Intramolecular hydrogen-bonded nitric oxide synthase inhibitors

Abstract

Compounds and related compositions and methods as can be used to selectively inhibit neuronal nitric oxide synthase and as can be employed in the treatment of various neurodegenerative diseases.

Claims

1. A compound selected from compounds of a formula ##STR00004## wherein Y is a covalent bond; Φ is a furanyl moiety; and X is an unshared electron pair on a heteroatom of said furanyl moiety, and salts thereof.

2. The compound of claim 1 comprising a cis diastereomeric configuration.

3. The compound of claim 1 wherein said compound is an ammonium salt.

4. The compound of claim 1 wherein said compound is an ammonium salt, and said salt has a counter ion that is a conjugate base of a protic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1. Chemical structure and inhibitory activity of prior art compounds 1 and 2.

(3) FIG. 2. Chemical structures of inhibitor compounds 3a-f, in accordance with several non-limiting embodiments of this invention.

(4) FIGS. 3A-B. A. Crystallographic binding conformations of compounds 3a-3d (overlaid). 3a (green), 3b cyan, 3c yellow, 3d magenta, heme is shown in light pink; H.sub.4B is blue. Residues in the structures of 3a-3c overlay nearly perfectly so only 3a is shown. Residues of 3d (the furan-containing inhibitor) are also shown (magenta) because of the difference in the configuration of Met336. B. Crystallographic binding conformations of 1 (3JWS, cyan), 2 (3NLK, magenta) and 3a (green), overlaid. Residues overlay nearly perfectly, so only those corresponding to the crystal structure of 3a are shown.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

(5) The design and synthesis of certain nNOS inhibitors (e.g., without limitation, compounds 3a-f, FIG. 2) of this invention can be considered 1) through replacement of the amino group on the chiral pyrrolidine ring of compound 1 by an oxygen atom, and 2) with a hydrogen bond acceptor/donor group (X) incorporated into the aromatic tail. It was hypothesized that an intramolecular hydrogen bond can influence inhibitor membrane and BBB permeability while not compromising inhibitory activity. Without limitation to any one theory or mode of operation in cell-membrane or the BBB, where the chemical environments are highly hydrophobic, the inhibitors can be considered to form an intramolecular hydrogen bond and adopt a “closed” conformation, which show lower overall polarity and improved BBB permeability by the mechanism of passive diffusion. Conversely, the inhibitors bind to nNOS with an “open” conformation, similar to parent compound 1, in which the secondary amino group is released from the intramolecular hydrogen bonding. A simple, colorimetric, cell-based assay using HEK293t cells stably transfected with rat nNOS was employed to assess the membrane permeability of such compounds, and crystal structures show that the “open” conformation is indeed achieved when these inhibitors are in the nNOS active site.

(6) The synthesis of inhibitors 3a-d used chiral precursor 4. (See, application Ser. No. 61/360,727 filed Jul. 1, 2010, the entirety of which is incorporated herein by reference, for preparation of the pyrrolidine 3,4-cis and -trans diastereomers; Scheme 1, below.) Allylation of (3S,4S) pyrrolidine intermediate 4 using allyl methyl carbonate in the presence of Pd(PPh.sub.3).sub.4 gave alkene 5 in good yield. Alkene ozonolysis provided aldehyde 6, which was then subjected to reductive amination with various methanamines to yield the corresponding secondary amines 7a-d. Finally, the three Boc-protecting groups were removed concurrently in TFA to generate inhibitors 3a-d. Likewise, as would be understood by those skilled in the art and made aware of this invention, aldehyde 6 can be reacted with suitably protected 2-oxoethanamine or 3-oxopropanamine en route to inhibitor compounds 3e and 3f, respectively. (See, e.g., FIG. 2 where, without limitation, R can represent one or more halo substituents (e.g., F, Cl, etc.) of the sort to affect NOS inhibition.)

(7) ##STR00003##

(8) While the present invention can be illustrated in the context of a 4-methylpyridine moiety conjugated with a pyrrolidine core, will be understood by those skilled in the art that heterocycle conjugation via alkylation and pyrrolidine epoxide ring-opening can be achieved with various other haloalkyl-pyridine and other haloalkylheterocyclic moieties. For example, without limitation, various other heterocyclic moieties including but not limited to substituted and unsubstituted thiazine, oxazine, pyrazine, oxazole and imidazole moieties are described in U.S. Pat. No. 7,470,790 issued Dec. 30, 2008 and co-pending application Ser. No. 11/906,283 filed Oct. 1, 2007, in the context of substructures I and III as discussed more fully therein, each of which is incorporated herein by reference in its entirety. The corresponding chiral pyrrolidine core compounds can be prepared using synthetic techniques of the sort described herein or straight forward modifications thereof, as would be understood by those skilled in the art and made aware of this invention. Such heterocycle-conjugated compounds, analogous to core compound 4, above, can be used en route to NOS inhibitors, including selective nNOS inhibitors, of the sort described in the aforementioned incorporated references.

(9) The potencies of these compounds were tested in vitro both in a purified enzyme assay and in a cell-based assay using 293t cells stably transfected with rat nNOS. The purified enzyme assay results demonstrate that compounds 3a-d maintain adequate potency for nNOS in comparison with previous inhibitors 1 and 2 (Table 1). Furthermore, these compounds retain some selectivity over the iNOS and eNOS isoforms.

(10) TABLE-US-00001 TABLE 1 K.sub.i values of 3a-3d with all three NOS isoforms.sup.a. selectivity.sup.b Compound nNOS (μM) eNOS (μM) iNOS (μM) n/e n/i 3a 0.41 40 44 98 105 3b 0.82 77 114 93 139 3c 0.39 37 57 93 143 3d 0.40 66 36 169 92 .sup.aThe K.sub.i values were calculated based on the directly measured IC.sub.50 values (see examples, below), which represent at least duplicate measurements with standard deviations of ±10%. .sup.bThe ratio of K.sub.i (eNOS or iNOS) to K.sub.i (nNOS).

(11) The cellular potencies of these compounds were measured from the inhibition of the production nitric oxide in HEK293t cells stably expressing nNOS (Table 2). Comparing the IC.sub.50 values of all compounds in the purified enzyme assay versus the cellular assay clearly indicates that permeability limits the effective concentration inhibitors within cells. However, in comparison to 1 and 2, 3c and 3d are similar in permeability while 3a and 3b clearly show improved penetration. Without limitation, the difference in membrane penetration between 3a-b and 3c-d, may also be a result of the hydrophilic nature of the phenol and the furan substituents of 3c and 3d, respectively, in comparison with the more lipophilic fluorophenyl and methoxyphenyl substituents of 3a and 3b, respectively.

(12) TABLE-US-00002 TABLE 2 IC.sub.50 values of NOS inhibitors in purified enzyme assay and cell-based assay. In vitro Cell-based Relationship Compound IC.sub.50 (μM) IC.sub.50 (μM) (fold higher in cells) 1 0.45 325 722 x 2 1.0 125 125 x 3a 3.6 120  33 x 3b 7.1 190  27 x 3c 3.5 520 150 x 3d 3.4 420 123 x Aminopyridine.sup.a 0.05 25  500x L-NNA.sup.b 5.0 5.0 1 .sup.aaminopyridine = 2-amino-4,6-dimethylaminopyridine hydrochloride. .sup.bL-NNA = L-N-nitroarginine, a NOS inhibitor known to be cell-permeable.

(13) Furthermore, Table 2 shows that while 2 and 3a have similar IC.sub.50 values in the cell-based assay, when compared to their potencies in the purified enzyme assay, the cell-based IC.sub.50 value of 3a is only 33 times higher than its in vitro IC.sub.50, while the cell-based IC.sub.50 value of 2 is 125 higher than its in vitro potency. Compounds 3a and 3b have slightly weaker inhibition of nNOS directly, as reported by their in vitro IC.sub.50 values, but a cell-based IC.sub.50 value close to that of 2. This suggests that 3a and 3b have significantly higher permeability. The aminopyridine fragment alone (2-amino-4,6-dimethylaminopyridine) was also tested to comparatively evaluate the membrane permeability of this moiety of our nNOS inhibitors. This aminopyridine has a pK.sub.a of ˜7, and will therefore be partially protonated at physiological pH. It has moderate cell-based potency, and a similar low micromolar in vitro potency, suggesting that this fragment is actually quite membrane-permeable.

(14) Crystal structures of all four compounds in the nNOS active site were obtained (FIG. 3). Like 1 and 2, compounds 3a-3d all bind the nNOS active site in a “normal binding mode”, with the 2-amino-4-methylpyridine binding over the heme iron and interacting with Glu592. The pyrrolidine nitrogen hydrogen bonds with Glu592, and interacts near Asp597, the residue that can be exploited for selectivity between nNOS and eNOS (Asn368). The nitrogen of the substituted phenylmethyamino (or furanmethylamino) hydrogen bonds with heme propionate D in all 4 inhibitors. The substituted phenyls or the furan occupy the pocket lined with hydrophobic residues Met336, Leu337, Tyr709. There are small differences in the orientation of the carbon chain between the pyrrolidine and the amine that engages in hydrogen bonding with the heme propionate. Another difference is that in the crystal structure of 3d, which contains the furan in place of the phenyl substituents of 3a-c, Met336 is rotated in closer to the inhibitor molecule which the smaller furan is able to accommodate. FIG. 3B shows the overlay of previously designed inhibitors 1 and 2 with 3a. Inhibitor 1 contains a nitrogen in place of the oxygen atoms off of the pyrrolidine moiety in 2 and 3a-d, but the more noticeable difference is between inhibitors 1 and 2 and inhibitor 3: in the latter, one less methylene unit in the carbon chain to the phenyl substituent. The hydrophobic pocket is large enough to accommodate either chain length, and although these inhibitors adapt slightly different conformations, there are no significant changes in the residues in this pocket. The slightly higher K.sub.i of compound 3b, the methoxy-substituted inhibitor, may be due to a slight steric clash with Met336 in this pocket.

(15) As demonstrated by the following representative examples, a new series of chiral nNOS inhibitors can be synthesized. Without limitation, through an intramolecular hydrogen bond, representative inhibitor compounds 3a-d can form a “closed” conformation, which provides enhanced lipophilicity and therefore, improved membrane and BBB permeability. When binding to a target nNOS, inhibitors 3a-d can release the secondary amino group to adopt an “open” conformation—to maintain the high potency and isozyme selectivity of corresponding parent compound.

EXAMPLES OF THE INVENTION

(16) The following non-limiting examples and data illustrate various aspects and features relating to the compounds, compositions and/or methods of the present invention, including the use of intramolecular hydrogen bonding to improve the efficacy of such compounds, as are available through the synthetic methodologies described herein. In comparison with the prior art, the present compounds, compositions and/or methods provide results and data which are surprising, unexpected and contrary thereto. While the utility of this invention is illustrated through the use of several compounds, moieties thereof and groups thereon, it will be understood by those skilled in the art that comparable results are obtainable with various other compounds and moieties/groups, as are commensurate with the scope of this invention.

Example 1

(17) (3S,4S)-tert-Butyl 3-(allyloxy)-4-((6-(bis(tert-butoxycarbonyl)amino)-4-methylpyridin-2-yl)methyl)pyrrolidine-1-carboxylate (5). To a solution of 4 (1.15 g, 2.0 mmol) and Pd(Ph.sub.3P).sub.4 (235 mg, 0.2 mmol) in dry THF (50 mL) was added allyl methyl carbonate (700 μL, 6.0 mmol). (See, Haight, A. R.; Stoner, E. J.; Peterson, M. J.; Grover, V. K. General method for the palladium-catalyzed allylation of aliphatic alcohols. J. Org. Chem. 2003, 68, 8092-8096.) The reaction mixture was allowed to stir at 45° C. for 5 h, and then concentrated. The resulting material was purified by flash column chromatography (silica gel, EtOAc/hexanes, 1:2, R.sub.f=0.40) to yield 5 (675 .g, 66%) as a colorless oil: .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.40-1.50 (m, 27H), 2.25-2.27 (m, 3H), 2.60-2.75 (m, 1H), 2.78-2.85 (dd, J=9.0, 13.5 Hz, 1H), 2.98-3.05 (dd, J=9.0, 13.5 Hz, 1H), 3.10-3.21 (m, 1H), 3.25-3.29 (dd, J=4.0, 12.5 Hz, 1H), 3.40-3.62 (m, 2H), 3.75-3.85 (m, 2H), 4.00-4.10 (td, J=5.5, 13.0 Hz, 1H), 5.15-5.17 (d, J=10.5 Hz, 1H), 5.25-5.29 (d, J=17.0 Hz, 1H), 5.84-5.91 (ddd, J=5.0, 10.5, 17.0 Hz, 1H), 6.85-6.95 (m, 2H); .sup.13CNMR (125 MHz, CDCl.sub.3) δ 20.9, 27.9, 28.4, 28.5, 34.7, 34.8, 42.7, 43.3, 48.9, 49.2, 50.4, 51.0, 70.2, 70.3, 77.8, 78.6, 79.1, 79.2, 82.8, 116.7, 116.9, 119.6, 122.9, 134.6, 134.7, 149.50, 149.52, 151.4, 151.5, 151.8, 154.5, 154.8, 159.2, 159.3; LC-TOF (M+H.sup.+) calcd for C.sub.29H.sub.46N.sub.307 548.3336. found 548.3339.

Example 2

(18) (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-4-methylpyridin-2-yl)methyl)-4-(2-oxoethoxyl)pyrrolidine-1-carboxylate (6). A solution of 5 (100 mg, 0.19 mmol) in CH.sub.2Cl.sub.2 (10 mL) was cooled to −78° C., to which O.sub.3 was charged until the reaction solution turned purple (˜10 min). The O.sub.3 flow was stopped, and the reaction was allowed to stir at the same temperature for 30 min. To the resulting solution was added Me.sub.2S (150 μL). The reaction mixture was then warmed to room temperature and kept stirring at room temperature for an additional 2 h. The solvent was removed by rotary evaporation and the resulting crude product was purified by flash column chromatography (EtOAc/hexanes, 1:1, R.sub.f=0.2) to yield 6 (87 mg, 87%) as a colorless oil: .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.45 (s, 27H), 2.22 (s, 3H), 2.70-2.85 (m, 111), 2.85-2.95 (m, 1H), 3.05-3.15 (m, 1H), 3.16-3.25 (m, 1H), 3.30-3.37 (m, 1H), 3.45-3.70 (m, 2H), 3.85-3.95 (m, 2H), 4.05-4.20 (t, J=10.0 Hz, 1H), 6.90-6.93 (m, 2H), 9.66 (s, −1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 20.9, 24.7, 27.9, 28.5, 29.7, 34.4, 42.5, 43.2, 48.8, 49.1, 50.3, 51.0, 74.6, 74.9, 79.4, 79.5, 80.4, 83.0, 119.66, 119.73, 122.8, 149.7, 149.8, 151.57, 151.60, 151.8, 154.4, 154.8, 159.87, 158.94, 200.2, 200.6; LC-TOF (M+H.sup.+) calcd for C.sub.28H.sub.44N.sub.3O.sub.8 550.3128. found 550.3130.

Example 3

(19) (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-4-methylpyridin-2-yl)methyl)-4-(2-(2-fluorobenzylamino)ethoxy)pyrrolidine-1-carboxylate (7a). To a solution of aldehyde 6 (100 mg, 0.18 mmol) in DCM (4 mL) was added (2-fluorophenyl)methanamine (45 mg, 0.36 mmol), triethylamine (11 μL, 0.36 mmol), followed by NaHB(OAc).sub.3 (100 mg, 0.45 mmol). The reaction mixture was stirred for an additional 3 h and then concentrated. The crude product was purified by flash column chromatography (EtOAc/hexanes, 1:1, R.sub.f=0.15) to yield 7a (115 mg, 91%) as a colorless oil: .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.43-1.44 (s, 27H), 2.29-2.30 (m, 3H), 2.60-2.75 (m, 1H), 2.76-2.85 (m, 2H), 2.92-3.00 (m, 1H), 3.05-3.14 (m, 1H), 3.20-3.50 (m, 5H), 3.52-3.70 (m, 2H), 3.72-3.80 (m, 1H), 3.91 (s, 2H), 6.84-6.85 (d, J=8.0 Hz, 1H), 6.89-6.91 (d, J=10.0 Hz, 1H), 7.02-7.06 (dd, J=9.0, 9.5 Hz, 1H), 7.10-7.13 (dd, J=7.0, 7.5 Hz, 1H), 7.20-7.30 (m, 1H), 7.35-7.40 (m, 1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 20.9, 24.7, 27.9, 28.47, 28.50, 29.7, 34.6, 34.7, 36.6, 42.6, 43.3, 46.8, 48.2, 48.8, 49.1, 50.3, 50.8, 68.0, 68.2, 78.7, 79.2, 79.3, 79.4, 82.8, 115.2, 115.3, 115.4, 115.5, 119.5, 119.6, 122.8, 124.17, 124.19, 128.89, 128.92, 128.95, 128.99, 130.46, 130.51, 130.55, 149.6, 151.4, 151.5, 151.8, 154.5, 154.8, 159.08, 159.14, 160.3, 162.2; LC-TOF (M+H.sup.+) calcd for C.sub.35H.sub.52FN.sub.4O.sub.7 659.3820. found 659.3818.

Example 4

(20) (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-4-methylpyridin-2-yl)methyl)-4-(2-(2-methoxybenzylamino)ethoxy)pyrrolidine-1-carboxylate (7b). Compound 7b was synthesized using a similar procedure to that of 7a (88%): .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.42-1.43 (s, 27H), 2.31-2.33 (m, 3H), 2.60-2.70 (m, 1H), 2.70-2.80 (m, 1H), 2.90-3.10 (m, 4H), 3.28-3.32 (m, 1H), 3.35-3.52 (m, 3H), 3.65-3.75 (m, 1H), 3.79-3.81 (m, 2H), 3.85-3.87 (m, 3H), 4.01-4.20 (m, 2H), 6.75-7.00 (m, 4H), 7.26-7.30 (m, 1H), 7.35-7.40 (m, 1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 20.9, 21.9, 27.9, 28.3, 28.4, 28.5, 29.7, 34.4, 34.5, 42.5, 43.2, 46.1, 47.5, 47.7, 48.7, 48.9, 50.3, 50.8, 55.4, 55.5, 55.6, 60.4, 65.2, 65.3, 79.3, 79.4, 79.8, 82.92, 82.94, 110.5, 110.6, 119.7, 119.8, 121.0, 121.2, 121.3, 122.7, 122.8, 130.50, 130.52, 131.4, 149.9, 151.47, 151.51, 151.77, 151.79, 154.5, 154.7, 157.7, 158.89, 158.91, 176.2; LC-TOF (M+H.sup.+) calcd for C.sub.36H.sub.55N.sub.4O.sub.8 671.4020. found 671.4016.

Example 5

(21) (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-4-methylpyridin-2-yl)methyl)-4-(2-(2-hydroxybenzylamino)ethoxy)pyrrolidine-1-carboxylate (7c). Compound 7c was synthesized using a similar procedure to that of 7a (55%): .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.45-1.46 (s, 27H), 2.34 (s, 3H), 2.60-2.75 (m, 2H), 2.75-2.85 (m, 1H), 2.85-3.00 (m, 2H), 3.00-3.07 (m, 1H), 3.07-3.20 (m, 1H), 3.20-3.33 (m, 1H), 3.33-3.51 (m, 3H), 3.51-3.65 (m, 1H), 3.65-3.74 (m, 2H), 3.74-3.90 (m, 1H), 4.00-4.18 (m, 2H), 6.75-6.85 (m, 2H), 6.93 (s, 1H), 7.02-7.10 (m, 1H), 7.15-7.20 (m, 1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 20.9, 21.1, 27.9, 28.5, 29.7, 34.1, 42.4, 43.1, 46.8, 47.0, 48.4, 48.9, 50.1, 50.3, 50.7, 50.8, 53.4, 60.3, 66.3, 66.4, 78.6, 79.3, 79.5, 79.6, 82.9, 83.2, 833, 116.1, 116.3, 118.9, 119.4, 119.7, 120.3, 120.5, 120.9, 122.9, 123.0, 123.2, 128.8, 128.9, 129.4, 129.6, 129.7, 150.3, 150.6, 151.4, 151.6, 151.7, 154.7, 154.9, 157.2, 157.3, 159.1, 159.2; LC-TOF (M+H.sup.+) calcd for C.sub.35H.sub.53N.sub.4O.sub.8 657.3863. found 657.3874.

Example 6

(22) (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-4-methylpyridin-2-yl)methyl)-4-(2-(furan-2-ylmethylamino)ethoxy)pyrrolidine-1-carboxylate (7d). Compound 7b was synthesized using a similar procedure to that of 7a (90%): .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.43-1.44 (s, 27H), 2.30-2.32 (m, 3H), 2.50-2.60 (m, 1H), 2.75-2.83 (m, 2H), 2.92-3.17 (m, 3H), 3.20-3.50 (m, 5H), 3.52-3.70 (m, 2H), 3.77-3.80 (m, 1H), 3.81 (s, 2H), 6.20-6.21 (d, J=3.0 Hz, 1H), 6.32 (s, 1H), 6.86-6.92 (m, 2H), 7.37 (s, 1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 19.1, 20.9, 21.0, 23.4, 24.7, 27.9, 28.39, 28.48, 28.51, 29.7, 30.6, 34.6, 34.7, 36.6, 42.6, 43.2, 45.8, 45.9, 48.2, 48.3, 48.8, 49.1, 50.3, 50.8, 64.4, 68.2, 68.4, 78.7, 79.2, 79.3, 79.4, 82.85, 82.86, 107.0, 107.2, 110.16, 110.24, 119.57, 119.61, 122.8, 141.90, 141.94, 141.97, 149.6, 151.45, 151.50, 151.8, 153.4, 154.5, 154.7, 159.1, 159.2, 171.3; LC-TOF (M+H.sup.+) calcd for C.sub.33H.sub.51N.sub.4O.sub.8 631.3703. found 631.3703.

Example 7

(23) 6-(((3S,4S)-4-(2-(2-Fluorobenzylamino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine (3a). To a solution of 7a (70 mg, 0.10 mmol) in MeOH (2 mL) was added 6 N HCl (4 mL) at room temperature. The mixture was stirred for 12 h and then concentrated. The crude product was purified by recrystallization (EtOH/H.sub.2O) to give inhibitor 3a (38 mg, 97%): .sup.1H NMR (500 MHz, D.sub.2O) δ 2.18 (s, 3H), 2.68-2.73 (m, 1H), 2.76-2.82 (dd, J=7.0, 15.5 Hz, 1H), 2.85-2.90 (dd, J=8.0, 15.0 Hz, 1H), 3.04-3.09 (t, J=11.5 Hz, 1H), 3.15-3.25 (m, 2H), 3.39-3.43 (dd, J=8.5, 11.5 Hz, 1H), 3.50-3.53 (d, J=13.5 Hz, 1H), 3.53-3.60 (m, 1H), 3.73-3.80 (m, 1H), 4.05-4.10 (m, 1H), 4.22 (s, 2H), 6.47 (s, 1H), 6.53 (s, 1H), 7.12-7.14 (dd, J=1.0, 8.5 Hz, 1H), 7.16-7.18 (dd, J=1.0, 8.0 Hz, 1H), 7.35-7.40 (m, 1H); .sup.13C NMR (125 MHz, D.sub.2O) δ 21.0, 28.8, 41.3, 44.67, 44.70, 46.4, 47.1, 49.3, 63.9, 78.0, 110.3, 114.0, 115.8, 115.9, 117.4, 117.5, 125.0, 125.1, 132.1, 132.2, 132.3, 132.4, 145.7, 153.8, 158.1, 160.1, 162.0; LC-TOF (M+H.sup.+) calcd for C.sub.20H.sub.28FN.sub.4O 359.2247. found 359.2253.

Example 8

(24) 6-(((3S,4S)-4-(2-(2-Methoxybenzylamino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine (3b). Compound 3b was synthesized using a similar procedure to that of 3a (96%): .sup.1H NMR (500 MHz, CDCl.sub.3) δ 2.18 (s, 3H), 2.65-2.85 (m, 3H), 3.03-3.10 (t, J=11.5 Hz, 1H), 3.17-3.32 (m, 3H), 3.40-3.47 (dd, J=8.5, 12.0 Hz, 1H), 3.50-3.57 (m, 2H), 3.72-3.80 (m, 4H), 4.05-4.17 (m, 3H), 6.43 (s, 1H), 6.49 (s, 1H), 6.91-6.94 (dd, J=7.0, 7.5 Hz, 1H), 6.97-6.99 (d, J=8.5 Hz, 1H), 7.23-7.25 (dd, J=1.0, 7.5 Hz, 1H), 7.34-7.38 (ddd, J=1.5, 8.5, 9.0 Hz, 1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 21.0, 38.7, 41.0, 46.2, 46.9, 47.1, 49.1, 55.4, 64.0, 77.8, 110.3, 111.2, 113.8, 118.2, 121.0, 131.6, 131.8, 145.6, 153.8, 157.6, 158.0; LC-TOF (M+H.sup.+) calcd for C.sub.21H.sub.31N.sub.4O.sub.2 371.2447. found 371.2450.

Example 9

(25) 2-((2-((3S,4S)-4-((6-Amino-4-methylpyridin-2-yl)methyl)pyrrolidin-3-yloxy)ethylamino)methyl)phenol (3c). Compound 3c was synthesized using a similar procedure to that of 3a (92%): .sup.1H NMR (500 MHz, CDCl.sub.3) δ 2.15 (s, 3H), 2.60-2.70 (m, 1H), 2.77-2.82 (m, 2H), 3.00-3.10 (t, J=11.5 Hz, 1H), 3.18-3.22 (m, 3H), 3.42-3.51 (dd, J=8.5, 11.5 Hz, 1H), 3.51-3.56 (m, 2H), 3.73-3.80 (m, 1H), 4.00-4.25 (m, 3H), 6.38 (s, 1H), 6.50 (s, 1H), 6.83-6.88 (m, 2H), 7.21-7.25 (m, 2H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 21.0, 28.8, 41.2, 46.0, 47.0, 47.1, 49.2, 63.7, 77.8, 110.3, 114.0, 115.4, 117.0, 120.6, 131.6, 131.7, 145.6, 153.8, 155.0, 158.0; LC-TOF (M+H.sup.+) calcd for C.sub.20H.sub.29N.sub.4O.sub.2 357.2291. found 357.2277.

Example 10

(26) 6-(((3S,4S)-4-(2-(Furan-2-ylmethylamino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine (3d). Compound 3b was synthesized using a similar procedure to that of 3a (96%): .sup.1H NMR (500 MHz, CDCl.sub.3) δ 2.32 (s, 3H), 2.65-2.73 (m, 1H), 2.76-2.83 (dd, J=7.5, 15.0 Hz, 1H), 2.88-2.93 (dd, J=7.5, 14.5 Hz, 1H), 3.04-3.09 (t, J=11.5 Hz, 1H), 3.17-3.24 (m, 3H), 3.39-3.43 (dd, J=9.0, 11.5 Hz, 1H), 3.48-3.51 (d, J=13.5 Hz, 2H), 3.51-3.56 (m, 1H), 3.70-3.75 (m, 1H), 4.05-4.10 (m, 1H), 4.22 (s, 2H), 6.38-6.40 (dd, J=2.0, 5.0 Hz, 1H), 5.48 (s, 1H), 6.52-6.53 (d, J=3.5 Hz, 1H), 6.55 (s, 1H); .sup.13C NMR (125 MHz, CDCl.sub.3) δ 21.0, 28.8, 41.4, 43.1, 46.0, 47.0, 49.4, 64.0, 78.1, 110.3, 110.8, 111.0, 113.0, 114.0, 144.1, 144.3, 144.9, 145.8, 153.9, 158.1; LC-TOF (M+H.sup.+) calcd for C.sub.18H.sub.27N.sub.4O.sub.2 331.2134. found 331.2136.

Example 11

(27) In Vitro Enzyme Assays.

(28) The three NOS isoforms, rat nNOS, murine iNOS and bovine eNOS were recombinant enzymes overexpressed in E. coli and purified as reported in the literature. (See, e.g., example 51 of the aforementioned incorporated '790 patent and the references cited therein.) The hemoglobin capture was used to measure nitric oxide production. (See, Hevel, J. M. and Marletta, M. A. Nitric Oxide Synthase Assays. Methods Enzymol. 1994, 133, 250-258.) Briefly, the assay was run at 37° C. in 100 mM HEPES buffer (10% glycerol; pH 7.4) in the presence of 10 μM L-arginine. The following NOS cofactors were also included in the assay: 100 μM NADPH, 10 μM tetrahydrobiopterin, 1 mM CaCl.sub.2, 11.6 μg/mL calmodulin and 3.0 μM oxyhemoglobin. For iNOS, calmodulin and CaCl.sub.2 were omitted. The assay was run in a high throughput manner, using the Synergry 4 by BioTek, at the Northwestern University HighThroughput Analysis Facility. The assay was run in triplicate; 48 100 uL reactions were ran at once in a 96 well plate. The addition of hemoglobin and NOS were automated with a maximum of a 30 second delay before the reactions could be recorded at 401 nm. The absorbance increase at 401 nm is due to the formation of NO via the conversion of oxyhemoglobin to methemoglobin.

(29) The IC.sub.50 values were obtained using non-linear regression in GraphPad Prism5 software. Subsequent K.sub.i values were determined using the well-known Cheng-Prusoff relationship: K.sub.i=IC.sub.50/(1+[S]/K.sub.m) The following known K.sub.m values were used: rat nNOS 1.3 μM; murine iNOS 8.3 μM; bovine eNOS 1.7 μM.

Example 12

(30) nNOSCell Based Assay

(31) HEK293t cells stably transfected with rat nNOS were cultured as previously described, and the nNOS inhibition assay was performed as previously reported (Fang, J.; Silverman, R. B. A cellular model for screening neuronal nitric oxide synthase inhibitors. Analytical Biochemistry 2009, 390, 74-78), with the following modifications: assays were performed in 96-well plates with a total volume of 100 μL, and 10 μM A23187 (Sigma Aldrich, St Louis, Mo., USA) in 50% DMSO was used in place of 5 μM. Nine concentrations of each inhibitor were tested, in at least triplicate wells. All inhibitors were assayed within the same experiment to assure consistencies in cell concentration and passage number. The entire assay was repeated at least twice for each inhibitor, and the IC.sub.50 values averaged. After 6 hours of activation (in the presence or absence of inhibitor, which was added 30 minutes before activation) 50 μL aliquots of the media were removed and nitrite production was quantified using Griess reagent. (See, Hevel, J. M.; White, K. A.; Marietta, M. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. J. Biol. Chem. 1991, 266, 22789-22791.)