Discovery and synthesis of a novel and selective drug-like P2X1 antagonist

Article abstract of DOI:10.1016/j.bmcl.2005.04.049

Although there is extensive literature to indicate that many different types of P2 purinoceptors are present in the lower urinary tract, the physiological role of these receptors in micturition is still uncertain. In part, this uncertainty has been caused by a lack of P2 subtype selective ligands. In this paper we report the discovery, gram scale synthesis, and binding results for 1, the first potent, drug-like, selective P2X₁ receptor antagonist described. Compound 1 was shown to be more than 30-fold selective over other purinergic receptor subtypes.

Full text of DOI:10.1016/j.bmcl.2005.04.049

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3292–3295
Discovery and synthesis of a novel and selective drug-like
P2X1 antagonist
S. Jaime-Figueroa,* R. Greenhouse, F. Padilla, M. P. Dillon,
J. R. Gever and A. P. D. W. Ford
Roche Palo Alto, 3431 Hillview Ave., Palo Alto, CA 94304, USA
Received 7 February 2005; revised 21 April 2005; accepted 21 April 2005
Available online 31 May 2005
Abstract—Although there is extensive literature to indicate that many different types of P2 purinoceptors are present in the lower
urinary tract, the physiological role of these receptors in micturition is still uncertain. In part, this uncertainty has been caused by a
lack of P2 subtype selective ligands. In this paper we report the discovery, gram scale synthesis, and binding results for 1, the first
potent, drug-like, selective P2X1 receptor antagonist described. Compound 1 was shown to be more than 30-fold selective over other
purinergic receptor subtypes.
ꢀ 2005 Published by Elsevier Ltd.
In this study, we report the discovery and preparation of
compound 1 and its in vitro affinity profile as a P2X1
antagonist. High-throughput screening of the Roche
compound library resulted in the discovery of 1 (a com-
pound originally prepared as a potential rennin inhibitor
but shown to be inactive5) as a validated hit with P2X1
affinity. For further biological characterization a syn-
thetic route was required to provide 1 in substantial
quantities.
1. Introduction
P2X receptors are plasma membrane, ligand gated ion
channels that open in response to the binding of extra-
cellular ATP. These purinergic receptors are abundantly
distributed in the body, and functional responses are
seen in neurons, glia, epithelia, bone, muscle, and hemo-
poietic tissues.1 Although reports indicate that many dif-
ferent types of P2 receptors are present in the lower
urinary tract, the role of these receptors in micturition
is still uncertain.2,3 In recent years considerable effort
has been dedicated to identifying subtype selective
P2X receptor ligands.3,4 However, the study of the
P2X receptors continues to be hampered by the lack
of drug-like, potent and selective antagonists.
The synthetic approach developed to 1 is presented in
Schemes 1 and 2.
Synthesis of 1 was initiated with the preparation of large
quantities of the chiral a-hydroxy-b-aminoacid 2 as a
key starting material (following the synthesis described
in the literature6) (Scheme 1). Thus, addition of an ex-
cess of cyclopropyllithium7 to the carboxylic acid 2 gave
the cyclopropyl ketone 3 in 94% yield (Scheme 1).
Stereoselective reduction6of ketone 3 with triacetoxy-
borohydride produced the corresponding (S)-hydroxy
compound in quantitative yield. Treatment of the crude
product from the previous step with KOH in MeOH/
water afforded the crucial chiral aminodiol compound
4 (52% over two steps). Coupling reaction between
aminodiol 4 and the commercially available N-boc-pro-
tected aminoacid 5 using PyBOP,8 furnished the amide 6
(Scheme 2). TFA induced deprotection of 6 gave the
corresponding a-aminoamide 7 in 98% yield. Finally,
the coupling reaction between this aminoacid 7 with
Keywords: P2X1 antagonist.
*
Corresponding author. Fax: +1 650 354 2442; e-mail:
saul.jaime@roche.com
0960-894X/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2005.04.049

S. Jaime-Figueroa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3292–3295
3293
Table 1. Shows the (1) IC50s from [CA2+]i measurements obtained
with FLIPRa
Receptor
IC50, lM
hP2X1
hP2X2
hP2X3
hP22/3
3
>100
>100
>100
a Flurometric imaging plate reader.
2. Experimental
The 1H NMR and 13C NMR spectra were measured on a
Bruker Avance DPX-300 NMR or on a Bruker AMX-
300 NMR spectrometer, operating at a proton (1H) fre-
quency of 300.13 MHz and carbon (13C) frequency of
75.43 MHz. The mass spectra were recorded on a Finn-
igan-MAT 8230 or on
spectrometer.
a
VG ZabSpec-QFPD
2.1. (1-(S)-Cyclohexylmethyl-3-cyclopropyl-2-(R)-
hydroxy-3-oxo-propyl)-carbamic acid ethyl ester (3)
Scheme 1. Reagents and conditions: (a) cyclopropyllithium, toluene/
THF/LiBr, 94%; (b) triacetoxyborohydride, heptane/MeCN; (c) KOH,
MeOH/water, 52% over the two steps.
Lithium bromide (2.22 g, 25.58 mmol) and a solution of
4-cyclohexyl-3-(S)-ethoxycarbonylamino-2-(R)-hydroxy-
butyric acid6 (2) (2.69 g, 9.84 mmol) in toluene were com-
bined and stirred under a nitrogen atmosphere. The
mixture was cooled to 5 ꢁC and 10 mL of THF was
added. The mixture was stirred for 30 min at this tem-
perature, and then cooled to À40 ꢁC. A solution of
cyclopropyllithium7 (0.88 M in ethyl ether, 4.6 mL,
40.54 mmol) was slowly added over 30 min while main-
taining the temperature at À40 ꢁC. The reaction was
stirred for 15 min at this temperature and then stirred
at 5 ꢁC for 2 h. The reaction was quenched at 5 ꢁC
by addition of a saturated aqueous solution of ammo-
nium chloride. After the layers were separated, the
2-imidazolylcarboxylic acid afforded the final product 1
(51% yield).
Biological evaluation in vitro of 1 shows high affinity for
P2X1 receptor and remarkable selectivity (more than 30-
fold) over other purinergic receptor subtypes (Table 1).
In summary we have described the discovery that com-
pound 1 is a selective P2X1 antagonist. In addition, we
have reported a new practical synthesis of this material,
which allowed us to prepare 1 in large quantities for its
biological characterization.
Scheme 2. Reagents and conditions: (a) PyBOP, Et3N, DMF, 77%; (b) TFA, dichloromethane, 98%; (c) PyBOP, Et3N, DMF, 51%.

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aqueous layer was extracted with dichloromethane
(4 · 40 mL). The combined organics were dried
(MgSO4) and concentrated under vacuum. The crude
product was used in the next step without further puri-
fication. Yield 2.76 g (94%). 1H NMR (500 MHz,
CDCl3): d 4.73 (br s, 1H, D2O), 4.69 (d, 1H,
J = 10.0 Hz), 4.48 (m, 1H), 4.32 (s, 1H), 4.05 (q, 2H,
J = 6.9 Hz), 2.10 (m, 1H), 1.91 (d, 1H, J = 12.5 Hz),
1.76–1.51 (m, 8H), 1.28 (m, 1H), 1.92–1.30 (m, 7H),
1.20 (t, 3H, J = 6.9 Hz). 13C NMR (75.47 MHz,
CDCl3): d 211.02, 156.04, 78.56, 60.86, 49.57, 40.80,
34.17, 33.51, 33.10, 26.51, 26.27, 26.14, 17.44, 14.56,
13.02, 12.44. MS m/z: 298 [M+H]+.
chromatography (SiO2, Hex/EtOAc, 1:1) to provide
1.62 g (77% yield) of the title compound, mp 133–
135 ꢁC. Calculated for C24H39N3O5S: 0.5 mol H2O; C,
59.85; H, 8.16; N, 8.72. Found: C, 58.85; H, 8.03; N,
8.81. 1H NMR (300 MHz, DMSO): d 9.0 (d, 1H,
J = 1.8 Hz), 7.38 (d, 1H, J = 8.5 Hz, D2O), 7.34 (d,
1H, J = 1.8 Hz), 7.02 (d, 1H, J = 8.1 Hz, D2O), 4.76
(br d, 1H, D2O), 4.40 (br s, 1H, D2O), 4.31 (m, 1H),
4.09 (m, 1H), 2.81–3.21 (m, 4H) 0.70–1.75 (m, 14H),
1.34 (s, 9H), 0.14–0.36 (m, 4H). 13C NMR
(75.47 MHz, DMSO): d 171.95, 155.10, 153.45, 153.30,
115.38, 78.23, 76.04, 70.89, 54.34, 46.87, 45.70 33.40,
33.05, 32.10, 28.07, 26.11, 25.78, 25.63, 13.71, 8.62,
1.95, 0.14. MS m/z: 482 [M+H]+.
2.2. 3-(S)-Amino-4-cyclohexyl-1-cyclopropyl-butane-1-
(S),2-(R)-diol (4)
2.4. 2-(R)-Amino-N-(1-(S)-cyclohexylmethyl-3-cyclopro-
pyl-2-(R),3-(S)-dihydroxy-propyl)amino]-3-thiazol-4-yl-
propionamide (7)
Sodium triacetoxyborohydride (1.92 g, 9.09 mmol) was
suspended in heptane (5 mL), and (1-(S)-cyclohexyl-
methyl-3-cyclopropyl-2-(R)-hydroxy-3-oxo-propyl)-car-
bamic acid ethyl ester (3) (2.76 g, 9.28 mmol) dissolved
in acetonitrile (5 mL) was added at room temperature.
The reaction mixture was stirred for 4 h at 20–24 ꢁC,
then cooled to 5–10 ꢁC, and quenched with 9% aqueous
sodium bicarbonate (18 mL). The product was extracted
with dichloromethane (4 · 30 mL). Combined organics
were dried (MgSO4) and concentrated under vacuum.
To the crude product dissolved in a mixture of metha-
nol/water (7:3) (25 mL) was added 45% aqueous potas-
sium hydroxide (4.6 g), and the reaction solution was
heated at reflux for 18 h. The reaction solution was
cooled to 10 ꢁC and heptane (7 mL) was added. After
bringing to reflux, again, the solution was cooled in an
ice bath, and the resultant solid was collected. The solid
product was washed with a mixture of methanol/water
(6:1) (10 mL) and water (2 · 15 mL) and vacuum dried
to obtain 1.09 g of off-white crystalline solid 4 (52%
yield), mp 139.1–140.9 ꢁC (Lit.5 mp 141–142 ꢁC). Calcu-
lated for C13H25NO2: 0.3 mol H2O; C, 67.09; H, 11.09;
To a solution of Boc-aminodiol 6 (1.8 g, 4.7 mmol) in
dichloromethane (100 ml) was added TFA (10 ml) at
0 ꢁC. The reaction mixture was stirred for 4 h at room
temperature. Then the mixture was poured into a satu-
rated aqueous solution of NaHCO3 (300 ml) and the
product
was
extracted
with
dichloromethane
(3 · 200 ml). Organic extracts were dried (Na2SO4) and
evaporated under vacuum. The crude product was used
in the next step without any further purification (1.4 g,
98% yield). MS m/z: 382 [M+H]+.
2.5. 1H-Benzoimidazole-2-carboxylic acid [1-(R)-(1-(S)-
cyclohexylmethyl-3-cyclopropyl-2-(R),3(S)-dihydroxy-
propylcarbamoyl)-2-thiazol-4-yl-ethyl]-amide (1)
To a mixture of aminothiazole 7 (1.5 g, 4.1 mmol), 1H-
benzimidazole-2-carboxylic acid (0.73 g, 4.5 mmol)
(Maybridge) and triethylamine (1.25 g, 12.3 mmol) in
DMF (50 ml) was added PyBOP (2.57 g, 4.9 mmol) at
room temperature. The reaction mixture was stirred
for 8 h at the same temperature. After that the reaction
was poured into water (300 ml) and the product crystal-
lized out from the mixture. The product was collected by
filtration and crystallized from ether to give 1.1 g of pure
product (51% yield) as a white solid. [a]D À39.0
1
N, 6.02. Found: C, 66.92; H, 10.69; N, 6.03. H NMR
(300 MHz, CDCl3): d 4.77 (br s, 1H, D2O), 3.44 (m,
1H), 3.26 (m, 1H), 3.15 (dd, 1H, J = 3.6, 7.6 Hz),
1.80–1.60 (m, 5H), 1.50–1.10 (m, 6H), 1.05–0.80 (m,
3H), 0.63–0.36 (m, 3H), 0.33–0.22 (m, 1H). 13C NMR
(75.47 MHz, CDCl3): d 78.92, 74.34, 48.62, 43.53,
34.05, 33.95, 32.91, 26.55, 26.36, 26.20, 13.96, 2.39,
1.79. MS m/z: 228 [M+H]+.
1
(c = 0.5 g/mL, MeOH). H NMR (300 MHz, DMSO):
d 13.29 (s, 1H, D2O), 9.92 (d, 1H, J = 1.9 Hz), 8.92 (d,
1H, J = 8.5, D2O), 7.53–7.50 (br d, 1H, J = 7.2 Hz),
7.75–7.57 (m, 2H), 7.40 (d, 1H, J = 1.9 Hz), 7.34–7.24
(m, 2H), 4.98–4.90 (m, 1H), 4.81 (d, 1H, J = 6.3 Hz,
D2O) 4.42 (d, 1H, J = 4.7 Hz, D2O), 4.18–4.08 (m,
1H), 3.44 (m, 1H), 3.33–315 (m, 2H) 3.16–3.11 (m,
1H), 2.92–2.86 (m, 1H), 1.75–0.77 (m, 14H), 0.33–0.17
(m, 4H). 13C NMR (75.47 MHz, DMSO): d 170.86,
158.39, 153.85, 153.11, 145.11, 142.50, 134.63, 124.37,
122.77, 120.07, 115.80, 112.68, 76.07, 70.92, 53.13,
47.24, 39.08, 33.65, 33.51, 32.27, 26.22, 25.88, 25.78,
13.88, 2.10, 0.22, 0.10. HRMS calcd for C27H36N5O4S
(M+H)+ 526.2488, found 526.2481.
2.3. [1-(R)-(1-(S)-Cyclohexylmethyl-3-cyclopropyl-2-
(R),3-(S)-dihydroxy-propylcarbamoyl)-thiazol-4-yl-
ethyl]-carbamic acid tert-butyl ester (6)
To a mixture of the amine 4 (1 g, 4.3 mmol), Boc-D-ami-
noacid 5 (1.19 g, 4.3 mmol) (Synthetech Inc.) and trieth-
ylamine (1.3 g, 12.9 mmol) in DMF (50 ml) was added
PyBOP8 (2.74 g, 5.2 mmol) (Calbiochem–Novabiochem
Corporation) at room temperature. The reaction mix-
ture was stirred for 8 h at the same temperature. The
mixture was then poured into water (200 ml), and
the product was extracted with EtOAc (3 · 100 ml).
The combined organic layers were washed with water
(4 · 100 ml), dried (Na2SO4), and evaporated under
vacuum. The crude product was purified by column
2.6. Intracellular calcium measurements (FLIPR)
Transfected CHO-K1 cells (expressing hP2X1 or hP2X3)
and 1321N1 astrocytoma cells (expressing hP2X2 or

S. Jaime-Figueroa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3292–3295
3295
hP2X2/3) were centrifuged and resuspended in nutrient
media at 2.5 · 105 cells/ml. The cells were aliquoted into
black-walled, clear bottom, 96-well FLIPR plates at a
density of 50,000 cells/well and incubated overnight in
7% CO2 at 37 ꢁC. After overnight incubation, the cells
were washed in calcium- and magnesium-free HankÕs
balanced salt solution supplemented with 10 mM
HEPES, 2 mM CaCl2 and 2.5 mM probenecid (FLIPR
buffer); the cells were then allowed to incorporate a cal-
cium-sensitive fluorescent dye (2 lM Fluo-3 AM;
Molecular Probes, Eugene, OR) at 37 ꢁC for 1 h in
100 lL FLIPR buffer. RO0437626 was dissolved in
DMSO at 10 mM and serially diluted with FLIPR buffer
to 4· concentrations ranging from 400 lM to 40 nM.
The cells were washed four times with FLIPR buffer,
leaving 75 lL in each well, and 25 lL of the 4X solu-
tions of RO0437626 or vehicle added to each well in or-
der to attain final concentrations ranging from 100 lM
to 10 nM. The cells were allowed to equilibrate with
antagonist at room temperature for 20 min before a
baseline fluorescence measurement was obtained (excita-
tion 488 nm, emission 510–570 nm) and the reactions
started with the addition of a,b-meATP (0.1 lM for
hP2X1, 1 lM for hP2X3, 5 lM for hP2X2/3; final con-
centrations) or ATP (3 lM for hP2X2; final concentra-
tion). Fluorescence was measured for 2 min at 1–20 s
intervals, with readings taken until either a plateau
phase was reached (hP2X2 and hP2X2/3) or the fluores-
cence returned to baseline (hP2X1 and hP2X3). Finally,
a calcium ionophore, ionomycin, was added to each well
(5 lM final concentration) to establish cell viability and
maximum fluorescence of dye-bound cytosolic calcium.
IC50s were determined by fitting the data using the four
parameter logistic equation utilized by GraphPad Prism
software (GraphPad Software Inc., San Diego, CA).
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