Chromenones as potent bradykinin B1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.10.068

A series of fused 6,6-bicyclic chromenones was investigated for activity against the bradykinin B1 receptor. SAR studies based on a pharmacophore model revealed compounds with high affinity for both human and rabbit B1. These compounds demonstrated favorable pharmacokinetic properties and 5-chlorochromenone 15 was efficacious in a carrageenan-induced mechanical hyperalgesia model for chronic pain.

Full text of DOI:10.1016/j.bmcl.2011.10.068

Bioorganic & Medicinal Chemistry Letters 22 (2012) 619–622
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chromenones as potent bradykinin B1 antagonists
Marian C. Bryan a, Kaustav Biswas a, , Tanya A.N. Peterkin a, Robert M. Rzasa a, Leyla Arik c,
⇑
Sonya G. Lehto c, Hong Sun c, Feng-Yin Hsieh b, Cen Xu c, Robert T. Fremeau c, Jennifer R. Allen a
a Department of Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Neuroscience Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fused 6,6-bicyclic chromenones was investigated for activity against the bradykinin B1 recep-
tor. SAR studies based on a pharmacophore model revealed compounds with high affinity for both human
and rabbit B1. These compounds demonstrated favorable pharmacokinetic properties and 5-chloro-
chromenone 15 was efficacious in a carrageenan-induced mechanical hyperalgesia model for chronic
pain.
Received 1 October 2011
Revised 18 October 2011
Accepted 20 October 2011
Available online 28 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Bradykinin B1 receptor
Chromenone
Inflammatory pain
Bradykinin (BK), kallidin (Lys-BK), and their active metabolites
form a group of naturally occurring peptides called kinins.1,2 Kinins
are important biological mediators in neutrophil trafficking, car-
diovascular homeostasis, contraction and relaxation of smooth
muscles, inflammation, and nociception.1,3,4 The endogenous, vaso-
active bradykinin and kallidin are produced at and act at the site of
tissue injury and inflammation.5–7 The peptides are then rapidly
degraded in the plasma by kininases to release two biologically ac-
tive metabolites: des-Arg9-bradykinin (DABK) and des-Arg10-kalli-
din (DAK).
The effect of kinins and their des-Arg metabolites are exerted
through their interactions with two G-protein-coupled receptors
named the bradykinin 1 and 2 receptors (B1 and B2).8 They differ
in their expression, function, and regulation. Upon binding to DABK
or DAK, the B1 receptor signals the release of inflammation medi-
geenan.11 Furthermore, induction of the B1 receptor has been sug-
gested to play a role in developing and maintaining chronic pain in
preclinical models.5,11
We have previously reported on peptidic and non-peptidic
chemotypes as potent B1 antagonists.12–14 Our work in this area
suggested a pharmacophore model (Fig. 1A) comprised of a hydro-
phobic region containing a hydrogen bond acceptor on one side
separated by a linker moiety from a basic domain on the other.
As reported recently, ongoing efforts to improve the pharmacoki-
netic and physicochemical profiles of our previous series has led
to a series of 6,6-fused bicycles, shown in Figure 1B.15 Herein, we
report the SAR surrounding chromenones 3 and 4, bicycles with
comparable potency and pharmacokinetic properties to the previ-
ously discussed phthalazinones 1 and 2.
Chromenones were generated via the synthetic route shown in
Scheme 1. Methyl Grignard addition to nitrile 5 with subsequent
deprotection of the methyl ether provided the ketophenol interme-
diate 6. Subsequent reaction with dimethylformamide dimethyl-
acetal (DMF–DMA) and bromine formed brominated chromenone
core 7. Suzuki coupling with either the meta- or para- substituted
(methoxycarbonylphenyl)boronic acid gave ester 8. The previously
identified privileged basic tetraline amine was then coupled to the
corresponding carboxylic acid, unmasked under acidic conditions,
using standard peptide coupling conditions to give desired final
products 9.
ators (e.g., IL-6, IL-8, IL-1b, and TNF-a), potentiates neutrophil traf-
ficking, and sensitizes primary nociceptors.9,10 The low basal
expression, rapid induction following tissue injury and inflamma-
tion, and resistance to desensitization suggests that antagonism
of the B1 receptor could be an interesting therapeutic target.1,5
B1 antagonists have proven efficacious in reversing hyperalgesia
in chronic inflammatory pain models and reversing pain and
inflammatory pain induced by capsaicin, UV irradiation, and carra-
Abbreviations: B1, bradykinin B1 receptor; B2, bradykinin B2 receptor; BK,
The activity of the resulting analogs was determined in human
binding and functional assays as well as a rabbit functional assay.
Along with confirming binding potency, the human B1 (hB1) func-
tional assay was used to verify the antagonistic profile of each
bradykinin; DABK, des-Arg9-bradykinin; DAK, des-Arg10-kallidin; IL, interleukin;
TNF-
a
, tumor necrosis factor-alpha.
⇑
Corresponding author. Tel.: +1 805 447 4836; fax: +1 805 480 1337.
E-mail address: kbiswas@amgen.com (K. Biswas).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.068

620
M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 619–622
H
N
A
B
C
N
N
A)
B)
O
N
N
O
N
H
N
N
H
O
N
O
O
A = Hydrophobic unit
B = H-bond acceptor
C = Linker
N
1
2
hB1 Ki (nM)
6,366 793
3,405 709
>6,465
43
7
9
hB1 cell IC50 (nM)
rabbit cell IC50 (nM)
2
98 18
O
O
O
O
N
H
N
N
H
N
O
O
3
4
hB1 Ki (nM)
64 11
112 22a
184 32
98 49
122 39
hB1 cell IC50 (nM)
rabbit cell IC50 (nM) 476 79a
Figure 1. (A) Pharmacophore model; (B) Fused 6,6-bicycles as potent B1 inhibitors.15 Data reported as the mean SD where n P 3 except (a) tested twice.
(HO)2B
OH
O
O
O
CO2Me
OMe
CN
R
R
a,b
c,d
R
R
Br
e
5
6
7
8
5
O
7
R
O
O
O
6
f,g
O
HN
CO2Me
8
9
N
Scheme 1. General synthesis of the substituted chromenones. Reagents and conditions: (a) MeMgBr, THF, reflux, 6 h; 2 N HCl, 75 °C, 20 h; (b) BBr3, DCM, À78 to 23 °C, 1 h; (c)
DMF–DMA, 100 °C, 2 h; (d) Br2, CHCl3, 0–23 °C, 5 min; (e) RB(OH)2, Pd(PPh3)4, K2CO3, toluene:H2O (2.6:1), 60 °C, 16 h; (f) 1 N HCl, dioxane, reflux, 16 h; (g) EDCI, HOBT, TEA,
H2NR, DMF, 23 °C, 16 h.
compound in an aequorin-based calcium flux assay. This assay was
performed in CHO cells expressing the hB1 receptor.11,12 The hB1
functional assay data closely followed the human B1 receptor bind-
ing affinity data. The rabbit functional assay (conducted in CHO
cells transfected with rabbit B1 receptor) was used to identify com-
pounds with optimal cross-species rabbit receptor activity, which
was requisite, as in vivo efficacy studies were conducted in
rabbits.13
Unlike phthalazinone 1, meta-linked chromenone 3 showed
comparable potency to both para-linked systems. As such, the
SAR surrounding the fused ring system of this linkage was ex-
plored. We had previously observed improved potency in the
para-linked phthalazinone series by incorporating hydrophobic
moieties in the benzo-fused ring of the phthalazinone. The SAR
surrounding the hydrophobic pocket of the meta-substituted
chromenone 3 was similarly investigated by studying the preferred
site for a chlorine substitution of the phenyl ring (Table 1). These
studies revealed 5-chlorochromenone 10 to be the most potent
for the hB1 receptor in both binding and functional assays (hB1
Ki = 29 nM, IC50 = 17 nM) with an order of magnitude increase in
activity over unsubstituted chromenone 3. Substitution of the C6
position (compound 11) with chlorine slightly reduced potency
for human B1, while 6-fluorochromenone (12) gave comparable
potency to 3 in the human functional assay for human B1 as did
8-chloro derivative 14. The remaining regioisomer, C7 substituted
chromenone 13, was less potent. This trend differs significantly
from the para-linked phthalazinone series, where substitution gave
marked improvement in potency in all but the 6-position when
compared to unsubstituted phthalazinone 2. Rabbit B1 receptor
potency was modest in this series, with the best potency seen with
5-chlorochromenone 10 (rabbit B1 IC50 = 248 nM).
Next, we examined the SAR in the para-linked series. This data
is shown in Table 2. As with the meta-series, 5-Cl analogue 15 (hB1
IC50 = 4 nM) was the most potent of the para-linked chlorochrome-
nones for the human B1 receptor. Interestingly, compound 15 also
displayed a fourfold improvement in the rabbit B1 cellular assay.
Further differentiating the SAR of the para-linked series from that
of the meta-linked series was the effect of substitution at C6 and
C7. Substitution at C6 (16) was poorly tolerated in both species,
whereas substitution of the para-linked chromenone at C7 (17)
was permitted in the case of human B1 (Ki = 42 nM; IC50 = 264 nM).
The 8-chlorochromenone exhibited similar potency to unsubstitut-

M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 619–622
621
Table 1
SAR around meta-linked chromenones
8
O
7
6
O
N
H
5
N
O
Compound #
SAR
hB1 Binding Ki (nM)
hB1 Functional IC50 (nM)a
112 22
Rabbit B1 functional IC50 (nM)a
3
H
64 11
476 79
248 18
>160
393 40
>800
10
11
12
13
14
5-Cl
6-Cl
6-F
7-Cl
8-Cl
29 12
17
6
767 225a
398 44a
ND
279 143
78 28
799 26
188 41
322 126a
>800
a
Data reported as the mean SD where n P 3 except (a) tested twice.
Table 2
SAR around para-linked chromenones
8
5
O
O
7
6
N
H
N
O
Compound
SAR
hB1 Binding Ki (nM)
hB1 Functional IC50 (nM)
98 49
Rabbit B1 functional IC50 (nM)
122 79
4
H
184 32
15
16
17
18
19
20
2115
5-Cl
6-Cl
7-Cl
8-Cl
4
2
4
2
30
9
1616 498
42
481 87
>4000a
264 25a
186a
>4000a
>800a
4
>4000a
24 4a
139 12a
5-F
5-Me
5-Cl-Phthalazinone
17
8
12 2a
66 <1a
1.0 0.4
15 2a
0.9 0.3
21
7
a
Data reported as the mean SD where n P 3 except (a) tested twice.
ed chromenone 4 for hB1 but not the rabbit B1 receptor, where a
significant loss in potency was observed. This trend follows that re-
ported for the para-linked phthalazinone series where C5 (com-
pound 21) was identified as the preferred substitution position,
followed by C7 and finally C6. The C8 analogue was not synthe-
sized in that series.
Table 3
Pharmacokinetic parameters of select chromenones
Compound #
Rat t (h)a
CL (L/h/kg)a
Vss (L/kg)a
F (%)
½
4
15
19
4.2
4.5
3.8
1.78
1.06
1.53
9.27
6.67
8.83
29.5b
43.6b
27.9c
To further investigate the nature of C5 substitution, other sub-
stituents were appended including a fluorine (19) and methyl
group (20). Compound 20 was equipotent to unsubstituted
chromenone 4 for both hB1 and rabbit B1 in the functional assays
(20 IC50 = 66 nM; 139 nM vs 4 IC50 = 98 nM; 122 nM, respectively).
5-Fluoro analogue 19 was comparable to 5-chlorochromenone 15
for hB1 (19 Ki = 17 nM, IC50 = 12 nM vs 15 Ki, IC50 = 4 nM) and rab-
bit B1 (19 IC50 = 24 nM vs 15 IC50 = 30 nM), possibly demonstrating
a preference for electron withdrawing groups at this position. The
comparable potency for the 5-fluoro and 5-chloro analogues mir-
rors that seen in the phthalazinone series. Both compounds 15
and 19 were taken into preliminary in vivo PK.
The PK properties of the potent chromenones in rats are shown
in Table 3. Fluorochromenone 19 and unsubstituted chromenone 4
gave comparable clearance, steady state volume, and bioavailabil-
ity. 5-Chlorochromenone 15 demonstrated a modest decrease in
clearance (CL = 1.06 L/h/kg) as well as improved bioavailability
(F = 43.6%). Comparable clearance and steady state volume with
improved bioavailability upon C5 chlorination was also seen with
compound 21 relative to compound 2 (F = 90% vs 51%).15 As such,
this compound was examined in a rabbit carrageenan efficacy
model of inflammatory pain.
a
b
c
Dosed i.v. at 2 mg/kg, formulated in 100% DMSO.
Dosed orally at 5 mg/kg, formulated in 1% Tween 80, 2% HPMC, pH = 2.2.
Dosed orally at 2 mg/kg, formulated in 1% Tween 80, 2% HPMC, 97% water,
pH = 2.2.
As mentioned previously, we and others have evaluated the ef-
fects of B1 antagonists in vivo in rabbits.12–14,16 In our efficacy
model of inflammatory pain, mechanical hyperalgesia was induced
in the rabbit hind paw by injection of carrageenan followed by the
compound.13 The maximum force tolerated on the hind paw prior
to withdrawal from a probe was then recorded.17 Rabbits treated
with 5-chlorochromenone 15 showed a 65% reduction in their re-
sponse to pain stimuli relative to vehicle as shown in Figure 2.
We previously reported a series of 6,6-bicycles with potent
activity against bradykinin B1.15 In addition to the phthalazinone
series described therein, meta- and para-linked chromenones 3
and 4 were identified with respectable potency in rabbit and hu-
man functional assays as well as acceptable pharmacokinetic prop-
erties for further investigation. Unlike the phthalazinone series,
both the meta- and para-linked chromenones demonstrated mod-
est potency in the human B1 binding assay. Further exploration

622
M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 619–622
while maintaining potency in the meta-series coupled with the
ability to reverse inflammatory pain in vivo with compound 15
of the para-series highlight the potential of the chromenones rela-
tive to the previously reported phthalazinone series. Additional
exploration of this viable alternate scaffold for the inhibition of
bradykinin B1-mediated pain is in progress.
References and notes
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Figure 2. Efficacy of chlorochromenone 15 in
a rabbit carrageenan model of
inflammatory pain. Vehicle: 25% captisol/water, pH 3.4; Cmpd 15 dosed s.c. at
10 mpk at 60 min before testing; ⁄⁄P < 0.01, one-way ANOVA, followed by Dunnett’s
post-hoc multiple comparison test, compared to vehicle; data are plotted as group
mean SEM. Plasma exposure measured at 379 133 nM at 10 mpk dose.
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of the meta-series demonstrated a preference for C5 substitution
while highlighting difference in the effect of substitution on po-
tency elsewhere on the ring. Investigation of the SAR around the
hydrophobic left-hand side of the para-linked chromenones gave
similar trends in potency relative to that of the phthalazinone ser-
ies as highlighted by compounds 15 and 21 compared to 4 and 2.
Furthermore, 5-substituted, para-linked chromenones 15 and
19 were identified as potent B1 antagonists. These analogs gave
over eightfold increase in activity in an hB1 functional assay and
over fourfold increase in activity in a rabbit B1 functional assay
as well as similar or improved pharmacokinetic properties when
compared to unsubstituted chromenone 4. 5-Chlorochromenone
15 additionally proved efficacious in a rabbit carrageenan model
for inflammatory pain. Beneficial SAR discovered outside of the
fused 6,6-ring system of the phthalazinone core is expected to fur-
ther improve both potency and pharmacokinetic properties of
these compounds. The potential to exploit alternate chemical space
17. Dong, H.; Sun, H.; Magal, E.; Ding, X.; Kumar, G. N.; Chen, J. J.; Johnson, E. J.;
Manning, B. H. J. Neurosci. Methods 2008, 168, 76.