1-(Phenyl)isoquinoline carboxamides: A novel class of subtype selective inhibitors of thyrotropin-releasing hormone (TRH) receptors

Article abstract of DOI:10.1016/j.bmcl.2004.11.017

We report the synthesis of and binding to the two subtypes of mouse thyrotropin-releasing hormone (TRH) receptors, TRH-R1 and TRH-R2, of several 1-(phenyl)isoquinoline carboxamide analogues. These analogues showed a degree of selectivity for binding at TR

Full text of DOI:10.1016/j.bmcl.2004.11.017

Bioorganic & Medicinal Chemistry Letters 15 (2005) 733–736
1-(Phenyl)isoquinoline carboxamides: a novel class of
subtype selective inhibitors of thyrotropin-releasing hormone (TRH)
receptors
Jian-kang Jiang,a Craig J. Thomas,a Susanne Neumann,b Xinping Lu,b
Kenner C. Ricec and Marvin C. Gershengornb,*
aChemical Biology Core Facility, National Institute of Diabetes and Digestive and Kidney Disorders, National Institutes of Health,
Bethesda, MD 20892-1818, USA
bClinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Disorders, National Institutes of Health,
Building 10, Room 9N-222, Bethesda, MD 20892-1818, USA
cLaboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Disorders, National Institutes of Health,
Bethesda, MD 20892-1818, USA
Received 17 September 2004; revised 4 November 2004; accepted 4 November 2004
Available online 25 November 2004
Abstract—We report the synthesis of and binding to the two subtypes of mouse thyrotropin-releasing hormone (TRH) receptors,
TRH-R1 and TRH-R2, of several 1-(phenyl)isoquinoline carboxamide analogues. These analogues showed a degree of selectivity
for binding at TRH-R2. These are the first ligands reported that show selective binding to these receptors.
Published by Elsevier Ltd.
1. Introduction
in humans. The two receptors likely mediate different ac-
tions of TRH because they exhibit marked differences in
tissue distribution in the rat3 and TRH-R2 displays
higher basal signaling activity than TRH-R1.4 However,
although the two subtypes of TRH receptors are only
approximately 50% homologous, they exhibit similar
binding affinities for TRH and numerous TRH ana-
logues.3 Ligands that could distinguish between TRH-
R1 and TRH-R2 would be useful in delineating the
physiological roles of these receptors.
Thyrotropin-releasing hormone (TRH) (1) (Fig. 1), a
tripeptide, plays important roles in the regulation of thy-
roid-stimulating hormone (TSH) and prolactin synthesis
and secretion in the pituitary gland, and within the cen-
tral and peripheral nervous systems.1 The actions of
TRH in rodents are mediated by G protein-coupled
receptors (GPCRs) designated TRH-R1 and TRH-
R2;2 a homologue of TRH-R2 has not been observed
The identification of small molecule ligands that modu-
late the activity of GPCRs continues to be of high inter-
est based upon established and expectant therapeutic
value. TRH has been tested in the clinic for the treat-
ment of several diseases including neurodegeneration,
however, the selectivity for the two receptor subtypes
has not been addressed and the rapid rate of TRH dis-
persal in circulation have limited these studies.5 To date
the use of nonpeptidic, small molecule ligands to modu-
late the activities of TRH receptors has been largely
ignored.
O
O
NH2
O
H
N
N
O
N
H
N
N
O
N
Cl
NH
1
2
Figure 1. Structures of TRH (1) and PK 11195 (2).
Benzodiazepines are among the few small molecules re-
ported to affect TRH receptors. Benzodiazepines act as
inverse agonists (or negative antagonists) at TRH-R1
Keywords: TRH; TRH receptors; PK 11195.
*
Corresponding author. Tel.: +1 301 496 4128; fax: +1 301 496
9943; e-mail: marving@intra.niddk.nih.gov
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.11.017

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J. Jiang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 733–736
and TRH-R22 but their selectivities have not been tested
and their affinities for the receptors are low. Moreover,
utilization of a benzodiazepine as a TRH receptor modu-
lator in an intact animal or human would be accompa-
nied by central nervous system depression thereby
limiting their usefulness as reagents to study TRH recep-
tor biology. Consequently, novel small molecule modu-
lators of TRH-R1 and TRH-R2 without these liabilities
are of interest. Herein, we report a series of novel inhibi-
tors of TRH-R1 and TRH-R2 based upon a 1-(phenyl)-
isoquinoline amide scaffold.
ylic carbon was accomplished through a two-step proce-
dure using selenium oxide to form aldehydes 5a,b, and
5c followed by silver nitrate oxidation to the carboxylic
acids 6a,b, and 6c. Attempts to oxidize directly to the
carboxylic acid via KMnO4 were unsuccessful. The
resulting carboxylic acids were derivatized via acid chlo-
ride formation followed by coupling with the appropri-
ate amine to yield compounds 7–16 (Table 1).8
Additionally, derivative 17 was achieved via the cou-
pling of the analogous acid and sec-butyl amine through
the acid chloride.9
Through random screening we found that analogues of
the peripheral benzodiazepine receptor ligand PK 11195
(2) (Fig. 1) had TRH inhibitory activity at both TRH-
R1 and TRH-R2. Jarvinen et al.6 had previously re-
ported that PK11195 could compete with binding to
TRH receptors in rat anterior pituitary gland, hypothal-
amus, cortex, and brainstem. Furthermore, we noted
that the affinities of these compounds were greater at
TRH-R2 than at TRH-R1, which is a transposition
from the affinities of centrally acting benzodiazepines
(unpublished observations). Aiming to both amplify
the affinity for TRH receptors and further establish a de-
gree of selectivity between the two receptors, we synthe-
sized several analogues of this compound examining
structure–activity relationships (SAR) at the amide site
most specifically.
3. Assay results
Having identified PK 11195 as an inhibitor of
[3H]MeTRH binding to TRH-R1 and TRH-R2 during
our initial screen, we studied the binding of a series of
1-(2-chlorophenyl) isoquinoline carboxamide analogues
at a single concentration (100lM) (Table 1).10 We found
that there were differences among the analogues in their
abilities to compete with [3H]MeTRH binding to TRH-
R1 versus TRH-R2. In general, analogues with rela-
tively bulky alkyl groups on the amide nitrogen were
more potent inhibitors and there appeared to be selecti-
vity in the binding for TRH-R2 versus TRH-R1. Inter-
estingly, this selectivity seemed to be reversed by the
inclusion of a nitro group at the 5 position of the 2-chloro-
phenyl ring. To demonstrate this, we further analyzed
the binding of four analogues that contained at least a 4-
carbon substituent on the amide nitrogen and with var-
ious substitutions on the phenyl ring. Figure 2 illustrates
the dose-dependent competition binding of these ana-
logues. Analogue 11, 13, and 14 showed greater affinity
for TRH-R2 than TRH-R1 whereas analogue 17 did in-
deed display reversed affinities for TRH-R2 and TRH-
R1.11 The influence of nitro group inclusion seemed to
depress the potency of these compounds (analogues
14, 15, and 16) and only in the case of the 2-chloro-5-
nitro substituted phenyl ring was the selectivity reversed
2. Chemistry
The synthesis employed was a combination of the re-
ported routes of Janin et al. and Manning et al. (Scheme
1).7 Intermediates 3a,b, and 3c were achieved via the
base catalyzed coupling of norephedrine with 2-chloro-
benzoyl chloride, 3-nitrobenzoyl chloride, or 4-nitro-
benzoyl chloride. Condensation to the isoquinoline
ring was accomplished through treatment with P2O5 to
provide compounds 4a,b, and 4c. Oxidation of the benz-
R'
R
O
R''
OH
COCl
R''
H
N
N
a
b
R
R
NH3Cl
OH
R'
R'
3a-c
R''
4a-c
O
CHO
OH
N
N
c
d
e
R
Analogues 7-16
R
R'
3-6a R = Cl, R' = H, R'' = H
3-6b R = H, R' = NO2, R'' = H
3-6c R = H, R' = H, R'' = NO2
R'
R''
R''
6a-c
5a-c
Scheme 1. Reagents and conditions: (a) 5% NaOH, CH2Cl2; (b) P2O5, o-dichlorobenzene; (c) SeO2, o-dichlorobenzene; (d) AgNO3, NaOH, EtOH,
H2O; (e) SOCl2, CH2Cl2, DMF (one drop) stirred for 5min followed by the addition of the appropriate amine.

J. Jiang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 733–736
735
Table 1. Competition of binding of [3H]MeTRH to TRH-R1 and TRH-R2 by 2, 7–17 at 100lM
Analogue #
R1
R2
R3
R4
R5
R6
% Competition
TRH R1 TRH R2
2 (PK 11195)
7
sec-Butyl
Methyl
Ethyl
Methyl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
44 3.5
33 5.5
55 16.8
78 4.0
73 7.8
26 2.7
84 5.2
49 3.2
26 1.2
21 2.9
16 0.3
65 5.6
56 7.0
29 3.2
82 6.9
91 1.9
90 1.5
68 2.9
92 2.9
85 2.9
61 2.2
31 3.0
40 2.6
33 2.4
H
H
H
O
8
H
H
H
H
R1
9
Propyl
H
H
H
H
N
10
11
12
13
14
15
16
17
Isopropyl
t-Butyl
Butyl
H
H
H
H
N
R2
H
H
H
H
R3
Methyl
H
H
H
H
sec-Butyl
Isobutyl
sec-Butyl
sec-Butyl
sec-Butyl
H
H
H
R6
R4
H
NO2
NO2
H
H
H
R5
H
H
H
H
H
H
Cl
NO2
H
H
NO2
H
H
100
75
100
75
50
25
0
TRH-R1
TRH-R2
50
TRH-R1
TRH-R2
25
0
-7
-6
-5
-4
-7
-6
-5
-4
Analogue # 11 (log M)
Analogue # 13 (logM)
100
75
100
75
50
25
0
TRH-R1
TRH-R2
50
TRH-R1
TRH-R2
25
0
-7
-6
-5
-4
-7
-6
-5
-4
Analogue # 14 (logM)
Analogue # 17 (log M)
Figure 2. Competition binding curves for 11, 13, 14, 17.
(Table 1). Studies to determine the generality of this ef-
fect are currently underway. An additional 20 analogues
of PK 11195 were synthesized and tested to explore
additional amide substitutions including larger alkyl
groups (i.e., adamantyl), positively charged groups
(i.e., spermine), negatively charged groups (i.e., propyl
sulfonic acid) and numerous heterocycles (i.e., propyl
imidazole); all of which eradicated TRH receptor bind-
ing activity (data not shown).
several showed selectivity for mouse TRH-R2 over
TRH-R1. These are the first compounds reported to dis-
tinguish between these two subtypes of TRH receptors.
In contrast, although a number of peptidic analogues of
TRH have been tested,3,11 none are noted to selectively
bind to either TRH receptor subtype. The low molecular
weight analogues described here and surveyed in subse-
quent studies will likely be better probes of TRH recep-
tor function in vivo as they would be predicted to cross
the blood–brain barrier and may allow for study of
TRH receptors within the central nervous system.
4. Conclusion
Small, nonpeptide molecules that selectively and po-
tently bind and modulate the TRH receptors would be
valuable research tools and may have therapeutic poten-
tial. In this study we synthesized a series of 1-(phenyl)-
isoquinoline carboxamide analogues and found that
Acknowledgements
The authors thank Carole Bewley for helpful discussions
during the preparation of this manuscript. Appreciation

736
J. Jiang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 733–736
3.14 (m, 6H), 3.34–2.48 (m, 2H), 3.51–3.62 (m, 2H), 7.40–
7.48 (m, 6H), 7.53–7.59 (m, 4H), 7.63–7.65 (m, 2H), 7.70–
7.76 (m, 2H), 7.95–7.97 (m, 2H), 8.10–8.11 (m, 2H), 8.64
(s, 1H); TOFMS m/e 353.1407 (M+H+) (calculated for
C21H22ClN2O+ 353.1415). Compound 13 1H NMR
is also expressed to Victor Livengood and Sonja Hess
for assistance in obtaining the HRMS data.
References and notes
(CDCl3):
d
0.96 (t, JHH = 7.8Hz, 3H), 1.26 (d,
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3. OÕDowd, B. F.; Lee, D. K.; Huang, W.; Nguyen, T.;
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A. J.; Winokur, A. J. Pharm. Exp. Ther. 2003, 305,
410.
JHH = 6.6Hz, 3H), 1.62 (m, 2H), 4.18 (m, 1H), 7.47–7.54
(m, 3H), 7.57–7.62 (m, 2H), 7.66–7.77 (m, 2H), 8.03–8.08
(m, 2H), 8.67 (s, 1H); TOFMS m/e 339.1276 (M+H+)
(calculated for C20H20ClN2O+ 339.1258). Compound 14
1H NMR (CDCl3): d 0.99 (d, JHH = 6.6Hz, 6H), 1.89–1.99
(m, 1H), 3.11–3.19 (m, 2H), 7.52–7.78 (m, 3H), 7.91–8.07
(m, 3H), 8.21 (br s, 1H), 8.33–8.35 (m, 1H), 8.54–8.61 (m,
1H); TOFMS m/e 350.1499 (M+H+) (calculated for
C20H20N3O+ 350.1499). Compound 15 1H NMR (CDCl3):
d 0.99 (t, JHH = 7.5Hz, 3H), 1.29 (d, JHH = 6.6Hz, 3H),
1.60–1.68 (m, 2H), 4.09–4.16 (m, 1H), 7.66–7.71 (m, 1H),
7.75–7.84 (m, 2H), 7.98–8.12 (m, 4H), 8.41–8.45 (m, 1H),
8.60 (s, 1H), 8.70 (s, 1H); TOFMS m/e 350.1489 (M+H+)
6. Jarvinen, A.; Paakkari, I.; Mannisto, P. T. Neuropeptides
1991, 19, 147.
1
(calculated for C20H20N3O+ 350.1499). Compound 16 H
7. (a) Manning, H. C.; Goebel, T.; Marx, J. N.; Bornhop, D.
J. Org. Lett. 2002, 4, 1075; (b) Janin, Y. L.; Roulland, E.;
Beurdeley-Thomas, A.; Decaudin, D.; Monneret, C.;
Poupon, M.-F. J. Chem. Soc., Perkin Trans. 1 2002, 529.
8. Within the synthesis of 13, 15, 16, and 17 racemic sec-butyl
amine was utilized. Spectroscopic information: 7 1H NMR
(CDCl3): d 3.06 (d, JHH = 4.8Hz, 3H), 7.45–7.51 (m, 3H),
7.57–7.63 (m, 2H), 7.67–7.69 (m, 1H), 7.73–7.79 (m, 1H),
8.05–8.08 (m, 1H), 8.21 (br s, 1H), 8.67 (s, 1H); TOFMS
m/e 297.0795 (M+H+) (calculated for C17H14ClN2O+
297.0789). Compound 8 1H NMR (CDCl3): d 1.27 (t,
JHH = 7.5Hz, 3H), 3.55 (q, JHH = 6.0Hz, 2H), 7.44–7.53
(m, 3H), 7.56–7.60 (m, 2H), 7.66–7.69 (m, 1H), 7.73–7.78
(m, 1H), 8.04–8.07 (m, 1H), 8.21 (br s, 1H), 8.68 (s, 1H);
TOFMS m/e 311.0940 (M+H+) (calculated for
NMR (CDCl3): d 0.98 (t, JHH = 7.5Hz, 3H), 1.28 (d,
JHH = 6.9Hz, 3H), 1.59–1.68 (m, 2H), 4.06–4.14 (m, 1H),
7.65–7.70 (m, 1H), 7.78–7.83 (m, 1H), 7.88–7.92 (m, 2H),
7.96–8.03 (m, 2H), 8.08–8.12 (m, 1H), 8.44–8.47 (m, 2H),
8.69 (s, 1H); TOFMS m/e 350.1503 (M+H+) (calcu-
lated for C20H20N3O+ 350.1499). Compound 17 1H
NMR (DMSO-d6): d 1.04 (t, JHH = 7.5Hz, 3H), 1.35
(d, JHH = 6.6Hz, 3H), 1.61–1.68 (m, 2H), 4.17–4.28 (m,
1H), 8.05–8.16 (m, 3H), 8.30–8.33 (m, 1H), 8.38–8.46
(m, 1H), 8.62–8.70 (m, 2H), 8.83–8.56 (m, 1H), 9.40 (s,
1H); TOFMS m/e 384.1100 (M+H+) (calculated for
+
C20H19ClN3O3 384.1109).
9. Newman, A. H.; Lueddens, H. W. M.; Skolnick, P.; Rice,
K. C. J. Med. Chem. 1987, 30, 1901.
10. Assay description: HEK293EM cells stably expressing
either TRH-R1 or TRH-R2 were grown in DulbeccoÕs
modified EagleÕs medium containing 10% fetal bovine
serum. Cells were selected to express TRH-R1 and TRH
R2 at similar concentrations. Competition binding exper-
iments were performed initially using 1nM [3H][methyl-
His]TRH ([3H]MeTRH, DuPont), an analogue of TRH
with 5- to10-fold higher affinity for TRH-R1, and a single
dose of competing ligand using intact cells at 37°C for 1h.
Apparent binding inhibitory constants (Kis) were meas-
ured at equilibrium using 1nM [3H]MeTRH and various
concentrations of competing ligands. Equilibrium binding
constants were derived from competition binding experi-
ments using the formula Ki = (IC50/(1+([L]/Kd)), where
IC50 is the concentration of unlabeled ligand that half-
competes with specifically bound [3H]MeTRH. Curves
were fitted by nonlinear regression analysis and drawn
with the PRISM program 3 (GraphPad Inc.).
C18H16ClN2O+ 311.0945). Compound
9
1H NMR
(CDCl3): d 0.98 (t, JHH = 7.5Hz, 3H), 1.67 (m, 2H), 3.47
(q, JHH = 6.3Hz, 2H), 7.45–7.53 (m, 3H), 7.57–7.62 (m,
2H), 7.67–7.70 (m, 1H), 7.73–7.78 (m, 1H), 8.04–8.07 (m,
1H), 8.26 (br s, 1H), 8.67 (s, 1H); TOFMS m/e 325.1097
(M+H+) (calculated for C19H18ClN2O+ 325.1102). Com-
pound 10 1H NMR (CDCl3): d 1.29 (d, JHH = 6.6Hz, 6H),
4.34 (m, 1H), 7.46–7.53 (m, 3H), 7.56–7.61 (m, 2H), 7.66–
7.69 (m, 1H), 7.72–7.77 (m, 1H), 8.04–8.07 (m, 2H), 8.67
(s, 1H); TOFMS m/e 325.1111 (M+H+) (calculated for
C19H18ClN2O+ 325.1102). Compound 11 1H NMR
(CDCl3): d 1.51 (s, 9H), 7.46–7.52 (m, 3H), 7.55–7.60
(m, 2H), 7.67–7.71 (m, 1H), 7.74–7.78 (m, 1H), 8.02–8.05
(m, 1H), 8.15 (br s, 1H), 8.64 (s, 1H); TOFMS m/e
339.1253 (M+H+) (calculated for C20H20ClN2O+
339.1258). Compound 12 (partial double bond effect
observed) 1H NMR (CDCl3): d 0.72 (t, JHH = 7.2Hz,
3H), 0.98 (t, JHH = 7.2Hz, 3H), 1.08–1.15 (m, 2H), 1.39–
1.47 (m, 2H), 1.48–1.61 (m, 2H), 1.63–1.71 (m, 2H), 3.10–
11. Hinkle, P. M.; Pekary, A. E.; Senanayaki, S.; Sattin, A.
Brain Res. 2002, 935, 59.