Discovery of Potent, orally bioavailable phthalazinone bradykinin B1 receptor antagonists

ARTICLE

pubs.acs.org/jmc

Discovery of Potent, Orally Bioavailable Phthalazinone Bradykinin B1

Receptor Antagonists

Kaustav Biswas,*^,†Tanya A. N. Peterkin,^†Marian C. Bryan,^†Leyla Arik,^§Sonya G. Lehto,^§Hong Sun,^§

Feng-Yin Hsieh,^‡Cen Xu,^§Robert T. Fremeau,^§and Jennifer R. Allen^†

Departments of ^†Chemistry Research and Discovery, ^‡Pharmacokinetics and Drug Metabolism, and ^§Neuroscience Research,

Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States

ABSTRACT:

The bradykinin B1 receptor is rapidly induced upon tissue injury and inﬂammation, stimulating the production of inﬂammatory

mediators resulting in plasma extravasation, leukocyte traﬃcking, edema, and pain. We have previously reported on sulfonamide and

sulfone-based B1 antagonists containing a privileged bicyclic amine moiety leading to potent series of 2-oxopiperazines. The

suboptimal pharmacokinetics and physicochemical properties of the oxopiperazine sulfonamides led us to seek B1 antagonists with

improved druglike properties. Using a pharmacophore model containing a bicyclic amine as anchor, we designed a series of amide

antagonists with targeted physicochemical properties. This approach led to a novel series of potent phthalazinone B1 antagonists,

where we successfully replaced a sulfonamide acceptor with a cyclic carbonyl unit. SAR studies revealed compounds with sub-

nanomolar B1 binding aﬃnity. These compounds demonstrate excellent cross-species PK properties with high oral bioavailability

and potent activity in a rabbit biochemical challenge pharmacodynamic study.

’ INTRODUCTION

agonist stimulation, the B2 receptor is rapidly desensitized and

internalized interrupting its function; this is in contrast to the B1

receptor that does not internalize upon activation.⁷

The kinins are a group of naturally occurring peptides,

including the nonapeptide bradykinin (BK), the decapeptide

kallidin (Lys-BK), and their active des-Arg^9/10metabolites.¹They

are formed by the proteolytic action of plasma kallikrein and

tissue kallikrein on precursors called high molecular weight and

low molecular weight kininogens, respectively.²Kinins are im-

portant biological mediators in cardiovascular homeostasis, con-

traction and relaxation of smooth muscles, inﬂammation, and

nociception.^3,4

Kinins exert their biological inﬂuences by binding to and

activating two cell surface G-protein-coupled receptors, the B1

and B2 receptors.⁵BK and Lys-BK activate the receptor B2

subtype, whereas des-Arg⁹-BK and Lys-des-Arg¹⁰-BK are inactive

on this receptor. Conversely, des-Arg⁹-BK and Lys-des-Arg¹⁰-BK

are ligands for the B1 receptor, with BK and Lys-BK being less

potent. Both receptors are expressed on cells that initiate, exacerbate,

and/or maintain inﬂammation and pain. They are linked via the

G-proteins G_qα/11/G_iα1,2to phospholipase C activation, leading

to intracellular Ca²⁺generation by inositol 1,4,5-phosphate.⁶

There are, however, temporal diﬀerences in their expression,

function, and regulation. B2 receptors are ubiquitously and con-

stitutively expressed. B1 receptors, on the other hand, are absent

or expressed at low levels in normal tissue but are induced/

up-regulated following tissue injury and/or inﬂammation. After

In vivo studies focusing on the role of B1 and B2 receptors in

pain are consistent with their expression/regulation patterns.

Preclinical pain models and knockout studies suggest that the B2

receptor plays a signiﬁcant role in acute pain processing and the

development of primary nociceptor sensitization and is the

principal kinin receptor involved in cardiovascular and renal

function. In contrast, the inducible nature of the B1 receptor

suggests a limited role in acute pain processing but a prominent

role in establishing and maintaining chronic pain. Evidence of

relationship of the B1 receptor to chronic pain has come from

model studies with synthetic antagonists. Peptide antagonists

have been shown to reduce neurogenic pain induced by capsaicin

as well as inﬂammatory pain induced by UV irradiation, carra-

geenan, complete Freund’s adjuvant, or lipopolysaccharide.⁸The

induction of functional B1 receptors and reversal of established

hyperalgesia by B1 antagonists also have been demonstrated

in models of neuropathic pain including streptozotocin-

induced diabetes, chronic constriction injury, and partial

sciatic nerve lesion.⁹These data suggest that the B1 receptor

Received: June 22, 2011

Published: September 08, 2011

r

2011 American Chemical Society

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Scheme 1. Preparation of Amide Targets^a

^aReagents and conditions: EDCI/HOBt, DMF, room temp.

properties. These include high PSA (99), multiple hydrogen

bond donors, and low lipophilicity (cLogD = 0.78). The relation-

ship between physicochemical properties and in vivo PK has

been the focus of elegant reviews.^15,16We therefore sought to

focus on new chemical matter that possessed druglike physico-

chemical properties, with PSA e 80 and cLogD > 1, in the hopes

of identifying B1 antagonists with improved PK properties.

Examination of the ﬁrst generation chemotypes suggested a

pharmacophore containing a hydrophobic unit on the left and

a hydrogen bond acceptor, a linker moiety, and a basic domain on

the right occupied by the bicyclic amine.¹⁷Using this as a design

template, we decided to employ the bicyclic amine in 4, (R)-

6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-amine

(compound 6, Scheme 1), as an anchoring element and prepared

a small 35-member amide library that contained linker units

joining the amine to a hydrogen bond acceptor and a hydro-

phobic moiety (Figure 2a). Testing this library in human B1

binding and cellular assays revealed a hit, albeit of modest

potency, compound 5 (hB1 binding K_i= 1681 nM, hB1 cellular

IC₅₀= 1371 nM, Figure 2b).¹⁸Importantly, this compound

displayed physicochemical properties that were in the range we

had sought in a new hit (PSA = 49, cLogD = 3.57). With this result

in hand, we embarked upon a lead identiﬁcation and optimiza-

tion eﬀort that led to potent phthalazinone-based B1 receptor

antagonists with excellent PK properties. This SAR eﬀort is described

herein, along with a PD study in rabbits with lead compounds.

Figure 1. Amgen’s ﬁrst generation B1 receptor antagonists.

is involved in the potentiation, propagation, and maintenance

of chronic pain. As such, the B1 receptor is an interesting

therapeutic target for the treatment of chronic inﬂammatory

and neuropathic pain conditions.

Along with studies with peptide antagonists, eﬀorts toward

developing small molecule antagonists of the B1 receptor by the

biopharmaceutical industry have also been published. This work

has been the subject of multiple recent reviews.¹⁰At Amgen,

initial discovery of potent arylsulfonamide based antagonists

containing a bicyclic amine motif (compound 1, Figure 1)¹¹

was followed by SAR studies seeking to improve the PK and PD

properties of the series (compounds 2 and 3, Figure 1).^12,13

These experiments culminated in the synthesis of sulfonamides

containing 2-oxopiperazine moieties as potent B1 antagonists

and in the identiﬁcation of compound 4 (Figure 1) as a lead

compound.¹⁴

Further proﬁling of these compounds identiﬁed deﬁciencies in

compound 4, which were consistent across the 2-oxopiperazine

class of B1 antagonists, making them undesirable for further

development. This was exempliﬁed by poor PK properties in rats,

from high clearance to poor oral bioavailability (CL = 7.4 L

’ RESULTS AND DISCUSSION

We adopted two strategies to increase the binding aﬃnity of

compound 5 for the human B1 receptor. One focused on the

long aliphatic linker moiety in the middle of the molecule. We

reasoned that decreasing the number of rotatable bonds in this

section would attenuate the loss in entropy upon receptor binding.

The other modiﬁcation was incorporation of hydrophobic substit-

uents onto the left phenyl ring, since we believed it occupied a

hydrophobic pocket on the receptor. Continuing with the initial

strategy of making small, focused libraries to advance the SAR

studies, we obtained 40 commercial carboxylic acids that in-

cluded one or both of the above structural features and coupled

them to amine 6(Scheme 1). The majority of this library was inactive

in the hB1 binding assay except for the compounds shown in Figure 3.

Compounds 7ꢀ9 shared the common feature of a phenyl ring in

the central linker unit. Rigidifying the core of the molecule had

led to a 3- to 5-fold increase in binding aﬃnity (Figure 3). A

carbonyl moiety, as the proposed required hydrogen bond acceptor,

was also present in all three structures, similar to compound 5.

With the success of applying the principle of reduction of

rotatable bonds to improving binding aﬃnity, we asked the

h

^ꢀ1kg^ꢀ1, F = 6% for compound 4). We initiated a second generation

discovery eﬀort to address the identiﬁed liabilities. In particular,

we sought B1 receptor antagonists that would possess improved rat

PK properties (CL < 2 L h^ꢀ1kg^ꢀ1; F > 25%) while main-

taining the antagonistic potency that distinguished the 2-oxopi-

perazine series. Since PD studies in our program have been

conducted in rabbits, these new compounds would also need to

display high binding aﬃnity at the rabbit B1 receptor.¹²

To seek new antagonists of the B1 receptor with optimal PK

properties, we employed a 2-fold approach of high-throughput

screening and rational design. Unfortunately, a high-throughput

rescreen of our corporate collection did not yield any validated

hits suitable for optimization. Hence, we focused on identifying a

new chemotype by design from ﬁrst principles. To establish

guidelines to drive the hit generation eﬀort, we turned our

attention to the physicochemical properties of compound 4

which could perhaps oﬀer a rationale for suboptimal PK

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Figure 2. (a) Library design. (b) Identiﬁed hit with desirable physicochemical properties.

Figure 3. Compounds with measurable hB1 binding aﬃnity from second amide library.

question if a further restriction in the degrees of freedom of the

structures in Figure 3 would “lock” the conformation into a better

binding mode. To address this, compounds 10ꢀ19 shown in

Table 1 were synthesized and tested in the hB1 binding assay.

These compounds possessed a 6,6-bicyclic unit on the left-hand

sector of the molecule, ﬁxing the location of the carbonyl H-bond

acceptor compared to the precursor molecules in Figure 3. The

amide linkages for each structure were examined in both the meta

(1,3-) and the para (1,4-) orientation for each bicyclic moiety.

As can be seen from Table 1, this approach led to further gains

in B1 receptor binding aﬃnity. Certain structures were more

preferred than the others; chromenones 10 and 11 (hB1 K_i= 64

and 183 nM, respectively), thiochromemones 14 and 15 (hB1

K_i= 159 and 18 nM, respectively), and phthalazinone 17 (hB1

K_i= 35 nM) were among the most potent compounds in this

series. Other bicycles like the 1-methyl-3-phenylquinolin-4(1H)-

ones (12 and 13) and 3-phenylquinazolin-4(3H)-ones (18 and

19) were less potent.

Table 1. Bicycle SAR: Cyclizing the Linker to the Hydro-

phobic Moiety^a

Since the key goal for this discovery eﬀort was to identify

compounds with improved PK properties, we decided to exam-

ine the more potent chemotypes from Table 1 in a rat PK study

to select the best opportunities for further optimization. These

data for compounds 11, 15, and 17 are shown in Table 2.

While all three compounds possessed acceptable PK proper-

ties in rat, phthalazinone 17 demonstrated the lowest clearance

(0.9 L h^ꢀ1kg^ꢀ1) and the highest oral bioavailability (51%) in this

group. Comparison of these data to the corresponding ﬁgures for

compound 4 (CL = 7.4 L h^ꢀ1kg^ꢀ1, F = 6%) shows a marked

improvement in the in vivo PK proﬁle for this new generation of

B1 antagonists. Encouraged by this result, we embarked upon

further SAR studies that focused on optimizing the potency of

phthalazinone 17 to identify candidates for further preclinical

evaluation.

^aValues represent the average of three or more determinations ( SD.

We ﬁrst modiﬁed the 4-position of the phthalazinone bicycle

to probe the eﬀect of substitution and increased steric bulk at this

site on receptor potency. These data are shown in Table 3. While

changing the 4-methyl group from compound 17 to hydrogen

(compound 20, hB1 K_i= 52 nM) or 4-ethyl (compound 21, hB1

K_i= 29 nM) had no signiﬁcant eﬀect on B1 potency, increasing

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the size of the 4-substituent led to loss in binding aﬃnity.

Isopropyl (compound 22, hB1 K_i= 214 nM), triﬂuoromethyl

(compound 23, hB1 K_i= 98 nM), and phenyl (compound 24,

hB1 K_i= 418 nM) substitutions led to less potent analogues. For

further explorations of the SAR, the methyl functionality was

selected as the 4-subsituent.

the binding assay. These data, with chlorine or ﬂuorine atom

substitution, are shown in Table 4. With the exception of the

7-position (compound 28, hB1 K_i= 198 nM), the resulting

compounds showed a marked improvement in potency com-

pared to unsubstituted phthalazinone 17. Of particular interest

was compound 27, with a chlorine atom at the 8-position of the

phthalazinone ring. This compound possessed subnanomolar

potency at the B1 receptor (hB1 K_i= 0.9 nM) and was now

comparable to some of our most potent B1 antagonists listed in

Figure 1. This exciting gain in binding aﬃnity allowed us to

progress into the next step in our optimization campaign of

identifying lead candidates for in vivo pharmacodynamic and

eﬃcacy studies.

Next, the eﬀect of substitutions at the 5, 6, 7, or 8 position of

the phthalazinone ring on receptor potency was examined in

Table 2. In Vivo PK Parameters of Select Compounds from

Table 1

rat

B1 receptor antagonism programs have been hampered by the

lack of receptor homology across species, especially when con-

sidering preclinical studies in animal models of eﬃcacy. The

rabbit B1 receptor has a higher sequence homology to the human

receptor (82%)¹⁹compared to the rat (71%).²⁰Hence, we and

others have evaluated the pharmacodynamic eﬀects of B1 antago-

nists in vivo in rabbits.^12ꢀ14,21For such studies to be feasible, the

compounds under consideration have to be potent antagonists at

compd

t_1/2^a(h)

CL ^a(L h^ꢀ1kg^ꢀ1

)

V_ss^a(L/kg)

F ^b(%)

11

15

17

4.2

2.9

6.1

1.78

2.93

0.9

9.27

11.47

8.09

29

27

51

^aDosed iv at 2 mg/kg, formulated in 100% DMSO. ^bDosed orally at 5

mg/kg, formulated in 1% Tween 80, 2% HPMC, pH 2.2; n = 3;

interanimal variability was less than 20%.

Table 3. SAR at the 4-Position of the Phthalazinones^a

^aValues represent the average of three or more determinations ( SD.

Table 4. SARs at the 5- to 8-Positions of the Phthalazinones^a

^aValues represent the average of three or more determinations ( SD.

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Table 5. Introduction of Heteroatoms and Eﬀect on Human and Rabbit B1 Potency^a

^aValues represent the average of three or more determinations ( SD. ^bn = 1.

the rabbit B1 receptor. As the next step in our SAR studies, we

probed the eﬀect of various heteroatom substitutions on potent

ﬂuoro- and chloro-substituted phthalazinones. In addition to

evaluating the antagonistic activity at the human B1 receptor, we

also measured the IC₅₀in a rabbit B1 cellular calcium-ﬂux assay

to ﬁnd compounds that would possess optimal activity across

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Table 6. Physicochemical and in Vivo PK Parameters of Select Compounds from Table 5

rat PK

dog PK

CL ^c(L h^ꢀ1kg^ꢀ1

compd

PSA/cLogD

t_1/2^a(h)

CL ^a(L h^ꢀ1kg^ꢀ1

)

V_ss^a(L/kg)

F ^b(%)

t_1/2^c(h)

)

V_ss^c(L/kg)

F ^d(%)

27

30

35

67/3.05

80/2.18

80/1.64

10.4

4.4

0.67

0.8

7.85

5.22

6.45

90

98

61

8.9

5.1

nd^e

1.14

1.42

nd^e

10.6

7.43

nd^e

60

44

nd^e

3.3

1.41

^aDosed iv at 2 mg/kg, formulated in 100% DMSO. ^bDosed orally at 5 mg/kg, formulated in 1% Tween 80, 2% HPMC, pH 2.2. ^cDosed iv at 1 mg/kg,

formulated in 10% Captisol, pH 4.0. ^dDosed orally at 1 mg/kg, formulated in 10% Captisol, pH 4.0. Interanimal variability was less than 20%. ^end = not

determined.

Scheme 2. Synthesis of Chromenone 11^a

^aReagents and conditions: (a) 4-(tert-butoxycarbonyl)phenylboronic acid, Pd(PPh₃)₄, 2.0 M sodium carbonate, dioxane, 140 °C (42%); (b) TFA; (c) 6,

EDCI, HOBt, DMF, room temp (31%, two steps).

species. These data are shown in Table 5. Interestingly, this set of

modiﬁcations led to unexpected gains in potency at the rabbit

receptor. The compounds were also tested in a functional assay in

CHO cells expressing the human B1 receptor, verifying the

antagonistic proﬁle of each compound in an aequorin-based

calcium ﬂux assay. The human B1 receptor functional potency

closely correlates with binding aﬃnity SAR.

Compound 27, which demonstrated subnanomolar potency

at the human receptor, inhibited rabbit B1 receptor signaling

with IC₅₀= 21 nM. Incorporation of a nitrogen atom in the core

phenyl ring ortho to the phthalazinone substituent aﬀorded the

’ CHEMISTRY

The compounds in this report were synthesized by the coupling

of (R)-6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-amine (compound 6) to various commercially available or

synthesized carboxylic acids, employing EDCI/HOBt mediated

amidation (Scheme 1).

Compounds 5, 7, 8, and 9 were prepared from commercially

available carboxylic acids as described in Scheme 1. Schemes 2

and 3 depict the syntheses of the para-substituted analogues

shown in Table 1. The meta-substituted compounds were prepared

in an analogous fashion, employing 3-(tert-butoxycarbonyl)phe-

nylboronic acid in the SuzukiꢀMiyaura coupling step.

The synthesis of chromenone 11 is shown in Scheme 2.

Commercially available 3-bromochromone (11a) was reacted

with 4-(tert-butoxycarbonyl)phenylboronic acid in a palladium-

(0)-mediated SuzukiꢀMiyaura coupling reaction to aﬀord the

arylated ester 11b. Deprotection of the tert-butyl ester with TFA

and amide coupling with amine 6 aﬀorded the ﬁnal product,

compound 11.

Quinolinone 13 was prepared as shown in Scheme 3. Con-

jugate addition of N-methylaniline to acrylic acid followed by

FreidelꢀCrafts acylation aﬀorded the quinolinone core 13c.

Bromination, SuzukiꢀMiyaura coupling, and deprotection of

the tert-butyl ester under acidic conditions gave acid 13f, which

was converted to the desired amide 13 as described previously.

Thiochromenone 15 was prepared in a sequence analogous to

that for chromenone 11, as shown in Scheme 2, beginning with

the known 3-bromo-4H-thiochromen-4-one.²²Phthalazinone

17 was prepared from commercially available 4-(4-methyl-1-

oxophthalazin-2(1H)-yl)benzoic acid, and 4-oxoquinazoline 19

was synthesized from commercially available 4-(4-oxoquinazo-

lin-3(4H)-yl)benzoic acid, using the amide coupling conditions

in Scheme 1.

potent nicotinamide 30 (hB1 K_i= 0.3 nM, rabbit B1 cell IC₅₀

=

1.1 nM). Addition of nitrogen to the other position of the core

phenyl moiety did not oﬀer any such gain with respect to rabbit

potency (picolinamide 31, hB1 K_i= 3.1 nM, rabbit B1 cell IC₅₀

=

31 nM). Compound 32, with nitrogen in the phthalazinone

moiety, lost activity against both species, while replacing the

tetralin amine in compound 27 with chroman in compound 33

had minimal aﬀect on B1 antagonism. Fluorine-substituted phtha-

lazinones when linked to the nicotinamide core (compounds 34

and 35) gave rise to very potent compounds at both human and

rabbit receptors, with compound 35 displaying subnanomolar

potency at both receptors (hB1 K_i= 0.6 nM, rabbit B1 cell IC₅₀

=

0.6 nM).

Select potent compounds were subsequently tested in rat and

dog PK studies. As seen in Table 6, the lead phthalazinones

possessed excellent oral bioavailability (61ꢀ98% in rats and

44ꢀ60% in dogs) and exhibited low clearance in both species.

The physicochemical properties of the lead compounds are also

included, demonstrating improvements over the 2-oxopiperazine

4 scaﬀold. Compounds 30 and 35 were tested against a 100-

kinase panel from Ambit Biosciences, a GPCR panel at CEREP

and the hERG, Kv1.5, and Nav1.5 ion channels, and showed no

activity at 1 μM.

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Scheme 3. Synthesis of Quinolinone 13^a

^aReagents and conditions: (a) acrylic acid, toluene, heptanes, 40ꢀ80 °C (99%); (b) P₂O₅, methanesulfonic acid, 110 °C (19%); (c) bromine, HCl,

dioxane, chloroform, room temp (64%); (d) 4-(tert-butoxycarbonyl)phenylboronic acid, Pd(PPh₃)₄, 2.0 M sodium carbonate, dioxane, 150 °C (10%);

(e) TFA (98%); (f) 6, EDCI, HOBt, DMF, room temp (52%).

Scheme 4. Synthesis of Phthalazinone 21^a

^aReagents and conditions: (a) 4-hydrazinobenzoic acid, conc sulfuric acid, MeOH, 75 °C (57%); (b) 1 N HCl, 1,4-dioxane, reﬂux (99%); (c) 6, EDCI,

HOBt, DMF, room temp (79%).

For the compounds shown in Table 3, the preparation of

phthalazinones 17, 20, and 24 followed the procedure outlined in

Scheme 1, from commercially available substituted benzoic acids.

Phthalazinone 21 was prepared from commercially available 2-

propionylbenzoic acid 21a as shown in Scheme 4. Acid-catalyzed

condensation with 4-hydrazinobenzoic acid in methanol aﬀorded

the 4-phthalazinobenzoate 21b, which was deprotected and

coupled to amine 6, aﬀording the desired amide 21. Compound

23 was synthesized by analogous route, employing 2-(2,2,2-

triﬂuoroacetyl)benzoic acid as the starting material.

Isopropylphthalazinone 22 was prepared from methyl

2-iodobenzoate as shown in Scheme 5. The iodide was con-

verted to the isopropyl ketone 22b using Grignard conditions,

which was then further processed to the ﬁnal product as

described in Scheme 4, using the same sequence of steps

described previously.

which was converted to the desired product 27 using the

chemistry described above.

The compounds in Table 5 were also prepared by a similar

procedure, either using 4-hydrazinobenzoic acid to construct the

phthalazinone core in compounds 27, 32, and 33 or using

6-hydrazinylnicotinic acid to synthesize the compounds 30, 34,

and 35. The synthesis of 5-hydrazinylpicolinate for making com-

pound 31 is shown in Scheme 7. 5-Aminopicolinic acid was

converted to the methyl ester 31b, followed by treatment with

sodium nitrite and hydrochloric acid. The resulting diazonium

intermediate was reduced in situ to the hydrazine using tin(II)

chloride, which was used to complete the synthesis in a similar

fashion.

’ PHARMACOLOGY

Phthalazinones in Table 4 were prepared by analogous keto

ester condensation with 4-hydrazinobenzoic acid. A representa-

tive example of compound 27 is shown in Scheme 6. 2-Bromo-6-

chlorobenzoic acid was methylated using dimethyl sulfate to give

ester 27b, followed by Pd(0)-mediated Stille coupling with

(1-ethoxyvinyl)tin. The resulting vinyl ether was hydrolyzed

in situ with 6 N hydrochloric acid, aﬀording the keto ester 27c,

Because of their desirable rabbit B1 potency and cross-species

PK properties, the nicotinamide phthalazinones 30 and 35 were

selected for evaluation in a rabbit preclinical pharmacology

experiment. B1 agonists like des-Arg-kallidin (DAK) cause a

drop in blood pressure in animals where receptor expression has

been stimulated by administration of an inﬂammation source, for

example, lipopolysaccharide. Treatment of these animals with B1

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Scheme 5. Synthesis of Phthalazinone 22^a

^aReagents and conditions: (a) ⁱPrMgCl, THF, ꢀ78 °C, then isobutyryl chloride, ꢀ78 °C to room temp (15%); (b) 4-hydrazinobenzoic acid, conc

sulfuric acid, methanol, 75 °C (22%); (c) 1 N HCl, 1,4-dioxane, reﬂux (99%); (d) 6, EDCI, HOBt, DMF, room temp (40%).

Scheme 6. Synthesis of Phthalazinone 27^a

^aReagents and conditions: (a) LiOH, Me₂SO₄, THF, 85 °C (97%); (b) (1-ethoxyvinyl)tin, PdCl₂(PPh₃)₂, 1,4-dioxane, 150 °C, then 6 N HCl, room

temp (78%); (c) 4-hydrazinobenzoic acid, conc sulfuric acid, MeOH, 80 °C (71%); (d) 1 N HCl, 1,4-dioxane, reﬂux (99%); (e) 6, EDCI, HOBt, DMF,

room temp (94%).

Scheme 7. Synthesis of Phthalazinone 31^a

^aReagents and conditions: (a) thionyl chloride, methanol, 70 °C (62%); (b) NaNO₂, 6 N HCl, 0 °C to room temp, then cool to 0 °C, SnCl₂, warm to

10 °C (67%); (c) 27c, conc sulfuric acid, methanol, 80 °C (99%); (d) 1 N HCl, 1,4-dioxane, reﬂux (99%); (e) 6, EDCI, HOBt, DMF, room temp (68%).

Figure 4. (a) Compound 30 and (b) compound 35 reverse DAK-induced hypotension in rabbits in plasma concentration-dependent fashion. The

compounds were dosed subcutaneously. See ref 12 for protocol details and the Experimental Section for administered doses. The x-axis is the measured

plasma concentration of each compound at the time of measurement of the PD eﬀect.

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Table 7. Correlation of in Vitro and in Vivo Activity for Select

Compounds

that both compounds potently reversed B1-agonist induced

hypotension in a proof of on-mechanism PD activity, being the

most potent in this setting among the compounds discovered in

our program. Further preclinical evaluation of phthalazinone B1

antagonists, including eﬃcacy studies in pain models, will be

reported shortly.

rabbit

a

B1 cell IC₅₀

(nM)

cell IC₅₀/F_u

PD plasma IC₅₀

(nM)

compd

plasma F_u

(nM)

30

35

3

1.1

0.6

0.09

0.24

0.21

0.28

12.0

2.4

16.2

2.5

127.0^b

10.3^c

’ EXPERIMENTAL SECTION

Unless otherwise noted, all materials were obtained from commercial

suppliers and used without further puriﬁcation. Anhydrous solvents

were obtained from EMD or Aldrich and used were directly. All reactions

involving air- or moisture-sensitive reagents were performed under a

nitrogen or argon atmosphere. Microwave-assisted reactions were con-

ducted with a Smith synthesizer from Personal Chemistry (Uppsala,

Sweden). Silica gel chromatography was performed using medium

pressure liquid chromatography (MPLC) on a CombiFlash Companion

(Teledyne Isco) with RediSep normal-phase silica gel (35ꢀ60 μm)

columns and UV detection at 254 nm. Preparative reversed phase HPLC

was performed using a Shimadzu Prominence system and Phenomenex

Gemini C18 column (30 μm, 150 mm ꢁ 30 mm i.d.), eluting with a binary

solvent system (A, H₂O with 0.1% TFA; B, CH₃CN with 0.1% TFA;

gradient elution) with UV detection at 254 nm. All ﬁnal compounds

were puriﬁed to g95% purity as determined by Agilent 1100 series high

performance liquid chromatography (HPLC) with UV detection at

254 nm using one of the following methods: Method A: Zorbax SB-C8

column (150 mm ꢁ 4.6 mm, 3.5 μm); mobile phase, A = H₂O with 0.1%

TFA, B = CH₃CN with 0.1% TFA; gradient: 5ꢀ95% B (0.0ꢀ15.0 min);

ﬂow rate, 1.5 mL/min. Method B: Zorbax analytical C18 column

(50 mm ꢁ 3 mm, 3.5 μm, 40 °C); mobile phase, A = H₂O with 0.1%

TFA, B = CH₃CN with 0.1% TFA; gradient, 5ꢀ95% B (0.0ꢀ3.6 min);

ﬂow rate, 1.5 mL/min. Method C: YMCODS-AM (100 mm ꢁ 2.1 mm,

5 μm, 40 °C); mobile phase, A = H₂O with 0.1% HCO₂H, B = CH₃CN

with 0.1% HCO₂H; gradient, 10% B (0.0ꢀ0.5 min), 10ꢀ100% B

(0.5ꢀ7.0 min), 100% B (7.0ꢀ10 min); ﬂow rate, 0.5 mL/min. Method

D: Phenomenex Synergi MAX-RP (50 mm ꢁ 2.0 mm, 4.0 μm, 40 °C);

mobile phase, A = H₂O with 0.1% TFA, B = CH₃CN with 0.1% TFA;

gradient, 10% B (0.0ꢀ0.2 min), 10ꢀ100% B (0.2ꢀ3.0 min), 100% B

(3.0ꢀ4.5 min), 100ꢀ10% B (4.5ꢀ5.0 min); ﬂow rate, 0.8 mL/min. Low

resolution mass spectrometry (MS) data were obtained using an Agilent

G1956B mass spectrometer operated in electrospray ionization (ESI)

mode (positive or negative). NMR spectra were obtained at ambient

temperature with a Bruker Avance II spectrometer operating at 300 or

400 MHz. Chemical shifts are reported in ppm downﬁeld of an internal

standard, tetramethylsilane (δ 0.00 ppm). Data are reported as follows:

chemical shift, number of protons, multiplicity (s = singlet, d = doublet,

t = triplet, q = quartet, br = broad, m = multiplet), and coupling constants.

(R)-6-(Piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphtha-

len-1-amine Dihydrochloride (6). Step 1. (R)-methyl CBS-ox-

aborolidine (7.4 mL of a 1 M solution in toluene, 7.4 mmol) in toluene

11.2

53.3

4

4.1

14.6

^aPD plasma IC₅₀: compound 30 CI_95%4.25ꢀ62.14 nM, R²= 0.6063;

compound 35 CI_95%1.67ꢀ3.95 nM, R²= 0.9267. ^bReference 13.

^cReference 14.

antagonists has been shown to reverse the observed hypotension

eﬀect.^23,24This PD assay is therefore a rapid way to establish

in vivo on-mechanism activity of new B1 antagonist chemotypes.

In this instance, treatment of rabbits with bacterial lipopolysac-

charide, followed by administration of DAK, caused a drop in

blood pressure, which was then reversed by increasing doses of

phthalazinones 30 and 35. The % inhibition of hypotensive

response was plotted against the measured plasma concentration

of the drug at two time points for each dose (30 and 75 min) to

obtain a quantitative measure of the inhibition of hypotension

(Figure 4).

We also measured the rabbit plasma protein binding of each

compound. Phthalazinone 35 displayed a larger free fraction

(F_u= 0.24) compared to phthalazinone 30 (F_u= 0.09). As shown

in Table 7, the in vivo IC₅₀values correlate very well with rabbit

cellular potency when corrected for free fraction. Comparison of

these values with our previously reported B1 antagonists 3 and 4

demonstrates that phthalazinone 35 is the most potent com-

pound identiﬁed in this PD assay in our laboratories, possessing a

desirable balance of in vitro potency and plasma free fraction.²⁵

’ CONCLUSION

Structureꢀactivity relationship studies on Amgen’s sulfonam-

ide based B1 antagonists had identiﬁed very potent scaﬀolds with

subnanomolar binding aﬃnity to the target. However, subopti-

mal PK properties of the ﬁrst generation sulfonamide lead,

2-oxopiperazine 4, compelled us to undertake a search for novel

chemotypes with improved physicochemical and PK properties

suitable for preclinical optimization. Using a pharmacophore-

based approach anchored by the bicyclic amine moiety, we

synthesized small, focused amide libraries with restricted phys-

icochemical properties. Starting from the initial hit compound 5,

multiple iterations of a strategy of reducing rotatable bonds

aﬀorded potent scaﬀolds, of which the phthalazinones series was

selected for further optimization based on superior rat PK

properties. It is interesting to note that a sulfonamide moiety

has been successfully replaced with a cyclic carbonyl unit in this

scaﬀold-modiﬁcation exercise, and this could be of use in other

medicinal chemistry programs where sulfonamides lead to un-

desirable properties.

(190 mL) was cooled to ꢀ10 °C and was treated with BH₃SMe₂

3

(17 mL, 180 mmol). A solution of methyl 5-oxo-5,6,7,8-tetrahydro-

naphthalene-2-carboxylate (30 g, 150 mmol)²⁶in THF (200 mL) was

added over 5 h using a syringe pump. After the addition was complete,

the mixture was stirred for an additional 1 h and then was poured into an

addition funnel. The reaction mixture was added to methanol (200 mL),

and the mixture was cooled in an iceꢀsalt bath over 30 min at such a rate

that the internal temperature was kept below 0 °C. The mixture was

concentrated in vacuo, diluted with diethyl ether (1 L), and washed with

1 M H₃PO₄, saturated sodium bicarbonate solution, and brine. The

organic layer was dried over MgSO₄and concentrated. The residue was

dissolved in diethyl ether again (500 mL) and washed with 1 M H₃PO₄,

saturated sodium bicarbonate solution, and brine. After the organic layer

was dried over MgSO₄, the mixture was concentrated in vacuo to give

Further SAR led to potent B1 antagonists like compounds 27,

30, and 35 with subnanomolar binding aﬃnity to the human B1

receptor and exceptional cross-species PK properties, exempli-

ﬁed by oral bioavailabilities in the 44ꢀ98% range in rats and dog.

On the basis of their potencies at the rabbit receptor, compounds

30 and 35 were tested in a rabbit PD study. We demonstrated

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methyl 5-(S)-hydroxy-5,6,7,8-tetrahydronaphthalene-2-carboxylate as a

white-yellow solid.

Step 7. (R)-tert-Butyl 6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydro-

naphthalen-1-ylcarbamate (388 g, 1.13 mol) was dissolved in methanol

(4.0 L). A solution of hydrogen chloride (4.0 N in 1,4-dioxane, 592 mL,

2.4 mol, 2.1 equiv) was added in a dropwise fashion via an addition

funnel. The reaction mixture was heated to 55 °C for 1 h, cooled to

25 °C, and concentrated in vacuo. The brown oil was dissolved in methanol

and treated with diethyl ether to cause a white solid to precipitate which

was collected by filtration and washed with diethyl ether. The solid was

dried under high vacuum, affording the title compound 6 as the dihydro-

chloride salt (297 g, 84% over four steps). ¹H NMR (400 MHz, D₂O) δ

ppm 1.34 (qt, J = 12.9, 3.9 Hz, 1H), 1.47ꢀ1.63 (m, 2H), 1.64ꢀ1.74 (m,

2H), 1.74ꢀ1.86 (m, 4H), 1.86ꢀ1.98 (m, 1H), 2.02ꢀ2.15 (m, 1H),

2.64ꢀ2.90 (m, 4H), 3.34 (bd, J = 12.4 Hz, 2H), 4.14 (s, 2H), 4.52 (t, J =

5.2 Hz, 1H), 7.23 (s, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz,

1H); ¹³C NMR (100 MHz, D₂O) δ ppm 17.7, 21.1, 22.7, 27.1, 28.0,

48.7, 52.8, 60.0, 128.9, 129.0, 129.3, 132.5, 132.9, 139.2; HRMS calcd for

C₁₆H₂₄N₂[M + H]⁺245.2018, found 245.2017. This amine was used in

peptide coupling steps as its salt or by converting to the free base before

addition to the reaction mixture. Conversion to the free base was

accomplished by washing it in a dichloromethane/saturated sodium

bicarbonate mixture, separating the organic layer, drying over MgSO₄,

and concentrating in vacuo.

(R)-3-(4-Oxo-4H-chromen-3-yl)-N-(6-(piperidin-1-ylmethyl)-

1,2,3,4-tetrahydronaphthalen-1-yl)benzamide (10). 10 was

prepared by a method analogous to that used for the preparation of

compound 11. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.30 (1 H, d, J = 7.5

Hz), 8.09 (1 H, s), 7.95 (1 H, s), 7.81 (1 H, d, J = 8.0 Hz), 7.65ꢀ7.78 (2

H, m), 7.51 (2 H, t, J = 7.5 Hz), 7.45 (1 H, t, J = 7.8 Hz), 7.21ꢀ7.33 (1 H,

m), 7.00ꢀ7.16 (2 H, m), 6.39 (1 H, d, J = 8.5 Hz), 5.40 (1 H, q, J = 6.5

Hz), 3.42 (2 H, s), 2.70ꢀ2.95 (2 H, m), 2.37 (4 H, s), 2.09ꢀ2.21 (1 H,

m), 1.80ꢀ2.01 (3 H, m), 1.50ꢀ1.65 (4 H, m), 1.42 (2 H, s); ESI MS

calcd for C₃₂H₃₂N₂O₃[M + H]⁺493.2, found 493.2.

(R)-4-(4-Oxo-4H-chromen-3-yl)-N-(6-(piperidin-1-ylmethyl)-

1,2,3,4-tetrahydronaphthalen-1-yl)benzamide (11). Step 1.

A mixture of 3-bromochromone (250 mg, 1.11 mmol), 4-(tert-butox-

ycarbonyl)phenylboronic acid (296 mg, 1.33 mmol), Pd(PPh₃)₄(257 mg,

0.222 mmol), and 2.0 M sodium carbonate (1.67 mL, 3.33 mmol) in 1,4-

dioxane (5 mL in a sealed 10 mL tube was evacuated and backfilled

with argon (three cycles). The reaction mixture was heated to 140 °C for

20 min in a microwave (Personal Chemistry, 300 W). The solution was

then diluted with EtOAc (50 mL) and washed with water and brine. The

organic solution was dried over magnesium sulfate, concentrated in

vacuo, and purified by flash column chromatography (silica, 0ꢀ100%

DCM/hexane) to give tert-butyl 4-(4-oxo-4H-chromen-3-yl)benzoate

(152 mg, 42.4%).

Step 2. A solution of methyl 5-(S)-hydroxy-5,6,7,8-tetrahydro-

naphthalene-2-carboxylate (29 g, 140 mmol) in toluene (280 mL) was

cooled in an iceꢀsalt bath. Diphenylphosphorylazide (36 mL, 170

mmol) was added. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 25 mL,

170 mmol) was added over 10 min at such a rate that the internal

temperature was kept below 1 °C. The ice in the bath was allowed to

melt, and the mixture was stirred for 23 h. The reaction contents were

poured into a 2 L separatory funnel, and the lower dark brown layer was

removed. Water (500 mL) was added to the remaining top layer, and the

mixture was extracted with Et₂O. The combined organic layers were

washed with 1 M H₃PO₄, water, saturated NaHCO₃, and brine. The

organic layer was dried over MgSO₄, filtered, and concentrated in vacuo.

Purification by flash column chromatography (silica, 50% hexane/

DCM) of the crude material provided methyl 5-(R)-azido-5,6,7,8-

tetrahydronaphthalene-2-carboxylate.

Step 3. A solution of lithium aluminum hydride (470 mL of a 1 M

solution in THF, 470 mmol) in THF (700 mL) was cooled in a iceꢀsalt

bath, and methyl 5-(R)-azido-5,6,7,8-tetrahydronaphthalene-2-carbox-

ylate (27 g, 120 mmol) in 100 mL of THF was added over 30 min. The

reaction mixture was warmed to 25 °C overnight, then cooled in an

iceꢀsalt bath the next morning. Water (18 mL) in THF (20 mL) was

added to the reaction mixture over 4 h. Vigorous gas evolution occurred.

Then 5 M NaOH (18 mL) was added over 30 min followed by 54 mL

of water. After being stirred for an additional 1 h, the mixture was filtered,

and the filtrate was concentrated in vacuo. The residue was reconsti-

tuted in MeOH and CH₃CN and concentrated in vacuo again to provide

(R)-(5-amino-5,6,7,8-tetrahydronaphthalen-2-yl)methanol as light-

brown solid.

Step 4. Methanol (4.0 L) was added to (R)-(5-amino-5,6,7,8-tetra-

hydronaphthalen-2-yl)methanol (321 g, 1.81 mol), followed by di-tert-

butyl dicarbonate (514 g, 2.36 mol) in methanol (500 mL). The reaction

mixture was stirred at 25 °C for 4 h, followed by concentration, in vacuo.

The residue was dissolved in 1,2-dichloroethane (2.0 L) and concen-

trated again to remove residual methanol. The crude product was used in

the next step.

Step 5. A solution of (R)-tert-butyl 6-(hydroxymethyl)-1,2,3,4-tetra-

hydronaphthalen-1-ylcarbamate (312.7 g, 1.13 mol) in dichloromethane

(4.0 L) was treated with 225 mL (112.8 mmol) of a 0.5 N KBr solution

(aq), followed by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl or (2,2,6,6-

tetramethylpiperidin-1-yl)oxidanyl (TEMPO, 1.76 g, 11.3 mmol). The

reaction mixture was cooled to 0ꢀ2 °C using an ice bath, and a bleach

solution (prepared from commercial bleach (1.6 L), water (2.18 L), and

sodium bicarbonate (236 g)) was added in a dropwise fashion. The

internal temperature was kept below 10 °C. After addition of a total of

3.4 L of bleach solution, the reaction was quenched with saturated

sodium bisulfite solution. The layers were separated, and the aqueous

phase was extracted with dichloromethane. The combined extracts were

dried over MgSO₄and concentrated to give (R)-tert-butyl 6-formyl-

1,2,3,4-tetrahydronaphthalen-1-ylcarbamate as a thick brown syrup,

which was used in the next step without purification.

Step 2. A solution of tert-butyl 4-(4-oxo-4H-chromen-3-yl)benzoate

(135 mg, 0.419 mmol) in TFA (5 mL, 67.3 mmol) was stirred at 25 °C

for 10 min. The solution was concentrated, azeotroped in DCM, and

dried in vacuo to give 4-(4-oxo-4H-chromen-3-yl)benzoic acid (127 mg;

crude material was advanced to the next step).

Step 3. 4-(4-Oxo-4H-chromen-3-yl)benzoic acid (100 mg, 0.376

mmol), 6 (110 mg, 0.451 mmol), HOBt (74.8 mg, 0.488 mmol), and

EDCI (93.6 mg, 0.488 mmol) were combined in DMF (2 mL). The

reaction mixture was stirred at 25 °C for 16 h and then quenched with

1 N sodium hydroxide solution (30 mL) and extracted with EtOAc. The

organic solution was washed with water, followed by brine. It was dried

over magnesium sulfate and concentrated in vacuo. Purification by flash

column chromatography (silica, 5ꢀ10% MeOH/DCM) gave 11 (58.2

mg, 31.5% over two steps) as an off-white solid. ¹H NMR (400 MHz,

DMSO-d₆) δ ppm 8.79 (1 H, d, J = 8.5 Hz), 8.65 (1 H, s), 8.17 (1 H, d,

J = 8.0 Hz), 7.99 (2 H, d, J = 8.0 Hz), 7.86 (1 H, t, J = 7.8 Hz), 7.68ꢀ7.76

(3 H, m), 7.55 (1 H, t, J = 7.5 Hz), 7.05ꢀ7.17 (2 H, m), 7.03 (1 H, s),

5.21ꢀ5.28 (1 H, m), 3.34 (2 H, s), 2.71ꢀ2.85 (2 H, m), 2.30 (4 H, s),

1.94ꢀ2.04 (2 H, m), 1.71ꢀ1.88 (2 H, m), 1.43ꢀ1.52 (4 H, m),

Step 6. A solution of (R)-tert-butyl 6-formyl-1,2,3,4-tetrahydro-

naphthalen-1-ylcarbamate (306 g, 1.13 mol), piperidine (558 mL, 5.64

mol), and catalytic acetic acid (12 mL in 1,2-dichloroethane (5.0 L) was

heated to 55 °C under nitrogen. After 30 min, NaBH(OAc)₃(598 g, 2.82

mol, 2.5 equiv) was added slowly. After 1 h, the reaction mixture was

cooled to 25 °C and was quenched with saturated sodium bicarbonate

solution (8.0 L) slowly (caution: effervescence). The organic layer was

separated, and the aqueous layer was extracted with dichloromethane).

The combined organic layers were dried (MgSO₄) and concentrated in

vacuo to afford crude (R)-tert-butyl 6-(piperidin-1-ylmethyl)-1,2,3,4-

tetrahydronaphthalen-1-ylcarbamate, which was carried forward to the

next step.

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1.32ꢀ1.42 (2 H, m); ESI MS calcd for C₃₂H₃₂N₂O₃[M + H]⁺493.2,

found 493.2.

addition of HOBt (38 mg, 0.28 mmol) and EDCI (45 mg, 0.23 mmol).

The reaction mixture was stirred at 23 °C under nitrogen. After 20 h, the

reaction mixture was diluted with DCM and washed with saturated

sodium bicarbonate solution and brine, dried over magnesium sulfate,

concentrated in vacuo, and purified by flash column chromatography

(silica, 1ꢀ9% MeOH/DCM), affording 13 (61 mg, 52%) as a light

yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.83 (1 H, d, J = 8.6

Hz), 8.48 (1 H, d, J = 2.3 Hz), 8.15 (1 H, dd, J = 9.0, 2.3 Hz), 8.06 (2 H, d,

J = 8.4 Hz), 8.00 (1 H, d, J = 7.6 Hz), 7.87 (2 H, d, J = 8.6 Hz), 7.79 (1 H,

d, J = 9.0 Hz), 7.15 (1 H, d, J = 8.0 Hz), 7.06 (1 H, d, J = 8.0 Hz),

6.99ꢀ7.05 (1 H, m), 6.09 (1 H, d, J = 7.8 Hz), 5.11ꢀ5.34 (1 H, m), 3.87

(3 H, s), 3.34 (2 H, s), 2.56ꢀ2.92 (2 H, m), 2.29 (4 H, s), 1.90ꢀ2.10

(2 H, m), 1.54ꢀ1.95 (2 H, m), 1.47(4 H, s), 1.29ꢀ1.41 (2 H, m); ESIMS

calcd for C₃₃H₃₅N₃O₂[M + H]⁺506.3, found 506.2.

(R)-3-(1-Methyl-4-oxo-1,4-dihydroquinolin-3-yl)-N-(6-

(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)-

benzamide (12). 12 was prepared by a method analogous to that used

for the preparation of compound 13. ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 9.00 (1 H, d, J = 8.6 Hz), 8.51 (1 H, d, J = 2.3 Hz), 8.30 (1 H, s), 8.15

(1 H, dd, J = 9.0, 2.3 Hz), 7.99 (1 H, d, J = 7.6 Hz), 7.93 (1 H, dd, J = 11.6,

7.9 Hz), 7.78 (1 H, d, J = 9.0 Hz), 7.60 (1 H, t, J = 7.7 Hz), 7.17 (1 H, d,

J = 7.8 Hz), 7.05ꢀ7.14 (2 H, m), 7.03 (1 H, s), 6.08 (1 H, d, J = 7.6 Hz),

5.14ꢀ5.38 (1 H, m), 3.86 (3 H, s), 3.34 (2 H, s), 2.64ꢀ2.85 (2 H, m),

2.23ꢀ2.36 (4 H, m), 1.91ꢀ2.08 (2 H, m), 1.67ꢀ1.93 (2 H, m), 1.43ꢀ

1.54 (4 H, m), 1.29ꢀ1.40 (2 H, m); ESI MS calcd for C₃₃H₃₅N₃O₂[M +

H]⁺506.2, found 506.2.

(R)-3-(4-Oxo-4H-thiochromen-3-yl)-N-(6-(piperidin-1-

ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide (14).

14 was prepared by a method analogous to that used for the preparation of

compound 15. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 8.79 (d, J = 8.48

Hz, 1H), 8.56 (s, 1H), 8.46 (d, J = 7.16 Hz, 1H), 8.06 (s, 1H), 7.94 (dd, J =

3.87, 7.53 Hz, 2H), 7.74ꢀ7.82 (m, 1H), 7.63ꢀ7.72 (m, 2H), 7.48ꢀ7.56

(m, 1H), 6.98ꢀ7.21 (m, 3H), 5.25 (br s, 1H), 3.32 (s, 2H), 2.76 (br s, 2H),

2.30 (br s, 4H), 1.98 (br s, 2H), 1.68ꢀ1.90 (m, 2H), 1.29ꢀ1.56 (m, 6H);

ESI MS calcd for C₃₂H₃₂N₂O₂S [M + H]⁺509.2, found 509.1.

(R)-4-(4-Oxo-4H-thiochromen-3-yl)-N-(6-(piperidin-1-

ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide

(15). Step 1. A vessel containing 3-bromo-4H-thiochromen-4-one

(250 mg, 1.037 mmol),²¹4-(tert-butoxycarbonyl)phenylboronic acid

(253 mg, 1.141 mmol), potassium carbonate (430 mg, 3.111 mmol), and

Pd(PPh₃)₄(24 mg, 21 μmol) was purged with nitrogen. A mixture of

toluene (2.72 mL)/H₂O (1.03 mL) (2.6:1) was then added, and the

reaction mixture was stirred at 60 °C. After 72 h, the reaction mixture

was cooled to 25 °C and diluted with DCM and saturated aqueous

sodium bicarbonate. The aqueous layer was washed with DCM, and the

combined organic layers were dried over sodium sulfate, concentrated in

vacuo, and purified by flash column chromatography (silica, 10% EtOAc/

hexanes) to give tert-butyl 4-(4-oxo-4H-thiochromen-3-yl)benzoate as a

light yellow solid. This was then washed with MeOH to give the desired

pure product as a white solid (282.4 mg, 80%).

(R)-4-(1-Methyl-4-oxo-1,4-dihydroquinolin-3-yl)-N-(6-

(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)-

benzamide (13). Step 1. A solution of N-methylaniline (1.476 mL,

13.63 mmol) and acrylic acid (982 mg, 13.63 mmol) in toluene (6 mL)

and heptanes (3 mL) was heated to 40 °C under nitrogen for 14 h. The

temperature was increased to 80 °C, and the reaction continued for

another 2 h. The reaction mixture was cooled to 23 °C, the solvents were

concentrated, and the residue was dried under vacuum, affording

3-(methyl(phenyl)amino)propanoic acid (2.44 g, 99.9%) as a dark yellow oil.

Step 2. A suspension of phosphorus pentoxide (3.865 g, 27.23 mmol)

and methanesulfonic acid (17.6 mL, 272.3 mmol) was stirred at 23 °C.

After 2 h, the reaction mixture was quickly filtered, and the filtrate was

added to 3-(methyl(phenyl)amino)propanoic acid (2.440 g, 13.62 mmol).

The reaction mixture was heated to 110 °C for 3 h. It was then cooled to

23 °C, poured into iceꢀwater, and extracted with DCM. The organic

layers were combined, washed with saturated sodium bicarbonate solution

and brine, dried over magnesium sulfate, and concentrated, affording

1-methyl-2,3-dihydroquinolin-4(1H)-one (425 mg, 19.4%) as an orange oil.

Step 3. A solution of 1-methyl-2,3-dihydroquinolin-4(1H)-one (153

mg, 0.949 mmol) in chloroform (8 mL) was treated with hydrogen

chloride, 4.0 M in 1,4-dioxane (0.26 mL, 1.04 mmol) at 23 °C under

nitrogen. After 5 min, the reaction mixture was cooled to 0 °C and was

treated with a solution of bromine (50 μL, 0.95 mmol) in chloroform

(2 mL) in a dropwise fashion over 20 min. The reaction mixture was

warmed to 23 °C and stirred under nitrogen for 2 h. The reaction

mixture was concentrated under vacuum and diluted with ice-cold water.

The precipitate was collected by filtration, washed with water, and dried

under vacuum, affording 3-bromo-1-methylquinolin-4(1H)-one (144 mg,

64%) as a dark yellow solid.

Step 4. A suspension of 3-bromo-1-methylquinolin-4(1H)-one(142mg,

0.596 mmol), 4-(tert-butoxycarbonyl)phenylboronic acid (265 mg,

1.193 mmol), 2.0 M sodium carbonate solution (0.59 mL, 1.193 mmol),

and Pd(PPh₃)₄(69 mg, 60 μmol) in 1,4-dioxane (5 mL) was degassed

and backfilled with argon in a sealed vial. The reaction mixture was

heated to 150 °C in a microwave for 20 min. The reaction mixture was

diluted with EtOAc (75 mL) and washed with saturated sodium bicar-

bonate solution and brine, dried over magnesium sulfate, concentrated

in vacuo, and purified by flash column chromatography (silica, 0.5ꢀ10%

MeOH/EtOAc), affording tert-butyl 4-(1-methyl-4-oxo-1,4-dihydroqui-

nolin-3-yl)benzoate (81 mg, 10%) as a white solid.

Step 5. A solution of tert-butyl 4-(1-methyl-4-oxo-1,4-dihydroquino-

lin-3-yl)benzoate (80 mg, 0.239 mmol) in TFA (4.43 mL, 59.63 mmol)

was stirred at 23 °C. After 30 min, the reaction mixture was concentrated

and azeotroped with toluene (3 ꢁ 10 mL), affording 4-(1-methyl-4-oxo-

1,4-dihydroquinolin-3-yl)benzoic acid trifluoroacetate salt (92 mg, 98%) as

a white solid.

Step 2. A solution of tert-butyl 4-(4-oxo-4H-thiochromen-3-yl)benzo-

ate (282.4 mg, 0.834 mmol) and TFA (1.7 mL) was stirred at 25 °C for

10 min. The reaction mixture was concentrated in vacuo and coevapo-

rated with DCM to give 4-(4-oxo-4H-thiochromen-3-yl)benzoic acid as

a white solid, which was used in the next step without purification.

Step 3. To a solution of 4-(4-oxo-4H-thiochromen-3-yl)benzoic acid

(264 mg, 0.935 mmol) and 6 (251 mg, 1.029 mmol) in DMF (9.35 mL)

was added TEA (0.156 mL, 1.122 mmol). The reaction mixture was

stirred at 25 °C for 10 min. EDCI (179 mg, 0.935 mμmol) and HOBt

(172 mg, 1.122 mmol) were then added, and the reaction mixture was

stirred at 25 °C for 16 h. The reaction mixture was then diluted with

brine and extracted with DCM. The combined organic layers were

washed with brine, dried over sodium sulfate, filtered, and concentrated

in vacuo. The resulting mixture was purified by flash column chroma-

tography (silica, 0.5ꢀ10% MeOH/DCM) to give the crude product as a

light yellow solid. This was washed with MeOH to give 15 as a white

1

solid (138.9 mg, 29% over two steps). H NMR (300 MHz, CDCl₃)

1.44ꢀ1.58 (m, 6H), 2.0ꢀ2.15 (m, 4H), 2.38 (m, 4H), 2.83 (m, 2H),

3.43 (s, 2H), 5.39 (m, 1H), 6.45 (m, 1H), 7.09 (m, 2H), 7.27 (m, 1H),

7.63 (m, 4H), 7.84 (m, 3H), 8.63 (s, 1H); ESI MS calcd for C₃₂H₃₂-

N₂O₂S [M + H]⁺509.2, found 509.1.

(R)-3-(4-Methyl-1-oxophthalazin-2(1H)-yl)-N-(6-(piperidin-

1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide

(16). 16 was prepared by a method analogous to that used for the

preparation of compound 17. ¹H NMR (400 MHz, DMSO-d₆) δ

ppm 8.87 (1 H, d, J = 8.5 Hz), 8.36 (1 H, d, J = 7.5 Hz), 8.16 (1 H, t,

Step 6. A suspension of 4-(1-methyl-4-oxo-1,4-dihydroquinolin-3-yl)-

benzoic acid trifluoroacetate salt (92 mg, 234 μmol) and 6 (63 mg,

257 μmol) in DMF (5 mL) was treated with TEA (39 μL, 281 μmol).

The reaction mixture was stirred at 23 °C for 10 min, followed by the

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J = 1.5 Hz), 7.91ꢀ8.07 (4 H, m), 7.73ꢀ7.79 (1 H, m), 7.59 (1 H, t, J =

8.0 Hz), 7.15 (1 H, d, J = 8.0 Hz), 7.06 (1 H, d, J = 8.0 Hz), 7.02 (1 H, s),

5.21ꢀ5.30 (1 H, m), 3.33ꢀ3.39 (2 H, m), 2.70ꢀ2.83 (2 H, m), 2.62

(3 H, s), 2.29 (4 H, s), 1.91ꢀ2.04 (2 H, m), 1.69ꢀ1.89 (2 H, m),

1.41ꢀ1.53 (4 H, m), 1.31ꢀ1.41 (2 H, m); ESI MS calcd for

C₃₂H₃₄N₄O₂[M + H]⁺507.2, found 507.2.

(R)-4-(4-Methyl-1-oxophthalazin-2(1H)-yl)-N-(6-(piperidin-

1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide

(17). Phthalazinone 17 was prepared from commercially available 4-

(4-methyl-1-oxophthalazin-2(1H)-yl)benzoic acid and amine 6. ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 8.32 (1 H, d, J = 8.4 Hz), 8.21

(1 H, d, J = 8.4 Hz), 8.17 (1 H, s), 7.95 (1 H, d, J = 8.4 Hz), 7.04 (2 H, s),

6.99 (1 H, s), 4.85ꢀ4.94 (1 H, m), 3.58 (1 H, t, J = 8.0 Hz), 3.47ꢀ3.55

(1 H, m), 3.36ꢀ3.45 (2 H, m), 3.33 (2 H, s), 2.98ꢀ3.09 (1 H, m), 2.69

(2 H, d, J = 7.2 Hz), 2.29 (4 H, s), 2.03ꢀ2.15 (2 H, m), 1.75ꢀ1.89 (2 H,

m), 1.54ꢀ1.72 (2 H, m), 1.42ꢀ1.50 (4 H, m), 1.32ꢀ1.42 (2 H, m); ESI

MS calcd for C₃₂H₃₄N₄O₂[M + H]⁺507.3, found 507.2.

(R)-3-(4-Oxoquinazolin-3(4H)-yl)-N-(6-(piperidin-1-ylmethyl)-

1,2,3,4-tetrahydronaphthalen-1-yl)benzamide (18). 18 was

prepared by a method analogous to that used for the preparation of

compound 19. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.85 (1 H, d, J =

8.4 Hz), 8.40 (1 H, s), 8.22 (1 H, dd, J = 8.0, 1.0 Hz), 8.04ꢀ8.11 (2 H,

m), 7.90 (1 H, td, J = 8.2, 7.0, 1.4 Hz), 7.71ꢀ7.79 (2 H, m), 7.66 (1 H, t,

J = 7.8 Hz), 7.61 (1 H, t, J = 7.6 Hz), 7.12ꢀ7.19 (1 H, m), 7.00ꢀ7.11

(2 H, m), 5.20ꢀ5.29 (1 H, m), 3.33ꢀ3.42 (2 H, m), 2.65ꢀ2.84 (2 H,

m), 2.30 (4 H, s), 1.90ꢀ2.06 (2 H, m), 1.70ꢀ1.88 (2 H, m), 1.41ꢀ1.55

(4 H, m), 1.31ꢀ1.41 (2 H, m); ESI MS calcd for C₃₁H₃₂N₄O₂[M + H]⁺

493.2, found 493.2.

(R)-4-(4-Oxoquinazolin-3(4H)-yl)-N-(6-(piperidin-1-ylmethyl)-

1,2,3,4-tetrahydronaphthalen-1-yl)benzamide (19). 4-Oxo-

quinazoline 19 was synthesized from commercially available 4-(4-

oxoquinazolin-3(4H)-yl)benzoic acid and amine 6. ¹H NMR (400

MHz, DMSO-d₆) δ ppm 8.90 (1 H, d, J = 8.6 Hz), 8.38 (1 H, s), 8.22

(1 H, dd, J = 8.0, 1.2 Hz), 8.08 (2 H, d, J = 8.4 Hz), 7.90 (1 H, dt, J = 8.4,

7.0, 1.6 Hz), 7.74ꢀ7.79 (1 H, m), 7.65 (2 H, d, J = 8.6 Hz), 7.61 (1 H, dt,

J = 8.0, 7.2, 1.0 Hz), 7.05ꢀ7.20 (2 H, m), 7.03 (1 H, s), 5.20ꢀ5.30 (1 H,

m), 3.35 (2 H, s), 2.71ꢀ2.83 (2 H, m), 2.30 (4 H, s), 1.94ꢀ2.06 (2 H,

m), 1.73ꢀ1.89 (2 H, m), 1.43ꢀ1.52 (4 H, m), 1.32ꢀ1.42 (2 H, m); ESI

MS calcd for C₃₁H₃₂N₄O₂[M + H]⁺493.3, found 493.2.

(R)-4-(1-Oxophthalazin-2(1H)-yl)-N-(6-(piperidin-1-ylmethyl)-

1,2,3,4-tetrahydronaphthalen-1-yl)benzamide (20). Phthala-

zinone 20 was synthesized from commercially available 4-(1-oxophtha-

lazin-2(1H)-yl)benzoic acid and amine 6. ¹H NMR (400 MHz, DMSO-

d₆) δ ppm 8.86 (1 H, d, J = 8.6 Hz), 8.62 (1 H, s), 8.35 (1 H, d, J = 7.8

Hz), 7.98ꢀ8.08 (4 H, m), 7.94 (1 H, td, J = 8.1, 6.4, 2.0 Hz), 7.74 (2 H, d,

J = 8.6 Hz), 7.16 (1 H, d, J = 8.0 Hz), 7.01ꢀ7.10 (2 H, m), 5.21ꢀ5.30

(1H, m), 3.34(2H, s), 2.69ꢀ2.86(2H, m), 2.30(4H, s), 1.92ꢀ2.08 (2 H,

m), 1.70ꢀ1.90 (2 H, m), 1.42ꢀ1.54 (4 H, m), 1.32ꢀ1.41 (2 H, m); ESI

MS calcd for C₃₁H₃₂N₄O₂[M + H]⁺493.3, found 493.2.

(R)-4-(4-Ethyl-1-oxophthalazin-2(1H)-yl)-N-(6-(piperidin-

1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide

(21). Step 1. A suspension of commercially available 2-propionylben-

zoic acid (422 mg, 2.36 mmol) and 4-hydrazinobenzoic acid (378 mg,

2.48 mmol) in methanol (10 mL) was treated with concentrated sulfuric

acid (5.26 mL, 9.473 mmol). The mixture was stirred at 75 °C in a sealed

vessel. After 17 h, the mixture was cooled to 23 °C and poured into

iceꢀwater (150 mL). The precipitated solid was collected by filtration,

washed with water (25 mL) and diethyl ether (10 mL), and dried under

vacuum, affording methyl 4-(4-ethyl-1-oxophthalazin-2(1H)-yl)benzo-

ate (417 mg, 57%) as a yellow solid.

under vacuum, affording 4-(4-ethyl-1-oxophthalazin-2(1H)-yl)benzoic

acid (198 mg, 99%) as a brown solid.

Step 3. A solution of 4-(4-ethyl-1-oxophthalazin-2(1H)-yl)benzoic

acid (190 mg, 646 μmol), 6 (174 mg, 710 μmol), HOBt (105 mg, 775

μmol), and EDCI (149 mg, 775 μmol) in DMF (10 mL) was stirred at

23 °C. After 17 h, the solution was diluted with EtOAc (100 mL) and

washed with saturated sodium bicarbonate solution (75 mL), water

(75 mL), and brine (75 mL), dried over MgSO₄, concentrated in vacuo,

and purified by flash column chromatography (silica, 2ꢀ6% MeOH/

1

DCM), affording 21 (264 mg, 79%) as a yellow solid. H NMR (400

MHz, CDCl₃) δ ppm 8.54 (1 H, d, J = 7.8 Hz), 7.84ꢀ7.94 (6 H, m),

7.75ꢀ7.82 (1 H, m), 7.28 (1 H, d, J = 7.8 Hz), 7.13 (1 H, d, J = 8.0 Hz),

7.09 (1 H, s), 6.35 (1 H, d, J = 8.2 Hz), 5.34ꢀ5.45 (1 H, m), 3.42 (2 H, s),

3.06 (2 H, q, J = 7.4 Hz), 2.71ꢀ2.93 (2 H, m), 2.38 (4 H, s), 2.08ꢀ2.24

(1 H, m), 1.78ꢀ2.00 (3 H, m), 1.51ꢀ1.67 (4 H, m), 1.33ꢀ1.50 (5 H,

m); ESI MS calcd for C₃₃H₃₆N₄O₂[M + H]⁺521.2, found 521.3.

(R)-4-(4-Isopropyl-1-oxophthalazin-2(1H)-yl)-N-(6-(piperidin-

1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide

(22). Step 1. A solution of methyl 2-iodobenzoate (0.8 mL, 5.45

mmol) in THF (20 mL) in a flame-dried round bottomed flask was

cooled to ꢀ20 °C under nitrogen. Isopropylmagnesium chloride, 2.0 M

solution in tetrahydrofuran (2.99 mL, 5.99 mmol), was added in a

dropwise fashion over 10 min. After 3 h, isobutyryl chloride (0.85 mL,

8.16 mmol) was added in a dropwise fashion. The reaction mixture was

warmed to 23 °C overnight. After 15 h, the reaction mixture was

quenched with MeOH (3 mL), diluted with EtOAc, and washed with

water (100 mL). The organic solution was washed with brine (100 mL),

dried over magnesium sulfate, concentrated in vacuo, and purified by

flash column chromatography (silica, 5ꢀ15% EtOAc/hexane), afford-

ing methyl 2-isobutyrylbenzoate (166 mg, 15%) as a light yellow oil.

Step 2. A solution of methyl 2-isobutyrylbenzoate (150 mg, 727

μmol) and 4-hydrazinobenzoic acid (116 mg, 764 μmol) in methanol

(10 mL) was treated with concentrated sulfuric acid (1.61 mL, 29.09

mmol). The mixture was stirred at 75 °C in a sealed vessel. After 2 days,

the mixture was cooled to 23 °C, diluted with water (50 mL), and

extracted with dichloromethane (2 ꢁ 100 mL). The organic layers were

combined, washed with brine (75 mL), dried over MgSO₄, concentrated

in vacuo, and purified by flash column chromatography (silica, 5ꢀ25%

EtOAc/hexane), affording methyl 4-(4-isopropyl-1-oxophthalazin-

2(1H)-yl)benzoate (51 mg, 22%) as a yellow oil.

Step 3. A solution of methyl 4-(4-isopropyl-1-oxophthalazin-2(1H)-

yl)benzoate (51 mg, 158 μmol) in 1,4-dioxane (8 mL) was treated with

1 N hydrochloric acid (7.91 mL, 7.91 mmol). The mixture was heated to

reflux. After 14 h, the mixture was cooled to 23 °C, concentrated, azeotroped

with toluene (10 mL), and dried under vacuum, affording 4-(4-isopropyl-1-

oxophthalazin-2(1H)-yl)benzoic acid (48 mg, 99%) as a yellow solid.

Step 4. A solution of 4-(4-isopropyl-1-oxophthalazin-2(1H)-yl)-

benzoic acid (49 mg, 159 μmol), 6 (42 mg, 173 μmol), HOBt (23

mg, 173 μmol), and EDCI (33 mg, 173 μmol) in DMF (5 mL) was

stirred under nitrogen at 23 °C. After 16 h, the mixture was diluted with

EtOAc (75 mL) and washed with saturated sodium bicarbonate solution

(50 mL), water (50 mL), and brine (50 mL), dried over MgSO₄,

concentrated in vacuo, and purified by flash column chromatography

(silica, 2.5ꢀ8% MeOH/DCM), affording 22 (31 mg, 40%) as a yellow

solid. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.52ꢀ8.59 (1 H, m),

7.82ꢀ7.95 (6 H, m), 7.75ꢀ7.82 (1 H, m), 7.29 (1 H, d, J = 7.8 Hz), 7.13

(1 H, d, J = 7.8 Hz), 7.09 (1 H, s), 6.37 (1 H, d, J = 8.2 Hz), 5.34ꢀ5.44

(1 H, m), 3.50ꢀ3.61 (1 H, m), 3.43 (2 H, s), 2.74ꢀ2.88 (2 H, m), 2.38

(4 H, s), 2.09ꢀ2.21 (1 H, m), 1.84ꢀ2.00 (3 H, m), 1.52ꢀ1.63 (4 H, m),

1.42ꢀ1.49 (2 H, m), 1.40 (6 H, d, J = 6.7 Hz); ESI MS calcd for

C₃₄H₃₈N₄O₂[M + H]⁺535.2, found 535.3.

Step 2. A suspension of methyl 4-(4-ethyl-1-oxophthalazin-2(1H)-

yl)benzoate (208 mg, 675 μmol) and 1 N hydrochloric acid (6.74 mL,

6.74 mmol) in 1,4-dioxane (10 mL) was heated to 100 °C. After 17 h, the

mixture was concentrated, azeotroped with toluene (10 mL), and dried

(R)-4-(1-Oxo-4-(trifluoromethyl)phthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)benzamide (23). Phthalazinone 23 was synthesized by analogous

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route to compound 21, employing 2-(2,2,2-trifluoroacetyl)benzoic acid as

the starting material. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.88 (1 H, d,

J = 9.0 Hz), 8.46 (1 H, d, J = 8.0 Hz), 7.95ꢀ8.21 (5 H, m), 7.75 (2 H, d, J =

8.5 Hz), 7.17 (1 H, d, J= 8.0 Hz), 6.95ꢀ7.12 (2 H, m), 5.19ꢀ5.32 (1 H, m),

3.35ꢀ3.53 (2 H, m), 2.68ꢀ2.86 (2 H, m), 2.30 (4 H, s), 1.93ꢀ2.07 (2 H,

m), 1.69ꢀ1.90 (2 H, m), 1.41ꢀ1.55 (4 H, m), 1.29ꢀ1.43 (2 H, m); ESI

MS calcd for C₃₂H₃₁F₃N₄O₂[M + H]⁺561.2, found 561.1.

affording methyl 2-acetyl-6-chlorobenzoate (1.895 g, 78.14%) as a

yellow solid.

Step 3. A solution of methyl 2-acetyl-6-chlorobenzoate (1.88 g, 8.88

mmol) and 4-hydrazinobenzoic acid (1.48 g, 9.76 mmol) in methanol

(75 mL) was treated slowly with concentrated sulfuric acid (17.26 mL,

621.2 mmol). The reaction mixture was heated to 80 °C in a sealed vessel

for 5 h. It was then cooled to 23 °C, and the precipitate was poured into an

iceꢀwater solution (100 mL). The solid was collected by filtration and

washed with water (25 mL), affording methyl 4-(8-chloro-4-methyl-1-

oxophthalazin-2(1H)-yl)benzoate (2.084 g, 71%) as off-white crystals.

Step 4. A solution of methyl 4-(8-chloro-4-methyl-1-oxophthalazin-

2(1H)-yl)benzoate (2.00 g, 6.08 mmol) and 5 N hydrochloric acid

(100 mL, 100 mmol) in 1,4-dioxane (100 mL) was stirred at 110 °C.

After 22 h, the solution was concentrated and dried in vacuo to give

4-(8-chloro-4-methyl-1-oxophthalazin-2(1H)-yl)benzoic acid (2.09 g;

crude material was used directly in the next step).

(R)-4-(1-Oxo-4-phenylphthalazin-2(1H)-yl)-N-(6-(piperidin-

1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-yl)benzamide

(24). Phthalazinone 24 was synthesized from commercially available

1

4-(1-oxo-4-phenylphthalazin-2(1H)-yl)benzoic acid and amine 6. H

NMR (400 MHz, DMSO-d₆) δ ppm 8.83 (1 H, d, J = 8.4 Hz), 8.39

(1 H, d, J = 8.0 Hz), 8.02 (1 H, t, J = 7.6 Hz), 7.81ꢀ7.88 (3 H, m), 7.60

(2 H, d, J = 8.6 Hz), 7.51 (2 H, dd, J = 6.7, 2.6 Hz), 7.37ꢀ7.43 (3 H, m),

7.35 (1 H, d, J = 8.2 Hz), 6.97ꢀ7.13 (3 H, m), 5.13ꢀ5.23 (1 H, m), 3.33

(2 H, s), 2.65ꢀ2.83 (2 H, m), 2.29 (4 H, br s), 1.88ꢀ2.01 (2 H, m),

1.67ꢀ1.84 (2 H, m), 1.42ꢀ1.52 (4 H, m), 1.33ꢀ1.41 (2 H, m); ESI MS

calcd for C₃₇H₃₆N₄O₂[M + H]⁺569.3, found 569.3.

(R)-4-(8-Fluoro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-

yl)benzamide (25). Phthalazinone 25 was prepared from 2-bromo-6-

fluorobenzoic acid using a procedure analogous to that for compound

27. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.80ꢀ7.88 (1 H, m), 7.84 (4 H,

q, J = 30.7, 8.6 Hz), 7.60 (1 H, d, J = 8.0 Hz), 7.46 (1 H, dd, J = 10.3, 8.5

Hz), 7.29 (1 H, d, J = 8.0 Hz), 7.15 (2 H, d, J = 7.8 Hz), 6.35 (1 H, d, J =

8.2 Hz), 5.35ꢀ5.45 (1 H, m), 3.49 (2 H, s), 2.72ꢀ2.92 (2 H, m), 2.64

(3 H, s), 2.45 (3 H, br s), 2.09ꢀ2.23 (1 H, m), 1.94ꢀ2.01 (1 H, m),

1.84ꢀ1.94 (2 H, m), 1.63 (5 H, br s), 1.43 (2 H, br s); ESI MS calcd for

C₃₂H₃₃FN₄O₂[M + H]⁺525.3, found 525.2.

(R)-4-(5,8-Difluoro-4-methyl-1-oxophthalazin-2(1H)-yl)-

N-(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)benzamide (26). Phthalazinone 26 was prepared from 2-bromo-

3,6-fluorobenzoic acid using a procedure analogous to that for compound

27. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.84ꢀ7.96 (2 H, m),

7.72ꢀ7.80 (2 H, m), 7.46ꢀ7.57 (1 H, m), 7.35ꢀ7.46 (1 H, m),

7.21ꢀ7.29 (1 H, m), 7.12 (1 H, d, J = 7.8 Hz), 7.07 (1 H, s), 6.39

(1 H, d, J = 8.2 Hz), 5.33ꢀ5.46 (1 H, m), 3.41 (2 H, s), 2.75ꢀ2.92 (2 H,

m), 2.73 (3 H, d, J = 7.6 Hz), 2.37 (4 H, br s), 2.07ꢀ2.21 (1 H, m),

1.82ꢀ2.01 (3 H, m), 1.52ꢀ1.64 (4 H, m), 1.33ꢀ1.48 (2 H, m); ESI MS

calcd for C₃₂H₃₂F₂N₄O₂[M + H]⁺543.2, found 543.2.

(R)-4-(8-Chloro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)benzamide (27). Step 1. A solution of 2-bromo-6-chloroben-

zoic acid (5.60 g, 23.8 mmol) and lithium hydroxide monohydrate (1.10

g, 26.2 mmol) in THF (50 mL) was stirred at 25 °C for 1 h. Dimethyl

sulfate (2.70 mL, 28.5 mmol) was added, and the mixture was stirred at

85 °C for 21 h. The solution was quenched with ammonium hydroxide/

water (1:1, 50 mL), stirred for 30 min, then concentrated in vacuo. It was

diluted with water (50 mL) and extracted with EtOAc. The organic

solution was washed with brine (75 mL), dried over magnesium sulfate,

concentrated, and dried in vacuo to give methyl 2-bromo-6-chloro-

benzoate (5.745 g, 96.8%).

Step 5. A suspension of 4-(8-chloro-4-methyl-1-oxophthalazin-

2(1H)-yl)benzoic acid (1.91 g, 6.07 mmol), 6 (1.78 g, 7.28 mmol),

HOBt (1.07 g, 7.89 mmol), and EDCI (1.51 g, 7.89 mmol) in DMF

(30 mL) was stirred at 25 °C for 4.5 h. The solution was diluted with

EtOAc (300 mL) and washed with 1 N sodium hydroxide solution and

brine. It was dried over magnesium sulfate, concentrated in vacuo, then

purified by flash column chromatography (silica, 2ꢀ8% MeOH/DCM)

to give 27 (3.08 g, 94% over two steps) as a white solid. ¹H NMR (400

MHz, CDCl₃) 7.88 (2 H, d, J = 8.6 Hz), 7.69ꢀ7.83 (5 H, m), 7.28 (1 H,

d, J = 7.8 Hz), 7.07ꢀ7.20 (2 H, m), 6.33 (1 H, d, J = 8.2 Hz), 5.34ꢀ5.46

(1 H, m), 3.39ꢀ3.55 (2 H, m), 2.73ꢀ2.92 (2 H, m), 2.63 (3 H, s), 2.42

(4 H, s), 2.10ꢀ2.23 (1 H, m), 1.81ꢀ2.01 (3 H, m), 1.61 (4 H, s), 1.45

(2 H, s); ESI MS calcd for C₃₂H₃₃ClN₄O₂[M + H]⁺541.2, found 541.2.

(R)-4-(7-Chloro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)benzamide (28). Phthalazinone 28 was prepared from 2-bromo-

5-chlorobenzoic acid using a procedure analogous to that for compound

1

27. H NMR (300 MHz, CDCl₃) δ ppm 8.49 (1 H, d, J = 1.8 Hz),

7.86ꢀ7.96 (2 H, m), 7.72ꢀ7.85 (4 H, m), 7.28 (1 H, d, J = 8.0 Hz), 7.14

(2 H, d, J = 8.0 Hz), 6.36 (1 H, d, J = 8.2 Hz), 5.34ꢀ5.44 (1 H, m), 3.48

(2 H, s), 2.77ꢀ2.89 (2 H, m), 2.65 (3 H, s), 2.46 (4 H, br s), 2.11ꢀ2.22

(1 H, m), 1.84ꢀ2.01 (3 H, m), 1.62 (4 H, s), 1.39ꢀ1.51 (2 H, m); ESI

MS calcd for C₃₂H₃₃ClN₄O₂[M + H]⁺541.2, found 541.2.

(R)-4-(6-Chloro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)benzamide (29). Phthalazinone 29 was prepared from 2-bromo-

4-chlorobenzoic acid using a procedure analogous to that for compound

27. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.46 (1 H, d, J = 8.4 Hz), 7.90

(2 H, d, J = 8.6 Hz), 7.73ꢀ7.83 (4 H, m), 7.28 (1 H, d, J = 8.2 Hz), 7.13

(1 H, d, J = 8.0 Hz), 7.09 (1 H, s), 6.35 (1 H, d, J = 8.2 Hz), 5.33ꢀ5.46

(1 H, m), 3.42 (2 H, s), 2.73ꢀ2.96 (2 H, m), 2.64 (3 H, s), 2.38 (4 H, br

s), 2.10ꢀ2.21 (1 H, m), 1.80ꢀ2.01 (3 H, m), 1.50ꢀ1.64 (4 H, m),

1.34ꢀ1.48 (2 H, m); ESI MS calcd for C₃₂H₃₃ClN₄O₂[M + H]⁺541.2,

found 541.2.

(R)-6-(8-Chloro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)nicotinamide (30). Phthalazinone 28 was prepared from 2-bro-

mo-6-chlorobenzoic acid and 6-hydrazinylnicotinic acid using a procedure

Step 2. A suspension of methyl 2-bromo-6-chlorobenzoate (2.846 g,

11.41 mmol), tributyl(1-ethoxyvinyl)tin (4.24 mL, 12.55 mmol), and

dichlorobis(triphenylphosphine)palladium(II) (0.40 g, 0.57 mmol) in

1,4-dioxane (18 mL) in a sealed microwave tube was degassed and

backfilled with argon. The reaction vessel was heated in a microwave at

150 °C for 30 min, and vinyl ether formation was confirmed by LCMS.

ESI MS calcd for C₁₂H₁₃ClO₃[M + H]⁺241.0, found 241.0. The

reaction mixture was diluted with 6 N hydrochloric acid (10 mL) and

stirred at 23 °C for 3 h. The reaction mixture was diluted with EtOAc

(250 mL) and washed with 10% hydrochloric acid solution (150 mL),

dried over magnesium sulfate, and concentrated in vacuo. It was purified

by flash column chromatography (silica, 10ꢀ25% EtOAc/hexane),

1

analogous to that for compound 27. H NMR (400 MHz, CDCl₃)

δ ppm 9.01 (1 H, d, J = 2.2 Hz), 8.26 (1 H, dd, J = 8.4, 2.3 Hz),

7.65ꢀ7.85 (4 H, m), 7.26 (1 H, d, J = 7.6 Hz), 7.14 (1 H, d, J = 7.8 Hz),

7.10 (1 H, s), 6.36 (1 H, d, J = 8.2 Hz), 5.34ꢀ5.46 (1 H, m), 3.37ꢀ3.45

(2 H, m), 2.72ꢀ2.93 (2 H, m), 2.65 (3 H, s), 2.38 (4 H, br s), 2.11ꢀ2.24

(1 H, m), 1.82ꢀ2.00 (3 H, m), 1.52ꢀ1.63 (4 H, m), 1.36ꢀ1.47 (2 H,

m); ESI MS calcd for C₃₁H₃₂ClN₅O₂[M + H]⁺542.2, found 542.2.

(R)-5-(8-Chloro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)picolinamide (31). Step 1. A suspension of 5-aminopicolinic

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acid (4.98 g, 36.07 mmol) in methanol (50 mL) was cooled to 0 °C

under nitrogen, and thionyl chloride (7.89 mL, 108.2 mmol) was added

in dropwise fashion over 10 min. The reaction mixture was stirred at

23 °C for a further 10 min and then heated to reflux under nitrogen for

48 h. The reaction mixture was cooled to 23 °C, concentrated in vacuo,

diluted with saturated sodium bicarbonate solution (150 mL), and

extracted with EtOAc. The combined organic layers were washed with

brine, dried over magnesium sulfate, and concentrated in vacuo, afford-

ing methyl 5-aminopicolinate (3.390 g, 61%) as a light yellow solid.

Step 2. A solution of methyl 5-aminopicolinate (980 mg, 6.44 mmol)

in 6 N hydrochloric acid (10.74 mL, 64.41 mmol) was cooled to 0 °C. A

solution of sodium nitrite (667 mg, 9.66 mmol) in water (5 mL) was

added in dropwise fashion from an addition funnel. The reaction mixture

was stirred at 0 °C for 15 min, followed by 23 °C for 30 min, followed by

cooling to 0 °C. Tin(II) chloride dihydrate (3.079 g, 13.53 mmol) was

added, followed by warming to 10 °C over 2 h. The reaction mixture was

quenched with 5 N sodium hydroxide solution (until the solution was

basic to pH paper) and was extracted with DCM. The combined organic

layers were dried over magnesium sulfate and concentrated in vacuo,

affording methyl 5-hydrazinylpicolinate (718 mg, 67%) as a yellow solid.

Step 3. A solution of methyl 2-acetyl-6-chlorobenzoate (0.825 g, 3.8

mmol) and methyl 5-hydrazinylpicolinate (0.713 mg, 4.2 mmol) in

methanol (15 mL) was treated with concentrated sulfuric acid (7.5 mL,

13.5 mmol). The mixture was heated to 80 °C in a sealed vessel. After 4

h, the mixture was cooled to 23 °C, and the precipitate was poured into

an iceꢀwater solution (100 mL). The solid was collected by filtration

and washed with water (25 mL), affording an off-white gel-like solid,

which was dissolved in dichloromethane/methanol (1:1), concentrated,

and azeotroped with toluene (5 mL), affording methyl 5-(8-chloro-4-

methyl-1-oxophthalazin-2(1H)-yl)picolinate (1.27 g, 99%) as a brown

solid.

Step 4. A suspension of crude methyl 5-(8-chloro-4-methyl-1-ox-

ophthalazin-2(1H)-yl)picolinate (1.2 g, 3.8 mmol) in 1,4-dioxane (20 mL)

was treated with 1 N hydrochloric acid (19.3 mL, 19.3 mmol). The mixture

was heated to reflux. After 15 h, the mixture was cooled to 23 °C and

concentrated in vacuo. The crude product was azeotroped with toluene

(2 ꢁ 5 mL) and dried under vacuum, affording 5-(8-chloro-4-methyl-1-

oxophthalazin-2(1H)-yl)picolinic acid (1.2 g, 99%) as a yellow solid.

Step 5. A suspension of 5-(8-chloro-4-methyl-1-oxophthalazin-

2(1H)-yl)picolinic acid (1.255 g, 3.98 mmol), TEA (1.1 mL, 7.95

mmol), 6 (1.069 g, 4.37 mmol), HOBt (0.591 g, 4.37 mmol), and

EDCI (0.838 g, 4.37 mmol) in DMF (25 mL) was stirred under nitrogen

at 23 °C for 15 h. The solution was diluted with EtOAc (250 mL) and

washed with saturated sodium bicarbonate solution (150 mL), water

(100 mL), and brine (100 mL). The organic solution was dried over

magnesium sulfate, concentrated in vacuo, and purified by flash column

chromatography (silica, 2.5ꢀ10% MeOH/DCM), affording 31 (1.469

g, 68%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.89

(1 H, d, J = 1.8 Hz), 8.36 (1 H, dd, J = 8.4, 0.6 Hz), 8.21ꢀ8.31 (2 H, m),

7.83 (1 H, dd, J = 7.2, 2.0 Hz), 7.71ꢀ7.80 (2 H, m), 7.18ꢀ7.34 (1 H, m),

7.06ꢀ7.14 (2 H, m), 5.32ꢀ5.47 (1 H, m), 3.44 (2 H, s), 2.72ꢀ2.94 (2 H,

m), 2.64 (3 H, s), 2.39 (4 H, s), 2.05ꢀ2.27 (1 H, m), 1.81ꢀ2.01 (3 H,

m), 1.54ꢀ1.67 (4 H, m), 1.36ꢀ1.47 (2 H, m); ESI MS calcd for

C₃₁H₃₂ClN₅O₂[M + H]⁺542.2, found 542.2.

m), 1.31ꢀ1.49 (2 H, m); ESI MS calcd for C₃₁H₃₃N₅O₂[M + H]⁺

508.2, found 508.2.

(R)-4-(8-Chloro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(7-(piperidin-1-ylmethyl)chroman-4-yl)benzamide (33). A

solution of 4-(8-chloro-4-methyl-1-oxophthalazin-2(1H)-yl)benzoic acid

(189 mg, 0.60 mmol, intermediate for compound 27) and(R)-7-(piperidin-

1-ylmethyl)-3,4-dihydro-2H-chromen-4-amine dihydrochloride (211 mg,

0.661 mmol)¹⁰in DMF (12 mL) was treated with TEA (0.21 mL, 1.50

mmol), and the mixture was stirred at 23 °C for 5 min. The addition of

HOBt (97.4 mg, 0.721 mmol) and EDCI (138 mg, 0.721 mmol) followed,

and the reaction mixture was then stirred for 5 h. The solution was diluted

with EtOAc (100 mL) and washed with saturated sodium bicarbonate

solution, water, and brine. It was dried over magnesium sulfate, concen-

trated in vacuo, and purified by flash column chromatography (silica, 2ꢀ8%

1

MeOH/DCM), affording 33 (232 mg, 71%) as a white solid. H NMR

(400 MHz, CDCl₃) δ ppm 7.84ꢀ7.95 (2 H, m), 7.65ꢀ7.83 (5 H, m), 7.20

(1 H, d, J = 8.0 Hz), 6.90 (1 H, dd, J = 7.8, 1.4 Hz), 6.84 (1 H, d, J = 1.4 Hz),

6.39 (1 H, d, J = 7.2 Hz), 5.34 (1 H, dd, J = 12.3, 7.0 Hz), 4.27ꢀ4.37 (1 H,

m), 4.14ꢀ4.26 (1 H, m), 3.41 (2 H, s), 2.63 (3 H, s), 2.28ꢀ2.47 (5 H, m),

2.12ꢀ2.25 (1 H, m), 1.51ꢀ1.67 (4 H, m), 1.31ꢀ1.48 (2 H, m); ESI MS

calcd for C₃₁H₃₁ClN₄O₃[M + H]⁺543.2, found 543.2.

(R)-6-(5,8-Difluoro-4-methyl-1-oxophthalazin-2(1H)-yl)-

N-(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-

1-yl)nicotinamide (34). Phthalazinone 28 was prepared from 2-bro-

mo-3,6-fluorobenzoic acid and 6-hydrazinylnicotinic acid using a procedure

1

analogous to that for compound 27. H NMR (400 MHz, CDCl₃)

δ ppm 9.02 (1 H, d, J = 2.2 Hz), 8.26 (1 H, dd, J = 8.4, 2.3 Hz), 7.77 (1 H,

d, J = 8.2 Hz), 7.46ꢀ7.60 (1 H, m), 7.34ꢀ7.48 (1 H, m), 7.21ꢀ7.30

(1 H, m), 7.14 (1 H, d, J = 7.8 Hz), 7.09 (1 H, s), 6.40 (1 H, d, J = 8.2 Hz),

5.34ꢀ5.46 (1 H, m), 3.42 (2 H, s), 2.77ꢀ2.92 (2 H, m), 2.75 (3 H, d, J =

7.4 Hz), 2.37 (4 H, br s), 2.10ꢀ2.24 (1 H, m), 1.78ꢀ2.01 (3 H, m),

1.49ꢀ1.68 (4 H, m), 1.33ꢀ1.50 (2 H, m); ESI MS calcd for

C₃₁H₃₁F₂N₅O₂[M + H]⁺544.2, found 544.2.

(R)-6-(8-Fluoro-4-methyl-1-oxophthalazin-2(1H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-

yl)nicotinamide (35). A solution of 6-(8-fluoro-4-methyl-1-ox-

ophthalazin-2(1H)-yl)nicotinic acid (6.9 g, 23.3 mmol, prepared from

2-bromo-6-fluorobenzoic acid in a procedure analogous to that for

compound 30), 6 (6.2 g, 25.7 mmol), HOBt (3.1 g, 23.3 mmol), and

EDCI (4.4 g, 23.3 mmol) in DMF (100 mL) was stirred at 23 °C under

nitrogen. After 15 h, the mixture was diluted with EtOAc (500 mL) and

washed with saturated sodium bicarbonate solution (200 mL), water

(200 mL), and brine (200 mL), dried over MgSO₄, concentrated in

vacuo, and purified by flash column chromatography (silica, 1ꢀ10%

MeOH/DCM), affording 35 (7.6 g, 62%) as an off-white solid. ¹H NMR

(400 MHz, CDCl₃) δ ppm 9.04 (1 H, d, J = 2.5 Hz), 8.25 (1 H, dd, J =

8.3, 2.3 Hz), 7.81ꢀ7.90 (1 H, m), 7.75 (1 H, d, J = 8.5 Hz), 7.61 (1 H, d,

J = 8.0 Hz), 7.39ꢀ7.50 (1 H, m), 7.21ꢀ7.29 (1 H, m), 7.13 (1 H, d, J =

7.5 Hz), 7.05 (1 H, s), 6.57 (1 H, d, J = 8.0 Hz), 5.35ꢀ5.46 (1 H, m), 3.40

(2 H, s), 2.71ꢀ2.90 (2 H, m), 2.66 (3 H, s), 2.37 (4 H, s), 2.10ꢀ2.22

(1 H, m), 1.81ꢀ2.05 (3 H, m), 1.51ꢀ1.66 (4 H, m), 1.32ꢀ1.50 (2 H,

m). ESI MS calcd for C₃₁H₃₂FN₅O₂[M + H]⁺526.2, found 526.2.

Biological Assays. Details of the in vitro binding and cellular assays

have been described previously in ref 11. The PK and PD assay methods

have been previously described in detail in ref 12.

(R)-4-(8-Methyl-5-oxopyrido[2,3-d]pyridazin-6(5H)-yl)-N-

(6-(piperidin-1-ylmethyl)-1,2,3,4-tetrahydronaphthalen-1-

yl)benzamide (32). Phthalazinone 32 was prepared from methyl

2-bromonicotinate using a procedure analogous to that used for the

preparation of compound 27. ¹H NMR (400 MHz, CDCl₃) δ ppm 9.12

(1H, dd,J=4.6, 1.9Hz), 8.77(1H, dd,J= 8.1, 1.9 Hz), 7.88ꢀ7.95(2H, m),

7.76ꢀ7.85 (2 H, m), 7.72 (1 H, dd, J = 8.1, 4.6 Hz), 7.28 (1 H, d, J = 7.8

Hz), 7.13 (1 H, d, J = 8.0 Hz), 7.09 (1 H, s), 6.37 (1 H, d, J = 8.2 Hz),

5.34ꢀ5.45 (1 H, m), 3.43 (2 H, s), 2.78ꢀ2.95 (2 H, m), 2.76 (3 H, s),

2.38 (4 H, s), 2.09ꢀ2.21 (1 H, m), 1.81ꢀ2.06 (3 H, m), 1.52ꢀ1.63 (4 H,

Doses of compound used in the rabbit blood pressure assay

(Figure 4) were as follows: 0.03, 0.1, 1, and 3 mg/kg for compound

30 and 0.003, 0.01, 0.03, 0.1, 0.3, and 1 mg/kg for compound 35.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: 805-447-4836. Fax: 805-480-1337. E-mail: kbiswas@

amgen.com.

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ARTICLE

’ ABBREVIATIONS USED

Mareska, D. A.; Zhan, J.; Clarke, D. E.; Toro, A.; Groneberg, R. D.;

Burgess, L. E.; Lester-Zeiner, D.; Biddlecome, G.; Manning, B. H.; Arik,

L.; Dong, H.; Huang, M.; Kamassah, A.; Loeloﬀ, R.; Sun, H.; Hsieh,

F.-Y.; Kumar, G.; Ng, G. Y.; Hungate, R. W.; Askew, B. C.; Johnson, E.

Potent nonpeptide antagonists of the bradykinin B1 receptor: structure

ꢀactivity relationship studies with novel diaminochroman carboxa-

mides. J. Med. Chem. 2007, 50, 2200–2212.

(13) Biswas, K.; Aya, T.; Qian, W.; Peterkin, T. A. N.; Chen, J. J.;

Human, J.; Hungate, R. W.; Kumar, G.; Arik, L.; Lester-Zeiner, D.;

Biddlecome, G.; Manning, B. H.; Sun, H.; Dong, H.; Huang, M.; Loeloﬀ,

R.; Johnson, E. J.; Askew, B. C. Aryl sulfones as novel bradykinin B1

receptor antagonists for treatment of chronic pain. Bioorg. Med. Chem.

Lett. 2008, 18, 4764–4769.

BK, bradykinin; PSA, polar surface area; SAR, structureꢀ activity

relationship; PK, pharmacokinetics; PD, pharmacodynamics;

DAK, des-Arg-kallidin; HPLC, high-performance liquid chroma-

tography; ESI MS, electrospray ionization mass spectrometry;

DCM, dichloromethane; DMF, N,N-dimethylformamide; EDCI,

N-(3-dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride;

EtOAc, ethyl acetate; HOBt, 1-hydroxybenzotriazole; HPMC, hy-

droxypropyl methylcellulose; MeOH, methanol; Pd(PPh₃)₄, tetrakis-

(triphenylphosphine)palladium(0); TEA, triethylamine; TFA,

triﬂuoroacetic acid; THF, tetrahydrofuran

(14) Chen, J. J; Nguyen, T.; D’Amico, D. C.; Qian, W.; Human, J.;

Aya, T.; Biswas, K.; Fotsch, C.; Han, N.; Liu, Q.; Nishimura, N.;

Peterkin, T. A.; Yang, K.; Zhu, J.; Riahi, B. B.; Hungate, R. W.; Andersen,

N. G.; Colyer, J. T.; Faul, M. M.; Kamassah, A.; Wang, J.; Jona, J.; Kumar,

G.; Johnson, E.; Askew, B. C. 3-Oxo-2-piperazinyl acetamides as potent

bradykinin B1 receptor antagonists for the treatment of pain and

inﬂammation. Bioorg. Med. Chem. Lett. 2011, 21, 3384–3389.

(15) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward,

K. W.; Kopple, K. D. Molecular properties that inﬂuence the oral

bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615–2623.

(16) Waring, M. J. Lipophilicity in drug discovery. Expert Opin. Drug

Discovery 2010, 5, 235–248.

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dx.doi.org/10.1021/jm200808v |J. Med. Chem. 2011, 54, 7232–7246

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Discovery of Potent, orally bioavailable phthalazinone bradykinin B1 receptor antagonists

DOI: 10.1021/jm200808v

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Article abstract of DOI:10.1021/jm200808v

Full text of DOI:10.1021/jm200808v

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