Discovery of a potent, orally bioavailable and highly selective human neuronal nitric oxide synthase (nNOS) inhibitor, N-(1-(piperidin-4-yl)indolin-5- yl)thiophene-2-carboximidamide as a pre-clinical development candidate for the treatment of migraine

European Journal of Medicinal Chemistry 55 (2012) 94e107

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Discovery of a potent, orally bioavailable and highly selective human neuronal

nitric oxide synthase (nNOS) inhibitor, N-(1-(piperidin-4-yl)indolin-5-yl)

thiophene-2-carboximidamide as a pre-clinical development candidate

for the treatment of migraine

,

a

Subhash C. Annedi ^a, Shawn P. Maddaford , Jailall Ramnauth , Paul Renton , Taras Rybak ,

Sarah Silverman ^a, Suman Rakhit ^a, Gabriela Mladenova ^a, Peter Dove ^a, John S. Andrews ^a,

Dongqin Zhang ^b, Frank Porreca ^b

*

^aNeurAxon Inc., 2395 Speakman Drive, Suite #1001, Mississauga, ON, Canada L5K 1B3

^bDepartment of Pharmacology, University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

We recently reported a series of 1,6-disubstituted indoline-based thiophene amidine compounds (5) as

Received 28 February 2012

Received in revised form

1 July 2012

Accepted 4 July 2012

Available online 14 July 2012

selective neuronal nitric oxide synthase (nNOS) inhibitors to mitigate the cardiovascular liabilities

associated with hERG K^þchannel inhibition (IC₅₀

¼

4.7

mM) with previously reported

tetrahydroquinoline-based selective nNOS inhibitors (4). The extended structureeactivity relationship

studies within the indoline core led to the identiﬁcation of 43 as a selection candidate for further

evaluations. The in vivo activity in two different pain (spinal nerve ligation and migraine pain) models,

the excellent physicochemical and pharmacokinetic properties, oral bioavailability (F_po¼ 91%), and the

in vitro safety proﬁle disclosed in this report make 43 an ideal candidate for further evaluation in clinical

applications related to migraine pain.

Keywords:

1,5-Disubstituted indoline derivatives

Nitric oxide

Selective neuronal nitric oxide synthase

inhibitors

Migraine

1. Introduction

success of triptan treatment was limited by the appearance of

cutaneous allodynia (pain resulting from a non-noxious stimulus to

Migraine is a complex central nervous system (CNS) related

disorder that involves multiple macromolecules such as 5HT_1D/1B

receptors, calcitonin gene related peptide (CGRP) receptors, and/or

nitric oxide synthase (NOS) and regulation of any of these macro-

molecules activity would offer a therapeutic beneﬁt. For example,

the development of the ﬁrst triptan (1, Fig. 1, selective 5HT_1D/1B

agonist) in 1988 was considered revolutionary and became

a benchmark for the treatment of acute migraine [1]. However, the

the skin) and the development of central sensitization (abnormal

neuronal activity) during treatment [2]. Although rare, triptans

were also associated with coronary vasospasm, an event that can be

life threatening in patients with coronary artery disease [3]. Recent

clinical trials of acute migraine with orally acting CGRP antagonist

(2, Fig. 1) showed improvement in pain relief relative to placebo

with modest consistency in pain freedom [4]. Unfortunately, the

development of 2 was suspended due to liver toxicity issues

observed in some patients [5]. Given the cardiovascular liabilities

and cutaneous allodynia associated with triptan treatment and

potential liver toxicity issues with CGRP antagonist, there is

a continued interest in new and effective treatment for migraine

pain.

Abbreviations: CNS, central nervous system; NO, nitric oxide; CGRP, calcitonin

gene related peptide; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide

synthase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide

synthase; CTTH, chronic tension type headache;

L-NMMA, N-monomethyl

L

-arginine; SAR, structureeactivity relationship studies; SNL, spinal nerve ligation;

NO is synthesized by three closely related, but separate nitric

oxide synthase (NOS) isoforms. Neuronal NOS (nNOS) and endo-

thelial NOS (eNOS) are constitutively expressed in the brain and

spinal cord and vascular tissue, respectively, while inducible NOS

(iNOS) is expressed in glial cells and immune cells during

IM, inﬂammatory mediator; hERG, human ether-a-go-go-related gene; PK, Phar-

macokinetic; CNS MPO, central nervous system multiparameter optimization.

* Corresponding author. Current address: Vibrant Pharma Inc., 45 Dalkeith Drive,

Unit #14, Brantford, ON, Canada N3P 1M1. Tel./fax: þ1 519 756 9550.

E-mail address: sannedi@vibrantpharma.com (S.C. Annedi).

http://dx.doi.org/10.1016/j.ejmech.2012.07.006

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

95

2. Results and discussion

2.1. Chemistry

The syntheses of all target compounds were carried out

according to the procedures described in Schemes 1e4. The alkyl-

ation at the 1-position of the indoline ring was carried out under

strong basic conditions to obtain 7 and 8, respectively (Scheme 1).

Reduction of the nitro group under hydrogenation conditions, fol-

lowed by coupling to the thiophene-2-carbimidothioate 9 [24]

provided the target compounds 10 and 11, respectively. N-Acyla-

tion of 6 with 2-chloroacetyl chloride [25] provided chloro inter-

mediate 12, which was substituted with two different amines

under basic conditions to obtain 13 and 14, respectively (Scheme 2).

The secondary amine in 14 was protected as the tert-butyl carba-

mate to obtain 15. The amide group in 15 was reduced into the

corresponding amine using borane in THF to obtain 16. Reduction

of the nitro group in 13 and 16 into the corresponding amine

followed by the coupling to thiophene-2-carbimidothioate 9 gave

17 and 18, respectively. Removal of the Boc-protecting group in 18

was carried out under acidic conditions to obtain the target

compound 19.

The reductive amination of 20 with ketones 22e24 gave

compounds 27, 31, and 35, and analog reaction of indolines 21 and 6

with ketones 25 and 26 gave 37 and 38, respectively (Scheme 3)

[26]. Bromination of 27, 31 and 35 under neutral conditions with

N-bromosuccinamide gave 5-bromoindoline derivatives 28, 32 and

36, respectively. Removal of the Boc-protecting group in 28 was

carried out under strong acidic conditions to obtain 29; subsequent

reductive amination of the obtained amine with formaldehyde in

presence of sodium triacetoxyborohydride gave 30. Similarly,

Boc-deprotection in 32, followed by the N-alkylation with

2-bromoethanol provided 34. The bromo in 28, 30, 32, 34, 36 and

37 was converted into the corresponding amine by standard

BuchwaldeHartwig amination conditions using LiHMDS as an

ammonia surrogate [27], followed by the coupling to thiophene-2-

carbimidothioate 9 gave 39, 41, 42, 44, 45 and 46, respectively.

Reduction of the nitro group in 38 to the corresponding amine was

carried out under transfer hydrogenation conditions followed by

coupling to thiophene-2-carbimidothioate 9 to give 48. The Boc-

protecting group in 39, 42, 46 and 48 was removed under strong

acidic conditions to obtain the target compounds 40, 43, trans-47,

cis-47 and 49, respectively. The enantiomeric mixture of cis-47 was

separated into pure enantiomers (ꢀ)-cis-47 and (þ)-cis-47 using

chiral HPLC techniques. The stereochemistry of cis-47 and trans-47

was conformed by various 2D NMR studies such as COSY, HSQC,

NOESY, HMBC experiments (Supporting information). The NOESY

between the 1-3 diaxial protons on C6 and C4 carbons for cis-47

and 1-4 diaxial protons on C7 and C4 for trans-47 is consistent with

the proposed structures (Supporting information). No attempts

were made to separate the enantiomeric mixtures of 40 and 41 at

this time and they were tested as racemic mixtures. At the same

time, no attempts were made to determine the stereochemistry of

(ꢀ)-cis-47 and (þ)-cis-47 at this time.

Fig. 1. Compound 1 is the ﬁrst triptan currently on the market for the treatment of

migraine. Compound 2 is a potent CGRP receptor antagonist developed for the treat-

ment of migraine. Compound 3 is a non-selective NOS inhibitor proven to be effective

in clinical trials of migraine. Compounds 4 and 5 are recently reported selective nNOS

inhibitors.

inﬂammation [6,7]. NO has been shown to be involved in the

development and maintenance of central sensitization and inhib-

itors of NOS have been shown to be effective in clinical trials of

acute migraine and chronic tension type headache (CTTH) [8e11].

For example, 3 (Fig. 1), a non-selective NOS inhibitor showed 67%

antimigraine effect compared to placebo in a randomized double-

blind clinical study with 15 migraine patients [11]. A similar

randomized double-blind, crossover clinical study was conducted

on 16 patients with CTTH using 3 (6 mg/kg) and signiﬁcantly

reduced the headache pain intensity on the visual analog scale

when compared to placebo [11]. However, a signiﬁcant increase in

blood pressure, most likely due to the inhibition of eNOS, was

observed in these patients. Therefore, the selective inhibition of

nNOS over the eNOS isoform is necessary in order to avoid the

cardiovascular liabilities associated with eNOS inhibition [12,13].

Numerous attempts have been made towards the design and

synthesis of selective NOS inhibitors and early NOS inhibitor

design, which was based on L-arginine (substrate), produced only

non-selective NOS inhibitors. However, subsequent designed

inhibitors based on the available crystal structures of NOS

enzymes, which targeted the arginine binding site or the (6R)-2-

amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydropteridin-

4(1H)-one (BH4) co-factor site as well as dimerization inhibitors

have been shown to be more potent and selective among the NOS

isoforms [14e17].

The simpliﬁed pharmacophore model adapted by our group for

competitively inhibiting NOS at the substrate binding site contains

a guanidine isosteric group and a basic amine group both attached

through a central aryl scaffold as described earlier [18]. We recently

reported a series of 1,6-disubstituted indoline-based selective

nNOS inhibitors (5) without any cardiovascular liabilities associated

with 4 due to hERG K^þchannel inhibition (IC₅₀¼ 4.7

mM) [19]. To

extend the structureeactivity relationship studies (SAR) of 5 as part

of the lead optimization process, a small focused library containing

various alkylamino groups on the 1-position of the indoline ring

with thiophene amidine attached at 5-position was synthesized.

From these studies, 43 was identiﬁed as a potent and selective

nNOS inhibitor with exceptional oral bioavailability (91%) without

any cardiovascular liabilities (hERG K^þchannel inhibition, IC₅₀

>100 mM). As part of our ongoing efforts to ﬁnd small molecule

selective nNOS inhibitors [18e23], herein we report the extended

SAR studies on indoline-based thiophene amidine compounds [19]

that led to the identiﬁcation of 43 as a pre-clinical development

candidate for the treatment of migraine.

Scheme 1.

96

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

Scheme 2.

The reductive amination of indoline (20) with 1,4-dioxaspiro

is difﬁcult to observe a clear NOE between these positions due to

certain degree of inter-conversion between boat and chair confor-

mations (Supporting information). However, it was expected that

the trans-conformation would permit the 1, 4 substituents to reside

in a more stable, equatorial position to stabilize the chair confor-

mation. In this conformation the protons in the 2, 3 (and 5, 6)

positions (Supporting information) would be predominantly in

equatorial and axial positions and experiencing the maximum

effect of chemical shift anisotropy from the carbonecarbon bonds

[28]. Therefore the trans-conformation would have the largest

chemical shift range (0.93 ppm) for protons in the 2, 3 (and 5, 6)

positions as opposed to the cis-compound (0.43 ppm).

[4.5]decan-8-one (50), followed by bromination under neutral

conditions as described above provided 52 (Scheme 4). The

deprotection of the acetal group was carried out under acidic

conditions to obtain 53, followed by reductive amination with

methylamine hydrochloride provided two diastereomers cis-54

and trans-54 in 1:1.2 ratio, which were separated by regular silica

gel column chromatography (the stereochemistry was conformed

with the ﬁnal compounds after the sequence was completed). The

protection of secondary amine in cis-54 and trans-54 was achieved

under basic conditions to obtain cis-55 and trans-55, respectively.

The aryl bromides in cis-55 and trans-55 were converted into the

corresponding amines by the BuchwaldeHartwig amination

procedure as described above. The intermediate anilines were then

coupled with thiophene-2-carbimidothioate 9 to provide cis-56

and trans-56, respectively. Finally the Boc-deprotection was carried

out under acidic conditions to obtain the ﬁnal compounds cis-57

and trans-57, respectively. Both cis-57 and trans-57 differ by the cis-

and trans-substitution at the 1, 4 positions of the cyclohexyl ring. It

2.2. Structureeactivity relationship studies (SAR)

All compounds were converted into their corresponding dihy-

drochloride salts and their inhibitory activities were measured

against all three human NOS isoforms (Table 1). Recombinant

human nNOS, eNOS and iNOS were produced in Baculovirus-260

Scheme 3.

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

97

selectivity over the eNOS isoform. Compound 43 with a piperidine

side chain showed excellent potency for nNOS (IC₅₀¼ 0.11 M) and

m

the best selectivity over eNOS (368 fold) and iNOS (3772 fold)

isoforms among the current series. Hydroxyethyl (44) and methyl

(45) substitutions of the secondary amine in 43 resulted up to 2 fold

weaker potency for the nNOS isoform and completely lost the

selectivity over iNOS (3772 fold for 43 compared to 10 fold for 44

and 23 fold for 45). Similarly, ortho-substitution to the basic amine

(trans-47, cis-47, (ꢀ)-cis-47 and (þ)-cis-47) resulted up to 9 fold

weaker potency for nNOS isoform and became non-selective over

the iNOS isoform (2e13 fold) when compared to 43. Moving the

basic amine to 3-position of the piperidine ring ((ꢁ)-49) also

resulted in weaker potency for nNOS isoform and decreased

selectivity over iNOS isoform (iNOS/nNOS ¼ 158) when compared

to 43. Moving the basic amine out of the ring by extending one

carbon (cis-57 and trans-57) did not yield any signiﬁcant difference

in nNOS potency, but the selectivity over iNOS was dramatically

decreased (20e50 fold compared to 3772 fold for 43). Overall the

functionalization or modiﬁcation of the piperidine ring in 43

resulted in weaker potency for nNOS isoform and reduced selec-

tivity over other isoforms particularly for iNOS isoform. The two

sets of diastereomers cis-47 and trans-47, cis-57 and trans-57 did

not show any signiﬁcant difference in nNOS potency or selectivity

over eNOS or iNOS isoforms. At the same time the separated

enantiomers (ꢀ)-cis-47 and (þ)-cis-47 did not show any signiﬁcant

difference in potency or selectivity among the NOS isoforms, when

compared to the racemate cis-47.

2.3. Cytochrome P450 enzyme inhibition studies

Cytochrome P450 enzyme inhibition studies were performed to

assess the potential for metabolism-based drugedrug interactions,

which is an inevitable hurdle during the drug development process.

This will also rule out the inhibitory activity with cytochrome P450

enzymes that are closely related to NOS [29]. Hence, 43 was tested

against the ﬁve major human cytochrome P450 enzymes (CYP1A2,

CYP2C9, CYP2C19, CYP2D6 and CYP3A4). Compound 43 was inac-

Scheme 4.

infected Sf9 cells. In a radiometric method, inhibitory activities

tive up to a maximum concentration of 100

major human cytochrome P450 enzymes, while an IC₅₀value of

0.75 M was determined for CYP2D6.

mM at four of the ﬁve

were measured by the conversion of [³H]- -arginine into [³H]-

L L-

citrulline. In our present design strategy, we extended the SAR

around substituted indoline-based thiophene amidine compounds

[19], where the indoline core acts as an aromatic linker with the

indoline nitrogen serving as an attachment point for various basic

amine side chains, while keeping the thiophene amidine group

ﬁxed at 5-position. All compounds showed sub-micromolar

potency for the nNOS isoform, good to excellent selectivity over

the eNOS isoform, and are comparable to previously reported

1,6-disubstituted indoline derivatives [19]. Compound 10 with

m

2.4. Spinal nerve ligation (SNL) model and migraine model studies

Evaluated as the most selective compound among the series, 43

was selected for further proﬁling in two different in vivo pain

models (Chung and migraine). The Chung or SNL model involves

ligation of both the L₅and L₆spinal nerves of one side of the rat,

which will produce thermal and mechanical allodynia of the

affected paw [30]. Withdrawal latency to application of radiant heat

to the paw was tested. Intraperitoneal administration of 43 at

30 mg/kg resulted in reversal of thermal hyperalgesia with

a maximum reversal effect of 71% observed at 60 min (Fig. 2). After

the positive results with 43 in the Chung model, the compound was

investigated in a rat model of migraine pain. This model involves

the application of an inﬂammatory mediator (IM) onto the dura via

a cannula with subsequent measurements in the development of

cutaneous allodynia in the hind paws [31]. After administration of

the IM, animals develop signs of allodynia which peaks approxi-

mately 3 h after cannulation. The appearance of cutaneous allo-

dynia occurring in face and paws after the administration of IM

parallels the delayed extra-cranial symptoms seen in patients after

the onset of a migraine attack. The oral administration of 43

(30 mg/kg) 15 min prior to IM attenuated the development of

allodynia between 1 and 5 h of post-dose, peaking at the 3 h time

point (Fig. 3).

N,N-dimethyl ethyl side chain was most potent (IC₅₀¼ 0.04

mM) for

nNOS isoform among the current series with excellent selectivity

(287 fold) over eNOS isoform. Compound 11 with a pyrrolidin-1-yl

side chain was also potent and selective for the nNOS isoform

(nNOS IC₅₀¼ 0.12 M, eNOS/nNOS ¼ 311). Compound 17 with an

m

amide functionality was 5 times less potent at the nNOS isoform

with moderate selectivity (17 fold) over eNOS isoform. This may be

due to the restricted ﬂexibility with an amide functionality, which

may be preventing the favorable binding orientation as in case of 11

with an amine function and is consistent with our previous

observation with 3,4-dihydroquinolin-2(1H)-one based nNOS

inhibitors [20].

Both (ꢁ)-40 and (ꢁ)-41 with pyrrolidine side chain showed very

good potency for nNOS and selectivity over eNOS isoform.

Compound (ꢁ)-41 with a tertiary amine with N-methyl substitu-

tion showed 5 times weaker potency for the nNOS isoform, when

compared to (ꢁ)-40 with a secondary amine while maintaining the

98

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

Table 1

Inhibition of human NOS enzymes by 1,5-disubstituted indoline derivatives.

Compound

R

Human NOS IC₅₀

nNOS

(

m

M)^a

Selectivity

eNOS

iNOS

eNOS/nNOS

iNOS/nNOS

10

11

0.04 (0.02e0.05)

0.12 (0.09e0.17)

11.4 (8.4e15.6)

37.3 (15.1e92.2)

4.07 (1.93e8.61)

0.41 (0.27e0.62)

287

311

102

3

17

0.62 (0.35e1.08)

10.5 (5.9e18.7)

0.77 (0.50e1.20)

17

1

19

0.35 (0.27e0.45)

0.08 (0.03e0.21)

10.1 (3.43e29.4)

5.45 (2.62e11.3)

>100^b

29

68

>286

(ꢁ)-40

NT^c

NC^d

(ꢁ)-41

43

0.43 (0.32e0.57)

0.11 (0.05e0.25)

0.16 (0.03e0.85)

21.2 (14.2e31.4)

40.5 (17.0e96.3)

39.3 (11.1e138.6)

NT^c

49

368

246

NC^d

3772

10

z415

44

1.58 (1.16e2.15)

45

0.2 (0.12e0.34)

0.37 (0.25e0.53)

58.2 (30.0e112.6)

36.7 (21.4e62.8)

4.67 (1.60e13.6)

4.75 (2.68e8.42)

291

99

23

13

trans-47

cis-47

0.69 (0.50e0.96)

0.71 (0.26e1.92)

0.91 (0.31e2.68)

0.33 (0.19e0.56)

124 (20.6e744)

53.7 (29.4e97.8)

51.7 (28.0e95.0)

94 (45.6e193.8)

1.17 (0.91e1.50)

1.09 (0.80e1.49)

1.80 (1.36e2.37)

52.2 (37.3e72.9)

180

76

2

(ꢀ)-cis-47

(þ)-cis-47

(ꢁ)-49

2

57

2

285

158

cis-57

trans-57

5^e

0.20 (0.11e0.36)

0.10 (0.02e0.56)

0.37 (0.07e1.93)

17.9 (11.2e28.3)

34.1 (12.2e94.7)

195 (146e259)

4.08 (2.37e7.04)

4.87 (2.56e9.29)

83 (32.3e212)

90

341

527

20

50

224

Values reported in parentheses are 95% conﬁdence intervals.

a

In a radiometric method, inhibitory activities were measured by the conversion of [³H]- -arginine into [³H]-

L L-citrulline.

b

c

>100 ¼ Not active at maximum test concentration of 100

mM.

NT ¼ Not tested.

d

e

NC ¼ Not calculable.

Compound 5 is a recently reported selective nNOS inhibitor, reported for comparison.

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

99

Table 2

Physicochemical properties of 43.

30

20

10

0

clogP PSA

LogD (pH 7.4) pK_a

Hb-A Hb-D FRB MW

326.46

CNS MPO

43 (30 mg/kg, i.p.)

3.58 79.39 ꢀ1.78

10.44

8.62

4

3

4

limitations and liabilities of the selection candidate associated with

off target activities to minimize the risks and overall cost associated

with the drug development process. This process is also a rapid and

cost effective way of prioritizing the most promising compound

such as 43 for further evaluation in various pre-clinical toxicology

studies during the selection process. Accordingly, 43 was tested at

a single concentration of 10 mM in 78 different pharmacological

BLPost-SNL30

60

90

120

180

assays to identify off target activities (Table 1 from Supporting

information). Compound 43 was found to be not active (very

weak inhibitor) for most of the targets tested (<50% inhibition at 71

out of 78 targets) and showed signiﬁcant inhibition (>50%) at the

following targets: alpha adrenergic receptor (a₁: 56%, a₂: 74%),

human muscarinic receptor (M₁: 68%, M₂: 76%, M₃: 86%, M₄: 91%)

and 5HT_1Btransporter (81%). As a follow-up to the high throughput

proﬁle, the IC₅₀values were determined for target assays that were

Time (min) after injection

Fig. 2. Compound 43 attenuates thermal hyperalgesia in the L₅/L₆SNL (Chung) model

of neuropathic pain. The reported mean values are from 5 animals.

2.5. hERG K^þchannel inhibition studies

Antiarrhythmic drugs that induce QT prolongation by inhibiting

the human ether-a-go-go-related gene (hERG) K^þchannel has been

linked to drug induced sudden cardiac death and is now the second

leading cause for withdrawing approved drugs from the market

[32]. The hERG binding as well as the functional activity (conven-

tional patch-clamp assay) of 43 was tested [33,34] in human

recombinant HEK-293 cell lines for its ability to inhibit the hERG K^þ

channel considering the moderate hERG activity with 5 (functional

showed signiﬁcant inhibition and the values are: a_1A(1.9

m

M), a_1B

M), a_2B(32 M), M₁

M) and 5HT_1B(2.2 mM). Overall 43

(1.8

(8.6

m

M), a_1D(2.2

M), M₂(3.6

m

M), a_2A(24

m

mM), a_2C(11 m

mM), M₃(4.5

m

possess an excellent in vitro safety proﬁle.

2.7. Physicochemical and pharmacokinetic (PK) properties

activity, IC₅₀¼ 13

(IC₅₀of 160 M) as well as functional activity (not active at the

maximum test concentration of 100

M) against hERG K^þchannel,

when compared to reference compound 5. The inactivity of 43 at

hERG channel can be correlated to its lower lipophilicity

(clogP ¼ 2.57) compared to 5 (clogP ¼ 3.15). The data suggests that

43 would be devoid of any cardiovascular liabilities associated with

hERG K^þchannel binding that are physiologically relevant based on

its 1455 fold weaker potency, when compared to its nNOS potency

mM). Compound 43 was inactive in both binding

The nNOS enzyme active site is polar and acidic and the

substrate (L-arginine) based nNOS inhibitors are polar and contains

m

basic functional groups. Being polar with a basic amine is not an

ideal combination for selective nNOS inhibitors for treating CNS

related targets due to the bloodebrain barrier and cell membrane

permeability issues. Various reports have already appeared in the

literature describing the important role of physicochemical prop-

erties on the in vivo behavior of marketed drugs and drug candi-

dates [36]. Recently, a central nervous system multiparameter

optimization (CNS MPO) algorithm was developed using six

important physicochemical properties to assess the druglikeness

characteristics [37]. Using this tool, a large number of marketed

drugs and drug candidates for CNS related targets were evaluated

and it was shown that most drugs/drug candidates have a CNS MPO

score of ꢂ4 on a scale of 0e6. To assess the druglike characteristics

of 43, the physicochemical properties and CNS MPO score were

determined as shown in Table 2. Compound 43 does not violate any

of the Lipinski’s rule of ﬁve (log P < 5, hydrogen bond donors <5,

hydrogen bond acceptors <10, molecular weight <500) and has an

ideal CNS MPO score of 4. The pKa of the secondary amine and the

amidine group were determined as 10.44 and 8.62, respectively for

43 in order to assess the basicity. At physiological pH of 7.4, this

compound exists as doubly protonated species to an appreciable

amount, which is correlated to its negative log D value of ꢀ1.78.

Based on its excellent nNOS inhibitory activity and selectivity, its

chemical stability (weak P450 inhibition) and favorable physico-

chemical properties (Table 2) for membrane permeability, 43 was

tested further to assess the pharmacokinetic (PK) properties. The

PK parameters estimated from non-compartmental analysis of the

plasma concentration vs time curves for 43 following intravenous

(3 mg/kg, iv) and oral (10 mg/kg, po) administration to male

Sprague-Dawley rats are summarized in Table 3. Maximum plasma

concentration (C_max¼ 101 ng/mL) for 43 was observed at 2 h

following the oral dosing and declined with a half-life of 12 h.

Compound 43 exhibited a large systemic clearance (CL ¼ 420 mL/

(IC₅₀¼ 0.11

mM).

2.6. High throughput proﬁle of 43

To identify off target activity, 43 was tested in 78 validated

in vitro pharmacological assays that cover a broad range of targets

[35]. This high throughput proﬁle is used to identify the potential

18

15

12

9

43 (30 mg/kg, p.o.)

Vehicle

6

3

0

BL

1

2

3

4

5

6

Time (h) dural stimulation

Fig. 3. Oral administration of 43 (30 mg/kg) attenuates tactile hyperesthesia of the

hind paw in rats with inﬂammation of the Dura. The reported mean values are from 6

animals for vehicle and 4 animals for 43.

100

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

reaction was heated to 90 ^ꢃC and stirred for 1.5 h. After allowing the

Table 3

Rat pharmacokinetic properties of 43.^a

reaction to cool to room temperature, water was added and the

product was extracted into EtOAc. The combined ethyl acetate layer

was washed with water, brine and dried (Na₂SO₄). Solvent was

evaporated and the crude was puriﬁed by ﬂash column chroma-

tography (2 M NH₃in MeOH:CH₂Cl₂, 2.5:97.5) to obtain the title

C_max(ng/mL)^b

T_max

t_1/2

AUC

(ng h/mL)

V_ss

(L/kg)

CL

F_po

(%)

(h)^b

(mL/min/kg)

101

2

12

95.2

330

420

91

a

3 mg/kg iv.

10 mg/kg po.

compound (0.4 g, 56%) as a solid. ¹H NMR (DMSO-d₆)

d 7.96 (dd, 1H,

b

J ¼ 2.1, 8.7 Hz), 7.79 (d, 1H, J ¼ 2.1 Hz), 6.49 (d, 1H, J ¼ 9.0 Hz), 3.72

(t, 2H, J ¼ 8.7 Hz), 3.39 (t, 2H, J ¼ 6.6 Hz), 3.04 (t, 2H, J ¼ 8.7 Hz), 2.44

(t, 2H, J ¼ 6.3 Hz), 2.18 (s, 6H).

min/kg) and volume of distribution (V_ss¼ 330 L/kg) with excep-

tional oral bioavailability (F_po¼ 91%).

PSA, polar surface area; Hb-A, sum of hydrogen bond acceptors;

Hb-B, sum of hydrogen bond donors; FRB, number of freely rotat-

able bonds; MW, molecular weight; CNS MPO, central nervous

system multiple parameter optimization score.

4.1.2. 5-Nitro-1-(2-(pyrrolidin-1-yl)ethyl)indoline (8)

Prepared from 5-nitroindoline (6) (0.5 g, 3.05 mmol) as

described for 7 to obtain the title compound (0.4 g, 50%) as a solid.

¹H NMR (DMSO-d₆)

J ¼ 2.1 Hz), 6.47 (d, 1H, J ¼ 9.0 Hz), 3.73 (t, 2H, J ¼ 9.0 Hz), 3.41 (t, 2H,

J ¼ 6.9 Hz), 3.04 (t, 2H, J ¼ 8.1 Hz), 2.62 (t, 2H, J ¼ 6.9 Hz), 2.58e2.48

(m, 4H), 1.70e1.64 (m, 4H); ESI-MS (m/z, %): 262 (MH^þ, 100).

d

7.96 (dd, 1H, J ¼ 2.4, 9.0 Hz), 7.80 (d, 1H,

3. Conclusions

In conclusion, a series of 1,5-disubstitued indoline-based thio-

phene amidine compounds were designed, synthesized and shown

to be selective inhibitors of human nNOS over the eNOS and iNOS

isoforms. By varying the basic amine at 1-position of the indoline

ring several potent and highly selective inhibitors of nNOS over

eNOS were identiﬁed. Compound 43, a potent and highly selective

inhibitor of nNOS having favorable physicochemical properties

with exceptional oral bioavailability (91%) was shown to be effec-

tive in two different in vivo pain models. At the same time 43 is

devoid of any cardiovascular liabilities associated with QT prolon-

gation with hERG K^þchannel binding. Compound 43 does not have

any off target activities (78 receptors/transporters/ion channels)

associated with the side effects, making it an ideal candidate to

carry forward through the pre-clinical development process to

investigate the role that excess NO produced by nNOS enzyme plays

in CNS related disorders such as migraine pain. Finally this report

addresses some of the formidable issues in developing selective

nNOS inhibitors for therapeutic use.

4.1.3. N-(1-(2-(Dimethylamino)ethyl)indolin-5-yl)thiophene-2-carbo-

ximidamide (10)

A

suspension of N,N-dimethyl-2-(5-nitroindolin-1-yl)ethan-

amine (7) (0.18 g, 0.77 mmol) and PdeC (0.08 g, 0.07 mmol, 10% wt)

in dry EtOH (5 mL) was purged with hydrogen gas. The reaction was

stirred at room temperature overnight under hydrogen atmosphere

(balloon pressure). The reaction was ﬁltered through a pad of celite

and washed with EtOH. The ﬁltrate was treated with methyl thio-

phene-2-carbimidothioate hydroiodide (9) (0.44 g, 1.55 mmol) and

stirred overnight at room temperature. The reaction was diluted

with saturated NaHCO₃solution and the product was extracted into

CH₂Cl₂. The combined organic layer was dried (Na₂SO₄) and

concentrated. The crude was puriﬁed by ﬂash column chromatog-

raphy (2 M NH₃in MeOH:CH₂Cl₂, 2.5:97.5 to 5:95) to obtain the title

compound (0.09 g, 40%) as a solid. ¹H NMR (DMSO-d₆)

d 7.68 (d, 1H,

J ¼ 2.7 Hz), 7.56 (dd, 1H, J ¼ 5.1, 1.2 Hz), 7.07 (dd, 1H, J ¼ 3.6, 5.1 Hz),

6.62 (s, 1H), 6.55e6.46 (m, 2H), 6.28 (s, 2H), 3.32e3.27 (m, 2H), 3.09

(t, 2H, J ¼ 6.6 Hz), 2.84 (t, 2H, J ¼ 8.1 Hz), 2.45 (t, 2H, J ¼ 6.9 Hz), 2.20

(s, 6H); ESI-MS (m/z, %): 315 (MH^þ, 100), 244 (29), 127 (38);

ESI-HRMS calculated for C₁₇H₂₃N₄S (MH^þ): 315.1637, observed:

315.1645.

4. Experimental section

4.1. General

All reactions were performed under an atmosphere of argon and

stirred magnetically unless otherwise noted. Commercial reagents

and anhydrous solvents were used as received without further

puriﬁcation. Reactions were monitored by analytical TLC using pre-

coated silica gel aluminum plates (SigmaeAldrich, 0.2 mm, 60 Å)

and were visualized with UV light or stained appropriately. Flash

column chromatography was performed using Silicycle Siliaﬂash

4.1.4. N-(1-(2-(Pyrrolidin-1-yl)ethyl)indolin-5-yl)thiophene-2-carbo-

ximidamide (11)

Prepared from 5-nitro-1-(2-(pyrrolidin-1-yl)ethyl)indoline (8)

(0.40 g, 1.53 mmol) as described for 10 to obtain the title compound

(0.23 g, 44%) as a solid. ¹H NMR (DMSO-d₆)

d

7.68 (dd, 1H, J ¼ 0.9,

3.6 Hz), 7.55 (dd, 1H, J ¼ 0.9, 5.1 Hz), 7.07 (dd, 1H, J ¼ 3.6, 4.8 Hz),

6.62 (s, 1H), 6.50e6.45 (m, 2H), 6.23 (s, 2H), 3.33e3.27 (m, 2H), 3.11

(t, 2H, J ¼ 6.9 Hz), 2.84 (t, 2H, J ¼ 8.1 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz),

2.55e2.45 (m, 4H), 1.70e1.65 (m, 4H); ESI-MS (m/z, %): 341 (MH^þ,

100), 244 (45), 127 (57); ESI-HRMS calculated for C₁₉H₂₅N₄S (MH^þ):

341.1794, observed: 341.1788.

F60 (40e63 m

m) silica gel. The ¹H NMR spectra were performed on

a Bruker 300 MHz spectrometer. Low and high resolution Mass

Spectra were performed on an applied Biosystems/MDS Sciex

QstarXL hybrid quadrupole/TOF instrument using electrospray

ionization. Chemical purity was determined using an Agilent 1100

HPLC system using Zorbax, SB-C18 and Waters XTerra RP8.5

mm

4.1.5. 2-Chloro-1-(5-nitroindolin-1-yl)ethanone (12)

reverse phase columns and the purity was determined to be ꢂ95%

until unless speciﬁed. Chiral purity was determined using Chiracel

OJ-H column and the chiral purity was determined to be >99%. No

attempts were made to optimize the yields.

A suspension of 5-nitroindoline (6) (1.0 g, 6.09 mmol) in toluene

(20 mL) was treated with 2-chloroacetyl chloride (0.97 mL,

12.18 mmol) and heated at 110 ^ꢃC for 15 min. The reaction was

cooled to room temperature, treated with sat. NaHCO₃solution and

extracted into ethyl acetate. The combined organic layer was dried

(Na₂SO₄) and concentrated. The crude was puriﬁed by ﬂash column

chromatography (EtOAc:hexanes, 1:9 to 1:1 then 2 M NH₃MeOH:

CH₂Cl₂, 1:9) to obtain the title compound (1.08 g, 74%). ¹H NMR

4.1.1. N,N-Dimethyl-2-(5-nitroindolin-1-yl)ethanamine (7)

A solution of 5-nitroindoline (6) (0.5 g, 3.05 mmol) in DMF

(10 mL) was treated with NaH (0.39 g, 9.75 mmol, 60% wt in mineral

oil) at 0 ^ꢃC resulting in an orange mixture. The reaction mixture was

then treated with 2-chloro-N,N-dimethylethanamine hydrochlo-

ride (0.87 g, 6.09 mmol) resulting in a dark red mixture. The

(CDCl₃)

d

8.30e8.27 (m, 1H), 8.14 (dd, 1H, J ¼ 2.1, 9.0 Hz), 8.06 (s,

1H), 4.30 (t, 2H, J ¼ 8.7 Hz), 4.18 (s, 2H), 3.33 (t, 2H, J ¼ 8.4 Hz);

ESI-MS (m/z, %): 241, 243 (MH^þ).

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

101

4.1.6. 1-(5-Nitroindolin-1-yl)-2-(pyrrolidin-1-yl)ethanone (13)

A pressure vessel containing 2-chloro-1-(5-nitroindolin-1-yl)

ethanone (12) (1.05 g, 4.38 mmol) in acetonitrile (30 mL) and water

(2 mL) was treated with potassium carbonate (3.03 g, 21.90 mmol)

followed by pyrrolidine (1.81 mL, 21.90 mmol) and the resulting

mixture was heated at 60 ^ꢃC for 1 h. The reaction was brought to

room temperature and product was extracted into CH₂Cl₂. The

CH₂Cl₂layer was washed with water and sat. sodium carbonate

solution. The organic layer was separated and the aqueous layer

(pH >9) was further extracted with CH₂Cl₂. The combined organic

layer was dried (Na₂SO₄) and concentrated. The crude product was

puriﬁed by ﬂash column chromatography (2 M NH₃MeOH:CH₂Cl₂,

5:95) to obtain the title compound (1.2 g, quantitative) as a solid. ¹H

(100), 133 (56); EI-HRMS calculated for C₁₉H₂₂N₄OS (M^þ): 354.1514,

observed: 354.1504.

4.1.11. tert-Butyl 2-hydroxyethyl (2-(5-(thiophene-2-carboximidamido)

indolin-1-yl) ethyl)carbamate (18)

Prepared from tert-butyl 2-hydroxyethyl(2-(5-nitroindolin-1-

yl)ethyl)carbamate (16) (3.98 g, 11.35 mmol) as described for 10

to obtain the title compound (4.56 g, 93%) as a foam. ESI-MS (m/z,

%): 431 (MH^þ, 100), 429 (46), 242 (16).

4.1.12. N-(1-(2-(2-Hydroxyethylamino)ethyl)indolin-5-yl)

thiophene-2-carboximidamide (19)

A solution of tert-butyl 2-hydroxyethyl (2-(5-(thiophene-2-

carboximidamido) indolin-1-yl) ethyl)carbamate (18) (4.56 g,

10.59 mmol) in methanol (50 mL) was treated with 1 N HCl solution

(50 mL) at rt. The resulting mixture was reﬂuxed for 30 min and

brought to rt. The reaction was basiﬁed with sat. NaHCO₃solution

and extracted into CH₂Cl₂. The solvent was evaporated and crude

NMR (CDCl₃)

d

8.31 (d, 1H, J ¼ 9.0 Hz), 8.16 (dd, 1H, J ¼ 2.1, 9.0 Hz),

8.08 (s, 1H), 4.31 (t, 2H, J ¼ 8.7 Hz), 4.20 (s, 2H), 3.34 (t, 2H,

J ¼ 8.4 Hz); ESI-MS (m/z, %): 276 (MH^þ, 100).

4.1.7. 2-(2-Hydroxyethylamino)-1-(5-nitroindolin-1-yl)ethanone (14)

Prepared from 2-chloro-1-(5-nitroindolin-1-yl)ethanone (12)

(6.06 g, 25.2 mmol) as described for 13 to obtain the title compound

was puriﬁed by ﬂash column chromatography (2

MeOH:CH₂Cl₂, 1:99 to 1:9) to obtain the title compound (0.435 g,

12%) as a foam. ¹H NMR (DMSO-d₆)

M NH₃

(3.86 g, 57%) as an oil. ¹H NMR (CDCl₃)

d

8.32 (d, 1H, J ¼ 9.3 Hz), 8.15

d

7.68 (d, 1H, J ¼ 3.3 Hz), 7.55 (d,

(dd, 1H, J ¼ 2.1, 9.0 Hz), 8.07 (s, 1H), 4.15 (t, 2H, J ¼ 8.4 Hz), 3.67 (t,

2H, J ¼ 4.8 Hz), 3.60 (s, 2H), 3.31 (t, 2H, J ¼ 8.7 Hz), 2.88 (t, 2H,

J ¼ 5.1 Hz), 2.23e1.96 (m, 2H); ESI-MS (m/z, %): 288 (36, M þ Na),

266 (MH^þ, 100).

1H, J ¼ 5.1 Hz), 7.07 (t, 1H, J ¼ 4.8 Hz), 6.62 (s, 1H), 6.55e6.47 (m,

2H), 6.36e6.04 (m, 2H), 4.59 (brs, 1H), 3.49e3.48 (m, 2H),

3.30e3.24 (m, 2H), 3.10 (t, 2H, J ¼ 6.6 Hz), 2.88e2.77 (m, 4H), 2.68

(t, 2H, J ¼ 5.7 Hz); ESI-MS (m/z, %): 331 (MH^þ, 100), 285 (26), 127

(22); ESI-HRMS calculated for C₁₇H₂₃N₄OS (MH^þ): 331.1585,

observed: 331.1587; HPLC purity: 92.45% by area.

4.1.8. tert-Butyl 2-hydroxyethyl(2-(5-nitroindolin-1-yl)-2-oxoethyl)

carbamate (15)

A solution of 2-(2-hydroxyethylamino)-1-(5-nitroindolin-1-yl)

ethanone (14) (3.81 g, 14.36 mmol) and triethylamine (4.04 mL,

28.7 mmol) in dioxane (55 mL) was treated with di-tert-butyl

dicarbonate (3.29 g, 15.08 mmol) and stirred for 1 h at room

temperature. The reaction was diluted with water and extracted

into CH₂Cl₂. The combined organic layer was concentrated and

puriﬁed by ﬂash column chromatography (2 M NH₃MeOH:CH₂Cl₂,

5:95) to obtain the title compound (4.54 g, 87%) as a viscous liquid.

4.1.13. tert-Butyl 3-(indolin-1-yl)pyrrolidine-1-carboxylate (27)

A solution of indoline (20) (1.88 mL, 16.78 mmol), tert-butyl

3-oxopyrrolidine-1-carboxylate (22) (3.73 g, 20.13 mmol) in dry

methanol (20 mL) was treated with AcOH (2.37 mL, 41.95 mmol)

followed by NaCNBH₃(1.26 g, 20.13 mmol) at 0 ^ꢃC. The reaction was

brought to room temperature and stirred for 3 h. The reaction was

basiﬁed with 1 N NaOH solution and product was extracted into

CH₂Cl₂. The combined CH₂Cl₂layer was dried (Na₂SO₄) and solvent

was evaporated to obtain crude product. The crude was puriﬁed by

column chromatography (EtOAc:hexanes, 1:9) to obtain the title

¹H NMR (CDCl₃)

d 8.39e8.31 (m, 1H), 8.23e8.10 (m, 1H), 8.04e7.96

(m, 1H), 4.31e4.13 (m, 4H), 3.85e3.73 (m, 2H), 3.57e3.49 (m, 2H),

3.39e3.29 (m, 2H), 3.18e3.09 (m, 1H), 1.50 (s, 9H); ESI-MS (m/z, %):

388 (43, MNa^þ), 366 (MH^þ, 9), 288 (27), 266 (100).

compound (4.2 g, 87%) as a syrup. ¹H NMR (CDCl₃)

d 7.08e7.02 (m,

2H), 6.66 (t, 1H, J ¼ 7.5 Hz), 6.49 (d, 1H, J ¼ 7.8 Hz), 4.17e4.05 (m,

1H), 3.70e3.51 (m, 2H), 3.47e3.37 (m, 4H), 2.95 (t, 2H, J ¼ 8.4 Hz),

2.18e1.99 (m, 2H), 1.46 (s, 9H); ESI-MS (m/z, %): 289 (MH^þ, 16), 233

(100).

4.1.9. tert-Butyl 2-hydroxyethyl(2-(5-nitroindolin-1-yl)ethyl)

carbamate (16)

A solution of tert-butyl 2-hydroxyethyl(2-(5-nitroindolin-1-yl)-

2-oxoethyl)carbamate (15) (4.42 g, 12.10 mmol) in anhydrous THF

(50 mL) was stirred at 0 ^ꢃC and 1 M borane in THF (36.3 mL,

36.3 mmol) was added drop-wise over a period of 15 min. The

reaction was brought to rt and stirred for 1 h. The reaction was

quenched with methanol (100 mL) and was stirred for 30 min. The

mixture was concentrated and was quenched once more with

methanol (100 mL). The crude product was concentrated and

ﬁltered through a pad of silica gel (EtOAc:hexanes, 1:1, then 2 M

NH₃in MeOH:CH₂Cl₂, 2.5:97.5 to 5:95) to obtain the title compound

(4.01 g, 95%) as an orange-red solid. ESI-MS (m/z, %): 390 (18,

M þ K), 374 (71, MNa^þ), 351 (MH^þ, 31), 296 (100), 252 (86).

4.1.14. tert-Butyl 3-(5-bromoindolin-1-yl)pyrrolidine-1-carboxylate (28)

A solution of tert-butyl 3-(indolin-1-yl)pyrrolidine-1-carboxylate

(27) (4.15 g, 14.390 mmol) in dry DMF (30 mL) was treated with

N-bromosuccinimide(2.56g,14.390mmol) in dryDMF(20mL)at0 ^ꢃC

and resulting solution was stirred at same temperature for 3 h. The

reaction was diluted with water and product was extracted into ethyl

acetate. The combined ethyl acetate layer was washed with water,

brine and dried (Na₂SO₄). Solvent was evaporated and crude was

puriﬁed by column chromatography (EtOAc:hexanes, 1:4) to obtain

the title compound (5.12 g, 97%) as a syrup. ¹H NMR (CDCl₃)

d

7.18e7.07 (m, 2H), 6.33 (d, 1H, J ¼ 8.7 Hz), 4.13e3.99 (m, 1H),

3.68e3.26 (m, 6H), 2.94 (t, 2H, J ¼ 8.1 Hz), 2.17e1.99 (m, 2H), 1.46 (s,

4.1.10. N-(1-(2-(Pyrrolidin-1-yl)acetyl)indolin-5-yl)thiophene-2-

carboximidamide (17)

9H); ESI-MS (m/z, %): 368 (MH^þ, 3), 313, 311 (100).

Prepared from 1-(5-nitroindolin-1-yl)-2-(pyrrolidin-1-yl)etha-

none (13) (1.17 g, 4.26 mmol) as described for 10 to obtain the title

4.1.15. 5-Bromo-1-(pyrrolidin-3-yl)indoline (29)

Prepared from tert-butyl 3-(5-bromoindolin-1-yl)pyrrolidine-1-

carboxylate (28) (3.0 g, 8.16 mmol) as described for 19 to obtain the

title compound (1.75 g, 80%) as a syrup. ¹H NMR (DMSO-d₆)

compound (0.19 g, 13%) as a foam. ¹H NMR (DMSO-d₆)

d 7.99 (d, 1H,

J ¼ 8.4 Hz), 7.71 (d, 1H, J ¼ 3.3 Hz), 7.59 (d, 1H, J ¼ 5.1 Hz), 7.08 (dd,

1H, J ¼ 3.9, 4.9 Hz), 6.73 (s, 1H), 6.62 (d, 1H, J ¼ 8.4 Hz), 6.37 (brs,

2H), 4.15 (t, 2H, J ¼ 8.1 Hz), 3.37 (s, 2H), 3.10 (t, 2H, J ¼ 8.1 Hz), 2.57

(brs, 4H), 1.71 (brs, 4H); EI-MS (m/z, %): 354 (M^þ, 14), 245 (43), 176

d

7.12e7.06 (m, 2H), 6.45 (d, 1H, J ¼ 8.1 Hz), 4.00e3.92 (m, 1H), 3.34

(t, 2H, J ¼ 8.4 Hz), 2.96e2.66 (m, 7H), 1.92e1.80 (m, 1H), 1.69e1.60

(m, 1H); ESI-MS (m/z, %): 267, 269 (MH^þ, 100).

102

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

4.1.16. 5-Bromo-1-(1-methylpyrrolidin-3-yl)indoline (30)

a syrup. ¹H NMR (DMSO-d₆)

d 7.14e7.06 (m, 2H), 6.37 (d, 1H,

Prepared from 5-bromo-1-(pyrrolidin-3-yl)indoline (29) (1.0 g,

3.74 mmol) and formaldehyde (0.36 g, 4.49 mmol, 37% in water) as

described for 27 to obtain the title compound (0.7 g, 67%) as

J ¼ 8.4 Hz), 3.35e3.24 (m, 3H, merged with water peak), 2.93e2.80

(m, 4H), 2.16 (s, 3H), 2.00e1.88 (m, 2H), 1.64e1.55 (m, 4H); EI-MS

(m/z, %): 293, 295 (M^þ, 47), 98 (59), 97 (100), 71 (47).

a syrup. ¹H NMR (CDCl₃)

d

7.12e7.08 (m, 2H), 6.35 (d,1H, J ¼ 9.0 Hz),

4.19e4.10 (m, 1H), 3.45e3.39 (m, 2H), 2.91 (t, 2H, J ¼ 8.1 Hz),

2.70e2.63 (m, 3H), 2.51e2.43 (m, 1H), 2.34 (s, 3H), 2.19e2.07 (m,

1H), 1.93e1.81 (m, 1H); ESI-MS (m/z, %): 281, 283 (MH^þ, 100).

4.1.23. tert-Butyl 4-(5-bromoindolin-1-yl)-2-methylpiperidine-1-

carboxylate (37)

Prepared from 5-bromoindoline (21) (0.464 g, 2.34 mmol) and

tert-butyl 2-methyl-4-oxopiperidine-1-carboxylate (25) (0.50 g,

2.34 mmol) as described for 27 to obtain the title compound (0.5 g,

54%) as a 7:3 mixture (cis: trans) of diastereomers. ¹H NMR (CDCl₃)

4.1.17. tert-Butyl 4-(indolin-1-yl)piperidine-1-carboxylate (31)

Prepared from indoline (20) (2.0 g, 16.78 mmol) and tert-butyl

4-oxopiperidine-1-carboxylate (23) (3.68 g, 18.46 mmol) as

described for 27 to obtain the title compound (5.0 g, 99%) as

d

7.16e7.08 (m, 2H), 6.29 (d, 0.7 H, J ¼ 8.7 Hz), 6.25 (d, 0.3H,

J ¼ 8.8 Hz), 4.71e4.45 (brs, 0.3H), 3.96e3.86 (m, 0.7H), 3.82e3.74

(m, 1H), 3.70e3.53 (m, 1H), 3.38e3.30 (m, 2H), 3.24e3.14 (m,

0.7H) 2.92 (m þ t, 2.3H, J ¼ 8.3 Hz), 2.10e1.98 (m, 1H), 1.87e1.54 (m,

3H), 1.48e1.47 (2s, 9H), 1.23 (d, 3H, J ¼ 6.4 Hz); ESI-MS (m/z, %):

395/393 (MH^þ, 7), 339/341 (100).

a syrup. ¹H NMR (CDCl₃)

d

7.03 (t, 2H, J ¼ 7.8 Hz), 6.59 (t, 1H,

J ¼ 7.2 Hz), 6.40 (d,1H, J ¼ 7.5 Hz), 4.25e4.20 (m, 2H), 3.55e3.43 (m,

1H), 3.33 (t, 2H, J ¼ 8.4 Hz), 2.93 (t, 2H, J ¼ 8.4 Hz), 2.76 (t, 2H,

J ¼ 12.3 Hz), 1.80e1.74 (m, 2H), 1.63e1.51 (m, 2H), 1.45 (s, 9H);

ESI-MS (m/z, %) 303 (MH^þ, 5), 247 (100).

4.1.24. tert-Butyl 3-(5-nitroindolin-1-yl)piperidine-1-carboxylate

(38)

4.1.18. tert-Butyl 4-(5-bromoindolin-1-yl)piperidine-1-carboxylate (32)

Prepared from tert-butyl 4-(indolin-1-yl)piperidine-1-carboxylate

(31) (2.0 g, 6.61 mmol) and N-bromosuccinimide (1.17 g, 6.61 mmol)

asdescribed for 28 to obtainthe titlecompound (2.5 g, 99%)asa syrup.

Prepared from 5-nitroindoline (6) (0.5 g, 3.05 mmol) and (26) as

described for 27 to obtain the title compound (0.585 g, 55%) as

a solid. ¹H NMR (CDCl₃)

6.35 (d, 1H, J ¼ 8.7 Hz), 4.15e4.07 (m, 2H), 3.76e3.66 (m, 2H),

3.52e3.44 (m, 1H), 3.06 (t, 2H, J ¼ 8.7 Hz), 2.77e2.65 (m, 2H),

2.04e1.45 (m, 13H); EIS-MS (m/z, %): 370 (MNa^þ, 100), 292 (100).

d

8.05 (dd, 1H, J ¼ 2.1, 8.8 Hz), 7.88 (s, 1H),

¹H NMR (CDCl₃)

d

7.13e7.10 (m, 2H), 6.27 (d,1H, J ¼ 9.0 Hz), 4.28e4.20

(m, 2H), 3.50e3.40 (m, 1H), 3.35 (t, 2H, J ¼ 8.4 Hz), 2.93 (t, 2H,

J ¼ 8.4 Hz), 2.75 (t, 2H, J ¼ 12.9 Hz), 1.78e1.72 (m, 2H), 1.60e1.49 (m,

2H), 1.46 (s, 9H); ESI-MS (m/z, %) 381, 383 (MH^þ, 3), 325, 327 (100).

4.1.25. tert-Butyl 3-(5-(thiophene-2-carboximidamido)indolin-1-

yl)pyrrolidine-1-carboxylate (39)

4.1.19. 5-Bromo-1-(piperidin-4-yl)indoline (33)

Prepared from tert-butyl 4-(5-bromoindolin-1-yl)piperidine-1-

carboxylate (32) (1.8 g, 4.72 mmol) as described for 19 to obtain

the title compound (1.3 g, 98%) as a syrup. ¹H NMR (CDCl₃)

A suspension of Pd₂(dba)₃(0.124 g, 0.13 mmol) and P^tBu₃

(1.65 mL, 0.54 mmol, 10% wt in hexane) in anhydrous THF (5 mL)

was treated with a solution of tert-butyl 3-(5-bromoindolin-1-yl)

pyrrolidine-1-carboxylate (28) (1.0 g, 2.72 mmol) in THF (15 mL)

followed by LiHMDS (5.44 mL, 5.44 mmol, 1 M solution in THF) at

room temperature. The resulting dark brown mixture was heated

to 100 ^ꢃC and stirred for 3 h in a sealed tube. The reaction was

cooled to room temperature and treated with TBAF (5 mL, 1 M

solution in THF) and stirred for 20 min. The reaction was diluted

with water and product was extracted into ether. The combined

organic layer was dried (Na₂SO₄) and concentrated to give a dark

brown residue. The crude product was puriﬁed by column chro-

matography (2 M NH₃in MeOH:CH₂Cl₂, 3:97) to obtain the inter-

mediate amine, tert-butyl 3-(5-aminoindolin-1-yl)pyrrolidine-1-

carboxylate (0.68 g, 82%) as a foam. ESI-MS (m/z, %): 304 (MH^þ,

16), 248 (100).

d

7.12e7.09 (m, 2H), 6.26 (d, 1H, H, J ¼ 8.7 Hz), 3.47e3.34 (m, 3H),

3.22e3.20 (m, 2H), 2.92 (t, 2H, J ¼ 8.4 Hz), 2.73e2.65 (m, 2H), 2.13

(s, 1H), 1.80e1.76 (m, 2H), 1.65e1.52 (m, 2H); ESI-MS (m/z, %): 283,

281 (MH^þ, 100).

4.1.20. 2-(4-(5-Bromoindolin-1-yl)piperidin-1-yl)ethanol (34)

A solution of 5-bromo-1-(piperidin-4-yl)indoline (33) (1.3 g,

4.62 mmol) in dry dioxane (20 mL) was treated with triethylamine

(3.25 mL, 23.12 mmol) followed by 2-bromoethanol (0.65 mL,

9.25 mmol) at room temperature and the resulting mixture was

reﬂuxed for 4 h. The reaction was brought to room temperature,

diluted with 1 N NaOH solution and product was extracted into

CH₂Cl₂. The combined CH₂Cl₂layer was washed with brine and

dried (Na₂SO₄). Solvent was evaporated and crude was puriﬁed by

column chromatography (2 M NH₃in MeOH:CH₂Cl₂, 5:95) to obtain

the title compound (1.42 g, 94%) as a solid. ¹H NMR (DMSO-d₆)

Prepared from the above amine intermediate tert-butyl 3-(5-

aminoindolin-1-yl)pyrrolidine-1-carboxylate (0.65 g, 2.142 mmol)

and methyl thiophene-2-carbimidothioate hydroiodide (9) (1.22 g,

4.28 mmol) as described for 10 to obtain the title compound (0.68 g,

d

7.10e7.06 (m, 2H), 6.37 (d, 1H, J ¼ 8.4 Hz), 4.37 (t, 1H, J ¼ 5.1 Hz),

3.50e3.44 (m, 2H), 3.35e3.30 (m, 3H, merged with water peak),

2.95e2.83 (m, 4H), 2.38 (t, 2H, J ¼ 6.3 Hz), 2.08e2.00 (m, 2H),

1.61e1.54 (m, 4H); ESI-MS (m/z, %): 325, 327 (MH^þ, 100).

77%) as a foam. ¹H NMR (DMSO-d₆)

d 7.42e7.36 (m, 2H), 7.06 (dd,

1H, J ¼ 3.6, 4.9 Hz), 6.79 (brs, 1H), 6.71 (t, 1H, J ¼ 5.1 Hz), 6.48 (d, 1H,

J ¼ 8.1 Hz), 4.89 (brs, 2H), 4.12e4.02 (m, 1H), 3.68e3.35 (m, 6H),

2.93 (t, 2H, J ¼ 8.1 Hz), 2.14e2.04 (m, 2H), 1.46 (s, 9H); ESI-MS (m/z,

%): 413 (MH^þ, 100).

4.1.21. 1-(1-Methylpiperidin-4-yl)indoline (35)

Prepared from indoline (20) (1.0 g, 8.39 mmol) and N-methyl-4-

piperidone (24) (1.23 mL, 10.06 mmol) as described for 27 to obtain

the title compound (1.15 g, 63.5%) as a syrup. ¹H NMR (DMSO-d₆)

4.1.26. N-(1-(Pyrrolidin-3-yl)indolin-5-yl)thiophene-2-carboximida-

mide (40)

d

7.06e7.01 (m, 2H), 6.59 (t, 1H, J ¼ 6.6 Hz), 6.40 (d, 1H, J ¼ 7.8 Hz),

Prepared from tert-butyl 3-(5-(thiophene-2-carboximidamido)

indolin-1-yl)pyrrolidine-1-carboxylate (39) (0.35 g, 0.84 mmol) as

described for 19 to obtain the title compound (0.3 g, 92%) as

3.40e3.34 (m, 3H), 2.97e2.91 (m, 4H), 2.30 (s, 3H), 2.09e2.00 (m,

2H), 1.80e1.72 (m, 4H); ESI-MS (m/z, %): 217 (MH^þ, 42). 98 (100).

a solid. ¹H NMR (DMSO-d₆)

d 11.25 (s, 1H), 9.82e9.60 (m, 3H), 8.63

4.1.22. 5-Bromo-1-(1-methylpiperidin-4-yl)indoline (36)

(s, 1H), 8.15e8.13 (m, 2H), 7.36 (t, 1H, J ¼ 4.5 Hz), 7.12e7.06 (m,

2H), 6.68 (d, 1H, J ¼ 8.4 Hz), 4.39e4.35 (m, 1H), 3.56e3.06 (m,

6H), 2.96 (t, 2H, J ¼ 8.1 Hz), 2.18e2.00 (m, 2H); ESI-MS (m/z, %):

313 (MH^þ, 100), 244 (61); ESI-HRMS calculated for C₁₇H₂₁N₄S

Prepared from 1-(1-methylpiperidin-4-yl)indoline (35) (1.12 g,

5.17 mmol) and N-bromosuccinimide (0.92 g, 5.176 mmol) as

described for 28 to obtain the title compound (1.15 g, 76%) as

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

103

(MH^þ): 313.1481; observed: 313.1473; HPLC purity: 94.05% by

area.

J ¼ 3.6 Hz), 7.54 (d,1H, J ¼ 5.1 Hz), 7.06 (t,1H, J ¼ 3.9 Hz), 6.61 (s,1H),

6.51 (d, 1H, J ¼ 8.1 Hz), 6.39 (d, 1H, J ¼ 8.1 Hz), 6.24 (brs, 2H),

3.30e3.24 (m, 3H), 2.86e2.80 (m, 4H), 2.16 (s, 3H), 2.00e1.92 (m,

2H), 1.66e1.56 (m, 4H); ESI-MS (m/z, %): 341 (MH^þ, 100), 244 (87);

ESI-HRMS calculated for C₁₉H₂₅N₄S (MH^þ): 341.1794; observed:

4341.1805.

4.1.27. N-(1-(1-Methylpyrrolidin-3-yl)indolin-5-yl)thiophene-2-

carboximidamide (41)

Prepared from 5-bromo-1-(1-methylpyrrolidin-3-yl)indoline

(30) (0.64 g, 2.276 mmol) as described for 39 and 10 to obtain the

title compound (0.43 g, 72%) as a solid. ¹H NMR (DMSO-d₆)

d

7.68

4.1.32. tert-Butyl 2-methyl-4-(5-(thiophene-2-carboximidamido)

indolin-1-yl)piperidine-1-carboxylate (46)

Prepared from tert-butyl 4-(5-bromoindolin-1-yl)-2-methy-

lpiperidine-1-carboxylate (37) (0.25 g, 0.63 mmol) as described for

39 and 10 to obtain the title compound (96 mg, 64.5%) (7:3 mixture of

(d, 1H, J ¼ 3.0 Hz), 7.58 (dd, 1H, J ¼ 1.2, 5.1 Hz), 7.07 (dd, 1H,

J ¼ 3.6, 4.9 Hz), 6.62 (s, 1H), 6.52e6.48 (m, 2H), 6.32 (brs, 2H),

4.16e4.08 (m, 1H), 3.32e3.24 (m, 2H), 2.81 (t, 2H, J ¼ 8.1 Hz),

2.63e2.55 (m, 3H), 2.38e2.31 (m, 1H), 2.24 (s, 3H), 2.09e1.97 (m,

1H), 1.82e1.71 (m, 1H); ESI-MS (m/z, %): 327 (MH^þ, 100), 244 (83);

ESI-HRMS calculated for C₁₈H₂₃N₄S (MH^þ): 327.1637; observed:

327.1650.

diastereomers) as a solid.¹H NMR (DMSO-d₆)

d

7.71 (d, 1H, J ¼ 3.3 Hz),

7.59 (d, 1H, J ¼ 4.9 Hz), 7.11e7.08 (m, 1H), 6.69 (s, 1H), 6.68e6.22 (m,

4H), 4.51e4.34 (m, 0.4H), 3.99e3.87 (m, 0.4H), 3.83e3.70 (m, 1H),

3.62e3.49 (m, 1H), 3.35e3.17 (m, 3H), 2.86e2.81 (m, 2H), 1.94e1.57

(m, 4H), 1.41, 1.40 (2s, 9H), 1.19 (d, 3H, J ¼ 6.3 Hz); EI-MS (m/z, %):

440 (M^þ, 20), 340 (100).

4.1.28. tert-Butyl 4-(5-(thiophene-2-carboximidamido)indolin-1-yl)

piperidine-1-carboxylate (42)

Prepared from tert-butyl 4-(5-bromoindolin-1-yl)piperidine-1-

carboxylate (32) (0.67 g, 1.757 mmol) as described for 39 and 10

to obtain the title compound (0.43 g, 85%) as a solid. ¹H NMR

4.1.33. N-(1-(2-Methylpiperidin-4-yl)indolin-5-yl)thiophene-2-

carboximidamide (cis-47 and trans-47)

(DMSO-d₆)

d

7.72 (d, 1H, J ¼ 3.0 Hz), 7.62 (d, 1H, J ¼ 4.8 Hz), 7.10 (dd,

Prepared from tert-butyl 2-methyl-4-(5-(thiophene-2-carboxi-

midamido)indolin-1-yl)piperidine-1-carboxylate (46) (0.140 g,

0.31 mmol) as described for 19 to obtain the title compounds (90 mg,

83%) as a separable mixture of diastereomers (cis:trans, 7:3). cis-47:

1H, J ¼ 3.6, 4.9 Hz), 6.68 (s, 1H), 6.59 (d, 1H, J ¼ 8.1 Hz), 6.47 (d, 1H,

J ¼ 8.4 Hz), 4.05 (d, 2H, J ¼ 12.3 Hz), 3.58e3.50 (m, 1H), 3.28e3.24

(m, 2H), 2.90e2.78 (m, 4H), 1.68 (d, 2H, J ¼ 11.1 Hz), 1.50e1.36 (m,

11H); ESI-MS (m/z, %) 427 (MH^þ, 100).

solid; ¹H NMR (DMSO-d₆)

d

7.67 (d, 1H, J ¼ 3.6 Hz), 7.55 (d, 1H,

J¼ 5.0 Hz), 7.08e7.05 (m,1H), 6.61 (s,1H), 6.53e6.50 (m,1H), 6.41e6.38

(m,1H), 6.20 (brs, 2H), 3.46e3.32 (m,1H), 3.31e3.21 (m, 2H), 3.04e2.95

(m, 1H), 2.83 (t, 2H, J ¼ 8.1 Hz), 2.66e2.56 (m, 2H), 2.20e1.86 (m, 1H),

1.69e1.55 (m, 2H), 1.39 (dq, 1H, J ¼ 11.9, 4.0 Hz),1.09 (q,1H, J ¼ 11.6 Hz),

1.00 (d, 3H, J ¼ 6.2 Hz); ESI-MS (m/z, %): 341 (MH^þ, 100), 244 (95); ESI-

HRMS calculated for C₁₉H₂₅N₄S (MH^þ): 341.1794; observed: 341.1802;

4.1.29. N-(1-(Piperidin-4-yl)indolin-5-yl)thiophene-2-carboximidamide

(43)

A solution of tert-butyl 4-(5-(thiophene-2-carboximidamido)

indolin-1-yl)piperidine-1-carboxylate (42) (0.23 g, 0.53 mmol) in

methanol (10 mL) was treated with 1 N HCl solution (10 mL) and

the resulting mixture was reﬂuxed for 30 min. The reaction was

brought to room temperature and solvent was evaporated. The

crude was dissolved into water (10 mL), ﬁltered and washed with

water (2 ꢄ 5 mL). The combined water layer was evaporated to

obtain the dihydrochloride salt of the title compound (0.18 g,

HPLC purity: 94.65% by area. trans-47: solid; ¹H NMR (DMSO-d₆)

d 7.67

(d,1H, J ¼ 3.5 Hz), 7.55 (d,1H, J ¼ 5.1 Hz), 7.08e7.05 (m,1H), 6.62 (s,1H),

6.53e6.50 (m,1H), 6.42e6.38 (m,1H), 6.21 (brs, 2H), 3.65e3.55 (m,1H),

3.31e3.21 (m, 4H), 2.89e2.75 (m, 4H), 1.77e1.40 (m, 4H), 1.14 (d, 3H,

J ¼ 6.8 Hz); ESI-MS (m/z, %): 341 (MH^þ, 100), 244 (75); ESI-HRMS

calculated for C₁₉H₂₅N₄S (MH^þ): 341.1794; observed: 341.1801; HPLC

purity: 91.91% by area.

84%) as a solid. ¹H NMR (DMSO-d₆)

d 11.21 (s, 1H), 9.64 (s, 1H),

9.20e9.04 (m, 2H), 8.60 (s, 1H), 8.15e8.12 (m, 2H), 7.36 (t, 1H,

J ¼ 4.5 Hz), 7.11e7.03 (m, 2H), 6.66 (d, 1H, J ¼ 8.4 Hz), 3.86e3.74

(m, 1H), 3.44e3.32 (m, 4H), 3.06e2.92 (m, 4H), 1.98e1.78 (m,

4.1.34. N-(1-(2-Methylpiperidin-4-yl)indolin-5-yl)thiophene-2-

carboximidamide [(ꢀ)-cis-(47) and (þ)-cis-(47)]

4H); ¹³C NMR (DMSO-d₆)

d 156.37, 149.67, 134.37, 131.61, 129.14,

128.47, 125.19, 124.28, 122.26, 107.90, 55.04, 50.22, 46.70, 42.65,

27.62, 24.00; ESI-MS (m/z, %) 327 (MH^þ, 100), 244 (81); ESI-

HRMS calculated for C₁₈H₂₃N₄S (MH^þ): 327.1637; observed:

327.1636.

Separation of compounds (ꢀ)-cis-(47) and (þ)-cis-(47) from

cis-47 was achieved by preparative HPLC techniques using Chir-

acel OJ (21 ꢄ 250 mm) column eluting with ethanol:hexane (0.1%

diethylamine), 18: 82, ﬂow rate: 15 mL/min, wavelength: 254 nm,

injection volume: 100

m

L of 50 mg/mL (MeOH) solution. (ꢀ)-cis-

4.1.30. N-(1-(1-(2-Hydroxyethyl)piperidin-4-yl)indolin-5-yl)

thiophene-2-carboximidamide (44)

(47): solid; ½a ²_D⁸

ꢅ

¼ ꢀ19.6 (c ¼ 0.1, MeOH); ¹H NMR (DMSO-d₆)

d

7.67 (dd, 1H, J ¼ 3.6, 0.8 Hz), 7.55 (d, 1H, J ¼ 5.0 Hz), 7.08e7.05

Prepared

from

2-(4-(5-bromoindolin-1-yl)piperidin-1-yl)

(m, 1H), 6.61 (s, 1H), 6.53e6.50 (m, 1H), 6.41e6.38 (m, 1H), 6.23

(brs, 2H), 3.46e3.32 (m, 1H), 3.31e3.21 (m, 2H), 3.04e2.94 (m,

1H), 2.83 (t, 2H, J ¼ 8.1 Hz), 2.66e2.56 (m, 2H), 2.20e1.86 (m, 1H),

1.69e1.54 (m, 2H), 1.39 (dq, 1H, J ¼ 12.0, 4.0 Hz), 1.09 (q, 1H,

J ¼ 11.3 Hz), 1.00 (d, 3H, J ¼ 6.2 Hz); ESI-MS (m/z, %): 341 (MH^þ,

100), 244 (95); ESI-HRMS calculated for C₁₉H₂₅N₄S (MH^þ):

ethanol (34) (1.15 g, 3.54 mmol) as described for 39 and 10 to obtain

the title compound (0.85 g, 75%) as a foam. ¹H NMR (DMSO-d₆)

d

7.67 (d, 1H, J ¼ 3.6 Hz), 7.54 (d, 1H, J ¼ 4.8 Hz), 7.06 (dd, 1H, J ¼ 3.9,

4.9 Hz), 6.61 (s, 1H), 6.51 (d, 1H, J ¼ 8.1 Hz), 6.39 (d, 1H, J ¼ 8.1 Hz),

6.22 (s, 2H), 4.37 (t,1H, J ¼ 5.1 Hz), 3.51e3.45 (m, 2H), 3.33e3.25 (m,

3H, merged with water peak), 2.96e2.92 (m, 2H), 2.83 (t, 2H,

J ¼ 8.4 Hz), 2.38 (t, 2H, J ¼ 6.3 Hz), 2.09e2.00 (m, 2H), 1.66e1.56 (m,

4H); ESI-MS (m/z, %): 371 (MH^þ, 100); ESI-HRMS calculated for

C₂₀H₂₇N₄OS (MH^þ): 371.1900, observed: 371.1917.

341.1794; observed: 341.1805. (þ)-cis-(47): solid; ½a _D²⁸

¼ þ18.6

ꢅ

(c ¼ 0.1, MeOH); ¹H NMR (DMSO-d₆)

d

7.67 (dd, 1H, J ¼ 3.6,

0.9 Hz), 7.55 (d, 1H, J ¼ 5.0 Hz), 7.08e7.05 (m, 1H), 6.61 (s, 1H),

6.53e6.50 (m, 1H), 6.41e6.38 (m, 1H), 6.23 (brs, 2H), 3.46e3.32

(m, 1H), 3.31e3.21 (m, 2H), 3.04e2.95 (m, 1H), 2.83 (t, 2H,

J ¼ 8.1 Hz), 2.66e2.56 (m, 2H), 2.20e1.86 (m, 1H), 1.69e1.55 (m,

2H), 1.39 (dq, 1H, J ¼ 11.9, 4.1 Hz), 1.09 (q, 1H, J ¼ 11.3 Hz), 1.00 (d,

3H, J ¼ 6.2 Hz); ESI-MS (m/z, %): 341 (MH^þ, 97), 244 (100);

ESI-HRMS calculated for C₁₉H₂₅N₄S (MH^þ): 341.1794; observed:

341.1811.

4.1.31. N-(1-(1-Methylpiperidin-4-yl)indolin-5-yl)thiophene-2-

carboximidamide (45)

Prepared from 5-bromo-1-(1-methylpiperidin-4-yl)indoline

(36) (0.5 g, 1.69 mmol) as described for 39 and 10 to obtain the title

compound (0.1 g, 57%) as a solid. ¹H NMR (DMSO-d₆)

d 7.67 (d, 1H,

104

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

4.1.35. tert-Butyl 3-(5-(thiophene-2-carboximidamido)indolin-1-yl)

piperidine-1-carboxylate (48)

Prepared from tert-butyl 3-(5-nitroindolin-1-yl)piperidine-1-

carboxylate (38) (0.55 g, 1.58 mmol) as described for 10 to obtain

the title compound (0.51 g, 79%) as a solid. ¹H NMR (DMSO-d₆)

(m, 3H), 2.85 (t, 2H, J ¼ 8.4 Hz), 2.30e2.16 (m, 5H), 1.95e1.91 (m,

2H), 1.69e1.65 (m, 2H), 1.45e1.32 (m, 2H), 1.32e1.02 (m, 2H);

ESI-MS (m/z, %): 311, 309 (MH^þ, 8), 280 (93), 278 (100).

4.1.41. tert-Butyl 4-(5-bromoindolin-1-yl)cyclohexyl(methyl)

carbamate (cis-55)

d

7.68 (d, 1H, J ¼ 3.6 Hz), 7.55 (d, 1H, J ¼ 4.2 Hz), 7.07 (t, 1H,

J ¼ 3.6 Hz), 6.64 (s, 1H), 6.53 (d, 1H, J ¼ 8.1 Hz), 6.45 (d, 1H,

J ¼ 8.1 Hz), 6.26 (brs, 2H), 4.00e3.86 (m, 2H), 3.78e3.56 (m, 1H),

3.39e3.22 (m, 2H), 2.84 (t, 2H, J ¼ 8.4 Hz), 2.76e2.60 (m, 2H),

1.90e1.23 (m, 13H); ESI-MS (m/z, %): 427 (MH^þ, 100).

Prepared from 4-(5-bromoindolin-1-yl)-N-methylcyclohexan-

amine (cis-54) (0.28 g, 0.90 mmol) as described for 15 to obtain the

title compound (0.35 g, 94%) as a syrup. ¹H NMR (CDCl₃)

d 7.14e7.10

(m, 2H), 6.32 (d, 1H, J ¼ 8.1 Hz), 4.02e3.98 (m, 1H), 3.70 (s, 2H), 3.56

(t, 2H, J ¼ 8.1 Hz), 3.32e3.30 (m, 1H), 2.94 (t, 2H, J ¼ 8.1 Hz),

2.79e2.74 (m, 3H), 2.15e2.10 (m, 2H), 1.81e1.67 (m, 4H), 1.46

(s, 9H); ESI-MS (m/z, %): 411 (61), 409 (M^þ, 58), 331 (100).

4.1.36. N-(1-(Piperidin-3-yl)indolin-5-yl)thiophene-2-

carboximidamide (49)

Prepared from tert-butyl 3-(5-(thiophene-2-carboximidamido)

indolin-1-yl)piperidine-1-carboxylate (48) (0.46 g, 1.078 mmol) as

described for 19 to obtain the title compound (0.22 g, 59%) as

4.1.42. tert-Butyl 4-(5-bromoindolin-1-yl)cyclohexyl(methyl)

carbamate (trans-55)

a solid. ¹H NMR (DMSO-d₆)

d

7.67 (d, 1H, J ¼ 3.0 Hz), 7.54 (dd, 1H,

Prepared from 4-(5-bromoindolin-1-yl)-N-methylcyclohexan-

amine (trans-54) (0.35 g, 1.14 mmol) as described for 15 to obtain

J ¼ 0.9, 5.1 Hz), 7.06 (dd, 1H, J ¼ 3.9, 5.1 Hz), 6.60 (s, 1H), 6.52 (d, 1H,

J ¼ 8.1 Hz), 6.39 (d, 1H, J ¼ 8.1 Hz), 6.20 (brs, 2H), 3.35e3.17 (m, 3H),

2.97e2.79 (m, 4H), 2.53e2.32 (m, 3H), 1.84e1.64 (m, 2H), 1.52e1.44

(m, 2H); ESI-MS (m/z, %): 327 (MH^þ, 80), 244 (100); ESI-HRMS

calculated for C₁₈H₂₃N₄S (MH^þ): 327.1637, observed: 327.1628;

HPLC purity: 94.38% by area.

the title compound (0.43 g, 92%) as a syrup. ¹H NMR (CDCl₃)

d 7.12

(brs, 2H), 6.27 (d, 1H, J ¼ 7.8 Hz), 3.96e3.92 (m, 1H), 3.70 (s, 2H),

3.37 (t, 2H, J ¼ 8.4 Hz), 3.32e3.23 (m, 1H), 2.93 (t, 2H, J ¼ 8.4 Hz),

2.80e2.74 (m, 4H), 1.91e1.73 (m, 5H), 1.52 (s, 9H); ESI-MS (m/z, %):

411 (MH^þ, 14), 331 (100), 156 (85).

4.1.37. 1-(1,4-Dioxaspiro[4.5]decan-8-yl)indoline (51)

4.1.43. tert-Butylmethyl(4-(5-(thiophene-2-carboximidamido)

indolin-1-yl)cyclohexyl)carbamate (cis-56)

Prepared from tert-butyl 4-(5-bromoindolin-1-yl)cyclo-

hexyl(methyl)carbamate (cis-55) (0.36 g, 0.87 mmol) as described

for 39 and 10 to obtain the title compound (0.25 g, 67%). ¹H NMR

Prepared from indoline (20) (2.0 g, 16.78 mmol) and 1,4-

dioxaspiro[4.5]decan-8-one (50) (3.15 g, 20.139 mmol) to obtain

the title compound (3.52 g, 81%) as a solid. ¹H NMR (CDCl₃)

d

7.06e7.02 (m, 2H), 6.62e6.57 (m, 1H), 6.42 (d, 1H, J ¼ 8.1 Hz), 3.46

(s, 4H), 3.46e3.35 (m, 3H), 2.93 (t, 2H, J ¼ 8.2 Hz), 1.87e1.60 (m,

(DMSO-d₆)

d

7.68 (d, 1H, J ¼ 3.6 Hz), 7.56 (d, 1H, J ¼ 5.1 Hz), 7.07 (dd,

8H); ESI-MS (m/z, %): 260 (MH^þ, 100), 120 (28).

1H, J ¼ 3.9, 4.8 Hz), 6.63 (s, 1H), 6.51 (d, 1H, J ¼ 7.5 Hz), 6. 42 (d, 1H,

J ¼ 8.4 Hz), 6.28e6.27 (m, 2H), 3.88e3.81 (m, 1H), 3.45 (t, 2H,

J ¼ 7.9 Hz), 3.26e3.24 (m, 1H), 2.84 (t, 2H, J ¼ 7.8 Hz), 2.72 (s, 3H),

2.10e2.06 (m, 2H), 1.81e1.74 (m, 2H), 1.66e1.57 (m, 2H), 1.51e1.46

(m, 2H), 1.40 (s, 9H); ESI-MS (m/z, %): 455 (MH^þ, 100).

4.1.38. 5-Bromo-1-(1,4-dioxaspiro[4.5]decan-8-yl)indoline (52)

Prepared from 1-(1,4-dioxaspiro[4.5]decan-8-yl)indoline (51)

(3.45 g, 13.30 mmol) as described for 27 to obtain the title

compound (4.05 g, 90%) as a syrup. ¹H NMR (CDCl₃)

d 7.12e7.10 (m,

2H), 6.27e6.24 (m, 1H), 3.95 (s, 4H), 3.38 (t, 3H, J ¼ 8.4 Hz), 2.91 (t,

2H, J ¼ 8.4 Hz), 1.86e1.57 (m, 8H); ESI-MS (m/z, %): 340, 338 (MH^þ,

100), 198 (18).

4.1.44. tert-Butylmethyl(4-(5-(thiophene-2-carboximidamido)

indolin-1-yl)cyclohexyl)carbamate (trans-56)

Prepared from tert-butyl 4-(5-bromoindolin-1-yl)cyclohexyl

(methyl)carbamate (trans-55) (0.42 g,1.02 mmol) as described for 39

and10 to obtain the title compound (0.37 g, 86%).¹H NMR (DMSO-d₆)

4.1.39. 4-(5-Bromoindolin-1-yl)cyclohexanone (53)

A solution of 5-bromo-1-(1,4-dioxaspiro[4.5]decan-8-yl)indo-

line (52) (4.0 g, 11.83 mmol) in acetone (50 mL) was treated with

10% HCl solution (50 mL) and the resulting mixture was stirred for

overnight (16 h). Acetone was evaporated, crude was basiﬁed with

2 N NaOH solution and product was extracted into CH₂Cl₂. The

combined CH₂Cl₂layer was washed with brine and dried (Na₂SO₄).

Solvent was evaporated and crude was puriﬁed by column chro-

matography (EtOAc:hexanes, 1:4) to obtain the title compound

d

7.68 (d, 1H, J ¼ 3.0 Hz), 7.56 (d, 1H, J ¼ 5.1 Hz), 7.07 (dd, 1H, J ¼ 3.9,

5.1 Hz), 6.62 (s, 1H), 6.54 (d, 1H, J ¼ 8.4 Hz), 6.41 (d, 1H, J ¼ 8.4 Hz),

6.32e6.28 (m, 2H), 3.81e3.77 (m,1H), 3.28e3.25 (m, 3H), 2.83 (t, 2H,

J ¼ 8.1 Hz), 2.68 (s, 3H), 1.78 (d, 2H, J ¼ 10.8 Hz), 1.64e1.62 (m, 4H),

1.50e1.46 (m, 2H), 1.40 (s, 9H); ESI-MS (m/z, %): 455 (MH^þ, 100).

4.1.45. N-(1-(4-(Methylamino)cyclohexyl)indolin-5-yl)thiophene-

2-carboximidamide (cis-57)

(2.9 g, 83%) as a solid. ¹H NMR (CDCl₃)

d

7.16e7.14 (m, 2H), 6.32 (d,

Prepared from tert-butylmethyl(4-(5-(thiophene-2-carboximid-

amido)indolin-1-yl)cyclohexyl)carbamate (cis-56) (0.23 g, 0.50 -

mmol) as described for 19 to obtain the title compound (0.145 g, 81%).

1H, J ¼ 8.7 Hz), 3.81 (tt, 1H, J ¼ 3.6, 11.7 Hz), 3.37 (t, 2H, J ¼ 8.4 Hz),

2.95 (t, 2H, J ¼ 8.2 Hz), 2.51e2.41 (m, 4H), 2.18e2.11 (m, 2H),

1.92e1.78 (m, 2H); ESI-MS (m/z, %): 296, 294 (MH^þ, 100), 200 (30).

¹H NMR (DMSO-d₆)

d

7.67 (d, 1H, J ¼ 3.0 Hz), 7.54 (dd, 1H, J ¼ 0.9,

5.1 Hz), 7.06 (dd,1H,J ¼ 3.9, 4.9 Hz), 6.60 (s,1H), 6.51 (d,1H, J ¼ 8.1 Hz),

6.38 (d, 1H, J ¼ 8.1 Hz), 6.21 (brs, 2H), 3.32e3.25 (m, 4H), 2.82 (t, 2H,

J¼8.4Hz), 2.62(brs,1H), 2.25(s, 3H),1.81e1.66(m, 4H),1.53e1.39(m,

4H); ESI-MS (m/z, %): 355 (MH^þ, 82), 324 (100); ESI-HRMS calculated

for C₂₀H₂₇N₄S (MH^þ): 355.1949, observed: 355.1950; HPLC purity:

91.42% by area.

4.1.40. 4-(5-Bromoindolin-1-yl)-N-methylcyclohexanamine (cis-54

and trans-54)

Prepared from 4-(5-bromoindolin-1-yl)cyclohexanone (53)

(0.5 g, 1.70 mmol) and methylamine hydrochloride (0.11 g,

1.70 mmol) as described for 27 to obtain the title compounds cis-54

and trans-54 (0.48 g, 91%) as partially separable mixture of dia-

stereomers (1:1.2). Cis-54: ¹H NMR (DMSO-d₆)

d

7.09e7.04 (m, 2H),

4.1.46. N-(1-(4-(Methylamino)cyclohexyl)indolin-5-yl)thiophene-

2-carboximidamide (trans-57)

Prepared from tert-butylmethyl(4-(5-(thiophene-2-carboximid-

amido)indolin-1-yl)cyclohexyl)carbamate (trans-56) (0.37 g, 0.82 -

mmol) as described for 19 to obtain the title compound (0.19 g, 66%).

6.35 (d, 1H, J ¼ 8.1 Hz), 3.36e3.26 (m, 2H), 2.85 (t, 2H, J ¼ 8.4 Hz),

2.59e2.58 (m, 1H), 2.23 (s, 3H), 1.80e1.63 (m, 5H), 1.51e1.33 (m,

5H); ESI-MS (m/z, %): 311, 309 (MH^þ, 100). trans-54: ¹H NMR

(DMSO-d₆)

d

7.09e7.05 (m, 2H), 6.36 (d, 1H, J ¼ 8.1 Hz), 3.34e3.29

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

105

¹H NMR (DMSO-d₆)

d

7.67 (d, 1H, J ¼ 3.3 Hz), 7.54 (dd, 1H, J ¼ 0.6,

4.5. Migraine model

5.1 Hz), 7.06 (dd, 1H, J ¼ 3.9, 4.9 Hz), 6.60 (s, 1H), 6.52 (d, 1H, J ¼ 8.1),

6.21 (brs, 2H), 3.31e3.24 (m, 4H), 3.16 (s, 1H), 2.82 (t, 2H, J ¼ 8.2 Hz),

2.31e2.19 (m, 4H), 1.95 (d, 2H, J ¼ 11.4 Hz), 1.73 (d, 2H, J ¼ 11.4 Hz),

1.47e1.35 (m, 2H), 1.16e1.04 (m, 2H); ESI-MS (m/z, %):

355 (MH^þ, 100), 324 (81), 133 (35); ESI-HRMS calculated for

C₂₀H₂₇N₄S (MH^þ): 355.1935, observed: 355.1950; HPLC purity:

93.05% by area.

Male Sprague-Dawley rats were anesthetized using ketamine/

xylazine (80 mg/kg, i.p.), the top of the head was shaved using

a rodent clipper (Oster Golden A5 w/size 50 blade), and the shaved

area was cleaned with betadine and 70% ethanol. Animals were

placed into a stereotaxic apparatus (Stoelting model 51600) and the

body core temperatures of 37 ^ꢃC were maintained using a heating

pad placed below the animals. Within the shaved and cleaned area

on the head, a 2 cm incision was made using a scalpel with a #10

blade and any bleeding was cleaned using sterile cotton swabs.

Location of bregma and midline bone sutures were identiﬁed as

references and a small hole 1 mm in diameter was made using

a hand drill without breaking the dura but deep enough to expose

the dura. Two additional holes (1 mm in diameter) 4e5 mm from

the previous site were made in order to mount stainless steel

screws securing the cannula through which an inﬂammatory

soup could be delivered to induce experimental migraine. A

modiﬁed intracerebroventricular (ICV) cannula was placed into

the hole without penetrating into or through the dura. The ICV

cannula was modiﬁed by cutting it to a length of 1 mm from the

bottom of the plastic threads using a Dremel mototool and a ﬁle

to remove any steel burrs. Once the modiﬁed migraine cannula

was in place, dental acrylic was placed around the migraine

cannula and stainless steel screws in order to assure that the

cannula was securely mounted. Once the dental acrylic was dry

(i.e., after 10e15 min) the cap of the cannula was secured on top

to avoid contaminants entering the cannula and the skin was

sutured back using 3-0 silk suture. Animals were given an antibiotic

injection (Amikacin C, 5 mg/kg, i.m.) and removed from the

stereotaxic frame and allowed to recover from anesthesia on

a heated pad. Animals were placed in a clean separate rat cage for

a 5 day recovery period. An injection cannula (Plastics One, C313I

4.2. General procedure for the conversion of the free base to the

dihydrochloride salt

To a solution of the free base (1.0 equiv) in methanol was added

1 M HCl in diethyl ether (3.0 equiv). The solution was stirred at

room temperature for 10 min then concentrated to dryness. The

residue was dried under reduced pressure to obtain the dihydro-

chloride salt as a solid. The chemical purity of the dihydrochloride

salts was similar to their corresponding free bases.

4.3. NOS enzyme assays

Recombinant human nNOS, eNOS and iNOS were produced in

Baculovirus-infected Sf9 cells. In a radiometric method, NOS

activity is determined by measuring the conversion of [³H]

L-

arginine to [³H]

enzyme is added to 100

2.4 mM CaCl₂, 1 mM MgCl₂, 1 mg/mL BSA, 1 mM EDTA, 1 mM

dithiothreitol, 1 M FMN, 1 M FAD, 10 M tetrahydrobiopterin,

M CaM. To measure iNOS, 10 L of

L of 100 mM HEPES, pH ¼ 7.4, con-

M FMN,

M NADPH, and

L

-citrulline. To measure eNOS and nNOS, 10

mL of

m

L of 40 mM HEPES, pH ¼ 7.4, containing

m

1 mM NADPH, and 1.2

enzyme is added to 100

m

taining 1 mM CaCl₂, 1 mM EDTA, 1 mM dithiothreitol, 1

M FAD, 10 M tetrahydrobiopterin, 120

100 nM CaM.

To measure enzyme inhibition, a 15

m

1

m

cut to ﬁt the modiﬁed ICV cannulas) connected to a 25

Syringe (1702SN) by tygon tubing was used to inject 10

solution onto the dura.

m

L Hamilton

L of the IM

m

L solution of a test

substance is added to the enzyme assay solution, followed by a pre-

incubation time of 15 min at rt. The reaction is initiated by addition

Naïve animals prior to the day of migraine surgery are placed in

suspended plexiglass chambers (30 cm L ꢄ 15 cm W ꢄ 20 cm H)

with a wire mesh bottom (1 cm²) and acclimated to the testing

chambers for 30 min. The paw withdrawal thresholds to tactile

stimuli were determined in response to probing with calibrated

von Frey ﬁlaments (Stoelting, 58011). The von Frey ﬁlaments were

applied perpendicularly to the plantar surface of the hind paw of

the animal until it buckles slightly, and is held for 3e6 s. A positive

response was indicated by a sharp withdrawal of the paw. The 50%

paw withdrawal threshold was determined by the non-parametric

method of Dixon (9). An initial probe equivalent to 2.00 g was

applied and if the response was negative the stimulus was

increased one increment, otherwise a positive response resulted in

a decrease of one increment. The stimulus was incrementally

increased until a positive response was obtained, then decreased

until a negative result was observed. This “up-down” method was

repeated until three changes in behavior were determined. The

pattern of positive and negative responses was tabulated. The 50%

paw withdrawal threshold is determined as (10^[XfþkM])/10,000,

where Xf ¼ the value of the last von Frey ﬁlament employed,

k ¼ Dixon value for the positive/negative pattern, and M ¼ the

mean (log) difference between stimuli. Only naïve animals with

baselines of 11e15 g were used in the experiment. Fifteen

grams was used as the maximal cut-off. Five days post migraine

surgery animals paw withdrawal thresholds were re-tested

using the same habituation and von Frey procedure as stated

above. Data were converted to % “antiallodynia” by the formula:

% activity ¼ 100 ꢄ (post-migraine value ꢀ baseline value)/

(15 g ꢀ baseline value). Only animals that demonstrated no

of 20

24

in every well. The reactions are carried out at 37 ^ꢃC for 45 min. The

mL

L-arginine containing 0.25

m

Ci of [³H] arginine/mL and

mM

L-arginine. The total volume of the reaction mixture is 150

mL

reaction is stopped by adding 20 mL of ice-cold buffer containing

100 mM HEPES, 3 mM EGTA, 3 mM EDTA, pH ¼ 5.5. [³H]

L-citrulline

is separated by DOWEX (ion-exchange resin DOWEX 50 W X 8-400,

SIGMA) and the DOWEX is removed by spinning at 12,000 g for

10 min in the centrifuge. 70

mL aliquot of the supernatant is added

to 100 L of scintillation ﬂuid and the radio activity is counted in

m

a liquid scintillation counter (1450 Microbeta Jet, Wallac). Speciﬁc

NOS activity is reported as the difference between the activity

recovered from the test solution and that observed in a control

sample containing 240 mM of 3. All assays are performed in

duplicate.

4.4. Efﬁcacy in the Chung model of neuropathic pain

Nerve ligation injury was performed according to the literature

procedure. Rats were anesthetized with halothane and the L₅and

L₆spinal nerves were exposed, carefully isolated, and tightly ligated

with 4-0 silk suture distal to the DRG. After ensuring homeostatic

stability, the wounds were sutured, and the animals allowed to

recover in individual cages. This technique produces signs of

neuropathic dysesthesias, including tactile allodynia, thermal

hyperalgesia, and guarding of the affected paw which begins on day

1 of the surgery and peaks on day 16. After a period of recovery

following the surgical intervention, rats show enhanced sensitivity

to painful and normally non-painful stimuli.

106

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

difference in their tactile hypersensitivity as compared to their pre-

migraine surgery values were used in all studies.

After establishing baseline paw withdrawal thresholds, indi-

vidual animals were removed from the testing chamber, the cap of

the migraine cannula was removed and animals received an

injection of either a mixture of IM (1 mM Histamine, 1 mM 5-HT

The degree of inhibition (%) was obtained by measuring the tail

current amplitude before and after drug superfusion (the difference

current was normalized to control and multiplied by 100 to obtain

the percent of inhibition). Concentration (log) response curves

were ﬁtted to a logistic equation to generate estimates of IC₅₀. The

concentrationeresponse relationship of 43 was constructed from

the percentage reductions of current amplitude by sequential

concentrations.

[Serotonin], 1 mM Bradykinin, 1 mM PGE₂) or vehicle at 10

mL

volume via the migraine cannula over a 5e10 s period. The IM

cocktail was made fresh on the day of each experiment. The cap

of the migraine cannula was replaced, individual animals were

placed back into their corresponding testing chamber and paw

withdrawal thresholds were measured at 1 h intervals over a 6 h

time course. Data were converted to % “antiallodynia” by the

formula: % activity ¼ 100 ꢄ (post-IM value ꢀ pre-IM baseline

value)/(15 g ꢀ pre-IM baseline value).

4.8. High throughput broad screen

In each experiment, the respective reference compound was

tested concurrently with 43, and the data were compared

with historical values determined (Table

1 from Supporting

information). Results showing an inhibition (or stimulation for

assays run in basal conditions) higher than 50% are considered to

represent signiﬁcant effect of 43. The speciﬁc ligand binding to the

receptor is deﬁned as the difference between the total binding and

the nonspeciﬁc binding determined in the presence of an excess of

unlabelled ligand. The results are expressed as a percent of control

speciﬁc binding ((measured speciﬁc binding/control speciﬁc

binding) ꢄ 100) and as a percent inhibition of control speciﬁc

4.6. hERG K^þchannel binding assay

The assay was carried with human recombinant HEK-293 cells

using [³H]astemizole as a ligand (2 nM) with incubation at 22 ^ꢃC for

75 min with reference to astemizole according to the literature

procedure. The speciﬁc ligand binding to the receptor is deﬁned as

the difference between the total binding and the nonspeciﬁc

binding determined in the presence of an excess of unlabelled

ligand. The results are expressed as a percent of control speciﬁc

binding (100

ꢀ

((measured speciﬁc binding/control speciﬁc

binding) ꢄ 100)) obtained in the presence of the test compound.

Acknowledgments

binding

((measured

speciﬁc

binding/control

speciﬁc

binding) ꢄ 100) obtained in the presence of 43. The IC₅₀values

(concentration causing a half-maximal inhibition of control speciﬁc

binding) and Hill coefﬁcients (nH) were determined by non-linear

regression analysis of the competition curves generated with

mean replicate values using Hill equation curve ﬁtting

We are grateful to NoAb BioDiscoveries Inc. (Mississauga, ON,

Canada) for performing the human NOS inhibition assays, Cyto-

chrome P450 enzyme inhibition studies and pharmacokinetic

studies, Asinex Ltd. (Moscow, Russia) for performing the human

iNOS inhibition assays and Cerep (France and USA) for hERG

binding assay, hERG patch-clamp assay and broad screen proﬁle.

We thank the Natural Sciences and Engineering Research Council

(NSERC) of Canada for providing an undergraduate student

research award (USRA) for T.R.

(Y ¼ D þ [(A ꢀ D)/(1 þ (C/C₅₀

)

^nH)], where Y ¼ speciﬁc binding,

D ¼ minimum speciﬁc binding, A ¼ maximum speciﬁc binding,

C ¼ compound concentration, C₅₀¼ IC₅₀, and nH ¼ slope factor).

4.7. hERG K^þchannel conventional patch-clamp assay

Appendix A. Supporting information

Cultured cells (1e7 days) were used for patch-clamp assay. The

cells were cultured in DMEM/GlutaMax-1 þ 10% FBS and were

planted on collagen-coated dishes at low density (w2 ꢄ 10⁴cells/

dish). The cell was held at ꢀ80 mV. A 50-ms pulse to ꢀ40 mV was

delivered to measure the leaking currents, which were subtracted

from the tail currents online. Then the cell was depolarized

to þ20 mV for 2 s, followed by a second pulse to ꢀ40 mV for 1 s to

reveal the tail currents. This paradigm was delivered once every 5 s

to monitor the current amplitude. After the current amplitude

stabilized, 43 was delivered to the extracellular medium by a rapid

solution changer perfusion system. During superfusion, the cell was

repetitively stimulated with the protocol described above, and the

current amplitude was continuously monitored. The experimental

conditions are described in Table 4.

Supplementary material associated with this article can be

found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.

2012.07.006.

References

[1] J.U. Adelman, E.J. Lewit, Clin. Cornerstone 4 (2002) 53e64.

[2] R. Burstein, B. Collins, M. Jakubowski, Ann. Neurol. 55 (2004) 19e24.

[3] S. Wasson, V.K. Jayam, Clin. Neuropharmacol. 27 (2004) 198e200.

[4] I.M. Bell, S.N. Gallicchio, M.R. Wood, A.G. Quigley, C.A. Stump, C.B. Zartman,

J.F. Fay, C.-C. Li, J.J. Lynch, E.L. Moore, S.D. Mosser, T. Prueksaritanont,

C.P. Regan, S. Roller, C.A. Salvatore, S.A. Kane, J.P. Vacca, H.G. Selnick, ACS Med.

Chem. Lett. 1 (2010) 24e29.

[5] C. Drahl, Chem. Eng. News 87 (2009) 31e33.

[6] G. Wei, V.L. Dawson, J.L. Zweier, Biochim. Biophys. Acta 1455 (1999) 23e34.

[7] D.O. Willenborg, S.A. Fordham, M.A. Staykova, I.A. Ramshawn, W.B. Cowden,

J. Immunol. 163 (1999) 5278e5286.

[8] F. Tao, Y.-X. Tao, P. Mao, C. Zhao, D. Li, W.J. Liaw, S.N. Raja, R.A. Johns,

Neuroscience 120 (2003) 847e854.

[9] Q. Lin, J. Palecek, V. Palecková, Y.B. Peng, J. Wu, M. Cui, W.D. Willis,

J. Neurophysiol. 81 (1999) 1075e1085.

Table 4

ꢀ

Experimental conditions for hERG K^þchannel conventional patch-clamp assay.

[10] J.H. Hao, X.J. Xu, Pain 66 (1996) 313e319.

[11] J. Olesen, Pharmacol. Ther. 120 (2008) 157e171.

Cells

Solutions

Incubation

Detection

HEK-293 cell Extracellular solutions:

line stably 137 NaCl, 4 KCl, 1.8 CaCl₂,

expressing 1 MgCl₂, 10

hERG

5e10 min for

concentration at whole-cell

rt (22e24 ^ꢃC)

Conventional

[12] P.L. Huang, Z. Huang, H. Mashimo, K.D. Bloch, M.A. Moskowitz, J.A. Bevan,

M.C. Fishman, Nature 377 (1995) 239e242.

[13] H.L. Lassen, H.K. Iverson, J. Olesen, Eur. J. Clin. Pharmacol. 59 (2003) 499e505.

[14] S. Maddaford, S.C. Annedi, J. Ramnauth, S. Rakhit, Annu. Rep. Med. Chem. 44

(2009) 27e50. and references within.

[15] E.P. Erdal, E.A. Litzinger, J. Seo, Y. Zhu, H. Ji, R.B. Silverman, Curr. Top. Med.

Chem. 5 (2005) 603e624.

[16] A.C. Tinker, A.V. Wallace, Curr. Top. Med. Chem. 6 (2006) 77e92.

[17] R.B. Silverman, Acc. Chem. Res. 42 (2009) 439e451.

D

(þ)-glucose,

patch clamp

(by Axopatch

200 B or HEKA

EPC9).

10 HEPES (pH 7.4 by NaOH).

Intracellular solutions: 130 KCl,

10 NaCl, 1 MgCl₂, 10 EGTA, 5

MgATP, 10 HEPES (pH 7.2

by KOH).

cumulatively.

S.C. Annedi et al. / European Journal of Medicinal Chemistry 55 (2012) 94e107

107

[18] J. Patman, N. Bhardwaj, J. Ramnauth, S.C. Annedi, P. Renton, S.P. Maddaford,

S. Rakhit, J.S. Andrews, Bioorg. Med. Chem. Lett. 17 (2007) 2540e2544.

[19] S.C. Annedi, J. Ramnauth, S.P. Maddaford, P. Renton, S. Rakhit, G. Mladenova,

P. Dove, S. Silverman, J.S. Andrews, M.D. Felice, F. Porreca, J. Med. Chem. 55

(2012) 943e955.

[27] X. Huang, S. Buchwald, Org. Lett. 3 (2001) 3417e3419.

[28] R.J. Abraham, J.J. Byrne, L. Grifﬁths, R. Koniotou, Magn. Reson. Chem. 43 (2005)

611e624.

[29] D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed, S.H. Snyder, Nature

351 (1991) 714e718.

[20] J. Ramnauth, J. Speed, S.P. Maddaford, P. Dove, S.C. Annedi, P. Renton, S. Rakhit,

J. Andrews, S. Silverman, G. Mladenova, S. Zinghini, S. Nair, C. Catalano,

D.K.H. Lee, M.D. Felice, F. Porreca, J. Med. Chem. 54 (2011) 5562e5575.

[21] S. Maddaford, P. Renton, J. Speed, S.C. Annedi, J. Ramnauth, S. Rakhit,

J. Andrews, G. Mladenova, L. Majuta, F. Porreca, Bioorg. Med. Chem. Lett. 21

(2011) 5234e5238.

[22] P. Renton, J. Speed, S. Maddaford, S.C. Annedi, J. Ramnauth, S. Rakhit,

J. Andrews, Bioorg. Med. Chem. Lett. 21 (2011) 5301e5304.

[23] S.C. Annedi, S.P. Maddaford, G. Mladenova, J. Ramnauth, S. Rakhit,

J.S. Andrews, D.K.H. Lee, D. Zhang, F. Porreca, D. Bunton, L. Christie, J. Med.

Chem. 54 (2011) 7408e7416.

[30] S.H. Kim, J.M. Chung, Pain 50 (1992) 355e363.

[31] R.M. Edelmayer, T.W. Vanderah, L. Majuta, E.T. Zhang, B. Fioravanti,

M.D. Felice, J.G. Chichorro, M.H. Ossipov, T. King, J. Lai, S.H. Kori,

A.C. Nelsen, K.E. Cannon, M.M. Heinricher, F. Porreca, Ann. Neurol. 65

(2009) 184e193.

[32] C. Jamieson, E.M. Moir, Z. Rankovic, G. Wilshart, J. Med. Chem. 49 (2006)

5029e5046.

[33] K. Finlayson, A.J. Pennington, J.S. Kelly, Eur. J. Pharmacol. 412 (2001)

203e212.

[34] Z. Zhou, Q. Gong, B. Ye, Z. Fan, J.C. Makielski, G.A. Robertson, C.T. January,

Biophys. J. 74 (1998) 230e241.

[24] M. Bercot-Vatteroni, Ann. Chim. 7 (1962) 303e337.

[35] http://www.cerep.fr/.

[25] V. Hasimbegovic, J. Slätt, J. Bergman, T. Janosik, J. Heterocycl. Chem. 44 (2007)

1213e1217.

[36] M.C. Wenlock, R.P. Austin, P. Barton, A.M. Davis, P.D. Leeson, J. Med. Chem. 46

(2003) 1250e1256. and references therein.

[26] A.F. Abdel-Magid, K.G. Carson, B.D. Harris, C.A. Maryanoff, R.D. Shah, J. Org.

Chem. 61 (1996) 3849e3862.

[37] T.T. Wager, X. Hou, P.R. Verhoest, A. Vilalobos, ACS. Chem. Neurosci. 1 (2010)

435e449.

Article Doi

Discovery of a potent, orally bioavailable and highly selective human neuronal nitric oxide synthase (nNOS) inhibitor, N-(1-(piperidin-4-yl)indolin-5- yl)thiophene-2-carboximidamide as a pre-clinical development candidate for the treatment of migraine

DOI: 10.1016/j.ejmech.2012.07.006

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