Discovery of a dihydropyrimidine series of molecules that selectively mimic the biological actions of calcitonin

Article abstract of DOI:10.1016/j.bmcl.2003.12.071

The use of a multiplex mimetic assay led us to identify 1,4-dihydropyrimidines with potent and selective calcitonin receptor mimetic activity. Subsequent modification of the dihydropyrimidine scaffold led to a series of molecules that were efficacious in a neonatal mouse calvaria in vitro model. Dihydropyrimidine 5h, in particular, was identified as a calcitonin mimetic (EC₅₀=6 μM), active in-vivo in the Weanling rat model when administered subcutaneously.

Full text of DOI:10.1016/j.bmcl.2003.12.071

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1155–1159
Discovery of a dihydropyrimidine series of molecules that
selectively mimic the biological actions of calcitonin
Jay M. Matthews,a,* Fina Liotta,b William Hageman,a Ralph A. Rivero,a,y
b
Lori Westover,b Maria Yang,b Jun Xub andKeith Demarest
aDrug Discovery, Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA 19477-0776, USA
bRaritan, New Jersey 08869, USA
Received26 September 2003; revised12 December 2003; accepted18 December 2003
Abstract—The use of a multiplex mimetic assay led us to identify 1,4-dihydropyrimidines with potent and selective calcitonin
receptor mimetic activity. Subsequent modification of the dihydropyrimidine scaffold led to a series of molecules that were effica-
cious in a neonatal mouse calvaria in vitro model. Dihydropyrimidine 5h, in particular, was identified as a calcitonin mimetic
(EC50=6 mM), active in-vivo in the Weanling rat model when administered subcutaneously.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
(GIP-1; clone #1), parathyroidhormone receptor 1
(EPTH-1-1; clone #1) andcalcitonin gene relatedpep-
tide-1 (CGRP1-7; cone 7)—were cloned onto the human
embryonic kidney (HEK 293) cell line and plated toge-
ther in one assay well.
Calcitonin, a 32 amino acidpolypeptied hormone
secretedby the thyroidandthymus glands, plays an
important role in inhibiting bone resorption through the
mediation of osteoclasts. By inhibiting bone resorption
andpromoting renal calcium excretion, calcitonin has
therapeutic applications in a variety of clinical dis-
The compounds (1–4) in Figure 1 are exemplars of an
active series of 1,4-dihydropyrimidines that stimulated
orders, including hypercalcemia associated with Paget’s
disease1 andosteoporosis.
2,3
Although, salmon-calcito-
nin (s-CT) has been effective in decreasing bone resorp-
tion via parenteral administration, its widespread use is
limitedbecause of a short half-life andlack of com-
pliance due to the need for multiple injections. To date,
6
only the Suntory group4,5 andthe Zymogenetics group
have reportednon-peptidic calcitonin mimetics. There-
fore, we sought to overcome the problems associated
with s-CT by identifying novel non-peptide mimetics of
calcitonin that are selective andorally bioavailable.
To screen our internal library, a multiplex mimetic cell
7
basedassay was designed for high-throughput screen-
ing. In an effort to differentiate activity amongst similar
G-protein coupledreceptors, 6 cell lines—calcitonin
receptor-2 (CTR-2; clone #33), glucogen-like peptide 1
(GLP1-7; clone #7), gastric inhibitory polypeptide
Keywords: Calcitonin; Dihydropyrimidine; GPCR.
* Corresponding author. Tel.: +1-215-628-5214; fax: +1-215-628-
3297; e-mail: jmatthew@pruis.jnj.com
y
Present address: GlaxoSmithKline, 709 Swedeland Road, PO Box
1539, King of Prussia, PA 19406, USA.
Figure 1. Structures of 1,4-dihyropyrimides (1–4) from the multiplex
mimetic assay.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.071

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J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1155–1159
Table 1. Foldincrease a data for compounds 1–4 in the multiplex cell-basedassay 7
b
CompdMuliplex
CTR-2HEK
GLP1-7HEK
GIP-1HEK
GLU-1HEK
EPTH1-1HEK
CGRP1-7HEK
(6 cell lines)
1
2
3
4
4.5/5.7
4.7/4.0
4.7/3.2
5.1/4.4
8
12
8
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
9
a pmol of cAMP accumulation simulated by the compound/basal level of cAMP produced by the cell line @ 25 mM.
b Data for multiple screens; NA=no activity.
cAMP accumulation in HEK 293 cells expressing the
CTR-2 ligand. The activity and selectivity of these
compounds are outlined in Table 1 with activity shown
as a measurement of fold-induction (FI).
boxylic acid( 6). Standard coupling conditions of (6)
with benzyl alcohols (7) provide target compounds (8),
as shown in Scheme 2.
In a single effort, we were able to identify a lead series
that demonstrated selectivity against the parental cells
and additional GPCR’s.
3. Results and discussion
To elucidate the structure–activity relationship of the
dihydropyrimidine series, modifications of the (R1) C-6
side-chain, the ester appendage (R2) andthe C-4 aryl
(R3) where examined. The cAMP accumulation in HEK
293 cells in the CTR-2 receptor was measuredand
recorded as fold increase.
Herein we report the synthesis andbiological activity of
a 1,4-dihydropyrimides series. The chemistry necessary
to develop structure–activity relationships was accom-
plishedusing the well-odcumentedBiginelli reaction,
shown in Schemes 1 and2 .
Compound 5a, shown in Table 2, was our first entry
into the 1,4-dihydropyrimidine series and by simply
modifying the ester linkage and the C-4 aryl moiety
(R3), we achieveda 4 to 5-foldimprovement over com-
pounds 1–4, in regards to fold-increase. Addition of a 3-
methyl phenyl (5b) onto R3 afforded a compound with a
modest increase in potency. Lengthening the R1 alkyl
chain from a methyl (5b) to a propyl (5c) resultedin a 2
to 3-foldincrease in activity, however, further increases
ledto compounds which were poorly soluble in the
biological media, exemplified by compound 5d. The
benzyl ester andthe ethyl ester ( 5e) analogues were
equipotent. Replacement of the 3-methyl phenyl moiety
(5e) with a 3-chloro-phenyl group (5f) or the 3,5-dime-
thyl phenyl group (5g) at R3 did not significantly alter
activity. Compounds with hydrogen bonding groups,
such as a 4-methoxy benzyl (5h) at R2 were synthesized
andequipotent to compound 5c, andfurther manipu-
lations ledto the 4-methylester benzyl analogue 5i, the
2. Chemistry
1,4-dihydropyrimidines (5) of interest were assembled
via a multi-component reaction of an aldehyde, urea,
and b-keto ester as shown in Scheme 1.8,9 Using parallel
synthesis, we utilizedthis reaction for the rapidsynth-
esis of diverse compounds. In the instance when
R2=benzyl (5c), catalytic hydrogenation afforded car-
Scheme 1. (a) ArCHO, urea, MeOH, cat. HCl, 65 ꢀC.
Scheme 2. (a) H2, Pd/C, EtOAc:EtOH (9:1), 25 ꢀC; (b) CH2Cl2:DMF (2:1), EDC, cat. DMAP, 7, 65 ꢀC; (c) CH2Cl2, EDC, R4R5NH, 25 ꢀC.

J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1155–1159
1157
first low micromolar compoundin the series. Replace-
ment of the ester in 5i with an acidmoiety ( 5j) did not
significantly change the activity.
response towardthe inhibition of bone resorption in the
calcium-45 labeledmouse calvaria.
Compound 5h was further evaluatedin a Weanling rat
study.11 Weanling rats are a species that undergo rapid
bone remodeling, thus have high rates of bone resorption.
The rats are placedon a low calcium diet prior to the
study to ensure that the source of calcium in the blood
will be dependent on bone resorption and not the diet.
After dosing the compounds, the urine is collected and
analyzedfor urinary deoxypyridinolines, a common bone
marker. Figure 3 depicts s-CT, dosed at 1 mpk, and
compound 5h, dosed at 100 mpk, both subcutaneously.
Several compounds were evaluated in a bone organ
culture assay. This assay is an in vitro bone resorption
assay in which the % inhibition of parathyroidhormone
(PTH) stimulatedbone resorption is measuredin neo-
natal mouse calvaria.10 Figure 2 demonstrates the effi-
cacy of compounds 5a, b, e, and h at 10, 30 and100 mM,
as well as s-CT as a control at 0.01, 1 and3 nM. Though
the dihydropyrimidines are several orders of magnitude
less potent than s-CT, there is a clear dose-dependent
Table 2. The foldincrease andEC 50 of 1,4-dihydropyrimidine 5
CompdR
R2
R3
FIa
EC50 (mM)a
1
5a
5b
5c
5d
5e
5f
5g
CH3
CH3
CH2C6H5
CH2C6H5
CH2C6H5
CH2CH3
CH2CH3
Ph
17
23
36
31
35
21
34
33
42
61
40
>20 (n=1)
15 (n=1)
5.2 (n=1)
NDb
7.5 (n=2)
6.8 (n=1)
9.5 (n=1)
5.8 (n=4)
1.0 (n=2)
3.4 (n=7)
0.003
3-CH3-Ph
3-CH3-Ph
3-CH3-Ph
3-CH3-Ph
3-Cl-Ph
3,5-CH3-Ph
3-CH3-Ph
3-CH3-Ph
3-CH3-Ph
(CH2)2CH3
(CH2)4CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH3)2CH
(CH2)2CH3
(CH2)2CH3
CH2CH3
CH2CH3
CH2C6H4-4-OCH3
CH2C6H4-4-CO2CH3
CH2C6H4-4-COOH
5h
5i
5j
sCalcitonin
a Activity of compounds (assessed at 75 mM) were expressedas pmol of cAMP andfoldincrease above vehicle ꢁ5%. Potency of the compounds
were determined by performing a dose–response curve starting with a concentration of 75 mM with 1:2 dilution (8 to 16 points curve) and reported
result as 50% of EMAX (EC50), ꢁ5%.
b ND=not determined due to lack of solubility.
Figure 2. % Inhibition of PTH stimulatedresponse for s-CT, 5a, 5b, 5e, 5h and 8b.

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J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1155–1159
Figure 3. Urinary deoxypyridinolines collected from Weanling rat for s-CT and compound 5h.
Though the dosage of s-CT and compound 5h are sig-
nificantly different, we were able to achieve efficacy
similar to s-CT in the Weanling rat, indicative that the
dihydropyrimidine series had potential as a calcitonin
mimetic.
Table 3. The foldincrease andEC 50 of dihydropyrimidine series 8
The low solubility of the dihydropyrimide series, exem-
plifiedby compound 5i (pH 2; 25 mg/mL; pH 7.4; 20 mg/
mL), andlow oral bioavailability in rats, exemplifiedby
compound 5e (2%, dosed at 100 mpk, po and 2 mpk,
iv), ledus to seek to improve the physiochemical prop-
erties of the series.
CompdR
R5
FIa
40
EC50 (mM)a
3.4 (n=1)
4
Basedon the SAR generated, the most amenable point
for modification in the molecule was at R2—the benzyl
ester. In an effort to address both the solubility and
potency issues, several compounds were prepared. As
shown in Table 3, attachments of an ethyl piperidine
(8b), dimethyl-aminopropyl (8c), or propyl piperidine (8d)
moieties afforded compounds equipotent to compound
5i. As desired, compounds in Table 3 also showed
improvedsolubility, with 184 mg/mL (pH 2) and274 mg/
mL (pH 7.4); and500 mg/mL (pH 2) and270 mg/mL
(pH 7.4) for compounds 8b and 8c, respectively. Com-
pound 8b was evaluatedfor oral bioavailability, and
showedmarkedimprovement as well, with 65% (30
mpk po, 1 mpk, iv) and13% (10 mpk, po, 1 mpk, iv) in
rats andrhesus monkeys, respectively.
8a
CH3
CH3
8b
H
H
51
55
52
55
50
55
40
1.3 (n=5)
1.4 (n=2)
1.9 (n=2)
4.3 (n=1)
4.3 (n=1)
4.4 (n=1)
0.003
8c
8d
H
8e
H
8f
H
Compound 8b was evaluatedfor its ability to inhibit
PTH stimulatedbone resorption in the mouse calvaria
model, as shown in Figure 2. As shown, 8b inhibits bone
resorption in a dose-dependent manner at concentra-
tions of 30 and100 mM.
8g
CH3
SCalcitonin
a Activity of compounds (assessed at 75 mM) were expressedas pmol of
cAMP andfoldincrease above vehicle ꢁ5%. Potency of the com-
pounds were determined by performing a dose–response curve start-
ing with a concentration of 75 mM with 1:2 dilution (8 to 16 points
curve) andreportedresult as 50% of EMAX (EC 50), ꢁ5%.
Compound 8b was next evaluatedorally in the Weanl-
ing rat model. Unfortunately, when 8b was adminis-
teredpo at 100 mpk, there was no significant decrease

J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1155–1159
1159
in the plasma calcium andserum pyriidnoline bone
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4. Conclusion
6. Baindur, N.; Labroo, U.; Stroop, S.; Beigel, S.; Martinez,
T.; Petrie, C.; Orme, M.W.; McKernan, P.A.; Moore,
E.E. WO Patent 98,37077
We have identified a novel series of calcitonin mimetics
containing a dihydropyrimidine nucleus. These com-
pounds stimulate cAMP accumulation in HEK 293 cells
expressing the CTR-2 receptor andare selective against
other G-protein coupledreceptors such as GLU-1 and
EPTH-1. Several analogues in the series inhibitedPTH
stimulatedbone resorption in a bone organ culture
assay in a dose-dependent manner and compound 5h
was efficacious in a Weanling rat model when adminis-
teredsubcutaneously. While we were able to significantly
improve the oral bioavailability andpotency, while
retaining efficacy in the mouse calvaria model, pre-
liminary oral evaluation of 8b in the Weanling rat study
showed no in vivo efficacy. While these compounds did
not demonstrate the in vivo efficacy we desired, they
may in fact serve as a template for future small molecule
calcitonin mimetic ligands to be developed.
7. A ‘multiplex’ cell basedassay was developedto increase
the screening throughput for non-peptide mimetics of
several G-protein coupledreceptors. Clonedstable
HEK293 cell lines mentionedin the text were combinedin
each assay well andassayedfor their ability to stimulate
cAMP accumulation in the cell mixture. All compounds
were testedin parallel in the HEK293s parental line that
did not contain target receptors. If a test compound sti-
mulatedcAMP in the multiplex cells andwas inactive in
HEK293s parental line, the test compoundwas deemed
active andmovedto a edconvolution assay. In the
deconvolution assay, the active compound was run again
in a cAMP accumulation assay using each of the six
cloned HEK293 cell lines individually. This ‘deconvolu-
tion’ assay determined activity and selectivity of the test
compound.
8. (a) Kappe, C. O.; Uray, G.; Roschger, P.; Lindner, W.;
Kratky, C.; Keller, W. Tetrahedron 1992, 48, 5473. (b)
Sadanandam, Y. S.; Shetty, M. M.; Diwan, P. V. Eur. J.
Med. Chem. 1992, 27, 87.
Acknowledgements
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26, 235.
We thank Mr. Eric Ericson, andMr. Pat Sasso for
assistance with the in vivo experiments andDr. Gary
Caldwell and Dr. John Masucci for assistance with
bioavailability studies and physiochemical property
determinations.
11. (a) Morgan, D. B.; Gasser, A.; Largiader, U.; Jung, A.;
Fleisch, H. Am. J. Physiol. 1975, 228, 1750. (b) Rader,
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12. While compound 8b is orally bioavailable, the PK analy-
sis (AUC 0.9 um-Hrs; Cmax 0.5 uM) suggests that, given
the modest potency, drug levels required for efficacy may
not have been achieved.
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