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1. INTRODUCTION
Article
Chart 1. Structures of 1 (Ticagrelor), 2 (Elinogrel), and 3 (BX 667) and Their Respective Anity in the P2Y12 Binding Assay
and Antiaggregatory Activity in the Human Platelet-Rich Plasma (hPRP) Assay Used Throughout This Investigation (See the
Experimental Section)
Figure 1. (a) Binding hypothesis of quinoline 3 in the P2Y12 receptor obtained from docking into a receptor homology model. (b) Threedimensional alignment of quinoline 3 and phenylpyrazolone 4.
Article
Figure 2. (a) Schematic representation of the regions to be investigated based on the phenylpyrazolone backbone. (b) Proposed binding mode of
pyrazole 4 into the P2Y12 homology model.
3. CHEMISTRY
Scheme 1 shows the synthesis of representative building blocks,
which were sequentially attached to the 3-carboxyphenylpyrazole, enabling a highly convergent and exible synthesis of the
nal ligands. The glutamic piperazinyl carbamate building block
7 was prepared from the commercially available Z-O-tert-butyl
protected 5 in two steps by standard amide coupling using O((ethoxycarbonyl)cyanomethyleneamino)-N,N,N,N-tetramethyluronium tetrauoroborate (TOTU) followed by the
hydrogenolytic removal of the CbZ-protecting group (sequence
a). The second key intermediate used throughout this
investigation was the fully decorated phenylpyrazole building
block 10 (sequence b). This fragment was synthesized by
subjecting the phenylpyrazole ester 8 to an O-alkylation with
benzyl 2-bromoacetate followed by selective hydrogenolytic
cleavage of the benzylester to give 9. This intermediate was
then converted to the acid 10 by a high-yielding sequence of
amide coupling and deprotection steps.
The syntheses of the fully decorated nal phenylpyrazolone
ligands with dierent substituents attached to the 5-position of
the pyrazole and all other side chain variations were carried out
by two optional routes starting from commercially available 3carboxy phenyl pyrazolones 8 and 11 (Scheme 2).
Phenylpyrazolone variations keeping the piperazinyl side
chain constant were synthesized according to route 1.
Phenylpyrazolones of type 14 were prepared by using standard
amide coupling conditions with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC)/1hydroxybenzotriazole (HOBt) followed by O-alkylation of the
phenylpyrazole with benzyl 2-bromoacetate in the presence of
cesium carbonate in DMF and hydrogenolytic deprotection to
the carboxylic acid 12. The diversifying amide variation step
was then conducted using EDC/HOBt or O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexauorophosphate
(HATU) in DMF as activating reagents.
Route 2 was used to explore the impact of other side chain
variations retaining the 5-O-alkyl amide substituent. The
preassembled phenylpyrazole 15 and the piperazinyl side
chain 16 were prepared according to the synthetic sequences
outlined for the respective building blocks (Scheme 1, synthesis
of 7 and 10) and then coupled using either EDC/HOBt or
HATU activation in DMF to give the nal derivatives 14. By
using these synthetic routes, we were able to systematically
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Reagents and conditions: (a) EDC, HOBt, EtNiPr2, 16, DMF, room
temp. (b) Benzyl 2-bromoacetate, Cs2CO3, DMF, room temp. (c) H2
(3 bar), Pd/C (10%), EtOAc, room temp. (d) EDC, HOBt, EtNiPr2,
DMF, room temp or HATU, EtNiPr2, DMF, room temp. (e) NaOH,
H2O/THF, room temp.
P2Y12 binding assay using cell membranes from CHO cells with
recombinant expression of human P2Y12 receptors employing [33P]2MeS-ADP as the radioligand. bAntiagreggatory eect on ADPstimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated hPRP according to the Born
method using single cuvettes.
Article
Table 2. Binding and hPRP Activity of Compounds Carrying Various Proline Variations
P2Y12 binding assay using cell membranes from CHO cells with recombinant expression of human P2Y12 receptors employing [33P]2-MeS-ADP as
the radioligand. bAntiagreggatory eect on ADP-stimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated
hPRP according to the Born method using single cuvettes.
Article
Table 3. Binding and hPRP Activity of Variations around the Piperazine Ethyl Carbamate
P2Y12 binding assay using cell membranes from CHO cells with recombinant expression of human P2Y12 receptors employing [33P]2-MeS-ADP as
the radioligand. bAntiagreggatory eect on ADP-stimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated
hPRP according to the Born method using single cuvettes.
Article
optimal trajectorial angle presenting the cyclobutyl carboxamide moiety as in compounds 18n or 18o turned out to be
deleterious for the activity [Ki (P2Y12): 238 and 613 nM; IC50
(hPRP): 3.13 and 2.94 M]. Next, we explored compounds
bearing a cyclic amide variation of increasing ring size. Again,
even small, incremental changes from the moderately active
pyrrolidine carboxamide 18p to the piperidine carboxamide
18q were sucient to obtain an acceptable binding anity and
potency in hPRP [Ki (P2Y12): 219 vs 56 nM; IC50 (hPRP):
0.60 vs 0.070 M]. The attempted bioisosteric replacement of
the carboxamide group with an imidazole moiety resulted in the
reasonably active compound 18r. Notably, the profound impact
of the lipophilic interaction with the receptor at this region was
also demonstrated when the entire alkyl carboxamide was
omitted and replaced by a phenyl ring. Despite the relatively
large structural change, this compound 18s displayed an
unexpectedly high binding anity and hPRP activity as
compared to several other more conservative variations of the
proline cyclobutyl amide motif [Ki (P2Y12): 33 nM; IC50
(hPRP): 0.29 M].
These results indicate that the 5-position of the pyrazole ring
directs the substituent to a lipophilic pocket, which exhibits a
steric connement, as illustrated by the signicant drop of
activity, if the ideal geometry is violated. A more concise
structure-based rationalization of the observed SAR is provided
in the section Development of 3D QSAR Models.
The results in Table 2 established the structural requirements
for the decoration of the 5-position of the phenylpyrazole with
the N-acetyl-(S)-proline cyclobutyl amide residue as the
optimal substituent. We next turned our attention toward a
potential optimization of the piperazine carbamate portion of
our preliminary lead compound (Table 3). Our docking
hypothesis (Figure 1) proposed that this substituent points to
the extracellular site of the receptor and establishes interactions
with residues from the ECs. Because we considered the
accuracy of our protein model in this region to be very low, a
reliable structure-based rationalization of the piperazine
carbamate SAR was not feasible. Nevertheless, the preference
for lipophilic residues of a certain length (e.g., butyl
substituents) was captured by the steric elds of the CoMSIA
model (see Figure 3). Introducing a 4-amino piperidine group
as a piperazine homologue resulted in the considerably less
active compound 19a (Table 3). Interestingly, 19b incorporating the inverse 4-amino piperidine spacer exhibited a higher
binding anity to the receptor and had a much higher activity
in hPRP than 19a [Ki (P2Y12): 22 vs >5000 nM; IC50 (hPRP):
0.055 vs >5.0 M]. The same trend was also observed for the
related analogues, the corresponding 3-amino pyrrolidine
compounds 19c and 19d, which conrmed that a C-terminal
attachment of the glutamic acid to a secondary amine is crucial
for high receptor anity. Any eort to replace the alkyl
carbamate unit by more rigid N-aryl piperazines like in 19e or
N-acyl piperazines like in 19g was unsuccessful and did not lead
to compounds of higher binding anity or hPRP activity. In
contrast, very conservative variations of the alkyl carbamate
moiety turned out to be most eective. Simple alkyl extension
from ethyl (18i) to n-propyl (19j) and n-butyl (19k) provided
derivatives that showed the highest binding anities and an
extraordinarily good hPRP activity [Ki (P2Y12): 3.1 and 0.8 nM;
IC50 (hPRP): 0.021 and 0.019 M]. An equal potency was
found for the cyclobutyl derivative 19l [Ki (P2Y12): 5.0 nM;
IC50 (hPRP): 0.014 M]. In an attempt to rigidify and restrict
the conformational exibility of the piperazine spacer, the
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18i
20n
20o
139 nM
>20 M
0.701 M
2.93 M
>2 M
>2 M
7.3 nM
>20 M
0.007 M
0.009 M
>2 M
>2 M
107 nM
ND
0.099 M
0.100 M
>2 M
>2 M
7.7 nM
>20 M
0.022 M
0.046 M
>2 M
>2 M
P2Y12 binding assay using cell membranes from CHO cells with
recombinant expression of human P2Y12 receptors employing [33P]2MeS-ADP as the radioligand. bP2Y1 binding assay using cell
membranes from CHO cells with recombinant expression of human
P2Y1 receptors employing [33P]2-MeS-ADP as the radioligand.
c
Antiagreggatory eect on ADP-stimulated human IPs according to
the Born method using single cuvettes. dAntiagreggatory eect on
ADP-stimulated hPRP according to the Born method using single
cuvettes. eAntiagreggatory eect on collagen (col)-stimulated human
IP according to the Born method using single cuvettes. fAntiagreggatory eect on thrombin (thr)-stimulated human IP according to the
Born method using single cuvettes.
P2Y12 binding assay using cell membranes from CHO cells with
recombinant expression of human P2Y12 receptors employing [33P]2MeS-ADP as the radioligand. bAntiagreggatory eect on ADPstimulated hPRP using a 96-well format. cIn parentheses: Antiagreggatory eect on ADP-stimulated hPRP according to the Born
method using single cuvettes.
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5. CONCLUSION
We have developed a series of highly active P2Y12 antagonists
based on a phenylpyrazolone scaold. Investigation of the
structural requirements at three dierent regions generated a
set of structural diverse P2Y12 antagonists. Notably, noncharged
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7.4 Hz, 2H), 3.52 (m, 4H), 3.39 (m, 4H), 2.27 (m, 2H), 1.80 (m, 1H),
1.67 (m, 1H), 1.39 (s, 9H), 1.19 (t, J = 7.4 Hz, 3H). tR (method j):
1.52 min. MS (ES) m/z: 478.3 (MH+).
6.1.1.2. 4-((S)-2-Amino-4-tert-butoxycarbonyl-butyryl)-piperazine-1-carboxylic Acid Ethyl Ester (7). To a solution of 4-((S)-2benzyloxycarbonylamino-4-tert-butoxycarbonyl-butyryl)-piperazine-1carboxylic acid ethyl ester (20.9 g, 43.9 mmol) in ethanol (120 mL)
was added Pd/C (2.0 g, 10% w/w), and the suspension was stirred
under an atmosphere of hydrogen (3 bar) for 12 h. The reaction
mixture was ltrated over a plug of Celite, washed with ethanol, and
concentrated to give the pure product 7 (13.6 g, 89%) as a colorless
oil, which was not further puried. 1H NMR (DMSO-d6): 4.06 (q, J
= 7.4 Hz, 2H), 3.57 (m, 1H), 3.49 (m, 4H), 3.37 (m, 4H), 2.30 (m,
2H), 1.65 (m, 2H), 1.39 (s, 9H), 1.19 (t, J = 7.4 Hz, 3H). tR (method
j): 0.82 min. MS (ES) m/z: 344.3 (MH+).
6.1.2. 5-Carboxymethoxy-1-phenyl-1H-pyrazole-3-carboxylic
Acid Ethyl Ester (9). 6.1.2.1. 5-Benzyloxycarbonylmethoxy-1phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a solution of
5-hydroxy-1-phenyl-1H-pyrazole-3-carboxylic acid ethyl ester (8)
(10.0 g, 43.1 mmol) in DMF (100 mL) were added benzyl
bromoacetate (7.1 mL, 43.1 mmol) and cesium carbonate (24.0 g,
86.1 mmol). After it was stirred for 16 h at room temperature, the
suspension was ltered, washed with DMF, and concentrated. The
residue was taken up with ethyl acetate and extracted with aqueous
LiCl (4% w/w). Evaporation of the solvent yielded the crude product
as a yellow oil (15.5 g, 95%), which was used in the subsequent
debenzylation reaction. tR (method j): 1.64 min. MS (ES) m/z: 381.2
(MH+).
6.1.2.2. 5-Carboxymethoxy-1-phenyl-1H-pyrazole-3-carboxylic
Acid Ethyl Ester (9). To a solution of 5-benzyloxycarbonylmethoxy1-phenyl-1H-pyrazole-3-carboxylic acid ethyl ester (15.5 g, 40.8 mmol)
in ethyl acetate (100 mL) was added Pd/C (1 g, 10%), and the
resulting suspension stirred under an atmosphere of hydrogen (3 bar)
for 5 h. The reaction mixture was ltered over a plug of Celite and
washed with ethyl acetate and methanol to give the crude product 9 as
colorless platelets (11.5 g, 98%) after evaporation of the solvents. 1H
NMR (DMSO-d6): 13.28 (s br, 1H), 7.74 (d, J = 7.9 Hz, 2H), 7.54
(t, J = 7.9 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 6.42 (s, 1H), 4.93 (s, 2H),
4.29 (q, J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H). tR (method j): 1.11
min. MS (ES) m/z: 291.2 (MH+).
6.1.3. Preparation of 5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid (10).
6.1.3.1. 5-[2-((S)-2-Benzyloxycarbonyl-pyrrolidin-1-yl)-2-oxoethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a
solution of 5-carboxymethoxy-1-phenyl-1H-pyrazole-3-carboxylic acid
ethyl ester (9) (9.5 g, 32.8 mmol) in DMF (100 mL) were added
HOBt (5.0 g, 32.8 mmol), EDC (6.3 g, 32.8 mmol), and DIPEA (10.9
mL, 65.7 mmol). After 5 min, L-proline benzyl ester hydrochloride (7.9
g, 32.8 mmol) was added, and the resulting solution was stirred for 16
h. The reaction mixture was concentrated, dissolved in dichloromethane, and extracted with aqueous LiCl (4% w/w) and saturated
NaHCO3. The crude product obtained after evaporation of the solvent
was puried by ash chromatography on silica using ethyl acetate/
heptane 1:1 as an eluent to give the product as colorless foam (13.2 g,
84%). tR (method j): 1.55 min. MS (ES) m/z: 478.3 (MH+).
6.1.3.2. 5-[2-((S)-2-Carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a solution of 5-[2((S)-2-benzyloxycarbonyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl1H-pyrazole-3-carboxylic acid ethyl ester (13.2 g, 27.6 mmol) in ethyl
acetate (100 mL) was added Pd/C (1 g, 10%), and the resulting
suspension was stirred under an atmosphere of hydrogen (3 bar) for 6
h. The reaction mixture was ltered over a plug of Celite and washed
with ethanol to give the crude product as colorless foam (8.9 g, 70%)
after evaporation of the solvents. 1H NMR (DMSO-d6): 12.61 (s br,
1H), 7.83 (d, J = 8.4 Hz, 2H), 7.57 (t, J = 7.8 Hz, 2H), 7.45 (t, J = 7.8
Hz, 1H), 6.48 (s, appears as 2 rotamers, 1H), 5.12 (s, 2H), 4.32 (m,
3H), 3.55 (m, 2H), 2.15 (m, 1H), 1.89 (m, 3H), 1.28 (t, J = 7.0 Hz,
3H). tR (method j): 1.10 min. MS (ES) m/z: 388.2 (MH+).
6.1.3.3. 5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxoethoxy]-1-phenyl-1H-pyrazole-3-carboxylic Acid Ethyl Ester. To a
solution of 5-[2-((S)-2-carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phe-
6. EXPERIMENTAL SECTION
6.1. Chemistry. Solvents and other reagents were used as received
without further purication. Normal phase column chromatography
was carried out on Merck silica gel 60 (230400 mesh). Reversed
phase high-pressure chromatography was conducted on an Agilent
1100 instrument using an Agilent Prep. C18 column (10 m, 30 mm
250 mm). Thin-layer chromatography (TLC) was carried out on
TLC aluminum sheets with silica gel 60F254 from Merck. Varying
ratios of acetonitrile and 0.1% triuoroacetic acid in water were used as
solvent systems. NMR spectra were recorded in CD3OD, CDCl3, or
DMSO-d6 either on a Bruker DRX 400 or a Bruker FN-ARX 500.
Chemical shifts are reported as values from an internal
tetramethylsilane standard. All nal compounds were puried by
normal phase or reversed phase chromatography. Purity and
characterization of compounds were established by a combination of
analytical HPLC, LC-MS, and NMR analytical techniques. All tested
compounds were found to be >95% pure by analytical HPLC and LCMS analysis.
LC-MS analyses were performed with an Agilent HPLC Series
1100. The following HPLC methods were used to obtain the reported
retention times. Method a: YMC Jsphere column 33 mm 2 mm; 4
M; ow rate, 1.0 mL/min. Gradient: water + 0.05% triuoroacetic
acid:acetonitrile + 0.05% triuoroacetic acid: 98 (1 min) to 95:5 (5.0
min) to 95:5 (6.25 min); Waters LCT-MUX, ESI+ mode. Method b:
YMC Jsphere column 33 mm 2 mm; 4 m; ow rate, 1.0 mL/min.
Gradient: water + 0.05% triuoroacetic acid:acetonitrile + 0.05%
triuoroacetic acid 95:5 (0 min) to 5:95 (3.7 min); Waters LCTMUX, ESI+ mode. Method c: Waters XBridge C18 column 4.6 mm
50 mm; 2.5 m; ow rate, 1.3 mL/min. Gradient: water + 0.1% formic
acid:acetonitrile + 0.08% formic acid 97:3 (0 min) to 40:60 (3.5 min)
to 2:98 (4 min) to 2:98 (5 min) to 97:3 (5.2 min) to 97:3 (6.5 min);
Waters Quattro Ultima, ESI+ mode. Method d: Waters XBridge C18
column 4.6 mm 50 mm; 2.5 m; ow rate, 1.3 mL/min. Gradient:
water + 0.05% triuoroacetic acid:acetonitrile + 0.05% triuoroacetic
acid 95:5 (0 min) to 95:5 (0.3 min) to 5:95 (3.5 min) to 5:95 (4 min);
Waters LCT, ESI+ mode. Method e: YMC Jsphere column 33 mm 2
mm; 4 m; ow rate, 1.3 mL/min. Gradient: water + 0.05%
triuoroacetic acid:acetonitrile + 0.05% triuoroacetic acid 95:5 (0
min) to 5:95 (2.5 min) to 95:5 (3.2 min); Waters LCT, ESI+ mode.
Method f: YMC Jsphere column 33 mm 2 mm; 4 m; ow rate, 1.0
mL/min. Gradient water + 0.05% triuoroacetic acid:AcN + 0.05%
triuoroacetic acid 95:5 (0 min) to 5:95 (3.7 min); Waters LCTMUX, ESI+ mode. Method g: YMC Jsphere column 33 mm 2 mm;
4 m; ow rate, 1.3 mL/min. Gradient: water + 0.05% formic
acid:acetonitrile + 0.05% formic acid 95:5 (0 min) to 95:5 (0.5 min) to
5:95 (3.5 min) to 5:95 (4 min); Waters LCT, ESI+ mode. Method h:
YMC Jsphere column 33 mm 2 mm; 4 m; ow rate, 1.3 mL/min.
Gradient: water + 0.05% formic acid:acetonitrile + 0.05% formic acid
95:5 (0 min) to 5:95 (2.5 min) to 5:95 (3 min); Waters LCT, ESI+
mode. Method i: YMC Jsphere column 33 mm 2 mm; 4 m; ow
rate, 1.3 mL/min. Gradient: water + 0.1% formic acid:acetonitrile +
0.08% formic acid 95:5 (0 min) to 5:95 (2.5 min); Waters Quattro
Ultima, ESI+ mode. Method j: YMC Jsphere column 20 mm 2 mm;
4 m; ow rate, 1.0 mL/min. Gradient: water + 0.05% triuoroacetic
acid:acetonitrile + 0.05% triuoroacetic acid 94:6 (0 min) to 5:95 (2.4
min) to 94:6 (2.45 min); Agilent series 1100 MSD, ESI+ mode.
6.1.1. 4-((S)-2-Amino-4-tert-butoxycarbonyl-butyryl)-piperazine1-carboxylic Acid Ethyl Ester (7). 6.1.1.1. 4-((S)-2-Benzyloxycarbonylamino-4-tert-butoxycarbonyl-butyryl)-piperazine-1-carboxylic
Acid Ethyl Ester. To a solution of (S)-2-benzyloxycarbonylaminopentanedioic acid 5-tert-butyl ester (5) (15.0 g, 44.5 mmol) in DMF
(75 mL) were added 1-ethoxycarbonylpiperazine (6) (6.9 mL, 44.5
mmol), N-ethylmorpholine (22.6 mL, 177.8 mmol), and TOTU (14.6
g, 44.5 mmol). After it was stirred for 12 h, the solution was diluted
with ethyl acetate and subsequently washed with aqueous LiCl (4% w/
w) and saturated aqueous NaHCO3. The crude product (20.9 g, 99%)
obtained after evaporation of the solvent was directly subjected to the
subsequent deprotection reaction without further purication. 1H
NMR (DMSO-d6): 7.52 (d, J = 8.3 Hz, 1H), 7.34 (m, 5H), 5.04 (d, J
= 12.6 Hz, 1H), 4.99 (d, J = 12.6 Hz, 1H), 4.47 (m, 1H), 4.06 (q, J =
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(t, J = 7.4 Hz, 1H), 6.35 (s, 1H, appears as 2 rotamers), 4.95 (m, 4H),
4.29 (s, 1H, appears as 2 rotamers), 4.05 (q, J = 6.9 Hz, 2H), 3.44 (m,
12H), 2.29 (m, 2H), 1.96 (m, 2H), 1.84 (m, 2H), 1.19 (t, J = 6.8 Hz,
3H). tR (method e): 1.22 min. MS (ES) m/z: 501.2 (MH+).
6.1.7. Representative Procedure: Preparation of 4-[(S)-4-Carboxy2-({5-[2-((S)-2-methylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1phenyl-1H-pyrazole-3-carbonyl}-amino)-butyryl]-piperazine-1-carboxylic Acid Ethyl Ester (18b) via Route 1 (Scheme 2). 6.1.7.1. 4-[(S)2-({5-[2-((S)-2-Benzyloxycarbonyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1phenyl-1H-pyrazole-3-carbonyl}-amino)-4-tert-butoxycarbonyl-butyryl]-piperazine-1-carboxylic Acid Ethyl Ester. To a solution of 4{(S)-4-tert-butoxycarbonyl-2-[(5-carboxymethoxy-1-phenyl-1H-pyrazole-3-carbonyl)-amino]-butyryl}-piperazine-1-carboxylic acid ethyl
ester (9.5 g, 16.2 mmol) in DMF (80 mL) were added HOBt (2.5
g, 16.2 mmol), DIPEA (5.7 mL, 32.4 mmol), and L-proline benzyl
ester hydrochloride (3.9 g, 16.2 mmol) at 0 C. At this temperature
was added EDC (3.1 g, 16.2 mmol) portionwise, and the suspension
was allowed to warm to room temperature over a period of 12 h. The
solvent was evaporated, and the residue was dissolved in ethyl acetate
and subsequently extracted with aqueous LiCl (4% w/w), aqueous
HCl (0.1 M), and aqueous NaHCO3. The organic layer was dried over
MgSO4, and the solvent was removed under reduced pressure to give
the crude product as amorphous solid (11.6 g, 92%). An analytically
pure sample could be obtained by purication of an aliquot by
preparative HPLC. 1H NMR (DMSO-d6): 8.13 (d, J = 7.8 Hz, 1H),
7.84 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz, 2H), 7.40 (t, J = 7.3 Hz,
1H), 7.33 (m, 5H), 6.38 (s, appears as rotamers, 1H), 5.11 (m, 4H),
4.95 (m, 1H), 4.42 (dd, J = 9.0 Hz, J = 4.3 Hz, 1H), 4.05 (q, J = 7.3
Hz, 2H), 3.56 (m, 10H), 2.331.74 (several multiplets, 8H), 1.38 (s,
9H), 1.18 (t, J = 7.0 Hz, 3H). tR (method j): 1.71 min. MS (ES) m/z:
775.3 (MH+).
6.1.7.2. 4-[(S)-4-tert-Butoxycarbonyl-2-({5-[2-((S)-2-carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-butyryl]-piperazine-1-carboxylic Acid Ethyl Ester. To a
solution of 4-[(S)-2-({5-[2-((S)-2-benzyloxycarbonyl-pyrrolidin-1-yl)2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-4-tert-butoxycarbonyl-butyryl]-piperazine-1-carboxylic acid ethyl ester (11.6 g,
14.9 mmol) in ethyl acetate (75 mL) was added under argon Pd/C (1
g, 10%), and the suspension was stirred under an atmosphere of
hydrogen (3 bar) for 24 h. The suspension was ltered over a plug of
Celite and washed with ethyl acetate. The crude product was obtained
after evaporation of the solvent (9.9 g, 89%) and used without further
purication. 1H NMR (DMSO-d6): 12.56 (s br, 1H), 8.11 (d, J = 7.8
Hz, 1H), 7.85 (d, J = 7.3 Hz, 2H), 7.52 (t, J = 7.3 Hz, 2H), 7.39 (t, J =
7.3 Hz, 1H), 6.37 (s, appears as rotamers, 1H), 5.06 (s, 2H), 4.94 (m,
1H), 4.28 (dd, J = 9.0 Hz, J = 4.1 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H),
3.57 (m, 5H), 3.42 (m, 5H), 2.311.74 (several multiplets, 8H), 1.37
(s, 9H), 1.18 (t, J = 7.0 Hz, 3H). tR (method j): 1.40 min. MS (ES) m/
z: 685.3 (MH+).
6.1.7.3. 4-[(S)-4-Carboxy-2-({5-[2-((S)-2-methylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)butyryl]-piperazine-1-carboxylic Acid Ethyl Ester (18b). To a
solution of 4-[(S)-4-tert-butoxycarbonyl-2-({5-[2-((S)-2-carboxy-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-butyryl]-piperazine-1-carboxylic acid ethyl ester (133 mg, 0.19
mmol) in DMF (5 mL) were added DIPEA (80 L, 0.49 mmol),
HOBt (45 mg, 0.29 mmol), and EDC (56 mg, 0.29 mmol). After 20
min, methylamine hydrochloride (20 mg, 0.29 mmol) was added, and
the reaction mixture was stirred for 12 h. After dilution with ethyl
acetate, the reaction mixture was extracted with aqueous LiCl (4% w/
w) and aqueous NaHCO3. The organic layer was dried over MgSO4,
and the solvent was removed under reduced pressure. The crude
product was dissolved in dichloromethane (4 mL) and treated with
triuoroacetic acid (128 L). After this was stirred for 12 h, the
solvents were removed under reduced pressure, and the residue was
puried by preparative reversed-phase HPLC, eluting with a gradient
of acetonitrile in water (+0.01% triuoroacetic acid). After
lyophilization, the product 18b (46 mg, 37%) was obtained as a
white solid. 1H NMR (DMSO-d6): 8.13 (d, J = 8.4 Hz, 1H), 7.85 (d,
J = 8.0 Hz, 2H), 7.76 (m, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4
Hz, 1H), 6.41 (s, 1H), 5.00 (m, 3H), 4.22 (dd, J = 8.5 Hz, J = 3.2 Hz,
1H), 4.06 (q, J = 6.9 Hz, 2H), 3.51 (m, 10H), 2.55 (d, J = 4.6 Hz, 3H),
2.29 (m, 2H), 1.99 (m, 2H), 1.89 (m, 2H), 1.81 (m, 2H), 1.19 (t, J =
6.8 Hz, 3H). tR (method e): 1.25 min. MS (ES) m/z: 642.3 (MH+).
6.1.8. Representative Procedure: Preparation of 4-[(S)-4-Carboxy2-({5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]1-phenyl-1H-pyrazole-3-carbonyl}-amino)-butyrylamino]-piperidine-1-carboxylic Acid Ethyl Ester (19a) via Route 2 (Scheme 2).
6.1.8.1. (S)-2-({5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-pentanedioic Acid 5-tert-Butyl Ester 1-Methyl Ester. To a solution of 5-[2((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl1H-pyrazole-3-carboxylic acid (1.1 g, 2.7 mmol) in DMF (20 mL)
were added DIPEA (1 mL, 6.1 mmol), HOBt (0.41 g, 2.7 mmol),
EDC (0.51 g, 2.7 mmol), and H-Glu(OtBu)-OMe hydrochloride (0.68
g, 2.7 mmol). After it was stirred for 24 h, the solution was
concentrated, taken up with dichloromethane, and subsequently
extracted with aqueous LiCl (4% w/w), aqueous HCl (0.1 M), and
saturated NaHCO3. The crude product was puried by ash
chromatography on silica using an ethyl acetate/heptane 50:50 to
100:0 gradient to give the title compound as a colorless foam (1.88 g,
100%). tR (method j): 1.55 min. MS (ES) m/z: 612.3 (MH+).
6.1.8.2. (S)-2-({5-[2-((S)-2-Cyclobutylcarbamoyl-pyrrolidin-1-yl)-2oxo-ethoxy]-1-phenyl- 1H-pyrazole-3-carbonyl}-amino)-pentanedioic Acid 5-tert-Butyl Ester. To a solution of (S)-2-({5-[2-((S)-2cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}-amino)-pentanedioic acid 5-tert-butyl ester 1-methyl
ester (1.84 g, 3.0 mmol) in THF (12 mL) was added LiOH (73 mg,
3.0 mmol) in water (4 mL). After 2 h, the reaction mixture was
neutralized with Amberlite IR-120, ltered, and washed with methanol.
After evaporation of the solvent, the title compound was obtained as a
colorless oil (1.80 g, 100%). 1H NMR (DMSO-d6): 8.24 (d, J = 7.5
Hz, 1H), 8.02 (d, J = 7.5 Hz, appears as rotamers, 1H), 7.91 (d, J = 8.3
Hz, 2H), 7.57 (t, J = 8.3 Hz, 2H), 7.43 (t, J = 8.3 Hz, 1H), 6.44 (s, 1H,
appears as rotamers), 5.09 (s, 2H), 4.44- 4.11 (m, 3H), 3.60 (m, 1H),
3.48 (m, 2H), 2.321.52 (several multiplets, 14H), 1.37 (s, 9H). tR
(method j): 1.38 min. MS (ES) m/z: 598.2 (MH+).
6.1.8.3. 4-[(S)-4-Carboxy-2-({5-[2-((S)-2-cyclobutylcarbamoyl-pyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-butyrylamino]-piperidine-1-carboxylic Acid Ethyl Ester
(19a). To a solution of (S)-2-({5-[2-((S)-2-cyclobutylcarbamoylpyrrolidin-1-yl)-2-oxo-ethoxy]-1-phenyl-1H-pyrazole-3-carbonyl}amino)-pentanedioic acid 5-tert-butyl ester (150 mg, 0.25 mmol) in
DMF (7 mL) were added DIPEA (62 L, 1.1 mmol), HATU (95 mg,
0.36 mmol), and ethyl 4-amino-1-piperidinecarboxylate (43 mg, 0.25
mmol). After it was stirred for 12 h, the saturated NaHCO3 solution
was added, and the mixture loaded on a chem elut cartridge, the crude
product being eluted with dichloromethane. The solution was
concentrated to a volume of 1 mL and stirred in the presence of
triuoroacetic acid (190 L). After it was stirred for 4 h, the solvents
were removed under reduced pressure, and the residue was puried by
preparative HPLC (C18 reverse phase column, elution with a water/
acetonitrile gradient with 0.1% triuoroacetic acid). After lyophilization, the product 19a (35 mg, 20%) was obtained as a white solid. 1H
NMR (DMSO-d6): 8.09 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.4 Hz,
1H), 7.85 (d, J = 8.4 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.6
Hz, 1H), 6.41 (s, 1H), 5.04 (s, 2H), 4.96 (m, 1H), 4.21 (dd, J = 8.4
Hz, J = 3.9 Hz, 1H), 4.14 (m, 1H), 4.05 (q, J = 6.9 Hz, 2H), 3.82 (m,
1H), 3.52 (m, 8H), 2.29 (m, 2H), 2.11 - 1.80 (m, 8H), 1.72 (m, 3H),
1.59 (m, 2H), 1.25 (m, 2H), 1.19 (t, J = 6.8 Hz, 3H). tR (method b):
1.59 min. MS (ES) m/z: 696.5 (MH+).
6.2. Biology: Human P2Y12 Recombinant Cell Membrane
Binding Assay.17 As a source of P2Y12, a membrane preparation was
prepared from CHO cells with recombinant expression of the human
P2Y12 receptor according to standard procedures. To a 96-well
microtiter plate, added were the following: (a) 24 L of assay buer
[10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), 138 mM NaCl, 2.9 mN KCl, 12 mM NaHCO3, 1 mM
EDTA-Na, and 0.1% BSA, pH 7.4], (b) 1 L of compound in DMSO,
(c) 50 L of P2Y12 CHO membrane (20 g/mL) and after 15 min at
room temperature, and (d) 25 L of 1.61 nM [33P]2-MeS-ADP
(Perkin-Elmer NEN custom synthesis, specic activity 2100 Ci/
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Article
mmol) made in assay buer. The nal concentration of [33P]2-MeSADP was 0.8 nM, and the Kd value determined for [33P]2-MeS-ADP
was 0.5 nM. After 20 min of incubation at room temperature, samples
were transferred and aspirated to 96-well microtiter lterplates
(Millipore HTS GF/B) and prewetted for 20 min with 300 L of
stop buer (10 mM HEPES, 138 mM NaCl, pH 7.4). After they were
washed four times with 400 L/well of stop buer, the lters were airdried overnight. After the addition of 0.1 mL of Microscint 20
Scintillation Fluid (Perkin-Elmer #6013621), the lter plates were
incubated for 2 h at room temperature and counted in a Microbeta
Scintillation Counter. The binding of compound is expressed as a %
inhibition of specic binding, dened by subtraction of the background
with 1 mM ADP. Ki values were calculated from the IC50 values using
the ChengPruso equation assuming binding to a single binding site
and were averaged from at least triplicate determination. The mean Ki
value of the positive control used for each run was 0.4 M, and the
standard error of the mean (SEM) for this standard compound was
0.016 M (n = 61).
6.2.1. Inhibition of Human Platelet Aggregation. 6.2.1.1. hPRP
Preparation. Human whole blood was from the Sano-Aventis inhouse blood donor service. The protocol was approved by the local
ethics committee, and all blood donors signed informed consent.
Whole blood was collected from healthy volunteers using 20 mL
syringes containing 2 mL of acidcitratedextrose solution (ACD-A)
(for 96-well assays) or 2 mL of buered citrate (for the light
transmission aggregometry, Born method). The anticoagulated whole
blood was transferred into 15 mL polypropylene conical tubes (10 mL
per tube). The tubes were centrifuged for 15 min at room temperature
at 150g (for 96-well assays) or at 340g (for the Born method), leading
to a supernatant of hPRP. The hPRP layer was collected from each
tube and pooled for each donor. The platelet concentration was
determined using a Coulter counter. The 15 mL tubes containing the
pellet of cellular components were centrifuged again for 10 min at
1940g, leaving a supernatant of platelet poor plasma (PPP). The PPP
was collected for each donor. The PRP was diluted with PPP to a nal
concentration of 3 108 platelets/mL with the PPP.
6.2.1.2. IPs Preparation. IPs were obtained by centrifugation of
PRP at 430g for 20 min at room temperature. The resulting platelet
pellet was dissolved in a modied Tyrode's buer containing 145 mM
NaCl, 5 mM KCl, 0.1 mM MgCl6H2O, 5.5 mM glucose, and 15 mM
Hepes. The buer was adjusted to pH 7.4. Platelets were counted and
adjusted to 300 103 platelets/L. To 320 L of IPs, 20 L of CaCl2
(nal concentration, 0.5 mM) was added and incubated at 37 C for 2
min before adding 20 L of brinogen (nal concentration, 1 mg/
mL). The respective P2Y12 antagonist was added (20 L) and
incubated, and the response to platelet activating agonists (20 L) was
recorded.
6.2.1.3. Human Platelet Aggregation 96-Well Assay.10 The
human platelet aggregation assay was performed in 96-well plates
using a microtiter plate reader (SpectraMax Plus 384 with SoftMax Pro
software from Molecular Devices). In the plate, 15 L of test
compound at 10 nal concentration in NaCl was mixed with 120 L
of fresh hPRP and incubated for 5 min. Following that incubation
period, 15 L of 40 M ADP was added to the reaction mix, leading to
a nal concentration of 4 M ADP. The plates were then transferred
to the microplate reader, and aggregation was measured over 20 min.
The instrument settings include the following: absorbance at 650 nm,
run time 20 min with readings in 1 min intervals, and 50 s of shaking
between readings, all performed at 37 C. Results of the assay are
expressed as % inhibition and are calculated using the AUC of the
absorbance over 20 min. The IC50 values were averaged from at least
triplicate determination. The mean value of the positive control used
for each run was 0.27 M, and the SEM for this standard compound
was 0.026 M (n = 96).
6.2.1.4. Human Platelet Aggregation Assay Light Transmission
Aggregometry (Born Method).10 The human platelet aggregation
assay was performed in single use cuvettes using the platelet
aggregation proler (PAP-8, Bio/Data corporation, Horsham, PA).
In the assay cuvette, 4 L of the test compound at 100 nal
concentration in DMSO was mixed with 392 L of fresh hPRP and
ASSOCIATED CONTENT
S Supporting Information
*
AUTHOR INFORMATION
Corresponding Author
*E-mail: Marc.Nazare@sano.com.
Notes
ACKNOWLEDGMENTS
We thank A. Sihorsch, M. Kammerer-Dienst, C. Prosser, S.
Herok, D. Thorn, H. Krause, G. Sibenhorn, S. Stamm, A.
Haber, F. Hopnger, M. Pfeier, and R. Katzenmeier for their
excellent technical assistance.
ABBREVIATIONS USED
ACD-A, acidcitratedextrose solution; ADP, adenosine
diphosphate; AUC, area under curve; CHO, Chinese hamster
ovary cell; Col, collagen; CoMFA, comparative molecular eld
analysis; CoMSIA, comparative molecular similarity index
analysis; CXCR4, C-X-C chemokine receptor type 4; EC,
extracellular loop; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU, O-(7-azabenzotriazol-1yl)-N,N,N,N-tetramethyluronium hexauorophosphate;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HOBt, 1-hydroxybenzotriazole; IP, isolated platelets; MARS,
multiple alignments by ROCS-based similarity; ND, not
determined; P2Y1, P2Y purinoceptor 1; P2Y12, P2Y purinoceptor 12; PI3K, phosphatidylinositol 3-kinase; hPRP, human
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