Identification, synthesis, and biological evaluation of novel pyrazoles as low molecular weight luteinizing hormone receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2006.12.062

In the course of a high throughput screening, a series of pyrazole compounds were identified with luteinizing hormone receptor (LH-R) agonist activity. A focused pyrazole library was produced by solid-phase synthesis and key pyrazole regioisomers were obt

Full text of DOI:10.1016/j.bmcl.2006.12.062

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2080–2085
Identification, synthesis, and biological evaluation of novel
pyrazoles as low molecular weight luteinizing
hormone receptor agonists
Catherine Jorand-Lebrun,a,* Bill Brondyk,b Jing Lin,b Sharad Magar,b Robert Murray,b
Adulla Reddy,b Hitesh Shroff,b Greg Wands,b Weishui Weiser,b
Qihong Xu,b Sean McKennab and Nadia Bruggerb,*
aMerck Serono Geneva Research Center, Bat. B3, 9, chemin des Mines, 1211 Geneva, Switzerland
bEMD Serono Research Institute, 1 Technology Road, Rockland, MA 02370, USA
Received 27 October 2006; revised 13 December 2006; accepted 16 December 2006
Available online 22 December 2006
Abstract—In the course of a high throughput screening, a series of pyrazole compounds were identified with luteinizing hormone
receptor (LH-R) agonist activity. A focused pyrazole library was produced by solid-phase synthesis and key pyrazole regioisomers
were obtained selectively in solution. Evaluation of those compounds in a cAMP assay in CHO cells transfected with h-LH receptor
allowed us to propose a structure–activity relationship model for this series and led to the identification of the first low molecular
weight molecule with in vitro activity in a Leydig cells assay (ED50 = 1.31 lM) and in vivo in a model of testosterone induction in
rats (significant effect at 32 mpk ip).
Ó 2007 Elsevier Ltd. All rights reserved.
Luteinizing hormone (LH), a member of the gonadotro-
pin family, plays an essential role in human reproduc-
tion. In women, LH promotes the maturation of
follicular cells in the ovary,1 while in men, it is involved
in spermatogenesis and stimulation of testosterone pro-
duction in Leydig cells.2 LH is a relatively large hetero-
dimeric glycoprotein (28–38 kDa). Its receptor (LH-R)
belongs to the super family of G protein-coupled recep-
tors (GPCRs). The signal transduction pathway is essen-
tially mediated by an increase in the level of cAMP
through Gs protein. Like all other members of the gon-
adotropin hormone receptor family, LH receptor can be
distinguished by an extremely long extracellular domain
containing several leucine-rich repeats.3 Although the
binding mode of LH to its receptor is currently still
not fully understood, it was demonstrated that LH
accesses the receptor via its extracellular domain in
the region of exons 1–8. With high affinity binding,
LH could induce conformational changes in the
extracellular loop of the receptor, activating the signal
transduction cascade of the transmembrane region.4,5
LH therapy is currently utilized in conjunction with fol-
licle stimulating hormone (FSH) to treat infertility and
spermatogenesis disorders. Historically the gonadotro-
pins FSH and LH were extracted from the urine of post-
menopausal women. The LH activity of urinary hMG
(human menopausal gonadotropin) was however pri-
marily provided by addition of urinary human chorionic
gonadotropin (hCG) due to the low amount of naturally
occurring LH in postmenopausal urine. Recombinant or
urinary gonadotropins are required to be injected paren-
terally, the availability of oral drugs would simplify the
treatment regimen and increase the level of comfort for
patients. In this paper, we report novel LMW (low
molecular weight) LH-receptor agonists exhibiting in
vivo activity in a model of testosterone induction in
rat. To date, only few LMW modulators of gonadotro-
pin receptors have been described6–10 and only one
example of a LH receptor agonist exhibiting in vivo
activity has been reported so far.11 A screening
campaign was performed in our laboratory, resulting
in the identification of a series of pyrazole positives. A
focused library of pyrazoles was then synthesized by a
Keywords: Gonadotropins; Luteinizing hormone receptor agonist;
Regioselective pyrazole synthesis.
*
Corresponding authors. Tel.: +41 22 414 9838; fax: +41 22 414 9565
(C.J.-L.); e-mail addresses: catherine.jorandlebrun@merckserono.
net; nadia.brugger@emdserono.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.062

C. Jorand-Lebrun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2080–2085
2081
solid-phase synthesis (SPS) strategy. The preliminary
structure–activity relationship (SAR) obtained from
these pyrazoles was further refined by the synthesis of
key compounds following the same solid-phase route.
A new solution-phase route was developed in order to
access the most active compounds in large amounts
and with the desired regioselectivity. The best agonists
were further evaluated in vitro in a cell assay measuring
testosterone production and in vivo in a testosterone
induction model in rats.
regioselective pyrazole syntheses are described in the lit-
erature and most of them lead to the 2,3,5-substituted
regioisomer. One new regiospecific approach for 1,3,5-
substituted pyrazoles was recently proposed by Katzen-
ellenbogen and co-workers,14 using the cyclization of a
hydrazone intermediate. However, when the hydrazone
formed by condensation of 3-acetylpyridine and 4-tert-
butylphenyl hydrazine was treated with propionic
anhydride, the cyclization failed in all conditions,
whether neutral, acidic or basic. Another regiospecific
route to access 1,3,5-substituted pyrazoles was de-
scribed by Murray.15 He observed that the presence
of a hydroxypropyl chain in the b-diketo precursor
could reverse the usual regioselectivity of the pyrazole
ring formation. Indeed, when applied to 3-pyridinyldik-
etone 12 and 4-tert-butyl hydrazine, those conditions
led to the formation of 2,3,5-substituted pyrazole 13
and 1,3,5-substituted pyrazole 14 in a 2:8 ratio, respec-
tively, with a good overall yield (Scheme 2). Pyrazole
alcohol 14 was a key intermediate for the synthesis of
numerous 1,3,5-substituted pyrazoles as illustrated by
the synthesis of compound 10. The mixture of the
two alcohols 13 and 14 was oxidized to the correspond-
ing aldehydes with Dess–Martin reagent. A two-carbon
homologation through a Wittig–Horner reaction with
tert-butyl diethylphosphonoacetate gave a,b-unsaturat-
ed esters 15 and 16. Separation of the two regioisomers
was achieved at this stage by flash chromatography.
Reduction of the double bond, followed by acidic
hydrolysis of the ester, led to the acid 17 in good yield.
The final compound 10 was easily obtained from that
intermediate by coupling with O-tert-butyl-L-tyrosina-
mide using standard coupling conditions and acidic
hydrolysis of the tert-butyl ether.
The pyrazole library was prepared on solid phase using
a rink amide linker (Scheme 1). After deprotection of
the linker, amino acids 1 were first loaded on the resin
using standard coupling conditions and subsequently
deprotected. The free amines were then coupled with
keto-acids 3 possessing diverse spacers L such as alkyl
or heteroalkyl chains or aryl or heteroaryl rings to give
the aceto-derivatives 4. b-Diketones 6 were obtained by
reaction of intermediates 4 with aromatic or heteroaro-
matic esters 5 and treated with alkyl or aryl hydrazines
7 to form the pyrazole ring. After cleavage from the
resin, a mixture of two regioisomers, 1,3,5- and 2,3,5-
substituted pyrazoles, was isolated with overall yields
higher than 90% in most examples. Both regioisomers
could be isolated by preparative HPLC. Generally,
the 2,3,5-substituted pyrazole is observed to be the
major isomer with this synthetic method.12 This was
proven to be the case for this library as well, when
the structure of compounds 10 and 11 was unambigu-
ously assigned by nuclear Overhauser experiments on
both regioisomers13 (Fig. 1). As the SPS route
gave too small quantities of the minor 1,3,5-isomer, a
novel, regioselective route was developed. Very few
O
O
a, b
a, c
H
H
N
N
H
L
fmoc
N
fmoc
N
Pol
N
H
Pol
Pol
R3
H
R3
O
O
2
4
d
R2
R2
O
O
O
H
f
e
H
H
N
N
R2
L
Pol
L
N
N
NH2
N
N
R1
N
Pol
H
R1
R3
H
L
N
N
R3
R3
O
H
H
O
O
O
O
9
8
6
Scheme 1. Solid-phase synthesis of pyrazoles. Reagents and conditions: (a) 20% piperidine in DMF, 25 °C, 2 h; (b) HOOCCHR3NHFmoc 1, HATU,
DIEA, DMF, 25 °C, 24 h; (c) HOOC-L-COMe 3, HATU, DIEA, DMF, 25 °C, 24 h; (d) R2COOMe 5, NaH, DMA, 85 °C, 24 h; (e) R1NHNH2 7,
DIEA, DMA, 80 °C, 24 h; (f) TFA/DCM 2:1, 25 °C, 2 h.
N
N
3
3
CONH2
CONH2
NOE
H
H
5
2 N
N
N
5
N
1 N
N
O
O
OH
OH
10
11
Figure 1. NOE cross peaks were observed between the ortho-protons of the pyridine ring and of the tert-butylphenyl ring for isomer 11 but not for
isomer 10.

2082
C. Jorand-Lebrun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2080–2085
N
N
N
N
N
OH
a
b
N
+
+
OH
N
O
OH
O
N
O
O
O
12
13
14
c
N
N
N
O
N
O
N
N
+
O
O
15
16
d
N
N
N
H
H
N
N
CONH2
f
e
N
N
CONH2
N
OH
N
N
N
O
O
O
OH
10
18
17
O
Scheme 2. Solution-phase synthesis of pyrazoles. Reagents and conditions: (a) NaH, THF/DMSO, 25 °C, 24 h, 65%; (b) p-tBuPheNHNH2ÆHCl, 2 M
HCl in ether, MeOH, 25 °C, 15 h, 87%; (c) i—Dess–Martin, DCM, 25 °C, 4 h, ii—(EtO)2POCH2COOtBu, NaH, THF, 0 °C, 1 h, 40%;
(d) i—HCOONH4, 10% Pd/C, MeOH, reflux, 1 h, ii—TFA/DCM, 25 °C, 4 h, 90%; (e) O-tert-butyltyrosinamide, EDC, HOBT, DIEA, 25 °C, 24 h,
80%; (f) TFA/DCM, 25 °C, 1 h, 82%.
The activity of compounds was assessed by an Amer-
sham cAMP specific SPA assay using hLH-R CHO
cells. The initial screening was designed to detect
agonists: production of cAMP in response to a 12lM
concentration of tested compound was measured.
Compounds showing more than 60% of the maximal
observed response normalized to the response obtained
without stimulation were considered as positives. Purity
of positives was controlled,16 and EC50 and efficacy were
measured with dose–response curves for selected
positives.17 Specificity for hLHR was demonstrated by
testing the compounds on untransfected CHO parental
cells. The screening of the pyrazole library allowed us
to define the main features required for the activity
(Fig. 2): the nitrogen substituent of the pyrazole ring
(R1) has to be a large lipophilic group, such as 4-tert-
butylphenyl or benzyl. Pentyl and heptyl chains were
more active than phenyl substituted with small non-po-
lar groups, like methyl, or polar groups, like Cl, COOH,
OMe. For the substituent R2 in position 3, pyridines and
quinolines gave the best activities amongst a selection of
aryl and heteroaryl groups. Lipophilic spacers L like
alkyl chains or phenyl rings were well tolerated between
the pyrazole ring and the amino acid moiety, but the
presence of a heteroatom either in the chain or in a cycle
led to a decrease in activity. More importantly, only
good activity with Pyridines/quinolines
no activity with aryl, furane, pyrrole
N
lipophilic spacer
CONH2
N
N
R
L
N
O
Large lypophilic pocket
Polar group not tolerated
OH
Tyrosine moiety
Figure 2. Preliminary SAR of the pyrazole series.

C. Jorand-Lebrun et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2080–2085
2083
compounds with tyrosine as the amino acid moiety gave
acceptable activity from a selection of nineteen diverse
amino acids. As the screening was performed using a
mixture of the two possible pyrazole regioisomers,
refinement of the initial SAR could be achieved by the
synthesis of some key compounds as single regioisomers.
Compounds 19 and 20, isolated as pure regioisomers
(Fig. 3), were tested in the SPA assay. The minor
and the position of the amino acid, as illustrated by the
activities found for compounds 25, 26, and 20. The
pyrazole substituent at position 3 can be a pyridine or
a quinoline, although pyridines are more active than
quinolines (data not shown) and 3-pyridine appears to
be optimal in terms of potencies and efficacies compared
to 2- and 4-pyridines (see compounds 20, 10 and 26, for
example). Finally, the carboxamido group does not
appear to be necessary and can be removed without
altering the activity (compounds 27 and 28).
regioisomer, the 1,3,5-substituted pyrazole 20 (EC50
=
250 nM) was more active than the 2,3,5-substituted
pyrazole 19 (EC50 = 2.3 lM) and this observation
entailed the necessity to optimize a route to access the
minor 1,3,5-regioisomer in higher yield. Results
obtained for representative 1,3,5-substituted pyrazoles
are summarized in Table 1. A four-carbon alkyl chain
spacer gives better activity compared to a shorter chain
(compounds 21 and 10), while a phenyl ring spacer
seems to decrease the activity (isoquinoline 22), as
well as the presence of a heteroatom in the alkyl
chain (compound 23). The hydroxy substituent of
the tyrosine moiety contributes to the high activity
(compound 10 vs 24). The distance between the pyrazole
ring and the hydroxy residue appears to be more impor-
tant for the activity than the arrangement of the spacer
Compound 10, which displayed one of the best potency
and efficacy, was selected for further pharmacological
characterization. It was tested in a rat Leydig cells assay
using testosterone as the end point measurement18 and
displayed an ED50 of 1.31 0.41 lM with a 78% effica-
cy. The lower activity in this bioassay (65-fold difference
as compared to the cAMP assay in hLH-transfected
CHO cells) may be explained by a species difference.
However, such differences in potency are often observed
between cellular assay using native cells and constructed
cells.9 In a binding assay, compound 10 was not able to
displace 125I-labeled hCG while LH was measured with
a Ki = 311 pM in the same assay. Only one LMW
N
N
CONH2
H
N
CONH2
H
N
N
N
N
O
N
OH
O
OH
19
20
Figure 3.
Table 1. Refined SAR of the 1,3,5-pyrazoles series
R4
R2
O
X
N
L
N
H
n
N
a
Compound
R2
L
X
R4
n
EC50 (lM) (%)
Purityb
10
20
21
22
23
24
25
26
27
28
3-Pyridine
4-Pyridine
3-Pyridine
3-Isoquinoline
4-Pyridine
3-Pyridine
2-Pyridine
2-Pyridine
4-Pyridine
3-Pyridine
(CH2)4
(CH2)4
(CH2)2
1,3-Ph
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CH2CONH2
H
4-OH
4-OH
4-OH
4-OH
4-OH
4-OtBu
4-OH
3-OH
4-OH
4-OH
1
1
1
1
1
1
0
1
1
1
0.02 0.01 (70%)
0.25 0.04 (55%)
>100
98
99
92
77
94
98
96
90
88
98
0.50 0.10 (19%)
>100
HN(CH2)3
(CH2)4
(CH2)5
(CH2)5
(CH2)4
(CH2)4
12.1 2.0 (72%)
0.17 0.02 (78%)
0.15 0.02 (41%)
0.19 0.06 (66%)
0.08 0.01 (45%)
a EC50 (lM) in the CHO-hLHR-cAMP assay (% max LH response). Data are presented as means SE (n = 3).
b % purity determined by LC/MS.

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3000
2500
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Murray, R.; Weiser, W.; Nabioullin, R.; Rosenthal, J.;
Buckler, D.; Cheng, S.; Liu, J.; McKenna, S.; Jiang, X.;
Evans, D.; Tepper, M.; El Tayar, N. Abstracts of Papers,
226th National Meeting of the American Chemical Soci-
ety, New York, NY, United States, September 7–11, 2003;
American Chemical Society: Washington, DC, 2003;
MEDI 365.
8. El Tayar, N.; Reddy, A.; Liao, Y.; Magar, S.; Murray, R.;
Kozack, R.; Weiser, W.; Nabioullin, R.; Rosenthal, J.;
Buckler, D.; Cheng, S.; Liu, J.; McKenna, S.; Jiang, X.;
Evans, D.; Tepper, M.; Goutopoulos, A. Abstracts of
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MEDI 355.
0
Vehicle
32 mg/kg
16 mg/kg
4 mg/kg
Figure 4. Two-month-old male rats testosterone-suppressed by a
subcutaneous pellet of DES were injected with compound 10 (32, 16,
and 4 mg/kg) or vehicle. Serum testosterone concentrations measured
by radioimmunoassay.
9. Palmer, S. S.; McKenna, S.; Arkinstall, S. RBM Online
2005, 10, 45.
10. Van Straten, N. C. R.; Van Berkel, T. H. J.; Demont, D.
R.; Karstens, W.-J. F.; Merkx, R.; Oosterom, J.; Schulz,
J.; Van Someren, R. G.; Timmers, C. M.; Van Zandvoort,
P. M. J. Med. Chem. 2005, 48, 1697.
modulator of gonadotropin receptors is described with
the ability to displace the corresponding gonadotropin6
while many others10,11 do not. It is assumed that for
most LMW modulators, the interaction with the recep-
tor takes place directly in the transmembrane region of
the receptor.
11. Van Straten, N. C. R.; Schoonus-Gerritsma, G. G.; Van
Someren, R. G.; Draaijer, J.; Adang, A. E. P.; Timmers,
C. M.; Hanssen, R. G. J. M.; Van Boeckel, C. A. A.
ChemBioChem 2002, 10, 1023.
12. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J.
S.; Collins, P. W.; Docter, S.; Graneto, M. J.; Lee, L. F.;
Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn,
J. N.; Gregory, S. A.; Koboldt, C. M.; Perkins, W. E.;
Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.; Isakson, P.
C. J. Med. Chem 1997, 40, 1347.
Finally, compound 10 was tested in a model of testoster-
one induction in rat.19 As shown in Figure 4, when
administered ip at doses between 4 and 32 mg/kg, com-
pound 10 induced increased testosterone concentrations
in a dose-dependent manner.
13. Compounds 10 and 11 were obtained in a 1:9 ratio and
separated by reversed-phase HPLC using a DELTAPAK
C18 column with a linear gradient of 0.1% TFA water/
acetonitrile 95:5 to 60:40 in one hour. Structures were
assigned by Nuclear Overhauser experiments performed
on a JEOL 400 MHz NMR apparatus. There were NOE
cross peaks displayed between protons of the pyridine ring
and protons of the tert-butylphenyl ring of isomer 11, and
no NOE effect observed between those protons for isomer
10. Compound 11 (more polar): N-{5-[2-(4-tert-butylphe-
nyl)-3-pyridin-3-yl-1H-pyrazol-5-yl]pentanoyl}-L-tyrosi-
namide trifluoroacetate salt, 1H NMR (400 MHz,
DMSO): d = 1.28 (s, 9H), 1.55 (m, 4H), 2.12 (t,
J = 7.3 Hz, 2H), 2.63 (m, 3H), 2.86 (dd, J = 13.9, 5.3 Hz,
1H), 4.35 (m, 1H), 5.60 (br s, 1H), 6.61 (s, 1H), 6.62 (d,
J = 8.8 Hz, 2H), 6.98 (m, 1H), 7.01 (d, J = 8.8 Hz, 2H),
7.19 (d, J = 8.8 Hz, 2H), 7.36 (m, 1H), 7.42 (d, J = 8.8 Hz,
2H), 7.51 (dd, J = 8.1, 5.1 Hz, 1H), 7.75 (dt, J = 8.1,
2.2 Hz, 1H), 8.52 (dd, J = 2.2, 0.7 Hz, 1H), 8.58 (dd,
J = 4.9, 1.5 Hz, 1H); MS (ESI, pos.) m/z: 540 (M+1).
Compound 10 (less polar): N-{5-[1-(4-tert-butylphenyl)-3-
pyridin-3-yl-1H-pyrazol-5-yl]pentanoyl}-L-tyrosinamide
trifluoroacetate salt, 1H NMR (400 MHz, DMSO):
d = 1.34 (s, 9H), 1.47 (m, 4H), 2.05 (t, J = 9.1 Hz, 2H),
2.61 (m, 1H), 2.64 (t, J = 6.9 Hz, 2H), 2.84 (m, 1H), 4.34
(m, 1H), 6.30 (br s, 1H), 6.59 (d, J = 8.4 Hz, 2H), 6.98 (m,
1H), 6.99 (d, J = 8.4 Hz, 2H), 7.02 (s, 1H), 7.37 (m, 1H),
7.48 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.86 (d,
J = 8.4 Hz, 1H), 7.92 (dd, J = 8.1, 5.5 Hz, 1H), 8.72 (dt,
J = 8.4, 1.5 Hz, 1H), 8.76 (dd, J = 5.5, 1.5 Hz, 1H), 9.24
In summary, we have identified, synthesized, and opti-
mized a new series of pyrazoles as LH receptor ago-
nists. Compound 10, one of the most potent and
efficient agonist in the cAMP assay, revealed a prom-
ising biological profile in cellular and in vivo LH acti-
vation models. This compound is the first LH agonist,
which showed in vivo activity in a testosterone induc-
tion model.
Acknowledgments
We gratefully acknowledge A. Bombrun and M. Sch-
warz for reviewing this manuscript. We would like to
dedicate this paper to Dr. Nabil El Tayar.
References and notes
1. Erickson, G. F.; Magoffin, D. A.; Dyer, C. A.; Hofeditz,
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2. Sharpe, R. M. Clin. Endocrinol. 1990, 33, 787.
3. Dufau, M. L. Annu. Rev. Physiol. 1998, 60, 461.
4. Jiang, X.; Dreano, M.; Buckler, D. L.; Cheng, S.; Ythier,
A.; Wu, H.; Hendrickson, W. A.; El Tayar, N. Structure
1995, 3, 1341.
2
(d, J = 2.2 Hz, 1H); MS (ESI, pos.) m/z: 540 (M+1); ½aꢀD
5. El Tayar, N. Mol. Cell. Endocrinol. 1996, 125, 65.
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16. Compounds with purity lower than 60% by LC/MS were
not considered.
collagenase-containing media (phenol red-free DMEM/
F12 + 0.1% collagenase) at 37 °C in 5% CO2 for 15 min.
Cells were washed, plated at a density of 350,000 cells/well
in 96-well plates (in phenol red-free DMEM/F12 media
with 0.1% BSA), and stimulated with test compounds for
3 h. The plate was centrifuged to bring down the cells and
50 lL of culture media transferred to an ELISA plate
(DSL) for testosterone measurement.
17. Production cAMP in response to LH or small molecules
was measured in CHO cells stably transfected with h-LH
receptor. The cells were plated at a density of 20,000 cells/
wells in growth medium (MEMalpha plus, 10% FBS, 1%
L-glutamine, and 600 lg/ml Geneticin) in 96-well plates 1
day prior to the assay. Stimulation was carried out in
assay buffer (phenol red-free DMEM/F12 containing 0.1%
BSA, 0.1 mmol/L IBMX, and 1% pen-strep) for 60 min
with increasing doses of test molecules. Following stimu-
lation, cells were lysed and cAMP in the lysate was
measured using a cAMP chemiluminescent assay kit
(Tropix, Bedford, MA, USA) as per manufacturer’s
instructions.
19. In vivo testosterone induction activity assay: on day 1,
adult Sprague–Dawley rats were implanted subcutane-
ously in the scalpular region with a single 7-day slow-
release pellet containing 7.5 mg of diethylstilbestrol
(DES). After two days, rats were injected intraperitone-
ally with compound 10 at 0, 4, 16, or 32 mg/kg. Three
rats were used in each treatment group. Blood samples
were removed from the rat by retro-orbital plexus
disruption after two hours following the administration.
Serum was collected from blood by centrifugation and
frozen at ꢁ80 °C until the time of assay. Serum testos-
terone concentrations and standard deviations were
determined by ELISA and analyzed using Microsoft
Excel software program.
18. LH-stimulated testosterone production or small molecule
LH-mimetic activity was measured in Leydig cells har-
vested from male Sprague–Dawley rats between the ages
of 21 and 40 days. Decapsulated testis was digested in