Discovery of a novel non-peptide somatostatin agonist with SST4 selectivity

Article abstract of DOI:10.1021/ja973325x

The discovery of novel non-peptide compounds with a high affinity for the peptide hormone somatostatin (SST) receptor is described. The compounds were tested for affinity at five human SST receptor subtypes individually expressed in mammalian cells. The c

Full text of DOI:10.1021/ja973325x

1368
J. Am. Chem. Soc. 1998, 120, 1368-1373
Discovery of a Novel Non-Peptide Somatostatin Agonist with SST4
Selectivity
Michael Ankersen,*,† Michael Crider,§ Shengquan Liu,§ Bin Ho,§ Henrik S. Andersen,† and
Carsten Stidsen‡
Contribution from MedChem Research and Molecular Pharmacology, NoVo Nordisk A/S,
NoVo Nordisk Park, DK-2760 MåløV, Denmark and DiVision of Basic Pharmaceutical Sciences,
Northeast Louisiana UniVersity, Monroe, Louisiana 71209-0470
ReceiVed September 22, 1997
Abstract: The discovery of novel non-peptide compounds with a high affinity for the peptide hormone
somatostatin (SST) receptor is described. The compounds were tested for affinity at five human SST receptor
subtypes individually expressed in mammalian cells. The compound NNC 26-9100 showed a Ki of 6 nM at
SST4 and more than 100 fold selectivity for SST4 over SST1, SST2, SST3, or SST5. Competition binding
studies and Scatchard analysis of the interaction by NNC 26-9100 with SST showed specificity at SST4.
Furthermore, NNC 26-9100 was highly selective for SST4 over a variety of other G protein-coupled receptors,
having affinities for M1 muscarinic acetylcholin and D3 dopamine receptors of around 500 and 1000 nM,
respectively. Finally, NNC 26-9100 was found to fully inhibit forskolin-induced accumulation of adenosine
3′,5′-cyclic monophosphate in baby hamster kidney cells, expressing the human SST4 receptor with an EC50
of 2 nM.
Somatostatin (somatotropin release inhibiting factor; SRIF),
protein-coupled receptors with seven putative membrane-
spanning regions.
a tetradecapeptide originally isolated from ovine hypothalamus
on the basis of its ability to inhibit growth hormone (GH) release
from anterior pituitary cells,1 has been shown to be present in
several other tissues.2 Somatostatin appears to have widespread
functions as a modulator of neuronal activity as well as of
endocrine and exocrine secretion. Inhibitory effects of this
peptide on the release of a variety of hormones such as GH,
prolactin, glucagon, insulin, gastrin, and thyroid-stimulating
hormone have been described.3 SRIF is best regarded as
belonging to a phylogenetically ancient, multigene family of
peptides with two important bioactive products, namely, SRIF-
14 and SRIF-28, a congener of SRIF extended at the N terminus.
Since the discovery of SRIF, the biological effects have
gained a lot of pharmacological attention in the development
of bioactive compounds with selective modes of action. Many
cyclic peptides have been synthesized (Figure 1) which bind
with high affinity to the SST receptors, but so far, no compound
has been reported to bind selectively to one of the five cloned
SST receptors.
The smallest SST analogue which retains biological activity
is the hexapeptide MK 678.8 The octapeptide analogue SMS
201-995 (Octreotide) is used clinically in long-acting prepara-
tions for the diagnosis and treatment of a variety of neuroen-
docrine tumors and gastrointestinal disorders.9
The regulatory functions of SRIF are mediated by specific
membrane receptors. Currently, only agonists are available to
study the pharmacology of SRIF receptors. High-affinity
saturable binding sites have been demonstrated in a number of
tissues, e.g., pituitary gland,4 brain,5 and pancreas,6 Within the
last few years, the cloning and isolation of five SST receptor
genes have been reported for various species (human, rat, and
mouse).7 Structural considerations of the encoded proteins
revealed that the SST receptor proteins (SST1-SST5) represent
a distinct receptor subfamily belonging to the superfamily of G
Recent works on the development of non-peptide structures
substituting the peptide backbone of small cyclic peptides with
â-D-glucose,10 xylofuranose,11 or mannitol scaffolds12 have
demonstrated low SST receptor affinity. However, these
(7) Human SST1-SST5 receptors reviewed by: Patel, Y. C.; Greenwood,
M. T.; Panetta, R.; Demchyshyn, L.; Niznik, H.; Srikant, C. B. Life Sci.
1995, 57, 1249. Rat SST1-SST5 receptors reported by: Meyerhof, W.; Paust,
H. J.; Schoenrock, C.; Richter, D. DNA Cell Biol. 1991, 10, 689. Kluxen,
F.-W.; Bruns, C.; Luebbert, H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4618.
Meyerhof, W.; Wulfsen, I.; Schoenrock, C.; Fehr, S.; Richter, D. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 10267. Bruno, J. F.; Xu, Y.; Song, J.;
Berelowitz, M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11151. O’Carroll,
A.-M.; Lolait, S. J.; Konig, M.; Mahan, L. C. Mol. Pharmacol. 1992, 42,
939. Mouse SST1-SST4 receptors reported by: Yamada, Y.; Post, S. R.;
Wang, K.; Tager, H. S.; Bell, G. I.; Seino, S. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 251. Yasuda, K.; Rens-Domiano, S.; Breder, C. D.; Law, S. F.;
Saper, C. B.; Reisine, T.; Bell, G. I. J. Biol. Chem. 1992, 267, 20422.
Schwabe, W.; Brennan, M. B.; Hochgeschwender, U. Gene 1996, 168, 233.
(8) Veber, D. F.; Saperstein, R.; Nutt, R. F.; Freidinger, R. M., Brady,
S. F.; Curley, P.; Perlow, D. S.; Paleveda, W. J.; Colton, C. D.; Zacchei,
A. G.; Tocco, D. J.; Hoff, D. R.; Vandlen, R. L.; Gerich, J. E.; Hall, L.;
Mandarino, L.; Cordes, E. H.; Anderson, P. S.; Hirschmann, R. Life Sci.
1984, 34, 1371.
† MedChem Research, Novo Nordisk A/S.
‡ Molecular Pharmacology, Novo Nordisk A/S.
§ Northeast Louisiana University.
(1) Brazeau, P.; Vale, W.; Burgus, R.; Ling, N.; Butcher, M.; Rivier, J.;
Guillemin, R. Science 1973, 179, 77.
(2) For review, see: Reichlin, S. N. Engl. J. Med. 1983, 309, 1495.
(3) For review, see: Wass, J. A. H., In Endocrinology; deGrott, L. J.,
Ed.; 1989; Vol. 1, p 152.
(4) Srikant, C. B.; Patel, Y. C. Endocrinology 1982, 110, 2138.
(5) Reubi, J. C.; Perrin, M. H.; Rivier, J. E.; Vale, W. Life Sci. 1981,
28, 2191.
(6) Zeggari, M.; Viguerie, N.; Susini, C.; Esteve, J. P.; Vaysse, N.; Rivier,
J.; Wunsch, E.; Ribet, A. Peptides 1986, 7, 953.
(9) Lamberts, S. W. J.; Krenning, E. P.; Reubi, J.-B. Endocr. ReV. 1991,
12, 450.
S0002-7863(97)03325-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/04/1998

Non-Peptide Somatostatin Agonist with SST4 SelectiVity
J. Am. Chem. Soc., Vol. 120, No. 7, 1998 1369
Figure 1.
structures are nonselective, displaying higher affinities for both
â2-adrenergic receptors and tachykinin receptors. Thus, there
have been no reports in the literature on the successful
development of a selective, competitive SST receptor ligand of
non-peptide origin.
The structure-activity relationships of SRIF have suggested
that the amino acid residues Phe7, Trp8, Lys9, and Thr10, which
comprise a â turn, are necessary for biological activity, with
residues Trp8 and Lys9 being essential, whereas Phe7 and Thr10
can undergo minor substitutions.13
We initiated a screening program to identify new non-peptide
chemical entities with SST1-SST5 affinities, based on a scaffold
containing two aromatic groups and a basic group. We now
report our findings and the subsequent optimization which led
to a small non-peptide SST receptor agonist with high affinity
and selectivity for SST4.
Results and Discussion
Our broad screening was based on a query consisting of two
aromatic groups, one which was heteroaromatic, postulated to
mimic the Trp8, and one nonheteroaromatic, mimicing the Phe7
of SRIF. On the basis of previous observations,10 we wanted
not only to mimic the Lys9 by primary amino groups but extend
the query to contain all sorts of nitrogen containing functional
groups. The initial screening identified the thiourea 1, which
showed moderate affinity at SST4 and a 20-fold selectivity for
SST4 over SST2 (Figure 2).
As seen in Table 1, our initial lead compound 1, containing
one heteroaromatic, one aromatic, and one imidazole group
connected through a thiourea scaffold, had a Ki of 118 nM at
SST4 and showed a 20 fold selectivity for SST4 over the other
subtypes. This finding prompted us to screen our in-house
databases for all similar compounds, and the most selective
compound turned out to be a simple S-methyl derivative of 1.
The S-methylated thiourea 2 was slightly more potent than 1 at
SST4, but more interestingly, it showed about a 100-fold
selectivity for SST4 over SST2, yet only a 3-fold selectivity for
SST4 over SST5. This encouraged us to derivatize 2, and a
series of thiourea analogues14 led us to our most potent
compound, NNC 26-9100 (3), in which we have replaced the
pyridinyl moiety with 5-bromopyridinyl and the 4-bromobenzyl
moiety with 3,4-dichlorobenzyl, respectively. NNC 26-9100
showed a 20 fold increase in affinity at SST4 (Ki ) 6 nM) and
was also the most potent compound at SST2, although NNC
26-9100 showed more than 100 fold selectivity for SST4 over
SST2. We compared the binding profile of NNC 26-9100 to
that of SRIF at SST4 as depicted in Figure 3. The shape of the
displacement curve with a Hill coefficient close to unity
Figure 2.
Table 1. Dissociation Constants for a Number of SST Analogues
Assayed by 125I-Tyr11-SRIF Radioligand Binding to Membranes
from Transfected BHK and HEK 293 Cells
compd
Ki(SST1)a Ki(SST2)b Ki(SST3)a Ki(SST4)a Ki(SST5)a
SRIF-14
SMS 201-995
MK 678
1
0.8
1 200
>10 000
0.2
0.3
0.3
0.6
108
20
7 000
12 000
1 400
1.4
1904
3200
118
50
0.3
1.2
3.4
2500
4600
621
2
145
1900
NNC 26-9100 (3)
1 800
6
a Ki (nM) in transfected BKH cells. b Ki (nM) in transfected HEK
293 cells.
Figure 3. Displacement by NNC 26-9100 (9) and SRIF ([) of 125I-
Tyr11-SRIF binding to membranes from BHK cells expressing human
SST4 receptors.
suggested competitive inhibition by NNC 26-9100 of 125I-Tyr11-
SRIF. This was further substantiated by Scatchard analysis of
125I-Tyr11-SRIF binding in the absence or presence of 5 nM
NNC 26-9100. The pKd of 125I-Tyr11-SRIF for SST4 was
unaffected (p > 0.10, students t test) by NNC 26-9100 (8.86 (
0.12 vs 8.55 ( 0.10, n ) 3), whereas Bmax was significantly
lower (data not shown).
NNC 26-9100 was further tested in a functional assay based
on inhibition of forskolin-induced accumulation of adenosine
cyclic 3′,5′-monophosphate (c-AMP) in BHK cells expressing
the human SST4 receptor. Like SRIF, NNC 26-9100 was able
(10) Hirshmann, R.; Nicolau, K. C.; Pietranico, S.; Leahy, S. M.; Salvino,
J.; Arison, B.; Cichy, M. A.; Spoors, P. G.; Shakespeare, W. C.; Sprengeler,
P. A.; Hamley, P.; Smith, A. B., III; Reisine, T.; Raynor, K.; Maechler, L.;
Donaldson, C.; Vale, W.; Freidinger, R. M.; Cascierie, M. R.; Strader, C.
D. J. Am. Chem. Soc. 1993, 115, 12550.
(11) Papageorgiou, C.; Haltiner, R.; Bruns, C.; Pletcher, T. J. Bioorg.
Med. Chem. Lett. 1992, 2, 135.
(12) Damour, D.; Barreau, M.; Blanchard, J.-C.; Burgevin, M.-C.; Doble,
A.; Herman, F.; Pantel, G.; James-Surcouf, E.; Vuilhorgne, M.; Mignani,
S.; Bioorg. Med. Chem. Lett. 1996, 6 (14), 1667-1672.
(13) Patel, Y. C.; Greenwood, M. T.; Panetta, R.; Demchyshyn, L.;
Niznik, H.; Srikant, C. B. Life Sci. 1995, 57, 1249.
(14) To be published.

1370 J. Am. Chem. Soc., Vol. 120, No. 7, 1998
Ankersen et al.
suggesting functional roles of somatostatin in the autonomic
nervous system in the anterior segments of the eye.16 Since
NNC 26-9100 shows such a superior selectivity to SST4, it now
should open up the possibility to further evaluate the physi-
ological role of this receptor.
The low molecular weight and non-peptide nature of NNC
26-9100 seem promising for the development of a drug
candidate for treatment of various diseases (e.g., glaucoma)
related to the malfunction of SST receptors.
Conclusion
We have described the synthesis and biological properties
of compounds 1, 2, and NNC 26-9100. These compounds bind
to five individual SST receptor subtypes with various affinities.
The most potent compound, NNC 26-9100, binds to SST4 with
an affinity of 6 nM and at least a 100 fold selectivity for SST4
over the other four SST receptor subtypes. In a functional assay,
NNC 26-9100 showed full SST receptor agonism (95% of SRIF)
with an EC50 of 2 nM.
Figure 4. Inhibition by NNC 26-9100 (9) and SRIF ([) of forskolin
induced accumulation of c-AMP in BHK cells expressing SST4. The
effects by NNC 26-9100 of c-AMP accumulation in wild-type BHK
cells are indicated by 2.
Table 2. Inhibition by NNC 26-9100 of Radioligand Binding on a
Variety of G Protein-Coupled Receptors
receptor
ligand
IC50, nM
angiotensin II
bradykinin B2
dopamine D3
CCKA
[3H]angiotensin II
[3H]bradykinin
[3H]spiperone
[3H]L-364718
[3H]CCK-8
>10 000
>10 000
1 000
>10 000
>10 000
>10 000
>10 000
>10 000
540
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
Chemistry
A convergent synthesis-strategy was chosen for the preparation of
compounds 1 and 3, using the amine 8 and the isothiocyanates 17 or
18, respectively, to form the thiourea in the last step. Compound 2
was prepared from 1 by simple S-methylation. The intermediate 3-[1-
(triphenylmethyl)imidazol-4-yl]propylamine (8; Scheme 1) was pre-
pared from the ester 4, which has previously been described by Sellier
et al.17 The imidazole of 4 was protected with trityl chloride and the
ester was reduced to the alcohol 6. The alcohol 6 was converted Via
a Mitsunobu reaction with DEAD, phthtalimide, and triphenylphosphine
to the phthalimide 7. By refluxing 7 in hydrazine hydrate, the
propylamine 8 was obtained in 85% yield.
The amines 15 and 16 were prepared by alkylation of 1,3-
diaminopropane with 2-bromopyridine (9) and 2,5-dibromopyridine
(10), respectively, followed by alkylation of the obtained secondary
amines 11 and 12 with 4-bromobenzyl bromide (13) and 3,4-
dichlorobenzyl bromide (14), respectively. The isothiocyanates 17 and
18 were obtained by a DCC coupling in carbon disulfide of 15 and 16,
respectively, similar to a procedure described by Jochims and Seeliger18
(Scheme 2).
CCKB
endothelin ETA
endothelin ETB
galanin
[
[
[
125I]endothelin
125I]endothelin
125I]galanin
muscarinic M1
muscarinic M2
neurokinin NK1
neurokinin NK1
neuropeptide Y2
serotonin 5-HT1A
serotonin 5-HT3
VIP
[3H]pirenzepine
[3H]NMS
[3H]substance P
[3H]substance P
[
125I]NPY
[3H]8-OH-DPAT
[3H]GR-65630
[
125I]VIP
to almost completely inhibit (efficacy 95%) the accumulation
of c-AMP with an EC50 of 2 nM (Figure 4). Mock-transfected
BHK cells, on the other hand, did not respond to NNC 26-
9100 upon forskolin stimulation. These data demonstrate NNC
26-9100 to be a full agonist at the human SST4 receptor.
NNC 26-9100 is also highly selective for SST4 compared to
other G protein-coupled receptors as outlined in Table 2. The
The desired compounds 1 and 3 were prepared by reacting 8 with
17 and 18, respectively, followed by deprotection in hydrochloric acid
(Scheme 3).
When compound 1 was treated with sodium hydride for 2 days
followed by methyl iodide, the S-methylated derivative 2 was obtained
(Scheme 4). This compound exhibited a singlet in 1H NMR at d 2.71,
which is characteristic for S-methylated thioureas.19
initial binding affinity for the muscarinic M1 receptor (IC50
)
540 nM) could not be confirmed using the rabbit vas deferens
relaxation model with NNC 26-9100 in concentrations up to
100 µM (Panlabs Inc., data not shown).
Previously, non-peptides with SST receptor affinity have been
reported.15 These binding studies have been employed on
pituitary and cerebral cortex membranes comprising mixtures
of several subtypes of SST receptors. To our knowledge, no
reports of non-peptides evaluated at the individual cloned SST
receptor subtypes have ever been reported.
Hirschmann et al.10 have previously postulated that the His7
analogue of c-(Phe-D-Trp-Lys-Thr-Phe-Pro) displayed a 1.6-
fold increase in potency when assayed using homogenates from
AtT-20 cells. This gives rise to speculation to whether the
imidazole in NNC 26-9100 mimics the Phe7 (His7) of SRIF or
the basic amine of Lys9.
Experimental Section
All reactions were carried out under a nitrogen atmosphere. Melting
points were determined on a Thomas Hoover melting point apparatus
and were not corrected. The NMR spectra were recorded on a JEOL
FX 90Q spectrometer or on a Bruker DPX200, and the chemical shifts
were recorded in parts per million (d) relative to tetramethylsilane.
Analytical data were obtained from Oneida Research Services, Inc.
Whitesboro, NY. All reagents and chemicals were used without further
purification except where indicated. Flash chromatography20 was
performed on Ace glass columns (i.d. ) 5 cm, length ) 45 cm) with
150-200 g of silica gel (40 mm). All solvents which were used for
recrystallizations were of analytical grade.
N-(Pyrid-2-yl)propane-1,3-diamine (11). To a solution of propane-
1,3-diamine (310 mL, 3.6 mol) in dry pyridine (75 mL) was added
The identification of SST4’s biological and pharmacological
properties has lagged behind due to the lack of selective SST4
ligands. Recently, in situ hybridization showed predominant
SST4 expression in the posterior iris epithelium and ciliary body,
(16) Mori, M.; Aihara, M.; Shimizu, T. Neurosci. Lett. 1997, 223, 185.
(17) Sellier, C.; Buschauer, A.; Elz, S.; Schunack, W. Liebigs Ann. Chem.
1992, 317.
(15) Damour, D.; Barreau, M.; Blanchard, J.-C.; Burgevin, M.-C.; Doble,
A.; Herman, F.; Pantel, G.; James-Surcouf, E.; Vuilhorgne, M.; Mignani,
S. Bioorg. Med. Chem. Lett. 1996, 6 (14), 1667.
(18) Jochims, J. C.; Seeliger, A. Angew Chem. 1967, 79, 151.
(19) Narayanan, K.; Griffith, O. W. J. Med. Chem. 1994, 37, 885.
(20) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.

Non-Peptide Somatostatin Agonist with SST4 SelectiVity
J. Am. Chem. Soc., Vol. 120, No. 7, 1998 1371
Scheme 1
Scheme 2
Scheme 3
Scheme 4
precipitate was filtered off and washed with tetrahydrofuran (0.5 L).
The solvent was evaporated in Vacuo and the residue purified by
distillation at 95-97 °C (0.02 mmHg) affording 83.4 g (76%) of 11. 1H
NMR (200 MHz, DMSO-d6) δ 1.20 (br s, 2 H, NH2), 1.74 (m, 2 H),
2-bromopyridine (9, 70 mL, 0.73 mol). The reaction mixture was
heated at reflux for 18 h and cooled, and the volatiles were evaporated
in Vacuo. To the residue was added tetrahydrofuran (1 L), and the

1372 J. Am. Chem. Soc., Vol. 120, No. 7, 1998
Ankersen et al.
2.82 (t, 2 H), 3.34 (q, 2 H, CH2NH), 4.86 (br s, 1 H, NH), 6.35 (dt, 1
H), 6.51 (ddd, 1 H), 7.37 (ddd, 1 H), 8.04 (ddd, 1 H).
sulfate, pH ) 2.5, over 10 min at room temperature). 1H NMR (90
MHz, MeOD-d3) δ 2.12 (m, 4 H), 2.71 (s, 3 H, SCH3), 2.85 (t, 2 H),
3.61 (m, 4 H), 3.84 (m, 2 H), 4.95 (s, 2 H, CH2Ph), 7.02 (t, 1 H), 7.22
(d, 2 H), 7.30 (d, 1 H), 7.42 (br s, 1 H), 7.52 (d, 2 H), 7.98 (d, 1 H),
8.05 (br s, 1 H), 8.80 (d, 1 H).
N-(4-Bromobenzyl)-N-(pyrid-2-yl)propane-1,3-diamine (15). To
a mixture of sodium hydride (5.9 g, 60% dispersion in mineral oil,
0.14 mol) in dry dimethyl sulfoxide (250 mL) was slowly added a
solution of 11 (20.0 g, 0.13 mol) in dry dimethyl sulfoxide (50 mL) at
room temperature. The reaction mixture was stirred until gas evolution
ceased. A solution of 4-bromobenzyl bromide (36.1 g, 0.14 mol) in
dry dimethyl sulfoxide (100 mL) was slowly added at room temperature.
The reaction mixture was stirred for 48 h at room temperature, and the
mixture was poured onto ice water (500 mL) and extracted with ethyl
acetate (3 × 250 mL). The combined organic extracts were washed
with water (3 × 150 mL), dried (MgSO4), filtered, and evaporated in
Vacuo. The residue (40.6 g) was washed with n-heptane (30 mL) which
afforded 36.8 g of 15. The crude product (20 g) was purified by column
chromatography on silica gel (200 g) using a mixture of dichlo-
romethane/methanol/triethylamine (90:5:5) as the eluent, affording 13.8
g (70%) of 15 as an oil. 1H NMR (200 MHz, CDCl3) δ 1.64 (s, 2 H,
NH2), 1.74 (t, 2 H), 2.72 (t, 2 H), 3.60 (t, 2 H, CH2N), 4.67 (s, 2 H,
CH2Ph), 6.41 (d, 1 H), 6.53 (dd, 1 H), 7.07 (d, 2 H), 7.33-7.41 (m, 3
H), 8.13 (dt, 1 H).
3-[N-(4-Bromobenzyl)-N-(pyrid-2-yl)amino]propyl Isothiocyanate
(17). To a solution of N,N-dicyclohexylcarbodiimide (2.1 g, 10 mmol)
in dry tetrahydrofuran (20 mL) was slowly added at -10 °C a solution
of 15 (3.2 g, 10 mmol) and carbon disulfide (4.3 mL, 70 mmol) in dry
tetrahydrofuran (20 mL). The mixture was stirred at -10 °C for 3 h
and for 48 h at room temperature. The reaction mixture was filtered
and evaporated in Vacuo. The residue (5.3 g) was extracted with diethyl
ether (3 × 20 mL), and the combined organic extracts were evaporated
in Vacuo, affording 3.2 g (88%) of 17 as an oil. TLC Rf ) 0.72 [SiO2;
ethyl acetate/n-heptane (1:1)]. 1H NMR (200 MHz, CDCl3) δ 2.00 (q,
2 H), 3.57 (t, 2 H), 3.68 (t, 2 H), 4.66 (s, 2 H), 6.41 (d, 1 H), 6.58 (dd,
1 H), 7.07 (d, 2 H), 7.35-7.43 (m, 3 H), 8.15 (dd, 1 H).
N-(5-Bromopyrid-2-yl)propane-1,3-diamine (12).21 A mixture of
2,5-dibromopyridine (12, 4.4 g, 18.6 mmol), pyridine (1.9 g, 23.6
mmol), and 1,3-diaminopropane (25 mL) was refluxed for 18 h. The
reaction mixture was evaporated in Vacuo to yield an oil, which was
vacuum distilled to give 2.9 g (63%) of 12 as an oil. Bp 135-139 °C
(0.1 mmHg). 1H NMR (90 MHz, CDCl3) δ 1.52 (br s, 2 H, NH2),
1.72 (m, 2 H), 2.89 (t, 2 H), 3.36 (m 2 H), 5.30 (br s, 1 H, NH), 6.29
(d, J ) 9 Hz, 1 H, pyridine H-3), 7.44 (dd, J ) 2.4, 8.8 Hz, 1 H,
pyridine H-4), 8.09 (d, J ) 2.4 Hz, 1 H, pyridine H-6). 13C NMR
(CDCl3) d 32.61, 39.98, 40.25, 106.45, 108.29, 139.50, 148.49, 157.48.
N-(5-Bromopyrid-2-yl)-1-(3,4-dichlorobenzyl)propane-1,3-di-
amine (16). A mixture of sodium hydride (0.9 g, 22.9 mmol, 60% in
mineral oil) and 12 (5.0 g, 21.7 mmol) in dimethyl sulfoxide (45 mL)
was stirred for 2 h. The suspension was cooled to 0-5 °C and treated
dropwise with a solution of 3,4-dichlorobenzyl chloride (14, 4.24 g,
21.7 mmol) in dimethyl sulfoxide (15 mL). The mixture was stirred
overnight at room temperature and poured into ice-water (200 mL).
The mixture was extracted with ethyl acetate (3 × 75 mL), and the
combined ethyl acetate extracts were washed with water (2 × 50 mL),
dried (Na2SO4), filtered, and evaporated to yield an oil. Flash
chromatography using a mixture of dichloromethane/methanol/triethy-
1
lamine (90:5:5) as the eluent afforded 4.9 g (58%) of 16 as an oil. H
NMR (90 MHz, CDCl3) δ 1.44 (s, 2 H, NH2), 1.80 (m, 2 H), 2.73 (t,
2 H), 3.56 (t, 2 H), 4.66 (s, 2 H, ArCH2), 6.37 (d, J ) 9 Hz, 1 H,
pyridine H-3), 7.31 (m, 4 H), 8.15 (d, J ) 2.5 Hz, 1 H, pyridine H-6).
13C NMR (90 MHz, CDCl3) d 30.93, 39.44, 46.21, 50.71, 106.67,
107.16, 126.23, 128.72, 130.51, 130.89, 132.62, 138.90, 139.66, 148.55,
156.46.
3-[N-(5-Bromopyrid-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl
Isothiocyanate (18). To a solution of N,N-dicyclohexylcarbodiimide
(1.9 g, 9.4 mmol) in dry tetrahydrofuran (20 mL) was slowly added at
-10 °C a solution of 16 (3.6 g, 9.4 mmol) and carbon disulfide (7.6
mL, 100 mmol) in dry tetrahydrofuran (25 mL). The mixture was
stirred at -10 °C for 3 h and for 48 h at room temperature. The reaction
mixture was filtered and the solvent evaporated in Vacuo. The residue
was purified on silica gel using a mixture of hexane/ethyl acetate/
triethylamine (70:30:1) as the solvent which afforded 3.2 g (79%) of
18 as an oil. 1H NMR (90 MHz, CDCl3) δ 2.06 (m, 2 H), 3.62 (m, 4
H), 4.66 (s, 2 H), 6.40 (m, 1 H), 7.35 (m, 4 H), 8.22 (m, 1 H). 13C
NMR (90 MHz, CDCl3) δ 28.06, 42.91, 46.10, 51.41, 107.21, 107.43,
126.17, 128.72, 130.72, 131.26, 138.31, 139.93, 148.76, 156.13. MS
(CI, CH4) 432 M+. Anal. Calcd for C16H14BrCl2N3S: C, 44.56; H,
3.28; N, 9.75. Found: C, 44.39; H, 3.42; N, 9.79.
1-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]pro-
pyl]-3-[3-(1H-imidazol-1-yl)propyl]thiourea (3). A suspension of
3-[1-(triphenylmethyl)imidazol-4-yl]propylamine (8, 1.3 g, 3.6 mmol)
in tetrahydrofuran (50 mL) was treated dropwise with 18 (1.6 g, 3.6
mmol) in THF (25 mL) at 0-5 °C under a nitrogen atmosphere. The
reaction mixture was allowed to warm to room temperature and was
stirred overnight. Removal of the solvent under reduced pressure
afforded an oil. Flash chromatography on silica gel using a mixture
of ethyl acetate/methanol/triethylamine (92:4:4) afforded 2.1 g (73%)
of the trityl-protected intermediate as an oil. The oil was suspended
in 1 N hydrochloric acid (50 mL) and refluxed for 7 h. The precipitated
triphenylmethanol was filtered off, and the filtrate was evaporated under
reduced pressure to yield a foam. The hygroscopic hydrochloride was
converted to the free base with 1 N sodium hydroxide, and the aqueous
layer was extracted with ethyl acetate (3 × 75 mL). The combined
extracts were washed with water (2 × 50 mL), dried (Na2SO4), filtered,
and evaporated to afford 600 mg of a foam. Purification by flash
chromatography on silica gel using a mixture of ethyl acetate/methanol/
concentrated ammoniun hydroxide (85:15:1) as solvent afforded 0.5 g
(27%) of 3 as a solid foam. 1H NMR (90 MHz, CDCl3) δ 2.05 (m, 4
1-[3-[N-(4-Bromobenzyl)-N-(pyrid-2-yl)amino]propyl]-3-[3-(1H-
imidazol-4-yl)propyl]thiourea Dihydrochloride (1). A mixture of
17 (1.0 g, 2.8 mmol) and 3-[(1-triphenylmethyl)imidazol-4-yl]propy-
lamine (8, 1.0 g, 2.8 mmol) in chloroform (10 mL) was heated at reflux
for 4 h. The solvent was removed by evaporation in Vacuo, and the
residue (3.1 g) was purified by column chromatography on silica gel
(400 mL) using a mixture of ethyl acetate/methanol/triethylamine (90:
5:5) as the eluent, affording 1.6 g (80%) of pure 3-[3-[(1-triphenylm-
ethyl)imidazol-4-yl]propyl]-1-[3-[N-(4-bromobenzyl)-N-(pyridin-2-yl)-
amino]propyl]thiourea. TLC Rf ) 0.59 (SiO2; ethyl acetate/methanol/
triethylamine ) 90:5:5). 1H NMR (200 MHz, CDCl3) δ 1.90 (m, 6
H), 2.62 (t, 2 H), 3.44-3.68 (m, 6 H), 4.55 (s, 2 H, CH2Ph), 6.33 (d,
1 H), 6.49 (dd, 1 H), 6.56 (s, 1 H), 6.98-7.11 (m, 9H), 7.30-7.39 (m,
11 H), 8.06 (d, 1 H).
To a solution of 3-[3-[(1-(triphenylmethyl)imidazol-4-yl]propyl]-1-
[3-[N-(4-bromobenzyl)-N-(pyridin-2-yl)amino]propyl]thiourea (1.6 g,
2.2 mmol) in ethanol (50 mL), 1 N hydrochloric acid (16 mL) was
added, and the reaction mixture was heated to 50 °C for 10 h. The
cooled reaction mixture was washed with diethyl ether (3 × 30 mL)
and the aqueous phase evaporated in Vacuo. The residue was extracted
with absolute ethanol (3 × 20 mL) and evaporated in Vacuo followed
by drying in Vacuo affording 1.2 g (99%) of 1 as an amorphous powder.
1H NMR (200 MHz, MeOD-d3) δ 2.0 (m, 4 H), 2.77 (t, 2 H), 3.57 (m,
4 H), 3.76 (t, 2 H), 4.90 (s, 2 H, CH2Ph), 6.99 (t, 1 H), 7.19 (d, 2 H),
7.26 (d, 1 H), 7.36 (s, 1 H), 7.50 (d, 2 H), 7.94 (d, 1 H), 8.02 (dd, 1
H), 8.78 (d, 1 H).
3-[3-(Imidazol-4(5)-yl)propyl]-1-[3-[N-(4-bromobenzyl)-N-(pyrid-
2-yl)amino]propyl]-S-methylisothiourea Hydroiodide Dihydrochlo-
ride (2). To a solution of 1 (0.58 g, 1.0 mmol) in absolute ethanol (50
mL) was added sodium hydride (0.10 mL, 1.28 mmol, 60% in mineral
oil), and the reaction mixture was stirred for 60 h at room temperature.
Iodomethane (0.025 mL, 0.32 mmol) was added, and the reaction
mixture was stirred at room temperature for an additional 5 h. The
volatiles were evaporated in Vacuo, affording 0.7 g (97%) of 2 as an
amorphous powder. TLC Rf ) 0.40 (SiO2; methanol/triethylamine )
75:25). HPLC retention time ) 12.58 min (5 µM C18 4- × 250-mm
column, eluting with a gradient of 15% acetonitrile/0.1 N aqueous
ammonium sulfate to 25% acetonitrile/0.1 N aqueous ammonium
(21) Ife, R. J.; Catchpole, K. W.; Durant, G. J.; Ganellin, C. R.; Harvey,
C. A.; Meeson, M. L.; Owen, D. A. A.; Persons, M. E.; Slingsby, B. P.;
Theobald, C. J. Eur. J. Med. Chem. 1989, 24, 249.

Non-Peptide Somatostatin Agonist with SST4 SelectiVity
J. Am. Chem. Soc., Vol. 120, No. 7, 1998 1373
H), 2.70 (m, 2 H), 3.59 (m, 6 H), 4.59 (s, 2 H, ArCH2), 6.30 (d, J )
9.3 Hz, 1 H), 6.50-8.11 (m, 9 H). 13C NMR (90 MHz, CDCl3) δ 23.73,
27.03, 28.82, 41.88, 43.29, 46.10, 106.99, 107.70, 115.93, 126.01,
128.50, 130.72, 131.10, 132.78, 134.41, 137.39, 138.20, 140.04, 148.33,
156.56, 181.38. Anal. Calcd for C22H25BrCl2N6S: C, 47.49; H, 4.54;
N, 15.11. Found: C, 47.48; H, 4.48; N, 14.96.
Biological Assay. Cell Lines Expressing SST Receptor Subtypes.
BHK cells (tk-ts13, ATCC CRL No. 1632) and HEK 293 cells (ATCC
CRL No. 1573) were grown to 20-40% confluency in a tissue culture
dish in DMEM containing 1% penicillin/streptomycin, 10% fetal bovine
serum, and 1% Glutamax. Prior to transfection, the cells were washed
twice with calcium-free PBS, after which 20 mL of serum-free DMEM
was added to the cells.
Transfection was carried out as described previously (product
description: lipofectamin, Gibco BRL Catalog No. 18324-012).
Briefly, 10 µg of c-DNA encoding a SST-receptor subtype inserted
into the mammalian expression vector pcDNA3 (Invitrogen) was diluted
in 300 µL of sterile water. Lipofectamin (30 µg) was diluted in 300
µL of sterile water. The c-DNA and lipofectamin solutions were mixed
and left at room temperature for 15 min. The lipofectamin/c-DNA
mixture was added dropwise to the cells (HEK 293 cells for SST2,
BHK for the other receptor subtypes) while the plates were gently
swirled. The cells were then incubated for 16-24 h, after which the
medium was replaced with standard medium containing 1 mg/mL
Geneticin (G-418 sulfate). Resistant colonies appearing after 1-2 weeks
were isolated and propagated for further characterization.
Binding Assay. Cells expressing individual SST receptor subtypes
were resuspended in buffer (50 mM Tris-HCl (pH 7.4), 1 mM EGTA,
5 mM MgCl2), and homogenized. Membranes were washed twice in
buffer by homogenization and centrifugation. The final membrane
pellets were resuspended at a protein concentration of 125 µg/mL in
buffer. Binding assays using 75 pM 125I-Tyr11-SRIF (Amersham, IM-
161) were done in duplicates in minisorb polypropylene tubes in a
volume of 250 µl. The assays were incubated at 30-37 °C for 30-90
min depending on the receptor subtype. Binding was terminated by
filtration through Whatman GF/B glass fiber filters presoaked for 4 h
in 0.5% poly(ethylenimine) and 0.1% BSA. The filters were washed
three times with 5 mL of ice-cold 0.9% saline and counted in a Packard
Cobra II gamma counter.
Assays for other G protein-coupled receptors were performed by
Panlabs Inc., Bothell, WA (PT No. 118422) with the radioligands
mentioned in Table 2 using plasma membrane preparations from rabbit
adrenal gland (ATII), guinea pig ileum (B2), rat pancreas (CCKA),
mouse brain (CCKB), human recombinant (D3), A10 cells (ETA), rat
cerebellum (ETB), rat brain (galanin), rat brain cortex (M1), rat heart
(M2), guinea pig submaxillary gland (NK1), rabbit kidney medulla (Y2),
rat brain cortex (5-HT1A), rabbit ileum (5-HT3), and guinea pig lung
(VIP).
Functional Assay. Cells expressing human SST receptors were
seeded in 24-well tissue culture multidishes at 200 000 cells/well and
grown for 16-20 h. The medium was removed, and fresh DMEM
medium, supplemented with (1) 3-isobutyl-1-methylxanthine (IBMX),
(2) forskolin or medium, and (3) medium, SRIF, SST analogue, or
compound, was added. The plates were incubated for 15-30 min at
37 °C, the reaction medium was removed, and the cells were lysed
with 0.1 M sodium hydroxide. Following neutralization with 0.1 M
hydrochloric acid, an aliquot was removed for c-AMP determination
using Amersham SPA RIA (RPA 538).
Acknowledgment. We acknowledge Marianne Schiødt and
Anja L. Almind for excellent technical assistence.
JA973325X