1-(2-Nitrophenyl)thiosemicarbazides: A novel class of potent, orally active non-peptide antagonist for the bradykinin B2 receptor [2]

Full text of DOI:10.1021/jm991155o

J . Med. Chem. 2000, 43, 769-771
769
1-(2-Nitr op h en yl)th iosem ica r ba zid es: A
Novel Cla ss of P oten t, Or a lly Active
Non -P ep tid e An ta gon ist for th e
Br a d yk in in B2 Recep tor
Edward K. Dziadulewicz,*,† Timothy J . Ritchie,†
Allan Hallett,† Christopher R. Snell,† Soo Y. Ko,†,#
Roger Wrigglesworth,†,∇ Glyn A. Hughes,†
Andrew R. Dunstan,†, Graham C. Bloomfield,†,
Gillian S. Drake,† Michael C. Brown,‡,| Wai Lee,‡
Gillian M. Burgess,‡,) Clare Davis,‡
Mohammed Yaqoob,‡ Martin N. Perkins,§,X
Elizabeth A. Campbell,§, Andrew J . Davis,§ and
Humphrey P. Rang§
Departments of Medicinal Chemistry, Biology, and
Pharmacology, Novartis Institute for Medical Sciences, 5
Gower Place, London WC1E 6BN, England
Received September 3, 1999
Bradykinin (BK) is an endogenous hormonal non-
apeptide (RPPGFSPFR) cleaved from high-molecular-
weight protein precursors (kininogens) by the action of
kallikrein enzymes under conditions of tissue trauma
or injury.1 The discovery of potent peptidic BK antago-
nists2 together with the availability of the cloned
receptor subtypes (B1 and B2)3,4 has led to an intense
exploration of the pharmacology of BK. The actions of
BK are mediated mainly by the B2 class of receptors,
which are constitutively expressed inter alia on nocice-
ptive neurons suggesting a principal role for this media-
tor in the perception of pain.5 Indeed, BK has been
described as the most potent algogenic substance
known.5,6 Consequently, a B2 antagonist may be of
therapeutic benefit in the symptomatic treatment of
chronic pain and various inflammatory disorders in
which elevated levels of BK are a feature.
To date, only two classes of structurally different non-
peptide B2 antagonists have been identified, the phos-
phonium-derived WIN 64338 (1)7 and the heteroaryl
benzyl ether FR 173657 (2)8 being the most thoroughly
studied representatives (Figure 1). Given the funda-
mental difficulty in projecting pharmacophoric informa-
tion from a peptide agonist onto a non-peptide scaffold,
we initiated a screening program which culminated in
the discovery of the thiosemicarbazide (TSC) derivative
3 with a Ki of 0.54 µM at the rat B2 receptor (expressed
in NG108-15 neuroblastoma-glioma hybrid cell mem-
F igu r e 1. Structures of BK B2 receptor antagonists WIN
64338 (1) and FR 173657 (2) and the thiosemicarbazide lead
3.
branes). Compound 3 was also able to antagonize the
functional response to BK with an IC50 of 2 µM in a
45Ca2+ efflux functional assay (NG108-15 cells).
An extensive structure-activity study was imple-
mented, part of which is summarized in Tables 1 and
2. The terminal furan rings of 3 could be replaced by
phenyl without loss of binding affinity (4, Ki ) 0.31 µM).
By gradual deletion of functionality from each terminus
in turn, the minimum pharmacophore required for
binding to the rat B2 receptor was established, micro-
molar affinity arising from the 1-(2-nitrophenyl)-4-
benzylthiosemicarbazide half-structure (5). Replacement
of the N(4)-benzyl moiety with a benzhydryl group
afforded the compound 6, resulting in a 42-fold gain in
affinity. Molecular recognition of the benzhydryl motif
in the field of G-protein-coupled receptor research has
been previously documented9 and supported the premise
that this part of the molecule is interacting with a
complementary hydrophobic binding site in the B2
receptor, as polar functionality is not tolerated in this
part of the molecule. With the benzhydryl group in
place, a significant advancement in the potency of the
series was observed in those analogues incorporating
simple functionality in the aryl ring para to the TSC
backbone (7 and 8).
* To whom correspondence should be addressed. Tel: 0044 171 333
2167. Fax: 0044 171 387 4116. E-mail: ed.dziadulewicz@pharma.
novartis.com.
† Department of Medicinal Chemistry.
‡ Department of Biology.
§ Department of Pharmacology.
#
Present address: Department of Chemistry, Ewha Woman’s
University, Seoul 120-750, South Korea.
3 Present address: Institut de Recherche J ouveinal, Parke-Davis,
3-9 Rue de la Loge, B.P. 100, 94265 Fresnes Cedex, France.
Present address: Novartis Horsham Research Centre, Wimble-
hurst Rd., Horsham, West Sussex RH12 5AB, England.
| Present address: Packard, Brook House, 14 Station Rd., Pang-
bourne, Berkshire RG8 7AN, England.
There were very few changes that could be made to
the TSC core which were not detrimental to binding
affinity (Table 2). Neither of the thiocarbonyl-flanking
nitrogen atoms, N(2) and N(4), could be replaced by
methylene without a complete loss in binding (9 and
10), presumably due to an unacceptable increase in
) Present address: Pfizer Central Research, Sandwich, Kent CT13
9NJ , England.
X Present address: AstraZeneca Research Center Montreal, 7171
Frederick Banting, St. Laurent, Quebec H1S 1B7, Canada.
10.1021/jm991155o CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/12/2000

770 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Communications to the Editor
Ta ble 1. BK B2 Receptor Binding Affinities (Ki) of Compounds
3-8 and 16-18a
TSCs, it was necessary to incorporate functionality
which could facilitate dissolution without compromising
receptor binding. The sulfonamide and carboxamide
groups of 7 and 8, respectively, were thus conveniently
located synthetic handles, potentially allowing further
structural elaboration. When the primary sulfonamide
group of 7 was exchanged for a bis(3-dimethylamino-
propyl)sulfonamido group, permitting salt formation
(16, Table 1) and increasing solubility (>9.7 mg/mL at
pH 7.4), a 37-fold loss in affinity was compensated by
the first demonstration of oral activity (ED50 ) 33 µmol/
kg po in the Freund’s adjuvant-induced mechanical
hyperalgesia model in the rat knee joint). Furthermore,
we found that the in vitro potency of such amphiphilic
molecules could be restored by altering the proximity
of the basic center(s) to the rest of the pharmacophore.
This was achieved by incorporation of an R-amino acid
backbone into the sulfonamide bond. Within the modest
number of amino acid residues explored, the (S)-(-)-
proline derivative 17 (Bradyzide13) emerged as the most
potent antagonist overall (Ki ) 0.5 ( 0.2 nM). Incorpo-
ration of the basic centers into a single chain, as opposed
to the bifurcated arrangement of 16, accorded 17 a
better overall in vitro profile albeit with a decreased
relative solubility (0.19-0.49 mg/mL at pH 7.4 in all
buffers tested). The synthesis of 17 is presented in
Scheme 1 and is illustrative of the approach adopted
for this class.
compdb
R1
R2
R3
R4
Ki (nM)c
3
4
5
6
7
8
-
-
-
-
-
-
-
NHCH2(2-furyl) SO2 CH2(2-furyl) 540 ( 160
NHCH2Ph
SO2 CH2Ph
310 ( 80
1600 ( 500
38 ( 8
-
H
H
CH2Ph
CHPh2
-
NH2
NH2
SO2 CHPh2
CO CHPh2
4 ( 1
1.8 ( 0.8
148 ( 70
0.5 ( 0.2
0.6 ( 0.12
16d
17d
18d
N[(CH2)3NMe2]2 SO2 CHPh2
DMEe (S)-prolyl
DMEe (R)-prolyl
SO2 CHPh2
SO2 CHPh2
a
[3H]BK binding assay in NG108-15 cell membranes expressing
b
the rat B2 receptor (see ref 13). All compounds gave satisfactory
1H NMR, mass spectra, and elemental microanalyses. c Ki values
were calculated on the basis of a one-site binding model using
LIGAND software (see ref 10). The values shown are the mean (
d
SEM (n g 3). Compound isolated as amorphous TFA salt after
preparative HPLC. e Abbreviation: DME ) 2-[(2-dimethylamino-
ethyl)methylamino]ethylamino (Me2N(CH2)2N(Me)(CH2)2NH-).
Ta ble 2. Effects of TSC Core Changes on BK B2 Receptor
Binding Affinitiesa
During the course of the present study it transpired
that the configuration of the proline C-2 stereocenter
was not an important determinant of receptor affinity.
The (R)-(+)-proline antipode 18 was found to be equi-
potent to 17, a lack of stereochemical discrimination
between the enantiomers suggesting that the proline
rings do not make intimate contact with the receptor
but nevertheless allow the conformationally flexible
amine chains to approach a common complementary
site.
compdb
R1
BnNHSO2 2-NO2
2-NO2
BnNHSO2 2-NO2
BnNHSO2 2-NO2
R2
R3
Ki (µM)c
4
5
9
10
11
12
13
14
15
NHNHCSNH
NHNHCSNH
NHCH2CSNH
NHNHCSCH2
CH2NHCSNH
CH2NHCONH
NHNHCSNH
NHNHCSNH
NHNHCSNH
0.31 ( 0.08
1.6 ( 0.5
>100
H
>100
H
H
H
H
H
2-NO2
2-NO2
2-H
3-NO2
2-CO2H
7.1 ( 4.9
>100
∼100
The presence of nitroaromatic and thiocarbonyl func-
tionality in this initial congeneric series made it impor-
tant to assess the mutagenic potential of 17. Incubation
of 17 with rat S9-liver homogenate in V79 Chinese
hamster cells did not increase the frequency of cells
containing membrane-encapsulated fragments (‘micro-
nuclei’) at any of the concentrations tested, irrespective
of the presence or absence of metabolic activation. The
compound was therefore not considered to be clastogenic
and/or aneugenic.
>100
>100
a-c See corresponding footnotes in Table 1.
backbone flexibility of these compounds although loss
of key hydrogen-bonding elements could not be ruled
out as a contributing factor. On the other hand, replace-
ment of N(1) by CH2 to afford the thiourea 11 resulted
in a tolerable 4-fold loss in affinity compared to 5. The
corresponding urea 12, however, was inactive.
Compound 17 exhibited competitive antagonism. It
inhibited the increase in 45Ca2+ efflux from NG108-15
cells caused by activation of the B2 receptor with a pA2
of 8.0 ( 0.16 (n ) 5) and inhibited B2 receptor-mediated
contractions of rat uterus smooth muscle with a pA2
value of 8.6 ( 0.13 (n ) 4). In each case the compound
caused a parallel shift in the log concentration-response
curve with no decrease in the maximum response.
Compound 17 also displayed in vivo functional antago-
nist activity against Freund’s adjuvant-induced me-
chanical hyperalgesia in rats with an ED50 value of 0.84
µmol/kg by oral administration. When dosed at 5 µmol/
kg po, compound 17 completely reversed the mechanical
hyperalgesia with a duration of action in excess of 4 h.
Preliminary pharmacokinetic studies in rats at this dose
and route of administration (n ) 4) revealed that 17
The presence of the o-nitro group in these early N(4)-
benzyl analogues was crucial to the level of binding
achieved. Deletion of the group to afford the known TSC
13,11 transposition to the meta position (14), or replace-
ment with a number of other functionalities,12 for
example, CO2H (15), afforded analogues which were all
devoid of activity. A measure of affinity could be restored
in the N(4)-benzhydryl series, but activities were gener-
ally in the 1-10 µM range, and although some trends
could be discerned (2-F > 2-Cl ≈ 2-CO2Me > 2-Br), the
nitro group remained the best ortho substituent (6, Ki
) 38 nM) and was retained for the present time.
A common physicochemical property of compounds
3-15 was their very low solubility in physiological
media, typically less than 0.0005 mg/mL at pH 7.4. To
improve the in vivo transport characteristics of the

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 771
Sch em e 1a
a
Reagents: (a) (i) ZOSu, EtOAc (85%), (ii) TsCl, py (80%); (b) N,N,N′-trimethylethylenediamine (93%); (c) HCO2NH4, Pd/C, MeOH
(95%); (d) (i) H-Pro‚OtBu, Et3N, THF, (ii) H2NNH2‚H2O (92%); (e) Ph2CHNdCdS (91%); (f) HBr, AcOH (95%); (g) iBuOCOCl, Et3N, THF,
22 (67%).
(3) (a) Hess, J . F.; Borkowski, J . A.; Young, G. S.; Strader, C. D.;
achieved only very low plasma levels (<100 nM 30 min
after dosing) suggesting rapid blood clearance. Follow-
Ransom, R. W. Cloning and Pharmacological Characterization
of a Human Bradykinin (BK-2) Receptor. Biochem. Biophys. Res.
ing iv injection of 10 µmol/kg, the compound was seen
to disappear rapidly from plasma, falling to a concen-
tration of 4.4 ( 0.48 µM (n ) 4) in 5 min. Its metabolic
fate in vivo remains to be ascertained.
To determine the selectivity of 17, this compound was
also evaluated in binding assays for the A1, A2, AT1, B1,
CCKA, CCKB, H3, kainate, M2, NK1, NMDA, 5-HT1A, and
5-HT3 receptors. At concentrations up to 10 µM, com-
pound 17 did not significantly affect (<20%) radioligand
binding to these receptors. When the binding activity
of 17 was evaluated against the human B2 receptor
expressed in Cos-7 cells, a Ki value of 772 ( 144 nM (n
) 3) was obtained.13
In conclusion, we have developed a novel series of
potent non-peptide antagonists of the rat BK B2 receptor
from a high-throughput screening lead 3, by appending
appropriate functionality onto the nitrophenylthiosemi-
carbazide framework. The subnanomolar-potent com-
pound 17 is a competitive and selective B2 antagonist
and is orally efficacious in a model of inflammatory
hyperalgesia. A full exploration of the SAR and optimi-
zation of this class of B2 antagonist for the human
receptor will be reported in subsequent papers.
Commun. 1992, 184, 260-268. (b) Hess, J . F.; Borkowski, J . A.;
MacNeil, T.; Stonesifer, G. Y.; Fraker, J .; Strader, C. D.; Ransom,
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Derrick, A. W.; Schneck, K. A.; Ransom, R. W.; Strader, C. D.;
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Ack n ow led gm en t. We are grateful to Derek Reid
(NIMS) and Anne Chabert (Novartis Pharma, Basel,
Switzerland) for their synthetic contributions and for
the solubility determinations (D.R.). We also thank Dr.
J ohn W. Davies (Novartis Corp., Summit, NJ ) for
undertaking QSAR studies.
(11) Walter, W.; Rohloff, C. Oxidation Products of Carbothioamides.
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J M991155O