme 1. Keto-
esters (30) were brominated by the addition of bromine in
chloroform at 0 °C. The initially formed α-brominated product is
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a
Scheme 3. General Synthesis Route to Targets 7−26
a
(a) EDC, HOAt, i-Pr2NEt, DMF; (b) chiral HPLC separation, (R,R)-Whelk-O column, eluent IPA/heptanes; (c) TFA, CH2Cl2.
LiOH in the presence of 30% H2O2 provided acids 43a−e, which
underwent Curtius rearrangement in the presence of 4-
methoxybenzyl alcohol to afford the Moz-protected amines
44a−e. Sonogashira coupling of 44a−e with 4-iodonitrobenzene
provided the 4-nitrophenylacetylenes 45a−e, which were
converted to ketones 46a−e using a procedure developed by
Fukuyama.19 Regioselective conjugate addition of pyrrolidine to
the acetylene afforded an intermediate enamine, which was then
hydrolyzed under acidic conditions to the ketone (46a−e).
Removal of the Moz protecting group by treatment with TFA
and concomitant cyclization afforded a 2,3-dihydropyrrole
intermediate, which was reduced to a mixture of cis and trans
pyrrolidine isomers using sodium cyanoborohydride. During the
reductive amination step, selective formation of the cis over the
trans pyrroline was always observed, with selectivities generally in
the 80:20 range, although selectivity as low as 60:40 was
observed. The desired cis pyrrolidine isomers 47a−e could be
readily separated from the trans isomers by silica gel
chromatography. Boc protection under standard conditions
gave the Boc-pyrrolidines 48a−e. Anilines 50a−d were prepared
by reduction of nitro intermediates 48a−d using either
hydrogenolysis or zinc in acetic acid, followed by TBS
deprotection. Hydrogenolysis of 48e under pressure in the
presence of potassium acetate reduced the nitro group and
removed the pyridyl chlorine and following TBS deprotection
afforded 50e. Aniline 50a was identical spectroscopically to the
same intermediate prepared by a different route previously, and
whose identity had been fully characterized by 2D NMR and X-
ray analysis.15
Standard EDC coupling of the anilines 50a−e with thiazole
acetic acids 33a−g, 35, and 38a−d (Scheme 3) afforded the
desired coupled products as a mixture of two diastereoisomers.
The diastereoisomers were readily separated utilizing chiral
preperative HPLC methodology, all mixtures being separated
using an (R,R)-Whelk-O column with mixtures of IPA and
heptanes as eluent. The resultant pure diastereoisomers were
Boc deprotected under standard conditions to give the target
agonists 7−26.
Assignment of an absolute configuration to the more active
acid component in the final derivatives was determined by the
following method. Separation of the enantiomers of acid 38a was
achieved utilizing chiral SFC HPLC. Coupling of the individual
acids to aniline 50e using standard EDC conditions (including
Hunig’s Base) resulted in complete racemization of the thiazole
component, however, EDC coupling with the omission of
Hunig’s Base proceeded smoothly with no sign of racemization.
Deprotection and biological evaluation of the individual
diastereoisomers allowed us to determine which acid resulted
in the more potent agonist. X-ray crystallographic analysis of this
acid determined the absolute configuration to be R. By inference,
given that the more potent agonist was always derived from the
protected precursor which eluted from the (R,R)-Whelk-O
column first, we have assigned this diastereoisomer as also having
the R configuration.
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a
Scheme 4. Synthesis Route to Pyrazole Derivatives
a
(a) Bis(trimethylsilyl)acetylene, AlCl3, CH2Cl2; (b) Boc-hydrazine, i-PrOH; (c) TBAF, THF; (d) benzyl bromide, K2CO3, DMF; (e) (i) LiHMDS,
TMS-Cl, THF, (ii) NBS, (iii) TFA, CH2Cl2, (iv) K2CO3, NaI, acetone 58 °C; (f) H2, Pd(C), EtOH; (g) EDC, HOAt, Hunig’s base, DMF; (h) chiral
HPLC separation, (R,R)-Whelk-O column, eluent IPA/heptanes; (i) TFA, DCM.
Table 1. SAR Results for Alkylated and Ring Constrained Amino-Thiazole Analogues
a
b
c
human EC50 (nM) (%Act)
human IC50 (nM)
compd
R1
R2
β3
β1
β2
4
H
H
0.98 0.59 (92)
0.18 0.04 (84)
0.67 0.02 (85)
0.83 0.13 (85)
0.88 0.01 (66)
1.60 0.01 (85)
0.15 0.03 (91)
44.2 6.00 (91)
0.21 0.03 (103)
1.86 0.16 (70)
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
7a isomer 1
7b isomer 2
Me
H
H
H
H
H
Me
d
8a isomer 1
9a isomer 1
Et
d
n-Pr
d
10a isomer 1
11a isomer 1
iso-Pr
−CH2CH2−
−CH2CH2−
−CH2CH2CH2−
−CH2CH2CH2CH2−
11b isomer 2
d
12a isomer 1
13a isomer 1
d
a
b
Unless otherwise noted, the results are shown as the mean SD (n ≥ 3) or presented as the average of two experiments. % Activation defined as
c
the fitted maximal value of the test compound concentration response expressed as a percent of the maximal response to R-(−)-isoproterenol. n = 1.
d
Single diastereoisomer with only more potent isomer indicated.
Preparation of the pyrazole derivatives 27−29 was achieved
using similar methodology utilizing commercially available acids
57a−b and the cyclic acid 56, which was prepared as outlined in
Scheme 4. Glutaric anhydride (51) and bis(trimethylsilyl)-
acetylene was treated with anhydrous AlCl3 to afford keto-acid
52. Condensation of 52 with Boc-hydrazine gave hydrazone 53,
which following TMS deprotection and benzyl ester formation
afforded pyrazole 54. Formation of a trimethylsilylketene acetal
followed by bromination and acidic removal of the Boc group,
and then base induced cyclization generated the cyclic pyrazole
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Table 2. SAR Results of LHS Aromatic Variation
a
b
c
human EC50 (nM) (%Act)
human IC50 (nM)
d
compd
Ar
Ph
β3
β1
β2
hERG IC50(μM)
11a
14
15
16
17
0.15 0.03 (103)
0.15 0.06 (93)
0.04 0.002 (94)
0.13 0.02 (72)
0.37 0.02 (101)
>20000
>20000
>20000
>20000
>20000
>20000
8451 2368
>20000
12.7
1.5
e
3-ClPh
3-FPh
4-FPh
3-Py
4.0
>20000
5.6
>20000
23.3
a
b
Unless otherwise noted, the results are shown as the mean SD (n ≥ 3) or presented as the average of two experiments. % Activation defined as
c
the fitted maximal value of the test compound concentration response expressed as a percent of the maximal response to R-(−)-isoproterenol. n = 1.
d
e
Single diastereoisomer with only more potent isomer indicated. n = 2.
Table 3. SAR Results for Substitution at the Thiazole-2-Position
a
b
c
human EC50 (nM) (%Act)
human IC50 (nM)
d
compd
X
R3
β3
β1
β2
18
19
20
21
22
23
24
25
26
CH
N
H
H
0.64 0.53 (101)
1.36 0.69 (103)
0.48 0.13 (96)
1.15 0.57 (89)
1.95 0.45 (37)
13.6 2.36 (49)
12.4 4.74 (43)
186 114.6 (76)
8.78 1.32 (15)
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
CH
N
Me
Me
CH
N
Et
Et
N
c-Pr
4-F-Ph
4-F-Ph
e
e
e
CH
N
4329 140
2513 539
2471 585
e
13340 1183
a
b
Unless otherwise noted, the results are shown as the mean SD (n ≥ 3) or presented as the average of two experiments. % Activation defined as
c
the fitted maximal value of the test compound concentration response expressed as a percent of the maximal response to R-(−)-isoproterenol. n = 1.
d
e
Single diastereoisomer with only more potent isomer indicated. n = 2.
55. Cleavage of the ester under hydrogenolyis conditions
afforded acid 56.
4. Of note is that in all cases the receptor demonstrated a distinct
stereochemical preference for one diastereomer over the other.
We next focused our attention on analogues in which a ring of
various sizes has been formed between the thiazole-5-position
and the α position. Constraining the side chain through
formation of a five-membered (11a) or six-membered ring
(12a) also improved β3-AR activity (4−5-fold) over the
unsubstituted compound while also maintaining the excellent
selectivity profile over the β1 and β2-AR. Increasing the ring size
to a seven-membered ring (13a) caused a 2-fold decrease in β3-
AR activity over the unsubstituted compound. While compounds
11a and 12a were comparable to 4 in terms of potency,
pharmacokinetic evaluation in the rat revealed that both
compounds displayed an inferior pharmacokinetic (PK) profile
compared to 4 (Table 5), with both demonstrating increased
clearance (Clp = 50 mL/min/kg for 11a and 185 mL/min/kg for
12a), decreased bioavailability (F = 6% for 11a and 0% for 12a),
and decreased unbound drug exposures. The five-membered
analogue 11a appeared slightly superior to the six-membered
analogue, and thus all further studies focused on fused five-
membered ring analogues.
RESULTS AND DISCUSSION
■
All final compounds were evaluated as human β3 adrenergic
receptor agonists by measuring their ability to stimulate increases
in intracellular cAMP in Chinese hamster ovary (CHO) cell lines
stably expressing the human β3 receptor. Selectivity over human
β1 and β2-AR was assessed by measuring the binding affinity of
compounds for these receptors using membranes prepared from
recombinant cells expressing either β1 or β2-AR.20
We initially investigated the effect of substitution with alkyl
groups at the position α to the carbonyl in compound 4. The
results of this study are summarized in Table 1. In line with
previous observations in the nonpyrrolidine containing series of
acetanilides, introduction of a methyl group α to the carbonyl
improved potency.15 For example, diasteroisomer 7a is 5-fold
more potent than the unsubstituted analogue 4. Increasing the
size of the alkyl group gives compounds with decreasing activity
over the methyl analogue; for example, the ethyl 8a and n-propyl
9a analogues are equipotent to the unsubstituted analogue 4, and
the iso-propyl analogue 10a shows a loss in activity compared to
We next explored replacement of the LHS phenyl group in 11a
with several substituted phenyl groups (Table 2). Addition of a
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Table 4. SAR Results for Pyrazole Derivatives
a
b
c
human EC50 (nM) (%Act)
human IC50 (nM)
d
compd
R1
R2
β3
β1
β2
27
28
29
H
H
H
76.7 13.68 (72)
16.7 10.33 (102)
3.7 2.86 (84)
>20000
>20000
>20000
>20000
>20000
>20000
Me
−CH2CH2−
a
b
Unless otherwise noted, the results are shown as the mean SD (n ≥ 3) or presented as the average of two experiments. % Activation defined as
c
the fitted maximal value of the test compound concentration response expressed as a percent of the maximal response to R-(−)-isoproterenol. n = 1.
d
Single diastereoisomer with only more potent isomer indicated.
a
Table 5. Mean Pharmacokinetic Parameters of Selected β3 Adrenergic Agonists in Non Clinical Species
Clp
T1/2
F
(%)
%
rat LM Clint
mL/min/kg
Papp (× 10−6
cm/s)
b
b
compd
species
mL/min/kg (h) PO AUC total (nM·h/mpk) PO AUC unbound (nM·h/mpk)
unbound
d
3
rat
62
20
19
11
50
185
32
80
33
27
36
87
25
34
24
34
36
8
14
9
70
290
50
1
6
17
27
4
2
2
ND
ND
dog
monkey
rat
1
2
4
8
440
40
26
7
12
6
6
38
59
5
5
11a
12a
rat
4
18
c
c
c
c
rat
3
<LQ
<LQ
60
<LQ
<LQ
6
0
14
63
8
c
17
rat
4
0
unstable
10
57
<1
24
18
19
rat
1
12
41
67
8
68
e
rat
2
480
980
90
91
686
90
2
19
<15
13
dog
monkey
rat
6
70
2
100
5
20
21
28
29
1
30
6
135
62
26
17
rat
1
700
420
590
77
ND
212
46
29
35
100
11
11
rat
2
ND
36
ND
55
ND
e
rat
3
6
dog
monkey
8
1680
120
1176
70
4
120
100
a
b
c
d
Dose: iv, 1 mpk; po, 2 mpk. Cassette dosing: iv, 0.13 mpk; po, 0.25 mpk. <LQ = below limits of quantification. Data taken from ref 24.
e
Transport was observed in control cells, hence permeability is likely overestimated. ND: not determined.
chlorine atom to the three position (such as that found in
salabegron) resulted in a compound (14) equipotent to the
phenyl analogue, however, selectivity over β2-AR and the human
(24) decreased potency further but more importantly rendered
these analogues partial agonists. Replacement with a 4-
fluorophenyl group either decreased potency still further (25)
or led to a dramatic loss in receptor activation (26) and in both
cases showed decreased selectivity over β1/2-AR.
̀
Ether-a-go-go Related Gene (hERG) potassium channel is
reduced. Replacement of the chlorine with a fluorine (15)
resulted in a 4-fold increase in β3-AR activity and also improved
selectivity over hERG. Shift of the fluoro group to the 4-position
(16) maintained β3-AR activity but also resulted in reduced
selectivity over hERG. Finally, replacement of the phenyl moiety
with a 3-pyridyl (17) group led to a slight reduction in potency
(EC50 = 0.37nM) with similar selectivity over hERG, however,
this compound demonstrated no bioavailability (F = 0%).
We sought to improve the bioavailability in both the phenyl
and pyridyl series by further examination of the thiazole RHS. Of
particular interest was the replacement of the amino-group from
the 2-position of the thiazole based upon the metabolism-
induced toxicity potential of this class of heterocycles.21
Transformation of the amino group to either a hydrogen (18
and 19) or a methyl group (20 and 21) resulted in a 3−4-fold loss
in activity in both the phenyl and pyridyl cases, however, this was
counterbalanced by much improved rat bioavailability in the
pyridyl LHS analogues (F = 41% for 19 and 46% for 21). Further
homologation to an ethyl (22 and 23) or a cyclopropyl group
Finally, we examined replacement of the thiazole with
alternative heterocycles (Table 4). In our previous work, we
demonstrated that a pyrazole was an effective replacement for the
thiazole.15 Given the improved rat bioavailability observed with
the pyridyl LHS in the thiazole series, we chose to explore
thiazole replacement in this series. The parent pyrazole
acetamide (27) showed significantly reduced β3-AR potency
(EC50 = 77nM) compared to 4, however, good selectivity over
β1-AR and β2-AR was maintained. As expected, the introduction
of a methyl group at the α position furnished a compound (28)
with improved β3-AR activity (EC50 = 16.7nM) and which also
had good rat bioavailability (F = 29%). Fusion of the α position
to the pyrazole 5-position through a five-membered ring resulted
in 29, which had a further gain in β3-AR activity (EC50 = 3.7nM)
and also maintained good rat bioavailability (F = 35%). To try
and understand what was driving the differences observed in
bioavailability with these analogues, we examined intrinsic
clearance (Clint) in liver microsomal (LM) incubations and
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Table 6. Off-Target Activities of Selected β3 Adrenergic Agonists
a
P450 inhibition IC50s (μM)
ion channel IC50s (μM)
b
c
d
d
compd
2C9
2D6
2.2
3A4
hERG
hCav1.2
hNav1.5
SERT IC50 (nM)
4
>100
>100
>100
>100
86
11.2
12.7
6.1
8.2
>30
>30
>30
>30
>30
5.6
1613
1218
422
89
11a
19
21
29
18.3
78.8
>100
>30
>30
>30
67.2
>100
>100
>100
41.8
>100
12
>30
898
a
Human liver microsome P450 marker enzyme activities with diclofenac α′-hydroxylation for 2C9, dextromethorphan O-demethylation for 2D6, and
b
c
d
testosterone 6β-hydroxylation for 3A4. IKr binding assay, ref 23. Binding assay with [3H]-diltiazem, ref 23. Reference 23.
passive permeability (Papp) in MDCKII cells (Table 5).
Examination of the in vitro intrinsic clearance alone did not
appear to explain the differences in oral bioavailability in rats.
Low passive permeability likely contributed to the low oral
bioavailability of several compounds, however, compounds 19
and 29 had improved oral bioavailability despite low passive
permeability, indicating the potential role of uptake transporters
in absorption. In support of this hypothesis, a recent paper
suggested mirabegron (1) also has low-to-moderate membrane
permeability and P-gp is likely to be involved in its efflux and that
it could be transported by intestinal influx transporters.22
On the basis of the β3-AR activity and rat pharmacokinetic
profiles, compounds 19, 21, and 29 were selected for further
evaluation. Further examination of these analogues against
ancillary targets (Table 6) demonstrated a clean P450 inhibition
profile for all three analogues, with compound 21 being the least
selective, showing an IC50 of 42 μM against CYP3A4. Binding at
the hERG, human cardiac calcium channel, and human cardiac
sodium channels was measured to examine possible cardiac
liabilities of these compounds. All three compounds were clean
(IC50 > 30 μM) at the calcium and sodium channels, however, 19
and 21 do show some inhibition of the hERG potassium channel
(IC50 = 6 and 12 μM, respectively). Additional in vitro profiling
in an extensive panel of receptor and ion channel binding and
enzyme inhibition assays showed these compounds possessed
activity at the serotonin transporter (SERT), the target of the
selective serotonin reuptake inhibitor (SSRI) class of anti-
depressants. Compound 21 proved to be the least selective (77-
fold), with compounds 19 and 29 being somewhat more selective
(310- and 243-fold respectively).
MS/MS. 29 was well absorbed, with 73% of the administered
dose recovered in urine and bile. 29 was eliminated renally
(∼50% of recovered dose) and through metabolism (∼50% of
recovered dose). This differs significantly from 3, which was
poorly absorbed and metabolized extensively, and hepatic
extraction was the major route for clearance of this compound
in rats.14,24
In comparison to our first-generation pyridylethanolamine β3-
AR agonist, 3, both 19 and 29 demonstrated significantly
different properties such as lower Log D (4.6 for 3, compared to
0.9−1.2 for 19 and 29 at pH 7.3) reduced plasma protein binding
(∼2% unbound for 3, compared to 20−100% unbound for 19
and 29) and reduced in vivo intrinsic clearance (in rats estimated
as ∼4000 mL/min/kg for 3 compared to less than 200 mL/min/
kg for 19 and 29). The lower intrinsic clearance results in
significantly improved unbound drug exposures in vivo for 19
and 29 compared to 3 (Table 5).
Compounds 19 and 29 were evaluated in an acetic acid
induced bladder hyperactivity model in anesthetized rats (Figure
1).25,26 Before conducting in vivo experiments, we confirmed the
in vitro potency of these compounds at β3-AR across a number of
species as shown in Table 8. We observed significant differences
between human and rat β3-AR activity with 19 and 29, which is
expected based on previous observations.26 The EC50 values for
the rat β3-AR of compounds 19 and 29 showed significantly
reduced potency (19, 20-fold; 29, 100-fold) relative to human β3-
AR activity. After intravenous dosing, both 19 and 29 caused a
reduction in micturition pressure with a trend for dose
dependence (Figure 3). Reductions in micturition pressure are
an indirect measure of detrusor muscle relaxation. As anticipated
by its greater potency at the rat β3-AR, the thiazole analogue 19
showed a reduction in micturation pressure (59%) at a lower
unbound plasma level (1.2 μM) than the pyrazole analogue 29.
Compound 29 was able to elicit a greater reduction in
micturation pressure overall (69%) but at an unbound plasma
level of 27 μM.
The dog pharmacokinetic profiles of 19 and 29 revealed good
oral bioavailabilities (Fdog = 67−100%). However, both
compounds exhibit poor biovailability in rhesus monkeys (8−
11%). Examination of the stability of these compounds in liver
microsomes across a range of species (Table 7) indicates that 19
Table 7. In Vitro Intrinisc Clearance of Compounds 19 and 29
in Liver Microsomes
CONCLUSIONS
■
In summary, we have investigated changes to both the LHS
aromatic and RHS heterocycle of a series of pyrrolidine
containing β3 adrenergic receptor agonists with the aim of
addressing issues related to pharmacokinetics (e.g., low rat oral
bioavailability) and potential metabolic/toxicological issues (e.g.,
toxicity potential of amino-thiazole, ion channel inhibition). To
accomplish these goals, we developed a novel route to the
pyrrolidine core to allow modification of the LHS aromatic ring.
We also expanded the SAR in the pyrrolidine series by preparing
conformationally restricted acetanilide RHS heterocycles.
Excellent on-target potency was maintained with a number of
examples; compounds 19 and 29 no longer contain an amino-
thiazole group and demonstrate marked improvements in rat in
intrinsic clearance (mL/min/kg body weight)
compd
human
rat
dog
monkey
19
29
<10
19
<15
55
<10
31
205
261
and 29 both have a much higher in vitro intrinsic clearance in
monkey liver microsomes than in rat, dog, and human liver
microsomes, which may lead to greater hepatic extraction in this
species.
A metabolism and excretion study was conducted in bile duct
cannulated rats after oral dosing with 3H-labeled 29 at 10 mg/kg.
Urine and bile were collected for up to 48 h and analyzed by LC/
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Table 8. β3-AR Activity of Selected in Compounds in Different Species
a
b
EC50 (nM) (%Act)
compd
human
rat
dog
Rhesus
c
c
11a
19
0.15 0.03 (103)
1.4 0.69 (103)
3.7 2.86 (84)
1.3 0.39 (100)
27.6 6.9 (89)
434 171 (98)
0.19 (102)
0.06 (78)
c
c
5.0 (82)
2.0 (98)
4.7 (93) 3.19
29
22 (86) 12.41
a
b
Unless otherwise noted, the results are shown as the mean SD (n ≥ 3) or presented as the average of two experiments. % Activation defined as
c
the fitted maximal value of the test compound concentration response expressed as a percent of the maximal response to R-(−)-isoproterenol. n = 1.
are of ≥95 HPLC area % purity, unless otherwise noted. Purity analysis
was carried out by LC/MS analysis using an Agilent 1100 system fitted
with a Waters Micromass ZQ2000 quadrupole spectrometer.28
(R)-2-(2-Aminothiazol-4-yl)-N-(4-(((2S,5R)-5-((R)-hydroxy-
(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)propanamide
(7a) and (S)-2-(2-Aminothiazol-4-yl)-N-(4-(((2S,5R)-5-((R)-
hydroxy(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)-
propanamide (7b). To a solution of (2S,5R)-tert-butyl 2-(4-
aminobenzyl)-5-((R)-hydroxy(phenyl)methyl)pyrrolidine-1-carboxy-
late (50a) (100 mg, 0.26 mmol) and 2-(2-((tert-butoxycarbonyl)-
amino)thiazol-4-yl)propanoic acid (33a) (71 mg, 0.26 mmol) in DMF
(3 mL) was added EDC (100 mg, 0.52 mmol), HOBt (60 mg, 0.39
mmol), and triethylamine (0.146 mL, 1.05 mmol), and the resulting
mixture stirred at room temperature for 4 h. The mixture was poured
into water (15 mL) and extracted with ethyl acetate (3 × 5 mL). The
combined extracts were washed with brine (15 mL), dried (MgSO4),
and evaporated. The residue was purified by silica gel column
chromatography (Biotage Horizon: SNAP 10g: elution with gradient
0−50% ethyl acetate in hexanes) to give (2S,5R)-tert-butyl 2-(4-(2-(2-
((tert-butoxycarbonyl)amino)thiazol-4-yl)propanamido)benzyl)-5-
((R)-hydroxy(phenyl)methyl)pyrrolidine-1-carboxylate. The diaster-
eoisomers were separated by preparative HPLC (Pirkle (R,R)-Whelk-
O column: eluent 25% isopropyl alcohol in heptanes) to give first eluting
isomer (2S,5R)-tert-butyl 2-(4-((R)-2-(2-((tert-butoxycarbonyl)-
amino)thiazol-4-yl)propanamido)benzyl)-5-((R)-hydroxy(phenyl)-
methyl)pyrrolidine-1-carboxylate (34 mg, 20%) and second eluting
isomer (2S,5R)-tert-butyl 2-(4-((S)-2-(2-((tert-butoxycarbonyl)-
amino)thiazol-4-yl)propanamido)benzyl)-5-((R)-hydroxy(phenyl)-
methyl)pyrrolidine-1-carboxylate (26 mg, 16%). A mixture of (2S,5R)-
tert-butyl 2-(4-((R)-2-(2-((tert-butoxycarbonyl)amino)thiazol-4-yl)-
propanamido)benzyl)-5-((R)-hydroxy(phenyl)methyl)pyrrolidine-1-
carboxylate (34 mg, 0.053 mmol) was dissolved in dichloromethane (0.5
mL) and trifluoroacetic acid (0.1 mL, 1.3 mmol) and the resulting
mixture stirred at room temperature for 2 h. The mixture was evaporated
and the free base formed by eluting through an SCX cartridge with 2 M
ammonia in methanol and concentrating the filtrate. The residue was
purified by preparative thin layer chromatography (elution with 0.5%
ammonium hydroxide and 10% methanol in dichloromethane) and
afforded the title compound 7a (9.3 mg, 40%). LC/MS m/e 437.1 (M +
H)+. 1H NMR (500 MHz, CD3OD) δ 1.49 (d, J = 7.1 Hz, 3H), 1.49−
1.60 (m, 3H), 1.78−1.85 (m, 1H), 2.78 (dd, J = 13.0 and 6.8 Hz, 1H),
2.85 (dd, J = 13.0 and 7.3 Hz, 1H), 3.30−3.40 (m, 2H), 3.70 (q, J = 7.1
Hz, 1H), 4.47 (d, J = 8.0 Hz, 1H), 6.35 (s, 1H), 7.19 (d, J = 8.5 Hz, 2H),
7.27 (t, J = 6.9 Hz, 1H), 7.31−7.37 (m, 4H), 7.50 (d, J = 8.5 Hz, 2H). In a
similar manner, (2S,5R)-tert-butyl 2-(4-((S)-2-(2-((tert-
butoxycarbonyl)amino)thiazol-4-yl)propanamido)benzyl)-5-((R)-
hydroxy(phenyl)methyl)pyrrolidine-1-carboxylate (26 mg, 0.04 mmol)
was deprotected to afford 8a (6 mg, 34%). LC/MS m/e 437.1 (M + H)+.
1H NMR (500 MHz, CD3OD) δ 1.49 (d, J = 7.1 Hz, 3H), 1.49−1.60 (m,
Figure 3. Reduction in micturation pressure produced by increasing
doses of 19 and 29 in acetic acid induced bladder hyperactivity model
with unbound plasma levels. *, P < 0.05, and **, P < 0.01, versus vehicle
group. Each bar represents mean SEM.
vivo intrinsic clearance and unbound drug exposures compared
to our original clinical candidate, 3. Both compounds also
demonstrated a good selectivity profile in vitro against a panel of
receptors, ion channels and enzymes, which made them suitable
candidates for additional in vivo preclinical studies. For the first
time in this series, we were able to demonstrate an in vivo
pharmocodynamic effect. In rats, compounds 19 and 29 were
able to dose-dependently reduce bladder pressure following
intravenous dosing, albeit at unbound plasma concentrations
above their intrinsic EC50s. This model is able to afford a rank
order of potency which agrees with differences in in vitro activity.
Additional SAR analysis, as well as in vivo efficacy of related
compounds, will be disclosed in future articles.
EXPERIMENTAL SECTION
■
General. Unless otherwise stated, all reactions were carried out
under an atmosphere of dry argon or nitrogen in dried glassware.
Indicated reaction temperatures refer to those of the reaction bath, while
room temperature (rt) is noted as 25 °C. All solvents and reagents were
obtained from commercial sources and were used as received. Analytical
thin layer chromatography (TLC) was performed with Whatman TLC
plates (Partisil K6F, 60 Å, 250 μm). Visualization was accomplished by
irradiation under a 254 nm UV lamp. Strong cation exchange was
performed using Varian Bond Elut SCX columns. Chromatography on
silica gel was carried out using prepacked silica gel cartridges using the
indicated solvent system on either a Biotage Horizon flash
chromatography system or a Teledyne ISCO Combiflash Companion
flash chromatography system. If needed, products were purified by
reverse phase chromatography, which was performed using a Gilson
purification system employing a YMC Pro-Pac C18 column (5 μm, 20
mm × 100 mm). The mobile phase was a mixture of acetonitrile and
H2O, each containing 0.1% trifluoroacetic acid. If needed, products were
purified by preparative TLC on silica gel gf precoated 20 cm × 20 cm
3H), 1.78−1.85 (m, 1H), 2.78 (dd, J = 13.0 and 6.8 Hz, 1H), 2.85 (dd, J
= 13.0 and 7.3 Hz, 1H), 3.30−3.40 (m, 2H), 3.70 (q, J = 7.1 Hz, 1H),
4.47 (d, J = 8.0 Hz, 1H), 6.35 (s, 1H), 7.19 (d, J = 8.5 Hz, 2H), 7.27 (t, J =
6.9 Hz, 1H), 7.31−7.37 (m, 4H), 7.50 (d, J = 8.5 Hz, 2H).
(R)-2-(2-Aminothiazol-4-yl)-N-(4-(((2S,5R)-5-((R)-hydroxy-
(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)butanamide
(8a). Starting from aniline 50a and intermediate 33b, the procedures
summarized above provided 8a. LC/MS m/e 451.2 (M + H)+. 1H NMR
(500 MHz, DMSO-d6) δ 0.87 (t, J = 7.5 Hz, 3H), 1.27−1.34 (m, 2H),
1.38−1.44 (m, 1H), 1.57−1.64 (m, 1H), 1.74−1.82 (m, 1H), 1.84−1.93
1
1000 μm from Analech. H NMR spectra were recorded on a Varian
Unity Inova AS500 500 MHz spectrometer. The reported compounds
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(m, 1H), 2.58 (dd, J = 13.0 and 7.0 Hz, 1H), 2.67 (dd, J = 13.0 and 6.5
Hz, 1H), 3.12 (q, J = 7.5 Hz, 1H), 3.22 (quint, J = 7.0 Hz, 1H), 3.45 (dd,
J = 9.0 and 6.5 Hz, 1H), 4.26 (d, J = 7.5 Hz, 1H), 6.26 (s, 1H), 6.88 (br s,
2H), 7.13 (d, J = 8.5 Hz, 2H), 7.22 (t, J = 7.0 Hz, 1H), 7.27−7.33 (m,
4H), 7.52 (d, J = 8.5 Hz, 2H), 9.94 (s, 1H).
(R)-2-(2-Aminothiazol-4-yl)-N-(4-(((2S,5R)-5-((R)-hydroxy-
(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)pentanamide
(9a). Starting from aniline 50a and intermediate 33c, the procedures
summarized above provided 9a. LC/MS m/e 465.2 (M + H)+. 1H NMR
(500 MHz, DMSO-d6) δ 0.89 (t, J = 7.0 Hz, 3H), 1.22−1.36 (m, 4H),
1.38−1.46 (m, 1H), 1.56−1.65 (m, 1H), 1.68−1.76 (m, 1H), 1.82−1.90
(m, 1H), 2.58 (dd, J = 13.0 and 6.5 Hz, 1H), 2.68 (dd, J = 13.0 and 7.0
Hz, 1H), 3.14 (q, J = 7.0 Hz, 1H), 3.22 (quint, J = 7.0 Hz, 1H), 3.56 (t, J
= 8.5 and 6.0 Hz, 1H), 4.27 (d, J = 7.5 Hz, 1H), 6.25 (s, 1H), 6.88 (br s,
2H), 7.12 (d, J = 8.5 Hz, 2H), 7.22 (t, J = 7.0 Hz, 1H), 7.27−7.34 (m,
4H), 7.51 (d, J = 8.5 Hz, 2H), 9.95 (s, 1H).
(R)-2-(2-Aminothiazol-4-yl)-N-(4-(((2S,5R)-5-((R)-hydroxy-
(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)-3-methylbuta-
namide (10a). Starting from aniline 50a and intermediate 33d, the
procedures summarized above provided 10a. LC/MS m/e 465.3 (M +
H)+. 1H NMR (500 MHz, DMSO-d6) δ 0.80 (d, J = 4.5 Hz, 3H), 0.93 (d,
J = 4.5 Hz, 3H), 1.32−1.41 (m, 2H), 1.41−1.49 (m, 1H), 1.60−1.68 (m,
1H), 2.23−2.32 (m, 1H), 2.63 (dd, J = 13.0 and 7.0 Hz, 1H), 2.76 (dd, J
= 13.0 and 6.0 Hz, 1H), 3.18−3.25 (m, 2H), 3.28 (t, J = 6.5 Hz, 1H),
4.36 (d, J = 7.0 Hz, 1H), 6.32 (d, J = 2.0 Hz, 1H), 6.90 (br s, 2H), 7.12 (d,
J = 8.0 Hz, 2H), 7.24 (t, J = 6.5 Hz, 1H), 7.27−7.35 (m, 4H), 7.51 (d, J =
8.0 Hz, 2H), 9.94 (s, 1H).
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-cyclopenta[d]-
thiazole-4-carboxamide (11a). Starting from aniline 50a and
intermediate 33e, the procedures summarized above provided 11a.
LC/MS m/e 449.1 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.25−
1.33 (m, 2H), 1.36−1.44 (m, 1H), 1.55−1.63 (m, 1H), 2.45−2.53 (m,
1H), 2.53−2.60 (m, 2H), 2.62−2.70 (m, 2H), 2.74−2.81 (m, 1H), 3.10
(q, J = 7.0 Hz, 1H), 3.21 (quint, J = 7.0 Hz, 1H), 3.78 (dd, J = 8.0 and 6.0
Hz, 1H), 4.23 (d, J = 7.5 Hz, 1H), 6.86 (br s, 2H), 7.12 (d, J = 8.5 Hz,
2H), 7.21 (t, J = 7.0 Hz, 1H), 7.27−7.32 (m, 4H), 7.51 (d, J = 8.5 Hz,
2H), 10.00 (s, 1H).
(S)-2-Amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-cyclopenta[d]-
thiazole-4-carboxamide (11b). Starting from aniline 50a and
intermediate 33e, the procedures summarized above provided 11b.
LC/MS m/e 449.1 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.20−
1.35 (m, 2H), 1.35−1.45 (m, 1H), 1.51−1.64 (m, 1H), 2.43−2.60 (m,
3H), 2.60−2.72 (m, 2H), 2.74−2.85 (m, 1H), 3.08 (q, J = 7.0 Hz, 1H),
3.19 (quint, J = 7.0 Hz, 1H), 3.78 (m, 1H), 4.21 (d, J = 7.5 Hz, 1H), 6.86
(br s, 2H), 7.12 (d, J = 8.5 Hz, 2H), 7.21 (t, J = 7.0 Hz, 1H), 7.27−7.32
(m, 4H), 7.51 (d, J = 8.5 Hz, 2H), 10.00 (s, 1H).
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-4,5,6,7-tetrahydrobenzo[d]-
thiazole-4-carboxamide (12a). Starting from aniline 50a and
intermediate 33f, the procedures summarized above provided 12a.
LC/MS m/e 463.0 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.27−
1.37 (m, 2H), 1.39−1.48 (m, 1H), 1.57−1.69 (m, 2H), 1.87−1.98 (m,
3H), 2.44−2.54 (m, 2H), 2.59 (dd, J = 13.0 and 7.5 Hz, 1H), 2.71 (dd, J
= 13.0 and 6.5 Hz, 1H), 3.17 (t, J = 7.5 Hz, 1H), 3.25 (quint, J = 7.0 Hz,
1H), 3.55 (m, 1H), 4.29 (d, J = 7.0 Hz, 1H), 6.74 (br s, 2H), 7.12 (d, J =
8.5 Hz, 2H), 7.22 (t, J = 6.5 Hz, 1H), 7.28−7.32 (m, 4H), 7.52 (d, J = 8.5
Hz, 2H), 10.03 (s, 1H).
cyclopenta[d]thiazole-4-carboxamide (14). Starting from aniline
50b and intermediate 33e, the procedures summarized above provided
14. LC/MS m/e 483.3/485.3 (M + H)+. 1H NMR (500 MHz, DMSO-
d6) δ 1.19−1.28 (m, 1H), 1.30−1.38 (m, 1H), 1.38−1.46 (m, 1H),
1.54−1.61 (m, 1H), 2.44−2.58 (m, 3H), 2.61−2.69 (m, 2H), 2.73−2.80
(m, 1H), 3.11 (dd, J = 7.0 Hz, 1H), 3.19 (quint, J = 6.5 Hz, 1H), 3.77 (t, J
= 6.0 Hz, 1H), 4.29 (d, J = 6.5 Hz, 1H), 6.85 (br s, 2H), 7.10 (d, J = 8.5
Hz, 2H), 7.25−7.33 (m, 3H), 7.38 (s, 1H), 7.50 (d, J = 8.5 Hz, 2H), 9.99
(s, 1H).
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-(3-fluorophenyl)(hydroxy)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (15). Starting from aniline
50c and intermediate 33e, the procedures summarized above provided
1
15. LC/MS m/e 467.3 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.25−1.38 (m, 2H), 1.41−1.48 (m, 1H), 1.56−1.63 (m, 1H), 2.44−2.60
(m, 3H), 2.62−2.70 (m, 2H), 2.73−2.80 (m, 1H), 3.12 (q, J = 7.0 Hz,
1H), 3.21 (quint, J = 7.5 Hz, 1H), 3.77 (m, 1H), 4.31 (d, J = 6.5 Hz, 1H),
6.85 (br s, 2H), 7.03 (td, J = 8.5 and 2.0 Hz, 1H), 7.11 (d, J = 8.5 Hz,
2H), 7.12−7.18 (m, 2H), 7.30−7.35 (m, 1H), 7.51 (d, J = 8.5 Hz, 2H),
10.01 (s, 1H).
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-(4-fluorophenyl)(hydroxy)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (16). Starting from aniline
50d and intermediate 33e, the procedures summarized above provided
1
16. LC/MS m/e 467.3 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.21−1.33 (m, 2H), 1.34−1.42 (m, 1H), 1.55−1.62 (m, 1H), 2.44−2.59
(m, 3H), 2.62−2.69 (m, 2H), 2.73−2.80 (m, 1H), 3.10 (q, J = 7.0 Hz,
1H), 3.21 (quint, J = 7.0 Hz, 1H), 3.77 (m, 1H), 4.27 (d, J = 7.0 Hz, 1H),
6.85 (br s, 2H), 7.07−7.13 (m, 4H), 7.34 (dd, J = 8.5 and 6.0 Hz, 2H),
7.50 (d, J = 8.5 Hz, 2H), 10.00 (s, 1H).
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-hydroxy(pyridin-3-yl)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (17). Starting from aniline
50e and intermediate 33e, the procedures summarized above provided
1
15. LC/MS m/e 450.1 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.19−1.26 (m, 1H), 1.30−1.44 (m, 2H), 1.56−1.62 (m, 1H), 2.45−2.58
(m, 3H), 2.62−2.70 (m, 2H), 2.74−2.81 (m, 1H), 3.15−3.25 (m, 2H),
3.77 (m, 1H), 4.34 (d, J = 7.0 Hz, 1H), 6.86 (br s, 2H), 7.11 (d, J = 8.5
Hz, 2H), 7.32 (dd, J = 8.0 and 5.0 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.72
(dt, J = 8.0 and 2.0 Hz, 1H), 8.43 (dd, J = 5.0 and 1.5 Hz, 1H), 8.53 (d, J =
2.0 Hz, 1H), 10.00 (s, 1H).
(R)-N-(4-(((2S,5R)-5-((R)-Hydroxy(phenyl)methyl)pyrrolidin-
2-yl)methyl)phenyl)-5,6-dihydro-4H-cyclopenta[d]thiazole-4-
carboxamide (18). Starting from aniline 50a and intermediate 35, the
procedures summarized above provided 18. LC/MS m/e 434.1 (M +
H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.26−1.34 (m, 2H), 1.38−1.45
(m, 1H), 1.57−1.64 (m, 1H), 2.58 (dd, J = 13.5 and 7.0 Hz, 1H), 2.66−
2.76 (m, 3H), 2.85−2.92 (m, 1H), 2.97−3.04 (m, 1H), 3.12 (q, J = 7.0
Hz, 1H), 3.23 (quin, J = 7.0 Hz, 1H), 4.06 (t, J = 7.5 Hz, 1H), 4.26 (d, J =
7.0 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 7.21 (t, J = 6.5 Hz, 1H), 7.27−7.33
(m, 4H), 7.53 (d, J = 8.5 Hz, 2H), 8.92 (s, 1H), 10.20 (s, 1H).
(R)-N-(4-(((2S,5R)-5-((R)-Hydroxy(pyridin-3-yl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-cyclopenta[d]-
thiazole-4-carboxamide (19). Starting from aniline 50e and
intermediate 35, the procedures summarized above provided 19. LC/
MS m/e 435.2 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.35−1.54
(m, 3H), 1.64−1.71 (m, 1H), 2.66 (dd, J = 13.0 and 7.5 Hz, 1H), 2.73 (q,
J = 7.0 Hz, 2H), 2.81 (dd, J = 13.0 and 5.5 Hz, 1H), 2.86−2.92 (m, 1H),
2.97−3.04 (m, 1H), 4.06 (t, J = 7.0 Hz, 1H), 4.51 (d, J = 7.0 Hz, 1H),
7.15 (d, J = 8.0 Hz, 2H), 7.35 (dd, J = 7.5 and 5.0 Hz, 1H), 7.55 (d, J = 8.0
Hz, 2H), 7.75 (d, J = 7.5 Hz, 1H), 8.46 (d, J = 5.0 Hz, 1H), 8.56 (s, 1H),
8.92 (s, 1H), 10.24 (s, 1H).
(R)-N-(4-(((2S,5R)-5-((R)-Hydroxy(phenyl)methyl)pyrrolidin-
2-yl)methyl)phenyl)-2-methyl-5,6-dihydro-4H-cyclopenta[d]-
thiazole-4-carboxamide (20). Starting from aniline 50a and
intermediate 38a, the procedures summarized above provided 20.
LC/MS m/e 448.1 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.25−
1.33 (m, 2H), 1.36−1.44 (m, 1H), 1.56−1.63 (m, 1H), 2.57 (dd, J = 13.5
and 6.5 Hz, 1H), 2.60 (s, 3H), 2.63−2.69 (m, 3H), 2.80−2.87 (m, 1H),
2.91−2.99 (m, 1H), 3.10 (q, J = 7.0 Hz, 1H), 3.21 (quint, J = 7.0 Hz,
1H), 3.98 (t, J = 7.5 Hz, 1H), 4.23 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz,
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[d]thiazole-4-carboxamide (13a). Starting from aniline
50a and intermediate 33g, the procedures summarized above provided
1
13a. LC/MS m/e 477.0 (M + H)+. H NMR (500 MHz, CD3OD) δ
1.46−1.60 (m, 3H), 1.60−1.69 (m, 1H), 1.77−1.95 (m, 5H), 2.29−2.37
(m, 1H), 2.61 (dd, J = 13.5 and 6.0 Hz, 1H), 2.73−2.81 (m, 2H), 2.85
(dd, J = 13.5 and 7.0 Hz, 1H), 3.32−3.40 (m, 2H), 3.85 (dd, J = 6.5 and
2.5 Hz, 1H), 4.47 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 8.5 Hz, 2H), 7.26 (t, J =
7.0 Hz, 1H), 7.31−7.36 (m, 4H), 7.48 (d, J = 8.5 Hz, 2H).
(R)-2-Amino-N-(4-(((2S,5R)-5-((R)-(3-chlorophenyl)(hydroxy)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
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2H), 7.21 (t, J = 7.0 Hz, 1H), 7.27−7.32 (m, 4H), 7.53 (d, J = 8.5 Hz,
2H), 10.16 (s, 1H).
(27). Starting from aniline 50e and 1H-pyrazol-1-ylacetic acid 57a,
the procedures summarized above provided 27. LC/MS m/e 392.1 (M +
H)+. 1H NMR (500 MHz, CD3OD) δ 1.81−1.90 (m, 1H), 1.90−2.00
(m, 2H), 2.05−2.14 (m, 1H), 2.96 (dd, J = 13.7 and 8.7 Hz, 1H), 3.13
(dd, J = 13.7 and 6.5 Hz, 1H), 3.73−3.81 (m, 1H), 3.84−3.91 (m 1H),
5.00 (m, 3H), 6.55 (t, J = 2.2 Hz, 1H), 7.24 (d, J = 8.5 Hz, 2H), 7.55 (m,
3H), 7.71 (d, J = 2.2, 1H), 7.86 (dd, J = 8.0 and 5.4 Hz, 1H), 8.41 (d, J =
8.0 Hz, 1H), 8.72 (dd, J = 5.4 and 1.0 Hz, 1H), 8.85 (d, J = 1.9 Hz, 1H).
(S)-N-(4-(((2S,5R)-5-((R)-Hydroxy(pyridin-3-yl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-2-(1H-pyrazol-1-yl)-
propanamide, TFA Salt (28). Starting from aniline 50e and 2-(1H-
pyrazol-1-yl)propanoic acid 57b, the procedures summarized above
(R)-N-(4-(((2S,5R)-5-((R)-Hydroxy(pyridin-3-yl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-2-methyl-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (21). Starting from aniline
50e and intermediate 38a, the procedures summarized above provided
1
21. LC/MS m/e 449.1 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.17−1.16 (m, 1H), 1.29−1.36 (m, 1H), 1.36−1.44 (m, 1H), 1.55−1.62
(m, 1H), 2.55 (dd, J = 13.0 and 7.0 Hz, 1H), 2.60 (s, 3H), 2.61−2.69 (m,
3H), 2.80−2.87 (m, 1H), 2.91−2.99 (m, 1H), 3.14−3.24 (m, 2H), 3.98
(t, J = 7.5 Hz, 1H), 4.34 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 2H), 7.32
(dd, J = 7.5 and 5.0 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 7.5 Hz,
1H), 8.43 (dd, J = 5.0 and 1.5 Hz, 1H), 8.53 (d, J = 1.5 Hz, 1H), 10.16 (s,
1H).
(R)-2-Ethyl-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-cyclopenta[d]-
thiazole-4-carboxamide (22). Starting from aniline 50a and
intermediate 38b, the procedures summarized above provided 22.
LC/MS m/e 462.0 (M + H)+. 1H NMR (500 MHz, CD3OD) δ 1.33 (t, J
= 7.5 Hz, 3H), 1.48−1.61 (m, 3H), 1.79−1.87 (m, 1H), 2.70−2.95 (m,
5H), 2.98 (q, J = 7.5 Hz, 2H), 3.01−3.09 (m, 1H), 3.34−3.43 (m, 2H),
3.98 (m, 1H), 4.49 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 7.26 (m,
1H), 7.31−7.38 (m, 4H), 7.54 (d, J = 8.5 Hz, 2H).
1
provided 27. LC/MS m/e 406.2 (M + H)+. H NMR (500 MHz,
CD3OD) δ 1.62−1.78 (m, 3H), 1.67 (d, J = 7.0 Hz, 3H), 1.82−1.90 (m,
1H), 2.84 (dd, J = 13.5 and 9.4 Hz, 1H), 3.07 (dd, J = 13.5 and 5.5 Hz,
1H), 3.56−3.66 (m, 1H), 3.68−3.78 (m 1H), 4.83 (d, J = 8.2 Hz, 1H),
5.19 (q, J = 7.0 Hz, 1H), 6.28 (t, J = 2.1 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H),
7.44 (d, J = 1.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.57 (dd, J = 8.0 and 5.1
Hz, 1H), 7.85 (d, J = 2.1 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 8.50 (q, J =
8.2 Hz, 1H), 8.62 (q, J = 5.1 and 1.4 Hz, 1H) 8.70 (d, J = 1.8 Hz, 1H),
9.47 (t, J = 4.8 Hz, 1H), 10.33 (s, 1H).
(S)-N-(4-(((2S,5R)-5-((R)-Hydroxy(pyridin-3-yl)methyl)-
pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-pyrrolo[1,2-b]-
pyrazole-6-carboxamide, TFA Salt (29). Starting from aniline 50e
and intermediate 56, the procedures summarized above provided 27.
LC/MS m/e 418.4 (M + H)+. 1H NMR (500 MHz, CD3OD) δ 1.81−
1.95 (m, 3H), 2.06−2.13 (m, 1H), 2.70−2.77 (m, 1H), 2.89−3.08 (m,
4H), 3.14 (dd, J = 13.5 and 6.1 Hz, 1H), 3.78 (quint, J = 7.1 Hz, 1H),
3.85 (q, J = 8.2 Hz, 1H), 4.94 (d, J = 8.0 Hz, 1H), 5.01 (dd, J = 8.2 and 4.4
Hz, 1H), 6.06 (d, J = 1.6 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H), 7.53 (d, 1.6
Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.71 (dd, J = 8.0 and 5.3 Hz, 1H), 8.22
(d, J = 8.0 Hz, 1H), 8.65 (dd, J = 5.3 and 1.4 Hz, 1H), 8.76 (d, J = 1.9 Hz,
1H).
General Procedure for the Synthesis of Thiazole Acids 33a−g.
Step A: Synthesis of 31a−g. Bromine was added in a dropwise manner
to a solution of keto-esters (30a−g) in chloroform at 0 °C and the
resulting mixture stirred at 0 °C for 30 min. Air was blown through the
mixture for 1 h, and the mixture was dried over MgSO4 and concentrated
under reduced pressure. The residue was dissolved in ethanol and
thiourea added and the resulting mixture heated at reflux overnight. The
mixture was cooled and concentrated under reduced pressure and the
residue partitioned between CH2Cl2 and satd NaHCO3, and the organic
layer was dried over MgSO4 and concentrated under reduced pressure.
The crude material was purified by flash chromatography on a Biotage
Horizon system (silica gel, 100% hexanes to 100% EtOAc gradient) to
afford thiazoles 31a−g.
Step B: Synthesis of Boc-amino-thiazoles (32a−g). To a solution of
aminothiazoles 31a−g in CH2Cl2 was added Boc-anhydride, Et3N, and
DMAP and the resulting mixture stirred at room temperature overnight.
The reaction mixture was washed with 1N HCl and satd NaCl, dried
over MgSO4, and concentrated under reduced pressure. The crude
residue was purified by flash chromatography on a Biotage Horizon
system (silica gel, 100% hexanes to 40% EtOAc gradient) to afford the
products 32a−g.
(R)-2-Ethyl-N-(4-(((2S,5R)-5-((R)-hydroxy(pyridin-3-yl)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (23). Starting from aniline
50e and intermediate 38b, the procedures summarized above provided
1
23. LC/MS m/e 463.3 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.19−1.27 (m, 1H), 1.23 (t, J = 7.5 Hz, 3H), 1.30−1.44 (m, 2H), 1.55−
1.62 (m, 1H), 2.55 (dd, J = 13.0 and 7.0 Hz, 1H), 2.62−2.68 (m, 3H),
2.79−2.86 (m, 1H), 2.91 (q, J = 7.5 Hz, 2H), 2.92−2.99 (m, 1H), 3.16−
3.25 (m, 2H), 3.98 (t, J = 7.0 Hz, 1H), 4.35 (d, J = 7.0 Hz, 1H), 7.12 (d, J
= 8.5 Hz, 2H), 7.31 (dd, J = 7.5 and 5.0 Hz, 1H), 7.52 (d, J = 8.5 Hz, 2H),
7.71 (dd, J = 7.5 and 2.0 Hz, 1H), 8.43 (dd, J = 5.0 and 1.5 Hz, 1H), 8.52
(d, J = 2.0 Hz, 1H), 10.16 (s, 1H).
(R)-2-Cyclopropyl-N-(4-(((2S,5R)-5-((R)-hydroxy(pyridin-3-yl)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (24). Starting from aniline
50e and intermediate 38c, the procedures summarized above provided
1
24. LC/MS m/e 475.3 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
0.81−0.88 (m, 2H), 1.00−1.06 (m, 2H), 1.18−1.30 (m, 2H), 1.31−1.46
(m, 2H), 1.56−1.64 (m, 1H), 2.25−2.32 (m, 1H), 2.52−2.71 (m, 3H),
2.76−2.83 (m, 1H), 2.87−2.96 (m, 1H), 3.18−3.27 (m, 2H), 3.93 (dd, J
= 7.5 and 6.0 Hz, 1H), 4.38 (d, J = 7.0 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H),
7.32 (dd, J = 8.0 and 5.0 Hz, 1H), 7.52 (d, J = 8.5 Hz, 2H), 7.72 (dt, J =
8.0 and 2.0 Hz, 1H), 8.43 (dd, J = 5.0 and 1.5 Hz, 1H), 8.53 (d, J = 2.0
Hz, 1H), 10.13 (s, 1H).
(R)-2-(4-Fluorophenyl)-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)-
methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (25). Starting from aniline
50a and intermediate 38d, the procedures summarized above provided
1
25. LC/MS m/e 528.1 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.22−1.31 (m, 2H), 1.35−1.42 (m, 1H), 1.54−1.62 (m, 1H), 2.56 (dd, J
= 13.0 and 6.5 Hz, 1H), 2.63 (dd, J = 13.0 and 6.5 Hz, 1H), 2.67−2.77
(m, 2H), 2.90−2.99 (m, 1H), 3.03−3.10 (m, 1H), 3.19 (quint, J = 6.5
Hz, 1H), 4.09 (t, J = 7.5 Hz, 1H), 4.21 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 8.5
Hz, 2H), 7.21 (t, J = 7.0 Hz, 1H), 7.26−7.32 (m, 6H), 7.55 (d, J = 8.5 Hz,
2H), 7.90 (dd, J = 8.5 and 5.5 Hz, 2H), 10.24 (s, 1H).
Step C: Synthesis of Thiazole Acids (33a−g). To a solution of ethyl
esters 32a−g in a mixture of THF and methanol was added lithium
hydroxide solution and the resulting mixture stirred at room
temperature overnight. The organics solvents were removed by
evaporation and the aqueous residue diluted with more water and
extracted with EtOAc. The aqueous layer acidified by the addition of
conc HCl, extracted with EtOAc, and the organic layer dried over
MgSO4 and concentrated under reduced pressure. The residue was
triturated with acetonitrile, filtered, and dried to give the products 33a−
g.
(R)-2-(4-Fluorophenyl)-N-(4-(((2S,5R)-5-((R)-hydroxy(pyridin-
3-yl)methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-
cyclopenta[d]thiazole-4-carboxamide (26). Starting from aniline
50e and intermediate 38d, the procedures summarized above provided
1
25. LC/MS m/e 529.0 (M + H)+. H NMR (500 MHz, CD3OD) δ
1.45−1.62 (m, 3H), 1.77−1.85 (m, 1H), 2.75 (dd, J = 13.0 and 7.0 Hz,
1H), 2.79−2.92 (m, 3H), 2.96−3.04 (m, 1H), 3.10−3.18 (m, 1H),
3.30−3.38 (m, 2H), 4.12 (t, J = 7.0 Hz, 1H), 4.56 (d, J = 7.5 Hz, 1H),
7.18 (t, J = 9.0 and 8.5 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 7.42 (dd, J = 7.5
and 5.0 Hz, 1H), 7.56 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 8.0 Hz, 1H), 7.92
(dd, J = 8.5 and 5.0 Hz, 2H), 8.44 (d, J = 5.0 Hz, 1H), 8.55 (s, 1H).
N-(4-(((2S,5R)-5-((R)-Hydroxy(pyridin-3-yl)methyl)pyrrolidin-
2-yl)methyl)phenyl)-2-(1H-pyrazol-1-yl)acetamide, TFA Salt
2-(2-((tert-Butoxycarbonyl)amino)thiazol-4-yl)propanoic Acid
(33a). Starting from ethyl 2-methyl-3-oxo-butanoate 30a and following
1
the procedures above provided compound 33a. H NMR (500 MHz,
CDCl3) δ 1.56 (s, 9H), 1.57 (d, J = 7.4 Hz, 3H), 3.84 (q, J = 7.4 Hz, 1H),
6.69 (s, 1H), 11.05 (br s, 1H).
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2-(2-((tert-Butoxycarbonyl)amino)thiazol-4-yl)butanoic Acid
cooled evaporated in vacuo and the residue partitioned between satd
NaHCO3 and CH2Cl2. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. Purification by flash chromatog-
raphy on a Biotage Horizon system (silica gel, 100% hexanes to 20%
EtOAc in hexanes gradient) afforded thiazoles 37a−d.
Step B. To a solution of esters 37a−d in a mixture of THF and
methanol was added lithium hydroxide solution and the resulting
mixture stirred at room temperature for 5 h. Organic solvents removed
by evaporation and the remaining aqueous extracted with diethyl ether
(100 mL). The aqueous layer was acidified by the addition of 1N HCl
and extracted with CH2Cl2 (3×); combined CH2Cl2 extracts dried over
MgSO4 and concentrated in vacuo. The residue was triturated with
acetonitrile, filtered, and dried to give thiazole acids 38a−d.
2-Methyl-5,6-dihydro-4H-cyclopenta[d]thiazole-4-carboxylic
Acid (38a). Starting from thioacetamide (36a) following the procedures
above provided compound 38a. 1H NMR (500 MHz, CDCl3) δ 2.73 (s,
3H), 2.82 (m, 3H), 3.04 (m, 1H), 4.03 (m, 1H), 8.53 (br s, 1H).
2-Ethyl-5,6-dihydro-4H-cyclopenta[d]thiazole-4-carboxylic Acid
(38b). Starting from thiopropionamide (36b) following the procedures
above provided compound 38b. 1H NMR (500 MHz, CDCl3) δ 1.38 (t,
J = 7.5 Hz, 3H), 2.84 (m, 3H), 2.82 (m, 3H), 3.06 (m, 3H), 4.04 (dd, J =
8.5 and 7.0 Hz, 1H), 10.14 (br s, 1H).
2-Cyclopropyl-5,6-dihydro-4H-cyclopenta[d]thiazole-4-carbox-
ylic Acid (38c). Starting from cyclopropanethiocarboxamide (36c)
following the procedures above provided compound 38c. 1H NMR (500
MHz, CDCl3) δ 1.04 (m, 2H), 1.14 (m, 2H), 2.32 (m, 1H), 2.80 (m,
2H), 2.88 (m, 1H), 3.02 (m, 1H), 4.01 (t, J = 8.0 and 6.0 Hz, 1H), 7.83
(br s, 1H).
(33b). Starting from ethyl 2-ethyl-3-oxobutyrate 30b and following
1
the procedures above provided compound 33b. H NMR (500 MHz,
CDCl3) δ 0.98 (t, J = 7.5 Hz, 3H), 1.54 (s, 9H), 1.92 (m, 1H), 2.13 (m,
1H), 3.67 (t, J = 7.5 Hz, 1H), 6.74 (s, 1H), 11.58 (br s, 1H).
2-(2-((tert-Butoxycarbonyl)amino)thiazol-4-yl)pentanoic Acid
(33c). Starting from ethyl 2-propyl-3-oxobutanoate 30c and following
1
the procedures above provided compound 33c. H NMR (500 MHz,
CDCl3) δ 0.93 (t, J = 7.5 Hz, 3H), 1.36 (m, 2H), 1.55 (s, 9H), 1.86 (m,
1H), 2.06 (m, 1H), 3.73 (t, J = 7.5 Hz, 1H), 6.72 (s, 1H), 11.70 (br s,
1H).
2-(2-((tert-Butoxycarbonyl)amino)thiazol-4-yl)-3-methylbutanoic
Acid (33d). Starting from ethyl 2-isopropyl-3-oxobutanoate 30d and
1
following the procedures above provided compound 33d. H NMR
(500 MHz, CDCl3) δ 0.88 (d, J = 6.5 Hz, 3H), 1.10 (d, J = 6.5 Hz, 3H),
1.54 (s, 9H), 2.41 (m, 1H), 3.48 (d, J = 9.5 Hz, 1H), 6.81 (s, 1H), 10.70
(br s, 1H).
2-((tert-Butoxycarbonyl)amino)-5,6-dihydro-4H-cyclopenta[d]-
thiazole-4-carboxylic Acid (33e). Starting from ethyl 2-oxo-cyclo-
pentanecarboxylate 30e and following the procedures above provided
compound 33e. 1H NMR (500 MHz, CDCl3) δ 1.55 (s, 9H), 2.71 (m,
1H), 2.88 (m, 2H), 3.05 (m, 1H), 3.97 (m, 1H), 10.98 (br s, 1H).
2-((tert-Butoxycarbonyl)amino)-4,5,6,7-tetrahydrobenzo[d]-
thiazole-4-carboxylic Acid (33f). Starting from ethyl 2-oxo-cyclo-
hexanecarboxylate 30f and following the procedures above provided
compound 33f. 1H NMR (500 MHz, CDCl3) δ 1.55 (s, 9H), 1.87 (m,
1H), 2.00 (m, 1H), 2.06 (m, 1H), 2.25 (m, 1H), 2.65 (m, 1H), 2.72 (m,
1H), 3.70 (t, J = 5.5 Hz, 1H), 11.53 (br s, 1H).
2-((tert-Butoxycarbonyl)amino)-5,6,7,8-tetrahydro-4H-
cyclohepta[d]thiazole-4-carboxylic Acid (33g). Starting from ethyl 2-
oxo-cycloheptanecarboxylate 30g and following the procedures above
provided compound 33g. 1H NMR (500 MHz, CDCl3) δ 1.54 (s, 9H),
1.57 (m, 1H), 1.85 (m, 1H), 2.00 (m, 3H), 2.31 (m, 1H), 2.74 (m, 1H),
2.86 (m, 1H), 4.00 (t, J = 4.6 Hz, 1H), 11.23 (br s, 1H).
2-(4-Fluorophenyl)-5,6-dihydro-4H-cyclopenta[d]thiazole-4-car-
boxylic Acid (38d). Starting from 4-fluorothiobenzamide (36d)
1
following the procedures above provided compound 38d. H NMR
(500 MHz, DMSO-d6) δ 2.62 (m, 1H), 2.68 (m, 1H), 2.87 (m, 1H), 2.98
(m, 1H), 4.04 (dd, J = 8.5 and 5.5 Hz, 1H), 7.29 (t, J = 9.0 and 8.5 Hz,
2H), 7.90 (dd, J = 9.0 and 5.5 Hz, 2H).
Ethyl 5,6-Dihydro-4H-cyclopenta[d]thiazole-4-carboxylate
(34). To a solution of amino-thiazole 31e (9g, 42.4 mmol) in anhydrous
DMF (100 mL) warmed at 50 °C was added dropwise a solution of tert-
butyl nitrite (5.5 mL, 46.6 mmol). After addition was complete, the
mixture was heated at 50 °C and continued for 30 min. The mixture was
cooled and partitioned between EtOAc (300 mL) and water (500 mL);
the aqueous layer was separated and washed with water (2 × 400 mL)
and satd NaCl (100 mL), dried over MgSO4, and concentrated in vacuo.
Purification flash chromatography on a Biotage Horizon system (silica
gel, 100% hexanes to 50% EtOAc in hexanes gradient) gave the desired
product (1.8 g, 21% yield). 1H NMR (500 MHz, CDCl3) δ 1.31 (t, J =
7.0 Hz, 3H), 2.87 (m, 2H), 2.94 (m, 1H), 3.10 (m, 1H), 4.05 (t, J = 7.0
Hz, 1H), 4.23 (q, J = 7.0 Hz, 2H), 8.69 (s, 1H).
5,6-Dihydro-4H-cyclopenta[d]thiazole-4-carboxylic Acid
(35). To a solution of thiazole ester 34 (1.8g, 9.13 mmol) in a mixture
of THF (30 mL) and methanol (10 mL) was added lithium hydroxide
(10 mL of a 1 M aqueous solution, 10 mmol) and the resulting mixture
stirred at room temperature for 2 h. The organics were removed by
evaporation in vacuo and the aqueous residue diluted with water (25
mL). The mixture was extracted with EtOAc (×2) and the organic layes
discarded. The remaining aqueous was acidified to pH ∼ 4 with 1N HCl
and extracted with EtOAc (×3). The combined EtOAc layers were
washed with satd NaCl, dried over MgSO4, and concentrated in vacuo.
The crude residue was triturated with acetonitrile, filtered, and dried to
give 900 mg (58%) as an off white solid. 1H NMR (500 MHz, DMSO-
d6) δ 2.64 (m, 1H), 2.74 (m, 1H), 2.86 (m, 1H), 2.96 (m, 1H), 3.86 (t, J
= 7.0 Hz, 1H), 8.92 (s, 1H), 12.44 (s, 1H).
(4S)-3-Hex-5-ynoyl-4-benzyl-1,3-oxazolidin-2-one (40). To a
stirred solution of 5-hexynoic acid (69 g, 0.62 mol) and triethylamine
(214 mL, 1.54 mol) in THF (1.0 L) at −25 °C was added neat 2,2-
dimethylpropanoyl chloride (83 mL, 0.68 mol) dropwise over 20 min.
The resulting mixture was stirred at −25 °C for 2 h, then solid lithium
chloride (28.7 g, 0.677 mol) was added followed by (S)-4-benzyl-1,3-
oxazolidin-2-one (101 g, 0.615 mol). The reaction mixture then allowed
to warm to room temperature, stirred for an additional 12 h, then
evaporated in vacuo to remove all volatiles. The off-white residue was
diluted with water (400 mL) and extracted with ethyl acetate (3 × 300
mL). The combined organic layers were washed with brine, dried over
magnesium sulfate, filtered, and evaporated in vacuo to afford an off-
white solid. The solid was recrystallized from an ethyl acetate and
hexanes mixture to afford the desired compound as a white solid (135 g,
85%). LC/MS (EI) m/e: 272.3 (M + H)+. 1H NMR (500 MHz, CDCl3)
δ 1.89−1.95 (m, 2H), 1.99 (t, J = 2.8 Hz, 1H), 2.32 (td, J = 7.1 and 2.8
Hz, 2H), 2.77 (dd, J = 13.5 and 9.6 Hz, 1H), 3.01−3.13 (m, 2H), 3.28
(dd, J = 13.2 and 3.2 Hz, 1H), 4.16−4.22 (m, 2H), 4.65−4.69 (m, 1H),
7.20 (d, J = 7.1 Hz, 2H), 7.27 (m, 1H), 7.33 (t, J = 7.6 and 6.8 Hz, 2H).
General Procedure for the Synthesis of Anilines 50a−d. Step
A: Synthesis of 41a−e. To a stirred solution of (4S)-3-hex-5-ynoyl-4-
benzyl-1,3-oxazolidin-2-one 40 in ethyl acetate at room temperature was
added sequentially triethylamine, solid magnesium(II) chloride,
benzaldehyde, and chlorotrimethylsilane. After complete addition, the
resulting mixture was stirred for 3 days. The reaction mixture was filtered
through silica gel, which was subsequently washed with ethyl acetate.
The filtrate was evaporated in vacuo, and the residue was suspended in a
a mixture of ethyl acetate and methanol and stirred for 5 h, during which
time all solids dissolved. The reaction mixture was concentrated in vacuo
and the residue purified by flash column chromatography on a Biotage
Horizon system (silica gel, 10% EtOAc in hexanes to 25% EtOAc in
hexanes gradient) to afford the pure addition products 41a−e as yellow/
orange oils.
General Procedure for the Synthesis of Thiazole Acids 38a−d.
Step A: Synthesis of 37a−d. To a solution of ethyl 2-oxocyclopenta-
necarboxylate in anhydrous chloroform cooled at 0 °C was added
bromine dropwise over 15 min. After complete addition the mixture was
allowed to warm to room temperature and stirred overnight. Air was
bubbled through mixture for 1 h and then washed with satd NaHCO3
and satd NaCl, dried over MgSO4, and concentrated under reduced
pressure. The residue was taken up in ethanol (100 mL) and
thioacetamide added and the resulting mixture stirred at room
temperature for 1 h then heated at reflux overnight. The mixture was
Step B: General Procedure for tert-Butyl-dimethylsilyl (TBS)
Protection. To a solution of alcohols 41a−e in CH2Cl2 cooled at 0
°C was added 2,6-lutidine followed by tert-butyldimethylsilyl trifluor-
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omethanesulfonate and the resulting mixture allowed to warm to room
temperature and stirred overnight, diluted with satd NH4Cl, and washed
with 1N HCl, water and satd NaCl, dried (MgSO4), filtered, and
evaporated. The residue was purified by column chromatography on a
Biotage Horizon system (silica gel, 5% EtOAc in hexanes to 10% EtOAc
in hexanes gradient) to give protected alcohols 42a−e as oils.
Step C: General Procedure for Oxazolidinone Cleavage. To a
solution of oxazolidinones 42a−e in a mixture of THF and water cooled
at 0 °C was added slowly 35% H2O2, keeping the internal temperature
below 5 °C, followed by slow addition of an aqueous solution of LiOH.
The resulting mixture was stirred overnight warming up to 15 °C in the
process. The reaction was quenched by the addition of Na2SO3. The
THF was removed by evaporation and the remaining aqueous cooled (a
precipitate formed on cooling) and acidified to pH = 5 with 1N HCl,
extracted with EtOAc (3×), combined EtOAc layers washed with satd
NaCl, dried (MgSO4), and evaporated in vacuo. A solid formed in the
residue on standing which was diluted with hexanes and filtered. The
filtrate evaporated and residue purified by column chromatography on a
Biotage Horizon system (silica gel, 10% EtOAc in hexanes + 3% AcOH)
to afford acids 43a−e as a colorless oils.
Step D: General Procedure for Curtius Rearrangement. To a
solution of acid 43a−e in toluene was added triethylamine then diphenyl
azidophosphate, and the resulting solution was stirred at ambient
temperature for 5 h. Neat (4-methoxyphenyl)methanol was then added,
and the reaction mixture was slowly heated to 80 °C for 1 h, during
which time a vigorous evolution of nitrogen gas was observed. The
reaction was then heated to 110 °C for 12 h, cooled to room
temperature, and diluted with a saturated aqueous sodium bicarbonate
solution. The phases were separated, and the aqueous layer was
extracted with ethyl acetate (2 × 250 mL). The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered, and
evaporated in vacuo. The crude residue was purified by flash column
chromatography on a Biotage Horizon system (silica gel, 100% hexanes
to 25% EtOAc in hexanes gradient) to afford the desired compounds
44a−e as colorless oils.
Step E: General Procedure for Sonagashira Coupling. To a solution
of acetylenes 44a−e and 1-bromo-4-nitrobenzene in DMF was added
triethylamine, tetrakis(triphenylphosphine)palladium(0), and copper
iodide. The mixture was degassed, flushed with nitrogen three times,
then heated to 80 °C for 2 h. The reaction mixture was cooled, diluted
with water, and extracted with ethyl acetate (3×). The combined organic
layers were washed with brine, dried over magnesium sulfate, filtered,
and evaporated in vacuo. The residue was purified by flash column
chromatography on a Biotage Horizon system (silica gel, 100% hexanes
to 25% EtOAc in hexanes, gradient) to afford the desired coupled
products 45a−e as a orange oils.
The combined organic phases were dried over MgSO4 and evaporated in
vacuo. The residue was purified by flash column chromatography on a
Biotage Horizon system (silica gel, 100 hexanes to 20% EtOAc in
hexanes gradient) to afford the desired pyrrolidines 47a−e as clear oils.
Step H: General Procedure for tert-Butyloxycarbonyl (Boc)-
Protection. To a solution of pyrrolidines 47a−e in dichloromethane
was added di-tert-butyl dicarbonate and N,N-diisopropylethylamine and
the resulting mixture stirred at room temperature overnight. The
reaction was then washed sequentially with a 1.0 M aqueous citric acid
solution, water, a saturated aqueous sodium bicarbonate solution, and
brine. The organic phase was dried over MgSO4 and evaporated in
vacuo. The residue was purified by flash column chromatography on a
Biotage Horizon system (silica gel, 100% hexanes to 15% EtOAc in
hexanes gradient) to afford the desired protected pyrrolidines 48a−e as
white foams.
Step I: General Procedure for Nitro Group Reduction. To a nitrogen
flushed solution of nitro compounds 44a,c−d in methanol was added
10% palladium on carbon and the resulting mixture stirred under a
balloon of hydrogen gas for 12 h. The reaction mixture was filtered
through a plug of Celite, and the plug was washed with methanol. The
combined filtrates were concentrated in vacuo to afford the desired
anilines 49a,c−d as a white foam.
Step J: General Procedure for tert-Butyl(dimethyl)silyl (TBS)
Deprotection. To a solution of tert-butyl(dimethyl)silyl protected
alcohols 49a−d in tetrahydrofuran was added a 1.0 M solution of
tetrabutylammonium fluoride in tetrahydrofuran and the resulting
mixture heated at 55 °C for 2 h. After cooling to room temperature, the
mixture was evaporated in vacuo and partitioned between ethyl acetate
and water. The aqueous phase was extracted with ethyl acetate (2×), and
the combined organic layers were washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by flash column
chromatography on a Biotage Horizon system (silica gel, 100% hexanes
to 75% EtOAc in hexanes gradient) to afford the desired alcohols 50a−d
as white foams.
(2S,5R)-tert-Butyl 2-(4-Aminobenzyl)-5-((R)-hydroxy(phenyl)-
methyl)pyrrolidine-1-carboxylate (50a). Starting from benzaldehyde,
the procedures summarized above provided compound 50a. LC/MS
(EI) m/e: 383.2 (M + H)+. 1H NMR (500 MHz, CDCl3) δ 1.49−1.48
(m, 2H), 1.55 (s, 9H), 1.70−1.63 (m, 2H), 2.51 (dd, J = 13.3 and 8.9 Hz,
1H), 3.01 (br d, J = 2.1 Hz, 1H), 3.62 (br s, 2H), 4.13−4.06 (m, 2H),
4.43 (br s, 1H), 6.19 (br s, 1 H), 6.67 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.2
Hz, 2H), 7.36−7.29 (m, 5H).
(2S,5R)-tert-Butyl 2-(4-Aminobenzyl)-5-((R)-(3-chlorophenyl)-
(hydroxy)methyl)pyrrolidine-1-carboxylate (50b). Starting from 3-
chlorobenzaldehyde, the procedures summarized above provided
1
compound 50b. H NMR (500 MHz, CDCl3) δ 1.46 (m, 2H), 1.55
Step F: General Procedure for Ketone Formation. To a solution of
acetylenes 45a−e in DMF was added pyrrolidine, and the resulting
mixture was heated to 80 °C for 2 h. The reaction mixture was cooled to
room temperature, diluted with a 10% aqueous acetic acid solution, and
stirred for 3 h. The reaction mixture was then added to water and
extracted with ethyl acetate (3×). The combined organic layers were
washed with water and brine, dried over MgSO4, filtered, and evaporated
in vacuo. The residue was purified by flash column chromatography on a
Biotage Horizon system (silica gel, 100% hexanes to 25% EtOAc in
hexanes, gradient) to afford the desired ketones 46a−e as yellow oils.
Step G: General Procedure for Pyrrolidine Formation. To a solution
of ketones 46a−e in dichloromethane was added trifluoroacetic acid and
the resulting mixture stirred at room temperature for 1.5 h. The resulting
mixture was evaporated in vacuo and the residue partitioned between
dichloromethane and a saturated aqueous sodium bicarbonate solution.
The phases were separated and the aqueous phase extracted with
dichloromethane (2×). The combined organic phases were dried over
MgSO4 and evaporated in vacuo. The residue was then dissolved in
dichloromethane cooled to 0 °C, and solid sodium triacetoxyborohy-
dride was then added. The resulting mixture was allowed to warm to
room temperature with stirring for 12 h. The reaction mixture was then
carefully quenched with a saturated aqueous sodium bicarbonate
solution, and the biphasic solution was stirred for 6 h. The phases were
separated, and the aqueous phase extracted with dichloromethane (2×).
(s, 9H), 1.67 (m, 2H), 2.51 (dd, J = 13.5 and 9.0 Hz, 1H), 2.96 (br d, J =
10.0 Hz, 1H), 3.64 (br s, 2H), 4.06 (m, 2H), 4.39 (m, 1H), 6.33 (br s, 1
H), 6.67 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 7.22−7.30 (m,
3H), 7.37 (s, 1H).
(2S,5R)-tert-Butyl 2-(4-Aminobenzyl)-5-((R)-(3-fluorophenyl)-
(hydroxy)methyl)pyrrolidine-1-carboxylate (50c). Starting from 3-
fluorobenzaldehyde, the procedures summarized above provided
1
compound 50c. LC/MS (EI) m/e: 401 (M + H)+. H NMR (500
MHz, CD3OD) δ 1.24−1.54 (m, 11H), 1.60−1.81 (m, 2H), 2.62 (m,
1H), 3.86 (s, 1H), 4.03 (d, J = 6.0 Hz, 2H), 4.84 (m, 1H), 6.57 (d, J = 7.8
Hz, 2H), 6.74 (d, J = 7.3 Hz, 2H), 6.96 (t, J = 8.4 Hz, 2H), 7.03 (d, J =
10.0 Hz, 1H) 7.10 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H).
(2S,5R)-tert-Butyl 2-(4-Aminobenzyl)-5-((R)-(4-fluorophenyl)-
(hydroxy)methyl)pyrrolidine-1-carboxylate (50d). Starting from 4-
fluorobenzaldehyde, the procedures summarized above provided
1
compound 50d. LC/MS (EI) m/e: 401 (M + H)+. H NMR (500
MHz, CD3OD) δ 1.49−1.55 (m, 11H), 1.57 (m, 2H), 1.98 (m, 1H),
2.70 (s, 1H), 3.93 (s, 1H), 4.07 (s, 1H), 4.80 (m, 1H), 6.63 (d, J = 7.0 Hz,
2H), 6.81 (d, J = 7.0 Hz, 2H), 7.05 (t, J = 8.6 Hz, 2H), 7.35 (t, J = 8.6 Hz,
2H).
(2S,5R)-tert-Butyl 2-(4-Aminobenzyl)-5-((R)-hydroxy(pyridin-3-yl)-
methyl)pyrrolidine-1-carboxylate (50e). To a 500 mL Parr bottle was
added 10% palladium on carbon (1.8g, 1.7 mmol) followed by a solution
of tert-butyl (2R,5S)-2-[(R)-{[tert-butyl(dimethyl)silyl]oxy}(6-chloro-
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pyridin-3-yl)methyl]-5-(4-nitrobenzyl)pyrrolidine-1-carboxylate 48e
(19.2g, 34.2 mmol) and potassium acetate (5.03 g, 51.3 mmol) in
ethanol (150 mL). The resulting mixture was agitated under a 50 psi
atmosphere of hydrogen gas for 12 h. The reaction mixture was then
filtered through a plug of Celite, the plug was washed with ethanol (200
mL), and the combined filtrates concentrated in vacuo. The residue was
dissolved in tetrahydrofuran (100 mL), and a 1.0 M solution of
tetrabutylammonium fluoride in tetrahydrofuran (67 mL, 67 mmol) was
then added. The resulting mixture was heated at 55 °C for 2 h. After
cooling to room temperature, the mixture was evaporated in vacuo and
partitioned between ethyl acetate and water. The aqueous phase was
extracted with ethyl acetate (2 × 150 mL), and the combined organic
layers were washed with brine (100 mL), dried over MgSO4, and
evaporated in vacuo. The residue was purified by flash column
chromatography on a Biotage Horizon system (silica gel, 100% hexanes
to 100% EtOAc in hexanes gradient) to afford the desired compound as
white foam (11 g, 84%). LC/MS (EI) m/e: 384.2 (M + H)+. 1H NMR
(500 MHz, CDCl3) δ 1.40−1.47 (m, 2H), 1.55 (s, 9H), 1.66−1.72 (m,
2H), 2.55 (dd, J = 13.5, 8.7, 1H), 2.95 (m, 1H), 2.95 (m, 1H), 3.64 (br s,
2H), 4.06−4.13 (m, 2H), 4.43 (br s, 1H), 6.45 (br s, 1H), 6.68 (d, J = 8.3
Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 7.30 (m, 2H), 8.53 (br d, J = 3.7 Hz,
1H), 8.54 (m, 2H).
7-(Trimethylsilyl)-5-oxohept-6-ynoic Acid (52). To a stirred
solution of glutamic anhydride (32g, 280 mmol) in CH2Cl2 (1500 mL)
at 0 °C was added bis(trimethylsilyl)acetylene (65.8 mL, 293 mmol)
followed by slow addition of solid aluminum chloride (39.3g, 294
mmol) over 25 min. The resulting suspension was stirred for 3 h at 0 °C
and then allowed to gradually warm to room temperature overnight.
After cooling to 0 °C, the reaction was quenched slowly with 1 N aq HCl
(500 mL) so as to keep the internal temperature below 10 °C. The layers
were then separated, and the aqueous phase was extracted with DCM
(2×). The combined organics were washed with 1 N HCl, water, and
then brine and dried over MgSO4 and evaporated in vacuo. The residue
was purified by flash column chromatography on a Biotage Horizon
system (silica gel, eluent 50% EtOAc in hexanes) to afford the title
compound (43.4g, 72.9% yield) as a yellow/brown oil. 1H NMR (500
MHz, CDCl3) δ 0.24 (s, 9H), 1.97 (m, 2H), 2.42 (t, J = 7.3 Hz, 2H), 2.66
(t, J = 7.3 Hz, 2H).
to 4% EtOAc in hexanes gradient) to afford the desired compound as a
yellow oil (56 g, 68%). 1H NMR (500 MHz, CDCl3) δ 1.63 (s, 9H), 2.02
(quintet, J = 7.5 Hz, 2H), 2.42 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz,
2H), 5.11 (s, 2H), 6.19 (d, J = 2.5 Hz, 1H), 7.34 (m, 5H), 7.95 (d, J = 2.5
Hz, 1H).
Benzyl 5,6-Dihydro-4H-pyrrolo[1,2-b]pyrazole-6-carboxy-
late (55). To a stirred solution of 54 (27.5g, 80.0 mmol) in THF
(250 mL) cooled to −78 °C was added a 1.0 M solution of sodium
bis(trimethylsilyl)amide in THF (88 mL, 88 mmol). The resulting dark-
yellow solution was stirred for 1 h at −78 °C and then
chlorotrimethylsilane (12 mL, 96 mmol) was added dropwise over 10
min, and the resulting mixture was stirred for 30 min. Solid N-
bromosuccinimide (16g, 88 mmol) was then added in one portion, and
the resulting mixture was stirred for 3 h at −78 °C followed by gradual
warming to 0 °C over 1 h. The reaction was quenched with a saturated
aqueous ammonium chloride solution and the aqueous phase extracted
with ethyl acetate (3 × 200 mL). The combined organics were washed
with brine, dried over MgSO4, and evaporated to dryness in vacuo. To a
stirred solution of the crude residue of in dichloromethane (50 mL) was
added trifluoroacetic acid (50 mL), and the resulting mixture was stirred
for 2 h at ambient temperature. All volatiles were then evaporated in
vacuo, and the residue was diluted with toluene (50 mL) and evaporated
again in vacuo to remove all residual trifluoroacetic acid. The crude
material was then dissolved in acetone (125 mL), and solid potassium
carbonate (14.0 g, 100 mmol) was slowly added over 15 min, followed
by sodium iodide (1.2 g, 8.0 mmol), and the resulting mixture was
heated at reflux for 16 h. The mixture was cooled to room temperature
and evaporated to dryness in vacuo. The residue was diluted with a
saturated aqueous ammonium chloride solution (100 mL) and then
extracted with EtOAc (3 × 100 mL). The combined organics layers were
washed with brine, dried over MgSO4, and evaporated in vacuo. The
residue was purified by flash column chromatography on a Biotage
Horizon system (silica gel, 10% EtOAc in hexanes to 80% EtOAc in
hexanes gradient) to afford the desired compound as clear gum (6.2 g,
64%). 1H NMR (500 MHz, CDCl3) δ 2.63−2.70 (m, 1H), 2.85−3.01
(m, 3H), 4.99 (d, J = 9.4 Hz, 1H), 5.21 (s, 2H), 5.99 (d, J = 1.6 Hz, 1H),
7.31−7.38 (m, 5H), 7.58 (d, J = 1.6 Hz, 1H).
5,6-Dihydro-4H-pyrrolo[1,2-b]pyrazole-6-carboxylic Acid
(56). A 100 mL round-bottom flask under an atmosphere of nitrogen
and charged with 10 wt % palladium on activated carbon (6 mg, 0.560
mmol), which was subsequently wet with ethanol (10 mL). A solution of
55 (6.0 g, 0.025 mol) in ethanol (40 mL) was then added and the
mixture placed under a balloon of hydrogen for 4 h. The reaction was
filtered through a pad of Celite, the pad was washed with ethanol (20
mL), and the filtrate evaporated in vacuo to afford the title compound as
a colorless solid (3.7 g, 100%). LC-MS: m/z (ES) 153 (MH)+. 1H NMR
(500 MHz, DMSO-d6): δ 2.55 (m, 1H), 2.88 (m, 3H), 4.87 (dd, J = 8.9,
3.7 Hz, 1H), 5.98 (s, 1H), 7.44 (s, 1H), 11.90 (br s, 1H).
(5Z)-5-[(tert-Butoxycarbonyl)hydrazono]-7-(trimethylsilyl)-
hept-6-ynoic Acid (53). To a stirred solution of 7-(trimethylsilyl)-5-
oxohept-6-ynoic acid 52 (54.0g, 0.254 mol) in isopropyl alcohol (750
mL) was added tert-butyl carbazate (33.6g, 0.254 mol). The reaction
mixture was stirred for 4 h at ambient temperature then evaporated in
vacuo to remove all volatiles. This afforded the title compound as a
yellow gum which was used without further purification (77g, 93%).
LC/MS (EI) m/e: 327.2 (M + H)+. 1H NMR (500 MHz, CDCl3) δ 0.27
(s, 9H), 1.51 (s, 9H), 1.95 (m, 2H), 2.40 (t, J = 7.5 Hz, 2H), 2.46 (t, J =
7.5 Hz, 2H), 8.42 (br s, 1H).
β3 cAMP Assay. CHO cells, stably transfected with the cloned β3-
adrenergic receptor were harvested after 3 days of subculturing.
Harvesting of cells is done with Enzyme-Free Dissociation Media
(Specialty Media). Cells are then counted and resuspended in assay
buffer (Hank’s Balanced Salt Solution supplemented with 5 mM
HEPES, 0.1%BSA) containing a phosphodiesterase inhibitor (IBMX,
0.6 mM). The reaction was initiated by mixing 6000 cells in with 6 μL of
Alexa Fluor labeled cAMP antibody (LANCE kit), which was then
added to an assay well containing 12 μL of compound (diluted in assay
buffer to 2× final concentration). The reaction proceeded for 30 min at
room temperature and was terminated by the addition of 24 μL of
detection buffer (LANCE kit). The assay plate was then incubated for 1
h at room temperature and time-resolved fluorescence measured on a
Perkin-Elmer Envision reader. The unknown cAMP level was
determined by comparing fluorescence levels to a cAMP standard
curve. The nonselective, full agonist β-adrenergic ligand isoproterenol
was used to determine maximal stimulation. The human β3-adrenergic
receptor selective ligand (S)-N-[4-[2[[2-hydroxy-3-(4-
hydroxyphenoxy)propyl]amino]ethyl]-phenyl]-4-iodobenzenesulfona-
mide was used as a control. Isoproterenol was titrated at a final
concentration in the assay of 10−10 to 10−5 M, and the selective ligand
(S)-N-[4-[2[[2-hydroxy-3-(4-hydroxyphenoxy)propyl]amino]ethyl]-
tert-Butyl 3-[4-(Benzyloxy)-4-oxobutyl]-1H-pyrazole-1-car-
boxylate (54). To a stirred solution of 53 (77.0g, 0.236Mol) in THF
(500 mL) was added a 1.0 M solution of tetrabutylammonium fluoride
in THF (350 mL, 0.35 mol) over 30 min, and the resulting mixture was
stirred at ambient temperature for 48 h. The reaction mixture was
evaporated to dryness in vacuo, and the residue was diluted with a 5%
aqueous acetic acid solution (1 L). The aqueous phase was extracted
with ethyl acetate (3 × 350 mL), and the combined organic layers were
washed with water (2 × 100 mL) and brine (150 mL). The organic
layers were dried over MgSO4 and evaporated to dryness in vacuo. The
crude residue was purified by flash column chromatography on a Biotage
Horizon system (silica gel, eluent 35% EtOAc in hexanes containing 3%
acetic acid) to afford 4-[1-(tert-butoxycarbonyl)-1H-pyrazol-3-yl]-
butanoic acid as (60 g, quantitative yield) as a yellow oil.
To a solution of the above acid (60.0 g, 0.236 mol) in DMF (200 mL)
was added potassium carbonate (48.9g, 0.354 mol) followed by
dropwise addition of benzyl bromide (40 mL, 0.30 mol) over 30 min.
The resulting mixture was stirred for 24 h, quenched with water (1 L),
and extracted with ethyl acetate (3 × 300 mL). The combined organic
layers were washed with brine (150 mL), dried over MgSO4, and
evaporated to dryness in vacuo. The residue was purified by flash column
chromatography on a Biotage Horizon system (silica gel, 100% hexanes
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phenyl]-4-iodobenzenesulfonamide was titrated at the β3 receptor at
concentration of 10−10 to 10−5 M. Unknown ligands were titrated at the
β3-adrenergic receptor at a final concentration in the assay of 10−10 to
10−5 M to determine the EC50. The EC50 is defined as the concentration
of compound that gives 50% activation of its own maximum. Data were
analyzed using Microsoft Excel and Graphpad Prism, v4.
ASSOCIATED CONTENT
■
S
* Supporting Information
Conditions for the separation of the isomers of 38a and X-ray
data collection on the isomer leading to more potent B3-AR
agonists, and procedures and characterization for compounds
31a−g, 32a−g, 33a, 37a−d, 38a, 41a−e, 42a−e, 43a−e, 44a−e,
45a−e, 46a−e, 47a−e, 48a−e, 49a−d, 50a. This material is
available free of charge via the Internet at http://pubs.acs.org.
β1 and β2 Binding Assays. CHO cells recombinantly expressing β1
and β2 receptors were grown for 3−4 days post splitting; the attached
cells were washed with PBS and then lysed in 1 mM Tris, pH 7.2 for 10
min on ice. The flasks are scraped to remove the cells then homogenized
using a Teflon/glass homogenizer. Membranes were collected by
centrifuging at 38000g for 15 min at 4 °C. The pelleted membranes were
resuspended in TME buffer (50 mM Tris, pH 7.4, 5 mM MgCl2, 2 mM
EDTA) at a concentration of 1 mg protein/mL. The binding assay was
performed by incubating together membranes (2−5 μg of protein), the
radiolabeled tracer 125I-cyanopindolol (125I-CYP, 45 pM), 200 μg of
WGA-PVT SPA beads (GE Healthcare), and the test compounds at final
concentrations from 10−10 to 10−5 M in a final volume of 200 μL of TME
buffer containing 0.1% BSA. The assay plate is incubated for 1 h with
shaking at room temperature and then placed in a Perkin-Elmer Trilux
scintillation counter. The plates were allowed to rest in the Trilux
counter for approximately 10 h in the dark prior to counting. Data were
analyzed using a standard four-parameter nonlinear regression analysis
using Graphpad Prism software, v4. The IC50 is defined as the
concentration of the title compound capable of inhibiting 50% of the
binding of the radiolabeled tracer (125I-CYP).
Serotonin Transporter Assay. HEK293 cells stably expressing the
human serotonin transporter (SERT) were plated at a density of 20000
cells/well in a 384-well plate (BD Biocoat no. 35663) and incubated
overnight at 37 °C. The following day, growth media was removed and
replaced with assay buffer (HBSS containing 20 mM HEPES and 0.1%
BSA; Sigma). Compounds (serially diluted in DMSO at 100× final
concentration) were added to appropriate assay wells and the plate
incubated for 30 min at 37 °C. Fluorescent transporter substrate was
prepared following the manufacturer’s instructions (Neurotransmitter
Uptake Kit, no. R8173, Molecular Probes) and added to the assay plate.
The plate was then incubated for an additional 30 min at 37 °C. Uptake
of the fluorescent dye was then determined by measuring fluorescence in
a bottom read mode fluorescence plate reader (440 nm excitation, 520
nm emission wavelengths). Each plate included 0% inhibition (DMSO
only) and 100% inhibition (10 μM fluoxetine) control wells which were
used to normalize the fluorescence data. Normalized data was then fit to
a standard four-parameter curve and IC50 determined.
Rat Bladder Hyperactivity Model. Adult female Sprague−Dawley
rats weighing 170−250 g (Charles River, Wilmington, MA) were used.
All procedures were approved by the Institutional Animal Care and Use
Committee at Merck and Co., Inc., Rahway, N.J. Cystometry was
performed as described previously.26,27 In brief, animals were
anesthetized with urethane (1.1 g/kg ip). A lower abdominal midline
incision was made to expose the bladder, and two polyethylene tubes
(PE-50) were inserted into the bladder dome, secured with a 4−0 silk
suture and connected to both a syringe pump (555920; Harvard
Apparatus, Holliston, MA) and a pressure transducer via a three way
stopcock. Physiological saline solution was intravesically infused at a rate
of 0.05 mL/min. The intravesical pressure signal was recorded using a
Powerlab unit (16/30; AD Instruments) at a sampling rate of 10 Hz. An
intravenous catheter (PE-100) was implanted into a femoral vein, and
the drug solutions were infused at a rate of 0.01 mL/min using an
infusion pump (BS-9000-8, Braintree Scientific, Braintree, MA). After a
stable micturition cycle was observed, a 0.5% acetic acid was
intravesically infused for 2 h. The drug solution was continuously
infused from 1 to 2 h after beginning the intravesical acetic acid infusion.
Peak micturition pressures were measured pre- and postdose. Data are
presented as the mean SEM. The mean values were compared with
one-way ANOVA with Bonferoni posthoc test. A probability value less
than or equal to 0.05 was considered significant.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 908-740-4618. Fax: 908-740-3131. E-mail:
christopher_moyes@merck.com. Address: Merck Research
Laboratories, Mail Code K15-A113B1, 2000 Galloping Hill
Road, Kenilworth, New Jersey 07033, United States.
Present Addresses
∥For A.K.: Saladax Biomedical Inc., Ben Franklin Techventures
Building, 116 Research Drive, Bethlehem, Pennsylvania 18015,
United States.
⊥For A.S.: Pfizer Global Research and Development, 401 N.
Middletown Road, Pearl River, New York 10965, United States.
#For H.N.: Extra Value Generation and General Medicine Drug
Discovery Unit, Pharmaceutical Research Division, Takeda
Pharmaceutical Co., Ltd., 2-26-1, Muraoka-Higashi, Fujisawa,
Kanagawa 251−8555, Japan.
∇For K.V.: Picower Institute for Learning and Memory,
Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Judy Morris, Regina Black, and Lisa Frey for assistance
with compound purification, Arlene McKeown and Richard Ball
for collecting diffraction data, and Donna Hrenuik for in vitro
assay support.
ABBREVIATIONS USED
■
AcOH, acetic acid; β-AR, β-adrenergic receptor; AUC, area
under the pharmacokinetic exposure curve; b, broad; Boc, t-butyl
carbamate; CHO cells, Chinese hamster ovary; CYP, cyto-
chrome P450; d, doublet; DMAP, 4-dimethylaminopyridine;
DMF, N,N-dimethylformamide; EDC, N-(3-dimethylamino-
propyl)-N′-ethylcabodiimide hydrochloride; EtOH, ethanol;
̀
hERG, human Ether-a-go-go Related Gene potassium channel;
HOAt, 1-hydroxy-7-azabenzotriazole; HPLC, high performance
liquid chromatography; IPA, propan-2-ol; LC/MS, liquid
chromatography−mass spectroscopy; LHS, left-hand side; m,
multiplet; MeOH, methanol; Moz, p-methoxybenzyl carbamate;
NMR, nuclear magnetic resonance; OAB, overactive bladder;
PK, pharmacokinetic; q, quartet; quin, quintet; RHS, right-hand
side; s, singlet; SAR, structure−activity relationship; SFC,
supercritical fluid chromatography; SSRI, selective serotonin
receptor uptake; t, triplet; TBS, t-butyldimethylsilyl; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; TMS, trimethylsilyl
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