Lead optimization of ethyl 6-aminonicotinate acyl sulfonamides as antagonists of the P2Y12 receptor. Separation of the antithrombotic effect and bleeding for candidate drug AZD1283

Article abstract of DOI:10.1021/jm400820m

Synthesis and structure-activity relationships of ethyl 6-aminonicotinate acyl sulfonamides, which are potent antagonists of the P2Y₁₂ receptor, are presented. Shifting from 5-chlorothienyl to benzyl sulfonamides significantly increased the pot

Full text of DOI:10.1021/jm400820m

Article
pubs.acs.org/jmc
Lead Optimization of Ethyl 6‑Aminonicotinate Acyl Sulfonamides as
Antagonists of the P2Y12 Receptor. Separation of the Antithrombotic
Effect and Bleeding for Candidate Drug AZD1283
,†
†
†
‡
†
̈
Peter Bach,* Thomas Antonsson, Ruth Bylund, Jan-Arne Bjorkman, Krister Osterlund,
̈
Fabrizio Giordanetto,† J. J. J. van Giezen,‡ Søren M. Andersen,§ Helen Zachrisson,‡
and Fredrik Zetterberg*,†
‡
§
†CVGI Medicinal Chemistry, CVGI Bioscience, and CVGI iMED Project Management, AstraZeneca R&D Molndal,
̈
Pepparedsleden 1, S-43183 Molndal, Sweden
̈
S
* Supporting Information
ABSTRACT: Synthesis and structure−activity relationships
of ethyl 6-aminonicotinate acyl sulfonamides, which are potent
antagonists of the P2Y12 receptor, are presented. Shifting from
5-chlorothienyl to benzyl sulfonamides significantly increased
the potency in the residual platelet count assay. Evaluation of
PK parameters in vivo in dog for six compounds showed a 10-
fold higher clearance for the azetidines than for the matched-
pair piperidines. In a modified Folts model in dog, both
piperidine 3 and azetidine 13 dose-dependently induced
increases in blood flow and inhibition of ADP-induced platelet
aggregation with antithrombotic ED50 values of 3.0 and 10 μg/kg/min, respectively. The doses that induced a larger than 3-fold
increase in bleeding time were 33 and 100 μg/kg/min for 3 and 13, respectively. Thus, the therapeutic index (TI) was ≥10 for
both compounds. On the basis of these data, compound 3 was progressed into human clinical trials as candidate drug AZD1283.
dines,10 thienopyrimidines,11 anthraquinones,12 adenosine
INTRODUCTION
■
analogs,13 dinucleoside polyphosphates and nucleotides,14 and
Acute arterial thrombosis is accountable for most cases of
phenylpyrazole derivatives.15
myocardial infarction and approximately 80% of strokes, which
We recently reported that a series of ethyl nicotinate
collectively are the most common causes of mortality and
derivatives are antagonists of the P2Y12 receptor. These were
morbidity in the developed world.1 By vascular damage, e.g.,
featured by urea16 (e.g., 1, Figure 1) or sulfonylurea17 (e.g., 2)
due to rupture of atherosclerotic plaque, platelets can get into
linkers. Further development of the urea series was hampered
contact with and adhere to subendothelial proteins such as
by low solubility and low microsomal stability. While the
collagen and von Willebrand factor.2 This activates the platelets,
sulfonylurea series improved these issues, it remained to
which in turn release ADP from their dense granules. ADP
identify one compound that integrated high potency with
activates the Gi-coupled P2Y12 receptors,3 which decrease the
desirable physicochemical and pharmacokinetic (PK) proper-
intracellular adenylyl cyclase activity and prolong intracellular
ties. In the present paper, we show how replacement of the
calcium signaling, thereby stabilizing the formed platelet
sulfonylurea linker with an acyl sulfonamide linker followed by
aggregates.4
structure−property and in vivo dog PK and PD investigations
of this new series led to the selection of compound 3 as a
candidate drug (named AZD1283) for clinical development.
The central role of the P2Y12 receptor in platelet function
makes it an attractive target for the development of novel
antiplatelet therapies.5 The “thienopyridines”, including ticlo-
pidine, clopidogrel, and prasugrel, are prodrugs, whose active
CHEMISTRY
■
metabolites bind irreversibly to the receptor, thus inhibiting the
The ethyl 6-chloronicotinates 4 and 517 (Scheme 1) were
treated with azetidine-3-carboxylic acid (3-aze) to give the
azetidinylpyridines 6 and 7 and with piperidine-4-carboxylic
acid (4-pip) to give the piperidinylpyridines 8 and 9. The
carboxylic acid functionalities of compounds 6−9 were
activated with a coupling reagent such as 1-ethyl-3-(3-
platelet for its entire life span.6 However, preclinical data
suggested that reversible binding could not only lead to a
recovery of platelet function, but also increase the separation
between antithrombotic effect and bleeding risk.7,8
Ticagrelor, which is the first reversibly binding, direct-acting
P2Y12 antagonist, was developed via a medicinal chemistry
program from ATP, the natural antagonist of the P2Y12
receptor, as the lead structure.9 Other series of P2Y12
antagonists include piperazinyl glutamate−pyridines/pyrimi-
Received: June 3, 2013
Published: July 30, 2013
© 2013 American Chemical Society
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Figure 1. Examples of ethyl nicotinate derivatives, featured by urea (1), sulfonylurea (2), and acyl sulfonamide (3) linkers.
a
Scheme 1. Synthesis of Ethyl 6-Azetidinylnicotinate and 6-Piperidinylnicotinate Derivatives
a
Reagents and typical reaction conditions: (a) (e.g., for 6) 4, azetidine-3-carboxylic acid, N,N-diisopropylethylamine (DIPEA), DMA, 120 °C, 18 h
(73%); (b) (e.g., for 10) 6, EDC, HOBT, DCM, rt, 30 min, then addition of 5-chlorothiophene-2-sulfonamide, DIPEA, rt, 18 h (56%).
a
Scheme 2. Synthesis of Ethyl 6-Piperidinylnicotinate Derivative 3 by the Reverse Method
a
Reagents and reaction conditions: (a) PhCH2SO2NH2, TBTU, triethylamine (TEA), THF, LiCl, rt, 22 h (72%); (b) HCOOH, rt, 26 h (76%); (c)
5, DIPEA, DMA, 60 °C,18 h (83%).
a
Table 1. SAR of Ethyl Nicotinate Derivatives with 5-Choro-2-thienyl Acyl Sulfonamides
compd
no.
central
ring
binding IC50
GTPγS IC50
(μM)
WPA IC50
RPC IC50
log D,
Caco-2 A to B
(10−6 cm/s)
CLint DLMs,
L/M/H
R2
R5
Cl
(μM)
(μM)
(μM)
pH 6.822
10
11
23
32
H
3-aze
3-aze
4-pip
4-pip
0.50
0.31
0.48
2.3
2.6
2.8
3.1
M (0.5)
M (0.2)
H (10)
H (6)
L
CH3
H
CN
Cl
0.059
0.20
0.099
0.13
0.068
0.74
>22
>22
L
H
M
CH3
CN
0.039
0.080
0.039
a
3-aze = 3-azetidinyl, 4-pip = 4-piperidinyl. In the washed platelet assay (WPA) the ability of the compounds to inhibit fibrinogen-induced
aggregation is determined. The residual platelet count (RPC) assay determines the number of single platelets that remain after induction of platelet
aggregation in whole blood using ADP as an agonist. Thus, the effect of an antagonist is determined by an increase in the residual platelet count.
Lipophilicity, given by log D (D = distribution coefficient), was determined by a chromatographic method. Caco-2 A to B = permeability in the apical
(A) to basolateral (B) direction in adenocarcinoma cells from human colon. CLint DLMs = intrinsic clearance, as a measure for metabolic stability,
in dog liver microsomes. L/M/H = low/moderate/high.
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a
Table 2. SAR of Ethyl Nicotinate Derivatives with Phenyl, Benzyl, Cyclohexylmethyl, and Phenethyl Acyl Sulfonamides
binding GTPγS
RPC
IC50
solubility,
pH 6.8
(μM)
CLint25 CYP 2C9 CYP 3A4
compd central
IC50
IC50
log D,
Caco-2 A to B CLint DLMs
HLMs,
IC50
IC50
no.
ring
R7
(μM)
(μM)
(μM) pH 6.8
(10−6 cm/s)
(μL/min/mg) L/M/H
(μM)
(μM)
12
13
14
15
16
17
18
19
20
21
22
24
3
3-aze
3-aze
3-aze
3-aze
3-aze
3-aze
3-aze
3-aze
3-aze
3-aze
3-aze
4-pip
4-pip
4-pip
4-pip
4-pip
4-pip
4-pip
Ph
Bn
0.20
0.84
1.7
0.2 (M)
0.3 (M)
0.3 (M)
3 (H)
<12 (L)
<12 (L)
M
L
0.017
0.22
0.033
2.6
2.0
2.2
2.6
2.2
120
>50
>50
−CH2CH2Ph
−CH2-c-hexyl
2-CH3Bn
3-CF3Bn
3-CH3Bn
4-ClBn
0.089
0.067
0.11
0.18
0.18
0.29
M
0.032
0.010
0.012
0.006
0.23
0.065
0.023
0.020
0.019
0.73
19
2.2
2.7
2.3
2.2
1.1
2.3
2.8
2.8
2.8
3.2
4.2
2.9
2.8
1.2
1.8
134
297
69
0.2 (M)
0.1 (L)
0.6 (M)
26 (M)
29 (M)
<12 (L)
M
L
7.1
>20
20
3.3
>20
20
4-FBn
4-CH3Bn
4-CH2OHBn
Ph
H
L
0.12
0.71
L
Bn
0.011
0.015
0.03
0.025
0.038
0.058
0.023
0.25
3.2
2.9
9.0
2.9
104
63
20 (H)
8 (H)
<12 (L)
17 (M)
L
3.3
5.9
20
4.5
25
26
27
28
29
4-FBn
M
M
H
H
H
4-OCH3Bn
4-CH3Bn
4-i-PrBn
2,4-di-FBn
17
40 (H)
40 (H)
40 (H)
5 (H)
15
>17
>17
>2
0.007
0.16
0.20
2.0
<12 (L)
>18
2.4
0.011
0.016
2.7
20 (M)
4.1
1.4
a
3-aze = 3-azetidinyl, 4-pip = 4-piperidinyl, and RPC = residual platelet count assay. Lipophilicity, given by log D (D = distribution coefficient), was
determined by a chromatographic method. Caco-2 A to B = permeability in the apical (A) to basolateral (B) direction in adenocarcinoma cells from
human colon. CLint = intrinsic clearance in DLMs (dog liver microsomes) or HLMs (human liver microsomes). L/M/H = low/moderate/high.
(dimethylamino)propyl)carbodiimide hydrochloride (EDC)/1-
hydroxybenzotriazole (HOBT),18 O-(7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluromium hexafluorophosphate (HATU), O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate (TBTU), or bromotrispyrrolidinophosphonium hexa-
fluorophosphate (PyBrop) and then treated with different
sulfonamides to produce the acyl sulfonamides 10−29.
Compound 3 (Scheme 2) was prepared by a reverse method
in which coupling of 30 with benzyl sulfonamide, followed by
boc deprotection with formic acid, provided intermediate 31,
which was treated with the ethyl 6-chloronicotinate derivative 5
to provide the candidate drug 3. By this sequence it was
possible to precipitate 3 from the reaction mixture, thus
enabling larger scale synthesis. Especially the coupling of 30
with benzyl sulfonamide was problematic due to the poor
nucleophilicity of benzyl sulfonamide, but it was found that in
THF, in the presence of lithium chloride (LiCl), a TBTU
coupling readily afforded 31. Compound 32 (Table 1) was
prepared by similar procedures.
thus, the effect of an added antagonist can be determined by
the fraction of unbound GTPγ35S. Potency in a residual platelet
count (RPC) assay20 was determined for most compounds.
This assay determines the number of single platelets that
remain after induction of platelet aggregation in whole blood
using ADP as an agonist. Thus, the effect of an antagonist can
be determined by an increase in the residual platelet count. A
few compounds from the early-phase project were tested for
potency in a functional human washed platelet aggregation
assay (WPA) that determines the ability of the compounds to
inhibit fibrinogen-induced aggregation.7
Ethyl 5-cyano-2-methylnicotinate derivatives (Table 1)
showed a 5−8-fold higher binding affinity and a 7−17-fold
higher potency in the WPA assay than the matched-pair ethyl
5-chloronicotinate derivatives (pairs 10/11 and 23/32). On the
basis of these differences in activity, the 2-methyl/5-
cyanopyridine substitution pattern was retained in further
structure−activity relationship (SAR) investigations. A similar
difference in activity as a function of the pyridine substitution
pattern was observed in the previously reported sulfonylurea
series.17 Another similarity with the sulfonylurea series was that
the carboxylic acid of 13, formed by hydrolysis of the ethyl
ester, was inactive in both the binding and GTPγS assays.
The higher lipophilicity (Δ log D = 0.5) of piperidinylpyr-
idines in comparison to the matched-pair azetidinylpyridines
(pairs 23/10 and 32/11) was reflected in the piperidinylpyr-
idines having higher permeability in Caco-2 monolayers21 and
higher intrinsic clearance (CLint) in dog liver microsomes
(DLMs).
RESULTS AND DISCUSSION
■
Compounds were tested in vitro for binding affinity for P2Y12
receptors in chinese hamster ovarian (CHO) cell membranes.7
The potency of the compounds was determined in a functional
guanosine 5′-O-[γ-thio]triphosphate (GTPγS)7,19 assay using a
similar membrane preparation. The GTPγS assay is based on
the principle that activation (by ADP) of P2Y12 favors binding
of the receptor to a G-protein. The formed ADP−P2Y12−G-
protein ternary complex induces the G-protein-bound GDP to
exchange with GTPγ35S, which is not hydrolyzed by GTPases;
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a
Table 3. In Vivo PK in Dog for a Selection of Potent Acyl Sulfonamides
RPC
compd central
IC50
Caco-2 A to B
(10−6 cm/s)
CLint DLMs
(μL/min/mg)
log D,
pH 6.8
dog PPB N,
dog CL
N,
dog t1/2, po
b
b
b
no.
ring
R7
(μM)
(% free)
iv (mL/min/kg)
po dog F (%)
(h)
13
20
21
3
3-aze
3-aze
3-aze
4-pip
4-pip
4-pip
Bn
2.4
1.1
1.8
3.1
2.8
2.7
M (0.3)
L (0.1)
M (0.6)
H (20)
H (40)
H (5)
<12 (L)
29 (M)
<12 (L)
<12 (L)
2.0
2.3
2.2
2.8
3.2
2.9
1.9
2
2
2
3
1
2
9.8 1.3
9.4 0.1
4.5 0.5
0.75 0.25
0.50
2
2
1
2
1
2
100 29
3.1
0
4-FBn
1.3
27
69
3
5.9 0.4
7.5
4-CH3Bn
Bn
0.82
0.35
0.59
0.14
100 11
92
8.0 0.5
13
27
29
4-CH3Bn
2,4-di-FBn
20 (M)
1.1 0.7
53 23
6.6 1.2
a
In vitro data have been included for reference. 3-aze = 3-azetidinyl, 4-pip = 4-piperidinyl, RPC = residual platelet count assay, Caco-2 A to B =
permeability in the apical (A) to basolateral (B) direction in adenocarcinoma cells from human colon, CLint = intrinsic clearance, and DLMs = dog
liver microsomes. Lipophilicity is given by log D (D = distribution coefficient). PPB = plasma protein binding, CL = clearance, F = bioavailability,
b
and L/M/H = low/moderate/high. The compounds were administered as solutions in TEG/DMA/H2O (1:1:1) or TEG/EtOH/H2O (50:5:45).
Surprisingly, the potency in the RPC assay was low for the 5-
chlorothienyl compounds 11 and 32 despite a high potency in
WPA. This was observed only for 5-chlorothienyl-containing
compounds. Further exploration of thienyl groups carrying
methyl and/or chloro substituents did not increase the potency
in RPC.
Further variations (Table 2) including phenyl (12), benzyl
(13), phenylethyl (14), and cyclohexyl (15) derivatives showed
superior binding affinity for the benzylic compound in the
azetidine subseries; this was also observed for the phenyl/
benzyl matched pair 24/3 in the piperidine subseries. Notably,
the benzylic compounds 3 and 13 were potent in the higher
order residual platelet count assay (RPC)20 in which the two
tested 5-chlorothienyls 11 and 32 showed low (IC50 >22 μM)
potency. Consequently, the effect of introducing substituted
benzyls in the azetidinyl- and piperidinylpyridines was
investigated.
In the azetidine subseries, methyl substitution led to a 6-fold
lower potency in the GTPγS assay when in the 2-position (16)
and unchanged potency when in the 3- or 4-position (18, 21),
compared to the unsubstituted benzyl compound 13. Also 4-
chloro (19, lipophilic and electron-withdrawing) and 4-fluoro
(20, electron-withdrawing) substituents led to unchanged
GTPγS potency. Substituents that led to a lower potency in
GTPγS are exemplified by the 3-trifluoromethyl substituent
(17, bulky, electron-withdrawing), which lowered the potency
6-fold compared to the 3-methyl (18), and 4-hydroxymethyl
(22, hydrophilic, electronically neutral),23 which lowered the
potency 38-fold compared to 4-methyl substitution (21).
In the piperidine subseries, the introduction of substituents
such as fluoro (25), methoxy (26), or methyl (27) in the
benzyl 4-position had no effect on the GTPγS potency
compared to an unsubstituted benzyl substituent (3).
Introduction of the sterically more demanding 4-isopropyl
(28) group led to a 10-fold decrease in potency compared to 4-
methyl (27). In both the azetidine and piperidine subseries, the
introduction of further substituents on the aromatic part of the
benzyl substituent, exemplified by the 2,4-difluorobenzyl
derivative 29, in the best cases led to no or very minor
increases in potency compared to mono- and unsubstituted
benzyl compounds.
The generally high aqueous solubility24 at pH 6.8 was likely
due to the acidity of the acyl sulfonamide linker (for example,
pKa(3) = 4.6, pKa(13) = 3.6, pKa(21) = 3.4, and pKa(27) =
4.6).24
The in vitro pharmacokinetic (PK) profile indicated in
general better permeability for 4-piperidines than for 3-
azetidines, as examplified by the matched pair 3/13. The
intrinsic clearance was low or moderate for all compounds in
DLMs and for most compounds in HLMs; however, no
correlation was observed between the clearance values in the
two different types of microsomes. The most striking example
is compounds 21 and 27, which showed low clearance in DLMs
but high clearance in HLMs. Metabolite identification in HLMs
indicated oxidation of the 4-methyl substituents as the main
metabolic pathway in addition to ester hydrolysis.
In Vivo PK Parameters in Dog. On the basis of their in
vitro parameters, a selection of compounds from Table 2 were
progressed for evaluation of PK parameters in dog. For some
compounds in Table 2, CYP450 inhibition was a possible
liability. This was considered when selecting compounds for
further evaluation. In total, six compounds (Table 3) were
selected. Two of these (3 and 13) were selected in spite of their
inhibition of two of the CYP450-metabolizing enzymes,
CYP2C9 and CYP3A4. Whether or not this was a risk factor
in later development (potential for drug−drug interactions)
would depend on the dose of the selected drug candidate and
should be weighted against the other properties of the
compounds.
The azetidines showed higher clearance in vivo than the
matched-pair piperidines, by comparison of the pairs 3/13 (13-
fold) and 21/27 (9-fold). Although the protein binding was
lower for the azetidines, this seems unlikely to explain their
higher clearance.26 No correlation was observed between the
intrinsic clearance in DLMs and the clearance in vivo. For
example, the two azetidines 13 (low intrinsic clearance in
DLMs) and 20 (moderate intrinsic clearance in DLMs) both
showed similar low to moderate clearance in vivo. This could
point to the possibility of 13 having additional metabolic
pathways in vivo.
On the basis of their favorable pharmacokinetic profiles and
high stabilities in HLMs, 3 and 13 were selected for further
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Figure 2. Effects of compounds 3 (N = 6) and 13 (N = 5) on blood flow, aggregation, bleeding time, and blood loss vs dose. In each graph, the left y-
axis describes the inhibition (%) of thrombosis (blood flow) and aggregation time, while the right y-axis describes the fold increase of bleeding time
and blood loss as a function of the dose given at the x-axis.
evaluation of their in vivo antithrombotic potential and
associated bleeding risk in a modified Folts dog model.
Evaluation of the in Vivo and ex Vivo Antithrombotic
Effect and Bleeding Time in a Modified Folts Dog
Model. In a modified Folts dog model8 the platelet/vessel wall
interactions were evaluated by measurement of femoral artery
blood flow after mechanical damage of the endothelium
followed by approximately 80% stenosis. A blood flow pattern
comprising thrombus growth followed by mechanical restora-
tion of artery is known as cyclic flow reductions (CFRs), and an
in vivo antithrombotic effect is seen as an abolition of the
CFRs. In vivo (blood flow) and ex vivo (platelet aggregation
measured with impedance aggregometry) antithrombotic
effects, bleeding time, and blood loss (incision in the tongue)
were measured during a 30 min control period followed by five
consecutive 30 min periods with increasing doses of the test
compounds. The results are summarized in Figure 2.
Both 3 and 13 induced dose-dependent increases in blood
flow and inhibition of ADP-induced platelet aggregation,
showing an antithrombotic ED50 for 3 and 13 of 3.0 and 10
μg/(kg × min), respectively. The two compounds displayed a
similar bleeding time pattern; the doses that induced a larger
than 3.5-fold increase in bleeding time (EDBT>3.5) were greater
than 33 and 100 μg/(kg × min) for 3 and 13, respectively.
Thus, the therapeutic index (TI), defined as antithrombotic
ED50/EDBT>3.5, was ≥10 for both compounds. This is at least
similar to the increase in TI previously reported for the
reversibly binding antagonist ticagrelor (ED50 = 1.02 μg/(kg ×
min); TI > 5.2) and the irreversibly binding antagonist
clopidogrel (ED50 = 15 mg/kg; TI = 2.3).
substitutents on the benzyl group gave no further improvement
in potency and in many cases led to higher microsomal
clearance. PK parameters in vivo in dog for six compounds
showed higher clearance for the azetidines than for the
matched-pair piperidines.
One azetidine (13) and one piperidine (3) were evaluated
for an antithrombotic effect in vivo and possible associated
bleeding risk in a modified Folts dog model. Both compounds
induced dose-dependent increases in blood flow and inhibition
of ADP-induced platelet aggregation, displaying a therapeutic
index ≥10 in the separation of efficacy and bleeding time. The
piperidine 3 showed the strongest antithrombotic effect and
was therefore progressed to human clinical trials as candidate
drug AZD1283.
EXPERIMENTAL SECTION
■
General Description. Commercially available chemicals were used
as provided by the commercial supplier. Noncommercial or expensive
sulfonamides were made by stirring the corresponding commercially
available sulfonyl chloride (0.75 mmol) at rt in a saturated solution of
ammonia in MeOH (5 mL) until completion of the reaction by LC/
MS detection. After evaporation of ammonia and MeOH, the crude
was dissolved in MeOH (5 mL). Optionally, DMF (2 mL) was added
to dissolve the crude. The solution were then filtered through an
ISOLUTE SCX-2 ion-exchange resin (while rinsing with MeOH) and
then concentrated to give a material which was used without further
purification. As a special case, the sulfonamide used in the synthesis of
19 was prepared from 4-(chloromethyl)benzyl alcohol in analogy to a
literature procedure.27
In general, reactions were performed under a nitrogen atmosphere.
Reactions in microwave reactors were performed by single-node
heating in a Smith Creator, a Smith Synthesizer, or an Emrys
Optimizer using microwave vials from Personal Chemistry (now
Biotage). Concentration of the solutions was done in vacuo on a rotary
evaporator at temperatures below 50 °C.
Thin-layer chromatography was performed on silica gel 60 F254
(Merck KGaA, Darmstadt). Flash chromatography was performed
with either standard glass or plastic columns using Merck silica gel
grade 9385, 60 Å (0.063−0.200 mm) from Sigma-Aldrich or on a
Biotage Horizon high-performance flash chromatography (HPFC)
system using Biotage silica gel. Preparative HPLC was performed on a
Waters YMC-ODS AQS-3 120 Å 3 × 500 mm. Gradient: 95% 0.1 M
CONCLUSIONS
■
In conclusion, a novel series of ethyl 6-aminonicotinate acyl
sulfonamides were discovered to be antagonists of the P2Y12
receptor. An advantage of this structural class was that
screening compounds could be prepared in a few synthetic
steps. SAR investigations showed an increase in potency in the
residual platelet count assay by replacement of a 5-
chlorothienyl with a benzyl substitutent. Introduction of
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aq NH4OAc buffer/5% CH3CN → 100% CH3CN. Alternatively,
preparative HPLC was performed on a Waters Fraction Lynx
purification system with a Kromasil C8 column (5 μm; 100 mm ×
20 mm i.d.) with gradients of CH3CN in 0.1 M aq NH4OAc buffer as
the mobile phase. MS-triggered fraction collection was used. Mass
spectra were recorded on either a Micromass ZQ single quadrupole or
a Micromass Quattro micro instrument, both equipped with a
pneumatically assisted electrospray interface. HRMS of isolated
compounds was performed on a Waters XEVO qToF instrument
operated in positive electrospray mode using a Waters Acquity iClass
UPLC system, with a 2.1 × 50 mm 1.7 μm CSH C18 column and a
linear gradient of 5−95% acetonitrile (1 mM ammonium formate, 10
mM formic acid, pH 3) in 2.5 min (system controlled by Waters
MassLynx 4.1 software). 1H NMR measurements were performed on a
m), 2.37−2.44 (1H, m), 2.73 (3H, s), 3.10−3.17 (2H, m), 4.33 (2H, q,
J = 7.0 Hz), 4.64−4.68 (4H, m), 7.36−7.41 (5H, m), 8.36 (1H, s).
MS: m/z 471 (M + 1). HRMS: m/z calcd for C23H26N4O5S (M + 1)+,
471.1702, found 471.1683.
1-[3-Chloro-5-(ethoxycarbonyl)pyridin-2-yl]azetidine-3-car-
boxylic Acid (6). 4 (3.68 g, 16.5 mmol) and azetidine-3-carboxylic
acid (2.50 g, 24.8 mmol) were suspended in DMA (50 mL). DIPEA
(6.38 g, 8.60 mL, 49.5 mmol) was added, and the reaction mixture was
heated at 120 °C for 18 h. The mixture was cooled to rt and
concentrated. The crude material was dissolved in DCM (300 mL),
and the solution was washed with 1 N HCl (150 mL), dried (MgSO4),
and concentrated. The crude was purified by flash chromatography
(1:3 EtOAc/hexanes to 1:3 EtOAc/hexanes, 0.5% AcOH). Yield: 3.44
g (73%) as a solid. 1H NMR (400 MHz, CDCl3): δ 1.37 (3H, t, J = 7.1
Hz), 3.54−3.61 (1H, m), 4.34 (2H, q, J = 7.1 Hz), 4.53−4.63 (4H, m),
8.00 (1H, s), 8.67 (1H, s). MS: m/z 283 (M − 1).
1
Varian Mercury VX 400 spectrometer, operating at a H frequency of
400 MHz, or on Varian UNITY plus 400, 500, and 600 spectrometers,
1
operating at H frequencies of 400, 500, and 600 MHz, respectively.
1-[3-Cyano-5-(ethoxycarbonyl)-6-methylpyridine-2-yl]-
azetidine-3-carboxylic Acid (7). A suspension of 5 (51.00 g, 227
mmol), azetidine-3-carboxylic acid (24.09 g, 238 mmol), and DIPEA
(88.22 g, 118.9 mL, 681 mmol) in EtOH (250 mL) was heated at
reflux for 1 h. The reaction mixture was cooled to rt and added
dropwise to a solution of KHSO4 (154.5 g, 1135 mmol) in water
(3000 mL). The formed solids were collected by filtration and dried
under vacuum. Yield: 65.33 g (100%). 1H NMR (400 MHz, CDCl3): δ
1.37 (3H, t, J = 7.1 Hz), 2.72 (3H, s), 3.59−3.68 (1H, m), 4.31 (2H, q,
J = 7.1 Hz), 4.55−4.68 (4H, m), 8.28 (1H, s). MS: m/z 290 (M + 1).
1-[3-Chloro-5-(ethoxycarbonyl)pyridin-2-yl]piperidine-4-
carboxylic Acid (8). 4 (5.00 g, 22.7 mmol) and piperidine-4-
carboxylic acid (4.40 g, 34.1 mmol) were suspended in DMA (50 mL).
DIPEA (8.83 g, 11.9 mL, 68.2 mmol) was added, and the reaction
mixture was heated at 120 °C for 2 h and then cooled to rt and
concentrated. The crude was diluted with DCM (300 mL), washed
with HCl (1 M, 150 mL), dried (MgSO4), concentrated, and purified
by flash chromatography (1:4 EtOAc/hexanes to 1:3 EtOAc/hexanes,
Chemical shifts are given in parts per million with the solvent as the
internal standard. Coupling constants are given in hertz.
For some samples with DMSO as the NMR solvent, a so-called wet
DMSO NMR experiment was used. This was conducted in the
following manner: A sample of a concentrated solution of the
compound in (CH3)2SO was diluted with (CD3)2SO. Since a
substantial amount of (CH3)2SO was present in the sample, a prescan
was run and analyzed to automatically suppress the (CH3)2SO and
H2O peaks at 2.54 and 3.30 ppm, respectively. In the wet DMSO
NMR experiment, the 1H NMR signal from the methine proton of the
piperidinyl group coincided with the suppressed (CH3)2SO signal at
2.54 ppm; however, the chemical shift for the methine proton could be
determined by H,H COSY, and this signal is reported for each
piperidinyl compound.
The purity of the screening compounds was determined by analytic
HPLC. Low-resolution mass spectra (electrospray ionization) were
acquired on a Waters ZQ quadrupole spectrometer coupled to an
Agilent Technologies 1100 series HPLC. The HPLC retention time
was recorded through a standard gradient from 5% to 95% CH3CN in
10 mM aq NH4OAc over 4 min using a Synergy MAX-RP C12 (4 μm;
50 mm × 3 mm i.d.) column with a flow rate of 2 mL/min. All
screening compounds had purity ≥95%.
1
0.5% AcOH). Yield: 6.36 g (90%) as a solid. H NMR (400 MHz,
CDCl3): δ 1.38 (3H, t, J = 7.1 Hz), 1.88−1.97 (2H, m), 2.03−1.12
(2H, m), 2.57−2.66 (1H, m), 2.99−3.09 (2H, m), 4.02−4.11 (2H, m),
4.36 (2H, q, J = 7.1 Hz), 8.12 (1H, s), 8.74 (1H, s). MS: m/z 311 (M
− 1).
Ethyl 6-(4-[(Benzylsulfonyl)carbamoyl]piperidin-1-yl)-5-
cyano-2-isopropylnicotinate (3). A suspension of 30 (7.73 g,
1.96 mmol) and TBTU (16.18 g, 50.4 mmol) in THF (80 mL) and
TEA (10.89 g, 15.0 mL, 107.7 mmol) was stirred at rt for 45 min. 1-
Phenylmethanesulfonamide (7.35 g, 42.92 mmol) and LiCl (0.46 g,
10.85 mmol) were added, and the reaction mixture was stirred at rt for
22 h. The mixture was concentrated, diluted with EtOAc (100 mL),
washed with diluted HCl, dried (MgSO4), and concentrated. Addition
of tert-butyl methyl ether (TBME) (100 + 50 mL) facilitated
precipitation. The mixture was stirred at rt for 1 h and filtered, and the
solids were washed with TBME (50 mL). Yield: 9.34 g (72%).
A suspension of tert-butyl 4-[(benzylsulfonyl)carbamoyl]piperidine-
1-carboxylate (6.45 g, 16.86 mmol) in formic acid (61.0 g, 50.0 mL,
46.0 mmol) was stirred at rt for 26 h, concentrated, and
coconcentrated with water (25 mL). Water (35 mL) was added, and
the pH was adjusted to 6 with NH4OH (saturated, aq solution). The
mixture was stirred at rt for 18 h and filtered, and the solids were
washed with cold water (35 mL) and dried in vacuo. Yield: 3.61 g
1-[3-Cyano-5-(ethoxycarbonyl)-6-methylpyridin-2-yl]-
piperidine-4-carboxylic Acid (9). A reaction mixture of 5 (3.00 g,
13.4 mmol), piperidine-4-carboxylic acid (1.90 g, 14.7 mmol), and
TEA (2.70 g, 2.0 mL, 26.7 mmol) was refluxed for 10 min. The
mixture was concentrated, water (50 mL) and EtOAc (50 mL) were
added, and the water phase was acidified to pH 3 with HCl. The
EtOAc phase was separated, and the water phase was extracted with
additional EtOAc (40 mL). The combined organic phases were dried
(Na2SO4) and concentrated. The crude material was purified by
preparative HPLC. Yield: 1.90 g (45%) as a white solid. 1H NMR (400
MHz, CDCl3): δ 1.38 (3H, t, J = 7.1 Hz), 1.82−1.94 (2H, m), 2.05−
2.13 (2H, m), 2.66−2.73 (1H, m), 2.74 (3H, s), 3.26−3.38 (2H, m),
4.33 (2H, q, J = 7.1 Hz), 4.54−4.65 (2H, m), 8.36 (1H, s). MS: m/z
318 (M + 1).
Ethyl 5-Chloro-6-[3-({[(5-chloro-2-thienyl)sulfonyl]amino}-
carbonyl)azetidin-1-yl]nicotinate (10). A mixture of 6 (0.150 g,
0.53 mmol), EDC (0.131 g, 0.68 mmol), and HOBT (0.093 g, 0.68
mmol) in DCM (5 mL) was stirred at rt for 30 min. 5-
Chlorothiophene-2-sulfonamide (0.208 g, 1.05 mmol) and DIPEA
(0.207 g, 0.280 mL, 1.58 mmol) were added, and the reaction mixture
was stirred at rt for 18 h. The mixture was diluted with DCM (70 mL),
washed with NH4Cl (2 × 40 mL, saturated, aq solution) and brine (40
mL), dried (MgSO4), and concentrated. The crude material was
purified by flash chromatography (EtOAc/hexanes (3:7), 0.5% AcOH,
to EtOAc/hexanes (7:3), 0.5% AcOH). Yield: 0.137 g (56%) as a
solid. 1H NMR (400 MHz, CDCl3): δ 1.37 (3H, t, J = 7.1 Hz), 3.40−
3.47 (1H, m), 4.34 (2H, q, J = 7.1 Hz), 4.46−4.53 (4H, m), 6.99 (1H,
d, J = 4.6 Hz), 7.72 (1H, d, J = 4.6 Hz), 8.01 (1H, s), 8.66 (1H, s). MS:
m/z 464 (M + 1). HRMS: m/z calcd for C23H26N4O5S (M + 1)+,
463.9908, found 463.9916.
1
(76%) of 31 as a white powder. H NMR (400 MHz, d6-DMSO): δ
1.57−1.72 (2H, m), 1.72−1.84 (2H, m), 2.08−2.19 (1H, m), 2.72−
2.85 (2H, m), 3.07−3.17 (2H, m), 4.24 (2H, s), 7.19−7.29 (5H, m),
8.12 (1H, br s). MS: m/z 283 (M + 1).
A suspension of 5 (0.671 g, 2.99 mmol), 31 (1.00 g, 3.14 mmol),
and DIPEA (1.56 mL, 8.96 mmol) in DMA (20 mL) was stirred at 60
°C for 18 h. The reaction mixture was cooled to rt and poured into a
mixture of EtOAc (200 mL) and NH4Cl (100 mL, saturated, aq
solution). The organics were washed with water (3 × 100 mL) and
brine (100 mL), dried (MgSO4), and concentrated to afford the crude
material. The crude was purified by flash chromatography (EtOAc/
1
hexanes (3:7) with 0.5% AcOH). Yield: 1.17 g (83%) as a solid. H
NMR (400 MHz, CDCl3): δ 1.38 (3H, t, J = 7.0 Hz), 1.77−1.91 (4H,
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Ethyl 6-[3-({[(5-Chloro-2-thienyl)sulfonyl]amino}carbonyl)-
azetidin-1-yl]-5-cyano-2-methylnicotinate (11). 11 was prepared
by the procedure of 10 from 7 (0.258 g, 0.89 mmol) and 5-
chlorothiophene-2-sulfonamide (0.353 g, 1.78 mmol). The crude
product was purified by flash chromatography (0−100% EtOAc in
heptane followed by 0−40% MeOH in DCM). Yield: 0.061 g (15%).
1H NMR (400 MHz, CDCl3): δ 1.29 (3H, t, J = 7.0 Hz), 2.60 (3H, s),
2.65 (3H, s), 3.53−3.62 (1H, m), 4.21−4.34 (4H, m), 4.39−4.47 (2H,
m), 4.72 (2H, s), 7.13−7.32 (4H, m), 8.33 (1H, s), 11.81 (1H, br s).
MS: m/z 457 (M + 1).
Ethyl 6-(3-{[(4-Chlorobenzyl)sulfonyl]carbamoyl}azetidin-1-
yl)-5-cyano-2-methylnicotinate (19). 19 was prepared by the
procedure of 13 from (4-chlorophenyl)methanesulfonamide (0.105 g,
1
0.38 mmol) and 7 (0.073 g, 0.25 mmol). Yield: 0.057 g (48%). H
NMR (400 MHz, d6-DMSO): δ 1.24 (3H, t, J = 7.1 Hz), 2.58 (3H, s),
3.45 (1H, m), 4.18 (2H, q, J = 7.1 Hz), 4.23 (2H, m), 4.36 (2H, m),
4.65 (2H, s), 7.29 (2H, d, J = 8.5 Hz), 7.37 (2H, d, J = 8.5 Hz), 8.25
(1H, s). MS: m/z 477 (M + 1).
3.55−3.68 (1H, m), 4.22 (2H, q, J = 7.0 Hz), 4.26−4.28 (2H, m),
4.37−4.46 (2H, m), 7.22 (1H, d, J = 4.2 Hz), 7.68 (1H, d, J = 4.2 Hz),
8.25 (1H, s). MS: m/z 469 (M + 1). HRMS: m/z calcd for
C18H17ClN4O5S2 (M + 1)+, 469.0407, found 469.0401.
Ethyl 5-Cyano-2-methyl-6-(3-{[(phenylsulfonyl)amino]-
carbonyl}azetidin-1-yl)nicotinate (12). 7 (0.072 g, 0.25 mmol)
and benzenesulfonamide (0.060 g, 0.38 mmol) were dissolved in DMF
(2 mL). DIPEA (0.162 g, 0.218 mL, 1.25 mmol) was added, followed
by a solution of HATU (0.100 g, 0.26 mmol) in DMF (2 mL). The
reaction mixture was stirred at rt for 16 h and then concentrated. The
crude material was purified by preparative HPLC. Yield: 0.075 g
Ethyl 6-(3-{[(4-Fluorobenzyl)sulfonyl]carbamoyl}azetidin-1-
yl)-5-cyano-2-methylnicotinate (20). 20 was prepared by the
procedure of 13 from (4-fluorophenyl)methanesulfonamide (0.088 g,
1
0.38 mmol) and 7 (0.073 g, 0.25 mmol). Yield: 0.050 g (43%). H
NMR (400 MHz, d6-DMSO): δ 1.24 (3H, t, J = 7.1 Hz), 2.57 (3H, s),
3.49 (1H, m), 4.20 (4H, m), 4.36 (2H, m), 4.65 (2H, s), 7.13 (2H, t, J
= 8.8 Hz), 7.32 (2H, m), 8.25 (1H, s). MS: m/z 461 (M + 1).
Ethyl 6-(3-{[(4-Methylbenzyl)sulfonyl]carbamoyl}azetidin-1-
yl)-5-cyano-2-methylnicotinate (21). Thionyl chloride (0.119 g,
1.0 mmol) was added to a solution of 7 (0.058 g, 0.20 mmol) in DCM
(1.0 mL) at 0 °C, and the reaction mixture was stirred at rt for 30 min.
The mixture was concentrated and coconcentrated with DCM. The
residue was dissolved in pyridine (1 mL) at 0 °C, and 1-(4-
methylphenyl)methanesulfonamide (0.044 g, 0.24 mmol) was added.
After the mixture was stirred at rt for 2 h, 4-(dimethylamino)pyridine
(DMAP) (a few crystals) was added, and stirring at rt was continued
for 19 h. 2-(tert-Butylimino)-2-(diethylamino)-1,3-dimethylperhydro-
1,3,2-diazaphosphorine (BEMP) (0.055 g, 0.20 mmol) was added, and
stirring at rt was continued for 22 h. LC/MS showed no conversion.
HATU (0.152 g, 0.40 mmol) and DIPEA (0.259 g, 2.0 mmol) were
added, and the mixture was stirred at rt for 20 h. The mixture was
concentrated, and the crude was purified by preparative HPLC. Yield:
0.019 g (15%). 1H NMR (400 MHz, d6-DMSO): δ 1.32 (3H, t, J = 7.0
Hz), 2.32 (3H, s), 2.66 (3H, s), 3.51−3.60 (1H, m), 4.20−4.37 (4H,
m), 4.38−4.47 (2H, m), 4.70 (2H, s), 7.17−7.28 (4H, m), 8.34 (1H,
s), 11.77 (1H, br s). MS: m/z 457 (M + 1).
1
(70%). H NMR (400 MHz, d6-DMSO): δ 1.24 (3H, t, J = 7.2 Hz),
2.55 (3H, s), 3.47−3.57 (1H, m), 4.11−4.22 (2H, m), 4.18 (2H, q, J =
7.2 Hz), 4.30−4.40 (2H, m), 7.56−7.62 (2H, m), 7.64−7.69 (1H, m),
7.87−7.92 (2H, m), 8.23 (1H, s), 12.40 (1H, s). MS: m/z 429 (M +
1).
Ethyl 6-(3-[(Benzylsulfonyl)carbamoyl]azetidin-1-yl)-5-
cyano-2-methylnicotinate (13). Phenylmethanesulfonamide
(0.068 g, 0.38 mmol) was added to a solution of 7 (0.073 g, 0.25
mmol) and DIPEA (0.164 g, 0.222 mL, 1.27 mmol) in DMF (2 mL).
A solution of HATU (0.079 g, 0.28 mmol) dissolved in DMF (1 mL)
was added, and the reaction mixture was stirred at rt for 16 h. The
mixture was concentrated, and the residue was purified by preparative
HPLC. Yield: 0.060 g (54%). 1H NMR (400 MHz, d6-DMSO): δ 1.23
(3H, t, J = 7.2 Hz), 2.57 (3H, s), 3.43 (1H, m), 4.17 (2H, q, J = 7.1
Hz), 4.23 (2H, t, J = 7.1 Hz), 4.34 (2H, t, J = 8.9 Hz), 4.68 (2H, s),
7.29 (5H, m), 8.33 (1H, s), 11.75 (1H, s). MS: m/z 443 (M + 1).
Ethyl 5-Cyano-2-methyl-6-(3-[(phenethylsulfonyl)-
carbamoyl]azetidin-1-yl)nicotinate (14). 14 was prepared by the
procedure of 13 from 2-phenylethanesulfonamide (0.078 g, 0.37
1
mmol) and 7 ((0.073 g, 0.25 mmol). Yield: 0.044 g (39%). H NMR
Ethyl 5-Cyano-6-(3-{[[4-(hydroxymethyl)benzyl]sulfonyl]-
carbamoyl}azetidin-1-yl)-2-methylnicotinate (22). 4-Chloro-
methyl benzyl alcohol (1.35 g, 8.60 mmol) and imidazole (0.763 g,
11.2 mmol) were dissolved in DCM (8.6 mL), and tert-
butyldimethylsilyl chloride (1.43 g, 9.50 mmol) was added in portions
at 0 °C. The reaction mixture was stirred at rt for 1 h and then
quenched with KHSO4 (0.5 M aq solution, 10 mL). The organic phase
was separated and concentrated to give a crude that was used in the
next step without further purification assuming a 100% conversion.
Sodium 3-methoxy-3-oxopropane-1-sulfinate27(1.76 g, 10.1 mmol)
was dissolved in DMSO (20 mL). A solution of tert-butyl{[4-
(chloromethyl)benzyl]oxy}dimethylsilane (2.40 g, 8.4 mmol) in
DMSO (5 mL) was added, and the reaction mixture was stirred at
rt for 16 h. Water (30 mL) was added, and the mixture was extracted
with EtOAc (2 × 30 mL). The combined organic phases were dried
(Na2SO4) and concentrated to give a product that was used in the next
step without further purification. Yield: 3.10 g (95%). MS: m/z 404
(ammonia adduct).
Methyl 3-{[4-[[(tert-butyldimethylsilyl)oxy]methyl]benzyl]-
sulfonyl}propanoate (3.10 g, 8.00 mmol) was dissolved in THF (20
mL), and sodium methoxide (3.2 M in methanol) was added until all
starting material had been consumed according to LC/MS. A solution
of hydroxylamine O-sulfonic acid (2.27 g, 20.0 mmol) and sodium
acetate (2.50 g, 30.0 mmol) in water (30 mL) was added, and the
reaction mixture was stirred at rt for 16 h. The mixture was extracted
with EtOAc (2 × 30 mL), dried (Na2SO4), and concentrated. Yield:
2.50 g (99%). MS: m/z 314 (M + 1).
(400 MHz, d6-DMSO): δ 1.23 (3H, t, J = 7.2 Hz), 2.48 (2H, m,
overlap with DMSO signal), 2.55 (3H, s), 2.95 (2H, t, J = 7.7 Hz),
3.45 (1H, m), 4.17 (4H, m), 4.34 (2H, m), 7.25−7.12 (5H, m), 8.23
(1H, s). MS: m/z 457 (M + 1).
Ethyl 5-Cyano-6-(3-{[(cyclohexylmethyl)sulfonyl]-
carbamoyl}azetidin-1-yl)-2-methylnicotinate (15). 15 was pre-
pared by the procedure of 13 from 1-cyclohexylmethanesulfonamide
(0.065 g, 0.37 mmol) and 7 (0.073 g, 0.25 mmol). Yield: 0.012 g
(11%). 1H NMR (400 MHz, d6-DMSO): δ 0.98−1.25 (8H, m), 1.60−
1.50 (3H, m), 1.74 (3H, m), 2.55 (3H, s), 3.26 (2H, d, J = 6.0 Hz),
3.58 (1H, m), 4.17 (2H, q, J = 7.1 Hz), 4.28 (2H, m), 4.41 (2H, m),
8.23 (1H, s). MS: m/z 449 (M + 1).
Ethyl 6-(3-{[(2-Methylbenzyl)sulfonyl]carbamoyl}azetidin-1-
yl)-5-cyano-2-methylnicotinate (16). 16 was prepared by the
procedure of 13 from 1-(2-methylphenyl)methanesulfonamide (0.010
1
g, 0.050 mmol) and 7 (0.073 g, 0.25 mmol). Yield: 0.002 g (2%). H
NMR (400 MHz, d6-DMSO): δ 1.24 (3H, t, J = 7.2 Hz), 2.32 (3H, s),
2.57 (3H, s), 3.40 (1H, m; overlapped by signal from water), 4.18 (2H,
m), 4.30 (2H, m), 4.39 (2H, m), 4.63 (2H, s), 7.15 (4H, m), 8.24 (1H,
s). MS: m/z 457 (M + 1).
Ethyl 5-Cyano-2-methyl-6-(3-{[[3-(trifluoromethyl)benzyl]-
sulfonyl]carbamoyl}azetidin-1-yl)nicotinate (17). 17 was pre-
pared by the procedure of 13 from (3-(trifluoromethyl)phenyl)-
methanesulfonamide (0.10 g, 0.38 mmol) and 7 (0.073 g, 0.25 mmol).
Yield: 0.050 g (39%). 1H NMR (400 MHz, d6-DMSO): δ 1.24 (3H, t,
J = 7.1 Hz), 2.57 (3H, s), 3.48 (1H, m), 4.18 (4H, m), 4.35 (2H, t, J =
8.8 Hz), 4.78 (2H, s), 7.57 (3H, m), 7.69 (1H, d, J = 6.6 Hz), 8.24
(1H, s). MS: m/z 511 (M + 1).
A mixture of 7 (0.100 g, 0.34 mmol), {4-[[(tert-butyldimethylsilyl)-
oxy]methyl]phenyl}methanesulfonamide (0.130 g, 0.41 mmol),
PyBrop (0.241 g, 0.52 mmol), and DIPEA (0.445 g, 0.60 mL, 3.45
mmol) in DCM (4.5 mL) was stirred at rt for 1 h. Water (4.5 mL) was
added, and the organic phase was separated and concentrated. Yield:
0.119 g (57%).
Ethyl 5-Cyano-2-methyl-6-(3-{[(3-methylbenzyl)sulfonyl]-
carbamoyl}azetidin-1-yl)nicotinate (18). 18 was prepared by the
procedure of 13 from (3-methylphenyl)methanesulfonamide (0.037 g,
1
0.20 mmol) and 7 (0.073 g, 0.25 mmol). Yield: 0.019 g (15%). H
NMR (400 MHz, d6-DMSO): δ 1.32 (3H, t, J = 7.1 Hz), 2.33 (3H, s),
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The crude was dissolved in TFA, and the reaction mixture was
stirred at rt for 15 min. Yield: 0.018 g (19%). 1H NMR (400 MHz, d6-
DMSO): δ 1.30 (3H, t, J = 7.1 Hz), 2.63 (3H, s), 3.34−3.45 (1H, m),
4.24 (2H, q, J = 7.1 Hz), 4.27−4.34 (2H, m), 4.34−4.43 (2H, m), 4.47
(2H, d, J = 5.4 Hz), 4.48−4.56 (2H, m), 5.16 (1H, t, J = 5.6 Hz), 7.24
(4H, s), 8.29 (1H, s), 11.69−11.91 (1H, m). MS: m/z 473 (M + 1).
Ethyl 5-Chloro-6-[4-({[(5-chloro-2-thienyl)sulfonyl]amino}-
carbonyl)piperidin-1-yl]nicotinate (23). 23 was prepared by the
procedure of 10 from 8 (0.250 g, 0.80 mmol) and 5-chlorothiophene-
Ethyl 6-[4-({[(2,4-Difluorobenzyl)sulfonyl]amino}carbonyl)-
piperidin-1-yl]-5-cyano-2-methylnicotinate (29). 29 was pre-
pared by the procedure of 25 from 9 (0.032 g, 0.10 mmol) and 1-(2,4-
difluorphenyl)methanesulfonamide (0.023 g, 0.11 mmol). Yield: 0.018
g (34%). 1H NMR (400 MHz, d6-DMSO): δ 1.31 (3H, t, J = 7.1 Hz),
1.59−1.69 (2H, m), 1.84−1.92 (2H, m), 2.65 (4H, m), 3.10−3.25
(2H, m), 4.25 (2H, q, J = 7.1 Hz), 4.49−4.60 (2H, m), 4.73 (2H, s),
7.13−7.23 (1H, m), 7.29−7.39 (1H, m), 7.40−7.50 (1H, m), 8.34
(1H, s), 11.75 (1H, s). MS: m/z 507 (M + 1).
Ethyl 6-[4-({[(5-Chloro-2-thienyl)sulfonyl]amino}carbonyl)-
piperidin-1-yl]-5-cyano-2-methylnicotinate (32). A mixture of
30 (7.00 g, 30.5 mmol), EDC (7.02 g, 36.6 mmol), and HOBT (4.95
g, 36.6 mmol) in DCM (200 mL) was stirred at rt for 30 min. 5-
Chlorothiophene-2-sulfonamide (7.54 g, 38.2 mmol) and DIPEA
(16.0 mL, 91.6 mmol) were added, and the reaction mixture was
stirred at rt for 20 h. The mixture was diluted with DCM (500 mL),
washed with NH4Cl (3 × 200 mL, saturated, aq solution), dried
(MgSO4), and concentrated. The crude was purified by flash
chromatography (EtOAc/hexanes (1:3), 1% AcOH). Yield: 11.3 g
(90%) as a solid. 1H NMR (400 MHz, CDCl3): δ 1.45 (9H, s), 1.55−
1.65 (2H, m), 1.79−1.82 (2H, m), 2.92−2.37 (1H, m), 2.73−2.80
(2H, m), 4.06−4.11 (2H, m), 6.96 (1H, d, J = 4.1 Hz), 7.69 (1H, d, J =
4.1 Hz), 8.11 (1H, br s).
A suspension of tert-butyl 4-{[(5-chlorothiophene-2-yl)sulfonyl]-
carbamoyl}piperidine-1-carboxylate (11.3 g, 27.6 mmol) in THF (500
mL) was treated with HCl (4 M in 1,4-dioxane, 138 mL, 552 mmol),
and the reaction mixture was stirred at rt for 20 h and then
concentrated. Yield: 9.52 g (100%) as a solid. 1H NMR (400 MHz, d6-
DMSO): δ 1.58−1.68 (2H, m), 1.87−1.90 (2H, m), 2.52−2.59 (1H,
m), 2.80−2.88 (2H, m), 3.22−3.25 (2H, m), 7.29 (1H, d, J = 4.1 Hz),
7.67 (1H, d, J = 4.1 Hz), 8.51 (1H, br s), 8.82 (1H, br s). MS: m/z 309
(M + 1).
Compound 5 (0.20 g, 0.89 mmol), N-(5-chlorothiophene-2-
ylsulfonyl)piperidine-4-carboxamide, hydrochloride (0.40 g, 1.3
mmol), and DIPEA (0.46 g, 0.62 mL, 3.6 mmol) were dissolved in
DMA (2 mL). The reaction mixture was heated at 160 °C for 30 min.
The mixture was cooled, diluted with EtOAc (75 mL), washed with
NH4Cl (2 × 40 mL, saturated, aq solution) and brine (40 mL), dried
(MgSO4), and concentrated. Yield: 0.198 g (45%) as a white solid. 1H
NMR (400 MHz, d6-DMSO): δ 1.30 (3H, t, J = 7.1 Hz), 1.50−1.59
(4H, m), 1.80 (2H, d, J = 11.0 Hz), 2.42−2.56 (1H, m), 2.63 (3H, s),
3.15 (2H, d, J = 11.9 Hz), 4.24 (2H, q, J = 7.1 Hz), 4.49 (2H, d, J =
13.5 Hz), 7.28 (1H, d, J = 4.1 Hz), 7.67 (1H, d, J = 4.1 Hz), 8.32 (1H,
s). MS: m/z 497 (M + 1).
1
2-sulfonamide (0.316 g, 1.60 mmol). Yield: 0.095 g (24%). H NMR
(400 MHz, CDCl3): δ 1.38 (3H, t, J = 7.1 Hz), 1.83−1.98 (4H, m),
2.42−2.50 (1H, m), 2.92−2.98 (2H, m), 4.09−4.13 (2H, m), 4.36
(2H, q, J = 7.1 Hz), 6.97 (1H, d, J = 4.1 Hz), 7.71 (1H, d, J = 4.1 Hz),
8.12 (1H, d, J = 1.7 Hz), 8.73 (1H, d, J = 1.7 Hz). MS: m/z 492 (M +
1).
Ethyl 5-Cyano-2-methyl-6-[4-[(phenylsulfonyl)carbamoyl]-
piperidin-1-yl]nicotinate (24). A mixture of 9 (0.067 g, 0.21
mmol), TBTU (0.080 g, 0.25 mmol), and DIPEA (0.136 g, 183 mL,
1.05 mmol) in DCM (2 mL) was stirred at rt for 10 min.
Benzenesulfonamide (0.039 g, 0.25 mmol) was added, and the
reaction mixture was stirred at rt for 16 h and then washed with
KHSO4 (2 mL, 0.1 M aq solution), separated, and concentrated. The
1
crude was purified by preparative HPLC. Yield: 0.047 g (49%). H
NMR (400 MHz, d6-DMSO): δ 1.29 (3H, t, J = 7.1 Hz), 1.38−1.54
(2H, m), 1.78−1.87 (2H, m), 2.47−2.54 (1H, m), 2.61 (3H, s), 3.07−
3.19 (2H, m), 4.24 (2H, q, J = 7.1 Hz), 4.40−4.52 (2H, m), 7.54−7.76
(3H, m), 7.90 (2H, d, J = 7.5 Hz), 8.31 (1H, s), 12.13−12.25 (1H, m).
MS: m/z 457 (M + 1).
Ethyl 6-[4-({[(4-Fluorobenzyl)sulfonyl]amino}carbonyl)-
piperidin-1-yl]-5-cyano-2-methylnicotinate (25). A mixture of 9
(0.350 g, 1.10 mmol), EDC (0.274 g, 1.43 mmol), HOBt (0.194 g,
1.43 mmol), and DIPEA (0.713 g, 5.51 mmol) in DCM (8 mL) was
stirred at rt for 30 min, and 1-(4-fluorophenyl)methanesulfonamide
(0.271 g, 1.43 mmol) was added. Stirring at rt was continued for 16 h.
KHSO4 (0.5 M aq solution, 2 mL) was added, and the aq phase was
extracted with DCM. Combined organic phases were concentrated to
give a crude which was purified by preparative HPLC. Yield: 0.429 g
1
(80%). H NMR (400 MHz, d6-DMSO): δ 1.30 (3H, t, J = 7.2 Hz),
1.56−1.69 (2H, m), 1.80−1.88 (2H, m), 2.57 (1H, m), 2.64 (3H, s),
3.13 (2H, m), 4.24 (2H, q, J = 7.2 Hz), 4.53 (2H, m), 4.68 (2H, s),
7.20−7.27 (2H, m), 7.30−7.35 (2H, m), 8.33 (1H, s), 11.60 (1H, br
s).
Ethyl 5-Cyano-6-[4-({[(4-methoxybenzyl)sulfonyl]amino}-
carbonyl)piperidin-1-yl]-2-methylnicotinate (26). 26 was pre-
pared by the procedure of 25 from 9 (0.040 g, 0.13 mmol) and 1-(4-
methoxyphenyl)methanesulfonamide (0.032 g, 0.16 mmol). Yield:
0.034 g (45%). 1H NMR (400 MHz, d6-DMSO): δ 1.32 (3H, d, J = 7.1
Hz), 1.55−1.68 (2H, m), 1.77−1.86 (2H, m), 2.27−2.36 (1H, m),
2.66 (3H, s), 3.16−3.25 (2H, m), 3.74 (3H, s), 4.22−4.30 (3H, m),
4.42−4.51 (2H, m), 6.85 (2H, br d, J = 8.5 Hz), 7.16 (2H, br d, J = 8.5
Hz), 8.33 (1H, s). MS: m/z 523 (M + 1).
ASSOCIATED CONTENT
* Supporting Information
■
S
Description of or references for the screening assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Ethyl 6-[4-({[(4-Methylbenzyl)sulfonyl]amino}carbonyl)-
piperidin-1-yl]-5-cyano-2-methylnicotinate (27). 27 was pre-
pared by the procedure of 25 from 9 (1.00 g, 3.15 mmol) and 1-(4-
methylphenyl)methanesulfonamide (0.67 g, 3.62 mmol). Yield: 0.69 g
■
*Phone: +46 738208612 (P.B.); +46 317761337 (F.Z.). Fax:
+46 317721394 (P.B.); +46 317763710 (F.Z.). E-mail: bach@
chalmers.se (P.B.); fredrik.zetterberg@astrazeneca.com (F.Z.).
1
(45%). H NMR (400 MHz, d6-DMSO): δ 1.30 (3H, t, J = 7.1 Hz),
1.56−1.68 (2H, m), 1.79−1.87 (2H, m), 2.29 (3H, s), 2.41−2.60 (1H,
m), 2.64 (3H, s), 3.09−3.18 (2H, m), 3.29 (1H, s), 4.24 (2H, q, J = 7.1
Hz), 4.48−4.56 (2H, m), 4.59 (2H, s), 7.13−7.21 (4H, m), 8.33 (1H,
s). MS: m/z 485 (M + 1).
Notes
The authors declare no competing financial interest.
Ethyl 6-[4-({[(4-Isopropylbenzyl)sulfonyl]amino}carbonyl)-
piperidin-1-yl]-5-cyano-2-methylnicotinate (28). 28 was pre-
pared by the procedure of 25 from 9 (0.159 g, 0.50 mmol) and 1-(4-
isopropylphenyl)methanesulfonamide (0.128 g, 0.60 mmol). Yield:
0.144 g (56%). 1H NMR (400 MHz, d6-DMSO): δ 1.18 (6H, d, J = 6.8
Hz), 1.32 (3H, t, J = 7.1 Hz), 1.54−1.67 (2H, m), 1.75−1.85 (2H, m),
2.20−2.30 (1H, m), 2.65 (3H, s), 2.80−2.89 (1H, m), 3.18−3.28 (2H,
m), 4.19 (2H, s), 4.26 (2H, q, J = 7.1 Hz), 4.38−4.47 (2H, m), 7.09−
7.18 (4H, m), 8.32 (1H, s). MS: m/z 513 (M + 1).
ACKNOWLEDGMENTS
■
We are grateful to Britt-Marie Wissing, Pia Berntsson, and
Maria Erichsson for performing screening experiments, to
Petter Svanberg and Maria Strimfors for in vivo experiments, to
Marie Ryden
NMR structure elucidation, to Kay Brickmann, Annika U.
Petersson, Johan Johansson, Kosrat Amin, Mikael Sellen, Lotta
́
-Landegren and Gunnar Gronberg for help with
̈
́
Hidestal, Laurence E. Burgess, David Clarke, Robert D.
̊
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Journal of Medicinal Chemistry
Article
istics and antithrombotic and bleeding effects in rat and dog models of
thrombosis/hemostasis. Thromb. Res. 2009, 124, 565−571.
Groneberg, Darren M. Harvey, Ellen R. Laird, Michael
O’Sullivan, and Jonas Bostrom for the medicinal chemistry
̈
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Leeson, P. D.; Leff, P.; Lewis, R. J.; Martin, B. P.; McGinnity, D. F.;
Mortimore, M. P.; Paine, S. W.; Pairaudeau, G.; Patel, A.; Rigby, A. J.;
Riley, R. J.; Teobald, B. J.; Tomlinson, W.; Webborn, P. J. H.; Willis, P.
A. From ATP to AZD6140: The discovery of an orally active reversible
P2Y12 receptor antagonist for the prevention of thrombosis. Bioorg.
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work leading up to and following the content of this paper, and
to Christina Olsson for proofreading.
ABBREVIATIONS USED
■
aze, azetidinyl; br, broad; BEMP, 2-(tert-butylimino)-2-
(diethylamino)-1,3-dimethylperhydro-1,3,2-diazaphosphorine;
CDI, N,N′-carbonyldiimidazole; CFR , cyclic flow reduction;
CHO, chinese hamster ovarian; CLint, intrinsic clearance;
DIPEA, N,N-diisopropylethylamine; DLMs, dog liver micro-
somes; DMAP, 4-(dimethylamino)pyridine; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide hydrochloride; GTPγS,
guanosine 5′-O-[γ-thio]triphosphate; HATU, O-(7-azabenzo-
triazol-1-yl)-1,1,3,3-tetramethyluromium hexafluorophosphate;
HLMs, human liver microsomes; HOBT, 1-hydroxybenzotria-
zole; log D, logarithm of the distribution coefficient; MW,
single-node heating in a microwave oven; pip, piperidinyl;
PyBroP, bromotrispyrrolidinophosphonium hexafluorophos-
phate; pyrr, pyrrolidinyl; RLMs, rat liver microsomes; RPC,
residual platelet count; TBME, tert-butyl methyl ether; TBTU,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetra-
fluoroborate; TEA, triethylamine; WPA, washed platelet assay
(10) (a) Parlow, J. J.; Burney, M. W.; Case, B. L.; Girard, T. J.; Hall,
K. A.; Harris, P. K.; Hiebsch, R. R.; Huff, R. M.; Lachance, R. M.;
Mischke, D. A.; Rapp, S. R.; Woerndle, R. S.; Ennis, M. D. Piperazinyl
glutamate pyridines as potent orally bioavailable P2Y12 antagonists for
inhibition of platelet aggregation. J. Med. Chem. 2010, 53, 2010−2037.
(b) Parlow, J. J.; Burney, M. W.; Case, B. L.; Girard, T. J.; Hall, K. A.;
Harris, P. K.; Hiebsch, R. R.; Huff, R. M.; Lachance, R. M.; Mischke,
D. A.; Rapp, S. R.; Woerndle, R. S.; Ennis, M. D. Part II: Piperazinyl-
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of platelet aggregation. Bioorg. Med. Chem. Lett. 2009, 19, 4657−4663.
(d) Parlow, J. J.; Burney, M. W.; Case, B. L.; Girard, T. J.; Hall, K. A.;
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