M. Maingot et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
to potent GHS-R1a ligands presenting good to high affinities
toward the receptor. Some compounds (8, 15, 17–23) exhibited a
nanomolar value of IC50 in two functional tests (calcium and CRE
reporter assays). Furthermore, the presence of an amino function
at the new chiral center allowed us to elongate the C-terminal part
of the molecule and to introduce the Leu-Leu dipeptide sequence
that is included in potent described inverse agonists. Unfortu-
nately, most of the compounds incorporating this dipeptide
behaved as agonists except compound 24 which is a neutral antag-
onist and compound 29 which exhibits a partial inverse agonist
1
1
behavior similar to the hexapeptide KwFwLL-NH
the literature. Some discrepancies between the functional assays
calcium mobilization, CRE-luciferase and IP3 turnover) were
2
described in
2
2
2. The purity assessed by analytical reversed phase C18 HPLC was found to be
greater than 98% for most of the target compounds and greater than 95% for
(
observed, indicating the complex signaling of this receptor which
is likely responsible for the diversity in biological effects elicited
by ghrelin. We are currently investigating to try to understand
how signaling efficacy and selectivity are regulated.27 In conclu-
sion, we demonstrated that the trisubstituted 1,2,4-triazole scaf-
fold bearing a second chiral center can be an alternative to
introduce more diversity and to obtain high affinity ligands.
2
the rest, and the structures of the compounds were confirmed by MS
13
(
electrospray), 1
H
and/or
C NMR.1 As an example: compound 8. White
powder, 51 mg (0.067 mmol, 32%). H NMR (300 MHz, DMSO-d
ppm) 1.62 (s, 3H, COCH ), 3.21–3.43 (m, 3H, CH Trp (2H), CH
3.47–3.62 (m, 4H, CH Trp (1H), OCH
.12–5.22 (m, 2H, CH (1H), CH Trp), 5.36–5.45 (m, 1H, CH Trp), 6.50 (d,
6
, 303 K): d
(
3
2
2
Trp (1H)),
2
3 2
), 4.95 (d, J = 16.8 Hz, 1H, CH U (1H)),
5
2
U
J = 8.7 Hz, 2H, CH aryl), 6.62 (d, J = 8.7 Hz, 2H, CH aryl), 6.76–6.90 (m, 2H, CH
aryl), 6.91–7.07 (m, 4H, CH aryl), 7.16 (d, J = 7.9 Hz, 1H, CH aryl), 7.22 (d,
J = 7.9 Hz, 1H, CH aryl), 7.24–7.31 (m, 2H, CH aryl), 7.51–7.58 (m, 1H, CH
pyridin-2-yl), 7.82 (d, J = 7.7 Hz, 1H, CH pyridin-2-yl), 7.91 (dt, J = 7.7,
J = 1.6 Hz, 1H, CH pyridin-2-yl), 8.53-8.61 (m, 2H, NH acetyl, CH pyridin-2-
Acknowledgments
The authors thank Æterna-Zentaris (Germany), the CNRS
(
France) and the University of Montpellier (France) for financial
13
yl), 8.95 (d, J = 8.7 Hz, 1H, NHCO), 10.72–10.79 (m, 2H, NH Trp). C NMR
support of this work, and for providing a research grant for the
Ph.D. thesis projects of M. Maingot and A.-L. Blayo.
(75 MHz, DMSO-d
6
, 303 K): d (ppm) 22.1, 28.6, 28.7, 44.2, 44.8, 45.2, 54.9,
1
1
1
09.3, 109.7, 111.2, 111.3, 113.6 (2C), 118.0 (2C), 118.2, 118.3, 120.7, 120.9,
21.9, 123.9, 124.0, 126.6, 126.7 (2C), 126.9, 127.0, 127.1, 135.9, 136.0, 137.6,
48.2, 148.8, 154.9, 155.8, 158.3, 163.2, 168.8. Purity >98% (reverse phase
+
+
References and notes
HPLC). LC–MS (ES): m/z 533.2 [M+H-4-methoxybenzyl] , 653.4 [M+H] .
3. In the CRE/Luc reporter gene assay, mouse LTK-cells were stably transfected
with a plasmid containing the CMV minimal promotor linked to three cAMP
response elements (CRE) followed by a luciferase reporter gene. Based on this
parental cell line, single cell clones stably overexpressing the human GHS-R1A
2
1
.
.
2
were established and characterized. The cells were incubated for 6 h with 1 lM
rolipram in the presence of different concentrations of the tested compounds
and saturating concentration of ghrelin. Subsequently, cells were lysed and
ATP bioluminescence was measured in the luminescence mode on FlexStation3
(Molecular Devices). All data were assessed in quadruplicate measurements
and calculated in % inhibition according to cells treated with saturating
concentrations of ghrelin (Polypeptide #sc1357) as negative (0% inhibition),
and non-treated cells as positive control (100%). IC50 values were determined
3.
4.
5.
6
7
8
9
.
.
.
.
25. The purity assessed by analytical reversed phase C18 HPLC was found to be
greater than 98% for most of the target compounds and greater than 95% for
the rest, and the structures of the compounds were confirmed by MS
1
13
(electrospray), H and/or C NMR. As an example: compound 29. White
1
powder, 38 mg (0.067 mmol, 27%). H NMR (400 MHz, DMSO-d
(ppm) 0.68 (d, J = 6.4 Hz, 3H, CH Leu), 0.70 (d, J = 6.4 Hz, 3H, CH Leu), 0.76 (d,
J = 6.4 Hz, 3H, CH Leu), 0.83 (d, J = 6.4 Hz, 3H, CH Leu), 0.97 (m, 2H, CH Lys),
1.22–1.33 (m, 3H, CH Lys, CH(CH ), 1.36–1.40 (m, 4H, 2 CH Leu), 1.52 (m,
1H, CH(CH ), 1.81 (s, 3H, COCH ), 2.51 (m, 2H, CH Lys), 3.09 (dd, J = 6.0 Hz,
J = 14.0 Hz, 1H, CH Trp (1H)), 3.18–3.29 (m, 2H, CH
3.38 (dd, J = 6.8 Hz, J = 14.0 Hz, 1H, CH Trp (1H)), 3.56 (m, 1H, CH
3.71 (m, 4H, OCH , CH
4.92 (d, J = 16.4 Hz, 1H, CH
6
, 303 K): d
3
3
3
3
2
2
3
)
2
2
3
)
2
3
2
2
2
Trp (1H), CH
2
Trp (1H),
1
2
2
Lys (1H)),
3
2
Lys (1H)), 4.15–4.27 (m, 3H, CH Lys, CH Leu, CH Leu),
(1H)), 5.06 (d, J = 16.4 Hz, 1H, CH (1H)), 5.17
2
U
2
U
(dd, J = 8.8 Hz, J = 14.8 Hz, 1H, CH Trp), 5.30 (dd, J = 8.0 Hz, J = 15.6 Hz, 1H, CH
Trp), 6.77 (d, J = 8.8 Hz, 2H, CH aryl), 6.81–6.91 (m, 4H, CH aryl), 6.98–7.06 (m,
5H, CH aryl), 7.26–7.32 (m, 3H, CH aryl), 7.56 (d, J = 8.4 Hz, 1H, NH-CO), 7.71 (s,
1
+
+
3H, NH
3 3
), 7.94 (s, 3H, NH ), 7.98 (d, J = 8.4 Hz, 1H, NH-CO), 8.63 (d, J = 8.8 Hz,
1
1H, NH-CO), 9.17 (d, J = 8.8 Hz, 1H, NH-CO), 10.76 (d, J = 8.4 Hz, 2H, NH Trp).
13
6
C NMR (100 MHz, DMSO-d , 303 K): d (ppm) 20.44, 21.31, 21.59, 22.37,
22.85, 22.89, 23.87, 24.06, 26.44, 28.78, 29.58, 30.07, 38.36, 40.21, 40.65, 43.95,
44.51, 45.00, 50.71, 50.95, 51.64, 54.99, 109.11, 109.67, 111.20, 111.24, 113.50,
114.00, 117.85, 118.00, 118.22, 118.26, 120.73, 120.93, 124.01, 124.20, 126.71,
126.96, 127.39, 127.49, 128.26, 135.88, 135.90, 154.63, 155.12, 158.73, 167.92,
169.28, 171.65, 171.72. Purity >96% (reverse phase HPLC). LC–MS (ES): m/z
1
1
4. Validation of macimorelin as a test for adult growth hormone deficiency (Phase
+
+
+
1