Nonpeptide bradykinin B2 receptor antagonists: Conversion of rodent-selective bradyzide analogues into potent, orally-active human bradykinin B2 receptor antagonists

Article abstract of DOI:10.1021/jm0111088

The 1-(2-nitrophenyl)thiosemicarbazide (TSC) derivative, (S)-l-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl] pyrrolidine-2-carboxylic acid {2-[(2-dimethylaminoethyl)methylamino]ethyl} amide (bradyzide; (S)-4), was recently disclosed as a novel, potent, orally active nonpeptide bradykinin (BK) B₂ receptor antagonist. The compound inhibited the specific binding of [³H]BK to NG108-15 cell membrane preparations (rodent neuroblastoma-glioma) expressing B₂ receptors with a K_i of 0.5 ± 0.2 nM. Compound (S)-4 also demonstrated oral efficacy against Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in rats with an ED₅₀ value of 0.84 μmol/kg. After we optimized the terminal binding determinants projecting from the TSC framework, we found that it was possible to replace the potentially toxicophoric nitro and divalent sulfur moieties with only a 15-fold loss in binding affinity ((S)-14a). However, bradyzide and its congeners were found to have much lower affinities for cloned human B₂ receptors, expressed in Cos-7 cells. The hitherto synthesized TSC series was screened against the human B₂ receptor, and the dibenzosuberane (DBS) pharmacophore emerged as the key structural requirement for potency. Incorporation of this group resulted in a series of derivatives ((S)-14d,e and 19b-d) with K_i ranges of 10.7-176 nM in NG108-15 cells (expressing the rodent B₂ receptor) and 0.79-253 nM in Cos-7 cells (expressing the human B₂ receptor). There was no evidence of agonist activity with any of the nonpeptides in any of the cell lines tested. In vivo, oral administration of compound 19c reversed FCA-induced and turpentine-induced mechanical hyperalgesia in rodents with ED₅₀ values of 0.027 and 0.32 μmol/kg, respectively. The selectivity profiles of compounds (S)-14f and (S)-14g were also assessed to determine the conformational and/or steric preferences of the double-ring arrangement. The affinity of (S)-14g for the human B₂ receptor suggested that it may be a hydrophobic interaction with the ethane bridge of the DBS moiety that accounts for the increased potency of compounds (S)-14d,e and 19b,c at this receptor, by favoring a binding mode inaccessible to the unsubstituted diphenylmethyl derivative, (S)-4.

Full text of DOI:10.1021/jm0111088

2160
J . Med. Chem. 2002, 45, 2160-2172
Non p ep tid e Br a d yk in in B2 Recep tor An ta gon ists: Con ver sion of
Rod en t-Selective Br a d yzid e An a logu es in to P oten t, Or a lly-Active Hu m a n
Br a d yk in in B2 Recep tor An ta gon ists1
Edward K. Dziadulewicz,* Timothy J . Ritchie, Allan Hallett, Christopher R. Snell, J ohn W. Davies,†
Roger Wrigglesworth,‡ Andrew R. Dunstan,§ Graham C. Bloomfield,§ Gillian S. Drake, Peter McIntyre,
Michael C. Brown,| Gillian M. Burgess, Wai Lee, Clare Davis, Mohammed Yaqoob, Steve B. Phagoo,
Elsa Phillips, Martin N. Perkins,# Elizabeth A. Campbell,∇ Andrew J . Davis,§ and Humphrey P. Rang
Novartis Institute for Medical Sciences, 5 Gower Place, London WC1E 6BS, England
Received November 21, 2001
The 1-(2-nitrophenyl)thiosemicarbazide (TSC) derivative, (S)-1-[4-(4-benzhydrylthiosemicarb-
azido)-3-nitrobenzenesulfonyl]pyrrolidine-2-carboxylic acid {2-[(2-dimethylaminoethyl)meth-
ylamino]ethyl}amide (bradyzide; (S)-4), was recently disclosed as a novel, potent, orally active
nonpeptide bradykinin (BK) B2 receptor antagonist. The compound inhibited the specific binding
of [3H]BK to NG108-15 cell membrane preparations (rodent neuroblastoma-glioma) expressing
B2 receptors with a Ki of 0.5 ( 0.2 nM. Compound (S)-4 also demonstrated oral efficacy against
Freund’s complete adjuvant (FCA)-induced mechanical hyperalgesia in rats with an ED50 value
of 0.84 µmol/kg. After we optimized the terminal binding determinants projecting from the
TSC framework, we found that it was possible to replace the potentially toxicophoric nitro and
divalent sulfur moieties with only a 15-fold loss in binding affinity ((S)-14a ). However, bradyzide
and its congeners were found to have much lower affinities for cloned human B2 receptors,
expressed in Cos-7 cells. The hitherto synthesized TSC series was screened against the human
B2 receptor, and the dibenzosuberane (DBS) pharmacophore emerged as the key structural
requirement for potency. Incorporation of this group resulted in a series of derivatives ((S)-
14d ,e and 19b-d ) with Ki ranges of 10.7-176 nM in NG108-15 cells (expressing the rodent B2
receptor) and 0.79-253 nM in Cos-7 cells (expressing the human B2 receptor). There was no
evidence of agonist activity with any of the nonpeptides in any of the cell lines tested. In vivo,
oral administration of compound 19c reversed FCA-induced and turpentine-induced mechanical
hyperalgesia in rodents with ED50 values of 0.027 and 0.32 µmol/kg, respectively. The selectivity
profiles of compounds (S)-14f and (S)-14g were also assessed to determine the conformational
and/or steric preferences of the double-ring arrangement. The affinity of (S)-14g for the human
B2 receptor suggested that it may be a hydrophobic interaction with the ethane bridge of the
DBS moiety that accounts for the increased potency of compounds (S)-14d ,e and 19b,c at this
receptor, by favoring a binding mode inaccessible to the unsubstituted diphenylmethyl
derivative, (S)-4.
In tr od u ction
large inactive precursor proteins (kininogens).2 BK itself
is produced in plasma from a high molecular weight
form of kininogen. The physiological actions of kinins
include vasodilation, increased vascular permeability,
stimulation of sensory neurones, vascular and bronchial
smooth muscle contraction, intestinal ion secretion,
release of prostaglandins and cytokines, and the pro-
duction of nitric oxide.2-5 Overproduction of kinins
under pathophysiological conditions such as tissue
trauma or injury is believed to underlie a number of
clinically relevant disorders including pain and inflam-
mation, hypotension, asthma, rhinitis, pancreatitis,
sepsis, and rheumatoid arthritis.6-8 BK has also been
implicated in the peripheral inflammatory processes
associated with Alzheimer’s disease9 and as a growth
stimulant for certain solid tumors.10
Bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-
Phe8-Arg9) and kallidin (KD; [Lys0]BK; Lys1-Arg2-Pro3-
Pro4-Gly5-Phe6-Ser7-Pro8-Phe9-Arg10) are the two major
mammalian representatives of the kinin family of
endogenous oligopeptide hormones. These linear pep-
tides are released predominantly by the action of
kallikrein enzymes (trypsin-like serine proteases) on
* To whom correspondence should be addressed. Tel.: 0044(0)20
7333 2167. Fax: 0044(0)20 7387 4116. E-mail: ed.dziadulewicz@
pharma.novartis.com.
† Present address: Novartis Pharmaceuticals Corp., 556 Morris
Avenue, Summit, NJ 07901.
‡ Present address: Pfizer Global Research and Development, 3-9
Rue de la Loge, Fresnes Cedex 94265, France.
§ Present address: Novartis Horsham Research Centre, Wimble-
hurst Road, Horsham, West Sussex RH12 5AB, England.
| Present address: Packard, Brook House, 14 Station Road, Pang-
bourne, Berkshire RG8 7AN, England.
Most of the effects of BK are mediated by the B2
receptor subtype,8,11 a constitutive receptor expressed
in many tissues.3 The chronic responses to kinins, on
the other hand, are mediated by B1 receptors,5,11 which
are inducible and recognize the C-terminally truncated
Present address: Pfizer Central Research, Sandwich, Kent CT13
9NJ , England.
#
Present address: AstraZeneca R&D Montre´al, 7171 Fre´de´rick-
Banting, Ville Saint-Laurent, Que´bec H4S 1Z9, Canada.
3 Present address: Celltech R&D Ltd., 208 Bath Road, Slough,
Berkshire SL1 3WE, England.
10.1021/jm0111088 CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/24/2002

Nonpeptide Bradykinin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2161
des-Arg active metabolites of BK and KD.12-14 Both
receptor subtypes are members of the G-protein-coupled
seven transmembrane receptor superfamily and have
been cloned and pharmacologically characterized.13,15,16
It follows that antagonism of B2 (and B1) receptor
activation could constitute a novel approach to the
treatment of pain17,18 and the other inflammatory
diseases listed above.6,19-21
Over the past decade or so, four generations of
sequence-related peptide BK receptor antagonists have
been described, building on the seminal work of Vavrek
and Stewart in the mid-1980s.22 Between them, NPC
56723 ([D-Arg0, Hyp3, D-Phe7]BK), HOE 14024 (icatibant;
[D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]BK), B-943025 ([D-Arg0,
Hyp3, Igl5, D-Igl7, Oic8]BK), and B-1005626 ([D-Arg0,
Hyp3, Igl5, D-f5f7, Igl8]BK) chart the evolution of highly
potent and stable peptide antagonists. While these may
find medical application in particular instances, agents
for oral administration are likely to be nonpeptidic in
nature and devoid of the limitations that hinder broader
therapeutic use of peptides.
The first competitive nonpeptide B2 antagonist was
published in 1993 by Salvino et al. at Sterling Win-
throp.27 WIN 64338 (1, Figure 1) inhibited [3H]BK
binding to B2 receptors in human IMR 90 fetal lung
fibroblasts with an Ki of 60 ( 10 nM. In 1997, Fujisawa
researchers disclosed a second class of nonpeptide B2
antagonist, exemplified by FR167344 (2) and FR173657
(3).28 These were not only potent (nanomolar IC50
values) and selective but also orally active. Subsequent
reports, principally from Fournier29 and Hoechst,30 have
described compounds very much patterned after the
Fujisawa model.
From our own research program, the 1-(2-nitrophen-
yl)thiosemicarbazide (TSC) derivative, (S)-1-[4-(4-benz-
h ydr ylt h iosem ica r ba zido)-3-n it r oben zen esu lfon yl]-
pyrrolidine-2-carboxylic acid {2-[(2-dimethylaminoethyl)-
methylamino]ethyl}amide (bradyzide; (S)-4), was re-
cently disclosed as a novel, potent, orally active non-
peptide BK B2 receptor antagonist.31,32 The compound
inhibited the specific binding of [3H]BK to NG108-15
cell membrane preparations (rodent neuroblastoma-
glioma) expressing B2 receptors with a Ki of 0.5 ( 0.2
nM. Compound (S)-4 also demonstrated oral efficacy
against Freund’s complete adjuvant (FCA)-induced me-
chanical hyperalgesia in rats with an ED50 value of 0.84
µmol/kg. While there was no evidence that (S)-4 pos-
sessed mutagenic activity, we found that it was possible
to replace the undesirable aromatic nitro and divalent
sulfur groups and retain nanomolar affinity. However,
compound (S)-4 and its analogues were more than 1000-
fold less potent at cloned human B2 receptors, expressed
in Cos-7 cells. A reevaluation of our compound library
revealed an apparently modest structural feature, which
had a significant impact on human BK B2 receptor
affinity once it was incorporated into the full TSC
structure. In this paper, we wish to document the
structural changes that enabled us to solve the problem
of species specificity in this compound series.
F igu r e 1. Structures of BK B2 receptor antagonists WIN
64338 (1), FR 167344 (2), FR 173657 (3), and bradyzide ((S)-
4).
9a or (R)-9a ) or (S)-prolinamide ((S)-9b) with com-
mercially available 4-chloro-3-nitrobenzenesulfonyl chlo-
ride or 3-chloro-4-fluorobenzenesulfonyl chloride in
aqueous sodium carbonate solution gave the sulfona-
mides 10a -c. This modification of our previously pub-
lished procedure31 allowed us to omit an ester depro-
tection step from the reaction sequence. In an analogous
fashion, the corresponding pyrrole derivatives 16a ,b
were prepared by sulfonylation of pyrrole-2-carboxylic
acid (15) following deprotonation with 2.2 equiv of
n-butyllithium in tetrahydrofuran (THF) at -20 °C.
Treatment of sulfonamides 10a -c and 16a ,b with
hydrazine monohydrate at reflux in THF in the presence
of triethylamine afforded the phenylhydrazine deriva-
tives 11a -c and 17a ,b, respectively. Generally, these
were not isolated but coupled in situ with isothiocyan-
ates (12a ,c,e,f)33 or isocyanates (12b,d )34 to give the
corresponding thiosemicarbazides 13a ,b,d ,f,g and 18a ,b
Ch em istr y
The compounds described herein are presented in
Table 1, and the methods for their syntheses are shown
in Schemes 1-3. Sulfonylation of (S)- or (R)-proline ((S)-

2162 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Dziadulewicz et al.
Ta ble 1. Binding Affinities (Ki) of Compounds at the Rat and Human BK B2 Receptors (B2R)
in vitro Ki (nM)
compda
R
X
Z
R1b
rat B2R NG108-15 cellsc
human B2R Cos-7 cellsd
(S)-4
(R)-4
Me2N(CH2)2N(Me)(CH2)2NH-
Me2N(CH2)2N(Me)(CH2)2NH-
H2N
NO2
NO2
NO2
Cl
S
S
S
O
S
S
S
O
S
S
S
S
O
O
DPM
DPM
DBS
DPM
DPM
DPM
DBS
DBS
FL
DCDPM
DPM
DBS
DBS
DBS
0.5 ( 0.2e
0.6 ( 0.12e
654 ( 65
7.3 ( 1.8
0.023 ( 0.007
0.31 ( 0.1
14.5 ( 2.5
10.7 ( 1.6
ND
5.46 ( 1.7
ND
64.5 ( 19.4
176 ( 51
ND
772 ( 144e
1430 ( 500
299 ( 33
NDf
119 ( 30
95 ( 10
4.67 ( 0.7
0.79 ( 0.14
645 ( 142
34.1 ( 6.5
237 ( 38
1.47 ( 0.34
2.79 ( 0.47
253 ( 33
(S)-13b
(S)-14a
(S)-14b
(S)-14c
(S)-14d
(S)-14e
(S)-14f
(S)-14g
19a
Me2N(CH2)2N(Me)(CH2)2NH-
Me2N(CH2)3N(Me)(CH2)2NH-
Me2N(CH2)3N(Me)(CH2)3NH-
Me2N(CH2)2N(Me)(CH2)2NH-
Me2N(CH2)3N(Me)(CH2)2NH-
Me2N(CH2)3N(Me)(CH2)2NH-
Me2N(CH2)2N(Me)(CH2)2NH-
Me2N(CH2)2N(Me)(CH2)2NH-
Me2N(CH2)2N(Me)(CH2)2NH-
Me2N(CH2)3N(Me)(CH2)2NH-
Me2N(CH2)3N(Me)(CH2)2NH-
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
Cl
19b
19c
19d
a
Unless otherwise noted, elemental microanalyses were within (0.4% of the theoretical values for the formulas given in the Experimental
Section. Abbreviations: DPM, diphenylmethyl; DBS, dibenzosuberyl; FL, 9-fluorenyl; DCDPM, 2,2′-dichlorodiphenylmethyl. c Concen-
b
tration required to inhibit specific binding of [3H]BK (1 nM) to B2 receptors by 50% in NG108-15 cell membranes. See the Experimental
Section for more details. Concentration required to inhibit specific binding of [3H]BK (1 nM) by 50% to cloned B2 receptors expressed in
d
Cos-7 cells. See the Experimental Section for further details. The results are expressed as the mean of at least three determinations (
SEM and calculated on the basis of a one site binding model using LIGAND software (see ref 46). e Previously published, see ref 31. f ND,
not determined.
Sch em e 1a
The desired products were mostly purified by pre-
parative high-performance liquid chromatography
(HPLC). Those incorporating the fluorenyl (FL) or
diphenylmethyl (DPM) moiety (4, (S)-14a -c,f,g and
19a ) could be isolated as amorphous trifluoroacetate
(TFA) salts by concentrating the column fractions under
reduced pressure. However, loss of the dibenzosuberyl
(DBS) group was experienced under the same condi-
tions. Compounds (S)-13b, (S)-14d ,e, and 19b-d were
therefore isolated by organic solvent extraction from the
neutralized HPLC fractions, followed by concentration
in vacuo to afford amorphous solids. The absolute
configurations of the final products were assigned by
extrapolation from whichever proline enantiomer was
employed. Enantiomeric pairs of compounds were not
routinely synthesized, but a nuclear magnetic resonance
(NMR)-based enantiomeric purity determination on one
such pair (data not shown) utilizing the chiral resolving
agent (R)-(+)-tert-butylphenylphosphinothioic acid39 in-
dicated that the stereochemical integrity of the final
compounds was not compromised by the reaction condi-
tions employed.
a
Reagents: (a) (i) ZOSu, EtOAc, (ii) TsCl, pyridine; (b) N,N,N′-
trimethylethylenediamine (for 7a ) or N,N,N′-trimethylpropylene-
diamine (for 7b,c); (c) HCO2NH4, Pd/C, MeOH or H2, Pd/C, MeOH.
or semicarbazides (S)-13c,e and 18c,d , respectively. In
a convergent approach, the final compounds were as-
sembled by mixed anhydride coupling of the carboxylic
acids 13a -g and 18a -d with the preformed triamines
8a -c35,36 using isopropyl- or isobutyl chloroformate in
the presence of N-methyl morpholine (Schemes 2 and
3). Isopropyl chloroformate was the reagent of choice
for the pyrrole series as this was found to minimize
attack at the “wrong” carbonyl group of the mixed
anhydride species. The triamines 8a -c were synthe-
sized in a two step procedure from tosylated N-Z
ethanolamine (6a )37 and tosylated N-Z propanolamine
(6b) (Scheme 1).38 The procedures described in the
Experimental Section for the displacement of the tosyl
group by the trimethyldiamines and for the hydro-
genolysis of the Z group could be used interchangeably.
Resu lts a n d Discu ssion
We have recently described a series of novel nonpep-
tide BK B2 receptor antagonists based on the TSC
framework.31 These compounds had nanomolar affini-
ties at the rat BK B2 receptor as measured in a
radioligand binding assay in NG108-15 cell membranes
using [3H]BK as the radiolabel. In particular, bradyzide
((S)-4) emerged as the most potent member of this series
with a Ki value of 0.5 ( 0.2 nM (Table 1).32 A stereo-
chemical preference for binding to the rat BK B2
receptor was not observed, with the (R) enantiomer ((R)-

Nonpeptide Bradykinin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2163
Sch em e 2a
a
Reagents: (a) Na2CO3, H2O, 4-chloro-3-nitrobenzenesulfonyl chloride (for 10a ,b) or 3-chloro-4-fluorobenzenesulfonyl chloride (for 10c);
(b) H2NNH2‚H2O, Et3N, THF, reflux; (c) R1NCS (R1 ) DPM, 12a ; DBS, 12c; FL, 12e; DCDPM, 12f), THF, room temperature or R1NCO
(R1 ) DPM, 12b; DBS, 12d ), THF, room temperature; (d) N-methyl morpholine, ClCO2iBu, THF, -20 °C, 8a (for 4, (S)-14a ,d ,g), 8b (for
(S)-14b,e,f) or 8c (for (S)-14c). See Table 1 footnotes for 12a -f abbreviations.
Sch em e 3a
a
Reagents: (a) nBuLi, THF, -20 °C, Ar, 4-chloro-3-nitrobenzenesulfonyl chloride (for 16a ) or 3-chloro-4-fluorobenzenesulfonyl chloride
(for 16b); (b) H2NNH2‚H2O, Et3N, THF, reflux; (c) RNCS (R ) DPM, 12a ; DBS, 12c), THF, reflux or RNCO (R ) DBS, 12d ), THF, room
temperature, 65 h; (d) N-methyl morpholine, ClCO2iPr, THF, -20 °C, 8a (for 19a ,b) or 8b (for 19c,d ). See Table 1 footnotes for 12a ,c,d
abbreviations.
4) eliciting a similar subnanomolar Ki value (0.6 ( 0.12
nM). Compound (S)-4 also demonstrated oral efficacy
against FCA-induced mechanical hyperalgesia in rats
with an ED50 value of 0.84 µmol/kg and a duration of

2164 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Dziadulewicz et al.
action in excess of 4 h. As a potential development
candidate, the presence of aromatic nitro and divalent
sulfur groups in (S)-4 was of obvious concern, despite
the absence of clastogenicity and/or aneugenicity in tests
to assess its mutagenic potential. When we optimized
the groups appended to the sulfonamide and thiosemi-
carbazide terminals of the TSC core, the nitro and sulfur
moieties could be replaced by chloro and oxygen, re-
spectively; this was a combined alteration that was not
previously tolerated in compounds with lower receptor
affinities. The resulting compound, (S)-14a , was ca. 15-
fold less potent at the rat B2 receptor but still displayed
a respectable Ki value of 7.3 ( 1.8 nM. However, these
compounds were found to have much lower affinities for
cloned human B2 receptors, expressed in Cos-7 cells,
despite the high sequence homology between the species
(ca. 80% identical at the amino acid level). The Ki values
of (S)-4 and (R)-4 for human B2 receptors were ca. 1500-
and 2400-fold higher, respectively, than those recorded
against rat B2 receptors, with only a marginal prefer-
ence for the (S) stereochemistry. While the affinity of
(S)-14a in Cos-7 cells was not determined, our structure-
activity relationship (SAR) experience of the series
suggested that the compound was very unlikely to
manifest any useful activity at human receptors. To
address the disparity between rat and human B2 recep-
tor affinities, the ca. 400 TSC compounds synthesized
to date were rescreened against the human B2 receptor
in order to identify structural elements responsible for
any improved binding characteristics at this subtype.
Table 1 shows a comparison of the binding affinities of
a core set of compounds for the rodent and human B2
receptor.
F igu r e 2. Potent rat BK B2 receptor antagonists.
human B2 receptors over the rat counterparts (Ki ) 654
nM). When the DBS moiety was incorporated into full
TSC structures containing the basic chain of (S)-4 and
19a , a ca. 160-fold increase in affinity at the human B2
receptor was achieved ((S)-14d , Ki ) 4.7 nM; 19b, Ki )
1.5 nM). In addition, affinity at the rat receptor was
maintained at a level below 20 nM for the pyrrolidine
(S)-14d and just above 60 nM for the pyrrole 19b. A
160-fold increase in affinity corresponds to a free energy
of interaction of 3.1 kcal/mol, which may reflect a
moderately strong ionic interaction with the receptor,
an interaction in which the basic chain could participate.
This result represents a divergence in human and rat
B2 binding SAR. In NG108-15 cells, low molecular
weight compounds such as 20 and 21 (the aforemen-
tioned DPM analogue of (S)-13b; Figure 2) still exhib-
ited high potencies (<10 nM) at the rat receptor as long
as the DPM motif was in place.31 In contrast, the human
receptor appears to be more discriminating with respect
to the ligands it accommodates, requiring the binding
energy contributions of both the DBS group and the
basic chain for full recognition. Interestingly, the need
for an extended molecular framework incorporating an
additional pharmacophore was also observed by Fujisa-
wa researchers in their efforts to overcome the species
difference between guinea pig and man.41
In the DBS series, we also examined the influence of
the basic chain and the five-membered ring on receptor
affinity. Increasing the length of the alkyl chain between
the basic nitrogen atoms, from ethylene ((S)-14d and
19b ) to propylene ((S)-14e and 19c, respectively),
resulted in a ca. 6-fold increase in human receptor
affinity for the pyrrolidine and a 2-fold decrease for the
pyrrole, with overall Ki values in a narrow range (0.8-
4.7 nM). The Ki values of these compounds at the rat
receptor were both higher and spread over a wider range
(10.7-176 nM). It should be noted that (S)-14e and 19c
incorporate a semicarbazide backbone, and in general,
the isosteric replacement of sulfur by oxygen was very
well-tolerated in the DBS series, leading to compounds
of very similar potencies at the human B2 receptor. (S)-
14e was confirmed as a functional antagonist at rodent
and human B2 receptors. It inhibited the increase in
45Ca2+ efflux from NG108-15 cells caused by activation
of the rat B2 receptor with an IC50 value of 105 ( 8 nM
Extending the terminal dimethylamino group by
increasing the length of the alkyl chain between the
basic nitrogen atoms from ethylene ((S)-4) to propylene
((S)-14b) was seen to afford a 6.5-fold increase in
binding affinity in the human Cos-7 cell system. Potency
at the rat receptor was increased ca. 22-fold, and with
a Ki value of 0.023 nM, (S)-14b approaches the potency
levels of many peptidic antagonists. Although further
extension of the remaining ethylene linker resulted in
little additional benefit ((S)-14c), an overall 8-fold
improvement in B2 binding affinity could be achieved
at the human receptor by manipulating the proline
amide side chain in this way. This result suggested that
the rat and human receptors prefer interaction with a
more basic chain, as an ethylene-to-propylene increase
in the separation of basic nitrogen centers is known to
raise their pKa values.40 In a separate observation,
removal of the C-2 stereogenic center by replacement
of the pyrrolidine ring of (S)-4 with a pyrrole ring (19a )
led to a 3-fold increase in affinity at the human B2
receptor. Given the low eudismic ratio observed with
the enantiomers of 4, such a replacement would have
been expected to maintain potency at the very least.
By far the most important result of our rescreening
exercise was that revealed by (S)-13b, a compound with
an unsubstituted prolinamide terminus and whose DPM
pharmacophore is formally adopted into the tricyclic
DBS moiety. This latter structural feature was identi-
fied as the key element responsible for human B2
receptor affinity (Ki ) 299 nM; the DPM analogue was
inactive) and for the 2-fold selectivity of (S)-13b for

Nonpeptide Bradykinin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2165
(n ) 3). It inhibited B2 receptor-mediated contractions
of rat uterus smooth muscle with an IC50 value of 2.4 (
0.7 nM (n ) 3). The IC50 values for antagonism of the
45Ca2+ efflux actions of BK at the human receptor lie
between 17 ( 3 nM (n ) 3; human WI38 fibroblasts)
and 91 ( 9 nM (n ) 3; human SK-N-SH neuroblastoma
cells). In vivo, (S)-14e reversed FCA-induced and tur-
pentine-induced mechanical hyperalgesia in rats with
ED50 values of 0.043 µmol/kg (0.03 mg/kg) and 0.11
µmol/kg (0.08 mg/kg), respectively, by oral administra-
tion, achieving maximum reversals of ca. 70%. However,
the dose-response curves for (S)-14e were bell-shaped
in both models of hyperalgesia, with a decrease in
response to the compound when the doses were in-
creased beyond 1 µmol/kg po in the FCA model and
beyond 0.5 µmol/kg po in the turpentine model. This loss
of activity at higher doses did not appear to be the result
of a prohyperalgesic effect, as (S)-14e had no effect in
na¨ıve animals up to 5 µmol/kg po. Nor was this a
consequence of poor solubility, as (S)-14e had a solubil-
ity of >6.2 mg/mL in pH 7.4 buffered saline solution.
Changing from the pyrrolidine ring to the pyrrole ring
had negligible in vitro effects at the human B2 receptor
((S)-14d vs 19b and (S)-14e vs 19c) but resulted in a
ca. 4- and 16-fold reduction in affinity at the rat
receptor, respectively. Compound 19c inhibited the
increase in 45Ca2+ efflux from NG108-15 cells caused
by activation of the rat B2 receptor with a similar IC50
value to that exhibited by (S)-14e (141 ( 15 nM, n )
3), and its inhibition of B2 receptor-mediated contrac-
tions of rat uterus smooth muscle was also of similar
magnitude (IC50 1.9 ( 0.4 nM, n ) 3). However, despite
a lower solubility (0.4 mg/mL at pH 7.4), 19c displayed
a marked improvement in in vivo activity compared to
the corresponding pyrrolidine, (S)-14e, with no fall off
in activity at higher doses. Compound 19c reversed
FCA-induced and turpentine-induced mechanical hy-
peralgesia in rats with ED50 values of 0.027 µmol/kg
(0.02 mg/kg) and 0.32 µmol/kg (0.22 mg/kg), respectively,
by oral administration and with maximum reversals of
ca. 67%. Following administration of 19c at 1 µmol/kg
po, withdrawal thresholds were significantly raised for
at least 4 h. Despite its long duration of action as an
antihyperalgesic agent, preliminary data suggested that
its antagonism of the hypotensive response to BK, when
given iv, is relatively short-lasting. Compound 19c may
therefore have a short plasma half-life, a pharmacoki-
netic trait it appears to share with (S)-4.31 The afore-
mentioned difference in rat and human B2 receptor
binding SAR was also evident upon attempting to
remove the nitro group from 19c. Replacement of this
moiety with a chloro group led to a much more rapid
decrease in binding affinity at the human receptor (91-
fold, 19d vs 19c) than previously observed in the rat
system (ca. 15-fold, (S)-14a vs (S)-4).
based system was conditional upon the satisfaction of
other steric parameters.42 For example, adoption of a
coplanar tricyclic arrangement, as afforded by the FL
derivative (S)-14f resulted in a drastic 816-fold reduc-
tion in affinity ((S)-14f vs (S)-14e), suggesting that the
tricyclic system must adopt a bent configuration in
which the lateral aromatic rings have at least a limited
ability to twist in relation to one another. Interestingly,
the 2,2′-dichlorodiphenylmethyl (DCDPM) analogue (S)-
14g exhibited a Ki value of 34 nM at the human
receptor, a ca. 23-fold improvement in affinity compared
to that of the unsubstituted DPM derivative, (S)-4. This
increase in affinity corresponds to 1.9 kcal/mol, which
is low for a full ionic interaction and may represent a
simple hydrogen bond. Substituents other than chloro
were also investigated but were found to be inferior,
with potency decreasing in the order 2,2′-dichloro (34
nM) > 2,2′-dimethyl (50 nM) > 2,2′-dimethoxy (92 nM)
> 2-chloro (147 nM) . 2-methyl (4100 nM). It is
intriguing that there is only a ca. 7-fold difference in
human B2 affinity between (S)-14g and (S)-14d , when
one would expect the aromatic rings of the DCDPM and
DBS groups to have markedly different angles of tor-
sion. If (S)-14g and (S)-14d make similar binding
contacts within the human receptor, as their affinities
imply, then it may be that the binding modes selected
by these ligands depend on the nature of the hydropho-
bic substituent at the N(4) position of the TSC core. We
explored the possible influence of the hydrophobic region
further by generating pharmacophore hypotheses using
the CATALYST43 software program. The best-computed
pharmacophore model to rationalize the observed activi-
ties of (S)-14d (DBS), (S)-4 (DPM), and (S)-14f (FL) is
shown in Figure 3. In this paradigm, the two carbon
bridging fragment of the DBS moiety (shown in red)
enhances affinity through an aliphatic hydrophobic
interaction in addition to the aromatic interactions
afforded by the two phenyl rings. This may favor a
binding mode that maximizes the binding contacts that
(S)-14d makes in the human receptor. The DPM group
(green) adopts a slightly different conformation with
respect to the phenyl groups and of course does not
possess the aliphatic bridge. The FL group (grey) is
unable to occupy the required pharmacophores to any
great extent. Also shown in Figure 3 is the correspond-
ing overlay, suggested by CATALYST, for the DCDPM
group (in black), which is able to map reasonably well
with the DBS group. In this case, one of the ortho-chloro
atoms is also able to act as the important aliphatic
hydrophobe, and the CATALYST program correctly
predicted the higher binding affinity of the DCDPM-
containing analogue (S)-14g, compared to that of the
DPM analogue (S)-4 (see Experimental Section for more
details). Thus, it may be that (S)-4 adopts a distinctly
different binding mode in the human receptor to that
of (S)-14d , which prevents it from exercising the ad-
ditional interactions it clearly enjoys in the rat receptor
environment.
To elucidate the steric requirements of the key DBS
pharmacophore for human B2 receptor affinity, we
synthesized a number of structurally related analogues.
Modifications to the tricyclic ring system, such as
deletion of one or both of the lateral aromatic rings or
transposing them to other fused positions around the
central seven-membered ring to afford isomeric tricyclic
systems, were all detrimental to activity (data not
shown). However, receptor preference for a benzhydryl-
Con clu sion
Potent, orally active rat BK B2 receptor antagonists
based on the TSC framework and exemplified by
bradyzide ((S)-4) were found to be much less potent at
cloned human B2 receptors. We restored affinity at
human receptors by replacing the DPM moiety of (S)-4

2166 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Dziadulewicz et al.
chromatography (230-400 mesh). Preparative reverse phase
HPLC was performed on a Waters Delta Prep 3000 liquid
chromatograph using a C18 bonded silica gel column (Dy-
namax-300 Å, 12 µm, 41.4 mm × 250 mm; Rainin Instrument
Co.). The mobile phase contained acetonitrile (10-100%, v/v)
in water containing 0.1% (v/v) TFA. Gradient elution at a flow
rate of 70 mL/min was employed; peak detection was at 254
nm. Chemical yields are not optimized. Melting points (mp)
were determined on a Reichert Thermovar hot stage melting
point apparatus and are uncorrected. Optical rotations were
determined with an AA-100 polarimeter (Optical Activity Ltd.,
U.K.). Infrared (IR) spectra were obtained on a Bruker IFS
66 infrared spectrophotometer using KBr disks, unless oth-
erwise noted, and are reported in cm-1. Proton NMR (1H NMR)
spectra were recorded on a Varian Gemini-200 spectrometer
(200 MHz), Bruker AM-360 spectrometer (360 MHz), or on a
Bruker Avance DPX 400 spectrometer (400 MHz) and are
referenced to residual solvent signals: CDCl3 (δ 7.26) or
DMSO-d6 (δ 2.49). Chemical shifts are reported in δ units
(parts per million) downfield from tetramethylsilane and are
assigned as singlets (s), doublets (d), doublet of doublets (dd),
triplets (t), quartet (q), quintet (quin), multiplets (m), broad
signals (br), or very broad signals (v br). Coupling constants
(J ) are reported in Hertz (Hz). Mass spectra (MS) were
measured on Finnigan MAT 8430 (for EI) or VG 70-SE (for
fast atom bombardment (FAB)) mass spectrometers. The
parent ion is given followed by peaks corresponding to major
fragment losses with relative intensities in parentheses.
Elemental analysis data for final compounds were obtained
from Novartis Pharma AG, Basel, and were within (0.4% of
the theoretical values for the formulas given, unless otherwise
noted.
Gen er a l P r oced u r e for Syn th esis of Tr ia m in es 8a -c.
N-(2-Am in oet h yl)-N,N′,N′-t r im et h yl-1,2-et h a n ed ia m in e
(8a ).35 Step 1. A solution of toluene-4-sulfonic acid 2-benzyl-
oxycarbonylaminoethyl ester (6a )37 (46.79 g, 133.9 mmol) in
N,N,N′-trimethylethylenediamine (180 mL) was stirred at
room temperature for 48 h. Excess diamine was removed by
evaporation under reduced pressure. The residue was diluted
with EtOAc (400 mL) and washed with 1 M NaOH solution (2
× 90 mL). The combined aqueous washings were reextracted
with EtOAc (2 × 100 mL), and the combined organic layers
were washed with brine (2 × 70 mL), dried over MgSO4, and
evaporated under reduced pressure to afford the Z-protected
triamine 7a as a straw-colored syrup (34.81 g, 93%), which
was used without further purification.
Step 2. A solution of the Z-protected triamine 7a (34.78 g,
125 mmol) in anhydrous MeOH (50 mL) was added to a
suspension of 10% Pd/C catalyst (4 g) in anhydrous MeOH (200
mL) under an argon atmosphere, followed by solid ammonium
formate (8.64 g, 137 mmol). After it was stirred at room
temperature for 24 h, the reaction mixture was filtered
through Celite and the filtrate was evaporated under reduced
pressure. A portion of the product (4.16 g) was purified by
distillation under reduced pressure to yield 8a as a colorless
oil (2.85 g, 68.5%): bp 120 °C (7 mmHg) [literature35 bp 38-
43 °C (0.2 mm Hg)].
N -(2-Am in oe t h yl)-N ,N ′,N ′-t r im e t h yl-1,3-p r op a n e d i-
a m in e (8b).36 Step 1. A solution of 6a (15 g, 42.9 mmol) in
N,N,N′-trimethyl-1,3-propanediamine (25 mL) was stirred at
140 °C for 4 h. After it was cooled to room temperature, excess
diamine was removed by evaporation under reduced pressure.
The residue was diluted with EtOAc (250 mL) and washed
with 1 M NaOH solution (2 × 100 mL). The combined aqueous
washings were reextracted with EtOAc (2 × 100 mL), and the
combined organic layers were washed with brine (100 mL),
dried over MgSO4, and evaporated under reduced pressure to
afford the Z-protected triamine 7b as a straw-colored syrup
(10.2 g, 81%), which was used without further purification.
F igu r e 3. CATALYST-generated pharmacophore model of
human BK B2 receptor antagonists. The DBS group (red) is
able to map all four pharmacophoric features generated in the
CATALYST hypothesis, whereas the DPM group (green) lacks
the aliphatic hydrophobe between the two aromatic interac-
tions and the FL group (grey) can only partially fit the
aromatic region. The DCDPM group (black) is able to overlay
with the DBS group reasonably well, with one of the ortho-
chloro atoms mimicking one of the methylene groups in the
DBS bridge. CATALYST correctly predicted the DCDPM group
to have higher affinity at human BK B2 receptors than the
DPM group.
with the DBS group, a modification revealed upon
screening our TSC compound collection against the
human receptor. Activity could be further tuned at the
sulfonamide terminus by manipulating the basic chain
and the five-membered heterocyclic ring, and this led
to compound 19c. Compound 19c is a potent and
selective44 inhibitor of human BK B2 receptors. The IC50
values for antagonism of the actions of BK at the human
receptor lie between 12 ( 2 nM (SK-N-SH cells) and 55
( 9 nM (WI38 cells). Furthermore, after oral adminis-
tration, the compound is highly potent in alleviating the
hyperalgesia associated with inflammation in two mod-
els of persistent inflammatory hyperalgesia. As such,
this compound represents a novel prototype for the
design of agents to treat the many painful inflammatory
conditions in which BK is an important mediator.
Further studies aimed at replacing the nitro group and
retaining human B2 receptor affinity will be reported
in a subsequent paper.45
Exp er im en ta l Section
(A) Ch em istr y. Commercial chemicals and solvents were
of reagent grade and used without further purification. The
following abbreviations are used for reagents and solvents:
DCM, dichloromethane; DMF, dimethyl formamide; DMSO,
dimethyl sulfoxide; EtOAc, ethyl acetate; Et2O, diethyl ether;
EtOH, ethanol; MeOH, methanol; and TFA, trifluoroacetic
acid. Merck silica gel (Kieselgel 60) was used for analytical
thin-layer chromatography (TLC; F254 plates) and flash column
Step 2. A solution of the Z-protected triamine 7b (10 g, 34.1
mmol) in anhydrous MeOH (75 mL) was stirred with 10% Pd/C
catalyst (0.5 g) under an atmosphere of hydrogen for 24 h. The
catalyst was removed by filtration through Celite, and the
filtrate was evaporated under reduced pressure to afford the

Nonpeptide Bradykinin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2167
title compound as a syrup (5.0 g, 93%), which was used without
further purification.
solvent was removed under reduced pressure to afford 12c as
a solid (3.3 g, 92%), which could be used without further
purification. 1H NMR (200 MHz, CDCl3): δ 7.5-7.35 (2H, m),
7.33-7.1 (6H, m), 6.34 (1H, s), 3.45-3.1 (4H, m). IR (KBr):
N-(3-Am in op r op yl)-N,N′,N′-t r im et h yl-1,3-p r op a n ed i-
a m in e (8c).36 The starting tosylated N-Z propanolamine 6b38
(4.0 g, 11.0 mmol) was converted to the title compound (1.6 g,
84% over 2 steps) as described for 8a . The syrup 8c was used
without further purification.
ν
max 2096 (NdCdS) cm-1. MS (EI): m/z 193 (M+ - NCS; 100%).
5-Isocya n a to-10,11-d ih yd r o-5H-d iben zo[a ,d ]cycloh ep -
ten e (12d ). A stirred solution of 5-chlorodibenzosuberane (2.0
g, 8.5 mmol) in anhydrous DMF (20 mL) was treated at room
temperature with sodium cyanate (0.57 g, 8.75 mmol) in one
portion. After 6.5 h, the solvent was evaporated under reduced
pressure, and the residue was taken up in EtOAc (20 mL) and
stored in a refrigerator overnight. The mixture was filtered,
and the clear filtrate was evaporated under reduced pressure
to afford 12d (2.2 g, ∼100%) as a straw-colored syrup, which
slowly solidified to a pale yellow wax upon storage in a
Dip h en ylm eth yl Isoth iocya n a te (12a ).33,34 A mixture of
bromodiphenylmethane (4.94 g, 20.0 mmol) and 18-crown-6
(0.15 g, 0.57 mmol) in 1,2-dichlorobenzene (10 mL) was heated
to 180 °C with stirring, whereupon potassium thiocyanate (2.5
g, 25.0 mmol) was added. The reaction was followed on TLC
and was complete after stirring at reflux for 30 min. After it
was cooled to room temperature, the mixture was washed with
water (2 × 25 mL) and brine (25 mL), dried over MgSO4, and
concentrated under reduced pressure. The resulting syrup was
purified by chromatography on silica gel (initial eluent 100%
hexane, final eluent hexane:Et2O 50:1) to afford 12a (3.03 g,
67%) as a white crystalline solid; mp 54-57 °C [literature33a
1
refrigerator. H NMR (200 MHz, CDCl3): δ 7.6-7.5 (2H, m),
7.4-7.15 (6H, m), 6.29 (1H, s), 3.48-3.17 (4H, m).
9-Isot h iocya n a t o-9H -flu or en e (12e).33a 9H-Fluoren-9-
ylamine (4.5 g, 24.8 mmol) was converted to the title compound
(1.8 g, 88%) as described for 12c. The solid compound 12e was
used without further purification. 1H NMR (200 MHz,
CDCl3): δ 7.8-7.6 (4H, m), 7.55-7.3 (4H, m), 5.75 (1H, s). IR
(KBr): νmax 2099 (NdCdS) cm-1. MS (EI): m/z 223 (M+; 15%),
165 (M+ - NCS; 100%).
1
mp 59 °C (EtOH)]. H NMR (400 MHz, CDCl3): δ 7.38-7.29
(10H, m), 5.97 (1H, s). IR (KBr): νmax 2103 (NdCdS) cm-1
MS (EI): m/z 167 (M+ - NCS; 100%).
.
Dip h en ylm eth yl Isocya n a te (12b).34 A stirred solution
of aminodiphenylmethane (0.92 g, 5 mmol) in anhydrous
toluene (25 mL) under a nitrogen atmosphere was treated
Bis-(2-ch lor op h en yl)m eth yl Isoth iocya n a te (12f). Step
1. A stirred solution of 2,2′-dichlorobenzophenone (3.06 g, 12.19
mmol) in anhydrous pyridine (25 mL) was treated with
hydroxylamine hydrochloride (3.39 g, 48.74 mmol), and the
mixture was heated at 100 °C overnight. The reaction was
followed on TLC (EtOAc:hexane 1:5), and upon completion, the
solvent was removed under reduced pressure. The residue was
diluted with EtOAc (150 mL) and washed sequentially with 2
M HCl solution (2 × 50 mL), water (50 mL), and brine (50
mL). The washings were combined and reextracted with EtOAc
(50 mL). The organic phases were combined and dried over
MgSO4, and the solvent was removed under reduced pressure
to afford the oxime intermediate (3.2 g, ∼100%) as a white
dropwise at room temperature with
a
∼20% solution of
phosgene in toluene (10.4 mL), whereupon a white suspension
was formed. The reaction mixture was heated to reflux, and
after a further 10 min, a colorless solution was observed. After
it was cooled to room temperature, the solvent was removed
under reduced pressure to afford 12b as an oil (1.0 g, ∼100%),
which was dried under high vacuum and used without
characterization or purification.
5-Isoth iocya n a to-10,11-d ih yd r o-5H-d iben zo[a ,d ]cyclo-
h ep ten e (12c). Step 1. A stirred solution of dibenzosuberone
(30 g, 144 mmol) in pyridine (30 mL) and EtOH (150 mL) was
treated with hydroxylamine hydrochloride (30.02 g, 432 mmol),
and the mixture was heated at 100 °C for 6 days under a
nitrogen atmosphere. The solvent was removed under reduced
pressure, and the residue was partitioned between EtOAc (200
mL) and water (200 mL). The aqueous layer was reextracted
with EtOAc (50 mL). The organic phases were combined and
dried over MgSO4, and the solvent was removed under reduced
pressure to afford the oxime intermediate as a pale yellow
solid. This was recrystallized from EtOAc-cyclohexane to
afford pure dibenzosuberone oxime (29.19 g, 91%) as a creamy-
white crystalline solid.
Step 2. A stirred suspension of dibenzosuberone oxime (20
g, 89.58 mmol) in EtOH (90 mL) and concentrated ammonia
solution (450 mL) was treated with ammonium acetate (3.45
g, 44.76 mmol) followed by portionwise addition of zinc powder
(29.28 g, 447.9 mmol). The mixture was heated to 50 °C, and
once effervescence had ceased to be vigorous, the mixture was
refluxed overnight. After it was cooled to room temperature,
the mixture was diluted with EtOAc (400 mL), stirred for 30
min, and filtered. The two phase filtrate was transferred to a
separating funnel, and the phases were separated. The aque-
ous phase was reextracted with EtOAc (100 mL). The com-
bined organic extracts were dried over MgSO4, and the solvent
was removed under reduced pressure to afford 5-aminodiben-
zosuberane as a pale yellow solid. The solid was purified by
chromatography on silica gel (EtOAc:cyclohexane, 1:9) to afford
pure 5-aminodibenzosuberane (17.95 g, 96%) as a white solid.
1H NMR (200 MHz, CDCl3): δ 7.44-7.3 (2H, m), 7.2-7.0 (6H,
m), 5.42 (1H, s), 3.5-3.28 (2H, m), 3.28-3.05 (2H, m), 1.8 (2H,
s). IR (KBr): νmax 3361, 3303, 2853, 1600, 1580, 1476, 1454,
1351 cm-1. MS (EI): m/z 209 (M+; 5%), 208 (16%), 192 (100%).
1
solid. H NMR (200 MHz, CDCl3): δ 8.62 (1H, br s), 7.55-7.2
(8H, m). IR (KBr): νmax 3224 (N-OH) cm-1. MS (EI): m/z 265
(M+; 47%), 248 (53%), 230 (100%).
Step 2. A stirred suspension of the oxime (3.0 g, 11.27 mmol)
in EtOH (11 mL) and concentrated ammonia solution (55 mL)
was treated with ammonium acetate (0.45 g, 5.86 mmol) and
zinc powder (3.97 g, 60.73 mmol). The mixture was heated at
reflux for 4 h. After it was cooled to room temperature, the
mixture was filtered and the solid residue was washed with
10 M NaOH solution (50 mL) and EtOAc (50 mL). The two
phase filtrate was transferred to a separating funnel and
washed with EtOAc (2 × 100 mL). The combined organic
extracts were washed with brine (2 × 30 mL) and dried over
MgSO4. The solvent was removed under reduced pressure to
afford bis-(2-chlorophenyl)methylamine (2.8 g, 99%) as a pale
yellow oil, which was used without further purification. 1H
NMR (200 MHz, CDCl3): δ 7.45-7.1 (8H, m), 5.87 (1H, s), 1.8
(2H, s). IR (film): νmax 3400-3300 (NH2) cm-1. MS (EI): m/z
252 (M+; 4%), 235 (17%), 140 (100%).
St ep 3. A stirred solution of bis-(2-chlorophenyl)methyl-
amine (1.0 g, 3.97 mmol) in anhydrous EtOAc (40 mL) at 0 °C
was treated with triethylamine (1.11 mL, 7.93 mmol) followed
by thiophosgene (0.3 mL, 3.97 mmol) whereupon the reaction
mixture immediately became opaque. After 3.5 h, the mixture
was diluted with EtOAc (100 mL) and washed with water (2
× 30 mL) and brine (30 mL). The combined washings were
reextracted with EtOAc (30 mL), and the combined organic
phases were dried over MgSO4. The solvent was removed
under reduced pressure to afford 12f (1.12 g, 96%) as a brown
syrup, which slowly solidified upon standing. Compound 12f
was used without further purification. 1H NMR (200 MHz,
CDCl3): δ 7.5-7.2 (8H, m), 6.8 (1H, s). IR (KBr): νmax 2048
(NdCdS) cm-1. MS (EI): m/z 235 (M+ - NCS; 75%), 199 (55%),
165 (100%).
Step 3. A stirred solution of thiophosgene (1.6 g, 14.3 mmol)
in EtOAc (200 mL) at -20 °C was treated with triethylamine
(2.9 g, 28.6 mmol) followed by a solution of 5-aminodibenzo-
suberane (3.0 g, 14.3 mmol) in EtOAc (100 mL). The reaction
was followed on TLC (Et2O:hexane 1:4) and was complete after
stirring for 10 min. The mixture was washed with water (2 ×
200 mL) and brine (100 mL) and dried over MgSO4. The
Gen er a l P r oced u r e for P r ep a r a tion of P yr r olid in es 4,
(S)-14a -g. (S)-1-[4-(4-Ben zh yd r yl-th iosem ica r ba zid o)-3-
n itr oben zen esu lfon yl]p yr r olid in e-2-ca r boxylic Acid {2-

2168 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Dziadulewicz et al.
[(2-Dim eth yla m in oeth yl)m eth yla m in o]eth yl}a m id e [(S)-
4]. Step 1. A stirred solution of (S)-proline ((S)-9a ) (7.5 g, 65.1
mmol) in water (100 mL) at 0 °C was treated with Na2CO3
(13.8 g, 130.2 mmol), followed by 4-chloro-3-nitrobenzenesulfon-
yl chloride (16.7 g, 65.1 mmol), which was added in portions.
The reaction was followed on TLC (EtOAc:hexane 1:3) and was
complete after stirring for 4 h. The mixture was treated with
2 M HCl solution [CAUTION: vigorous effervescence] until
acidic (pH 2). The mixture was washed with EtOAc (3 × 100
mL), and the combined organic phases were washed with
water (2 × 100 mL) and brine (100 mL) and dried over MgSO4.
The solvent was removed under reduced pressure, and the
resulting yellow oil solidified on standing at ambient temper-
ature to afford (S)-10a (19.4 g, 89%), which could be used
(R)-1-[4-(4-Ben zh yd r yl-th iosem ica r ba zid o)-3-n itr oben -
zen esu lfon yl]pyr r olidin e-2-car boxylic Acid {2-[(2-Dim eth -
yla m in oeth yl)m eth yla m in o]eth yl}a m id e [(R)-4]. All spec-
tral data for (R)-4 were identical with those described for (S)-
4; [R]D ) +48.8° (c ) 0.33, MeOH). Anal. (C32H42N8O5S2‚
3.24TFA) C, H, N.
22
(S)-1-(4-[4-(10,11-Dih ydr o-5H-d iben zo[a ,d ]cycloh ep ten -
5-yl)th iosem ica r ba zid o]-3-n itr oben zen esu lfon yl)p yr r o-
22
lid in e-2-ca r boxylic Acid Am id e [(S)-13b]. [R]D ) -44.7°
(c ) 0.51, MeOH); mp 155-159 °C. 1H NMR (400 MHz,
CDCl3): δ 9.12 (1H, s), 8.58 (1H, d, J ) 2 Hz), 7.77 (1H, dd, J
) 8.9, 1.9 Hz), 7.45 (1H, d, J ) 9 Hz), 7.39-7.38 (3H, apparent
d), 7.13-7.05 (7H, m), 6.66 (1H, d, J ) 7.8 Hz), 6.57 (1H, br
s), 5.52 (1H, br s), 3.97 (1H, dd, J ) 8.7, 2.7 Hz), 3.49 (1H, m),
3.12-3.06 (3H, m), 2.98-2.95 (2H, m), 2.15 (1H, m), 1.8 (1H,
m), 1.68-1.60 (2H, m). IR (KBr): νmax 3463, 3330, 1680, 1614,
1569, 1518, 1344, 1160 cm-1. MS (FAB): m/z 581 (MH+; 5%),
193 (100%). Anal. (C27H28N6O5S2‚0.125H2O) C, H, N.
1
without further purification. H NMR (200 MHz, DMSO-d6):
δ 8.5 (1H, d, J ) 2.6 Hz), 8.13 (1H, dd, J ) 7.7, 2.6 Hz), 8.0
(1H, d, J ) 7.7 Hz), 4.3 (1H, dd, J ) 10.2, 4.6 Hz), 3.5-3.2
(2H, m), 2.2-1.6 (4H, m). IR (KBr): νmax 3500-2500, 1712,
1536, 1352 cm-1. MS (FAB): m/z 337 (MH+; 18%), 335 (MH+;
44%), 291 (M+ - CO2H; 36%), 289 (M+ - CO2H; 100%).
(S)-1-[4-(4-Ben zh yd r yl-sem ica r b a zid o)-3-ch lor ob en -
zen esu lfon yl]p yr r olid in e-2-ca r b oxylic Acid {2-[(2-Di-
m eth yla m in oeth yl)m eth yla m in o]eth yl}a m id e [(S)-14a ].
[R]D22 ) -51.4° (c ) 0.55, MeOH). 1H NMR ((S)-14a ‚TFA, 400
MHz, DMSO-d6): δ 8.28 (2H, s and br s), 8.15 (1H, s), 7.69
(1H, d, J ) 2 Hz), 7.63 (1H, dd, J ) 8.7, 1.8 Hz), 7.46 (1H, d,
J ) 8.4 Hz), 7.4-7.15 (10H, m), 6.94 (1H, d, J ) 8.7 Hz), 5.98
(1H, d, J ) 8.4 Hz), 3.95 (1H, dd, J ) 7.8, 3.8 Hz), 3.5-3.35
(6H, m), 3.2-3.05 (2H, m), 2.84 (6H, s), 2.8 (2H, br s), 2.5 (3H,
s), 1.78-1.75 (3H, m), 1.53 (1H, m). IR (KBr): νmax 3362, 1678,
1594, 1539, 1496, 1344, 1160 cm-1. MS (FAB): m/z 658 (M+;
28%), 656 (M+; 58%), 167 (100%). Anal. (C32H42ClN7O4S‚
3.5TFA‚H2O) C, H, N.
Step 2. A stirred solution of (S)-10a (1.0 g, 2.99 mmol) in
anhydrous THF (25 mL) was treated at room temperature with
triethylamine (0.61 g, 5.98 mmol), followed by hydrazine
monohydrate (0.15 g, 2.99 mmol). The reaction mixture was
heated at reflux for 8 h, whereupon TLC analysis indicated
complete reaction. On cooling, 12a (0.81 g, 3.6 mmol) was
added and the reaction mixture was stirred overnight at room
temperature. The mixture was poured into water (600 mL),
and 2 M HCl solution was added until a pH of 2 was achieved.
The resulting precipitate was isolated by filtration and dried
1
in vacuo to afford (S)-13a (1.38 g, 81%). H NMR (200 MHz,
DMSO-d6): δ 10.0 (2H, s,), 9.1 (1H, d, J ) 10.2 Hz), 8.42 (1H,
d, J ) 1.7 Hz), 8.04 (1H, dd, J ) 10.2, 1.7 Hz), 7.44-7.06 (11H,
m), 7.0 (1H, d, J ) 10.2 Hz), 4.18 (1H, dd, J ) 7.5, 5.4 Hz),
3.38 (1H, m), 3.2 (1H, m), 2.1-1.75 (3H, m), 1.66 (1H, m). IR
(KBr): νmax 3650-2900, 3346, 3290, 1749, 1613, 1524, 1493,
1342, 1161 cm-1. MS (FAB): m/z 556 (MH+; 29%), 167 (100%).
(S)-1-[4-(4-Ben zh yd r yl-th iosem ica r ba zid o)-3-n itr oben -
zen esu lfon yl]p yr r olid in e-2-ca r b oxylic Acid {2-[(3-Di-
m eth ylam in opr opyl)m eth ylam in o]eth yl}am ide [(S)-14b].
1H NMR ((S)-14b‚TFA, 200 MHz, DMSO-d6): δ 10.01 (1H, s),
9.98 (1H, v br s), 9.09 (1H, d, J ) 9 Hz), 8.43 (1H, d, J ) 3
Hz), 8.40 (1H, br t), 8.04 (1H, dd, J ) 9, 3 Hz), 7.40-7.08 (11H,
m), 6.99 (1H, d, J ) 9 Hz), 4.03 (1H, m), 3.6-3.36 (4H, m),
3.34-2.98 (6H, m), 2.86 (3H, s), 2.81 (6H, s), 2.06 (2H, br quin),
1.94-1.67 (3H, m), 1.57 (1H, m). IR (KBr): νmax 3372, 3033,
1677, 1616, 1523, 1347, 1203, 1164 cm-1. MS (FAB): m/z 697
(MH+; 63%), 257 (25%), 167 (100%). Anal. (C33H44N8O5S2‚
2.5TFA‚2H2O) C, H, N.
Step 3. A stirred solution of (S)-13a (1.0 g, 1.8 mmol) in
anhydrous THF (50 mL) at -20 °C was treated with N-methyl
morpholine (0.18 g, 1.8 mmol) and isobutyl chloroformate (0.25
g, 1.8 mmol). After it was stirred for 1 h at -20 °C, the reaction
mixture was treated dropwise with a solution of 8a (0.26 g,
1.8 mmol) in anhydrous THF (25 mL). After a further 1.5 h
period, TLC analysis (DCM:MeOH:TFA 20:2:0.5) indicated a
complete disappearance of (S)-13a and formation of a more
polar compound. The solvent was removed under reduced
pressure, and the residue was purified by preparative HPLC.
All fractions containing pure product were combined, and the
solvent was removed under reduced pressure to afford the title
compound as an amorphous orange solid, which was dried
(S)-1-[4-(4-Ben zh yd r yl-th iosem ica r ba zid o)-3-n itr oben -
zen esu lfon yl]p yr r olid in e-2-ca r b oxylic Acid {3-[(3-Di-
m et h yla m in op r op yl)m et h yla m in o]p r op yl}a m id e [(S)-
22
14c]. [R]D ) -37.3° (c ) 0.89, MeOH). 1H NMR ((S)-14c‚
TFA, 200 MHz, CDCl3): δ 9.59 (1H, s), 9.52 (1H, br s), 8.59
(2H, br s and d, J ) 3 Hz), 8.18 (1H, br t), 7.9 (1H, dd, J ) 9,
3 Hz), 7.4-7.15 (11H, m), 6.85 (1H, d, J ) 9 Hz), 4.0 (1H, m),
3.7-3.0 (10 H, m), 2.8 (9H, s), 2.35 (2H, br quin), 2.2-1.8 (5H,
m), 1.69 (1H, m). IR (KBr): νmax 3400, 3250-2700, 1664, 1613,
1493, 1436, 1383, 1339, 1295, 1239, 1196, 1153 cm-1. MS
(FAB): m/z 711 (MH+; 18%), 274 (100%), 167 (59%). Anal.
(C34H46N8O5S2‚2.5TFA‚H2O) C, H, N, O, S, F.
22
under high vacuum (0.8 g, 49%); [R]D ) -43° (c ) 0.47,
1
MeOH). H NMR ((S)-4‚TFA, 360 MHz, DMSO-d6 at 120 °C):
δ 9.62 (1H, br s), 8.67 (1H, v br s), 8.42 (1H, d, J ) 3 Hz), 7.92
(1H, dd, J ) 9, 3 Hz), 7.65 (1H, br t), 7.33-7.13 (12H, m), 6.85
(1H, br s), 4.12 (1H, dd, J ) 9, 6 Hz), 3.41 (1H, m), 3.35-3.22
(3H, m), 3.2-3.1 (2H, t, J ) 7.2 Hz), 2.93-2.83 (2H, t, J ) 7.2
Hz), 2.78 (6H, s), 2.75-2.67 (2H, t, J ) 7.2 Hz), 2.44 (3H, s),
1.92-1.77 (3H, m), 1.66 (1H, m). IR (KBr): νmax 3337, 1676,
1615, 1522, 1346, 1202, 1163, 1131 cm-1. MS (FAB): m/z 683
(MH+; 55%), 241 (17%), 167 (100%). Anal. (C32H42N8O5S2‚
2.5TFA‚0.5H2O) C, H, N, O, S, F.
(S)-1-(4-[4-(10,11-Dih ydr o-5H-d iben zo[a ,d ]cycloh ep ten -
5-yl)th iosem ica r ba zid o]-3-n itr oben zen esu lfon yl)p yr r o-
lid in e-2-ca r b oxylic Acid {2-[(2-Dim et h yla m in oet h yl)-
m eth yla m in o]eth yl}a m id e [(S)-14d ]. 1H NMR (360 MHz,
DMSO-d6 at 120 °C): δ 8.32 (1H, d, J ) 3 Hz), 7.55 (1H, dd,
J ) 9, 3 Hz), 7.47 (1H, d, J ) 9 Hz), 7.45-7.05 (8H, m), 6.77
(1H, br s), 4.05 (1H, m), 3.4-3.1 (8H, m), 2.55-2.4 (4H, m),
2.28 (3H, s), 2.25 (6H, s), 2.22 (2H, m), 1.9-1.72 (3H, m), 1.72-
1.6 (1H, m). IR (KBr): νmax 3400, 2943, 1675, 1614, 1522, 1496,
1341, 1296, 1240, 1201, 1155 cm-1. MS (FAB): m/z 709 (MH+;
37%), 193 (100%). Anal. (C34H44N8O5S2‚0.75TFA) C, H, N.
The following compounds were prepared by the general
procedure described above for the preparation of (S)-4, except
that the isolation procedure in step 4 was used for compounds
(S)-13b and (S)-14d ,e after HPLC purification.
Step 4. After preparative HPLC purification, all fractions
containing pure product were combined, and acetonitrile
solvent was removed under reduced pressure. The pH of the
solution was adjusted to 8 with saturated aqueous NaHCO3
solution. The aqueous solution was washed with EtOAc (3 ×
50 mL), and the combined organic extracts were washed with
brine (50 mL) and dried over Na2SO4. After it was filtered,
the solvent was removed under reduced pressure to afford the
final product as an amorphous solid.
(S)-1-(4-[4-(10,11-Dih ydr o-5H-d iben zo[a ,d ]cycloh ep ten -
5-yl)sem ica r ba zido]-3-n itr oben zen esu lfon yl)p yr r olid in e-
2-ca r boxylic Acid {2-[(3-Dim eth yla m in op r op yl)m eth yl-
22
a m in o]eth yl}a m id e [(S)-14e]. [R]D ) -7.5° (c ) 1.1,
MeOH). 1H NMR (400 MHz, CDCl3): δ 9.2 (1H, br s), 8.58 (1H,
d, J ) 2 Hz), 7.8 (1H, dd, J ) 9, 2.1 Hz), 7.45-7.3 (4H, m),
7.25-7.1 (7H, m), 6.45 (1H, d, J ) 7.2 Hz), 6.13 (1H, d, J )
7.4 Hz), 4.11 (1H, m), 3.49 (1H, m), 3.39 (1H, m), 3.32-3.0

Nonpeptide Bradykinin Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2169
(6H, m), 2.8 (2H, m), 2.54 (6H, s), 2.52-2.43 (4H, m), 2.22 (3H,
s), 2.15 (1H, m), 1.9-1.6 (5H, m). IR (KBr): νmax 3363, 2947,
1672, 1615, 1525, 1348, 1202, 1162 cm-1. MS (FAB): m/z 707
(MH+; 49%), 193 (100%). Anal. (C35H46N8O6S‚0.5TFA‚1.5H2O)
C, H, N, S, F.
anhydrous DMF (7 mL) and anhydrous THF (13 mL) was
treated with N-methyl morpholine (0.09 g, 0.87 mmol). The
reaction mixture was cooled to -20 °C and treated with a 1 M
solution of isopropyl chloroformate in toluene (0.87 mL, 0.87
mmol). After it was stirred for 45 min at -20 °C, the reaction
mixture was treated dropwise with a solution of 8a (0.13 g,
0.87 mmol) in anhydrous THF (10 mL). After 24 h, the solvent
was removed under reduced pressure and the residue was
purified by preparative HPLC. All fractions containing pure
product were combined, and the solvent was removed under
reduced pressure to afford 19a as an amorphous orange solid,
(S)-1-(4-[4-(9H -F lu or en -9-yl)t h iosem ica r b a zid o]-3-n i-
tr oben zen esu lfon yl)p yr r olid in e-2-ca r boxylic Acid {2-[(3-
Dim eth yla m in op r op yl)m eth yla m in o]eth yl}a m id e [(S)-
14f]. [R]D22 ) -53.1° (c ) 0.52, MeOH). 1H NMR ((S)-14f‚TFA,
360 MHz, DMSO-d6): δ 10.2 (1H, s), 9.96 (1H, s), 8.87 (1H, d,
J ) 9 Hz), 8.41 (1H, br t), 8.29 (1H, d, J ) 3 Hz), 8.1 (1H, dd,
J ) 9, 3 Hz), 7.81 (2H, d, J ) 9 Hz), 7.53-7.42 (2H, m), 7.38
(2H, t, J ) 7.2 Hz), 7.33-7.18 (2H, m), 7.22 (1H, d, J ) 9 Hz),
6.94 (1H, d, J ) 9 Hz), 3.95 (1H, dd, J ) 9, 3 Hz), 3.56-3.35
(3H, m), 3.35-2.96 (7H, m), 2.87 (3H, s), 2.82 (6H, s), 2.15-
1.92 (2H, m), 1.87-1.58 (3H, m), 1.56-1.4 (1H, m). IR (KBr):
νmax 3320, 1677, 1615, 1525, 1347, 1202, 1164 cm-1. MS
(FAB): m/z 695 (M+ - 1; 72%), 257 (59%), 165 (100%). Anal.
(C33H42N8O5S2‚2.7TFA‚1.3H2O) C, H, N, O, S, F.
1
which was dried under high vacuum (0.47 g, 63%). H NMR
(19a ‚TFA, 400 MHz, DMSO-d6): δ 10.01 (1H, s), 9.91 (1H, v
br s), 9.01 (1H, v br d, J ) 6.4 Hz), 8.76 (1H, d, J ) 2.2 Hz),
8.46 (1H, v br t), 8.10 (1H, dd, J ) 9.1, 2 Hz), 7.54 (1H,
apparent s), 7.2-7.1 (6H, br s), 7.1-7.0 (4H, br s), 6.97 (1H,
d, J ) 9.3 Hz), 6.84 (1H, v br s), 6.64 (1H, apparent s), 6.30
(1H, t, J ) 3.4 Hz), 3.7-3.2 (6H, m), 2.71 (6H, s), 2.6-2.45
(2H, m), 2.41 (3H, s). IR (KBr): νmax 3416, 3200-2700, 1650,
1613, 1555, 1494, 1435, 1363, 1299, 1242, 1172, 1150 cm-1
.
(S)-1-(4-{4-[Bis-(2-ch lor o-p h en yl)m eth yl]th iosem ica r -
ba zid o}-3-n itr oben zen esu lfon yl)p yr r olid in e-2-ca r boxy-
lic Acid {2-[(2-Dim eth yla m in oeth yl)m eth yla m in o]eth yl}-
MS (FAB): m/z 679 (MH+; 100%). Anal. (C32H38N8O5S2‚3TFA‚
2H2O) C, H, N, F.
22
1
Compounds 19b-d were prepared following the general
procedure described above for the preparation of 19a , except
that after HPLC purification the procedure in step 4 was used
to isolate these DBS-containing compounds.
Step 4. After preparative HPLC purification, all fractions
containing pure product were combined and acetonitrile
solvent was removed under reduced pressure. The pH of the
solution was adjusted to 8 with saturated aqueous NaHCO3
solution. The aqueous solution was washed with EtOAc (3 ×
50 mL), and the combined organic extracts were washed with
brine (50 mL) and dried over Na2SO4. After filtration, the
solvent was removed under reduced pressure to afford the final
product as an amorphous solid.
1-(4-[4-(10,11-Dih yd r o-5H-d iben zo[a ,d ]cycloh ep ten -5-
yl)th iosem ica r ba zid o]-3-n itr oben zen esu lfon yl)-1H-p yr -
r ole-2-ca r boxylic Acid {2-[(2-Dim eth yla m in oeth yl)m eth -
yla m in o]eth yl}a m id e (19b). 1H NMR (400 MHz, DMSO-
d6): δ 8.61 (1H, br s), 8.26 (1H, br t), 7.65 (1H, br d), 7.57 (1H,
br s), 7.53 (1H, br s), 7.46 (2H, br m), 7.15-7.1 (6H, br s), 6.72
(1H, v br s), 6.60 (1H, br s), 6.47 (1H, v br s), 6.31 (1H, t, J )
3.3 Hz), 3.35-3.05 (6H, m), 2.7-2.42 (6H, m), 2.40 (6H, br s),
2.24 (3H, s). IR (KBr): νmax 3400, 3220-2680, 1660, 1614, 1555,
1523, 1492, 1426, 1360, 1299, 1173, 1151 cm-1. MS (FAB): m/z
705 (MH+; 80%), 346 (100%). Anal. (C34H40N8O5S2‚0.2EtOAc‚
H2O) C, H, N.
a m id e [(S)-14g]. [R]D ) -42.8° (c ) 0.49, MeOH). H NMR
((S)-14g‚TFA, 400 MHz, DMSO-d6): δ 9.97 (2H, s), 8.89 (1H,
v br s), 8.38 (1H, d, J ) 2.1 Hz), 8.32 (1H, v br t), 8.05 (1H, dd,
J ) 9.1, 2.1 Hz), 7.43 (2H, d, J ) 7.8 Hz), 7.34-7.26 (4H, m),
7.18-7.15 (2H, m), 7.02 (2H, v br s), 4.02 (1H, dd, J ) 7.9, 3.4
Hz), 3.5-3.05 (8H, m), 2.84 (6H, s), 2.78 (2H, br m), 2.5 (3H,
s), 1.84-1.75 (3H, m), 1.55 (1H, m). IR (KBr): νmax 3380, 3250-
2700, 1670, 1614, 1552, 1496, 1340, 1296, 1240, 1195, 1154
cm-1. MS (FAB): m/z 755 (5%), 753 (MH+; 14%), 751 (MH+;
19%), 237 (18%), 235 (24%). Anal. (C32H40Cl2N8O5S2‚2.9TFA‚
0.9H2O) C, H, N, O, Cl, F.
Gen er a l P r oced u r e for P r ep a r a tion of P yr r oles 19a -
d . 1-[4-(4-Ben zh yd r yl-th iosem ica r ba zid o)-3-n itr oben ze-
n esu lfon yl]-1H-p yr r ole-2-ca r boxylic Acid {2-[(2-Dim eth -
yla m in oeth yl)m eth yla m in o]eth yl}a m id e (19a ). Step 1. A
stirred solution of pyrrole-2-carboxylic acid (15) (2.5 g, 22.5
mmol) in anhydrous THF (100 mL) at -20 °C under an argon
atmosphere was treated with a solution of n-BuLi (19.8 mL,
49.5 mmol, 2.5 M solution in hexanes). After 1 h, the mixture
was recooled to -20 °C and treated with a solution of 4-chloro-
3-nitrobenzenesulfonyl chloride (7.2 g, 28.1 mmol) in anhy-
drous THF (5 mL) and allowed to warm to ambient temper-
ature. After it was stirred for 20 h, the mixture was diluted
with EtOAc (100 mL) and washed with 2 M HCl solution (2 ×
50 mL) followed by brine (50 mL). The EtOAc solution was
dried over MgSO4, and the solvent was removed under reduced
pressure. The residue was dissolved in hot 2-propanol-water
(1:1, 100 mL), and the stirred solution was treated with
activated charcoal (1.0 g). After 5 min, the mixture was filtered
and allowed to cool to ambient temperature. The resulting
precipitate was collected by filtration and dried under high
vacuum to give 16a as a crystalline solid (4.1 g, 55%). 1H NMR
(200 MHz, DMSO-d6): δ 8.74 (1H, d, J ) 2.2 Hz), 8.26 (1H,
dd, J ) 8.5, 2.3 Hz), 8.08 (1H, d, J ) 8.5 Hz), 7.86 (1H, dd, J
) 3.2, 1.75 Hz), 7.12 (1H, dd, J ) 3.7, 1.83 Hz), 6.51 (1H,
apparent t, J ) 3.5 Hz). IR (KBr): νmax 3350-2500, 1708, 1680,
1540, 1444, 1379, 1359, 1267, 1190, 1150 cm-1. MS (FAB): m/z
331 (MH+; 50%), 313 (100%).
Step 2. A stirred solution of 16a (4.1 g, 12.4 mmol) in
anhydrous THF (70 mL) was treated at room temperature with
triethylamine (2.76 g, 27.3 mmol), followed by hydrazine
monohydrate (0.68 g, 13.6 mmol). The reaction mixture was
heated at 70 °C for 6 h and then allowed to cool to ambient
temperature. On cooling, 12a (3.35 g, 14.9 mmol) was added
and the reaction mixture was heated at reflux for 4 h. The
mixture was poured into water (500 mL), and 2 M HCl solution
was added until a pH of 2 was achieved. The resulting
precipitate was isolated by filtration and dried in vacuo to
afford 18a (5.3 g, 78%). IR (KBr): νmax 3400-2650, 1703, 1613,
1518, 1494, 1449, 1369, 1345, 1277, 1181, 1144 cm-1. MS
(FAB): m/z 552 (MH+; 21%), 167 (100%).
1-(4-[4-(10,11-Dih yd r o-5H-d iben zo[a ,d ]cycloh ep ten -5-
yl)sem ica r ba zid o]-3-n itr oben zen esu lfon yl)-1H-p yr r ole-
2-ca r boxylic Acid {2-[(3-Dim eth yla m in op r op yl)m eth yl-
1
a m in o]eth yl}a m id e (19c). H NMR (360 MHz, DMSO-d6 at
120 °C): δ 8.70 (1H, d, J ) 3 Hz), 8.0 (1H, dd, J ) 9, 3 Hz),
7.75 (1H, br t), 7.45 (1H, m), 7.37 (1H, d, J ) 9 Hz), 7.34 (2H,
d, J ) 7.2 Hz), 7.23 (1H, d, J ) 9 Hz), 7.18-7.05 (8H, m), 6.63
(1H, m), 6.3 (1H, t, J ) 3 Hz), 6.22 (1H, d, J ) 9 Hz), 3.26
(2H, apparent q), 3.23-3.1 (4H, m), 2.50 (2H, t, J ) 7.2 Hz),
2.40 (2H, t, J ) 7.2 Hz), 2.34 (2H, t, J ) 7.2 Hz), 2.23 (3H, s),
2.20 (6H, s), 1.55 (2H, quin, J ) 7.2 Hz). IR (KBr): νmax 3350,
1668, 1614, 1558, 1527, 1373, 1360, 1278, 1179, 1151 cm-1
.
MS (FAB): m/z 703 (MH+; 72%), 193 (100%). Anal. (C35H42N8-
O6S‚0.2TFA) C, H, N, F.
1-(3-Ch lor o-4-[4-(10,11-d ih yd r o-5H-d iben zo[a ,d ]cyclo-
h epten -5-yl)sem icar bazido]ben zen esu lfon yl)-1H-pyr r ole-
2-ca r boxylic Acid {2-[(3-Dim eth yla m in op r op yl)m eth yl-
a m in o]eth yl}a m id e (19d ). 1H NMR (400 MHz, DMSO-d6):
δ 8.48 (1H, s), 8.28 (1H, br t, J ) 5.4 Hz), 8.2 (1H, s), 8.09
(1H, d, J ) 2 Hz), 7.88 (1H, dd, J ) 9, 2 Hz), 7.64 (1H, d, J )
8.4 Hz), 7.55 (1H, m), 7.40-7.38 (2H, m), 7.22-7.19 (6H, m),
6.91 (1H, d, J ) 8.7 Hz), 6.63 (1H, m), 6.35-6.31 (2H, m),
3.45-3.15 (6H, m), 2.48 (2H, t, J ) 6.7 Hz), 2.39 (2H, t, J )
7.1 Hz), 2.25 (2H, t, J ) 7.1 Hz), 2.23 (3H, s), 2.14 (6H, s),
1.57 (2H, quin, J ) 7.1 Hz). IR (KBr): νmax 3373, 3000-2750,
1666, 1591, 1554, 1524, 1460, 1452, 1370, 1152 cm-1. MS
(FAB): m/z 692 (MH+; 100%), 253 (40%), 193 (72%). Anal.
Step 3. A stirred solution of 18a (0.4 g, 0.73 mmol) in

2170 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Dziadulewicz et al.
(C35H42ClN7O4S‚0.3TFA‚0.75H2O) C, H, N; F, calcd, 2.3; found,
ments were taken, and test compounds were administered
intravenously, subcutaneously, or orally after three control
readings. The increase in load tolerated by the injected leg
resulting from a test compound was expressed as a percentage
maximal reversal of the hyperalgesia (with 100% reversal
representing an equal load tolerated by the control and injected
leg). The ED50 value (defined as the dose that reversed the
hyperalgesia by 50%) for groups of 8-10 animals was calcu-
lated from the peak response. For determination of maximum
efficacy, the maximum reversal produced by the test compound
was expressed as a percentage of the maximal possible reversal
of hyperalgesia in this model. Statistical analysis was per-
formed by two way analysis of variance (ANOVA) to compare
pre- and posttreatment values.
Tu r p en tin e-In d u ced Mech a n ica l Hyp er a lgesia in th e
Ra t P a w . The nociceptive pressure threshold was determined
on the left hind paw (Analgesymeter, Ugo Basile, Milan) of
male Sprague-Dawley rats (180-220 g), and the end point
was taken as paw withdrawal. The mean of two readings was
taken as the withdrawal threshold. Immediately after the
control reading, the rats received an intraplantar injection in
the left hind paw of 50 µL of a turpentine/paraffin mixture
(1:1). Three days later, the test compounds or vehicle were
administered orally or intravenously. A further threshold
reading was then taken 1 h later. For potency estimation, an
ED50 value was calculated as the dose producing 50% of the
maximum reversal of hyperalgesia for each test compound. The
efficacy of each test compound was the maximum reversal of
hyperalgesia obtained, expressed as a percentage of the
withdrawal threshold of the animals before injection with
turpentine; 100% represented complete reversal of hyperal-
gesia. The duration of action of a test compound was assessed
following a dose of 1 µmol/kg po of the compound or of vehicle.
The withdrawal threshold of both test compound- and vehicle-
treated animals (n ) 10) were then measured every hour for
4 h after administration of test compound.
1.8.
Molecu la r Mod elin g. The modeling calculations described
herein were performed using the CATALYST43 software pack-
age (version 4.6) running on
a Silicon Graphics Octane
workstation (IRIX64 6.5 operating system). The models were
built starting from a generic monosubstituted thiourea struc-
ture, R-NHCSNH2, in accordance with CATALYST standard
bond lengths and valence angles, where R ) DBS, DPM, FL,
and DCDPM. Conformers were generated using the standard
CATALYST method (“Best Quality” mode, a maximum of 250
conformers within a 20 kcal/mol energy range).
In the calculation of the pharmacophore model, the chemical
features selected for hypothesis generation were hydrogen
bond donors, hydrogen bond acceptors, hydrophobic aliphatic
regions, hydrophobic aromatic regions, and aromatic ring
interactions, as defined in the CATALYST software. Hydro-
phobic aromatic regions are placed at the centroids of the
aromatic rings. Hydrogen bond acceptors are characterized by
electronic lone pairs of nitrogen, oxygen, or sulfur atoms.
Pharmacophore hypotheses were generated using the standard
CATALYST method, employing the human B2 receptor binding
affinities of the full structures (S)-14d (DBS), (S)-4 (DPM),
and (S)-14f (FL). Subsequently, CATALYST was asked to map
the DCDPM group (present in (S)-14g) to the best hypothesis,
resulting in an estimate of predicted activity (Ki ) 19 nM) close
to that found experimentally (Ki ) 34 nM).
(B) Biology. Recep tor Bin d in g Assa ys. Standard binding
assays were performed using the membrane preparations
described in our previously published procedures.32 NG108-
15 cell membranes were thawed, homogenized, and diluted
with binding buffer (composition: 25 mM potassium phos-
phate, pH 6.5; 1 mM ethyleneglycolbis(aminoethyl ether)-
tetraacetate (EGTA); 0.1 mM bacitracin; 54 mg/mL chymo-
statin; and 2 mg/mL bovine serum albumin (BSA)) to give a
membrane protein concentration of about 0.3 mg/mL. The
assay mixture comprised 100 µL of [3H]BK (specific activity
65 Ci/mmol; Amersham Corp.), 50 µL of DMSO (total binding),
or 50 µL of the compound to be investigated in DMSO and
100 µL of binding buffer. The assay was started by the addition
of 750 µL of membrane suspension.
Cos-7 cell membranes were thawed, homogenized, and
diluted with binding buffer with composition 10 mM N-tris-
(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), pH
7.4; 1 mM EGTA; 0.14 g/L bacitracin; 54 µg/mL chymostatin;
and 0.2% BSA. The assay mixture comprised 100 µL of [3H]-
BK in binding buffer, 50 µL of DMSO (total binding), or 50 µL
of the test compound in DMSO. The assay was started by
addition of 750 µL of membrane suspension to give a mem-
brane protein concentration of 5 µg/mL.
For all binding assays, nonspecific binding was determined
in the presence of 10 µM BK and the concentration of [3H]BK
in displacement assays was 1 nM. Samples were incubated
for 60 min at 4 °C and then filtered through GF/B filters and
presoaked in polyethylenimine (6 g/L), using a Brandel
Harvester. The filters were washed with ice cold wash buffer
(25 mM potassium phosphate, pH 6.5, for the NG108-15 cells
and 50 mM tris(hydroxymethyl)aminomethane (TRIS), pH 7.4,
for the Cos-7 cells), placed in scintillation vials, and counted
in “Ready-Micro” liquid scintillation fluid. Binding parameters
were calculated using LIGAND.46 Ki values of compounds were
calculated from the equation Ki ) (IC50)/1 + S where S )
(concentration of radioligand)/(Kd of radioligand).47 Data are
the means ( SEM from at least three experiments performed
in triplicate.
F CA-In d u ced Mech a n ica l Hyp er a lgesia in th e Ra t
Kn ee J oin t.32 Female Sprague-Dawley rats (100-120 g) were
lightly anaesthetized with Enflurane, and 100 µL of FCA was
injected into one knee. Three to six days later, the animal was
placed with each hind paw on a pressure transducer, and a
downward force was exerted until the uninjected leg was
bearing 100 g. The force the animal would bear on the injected
leg was determined, and the reduction in the load tolerated
indicated the degree of hyperalgesia.48 Half-hourly measure-
Ack n ow led gm en t. We thank Derek Reid for tech-
nical assistance and for conducting solubility determi-
nations. We are grateful to Dr. Tangqing Li (Novartis
Corp., Summit, NJ ) for communicating the result of the
enantiomeric purity determination of a representative
pyrrolidine TSC analogue.
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