Discovery and characterization of novel, potent, non-peptide parathyroid hormone-1 receptor antagonists

Article abstract of DOI:10.1021/jm0707626

A 1,3,4-benzotriazepine was identified as a suitable lead in our effort toward obtaining a non-peptide parathyroid hormone-1 receptor (PTH₁R) antagonist. A process of optimization afforded derivatives displaying nanomolar PTH₁R affinity, a representative example of which behaved as a PTH₁R antagonist in cell-based cyclic adenosine monophosphate (cAMP) assays, with selectivity over PTH₂ receptors.

Full text of DOI:10.1021/jm0707626

© Copyright 2007 by the American Chemical Society
Volume 50, Number 20
October 4, 2007
Letters
Table 1. Biological Data for 1,3,4-Benzotriazepine-Based Compoundsa
Discovery and Characterization of Novel, Potent,
Non-Peptide Parathyroid Hormone-1 Receptor
Antagonists
Iain M. McDonald,* Carol Austin, Ildiko M. Buck,
David J. Dunstone, John Gaffen, Eric Griffin,
Elaine A. Harper, Robert A. D. Hull, S. Barret Kalindjian,
Ian D. Linney, Caroline M. R. Low, Dipa Patel,
Michael J. Pether, Michelle Raynor, Sonia P. Roberts,
Mark E. Shaxted, John Spencer, Katherine I. M. Steel,
David A. Sykes, Paul T. Wright, and Wei Xun
b
compd
R
PTH1
1
2
3
4
5
6
4-C6H4NHC(dNH)NH2
4-C6H4NH2
6.15 ( 0.12
39% (1 µM)c
<4
4-C6H4CO2H
3-C6H4NHC(dNH)NH2
(CH2)3NHC(dNH)NH2
4-CH2C6H4NHC(dNH)NH2
5.55
James Black Foundation, 68 Half Moon Lane,
Dulwich, London, SE24 9JE, U.K.
51% (30 µM)c
6.03 ( 0.12
ReceiVed June 27, 2007
a Data were generally obtained from at least three separate experiments.
Where no SEM is recorded, the data were obtained from two experiments.
b pKI ( SEM values obtained from competition with [125I]-[Nle,8,18 Tyr34]-
hPTH(1-34) for recombinant hPTH1Rs in HEK293 cell membranes.
c Where pKI could not be determined, the percentage inhibition achieved
at the given concentration is recorded.
Abstract: A 1,3,4-benzotriazepine was identified as a suitable lead in
our effort toward obtaining a non-peptide parathyroid hormone-1
receptor (PTH1R) antagonist. A process of optimization afforded
derivatives displaying nanomolar PTH1R affinity, a representative
example of which behaved as a PTH1R antagonist in cell-based cyclic
adenosine monophosphate (cAMP) assays, with selectivity over PTH2
receptors.
lytic osteoclasts. Not only does the resultant osteolysis exac-
erbate tumor cell metastasis but the attendant bone matrix
cytokine release and local elevated Ca2+ also promote tumor
cell proliferation in addition to stimulating further release of
tumor-derived PTHrP.1
There is now credible evidence of a beneficial role in MAHC
and in osteolytic metastasis for therapeutic interventions that
block the interaction of PTHrP with its receptor. Unlike
parathyroid hormone (PTH) (an 84 amino acid peptide circulat-
ing hormone produced by the parathyroid glands), which
interacts with two distinct type B G-protein-coupled receptors
(GPCRs), PTH1 and PTH2, PTHrP is a selective stimulant of
PTH1 receptors.
The hypercalcaemia and skeletal metastasis induced by
inoculation of animals with PTHrP-producing cancer cells was
prevented by using an anti-PTHrP antibody (Ab).2,3 The pivotal
role played by PTHrP in this setting was apparent, since the
oncological phenotype was more pronounced in animals inocu-
lated with cancer cells expressing PTHrP than in those with
cancer cells that expressed little or none.4,5 Moreover, anti-
PTHrP Ab treatment did not have a significant effect on the
metastasis to visceral organs, indicating the importance of the
bone environment in this pathology.6 Anti-PTHrP Ab treatment
End-stage cancer is often accompanied by hypercalcaemia.
This condition, known as malignancy-associated hypercalcaemia
(MAHCa), is caused by parathyroid hormone related peptide
(PTHrP), otherwise not normally present in the circulation, being
secreted by tumor cells and acting on PTH1 receptors (PTH1-
Rs) on the kidney and/or on osteoblasts. Stimulation of this latter
target results in particularly debilitating sequelae, since bone is
a fertile environment for metastasized tumor cells including
those from breast, lung, prostate, kidney, and multiple myeloma.
Osteoblasts, once activated by tumor cell derived PTHrP, are
bone forming, but their stimulation results in production of
secondary signaling molecules that promote maturation of bone
* To whom correspondence should be addressed. Phone: +44-(0)20-
7737-8282. Fax: +44-(0)20-7274-9687. E-mail: iain.mcdonald@kcl.ac.uk.
a Abbreviations: MAHC, malignancy-associated hypercalcaemia; PTHrP,
parathyroid hormone related peptide; PTH1Rs, PTH1 receptors; PTH,
parathyroid hormone; GPCRs, G-protein-coupled receptors; Ab, antibody;
hPTH1Rs, human PTH1 receptors; CGRP, calcitonin gene-related peptide;
CRLR, calcitonin receptor-like receptor; RAMP, receptor activating modify-
ing protein; ADME, absorption, distribution, metabolism, and excretion;
cAMP, cyclic adenosine monophosphate.
10.1021/jm0707626 CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/12/2007

4790 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Scheme 1. Synthesis of 1,3,4-Benzotriazepinesa
Letters
a
Reagents and conditions: (i) (a) (Cl3CO)2CO, NEt3, R1NHNH2/DCM; (b) p-TSA/i-PrOH; (ii) (a) Cl2CS, NEt3, R1NHNH2/DCM, then p-TSA/i-PrOH;
(b) NaOMe, H2O2/MeOH-THF; (iii) (a) (Cl3CO)2CO, NEt3, 39a-d/DCM; (b) CF3CO2H/DCM; (iv) KNO3, H2SO4; (v) SnCl2‚H2O /EtOAc; (vi) phthalic
anhydride/DMF; (vii) Cu(OAc)2, Cu, R′′′F, K2CO3/DMF; (viii) NaH, R′′′Br/DMF; (ix) CF3CO2H, ∆; (x) NaH, R1Br/DMF; (xi) Br2, HOAc/DCM; (xii)
piperidine/DCM; (xiii) BBr3/DCM; (xiv) K2CO3, BnBr/DMF; (xv) NaOH/EtOH; (xvi) N2H4‚H2O/EtOH; (xvii) 43a, 43b, HgCl2/DCM; (xviii) 43c, 43d,
EDCI, HOBt, DMAP/DMF; (xix) 43e-g, NaBH(OAc)3, HOAc/DCE; (xx) PhCHO, NaBH(OAc)3, HOAc/DCE; (xxi) HCl/dioxane; (xxii) CF3CO2H, room
temp; (xxiii) AcOH/MeOH. b See Supporting Information for definitions of R′′′, R′′, R′′, R, R1, R2, R3, R4. c See Tables 1 and 2 for definitions of R, R1, R2,
R3, R4.
also stimulated lean body growth and inhibited hypercalcaemia
in a mouse model of lung cancer-induced cachexia.7 There is
also evidence of direct proliferative effects of PTHrP on tumor
cell growth,8,9 suggesting that PTH1R stimulation may also
contribute to the progression of certain primary tumors.
Inhibition of the effects of PTHrP has also been achieved
with PTH1R antagonists.10 Until recently, these have been
limited to peptide-based compounds derived largely from
truncation of the N-terminal region of PTH,11 PTHrP,12 or TIP39
(a selective stimulant of PTH2 receptors).13,14 We considered
that a small molecule, non-peptide PTH1R antagonist, may have
greater therapeutic potential. The first compound of this type
has only been described recently15 and follows the first report
of a non-peptide PTH1R agonist.16 Herein, we describe our work
in the discovery and characterization of a novel series of small-
molecule, non-peptide PTH1R antagonists.
When a selected subset of compounds from our collection
were screened in a radioligand binding assay using recombinant
human PTH1 receptors (hPTH1Rs) expressed in HEK293 cells,
the 1,3,4-benzotriazepine-based compound 1 was found to
display micromolar affinity (Table 1). Using 1 as a lead, we
embarked on a program of optimization aimed at obtaining
derivatives (Scheme 1) with suitable potency and physicochem-
ical and ADME characteristics to explore in osteolytic metasta-
sis.
derivative was less potent than 1. The difference in affinity was
smaller for the phenyl-substituted compound (8) and absent
when a cyclohexylmethyl group (9) was present. Tolerance of
this latter substituent allowed introduction of more polar isosteric
groups, such as piperidin-1-ylmethyl (10), but the resulting
compounds were generally less active.
While initial examples had indicated a requirement for a
suitably located guanidine substituent, other basic groups were
at least as effective. For instance, 11, obtained on acylation of
the N-1 aniline with glycine, was not significantly different in
affinity from 1. Marginally higher affinity was achieved for the
valine derivative 12. The cyclic guanidine-containing 13 showed
a similar trend toward greater PTH1R affinity.
The synthetic route readily allowed introduction of substit-
uents on the benzo-fused ring, with location of a methyl group
in the C-8 position (14-16) proving particularly beneficial.
Around 5-fold greater affinity relative to the unsubstituted
analogues (1, 12, and 13) was realized with this change,
irrespective of the nature of the basic group on the N-1
substituent. The 8-methyl substituent was retained while explor-
ing the effect of alternative basic groups, leading to the
2-imidazolylalkyl-containing derivative 17, wherein the PTH1R
affinity was around 4 nM. Still higher affinity was achieved by
increasing the lipophilic character of the N-3 substituent, but
the high molecular weight and physicochemical properties of
derivatives of this type such as 18 did not satisfy our preferred
compound profile. Accordingly, we considered less bulky groups
while retaining substituents elsewhere that had conferred high
affinity in earlier compounds. For instance, the methyl analogue
19, in which the bulk in this position was greatly reduced relative
to the benzyl substituent present in 17, was only 3-fold less
potent. Altering the length of carbon chain and linking the
imidazol-2-yl group of 19 with the anilide did not offer any
particular advantage, as judged by lower affinity evident for
Initial derivatives of 1 (Table 1) suggested that a p-
guanidinylphenyl N-1 substitutuent was preferred, since com-
pounds obtained on replacement of guanidine by amino (2) and
carboxy (3) were less potent. Lower affinity also resulted when
guanidine was located in the meta position (4) and when the
phenyl ring was replaced by propyl (5). Only the p-guanidi-
nylbenzyl derivative (6) displayed comparable affinity to 1.
A minimum size requirement for the substituent at the C-5
position was also apparent (Table 2), since the isopropyl (7)

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4791
Table 2. Biological Data for N-1 Anilino-Substituted 1,3,4-Benzotriazepinesa
b
compd
R1
R2
R3
R4
X
PTH1
7
8
9
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
i-Pr
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
C(dNH)NH2
C(dNH)NH2
C(dNH)NH2
C(dNH)NH2
COCH2NH2
COCH(i-Pr)NH2
4,5-dihydro-1H-imidazol-2-yl
C(dNH)NH2
43% (10µM)c
5.47
6.25
c-C6H11CH2
piperidin-1-yl-CH2
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
c-C6H11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
27% (10µM)c
6.17 ( 0.10
6.42 ( 0.12
6.54 ( 0.06
6.81 ( 0.03
6.95 ( 0.05
7.15 ( 0.02
8.42 ( 0.10
9.04 ( 0.06
7.88 ( 0.16
7.30 ( 0.08
7.69 ( 0.04
8.17 ( 0.14
8.45 ( 0.13
8.84 ( 0.11
8.74 ( 0.05
8.17 ( 0.06
6.21
Bn
Bn
Bn
COCH(i-Pr)NH2
4,5-dihydro-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
CH2-1H-imidazol-2-yl
(CH2)3-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
(CH2)2-1H-imidazol-2-yl
3-benzosuberanyl
Me
Me
Me
n-Pr
i-Pr
Me
Me
3-benzosuberanyl
Me
3,4-dihydro-2H-1,5-benzodioxepin-3-yl
MeO(CH2)2
BnNH
BnO
H
H
H
4-tetrahydropyranyl
4-tetrahydropyranyl
c-C6H11
c-C6H11
c-C6H11
8.06 ( 0.31
7.08 ( 0.02
8.18 ( 0.11
H
H
4-tetrahydropyranyl
a Data were generally obtained from at least three separate experiments. Where no SEM is recorded, the data were obtained from two experiments. b pKI
( SEM values obtained from competition with [125I]-[Nle,8,18 Tyr34]-hPTH(1-34) for recombinant hPTH1Rs in HEK293 cell membranes. c Where pKI
could not be determined, the percentage inhibition achieved at the given concentration is recorded.
Table 3. Biological Activity at PTH Receptors from Cell-Based
20 and 21. The reduced affinity encountered on replacing benzyl
(17) by methyl (19) was largely restored when bulkier aliphatic
Assaysa
b
c
d
compd
PTH1
PTH1
PTH2
groups were used (22, 23), with the isopropyl-containing 23
being as potent as 17. An alternative approach involving further
substitution on the benzo-fused ring (24, 25) provided com-
pounds that were at least as potent, even though only an N-3
methyl group was present. Moreover, the beneficial effect on
affinity from changes of this type appeared to be over and above
that resulting from location of a methyl group at the C-8
position.
Compounds were now available, up to 1000-fold more potent
at hPTH1Rs than our initial lead (1), that had mostly stemmed
from the introduction of relatively more lipophilic substituents.
However, this increase in lipophilicicity was tempered by
exploring the effects of analogous heteroatom-containing groups.
Of the compounds containing a C-5 tetrahydropyran-4-yl group
(26, 27), the N-3 methyl derivative 27 suffered a relatively larger
reduction in affinity with respect to its carbon counterpart (19)
in comparison with 26 and 18. For substituents in the N-3
position, comparison with a more lipophilic analogue 18 was
only possible in the case of 28, whereby the latter compound
was evidently 10-fold less potent. Of the remaining compounds
of this type (29, 30) the tetrahydropyran-4-yl-containing com-
pound 30 was the more effective, displaying <10 nM affinity
at hPTH1Rs.
PTH(1-34)
PTHrP(1-34)
TIP(1-39)
[Nle30]-TIP(7-39)
19
7.95 ( 0.07e
8.60 ( 0.19e
<5e
7.98 ( 0.16e
8.70 ( 0.22e
<5e
8.59 ( 0.38e
<5e
9.62 ( 0.09e
7.55 ( 0.28g
5.54 ( 0.12g
8.35 ( 0.19f
8.18 ( 0.35f
7.71 ( 0.33f
6.29 ( 0.12f
a cAMP accumulation in cells expressing PTH receptors. Data were
obtained from at least three separate experiments. b SaOS-2 cells expressing
wild-type, hPTH1Rs. c MC3T3-E1 cells expressing wild-type, mouse PTH1Rs.
d CHO-K1 cells expressing recombinant, hPTH2Rs. e pEC50 ( SEM values.
f pA2 ( SEM determined from shift of PTHrP(1-34) concentration-effect
curve at 3 µM. g pA2 ( SEM determined from shift of TIP(1-39)
concentration-effect curve at 10 µM.
µM in SaOS-2 cells (a human osteocarcoma cell line expressing
endogenous PTH1Rs) but produced a dose-dependent rightward
shift of the PTHrP(1-34) concentration-effect curve (pKB )
7.55 ( 0.40). Similarly, with MC3T3-E1 cells (a mouse
osteoblastic cell line expressing endogenous PTH1Rs), 19 did
not stimulate cAMP output at 3 µM but antagonized PTHrP-
evoked cAMP production, albeit with around 20-fold lower
potency than in the SaOS-2 cell-based assay (Table 3).
Moreover, 19 was shown to be at least 100-fold selective toward
PTH1Rs over PTH2Rs, based on comparison of its potency in
inhibiting TIP(1-39) stimulated cAMP production in CHO-
K1 cell membranes bearing recombinant hPTH2Rs and PTH-
(1-34)-stimulated cAMP in HEK293 cell membranes express-
ing recombinant hPTH1Rs (pA2 ) 7.77 ( 0.20).
On the basis of the PTH1R affinity displayed by many
examples of this class of compounds, we had made considerable
progress toward accomplishing our initial aims. Where exam-
ined, the compounds also behaved as antagonists as judged by
their activity profile in cell-based functional assays. For instance,
19 had no effect on cAMP production on its own at up to 10
Species-dependent activity has previously been encountered
for other non-peptide antagonists of type B GPCRs, including
those for the calcitonin gene-related peptide (CGRP) receptor17

4792 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Letters
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(15) Carter, P. H.; Liu, R. Q.; Foster, W. R.; Tamasi, J. A.; Tebben, A.
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(16) Rickard, D. J.; Wang, F. L.; Rodriguez-Rojas, A. M.; Wu, Z.; Trice,
W. J.; Hoffman, S. J.; Votta, B.; Stroup, G. B.; Kumar, S.; Nuttall,
M. E. Intermittent Treatment with Parathyroid Hormone (PTH) as
Well as a Non-Peptide Small Molecule Agonist of the PTH1 Receptor
Inhibits Adipocyte Differentiation in Human Bone Marrow Stromal
Cells. Bone 2006, 39, 1361-1372.
(17) Williams, T. M.; Stump, C. A.; Nguyen, D. N.; Quigley, A. G.; Bell,
I. M.; Gallicchio, S. N.; Zartman, C. B.; Wan, B. L.; Penna, K. D.;
Kunapuli, P.; Kane, S. A.; Koblan, K. S.; Mosser, S. D.; Rutledge,
R. Z.; Salvatore, C.; Fay, J. F.; Vacca, J. P.; Graham, S. L. Non-
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(18) McLatchie, L. M.; Fraser, N. J.; Main, M. J.; Wise, A.; Brown, J.;
Thompson, N.; Solari, R.; Lee, M. G.; Foord, S. M. RAMPs Regulate
the Transport and Ligand Specificity of the Calcitonin-Receptor-Like
Receptor. Nature 1998, 393, 333-339.
(19) Sexton, P. M.; Morfis, M.; Tilakaratne, N.; Hay, D. L.; Udawela,
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(a heterodimeric receptor comprising the calcitonin receptor-
like receptor (CRLR) and receptor activating modifying protein
(RAMP) 1). Although RAMPs have not so far been strongly
associated with PTH1Rs, it is becoming increasingly clear that
they are often coexpressed with type B GPCRs18,19 and can have
a considerable influence on receptor pharmacology,20 including
ligand binding.21 Thus, the activity of compound 19 in the
PTH1R cell-based assays may be due to differences in the
sequences of the respective PTH1Rs and/or associated RAMPs.
While the wider receptor selectivity and ADME character-
istics are beyond the scope of this Letter, we have shown that
1,3,4-benzotriazepines are potent PTH1R antagonists. The SAR
that has been established so far has guided our subsequent effort
in pursuit of compounds having the appropriate balance of
interspecies PTH1R affinity and ADME properties to establish
proof of concept for a non-peptide PTH1R antagonist as a novel
treatment for bone metastases, hypercalcaemia, cachexia, and
hyperparathyroidism.
Supporting Information Available: Biological testing methods,
1H NMR data, experimental procedures for the preparation of
compounds, and elemental analysis results of compounds 1-30.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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JM0707626