Development of a high specific activity sulfur-35-labeled sulfonamide radioligand that allowed the identification of a new growth hormone secretagogue receptor

Full text of DOI:10.1021/jm960054c

J . Med. Chem. 1996, 39, 1767-1770
1767
Develop m en t of a High Sp ecific Activity
Su lfu r -35-La beled Su lfon a m id e
Ra d ioliga n d Th a t Allow ed th e
Id en tifica tion of a New Gr ow th Hor m on e
Secr eta gogu e Recep tor
Dennis C. Dean,*,† Ravi P. Nargund,*,‡
Sheng-Shung Pong,§ Lee-Yuh P. Chaung,§
Patrick Griffin,§ David G. Melillo,†
Robert L. Ellsworth,† Lex H. T. Van Der Ploeg,§
Arthur A. Patchett,‡ and Roy G. Smith§
Departments of Drug Metabolism, Medicinal Chemistry, and
Biochemistry, Merck Research Laboratories, Merck & Co.,
P.O. Box 2000, Rahway, New J ersey 07065
Received J anuary 17, 1996
Growth hormone (GH) secretagogues have received
considerable attention in the last several years based
on promising, ever-widening investigational applications
of recombinant growth hormone in animals and in
humans. The peptidyl GH secretagogue 1, GHRP-6
(His-D-Trp-Ala-Trp-D-Phe-Lys-NH2),1,2 the benzolactam
2a , L-692,429,3,4 and the recently disclosed orally active
spiropiperidine-based GH secretagogue 3a (MK-0677,
Figure 1),5 have been shown to elevate GH in several
species, including human. The mechanism(s) of action
of these three classes of secretagogues is not clearly
understood. Identification of a discrete receptor to
which these compounds bind and exert their action has
thus far been elusive.
The experimental evidence generated with rat pri-
mary pituitary cells shows that the two structurally
different secretagogues 3a and 2a are mechanistically
indistinguishable from the hexapeptide 1 and, hence,
are peptidomimetic agonists.3,6 This is remarkable
because small molecule agonists for peptide ligands are
rare, presumably due to multiple and precise functional
group requirements for agonist activity. We have
sought direct evidence to support the contention that
3a and 2a are indeed peptidomimetics of the hexapep-
tide 1. Competition binding studies with the GH
secretagogue receptor would provide strong evidence to
show that 1, 2a , and 3a are agonist mimetics of one
another. These binding studies may require a high
specific activity radioligand depending on the receptor
abundance and binding affinity. Previous attempts to
develop a binding assay using [3H]-7 and [125I]-labeled8
peptide ligands derived from GHRP-6 have met with
limited success. Generally, the binding was of low
affinity and of excessively high capacity. Moreover, the
binding affinities did not appear to correlate with the
GH secretory activity of the peptides. The lack of
correlation of binding affinity and GH secretory activity
most likely resulted from the relatively low specific
activity (in the case of [3H]GHRP-6) and nonspecific
binding properties of the radioligands. Therefore, we
explored the synthesis and utility of labeled peptidomi-
metics such as MK-0677 (3a ) and its analogs.
F igu r e 1.
choice for the study of receptors.9 Hence, we initially
evaluated the effect of halogen substitution on the
phenyl rings of 3a on GH release from rat pituitary cells.
However, incorporation of halogens (e.g. Cl, Br) at the
para position of the benzyl and in the 5-position of the
spiroindoline phenyl groups lead to an >20-fold loss in
intrinsic activity. This indicated that iodine substitu-
tion at these positions would not afford a high-potency
ligand. In addition, the amino functionality was es-
sential for biological activity, and so conjugation with
the widely used Bolton-Hunter reagent9 did not appear
a viable alternative. Therefore, we explored other high
specific activity sulfur-35 probes to identify the GH
secretagogue receptor.
In this article we detail the synthesis of a high specific
activity (700-1100 Ci/mmol) [35S]MK-0677 radioli-
gand.10 The availability of this [35S]sulfonamide-based
radioligand provided the key breakthrough in identify-
ing a saturable, specific, and high-affinity binding site
in porcine and rat anterior pituitary membranes which
has been linked to the activity of these secretagogues.6
We have previously reported that a methanesulfonyl
unit on the indoline nitrogen as in 3a was important in
order to obtain good intrinsic activity.5 Suprisingly,
equal activities were obtained using the benzenesulfonyl
substitution 3b. Initially, we chose to explore the
preparation of high specific activity benzene[35S]sulfonyl
chloride due to the relative ease of synthesis. However,
all attempts to sulfonate benzene with carrier free [35S]-
sulfuric acid (obtained from NEN at 1300 Ci/mmol) were
frustrated by substantial dilution of the specific activity
for the resulting benzene[35S]sulfonic acid. This dis-
crepancy was apparently the result of contamination by
trace amounts of ubiquitous sulfates in various reagents
and/or reaction vessels.
In situations where a specific activity of >100 Ci/
mmol is required, radioiodine has been the label of
Therefore, we turned our attention to the synthesis
of methane[35S]sulfonyl chloride. Much better results
were realized using the Strecker reaction11 to prepare
methane[35S]sulfonate from sodium [35S]sulfite. High
* Corresponding authors.
† Department of Drug Metabolism.
‡ Department of Medicinal Chemistry.
§ Department of Biochemistry.
0022-2623/96/1839-1767$12.00/0 © 1996 American Chemical Society

1768 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Communications to the Editor
Sch em e 1a
a
Reagents: (a) CBZCl, Et3N, CH2Cl2, 0 °C, 30 min; (b) ClCO2CHClCH3, ClCH2CH2Cl, reflux, 1 h; MeOH, reflux, 1 h (80%); (c) EDC,
HOBT, N-methylmorpholine, room temperature, 12 h; (d) TFA; aqueous Na2CO3 workup; (e) HOOCC(CH3)2NHtBOC, EDC, HOBT, CH2Cl2,
room temperature, 12 h (74%); (f) 10% Pd/C, H2, EtOH, Et3N(cat.); (g) Me[35S]SO2Cl, Et3N, CH2Cl2, room temperature, 30 min; (h) TFA,
CH2Cl2, room temperature, 30 min; (i) HPLC purification.
specific activity sodium [35S]sulfite could be obtained by
reduction of [35S]sulfuric acid on a copper surface with
trapping of the generated [35S]sulfur dioxide in sodium
hydroxide solution (eq 1). Because the sodium [35S]-
sulfite rapidly oxidized to [35S]sulfate, a one-pot reaction
system was devised in which excess methyl iodide was
added to the sodium hydroxide trap to directly obtain
sodium methane[35S]sulfonate (5-10% yield from [35S]-
sulfuric acid) along with varying amounts of sodium
[35S]sulfate, which was easily recycled (eq 2). Conver-
sion to methane[35S]sulfonyl chloride was best effected
(70-75% radiochemical yield) using oxalyl chloride with
dimethylformamide in dichloromethane.12
F igu r e 2. Saturation isotherm of specific [35S]MK-0677
6
binding in rat anterior pituitary membranes analyzed by
Scatchard plot.
chloride solution to a near saturated solution of 7 in
dichloromethane in the presence of triethylamine
The synthesis of the necessary amine precursor 7 is
illustrated in Scheme 1. Our original four-step synthe-
sis of compounds in the MK-0677 series commenced
with an appropriate spiroindoline sulfonamide.5 For the
expedient synthesis of [35S]sulfonamides, we devised a
new synthesis of 3a wherein the sulfonyl unit was
incorporated in the penultimate step. Protection of the
known spiroindoline 4 as a benzyl carbamate followed
by N-demethylation according to the procedure of Olof-
son13 gave the spiropiperidine 5 in 80% yield. Standard
peptide type coupling of 5 with 6, removal of the BOC
protecting group with strong acid followed by a second
coupling with N-t-BOC-aminoisobutyric acid, and sub-
sequent selective hydrogenolysis of the N-CBZ group
(Pd/C in ethanol containing a trace of triethylamine)
gave 7 in excellent yield.
(Scheme 1). The instantaneous reaction typically pro-
duced the desired [35S]sulfonamide in 60-70% radio-
chemical yields. The remaining radioactivity corre-
sponded to methane[35S]sulfonic acid, formed through
hydrolysis by residual water in the presence of amine
base. The utilization of more dilute solutions of 7, even
when rigorously dried dichloromethane and triethyl-
amine were used, resulted in almost exclusive formation
of the sulfonic acid.14 Subsequent BOC removal and
HPLC purification produced [35S]-3a of >99% radio-
chemical purity.15
Determination of [35S]MK-0677 specific activity by
standard UV/concentration measurement proved dif-
ficult and unreliable. However, accurate values were
readily obtained using µCi amounts of ligand by capil-
lary LC/MS/MS analysis to directly observe the isotopic
ratio of 32S/35S. Specific activities ranging from 700 to
1100 Ci/mmol were obtained using different batches of
Optimal conditions for sulfonylation of 7 consisted of
adding a maximally concentrated methane[35S]sulfonyl

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1769
development of methane[35S]sulfonic acid synthesis. We
also thank Dr. Allen J ones and Mr. Steve Staskiewicz
of Merck Drug Metabolism for providing analytical
support.
Ta ble 1. Correlation of Binding Affinities of GH Secretagogues
to [35S]MK-0677 Receptor (Swine) and GH Secretory Activity
(Rat)
binding affinity
EC50 secretion
(nM)b
compound
(Ki, nM)a
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization of compounds 5, 7, and unlabeled
MK-0677; the preparation of methane[35S]sulfonyl chloride;
and HPLC radiochromatographs and MS spectra of [35S]MK-
0677 (7 pages). Ordering information is given on any current
masthead page.
3a (MK-0677)
1 (GHRP-6)
2a (L-692,429)
2b (L-692,428)
0.24
6
63
1.3
10
60
>5000
>5000
a
Ki values were determined by using the formula Ki ) IC50
/
b
(1+[L]/KD); ligand concentration L ) 100 nM. Data form rat
pituitary cell culture assay4.
Refer en ces
(1) Momany, F. A.; Bowers, C. Y.; Reynolds, G. A.; Hong, A.;
Newlander, K. Conformational energy studies and in vitro and
in vivo activity data on growth hormone-releasing peptides.
Endocrinology 1984, 114, 1531-1536.
(2) Bowers, C. Y.; Momany, F. A.; Reynolds, G. A.; Hong, A. On the
in vitro and in vivo activity of a new synthetic hexapeptide that
acts on the pituitary to specifically release growth hormone.
Endocrinology 1984, 114, 1537-1545.
(3) Smith, R. G.; Cheng, K.; Schoen, W. R.; Pong, S. S.; Hickey, H.;
J acks, T.; Butler, B.; Chan, W. W.-S.; Chaung, L.-Y. P.; J udith,
F.; Taylor, J .; Wyvratt, M. J .; Fisher, M. H. A nonpeptidyl growth
hormone secretagogue. Science 1993, 260, 1640-1643.
(4) Schoen, W. R.; Pisano, J . M.; Prendergast, K.; Wyvratt, M. J .;
Fisher, M. H.; Cheng, K.; Chan, W. W.-S.; Butler, B.; Smith, R.
G.; Ball, R. G. A Novel 3-Substituted Benzazepinone Growth
Hormone Secretagogue (L-692,429). J . Med. Chem. 1994, 37,
897-906.
(5) Patchett, A. A.; Nargund, R. P.; Tata, J . R.; Chen, M.-H.;
Barakat, K. J .; J ohnston, D. B. R.; Cheng, K.; Chan, W. W.-S.;
Bulter, B.; Hickey, G.; J acks, T.; Schleim, K.; Pong, S.-S.;
Chaung, L.-Y. P.; Chen, H. Y.; Frazier, E.; Leung, K. H.; Chiu,
S.-H. L.; Smith, R. G. Design and biological activity of L-163,-
191 (MK-0677): A potent, orally active growth hormone secre-
tagogue. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7001-7005.
(6) For characterization of the MK-0677 receptor, see: Pong, S.-S.;
Chaung, L.-Y. P.; Dean, D. C.; Nargund, R. P.; Patchett, A. A.;
Smith, R. G. Identification of a new receptor for growth hormone
secretagugues. Mol. Endocrinol. 1996, 10, 57-61.
(7) Walker, R. F.; Shu, A. Y.; Codd, E. E. Binding of a growth
hormone releasing hexapeptide to specific hypothalamic and
pituitary binding sites. Neuropharmacology 1989, 28, 1139-
1144.
(8) Bowers, C. Y.; Veeraragavan, K.; Sethumadhavan, K. Demon-
stration and characterization of the specific binding of growth
hormone-releasing peptide to rat anterior pituitary and hypo-
thalamic membranes. Biochem. Biophys. Res. Commun. 1991,
178, 31-37.
(9) McFarthing, K. G. Selection and synthesis of receptor-specific
radioligands. In Receptor-Ligand Interactions: A Practical Ap-
proach; Hulme, E. C., Ed.; Oxford University Press: Oxford,
1992; pp 1-18.
(10) While sulfur-35 has found widespread use in the nucleotide and
peptide fields (for a general overview, see ref 17, pp 1-5), to the
best of our knowledge this paper details the first synthesis of
high specific activity methane[35S]sulfonyl chloride and its use
to prepare corresponding sulfonamides.
methane[35S]sulfonic acid, presumably a consequence
of variable isotopic purity of [35S]sulfate precursor and/
or contamination during the preparation of methane-
[35S]sulfonic acid.
With high specific activity methane[35S]sulfonamide
ligand 3a , saturable, stereoselective high-affinity bind-
ing sites were detected in porcine and rat anterior
pituitary membranes.6 No binding sites were identified
in posterior pituitary membranes. Scatchard analysis
(Figure 2) indicated the presence of a single class of
high-affinity sites with a dissociation constant (KD) of
161 ( 11 pM which is close to the inhibition constant
(Ki) of 240 pM, as determined by displacement of [35S]-
3a with unlabeled 3a in competition binding studies.
The binding affinities as Kis of the hexapeptide GHRP-6
(1) and the structurally diverse small molecules 3a , 2a ,
and 2b (the inactive stereoisomer) correlated tightly
with their GH secretory activities (Table 1). Binding,
which required Mg2+, was inhibited by GTP-γ-S, indi-
cating that this newly characterized receptor is G-
protein coupled. The GHS receptor was found in
exceedingly low concentration (Bmax ) 2.5-7.00 fmol/
mg of protein) which underscored the importance of
having used the [35S]MK-0677 radioligand 3a as de-
scribed in these studies. These results strongly support
the designation of 3a and 2a as peptidomimetic agonists
of GHRP-6.
In conclusion, we have described an efficient synthesis
of a high specific activity sulfur-35 labeled sulfonamide
for identification of a GH secretagogue receptor. [35S]-
MK-0677 was found to possess the necessary combina-
tion of high affinity, selectivity, and specific activity
required for utilization as a radioligand in the study of
this newly discovered receptor. Receptor characteriza-
tion and use of [35S]MK-0677 may also be of value in
the search for the presumed natural ligand of this GH
secretagogue receptor. The synthesis of [35S]MK-0677
was accomplished using new methodology to prepare
methane[35S]sulfonyl chloride at near theoretical spe-
cific activity. This reagent can be coupled to an ap-
propriate amine to provide methane[35S]sulfonamides
which possess excellent radiochemical purity and stabil-
ity.16 Importantly, this functionality should be consid-
ered as an alternative to 125I and may be particularly
useful in the synthesis of peptide and non-peptide
radiolabeled ligands with long radioactive half-lives and
(11) Gilbert, E. E. Sulfonation and Related Reactions; Wiley: New
York, 1965; pp 136-146.
(12) Of critical importance to the success of subsequent coupling,
excess oxalyl chloride was effectively removed by washing the
dichloromethane solution with 0.5% sodium bicarbonate solution.
This was accompanied by small amounts of methane[35S]sulfonyl
chloride hydrolysis (10-15%). The solution was then dried over
sodium sulfate and concentrated by atmospheric distillation.
(13) Olofson, R. A.; Martz, J . T.; Senet, J .-P.; Piteau, M.; Malfroot,
T. A new reagent for the selective, high-yield N-dealkylation of
tetriary amines: improved syntheses of naltrexone and nalbu-
phine. J . Org. Chem. 1984, 49, 2081-2082.
(14) In the presence of triethylamine, formation of [35S]sulfonamide
via minute amounts of a highly reactive sulfene intermediate
apparently requires high concentrations of 7 (with an attentu-
ated indoline nitrogen nucleophile) in order to compete effectively
with trace amounts of water. For a review of sulfenes, see: King,
J . K. The return of sulfenes. Acc. Chem. Res. 1975, 8, 10-17.
(15) Preparation of [35S]MK-0677 (3a ): A solution methane[35S]-
sulfonyl chloride (1.5 mCi) in dichloromethane was carefully
concentrated via atmospheric distillation in a pear-shaped flask
to a minimal volume (∼100 µL). This concentrate was added to
a mixture of the amine 7 (10 mg, 0.0182 mmol) and triethyl-
amine (5 µL) in 20 µL of dichloromethane at room temperature.
The distillation flask was rinsed with dichloromethane (2 × 100
µL), which was added to the amine solution. The mixture was
stirred at room temperature for 15 min at which point analysis
by HPLC (RX-C8 column, 30% MeCN-0.1% aqueous TFA to
with reduced lipophilicity17 (π value of NHSO2CH3
)
-1.1818) as compared with 125I congeners (π value of I
) 1.1218). Other interesting and potentially useful
observations involving high specific activity sulfur-35
radioligands will be the subject of future publications.
Ack n ow led gm en t. We gratefully acknowledge the
assistance of Dr. Roger Shaw of NEN-Du Pont in the

1770 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Communications to the Editor
100% MeCN over 30 min, 1 mL/min, radioactivity flow monitor)
indicated ∼70% N-BOC-[35S]MK-0677 (tR ) 20 min) along with
∼30% methane[35S]sulfonic acid. The reaction mixture was
diluted with 0.5 mL of dichloromethane, washed with saturated
sodium bicarbonate solution (2 × 0.5 mL), and dried over sodium
sulfate. Measurement of the radioactivity by scintillation count-
ing of the resulting solution indicated 950 µCi (63% yield) to be
present with a radiochemical purity of 93% by HPLC analysis
(above system). The solution of N-BOC-[35S]MK-0677 was con-
centrated to ∼1 mL under a stream of nitrogen at 40 °C, and
dimethyl sulfide (100 µL) followed by trifluoroacetic acid (300
µL) was added. The mixture was stirred at room temperature
for 1 h at which point analysis by HPLC (above system) indicated
complete conversion to [35S]MK-0677 (tR ) 10.5 min). The
mixture was concentrated in vacuo to near dryness and the
resulting residue partitioned between 1% sodium bicarbonate
solution (2 mL) and dichloromethane (3 mL). After vigorous
stirring, the dichloromethane layer was separated and dried over
sodium sulfate. Measurement of the radioactivity by counting
the resulting solution indicated 835 µCi (∼88% yield) of [35S]-
MK-0677 to be present with a radiochemical purity of 92%.
Purification was effected by sequential preparative HPLC with
attached radioactivity detector using (1) a semiprep RX-C8
column (9.4 mm × 25 cm, 500 µL loop) eluting with a gradient
system of 50% MeCN-0.1% aqueous TFA to 70% MeCN over
30 min at a flow rate of 3.5 mL/min (tR ) ∼24 min) followed by
(2) a analytical PRP-1 column (4.6 mm × 25 cm, 200 µL loop)
eluting with a gradient system of 50% MeOH-water with 1 mM
of HCl to 90% MeOH-water with 1 mmol HCl over 30 min at a
flow rate of 1 mL/min (tR) ∼22 min). The structural identity
was established via HPLC coelution with authentic MK-0677.
Additional verification was also obtained through analysis of ∼20
µCi of the radioligand by capillary LC/MS/MS which also
indicated a 10:3.5 ratio of 35S/32S corresponding to a specific
activity of 1110 Ci/mmol.
(16) [35S]-3a demonstrated remarkable stability (<0.5% loss of ra-
diochemical purity/month) when stored at -20 °C as a solution
in 15% methanol-water solution containing a trace of hydro-
chloric acid.
(17) Enhanced hydrophilicity (water solubility) is usually inversely
related to the observed “stickiness” of the radioligand which often
severely restricts its usefulness in various receptor preparations,
see: Bailey, G. S. In vitro labelling of proteins. In Radioisotopes
in Biology: A Practical Approach; Slater, R. J ., Ed.; Oxford
University Press: Oxford, 1990; pp 191-206.
(18) Hansch, C.; Leo, A.; Unger, S. H.; Kim, K. H.; Nikaitam, D.; Lien,
E. J . “Aromatic” substiturent constants for structure-activity
correlations. J . Med. Chem. 1973, 16, 1207-1216.
J M960054C