Pyrrolidine-carboxamides and oxadiazoles as potent hNK1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.08.028

The preparation and structure-activity-relationships of novel pyrrolidine-carboxamides and oxadiazoles are described. Compounds in this series were found to be potent hNK₁ antagonists in vitro and efficacious in vivo with minimal interactions with P₄₅₀ liver enzymes. Oxadiazole analog 22 was determined to have excellent hNK₁ binding affinity, functional activity, and a good PD response in vivo.

Full text of DOI:10.1016/j.bmcl.2007.08.028

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5310–5315
Pyrrolidine-carboxamides and oxadiazoles as potent
hNK1 antagonists
Jonathan R. Young,a,* Ronsar Eid,a Cherilyn Turner,a Robert J. DeVita,a
Marc M. Kurtz,b Kwei-Lan C. Tsao,b Gary G. Chicchi,b Alan Wheeldon,c
Emma Carlsonc and Sander G. Millsa
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO 2000, Rahway, NJ 07065-0900, USA
bDepartment of Immunology, Merck Research Laboratories, PO 2000, Rahway, NJ 07065-0900, USA
cMerck Sharpe & Dohme Research Laboratories, Harlow, UK
Received 11 July 2007; revised 9 August 2007; accepted 13 August 2007
Available online 16 August 2007
Abstract—The preparation and structure–activity-relationships of novel pyrrolidine-carboxamides and oxadiazoles are described.
Compounds in this series were found to be potent hNK1 antagonists in vitro and efficacious in vivo with minimal interactions with
P450 liver enzymes. Oxadiazole analog 22 was determined to have excellent hNK1 binding affinity, functional activity, and a good PD
response in vivo.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Neurokinin-1 (NK1) is a member of the superfamily of
G-protein-coupled-receptors whose natural ligand is
Substance P. The receptor is highly expressed in both
the CNS and also peripheral tissues. Antagonism of
the NK1 receptor has been proposed as a therapeutic
strategy for conditions such as cystitis,1 emesis,2 and
major CNS disorders3 due to effects of NK1 agonists
in animal models of those disorders.
CF3
F3C
CF3
F3C
R1
CF3
CF3
HN
N
O
N
O
&
BOC
O
O
N
BOC N
H
N
F
O
N
HN
1: Aprepitant
2 ( ):
NK1 IC50 = 450 nM
3a ( ): R1 = H
NK1 IC50 = 142 nM
3b: R1 = Me
NK1 IC50 = 0.09 nM
Cyp 3A4 IC50 = 46 nM
Previous efforts from these laboratories produced
Aprepitant (1)4 which is approved for use in chemother-
apy-induced nausea and vomiting and for post-opera-
tive nausea and vomiting (Fig. 1). The goals of our
backup program focused on achieving sufficient brain
penetration and pharmacokinetics consistent with once
daily oral dosing, while minimizing the potential for
cytochrome P450 interactions. This paper will describe
the discovery of novel pyrrolidine scaffold 3 and a sub-
sequent article will describe the piperidine scaffold 2,
both of which led to potent and efficacious NK1 antag-
onists with insignificant inhibition of the P450 class of
enzymes.
NK1 IC50 = 11 nM
Figure 1. From Aprepitant (1) to rationally derived leads 2 and 3.
An essential structural component to Aprepitant (1) and
other NK1antagonists has been the bis-phenyl motif,
typically a tethered-bis-CF3-phenyl moiety adjacent to
a phenyl group appended to a central core ring. While
maintaining this crucial feature, we were interested in
replacing the ether tether found in 1 with an amide
side-chain. To generate diversity, a systematic survey
of disubstituted pyrrolidine and piperidine scaffolds
employing an amide linkage to the bis-CF3-phenyl was
undertaken in which all regio- and stereochemical com-
binations were prepared and evaluated. Two promising
leads emerged from this initial study, specifically piper-
idine 2 and pyrrolidine 3a (Fig. 1).
Keywords: NK1 antagonists; Substance P.
*
Corresponding author. Present address: Merck & Co., Inc., 33
Avenue Louis Pasteur, Boston MA 02115, USA. Tel.: +1
6179922331; fax: +1 6179922406; e-mail: jonathan_young@
merck.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.028

J. R. Young et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5310–5315
5311
isocyanates), or alkylamines 10 (reductive amination)
to yield the targeted analogs. Cis-pyrrolidine 14 was
prepared from ester 13 analogously to the sequence
from 5 to 9.
a
Synthesis of racemic trans-pyrrolidines 8-10
OMe
OH
O
MeO2C
O
R1N
R1N
a,b,c
d
X
X
X
The pure enantiomers found in Table 2 were prepared
from acid 6 by conversion to the separable acyl-oxa-
zolidinone diastereomers 15 and 166 under the condi-
tions reported by Ho and Mathre.7 Hydrolysis of
intermediates 15 and 16 using LiOOH furnished chiral
acids 17 and 18 which were transformed to 27–29 (see
Table 2) according to the procedures described for
racemic acid 6.
4
5: R1 = BOC
CF3
6: R1 = BOC
F3C
F3C
CF3
g,h,
or i
N
N
e,f
O
O
X
HN
R1N
X
7
8: R1 = COR
9: R1 = CONHR
10: R1 = R
Oxadiazoles 20, 22, 25, and 26 were prepared from
amide-oximes 19, 21, and 24 by methodology reported
from these laboratories,8 wherein the requisite acids
were coupled to the amide-oximes (EDC/HOBT) fol-
lowed by thermal cyclodehydration (Scheme 2).
Amide-oximes 19, 23, and 24 in turn were prepared in
three steps from acids 17a, 18a, and 23,9 respectively,
by conversion to the primary amides, dehydration to
the nitrile, followed by treatment with hydroxylamine.
Deprotection of the tert-butylcarbamate group under
acidic conditions followed by coupling to 1-acetyl-4-pip-
eridinecarboxylic acid furnished targeted amides 20, 22,
25, and 26.
b
Synthesis of racemic cis-pyrrolidine 14
F3C
CF3
OMe
O
(1)d,e,f
CO2Me
O
(2)g,h, or i
R1N
j
a,b,c
N
H
O
R1N
11
12
13: R1 = BOC
14
c
Synthesis of chiral trans-pyrrolidine carboxylic acids 17-18
O
N
O
O
N
O
OH
O
Bn
R1N
O
Bn
R1N
O
The hNK1 binding affinities of carboxamides 3 (Fig. 1)
were found to be sensitive to amide substitution. For
example, N-methyl-amide 3b led to a >10-fold improve-
ment in receptor binding affinity over 3a. We therefore
maintained this potency-enhancing feature in subse-
quent analogs.
R1N
k
+
X
X
X
6: R1 = BOC
15: R1 = BOC 16: R1 = BOC
l
l
OH
O
OH
O
We next focused on N-pyrrolidine variations (Table 1)
and found that amides (8) were generally well tolerated
and preferred over amines (10). For example, the hNK1
antagonist activity of cyclohexylamide 8a was 2-orders
of magnitude more potent than the corresponding
amine 10a. Urea 9a which is isosteric with amide 8a
was substantially less potent than 8a. The homologated
urea 9b on the other hand was equipotent to 8a. Incor-
poration of basic substituents, such as piperidine 8b, led
to decreased receptor binding affinity compared to 8a.
However, acylated derivatives of 8b, such as amide 8c,
retained the potency of 8a. The receptor binding affinity
of the pyrimidine amides was sensitive to the substitu-
tion pattern in which the IC50’s were in the order:
8d < 8f ꢀ 8g. Pyrazines 8e and 8h and pyrimidine 8d
were the most potent heteroaromatic carboxamide ana-
logs identified.
R1N
R1N
+
X
X
17: R1 = BOC 18: R1 = BOC
Scheme 1. Reagents and conditions: (a) N-(methoxymethyl)-N-(trim-
ethylsilylmethyl)benzylamine, TFA, DCM; (b) ACE-Cl, PhMe,
110 ꢁC; MeOH, 80 ꢁC; (c) BOC2O, DMAP, Et3N, DCM; (d) NaOH,
THF; (e) EDC, HOBT, DMAP, N-methyl-3,5-bis-trifluoromethylben-
zylamine, DCM; (f) HCl-EtOAc; (g) EDC, DMAP, R1CO2H, DCM;
(h) 1-piperidinecarbonyl chloride, DCM, Et3N, DMAP or cyclohexy-
lisocyanate; (i) cyclohexanecarboxaldehyde, Na(OAc)3BH, HOAc,
DCE; (j) (F3CCH2O)2POCH2CO2Me, NaHMDS, 18-C-6, THF;
(k) pivoyl chloride, Et3N, DCM; (S)-benzyloxazolidine, LiCl, Et3N,
THF; separate diastereomers; (l) LiOOH, THF.
Azomethine ylide [3+2] cycloadditions afforded conve-
nient access, after protecting group manipulations, to
both trans and cis pyrrolidine derivatives 5 and 13 from
the corresponding E and Z5 cinnamate esters 4 and 12,
respectively, as key synthetic intermediates (Scheme 1).
Intrigued by the potent hNK1 binding activity of 8c, we
prepared the single enantiomer and determined that the
active component was the 3(R)-carboxamide-4(S)-phe-
nyl isomer 27 (Table 2). As a major indication for
NK1 antagonists is the treatment of chemotherapy-in-
duced emesis, it is beneficial to design NK1 antagonists
that lack the potential to participate in drug-drug inter-
actions. Accordingly, we were gratified to observe that
27 along with all analogs in Table 2 were only weak
inhibitors of the P450 liver enzymes.
The racemic analogs in Table 1 required preparation
of the common pyrrolidine intermediate 7 which was
readily prepared from ester 5 after saponification,
amide coupling, and pyrrolidine deprotection. Pyrroli-
dine 7 was converted to amides 8 (EDC, HOBT, car-
boxylic acids), ureas
9 (carbamoyl chlorides or

5312
J. R. Young et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5310–5315
Table 1. Optimization of the nitrogen substituent
Receptor binding affinity was also sensitive to the ste-
reochemistry of the pyrrolidine scaffold. In contrast to
the morpholine scaffold present in Aprepitant 1 wherein
the 1,2-cis relationship was favored (10-fold),4a the 1,2-
trans stereochemistry of 8c was > 10-fold more potent
than that of the cis-isomer 14a in the pyrrolidine class
of hNK1 inhibitors. This finding was consistent with
the cyclopentane analogs described from these
laboratories.10
F3C
Me
CF3
N
O
R1
N
Entrya
R1
b
hNK1 IC50 (nM)
O
Administration of an NK1 agonist (icv) has previously
been reported to elicit a rapid and robust foot tapping
response in gerbils and provides the basis for an impor-
tant in vivo model of functional antagonist activity.11
The assay was performed by dosing the test compound
by iv infusion, followed by the central administration
of the NK1 agonist at a specified time post-drug infusion
and monitoring the ability of the compound to block the
agonist effect.
3ac
142
11
O
O
O
3b
8a
O
0.68
10a
9a
62
We evaluated 8c in the in vivo gerbil foot tapping (GFT)
model and found that 8c demonstrated rapid brain pen-
etration and was able to effectively block the agonist-in-
duced foot-tapping response in the gerbil at an early
time point (t = 0; ID50 = 0.41 mpk), but showed a poor
duration of action (24 h pre-dose prior to agonist
administration). This was consistent with the short
in vivo half-life determined in rats (t1/2 = 0.7 h). Amide
8c showed a 10-fold shift in receptor-antagonism when
the binding assay was performed in the presence of
50% human serum, which often correlates with in vivo
plasma protein binding, and should have a significant
impact on in vivo activity.
O
N
13
O
9b
0.79
NH
O
8b
8c
4.1
HN
O
O
0.42
N
N
O
Another potential contributing factor to the poor dura-
tion of the pharmacodynamic response could be due to a
rapid dissociation from the receptor. We have recently
developed a functional binding assay (IP-1) that mea-
sures the ability of an hNK1 antagonist (100 nM) to
block the biochemical response (inositol phosphate gen-
eration) to Substance P (at a concentration of 10 lM) in
CHO cells engineered to overexpress hNK1.12 Signifi-
cant suppression of the maximal response can be inter-
preted as a desirable property for an antagonist,
insofar as it implies that the compound can maintain
receptor blockade even in the presence of a large excess
of agonist. The data are reported as the percent of the
Substance P response remaining (% SPRR) after a
30 min incubation time between the drug (100 nM)
and hNK1 cells followed by treatment with the agonist
(10 lM). Pyrrolidine 8c was found to be functionally
ineffective (81% SPRR) at blocking the effect of Sub-
stance P in this assay.
14ad
3.7
O
O
8d
8e
8f
0.84
0.74
3.4
N
N
N
O
N
O
N
N
N
O
8g
8h
24
N
O
0.58
N
N
During the course of our optimization studies of the 4-
phenylpyrrolidine substituent, a key discovery that led
to improved IP-1 functional activity across this series
was incorporation of an ortho-methyl on the phenyl
ring. For example, 2-methyl-4-fluorophenyl analog 28
was able to block 97% (3% SPRR) of the Substance re-
sponse despite showing only a modest twofold improve-
ment in intrinsic binding activity over analog 27. Amide
28 showed good in vivo activity for up to 4 h in the GFT
a All compounds are racemic and gave satisfactory 1H NMR and mass
spectral data.
b Displacement of [125I] SP from the human hNK1 receptor expressed
in CHO cells. All values represent the average of 2–6 independent
experiments. Interexperimental variability was typically less than
twofold. See Ref. 4 and references cited therein.
c 3a is a secondary amide (see Fig. 1).
d 14a was a cis-3,4 pyrrolidine.

J. R. Young et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5310–5315
Table 2. SAR for enantiomerically pure pyrrolidine carboxamides and oxadiazles
5313
Compounda
Structurea
IP-112 % SPRR
P450 Inh Isozyme:
IC50 (lM)
Gerbil Foot Tapping11
b
hNK1 IC50
(nM)/50% HS
CF3
N
O
N
O
CF3
8c
0.42/4.5
81%
3A4: 18
2C9: 26
2D6: 24
ED50 = 0.41 mpk at (0 h)
29% inh at 3 mpk (24 h)
N
O
27
ent-27
0.12/ND
0.97/ND
87%
NDc
ND
ND
CF3
N
N
O
N
O
O
CF3
F
28
3A4: 11
2C9: 69
2D6: 92
100% inh at 2 mpk (4 h)
24% inh at 6 mpk (24 h)
0.05/0.21
3%
N
O
CF3
O
N
CF3
F
29
30
3A4: 49
2C9: >100
2D6: 57
100% inh at 2 mpk (4 h)
2% inh at 6 mpk (24 h)
0.05/0.51
0.09/8.0
6%
N
O
CF3
CF3
N
O
N
O
3A4: 11
2C9: 43
2D6: 73
23%
ED50 = 6 mpk at (24 h)
N
N
F
N
O
CF3
N
O
N
N
24
3A4: 23
2C9: 47
2D6: >100
92% inh at 2 mpk (4 h)
1% inh at 6 mpk (24 h)
CF3
0.07/2.8
4%
F
F
N
O
N
O
CF3
N
O
25
3A4: 28
2C9: 89
2D6: >100
CF3
0.17/20
91%
1% inh at 6 mpk (24 h)
N
O
O
N
N
CF3
CF3
N
O
O
N
N
20
22
3A4: 100
2C9: 9
2D6: 63
CF3
CF3
0.07/6
66%
13%
ND
N
O
O
N
0.03/0.17
ND
60% inh at 6 mpk (24 h)
F
N
O
a All compounds, except 8 c, are enantiomerically pure and gave satisfactory 1H NMR and mass spectral data.
b Displacement of [125I]SP from the human hNK1 receptor expressed in CHO cells. All values represent the average of 2–6 independent experiments.
Interexperimental variability was typically less than twofold. See Ref. 4 and references cited therein.
c ND stands for no data obtained.
model but was only marginally efficacious after 24 h.
Analog 29, bearing a geminal-dimethyl group at the
benzylic position, was equipotent to 28 but resulted in
no improvement in in vivo activity. The 4-methylpyr-
azine carboxamide 30 had good intrinsic receptor-bind-
ing affinity that was substantially diminished in the

5314
J. R. Young et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5310–5315
ward the hNK1 receptor and showed reduced potential
CF3
for inhibition of CYP450 metabolizing enzymes com-
pared to Aprepitant. Acylation of the pyrrolidine core
led to increased hNK1 binding affinity. The ortho substi-
tuent on the 4-phenyl group was important for IP-1
functional activity. Replacement of the amide tether
with an oxadiazole was well-tolerated and equal to the
amide in all in vitro respects. Finally, carboxamide 30
and oxadiazole 22 were capable of inhibiting the effect
of an agonist 24 h after an initial iv dose in the gerbil
foot tapping assay.
F3C
OH
OH
O
N
N
R1N
NH2
R1N
a,b,c
d,e,f
O
O
N
N
17a: R1 = BOC
X = 2-methyl
19: R1 = BOC
N
O
20
CF3
F3C
OH
N
NH2
OH
O
N
R1N
R1N
a,b,c
d,e,f
O
O
N
Acknowledgments
N
F
F
18a: R1 = BOC
X = 2-methyl-
4-fluoro
21: R1 = BOC
The authors thank Sanjeev Kumar and the Drug Metab-
olism group for P450 counter-screening and the Synthetic
Services team for scale-up and large scale separation of
synthetic intermediates. Help from Paul Finke in pre-
paring this manuscript is also gratefully acknowledged.
N
F
O
22
CF3
F3C
N
CF3
CF3
N
F3C
a,b,c
d,e,f
N
F3C
O
O
H2N
References and notes
HO2C
N
HO
N
F
1. Saban, R.; Saban, M. R.; Nguyen, N.-B.; Lu, B.; Gerard,
C.; Gerard, N. P.; Hammaond, T. G. Am. J. Pathol. 2000,
156, 775.
2. Andrews, P. L. R.; Rapeport, W. G.; Sanger, W. G.
Trends Pharmacol. Sci. 1988, 9, 334.
23
24
O
25: X = 2-methyl-4-fluoro
26 (enantiomer)
Scheme 2. Reagents and conditions: (a) EDC, HOBT, H4NCl,
N-methylmorpholine; (b) 2,4,6-trichlorotriazine, DMF; (c) H2NOH,
EtOH–H2O, 80 ꢁC; (d) EDC, DMAP, 23; toluene 110 ꢁC; (e) HCl–
EtOAc; (f) EDC, DMAP, 1-acetyl-4-piperidinecarboxylic acid, DCM;
(g) EDC, DMAP, 17 or 18 (X = 2-methyl-4-fluoro); toluene 110 ꢁC.
3. Kramer, M. S.; Cutler, N.; Feighner, J. Shrivastava, R.;
Carman, J.; Sramek, J. J.; Reines, S. A.; Liu, G.; Snavely,
D.; Wyatt-Knowles, E.; Hale, J. J.; Mills, S. G.; MacCoss,
M.; Swain, C. J.; Harrison, T.; Hill, R. G.; Hefto, F;
Scolnick, E. M.; Cascieri, M. A.; Chicchi, G. G.; Sadow-
ski, S.; Williams, A. R.; Hewson, L.; Smith, D.; Carlson,
E. J.; Hargreaves, R. J.; Rupniak, N. M. J. Science. 1998,
281, 22, 1640.
4. (a) Hale, J. J.; Mills, S. G.; MacCoss, M.; Shah, S. K.; Qi,
H.; Mathre, D. J.; Cascieri, M. A.; Sadowski, S.; Strader,
C. D.; MacIntyre, D. E.; Metzger, J. M. J. Med. Chem.
1996, 39, 1760; (b) Hale, J. J.; Mills, S. G.; MacCoss, M.;
Finke, P. E.; Cascieri, M. A.; Sadowski, S.; Ber, E.;
Chicchi, G. G.; Kurtz, M.; Metzger, J. M.; Eiermann, G.;
Tsou, N. N.; Tattersall, F. D.; Rupniak, N. M. J.;
Williams, A. R.; Rycroft, W.; Hargreaves, R.; MacIntyre,
D. E. J. Med. Chem. 1998, 41, 4607.
5. Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 24, 4405.
6. Hale, J. J.; Budhu, R. J.; Mills, S. G.; MacCoss, M.;
Malkowitz, L.; Siciliano, S.; Gould, S. L.; DeMartino, J.
A.; Springer, M. S. Bioorg. Med. Chem. Lett. 2001, 11,
1437.
7. Ho, G. J.; Mathre, D. J. J. Org. Chem. 1995, 60, 2271.
8. Liang, G.-B.; Feng, D. D. Tetraheron Lett. 1996, 37, 6627.
9. Acid 23 was prepared in 2 steps from methyl 3,5-bis-
trifluoromethylphenylacetate: (1) LHMDS (3 eq), THF,
MeI (3 eq); (2) NaOH, MeOH.
presence of human serum (90-fold), yet 30 had only
slightly reduced IP-1 functional potency compared to
the acetylpiperidine analogs. Interestingly, compound
30 did provide 24 h efficacy in the gerbil assay. Based
on these data, we cannot rule out the possibility that
the in vivo activity was driven in part due to an active
metabolite.
We next examined amide replacements embedded in the
tether to see if improved metabolic stability would trans-
late to longer duration of in vivo activity. We prepared
the enantiomeric oxadiazoles 24 and 25, which were sur-
prisingly equipotent in terms of intrinsic receptor affin-
ity, but only isomer 24 demonstrated substantial
functional receptor blockade. Although oxadiazole 24
showed in vivo activity out to 4 h, this modification
did not provide the 24 h coverage that was desired.
The regioisomeric oxadiazoles 20 and 22 had exquisite
intrinsic activity, but only 22 had a low shift in the pres-
ence of 50% human serum (sixfold) and good functional
activity. An interesting and surprising feature was that
22 possessed the opposite absolute configuration to the
other carboxamides described thus far. Oxadiazole 22
showed improved (24 h) in vivo activity compared to
amide 28 presumably due to enhanced metabolic
stability.
10. (a) Finke, P. E.; Meurer, L. C.; Levorse, D. A.; Mills, S.
G.; MacCoss, M.; Sadowski, S.; Cascieri, M. A.;
Tsao, K.-L.; Chicchi, G. G.; Metzger, J. M.; MacIntyre,
D. E. Bioorg Med. Chem. Lett. 2006, 16, 4497; (b) Meurer,
L. C.; Finke, P. E.; Owens, K. A.; Tsou, N. N.; Ball, R.
G.; Mills, S. G.; MacCoss, M.; Sadowski, S.; Cascieri, M.
A.; Tsao, K.-L.; Chicchi, G. G.; Egger, L. A.; Luell, S.;
Metzger, J. M.; MacIntyre, D. E.; Rupniak, N. M. J.;
Williams, A. R.; Hargreaves, R. J. Bioorg. Med. Chem.
Lett. 2006, 16, 4504.
We have described a series of pyrrolidine carboxamides
and oxadiazoles that displayed potent antagonism to-

J. R. Young et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5310–5315
5315
11. Rupniak, N. M. J.; Tattersall, F. D.; Williams, A. R.;
Rycroft, W.; Carlson, E. J.; Cascieri, M. A.; Sadowski, S.;
Ber, E.; Hale, J. J.; Mills, S. G.; MacCoss, M.; Seward, E.;
Huscroft, I.; Owen, S.; Swain, C. J.; Hill, R. G.;
Hargreaves, R. J. Eur. J. Pharmcol. 1997, 326, 201.
phatases, in Hepes-buffered inositol-free (DMEM) medium
with 0.02% BSA for 15 min. Antagonists (100 nM) or
DMSO were added for 30 min then Substance P (10 lM)
was added for another 30 min, all at 37 ꢁC. The reaction
was terminated by removal of the medium and addition of
60 lL of 10 mM formic acid for 60 min. at 22 ꢁC. Lysates
(15 lL) were incubated with 1 mg RNA Binding Yittrium
Silicate (2–5 lm) SPA beads (Amersham) in Optiplates
with shaking for 2 h at 22 ꢁC. [3H]-inositol phosphate
generation was quantitated on a Packard Topcount and the
data were analyzed using Prism (GraphPad).
12. Briefly, CHO cells (2.5 · 104) stably expressing recombi-
nant human NK1 receptors were plated in 96-well tissue
culture dishes overnight followed by prelabeling overnight
with [3H]-myo-inositol (10–25 Ci/mmol, 1 lCi/well) in ino-
sitol-free medium with 0.02% BSA at 37 ꢁC. The cells were
incubated with 10 mM LiCl, to inhibit inositol monophos-