Novel small molecule bradykinin B1 receptor antagonists. Part 1: Benzamides and semicarbazides

Article abstract of DOI:10.1016/j.bmcl.2009.11.119

The synthesis and SAR of two series of bradykinin B₁ receptor antagonists is described. The benzamide moiety proved to be a suitable replacement for the aryl ester functionality of biaryl based antagonists. In addition, it was found that semica

Full text of DOI:10.1016/j.bmcl.2009.11.119

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1225–1228
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel small molecule bradykinin B1 receptor antagonists. Part 1:
Benzamides and semicarbazides
Marco Schaudt, Elsa Locardi, Gunther Zischinsky, Roland Stragies, Jochen R. Pfeifer, Christoph Gibson,
*
Dirk Scharn, Uwe Richter, Holger Kalkhof, Klaus Dinkel, Karsten Schnatbaum
Jerini AG, Invalidenstraße 130, 10115 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of two series of bradykinin B1 receptor antagonists is described. The benzamide
moiety proved to be a suitable replacement for the aryl ester functionality of biaryl based antagonists.
In addition, it was found that semicarbazides can effectively replace cyclopropyl amino acids. The com-
pounds with the best overall profile were biaryl semicarbazides which display high antagonistic activity,
low Caco-2 efflux and high oral bioavailability in the rat.
Received 22 October 2009
Revised 23 November 2009
Accepted 24 November 2009
Available online 2 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Bradykinin B1
Antagonist
Oral bioavailability
Pharmacokinetic profile
Kinin peptides are released from kininogen precursors by the
action of plasma and tissue kallikreins in response to traumatic
injury and infection.1 The biological actions of kinins are mediated
by two major G-protein coupled receptors designated as bradyki-
nin B1 and bradykinin B2 receptor. The B2 receptor is constitutively
expressed under physiological conditions in a variety of cells while
the B1 receptor is induced under pathological conditions such as
tissue damage or inflammation in several cell types including
endothelial, smooth muscle cells, blood cells, and neurons.2,3 This
difference in expression makes the B1 receptor a particularly
attractive drug target. Activation of the B1 receptor produces a
range of pro-inflammatory effects including edema, pain, and pro-
motion of blood-borne leukocyte trafficking.4,5
Recently, a number of publications and patent applications de-
scribed the identification and optimization of biaryl-based brady-
kinin B1 receptor antagonists such as 1 (MK-686, Fig. 1).6,7 It was
reported that 1 revealed certain metabolic liabilities related to
the methyl ester moiety8 and showed low aqueous solubility.9 To
resolve these issues, several studies were undertaken in which
the biaryl region, as well as the N-terminus of the amino acid, were
modified.8–11 Here we report an alternative strategy to replace the
biaryl unit and the cyclopropyl amino acid moiety (Fig. 1). Using a
benzamide moiety instead of the aryl ester resulted in a novel class
of B1 receptor antagonists (2a–g). Additionally, semicarbazides are
shown to serve as suitable replacement for the cyclopropyl amino
acid moiety. Highly active compounds were thus obtained by
combination of semicarbazides with benzamides (3a–f) or biaryls
(4a–k).
The general synthesis of target compounds 2a–g and 3a–f is
illustrated in Scheme 1. Ester 6 was prepared from 57,12 by cyana-
tion of the aryl bromide, hydrolysis and ester formation. The intro-
duction of the semicarbazide group was achieved by coupling Boc-
protected alkyl hydrazines13 with phosgene followed by Boc
O
O
H
N
H
N
N
H
CF3
N
H
R2
F
O
O
R1
O
X
X = CH, N
2a-g
O
O
Amine
Cl
alkyl O
1
H
alkyl O
N
N
H
N
N
H
N
N
H
R2
R2
R1
R5
O
R1
O
X
X
R3
R4
3a-f
O
Amine
4a-k
* Corresponding author. Correspondence should be addressed to: Pericles Calias,
Shire HGT, 125 Spring Street, Lexington, MA 02421, USA. Tel.: +1 781 482 0701; fax:
+1 781 482 2961. E-mail: pcalias@shire.com.
Figure 1. Design of new bradykinin B1 receptor antagonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.119

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M. Schaudt et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1225–1228
Table 1
alkyl O
H
N
B1 receptor antagonist activity of benzamides 2a–g
NH2
X
NH2
X
N
N
R2
O
R1
R1
R1
O
R3
H
N
c-e
a, b
X
N
H
R2
R1
O
X
Br
5a-c
X = CH, N
O
O
6a-c
O
O
7a-f
f,d,e
g,h
O
Amine
R1
R2
X
CAM IC50 (nM)
a
Compound
Amine
NHEt
O
O
H
N
H
N
L
N
N
N
N
N
H
R2
N
R2
2a
H
H
H
H
CH
>10,000
3100
500
R1
O
R1
O
R3
N
X
X
g,h
2b
2c
2d
NEt2
CH
CH
CH
8a-d
O
O
O
Amine
N
N
N
2a-g (L = cyclopropyl)
3a-f (L = N-alkyl)
NiPr2
Scheme 1. Reagents and conditions: (a) (i) AcCl, TEA, Et2O; (ii) CuCN, DMF, 160 °C;
(b) (i) 6 N HCl, 110 °C; (ii) MeOH, cat. HCl, 70 °C; (c) COCl2, N0-alkyl-hydrazine-
carboxylic acid tert-butyl ester, DIPEA, THF; (d) HCl/Methanol; (e) R2CO2H, HATU,
DIPEA, DMF; (f) tert-butoxycarbonyl-aminocyclopropane carboxylic acid, HATU,
DIPEA, DMF; (g) LiOH, dioxane/H2O; (h) amine, HATU, DIPEA, DMF.
N
125
CF3
deprotection. Coupling reactions with carboxylic acid derivatives
formed precursor 7. Applying a cyclopropyl amino acid derivate
in this synthesis pathway yielded precursor 8. The final com-
pounds 2a–g and 3a–f were obtained by hydrolysis of the methyl
ester and HATU coupling reactions with the corresponding amines.
After surveying a variety of primary and secondary amines in
combination with different R2s, a focused series of benzamides
was prepared (Table 1). The in vitro functional activity of the com-
pounds at the B1 receptor was assessed in a DAKD-mediated cal-
cium mobilization (CAM) assay using IL-1 beta pretreated human
lung fibroblasts IMR-90. The SAR of the pyrimidyl benzamides 2a
(IC50 = >10,000 nM) and 2b (IC50 = 3100 nM) demonstrates that
the secondary amine is advantageous to provide B1 receptor antag-
onist activity. Increasing steric bulk from diethyl (2b,
IC50 = 3100 nM) to diisopropyl (2c, IC50 = 500 nM) led to a sixfold
increase in activity. Encouraged by this result, we synthesized
the 2,5-dimethylpyrrolidyl amide 2d (IC50 = 125 nM) which
showed a fivefold increase in potency compared to the diisopropyl
amide 2c. Furthermore, switching of R2 from the polar pyrimidine
moiety (2d) to the bulky hydrophobic 3-fluoro-5-(trifluoro-
methyl)benzoic acid (2e) resulted in a drastic 25-fold increase of
activity (2e, IC50 = 5 nM). Employing the enantiomerically pure
2,5-dimethylpyrrolidyl and fluoro substitution at R1 led to 2f
(IC50 = 1.3 nM). Interestingly, and in contrast to biphenyl based
B1 receptor antagonists,6 modification of the phenyl core to a pyr-
idyl was not tolerated by the B1 receptor (2f, IC50 = 1.3 nM vs 2g,
IC50 = 28 nM).
N
N
N
2e
2f
H
F
CH
CH
N
5.0
1.3
28
F
F
F
CF3
CF3
2g
F
a
Numerical average of at least two experiments.
Table 2
B1 receptor antagonist activity of semicarbazides 3a–f
R1
N
O
H
N
CF3
N
F
O
R2
F
Compound 2f exhibited a high aqueous solubility of 42 lM as
well as a reasonable metabolic stability14 (45% remaining after
1 h in rat microsomes) and a reasonable pharmacokinetic profile
in male Wistar rat (F = 43%, t1/2 = 39 min, CL = 34 mL/min/kg).
However, the human microsomal stability of 2f proved to be low
(9% remaining after 1 h in human microsomes). Consequently, 2f
was not considered for further characterization.
O
N
a
Compound
R1
R2
CAM IC50 (nM)
3a
3b
3c
3d
3e
3f
H
H
>1000
>1000
>1000
1.0
4.4
15
Me
H
Me
Et
H
At this stage we decided to focus on the exploration of the ami-
no acid moiety. As it is well-known that aza amino acids can serve
as suitable replacement for amino acids15 we hypothesized that
semicarbazides might provide viable analogues. Therefore the
compounds 3a–f were synthesized (Table 2).
Et
Me
i-Pr
H
H
a
Numerical average of at least two experiments.

M. Schaudt et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1225–1228
1227
Table 3
O
Antagonistic activity of semicarbazides 4a–e
H
H
N
H
N
R1
N
R1
R1
N
Boc
a, b
Boc
N
H
R5
O
H
F
F
F
O
R1
N
N
X
X
c-f
X
O
N
H
N
O
F
O
X
Br
R2
R4
R2
R4
X = CH, N
R3
R3
R2
F
9a-c
10a-j
4a-k
Cl
Scheme 2. Reagents and conditions: (a) bis(pinacolato) diborane, Pd(dppf)Cl2,
KOAc, DMF, 80 °C; (b) aryl bromide, Pd(dppf)Cl2, K2CO3, DMF, 80 °C; (c) HCl/
methanol; (d) COCl2, N0-ethyl-hydrazinecarboxylic acid tert-butyl ester, DIPEA, THF;
(e) HCl/Methanol; (f) R5CO2H, HATU, DIPEA, DMF.
Compound
R1
H
X
R2
CAM IC50, nMa
0.43
Efflux ratiob
2.0
O
N
O
N
N
N
N
N
N
N
N
4a
CH
the introduction of a methyl group into the benzyl position (4e, ra-
tio 4.6 vs 4d, ratio 7.2).
N
N
N
N
4b
4c
4d
4e
H
N
0.26
0.13
0.20
0.14
4.7
3.8
7.2
4.6
Further exploration of more than 70 different aliphatic and het-
erocyclic amide derivatives revealed that the trifluoroacetic acid
(TFA) amide—present in 1—served as the best functional group
with respect to activity and Caco-2 efflux. In Table 4, representa-
tive examples of semicarbazides with the TFA amide are shown.
In both classes with the oxadiazole and the tetrazole moiety, the
compounds with a Cl/F-substitution pattern (4g, IC50 = 0.48 nM
and 4i, IC50 = 0.28 nM) were found to be more active than those
with Cl/Cl-substitution (4f, IC50 = 1.3 nM and 4h, IC50 = 0.64 nM).
However, 4g and 4i still showed marginal Caco-2 efflux ratios of
2.4 and 3.9, respectively. Inspired by researchers from Merck,8
we introduced a difluoroethyl ether into the phenyl ring of the
semicarbazide derivatives. For the compounds with the Cl/F (4k,
H
CH
N
N
N
N
H
CH3
N
a
Numerical average of at least two experiments.
Caco-2 directional transport ratio (B to A)/(A to B).
b
Table 4
Antagonistic activity of semicarbazides 4f–k
We found that the potency of both the N-unsubstituted semi-
carbazide 3a, as well as the bis-N-methylated analogue 3b, was
less than IC50 = 1000 nM. Installation of an ethyl group at the R2
position (3c, IC50 >1000 nM) did not lead to any measurable activ-
ity at the B1 receptor. In contrast, a highly active compound was
obtained (3d, IC50 = 1.0 nM) when R1 only was an ethyl group. Fur-
ther evaluation of the R1 alkyl substituent demonstrated that
methyl (3e, IC50 = 4.4 nM) and isopropyl (3f, IC50 = 15 nM) substi-
tution led to lower activity compared to 3d, suggesting the ethyl
moiety represents an optimal small alkyl substituent for R1 in this
series. Unfortunately, this class of compounds (3a–f) proved to be
compromised by low human microsomal stability, as for benzam-
ides 2a–g.
To improve the microsomal stability, the biaryl core was revis-
ited and SAR of the corresponding semicarbazide derivatives was
undertaken (Table 3). A 3-methoxyisoxazole-5-carbonyl group
was incorporated adjacent to the semicarbazide, based on the
observation that in similar biaryl cyclopropyl amino acid-based
antagonists, this group was found to be optimal.8 The general syn-
thesis of the compounds is depicted in Scheme 2. Transformation
of phenyl or pyridine bromides 97,16 gave rise to the respective
pinacol boron esters which were coupled with the selected aryl
bromides8,16,12 to furnish the biaryl compounds 10. The final com-
pounds 4a–k were obtained by removal of the Boc group and sub-
sequent HATU coupling with the corresponding carboxylic acids.
Compound 4a, with an oxadiazole ring attached to the distal
ring of the biaryl moiety, showed very good activity in the subn-
anomolar range (IC50 = 0.43 nM). A Caco-2 efflux ratio17 of 2.0 indi-
cates that the compound is likely to be a weak P-gp substrate.
Activity, as well as solubility, could be increased by switching to
the pyridine derivative 4b (IC50 = 0.26 nM). However, in the pyri-
dine series, Caco-2 efflux was higher as also shown for the tetra-
zoles 4c and 4d. This effect could be reduced to some extent by
O
H
N
N
N
H
CF3
F
O
N
R2
R4
Cl
a
Compound
R4
Cl
R2
CAM IC50 (nM)
1.3
Efflux ratio
n.d.
O
N
N
N
N
4f
N
O
4g
4h
4i
F
0.48
0.64
0.28
1.5
2.4
n.d.
3.9
1.2
1.1
N
N
Cl
F
N
N
N
N
F
N
4j
Cl
F
O
O
F
F
F
4k
1.8
a
Numerical average of at least two experiments.

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M. Schaudt et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1225–1228
2. Regoli, D.; Barabe, J. Pharmacol. Rev. 1980, 32, 1.
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M. M. Br. J. Pharmacol. 2004, 348, 803.
Pharmacokinetic data for selected compounds (Wistar rat)
a
Compound
t1/2 (min)
CLa (mL/min/kg)
Fb (%)
5. Leeb-Lundberg, L. M. F.; Marceau, F.; Mueller-Esterl, W.; Pettibone, D. J.; Zuraw,
B. L. Pharmacol. Rev. 2005, 90, 8234.
4b
4c
4j
24
93
135
121
14
5
7
>45
20
>44
50
6. Kuduk, S. D.; Bock, M. G. Curr. Top. Med. Chem. 2008, 8, 1420.
7. Kuduk, S. D.; Di Marco, C. N.; Chang, R. K.; Wood, M. R.; Schirripa, K. M.;
Kim, J. J.; Wai, J. C.; DiPardo, R. M.; Murphy, K. L.; Ransom, R. W.; Harrell, C.
M.; Reiss, D. R.; Holahan, M. A.; Cook, J. L.; Hess, J. F.; Sain, N.; Urban, M. O.;
Tang, C.; Prueksaritanont, T.; Pettibone, D. J.; Bock, M. G. J. Med. Chem. 2007,
50, 272.
4k
2
a
1 mg/kg iv.
1 mg mg/kg po.
b
8. Feng, D.-M.; DiPardo, R. M.; Wai, J. M.; Chang, R. K.; Di Marco, C. N.; Murphy, K.
L.; Ransom, R. W.; Reiss, D. R.; Tang, C.; Prueksaritanont, T.; Pettibone, D. J.;
Bock, M. G.; Kuduk, S. D. Bioorg. Med. Chem. Lett. 2008, 18, 682.
9. Kuduk, S. D.; DiPardo, R. M.; Chang, R. K.; DiMarco, C. N.; Murphy, K. L.;
Ransom, R. W.; Reiss, D. R.; Tang, C.; Prueksaritanont, T.; Pettibone, D. J.; Bock,
M. G. Bioorg. Med. Chem. Lett. 2007, 17, 3608.
10. Wood, M. R.; Schirripa, K. M.; Kim, J. J.; Kuduk, S. D.; Di Marco, C. N.; Chang, R.
K.; Wai, J. C.; DiPardo, R. M.; Wang, B. L.; Cook, J. L.; Holahan, M. A.; Lemaire,
W.; Mosser, S. D.; Bednar, R. A.; Wallace, A. A.; Mei, Q.; Yu, J.; Bohn, D. C.;
Clayton, F. D.; Adarayan, E. A.; Sitko, G. R.; Leonard, Y. M.; Murphy, K. L.;
Ransom, R. W.; Harrell, C. M.; Tang, C.; Prueksaritanont, T.; Pettibone, D. J.;
Bock, M. G. Bioorg. Med. Chem. Lett. 2008, 18, 716.
11. Kuduk, S. D.; Chang, R. K.; Dipardo, R. M.; Di Marco, C. N.; Murphy, K. L.;
Ransom, R. W.; Reiss, D. R.; Tang, C.; Prueksaritanont, T.; Pettibone, D. J.; Bock,
M. G. Bioorg. Med. Chem. Lett. 2008, 18, 5107.
12. Menzel, K.; Machrouhi, F.; Bodenstein, M.; Alorati, A.; Cowden, C.; Gibson, A.
W.; Bishop, B.; Ikemoto, N.; Nelson, T. D.; Kress, M. H.; Frantz, D. E. Org. Process
Res. Dev. 2009, 13, 519.
IC50 = 1.8 nM) and the Cl/Cl (4j, IC50 = 1.5 nM) pattern, the activity
as well as the Caco-2 efflux values, were found to be excellent.
Pharmacokinetic data for selected compounds in male Wistar
rats are shown in Table 5.18 Compound 4b revealed a satisfactory
PK profile as it is modestly cleared after iv administration
(CL = 14 mL/min/kg) and has an oral bioavailability of F >45%. Com-
pound 4c showed a better terminal plasma half life after iv admin-
istration (t1/2 = 93 min) and lower clearance but oral bioavailability
proved to be only 20%. The difluoro ethyl ether analogues 4j and 4k
exhibited the best PK profiles in rat with low clearances
(CL 6 7 mL/min/kg), long plasma half lifes (t1/2 > 120 min) and
good oral bioavailabilities (F P 44%). There was no indication of
any toxicity of semicarbazides in a cytotoxicity assay in human
13. (a) Dyker, H.; Scherkenbeck, J.; Gondol, D.; Goehrt, A.; Harder, A. J. Org. Chem.
2001, 66, 3760; (b) Bailey, M. D.; Halmos, T.; Goudreau, N.; Lescop, E.; Llinas-
Brunet, M. J. Med. Chem. 2004, 47, 3788; (c) Melendez, R. E.; Lubell, W. D. J. Am.
Chem. Soc. 2004, 126, 6759.
hepatocytes up to a concentration of 100
l
M19 and no mutagenic
liability was determined in an Ames test.20
14. Determined as described in: Kunz, W.; Gieschen, H. Drug. Metab. Dispos. 1998,
26, 1120. Microsomal preparations from different species were obtained from
Tebu-bio, Offenbach, Germany.
15. Gibson, C.; Goodman, S. L.; Hahn, D.; Hölzemann, G.; Kessler, H. J. Org. Chem.
1999, 64, 7388.
16. Kuduk, S. D.; Wood, M. R. WO Patent 2006/132837, 2006; Chem. Abstr. 2006,
143, 133188.
17. Absorption systems express bidirectional permeability assay through Caco-2
cell monolayers: Artursson, P.; Palma, K.; Luthmanb, K. Adv. Drug Delivery Rev.
2001, 46, 27; Sambuy, Y.; De Angelis, I.; Ranaldi, G.; Scarino, M. L.; Stammati,
A.; Zucco, F. Cell Biol. Toxicol. 2005, 21, 1.
18. The compounds were administered to male Wistar rats at 1 mg/kg iv (group
1) and 1 mg/kg. po (group 2). Aliquots of plasma samples were precipitated
with methanol containing a structural analogue of the analyte as internal
standard and analyzed by HPLC–MS/MS to determine time/concentration
values. The pharmacokinetic parameters were calculated by using Winnonlin
5.1.
19. Cell viability assay with human plateable cryopreserved hepatocytes.
Incubation was performed with different concentrations of the
semicarbazide for 24 h at 37 °C with resazurin as substrate and detection of
resorufin as reaction product by fluorimetry: Nociari, M. M.; Shalev, A.; Benias,
P.; Russo, C. J. Immunol. Meth. 1998, 213, 157.
In summary, we describe the synthesis of two series of bradyki-
nin B1 receptor antagonists. The benzamide series showed high
potency but suffered from low microsomal stabilities. This issue
was resolved by the introduction of the semicarbazide functional-
ity in combination with a biaryl scaffold. The resulting compounds
(e.g., 4j–k) expressed high B1 receptor antagonistic activity, low
Caco-2 efflux ratios, and excellent pharmacokinetic profiles.
Acknowledgements
We thank Pericles Calias and Brigitte Hoch for proofreading the
manuscript and for their consent to handle any future correspon-
dence. We also thank Jessica Bald, Anja Borrmann, Antje Burian,
Anett Hauser, Birgit Hollmann, Bianka Knopp, Stephanie Köpke,
Ricarda Lange, Nicole Liebelt, Corinna Mewes, Daniel Ohlendorf,
Katy Pesta, Frank Polster, Dagmar Riexinger, Beatrice Rosskopp,
Jana Rother, Edith Weigt, Rocco Weise, Daniela Wulf and Ariane
Zwintscher for their skillful technical assistance.
20. Pre-incubation test using salmonella typhimurium strains TA98, TA100, TA1535,
TA1537 and TA102. The assay was conducted with and without metabolic
activation by S9. No toxic effects were noted at any concentration of the test
item (up to 100
lM) in any of the tester strains. This means that no gene
References and notes
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