GPR103 antagonists demonstrating anorexigenic activity in vivo: Design and development of pyrrolo[2,3- c ]pyridines that mimic the c-terminal arg-phe motif of QRFP26

Article abstract of DOI:10.1021/jm401951t

GPR103, a G-protein coupled receptor, has been reported to have orexigenic properties through activation by the endogenous neuropeptide ligands QRFP26 and QRFP43. Recognizing that central administration of QRFP26 and QRFP43 increases high fat food intake in rats, we decided to investigate if antagonists of GPR103 could play a role in managing feeding behaviors. Here we present the development of a new series of pyrrolo[2,3-c]pyridines as GPR103 small molecule antagonists with GPR103 affinity, drug metabolism and pharmacokinetics and safety parameters suitable for drug development. In a preclinical obesity model measuring food intake, the anorexigenic effect of a pyrrolo[2,3-c]pyridine GPR103 antagonist was demonstrated. In addition, the dynamic 3D solution structure of the C-terminal heptapeptide of the endogenous agonist QRFP26 _(20-26) was determined using NMR. The synthetic pyrrolo[2,3-c] pyridine antagonists were compared to this experimental structure, which displayed a possible overlay of pharmacophore features supportive for further design of GPR103 antagonists.

Full text of DOI:10.1021/jm401951t

Article
pubs.acs.org/jmc
GPR103 Antagonists Demonstrating Anorexigenic Activity in Vivo:
Design and Development of Pyrrolo[2,3‑c]pyridines That Mimic the
C‑Terminal Arg-Phe Motif of QRFP26
†
‡
,†
§
§
̈
Jennie Georgsson, Fredrik Bergstrom, Anneli Nordqvist,* Martin J. Watson, Charles D. Blundell,
†
⊥
†
#
+
Magnus J. Johansson, Annika U. Petersson, Zhong-Qing Yuan, Yiqun Zhou, Lisbeth Kristensson,
∥
⊥
†
†
∥
Dorota Kakol-Palm, Christian Tyrchan, Eric Wellner, Udo Bauer, Peter Brodin,
and Anette Svensson Henriksson†
†
‡
∥
+
⊥
CVMD Medicinal Chemistry, CVMD DMPK, CVMD Bioscience, Discovery Sciences, and RIA Medicinal Chemistry,
AstraZeneca, Pepparedsleden 1, 431 83 Molndal, Sweden
̈
§
C4X Discovery Ltd., Unit 310 Ducie House, Ducie Street, Manchester M1 2JW, U.K.
#
Pharmaron Beijing, Co. Ltd., 6 Taihe Road, BDA, Beijing, 100176, P. R. China
*
S Supporting Information
ABSTRACT: GPR103, a G-protein coupled receptor, has
been reported to have orexigenic properties through activation
by the endogenous neuropeptide ligands QRFP26 and
QRFP43. Recognizing that central administration of
QRFP26 and QRFP43 increases high fat food intake in rats,
we decided to investigate if antagonists of GPR103 could play
a role in managing feeding behaviors. Here we present the
development of a new series of pyrrolo[2,3-c]pyridines as
GPR103 small molecule antagonists with GPR103 affinity,
drug metabolism and pharmacokinetics and safety parameters
suitable for drug development. In a preclinical obesity model
measuring food intake, the anorexigenic effect of a pyrrolo[2,3-
c]pyridine GPR103 antagonist was demonstrated. In addition,
the dynamic 3D solution structure of the C-terminal heptapeptide of the endogenous agonist QRFP26(20−26) was determined
using NMR. The synthetic pyrrolo[2,3-c]pyridine antagonists were compared to this experimental structure, which displayed a
possible overlay of pharmacophore features supportive for further design of GPR103 antagonists.
INTRODUCTION
Obesity is an increasing health concern related to cardiovas-
cular diseases and diabetes. The pyroglutamylated RF amide
peptide receptor 103 (GPR103), and its endogenous neuro-
The GPR103 neuropeptides QRFP26 and QRFP43 belong
to the RF amide group of peptides, including NPFF, NPAF and
PrPK. These peptides are characterized by the conserved C-
■
1
,2
4
3
terminal sequence motif Arg-Phe-NH2. Structure−activity
peptide agonist ligands QRFP26 and QRFP43 (Figure 1) are
relationship (SAR) studies on QRFP26 have demonstrated
that the C-terminal amino acids Phe24-Arg25-Phe26 are
11
important for biological activity. The conformation of this
C-terminal motif could be supportive in the design of new
GPR103 ligands. So far attempts to restrict the conformation of
the three C-terminal amino acids Phe24-Arg25-Phe26 via aza-
β3 amino acids have proven unsuccessful, but other turn
Figure 1. Primary structure of human QRFP26 and QRFP43.
present in brain areas involved in feeding and body weight
control in both rodents and man.
gene there are 46 orthologues annotated. In all these species
there is a one-to-one gene relationship with the exception of
mouse and rat, where two closely related genes are present,
12
4
−8
structures cannot be excluded. NMR studies of QRFP26 have
For the human GPR103
shown that the C-terminal residues are relatively unstruc-
13
tured.
Preclinical data suggest that endogenous GPR103 neuro-
peptide agonists have an important regulatory role in energy
homeostasis by promoting increases in food intake and body
9
GPR103A and GPR103B. Human GPR103 displays 85%
amino acid identity with mouse GPR103A and 79% identity
with GPR103B, while the rat GPR103A and GPR103B share
respectively 84% and 82% amino acid identity with human
Received: December 19, 2013
Published: June 17, 2014
9
,10
GPR103.
©
2014 American Chemical Society
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Journal of Medicinal Chemistry
Article
7
,8,12,14
weight.
Clinical data show increased levels of QRFP26 in
EDC/HOBt as coupling reagent to generate the library with a
undernourished anorexia nervosa patients compared to healthy
variety of carboxylic acids (3) and the THIQ dimethyl-
methylene amine (4). Among the newly identified com-
pounds, 1-methylpyrrolo[2,3-c]pyridine-2-carboxamide with
lipophilic ligand efficiency (LLE) 4.8, was taken further into
optimization by investigation of the substitution pattern of the
pyridine ring and substitutions on the THIQ.
1
5
24
controls. Activation of GPR103 also affects nociception
1
6
(
inflammatory pain response), heart rate, and blood pressure
8
in preclinical models. The orexigenic effect of GPR103
activation is at least partially mediated by neuropeptide Y
(
NPY) signaling, and it has been demonstrated that NPY1 and
NPY5 antagonists abolish the effect of QRFP26. Also, QRFP26
Synthesis of compounds with variation in the 5-position of
the pyrrolo[2,3-c]pyridine ring is outlined in Scheme 2, starting
inhibits the elevation of pro-opiomelanocortin (POMC) and
17
the anorectic effect of leptin. These data suggest that
inhibition of the orexigenic effect of GPR103 activation can
promote reduced body weight in overweight and obese patients
by appetite regulation.
Scheme 2. Synthesis of Compounds with Variation in the 5-
a
Position of the Pyrrolo[2,3-c]pyridine Ring (9a−9h)
Only a few GPR103 antagonists from the public domain are
1
8−21
available
inhibition of the orexigenic effect of GPR103 activation. In
010, 3-aryl- or heteroaryl-substituted indole derivatives were
claimed to antagonize the GPR103 receptor by Banyu
to validate the impact on energy homeostasis by
2
18−20
Pharmaceutical Co Ltd.
Two of the disclosed GPR103
antagonists are shown in Figure 2. A physicochemical
a
Reagents: (a) CH I, Cs CO , DMF, rt, 16 h. (b) Method A: NaOH,
3
2
3
EtOH:H O (10:1), rt followed by acid (7a, 7c, 7d, 8b and 8c) (1
2
equiv), 4 (1 equiv), TBTU (1.1 equiv), DIEA, DMF, rt, 18 h. Method
B: ester (7b, 8d) (1 equiv), 4 (1.2 equiv), Al(CH ) (4.0 equiv), TEA,
3
3
toluene, reflux, 16 h. (c) NaI, CuI, N1,N2-dimethylethanediamine, 1,4-
dioxane, 110 °C. (d) Ethynyltrimethylsilane, Pd(PPh ) Cl , CuI, 1,4-
3
2
2
−
+
Figure 2. Two examples of disclosed GPR103 antagonists.
dioxane, rt, 24 h followed by TBAF, THF, rt, 2 h; (e) CF
3 3
SO S-
(
CF )(Ph) , Cu, DMF, 60 °C, 18 h; (f) PhB(OH) , PdCl (dbpf),
3
2
2
2
K CO , DME:H O:EtOH (5:2.5:1), microwave, 110 °C, 20 min.
2
3
2
assessment of these lipophilic bases was made that displayed
liabilities on metabolic stability, solubility, and CYP inhibition
in addition to narrow margins to cardiac liability targets.
Improved compound properties with retained GPR103 potency
would provide high-quality leads amenable to in vivo
assessment of anorexigenic efficacy.
In this paper we describe the development of a new series of
GPR103 antagonists with drug metabolism and pharmacoki-
netics (DMPK) and safety properties suitable for drug
development. From this new series, one representative
compound was evaluated for modulation of appetite. An
NMR study to investigate the solution conformations of the C-
terminal heptapeptide QRFP26(20−26) is also presented, with
pharmacophore features of the herein developed GPR103
antagonists related to the experimental conformations of
from commercially available ethyl esters (6a−6d). The N-
methylated pyrrolo[2,3-c]pyridines (7a−7d) were prepared
2
2,23
from the ethyl esters (6a−6d) by treatment with CH I.
3
Palladium- or copper-catalyzed coupling reactions were utilized
to introduce iodo, acetylene, trifluoromethyl, and phenyl
substituents in the 5-position of the pyrrolo[2,3-c]pyridine
ring. The iodo functionality (8a) was introduced by reacting
the bromo-substituted ester (7c) with sodium iodide using
copper(I) iodide as catalyst together with N,N-dimethylethene-
25,26
1
,2-diamine.
Acetylene was introduced, starting from 8a, using a catalytic
system of Pd(PPh ) Cl and copper(I) iodide reacting with
3
2
2
27
ethynyltrimethylsilane as first described by Sonogashira. The
TMS group was cleaved using TBAF to give 8b. To generate
the trifluoromethyl-substituted pyrrolo[2,3-c]pyridine (8c),
compound 8a was treated with copper and diphenyl-
QRFP26(20−26)
.
CHEMISTRY
■
(
trifluoromethyl)sulfonium trifluoromethanesulfonate in
28
To explore replacements for the indole motif, the tetrahy-
droisoquinoline (THIQ) dimethylmethylene amine was used as
starting point for the synthesis of 177 diverse compounds.
Outlined in Scheme 1 is the standard amide formation using
DMF. The phenyl analogue 8d was synthesized by a standard
Suzuki−Miyaura coupling from the phenylboronic acid and
8
29
a.
The esters were hydrolyzed to the acids, and the final
products of 5-substituted 1-methylpyrrolo[2,3-c]pyridine-2-
carboxamides (9a, 9c, 9d, 9f, and 9g) were synthesized from
the corresponding acids (7a, 7c, 7d, 8b, and 8c) and the THIQ
dimethylmethylene amine (4) using a standard TBTU amide
Scheme 1. Library Synthesis of THIQ Dimethylmethylene
Amine Derivatives by Amide Coupling
a
30
coupling. An alternative amide formation was used to make
b and 9h, where the esters (7b and 8d) were treated with
trimethylaluminum and triethylamine (TEA) and reacted with
9
31
4
.
To map the available space in the receptor covered by the
THIQ part of compound 9c, a methyl group was introduced
a
32
Reagents: (a) EDC, HOBt, TEA, DMF.
5
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Scheme 3. Synthesis of 5-Bromo-1-methylpyrrolo[2,3-c]pyridine-2-carboxamide Derivatives Comprising Aliphatic Methyl
a
Substitutions on the THIQ Dimethylmethylene Amine
a
− +
Reagents: (a) (CH ) NCH BF K , Pd(OAc) , S-Phos, K PO , 1,4-dioxane:H O, 100 °C. (b) H , Raney-Ni, MeOH. (c) HCOCH , rt, 4.5 h. (d)
3
2
2
3
2
3
4
2
2
3
(
COCl) , FeCl , DCM, −78 to 25 °C. (e) NaBH , MeOH. (f) 5-Bromo-1-methyl-pyrrolo[2,3-c]pyridine-2-carboxylic acid (from hydrolysis of 7c)
2
3
4
EDC, HOBt, TEA, DMF, 25 °C, 3 h. (g) CH COCl, pyridine:DCM (1:1), 2 h, 25 °C; (h) H , PtO , HCl, MeOH. (i) Ghosez reagent, Weinreb
3
2
2
amine, TEA, DCM, rt, 14 h. (j) CH MgCl, THF, rt, 1 h. (k) 10% TFA in DCM, followed by T3P, TEA, EtOAc, 0 to 60 °C. (l) MeNH in EtOH,
3
2
NaBH , rt to 50 °C followed by formaldehyde (aq), NaBH(OAc) .
4
3
in all aliphatic positions of the THIQ (25a−25d) (see Scheme
). The syntheses of the different THIQ analogues followed
two principal routes, where the THIQ scaffold was either built
up from a phenethylamine analogue (10 or 14) or from the
aromatic isoquinoline 18 (Scheme 3). The first step in the
synthesis of 20a was to react the commercial building block 2-
enantiomers of 25a were separated using reversed phase chiral
chromatography to give 25e and 25f. For synthesis of 25d,
commercially available 21 was converted to the acid chloride
and treated with dimethylhydroxylamine to form the
3
36
corresponding Weinreb amide (22). To make the methyl
ketone (23), the Weinreb amide was treated with methyl
magnesium chloride. After Boc deprotection the amine was
coupled to 5-bromo-1-methylpyrrolo[2,3-c]pyridine-2-carbox-
(
3-bromophenyl)propanenitrile with dimethylaminomethyltri-
fluoroborate using a palladium-catalyzed Suzuki−Miyaura
37
33
ylic acid using a standard T3P coupling, giving compound 24.
Two consecutive reductive aminations, first on the ketone using
methyl amine followed by formaldehyde on the resulting
secondary amine, formed the final compound 25d.
protocol to give 11. The nitrile in 11 was reduced using
Raney-nickel and hydrogen gas to give the amine (12), which
was formylated in neat methyl formate to give 13. The
intramolecular condensation of compound 13 to give 17a was
34
conducted using oxalyl choride and FeCl3. All attempts to
RESULTS AND DISCUSSION
form the THIQ using polyphosphoric acid or p-toluenesulfonic
■
3
5
acid were unsuccessful. The imine 17a was reduced using
sodium borohydride to form the THIQ 20a.
It is well-established that high lipophilicity and high molecular
weight are properties that may hamper development of oral
38
Synthesis of 20b started from the commercially available
phenethylamine 14 that was acetylated using acetyl chloride to
drugs. To address liabilities profiled in 1, alternatives to the
indole scaffold were explored in a designed set of 177
compounds (Scheme 1). For all compounds synthesized, the
in vitro functional activity in a GPR103 inositol-1-phosphate
give 15. Compound 15 was then treated with FeCl and oxalyl
3
34
choride to form the ring 16. The methylene-dimethylamine
was introduced as above with a Suzuki−Miyaura coupling on
(
IP-1) assay and log D were measured. LLE was calculated
7.4
39
3
3
according to eq 1. A higher LLE may indicate less lipophilic
contribution to potency and is therefore a suitable way to
identify lead compounds with specific receptor interactions.
the bromide 16, and the imine was reduced to give 20b using
sodium borohydride in the same way as described for 13.
Starting from the aromatic isoquinoline 18, compound 20c
was accomplished by introduction of methylene-dimethylamine
39
In Figure 3, 45 compounds with confirmed GPR103 antagonist
activity are shown.
33
via a Suzuki−Miyaura coupling on the chloride to give 19. To
generate 20c, the isoquinoline was reduced to THIQ by
hydrogenation.
LLE = pIC − log D
7
.4
(1)
5
0
To generate the final compounds (25a−25c) the THIQ
derivatives (20a−20c) were coupled to the 5-bromo-1-
methylpyrrolo[2,3-c]pyridine-2-carboxylic acid (from hydrol-
Three compounds with antagonistic activity below 1 μM and
lipophilicity in the range of 0.5 < log D7.4 < 2.5 were identified.
The reduced lipophilicity achieved from exchanging the indole-
scaffold improved the LLE (range 5.6−4.6) compared to
24
ysis of 7c) using a standard EDC/HOBt amide coupling. The
5
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Data from functional assessment of GPR103 antagonism and
log D7.4 measurements are shown in Table 1. Halogenation in
the 5-position of the pyrrolo[2,3-c]pyridine as in compounds
9a, 9c, and 9e gave an increase in antagonistic effect on
GPR103 compared to the unsubstituted 9b. With substitution
of the halogen with a methyl substituent (compound 9d), the
potency decreased 100-fold compared to 9c. For larger
substituents, such as alkyne (9f) and trifluoromethyl (9g),
the potency dropped approximately 10 times, and phenyl (9h)
was not tolerated at all. From calculated pK values it is
a
speculated that electronic properties of the pyrrolo[2,3-
c]pyridine nitrogen could be of importance for antagonistic
activity (5-chloro substitution, pKa,calcd = 2.9; 5-bromo-
substitution, pKa,calcd = 4.1; 5-hydrogen-substitution, pKa,calcd
=
6
.6; 5-methyl substitution, pKa,calcd = 7.1). Compound 9c was
further evaluated when exploring SAR of the THIQ with a set
of compounds introducing a methyl group in all aliphatic
3
2
3
9
positions. These analogues were initially screened as
racemates. Methyl substitution in the 1- and 3-positions
(
Figure 3. GPR103 pIC50 vs LLE displaying identification of three
new scaffolds (marked as ▲) to explore in lead identification.
25b, 25c), as well as on the methylene linker in the 6-
position of the THIQ (25d), gave compounds equipotent to
a. The racemate 25a was 5 times more potent than 9a.
Separation of the enantiomers of 25a identified 25e with a
9
compound 1 (LLE = 4.3). Among the newly identified
compounds, 1-methylpyrrolo[2,3-c]pyridine-2-carboxamide 9a
with LLE = 4.8 was selected as the indole replacement suitable
for further exploration. A variety of regioisomers of 1-
methylpyrrolo[2,3-c]pyridine-2-carboxamide were part of the
initial library, and SAR assessment suggested that the
substitution at the 5-position and the pyridine nitrogen in the
m
e
a
s
u
r
e
d
I
C
o
f
4
6
n
M
c
o
m
p
a
r
e
d
t
o
e
n
a
n
t
i
o
m
e
r
2
5
f
(
I
C
=
5
0
5
0
870 nM).
During the course of compound exploration, compounds
were further profiled for DMPK and safety properties. Data for
1, 9c, and 25e are shown in Table 2. In general, the change
from the indole to the pyrrolo[2,3-c]pyridine scaffold lowered
the log D7.4 about 1.5 units and led to a decrease in intrinsic
clearance corrected for incubational binding in both human
hepatocytes and human liver microsomes. Metabolism
identification in human liver microsome incubations on indole
6
-position were crucial for potency (data displayed in Figure 3,
structures not shown). Therefore, further exploration was
focused on the 5-position of the pyrrolo[2,3-c]pyridine to
enhance potency and explore size restrictions.
Table 1. SAR of Position 5 of the Pyrrolo[2,3-c]pyridines and Methylation in the THIQ Substructure
a
b
compd
R′
Cl
R″
R‴
log D7.4
1.5 (1)
hGPR103 IP-1 IC50 (μM)
9
9
9
9
9
9
9
9
2
2
2
2
2
2
a
H
H
H
H
H
H
H
H
H
0.57 ± 0.09 (2)
2.9 ± 0.6 (2)
0.24 ± 0.08 (4)
18.9 ± 0.7 (2)
0.13 ± 0.04 (2)
6.0 ± 0.7 (2)
b
H
H
0.9 ± 0 (3)
c
Br
H
1.7 ± 0.2 (3)
1.3 ± 0.1 (3)
2.0 ± 0.1 (3)
1.2 ± 0.1 (3)
1.8 ± 0.2 (3)
3.0 ± 0.1 (4)
2.1 ± 0.1 (2)
2.2 ± 0.1 (2)
2.1 ± 0.1 (3)
1.8 ± 0.2 (3)
2.1 ± 0.1 (3)
1.9 ± 0.3 (3)
d
CH3
I
H
e
H
f
CCH
CF3
Ph
Br
H
g
H
3.3 ± 0.2 (3)
>41 (3)
h
H
5a
5b
5c
5d
5e
5f
3-CH3
H
0.11 ± 0.005 (2)
0.5 ± 0.1 (3)
0.71 ± 0.02 (2)
0.46 ± 0.04 (5)
0.046 ± 0.004 (2)
0.88 ± 0.017 (2)
Br
1-CH3
H
Br
4-CH3
H
Br
H
CH3
H
Br
(+)-7-CH3
(−)-7-CH3
Br
H
a
Values are reported as a single measurement, or where more than one independent experiment was done, the mean ± SD is given with the number
b
of independent experiments (N) in parentheses. IC50 is reported as mean ± SD with the number of experiments (N) in parentheses. The
confidence interval ratio (CIR) was 2.0 for the hGPR103 IP-1 assay with minimum discriminatory ratio (MDR) of 2.8. The 95% confidence interval
for the true IC50 of the compound is the single calculated value of IC50 ×/÷ CIR. Two compounds are considered different if their IC50 differ by a
ratio >MDR (p = 0.05).
5
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Journal of Medicinal Chemistry
Article
Table 2. DMPK Properties and Cardiac Safety Profile for Selected Compounds
assay
1
9c
25e
a
hGPR103 IP-1 IC50 (μM)
0.07 ± 0.02 (52)
0.07 ± 0.02 (5)
0.09 ± 0.02 (7)
0.08 ± 0.02 (7)
3.0 ± 0.1 (3)
62 ± 10 (3)
40 ± 3 (3)
0.24 ± 0.08 (4)
0.28 ± 0.02 (4)
0.33 ± 0.08 (5)
0.10 ± 0.01 (5)
1.7 ± 0.2 (3)
>1000 (2)
0.046 ± 0.004 (2)
0.04 ± 0.02 (2)
0.058 (1)
a
hGPR103 RLB IC50 (μM)
a
mGPR103A IP-1 IC50 (μM)
a
mGPR103B IP-1 IC (μM)
0.015 (1)
5
0
b
log D7.4
solubility (μM)
2.1 ± 0.1 (3)
>1000 (2)
b
6
c
intrinsic clearance in vitro in human hepatocytes (μL/min/10 cells) (CIR = 1.4)
5.3 ± 0.9 (3)
16 ± 2 (3)
13.2 (1)
6.0 ± 0.6 (3)
26 ± 5 (3)
11.8 (1)
c
intrinsic clearance in vitro in human liver microsomes (μL/min/mg) (CIR = 1.7)
Caco-2 apparent permeability (10− cm/s)
156 ± 22 (3)
7.8 (1)
6
b
b
CYP inhibition (IC 2D6) (μM)
2.9 ± 0.8 (2)
4.9 ± 1.7 (2)
10.3 (1)
>20 (2)
>20 (3)
5
0
d
hERG IC50 (μM)
31.2 (1)
12.9 (1)
d
e
hNav1.5 IC50 (μM)
>33 (1)
>33 (3)
d
e
hIKs IC50 (μM)
10.5 (1)
>33 (1)
>33 (1)
a
IC50 is reported as mean ± SD with the number of experiments (N) in parentheses. The confidence interval ratio (CIR) is reported as a statement
of the assay quality. The 95% confidence interval for the true IC50 of the compound is the single calculated value of IC50 ×/÷ CIR. hGPR103 IP-1
CIR = 2.0; hGPR103 RLB CIR = 1.7. The minimum discriminatory ratio (MDR) for the IP-1 assay was 2.8 and MDR = 2.1 for the radioligand
b
binding assay. Two compounds are considered different if their IC50 differ by a ratio >MDR (p = 0.05). Values are reported as a single
measurement, or where more than one independent experiment was done, the mean ± SD is given with the number of independent experiments
c
d
(
N) in parentheses. Measured intrinsic clearance corrected for unspecific binding in incubations. IC is reported as mean ± SD with the number
50
of experiments (N) in parentheses. Experiments were run using four replicates at each concentration. The confidence interval ratio (CIR) is reported
as a statement of the assay quality. The 95% confidence interval for the true IC50 of the compound is the single calculated value of IC50 ×/÷ CIR.
e
hERG CIR = 2.0; hNav1.5 CIR = 2.0; hIKs CIR = 2.3. Measured for the racemate 25a.
and pyrrolo[2,3-c]pyridine compounds revealed that the major
metabolites were the same for the two scaffolds (data not
shown). Hence, the improved metabolic stability was correlated
to the structural modification and was not a consequence of
altered metabolic pathways. Compounds 9c and 25e displayed
no inhibition toward any of the six tested isoforms of
cytochrome P450 enzymes (CYP3A4, CYP1A2, CYP2D6,
CYP2C8, CYP2C9, and CYP2C19) in a high-throughput
fluorescence assay. The solubility was dramatically increased
and, in combination with the high apparent permeability
measured in an in vitro Caco-2 assay, the novel compounds are
expected to have a high fraction absorbed in vivo. Improved
physicochemical properties accomplished with the pyrrolo[2,3-
c]pyridine scaffold also increased margins to cardiac liability
targets such as hERG, human cardiac sodium channel 1.5
Figure 4. Compound 25e administered by oral gavage at 75 μmol/kg
BID (gray bars, N = 8) and 125 μmol/kg BID (black bars, N = 7)
decreased food intake dose dependently in obese C57Bl/6 mice, but
vehicle (N = 8) did not. Results are presented as cumulative food
intake in grams over 24 h periods for 3 days. Data are presented as
mean ± SEM, *p < 0.05, ***p < 0.001.
(
hNav1.5), and human cardiac slowly activating delayed
rectifier K+ channel (hIKs) (Table 2). Investigated compounds
, 9c, and 25e were confirmed to bind human GPR103 using a
1
radioligand binding assay (RLB). Species crossover was
confirmed in mouse GPR103A and B IP-1 assays, which
showed that both compounds 9c and 25e had similar potency
range in mouse compared to human GPR103, but that they
were slightly more potent on mGPR103B (3.3 and 3.8 times
respectively) compared to mGPR103A.
To investigate the ability of a GPR103 antagonist to
modulate appetite, obese female mice on a high-fat diet were
administered 25e by oral gavage during automated food intake
measurement (FIA). The study was initiated with four basal
acclimatization days in the FIA monitoring system, measuring
food intake and meal frequency. Once acclimatized, mice were
given either vehicle or 25e at 75 and 125 μmol/kg twice daily
compared to vehicle-treated animals, indicating that GPR103
antagonist 25e reduces appetite without introducing malaise.
Also, an indication of body weight reduction was observed in
the compound-treated animals, but the duration of the study
was too short to show significance in body weight reduction
(Supporting Information, Supplementary Figure S9).
Data from [1 I]QRFP43 binding studies suggest that the
antagonists presented in this paper are competitive with the
endogenous agonist. To elucidate the bioactive pharmacophore,
the free solution structure of QRFP26(20−26) was determined
using a methodology specifically designed to characterize
25
40
flexible systems and was compared to compound 9c. Overall,
QRFP26(20−26) exhibited a high degree of conformational
flexibility in solution (Figure 5A), consistent with a previous
(
BID) for 3 days, with continuous measurements on the food
intake and meal frequency. As shown in Figure 4, animals
treated with 25e reduced their food intake compared to vehicle
treated animals in a dose-dependent manner. The highest dose
13
study.
Moreover, the detailed experimental results show that the
peptide backbone mainly adopts an extended (β-strand-like)
conformation that accounts for 60% of the conformations
observed in solution (Figure 5B). No other single backbone
(
125 μmol/kg) gave a significant reduction of 86% (p < 0.001)
in cumulative food intake compared to vehicle-treated animals
at day 3. Treatment with 25e did not change the eating pattern
5
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(
these consist of two staggered rotamers at χ , extended trans
1
rotamers at χ and χ , and two approximately orthogonal
2
3
rotamers at χ ; Supporting Information, Supplementary Figure
4
S5). Full details describing the conformational parameters for
each rotatable bond are given in the Supporting Information
(
Supplementary Table S9). Recently, the first structural study
of QRFP26 conformation by NMR was published by
(
20−26)
4
1
Pierry et al. The data presented in this publication differ from
our findings. We observed a number of pieces of data that are
clearly incompatible with a substantial helical content in the
structural ensemble (scalar couplings, diagnostic NOE
patterns), whereas they report helix or helixlike secondary
structure. In particular, the diagnostic medium intensity α,N(i,i
+
3) NOE crosspeak between residues 22 and 25, which is
shown in their Figure 6, is clearly absent in our own NOESY
spectrum. The source of the difference could lie in their use of
DPC micelles, whereas the study presented in this paper was
performed in aqueous solution. DPC micelles are commonly
seen to induce secondary structures in short peptides, due to
the requirement of burying hydrophilic moieties in a hydro-
phobic environment.
11
For the three key C-terminal amino acid residues of
QRFP26(20−26), eight dominant conformational families in
solution were found (Figure 5C), and we hypothesize that
the bioactive conformation of the peptide lies within this set of
4
0,42
conformations.
These eight conformational families were
simplified by setting each torsion to its mean position, leaving
eight idealized peptide conformations (Figure 5D) for
comparison with low-energy conformations of 9c. An excellent
overlay of key pharmacophore features of the terminal Arg-Phe-
NH in QRFP26
and 9c was observed (Figure 6) using
2
(20−26)
Figure 5. (A) Main structure overview, showing the complete
ensemble generated. The peptide backbone is highlighted in dark
gray for clarity. The overlay is on the backbone of Ser23, and the
additional flexibility of the N-terminal glycines can be seen. (B) The
dominant overall shape of the molecule as determined by the
dominant backbone conformation. This shows the overall amphiphilic
pattern of the molecule and the relatively well-defined gross shape. (C)
Overlay focusing on the key C-terminal residues and their most
dominant conformational families. (D) Idealized conformations
representing the dominant conformational families (macrostates) of
the key C-terminal residues.
conformation accounts for more than 20% of the conforma-
tions, and most of them are much less common. With the
backbone in the dominant extended β-strand-like conforma-
tion, the hydrophobic Phe22, Phe24, and Phe26 side chains
group together on one side of the molecule while the
hydrophilic Ser23 and Arg25 residues sit on the opposite side
Figure 6. Structural overlay of one of the idealized conformations of
QRFP26(20−26) (gray carbons) and a low-energy conformation of 9c
(magenta carbons). Pharmacophore features including the aromatic
ring plane, the Phe26 carbonyl oxygen, and the positively charged
Arg25 side chain were captured by the ligand, in addition to the overall
shape envelope of the two C-terminal amino acids.
(
Figure 5B). The second most populated backbone con-
formations are those in which single residues adopt a
conformation in the α-helical region of the Ramachandran
plot (Supporting Information, Supplementary Figure S4).
Conformations with multiple residues in α-helix-like con-
formations are rare (<2% of the ensemble): there is no
evidence of incipient helix formation in the peptide. The N-
terminal glycine residues adopt largely extended conformations
and are less ordered than the rest of the molecule. All three
phenylalanine side chains are best described by a mixture of two
one of the most populated conformations of the agonist
peptide. This conformation of compound 9c used in this
overlay was found to be 0.8 kcal/mol higher than the lowest
energy conformation identified after optimization with
quantum mechanics and was considered a reasonable
conformation consistent with the current understanding of
modeling energy calculations. Specifically, the relationship of
the positively charged amine, the aromatic pyrrolo[2,3-
c]pyridine, and the central carbonyl moieties of the antagonist
closely matched the relationship of the Arg25 and Phe26 side
chains and the C-terminal amide carbonyl of the agonist. It is
staggered rotamers at χ and adopt conformations close to
1
orthogonal at χ2 (Supporting Information, Supplementary
Figure S5). The side chain of Arg25 adopts a large number
of conformations, but four extended conformations account for
approximately 90% of the conformations observed in solution
5
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known that small structural differences can make an agonist
using a SiO
2
column with UV detection. LC/MS/MS for DMPK and
physicochemical assays were performed on an Agilent HPLC 1100
series binary pump and a CTC injector in combination with a Waters
Quattro Premier or a Waters Acquity UPLC coupled to a Waters
Micromass TQD. The columns used were either an Atlantis T3 C18 (3
μm, 2.1 × 30 mm) or an Acquity UPLC HSS T3 C18 (1.8 μm, 2.1 ×
into an antagonist (and vice versa) and that both agonists and
antagonists can share common pharmacophore motifs.
The harmony between these scaffolds is suggestive that the
presently described compounds and QRFP26(20−26) bind with
overlapping pharmacophores and that the selected conforma-
tion of the peptide may be similar to the bioactive.
43−45
5
0 mm). Gradient elution was used with MeCN/H O containing 0.2%
2
formic acid. Purity of all test compounds was determined by LC/MS.
All screening compounds had purity >95%.
CONCLUSIONS
Ethyl 5-Chloro-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate
■
(
7a). Compound 6a (1.60 g, 7.14 mmol), iodomethane (0.53 mL, 8.56
In summary, we have reported the development and synthesis
of a series of GPR103 small molecule antagonists with lead
compounds demonstrating DMPK and safety properties
amenable to drug development. Drivers for lipophilicity were
identified within the indole motif and, through replacement
with pyrrolo[2,3-c]pyridine, lead compounds with increased
metabolic stability and solubility as well as improved margins to
CYP inhibition and cardiac liability targets were achieved. We
also report unique preclinical data on the anorexigenic efficacy
of a GPR103 antagonist. Compound 25e demonstrated a
significant and dose-dependent reduction on food intake
compared to vehicle-treated animals as assessed in a 3 day
automated food intake measurement study.
To support further design on GPR103 antagonists, the free
solution structure of the C-terminal motif of the endogenous
GPR103 ligand QRFP26 was studied using the heptapeptide
QRFP26(20−26). The solution structure of QRFP26(20−26)
exhibited a high degree of conformational flexibility, in which
the peptide backbone mainly adopted an extended conforma-
tion where the overall placement of the amino acid side chains
gave the peptide an amphiphilic character. Considering the
NMR data in combination with conformational studies on the
antagonists, we were able to elucidate that QRFP26(20−26) and
this class of GPR103 antagonists could share overlapping
pharmacophores and may be able to bind accordingly. We
therefore hypothesize that the selected C-terminal conforma-
tion of the peptide is similar to the bioactive one and that the
synthesized antagonists mimic the C-terminal Arg25-Phe26
residues of the endogenous ligand.
mmol), and cesium carbonate (3.0 g, 9.28 mmol) were mixed in DMF
(
20 mL) under N , and the reaction mixture was stirred for 16 h at rt.
2
The crude product was precipitated by addition of H O (200 mL).
2
The solid was filtered, washed with H O, and then dissolved in DCM.
2
The organic solution was washed with H O, separated, and
2
1
concentrated to yield 7a (1.44 g, 84%) as a yellow solid. H NMR
(500 MHz, DMSO-d
q, J = 7.1 Hz, 2H), 7.23 (s, 1H), 7.76 (s, 1H), 8.90 (s, 1H). MS (ESI)
): δ 1.35 (t, J = 7.1 Hz, 3H), 4.11 (s, 3H), 4.36
6
(
+
m/z [M + H] : 239.1.
Ethyl 1-Methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (7b).
This compound was synthesized using the protocol as described for
a. Compound 6b (150 mg, 0.79 mmol) was used in the reaction.
Instead of precipitation, the solvent was evaporated and the residue
7
was purified by preparative HPLC using basic conditions to yield 7b
1
(
32 mg, 20%). H NMR (500 MHz, DMSO-d ): δ 1.35 (t, J = 7.1 Hz,
6
3H), 4.13 (s, 3H), 4.36 (q, J = 7.1 Hz, 2H), 7.26 (d, J = 0.9 Hz, 1H),
7.64 (dd, J = 0.9, 5.5 Hz, 1H), 8.22 (d, J = 5.5 Hz, 1H), 9.06 (s, 1H).
+
MS (ESI) m/z [M + H] : 205.2.
Ethyl 5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate
7c). This compound was synthesized using the protocol as described
(
for 7a. Compound 6c (992 mg, 3.69 mmol) was used to yield 7c (965
1
mg, 92%) as a yellow solid. H NMR (400 MHz, DMSO-d ): δ 1.33 (t,
6
J = 7.1 Hz, 3H), 4.08 (s, 3H), 4.34 (q, J = 7.1 Hz, 2H), 7.19 (d, J = 0.8
Hz, 1H), 7.88 (d, J = 0.8 Hz, 1H), 8.87 (s, 1H). MS (ESI) m/z [M +
+
H] : 284.8.
Ethyl 1,5-Dimethyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (7d).
Sodium hydride (17 mg, 0.40 mmol) was added to a solution of 6d
(82 mg, 0.40 mmol) in dry DMF (5 mL) under N at rt. After being
2
stirred for 30 min, iodomethane (0.025 mL, 0.40 mmol) dissolved in
dry DMF (1 mL) was added dropwise. The reaction mixture was
stirred for 16 h at rt under N . The solvent was removed and the crude
2
+
was used as such in the next step. MS (ESI) m/z [M + H] : 219.1.
Ethyl 5-Iodo-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate
EXPERIMENTAL SECTION
General Description. H NMR spectra were recorded on a Bruker
Avance II or III spectrometer at a proton frequency of 300, 400, 500,
or 600 MHz at 25 °C, with the frequency stated for each experiment.
The chemical shifts (δ) are reported in ppm and referenced indirectly
to TMS via the solvent signals (CDCl at 7.26 ppm, DMSO-d at 2.50
(
8a). A mixture of 7c (1.08 g, 3.81 mmol), copper(I) iodide (0.15
■
1
g, 0.76 mmol), N1,N2-dimethylethane-1,2-diamine (0.082 mL, 0.76
mmol), and sodium iodide (1.14 g, 7.63 mmol) in 1,4-dioxane (17.5
mL) was added to a microwave vial, which was flushed with N and
2
sealed. The reaction mixture was heated at 110 °C in an oil bath for 72
h. The reaction mixture was then diluted with DCM (20 mL) and
filtered through a pad of Celite, and the filtrate was concentrated. The
residue was purified by automated flash chromatography using a
3
6
ppm, CD OD at 3.31 ppm). All microwave-assisted synthesis was
3
carried out in an Initiator synthesizer single mode cavity instrument
producing controlled irradiation with a power range 0−400 W at 2450
MHz (Biotage AB, Uppsala, Sweden). UHPLC−MS experiments were
performed using a Waters Acquity UHPLC system combined with a
SQD mass spectrometer. The UHPLC system was equipped with both
a BEH C18 column (1.7 μm, 2.1 × 50 mm) in combination with a 46
mM (NH ) CO /NH buffer at pH 10 and a HSS C column (1.8
gradient of 0−40% of EtOAc in heptane as mobile phase to yield 8a
1
(1.02 g, 81%). H NMR (500 MHz, DMSO-d
): δ 1.34 (t, J = 7.1 Hz,
6
3H), 4.09 (s, 3H), 4.36 (q, J = 7.1 Hz, 2H), 7.19 (d, J = 0.8 Hz, 1H),
+
8.12 (d, J = 0.8 Hz, 1H), 8.89 (s, 1H). MS (ESI) m/z [M + H] :
331.1.
Ethyl 5-Ethynyl-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxy-
late (8b). N-Ethyl-N-isopropylpropan-2-amine (0.129 mL, 0.74
mmol) was added to a mixture of 8a (163 mg, 0.49 mmol),
bis(triphenylphosphine)palladium(II) chloride (35 mg, 0.05 mmol),
and copper(I) iodide (9 mg, 0.05 mmol) in 1,4-dioxane (9 mL) under
4
2
3
3
18
μm, 2.1 × 50 mm) in combination with 10 mM formic acid, 1 mM
ammonium formate buffer at pH 3. The flow rate was 1 mL/min. The
mass spectrometer used ESI± as the ion source. Preparative HPLC
was performed by a Waters Fraction Lynx med ZQ MS detector on
either a Waters Xbridge C18 OBD 5 μm column (19 × 150 mm, flow
rate 30 mL/min, or 30 × 150 mm, flow rate 60 mL/min) using a
N . The reaction mixture was purged with N for 5 min.
2
2
Ethynyltrimethylsilane (0.106 mL, 0.74 mmol) was added dropwise
and the color of the reaction mixture changed from light yellow to
dark blue to brown. The resulting reaction mixture was stirred at rt for
gradient of 5−95% MeCN with 0.2% NH at pH 10 or a Waters
3
SunFire C18 OBD 5 μm column (19 × 150 mm, flow rate 30 mL/min,
or 30 × 150 mm, flow rate 60 mL/min) using a gradient of 5−95%
MeCN with 0.1 M formic acid. Molecular mass (HR-ESI-MS) was
determined on a mass spectrometer equipped with an electrospray ion
source. Column chromatography was performed on a Biotage system
24 h under N . The reaction mixture was poured into H O and
2
2
extracted with EtOAc (3×). The combined organic layers were washed
with brine, dried over MgSO , filtered, and concentrated in vacuo. The
4
residue was purified by automated flash chromatography using a
5
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gradient of 5−40% of EtOAc in heptane as mobile phase to yield ethyl
(5-Chloro-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-
[(dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}-
methanone (9a). In a 25 mL round-bottomed flask, 7a (0.21 g, 0.93
mmol) and sodium hydride (0.05 mL, 1.03 mmol) were dissolved in
1
-methyl-5-((trimethylsilyl)ethynyl)-1H-pyrrolo[2,3-c]pyridine-2-car-
1
boxylate (88 mg, 59%). H NMR (500 MHz, DMSO-d ): δ 0.25 (s,
6
9
7
+
H), 1.35 (t, J = 7.1 Hz, 3H), 4.13 (s, 3H), 4.36 (q, J = 7.1 Hz, 2H),
dry DMF (3 mL) under N at rt to give a yellow solution. After being
.26 (s, 1H), 7.87 (d, J = 1.1 Hz, 1H), 9.02 (s, 1H). MS (ESI) m/z [M
2
+
stirred for 1 h, iodomethane (0.058 mL, 0.93 mmol) was added and
the reaction was continued for 2 h. LC/MS indicated remaining
starting material. Additional sodium hydride (0.024 mL, 0.93 mmol)
was added and after being stirred for 30 min, additional iodomethane
H] : 301.1.
A 1 M TBAF solution in THF (0.337 mL, 0.34 mmol) was added
dropwise to a solution of ethyl 1-methyl-5-((trimethylsilyl)ethynyl)-
H-pyrrolo[2,3-c]pyridine-2-carboxylate (88 mg, 0.29 mmol) in THF
1 mL) at rt under N and the reaction mixture was stirred for 2 h.
1
(0.058 mL, 0.93 mmol) was added. The mixture was stirred for 1 h.
(
2
The reaction was quenched by addition of ice and stirred for 20 min.
Solvents were evaporated, and the residue was purified by preparative
The reaction mixture was treated with NH Cl solution (sat. aq, 5 mL)
4
and extracted with EtOAc (3×). The combined organic layers were
HPLC using acidic conditions to yield 5-chloro-1-methyl-1H-pyrrolo-
washed with H O and brine, dried over Mg SO , filtered, and
2
2
4
1
[
2,3-c]pyridine-2-carboxylic acid (150 mg, 76%) as white solid. H
concentrated. The residue was purified by automated flash
chromatography using a gradient of 30−50% of EtOAc in heptane
NMR (500 MHz, DMSO-d ): δ 4.11 (s, 3H), 7.18 (s, 1H), 7.74 (s,
6
+
1
H), 8.88 (s, 1H). MS (ESI) m/z [M + H] : 211.0.
as mobile phase to yield 8b (62 mg, 93%) as a white solid after
1
General coupling method A was used with 5-chloro-1-methyl-1H-
pyrrolo[2,3-c]pyridine-2-carboxylic acid (70 mg, 0.33 mmol). The
evaporation of the solvent. H NMR (500 MHz, DMSO-d ): δ 1.35 (t,
6
J = 7.1 Hz, 3H), 4.08 (s, 1H), 4.13 (s, 3H), 4.36 (q, J = 7.1 Hz, 2H),
residue was purified by preparative HPLC using acidic conditions to
7
+
.27 (s, 1H), 7.88 (d, J = 1.0 Hz, 1H), 9.04 (s, 1H). MS (ESI) m/z [M
H] : 209.3.
1
+
yield 9a (25 mg, 19%). H NMR (600 MHz, DMSO-d
6
): δ 2.14 (s,
6
4
1
H), 2.85−2.98 (m, 2H), 3.30−3.40 (m, 2H), 3.66−3.99 (m, 5H),
Ethyl 1-Methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-c]pyridine-2-
.61−4.95 (m, 2H), 6.67−6.88 (m, 1H), 6.96−7.33 (m, 3H), 7.68 (s,
carboxylate (8c). Copper (89 mg, 1.40 mmol) was added to a
solution of diphenyl(trifluoromethyl)sulfonium trifluoromethanesulfo-
nate (377 mg, 0.93 mmol) and 8a (154 mg, 0.47 mmol) in DMF (4.7
1
H), 8.79 (s, 1H). H NMR indicates the existence of rotamers.
+
HRMS (ESI) m/z: [M + H] calcd for C H ClN O 383.1638, found
21 23
4
3
83.1643.
mL) under N in a microwave vial. The vial was sealed and stirred at
2
{
6-[(Dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}(1-
60 °C for 18 h. The reaction mixture was cooled down to rt, diluted
methyl-1H-pyrrolo[2,3-c]pyridin-2-yl)methanone (9b). General cou-
with EtOAc, and washed with H O (3×). The combined organic layers
2
pling method B was used to generate 9b (11 mg, 8% over three steps).
were washed with brine, dried over Na SO , filtered, and concentrated.
2
4
1
H NMR (600 MHz, DMSO-d ): δ 2.15 (s, 6H), 2.84−3.03 (m, 2H),
6
The residue was purified by automated flash chromatography using a
3
.30−3.40 (m, 2H), 3.68−4.01 (m, 5H), 4.67−4.93 (m, 2H), 6.68−
gradient of 0−35% of EtOAc in heptane as mobile phase to yield 8c
1
6.86 (m, 1H), 6.95−7.3 (m, 3H), 7.60 (d, J = 5.2 Hz, 1H), 8.21 (d, J =
5
(
102 mg, 80%). H NMR (500 MHz, DMSO-d ): δ 1.36 (t, J = 7.1 Hz,
6
+
.4 Hz, 1H), 8.96 (s, 1H). HRMS (ESI) m/z: [M + H] calcd for
3
9
H), 4.19 (s, 3H), 4.39 (q, J = 7.1 Hz, 2H), 7.43 (s, 1H), 8.23 (s, 1H),
.21 (s, 1H). 19F NMR (470 MHz, DMSO-d ): δ −64.44 (s).
C
21
H
24
N
4
O 349.2028, found 349.2007.
6
(
5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-
Ethyl 1-Methyl-5-phenyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate
[
(dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}-
(8d). Compound 8a (120 mg, 0.36 mmol), phenylboronic acid (58
methanone (9c). NaOH (355 mg, 8.88 mmol) was added to a
solution of 7c (710 mg, 2.51 mmol) in EtOH (absolute, 99.5%) (16
mg, 0.47 mmol), 1,1′-bis(di-tert-butylphosphino)ferrocene palladium
Pd-118) (23 mg, 0.04 mmol), and potassium carbonate (50 mg, 0.36
mmol) were mixed with DME (2.5 mL), H O (1.25 mL), and EtOH
(
mL) and H O (1.6 mL). The reaction mixture was stirred for 16 h at rt
2
2
before acetic acid (1.5 mL) was added to neutralize excess NaOH. The
(
absolute, 99.5%, 0.5 mL) in a microwave vial under N . The mixture
2
solvent was evaporated followed by addition of H O and then the pH
2
was flushed with N , sealed, and heated under microwave irradiation at
2
was adjusted to 2−3 by addition of HCl (aq, 1 M). The precipitated
110 °C for 20 min. The mixture was diluted with DCM (10 mL),
solid was filtered, washed with H O, and dried to provide 5-bromo-1-
2
filtered and the solvent evaporated. The residue was purified by
methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (600 mg, 94%).
preparative HPLC using acidic conditions to yield 8d (42 mg, 41%).
1
H NMR (400 MHz, DMSO-d ): δ 4.08 (s, 3H), 7.15 (d, J = 0.8 Hz,
1
6
H NMR (500 MHz, DMSO-d ): δ 1.36 (t, J = 7.1 Hz, 3H), 4.16 (s,
6
1
H), 7.87 (d, J = 0.8 Hz, 1H), 8.85 (s, 1H), 13.60 (s, 1H). MS (ESI)
3
H), 4.38 (q, J = 7.1 Hz, 2H), 7.30 (d, J = 0.8 Hz, 1H), 7.34−7.39 (m,
+
m/z [M + H] : 255.0.
1H), 7.47 (t, J = 7.7 Hz, 2H), 8.08 (d, J = 1.1 Hz, 1H), 8.10 (d, J = 1.3
General coupling method A was used with 5-bromo-1-methyl-1H-
pyrrolo[2,3-c]pyridine-2-carboxylic acid (388 mg, 1.52 mmol). The
Hz, 1H), 8.20 (d, J = 1.1 Hz, 1H), 9.14 (s, 1H). MS (ESI) m/z [M +
+
H] : 281.1.
residue was purified by preparative HPLC using basic conditions to
General Coupling Method A. 2-(1H-Benzo[d][1,2,3]triazol-1-
yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (TBTU, 1.1
equiv) was added to a solution of 5-substitued-1-methyl-1H-pyrrolo-
1
yield 9c (532 mg, 82%). H NMR (600 MHz, DMSO-d ): δ 2.14 (s,
6
6
4
7
H), 2.85−2.98 (m, 2H), 3.30−3.40 (m, 2H), 3.64−4.01 (m, 5H),
.57−4.93 (m, 2H), 6.68−6.88 (m, 1H), 6.94−7.36 (m, 3H), 7.76−
[
2,3-c]pyridine-2-carboxylic acid (1 equiv), 4 (1.01 equiv), and N-
ethyl-N-isopropylpropan-2-amine (4 equiv) in DMF (c = 0.2 M) at rt
under N . After being stirred for 18 h, the reaction mixture was poured
.98 (m, 1H), 8.70−8.90 (m, 1H). NMR indicates the existence of
+
rotamers. HRMS (ESI) m/z: [M + H] calcd for C H BrN O
2
1
24
4
2
427.1133, found 427.1165.
{6-[(Dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}-
(1,5-dimethyl-1H-pyrrolo[2,3-c]pyridin-2-yl)methanone (9d). LiOH
into NaHCO (sat. aq) solution and was extracted with DCM (3×).
3
The combined organic layers were washed with brine, dried through a
phase separator, and concentrated. The residue was purified by
preparative HPLC using basic conditions.
(38 mg, 1.6 mmol) in H O (0.5 mL) was added dropwise to a solution
2
of 7d (87 mg, 0.4 mmol, crude) in EtOH (5 mL) at rt under N . The
2
General Coupling Method B. Trimethylaluminum (2 M) in
toluene (4 equiv) was added to a solution of ethyl 1-methyl-5-
substitued-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (1 equiv), N,N-
dimethyl-1-(1,2,3,4-tetrahydroisoquinolin-6-yl)methanamine dihydro-
chloride (1.2 equiv), and TEA (6 equiv) in toluene (c = 0.06 M) under
reaction was heated at 40 °C for 4 h. The solvent was removed and the
residue 1,5-dimethyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid was
+
used as such in the next step. MS (ESI) m/z [M + H] : 191.1.
General coupling method A was used with 1,5-dimethyl-1H-
pyrrolo[2,3-c]pyridine-2-carboxylic acid (76 mg, 0.4 mmol, crude).
N at rt. After being refluxed for 16 h, the reaction mixture was poured
The residue was purified by preparative HPLC using basic conditions
2
1
into H O and extracted with DCM (3×). The combined organic layers
to provide 9d (23 mg, 16%). H NMR (600 MHz, DMSO-d ): δ 2.15
2
6
were washed with brine, dried, filtered, and concentrated. The residue
was purified by preparative HPLC using basic conditions. Note: NMR
indicated the existence of the rotamers for all the final products, but
shifts were not assigned to specific rotamers.
(s, 6H), 2.82−3.00 (m, 2H), 3.30−3.40 (m, 2H), 3.69−3.98 (m, 5H),
4.66−4.92 (m, 2H), 6.58−6.77 (m, 1H), 6.93−7.32 (m, 3H), 7.41 (s,
+
1H), 8.81 (s, 1H). HRMS (ESI) m/z: [M + H] calcd for C H N O
2
2
27
4
363.2185, found 363.2196.
5
942
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Journal of Medicinal Chemistry
Article
{
6-[(Dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}(5-
g, 76.46 mmol). The resulting solution was stirred overnight at 100 °C
iodo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl)methanone (9e). A
mixture of 9c (430 mg, 1.01 mmol), copper(I) iodide (38 mg, 0.20
mmol), N1,N2-dimethylethane-1,2-diamine (0.022 mL, 0.20 mmol),
and sodium iodide (302 mg, 2.01 mmol) in 1,4-dioxane (4.5 mL) in a
under N . After cooling to rt, the solids were filtered out. The resulting
2
mixture was concentrated under vacuum. The residue was applied
onto a silica gel column eluting with chloroform:MeOH (100:1). This
1
resulted in 11 (935 mg, 52%) as a colorless oil. H NMR (400 MHz,
microwave vial was flushed with N and sealed. The reaction mixture
was heated at 110 °C in an oil bath for 53 h. The reaction mixture was
diluted with DCM (20 mL), filtered, and concentrated. The residue
CDCl ): δ 1.65 (s, 3H), 2.24 (s, 6H), 3.27−3.64 (m, 2H), 3.89 (q, J =
2
3
+
7.3 Hz, 1H), 7.15−7.46 (m, 4H). MS (ESI) m/z [M + H] : 189.
HPLC: t = 0.61 min.
R
was purified by preparative HPLC using basic conditions to afford 9e
2-[3-[(Dimethylamino)methyl]phenyl]propan-1-amine (12). To a
solution of 11 (800 mg, 4.25 mmol) in MeOH (60 mL), was added
Raney-Ni (1.0 g). The resulting reaction mixture was hydrogenated
overnight at 25 °C under 20 psi of hydrogen pressure. After the
completion of the reaction, the solids were filtered out. The resulting
mixture was concentrated under vacuum. This resulted in 12 (770 mg,
1
(
380 mg, 80%). H NMR (600 MHz, DMSO-d ): δ 2.14 (s, 6H),
6
2
2
.86−2.95 (m, 2H), 3.34−3.31 (m, 2H), 3.70−3.92 (m, 5H), 4.76 (m,
H), 6.74 (s, 1H), 6.96−7.27 (m, 3H), 8.04 (s, 1H), 8.79 (s, 1H).
+
HRMS (ESI) m/z: [M + H] calcd for C H IN O 475.0995, found
21
24
4
475.0971.
{
6-[(Dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}(5-
94%) as a yellow oil, which was used directly in the next step without
1
ethynyl-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl)methanone (9f).
NaOH (37 mg, 0.93 mmol) was added to a solution of ethyl 5-
ethynyl-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (60 mg,
further purification. H NMR (400 MHz, CDCl ): δ 1.26 (d, J = 6.9
3
Hz, 3H), 2.25 (s, 6H), 2.75 (h, J = 6.8 Hz, 1H), 3.42 (d, J = 2.8 Hz,
2H), 7.07−7.19 (m, 3H), 7.20−7.30 (m, 2H). MS (ESI) m/z [M +
+
0
.26 mmol) in EtOH (absolute, 99.5%) (1.6 mL) and H O (0.160
H] : 193. HPLC: t = 0.86 min.
2
R
mL). The mixture was stirred for 7 h at rt, followed by addition of
acetic acid (0.2 mL) to neutralize an excess of NaOH. The solvent was
N-(2-[3-[(Dimethylamino)methyl]phenyl]propyl)formamide (13).
Into a 100 mL round-bottom flask was placed a solution of 12 (400
mg, 2.08 mmol) in methyl formate (20 mL). The resulting solution
was stirred for 4.5 h at rt. The resulting mixture was concentrated
evaporated followed by addition of H O and then the pH was adjusted
2
to 2−3 by addition of HCl (aq, 1 M). The precipitated solid was
filtered, washed with H O, and dried to provide 5-ethynyl-1-methyl-
under vacuum. The residue was purified by preparative TLC to yield
2
1
1
1
H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (34 mg, 65%). H NMR
13 (396 mg, 86%) as a yellow oil. H NMR (500 MHz, CDCl ): δ
3
(
500 MHz, DMSO-d ): δ 4.06 (s, 1H), 4.13 (s, 3H), 7.21 (s, 1H), 7.86
1.30 (d, J = 7.0 Hz, 3H), 2.25 (s, 6H), 2.97 (m, 1H), 3.29 (ddd, J =
4.9, 9.0, 13.7 Hz, 1H), 3.42 (d, J = 2.9 Hz, 2H), 3.72 (dt, J = 6.8, 13.5
6
+
(
s, 1H), 9.00 (s, 1H). MS (ESI) m/z [M + H] : 201.1.
General coupling method A was used with 5-ethynyl-1-methyl-1H-
pyrrolo[2,3-c]pyridine-2-carboxylic acid (33 mg, 0.16 mmol). The
Hz, 1H), 7.1−7.2 (m, 3H), 7.27−7.33 (m, 1H), 8.10 (s, 1H). MS
+
(ESI) m/z [M + H] : 221. HPLC: t = 1.01 min.
R
4
6
residue was purified by preparative HPLC using basic conditions to
N-[2-(3-Bromophenyl)ethyl]acetamide (15).
To a stirred
1
provide 9f (27 mg, 44%). H NMR (600 MHz, DMSO-d ): δ 2.15 (s,
solution of 14 (2.0 g, 10 mmol) in DCM (10 mL) was added
pyridine (10 mL) followed by slow addition of acetic chloride (799
mg, 10.2 mmol). The resulting solution was stirred for 2 h at 25 °C.
The solids were filtered out. The resulting mixture was concentrated
under vacuum. This resulted in 15 (2.0 g, 83%) as brown oil, which
6
6
4
3
H), 2.86−2.96 (m, 2H), 3.35−3.38 (m, 2H), 3.69−3.97 (m, 5H),
.06 (s, 1H), 4.86−4.70 (m, 2H), 6.75−6.85 (m, 1H), 6.98−7.27 (m,
+
H), 7.83 (s, 1H), 8.94 (s, 1H). HRMS (ESI) m/z: [M + H] calcd for
C H N O 373.2028, found 373.2040.
2
3
25
4
{
6-[(Dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}[1-
was used directly in the next step without further purification. MS
+
methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-c]pyridin-2-yl]-
methanone (9g). NaOH (53 mg, 1.33 mmol) was added to a solution
of 8c (102 mg, 0.37 mmol) in EtOH (absolute, 99.5%) (2.4 mL) and
(ESI) m/z [M + H] : 242. HPLC: t = 1.36 min.
R
46
6
-Bromo-1-methyl-3,4-dihydroisoquinoline (16). Oxalyl chlor-
ide (30.0 g, 238 mmol) was added to a stirred solution of 15 (5.00 g,
20.7 mmol) in DCM (50 mL) at 0 °C. The reaction mixture was then
warmed to rt and stirred for 2 h. The mixture was cooled to −78 °C.
H O (0.4 mL). The mixture was stirred for 2.5 h at rt before addition
2
of acetic acid (0.2 mL) to neutralize excess NaOH. The solvent was
evaporated followed by addition of H O and then the pH was adjusted
to 3 by addition of HCl (aq, 1 M). The precipitate was filtered, washed
with H O, and dried to provide 1-methyl-5-(trifluoromethyl)-1H-
pyrrolo[2,3-c]pyridine-2-carboxylic acid (71 mg, 78%). H NMR (500
MHz, DMSO-d ): δ 4.19 (s, 3H), 7.37 (s, 1H), 8.20 (s, 1H), 9.18 (s,
H). MS (ESI) m/z [M + H] : 245.1.
General coupling method A was used with 1-methyl-5-(trifluor-
2
FeCl (20.0 g, 123 mmol) was added in. The resulting solution was
3
warmed to 25 °C and stirred overnight. The solids were filtered out.
The resulting mixture was concentrated under vacuum. The residue
was dissolved in H SO :MeOH (1:10, 20 mL). The resulting solution
2
1
2
4
6
was heated to 80 °C and stirred overnight. The resulting mixture was
+
1
concentrated under vacuum and diluted with H O (100 mL). The pH
2
was adjusted to 7 with ammonia (30%). The solids were filtered out.
The resulting solution was extracted with DCM (3 × 200 mL). The
organic layers were combined and concentrated under vacuum. This
omethyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (35 mg, 0.14
mmol). The residue was purified by preparative HPLC using basic
conditions to provide 9g (46 mg, 76%). H NMR (600 MHz, DMSO-
1
1
resulted in 16 (4.0 g, 86%) as yellow oil. H NMR (400 MHz, DMSO-
d ): δ 2.15 (s, 6H), 2.86−2.98 (m, 2H), 3.31−3.34 (m, 2H), 3.74 (s,
d , 26 °C): δ 2.28 (s, 3H), 2.65 (t, J = 7.6 Hz, 2H), 3.49−3.56 (m,
6
6
+
1
8
H), 3.88−3.98 (m, 4H), 4.68−4.79 (m, 2H), 6.92−7.28 (m, 4H),
2H), 7.54−7.47 (m, 3H); MS (ESI) m/z [M + H] : 224. HPLC: t =
1.29 min.
R
+
.16 (d, J = 9.6 Hz, 1H), 9.11 (s, 1H). HRMS (ESI) m/z: [M + H]
calcd for C H F N O 417.1902, found 417.1924.
{
methyl-5-phenyl-1H-pyrrolo[2,3-c]pyridin-2-yl)methanone (9h).
General coupling method B was used with 8d (42 mg, 0.15 mmol).
The residue was purified by preparative HPLC using basic conditions
to provide 9h (14 mg, 22%). H NMR (600 MHz, DMSO-d ): δ
Dimethyl[(4-methyl-3,4-dihydroisoquinolin-6-yl)methyl]amine
(17a). Oxalyl chloride (6.21 g, 49 mmol) was added dropwise to a
stirred solution of 13 (1.09 g, 4.95 mmol) in DCM (300 mL) at 0 °C.
The reaction mixture was then warmed to rt and stirred for 2 h. Then
22
24
3
4
6-[(Dimethylamino)methyl]-3,4-dihydroisoquinolin-2(1H)-yl}(1-
the mixture was cooled to −78 °C. FeCl (4.0 g, 25 mmol) was added.
3
1
6
The resulting solution was warmed to 25 °C and stirred overnight.
The solids were filtered out. The resulting mixture was concentrated
under vacuum. The residue was dissolved in H SO :MeOH (1:10, 20
2
4
1
8
.31−2.49 (m, 6H), 2.91−2.98 (m, 2H), 3.64−3.99 (m, 7H), 4.77−
.91 (m, 2H), 6.78−6.86 (m, 1H), 7.05−7.34 (m, 3H), 7.35−7.4 (m,
H), 7.45−7.51 (m, 2H), 8.12 (dd, J = 1.2, 8.4 Hz, 2H), 8.15 (d, J =
2
4
mL). The resulting solution was heated to 80 °C and stirred overnight.
The resulting mixture was concentrated under vacuum and diluted
+
.0 Hz, 1H), 9.04 (s, 1H). HRMS (ESI) m/z: [M + H] calcd for
C H N O 425.2341, found 425.2378.
with H O (10 mL). The pH value of the solution was adjusted to 7
27
29
4
2
2
-[3-[(Dimethylamino)methyl]phenyl]propanenitrile (11). To a
with ammonia (30%). The solids were filtered out. The resulting
mixture was extracted with DCM (3 × 200 mL). The organic layers
were combined and concentrated under vacuum. The residue was
solution of 10 (2.0 g, 9.5 mmol) in 1,4-dioxane (100 mL) was added a
solution of potassium [(dimethylamino)methyl]trifluoroborate (2.05
g, 12.4 mmol) in H O (20 mL), followed by addition of Pd(OAc)2
purified by preparative TLC, this resulted in 17a (550 mg, 55%) as a
2
+
(
430 mg, 1.92 mmol), S-Phos (1.57 g, 3.83 mmol), and K PO (16.23
yellow oil. MS (ESI): m/z [M + H] : 203. HPLC: t = 0.28 min.
3
4
R
5
943
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Journal of Medicinal Chemistry
Article
Dimethyl[(1-methyl-3,4-dihydroisoquinolin-6-yl)methyl]amine
The organic layers were combined and concentrated under vacuum.
(
17b). To a solution of 16 (100 mg, 0.45 mmol) in 1,4-dioxane (15
The residue was purified by preparative TLC with DCM:MeOH
1
mL) was added potassium [(dimethylamino)methyl]-
trifluoroboranuide (96 mg, 0.58 mmol), Pd(OAc)2 (20 mg, 0.09
mmol), S-Phos (73 mg, 0.18 mmol), and a solution of K PO (757
(10:1). This resulted in 25a (38 mg, 24%) as a white solid. H NMR
(DMSO-d , 300 MHz): δ 1.29−1.13 (m, 3H), 2.13 (s, 6H), 3.15−2.98
6
3
4
(m, 1H), 3.55−3.40 (m, 1H), 3.97−3.72 (m, 5H), 4.97−4.72 (m, 2H),
6.73 (s, 1H), 7.30−6.95 (m, 3H), 7.83 (s, 1H), 8.77 (s, 1H). HRMS
mg, 3.57 mmol) in H O (3 mL). The resulting solution was stirred for
2
+
2
h at 100 °C under N . After cooling to rt, the solids were filtered out.
(ESI) (m/z): [M + H] calcd for C H BrN O 441.1290, found
2
22 26
4
The resulting solution was extracted with DCM (5 × 5 mL). The
organic layers were combined and concentrated under vacuum. The
crude product was purified by preparative TLC with DCM:MeOH
441.1316.
25e, (+)-isomer: [α]D +23 (c 1.0, ACN); column, Chiralcel OJ, 250
20
× 4.6; flow rate, 4 mL/min; wavelength, PDA 240 nm; t = 2.70 min.
R
D
0
(
10:1) to yield 17b (66 mg, 73%) as a brown oil. MS (ESI) m/z [M +
25f, (−)-isomer: [α]2 −24 (c 1.0, ACN); column: Chiralcel OJ,
+
H] : 203. HPLC: t = 0.18 min.
250 × 4.6; flow rate, 4 mL/min; wavelength, PDA 240 nm; t = 3.85
R
R
tert-Butyl 6-(Methoxy(methyl)carbamoyl)-3,4-dihydroisoquino-
line-2(1H)-carboxylate (22). Compound 21 (2.00 g, 7.21 mmol)
was dissolved in DCM (40 mL), 1-chloro-N,N,2-trimethylprop-1-en-1-
amine (1.15 mL, 8.65 mmol) was added in one portion, and the
mixture stirred at rt for 2 h. N,O-Dimethylhydroxylamine hydro-
chloride (1.41 g, 14.4 mmol) and TEA (4.0 mL, 29 mmol) were
added, and the reaction mixture was stirred at rt for 14 h. Then the
mixture was evaporated to dryness, redissolved in EtOAc, and washed
sequentially with sodium bicarbonate (sat., 50 mL), brine (50 mL),
and H O (50 mL). The organic phase was dried using Na SO ,
min.
(
5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-[(dimethyla-
mino)-methyl]-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl}-
methanone (25b). NaBH (18 mg, 0.50 mmol) was added in portions
4
to a stirred solution of 17b (90 mg, 0.44 mmol) in MeOH (15 mL) at
0 °C. The solution was stirred for 1 h at 0 °C. The reaction mixture
was diluted with H O (5 mL) and concentrated under vacuum. The
remaining solution was extracted with DCM (4 × 5 mL). The organic
layers were combined and concentrated under vacuum. This resulted
in 20b (60 mg, 59%) as a yellow oil, which was used in the next step
without further purification. MS (ESI) m/z [M + H] : 205. HPLC: t
= 1.61 min.
2
2
2
4
1
+
filtrated, and evaporated to dryness to give 22 (2.1 g, 91%). H NMR
R
(
400 MHz, CDCl ): δ 1.50 (s, 9H), 2.87 (t, J = 5.6 Hz, 2H), 3.36 (s,
3
3
7
H), 3.57 (s, 3H), 3.66 (t, J = 5.3 Hz, 2H), 4.61 (s, 2H), 7.14 (d, J =
.9 Hz, 1H), 7.44−7.56 (m, 2H).
To a solution of 5-bromo-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-
carboxylic acid (180 mg, 0.71 mmol) in DMF (20 mL) were added
EDC (226 mg, 1.18 mmol), HOBt (159 mg, 1.18 mmol), and TEA
(298 mg, 2.95 mmol). The mixture was stirred for 30 min, and then
20b (121 mg, 0.59 mmol) was added. The resulting solution was
stirred overnight at 25 °C, concentrated under vacuum, and finally
tert-Butyl 6-Acetyl-3,4-dihydroisoquinoline-2(1H)-carboxylate
(23). Compound 22 (466 mg, 1.45 mmol) was dissolved in THF
(
10 mL) and then CH MgCl in THF (0.58 mL, 1.75 mmol) was
3
added dropwise over 15 min at rt followed by stirring under nitrogen
for 1 h. The reaction was quenched by addition of H O followed by
diluted with DCM (50 mL). The organic phase was washed with H O
2
2
extraction with MTBE (3 × 50 mL). The organic product was dried
(3 × 10 mL). The organic layer was concentrated under vacuum. The
crude product was purified by preparative TLC with DCM:MeOH
(10:1) to yield [[2-([5-bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-
yl]carbonyl)-1-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl]methyl]-
using Na SO and evaporated to dryness. A clear oil of 23 (400 mg,
2
4
1
quantitative yield) remained. H NMR (400 MHz, CDCl ): δ 1.50 (s,
3
9
4
H), 2.59 (s, 3H), 2.90 (t, J = 5.7 Hz, 2H), 3.67 (t, J = 5.6 Hz, 2H),
.63 (s, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.69−7.82 (m, 2H).
1
dimethylamine (160 mg, 51%) as a white solid. H NMR (300 MHz,
1
-(2-(5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carbonyl)-
CD OD, 26 °C): δ 1.61−1.63 (m, 3H), 2.24 (s, 6H), 2.78−3.10 (m,
3
1
,2,3,4-tetrahydroisoquinolin-6-yl)ethanone (24). The amine 23 (50
2H), 3.45−3.88 (m, 7H), 4.65−4.80 (m, 0.2H), 5.00−5.10 (m, 0.2H),
5.69−5.80 (m, 0.5H), 6.73 (s, 1H), 6.90−7.28 (m, 3H), 7.82 (s, 1H),
mg, 0.29 mmol) was deprotected by dissolving the amine salt in 10%
TFA in DCM for 6 h at rt and then evaporated to dryness. The
resulting amine and 5-bromo-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-
carboxylic acid (80 mg, 0.31 mmol) were suspended in EtOAc (1070
μL) and TEA (119 μL, 0.86 mmol). The solution was cooled to 0 °C
and T3P (182 mg, 0.29 mmol) was added dropwise to the cooled
suspension. The reaction was then heated to 60 °C overnight. Then
+
8.64−8.68 (m, 1H); HRMS (ESI) (m/z): [M + H] calcd for
C H BrN O 441.1290, found 441.1307.
22
26
4
(5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-
[(dimethylamino)methyl]-3-methyl-3,4-dihydroisoquinolin-2(1H)-
yl}methanone (25c). Into a 250 mL three-necked round-bottom flask,
purged and maintained with an inert atmosphere of nitrogen, was
H O was added, followed by extraction with EtOAc. The organic
placed a solution of 18 (2.00 g, 11.3 mmol) in 1,4-dioxane:H
60 mL). Potassium ((dimethylamino)methyl)trifluoroborate (2.78 g,
16.8 mmol), Pd(OAc) (504 mg, 2.24 mmol), S-Phos (1.84 g, 4.49
mmol), and Cs CO (36.52 g, 112.1 mmol) were added in. The
2
O (4:1,
2
phase was washed with brine, H O, sat. bicarbonate, and HCl (0.1 M)
2
then dried using MgSO . The organic layer was evaporated to dryness
2
4
1
to yield 24 (280 mg, 85%), which was used as is in the next step. H
2
3
NMR (500 MHz, DMSO-d ): δ (mixture of rotamers) 2.57 (s, 3H),
resulting solution was stirred under inert atmosphere for 3 h at 100 °C.
After cooling to rt, the solids were filtered out. The resulting solution
was extracted with EtOAc (3 × 50 mL), and the organic layers were
combined and concentrated under vacuum. The residue was purified
on a silica gel column with EtOAc:hexane (1:2). This resulted in 19
6
2
=
.97 (s, 2H), 3.57−4.04 (m, 5H), 4.79 (s, 1H), 4.92 (s, 1H), 6.76 (d, J
19.1 Hz, 1H), 7.45 (d, J = 6.6 Hz, 1H), 7.82 (d, J = 9.3 Hz, 3H), 8.78
+
(
s, 1H). MS (ESI) m/z [M + H] : 412.0.
5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-
(
+
[
(dimethylamino)methyl]-4-methyl-3,4-dihydroisoquinolin-2(1H)-
(650 mg, 29%) as a yellow solid. MS (ESI) m/z [M + H] : 201.
yl}methanone (25a). To a solution of 17a (200 mg, 0.99 mmol) in
HPLC: t = 0.31 min.
R
MeOH (20 mL) was added NaBH (42 mg, 1.10 mmol, 1.10 equiv) at
To a stirred solution of 19 (600 mg, 3.00 mmol) in MeOH (8 mL)
4
0
°C. The resulting solution was stirred for 1 h at 0 °C. The resulting
was added aq concd HCl (320 mg) and PtO (90 mg, 0.40 mmol)
2
mixture was concentrated under vacuum. The resulting solution was
under N atmosphere. The resulting solution was hydrogenated for 5 h
2
diluted with H O (10 mL) and extracted with DCM (3 × 5 mL). The
organic layers were combined and concentrated under vacuum to yield
at rt under 20 psi hydrogen pressure. The solids were filtered out. The
resulting mixture was concentrated under vacuum. This resulted in 20c
2
2
0a (190 mg, 94%) as a colorless oil, which was used directly in the
(500 mg, 82%) as a yellow solid, which was used in the next step
+
next step without further purification.
without further purification. MS (ESI) m/z [M + H] : 205. HPLC: t =
R
To a stirred solution of 5-bromo-1-methyl-1H-pyrrolo[2,3-c]-
pyridine-2-carboxylic acid (99 mg, 0.39 mmol) in DMF (10 mL)
was added EDC (169 mg, 0.88 mmol), HOBt (72 mg, 0.53 mmol),
and TEA (178 mg, 1.76 mmol). After being stirred for 30 min, 20a (72
mg, 0.35 mmol) was added. The resulting reaction mixture was stirred
for 3 h at 25 °C and then concentrated under vacuum. The solution
0.21 min.
To a stirred solution of 20c (73 mg, 0.36 mmol) in DMF (15 mL)
were added 5-bromo-1-methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylic
acid (100 mg, 0.39 mmol), HATU (150 mg, 0.39 mmol), TEA (43.8
mg, 0.43 mmol). The resulting solution was stirred for 3 h at rt. The
reaction mixture was diluted with EtOAc (15 mL) and washed with
was diluted with DCM (10 mL) and washed with H O (3 × 3 mL).
NaHCO (sat. 2 × 15 mL) and brine (2 × 15 mL). The resulting
2
3
5
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Journal of Medicinal Chemistry
Article
mixture was concentrated under vacuum. The crude product was
acclimatization purposes. Food intake was monitored using an in-
house-built automatic monitoring system (AstraZeneca, R&D
Molndal, Sweden) allowing continuous measurement of food intake
̈
purified by preparative HPLC to give 25c (90 mg, 57%) as a white
1
solid. H NMR (DMSO-d , 300 MHz): δ 1.05−1.15 (m, 3H), 2.15 (s,
6
6
3
7
H), 2.61−2.75 (m, 1H), 3.13−3.16 (m, 1H), 3.32−3.45 (m, 2H),
in undisturbed animals housed in their home cage environment. The
mice had free access to two food hoppers and water. A feeding bout
was defined as continuously feeding for more than five seconds, a
minimum food intake (bout weight) of 10 mg, and a relapse time of 10
s between the bouts. The food intake was binned into hours and
cumulative food intake was calculated. All data were collected using
Microsoft Excel, and analysis was performed using Excel and Spotfire
(TIBCO, Spotfire3.1.1, TIBCO Software Inc.) software. After 4 days
of basal food intake registration, the animals (body mass 50−65 g)
were dosed by oral gavage (5 mL/kg) with compound (N = 7−8) or
5% mannitol vehicle (N = 8), twice daily for 3 days (first dose at the
beginning of the dark phase and the second dose 7 h later). Food
intake registration was performed during the whole period of the
experiment. Compound-treated and vehicle groups were compared
using ANOVA followed by Dunnett’s multiple comparisons test.
Peptide Conformational Analysis: NMR Data Collection.
QRFP26(20−26) was purchased from Peptide Protein Research Ltd.
(Fareham, UK). The peptide was dissolved to 100 mM in DMSO-d6:
aliquots of this stock solution were used to prepare samples for data
acquisition. Samples were prepared at a range of concentrations
(0.05−10 mM) in solutions of D O or H O, containing up to 10%
.81 (s, 3H), 4.36−4.57 (m, 2H), 4.90−5.28 (m, 1H), 6.74 (s, 1H),
.04−7.24 (m, 3H), 7.82 (s, 1H), 8.77 (s, 1H); HRMS (ESI) (m/z):
+
[
M + H] calcd for C H BrN O 441.1290, found 441.1302.
22
26
4
(
5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl){6-[1-
(
(
dimethylamino)ethyl]-3,4-dihydroisoquinolin-2(1H)-yl}methanone
25d). Compound 24 (40 mg, 0.10 mmol) was dissolved in a 33%
MeNH in ethanol solution (6 mL), and then 4 Å molecular sieves
2
(
4
500 mg) were added. The reaction mixture was heated to 50 °C for
0 min. Then sodium borohydride (3.67 mg, 0.10 mmol) was added
followed by stirring for 4 h. The mixture was then quenched with H O
2
and thereafter extracted with EtOAc (3 × 20 mL) followed by washing
with brine and H O. The organic layer was dried over MgSO and
2
4
then evaporated to dryness. Then the second alkylation was
performed. (5-Bromo-1-methyl-1H-pyrrolo[2,3-c]pyridin-2-yl)(6-(1-
(
methylamino)ethyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (6
mg, 0.01 mmol) was dissolved in DCM (3 mL) and then
formaldehyde (1.0 mL, 12 mmol) was added to this mixture. Sodium
triacetoxyhydroborate (12 mg, 0.06 mmol) was added in one portion,
followed by stirring at rt for 20 min. The reaction mixture was
extracted using DCM (50 mL), washed with brine and H O, and dried
2
2
2
1
over MgSO to yield 25d (6 mg, 21%). H NMR (500 MHz, DMSO-
residual DMSO-d6 at pH 4, with 1 mM DSS-d6 as an internal
reference. 1D- and 2D-NMR spectra for the measurement of chemical
shifts, temperature coefficients, and coupling constants were acquired
at 500, 600, or 800 MHz at a range of temperatures (10−35 °C) with
4
d6): δ (mixture of rotamers) 1.24 (d, J = 6.2 Hz, 3H), 2.08 (s, 6H),
2
4
7
.90 (d, J = 21.7 Hz, 2H), 3.21 (d, J = 5.7 Hz, 1H), 3.29 (s, 1H), 3.61−
.03 (m, 5H), 4.69 (s, 1H), 4.82 (s, 1H), 6.75 (d, J = 16.5 Hz, 1H),
1
3
15
.14 (d, J = 24.5 Hz, 3H), 8.77 (s, 1H). HRMS (ESI) (m/z): [M +
C- and N-edited experiments being performed at natural
+
3
H] calcd for C H BrN O 441.1290, found 441.1308.
22
25
4
abundance. NOE restraints and coupling constants ( JHH) used for
Functional Assay (Inositol-1-phosphate). Cryopreserved,
human QRFP (peptide P518/GPR103) receptor cell line (Perki-
nElmer) or CHO cells overexpressing either mouse GPR103A or
structure determination were recorded using a 10 mM 90% H O:10%
2
DMSO-d sample at 800 MHz and 10 °C. The NOESY mixing time
6
was 700 ms. Residual dipolar couplings were obtained using strained
8
47
GPR103B receptor (in house) were thawed in stimulation buffer and
gels prepared using 7% (w/v) polyacrylamide as described previously
preincubated with compound at 37 °C in a humidified incubator with
and soaked in a solution of 5 mM QRFP26(20−26) in 85% H O:10%
2
5
% CO . The cells were treated with hQRFP43 (Phoenix
D O:5% DMSO-d pH 4, before insertion into a suitably prepared 5
2
2
6
1
1
Pharmaceuticals) at EC80 in stimulation buffer and incubated at 37
C. After 50 min, reagents from the IP-1 Tb HTRF kit (CisBio
mm NMR tube. Coupling constants ( J , JNH) were measured from
[ H, C]-HSQC and [ H, N]-HSQC spectra recorded without
CH
1
13
1
15
°
Bioassays) were added and the resulting signal ratio (665 nm/620 nm)
was measured in Pherastar (BMG). Reported values for hGPR103 IP-1
IC50 are the mean of N = 2−52 determinations. Within each
determination of hGPR103 IP-1 IC , between one and three
broadband heteronuclear decoupling during acquisition.
Chemical Shifts and Coupling Constants. All H, 15N, and 13
1
C
chemical shifts and pertinent coupling constants of QRFP26(20−26) in
aqueous solution at pH 4 were assigned and measured using standard
1D- and 2D-NMR experiments (See Supporting Information,
Supplementary Tables S1−S3).
Additional Peptide Characterization. Chemical shifts were
measured at several solute concentrations (0.05−10 mM
50
replicates at each concentration were used. The confidence interval
ratio (CIR) was calculated to 2.0. Reported values for mGPR103A and
B IP-1 IC50 are a single determination or the mean of N = 5−7
determinations. Within each determination of IC , between two or
50
three replicates at each concentration were used.
QRFP26(
) and at different temperatures (10−35 °C). Since the
20−26)
1
Radioligand Binding Assay (RLB). Membranes from HEK cells
overexpressing the human GPR103 receptor were incubated in 96-well
microplates with compound and 0.25 nM radioligand 1 I-hQRFP43
differences in H chemical shifts between these conditions are small
(<0.1 ppm) and all temperature-induced perturbations occur linearly
(Supporting Information, Supplementary Table S3), it was concluded
that QRFP26(20−26) did not extensively self-associate in aqueous
solution and that its solution structure was insensitive to changes in
temperature in the range used in the present study. The N-terminal
25
(
NEX408, PerkinElmer) in binding buffer (50 mM Tris, pH 7.4, 1 mM
EDTA, and 0.1% BSA) at 30 °C. After 90 min incubation, the reaction
was filtered through filter plates (Whatman GF/C, 7700−4301) under
vacuum; the plates were dried, treated with scintillation liquid, and
counted in Trilux Microbeta (Wallac). Reported values are the mean
of N = 2−5 determinations with one replicate at each concentration.
The confidence interval ratio (CIR) was calculated to 1.7.
amine (Gly20 N) pK value was measured to be 8.0 ± 0.1, indicating
a
that the dominant state of this moiety under physiological conditions
was protonated; therefore, the peptide (including the arginine
guanidinium group) carried a +2 charge under physiological
conditions. Moreover, conformation-dependent coupling constants
compared at pH 4 and 10 indicated that the molecule adopted the
same shape in both charge states of the amine. Samples for
measurement of structural restraints were therefore prepared using a
pH value of 4, which maintained the molecule in its physiological
charge state while minimizing the amide hydrogen exchange rate,
thereby making them more amenable to NOE cross-peak observation
(Supporting Information, Supplementary Table S2 and Supplementary
Figures S1 and S2).
Animals and Housing. Twenty-three female C57Bl/6 mice were
used in the studies. The mice were housed in groups of at most six in
Macrolon 2L cages (Scanbur, Karslunde, Denmark) containing aspen
wood chip bedding and an enriched environment with free access to
food and water. The animal room had a regulated temperature (18−22
°
C), humidity (50%), and a 12:12 h light−dark cycle. The mice were
allowed at least 1 week of acclimatization before study initiation. To
induce obesity mice were assigned to a high caloric diet (D12492 diet,
6
2
0% kcal as fat; Research Diets) at the age of 10 weeks for a period of
1 weeks. The study was approved by the local Animal Research Ethics
Measurement of Structural Restraints. Distance-restraint
information was extracted by measuring the absolute values of NOE
cross-peak heights. All possible NOE cross-peaks were measured,
including overlapping peaks and those that were found to be
Board Committee (Gothenburg, Sweden).
Food Intake Measurement. Obese female mice were housed
individually for 7 days prior to the start of the experiment for
5
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dx.doi.org/10.1021/jm401951t | J. Med. Chem. 2014, 57, 5935−5948

Journal of Medicinal Chemistry
Article
unambiguously zero. Peaks that could not be measured due to NMR
artifacts were excluded. Scalar couplings were measured from
resonance multiplet patterns in a high-resolution (800 MHz) 1D H
ASSOCIATED CONTENT
■
* Supporting Information
S
1
Tables of the NMR data and the complete dynamic structural
NMR experiment. Residual dipolar couplings were calculated from the
parameters of QRFP26(20−26), additional figures relating to
structure determination, and a detailed description of pKa
calculation, as well as assay descriptions for DMPK, hERG,
Nav1.5, and IKs. This material is available free of charge via the
Internet at http://pubs.acs.org.
difference in observed H−C or H−N splitting between sets of
1
13
1
15
[
H, C]-HSQC and [ H, N]-HSQC spectra recorded in the free
solution and aligned phase. In total, 809 structural restraints were used
to define the dynamic 3D-structure of QRFP26(20−26) (Supporting
Information, Supplementary Tables S4−S7), comprising nuclear
Overhauser enhancements (NOEs), scalar couplings, and residual
dipolar couplings.
AUTHOR INFORMATION
■
Structure Calculations. The structure of QRFP26(20−26) was
determined on the basis of the data described above, using a previously
Corresponding Author
40
*E-mail: Anneli.Nordqvist@astrazeneca.com. Tel: +46 31 776
described method. Briefly, an initial 3D-structure for QRFP26(20−26)
was built with fixed bond lengths and angles (based on geometrical
constraints rather than by minimizing a computational chemistry
energy function) which when compared with the Cambridge
2
772.
Notes
The authors declare no competing financial interest.
48
49
Structural Database (CSD) using Mogul had Z-scores less than
. A structural ensemble for QRFP26(20−26) was constructed by
keeping all bonds and angles fixed and rotating the 24 rotatable bonds
see Supporting Information, Supplementary Figure S3). The rotation
3
ACKNOWLEDGMENTS
■
The authors would like to thank the Separation Science Group
(
at AstraZeneca Mo
and Global DMPK for support with DMPK and physicochem-
̈
lndal for help with compound purification
of torsions were matched to a set of mean angles, librations, and modal
probabilities for each bond (termed the dynamic model). The
ensemble thereby produced was used to predict the experimental
ical profiling.
2
data, and the fitness function χ was calculated from comparison of
these predictions with the measured experimental data. The dynamic
model was iteratively refined to improve agreement between the
predicted and measured NMR data until no further improvement was
achieved (see Supporting Information, Supplementary Tables S4−S7
ABBREVIATIONS USED
■
BID, twice daily; confidence interval ratio, CIR; CHO, Chinese
hamster ovarian; DIEA, ethyldiisopropylamine; DMPK, drug
metabolism and pharmacokinetics; EC , 80% of maximal
2
80
for final χ -scores for each restraint). Pro-chiral stereoassignments
effective concentration; EDC, 3-(ethyliminomethyleneamino)-
N,N-dimethylpropan-1-amine; EtOAc, ethyl acetate; EtOH,
ethanol; FIA, automated food intake measurement; GPR103,
pyroglutamylated RF amide peptide receptor 103; h, human;
HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; hIKs, human cardiac slowly activating
were determined during structure calculations. Quality of fit measures
were comparable with previous implementations of this structure
40
determination method. The conformational parameters for the final
structure are detailed in the Supporting Information (Supplementary
Table S9).
An ensemble, comprising 250 individual conformational states, was
generated which can be considered representative of the conforma-
tional space occupied by the QRFP26(20−26) molecule in solution.
Modeling. A conformational search on compound 9c was done
+
delayed rectifier K channel; hNav1.5, human cardiac sodium
channel 1.5; HOBt, 1-hydroxybenzotriazole; IP-1, inositol-1-
phosphate; LLE, lipophilic ligand efficiency; log D7.4, logarithm
of the distribution coefficient in octanol/water at pH 7.4; m,
mouse; MDR, minimum discriminatory ratio; MeCN, acetoni-
trile; MeOH, methanol; MTBE, methyl tert-butyl ether; PBS,
phosphate-buffered saline; POMC, Pro-opiomelanocortin;
RLB, radioligand binding assay; SEM, standard error of the
mean; T3P, 1-propanephosphonic anhydride; TBTU, 2-(1H-
benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tet-
rafluoroborate; TEA, triethylamine; THIQ, tetrahydroisoquino-
line
5
0
using mixed torsional/low-mode sampling in MacroModel with
default settings. 9c was chosen as a substitute for 25e due to the fact
that the correct stereochemistry of 25e was not assigned. The energy
window for saving structures was set to 42.0 kJ/mol, and 100 steps/
rotatable bond were used. Minimization was done using the
OPLS2005 force field in water solvent with the PRCG method
convergence gradient with the threshold set to 0.05. The global
minimum according to molecular mechanics was found at least 10
times. The obtained conformations were superpositioned on the
pyrrolo[2,3-c]pyridine scaffold of the global minima, and a full distance
rmsd matrix was calculated using rmsd.py from Schro
matrix was clustered using the hierarchical clustering in spotfire
TIBCO, Spotfire3.1.1, TIBCO Software Inc.). The lowest energy
̈
dinger. The rmsd
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