Design and synthesis of potent and orally active GPR4 antagonists with modulatory effects on nociception, inflammation, and angiogenesis

Article abstract of DOI:10.1016/j.bmc.2017.06.050

GPR4, a G-protein coupled receptor, functions as a proton sensor being activated by extracellular acidic pH and has been implicated in playing a key role in acidosis associated with a variety of inflammatory conditions. An orally active GPR4 antagonist 39c was developed, starting from a high throughput screening hit 1. The compound shows potent cellular activity and is efficacious in animal models of angiogenesis, inflammation and pain.

Full text of DOI:10.1016/j.bmc.2017.06.050

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of potent and orally active GPR4 antagonists with
modulatory effects on nociception, inflammation, and angiogenesis
a
b
b
c
Wolfgang Miltz a, , Juraj Velcicky , Janet Dawson , Amanda Littlewood-Evans , Marie-Gabrielle Ludwig ,
Klaus Seuwen c, Roland Feifel d, Berndt Oberhauser a, Arndt Meyer a, Daniela Gabriel c, Mark Nash e,
Pius Loetscher b
⇑
a Global Discovery Chemistry, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
b Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
c Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
d PK Sciences, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
e Muscoloskeletal Diseases, Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
GPR4, a G-protein coupled receptor, functions as a proton sensor being activated by extracellular acidic
pH and has been implicated in playing a key role in acidosis associated with a variety of inflammatory
conditions. An orally active GPR4 antagonist 39c was developed, starting from a high throughput screen-
ing hit 1. The compound shows potent cellular activity and is efficacious in animal models of angiogen-
esis, inflammation and pain.
Received 9 May 2017
Revised 26 June 2017
Accepted 28 June 2017
Available online xxxx
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
GPR4
Amino-pyrimidine derivatives
Imidazo-pyridine derivatives
Angiogenesis
Inflammation
Pain
1. Introduction
up-regulates the expression of the adhesion molecules VCAM-1
and ICAM-1 in association with cAMP accumulation.4 Furthermore,
GPR4 is a G-protein coupled receptor (GPCR) and belongs to the
family of proton sensing receptors comprising also ovarian cancer
G protein-coupled receptor 1 (OGR1; GPR68), and T-cell death-
associated gene 8 (TDAG8; GPR65).1 The sensing of extracellular
protons and the stimulation of intracellular secondary messengers
upon exposure to acidic pH have been reported.2 The pH-depen-
dent activation of GPR4 involves the protonation of histidine resi-
dues of the receptor located at the cellular surface leading to a
conformational change of the receptor and formation of cAMP via
GPR4 is suggested to be involved in acidic pH-induced expression
of a number of inflammatory genes, including chemokines, cytoki-
nes, NF-j
B pathway genes, COX-2 and stress response genes.7 An
acidic milieu is also characteristic for the tumor microenviron-
ment. It has been reported that GPR4-deficient mice show strongly
reduced pathological angiogenesis and tumor growth.3 An involve-
ment of GPR4 in the modulation of glucose homeostasis has been
demonstrated. GPR4 deficiency improves glucose tolerance and
insulin sensitivity.8 Moreover, GPR4 has very recently been impli-
cated in the regulation of breathing by CO2.9 Recently, it has been
reported that blocking GPR4 reduced the myocardial infarction-
induced injury in mice which is characterized by decreased tissue
pH.10 Furthermore, the pro-inflammatory properties of GPR4
within inflamed intestinal tissues have been demonstrated by
studies with GPR4-deficient mice.11 Also, GPR4 has been reported
as a novel mediator for endoplasmic reticulum (ER) stress in
response to acidosis in endothelial cells.12
Gas. GPR4 is expressed in a wide range of tissues including vascu-
lature, lung, kidney, heart and liver.3–5
GPR4 has been linked to pH sensor function in a number of tis-
sues and organs under physiological and pathological conditions.
For instance, extracellular acidification occurs at sites of inflamma-
tion.6 It has been shown that activation of GPR4 by acidic pH
increases endothelial cell adhesion with leukocytes and
Here we report the development of small molecules targeting
GPR4 that are expected to offer a new treatment option for
⇑
Corresponding author.
E-mail address: wolfgang.miltz@novartis.com (W. Miltz).
http://dx.doi.org/10.1016/j.bmc.2017.06.050
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 2a
Propargylamine analogues without cyano group.
Fig. 1. GPR4 antagonists identified by HTS.
inflammatory disorders, starting from the HTS-hits 1 and 2 (Fig. 1,
Table 1). In addition, we have just published the further develop-
ment and optimization of the compounds.13
R1
R2
7
cAMP (HeLa) IC50 [lM]
H
H
Me
Cl
OMe
Me
a
b
c
0.30 0.09 (n = 10)
0.14 0.02 (n = 6)
0.80 0.04 (n = 3)
2. Results and discussion
acid gave chloride 17 which was converted into the dimethylamino
derivative 18 by its reaction with dimethylamine (Scheme 4).
Dimethylpyrimidine derivative with triazole linker (21, Table 3)
was also synthesized via the common bromo-intermediate 8c
being first converted into the azide 19 by copper mediated cou-
2.1. Chemistry
The aminopyrimidine derivatives with a propargyl linker (com-
pounds 7a–c, Table 2a) were synthesized as outlined in Scheme 1.
Starting from the corresponding 4-chloro pyrimidine (3), the chlo-
rine was substituted by neopentyl amine. The amino group in 4
was further alkylated with 4-iodobenzylbromide to give com-
pound 5. The 2-methoxy group in 5b was introduced by nucle-
ophilic substitution of chlorine atom in the 2-chloro derivative
5a with sodium methylate.14 In the next step, the compounds
6a-c were synthesized from 5a-c by reaction with propargylalco-
hol under Sonogashira coupling conditions.15 The final products
7a–c were prepared by mesylation of the alcohol 6 and subsequent
reaction with N-isopropylpiperazine.
Derivatives with the allyl linker (compounds 11a–f, Table 2b;
and compound 12, Table 3) were prepared according to Scheme 2.
N-alkylation of intermediates 4a and 4c–e with 4-bromo-benzyl-
bromide led to the corresponding bromointermediates 8a and
8c–e. The methoxy derivative 8b was again prepared by reaction
of chloro-derivative 8a with sodium methoxide, while dimethy-
lamino-analog 8f was prepared from 8a by reaction with dimethy-
lamine. Heck reaction of products 8a–f with methyl acrylate16 and
subsequent reduction of the ester group in 9a–f led to allyl alcohols
10a–f. Introduction of the basic head groups was achieved by
direct reaction of alcohols 10a–f with the Zaragoza reagent (cyano-
methyl-trimethylphosphonium iodide)17 leading to compounds
11a–f and 12.
19
pling with NaN3. Subsequent Huisgen 1,3-dipolar cycloaddition
(Click-chemistry)20 with 1-bromo-3-butyne gave the bromoethyl-
triazole 20 which could further be converted by reaction with
4-hydroxypiperidine to derivative 21 (Scheme 5).
Pyrrolopyrimidine derivative 29 (Table 4) was accessed via
intermediate 26 (Scheme 6). This compound was prepared starting
from acetylacetate 22 by its alkylation with 1-bromo-2-
butanone,21 followed by cyclization with acetamidine,22 conver-
sion of the formed pyrimidinone 24 to the chloropyrimidine 25
and its final cyclization with 4-bromo benzylamine.23 It was then
submitted to Heck reaction with methyl acrylate leading to methyl
cinnamate 27. The ester in 27 was then reduced with DIBAL to allyl
alcohol 28 which was transformed to the analog 29 by its reaction
with N-isopropylpiperazine using Zaragoza reagent.17
Synthesis of the imidazopyridine derivatives 39 (Table 4) was
performed through the common intermediates 35a–f which could
be prepared by two different routes depending on the 2-sub-
stituent (Scheme 7). Method A was employed for synthesis of
derivatives 39b,c,e and the key-intermediates 35b,c,e were synthe-
sized from imidazolone 32 obtained from malonamidine by its
cyclization with acetylacetone24 followed by Hoffmann rearrange-
ment/cyclization.25 Reaction of 32 with the corresponding car-
boxylic anhydride in the presence of MgCl2 gave intermediates
35b,c,e.26 A different route (method B) was used for analogs 35a,
d,f. This method is based on cyclization of the pyridinediamines
34a,b, obtained from 3-nitro-2-aminopyridines 33a,b by nitro-
group reduction, with the corresponding carboxylic acid under
dehydration conditions.27
Amide derivative 15 (Table 3) was synthesized starting from the
common N-neopentyl intermediate 4c by its alkylation with ethyl
p-bromomethyl benzoate, followed by saponification of the ester
and coupling of the formed acid with 4-dimethylaminobutylamine
(Scheme 3).
For the synthesis of the corresponding inverse amide 18
(Table 3), bromo intermediate 8c was transferred into aniline 16
by the Buchwald amination using diphenylmethanimine as an
ammonia equivalent.18 Amide coupling with 5-chloropentanoic
Benzyl acrylates 37a–f could be reached from intermediates
35a–f either by a two-steps procedure (alkylation with p-bro-
mobenzylbromide followed by the Heck reaction with methylacry-
Table 1
HTS hits.
1
2
cAMP (HeLa); IC50
human TDAG8; IC50
h Cat K; IC50 M]
[
l
[
M]
M]
1.93 0.63 (n = 4)
>30 (n = 1)
0.49 0.03 (n = 112)
>23 (n = 1)
l
[l
0.003 (n = 1)
0.001 (n = 1)
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 1. Synthesis of the propargyl derivatives 7a–c. Reagents and conditions: a) K2CO3, DMF, 100 °C, 2 h (45–93%); (b) NaH, DMF, rt, 10 min (48–83%); (c) NaH, MeOH,
60 C, 16 h (100%); (d) CuI, PdCl2(PPh3)2, Et3N, DMF, 100 °C, 1.5 h (39–90%); (e) MsCl, Et3 N, rt, 5 min; then N-isopropylpiperazine, K2CO3, DMF, 80 °C, 15 min (30–59%).
late) or by a direct one-step synthesis for derivative 37b using
Table 2b
(E)-methyl 3-(4-(bromomethyl) phenyl) acrylate. The final products
39a–f were synthesized by reduction of 37a–f with DIBAL followed
by Zaragoza-type amination of the obtained allyl alcohol 38a–f.
Allylamine analogues without cyano group.
2.2. Lead optimization
2.2.1. Aminopyrimidines
Two 4-aminopyrimidine derivatives 1 and 2 were identified by
HTS as potential GPR4 inhibitors showing potency in a pH-depen-
dent cAMP release assay (HeLa cells) in a micromolar range. Inter-
estingly, such compounds displayed a selectivity against close pH
sensing receptors such as TDAG8 (Table 1). Both compounds are
highly potent Cathepsin K inhibitors with the cyano group being
the active principle.28
R1
R2
11
cAMP (HeLa) IC50 [lM]
H
H
H
Me
H
Cl
a
b
c
d
e
f
0.36 0.05 (n = 3)
0.70 0.14 (n = 3)
0.41 0.05 (n = 3)
0.38 0.06 (n = 4)
2.97 0.70 (n = 3)
4.80 0.42 (n = 3)
OMe
Me
Me
NMe2
H
Therefore, removal of the cyano group in 2-position, being cru-
cial for the activity on cysteine proteases such as Cathepsin K, was
CF3
Table 3
Linker variations.
cAMP IC50
[lM]
human cAMP (HeLa cells)
mouse cAMP (HEK cells)
rat cAMP
(HEK cells)
12
0.33 0.06 (n = 7)
1.63 0.42 (n = 7)
1.59 0.57(n = 5)
15
18
0.16 0.03 (n = 11)
0.20 0.03 (n = 3)
0.56 0.07(n = 11)
0.76 0.35 (n = 3)
1.26 0.30 (n = 7)
1.99 0.43 (n = 3)
21
0.05 0.01 (n = 6)
0.18 0.04 (n = 3)
0.48 0.15 (n = 3)
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 2. Preparation of the allyl series. Reagents and conditions: (a) NaH, DMF, 2 h, rt (26–69%); (b) NaOMe/MeOH, reflux, 16 h (100%); (c) Me2NH/EtOH, 6 h, 60 °C (91%);
(d) Pd(t-Bu3P)2, methyldicyclohexyl amine, dioxane, 130 °C, 5 min (52–99%); (e) DIBAH, CH2Cl2, ꢀ78 °C, 3 h (36–64%); (f) amine, cyanomethyl-trimethylphosphonium iodide,
DIPEA, CH3CH2CN, 120 °C, 2 h (21–66%).
Scheme 3. Preparation of amide derivative 15. Reagents and conditions: (a) NaH, DMF, 30 min, rt (58%); (b) 6 M HCl, 2 h, 100 °C (52%); (c) EDC, HOBT, DIPEA, CH2Cl2, 16 h, rt
(54%).
Scheme 4. Preparation of amide 18. Reagents and conditions: (a) NaOMe, Pd2(dba)3, rac-BINAP, PhMe, 2 h, 100 °C (68%); (b) EDC, DIPEA, CH2Cl2, 1 h, rt (81%); (c) Me2NH, THF,
4 d, 60 °C (98%).
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Scheme 5. Preparation of triazole 21. Reagents and conditions: (a) NaN3, CuI, N,N0-dimethylethylenediamine, sodium ascorbate, EtOH/H2O, 1 h, 100 °C (75%); (b) CuI, CH3CN,
18 h, rt (74%); (c) DMF, 30 min, 120 °C (44%).
Table 4
SAR of imidazopyridines.
Nr.
R1
R2
R3
X
Y
cAMP IC50 [lM]
Human (HeLa)
Rat (HEK)
Mouse (HEK)
29
39a
Me
H
Me
H
Et
Et
N
CH
CH
N
11.6 3.3 (n = 6)
9.10 3.97
(n = 3)
0.41 0.28
(n = 3)
0.11 0.01
(n = 427)
0.76 0.47
(n = 2)
2.44 1.28
(n = 7)
nd
>20
nd
>20
(n = 4)
6.10 1.83
(n = 3)
1.29 0.17
(n = 108)
3.31
(n = 1)
16.5 3.48
(n = 3)
>20
(n = 4)
1.84 0.39
(n = 3)
0.8 0.12
(n = 110)
5.22
(n = 1)
17.24 2.76
(n = 3)
>20
39b
39c
39d
39e
39f
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
CH
CH
CH
CH
CH
N
N
N
N
N
Et
Prop
iProp
iBu
7.31 1.99
(n = 2)
(n = 2)
(n = 2)
tried initially. Interestingly, compounds with other groups than
cyano in this position (7a–c or 11a–f; tables 2a,b) proved to have
improved potency in inhibiting GPR4. While most of these deriva-
tives showed good potency, analogs with larger substituents such
as dimethylamino (11e) or trifluoromethyl (11f) were less toler-
ated by GPR4 receptor.
In a second step, the derivatization was focused on the replace-
ment of the potentially labile propylene linker between the
aminopyrimidine and the basic tail groups (12, Table 3). Interest-
ingly, all derivatives with an increased polarity such as amides
15 and 18 or triazole 21 showed improved activity in both human
and mouse cells.
on activity. While pyrrolopyrimidine derivative 29 shows dramatic
decrease in potency on GPR4 compared to its open version (12),
the corresponding imidazopyridine derivative 39c proved to be
100-fold more potent than 29 and also more potent than 12.
Furthermore, the two methyl groups in 39c were found to con-
tribute significantly to the binding into GPR4, since the des-methyl
compound 39a is 90-fold less potent than 39c. The substituent in
the 2-position of imidazole ring was found to be of great impor-
tance as well. The ethyl group in compound 39c appeared to have
the optimal size. While methyl (39b) is tolerated, but is binding
with less efficiency to GPR4, larger substituents in this position
such as n-Pr (39d) and especially the branched ones like i-Pr
(39e) or i-Bu (39e) are much less accepted by the receptor.
2.2.2. Imidazopyridines
The PK profiles of two pyrimidine compounds 15 and 21 were
compared with compound 39c, the most potent imidazopyridine
analog (Table 5). Derivative 39c shows the most promising PK
properties (low clearance, good bioavailability and oral exposure)
The aminopyrimidine series was morphed into compounds
with bicyclic scaffolds such as pyrrolopyrimidines or imidazopy-
ridines. The nature of the bicyclic group seems to have high impact
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 6. Preparation of pyrrolopyrimidine analog 29. Reagents and conditions: (a) NaH, THF, 0 °C to rt, 16 h (87%); (b) Na/MeOH, 16 h, reflux (61%); (c) POCl3, 15 min, reflux
(70%); (d) CHCl3, 12 h, 100 °C (100%); (e) Et3N, Pd(OAc)2, DMF, 15 min, 150 °C (56%); (f) DIBAL, CH2Cl2, 1 h, ꢀ78 °C (70%); (g) cyanomethyl trimethyl phosphonium iodide,
DIPEA, CH3CH2CN, 1 h, 120 °C (59%).
Scheme 7. Preparation of the imidazopyridines. Reagents and conditions: (a) KOH, MeOH, 5 h, rt (100%); (b) KOH, MeOH, 16 h, rt (98%); (c) MgCl2, 16 h, 100 °C (45–80%); (d)
H2, Pd/C, EtOH, 4 h, rt (99%); (e) PPA, 2 h, 190 °C (21–80%); (f) NaH, DMF, 4-bromobenzylbromide, 2 h, rt (20–70%); (g) Pd(t-Bu3P)2, dicyclohexylmethylamine, 5 min, 130 °C
(59–92%); (h) NaH, methyl 4-bromomethylphenylacrylate, DMF, 2 h, rt (39%); (i) DIBAH, CH2Cl2, 3 h, ꢀ78 °C (65–97%); (j) amine, cyanomethyl-trimethylphosphonium iodide,
DIPEA, CH3CH2CN, 120 °C, 2 h (59–86%).
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Table 5
Pharmacokinetics in female Sprague Dawley rats.
Rat PK (1 mg/kg iv, 3 mg/kg po)
Nr.
CL
T ½
[h]
AUC po dn
[ug.min/ ml], SD
Cmax dn
[nM], SD
BAV
[%], SD
[ml/ min.kg]
15
21
39c
48
56
15
34
23
5.6 1.5
424 83
2051 132
2196 298
73 25
94 54
193 39
17
96
86 12
3
2
ber into mice, the compound was applied for three days at doses of
0.3, 1, 3 and 10 mg/kg po/bid. At day 4 the chamber was explanted
and the weight of the tissue in the chamber was determined. Com-
pound 39c dose dependently inhibited the VEGF-induced angio-
genesis showing a very strong effect already at 3 mg/kg po/bid
dose.
2.3.2. Rat antigen-induced arthritis
For the evaluation of anti-inflammatory activity of GPR4 inhibi-
tors, 39c was tested in the rat antigen-induced arthritis model
(Fig. 3). It was dosed for seven days after the day of intra-articular
challenge with methylated BSA (mBSA) into the right knees and
vehicle only into the left knees of Lewis rats being immunized with
mBSA on day ꢀ21 and ꢀ14 (i.d.) on the back of the animal. Knee
swelling was measured by digital calipers and the swelling ratio
(R/L) was calculated. In addition, histology (inflammatory cell infil-
tration, joint damage and proteoglycan loss) was determined at
day seven (Fig. 4).
Again, compound 39c dose-dependently inhibited knee swel-
ling in this model. Significant effect was observed already at
10 mg/kg po/bid dose even if 90 mg/kg po/bid dose was needed
to reach the efficacy comparable to that of dexamethasone. In addi-
tion, inhibition of joint damage/erosions was also seen at 90 mg/kg
with 39c. Dexamethasone at 0.3 mg/kg represents the effective
positive control compound and dose in this model (Fig. 4).
Fig. 2. Inhibition of VEGF-induced angiogenesis by compound 39c in mouse
chamber implant model. Statistics are One-Way ANOVA followed by Dunnett’s test
for multiple comparisons (** p < 0.01).
and was therefore chosen for further profiling. In order to assess
the pharmacological effects described for GPR4 inhibition, com-
pound 39c was tested in animal models of angiogenesis, arthritis
and pain.
2.3.3. Rat hyperalgesia
Finally, the activity of the GPR4 inhibitor 39c was tested for its
antinociceptive effect using rat hyperalgesia model (Fig. 5). In this
model, an injection of complete Freund’s Adjuvant (CFA) into the
hind paw on day 0 was followed by compound application (po)
on day 3. Paw withdrawal measurements were performed at 1, 3
and 6 h. Compound 39c showed a significant analgesic effect at
2.3. In vivo pharmacology
2.3.1. Angiogenesis
Compound 39c was first tested in a VEGF-induced angiogenesis
model (Fig. 2). After implantation of a VEGF-containing agar cham-
Fig. 3. Inhibition of swelling by compound 39c in rat antigen-induced arthritis model and compound levels in blood at trough on day 3. Fold above or below the IC50 value of
compound 39c in the rat cAMP assay is indicated. Statistics are One-Way ANOVA followed by Dunnett’s test for multiple comparisons (*p < 0.05, **p < 0.01). dexa,
dexamethasone.
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8
W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
receptors as assessed by its screening in our internal receptor
panel, except its activity on the histaminic 3 receptor (H3-antago-
nist: IC50 of 0.93 0.07 M). Also, 39c showed inhibition of the
HERG channel (IC50 of 1.00 0.32 M was determined by binding
l
l
assay using radiolabeled 3H-dofetilide binding to HEK293 cell
membranes expressing human recombinant HERG K+ channels).
While the compound was well tolerated during the course of ani-
mal models, the two mentioned liabilities prevented this com-
pound to be further considered as a clinical candidate and
required further optimization to find H3 and hERG selective com-
pounds with similar properties as 39c.
Compound 39c16 is now commercially available as a tool GPR4
inhibitor29 and has been used to demonstrate the suppression of
acidosis stimulated expression of inflammatory genes in endothe-
lial cells.7 The imidazopyridine seems to be a privileged structure
in medicinal chemistry field. It not only is an important motif for
binding into GPR4 receptor as shown by other research groups
(Fig. 6),30–37 but was also used e.g. in the field of angiotensin 2
receptor antagonist reasearch.38
Fig. 4. Histological analysis of rat antigen-induced arthritis model. Inhibition of
inflammatory cell infiltration, joint damage/erosions and proteoglycan loss was
determined at day seven. Statistics are One-Way ANOVA followed by Dunnett’s test
for multiple comparisons (*p < 0.05, **p < 0.01). dexa, dexamethasone.
4. Experimental section
4.1. Chemistry
All reagents and solvents were purchased from commercial
suppliers and used without further purification. All reactions were
performed under inert conditions (argon) unless otherwise stated.
1H NMR spectra were recorded on a Bruker 400 MHz or a Bruker
600 MHz NMR spectrometer. Chemical shifts are reported in parts
per million (ppm) relative to an internal solvent reference. Signifi-
cant peaks are tabulated in the order multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; quintet; m, multiplet; br, broad), cou-
pling constants, and number of protons. Final compounds were
purified to ꢁ95% purity as assessed by analytical liquid chromatog-
raphy with the following method: Waters Acquity UPLCꢀMS; col-
umn HSS T3 1.8
l
m, 2.1 ꢂ 50 mm; A, water + 0.05% formic acid
+ 3.75 mM ammonium acetate; B, acetonitrile + 0.04% formic
acid5ꢀ98% B in 1.4 min (short method) and 9.4 min (long method),
flow 1.0 (short method) and 0.8 (long method) mL/min, flow 1.0
(short method) and 0.8 (long method) mL/min; column tempera-
ture 60 °C. The analyses were performed by using electrospray ion-
ization in positive ion modus after separation by liquid
chromatography (Nexera from Shimadzu). The elemental composi-
tion was derived from the mass spectra acquired at the high reso-
lution of about 30’000 on an LTQ Orbitrap XL mass spectrometer
(Thermo Scientific). The high mass accuracy below 1 ppm was
obtained by using a lock mass.
Fig. 5. Anti-nociceptive effect of compound 39c. Statistics are One-Way ANOVA
followed by Dunnetts test compared to matched vehicle group **p < 0.01,
***p < 0.001.
10 and 30 mg/kg po dose. With the latter dose, an effect close to
that of the NSAID diclofenac could be reached in this model.
3. Conclusion
4.1.1. Synthesis of 2-chloro-N-(4-(3-(4-isopropylpiperazin-1-yl)prop-
1-yn-1-yl)benzyl)-N-neopentyl pyrimidin-4-amine (7a)
(The synthesis of compounds 7b-c is described in the supple-
mentary data file).
In this paper we have described the discovery of a novel GPR4
antagonist 39c starting from HTS hits 1 and 2. Despite the high
species specificity between human and rodent, which was
reflected by lower activity in mouse and rat cellular assays, com-
pound 39c showed a strong efficacy in three different animal mod-
els: mouse VEGF-induced angiogenesis model, rat antigen-induced
arthritis model and rat hyperalgesia model. In vitro this compound
showed no cross-activity in a panel of kinase and proteases assays
including Cathepsin D. It was also highly selective over other
4.1.1.1.
Synthesis
of
2-chloro-N-neopentylpyrimidin-4-amine
(4a). 2,4-Dichloropyrimidine (5 g, 33.2 mmol) was dissolved in
10 ml THF and after addition of neopentylamine (3.48 g,
39.9 mmol) and potassium carbonate (6.96 g, 49.9 mmol) the mix-
Fig. 6.
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W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
ture was stirred at 75 °C for 2 h min. The TLC showed 2 products
(regioisomers). The mixture was diluted with ethyl acetate,
washed with water and brine, dried over Na2SO4, filtrated and con-
centrated under reduced pressure. The crude product was purified
by MPLC (silica gel, ethyl acetate/hexanes, 0–50%) to give 2.37 g
(35.7%). 1H NMR (400 MHz, CDCl3): d 8.03 (br, 1H), 6.28 (d,
J = 6 Hz, 1H), 5.25 (br, 1H), 3.1 (br, 2H), 0.94 (s, 9H). MS (ESI):
200.2 [M+H]+.
portions, the mixture was stirred for 20 min at rt. 4-bromobenzyl-
bromide (3.88 g, 15.5 mmol) was added slowly and the mixture
was stirred for 2 h at rt. Then the mixture was concentrated under
reduced pressure. The residue was diluted with ethyl acetate,
washed with saturated NaHCO3 solution and brine, dried over
Na2SO4 and concentrated. The crude product was purified by MPLC
(silica gel, ethyl acetate/cyclohexane, 1:9) to yield 1.5 g (26%) of 8a
as a white solid. 1H NMR (400 MHz, DMSO-d6): d 8.02 (br, 1H), 7.52
(d, J = 8 Hz, 2H), 7.15 (d, J = 8 Hz, 2H), 6.94 and 6,53 (br, 1H), 4.81
(s, 2H), 3.61 and 3.43 (br s, 2H), 0.99 (s, 9H); MS (ESI):368.6,
370.6 [M+H]+.
4.1.1.2. Synthesis of 2-chloro-N-(4-iodobenzyl)-N-neopentylpyrim-
idin-4-amine (5a). To a solution of 2-chloro-N-neopentylpyrim-
idin-4-amine (4a) (0.5 g, 2.48 mmol) and 4-iodobenzylbromide
(810 mg, 2.73 mmol) in 10 ml of DMF was added sodium hydride
(60% in mineral oil, 119 mg, 5 mmol) in portions. The mixture
was stirred for 10 min at rt TLC indicated complete disappearance
of 4a. Then the mixture was diluted with ethyl acetate, washed
with saturated NaHCO3 solution and brine, dried over Na2SO4
and concentrated. The crude product was purified by MPLC (silica
gel, ethyl acetate/hexanes, 0–50%) to give 1.01 g (83%). 1H NMR
(400 MHz, CDCl3): d 8.0 (d, J = 6 Hz, 1H), 7.68 (d, J = 8 Hz, 2H), 6.9
(d, J = 8 Hz, 2H), 6.32 (br, 1H), 4.8 (br s, 2H), 3.45 (br s, 2H), 0.96
(s, 9H); MS (ESI): 416.1 [M+H]+.
4.1.2.2. Synthesis of (E)-methyl 3-(4-(((2-chloropyrimidin-4-yl)
(neopentyl) amino)methyl)phenyl)acrylate (9a). N-(4-Bromoben-
zyl)-2-chloro-N-neopentylpyrimidin-4-amine (8a) (1.5 g, 4.07
mmol) was dissolved in 10 ml of dioxane and after addition of
methyl acrylate (420 mg, 4.9 mmol), dicyclohexylmethylamine
(1.76 ml, 8.1 mmol) and Pd(tBu3P)2 (41 mg, 0.08 mmol) the mix-
ture was heated for 5 min at 130 °C in a microwave. Then the mix-
ture was concentrated under reduced pressure. The residue was
diluted with ethyl acetate, washed with saturated NaHCO3 solution
and brine, dried over Na2SO4 and concentrated to give 9a in 1.36 g
(97%) yield after purification by MPLC (silica gel, ethyl acetate/
cyclohexane, 1:9). 1H NMR (400 MHz, DMSO-d6): d 8.02 (br, 1H),
7.68 (d, J = 8 Hz, 2H), 7.65 (d, J = 16 Hz, 1H), 7.21 (d, J = 8 Hz, 2H),
6.61 (d, J = 16 Hz, 1H), 6.5 (br, 1H), 4.85 (br s, 2H), 3.73 (s, 3H),
3.66 (br s, 2H), 0.98 (s, 9H); MS (ESI): 374.7 [M+H]+.
4.1.1.3. Synthesis of 3-(4-(((2-chloropyrimidin-4-yl)(neopentyl)
amino)methyl)phenyl)prop-2-yn-1-ol (6a). 640 mg (1.54 mmol) of
2-chloro-N-(4-iodobenzyl)-N-neopentylpyrimidin-4-amine
was dissolved in 10 ml of DMF and after addition of propargyl alco-
hol (90 l, 1.54 mmol), CuI (14.7 mg, 0.077 mmol), PdCl2(PPh3)2
(5a)
l
(54 mg, 0.077 mmol) and triethylamine (2.15 ml, 15.4 mmol) the
mixture was heated for 1.5 h at 100 °C in a sealed tube. The mix-
ture was partitioned between ethyl acetate and water. The organic
layer was dried over Na2SO4 and concentrated to give 250 mg
(47%) after purification by MPLC (silica gel, ethyl acetate/hexanes,
0–80%). 1H NMR (400 MHz, CDCl3): d 7.95 (br d, J = 6 Hz, 1H), 7.37
(d, J = 8 Hz, 2H), 7.07 (d, J = 8 Hz, 2H), 6.32 (br, 1H), 4.8 (br s, 2H),
4.48 (d, J = 6 Hz, 2H), 3.45 (br s, 2H), 2.77 (m, 1H), 1.0 (s, 9H); MS
(ESI): 343.9, 345.8 [M+H]+.
4.1.2.3. Synthesis of (E)-3-(4-(((2-chloropyrimidin-4-yl)(neopentyl)
amino) methyl)phenyl)prop-2-en-1-ol (10a). (E)-Methyl 3-(4-(((2-
chloropyrimidin-4-yl)(neopentyl) amino)methyl)phenyl)acrylate
(9a) (1.36 g, 3.64 mmol) was dissolved in 25 ml of dichloro-
methane and cooled to ꢀ78 °C. After slow addition of DIBAL
(9.09 ml of a 1.2 M solution in toluene, 10.9 mmol) the mixture
was stirred at ꢀ78 °C for 3 h. Then the mixture was quenched with
water and concentrated under reduced pressure. The residue was
diluted with ethyl acetate, washed with water and brine, dried
over Na2SO4 and concentrated. The crude product was purified
by MPLC (silica gel, ethyl acetate/cyclohexane, 1:1) to give 600
mg (36%) of 10a as a white solid. 1H NMR (400 MHz, DMSO-d6):
d 7.99 (br, 1H), 7.38 (d, J = 8 Hz, 2H), 7.13 (d, J = 8 Hz, 2H), 6.97
(br, 1H), 6.52 (d, J = 16 Hz, 1H), 6.35 (dt, J = 16 and 6 Hz, 1H),
4.87 (t, J = 8 Hz, 1H), 4.81 (br s, 2H), 4.11 (m, 2H), 3.63 (br s, 2H),
0.98 (s, 9H); MS (ESI): 346.6 [M+H]+.
4.1.1.4. Synthesis of 2-chloro-N-(4-(3-(4-isopropylpiperazin-1-yl)
prop-1-yn-1-yl)benzyl)-N-neopentyl pyrimidin-4-amine (7a). To a
solution of 3-(4-(((2-chloropyrimidin-4-yl)(neopentyl)amino)
methyl)phenyl) prop-2-yn-1-ol (6a) (125 mg, 0.364 mmol) in
90 ml of dichloromethane was added mesyl chloride (57
ll,
0.727 mmol) followed by triethylamine (150 l, 1.09 mmol). After
l
5 min, 1-isopropylpiperazine (93 mg, 0.727 mmol) was added, fol-
lowed by 10 ml of DMF and K2CO3 (150 mg, 1.09 mmol). The mix-
ture was stirred for 15 min at 80 °C, then partitioned between ethyl
acetate and water. The organic layer was dried over Na2SO4 and
concentrated, which provided 97.6 mg (59%) after purification by
MPLC (silica gel, ethyl acetate/hexanes, 0–80%). 1H NMR
(400 MHz, CD3OD): d 7.9 (br, 1H), 7.37 (d, J = 8 Hz, 2H), 7.18 (d,
J = 8 Hz, 2H), 6.55 (br, 1H), 4.87 (br s, 2H), 3.57 (br, 2H), 3.52 (s,
2H), 2.55–2.8 (m, 9H), 1.08 (d, J = 6 Hz, 6H), 1.0 (s, 9H); HRMS
(ESI): calcd. For C26H36ClN5 [M+H]+ 454.27320, found 454.27313;
HPLC (long method): tR=4.68 min (91%).
4.1.2.4. Synthesis of (E)-N-(4-(3-([1,40-bipiperidin]-10-yl)prop-1-en-
1-yl)benzyl)-2-chloro-N-neopentyl pyrimidin-4-amine (11a). (E)-3-
(4-(((2-Chloropyrimidin-4-yl)(neopentyl)amino)methyl)phenyl)
prop-2-en-1-ol (10a) (150 mg, 0.434 mmol) was dissolved in 5 ml
of propionitrile and after addition of 4-piperidinyl piperidine
(73 mg,
iodide (freshly recrystallized from acetonitrile, 248 mg, 1.09 mmol)
and diisopropylethylamine (354 l, 2.17 mmol) the mixture was
0.434 mmol),
cyanomethyl-trimethyl-phosphonium
l
stirred for 2 h at 120 °C. Then the mixture was concentrated under
reduced pressure. The residue was diluted with ethyl acetate,
washed with 10% aqueous K2CO3 solution and brine, dried over
Na2SO4 and concentrated. The crude product was purified by MPLC
(silica gel, methanol/ethyl acetate, 40:60) to give 102 mg (48%) of
the product as a brown oil. 1H NMR (400 MHz, DMSO-d6): d 8.00
(br, 1H), 7.39 (d, J = 8 Hz, 2H), 7.11 (d, J = 8 Hz, 2H), 6.5 (br, 1H),
6.47 (d, J = 16 Hz, 1H), 6.25 (dt, J = 16, 6 Hz, 1H), 4.80 (br s, 2H),
3.63 (br s, 2H), 3.03 (d, J = 6 Hz, 2H), 2.88 (m, 2H), 2.42 (m, 4H),
2.14 (m, 1H), 1.88 (m, 2H), 1.65 (m, 2H), 1.3–1.5 (m, 8H), 0.97 (s,
9H); HRMS (ESI): calcd. For C29H42ClN5 [M+H]+ 496.32015, found
496.32010.
4.1.2. Synthesis of (E)-N-(4-(3-([1,40-bipiperidin]-10-yl)prop-1-en-1-
yl)benzyl)-2-chloro-N-neopentyl pyrimidin-4-amine (11a)
(The synthesis of compounds 11b–f and 12 is described in the
Supplementary data file).
4.1.2.1. Synthesis of N-(4-bromobenzyl)-2-chloro-N-neopentylpyrim-
idin-4-amine (8a). 2-Chloro-N-neopentylpyrimidin-4-amine (4a)
(3.1 g, 15.5 mmol) was dissolved in 50 ml of DMF. After addition
of sodium hydride (60% in mineral oil, 447 mg, 18.6 mmol) in
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10
W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.1.3. Synthesis of N-(4-(dimethylamino)butyl)-4-(((2,6-dimethylpyri-
midin-4-yl)(neopentyl)amino) methyl) benzamide (15)
4.1.4. Synthesis of 5-(dimethylamino)-N-(4-(((2,6-dimethylpyrimidin-
4-yl)(neopentyl)amino)methyl) phenyl)pentanamide (18)
4.1.3.1. Synthesis of 2,6-dimethyl-N-neopentylpyrimidin-4-amine
(4c). 4-Chloro-2,6-dimethylpyrimidine (0.5 g, 3.47 mmol) was dis-
solved in 10 ml DMF and after addition of neopentylamine
(363 mg, 4.16 mmol) and potassium carbonate (727 mg,
5.21 mmol) the mixture was stirred at 100 °C for 2 h. Then the mix-
ture was concentrated under high vacuum. The residue was diluted
with ethyl acetate, washed with water and brine, dried over
Na2SO4, filtrated and concentrated under reduced pressure. The
crude product was purified by MPLC (silica gel, MeOH/CH2Cl2,
0–10%) which provided 384 mg (57%). 1H NMR (400 MHz,
DMSO-d6): d 6.96 (br t, J = 8 Hz, 1H), 6.21 (s, 1H), 3.15 (br, 2H),
2.27 (s, 3H), 2.15 (s, 3H), 0.90 (s, 9H); MS (ESI): 194 [M+H]+.
4.1.4.1. Synthesis of N-(4-bromobenzyl)-2,6-dimethyl-N-neopentyl-
pyrimidin-4-amine
(8c). 2,6-Dimethyl-N-neopentylpyrimidin-4-
amine (4c) (1.53 g, 7.92 mmol) was dissolved in 50 ml of DMF.
After addition of sodium hydride (60% in mineral oil, 228 mg,
9.5 mmol) in portions, the mixture was stirred for 20 min at rt.
4-Bromobenzylbromide (1.98 g, 7.92 mmol) was added slowly
and the mixture was stirred for 2 h at rt. Then the mixture was
concentrated under reduced pressure. The residue was diluted
with ethyl acetate, washed with saturated NaHCO3 solution and
brine, dried over Na2SO4 and concentrated. Purification by MPLC
(silica gel, ethyl acetate/cyclohexane, 1:1) provided 841 mg
(29%). 1H NMR (400 MHz, DMSO-d6): d 7.5 (d, J = 8 Hz, 2H), 7.1
(d, J = 8 Hz, 2H), 6.4 (br s, 1H), 4.8 (br s, 2H), 3.44 (br s, 2H), 2.31
(s, 3H), 2.18 (s, 3H), 0.96 (s, 9H); MS (ESI): 362, 364 [M+H]+.
4.1.3.2. Synthesis of ethyl 4-(((2,6-dimethylpyrimidin-4-yl)
(neopentyl)amino)methyl)benzoate (13). Sodium hydride (902 mg,
35.7 mmol) was added portionwise to a solution of 2,6-dimethyl-
N-neopentylpyrimidin-4-amine (4c) (5 g, 23.8 mmol) in 90 ml of
DMF and the mixture was stirred for 15 min at rt. Then a solution
of ethyl bromomethyl benzoate (6.63 g, 26.2 mmol) in 15 ml DMF
was added dropwise over 30 min. The dark brown reaction mixture
was quenched with 400 ml ice water and extracted six times with
ethyl acetate. The combined organic layers were washed with
brine, dried over Na2SO4, filtered and concentrated to give 12 g of
a yellow oil, which was further purified by filtration over a silica
pad (ethyl acetate/hexane 0–40%). Yield: 5.46 g (58%) of a yellow
oil. 1H NMR (400 MHz, CDCl3): d 7.95 (d, J = 8 Hz, 2H), 7.15 (d,
J = 8 Hz, 2H), 6.1 (br s, 1H), 4.92 (br s, 2H), 4.81 (br s, 2H), 4.35
(q, J = 8 Hz, 2H), 2.44 (s, 3H), 2.25 (s, 3H), 1.37 (t, J = 8 Hz, 3H),
0.96 (s, 9H); MS (ESI): 356.2 [M+H]+.
4.1.4.2. Synthesis of N-(4-aminobenzyl)-2,6-dimethyl-N-neop-
entylpyrimidin-4-amine (16). N-(4-Bromobenzyl)-2,6-dimethyl-N-
neopentylpyrimidin-4-amine (8c) (1.5 g, 4.14 mmol) was dissolved
in 30 ml of toluene and after addition of benzophenone imine
(831 ll, 4.97 mmol), sodium methylate (858 mg, 6.21 mmol),
Pd2(dba)3 (94.8 mg, 0.103 mmol) and rac-BINAP (193 mg,
0.31 mmol) the mixture was stirred at 100 °C for 2 h. Then the mix-
ture was cooled to rt and after addition with 30 ml of 2 M aqueous
HCl the mixture was stirred at rt for 30 min. The mixture was fil-
trated over Celite and partitioned between ethyl acetate and 2 N
HCl. The water layer was neutralized by slowly addition of Na2CO3
(pH >7), followed by extraction with ethyl acetate. The organic
layer was dried over Na2SO4 and concentrated to give 840 mg
(68%) of a colorless resin, which was used in the next step without
further purification. MS (ESI): 299.2.
4.1.4.3. Synthesis of 5-chloro-N-(4-(((2,6-dimethylpyrimidin-4-yl)
4.1.3.3. Synthesis of 4-(((2,6-dimethylpyrimidin-4-yl)(neopentyl)
(neopentyl)amino)methyl)
phenyl)-pentanamide
(17). N-(4-
amino) methyl)benzoic acid (14). Ethyl 4-(((2,6-dimethylpyrim-
Aminobenzyl)-2,6-dimethyl-N-neopentylpyrimidin-4-amine (16)
idin-4-yl)(neopentyl)amino)methyl)benzoate
(13)
(3.32 g,
(300 mg, 1.01 mmol), 5-chloro valeric acid (165 mg, 1.21 mmol),
9.34 mmol) was dissolved in 10 ml of methanol. After addition of
4 N NaOH solution (23.3 ml, 93.4 mmol) the mixture was stirred
for 1 h at 40 °C. The reaction mixture was concentrated, diluted
with 10 ml ice water and then 6 N HCl solution was added until
a pH of 6 was reached. The organic layer was separated, dissolved
in acetone, dried over Na2SO4. Evaporation gave 2.1 g (62%) of a
light yellow foam, which was used in the next step without further
purification. MS (ESI): 328.1 [M+H]+.
EDCꢃHCl (295 mg, 1.51 mmol) and diisopropylethylamine (527
ll,
3.02 mmol) were dissolved in 6 ml of CH2Cl2 and the mixture
was stirred for 1 h at rt. The reaction mixture was directly purified
by MPLC (silica gel, ethyl acetate/hexanes 0–50%). Yield: 340 mg
(81%) as a colorless resin. MS (ESI): 417.2 [M]+.
4.1.4.4. Synthesis of 5-(dimethylamino)-N-(4-(((2,6-dimethylpyrim-
idin-4-yl)(neopentyl)amino)methyl)
phenyl)pentanamide
(18).
5-Chloro-N-(4-(((2,6-dimethylpyrimidin-4-yl)(neopentyl)amino)-
methyl)-phenyl)pentanamide (17) (50 mg, 0.12 mmol) and a 2 M
solution of dimethylamine in THF (3 ml, 6 mmol) were dissolved
in THF in a closed vial and stirred for 4 d at 60 °C. The reaction mix-
ture was concentrated and purified by HPLC (RP-18, acetone/water
gradient). Yield: 50 mg (98%) as a colorless oil. 1H NMR (400 MHz,
CD3OD): d 7.58 and 7.54 (d, J = 8 Hz, 2H), 7.22 and 7.18 (d, J = 8 Hz,
2H), 6.99 and 6.62 (s, 1H), 5.14 and 4.95 (s, 2H), 3.9 and 3.53 (s,
2H), 3.2 (m, 2H), 2.9 (s, 6H), 2.6, 2.55, 2.45 and 2.35 (s, 6H), 2.45
(m, 2H), 1.75 (m, 4H), 1.1 and 1.05 (s, 9H); HRMS (ESI): calcd.
For C25H39N5O [M+H]+ 426.32274, found 426.32275; HPLC (short
method): tR = 0.72 (90%).
4.1.3.4.
Synthesis
of
N-(4-(dimethylamino)butyl)-4-(((2,6-
dimethylpyrimidin-4-yl)(neopentyl)amino)methyl)benzamide (15). A
solution of 4-(((2,6-dimethylpyrimidin-4-yl)(neopentyl)amino)
methyl)-benzoic acid (14) (1.95 g, 4.98 mmol) in 70 ml of CH2Cl2
was treated with EDCꢃHCl (1.91 g, 9.97 mmol), HOBT (1.91 g,
12.5 mmol) and diisopropylethylamine (4.35 ml, 24.9 mmol). Then
4-dimethylaminobutylamine (0.984 ml, 5.23 mmol) was added
and the light yellow solution was stirred overnight at room tem-
perature. The reaction mixture was filtrated, concentrated, diluted
with 150 ml ethylacetate and washed with 100 ml of water and
50 ml of brine. The organic layer was dried over Na2SO4 and con-
centrated. The crude product was purified by HPLC (C18, acetoni-
trile/water gradient). Yield: 1.3 g (54%) of a white foam. 1H NMR
(400 MHz, CD3OD): d 7.74 (d, J = 8 Hz, 2H), 7.23 (d, J = 8 Hz, 2H),
6.32 (br s, 1H), 4.92 (br s, 2H), 3.6 (br s, 2H), 3.38 (t, J = 6 Hz, 2H),
2.41 (t, J = 6 Hz, 2H), 2.38 (s, 3H), 2.3 (s, 6H), 2.22 (s, 3H), 1.6 (m,
4H), 1.0 (s, 9H). %); HRMS (ESI): calcd. For C25H39N5O [M+H]+
426.32274, found 426.32266; HPLC (long method): tR = 2.39 min
(100%).
4.1.5. Synthesis of 1-(2-(1-(4-(((2,6-dimethylpyrimidin-4-yl)(neopentyl)
amino)methyl)phenyl)-2,3-dihydro-1H-1,2,3-triazol-4-yl)ethyl)piperidin-
4-ol (21)
4.1.5.1.
Synthesis
of
N-(4-azidobenzyl)-2,6-dimethyl-N-
neopentylpyrimidin-4-amine (19). A vial containing a solution of
N-(4-bromobenzyl)-2,6-dimethyl-N-neopentylpyrimidin-4-amine
(8c) (12.02 g, 31.5 mmol), sodium azide (4.1 g, 63 mmol), copper
(I)iodide (600 mg, 3.15 mmol), N,N0-dimethylethylenediamine
Please cite this article in press as: Miltz W., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.050

W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
(509
l
l, 4.73 mmol) and sodium ascorbate (312 mg, 1.58 mmol) in
25 ml of methanol, followed by acetamidine hydrochloride
(1.19 g, 12.2 mmol) and ethyl 2-acetyl-4-oxohexanoate (23)
(2.45 g, 12.25 mmol). The reaction mixture was refluxed for 16 h.
The methanol was evaporated, 13 ml of water was added and the
resulting solution was neutralized with 1 ml of acetic acid. The
mixture was extracted with chloroform and the organic layer
was concentrated. The crude product was recrystallized from
toluene to give 1.45 g (61%) of the product as colorless needles.
1H NMR (400 MHz, DMSO-d6): d 12.3 (s, 1H), 3.52 (s, 2H), 2.5 (m,
2H), 2.22 (s, 3H), 2.08 (s, 3H), 0.93 (t, J = 8 Hz, 3H); MS (ESI):
195.16 [M+H]+.
ethanol (47 ml) and water (15.7 ml) was heated for 1 h at 100 °C
in a microwave oven. The reaction mixture was diluted with ethyl
acetate and washed with saturated aqueous NaHCO3 solution,
water and brine. The organic layer was dried over Na2SO4, concen-
trated
and
purified
by
MPLC
(ethyl
acetate/
heptane 5–40%). Yield: 8.09 g (75%) as an yellow oil. 1H NMR
(400 MHz, CD3OD): d 7.68 (d, J = 8 Hz, 2H), 7.35 (d, J = 8 Hz, 2H),
6.4 (br s, 1H), 5.01 (s, 2H), 3.56 (s, 2H), 2.4 (s, 3H), 2.25 (s, 3H),
1.05 (s, 9H); MS (ESI): 309.5 [M+H]+.
4.1.5.2. Synthesis of N-(4-(4-(2-bromoethyl)-2,3-dihydro-1H-1,2,
3-triazol-1-yl)benzyl)-2,6-dimethyl-N-neopentylpyrimidin-4-amine
(20). A solution of N-(4-azidobenzyl)-2,6-dimethyl-N-neopentyl-
pyrimidin-4-amine (19) (530 mg, 1.63 mmol), 4-bromo-1-butyne
(0.237 ml, 2.45 mmol) and copper (I) iodide (622 mg, 3.27 mmol)
in 15 ml of acetonitrile was stirred at rt for 18 h. The reaction mix-
ture was diluted with ethyl acetate and washed with saturated
NaHCO3 solution and brine. The organic layer was dried over
Na2SO4, filtrated and concentrated under reduced pressure. The
crude product was purified by MPLC (silica gel, ethyl acetate/
CH2Cl2, 30–80%). Yield: 635 mg (74%) of a brown solid. 1H NMR
(400 MHz, CDCl3): d 7.9 (br, 1H), 7.7 (d, J = 8 Hz, 2H), 7.31 (d,
J = 8 Hz, 2H), 6.21 (br s, 1H), 4.98 (br s, 2H), 3.75 (t, J = 8 Hz, 2H),
3.5 (br, 2H), 3.42 (t, J = 8 Hz, 2H), 2.56 (s, 3H), 2.4 (s, 3H), 1.05 (s,
9H); MS (ESI): 457, 459 [M+H]+.
4.1.6.3. Synthesis of 1-(4-chloro-2,6-dimethylpyrimidin-5-yl)butan-2-
one (25). A mixture of 2,6-dimethyl-5-(2-oxobutyl)pyrimidin-4
(3H)-one (24) (950 mg, 4.89 mmol) and 10 ml of POCl3 was
refluxed (110 °C) for 15 min. Then the mixture was concentrated.
20 ml of cold water was added, followed by 20 ml of CHCl3. The
mixture was the neutralized by addition of Na2CO3 solution. The
organic layer was washed with saturated NaHCO3 solution and
brine, dried over Na2SO4, filtrated and concentrated to give the
product in 724 mg (70%) yield as yellow needles. MS (ESI):
213.13 [M+H]+.
4.1.6.4. Synthesis of 7-(4-bromobenzyl)-6-ethyl-2,4-dimethyl-7H-pyr-
rolo[2,3-d]pyrimidine (26). A solution of 1-(4-chloro-2,6-
dimethylpyrimidin-5-yl)butan-2-one (25) (150 mg, 0.705 mmol)
and 4-bromobenzylamine (273 mg, 1.41 mmol) in 5 ml of CHCl3
was heated for 12 h at 100 °C in a microwave oven. Evaporation
of solvent gave 242 mg (100%) of a yellow powder, which was used
in the next step without further purification. MS (ESI): 345.8 [M
+H]+.
4.1.5.3. Synthesis of 1-(2-(1-(4-(((2,6-dimethylpyrimidin-4-yl)
(neopentyl)amino)methyl)phenyl)-2,3-dihydro-1H-1,2,3-triazol-4-yl)
ethyl)piperidin-4-ol trifluoroacetyl salt (21). A solution of N-(4-
(4-(2-bromoethyl)-2,3-dihydro-1H-1,2,3-triazol-1-yl)benzyl)-2,6-
dimethyl-N-neopentylpyrimidin-4-amine
(20)
(74 mg,
0.162 mmol) and 4-hydroxypiperidine (50 mg, 0.485 mmol) in
0.6 ml of DMF was heated for 30 min at 120 °C in a microwave
oven. Then, the reaction mixture was diluted with ethyl acetate
and washed with brine. The organic layer was dried over Na2SO4,
filtrated, concentrated. The crude product was purified by HPLC
(RP-18, acetonitrile/water gradient). Yield: 50.8 mg (44%). 1H
NMR (400 MHz, CD3OD, 2 rotamers): d 8.48 and 8.47 (s, 1H),
7.88 and 7.83 (d, J = 8 Hz, 2H), 7.5 and 7.46 (d, J = 8 Hz, 2H), 7.06
and 6.65 (s, 1H), 5.25 and 5.08 (s, 2H), 4.18 and 4.0 (m, 1H), 3.9
(m, 1H), 3.78 (m, 1H), 3.65 (m, 1H), 3.58 (m, 2H), 3.53 (m, 4H),
3.4 (m, 1H), 3.18 (m, 1H), 2.65, 2.55, 2.5 and 2.4 (s, 6H), 2.22 (m,
1H), 2.03 (m, 2H), 1.8 (m, 1H), 1.12 and 1.1 (s, 9H); HRMS (ESI):
calcd. For C27H39N7O [M+H]+ 478.32888, found 478.32886; HPLC
(6 min method): tR = 2.60 min (100%).
4.1.6.5. Synthesis of (E)-methyl 3-(4-((6-ethyl-2,4-dimethyl-7H-pyr-
rolo[2,3-d]pyrimidin-7-yl)methyl)-phenyl) acrylate (27). Methyl
acrylate (39.6 ll, 0.435 mmol) and triethyl amine (122 ll,
0.871 mmol) were added under argon to a solution of 7-(4-bro-
mobenzyl)-6-ethyl-2,4-dimethyl-7H-pyrrolo[2,3-d]pyrimidine
(26) (150 mg, 0.436 mmol) and Pd(OAc)2 (20.8 mg, 0.044 mmol) in
1.6 ml of DMF. The mixture was heated for 15 min at 150 °C in a
microwave oven. After cooling to rt, the reaction mixture was
diluted with ethyl acetate and filtrated over Celite. The filtrate
was washed with water, saturated NaHCO3 solution and brine,
dried over Na2SO4, filtered and concentrated. The crude product
was purified by MPLC (silica gel, ethyl acetate/heptane gradient).
Yield: 85.4 mg (56%) as an yellow solid. MS (ESI): 350.9 [M+H]+.
4.1.6.6. Synthesis of (E)-3-(4-((6-ethyl-2,4-dimethyl-7H-pyrrolo
4.1.6. Synthesis of (E)-6-ethyl-7-(4-(3-(4-isopropylpiperazin-1-yl)
prop-1-en-1-yl)benzyl)-2,4-dimethyl-7H-pyrrolo[2,3-d]pyrimidine
(29)
[2,3-d]pyrimidin-7-yl)methyl)phenyl)prop-2-en-1-ol
(28). (E)-
Methyl 3-(4-((6-ethyl-2,4-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-
7-yl)methyl)phenyl) acrylate (27) (85 mg, 0.244 mmol) was dis-
solved in 25 ml of dichloromethane and cooled to -78 °C. After
4.1.6.1. Synthesis of ethyl 2-acetyl-4-oxohexanoate (23). A suspen-
sion of sodium hydride (60% in mineral oil, 690 mg, 17.2 mmol)
in 80 ml THF was cooled to 0 °C and ethyl acetoacetate (22)
(2 ml, 16 mmol) was added dropwise. The reaction mixture was
stirred for 30 min at 0 °C. Then 1-bromo-2-butanone (1.85 ml,
17.2 mmol) was added. The mixture was stirred another 2 h at
0 °C and then allowed to warm up to rt. The mixture was quenched
with 1 N HCl and extracted with diethyl ether. The organic layer
was washed with saturated aqueous NaHCO3 solution and brine,
dried over Na2SO4, filtrated and concentrated. Yield: 2.72 g (87%)
of a light yellow oil. 1H NMR (400 MHz, DMSO-d6): d 4.12 (m,
2H), 4.0 (t, J = 8 Hz, 1H), 2.98 (m, 2H), 2.48 (q, J = 8 Hz, 2H), 2.25
(s, 3H), 1.19 (t, J = 8 Hz, 3H), 0.92 (t, J = 8 Hz, 3H).
slow addition of DIBAL (1 M in THF, 730 ll, 0.73 mmol) the mix-
ture was stirred at -78 °C for 3 h. Then, the mixture was quenched
with water and concentrated under reduced pressure. The residue
was diluted with ethyl acetate, washed with water and brine, dried
over Na2SO4 and concentrated to give 55 mg (70%) of the product
as off-white crystals. MS (ESI): 322 [M+H]+.
4.1.6.7. Synthesis of (E)-6-ethyl-7-(4-(3-(4-isopropylpiperazin-1-yl)
prop-1-en-1-yl)benzyl)-2,4-dimethyl-7H-pyrrolo[2,3-d]pyrimidine (29).
(E)-3-(4-((6-Ethyl-2,4-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
methyl)phenyl)-prop-2-en-1-ol (28) (28 mg, 0.087 mmol) was
dissolved in 5 ml of propionitrile and after addition of 1-isopropyl-
piperazine (13
nium iodide (50 mg, 0.217 mmol, freshly recrystallized from acetoni-
trile) and diisopropylethylamine (76 l) the mixture was stirred for
ll, 0.087 mmol), cyanomethyl-trimethyl-phospho-
4.1.6.2. Synthesis of 2,6-dimethyl-5-(2-oxobutyl)pyrimidin-4(3H)-one
(24). Sodium (564 mg, 24.5 mmol) was added under argon to
l
Please cite this article in press as: Miltz W., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.050

12
W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
2 h at 120 °C. Then the mixture was concentrated under reduced
pressure. The residue was diluted with ethyl acetate, washed with
10% aqueous K2CO3 solution and brine, dried over Na2SO4 and con-
centrated. The crude product was purified HPLC (RP-18, acetoni-
trile/water gradient) to give 22 mg (59%) of a white powder. 1H
NMR (400 MHz, DMSO-d6): d 7.42 (d, J = 8 Hz, 2H), 7.07 (d, J = 8 Hz,
2H), 6.92 (s, 1H), 6.66 (d, J = 16 Hz, 1H), 6.26 (dt, J = 16 and 6 Hz,
1H), 5.57 (s, 2H), 2.9–4.0 (m, 11H), 2.87 (s, 3H), 2.78 (s, 3H), 2.7
(q, J = 7 Hz, 2H), 1.2–1.25 (m, 9H); HRMS (ESI): calcd. For C27H37N5
[M+H]+ 432.31217, found 432.31232.
4.1.7.5. Synthesis of (E)-methyl 3-(4-((2-ethyl-5,7-dimethyl-3H-imi-
dazo[4,5-b]pyridin-3-yl)methyl) phenyl)acrylate (37c). 3-(4-Bro-
mobenzyl)-2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (36c)
(500 mg, 1.45 mmol) was dissolved in 15 ml of dioxane and after
addition of methyl acrylate (654 mg, 7.26 mmol), dicyclohexyl-
methylamine (630 ll, 2.90 mmol) and Pd(tBu3P)2 (14.8 mg,
0.029 mmol) the mixture was heated for 5 min at 130 °C in a
microwave. Then the mixture was concentrated under reduced
pressure. The residue was diluted with ethyl acetate, washed with
saturated NaHCO3 solution and brine, dried over Na2SO4 and con-
centrated to give 300 mg (59%) of a colorless solid, which was used
in the next step without further purification. 1H NMR (400 MHz,
DMSO-d6): d 7.67 (d, J = 8 Hz, 2H), 7.62 (d, J = 16 Hz, 1H), 7.15 (d,
J = 8 Hz, 2H), 6.96 (s, 1H), 6.6 (d, J = 16 Hz, 1H), 5.49 (s, 2H), 3.72
(s, 3H), 2.77 (q, J = 8 Hz, 2H), 2.52 (s, 3H), 2.50 (s, 3H), 1.23 (t,
J = 8 Hz, 3H); MS (ESI): 350.8 [M+H]+.
4.1.7. Synthesis of (E)-2-ethyl-3-(4-(3-(4-isopropylpiperazin-1-yl)
prop-1-en-1-yl)benzyl)-5,7-dimethyl-3H-imidazo[4,5-b]pyridine
(39c)
(The synthesis of compounds 39a,b and d–f is described in the
supplementary data file).
4.1.7.6. Synthesis of (E)-3-(4-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-
b]pyridin-3-yl)methyl)phenyl)prop-2-en-1-ol (38c). (E)-Methyl 3-
(4-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)
phenyl)acrylate (37c) (500 mg, 1.43 mmol)) was dissolved in 25 ml
of dichloromethane and cooled to ꢀ78 °C. After slow addition of
DIBAL (3.05 ml of a 1.2 M solution in toluene, 4.29 mmol) the mix-
ture was stirred at ꢀ78 °C for 3 h. Then the mixture was quenched
with water and concentrated under reduced pressure. The residue
was diluted with ethyl acetate, washed with water and brine, dried
over Na2SO4 and concentrated to give 38c in 300 mg (65%) yield as
a colorless solid, which was used in the next step without further
purification. 1H NMR (400 MHz, DMSO-d6): d 7.32 (d, J = 8 Hz,
2H), 7.03 (d, J = 8 Hz, 2H), 6.91 (s, 1H), 6.47 (d, J = 16 Hz, 1H),
6.33 (dt, J = 16, 6 Hz, 1H), 5.39 (s, 2H), 4.82 (t, J = 6 Hz, 1H), 4.06
(m, 2H), 2.73 (q, J = 8 Hz, 2H), 2.49 (s, 6H), 1.20 (t, J = 8 Hz, 3H);
MS (ESI): 322 [M+H]+.
4.1.7.1.
Synthesis
of
2-amino-4,6-dimethylnicotinamide
(31b). Malonamidine hydrochloride (30) (20.4 g, 148.4 mmol)
was dissolved in 1 L of methanol. After addition of acetylacetone
(15.3 ml, 148 mmol) and KOH pellets (9.99 g, 178 mmol) the mix-
ture was stirred for 24 h at rt. Evaporation gave 24.5 g (100%) of the
product as a white solid. MS (ESI): 166.6 [M+H]+.
4.1.7.2. Synthesis of 5,7-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one (32b). 2-Amino-4,6-dimethyl-nicotinamide (31b) (24.5 g,
148.3 mmol) was dissolved in 100 ml of methanol and after addi-
tion of a solution of 20.8 g (370.8 mmol) KOH pellets in 100 ml of
methanol the mixture was stirred at rt for 30 min. Then, the mix-
ture was cooled to -5 °C and iodobenzene diacetate (47.8 g,
148.3 mmol) was added within 30 min. Stirring was continued
overnight at rt. Then, the product was filtered off, washed with
methanol and dried under HV. Yield: 23.8 g (98%) of a colorless
solid. 1H NMR (400 MHz, DMSO-d6): d 6.34 (s, 1H), 5.0 (br, 2H),
2.28 (s, 3H), 2.17 (s, 3H); MS (ESI): 164.4 [M+H]+.
4.1.7.7. Synthesis of (E)-2-ethyl-3-(4-(3-(4-isopropylpiperazin-1-yl)
prop-1-en-1-yl)benzyl)-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (39c).
(E)-3-(4-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)
methyl)phenyl)prop-2-en-1-ol (38c) (200 mg, 0.622 mmol) was
dissolved in 6 ml of propionitrile and after addition of
4.1.7.3. Synthesis of 2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine
(35c). A mixture of 5,7-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-
one (32b) (10 g, 61.3 mmol), propionic acid (87 ml, 1.18 mol),
propionic anhydride (88 ml) and MgCl2 (5.83 g, 61.3 mmol) was
stirred overnight at 145 °C. Then the mixture was cooled to rt,
50 ml of methanol was added followed by 25% ammonia until a
pH of 9 was reached. The mixture was extracted by ethyl acetate,
the organic layers were washed with brine, dried over Na2SO4 and
concentrated. The crude product was purified by MPLC (silica gel,
methanol/CH2Cl2, 2–5%). Yield: 4.85 g (45%). 1H NMR (400 MHz,
DMSO-d6): d 12.4 (br s, 1H), 6.85 (s, 1H), 2.82 (q, J = 8 Hz, 2H)
2.52 (s, 6H), 1.32 (t, J = 8 Hz, 3H); MS (ESI): 176.1 [M+H]+.
N-isopropyl-piperazine
trimethyl-phosphonium iodide (206 mg, 1.56 mmol) and diiso-
propylethylamine (534 l, 3.1 mmol) the mixture was stirred for
(89
ll,
0.622 mmol),
cyanomethyl-
l
2 h at 120 °C. Then the mixture was concentrated under reduced
pressure. The residue was diluted with ethyl acetate, washed with
10% aqueous K2CO3 solution and brine, dried over Na2SO4 and con-
centrated. The crude product was purified by MPLC (silica gel,
methanol/ethyl acetate 1:9) which provided 244 mg (73%) of the
product as a yellow oil. 1H NMR (400 MHz, DMSO-d6): d 7.37 (d,
J = 8 Hz, 2H), 7.05 (d, J = 8 Hz, 2H), 6.94 (s, 1H), 6.46 (d, J = 16 Hz,
1H), 6.23 (dt, J = 16, 6 Hz, 1H), 5.43 (s, 2H), 3.03 (d, J = 6 Hz, 2H),
2.77 (q, J = 8 Hz, 2H), 2.59 (sept, J = 6 Hz, 1H), 2.25–2.5 (m, 8H),
1.23 (t, J = 8 Hz, 3H), 0.96 (d, J = 6 Hz, 6H); HRMS (ESI): calcd. For
4.1.7.4. Synthesis of 3-(4-bromobenzyl)-2-ethyl-5,7-dimethyl-3H-imi-
dazo[4,5-b]pyridine (36c). 2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]
pyridine (35c) (3 g, 17.1 mmol) was dissolved in 50 ml of DMF.
After addition of sodium hydride (60% in mineral oil, 493 mg,
20.5 mmol) in portions, the mixture was stirred for 20 min at rt.
4-Bromobenzylbromide (4.28 g, 17.1 mmol) was added slowly
and the mixture was stirred for 2 h at rt. Then the mixture was
concentrated under reduced pressure. The residue was diluted
with ethyl acetate, washed with saturated NaHCO3 solution and
brine, dried over Na2SO4 and concentrated. The crude product
was purified by MPLC (silica gel, ethyl acetate/cyclohexane 2:3)
which provided 2.5 g (42%) of the product as a colorless solid. 1H
C
27H37N5 [M+H]+ 432.31217, found 432.31219; HPLC (long
method): tR = 3.01 min (100%).
4.2. Biology
4.2.1. High throughput screen
The assay was performed with COS-7 cells transiently trans-
fected with human GPR4 receptor cDNA with Lipofectamine.
900,000 compounds (Novartis owned library comprising diversity
set and natural product compounds) were screened at a final com-
NMR (400 MHz, DMSO-d6):
d
7.5 (d, J = 8 Hz, 2H), 7.05 (d,
pound concentration of 5 lM in 1536 well plates. pH-induced for-
J = 8 Hz, 2H), 6.93 (s, 1H), 5.41 (s, 2H), 2.76 (q, J = 8 Hz, 2H), 2.51
(s, 3H), 2.49 (s, 3H), 1.22 (t, J = 8 Hz, 3H); MS (ESI): 344, 346
[M+H]+.
mation of cAMP was determined using the homogeneous time
resolved fluorescence (HTRF) technology as provided by CisBio
Inc. 1605 primary hits (threshold >40% inhibition) were tested in
Please cite this article in press as: Miltz W., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.050

W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
a concentration-dependent manner of which 13% confirmed with
an IC50 value below 23 M. Compound specificity and selectivity
were tested against TDAG8 and the endogenous beta2-adrenergic
4.2.4. hERG binding assay
l
HEK293 cells stably transfected with the HERG-1 gene (Swiss-
Prot: Q12809) were obtained under license from the Wisconsin
Alumni Research Foundation. Membranes prepared from these
cells were used in the binding assay using the radioligand: [3H]
dofetilide with specific activity of 46.5 Ci/mmol. The assay contains
receptor in COS-7 cells.
The cells were grown in Dulbecco’s Modified Eagle Medium
(DMEM)/HAM’s tissue culture medium F12 (HAM’s F12) medium
supplemented with 10% fetal calf serum (FCS), 100 u/ml penicillin,
100 mg/ml streptomycin and 10 mM Hepes pH 8.2. Transiently
transfected cells were harvested in Hepes buffer (pH = 8.2) (Hepes
buffered saline (HBS), 20 mM Hepes). 100 nl compound was trans-
ferred prior to cell addition to the assay plate. 2 ml cells were
seeded in 1536-well plates at a density of 2000 cells/well. 2 ml buf-
fer (Hepes buffered saline (HBS), 8 mM Hepes/EPPS/MES, 2 mM 3-
isobutyl-1-methylxanthin (IBMX)) at appropriate pH were added
to the compound wells to reach the final pH of 6.5 for stimulation
and pH 8.2 in control wells. Cells were incubated for 30 min at
room temperature. 2 ml of cAMP-XL 665 and 2 ml anti cAMP-cryp-
tate were dispensed and plates were read on a Viewlux (Perkin
Elmer) reader after 90 min incubation at rt in the dark. Data were
calculated from the 665 nm/620 nm ratio and% activity was nor-
malized according to values at minimum and maximum of GPR4
activation.
in a 96-well Millipore GF/C filter plate 119
pound, 40 l crude membrane sus-
l 12.5 nM [3H]dofetilide, 40
pension (15 g protein). After for 90 min at room temperature
incubations are terminated by rapid filtration, three washes with
ll buffer, 1 ll com-
l
l
l
ice-cold buffer. The dried including 40
Wallac MicroBeta Trilux beta-counter.
ll scintillant was read in a
4.2.5. Histamine H3 receptor binding assay
Lyophilized PVT PEI-treated wheat germ agglutinin SPA beads,
type A (RPNQ0003) were purchased from GE Healthcare (Bucking-
hamshire, UK). [3H]-R(ꢀ)-alpha-Methyl[imidazole-2.5(n)]his-
tamine, an agonist radioligand, was purchased from GE
Healthcare, TRK1017, specific activity 34 Ci/mmol, 1 mCi/mL).
Membrane prepared from CHO-K1 cells stably expressing the
human H3 histamine receptor were purchased from Euroscreen
(ES-392-M). The SPA assay was performed in a final volume of 50
mL in 384-well polystyrene plate (10 mL test compounds, total bind-
ing was determined by adding 10 mL water and non-specific bind-
ing was determined by the addition of 10 mL Clobenpropit, 20 mL
7.5 nM [3H]-R-alpha-Methylhistamine in assay buffer [50 mM
Tris-HCl, 5 mM EDTA, 1 mM EDTA, pH 7.4], 20 mL beads and mem-
branes mixed suspension in assay buffer). The plates were shaken
at room temperature, then allowed to stand for at least 1 h and
then counted using a Perkin Elmer TopCount reader.
4.2.2. pH-dependent cAMP release assay (human GPR4 in HeLa, rat
and mouse GPR4 in HEK cells)
HeLa and HEK cells stably expressing human, rat and mouse
GPR4, respectively, were established by transfecting the cells with
a construct containing the respective GPR4 coding sequence. The
cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)/
HAM’s tissue culture medium F12 (HAM’s F12) medium supple-
mented with 10% fetal calf serum (FCS), 100 u/ml penicillin,
100 mg/ml streptomycin and 400 mg/ml G418 and 10 mM Hepes
pH 8.0. pH-induced formation of cAMP was determined using the
homogeneous time resolved fluorescence (HTRF) technology as
provided by CisBio Inc. The cells were seeded in 384-well plates
and cultured for 24 h at 37 °C, 5% CO2 before performing the assay.
Medium was removed and 10 ml buffer A (Hepes buffered saline
(HBS), 10 mM Hepes, pH 8, 2 mM 3-isobutyl-1-methylxanthin
(IBMX)) was added. For compound testing, buffer A with 2ꢂ con-
centrated compounds was used. Cells were incubated for 15 min
at room temperature. 10 ml buffer B (HBS, 30 mM Hepes, specific
pH) was added to reach the appropriate final pH for stimulation
and incubation was continued for 15 min at room temperature.
Finally, 10 ml of cAMP-XL 665 and 10 ml anti cAMP-cryptate were
dispensed and plates were read on a Pherastar reader after
60 min incubation at rt. Data were calculated from the
665 nm/620 nm ratio and% activity was normalized according to
values at minimum and maximum of GPR4 activation.
4.2.6. Mouse VEGF-induced angiogenesis model
The experiments were performed in accordance with the local
VET authorities under license BS-1325. Porous tissue chambers
made of perfluoro-alkoxy-Teflon (Teflon-PFA, 21 ꢂ 8 mm diameter,
550
ll volume) were filled with 0.8% agar and 20 U/ml heparin
supplemented with or without
2 lg/ml recombinant human
VEGF165. Female FVB mice of 8–10 weeks old were anesthetized
using 3% Isoflurane inhalation. For subcutaneous implantation, a
small skin incision was made on the flank and one chamber per
animal was implanted under aseptic conditions onto the back of
the animal. The skin incision was closed by wound clips. Animals
were placed in groups of 6. On the 4th day after implantation, ani-
mals were sacrificed using CO2. Chambers were excised and the
vascularized fibrous tissue formed around each implant carefully
removed and weighed. Body weight was used to monitor the gen-
eral condition of the mice. Compound 39c was dosed orally twice a
day at 10, 3, 1 and 0.3 mg/kg for 4 days and was started 5 h prior to
implantation of the chamber. The experiment was repeated twice
for 30 mg/kg, 5 times for 10 mg/kg, 3 times for 3 mg/kg and was
performed once for 1 and 0.3 mg/kg.
4.2.3. TDAG8 cAMP formation assay
Confluent TDAG8-transfected HeLa cells were grown in 24-well
plates and labelled with [3H]adenine (100 MBq/ml) for 4 h in
serum-free DMEM medium. Cells were then incubated at 37 °C in
a buffered salt solution containing 130 mM NaCl, 0.9 mM NaH2PO4,
5.4 mM KCl, 0.8 mM MgSO4, 1.0 mM CaCl2, 25 mM glucose, 20 mM
Hepes. The pH of the solutions was adjusted to pH 7.9, pH 7.0 and
pH 6.8 at room temperature. The phosphodiesterase inhibitor
isobutylmethylxanthine (1 mM, IBMX) was added to allow accu-
mulation of cAMP. Antagonist compounds were added 10 min
prior to IBMX addition. Incubation was continued for 15 min. Cells
were then extracted with ice-cold trichloroacetic acid and cAMP
separated from free adenine and ATP using batch column
chromatography.
4.2.7. Rat antigen-induced arthritis
Female Lewis rats (120–150 g) were sensitized intradermally on
the back at two sites with methylated bovine serum albumin
(mBSA) homogenised 1:1 with complete Freund’s adjuvant on days
ꢀ21 and ꢀ14 (0.1 ml containing 1 mg/ml mBSA). On day 0, the
right knee received 50
(antigen injected knee), while the left knee received 50
l
l of 10 mg/ml mBSA in 5% glucose solution
l of 5%
l
glucose solution alone (vehicle injected knee). The diameters of
the left and right knees were then measured using calipers imme-
diately after the intra-articular injections and again on days 2, 4
and 7. Treatments were administered daily as follows by oral gav-
age; vehicle (saline) at 5 ml/kg, compounds as indicated. Right
Please cite this article in press as: Miltz W., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.050

14
W. Miltz et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
knee swelling was calculated as a ratio of left knee swelling, and
the R/L knee swelling ratio plotted against time to give Area Under
the Curve (AUC) graphs for control and treatment groups. Right
and left knees were fixed, embedded and processed for histological
analysis. Sections were stained for both nuclear morphology (Hae-
matoxylin and Eosin) and proteoglycan content (Giemsa).
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intra-plantar injection of 25 ll of Complete Freund’s Adjuvant
(CFA) into one hindpaw with the contralateral paw acting as the
control. After 3 days, compound 39c (3, 10, and 30 mg/kg) or
diclofenac (30 mg/kg) were administered by gavage as suspension
in methylcellulose 5%. One hour later, paw withdrawal thresholds
were re-measured on both the ipsilateral (CFA-injected) and con-
tralateral (uninjected) paw (one measurement per paw and time
point); measurements were repeated at 3 h and 6 h post dosing.
The reversal of hyperalgesia was calculated using the following for-
mula: Reversal (%) = 100 ꢂ (postdose ipsilateral threshold ꢀ pre-
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Acknowledgements
The authors thank Dr. Ivan Cornella-Taracido, Dr. Timothy Hur-
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.050.
Please cite this article in press as: Miltz W., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.050