Hydrophilic, Potent, and Selective 7-Substituted 2-Aminoquinolines as Improved Human Neuronal Nitric Oxide Synthase Inhibitors

Article

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Hydrophilic, Potent, and Selective 7‑Substituted 2‑Aminoquinolines

as Improved Human Neuronal Nitric Oxide Synthase Inhibitors

Anthony V. Pensa,^†,⊥Maris A. Cinelli,^†,⊥Huiying Li,^‡Georges Chreiﬁ,^‡Paramita Mukherjee,^†

Linda J. Roman,^§Pavel Martasek,^§,∥Thomas L. Poulos,*^,‡and Richard B. Silverman*^,†

́

^†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular

Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208-3113, United States

^‡Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,

California 92697-3900, United States

^§Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States

^∥Department of Pediatrics and Center for Applied Genomics, First School of Medicine, Charles University and BIOCEV 121 08

Prague, Czech Republic

S

* Supporting Information

ABSTRACT: Neuronal nitric oxide synthase (nNOS) is a

target for development of antineurodegenerative agents. Most

nNOS inhibitors mimic ^L-arginine and have poor bioavail-

ability. 2-Aminoquinolines showed promise as bioavailable

nNOS inhibitors but suﬀered from low human nNOS

inhibition, low selectivity versus human eNOS, and signiﬁcant

binding to other CNS targets. We aimed to improve human

nNOS potency and selectivity and reduce oﬀ-target binding by

(a) truncating the original scaﬀold or (b) introducing a

hydrophilic group to interrupt the lipophilic, promiscuous

pharmacophore and promote interaction with human nNOS-

speciﬁc His342. We synthesized both truncated and polar 2-aminoquinoline derivatives and assayed them against recombinant

NOS enzymes. Although aniline and pyridine derivatives interact with His342, benzonitriles conferred the best rat and human

nNOS inhibition. Both introduction of a hydrophobic substituent next to the cyano group and aminoquinoline methylation

considerably improved isoform selectivity. Most importantly, these modiﬁcations preserved Caco-2 permeability and reduced oﬀ-

target CNS binding.

shown promise in treating or preventing neuronal damage in

animal models.^7,8

INTRODUCTION

■

Neurodegenerative disorders (such as Alzheimer’s and

Parkinson’s diseases, among others) result in severe neuro-

logical, cognitive, and motor deﬁcits. The prevalence of these

diseases has increased over the past century, and this upward

trend is predicted to continue, especially with an increasingly

aged population,¹making the development of therapeutic

agents to treat these disorders a priority. In addition, the

neuronal damage observed in these diseases can also be

observed in patients with neuropathic pain, stroke, cerebral

palsy, and head trauma.

We have been investigating the enzyme neuronal nitric oxide

synthase (nNOS) as a potential target for antineurodegener-

ative therapeutics. In the brain, nitric oxide (NO, produced by

nNOS) is needed for neuronal signaling, but under conditions

of neurodegeneration, NO levels are high, owing to overex-

pressed or unregulated nNOS. This increased NO can form

reactive species such as peroxynitrite, leading to neuronal cell

damage.²Indeed, nNOS³⁻⁶has been implicated in the

pathogenesis of neurodegeneration and peripheral nerve

dysfunction (neuropathic pain), and inhibition of nNOS has

NO is formed when homodimeric nNOS converts L-arginine

to ^L-citrulline, which occurs in the enzyme’s oxygenase domain.

Electrons (from NADPH) are shuttled through redox cofactors

(FAD and FMN) in the reductase domain and then move from

one monomer’s reductase domain to the other’s oxygenase

domain,⁹where electron transfer proceeds to (6R)-5,6,7,8-

tetrahydrobiopterin (H₄B) and ﬁnally to heme, which then

oxidizes the bound ^L-arginine.¹⁰Along with nNOS, two other

isoforms of NOS (inducible NOS, or iNOS, and endothelial

NOS, or eNOS) make NO in humans. iNOS is involved in the

immune response, whereas the NO produced by eNOS

regulates smooth muscle tone and blood pressure.

Over the years, several classes of nNOS inhibitors have been

developed in our laboratories (1−6 are representative

examples, Figure 1).¹¹⁻¹⁵These compounds are all competitive

with ^L-arginine; compound 3 is also a heme-iron coordinator.¹²

Received: June 7, 2017

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Figure 1. Previously designed human nNOS inhibitors.

Figure 2. Overall strategy for design of hydrophilic 2-aminoquinolines based on lead 5.

A general challenge in development of arginine-mimetic

inhibitors is that molecules that resemble arginine often possess

chemical properties (basicity, H-bonding potential, and high

polar surface area, or PSA) that impede oral bioavailability and

passage through the blood−brain barrier (BBB; compounds 1,

2, and 6, for example, have poor Caco-2 permeability).

In addition to having good BBB permeability, nNOS

inhibitors must be selective for the neuronal isoform over

iNOS and eNOS. For example, eNOS inhibition can cause

cardiovascular problems and hypertension.¹⁶Nonspeciﬁc iNOS

inhibition could interfere with immune defense, but its role in

the brain is more complex; one study indicated that mice

lacking iNOS develop enhanced Alzheimers’ pathology,¹⁷while

others indicate that iNOS inhibition could actually be

neuroprotective.¹⁸A more recent challenge has been designing

inhibitors with high potency against human nNOS (hnNOS).

Historically, many structure-based design eﬀorts have utilized

rat nNOS (rnNOS), so many compounds (such as most shown

in Figure 1) are selective for the rat enzyme, leading to weaker

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hnNOS inhibitors with low selectivity over human eNOS

(heNOS).

that could act as H-bond acceptors: homologated N,N-

dimethylamino derivatives (12−15), pyridine derivatives

(16−19), and nitriles 20−21. Finally, structure-based mod-

iﬁcations were performed to improve the hn/he selectivity of

nitrile 21. The o-methyl group (of 22) and o-chlorine (of 23)

were installed on the benzonitrile ring to make additional

contact with Met336 (in rnNOS; Met341 in hnNOS). This

residue is absent in eNOS isoforms (both bovine and human

eNOS), replaced by a smaller valine, and contact between

methionine residues (vs valine residues) and inhibitors has

previously been implicated in improved n/e selectivity.^14,15,22

Finally, two active nitrile-containing molecules (22 and 23)

were methylated at the 4-position of the quinoline. This

modiﬁcation was previously reported to improve potency (and

sometimes n/e selectivity) for the analogous 2-aminopyridines

by the interaction of the 4-methyl group with a small

hydrophobic pocket located near the “back wall” of the

heme-binding pocket.²⁴

In addition to possibly improving hnNOS activity, it was

hypothesized that the addition of more hydrophilic groups

(such as amines, pyridines, or nitriles) could reduce oﬀ-target

binding. For example, the addition of a nitrile (relative to an

aromatic chloride, as in 5) slightly increases the PSA and

decreases the overall hydrophobicity of the molecule,²⁵which

may (a) disfavor binding to GPCRs and other CNS targets,

many of which depend on extensive hydrophobic interactions

for ligand binding, and (b) decrease nonspeciﬁc hydrophobic

interactions with other proteins. As very high PSA (>100 Å²)

could hamper brain permeation or oral bioavailability, however,

we were careful to keep the PSA of all analogues below 80 Å².

All synthesized compounds were assayed against rnNOS and

hnNOS to determine their selectivity for the human isoform

(Table 1). To determine selectivity, murine iNOS and bovine

eNOS were used, and select compounds were also assayed

against human eNOS in an attempt to begin translating our

work to more “humanized” systems (Table 2). Finally, the most

selective compound (25) was assayed in a Caco-2 assay to

approximate cellular permeability and in the PDSP counter-

screen to determine the eﬀects of the new modiﬁcations on oﬀ-

target binding.

The 2-aminoquinolines (compounds 4−6) have shown

considerable promise as a scaﬀold for further nNOS inhibitor

development. Compounds 4 and 5 have good potency,

selectivity, and cellular permeability.¹⁴They also exhibit good

oral bioavailability in mice (4) and brain penetration in rats (5).

Unfortunately, compound 5, considered our best lead for

further development, had an unfavorable safety proﬁle in rats,

where it caused neurological and cardiovascular side eﬀects at

the doses required for eﬀective nNOS inhibition. Counter-

screening against a variety of CNS targets via the Psychoactive

Drug Screening Program’s (PDSP’s) radioligand binding

assay¹⁹revealed that 5, with its GPCR-ligand-like pharmaco-

phore, is a promiscuous binder (aﬃnity <100 nM) at serotonin,

opioid, and histamine receptors, and its scaﬀold also resembles

known human ether-a-go-go-related-gene (hERG) channel

blockers. It is imperative that speciﬁc CNS-acting NOS

inhibitors not strongly bind numerous other targets in the

brain, as this could cause neurological, psychological, or other

side eﬀects. Additionally, compound 5 (and 4) has poor

hnNOS activity and low hnNOS/heNOS (hn/he) selectivity.

One strategy for reducing the oﬀ-target binding of these

compounds involved rearranging the pharmacophore of 5 to

disrupt the GPCR ligand-like core, leading to compounds such

as 6,¹⁵where the position of the polar amine and hydrophobic

aryl group are “inverted” relative to 5. Unfortunately, this

compound had much lower cellular permeability and its

hnNOS activity, while improved from 5, was still low, as was

its hn/he selectivity. We therefore, in addition to our newer

studies on phenyl-ether linked aminoquinolines,^15,20sought

alternative (and parallel) strategies for reducing the oﬀ-target

binding of 5 and improving its hnNOS activity and hn/he

selectivity.

One such strategy, detailed therein, is based on a key

structural diﬀerence between rnNOS and hnNOS. While

rnNOS possesses an isoform-speciﬁc hydrophobic pocket,

consisting of Tyr706, Leu337, and Met336, hnNOS lacks the

leucine residue, which is replaced by His342.²¹This pocket in

hnNOS is smaller and more polar,²²and preliminary

crystallography studies indicated that the hydrophobic haloaryl

tail of 4, responsible for much of this compound’s stabilization

when bound to the hydrophobic pocket of rnNOS,¹⁴is not

well-accommodated in the analogous region of hnNOS

(possibly because of the bulky and polar histidine), resulting

in the loss of hydrophobic contacts and much lower hnNOS

activity. Our initial strategy consisted of simply truncating the

linker chain between the quinoline and the hydrophobic aryl

ring, hoping that lack of repulsion between the aryl ring and

His342 might improve the selectivity for hnNOS over rnNOS

(as observed for truncated 2-aminopyridines).²³

CHEMISTRY

■

Preparation of 7-substituted 2-aminoquinolines 7−13 pro-

ceeded through bromide 26, which was prepared according to

literature procedures.^14,15To prepare truncated analogues 7−

11 (Scheme 1) bromide 26 was treated with an excess of

commercially available anilines 27−31 and catalytic potassium

iodide under microwave conditions²⁶to aﬀord the acetamide-

protected intermediates 32−36 in good yields. These

intermediates were not characterized extensively but were

immediately deacetylated with K₂CO₃in reﬂuxing methanol,²⁷

followed by treatment with methanolic HCl to yield desired

analogues 7−11 as hydrochloride salts. The homologated

compounds (12 and 13) were prepared by treatment of 26 with

commercially available benzylamines 37 or 38 in the presence

of Cs₂CO₃(Scheme 2).²⁸The desired products were Boc-

protected at the secondary amine to aid in puriﬁcation,

providing protected intermediates 39 (from 37) and 40 (from

38). Subsequent deacetylation, workup, and cleavage of the Boc

group under acidic conditions aﬀorded 12 and 13, respectively.

To synthesize aminoquinolines possessing more than one

methylene unit between the aryl ring and the secondary amine

(analogues 14−16, 20, and 21), the requisite primary amines

To this end, compounds 7−11 (Figure 2), bearing

substituents on the aryl ring capable of a wide variety of

interactions, were prepared and assayed against rnNOS and

hnNOS. Although less active against these isoforms compared

to compounds 4−6 (Table 1, vide infra), the good rat/human

(rn/hn) selectivity of dimethylamines 10 and 11 encouraged

the development of a second strategy, to directly interact with

His342 itself.

The pyridine of compound 3 was reported to H-bond with

His342 (discovered via X-ray crystallographic analysis),¹³

improving this scaﬀold’s hnNOS potency. To this end, we

prepared several series of compounds with hydrophilic groups

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a

prepare isomeric amine 47 (Scheme 3B), commercially

available aldehyde 45 was condensed with nitromethane to

yield unstable nitrostyrene 46,³⁰which was immediately

reduced to 47 with LiAlH₄.³¹Pyridinepropanamine 50

(Scheme 3C) was prepared by the method of Mukherjee et

al.,¹³starting with the Mitsunobu conversion of primary alcohol

48 to azide 49, followed by Staudinger reduction to the amine

(and acidic hydrolysis to yield dihydrochloride salt 50).

Finally, cyanophenethylamine 53 (Scheme 3D) was prepared

by Boc-protection of commercially available 3-bromophene-

thylamine (51) followed by palladium-catalyzed cyanation of

intermediate carbamate 52. Removal of the Boc group yielded

hydrochloride 53.³²

Scheme 1

From these amines, as well as commercially available 4-

cyanophenethylamine (55), analogues 14−16, 20, and 21 were

prepared by an indirect reductive amination (Scheme 4) where

aldehyde 54 (prepared by literature procedures)¹⁵was treated

with the required primary amine under mildly acidic,

dehydrating conditions (cat. AcOH and anhydrous Na₂SO₄),

and the resulting imine was reduced with NaBH₄(and the

secondary amine protected with Boc₂O to aid in puriﬁcation as

described above) to yield intermediates 56−60. These were

deprotected to yield ﬁnal compounds 14−16, 20, and 21.

To prepare substituted pyridine compounds 17−19, the

necessary pyridinepropanamines were prepared from bromo-

pyridines 61−63 (Scheme 5). Sonogashira coupling with N-

Boc-propargylamine yielded alkynes 64−66, which were

hydrogenated and deprotected to give dihydrochloride salts

67−69. Subsequent indirect reductive amination between these

amines and 54, followed by reduction and protection, gave the

intermediate acetamides 70−72. Deacetylation and Boc group

removal aﬀorded pyridine derivatives 17−19 as water-soluble

trihydrochloride salts.

The substituted cyanophenethylamines (81 and 82) and

their respective aminoquinoline derivatives 22 and 23 were

prepared as shown in Scheme 6. Starting with commercially

available methyl 4-bromo-3-methylbenzoate (73, for 81) and 4-

bromo-3-chlorobenzoic acid (74, for 82), reduction with either

LiAlH₄or BH₃−THF, respectively, resulted in primary alcohols

75 and 76.^33,34Subsequent bromination of the alcohol under

Appel conditions and displacement of the bromide with KCN

resulted in benzonitrile intermediates 77³⁵and 78 in good

yield. BH₃−THF reduction³⁶and protection with Boc₂O

aﬀorded 79 and 80.

a

Reagents and conditions: (a) cat. KI, MeCN, 110 °C, μ-wave; (b) (i)

K₂CO₃, MeOH, reﬂux, (ii) MeOH/HCl, rt (after isolation).

a

Scheme 2

Palladium-catalyzed cyanation of 79 followed by depro-

tection (vide supra) yielded the hydrochloride salt 81. Likely

because of the bulk of the o-chloro substituent, the analogous

cyanation was very low-yielding, and 82 was instead prepared

by reaction with CuCN in reﬂuxing DMF followed by acidic

deprotection (to yield the crude hydrochloride salt). Sub-

sequent indirect reductive amination with 54 and either 81 or

82, followed by NaBH₄reduction and Boc-protection, yielded

protected intermediates 83 and 84.

Deprotection, as described above, aﬀorded 22 and 23 in

moderate yield. Synthesis of 4-methylated aminoquinolines³⁷

24 and 25 (Scheme 7) began with Doebner−Miller

condensation of 3-bromoaniline 85 and 3-buten-2-one to

yield 86, and oxidation with m-CPBA resulted in N-oxide 87.

Deoxygenative amination (and debutylation of the intermediate

t-butylaminoquinoline with TFA)³⁸yielded 88. Acetylation

with N-acetylimidazole yielded 89, followed by Pd-catalyzed

formylation³⁹to yield 90. A similar indirect reductive amination

between 90 and either 55 or 81, followed by reduction and

a

Reagents and conditions: (a) (i) Cs₂CO₃, DMF, rt, (ii) Boc₂O, THF,

rt; (b) (i) K₂CO₃, MeOH, reﬂux, (ii) MeOH/HCl, rt (after isolation).

44, 47, 50, and 53 were ﬁrst prepared. To prepare

phenethylamine 44, nitroarene 41 (Scheme 3A) was ﬁrst

hydrogenated to yield aniline 42,²⁹followed by Eschweiler−

Clarke methylation and hydrogenation of 43 to aﬀord 44. To

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a

Scheme 3

a

Reagents and conditions: (a) H₂, Pd/C, MeOH, rt; (b) formalin, HCO₂H, DMF, 0 °C−reﬂux; (c) H₂, Raney Ni, MeOH/NH₃:EtOH (1:1), rt; (d)

NO₂CH₃, NH₄OAc, AcOH, reﬂux; (e) LiAlH₄in THF. 0 °C−reﬂux; (f) PPh₃, diphenylphosphoryl azide, DIAD, THF, 0 °C−rt; (g) (i) P(OEt)₃,

benzene, reﬂux, (ii) MeOH/HCl, rt (after isolation); (h) Boc₂O, THF, rt; (i) (i) K₄[Fe(CN)₆], t-BuXPhos Pd G₃, t-BuXPhos, dioxane, KOAc (0.1

M in H₂O), reﬂux, (ii) MeOH/HCl, rt (after isolation).

a

Scheme 4

Scheme 5

a

Reagents and conditions: (a) N-Boc-propargylamine, Cul, PPh₃,

Pd(PPh₃)₂Cl₂, Et₃N, 90 °C; (b) (i) H₂, Pd/C, MeOH, rt, (ii) MeOH/

HCl, rt (after isolation); (c) (i) AcOH, Na₂SO₄, CHCl₃, rt, (ii)

NaBH₄, MeOH, 0 °C−rt, (iii) Boc₂O, THF, rt; (d) (i) K₂CO₃,

MeOH, reﬂux, (ii) MeOH/HCl, rt (after isolation).

a

Reagents and conditions: (a) (i) Et₃N ﬁrst, or AcOH, Na₂SO₄,

CHCl₃, rt, (ii) NaBH₄, MeOH, 0 °C−rt, (iii) Boc₂O, THF, rt; (b) (i)

K₂CO₃, MeOH, reﬂux, (ii) MeOH/HCl, rt (after isolation).

RESULTS AND DISCUSSION

■

protection, aﬀorded protected intermediates 91 and 92, which

were then deprotected to yield 24 and 25 as the

dihydrochloride salts.

Historically, rat nNOS (rnNOS) and bovine eNOS (beNOS)

were the ﬁrst NOS enzymes to be expressed and crystallized

and to date have provided a majority of the structural data for

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a

Scheme 6

a

Reagents and conditions: (a) (for 73) LiAlH₄in THF, THF, −15 °C; (b) (for 74) BH₃−THF, THF, 0 °C−rt; (c) (i) CBr₄, PPh₃, DCM, 0 °C, (ii)

KCN, Bu₄NBr, CH₂Cl₂, H₂O, rt; (d) (i) BH₃−THF, THF, reﬂux, (ii) Boc₂O, THF, rt; (e) (for 78) K₄[Fe(CN)₆], t-BuXPhos Pd G3, t-BuXPhos,

dioxane, KOAc (0.1 M in H₂O), 100 °C; (f) (for 79) CuCN, DMF, reﬂux; (g) MeOH/HCl, rt (after isolation); (h) (i) 54, AcOH, Na₂SO₄, CHCl₃,

rt, (ii) NaBH₄, MeOH, 0 °C−rt, (iii) Boc₂O, THF, rt; (i) (i) K₂CO₃, MeOH, reﬂux, (ii) MeOH/HCl, rt (after isolation).

a

Scheme 7

a

Reagents and conditions: (a) 3-buten-2-one, FeCl₃·6H₂O, AcOH, 60 °C−140 °C; (b) m-CPBA, CH₂Cl₂, rt; (c) (i) t-BuNH₂, Ts₂O, PhCF₃/

CH₂Cl₂, 0 °C, (ii) TFA, reﬂux; (d) N-acetylimidazole, THF, reﬂux; (e) N-formylsaccharin, Et₃SiH, Pd(OAc)₂, dppb, Na₂CO₃, DMF, 75 °C; (f) (i)

55 or 81, AcOH, Na₂SO₄, CHCl₃, rt, (ii) NaBH₄, MeOH, 0 °C, rt, (iii) Boc₂O, THF, rt; (g) (i) K₂CO₃, MeOH, reﬂux, (ii) MeOH/HCl, rt (after

isolation).

isoform-selective inhibitor design. Therefore, compounds 7−25

were ﬁrst assayed against rnNOS, murine macrophage iNOS

(miNOS), and beNOS, using the previously described

hemoglobin capture assay (see Experimental Section). These

isoforms are also used to approximate isoform selectivity for

rnNOS because the amino acids that generally interact with our

inhibitors are identical in rat and murine iNOS, as well as in rat

and bovine eNOS, and are unlikely to result in signiﬁcant

species diﬀerences. Because of our 2-fold goal of decreasing the

oﬀ-target binding and improving the human nNOS (hnNOS)

activity of the previous leads, the studied compounds were also

assayed against puriﬁed hnNOS, and six of the most potent

compounds against hnNOS were also assayed against puriﬁed

human eNOS (heNOS). Table 1 summarizes the apparent K_i

values and isoform selectivities for 7−25, and the activity and

selectivity for hnNOS and heNOS are summarized in Table 2.

Values for 4−6 are included for comparison. We have found

human iNOS very diﬃcult to express and purify in our

laboratories, so it is not included in this discussion. As such,

most of the following discussion will focus on nNOS and

eNOS, as achieving high n/e selectivity was a primary goal of

this study. Additionally, iNOS isoforms do not easily grow

diﬀraction-quality crystals, the role of iNOS in neurodegenera-

tion is complicated, and n/e selectivity tends to be much harder

to achieve than n/i selectivity for 2-aminoquinolines.

against rnNOS, with the most potent derivative (7) showing

approximately 10-fold less activity than 4 and 5 (Table 1).

Initial Structural Analysis of Truncated and Aniline

Analogues. The X-ray crystal structure of compound 8 (which

was used for crystallographic analysis because of the poor

solubility of 7) reveals the reason for the K_iincrease for these

truncated inhibitors (Figure 3A). While the aminoquinoline

portion mimics arginine and binds to Glu592 (rnNOS), there

are no interactions with the nNOS-speciﬁc hydrophobic pocket

(Leu337, Met336, and Tyr706) that provided a major source of

stabilization upon binding of 4 and 5. Instead, the ﬂuorophenyl

ring points upward toward the roof of the active site near

Arg481, where the ﬂuorine interacts with Gln478. This “bent-

up” binding mode was previously observed for aniline-linked

aminoquinoline derivatives,¹⁵reﬂecting the geometry required

for the aniline to interact with the nearby heme propionate.

The larger methoxyl group of 9 and the N,N-dimethylamine of

10 should be similarly positioned, but owing to the larger size

of these substituents (relative to the ﬂuorine in 8), the potency

for 9 and 10 is lower.

Not too surprisingly, 8 has similar potency against rnNOS

and hnNOS, given that the binding mode is the same (see the

hnNOS-8 structure in Figure 3B). The rnNOS/hnNOS

selectivity approaches 1:1, and in addition to an identical

binding mode to rnNOS, there are no direct interactions with

His342 in the hnNOS-8 structure. Despite their low potency,

similar rnNOS/hnNOS activity is also observed for 10 and 11,

and this, along with the increased hydrophilicity, prompted

further investigation of the N,N-dimethylaniline-containing

scaﬀold.

Our initial attempts at improving the hnNOS activity of the

2-aminoquinoline scaﬀold involved truncation of the amino-

alkyl linker to remove the clash between His342 in hnNOS and

the bulky haloaryl groups of 4 and 5. The truncated derivatives

(7−10), however, were signiﬁcantly less potent than 4 and 5

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nNOS enzymes, although a greater increase in potency was

observed for homologation of 10 to 12. The rnNOS-13 and

hnNOS-13 structures (Figure 4A and B, respectively; the

rnNOS-12 and hnNOS-12 structures are shown in Supporting

Information, Figures S1A and B, respectively) indicate that

homologation of 11 to 13 does result in additional interactions

between the inhibitor and the enzyme.

For example, although the electron density is weak in this

area, one N-methyl group of 13 can potentially interact with

Leu337 and Met336 in rnNOS (Figure 4A). In hnNOS, the

N,N-dimethylamino group prefers an orientation facing away

from the polar and bulkier His342 (Figure 4B), instead making

contacts with Met341. The linker amine H-bonds with the

heme propionates in hnNOS but bends away from the

propionates in rnNOS. This diﬀerence is likely the result of

the diﬀerent nature of the interactions between the tail N,N-

dimethylamino group and either the polar His342 (hnNOS) or

nonpolar Leu337 (rnNOS). Nonetheless, there are some

deleterious interactions that may be present as well. The steric

crowding around the linker amino group and repulsion of the

heme propionates by the nearby phenyl ring make this ring and

its substituent partially disordered, as evidenced by the rather

poor density in the region (Figure 4B).

Unfortunately, this singly homologated scaﬀold does not

appear to oﬀer signiﬁcant advantages, potency- or selectivity-

wise, over a compound such as 8 (Table 1). Compared to the

truncated (7−11) or singly homologated (12 and 13)

analogues, the doubly homologated compounds 14 and 15

are signiﬁcantly improved rnNOS and hnNOS inhibitors. In the

rnNOS-15 structure (Figure 5A), the good potency of 15 is

visible in the clean electron density throughout the inhibitor

except for the N,N-dimethylamino group. The main reasons for

increased potency are that the elongated tail relieves the clash

and crowding around the linker amino group, allowing the H-

bond with heme propionate D, and there are nonpolar

interactions between the aryl ring and N,N-dimethylamino

group and Met336 and Trp306. The hnNOS-15 structure is

very much the same, except the aryl ring moves over slightly so

that it is farther away from Met341 (>4.2 Å) while the N,N-

dimethylamino group interacts with His342. Compound 15

thus provides another example where the His342/Leu337

diﬀerence between hnNOS and rnNOS, respectively, controls

diﬀerences in aﬃnity. Compound 15 makes better nonpolar

contacts with Leu337 and Met336 in rnNOS than His342 and

Met341 in hnNOS, which explains why it is still a better rnNOS

inhibitor.

Compound 15 has the best rn/be selectivity of the N,N-

dimethylaniline compounds tested. Like the nNOS structures,

the beNOS-15 structure is well ordered (Figure 5C) and

retains the H-bonds from the linker amine to both heme

propionates. The 44-fold rn/be selectivity is mainly the result of

the smaller Val106 in beNOS that replaces Met336 of rnNOS.

A glycerol molecule is bound next to Val106 (common in many

eNOS structures). The aryl ring moves slightly to avoid the

glycerol, and as such, any contacts less than 5.2 Å between the

N,N-dimethylamine and the small Val106 cannot be made,

although some hydrophobic contacts with Trp76, Leu107, and

Val106 are retained with the aryl ring itself. In contrast, the

close hydrophobic contacts from both the aryl ring and N,N-

dimethylamino group to the nearby Trp306 and bulkier

Met336 are more extensive and at shorter distances in

rnNOS. Therefore, the rn/be selectivity is likely a true isoform

diﬀerence (due to Met/Val variation) despite the binding being

Table 1. Inhibition of NOS Enzymes by Aminoquinoline

Analogues 7−25

a

K_i(μM)

selectivity

compd

rnNOS

hnNOS

miNOS

beNOS

rn/mi

rn/be

4

0.049

0.066

0.058

0.529

0.575

1.32

0.318

0.440

0.295

2.05

44.0

28.4

27.7

15.3

NT

11.2

7.24

12.5

7.95

NT

899

431

478

29

228

110

216

15

5

6

7

8

0.768

2.17

ND

71

ND

16

9

NT

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

4.83

4.30

NT

1.82

1.10

NT

0.421

0.522

0.085

0.036

0.038

0.090

0.050

0.216

0.041

0.037

0.021

0.031

0.019

0.025

1.02

30.1

42.2

13.3

10.0

11.2

13.8

17.2

84.2

25.0

21.3

10.3

5.15

4.70

4.83

6.53

8.42

NT

0.764

0.076

0.274

0.108

0.130

0.073

0.164

0.050

0.032

0.020

0.021

0.052

0.030

81

16

156

278

295

153

344

390

609

575

492

166

247

193

ND

44

1.57

1.04

0.562

1.86

NT

27

7

37

ND

7

0.273

0.581

0.092

0.115

1.22

0.468

16

4

64

19

a

Compounds 7−25 were assayed in vitro against four puriﬁed NOS

isoforms: rat nNOS, human nNOS, bovine eNOS, and murine iNOS,

using known literature methods, and K_ivalues were calculated directly

from IC₅₀values using the Cheng−Prusoﬀ equation (see Experimental

Section for details). IC₅₀values are the average of at least two

replicates from 7 to 9 data points; all experimental standard error

values are less than 16%, and all correlation coeﬃcients are >0.83.

Selectivity values are ratios of respective K_ivalues. NT = not tested;

ND = not determined.

Table 2. Inhibition of hnNOS and heNOS by Selected

a

Compounds

a

K_i(μM)

compd

human nNOS

human eNOS

selectivity (hn/he)

4

0.318

0.440

0.295

0.073

0.032

0.020

0.021

0.052

0.030

9.49

11.8

7.41

1.58

1.03

2.08

2.70

5.79

5.76

30

27

5

6

25

18

21

22

23

24

25

22

32

104

129

111

192

a

Compounds 18 and 21−25 were assayed in vitro against human

eNOS, using known literature methods and K_ivalues were calculated

directly from IC₅₀values using the Cheng−Prusoﬀ equation (see

Experimental Section for details). IC₅₀values are the average of at least

two replicates from 7 to 9 data points; all experimental standard error

values are less than 15%, and all correlation coeﬃcients are >0.94.

Selectivity values are ratios of respective K_ivalues; human nNOS K_i

values are from Table 2.

Further modiﬁcation of 10 and 11 (via homologation)

resulted in meta-substituted compound 12 and para-substituted

13. Both compounds displayed improved potency against

rnNOS and hnNOS, with similar K_ivalues between the two

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Figure 3. Active site structures of rnNOS-8 (A) and hnNOS-8 (B). For clarity, the residues on the roof of the rnNOS active site, Glu478 and

Arg481, are not shown. In this ﬁgure and all the following structural ﬁgures, major H-bonds are depicted with dashed lines. The omit F_o− F_c

electron density maps for the bound inhibitor are displayed at 2.5σ contour level. All structural ﬁgures were prepared with PyMol (www.pymol.org).

Figure 4. Active site structures of rnNOS-13 (A) and hnNOS-13 (B). Note that only in hnNOS can the linker amine of 13 make H-bonds with the

heme propionates; however, the N,N-dimethylaniline is more disordered in hnNOS than in rnNOS (indicated by density quality).

slightly disturbed by glycerol. Compound 15 also has the

highest rn/mi selectivity out of the aniline series. Although

structural data are not available for miNOS-15, miNOS has an

asparagine (Asn115) in place of Leu337 in rnNOS, and this

residue was previously shown to be a crucial determinant of rn/

mi selectivity,⁴⁰as it strongly repels hydrophobic moieties such

as aryl rings (or possibly 15’s aniline methyl groups).

Despite the increased hnNOS activity of these compounds, it

is worth noting that there are several drawbacks associated with

N,N-dimethylanilines such as 14 and 15. Often ﬂagged as

metabolic liabilities in drug discovery (because of potential

oxidation into toxic species),⁴¹anilines can also be air or light

sensitive. Although we handled all anilines carefully, storing

stocks and solutions in the dark and cold and assaying all

compound dilutions within 48 h of preparation, it was found

that solutions of 14 lost their nNOS inhibitory activity when

stored for longer periods of time (ca. 1 week), and a N,N-

diethyl analogue of 11 (not reported here) darkened rapidly in

solution. Both of these ﬁndings indicated possible instability of

the aniline and highlighted the need for other histidine-

interacting groups.

zole inhibitors.¹³Compound 16 is not only an excellent rnNOS

inhibitor (36 nM), but its hnNOS inhibitory activity is

improved 4-fold relative to 5. In both the rnNOS-16 (Figure

6A) and hnNOS-16 structures (Figure 6B), the linker amino

group H-bonds with heme propionate D, leaving the tail

pyridino group near a mainly hydrophobic pocket. In both

structures, the tail pyridino group shows ﬂexibility, indicated by

weaker electron density. However, the position of the pyridine

ring can be identiﬁed at lower contour levels, and the crystal

structures indicate that the pyridine can have two functions. In

the rnNOS-16 structure, the pyridine acts as a hydrophobic

group, with its aryl hydrogens making van der Waals contacts

with Met336, Leu337, and Trp306 (Figure 6A), while in the

hnNOS-16 structure, the pyridine nitrogen forms a 2.8 Å H-

bond with His342, as well as hydrophobic contacts with

Met341 and Trp311 (Figure 6B). The same binding mode is

maintained in beNOS (Figure 6C). As with other inhibitors,

the rn/be selectivity (27-fold) may stem from Val106 versus

Met336 in rnNOS, as contacts with Val106 in beNOS are less

extensive and farther away than those with Met336 in rnNOS.

The substituted pyridines (17 and 18) were designed to

strengthen the H-bond acceptor potential of the pyridine,

whereas 19 was meant to mimic the haloaryl group of 4 and 5.

However, neither the rnNOS nor hnNOS activities correlate

with the electronics of the pyridine, suggesting other factors are

Pyridine Analogues. As alternative hydrophilic groups,

pyridine analogues 16−19 were designed to mimic 3, which

was shown by X-ray crystallography to H-bond with His342

and resulted in good hnNOS inhibiton for pyrimidinylimida-

H

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Figure 5. Active site structures of rnNOS-15 (A), hnNOS-15 (B), and

beNOS-15 (C). Important van der Waals contacts are marked with red

arrows, and distances are labeled in Å. The Trp residue from the other

subunit is labeled with a B in parentheses. The glycerol in beNOS is

labeled as Gol.

Figure 6. Active site structures of rnNOS-16 (A), hnNOS-16 (B), and

beNOS-16 (C). Major van der Waals contacts are marked with red

arrows. Gol is a glycerol molecule bound next to H₄B in beNOS.

is obviously a favorable binding mode. In the rnNOS-18

structure, the methoxyl group is oriented in the opposite

direction, where it forms favorable contacts with Leu337

(Figure 7B).

Inhibitor 19 contains a ﬂuorine, and in the hnNOS-19

structure (Supporting Information, Figure S3B), this ﬂuorine

points toward Met341, while in rnNOS the ﬂuorine faces the

opposite direction, toward Leu337. This further underscores

that interactions with Met341 are favored in hnNOS, while

interactions with Leu337 are favored in rnNOS. As in 16, a H-

bond exists (3.0 Å between the pyridine and His342), although

it may be a weaker interaction than in the hnNOS-16 structure

because of the ﬂuorine. Unfortunately, the n/e selectivity for

at play, as revealed by crystallography. In the rnNOS-17

structure (Supporting Information, Figure S2A), the extra

methyl group in 17 contacts Met336, Leu337, and Trp306. In

the hnNOS-17 structure (Supporting Information, Figure

S2B), the pyridino group cannot adopt the same orientation,

owing to potential close contacts with His342 and thus moves

closer to the H₄B site. Similarly, the pyridine ring is oriented

diﬀerently in the hnNOS-18 and rnNOS-18 structures (Figure

7). In the hnNOS-18 structure, the methoxyl group orients

away from His342 and interacts with Met341, whose side chain

is repositioned to maximize interactions with the methoxypyr-

idino group (Figure 7A). As 18 is a good hnNOS inhibitor, this

I

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Figure 7. Active site structures of hnNOS-18 (A) and rnNOS-18 (B). The distance from the tail pyridino group to His342 is more than 5.0 Å. The

methoxyl group makes hydrophobic contact (red arrow) with Leu337 in rnNOS.

these compounds remains quite low. Additionally, compound

18 has very similar K_ivalues between beNOS and heNOS,

indicating that pyridines may bind similarly to the eNOS

enzymes of these two species. The pyridine series, however, has

good n/i selectivity, suggesting that the pyridino group may

also behave as a hydrophobic group in iNOS, clashing with

Asn115. Compound 19 is an extremely weak iNOS inhibitor

that closely resembles 4, an analogue that is a similarly poor

binder to iNOS.

Benzonitrile Analogues. We also investigated the use of a

nitrile as a potential hydrogen-bond acceptor from His342.

Compounds 20 and 21 are both potent dual rnNOS and

hnNOS inhibitors. However, the hnNOS-21 (Figure 8A) and

rnNOS-21 structures (Figure 8B) are nearly identical. While

the electron density for the cyano group is weak, precluding

precise positioning, it is clear that the cyano group does not

prefer an orientation that enables strong H-bonding with

His342. Instead, in both the rnNOS and rhNOS, the

benzonitrile group points toward and contacts Trp311

(hnNOS) or Trp306 (rnNOS). We do, however, observe

electron density consistent with a minor conformer where the

benzonitrile faces toward His342 (hnNOS) or Leu337

(rnNOS). Computer modeling of the other conformation

conﬁrmed that, because of the length of the linker and the

bulkiness of the benzonitrile in 21, even when the cyano group

points toward His342 in hnNOS, a H-bond between the two

cannot be easily established.

Unfortunately, the rnNOS/beNOS selectivity for 20 and 21

is, like the pyridines, still fairly low. This is mainly because of

the tight binding of 21 to beNOS (Table 1). To our surprise, in

beNOS the benzonitrile ring actually makes more extensive

close contacts with Trp76 and Leu107, as well as the smaller

Val106 (Figure 8C). These benzonitrile compounds may

simply bind to beNOS too strongly to allow for high rn/be

selectivity.

Given the high potency of 20 and 21, we decided to try

several structure-based modiﬁcations to improve the n/e

selectivity. The o-methyl group of 22 and o-chlorine of 23

were introduced to provide possible contacts with Met336/

Met341 (rnNOS/hnNOS). Interestingly, while this modiﬁca-

tion does result in a slight improvement in rnNOS and hnNOS

potency, it does not improve selectivity for rnNOS over beNOS

relative to the unsubstituted benzonitrile compounds (20 and

21), but it does substantially improve selectivity for hnNOS

over heNOS. Both 22 and 23 exhibit hn/he selectivity >100-

fold because they bind >20-fold weaker to heNOS than beNOS

(Table 1). Examination of the crystal structures revealed a

potential reason for this disparity.

In the rnNOS-22 structure (Figure 9A), the o-methyl group

turns inward toward Leu337, the phenyl ring and cyano group

are approximately 4.2−4.5 Å from Met336, and the cyano

group maintains close contacts with Trp306. In the beNOS-22

structure (Figure 9B), the aryl ring ﬂips over by 180°, where the

aryl group contacts (∼4.0 Å) Val106 while the cyano group

makes similar contacts as in the rnNOS-22 structure. The

similarity in these nonbonded contacts might explain why 22 is

the strongest beNOS inhibitor (92 nM) in the series, with a rn/

be selectivity of only 4 (Table 1). Similar to observations in

other structures, in the hnNOS-22 structure (Figure 9C), the

benzonitrile has turned inward to form a weak H-bond with

His342, which places both the aryl ring and extra o-methyl

group 3.9−4.2 Å from Met341. In the heNOS-22 structure

(Figure 9D), not only is the His342−benzonitrile interaction

missing (as His342 is replaced by Phe105), but there is also no

interaction between Val104 and the o-methyl group of the

inhibitor, although the aryl ring does make some contact with

Val104. The only obvious diﬀerence between heNOS and

beNOS that could explain why 22 is a much poorer inhibitor of

heNOS than of beNOS is the Phe105 (heNOS) vs Leu107

(beNOS) diﬀerence. A similar phenomenon could occur with

compound 23 and account for its diﬀerent potency against

beNOS and heNOS (Table 2, Supporting Information, Figure

S4). These results suggest that diﬀerences in a single residue

among the same isoform from diﬀerent species, such as

Leu337/His342 in rnNOS vs hnNOS (and Leu107/Phe105 in

beNOS vs heNOS), or even, more importantly, between

diﬀerent isoforms, as Met336/Val104 in hnNOS vs heNOS,

can have signiﬁcant eﬀects on binding of the same compound.

The Eﬀect of Methylation. Finally, a methyl group was

introduced at the 4-position of the aminoquinoline to yield

compounds 24 and 25 (methylated versions of 21 and 22,

respectively). Previously, this modiﬁcation was reported to

improve potency for 2-aminopyridines²⁴by providing an extra

hydrophobic contact along the back wall of the heme-binding

pocket (an area known as the “S-pocket”). Interestingly,

possibly because of the larger size and increased interacting

surface area of 2-aminoquinoline in comparison of 2-amino-

pyridine, the 4-methyl has little eﬀect on potency of these 2-

aminoquinoline compounds for nNOS despite the extra

hydrophobic contact visible in the crystal structures, such as

in the hnNOS-25 crystal structure (Figure 10A; for the rnNOS-

25 structure see Supporting Information, Figure S5A).

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It is worth noting that the 4-methyl group increases n/e

selectivity for rnNOS and beNOS, and to a more signiﬁcant

degree, hnNOS and heNOS (the hn/he selectivity for 21 and

22 more than tripled and nearly doubled upon methylation in

24 and 25, respectively). This increase in selectivity was also

reported for 2-aminopyridines²⁴although it is not obvious why.

For this 2-aminoquinoline scaﬀold, the improved selectivity is

mainly the result of poorer eNOS binding upon adding the 4-

methyl group (22 vs 25, Table 1). Comparing the heNOS-25

with the heNOS-22 structure (Figure 10C) indicates that in

addition to the weakened H-bonds with the propionates and

the extra contact made by the 4-methyl group (vide supra),

there is a diﬀerence in the orientation of the o-methyl group on

the benzonitrile ring, which points toward Phe105 for 25 but

away from this residue for 22. When the 4-methyl group is

present, the position of the entire inhibitor (as in 25) is more

sequestered inside the NOS active site due to the extra contact

with the protein, and this could introduce steric hindrance and

torsional strain in the linker chain or hinder ﬂexibility required

to achieve maximal contact. The global eﬀects of all these

structural diﬀerences lead to poorer binding of the methylated

compounds to eNOS, resulting in higher n/e selectivity in

general. The trend of increasing selectivity is observed from 21

to 24 as well. The rnNOS-24, beNOS-24, and heNOS-24

structures, and the superimposition of 24 and 21 in beNOS, are

shown in Supporting Information, Figure S6.

Oﬀ-Target Proﬁling and Cellular Permeability Assay.

To assess the eﬃcacy of our modiﬁcations in reducing oﬀ-target

binding, the compound with the highest hn/he selectivity (25)

was tested in the PDSP (Table 3). In this assay,¹⁹compounds

were tested for binding to 45 diﬀerent CNS targets and

receptors via radioligand displacement. Initially, compounds

were tested at a primary high dose (10 μM) and then a

secondary K_idetermination was performed for compounds

showing >50% binding in the primary assay. We have classiﬁed

oﬀ-target binding using the following rubric: concerning (K_i<

100 nM, or < ∼2× nNOS K_ivalue), moderate (100−300 nM, or

∼2−5× nNOS K_ivalue), weak (>300 nM, or > ∼5× nNOS K_i

value, typically ∼1 μM), and insignificant (<50% at 10 μM).

The oﬀ-target proﬁle for 5 reveals concerning or moderate

binding at 15/45 targets (mostly serotonin and histamine

receptors). There were 22/45 targets that were classiﬁed as

weak and 8/45 as insigniﬁcant for this compound. Conversely,

for rearranged compound 6,¹⁵the fraction ﬂagged as

concerning or moderate decreased, with a concomitant increase

in “insigniﬁcant” binding. The PDSP results for 25 indicate that

the installation of the polar nitrile group is also eﬀective at

reducing oﬀ-target binding (despite two extra hydrophobic

methyl groups on the molecule). Although it is not as eﬀective

as 6’s structural rearrangement, 25 shows only three concerning

targets (the 5-HT1a receptor, alpha1A adrenergic receptor, and

dopamine D3 receptor) and less overall binding to serotonin

receptors, indicating that the polar group may be successfully

interrupting the GPCR-ligand-like pharmacophore of 5 or

decreasing nonspeciﬁc hydrophobic binding. Indeed, it is

reported that the installation of polar nitriles reduce overall

ligand lipophilicity⁴²and, by proxy, may reduce promiscuity

and negative toxicological observations relative to isosteric aryl

chlorides.²⁵

Figure 8. Active site structures of hnNOS-21 (A), rnNOS-21 (B), and

beNOS-21 (C). Close van der Waals contacts are marked with red

arrows.

How can the 4-methyl group not improve potency with this

extra contact? In the crystal structures of most of these

compounds, a stabilizing H-bond is present between the linker

amino group and nearby heme propionate.¹³With the 4-methyl

group present, as in the hnNOS-25 structure, the aminoquino-

line’s orientation is more parallel with the heme than that in an

unmethylated analogue such as 22 (Figure 10C), which

possibly strains the linker chain and weakens the H-bond

between the amine and propionate. At least for these 2-

aminoquinoline compounds, any favorable extra contact with

the 4-methyl group may be oﬀset by the weakened H-bond

between the linker amine and propionate.

Finally, compound 25 was tested for permeability in a Caco-

2 assay (Table 4) to estimate its membrane permeability (such

as through the intestinal lumen or the blood−brain barrier,

although the eﬃcacy of using Caco-2 to approximate passage

K

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Figure 9. Active site structures of rnNOS-22 (A), beNOS-22 (B), hnNOS-22 (C), and heNOS-22 (D). van der Waals contacts discussed in the text

are marked with red arrows. The weak H-bond mentioned in the text for hnNOS has a distance of 3.4 Å in one subunit but is farther in the other.

through the latter is usually less accurate⁴³due to fundamental

diﬀerences in the two membranes).

binding modes were also observed in the crystal structures. The

most potent dual rat/human nNOS inhibition (with selectivity

of approximately 1:1 between the nNOS enzymes of these two

species) was achieved by installation of a nitrile, and selectivity

for hnNOS over heNOS was improved to >100-fold by

placement of a hydrophobic substituent ortho- to the nitrile to

interact with Met341 (which is replaced by a smaller valine in

heNOS). Interestingly, this modiﬁcation did not improve

selectivity for rnNOS over beNOS. Finally, we found that

introduction of a methyl group at position 4 of the

aminoquinoline of our most potent benzonitrile-containing

leads, while not able to improve potency much toward nNOS,

was able to enhance n/e selectivity (both rnNOS/beNOS and

hnNOS/heNOS). One compound, the doubly methylated

benzonitrile 25, had nearly 200-fold hnNOS/heNOS selectiv-

ity, although, based on the crystal structures, interpretation of

this observation is not that straightforward. Compound 25,

when assayed in the PDSP screen, displayed a marked

reduction in oﬀ-target binding (relative to 5) without

compromising the excellent Caco-2 permeability of the ﬁrst-

generation compounds, indicating that cyanated tail moieties of

this type may be useful for designing new bioavailable dual

rnNOS/hnNOS inhibitors with improved safety proﬁles.

Compound 5 is extremely permeable in this assay, has high

compound recovery values and a low eﬄux ratio [ratio of

membrane permeability (A → B) to eﬄux (B → A), <3 is

considered favorable]. Pleasingly, despite the hydrophilic

modiﬁcation and increase in total polar surface area (from

50.4 Å²for 5 to 74.2 Å²for 25), compound 25 maintains very

good permeability, good recovery, and low eﬄux (indicating

that it is unlikely to be a signiﬁcant substrate for P-gp or other

drug eﬄux transporters).

CONCLUSIONS

■

In summary, we sought to optimize our ﬁrst generation of

potent and selective 2-aminoquinolines (4 and 5) to both (a)

improve their binding to hnNOS and selectivity over heNOS,

and (b) reduce oﬀ-target binding in CNS counter-screening

assays by disrupting the GPCR-ligand-like pharmacophore with

a hydrophilic group. Although truncation of the scaﬀold to

remove repulsion between the haloaryl moiety and the hnNOS-

speciﬁc residue His342 did not result in improved potency or

selectivity against rnNOS or hnNOS, N,N-dimethylaniline

containing inhibitors had very similar activities against rat and

human nNOS. Singly homologating the truncated inhibitors

improved this activity slightly, and double homologation

signiﬁcantly improved human nNOS potency. Crystal

structures indicated that the N,N-dimethylaniline could form

a weak H-bond with His342. Further improvements in potency

were achieved by introduction of a 3-pyridine-containing tail,

which could act as both a hydrophobic group (in rnNOS) and

H-bond acceptor for His342 (hnNOS), although alternative

EXPERIMENTAL SECTION

■

General Procedures. Anhydrous solvents (MeOH, DMF,

CH₂Cl₂, and THF) were distilled prior to use. All remaining solvents

and reagents were purchased from commercial vendors and were used

without further puriﬁcation. Methanolic HCl (3 M, for Boc-

deprotection and ammonium salt formation) was prepared fresh by

the reaction of acetyl chloride and anhydrous MeOH at 0 °C. Air- or

moisture-sensitive reactions were run under an atmosphere of dry

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a

Table 3. PDSP Binding Summary for Selected Compounds

compd

concerning

moderate

weak

insigniﬁcant

total

5

8

3

7

3

6

22

17

22

8

22

14

45

6

25

a

Oﬀ-target binding is classiﬁed into four categories: concerning (K_i<

100 nM, or < ∼2× nNOS K_ivalue), moderate (100−300 nM, or ∼2−

5× nNOS K_ivalue), weak (>300 nM, or > ∼5× nNOS K_ivalue,

typically ∼1 μM), and insignificant (<50% bound at 10 μM), for a total

of 45 receptors as assayed by the PDSP’s “comprehensive screen” (see

ref 17).

Table 4. Caco-2 Permeability Data for Selected

a

Compounds

apparent permeability (P_app

,

b

10⁻⁶cm s⁻¹

mean A→B mean B→A eﬄux ratio

)

recovery (%)

c

compd

A→B

B→A

5

30.3

20.8

23.2

24.5

20.1

22.5

1.2

0.81

0.97

8.0

98.0

58.5

67.0

83.0

25

d

warfarin

e

ranitidine

0.15

0.066

f

talinolol

3.4

51.5

a

All assays were performed over 2 h at a concentration of 10 μM. See

b

Experimental Section for details. P_app: apparent permeability rate

value. Eﬄux ratio: P_app(B → A)/P_app(A → B). High permeability

control. Low permeability control. High eﬄux control.

c

d

e

f

spectrometer in positive ion mode using ESI, with an Agilent

G1312A HPLC pump and an Agilent G1367B autoinjector. Data were

processed using MassHunter software version B.02.00. Analytical

HPLC was performed using an Agilent Inﬁnity 1260 HPLC system

with an injection volume of 10 μL. A Phenomenex Luna 5 μm C-8(2)

100 Å column, 50 mm × 4.60 mm, was used for all analytical HPLC

experiments. The purity of all ﬁnal target compounds was found to be

≥95% by HPLC, using a 10 min gradient of 95% H₂O/5% acetonitrile

+ 0.05% TFA to 95% acetonitrile/5% H₂O + 0.05% TFA, at 1.5 mL/

min. Preparative HPLC was performed at the Center for Molecular

Innovation and Drug Discovery ChemCore laboratory (Northwestern

University), using an Agilent 1200 series HPLC, an Agilent 6120

quadrupole mass spectrometer (API-MS mode), and a Phenomenex

Luna 5 μm C-8(2) 100 Å preparative HPLC column, 150 mm × 21.2

mm (gradients used are described under subheadings of individual

compounds). Microwave chemistry was performed using a Biotage

Initiator Sixty research microwave in Biotage vials. Analytical thin-layer

chromatography was performed on Silicycle extra-hard 250 μm TLC

plates. Intermediates and ﬁnal analogues were visualized with short-

wavelength UV light, KMnO₄, and ninhydrin stain, where relevant.

Compounds 26,¹⁵42,²⁹46−50,^13,30,3152,³⁷54,¹⁵75−77,³³⁻³⁵and

85−90^20,37were prepared by known literature procedures, and their

spectral data are consistent with those data reported for the same; their

preparations are not reported here. The preparations of 43, 44, 53,

64−69, and 78−82 are described in the Supporting Information.

Figure 10. Active site structures of hnNOS-25 (A) and heNOS-25

(B). To illustrate the diﬀerent binding modes of the 4-methylated vs

unmethylated 2-aminoquinolines in heNOS, the structural model of

25 (yellow) is overlaid (C) with that of 22 (cyan). To better visualize

the inhibitors, part C has been rotated relative to part B about an axis

perpendicular to the ﬁgure plane by 180°.

General Procedure 1: Synthesis and Deprotection of

26

Truncated Aniline-Linked 2-Aminoquinolines.

Step 1: A 5 mL sealable microwave vial was charged with

intermediate 26 (1 equiv) and KI (10−15 mol %), which were then

diluted with anhydrous MeCN (0.5−1 mL). A solution of the requisite

aniline (3 equiv) in MeCN (1−1.5 mL) was added. The vial was

sealed and the reaction stirred under microwave heating at 110 °C for

20−25 min. The resulting solution was diluted with CH₂Cl₂(∼20−30

mL) and washed with satd aq NaHCO₃(3 × 20 mL). The aqueous

phase was extracted with EtOAc (3 × 25 mL), and the combined

organics were washed with H₂O (20 mL) and satd aq NaCl (20 mL),

dried over anhydrous sodium sulfate, and concentrated. The residue

was puriﬁed by ﬂash column chromatography (SiO₂; the gradient is

described below for individual compounds) to yield the desired

argon. An Agilent 971-FP automated ﬂash puriﬁcation system with

SiliaSep (Silicycle, 40−63 μm, 60 Å, 12−80 g) prepacked silica

cartridges was used in puriﬁcation of all compounds that required it.

Melting points were determined in capillary tubes using a Buchi

melting point B-540 apparatus and are uncorrected. ¹H and ¹³C NMR

spectra were determined using a Bruker Avance-III 500 (direct

cryoprobe) instrument at 500 and 126 MHz, respectively. Low-

resolution ESIMS was performed using a Bruker AmaZonSL ion trap

mass spectrometer. High-resolution mass spectral data were obtained

at the Integrated Molecular Structure Education and Research Center

(Northwestern University) on an Agilent 6210A TOF mass

M

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secondary amine. Step 2: The protected intermediate was immediately

diluted with MeOH (9−10 mL) and K₂CO₃(2 equiv) was added. The

mixture was heated at reﬂux for 2−2.5 h, cooled, and concentrated,

and the residue was partitioned between EtOAc (10 mL) and H₂O/

satd aq NaCl (1:1, 10 mL). The layers were separated, the aqueous

phase was extracted with EtOAc (3 × 20 mL), and the organics were

combined, washed with satd aq NaCl (20 mL), dried over anhydrous

sodium sulfate, and concentrated. Step 3: The resulting free 2-

aminoquinoline was dissolved in MeOH (10 mL) and treated with

methanolic HCl (∼3 M, 1.5 mL). The mixture was stirred at room

temperature for 20 min, and ether (15 mL) was added, which aﬀorded

the desired compound after ﬁltration, washing with ether, and drying

in vacuo.

3-(((2-Aminoquinolin-7-yl)methyl)amino)benzonitrile Dihydro-

chloride (7). Prepared from 26 (0.100 g, 0.36 mmol) and 3-

aminobenzonitrile (27, 0.126 g, 1.07 mmol), using general procedure

1, step 1. After workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of 5% EtOAc in CH₂Cl₂to

35% EtOAc in CH₂Cl₂, intermediate 32 (0.094 g, 88%) was

deprotected with K₂CO₃(0.082 g, 0.59 mmol), using general

procedure 1, step 2. Following workup, the free aminoquinoline was

converted to the dihydrochloride salt, following general procedure 1,

step 3, to give the title compound as a white solid (0.093 g, 90% from

32): mp 219−223 °C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.07 (s, 1

H), 9.14 (br s, 1 H), 8.34 (d, J = 9.3 Hz, 1 H), 8.17 (br s, 1 H), 7.90

(d, J = 8.2 Hz, 1 H), 7.61 (br s, 1 H), 7.48 (dd, J = 8.2, 1.1 Hz, 1 H),

7.24 (t, J = 7.9 Hz, 1 H), 7.06 (d, J = 9.3 Hz, 1 H), 6.94−6.89 (m, 3

H), 4.53 (s, 2 H); the anilinium protons are broadened into residual

water and appear as a broad hump at 5.78 ppm. ¹³C NMR (126 MHz;

DMSO-d₆): δ 154.5, 148.9, 145.5, 143.1, 136.1, 130.3, 129.1, 124.3,

120.0, 119.6, 119.5, 117.4, 114.7, 114.2, 113.3, 111.8, 45.8. ESIMS m/z

(rel intensity) 275 (MH⁺, 100). HRMS calcd for C₁₇H₁₅N₄, 275.1296;

found, 275.1291.

113.1, 105.8, 101.9, 98.7, 54.6, 46.5. ESIMS m/z (rel intensity) 280

(MH⁺, 40). HRMS calcd for C₁₇H₁₈N₃O, 280.1450; found, 280.1447.

N-((2-Aminoquinolin-7-yl)methyl)-N,N-dimethylbenzene-1,3-dia-

mine Trihydrochloride (10). Prepared from 26 (0.100 g, 0.36 mmol)

and N,N-dimethylbenzene-1,3-diamine (30, 0.225g, 1.07 mmol), using

general procedure 1, step 1. After workup and puriﬁcation by ﬂash

column chromatography, eluting with a gradient of 5% EtOAc in

CH₂Cl₂to 50% EtOAc in CH₂Cl₂, intermediate 35 (0.057 g, 48%) was

deprotected with K₂CO₃(0.047 g, 0.34 mmol), using general

procedure 1, step 2. Following workup, the free aminoquinoline was

converted to the trihydrochloride salt, following general procedure 1,

step 3, to give the title compound as a white solid (0.043 g, 63% from

35): mp 216−218 °C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.16 (s, 1

H), 9.10 (s, 1 H), 8.34 (d, J = 9.0 Hz, 1 H), 8.16 (s, 1 H), 7.89 (d, J =

8.0 Hz, 1 H), 7.65 (s, 1 H), 7.49 (d, J = 8.0 Hz, 1 H), 7.17 (dd, J = 8.0,

8.0 Hz, 1 H), 7.06 (d, J = 9.0 Hz, 1 H), 6.90 (br s, 1 H), 6.81 (br s, 1

H), 6.57 (br s, 1 H), 4.50 (s, 2 H), 3.02 (s, 6 H); the anilinium protons

are broadened into residual water and appear as a broad hump at 3.48

ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 154.5, 149.5, 145.4, 143.3,

136.3, 130.9, 129.3, 124.9, 120.4, 115.5, 113.7, 113.0, 108.0, 104.6,

46.8, 45.7; one of the aminoquinoline carbons is not visible due to

baseline broadening. ESIMS m/z (rel intensity) 293 (MH⁺, 100).

HRMS calcd for C₁₈H₂₁N₄, 293.1766; found, 293.1759.

N-((2-Aminoquinolin-7-yl)methyl)-N,N-dimethylbenzene-1,4-dia-

mine Trihydrochloride (11). Prepared from 26 (0.056 g, 0.20 mmol)

and N,N-dimethylbenzene-1,4-diamine (31, 0.127g, 0.60 mmol), using

general procedure 1, step 1. After workup and puriﬁcation by ﬂash

column chromatography, eluting with a gradient of 5% EtOAc in

CH₂Cl₂to 50% EtOAc in CH₂Cl₂, intermediate 36 (0.052 g, 77%) was

deprotected with K₂CO₃(0.044 g, 0.32 mmol), using general

procedure 1, step 2. Following workup and puriﬁcation by ﬂash

column chromatography, eluting with a gradient of EtOAc to 15%

MeOH in EtOAc, the free aminoquinoline was converted to the

trihydrochloride salt, following general procedure 1, step 3, to give the

title compound as an oﬀ-white solid (0.027 g, 41% from 36): mp 242−

244 °C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.02 (s, 1 H), 12.36 (s, 1

H), 9.09 (s, 1 H), 8.33 (d, J = 9.0 Hz, 1 H), 8.13 (s, 1 H), 7.88 (d, J =

8.0 Hz, 1 H), 7.60 (s, 1 H), 7.47 (d, J = 8.0 Hz, 1 H), 7.43 (d, J = 6.0

Hz, 2 H), 7.03 (d, J = 9.0 Hz, 1 H), 6.64 (d, J = 6.0 Hz, 2 H), 4.50 (s, 2

H), 3.01 (s, 6 H); the anilinium protons are broadened into residual

water and appear as a broad hump at 4.09 ppm. ¹³C NMR (126 MHz;

DMSO-d₆): δ 154.9, 149.3, 146.1, 143.3, 136.3, 129.4, 124.6, 122.1,

120.4, 115.1, 113.7, 112.8, 46.3; the methyl carbon signal overlaps with

the solvent signal, and one of the aminoquinoline carbons is not visible

due to baseline broadening. ESIMS m/z (rel intensity) 293 (MH⁺,

100). HRMS calcd for C₁₈H₂₁N₄, 293.1766; found, 293.1764.

7-(((3-Fluorophenyl)amino)methyl)quinolin-2-amine Dihydro-

chloride (8). Prepared from 26 (0.100 g, 0.36 mmol) and 3-

ﬂuoroaniline (28, 0.119 g, 1.07 mmol), using general procedure 1, step

1. After workup and puriﬁcation by ﬂash column chromatography,

eluting with a gradient of 5% EtOAc in CH₂Cl₂to 35% EtOAc in

CH₂Cl₂, intermediate 33 (0.088 g, 84%) was deprotected with K₂CO₃

(0.078 g, 0.57 mmol), using general procedure 1, step 2. Following

workup, the free aminoquinoline was converted to the dihydrochloride

salt, following general procedure 1, step 3, to give the title compound

1

as a white solid (0.084 g, 87% from 33): mp 209−212 °C. H NMR

(500 MHz; DMSO-d₆): δ 14.09 (s, 1 H), 9.13 (br s, 1 H), 8.33 (d, J =

9.3 Hz, 1 H), 8.15 (br s, 1 H), 7.88 (d, J = 8.2 Hz, 1 H), 7.61 (s, 1 H),

7.47 (dd, J = 8.2, 0.9 Hz, 1 H), 7.07−7.02 (m, 2 H), 6.41 (dd, J = 8.2,

1.4 Hz, 1 H), 6.33−6.28 (m, 2 H), 4.47 (s, 2 H); the anilinium protons

are broadened into residual water and appear as a broad hump at 5.65

ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ (164.5 + 162.6, 1 C), 154.5,

(150.5 + 150.4, 1 C), 145.9, 143.1, 136.1, (130.53 + 130.45, 1 C),

129.0, 124.3, 120.0, 114.8, 113.3, 108.9, (102.4 + 102.2, 1 C), (98.8 +

98.6, 1 C), 46.2. ESIMS m/z (rel intensity) 268 (MH⁺, 100). HRMS

calcd for C₁₆H₁₅FN₃, 268.1250; found, 268.1244.

7-(((3-(Dimethylamino)benzyl)amino)methyl)quinolin-2-amine

25

Trihydrochloride (12).

3-(Aminomethyl)-N,N-dimethylaniline (37, 0.044 g, 0.29 mmol)

and Cs₂CO₃(0.094 g, 0.29 mmol) were diluted with anhydrous DMF

(3 mL) and stirred for 30 min at room temperature. A solution of 26

(0.070 g, 0.25 mmol) in anhydrous DMF (1 mL) was added dropwise

over 5 min while stirring, and the resulting yellow solution was stirred

at room temperature for 16 h and concentrated. The residue was

diluted with EtOAc (50 mL) and washed with H₂O (2 × 50 mL) and

satd aq NaCl (50 mL). The organics were dried with anhydrous

sodium sulfate, concentrated, and diluted with anhydrous THF (5

mL). A solution of Boc₂O (0.073 g, 0.34 mmol) in anhydrous THF (2

mL) was added dropwise, and the mixture was stirred at room

temperature for 18 h. The mixture was concentrated, diluted with

CH₂Cl₂(25 mL), and washed with satd aq NaHCO₃(30 mL), H₂O

(30 mL), and satd aq NaCl (30 mL). The organic layer was dried over

anhydrous sodium sulfate, concentrated, and puriﬁed by ﬂash column

chromatography, eluting with a gradient of CH₂Cl₂to 40% EtOAc in

CH₂Cl₂to aﬀord 39 as a clear oil (0.050 g, 45%). This was

deprotected using K₂CO₃(0.031 g, 0.22 mmol), using general

procedure 1, step 2. After workup, the resulting clear oil was treated

with methanolic HCl (3 mL), the mixture was stirred at room

temperature for 16 h, and ether (15 mL) was added, aﬀording an oﬀ-

white solid (0.030 g, 65% from 39) after ﬁltration and washing with

7-(((3-Methoxyphenyl)amino)methyl)quinolin-2-amine Dihydro-

chloride (9). Prepared from 26 (0.100 g, 0.36 mmol) and 3-

methoxyaniline (29, 0.132g, 1.07 mmol), using general procedure 1,

step 1. After workup and puriﬁcation by ﬂash column chromatography,

eluting with a gradient of 5% EtOAc in CH₂Cl₂to 35% EtOAc in

CH₂Cl₂, intermediate 34 (0.082 g, 71%) was deprotected with K₂CO₃

(0.070 g, 0.51 mmol), using general procedure 1, step 2. Following

workup, the free aminoquinoline was converted to the dihydrochloride

salt, following general procedure 1, step 3, to give the title compound

as a cream-colored solid (0.069 g, 77% from 34): mp 237−240 °C. ¹H

NMR (500 MHz; DMSO-d₆): δ 14.00 (s, 1 H), 9.09 (s, 1 H), 8.34 (d,

J = 9.3 Hz, 1 H), 8.16 (s, 1 H), 7.88 (d, J = 8.2 Hz, 1 H), 7.63 (s, 1 H),

7.49 (dd, J = 8.2, 1.2 Hz, 1 H), 7.05 (d, J = 9.3 Hz, 1 H), 6.96 (t, J =

8.0 Hz, 1 H), 6.21−6.14 (m, 3 H), 4.46 (s, 2 H), 3.63 (s, 3 H); the

anilinium protons are broadened into residual water and appear as a

broad hump at 4.76 ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 160.3,

154.2, 149.1, 146.1, 142.9, 135.9, 129.7, 128.8, 124.2, 119.8, 114.7,

N

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ether: mp 294−295 °C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.61 (s,

1 H), 10.08 (s, 2 H), 9.39 (br s, 1 H), 8.39 (d, J = 9.5 Hz, 1 H), 8.36

(br s, 1 H), 7.97 (d, J = 8.5 Hz, 1 H), 7.87 (s, 1 H), 7.71 (d, J = 8.5 Hz,

1 H), 7.49−7.31 (m, 2 H), 7.18 (d, J = 9.5 Hz, 1 H), 7.11 (br s, 2 H),

4.32 (t, J = 5.0 Hz, 2 H), 4.16 (t, J = 5.0 Hz, 2 H), 2.99 (s, 6 H); the

anilinium proton is broadened into residual water and appears as a

broad hump at 4.90 ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 155.2,

143.1, 137.0, 135.8, 133.4, 130.0, 129.4, 127.2, 121.3, 119.3, 117.2,

115.0, 50.6, 49.8, 42.7; one of the aminoquinoline carbons and two of

the aryl carbons are not visible due to baseline broadening. ESIMS m/

z (rel intensity) 307 (MH⁺, 100). HRMS calcd for C₁₉H₂₃N₄,

307.1923; found, 307.1915.

compounds) to yield the Boc-protected amine. Step 3: This

intermediate was not characterized but was instead deprotected as in

general procedure 1, step 2. Step 4: After workup, the resulting

unprotected aminoquinoline was treated with methanolic HCl (1.5

mL) in ether (5−10 mL), and the mixture was stirred at room

temperature for 16 h. Ether (15−20 mL) was then added, which

aﬀorded the desired compound after ﬁltration and washing with ether.

If one ether wash was insuﬃcient to remove impurities, compounds

were precipitated from methanol (1 mL) with ether (15 mL), washed

with ether, and dried in vacuo.

3-(Dimethylamino)phenethyl)amino)methyl)quinolin-2-amine

Trihydrochloride (14). Prepared from aldehyde 54 (0.080 g, 0.37

mmol) and phenethylamine 44 (0.073 g, 0.44 mmol), using general

procedure 2, step 1. After concentration, reduction with NaBH₄(0.021

g, 0.55 mmol), and workup, the secondary amine was protected with

Boc₂O (0.089 g, 0.41 mmol), following general procedure 2, step 2.

Workup and puriﬁcation by ﬂash column chromatography, eluting

with a gradient of CH₂Cl₂to 15% EtOAc in CH₂Cl₂, aﬀorded the

protected intermediate 56 (0.153 g, 90%). This was immediately

deprotected with K₂CO₃(0.091 g, 0.66 mmol) following general

procedure 2, step 3. Following workup, the Boc group was removed,

following general procedure 2, step 4. An analytically pure sample for

assay was prepared by preparative LC-MS, using the instrument and

column detailed in the General Procedures section, eluting with a

gradient of H₂O + 0.1% formic acid to 10% MeOH + 0.1% formic

acid/90% H₂O + 0.1% formic acid. The puriﬁed compound was

converted to the trihydrochloride salt following general procedure 1,

step 3, to give the title compound as an oﬀ-white solid (0.016 g, 10%

from 56): mp 276−278 °C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.50

(s, 1 H), 9.68 (s, 2 H), 9.31 (br s, 1 H), 8.39 (d, J = 9.0 Hz, 1 H), 8.28

(br s, 1 H), 7.99 (d, J = 8.0 Hz, 1 H), 7.88 (s, 1 H), 7.69 (d, J = 8.0 Hz,

1 H), 7.27 (br s, 1 H), 7.16 (d, J = 9.0 Hz, 1 H), 6.86 (br s, 3 H), 4.36

(t, J = 6.0 Hz, 2 H), 3.22−3.16 (m, 2 H), 3.01−2.90 (m, 8 H); the

anilinium proton is broadened into residual water and appears as a

broad hump at 4.41 ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 155.4,

143.2, 139.0, 137.1, 135.9, 130.2, 129.6, 127.0, 121.4, 119.2, 115.0,

49.9, 48.0, 32.1; four of the aryl carbons are not visible due to baseline

broadening and the methyl carbon signal overlaps with the solvent

signal. ESIMS m/z (rel intensity) 321 (MH⁺, 100). HRMS calcd for

C₂₀H₂₅N₄, 321.2079; found, 321.2072.

7-(((4-(Dimethylamino)phenethyl)amino)methyl)quinolin-2-

amine Trihydrochloride (15). Prepared from aldehyde 54 (0.080 g,

0.37 mmol) and phenethylamine 47 (0.073 g, 0.44 mmol), using

general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.021 g, 0.55 mmol), and workup, the secondary amine was

protected with Boc₂O (0.089 g, 0.41 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of CH₂Cl₂to 15% EtOAc in

CH₂Cl₂, aﬀorded the protected intermediate 57 (0.069 g, 40%). This

was immediately deprotected with K₂CO₃(0.041 g, 0.30 mmol)

following general procedure 2, step 3. Following workup, the Boc

group was removed using general procedure 2, step 4. An analytically

pure sample for assay was prepared by preparative LC-MS, using the

instrument and column detailed in the General Procedures section,

eluting with a gradient of H₂O + 0.1% formic acid to 20% MeOH +

0.1% formic acid/80% H₂O + 0.1% formic acid. The puriﬁed

compound was converted to the trihydrochloride salt following

general procedure 1, step 3, to give the title compound as a white solid

(0.008 g, 13% from 57): mp 243−245 °C. ¹H NMR (500 MHz;

DMSO-d₆): δ 14.37 (s, 1 H), 9.48 (s, 2 H), 9.24 (s, 1 H), 8.39 (d, J =

9.0 Hz, 1 H), 8.25 (s, 1 H), 7.99 (d, J = 8.0 Hz, 1 H), 7.85 (s, 1 H),

7.65 (d, J = 8.0 Hz, 1 H), 7.54 (d, J = 9.0 Hz, 1 H), 6.84 (br s, 4 H),

4.35 (t, J = 5.5 Hz, 2 H), 3.12 (br s, 2 H), 2.92 (br s, 8 H); the

anilinium proton is broadened into residual water and appears as a

broad hump at 3.81 ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 155.1,

143.2, 137.0, 135.9, 129.8, 129.6, 127.0, 126.9, 121.4, 119.2, 115.0,

49.9, 48.5, 31.1, 29.0; two of the aminoquinoline carbons are not

visible due to baseline broadening. ESIMS m/z (rel intensity) 321

(MH⁺, 100). HRMS calcd for C₂₀H₂₅N₄, 321.2079; found, 321.2071.

7-(((4-(Dimethylamino)benzyl)amino)methyl)quinolin-2-amine

25

Trihydrochloride (13).

4-(Aminomethyl)-N,N-dimethylaniline dihydrochloride (38, 0.041

g, 0.41 mmol) and Cs₂CO₃(0.370 g, 1.13 mmol) were diluted with

anhydrous DMF (3.5 mL) and stirred for 30 min at room temperature.

A solution of 26 (0.100 g, 0.36 mmol) in anhydrous DMF (1 mL) was

added dropwise over 5 min while stirring. The resulting pale-yellow

solution was stirred at room temperature for 16 h and concentrated.

The residue was diluted with EtOAc (50 mL) and washed with H₂O

(2 × 50 mL) and satd aq NaCl (50 mL). The organic layer was dried

over anhydrous sodium sulfate, concentrated, and diluted with

anhydrous THF (5 mL). A solution of Boc₂O (0.103 g, 0.47 mmol)

in anhydrous THF (3 mL) was added dropwise, and the mixture was

stirred at room temperature for 18 h. The mixture was concentrated,

diluted with CH₂Cl₂(25 mL), and washed with satd aq NaHCO₃(30

mL), H₂O (30 mL), and satd aq NaCl (30 mL). The organic layer was

dried over anhydrous sodium sulfate, concentrated, and puriﬁed by

ﬂash column chromatography, eluting with a gradient of CH₂Cl₂to

40% EtOAc in CH₂Cl₂to aﬀord 40 as a clear oil (0.071 g, 37%). This

was deprotected using K₂CO₃(0.031 g, 0.22 mmol), using general

procedure 1, step 2. After workup, the resulting clear oil was treated

with methanolic HCl (3 mL), the mixture was stirred at room

temperature for 16 h and ether (15 mL) was added, aﬀording a white

solid (0.042 g, 64% from 40) after ﬁltration and washing with ether:

1

mp 366−368 °C. H NMR (500 MHz; DMSO-d₆): δ 14.65 (s, 1 H),

10.01 (s, 2 H), 9.43 (s, 1 H), 8.38 (d, J = 9.0 Hz, 1 H), 8.36 (br s, 1

H), 7.95 (d, J = 8.5 Hz, 1 H), 7.85 (s, 1 H), 7.70 (d, J = 8.5 Hz, 1 H),

7.57 (br s, 2 H), 7.24 (br s, 2 H), 7.18 (d, J = 9.0 Hz, 1 H), 4.28 (t, J =

5.5 Hz, 2 H), 4.13 (t, J = 5.5 Hz, 2 H), 3.00 (s, 6 H); the anilinium

proton is broadened into residual water and appears as a broad hump

at 4.62 ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 155.2, 148.0, 143.1,

137.1, 135.7, 132.0, 129.4, 127.1, 121.3, 119.2, 116.5, 114.9, 50.0, 49.6,

42.9; one of the aminoquinoline carbons is not visible due to baseline

broadening. ESIMS m/z (rel intensity) 307 (MH⁺, 100). HRMS calcd

for C₁₉H₂₃N₄, 307.1923; found, 307.1914.

General Procedure 2: Synthesis of 2-Aminoquinolines

Containing a Phenethylamine or Phenylpropylamine-Derived

Tail Portion. Step 1: The requisite phenethylamine or propylamine

(1.1−1.2 equiv) was diluted with anhydrous CHCl₃(6−9 mL) or

CHCl₃/MeOH (10:1−5:1). If the amine was a hydrochloride salt,

Et₃N (1.5−2 equiv) was added and the mixture was stirred until clear.

Aldehyde 54 or 90 (1 equiv, as a solution in minimal CHCl₃) and

anhydrous sodium sulfate (∼1 g) were added to the reaction mixture,

and the resulting suspension was stirred at room temperature for 1 h.

Acetic acid (10 μL/10 mg amine) was added, and the mixture was

stirred at room temperature for 16 h. The resulting solution was

ﬁltered and concentrated to give the crude imine, which was diluted

with MeOH (4−7 mL) and cooled to 0 °C. NaBH₄(1.5 equiv) was

added while stirring, and the mixture was warmed to room

temperature, stirred for 20 min, and concentrated. The residue was

diluted with EtOAc (30 mL) and washed with satd aq NaHCO₃(25

mL), H₂O (25 mL), and satd aq NaCl (25 mL). The organic layer was

dried over anhydrous sodium sulfate and concentrated. Step 2: The

crude amine was diluted with anhydrous THF (5−7 mL) and Boc₂O

(1.1−1.2 equiv, as a solution in minimal anhydrous THF) was added.

The mixture was stirred at room temperature for 4−18 h and

concentrated, and the residue was puriﬁed by ﬂash column

chromatography (SiO₂; the gradient is described below for individual

O

DOI: 10.1021/acs.jmedchem.7b00835

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7-(((3-(Pyridin-3-yl)propyl)amino)methyl)quinolin-2-amine Trihy-

drochloride (16). Prepared from aldehyde 54 (0.080 g, 0.37 mmol)

and phenpropylamine 50 (0.086 g, 0.41 mmol), using general

procedure 2, step 1. After concentration, reduction with NaBH₄

(0.021 g, 0.55 mmol), and workup, the secondary amine was protected

with Boc₂O (0.088 g, 0.40 mmol), following general procedure 2, step

2. Workup and puriﬁcation by ﬂash column chromatography, eluting

with a gradient of EtOAc to 5% MeOH in EtOAc, aﬀorded the

protected intermediate 58 (0.110 g, 68%). This was immediately

deprotected with K₂CO₃(0.070 g, 0.51 mmol) following general

procedure 2, step 3. Following workup, the Boc group was removed

using general procedure 2, step 4, to give the title compound as a white

ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 154.9, 143.1, 142.8, 141.3,

136.7, 135.7, 129.2, 127.8, 126.7, 121.1, 118.9, 114.7, 109.3, 58.0, 49.6,

46.0, 24.3, 23.9; one of the aryl carbons is not visible due to baseline

broadening. ESIMS m/z (rel intensity) 323 (MH⁺, 100). HRMS calcd

for C₁₉H₂₃N₄O, 323.1872; found, 323.1867.

7-(((3-(5-Fluoropyridin-3-yl)propyl)amino)methyl)quinolin-2-

amine Trihydrochloride (19). Prepared from aldehyde 54 (0.065 g,

0.30 mmol) and phenpropylamine 69 (0.082 g, 0.36 mmol), using

general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.017 g, 0.45 mmol), and workup, the secondary amine was

protected with Boc₂O (0.072 g, 0.33 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with EtOAc, aﬀorded the protected

intermediate 72 (0.108 g, 79%). This was immediately deprotected

with K₂CO₃(0.066 g, 0.48 mmol) following general procedure 2, step

3. Following workup, the Boc group was removed using general

procedure 2, step 4, to give the title compound as a white solid (0.039

g, 40% from 72): mp 236−237 °C. ¹H NMR (500 MHz; DMSO-d₆):

δ 14.60 (s, 1 H), 9.80 (s, 2 H), 9.43 (s, 1 H), 8.57 (s, 1 H), 8.46 (s, 1

H), 8.38 (d, J = 9.5 Hz, 1 H), 8.35 (br s, 1 H), 7.96 (d, J = 8.0 Hz, 1

H), 7.87 (s, 1 H), 7.85 (s, 1 H), 7.73 (d, J = 8.0 Hz, 1 H), 7.19 (d, J =

9.5 Hz, 1 H), 4.30 (t, J = 5.5 Hz, 2 H), 2.97−2.87 (m, 2 H), 2.80 (t, J =

7.5 Hz, 2 H), 2.10−2.04 (m, 2 H); the pyridinium proton is broadened

into residual water and appears as a broad hump at 5.29 ppm. ¹³C

NMR (126 MHz; DMSO-d₆): δ (160.7 + 158.7, 1 C), 155.2, 144.9,

143.1, 140.0, 137.2, 135.8, (134.8 + 134.6, 1 C), 129.5, 127.0, (125.4 +

125.3, 1 C), 121.3, 119.1, 115.0, 49.8, 46.2, 28.9, 26.7. ESIMS m/z (rel

intensity) 311 (MH⁺, 100). HRMS calcd for C₁₈H₂₀FN₄, 311.1672;

found, 311.1669.

1

solid (0.075 g, 74% from 58): mp: 260−262.5 °C. H NMR (500

MHz; DMSO-d₆): δ 14.57 (br s, 1 H), 9.72 (s, 2 H), 9.33 (br s, 1 H),

8.82 (s, 1 H), 8.74 (d, J = 4.7 Hz, 1 H), 8.40−8.34 (m, 3 H), 7.98 (d, J

= 8.2 Hz, 1 H), 7.91−7.88 (m, 2 H), 7.71 (dd, J = 8.2, 1.4 Hz, 1 H),

7.16 (d, J = 9.3 Hz, 1 H), 4.31 (t, J = 5.6 Hz, 2 H), 2.98−2.93 (m, 2

H), 2.89 (t, J = 7.6 Hz, 2 H), 2.09 (quintet, J = 7.5 Hz, 2 H); the

pyridinium proton is broadened into residual water and appears as a

broad hump at 4.52 ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 154.8,

144.0, 143.2, 142.9, 141.6, 139.9, 136.8, 135.5, 129.2, 126.7, 126.4,

121.0, 118.9, 114.7, 49.5, 45.8, 28.8, 26.3. ESIMS m/z (rel intensity)

293 (MH⁺, 100). HRMS calcd for C₁₈H₂₁N₄, 293.1766; found,

293.1759.

7-(((3-(4-Methylpyridin-3-yl)propyl)amino)methyl)quinolin-2-

amine Trihydrochloride (17). Prepared from aldehyde 54 (0.080 g,

0.37 mmol) and phenpropylamine 67 (0.092 g, 0.41 mmol), using

general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.021 g, 0.55 mmol), and workup, the secondary amine was

protected with Boc₂O (0.088 g, 0.40 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of EtOAc to 5% MeOH in

EtOAc, aﬀorded the protected intermediate 70 (0.118 g, 71%). This

was immediately deprotected with K₂CO₃(0.072 g, 0.53 mmol)

following general procedure 2, step 3. Following workup, the Boc

group was removed using general procedure 2, step 4, to give the title

compound as a white solid (0.075 g, 69% from 70) after precipitation

3-(2-(((2-Aminoquinolin-7-yl)methyl)amino)ethyl)benzonitrile

Dihydrochloride (20). Prepared from aldehyde 54 (0.065 g, 0.30

mmol) and phenethylamine 53 (0.061 g, 0.33 mmol), using general

procedure 2, step 1. After concentration, reduction with NaBH₄(0.016

g, 0.42 mmol), and workup, the secondary amine was protected with

Boc₂O (0.072 g, 0.33 mmol), following general procedure 2, step 2.

Workup and puriﬁcation by ﬂash column chromatography, eluting

with a gradient of 5% EtOAc in CH₂Cl₂to 30% EtOAc in CH₂Cl₂,

aﬀorded the protected intermediate 59 (0.120 g, 89%). This was

immediately deprotected with K₂CO₃(0.078 g, 0.54 mmol) following

general procedure 2, step 3. Following workup and puriﬁcation by

ﬂash column chromatography, eluting with a gradient of EtOAc to 5%

MeOH in EtOAc, the Boc group was removed using general procedure

2, step 4, to give the title compound as a white solid (0.076 g, 75%

1

from hot MeOH (1 mL) using ether (15 mL): mp: 287−290 °C. H

NMR (500 MHz; DMSO-d₆): δ 14.73 (br s, 1 H), 9.81 (s, 2 H), 9.34

(br s, 1 H), 8.74 (s, 1 H), 8.67 (d, J = 5.9 Hz, 1 H), 8.39 (d, J = 9.3 Hz,

1 H), 8.30 (br s, 1 H), 7.98 (d, J = 8.2 Hz, 1 H), 7.88 (s, 1 H), 7.86 (d,

J = 5.8 Hz, 1 H), 7.74 (dd, J = 8.2, 1.4 Hz, 1 H), 7.17 (d, J = 9.3 Hz, 1

H), 4.32 (t, J = 5.4 Hz, 2 H), 3.01−2.98 (m, 2 H), 2.89 (t, J = 7.8 Hz, 2

H), 2.55−2.53 (m, 3 H), 2.08−2.02 (m, 2 H); the pyridinium proton

is broadened into residual water and appears as a broad hump at 3.65

ppm. ¹³C NMR (126 MHz; DMSO-d₆): δ 154.7, 142.7, 140.5, 139.41,

139.40, 138.9, 136.7, 135.4, 129.1, 127.8, 126.6, 120.9, 118.7, 114.5,

49.4, 45.9, 26.5, 24.6, 19.5. ESIMS m/z (rel intensity) 307 (MH⁺,

100). HRMS calcd for C₁₉H₂₃N₄, 307.1923; found, 307.1923.

1

from 59): mp 268−269 °C (softens), 290−293 °C (melts). H NMR

(500 MHz; DMSO-d₆): δ 14.52 (s, 1 H), 9.72 (s, 2 H), 9.31 (br s, 1

H), 8.38 (d, J = 9.3 Hz, 1 H), 8.30 (br s, 1 H), 7.97 (d, J = 8.2 Hz, 1

H), 7.87 (s, 1 H), 7.78 (s, 1 H), 7.74 (dt, J = 7.7, 1.3 Hz, 1 H), 7.68

(dd, J = 8.2, 1.0 Hz, 1 H), 7.64 (d, J = 8.0 Hz, 1 H), 7.56 (t, J = 7.7 Hz,

1 H), 7.15 (d, J = 9.3 Hz, 1 H), 4.34 (s, 2 H), 3.24−3.23 (m, 2 H),

3.11 (t, J = 7.9 Hz, 2 H). ¹³C NMR (126 MHz; DMSO-d₆): δ 154.7,

142.6, 138.9, 136.4, 133.9, 132.3, 130.7, 129.8, 129.1, 126.5, 120.9,

118.8, 118.7, 114.5, 111.5, 49.5, 47.1, 30.8; one of the quinoline

carbons is not visible due to baseline broadening. ESIMS m/z (rel

intensity) 303 (MH⁺, 100). HRMS calcd for C₁₉H₁₉N₄, 303.1610;

found, 303.1602.

7-(((3-(4-Methoxypyridin-3-yl)propyl)amino)methyl)quinolin-2-

amine Trihydrochloride (18). Prepared from aldehyde 54 (0.080 g,

0.37 mmol) and phenpropylamine 68 (0.107 g, 0.45 mmol), using

general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.021 g, 0.55 mmol), and workup, the secondary amine was

protected with Boc₂O (0.098 g, 0.45 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of EtOAc to 7% MeOH in

EtOAc, aﬀorded the protected intermediate 71 (0.133 g, 77%). This

was immediately deprotected with K₂CO₃(0.072 g, 0.53 mmol)

following general procedure 2, step 3. Following workup, the Boc

group was removed using general procedure 2, step 4, to give the title

compound as a white solid (0.100 g, 81% from 71) after precipitation

4-(2-(((2-Aminoquinolin-7-yl)methyl)amino)ethyl)benzonitrile

Dihydrochloride (21). Prepared from aldehyde 54 (0.070 g, 0.33

mmol) and 4-cyano-phenethylamine hydrochloride (55, 0.071 g, 0.39

mmol), using general procedure 2, step 1. After concentration,

reduction with NaBH₄(0.019 g, 0.50 mmol), and workup, the

secondary amine was protected with Boc₂O (0.078 g, 0.36 mmol),

following general procedure 2, step 2. Workup and puriﬁcation by ﬂash

column chromatography, eluting with a gradient of CH₂Cl₂to 10%

EtOAc in CH₂Cl₂, aﬀorded the protected intermediate 60 (0.118 g,

79%). This was immediately deprotected with K₂CO₃(0.072 g, 0.52

mmol) following general procedure 2, step 3. Following workup, the

Boc group was removed using general procedure 2, step 4, to give the

title compound as a white solid (0.057 g, 57% from 60): mp 292−294

°C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.57 (s, 1 H), 9.85 (s, 2 H),

9.36 (s, 1 H), 8.38 (d, J = 9.5 Hz, 1 H), 8.33 (br s, 1 H), 7.97 (d, J =

1

from hot MeOH (1 mL) using ether (15 mL): mp: 225−228 °C. H

NMR (500 MHz; DMSO-d₆): δ 9.63 (br s, 2 H), 8.77 (d, J = 6.7 Hz, 1

H), 8.68 (s, 1 H), 8.39 (d, J = 9.4 Hz, 1 H), 7.98 (d, J = 8.2 Hz, 1 H),

7.87 (s, 1 H), 7.69 (dd, J = 8.2, 1.4 Hz, 1 H), 7.61 (d, J = 6.8 Hz, 1 H),

7.15 (d, J = 9.3 Hz, 1 H), 4.32−4.30 (m, 2 H), 4.11 (s, 3 H), 2.98−

2.93 (m, 2 H), 2.76 (t, J = 7.5 Hz, 2 H), 2.04−1.98 (m, 2 H). The

pyridinium, quinolinium, and aminoquinoline N−H protons are

broadened into residual water and appear as a broad hump at 3.39

P

DOI: 10.1021/acs.jmedchem.7b00835

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Journal of Medicinal Chemistry

Article

8.0 Hz, 1 H), 7.87 (s, 1 H), 7.81 (d, J = 8.0 Hz, 2 H), 7.71 (d, J = 9.5

Hz, 1 H), 7.49 (d, J = 8.0 Hz, 2 H), 7.17 (d, J = 8.0 Hz, 1 H), 4.34 (s, 2

H), 3.21 (t, J = 5.0 Hz, 2 H), 3.18−3.12 (m, 2 H). ¹³C NMR (126

MHz, DMSO-d₆): δ 159.9, 148.5, 147.9, 147.8, 141.7, 140.7, 137.8,

135.0, 134.3, 131.8, 126.1, 124.0, 119.7, 114.9, 54.7, 52.2, 36.6. ESIMS

m/z (rel intensity) 303 (MH⁺, 100). HRMS calcd for C₁₉H₁₉N₄,

303.1610; found, 303.1603.

4-(2-(((2-Aminoquinolin-7-yl)methyl)amino)ethyl)-2-methylben-

zonitrile Dihydrochloride (22). Prepared from aldehyde 54 (0.037 g,

0.17 mmol) and phenethylamine 81 (0.040 g, 0.20 mmol), using

general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.010 g, 0.26 mmol), and workup, the secondary amine was

protected with Boc₂O (0.041 g, 0.19 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of CH₂Cl₂to 15% EtOAc in

CH₂Cl₂, aﬀorded the protected intermediate 83 (0.043 g, 55%). This

was immediately deprotected with K₂CO₃(0.026 g, 0.19 mmol)

following general procedure 2, step 3. Following workup, the Boc

group was removed using general procedure 2, step 4, to give the title

compound as a white solid (0.019 g, 54% from 83): mp 316−317 °C.

¹H NMR (500 MHz; DMSO-d₆): δ 14.43 (s, 1 H), 9.65 (s, 2 H), 9.24

(s, 1 H), 8.38 (d, J = 9.0 Hz, 1 H), 8.28 (br s, 1 H), 7.98 (d, J = 8.0 Hz,

1 H), 7.86 (s, 1 H), 7.74 (d, J = 8.0 Hz, 1 H), 7.65 (d, J = 8.0 Hz, 1 H),

7.37 (s, 1 H), 7.29 (d, J = 8.0 Hz, 1 H), 7.14 (d, J = 9.0 Hz, 1 H), 4.35

(s, 2 H), 3.26−3.17 (m, 2 H), 3.12−3.08 (m, 2 H), 2.47 (s, 3 H). ¹³C

NMR (126 MHz; DMSO-d₆): δ 154.6, 152.5, 143.7, 137.7, 135.8,

133.0, 130.3, 126.8, 121.7, 119.6, 119.3, 113.7, 110.2, 49.8, 47.4, 31.9,

19.5; three of the aminoquinoline carbons are not visible due to

baseline broadening. ESIMS m/z (rel intensity) 317 (MH⁺, 100).

HRMS calcd for C₂₀H₂₁N₄, 317.1766; found, 317.1759.

(m, 2 H), 2.64 (s, 3 H). ¹³C NMR (126 MHz; DMSO-d₆): δ 154.6,

152.5, 143.7, 136.7, 133.0, 130.3, 126.8, 126.4, 121.7, 119.5, 119.3,

113.7, 110.2, 49.8, 47.4, 31.9, 19.5; one of the aminoquinoline carbons

is not visible due to baseline broadening. ESIMS m/z (rel intensity)

317 (MH⁺, 100). HRMS calcd for C₂₀H₂₁N₄, 317.1766; found,

317.1761.

4-(2-(((2-Amino-4-methylquinolin-7-yl)methyl)amino)ethyl)-2-

methylbenzonitrile Dihydrochloride (25). Prepared from aldehyde 90

(0.072 g, 0.315 mmol) and phenethylamine 81 (0.065 g, 0.33 mmol),

using general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.017 g, 0.45 mmol), and workup, the secondary amine was

protected with Boc₂O (0.072 g, 0.33 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of CH₂Cl₂to 35% EtOAc in

CH₂Cl₂, aﬀorded the protected intermediate 92 (0.141 g, 95%). This

was immediately deprotected with K₂CO₃(0.082 g, 0.60 mmol)

following general procedure 2, step 3. Following workup, the Boc

group was removed using general procedure 2, step 4, to give the title

compound as a white solid (0.096 g, 80%): mp 300−301 °C. ¹H NMR

(500 MHz; DMSO-d₆): δ 14.15 (s, 1 H), 9.58 (s, 2 H), 9.02 (br s, 1

H), 8.20 (br s, 1 H), 8.05 (d, J = 7.5 Hz, 1 H), 7.82 (s, 1 H), 7.75 (d, J

= 8.0 Hz, 1 H), 7.65 (br s, 1 H), 7.37 (s, 1 H), 7.29 (d, J = 8.0 Hz, 1

H), 6.95 (s, 1 H), 4.35 (s, 2 H), 3.26−3.15 (m, 2 H), 3.07 (t, J = 9.0

Hz, 2 H), 2.63 (s, 3 H), 2.47 (s, 3 H). ¹³C NMR (126 MHz; DMSO-

d₆): δ 143.4, 142.3, 133.3, 131.2, 127.5, 126.3, 121.9, 118.4, 113.7,

110.6, 49.9, 47.4, 31.8, 20.4, 19.4; four of the quinoline carbons and

two of the aryl carbons are not visible due to baseline broadening.

ESIMS m/z (rel intensity) 331 (MH⁺, 100). HRMS calcd for

C₂₁H₂₃N₄, 331.1922; found, 331.1924.

Puriﬁed NOS Enzyme Assays. Rat and human nNOS, murine

macrophage iNOS, and human and bovine eNOS were recombinant

enzymes (expressed in Escherichia coli and puriﬁed as reported

previously).⁴⁴⁻⁴⁶The hemoglobin capture assay was used to measure

nitric oxide production (to test for NOS inhibition). The assay was

performed at 37 °C in HEPES buﬀer (100 mM with 10% glycerol, pH

7.4) in the presence of 10 μM ^L-arginine. NADPH (100 μM), CaCl₂

(0.83 mM), calmodulin (approximately 320 units/mL), H₄B (10 μM),

and human oxyhemoglobin (3 μM) were also included in the assay

mixture. For iNOS, the CaCl₂and calmodulin were omitted and

replaced with HEPES buﬀer (as neither are required for iNOS

function). The assay was performed in 96-well plates using a Synergy 4

BioTek hybrid reader. Each well contained approximately 100 nM

enzyme. The dispensing of NOS enzyme and hemoglobin were

automated, and after 30 s (maximum delay), NO production was read

by monitoring the absorbance at 401 nm (resulting from conversion of

oxyhemoglobin to methemoglobin). Kinetic readouts were performed

for 5 min. Each compound was assayed at least in duplicate, and seven

to nine concentrations (500 μM to 50 nM or 100 μM to 10 nM for

eNOS and iNOS; 50 μM to 5 nM for rat and human nNOS) were

used to construct dose−response curves. IC₅₀values were calculated

by nonlinear regression (variable slope, four parameters, bottom

constraint set to 0 and top to 100) using GraphPad Prism software

(reported standard error is reported for the LogIC₅₀), and K_ivalues

were obtained from IC₅₀values, using the Cheng−Prusoﬀ⁴⁷equation

[K_i= IC₅₀/(1 + [S]/K_m)] with the following K_mvalues: 1.3 μM (rat

nNOS), 1.6 μM (human nNOS), 8.2 μM (murine macrophage iNOS),

1.7 μM (bovine eNOS), and 3.9 μM (human eNOS).⁴⁸

Inhibitor Complex Crystal Preparation. The sitting drop vapor

diﬀusion methods were used to grow crystals at 4 °C for the heme

domains of rat nNOS (8 mg/mL containing 20 mM histidine), the

human nNOS K301R/R354A/G357D mutant (10 mg/mL), and

human eNOS (7 mg/mL). The crystal growth conditions are as

described previously.²⁰Fresh crystals were ﬁrst passed stepwise

through cryoprotectant solutions and then soaked with 5−10 mM

inhibitor for 3−4 h at 4 °C before being ﬂash cooled with liquid

nitrogen and stored until data collection. The presence of an acetate

ion near the heme active site in bovine eNOS had caused interference

in the binding mode of some phenyl ether-linked aminoquinoline

compounds.^15,20The high concentration of magnesium acetate in the

heNOS growth conditions may also introduce an acetate near the

4-(2-(((2-Aminoquinolin-7-yl)methyl)amino)ethyl)-2-chloroben-

zonitrile Dihydrochloride (23). Prepared from aldehyde 54 (0.029 g,

0.13 mmol) and phenethylamine 82 (0.035 g, 0.16 mmol), using

general procedure 2, step 1. After concentration, reduction with

NaBH₄(0.008 g, 0.20 mmol), and workup, the secondary amine was

protected with Boc₂O (0.031 g, 0.14 mmol), following general

procedure 2, step 2. Workup and puriﬁcation by ﬂash column

chromatography, eluting with a gradient of 5% EtOAc in CH₂Cl₂to

35% EtOAc in CH₂Cl₂, aﬀorded the protected intermediate 84 (0.046

g, 72%). This was immediately deprotected with K₂CO₃(0.027 g, 0.19

mmol) following general procedure 2, step 3. Following workup, the

Boc group was removed using general procedure 2, step 4, to give the

title compound as a cream-colored solid (0.022 g, 56% from 84): mp

309−311 °C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.37 (s, 1 H), 9.58

(s, 2 H), 9.24 (br s, 1 H), 8.38 (d, J = 8.0 Hz, 1 H), 8.24 (br s, 1 H),

8.02−7.94 (m, 2 H), 7.85 (s, 1 H), 7.77 (s, 1 H), 7.64 (d, J = 8.0 Hz, 1

H), 7.49 (dd, J = 9.0 Hz, 1.5 Hz, 1 H), 7.14 (d, J = 9.0 Hz, 1 H), 4.39−

4.32 (m, 2 H), 3.28 (s, 2 H), 3.17−3.12 (m, 2 H). ¹³C NMR (126

MHz; DMSO-d₆): δ 155.1, 145.9, 143.2, 135.9, 135.2, 130.8, 129.6,

129.2, 126.9, 121.5, 119.3, 116.5, 115.0, 110.8, 60.1, 47.1, 31.6; two of

the aminoquinoline carbons are not visible due to baseline broadening.

ESIMS m/z (rel intensity) 337/339 (MH⁺, 100/35). HRMS calcd for

C₁₉H₁₈ClN₄, 337.1220; found, 337.1218.

4-(2-(((2-Amino-4-methylquinolin-7-yl)methyl)amino)ethyl)-

benzonitrile Dihydrochloride (24). Prepared from aldehyde 90 (0.060

g, 0.26 mmol) and 4-cyanophenethylamine hydrochloride (55, 0.058

g, 0.32 mmol), using general procedure 2, step 1. After concentration,

reduction with NaBH₄(0.015 g, 0.39 mmol), and workup, the

secondary amine was protected with Boc₂O (0.063 g, 0.29 mmol),

following general procedure 2, step 2. Workup and puriﬁcation by ﬂash

column chromatography, eluting with a gradient of CH₂Cl₂to 12%

EtOAc in CH₂Cl₂, aﬀorded the protected intermediate 91 (0.071 g,

62%). This was immediately deprotected with K₂CO₃(0.044 g, 0.32

mmol) following general procedure 2, step 3. Following workup, the

Boc group was removed using general procedure 2, step 4, to give the

title compound as a white solid (0.029 g, 47% from 91): mp 304−306

°C. ¹H NMR (500 MHz; DMSO-d₆): δ 14.28 (s, 1 H), 9.73 (s, 2 H),

9.11 (br s, 1 H), 8.18 (br s, 1 H), 8.05 (d, J = 8.0 Hz, 1 H), 7.85 (s, 1

H), 7.82 (d, J = 8.0 Hz, 2 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.49 (d, J = 8.0

Hz, 2 H), 6.98 (s, 1 H), 4.35 (s, 2 H), 3.27−3.20 (m, 2 H), 3.16−3.12

Q

DOI: 10.1021/acs.jmedchem.7b00835

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Journal of Medicinal Chemistry

Article

active site that may inﬂuence the binding mode of inhibitors. To avoid

having this acetate in the structure, the magnesium acetate in the

cryoprotectant solution was replaced with MgCl₂.

5VV1; hnNOS-19, 5VV2; hnNOS-21, 5VV3; hnNOS-22,

5VV4; hnNOS-25, 5VV5; beNOS-15, 5VV6; beNOS-16,

5VV7; beNOS-21, 5VVN; beNOS-22, 5VV8; beNOS-24,

5VV9; beNOS-23, 5VVG; beNOS-25, 5VVA; heNOS-22,

5VVB; heNOS-24, 5VVD; heNOS-25, 5VVC.

X-ray Diﬀraction Data Collection, Data Processing, and

Structural Reﬁnement. The cryogenic (100 K) X-ray diﬀraction

data were collected remotely at the Stanford Synchrotron Radiation

Lightsource (SSRL) or Advanced Light Source (ALS) through the

data collection control software Blu-Ice⁴⁹and a crystal-mounting

robot. When a Q315r CCD detector was used, 100−125° of data were

typically collected with 0.5° per frame. If a Pilatus pixel array detector

was used, 140−160° of ﬁne-sliced data were collected with a 0.2° per

frame. Raw CCD data frames were indexed, integrated, and scaled

using iMOSFLM,⁵⁰but the pixel array data were processed with

XDS⁵¹and scaled with Aimless.⁵²The binding of inhibitors was

detected by initial diﬀerence Fourier maps calculated with REFMAC.⁵³

The inhibitor molecules were then modeled in Coot⁵⁴and reﬁned

using REFMAC or PHENIX.⁵⁵The crystal packing of the MgCl₂-

soaked heNOS crystals was changed slightly, resulting in a symmetry

change from the orthorhombic P2₁2₁2₁reported previously¹⁹to

monoclinic P2₁, with a β angle only 0.6−0.7° oﬀ compared to the

original 90°. Therefore, a molecular replacement calculation with

PHASER-MR⁵⁶was needed to solve the structure. In the P2₁space

group, there are two heNOS dimers in the asymmetric unit.

Disordering in portions of inhibitors bound in the NOS active sites

was often observed, sometimes resulting in poor density quality.

However, partial structural features were usually still visible if the

contour level of the σA weighted 2m|F_o| − D|F_c| map was dropped to

0.5σ, which aﬀorded the building of reasonable models into the

disordered regions. Water molecules were added in PHENIX and

checked by Coot. The TLS⁵⁷protocol was implemented in the ﬁnal

stage of reﬁnements with each subunit as one TLS group. The omit F_o

− F_cdensity maps were calculated by removing inhibitor coordinates

from the input PDB ﬁle before running one more round of TLS

reﬁnement in PHENIX (simulated annealing protocol with a 2000 K

initial temperature). The resulting map coeﬃcients DELFWT and

PHDELWT were used to generate maps. The reﬁned structures were

validated in Coot before deposition in the Protein Data Bank.

Caco-2 Permeability Assay. Caco-2 monolayer assays were

performed by Cyprotex US, LLC (Watertown, MA), using standard

procedures, as previously reported for aminoquinolines.¹⁵

AUTHOR INFORMATION

■

Corresponding Authors

*For R.B.S.: phone, +1 847 491 5653; fax, +1 847 491 7713; E-

mail, Agman@chem.northwestern.edu.

*For T.L.P.: phone, +1 949 824 7020; E-mail, poulos@uci.edu.

ORCID

Thomas L. Poulos: 0000-0002-5648-3510

Richard B. Silverman: 0000-0001-9034-1084

Author Contributions

^⊥A.V.P. and M.A.C. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the National Institutes of Health (R01GM049725, to

R.B.S., GM057353 to T.L.P., and F32GM109667 to M.A.C.)

for generous support of this work. A.P. was supported by a

Lambert Fellowship (Chemistry of Life Processes Institute,

Northwestern University) and by the Katherine L. Kreighbaum

Scholarship (Northwestern University). L.J.R. is currently

supported on NIH GM081568 and NSF grant 13-573. P.M.

is supported by grants UNCE 204011 and PRVOUK P24/

LF1/3 from Charles University, Prague, Czech Republic. A.P.

and M.A.C. thank Saman Shafaie and Dr. S. Habibi Goudarzi

for assistance with HRMS experiments, and Drs. Arsen Gaisin

and Neha Malik of the Center for Molecular Innovation and

Drug Discovery (Northwestern University) for valuable

assistance with preparative HPLC. This work made use of

IMSERC at Northwestern University, which has received

support from the Soft and Hybrid Nanotechnology Exper-

imental (SHyNE) Resource (NSF NNCI-1542205), the State

of Illinois, and the International Institute for Nanotechnology

(IIN). H.L. thanks Carla Plaza for her assistance in NOS

protein expression and puriﬁcation; the puriﬁed samples were

used in both crystallography and enzyme assays. We also thank

the SSRL and ALS beamline staﬀ for their support during

remote X-ray diﬀraction data collection. Oﬀ-target K_i

determinations (CNS counterscreening) were generously

provided by the National Institute of Mental Health’s

Psychoactive Drug Screening Program, (contract no. HHSN-

271-2013-00017-C, NIMH PDSP), directed by Dr. Bryan L.

Roth (University of North Carolina at Chapel Hill) and project

oﬃcer Jamie Driscoll (NIH).

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b00835.

Crystallographic data collection and reﬁnement statistics

for rat and human nNOS and bovine eNOS-inhibitor

crystal structures: rnNOS-12 and hnNOS-12, rnNOS-17

and hnNOS-17, rnNOS-19 and hnNOS-19, rnNOS-23

and beNOS-23, rnNOS-25 and beNOS-25, rnNOS-24,

beNOS-24, and heNOS-24 crystal structures, and

synthesis and analytical data for compounds 43, 44, 53,

64−69 and 78−82 (PDF)

Molecular formula strings (CSV)

Accession Codes

X-ray crystal structures described in this study have been

deposited in the Protein Data Bank. We will release the atomic

coordinates and experimental data upon article publication

under the following accession codes: rnNOS-8, 5VUI; rnNOS-

12, 5VUJ; rnNOS-13, 5VUK; rnNOS-15, 5VUL; rnNOS-16,

5VUM; rnNOS-17, 5VUN; rnNOS-18, 5VUO; rnNOS-19,

5VUP; rnNOS-21, 5VUQ; rnNOS-22, 5VUR; rnNOS-23,

5VUS; rnNOS-24, 5VUT; rnNOS-25, 5VUU; hnNOS-8,

5VUV; hnNOS-12, 5VUW; hnNOS-13, 5VUX; hnNOS-15,

5VUY; hnNOS-16, 5VUZ; hnNOS-17, 5VV0; hnNOS-18,

ABBREVIATIONS USED

■

NO, nitric oxide; nNOS, neuronal nitric oxide synthase; eNOS,

endothelial nitric oxide synthase; iNOS, inducible nitric oxide

synthase; rnNOS, rat nNOS; hnNOS, human nNOS; beNOS,

bovine eNOS; heNOS, human eNOS; miNOS, murine

macrophage iNOS; P_app, apparent permeability; HEPES, 4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid; PDSP, psycho-

active drug screening program; FMN, ﬂavin mononucleotide;

H₄B, (6R)-5,6,7,8-tetrahydrobiopterin; PSA, polar surface area

R

DOI: 10.1021/acs.jmedchem.7b00835

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

L.; Hopkins, A. L. Automated design of ligands to polypharmacological

profiles. Nature 2012, 492, 215−220.

(20) Cinelli, M. A.; Li, H.; Chreifi, G.; Poulos, T. L.; Silverman, R. B.

Nitrile in the hole: discovery of a small auxiliary pocket in neuronal

nitric oxide synthase leading to the development of potent and

selective 2-aminoquinoline inhibitors. J. Med. Chem. 2017, 60, 3958−

3978.

REFERENCES

■

(1) Hebert, L. E.; Scherr, P. A.; Bienias, J. L.; Bennett, D. A.; Evans,

D. A. Alzheimer disease in the US population: prevalence estimates

using the 2000 census. Arch. Neurol. 2003, 60, 1119−1122.

(2) Torreilles, F.; Salman-Tabcheh, S.; Guerin, M.; Torreilles, J.

Neurodegenerative disorders: the role of peroxynitrite. Brain Res. Rev.

1999, 30, 153−163.

(3) Zhang, L.; Dawson, V. L.; Dawson, T. M. Role of nitric oxide in

Parkinson’s disease. Pharmacol. Ther. 2006, 109, 33−41.

(4) Huang, Z.; Huang, P. L.; Panahian, N.; Dalkara, T.; Fishman, M.

C.; Moskowitz, M. A. Effects of cerebral ischemia in mice deficient in

neuronal nitric oxide synthase. Science 1994, 265, 1883−1885.

(5) Dorheim, M.-A.; Tracey, W. R.; Pollock, J. S.; Grammas, P. Nitric

oxide synthase activity is elevated in brain microvessels in Alzheimer’s

disease. Biochem. Biophys. Res. Commun. 1994, 205, 659−665.

(6) Kim, K. H.; Kim, J. I.; Han, J. A.; Choe, M. A.; Ahn, J. H.

Upregulation of neuronal nitric oxide synthase in the periphery

promotes pain hypersensitivity after peripheral nerve injury. Neuro-

science 2011, 190, 367−378.

(21) Li, H.; Jamal, J.; Plaza, C.; Pineda, S. H.; Chreifi, G.; Jing, Q.;

Cinelli, M. A.; Silverman, R. B.; Poulos, T. L. Crystal structures of

human constitutive nitric oxide synthases. Acta Crystallogr., Sect. D:

Biol. Crystallogr. 2014, 70, 2667−2674.

́

(22) Xue, F.; Li, H.; Fang, J.; Roman, L. J.; Martasek, P.; Poulos, T.

L.; Silverman, R. B. Peripheral but crucial: A hydrophobic pocket

(Tyr706, Leu337, and Met336) for potent and selective inhibition of

neuronal nitric oxide synthase. Bioorg. Med. Chem. Lett. 2010, 20,

6258−6261.

́

(23) Kang, S.; Li, H.; Tang, W.; Martasek, P.; Roman, L. J.; Poulos,

T. L.; Silverman, R. B. 2-Aminopyridines with a truncated side chain to

improve human neuronal nitric oxide synthase inhibitory potency and

selectivity. J. Med. Chem. 2015, 58, 5548−5560.

(7) Maccallini, C.; Amoroso, R. Targeting neuronal nitric oxide

synthase as a valuable strategy for the therapy of neurological

disorders. Neural Regener. Res. 2016, 11, 1731−1734.

(8) Mukherjee, P.; Cinelli, M. A.; Kang, S.; Silverman, R. B.

Development of nitric oxide synthase (NOS) inhibitors for neuro-

degenerative diseases and neuropathic pain. Chem. Soc. Rev. 2014, 43,

6814−6838.

́

(24) Ji, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.;

Silverman, R. B. Discovery of highly potent and selective inhibitors of

neuronal nitric oxide synthase by fragment hopping. J. Med. Chem.

2009, 52, 779−797.

(25) Jones, L. H.; Summerhill, N. W.; Swain, N. A.; Mills, J. E.

Aromatic chloride to nitrile transformation: medicinal and synthetic

chemistry. MedChemComm 2010, 1, 309−318.

(9) Siddhanta, U.; Presta, A.; Fan, B.; Wolan, D.; Rousseau, D. L.;

Stuehr, D. J. Domain swapping in inducible NO synthase: electron

transfer occurs between flavin and heme groups located on adjacent

subunits in the dimer. J. Biol. Chem. 1998, 273, 18950−18958.

(10) Rosen, G. M.; Tsai, P.; Pou, S. Mechanism of free-radical

generation by nitric oxide synthase. Chem. Rev. 2002, 102, 1191−1199.

(11) Delker, S. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, F.; Silverman,

R. B.; Poulos, T. L. Unexpected binding modes of nitric oxide synthase

inhibitors effective in the prevention of cerebral palsy. J. Am. Chem.

Soc. 2010, 132, 5437−5442.

(26) Romera, J. L.; Cid, J. M.; Trabanco, A. A. Potassium iodide

catalysed monoalkylation of anilines under microwave irradiation.

Tetrahedron Lett. 2004, 45, 8797−8800.

(27) Inglis, S.; Jones, R.; Fritz, D.; Stojkoski, C.; Booker, G.; Pyke, S.

Synthesis of 5-, 6- and 7-subsituted -2-aminoquinolines as SH3 domain

ligands. Org. Biomol. Chem. 2005, 3, 2543−2557.

(28) Salvatore, R. N.; Nagle, A. S.; Jung, K. W. Cesium Effect: High

chemoselectivity in direct N-alkylation of amines. J. Org. Chem. 2002,

67, 674−683.

(29) Ahmed, G.; Bohnstedt, A.; Breslin, H. J.; Burke, J.; Curry, M. A.;

Diebold, J. L.; Dorsey, B.; Dugan, B. J.; Feng, D.; Gingrich, D. E.; Guo,

T.; Ho, K.-K.; Learn, K. S.; Lisko, J. G.; Liu, R.-Q.; Mesaros, E. F.;

Milkiewicz, K.; Ott, G. R.; Parrish, J.; Theroﬀ, J. P.; Thieu, T. V.;

Tripathy, R.; Underiner, T. L.; Wagner, J. C.; Weinberg, L.; Wells, G.

J.; You, M.; Ziﬁcsak, C. A. Fused Bicyclic Derivatives of 2,4-

Diaminopyrimidine as ALK and c-Met Kinase Inhibitors. WO2008/

051547, May 2, 2008.

(30) Lopchuk, J. M.; Hughes, P.; Gribble, G. W. What controls

regiochemistry in 1,3-dipolar cycloadditions of munchnones with

nitrostyrenes? Org. Lett. 2013, 15, 5218−5221.

(31) Guedat, P.; Berecibar, A.; Ciapetti, P.; Vekata Pithani, S.;

Trouche, N.; Vivalis, F. Hydantoin and Thiohydantoin Derivatives as

Antiviral Drugs. EP2664616, November 20, 2013.

(32) Senecal, T. D.; Shu, W.; Buchwald, S. L. A general, practical

palladium-catalyzed cyanation of (hetero)aryl chlorides and bromides.

Angew. Chem., Int. Ed. 2013, 52, 10035−10039.

(33) Charrier, J.-D.; Binch, H. M., Hurley, D. J.; Cleveland, T.; Joshi,

P.; Fanning, L. T. D.; Pinder, J.; O’Donnell, M.; Virani, A. N.; Knegtel,

R. M. A.; Durrant, S. J.; Young, S. C.; Storck, P.-H.; Kay, D.; Reaper, P.

M. Compounds Useful as Inhibitors of ATR Kinase. WO2011/

143426, November 17, 2011.

́

(12) Wang, H.-Y.; Qin, Y.; Li, H.; Roman, L. J.; Martasek, P.; Poulos,

T. L.; Silverman, R. B. Potent and selective human neuronal nitric

oxide synthase inhibition by optimization of the 2-aminopyridine-

based scaffold with a pyridine linker. J. Med. Chem. 2016, 59, 4913−

4925.

́

(13) Mukherjee, P.; Li, H.; Sevrioukova, I.; Chreifi, G.; Martasek, P.;

Roman, L. J.; Poulos, T. L.; Silverman, R. B. Novel 2,4-disubstituted

pyrimidines as potent, selective, and cell-permeable inhibitors of

neuronal nitric oxide synthase. J. Med. Chem. 2015, 58, 1067−1088.

́

(14) Cinelli, M. A.; Li, H.; Chreifi, G.; Martasek, P.; Roman, L. J.;

Poulos, T. L.; Silverman, R. B. Simplified 2-aminoquinoline-based

scaffold for potent and selective neuronal nitric oxide synthase

inhibition. J. Med. Chem. 2014, 57, 1513−1530.

́

(15) Cinelli, M. A.; Li, H.; Pensa, A. V.; Kang, S.; Martasek, P.;

Roman, L. J.; Poulos, T. L.; Silverman, R. B. Phenyl ether- and aniline-

containing 2-aminoquinolines as potent and selective inhibitors of

neuronal nitric oxide synthase. J. Med. Chem. 2015, 58, 8694−8712.

(16) Kobayashi, Y.; Ikeda, K.; Shinozuka, K.; Nara, Y.; Yamori, Y.;

Hattori, K. L-nitroarginine increases blood pressure in the rat. Clin.

Exp. Pharmacol. Physiol. 1991, 18, 397−399.

(17) Wilcock, D. M.; Lewis, M. R.; Van Nostrand, W. E.; Davis, J.;

Previti, M. L.; Gharkholonarehe, N.; Vitek, M. P.; Colton, C. A.

Progression of amyloid pathology to Alzhemer’s disease pathology in

an amyloid precursor protein transgenic mouse model by removal of

nitric oxide synthase 2. J. Neurosci. 2008, 28, 1537−1545.

(34) Adams, N. D.; Chuadhari, A. M.; Kiesow, T. J.; McSherry, A. K.;

Moore, M. L.; Parrish, C. A.; Reif, A. J.; Ridgers, L. H. Spirocyclic

Piperidine Derivatives for Use as Fatty Acid Synthase Inhibitors.

WO2014/008223, January 9, 2014.

(18) Li, M.; Dai, F.; Du, X.; Yang, Q.; Chen, Y. Neuroprotection by

silencing iNOS expression in a 6-OHDA model of Parkinson’s disease.

J. Mol. Neurosci. 2012, 48, 225−233.

(19) Besnard, J.; Ruda, G. F.; Setola, V.; Abecassis, K.; Rodriguiz, R.

M.; Huang, X. P.; Norval, S.; Sassano, M. F.; Shin, A. I.; Webster, L. A.;

Simeons, F. R.; Stojanovski, L.; Prat, A.; Seidah, N. G.; Constam, D.

B.; Bickerton, G. R.; Read, K. D.; Wetsel, W. C.; Gilbert, I. H.; Roth, B.

(35) Brown, W.; Johnstone, S.; Larecque, D. Benzimidiazole

Derivatives as Vanilloid Receptor Antagonists, Their Preparation,

Pharmaceutical Compositions, and Use in Therapy. WO2008/018827,

February 14, 2008.

(36) Runyon, S. P.; Mosier, P. D.; Roth, B. L.; Glennon, R. A.;

Westkaemper, R. B. Potential modes of interaction of 9-aminomethyl-

9,10-dihydroanthracene (AMDA) derivatives with the 5-HT2A

S

DOI: 10.1021/acs.jmedchem.7b00835

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

receptor: ligand structure-affinity relationship, receptor mutagenesis

and receptor modeling investigation. J. Med. Chem. 2008, 51, 6808−

6828.

(37) Silverman, R. B.; Cinelli, M. A.; Pensa, A. V. 2-Aminoquinoline

Compounds for Potent and Selective Neuronal Nitric Oxide Synthase

Inhibition. US Patent 9,663,468, May 30, 2017.

(38) Yin, J.; Xiang, B.; Huffman, M. A.; Raab, C. E.; Davies, I. W. A

general and efficient 2-amination of pyridines and quinolines. J. Org.

Chem. 2007, 72, 4554−4557.

structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66,

213−221.

(56) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.

D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.

Crystallogr. 2007, 40, 658−674.

(57) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Use of TLS

parameters to model anisotropic displacements in macromolecular

refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2001, 57, 122−

133.

(39) Ueda, T.; Konishi, H.; Manabe, K. Palladium-catalyzed reductive

carbonylation of aryl halides with N-formylsaccharin as a CO source.

Angew. Chem., Int. Ed. 2013, 52, 8611−8615.

(40) Fedorov, R.; Vasan, R.; Ghosh, D.; Schlichting, I. Structures of

nitric oxide synthase isoforms complexed with the inhibitor AR-

R17477 suggest a rational basis for specificity and inhibitor design.

Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 5892−5897.

(41) Smith, G. F. Designing drugs to avoid toxicity. Prog. Med. Chem.

2011, 50, 1−47.

(42) Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C.

Nitrile-containing pharmaceuticals: efficacious roles of the nitrile

pharmacophore. J. Med. Chem. 2010, 53, 7902−7917.

(43) Lundquist, S.; Renftel, M.; Brillault, J.; Fenart, L.; Cecchelli, R.;

Dehouck, M. P. Prediction of drug transport through the blood-brain

barrier in vivo: a comparison between two in vitro cell models. Pharm.

Res. 2002, 19, 976−981.

́

(44) Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.;

Masters, B. S. S. High-level expression of functional rat neuronal nitric

oxide synthase in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 1995,

92, 8428−8432.

(45) Hevel, J. M.; White, K. A.; Marletta, M. A. Purification of the

inducible murine macrophage nitric oxide synthase: identification as a

flavoprotein. J. Biol. Chem. 1991, 266, 22789−22791.

(46) Gerber, N. C.; Ortiz de Montellano, P. R. Neuronal nitric oxide

synthase: expression in Escherichia coli, irreversible inhibition by

phenyldiazene, and active site topology. J. Biol. Chem. 1995, 270,

17791−17796.

(47) Cheng, Y.-C.; Prusoff, W. H. Relationship between the

inhibition constant (K_i) and the concentration of the inhibitor

which causes 50% inhibition (IC50) of an enzymatic reaction. Biochem.

Pharmacol. 1973, 22, 3099−3108.

(48) Leber, A.; Hemmens, B.; Klosch, B.; Goessler, W.; Raber, G.;

Mayer, B.; Schmidt, K. Characterization of recombinant human

endothelial nitric-oxide synthase purified from the yeast Pichia pastoris.

J. Biol. Chem. 1999, 274, 37658−37665.

(49) McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.;

Deacon, A. M.; Ellis, P. J.; Garman, E.; Gonzalez, A.; Sauter, N. K.;

Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. Blu-Ice and the Distributed

Control System: software for data acquisition and instrument control

at macromolecular crystallography beamlines. J. Synchrotron Radiat.

2002, 9, 401−406.

(50) Battye, T. G. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.;

Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-

image processing with MOSFLM. Acta Crystallogr., Sect. D: Biol.

Crystallogr. 2011, 67, 271−281.

(51) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 125−132.

(52) Evans, P. R. Scaling and assessment of data quality. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72−82.

(53) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of

macromolecular structures by the maximum-likelihood method. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.

(54) Emsley, P.; Cowtan, K. Coot: model-building tools for

molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,

60, 2126−2132.

́

(55) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis,

I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-

Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.

J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.

PHENIX: a comprehensive Python-based system for macromolecular

T

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Hydrophilic, Potent, and Selective 7-Substituted 2-Aminoquinolines as Improved Human Neuronal Nitric Oxide Synthase Inhibitors

DOI: 10.1021/acs.jmedchem.7b00835

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