Enhancement of potency and selectivity of 2-aminoquinoline based human neuronal nitric oxide synthase inhibitors

Abstract

Neuronal nitric oxide synthase (nNOS) is a key enzyme in neurodegenerative diseases and melanoma, making it an important therapeutic target. We previously reported 2-aminoquinoline-based nNOS inhibitors with promising activity but limited by suboptimal potency, isoform selectivity, and off-target effects. To address these issues, we designed and synthesized a new series of 7-aryl-6-fluoro-4-methyl-2-aminoquinoline derivatives. Compound 16 showed excellent potency against human nNOS (K_i 16 nM), with ~1800-fold selectivity over human endothelial NOS (eNOS) and ~2900-fold over human inducible NOS (iNOS). PAMPA-BBB experiments indicated high effective permeability (P_e = 13.04 × 10⁻⁶ cm/s), suggesting strong CNS drug potential. In vivo pharmacokinetic studies in mice further demonstrated sustained systemic exposure, low clearance, and robust brain penetration. In contrast, compound 24, the N-Me analogue, was inactive. Molecular dynamics simulations indicated that N-methylation disrupted the favorable solvation of the tail amino group, likely contributing to its loss of activity and nNOS affinity.

Graphical Abstract

Introduction

The nitric oxide (NO) radical is an important cell signaling molecule in the mammalian central nervous system (CNS) and other parts of the body.^1,2 The role of NO in human physiology is to regulate neurotransmission,³ immune response,⁴ and vasodilation.⁵ In mammals, NO is produced by a family of enzymes known as nitric oxide synthase (NOS) by a mechanism in which L-arginine is converted to L-citrulline with a requirement for NADPH and molecular oxygen.⁶ NOSs exist in three isoforms in humans: neuronal NOS (nNOS), which acts as a neurotransmitter,^7-9 endothelial NOS (eNOS), which regulates the blood pressure by relaxing muscles,¹⁰ and inducible NOS (iNOS), which generates cytotoxic NO to kill pathogenic microorganisms.¹¹ The production of NO by nNOS at physiological levels is essential for the healthy functioning of the brain,¹² but its overproduction leads to the formation of highly reactive species such as peroxynitrite (ONOO⁻).^13-15 Peroxynitrite can cause nitration and nitrosylation of proteins, which leads to their misfolding and degradation.¹⁶

In recent years, it has been shown that inhibition of nNOS was influential in treating neuronal damage in animal models.^17,18 However, eNOS inhibition may lead to cardiovascular problems, whereas iNOS inhibition can result in compromising the immune defense system.^19,20 Since the three NOS isoforms have similar active sites, designing inhibitors selective for nNOS over eNOS and iNOS for treating neurodegenerative disorders is quite challenging.^21,22 Another difficulty associated with a potential nNOS inhibitor drug is that it must be able to cross the blood-brain barrier (BBB) for successful delivery to the CNS.^23-27 Because melanocytes in the skin are derived from the neural crest,^28,29 it has been found that nNOS inhibitors also inhibit the growth of melanoma, suggesting an additional indication for nNOS-inhibiting drugs.^30,31

Over the past decade, our research groups have been involved in developing selective nNOS inhibitors, work that has resulted in compounds with excellent nNOS potency, high isoform selectivity, and BBB permeability.^32-37 We have previously reported a series of aminoquinoline-based inhibitors that are isoform-selective arginine bioisosteres with modest potency and selectivity toward nNOS. The first series of inhibitors, such as 1 (Figure 1), showed 6-fold weaker potency for human nNOS (hnNOS) compared to rat nNOS (rnNOS), and low selectivity for human nNOS over human eNOS, with toxic side effects mainly due to its off-target promiscuity.³⁸ The second series of inhibitors we developed included a phenyl ether linkage, such as 2, that maintains the potency and selectivity of 1 with decreased off-target binding.³⁹ However, 2 exhibited low permeability in a Caco-2 membrane permeability assay (apical to basolateral (A → B) direction gave a mean P_app of 2.3 × 10^–6 cm s^–1) and a high efflux ratio of 5.5 (ratio of membrane permeability (A → B) to efflux (B → A); < 2 is considered favorable).

Figure 1.

Previously reported 2-aminoquinolines as nNOS inhibitors

Further investigation within the 2-aminoquinoline series was carried out by introducing a methyl group at the 4-position (3) and a cyano substituent on the phenyl ring (4).^40,41 These compounds showed higher hnNOS potency, moderate hn/heNOS selectivity, and enhanced membrane permeability in comparison to 1 and 2. Further modification of the 2-aminoquinoline series led to the 7-phenyl-2-aminoquinoline class of inhibitors, such as 5 and 6, which were prepared by attaching a phenyl group at the 7-position, providing rigidity to the molecules in contrast to the more flexible linear chain present in 1-4.⁴² Compounds 5 and 6 had good potency toward human nNOS (K_i 46 nM and 60 nM, respectively) with 457- and 877-fold selectivities over human eNOS, respectively (Figure 1). Here, we describe the modification of lead compound 6 to further enhance the potency and selectivity of the 2-aminoquinoline class of nNOS inhibitors (Figure 2).

Figure 2.

Molecules synthesized in this study

Chemistry

The synthesis initially focused on 33 as the head part so that different linkers could be attached to it with various types of chains as the tail (Scheme 1). Commercially available 3-bromo-4-fluoroaniline (25) was refluxed with methyl acetoacetate, followed by its treatment with sulfuric acid, leading to intermediate 27. Compound 27 reacted with POCl₃ producing 2-chloro derivative 29. Compound 29 underwent an addition-elimination reaction with 4-methoxybenzylamine in the presence of Et₃N in n-butanol under refluxing conditions to give 31, which was subsequently treated with TFA at 60 ^oC, providing the 2-amino derivative, which was di-Boc protected using Boc anhydride to give 33. Similarly, starting from commercially available 3-bromo-4-chloroaniline (26) and following the same sequence of reactions, we achieved 34.

Scheme 1. Synthetic route to 33 and 34^a.

^aReagents and conditions: (a) i) methyl 3-oxobutanoate, KOH, toluene, 100 °C, 16 h; ii) H₂SO₄, 120 °C, 3 h; (b) POCl₃, 120 °C - rt, 14 h; (c) 4-methoxybenzylamine, Et₃N, n-Bu₄NI, n-butanol, 150 °C, 10 h; (d) i) TFA, 70 °C, 2 h; ii) (Boc)₂O, DMAP, CH₃CN, 70 °C.

The synthetic route to inhibitors 7 and 8 requires commercially available linker 35. Fluoro-substituted head molecule 33 was coupled with 35 to give Suzuki adduct 36 (Scheme 2). The nitrile functionality in 36 was reduced to the primary amine using a combination of NaBH₄ and NiCl₂.6H₂O, followed by in situ Boc protection to produce 38. Finally, treating Boc-protected amine derivatives 38 with 4 N HCl/1,4-dioxane gave 7. Similarly, coupling 34 and 35 and following the same sequence of reactions produced chloro-substituted compound 8.

Scheme 2. Synthetic route to 7 and 8^a.

^aReagents and conditions: (a) Pd(dppf)Cl₂, B₂(Pin)₂, KOAc, K₂CO₃, H₂O, 1,4-dioxane, 130 °C, microwave; (b) NiCl₂.6H₂O, NaBH₄, Et₃N, (Boc)₂O, CH₃OH, 25 °C, 2 h; (c) HCl/1,4-dioxane, 0 °C – 25 °C, 12 h.

Various linkers (41, 44, 45, 48, 49, 54, and 58) with polar chains were synthesized for inhibitors 9-24 and outlined in Scheme 3.

Scheme 3. Synthesis of 41, 44, 45, 48, 49, 54, and 58^a.

^aReagents and conditions: (a) C₄H₇Br, Et₃N, CHCl₃, 65 °C, 12 h; (b) MOMCl, Et₃N, CHCl₃, 65 °C, 12 h; (c) LiAlH₄, THF, 0 °C, 30 min; (d) CBr₄, PPh₃, DCM, 25 °C, 12 h; (e) KCN, n-Bu₄NI, DCM:H₂O (1:1), 25 °C, 12 h; (f) BH₃.SMe₂, THF, 0 °C – 25 °C, 20 h.

The commercially available hydroxy-substituted benzonitrile (40) was subjected to O-alkylation using cyclopropyl methyl bromide and Et₃N, producing 41 (Scheme 3). Similarly, (42, 43, 46, and 47) were subjected to O-alkylation with MOMCl to give 44, 45, 48, and 49, respectively. The synthetic approach for linker 54 started from commercially available methyl 4-bromo-3-hydroxybenzoate (50), which underwent O-alkylation with MOMCl to give 51. Ester 51 was reduced to alcohol 52 using LiAlH₄ and further transformed into bromide 53 via the Appel reaction. Compound 53 was converted to the corresponding nitrile (54) using KCN. In the same way, linker 58 was synthesized starting from commercially available carboxylic acid 55 by reducing it to alcohol 56 with BH₃. Alcohol 56 was transformed into nitrile 58 via the same synthetic route that 52 was converted to 54 (Scheme 3).

The synthetic route to inhibitors 9-12 requires different linkers (44, 45, 54, and 58), and each was coupled with head molecule 33 following the same synthetic sequence as in Scheme 2, producing 9-12 (Scheme 4).

Scheme 4. Synthesis of 9-12^a.

^aReagents and conditions: (a) Pd(dppf)Cl₂, B₂(Pin)₂, KOAc, K₂CO₃, H₂O, 1,4-dioxane, 130 °C, microwave; (b) NiCl₂.6H₂O, NaBH₄, Et₃N, (Boc)₂O, CH₃OH, 25 °C, 2 h; (c) HCl/1,4-dioxane, 0 °C – 25 °C, 12 h.

Molecules 13 and 14 were synthesized with a two-carbon chain between the head and the linker to increase the flexibility of the molecule and the basicity of the 2-amino pyridine moiety. The synthesis was initiated from 58 by subjecting it to Sonogashira coupling with trimethylsilylacetylene to give 67 under microwave conditions (Scheme 5). The trimethylsilyl group in 67 was deprotected using K₂CO₃ at room temperature to give alkyne 68. Head molecule 33 was connected to alkyne 68 via Sonogashira coupling to give 69, which was further hydrogenated with H₂, Pd/C, giving 70. The nitrile in 70 was reduced to a primary amine using NaBH₄ and NiCl₂.6H₂O, followed by in situ Boc protection, producing 71, which was methylated using a combination of NaH and MeI, followed by acidolysis with HCl/1,4-dioxane to give 13. Similarly, direct acidolysis of 71 gave 14.

Scheme 5. Synthesis of 13 and 14^a.

^aReagents and conditions: (a) ethynyltrimethylsilane, Pd(dppf)Cl₂, CuI, Et₃N, PPh₃, 120 °C, CH₃CN, microwave, 1 h; (b) K₂CO₃, MeOH, 25 °C, 2 h; (c) 33, Pd(dppf)Cl₂, CuI, Et₃N, PPh₃, 90 °C, CH₃CN, microwave, 1 h; (d) H₂, Pd/C, MeOH, 25 °C, 12 h; (e) NiCl₂.6H₂O, NaBH₄, Et₃N, (Boc)₂O, CH₃OH, 25 °C, 2 h; (f) i) NaH, MeI, THF 0 °C – 25 °C, 12 h; ii) HCl/1,4-dioxane, 25 °C, 12 h (g) HCl/1,4-dioxane, 25 °C, 12 h.

We also explored the meta- and para-positions of the central phenyl ring. The 6-fluoro analogue of 5 was synthesized by coupling 33 with linker 41, giving 15 (Scheme 6) using the same sequence of reactions to make 9-12 (Scheme 4). The hydroxyl group at the 4-position of linker 44 was moved to the 2-position (48) and further transformed into 16.

Scheme 6. Synthesis of 15-19^a.

^aReagents and conditions: (a) Pd(dppf)Cl₂, B₂(Pin)₂, KOAc, K₂CO₃, H₂O, 1,4-dioxane, 130 °C, microwave; (b) NiCl₂.6H₂O, NaBH₄, Et₃N, (Boc)₂O, CH₃OH, 25 °C, 2 h; (c) HCl/1,4-dioxane, 0 °C – 25 °C, 12 h.

Commercially available linker 72 was introduced to incorporate a fluorine at the 2-position as a bioisostere that decreases the metabolism and increases the potency of the inhibitor.⁴³ Compound 17 was synthesized following the same sequence of reactions as in Scheme 4. The position of the aminomethyl group and hydroxyl or fluorine in 16 and 17, respectively, was interchanged to give 18 and 19 by coupling head 33 with the corresponding linkers 49 and 73, respectively (Scheme 6).

A structural modification to the tail was made by methylation of the primary amine in 9-11, 15, and 16. The Boc-protected amine functionality (63-65, 79, and 80) was methylated using NaH and MeI, followed by hydrolysis to 20-24 (Scheme 7).

Scheme 7. Synthesis of 20-24^a.

^aReagents and conditions: (a) i) NaH, MeI, THF 0 °C – 25 °C, 12 h; ii) HCl/1,4-dioxane, 25 °C, 12 h.

Results and Discussion

Surface Electrostatic Potential (ESP) analysis

The interaction between hnNOS and arginine, the natural substrate of NOS, has been well-studied.^44,45 The reported 2-aminoquinoline nNOS inhibitors interact with the main chain carbonyl of Trp592 and interact with the side chain carboxylate of Glu597 in hnNOS through hydrogen bond and charge interactions (Figure 3), similar to arginine.⁴² To enhance the key interactions of 2-aminoquinoline derivatives, the surface electrostatic potential (ESP) of the 6-fluoro and 6-chloro derivatives was calculated for each compound using quantum mechanical calculations with density functional theory (DFT). Upon performing DFT calculations at the B3LYP/6–31G+** theory level, both fluorine and chlorine substitution at the 6-position increased the positive surface ESP around the hydrogen atoms participating in hydrogen bonds and charge interactions (Figure 4), as expected. This result suggested that 6-halogen substitution could enhance the binding affinity of 2-aminoquinoline derivatives in hnNOS.

Figure 3.

The binding mode of a representative 2-aminoquinoline derivative with human nNOS. (PDB ID: 6PO7). For clearer visualization, parts of the ligand other than the 2-aminoquinoline ring and most of the enzyme residues have been omitted.

Figure 4.

The theoretical surface electrostatic potential (ESP) of the ligand extracted from the minimized crystal structure (PDB ID: 6PO7) and its 6-halogen substituted derivatives from DFT calculation (B3LYP/6-31G+**). (A) The atom numbering for the 2-aminoquinoline atoms participating in key interactions and calculated surface ESPs for each atom. The top and bottom of the box indicate the minimum ESP and the maximum ESP, respectively. The line in the box represents the mean ESP. (B) The relative surface ESP of each derivative compared to when X = H. Min, minimum surface ESP; Max, maximum surface ESP.

In addition to hydrogen bonds and charge interactions with Trp592 and Glu597, the 2-aminoquinoline ring forms π-stacking with heme (Figure 3).

Biochemical activity of the nNOS inhibitors

A study of the surface electrostatic potential (ESP) indicated that the substitution of a halogen atom at the 6-position (in 7 and 8, Figure 2) may enhance the binding affinity of 2-aminoquinoline derivatives in hnNOS (Figure 4). To determine which halogen substitution should be made at the 6-position of the aminoquinoline moiety, the potency and selectivity of 7 and 8 were determined using the NO hemoglobin (Hb) capture assay (see the Supporting Information for details).^48,49 Of the two, fluoro-substituted compound 7 was much more potent and selective than chloro-substituted compound 8 (Table 1).

Table 1. Inhibition of NOS enzyme by synthesized compounds 7-24.

Compound	Ki (nM)	Ki (nM)	Ki (nM)	Ki (nM)	Selectivity	Selectivity
	hnNOS	rnNOS	heNOS	hiNOS	hn/he	hn/hi
7	68	61	51177	14484	752	213
8	294	142	3268	6693	11	23
9	1072	N.T.	N.T.	N.T.	N.D.	N.D.
10	60	33	13257	20328	221	338
11	28	32	17954	7146	641	255
12	1258	N.T.	N.T.	N.T.	N.D.	N.D.
13	375	N.T.	N.T.	N.T.	N.D.	N.D.
14	800	N.T.	N.T.	N.T.	N.D.	N.D.
15	66	36	11503	13355	174	202
16	16	34	29741	46711	1858	2919
17	69	112	54936	14875	796	216
18	32	27	14834	7626	467	238
19	41	28	22864	13280	558	323
20	4356	N.T.	N.T.	N.T.	N.D.	N.D.
21	2387	N.T.	N.T.	N.T.	N.D.	N.D.
22	19089	N.T.	N.T.	N.T.	N.D.	N.D.
23	3452	N.T.	N.T.	N.T.	N.D.	N.D.
24	4623	N.T.	N.T.	N.T.	N.D.	N.D.

The compounds (7-24) were assayed for in vitro inhibition against four purified NOS isoforms: hnNOS, rnNOS, heNOS, and hiNOS, using previously reported methods.^37,41,42 K_i values were calculated from the IC₅₀ values of a dose–response curve using the Cheng–Prusoff equation. All experimental standard error values (for the log IC₅₀) were less than 10%, and all correlation coefficients corresponded tor² > 0.87. Selectivity values are ratios of respective K_i values. N.T. = not tested, N.D. = not determined.

With fluoro-substituted compound 7 as a key molecule, we designed a few closely related compounds, 9-24 (Figure 2), and assayed them against purified hnNOS. We investigated the effect of different electronic changes by various structure-based modifications. First, we employed a hydroxyl group at the ortho-position along with an aminoalkyl tail at the meta/para-positions of the central phenyl ring attached as a linker (9-11). To enhance lipophilicity, difluoro-substituted phenyl rings (12-14) were also synthesized. We explored the ortho-position with the hydroxyl/fluoro substituents with a polar primary amine tail (15-19). Fluoro-substituted scaffolds generally provide more profound insights, better outcomes on metabolic stability, and enhanced potency.⁴³ Lastly, the SAR of the secondary amines (20-24) on various positions of the phenyl ring, along with a meta- or ortho-positioned hydroxyl or O-alkyl group, were explored to analyze steric and polar effects. Whenever possible, the crystal structures of these inhibitors bound to hnNOS, rnNOS, and heNOS were determined to enhance our understanding of the SAR of these compounds.

The potency and selectivity of the newly synthesized compounds (7-24) were initially measured using only purified human nNOS as a prescreen. The most potent compounds were further assayed using purified human eNOS, human iNOS, and rat nNOS to determine isoform selectivity. The isoform selectivity is determined by the ratio of the K_i values of hnNOS over heNOS (hn/he) or over hiNOS (hn/hi). The ratio of hn/rn is also important to characterize the inhibitors for potential preclinical and clinical studies.

Surprisingly the di-HCl salts of all N-Me derivatives 20-24 were largely inactive compared to the corresponding primary amine derivatives (Table 1). In addition, compounds with a difluorophenyl central ring (12 and 14) were much poorer inhibitors of hnNOS than compounds 15-19.

Assessment of membrane permeability of the nNOS inhibitors

The most potent inhibitors (10, 11, and 15-19) from the NOS inhibition assay were further tested for their membrane permeability using the parallel artificial membrane permeability for the blood-brain barrier (PAMPA–BBB) assay. An effective permeability (P_e) greater than 4.0 × 10⁻⁶ cm s⁻¹ is considered an indication of good BBB penetration and is classified as “CNS (+)”.^24,50,51 In this assay, we used porcine brain lipid as an artificial membrane for analyzing the passive permeability of the selected compounds. Two commercial drugs, theophylline and verapamil, were used as negative and positive controls, respectively, during each permeability test of the selected NOS inhibitors (Table 2). Compound 15, a 6-fluoro substituted analogue of compound 5, displayed a similar value to 5, with high permeability (P_e = 14.37 ± 2.09 × 10⁻⁶ cm s⁻¹).⁴² Compounds 10 and 11 displayed the lowest permeability values among the selected compounds, presumably because of the presence of the hydroxyl group, which increases polarity of the molecule and are predicted to be CNS (−). However, 16 and 18 also have hydroxyl group substitutions, but they displayed high permeability and are predicted to be CNS (+). This may arise because their hydroxyl groups can undergo intramolecular hydrogen bonding with the adjacent aminomethyl substituent, which increases lipophilicity.⁵² Compound 16 is substantially more permeable than 18, even though both can participate in intramolecular hydrogen bonding. The para-hydroxyl group of 16 is in resonance with the nitrogen atom of the quinoline ring, thereby lowering the pK_a of the hydroxyl group. This may allow for a more stable intramolecular hydrogen bond than for 18, whose meta-hydroxyl group does not have this effect. Compound 17 has the highest P_e, greater than isomer 19, again likely because the polarity of the fluorine atom is decreased by a para-resonance effect.

Table 2. Effective permeability (P_e) of commercial drugs and the synthesized nNOS inhibitors in the PAMPA-BBB assay.

Compounds	Reported Pe (10−6 cm s−1)a	Determined Pe (10−6 cm s−1)b	Predictionc
theophylline	0.12	0.21 ± 0.12	CNS (−)
(±)-verapamil	16	22.19 ± 3.14	CNS (+)
10		1.92 ± 1.48	CNS (−)
11		0.43 ± 3.81	CNS (−)
15		14.37 ± 2.09	CNS (+)
16		13.04 ± 2.01	CNS (+)
17		17.01 ± 2.56	CNS (+)
18		6.29 ± 0.22	CNS (+)
19		12.08 ± 0.95	CNS (+)

^aEffective permeability (P_e) values from the literature.⁴²

^bAll assays were performed over 17 h at a concentration of 200 μM; see the Supporting Information for details.

^cCNS (+) = likely high BBB permeation. CNS (−) = likely low BBB permeation. The “predictions” for the two literature-based compounds listed have been experimentally tested.

SAR studies using crystal structures for compounds 7, 10, 11, and 15

Starting from lead compound 6, we introduced a halogen (F and Cl) at the 6-position of the 2-aminoquinoline in 7 and 8, respectively. Activity assays using hnNOS indicated that 7 is more potent than 8, so we determined the structures of 7 bound to three NOS isoforms, as shown in Figure 5. As expected for the 2-aminoquinoline series, 7 binds to the active site of all three NOSs with nitrogen atoms from its 2-aminoquinoline moiety hydrogen bonded to the active site glutamate (Glu597 in hnNOS, Glu592 in rnNOS, and Glu361 in heNOS) and the backbone carbonyl group from a nearby Trp residue (Trp592 in hnNOS, Trp587 in rnNOS, and Trp356 in heNOS). The quinoline ring is more or less parallel to the heme plane with the 6-fluorine atom in a van der Waals distance (4.0 - 4.3 Å) to a Met residue (Met575 in hnNOS, Met570 in rnNOS, and Met339 in heNOS) flanking the substrate binding pocket. The central phenyl ring sits on top of the heme propionates. The tail aminomethyl group points away from the pterin site water molecule in both hnNOS and rnNOS but displaces the water in the heNOS-7 structure, making a hydrogen bond with propionate A. The electron density for the tail aminomethyl group in both hnNOS and rnNOS is weak, but it is clear that the pterin site water is not displaced. In both rat and human nNOS structures, there is a water molecule between the tail aminomethyl group of 7 and a nearby Asn residue bridging the hydrogen bonding interactions. The almost identical binding mode of 7 to hnNOS and rnNOS (Figure 5) explains its similar potency for both nNOSs.

Figure 5.

Compound 7 bound to hnNOS (A, PDB Code: 9MYU), rnNOS (B, PDB Code: 9MYX), and heNOS (C, PDB Code: 9MZ1). In this and other structural figures, the major hydrogen bonds that are key to the inhibitor binding are highlighted with dashed lines. The distances are labeled in Å. The omit difference Polder density for the inhibitor is displayed at the 2.5-3.0 σ contour level.

In addition to 6-chloro substituted compound 8, we tested two other compounds with either an ethyl or a propyl group at the 6-position of the 2-aminoquinoline ring. While 8 showed weaker potency than 7, the other two compounds with bulkier 6-substituents (6-ethyl/n-propyl) did not bind to hnNOS in the NOS crystal preparations. Based on the known van der Waals contact from the 6-fluorine in the crystal structures (Figure 5), the bulkier substituents, Cl, ethyl, and propyl groups, would likely clash with the nearby Met residue. Because of its better potency against nNOS, 7 was used as the standard scaffold to further develop the 2-aminoquinoline-based inhibitor series.

The next modification was the introduction of two substituents on the central phenyl ring at either ortho- and meta-positions (compound 9) or ortho- and para-positions (compounds 10 and 11). Compounds 10 and 11 showed better potency than 9, which encouraged us to obtain crystal structures for both. The binding mode of 10 was found to be quite similar in all three NOS structures (Figure 6). The ortho-hydroxyl group fits in between the two propionates pointing downward and hydrogen bonds with propionate D in both rnNOS and heNOS. The tail aminomethyl group at the para-position hydrogen bonds with the pterin site water molecule in all three structures. Apparently, the aminomethyl group at the para-position in 10 makes it easier to interact with the pterin site water molecule than the meta-position aminomethyl in 7. Based on the known structural information, the dual ortho- and meta-substituents in 9 would have had to adopt an awkward position for the aminomethyl group to make any favorable interactions with the protein, which may explain its poor potency.

Figure 6.

Compound 10 bound to hnNOS (A, PDB Code: 9MYV), rnNOS (B, PDB Code: 9MYY), and heNOS (C, PDB Code: 9MZ2)

When the aminomethyl group in 10 was changed to an aminoethyl in 11, it was able to form dual hydrogen bonds to propionate A and BH₄ in both hnNOS and rnNOS, as shown in Figures 7A and 7B. The strong hydrogen bonds may account for the better affinity of 11 than 10 for hnNOS. However, the tail aminoethyl group of 11 can only make a hydrogen bond with the pterin site water molecule in heNOS (Figure 7C). In heNOS-11, the loss of direct hydrogen bonds with propionate A and BH₄ observed in the heNOS-10 structure might explain the poorer potency of 11 than 10. The central phenyl ring of 11 still sits in between the two propionates, with the ortho-hydroxyl group pointing downward, making a hydrogen bond with propionate D in all three structures. The gain in potency for hnNOS and loss of potency for heNOS is reflected in the better hn/he selectivity for 11.

Figure 7.

Compound 11 bound to hnNOS (A, PDB Code: 9MYW), rnNOS (B, PDB Code: 9MYZ), and heNOS (C, PDB Code: 9MZ3)

When a difluorobenzene was introduced in 12-14, the potency with hnNOS worsened significantly (Table 1). Difluorobenzene was previously used in the 2-aminopyridine-based nNOS inhibitor series with good success.³³ For these compounds, the difluorobenzene ring sits on top of the heme plane. However, the bulkier 2-aminoquinoline compounds push the difluorobenzene ring onto the propionates, causing unfavorable clashes.

Next, we attempted dual substituents at both the meta- and para-positions of the phenyl ring in compounds 15-19, with the primary aminomethyl group as one of the two substituents. As shown in Figure 8, the dual substituents on the phenyl ring of 15 cause the ring to rotate toward propionate D, forcing the latter into a downward conformation in both the rnNOS-15 and heNOS-15 structures. The meta-aminomethyl of 15 makes a hydrogen bond with the pterin site water molecule in rnNOS. In contrast, the same aminomethyl group of 15 in the heNOS-15 structure directly displaces the pterin site water, forming hydrogen bonds with both propionate A and BH₄. This difference results from the interactions between the O-alkyl tail and a different hydrophobic residue in the two NOS isoforms. The bulkier Phe105 in heNOS makes tighter van der Waals contacts with the O-alkyl tail of 15, thereby pushing the aminomethyl group on the phenyl ring closer to propionate A for a hydrogen bond and displacing the pterin site water molecule (Figure 8B). Leu337 in rnNOS makes looser contacts with the O-alkyl tail of 15, which instead of displacing the pterin site water molecule, pushes the aminomethyl group to make a hydrogen bond with the water molecule (Figure 8A).

Figure 8.

Compound 15 bound to rnNOS (A, PDB Code: 9MY0) and heNOS (B, PDB Code: 9MZ4)

The crystal structures of 15 bound to rnNOS and heNOS provide evidence that the dual meta- and para-substituents on the phenyl ring with a primary aminomethyl group fit in the NOS active site rather favorably, which agrees with the good potency of 15-19 for nNOS (Table 1). However, methylation of the primary amine as in 20-24 diminished binding affinity for hnNOS by >62500 fold. To rationalize these unexpected result, we carried out molecular dynamics simulations, as crystallography using these weak binders was impractical.

In vivo pharmacokinetics of lead compound 16 in C57BL/6 Mice

The pharmacokinetics of 16 were carried out in male C57BL/6 mice after single intravenous (IV) and oral (PO) doses. Plasma and brain concentrations were measured over 48 h using a tested LC-MS/MS method (LLOQ 2.5 ng/mL). After intravenous dosing (iv, 3 mg/kg), 16 showed a high initial plasma concentration (C_max) of 654.08 ng/mL with an early T_max of 0.083 h. The AUC_last was 654.03 h·ng/mL, and the terminal half-life (t_1/2) was relatively short at 2.49 h (Table 3). Systemic clearance was high at 74.6 mL/min/kg, and the steady-state volume of distribution (V_ss) was large 16.08 L/kg. This indicates rapid elimination from plasma and extensive distribution in tissues. After oral dosing (po, 10 mg/kg), 16 had a lower plasma C_max of 86.51 ng/mL at 1.0 h. The AUC_last was 356.54 h·ng/mL, with a longer terminal half-life (t_1/2) of 7.96 h and a high apparent distribution volume (V_ss) of 222.69 L/kg. This resulted in a low oral bioavailability (%F) of 16.4% and suggests limited systemic availability even with measurable plasma levels (Table 3). In the brain, oral dosing showed a delayed T_max of 12.0 h and a peak concentration of 172.0 ng/g, with 24 brains contributing over various time points. This supports a time-dependent accumulation in the CNS rather than quick penetration. Overall, these findings show that 16 has high clearance, low oral bioavailability, and a large distribution volume, with moderate and delayed exposure in the brain. This is consistent with extensive distribution in the body and limited CNS exposure after oral dosing in mice.

Table 3: Key Pharmacokinetic Parameters of lead compound 16 in Male C57BL/c Mice.

Matrix	Route	Dose (mg/kg)	Tmax (h)	aC0/Cmax (ng/mL)	AUClast (h*ng/mL)	T1/2 (h)	CL (mL/min/kg)	Vss (L/kg)	%F
Plasma	iv	3	0.083	654.08	654.03	2.49	74.6	16.08	-
	po	10	1.00	86.51	356.54	7.96	-	222.69	16.4%
Matrix	Route	Dose (mg/kg)	Tmax (h)	Cmax (ng/g)	AUClast (h*ng/g)	Brain-Kp (Cmax)	Brain-Kp (AUClast)
#Brain	iv	-	-	-	-	-	-	-	-
	po	10	12.0	172.0	-	-	24.0	-	-

^#Assuming brain density = 1.00 g/mL (1 ng/g ≈ 1 ng/mL)

Binding analysis of 16 and 24 via molecular dynamics (MD) simulations

The binding modes of 16 and 24 were predicted by a docking study designed to investigate the effect of the methyl group on binding. Based on docking in the crystal structure of the hnNOS-10 complex (PDB ID: 9MYV), the binding modes of the two molecules were highly similar (root-mean-square deviation, RMSD, for maximum common structure = 0.28 Å, Supplementary Figure 1). As observed earlier for compounds with the same head group,^41,42 the 2-aminoquinoline head groups of both compounds interact with the Glu597 side chain and Trp592 main chain carbonyl group by charge and hydrogen bond interactions as well as with the heme porphyrin ring, forming multiple π-interactions (Figure 9). Additionally, the tail parts of both molecules showed identical interactions with the heme propionate (charge interaction by the tail primary amine in 16 or the secondary amine in 24), Tyr711 (hydrogen bond by p-hydroxyl), and Trp683 (edge-to-face π-interaction by phenyl ring), except for additional hydrogen bonds with BH₄ and solvent water in the binding mode of 16 (Figure 9). However, the docking scores (−12.76 and −11.60 kcal/mol for compounds 16 and 24, respectively) failed to explain their experimental binding affinity difference (ΔΔG_Bind = 3.4 kcal/mol based on their K_i values). Possibly, the docking calculation underestimated the contribution from the additional hydrogen bonds with BH_4, and solvent water seen with 16.

Figure 9.

The predicted binding mode of (A) 16, (B) 24, and (C, D) in 2D representations of docking results using the hnNOS-10 structure (PDB ID: 9MYV). Gray, ivory, orange, and blue sticks represent binding site residues, cofactors, and each indicated ligand. Water molecules are shown in red spheres. Green, yellow, and pink-dotted lines indicate hydrogen bond, charge interaction, and π-interaction, respectively.

Because of the discrepancy between the experimental binding affinities and the predicted values from docking results, a 100-ns molecular dynamics (MD) simulation of each complex in the SPC solvent model box was conducted. In the docking-generated binding mode of 24, the methyl group on the tail amino group pointed toward a solvent-exposed vestibule of the binding site, preventing its solvation by water molecules (Figure 9B). However, since the conventional docking protocol only allows binding mode predictions with fixed water molecules, its scoring function is unreliable for accurately predicting solvation effects. Therefore, we hypothesized that the MD-generated trajectories enabling flexible water positions could be useful for the theoretical quantification of solvation effects on the tail amino groups.

As most interactions of both molecules were strong polar interactions, and due to the head groups’ well-known tight interactions in the substrate binding site,^41,42 the binding modes of the ligands were highly stable based on the RMSD during the simulations (Figure 10D). The head part RMSD was less than 0.8 Å during each simulation in every frame (Supplementary Figures 2B and 2D), highlighting its extremely stable binding mode. Additionally, the tail part of 16 maintained a stable RMSD (Supplementary Figure 2C) and distances for interactions (Figures 10E-G) in its binding mode from the docking result (Figure 9A and 9C). However, compared to its docking-predicted binding mode (Figure 9B and 9D), the tail secondary amino group of 24 formed an additional hydrogen bond with BH₄ and kept the distance (Figures 10B and 10G). At the same time, the charge interaction with the heme propionate showed slightly unstable distances after ~20 ns of simulation (Figure 10F). As a result of replica studies of the same simulations, 16 maintained relatively stable interaction distances compared to 24 (Supplementary Figure 3). Although one replica of 16 showed a transient loss of the hydrogen bond with Tyr711, the overall interaction pattern remained consistent across replicas.

Figure 10.

The molecular dynamics (MD) simulation results of 16 and 24 in hnNOS. (A, B) The binding modes of (A) 16, (B) 24, and their tail amino groups (in each box) in the representative frame of each MD trajectory. Gray, ivory, orange, and blue sticks represent binding site residues, cofactors, and compounds 16 and 24, respectively. Water molecules are shown in red spheres. Green, yellow, and pink-dotted lines indicate hydrogen bond, charge interaction, and π-interaction, respectively. (C) Geometric measurements for tail part binding interactions, (D) Root-mean-square deviation (RMSD) of the ligand and distances of (E) Hydrogen bond with Tyr711, (F) charge interaction with heme propionate, and (G) Hydrogen bond with BH₄ cofactor during each MD simulation.

To enable the interactions of the tail amino group with the heme propionate and BH₄, the methyl group of 24 was exposed to the solvent-accessible area of the binding site (Figure 10B), pointing away from the phenyl ring (dihedral angle C1-C2-N-C3 = ~180° in Figure 11B). Thus, based on the radial distribution function (g(r) in Figure 11C) of water molecules from the amine nitrogen, 24 showed a disrupted solvent shell with a major g(r) peak at 4.7 Å compared to that of 16 at 3.1 Å, implying a well-established hydrated shell around the amine cation in 16 but not in 24. Notably, considering the gap between the two major peaks (1.6 Å) and the typical C-N bond length (1.5 Å), it is obvious that methyl substitution mainly contributes to the disruption of the hydration shell around the tail amino group in 24.

Figure 11.

The effect of methyl substitution on the hydrated shell formation around the tail amino group of 24 during MD simulation in hnNOS. (A) The extracted representative conformation of 24 in the hnNOS binding site from MD trajectories and its atom numbering for dihedral angle measurement. (B) Dihedral angle measurement for C1-C2-N-C3 atoms during the simulation. (C) The radial distribution function, g(r), of water molecules around the amine nitrogen atom during MD simulation of each indicated molecule. r, distance from the amine nitrogen atom. The dotted lines indicate results from replicas.

Due to the positional instability of the amine potentially caused by the solvent-exposed methyl group, the distance between the amino group and the heme propionate ion, which is a critical determinant for their charge interaction, was relatively unstable throughout most 24 trajectories (Figure 10F). Moreover, starting from 73 ns of the simulation, the terminal methyl group on the tail amino group in 24 rotated with a dihedral angle of ~60° for 1.5 ns (Figure 11B), exposing its proton to the solvent (Supplementary Figure 4). Although this rotation enables additional solvation of the tail amino group, the rotated methyl group disrupts the charge interaction with heme by staying away from the heme propionate ion by up to ~6 Å (Figure 10F), which makes it no longer available for charge interaction.

Conclusion

In this study, we developed a series of inhibitors with a 2-aminoquinoline head and a tail of a phenyl ring with two substituents at various positions. After ruling out bulkier alkyl groups, initial testing with different substituents at the 6-position of the 2-aminoquinoline led to fluorine as the favored group for further trials focusing on where and what substituents are most effective on the phenyl ring. We concluded that (1) an ortho-substituent is too close to the heme propionates in nNOS and generates potential steric clashes, although the ortho-hydroxyl group forces the phenyl ring into a vertical orientation relative to the heme plane, thereby avoiding clashes with propionates; (2) dual meta- and para-substituents showed strong binding affinity because both substituents have room to extend beyond the heme binding active site, interacting with propionate or other protein residues; and (3) a primary aminomethyl group as one of the two substituents is essential for good potency, as it can form strong hydrogen bonds with propionate A and/or BH₄, while a secondary amino group leads to poor (or no) binding affinity. A molecular mechanics calculation with the generalized Born and surface area (MM/GBSA) technique indicated that the extra methyl group on the tail aminomethyl group disturbed solvation around the amine. Compound 16, with meta-aminomethyl and para-hydroxyl groups is the most favorable inhibitor in this series with K_i = 16 nM against hnNOS, hn/he selectivity of 1858, hn/hi selectivity of 2919, and good membrane permeability. A docking study using hnNOS revealed extensive favorable interactions from both substituents of 16 to propionate A, BH₄, a water molecule, and Tyr711. In vivo pharmacokinetic profiling in mice revealed that 16 has a moderate half-life, low clearance, and durable systemic exposure, together with pronounced brain penetration, supporting attainment of pharmacologically relevant CNS concentrations; however, its oral bioavailability is low.

In conclusion, this work presents one of the most potent and selective hnNOS inhibitors developed to date, 16, which has the additional favorable property of likely being CNS penetrant. In addition, this study also presents interpretable SAR data on the (7-24) nNOS inhibitor series that shed light on the observed binding behavior of representative compounds and that could be used to inform future efforts in nNOS inhibitor design.

Experimental Section

Chemistry

General procedures

All reagents were purchased from Sigma-Aldrich, Combi-blocks, and Oakwood. Anhydrous solvents (THF, 1,4-dioxane, CH₂Cl₂, MeOH, Et₃N, and DMF) were distilled before use. Metal catalysts bis(triphenylphosphine)palladium(II) dichloride, copper(I) iodide, and Pd/C [10 wt % loading (dry basis), matrix carbon powder, and wet support] were purchased from Sigma-Aldrich. Thin layer chromatography (TLC) was performed on silica gel 60 F254 precoated plates (0.25 mm) from Silicycle, and components were visualized by ultraviolet light (254 nm) and/or KMnO₄, ninhydrin stain, and phosphomolybdic acid stain. ¹H NMR spectra were recorded at 500 MHz, using a Bruker Avance III 500 (direct cryoprobe), and ¹³C NMR spectra were obtained at 126 MHz using the same instrument in CDCl₃, CD₃OD, or DMSO. Chemical shifts are reported in parts per million, and multiplicities are indicated by s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, dd = doublet of doublet, dt = doublet of triplet, and m = multiplet. Coupling constants J were reported in Hz. High resolution mass spectral data were obtained on an Agilent 6210 LC-TOF spectrometer in the positive ion mode using electrospray ionization with an Agilent G1312A HPLC pump and an Agilent G1367B autoinjector at the Integrated Molecular Structure Education and Research Center (IMSERC), Northwestern University. All final products used in assays were ≥ 95% pure by Shimadzu high-performance liquid chromatography (HPLC). Flash column chromatography was performed on an Agilent 971-FP automated flash purification system with a Varian column station and various Silicycle cartridges (4–80 g, 40–63 μm, 60 Å).

7-(3-(Aminomethyl)phenyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (7).

Compound 38 (0.14 g, 0.24 mmol) was treated with excess hydrogen chloride solution (1 M, 4.0 M in 1,4-dioxane) at 0 ^oC. The reaction mixture was stirred for 16 h at 25 °C. The solvent was removed under reduced pressure to afford the dihydrochloride salt 7 as a white solid (0.06 g, 68%). ¹H NMR (500 MHz, CD₃OD) δ 7.97 – 7.84 (m, 3H), 7.77 (d, J = 9.0 Hz, 1H), 7.71 – 7.62 (m, 2H), 7.05 (s, 1H), 4.29 (s, 2H), 2.75 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 158.1 (d, J = 248.2 Hz), 155.3, 154.2 (d, J = 3.7 Hz), 136.2, 135.2, 135.1 (d, J = 17.6 Hz), 133.8, 131.0 (d, J = 2.5 Hz), 130.8 (d, J = 2.5 Hz), 130.7, 130.6, 123.6 (d, J = 10.0 Hz), 120.5 (d, J = 3.7 Hz), 114.5, 112.7 (d, J = 26.4 Hz), 44.1, 19.4. HRMS calcd for C₁₇H₁₇FN₃⁺: 282.1401; found, 282.1408.

7-(3-(Aminomethyl)phenyl)-6-chloro-4-methylquinolin-2-amine dihydrochloride (8).

The same procedure as for 38 was followed, giving 8 as a white solid. (0.08 g, 77%) ¹H NMR (500 MHz, CD₃OD) δ 8.21 (s, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 7.62 (q, J = 2.8 Hz, 3H), 7.02 (s, 1H), 4.25 (s, 2H), 2.74 (s, 3H). ¹H NMR (500 MHz, CD₃OD) δ 8.21 (s, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 7.62 (q, J = 2.8 Hz, 3H), 7.02 (s, 1H), 4.25 (s, 2H), 2.74 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 154.3, 152.4, 143.7, 138.7, 134.5, 133.5, 129.8, 129.6, 129.2, 128.9, 128.8, 126.1, 122.0, 119.7, 113.3, 42.7, 17.9. HRMS calcd for C₁₇H₁₇ClN₃⁺: 298.1106; found, 298.1115.

General Procedure for conversion to HCl salt (Compounds 9-12).

The same procedure as for the conversion of 38 to 7 was used.

2-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-4-(aminomethyl)phenol dihydrochloride (9).

(0.07 g, 76%) ¹H NMR (500 MHz, CD₃OD) δ 7.85 (d, J = 10.4 Hz, 1H), 7.79 (d, J = 6.2 Hz, 1H), 7.48 (d, J = 2.9 Hz, 1H), 7.45 (dd, J = 8.3, 2.3 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 4.13 (s, 2H), 2.74 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 157.3 (d, J = 246.9 Hz), 155.7, 153.8, 152.9, 131.9, 131.8, 131.0, 123.9, 122.0 (d, J = 8.8 Hz), 121.7, 120.3, 116.0, 112.8, 110.3 (d, J = 25.2 Hz), 42.4, 18.0. HRMS calcd for C₁₇H₁₇FN₃O⁺: 298.1350; found, 298.1358.

2-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-5-(aminomethyl)phenol dihydrochloride (10).

(0.08 g, 80%) ¹H NMR (500 MHz, CD₃OD) δ 7.83 (d, J = 10.3 Hz, 1H), 7.74 (d, J = 6.2 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.09 (d, J = 7.7 Hz, 2H), 7.00 (d, J = 1.2 Hz, 1H), 4.14 (s, 2H), 2.72 (d, J = 1.1 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 158.2, 156.3, 155.4, 153.8, 152.9, 152.9, 135.4, 131.9, 131.9, 131.8, 131.5, 122.1, 122.0 (d, J = 26.4 Hz), 120.3 (d, J = 3.7 Hz), 119.3, 115.9, 112.8, 110.4 (d, J = 25.2 Hz), 42.6, 18.8. HRMS calcd for C₁₇H₁₇FN₃O⁺: 298.1350; found, 298.1360.

2-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-5-(2-aminoethyl)phenol dihydrochloride (11).

(0.08 g, 80%) ¹H NMR (500 MHz, CD₃OD) δ 7.83 (d, J = 10.3 Hz, 1H), 7.74 (d, J = 6.2 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.00 (s, 1H), 6.96 – 6.91 (m, 2H), 3.26 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.8 Hz, 2H), 2.73 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 157.4 (d, J = 248.2 Hz), 155.2, 153.7, 152.9 (d, J = 3.7 Hz), 139.2, 132.3, 131.9, 131.3, 121.8 (d, J = 8.8 Hz), 120.3 (d, J = 7.5 Hz), 120.1, 119.5, 115.7, 112.6, 110.3 (d, J = 26.4 Hz), 40.3, 33.0, 18.0. HRMS calcd for C₁₈H₁₉FN₃O⁺: 312.1507; found, 312.1521.

7-(5-(2-Aminoethyl)-2,3-difluorophenyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (12).

(0.07 g, 81%) ¹H NMR (500 MHz, CD₃OD) δ 7.95 (d, J = 10.4 Hz, 1H), 7.86 (d, J = 6.1 Hz, 1H), 7.50 – 7.43 (m, 1H), 7.36 (q, J = 2.9 Hz, 1H), 7.08 (d, J = 1.3 Hz, 1H), 3.30 (t, J = 7.6 Hz, 2H), 3.10 (t, J = 7.7 Hz, 2H), 2.75 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 156.6 (d, J = 248.2 Hz), 154.0, 152.8, 149.6, 147.9, 145.9, 139.13, 134.2, 132.1, 127.5 (d, J = 20.1 Hz), 126.5, 124.1 (d, J = 12.6 Hz), 123.0 (d, J = 8.8 Hz), 120.1, 118.3 (d, J = 17.6 Hz), 113.6, 111.0 (d, J = 25.2 Hz), 40.0, 32.2, 18.1. HRMS calcd for C₁₈H₁₇F₃N₃⁺: 332.1369; found, 332.1380.

7-(2,3-Difluoro-5-(2-(methylamino)ethyl)phenethyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (13).

To a solution of 71 (0.14 g, 0.21 mmol) in dry THF (3 mL) at 0 °C under a nitrogen atmosphere was added NaH (60% dispersion in mineral oil 0.02 g, 0.42 mmol) followed by iodomethane (0.04 g, 0.31 mmol). The reaction mixture was elevated to room temperature and was stirred for 16 h. After the completion of reaction, it was quenched with a saturated solution of NH₄Cl (5 mL), and the product was extracted with EtOAc (3 × 10 mL). The solution was dried over anhydrous Na₂SO₄, and the solvents were removed under reduced pressure. The product obtained was treated with excess hydrogen chloride solution (4.0 M in EtOAc) at 0 °C. The reaction mixture was stirred for 24 h at 25 °C. The solvent was removed under reduced pressure to afford dihydrochloride salt 13 as a white solid (0.07 g, 73%). ¹H NMR (500 MHz, CD₃OD) δ 7.79 (s, 1H), 7.73 (d, J = 10.4 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 6.0 Hz, 1H), 6.92 (s, 1H), 3.19 (d, J = 6.1 Hz, 7H), 3.15 – 3.10 (m, 2H), 2.93 (t, J = 7.8 Hz, 2H), 2.66 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 158.3 (d, J = 245.7 Hz), 150.6, 150.3 (dd, J = 246.9, 13.8 Hz), 147.9 (dd, J = 244.4, 12.6 Hz), 133.4 (t, J = 5.0 Hz), 132.5, 130.2 (d, J = 12.6 Hz), 125.8 (t, J = 3.15 Hz), 121.6, 119.4, 115.4 (d, J = 17.6 Hz), 113.2, 110.1 (d, J = 25.2 Hz), 40.2, 32.2, 29.5, 28.4, 27.9, 17.9. HRMS calcd for C₂₁H₂₃F₃N₃⁺: 374.1839; found, 374.1848.

7-(5-(2-Aminoethyl)-2,3-difluorophenethyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (14).

The same procedure as for 38 was followed giving 7 as a colorless solid dihydrochloride salt. (0.08 g, 81%) ¹H NMR (500 MHz, CD₃OD) δ 7.76 (d, J = 10.4 Hz, 1H), 7.54 (d, J = 6.4 Hz, 1H), 7.12 (dd, J = 8.7, 7.2 Hz, 1H), 7.06 (d, J = 5.9 Hz, 1H), 6.96 (s, 1H), 3.19 (q, J = 7.8 Hz, 4H), 3.15 – 3.09 (m, 2H), 2.94 (t, J = 7.8 Hz, 2H), 2.69 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 158.3 (d, J = 245.7 Hz), 153.7, 153.0, 150.3 (d, J = 246.9 Hz), 147.9 (d, J = 233.1 Hz), 134.7 (d, J = 20.1 Hz), 133.3, 132.2, 130.2 (d, J = 12.6 Hz), 125.7, 121.4 (d, J = 8.82 Hz), 119.0 (d, J = 5.0 Hz), 115.4 (d, J = 17.6 Hz), 112.5, 110.1 (d, J = 26.4 Hz), 40.1, 32.3, 29.5, 28.4, 18.0. HRMS calcd for C₂₀H₂₁F₃N₃⁺: 360.1682; found, 360.1693.

General Procedure for conversion to HCl salt (15-19).

The same procedure as for the conversion of 38 to 7 was used.

7-(3-(Aminomethyl)-4-(cyclopropylmethoxy)phenyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (15).

(0.07 g, 79%) ¹H NMR (500 MHz, CD₃OD) δ 7.93 – 7.73 (m, 4H), 7.28 (d, J = 8.3 Hz, 1H), 7.01 (s, 1H), 4.28 (s, 2H), 4.09 (d, J = 7.1 Hz, 2H), 2.73 (d, J = 1.2 Hz, 3H), 1.49 – 1.38 (m, 1H), 0.76 – 0.68 (m, 2H), 0.52 – 0.44 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 155.4 (d, J = 239.4 Hz), 152.4, 151.3 (d, J = 3.7 Hz), 132.0 (d, J = 16.3 Hz), 130.9, 130.1, 129.6, 124.7, 120.1 (d, J = 6.3 Hz), 116.9, 111.3, 110.6, 109.6 (d, J = 26.4 Hz), 71.8, 37.2, 16.5, 8.0, 0.7. HRMS calcd for C₂₁H₂₃FN₃O⁺: 352.1820; found, 352.1836.

4-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-2-(aminomethyl)phenol dihydrochloride (16).

(0.09 g, 75%) ¹H NMR (500 MHz, CD₃OD) δ 7.88 (d, J = 11.2 Hz, 1H), 7.79 (d, J = 6.8 Hz, 1H), 7.70 (t, J = 2.1 Hz, 1H), 7.64 (dt, J = 8.4, 2.0 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 1.3 Hz, 1H), 4.23 (s, 2H), 2.72 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 157.8, 157.0, 155.8, 153.9, 152.9, 152.8, 133.9 (d, J = 16.3 Hz), 132.5, 131.4 (d, J = 2.5 Hz), 125.3, 121.5 (d, J = 8.8 Hz), 119.9, 118.3 (d, J = 3.7 Hz), 115.2, 112.7, 111.1 (d, J = 26.4 Hz), 39.1, 18.0. HRMS calcd for C₁₇H₁₇FN₃O⁺: 298.1350; found, 298.1355.

7-(3-(Aminomethyl)-4-fluorophenyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (17).

(0.07 g, 83%) ¹H NMR (500 MHz, CD₃OD) δ 7.92 (t, J = 8.6 Hz, 2H), 7.85 (dd, J = 18.3, 5.3 Hz, 2H), 7.46 (t, J = 9.2 Hz, 1H), 7.04 (s, 1H), 4.34 (s, 2H), 2.73 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 161.4 (d, J = 250.7 Hz), 156.6 (d, J = 248.2 Hz), 154.0, 152.7, 132.7, 132.6 (d, J = 3.7 Hz), 132.2 (d, J = 8.8 Hz), 132.0, 130.9, 122.3 (d, J = 10.0 Hz), 120.9 (d, J = 15.1 Hz), 119.1, 116.1 (d, J = 22.6 Hz), 113.2, 111.3 (d, J = 25.2 Hz), 36.5 (d, J = 3.7 Hz), 18.0. HRMS calcd for C₁₇H₁₆F₂N₃⁺: 300.1307; found, 300.1320.

5-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-2-(aminomethyl)phenol dihydrochloride (18).

(0.08 g, 78%) ¹H NMR (500 MHz, CD₃OD) δ 7.90 (d, J = 11.1 Hz, 1H), 7.79 (d, J = 6.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.26 (t, J = 1.8 Hz, 1H), 7.21 (dt, J = 7.8, 1.7 Hz, 1H), 7.02 (s, 1H), 4.22 (s, 2H), 2.73 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 156.6 (d, J = 246.9 Hz), 156.1, 154.0, 152.7, 136.5, 133.8 (d, J = 17.6 Hz), 132.6, 130.7, 122.2 (d, J = 10.0 Hz), 120.2 (d, J = 13.8 Hz), 119.0, 115.4 (d, J = 2.5 Hz), 113.1, 111.2 (d, J = 26.4 Hz), 38.9, 18.0. HRMS calcd for C₁₇H₁₇FN₃O⁺: 298.1350; found, 298.1356.

7-(4-(Aminomethyl)-3-fluorophenyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (19).

(0.10 g, 82%) ¹H NMR (500 MHz, CD₃OD) δ 7.94 (d, J = 11.2 Hz, 1H), 7.86 (d, J = 6.6 Hz, 1H), 7.73 (t, J = 7.9 Hz, 1H), 7.66 – 7.59 (m, 2H), 7.05 (s, 1H), 4.33 (s, 2H), 2.74 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 161.0 (d, J = 248.2 Hz), 156.5 (d, J = 248.2 Hz), 154.0, 152.7 (d, J = 3.7 Hz), 137.4 (d, J = 7.5 Hz), 132.5, 132.1 (d, J = 16.3 Hz), 131.4 (d, J = 3.7 Hz), 125.4, 122.6 (d, J = 8.8 Hz), 120.9 (d, J = 16.3 Hz), 119.1 (d, J = 2.5 Hz), 116.2 (dd, J = 22.6, 3.7 Hz), 113.4, 111.5 (d, J = 26.4 Hz), 36.4, 18.0. HRMS calcd for C₁₇H₁₆F₂N₃⁺: 300.1307; found, 300.1319.

General Procedure for conversion to N-Me substituted HCl salt (20-24).

The same procedure as for the conversion of 71 to 13 was used.

2-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-4-((methylamino)methyl)phenol dihydrochloride (20).

(0.08 g, 76%) ¹H NMR (500 MHz, CD₃OD) δ 8.07 (s, 1H), 7.81 (d, J = 10.3 Hz, 1H), 7.48 (d, J = 3.2 Hz, 1H), 7.44 (dd, J = 8.4, 2.4 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.97 (s, 1H), 4.12 (s, 2H), 3.21 (s, 3H), 2.70 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 157.2 (d, J = 246.9 Hz), 155.7, 153.1, 132.3, 131.8 (d, J = 2.5 Hz), 130.9, 123.9, 122.2, 121.8, 120.8, 116.0, 113.5, 110.3 (d, J = 26.4 Hz), 42.4, 27.9, 17.9. HRMS calcd for C₁₈H₁₉FN₃O⁺: 312.1507; found, 312.1500.

2-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-5-((methylamino)methyl)phenol dihydrochloride (21).

(0.11 g, 80%) ¹H NMR (500 MHz, CD₃OD) δ 8.00 (s, 1H), 7.81 (d, J = 10.3 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.96 (s, 1H), 4.15 (s, 2H), 3.20 (s, 3H), 2.69 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 157.2 (d, J = 250.7 Hz), 155.4, 153.1, 150.6, 135.3, 132.3, 131.6 (d, J = 2.5 Hz), 131.3, 122.2, 120.7, 119.3, 115.9, 113.4, 110.3 (d, J = 26.4 Hz), 42.6, 27.76, 17.9. HRMS calcd for C₁₈H₁₉FN₃O⁺: 312.1507; found, 312.1503.

2-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-5-(2-(methylamino)ethyl)phenol dihydrochloride (22).

(0.08 g, 83%) ¹H NMR (500 MHz, CD₃OD) δ 8.00 (s, 1H), 7.80 (d, J = 10.4 Hz, 1H), 7.33 (dd, J = 8.3, 1.3 Hz, 1H), 6.94 (d, J = 6.2 Hz, 3H), 3.26 (t, J = 7.8 Hz, 2H), 3.21 (s, 3H), 3.01 (t, J = 7.8 Hz, 2H), 2.70 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 157.3 (d, J = 248.2 Hz), 155.2, 153.1, 139.1, 132.3, 131.3, 122.1, 120.7, 120.3, 119.5, 115.7, 113.3, 110.2 (d, J = 26.4 Hz), 40.3, 33.0, 27.7, 17.9. HRMS calcd for C₁₉H₂₁FN₃O⁺: 326.1663; found, 326.1668.

7-(4-(Cyclopropylmethoxy)-3-((methylamino)methyl)phenyl)-6-fluoro-4-methylquinolin-2-amine dihydrochloride (23).

(0.07 g, 79%) ¹H NMR (500 MHz, CD₃OD) δ 8.10 (s, 1H), 7.86 (d, J = 11.2 Hz, 1H), 7.81 – 7.73 (m, 2H), 7.29 (d, J = 8.6 Hz, 1H), 6.97 (s, 1H), 4.28 (s, 2H), 4.10 (d, J = 7.1 Hz, 2H), 3.24 (s, 3H), 2.70 (s, 3H), 1.48 – 1.39 (m, 1H), 0.72 (d, J = 8.2 Hz, 2H), 0.48 (d, J = 4.9 Hz, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 158.3, 157.1 (d, J = 246.9 Hz), 153.6, 133.3, 132.0, 131.6 (d, J = 3.7 Hz), 128.9, 126.9, 122.4, 122.1, 119.3, 112.7, 111.6 (d, J = 25.2 Hz), 73.7, 39.2, 28.3, 18.3, 10.0, 2.7. HRMS calcd for C₂₂H₂₅FN₃O⁺: 366.1976; found, 366.1998.

4-(2-Amino-6-fluoro-4-methylquinolin-7-yl)-2-((methylamino)methyl)phenol dihydrochloride (24).

(0.09 g, 72%) ¹H NMR (500 MHz, CD₃OD) δ 8.07 (s, 1H), 7.85 (d, J = 11.2 Hz, 1H), 7.71 (s, 1H), 7.64 (dt, J = 8.4, 2.0 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 6.96 (s, 1H), 4.23 (s, 2H), 3.24 (s, 3H), 2.69 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 156.9, 156.8 (d, J = 246.9 Hz), 153.2, 133.0, 131.38 (d, J = 3.7 Hz), 131.32 (d, J = 3.7 Hz), 125.5, 121.8, 119.9, 118.8, 115.3, 113.4, 111.1 (d, J = 25.2 Hz), 39.2, 27.8, 17.8. HRMS calcd for C₁₈H₁₉FN₃O⁺: 312.1507; found, 312.1503.

Pharmacokinetics

Animal pharmacokinetic experiments were conducted at TheraIndx Lifesciences Pvt. Ltd. (in vivo test facility, Bangalore, India) in accordance with the guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India, and were approved by the Institutional Animal Ethics Committee of TheraIndx Lifesciences Pvt. Ltd. (Study number: TI-2025-1532 A) prior to initiation of the study.

Supplementary Material

NOS enzyme inhibition assay, PAMPA-BBB assay, computational details, crystallographic data collection and refinement statistics for rat nNOS, human nNOS, and human eNOS, synthetic details and characterization for the key intermediates and the final compounds; copies of NMR spectra and HPLC traces for the final compounds (PDF)

Molecular formula strings (CSV)

Abbreviations Used

AUC_0-∞

Area under the plasma concentration-time curve (to infinity)

BBB

blood–brain barrier

C₀

initial plasma concentration

Caco-2

cancer coli-2

CL

clearance

C_max

Maximum plasma concentration

CNS

central nervous system

DFT

density functional theory

eNOS

endothelial nitric oxide synthase

ESP

electrostatic potential

FMN

flavin mononucleotide

GBSA

generalized Born and surface area

H4B

(6R)-5,6,7,8-tetrahydrobiopterin

heNOS

human endothelial nitric oxide synthase

hiNOS

human inducible nitric oxide synthase

hnNOS

human neuronal nitric oxide synthase

iNOS

inducible nitric oxide synthase

LLOQ

Lower Limit of Quantification

MD

molecular dynamics

MM

molecular mechanics

MOMCl

methoxymethyl chloride

NADPH

reduced nicotinamide adenine dinucleotide phosphate

nNOS

neuronal nitric oxide synthase

NO

nitric oxide

PAMPA

parallel artificial membrane permeability assay

P _e

effective permeability

rnNOS

rat neuronal nitric oxide synthase

RMSD

root-mean-square deviation

SAR

structure–activity relationship

TLC

thin-layer chromatography

t_1/2

Elimination half-life

T_max

Time to reach maximum concentration