Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1021/jm101071n

We report novel neuronal nitric oxide synthase (nNOS) inhibitors based on a symmetric double-headed aminopyridine scaffold. The inhibitors were designed from crystal structures of leads 1 and 2 (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) and synthesized using a highly efficient route. The best inhibitor, 3j, showed low nanomolar inhibitory potency and modest isoform selectivity. It also exhibited enhanced membrane permeability. Inhibitor 3j binds to both the substrate site and the pterin site in nNOS but only to the substrate site in eNOS. These compounds provide a basis for further development of novel, potent, isoform selective, and bioavailable inhibitors for nNOS.

Full text of DOI:10.1021/jm101071n

ARTICLE
pubs.acs.org/jmc
Symmetric Double-Headed Aminopyridines, a Novel Strategy for
Potent and Membrane-Permeable Inhibitors of Neuronal Nitric Oxide
Synthase
Fengtian Xue,†,b Jianguo Fang,†,3 Silvia L. Delker,§ Huiying Li,§ Pavel Martꢀasek,#,f Linda J. Roman,#
Thomas L. Poulos,*,§,2 and Richard B. Silverman*,†,‡
†Department of Chemistry and ‡Department of Molecular Biosciences, Center for Molecular Innovation and Drug Discovery, Chemistry
of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
§Department of Molecular Biology and Biochemistry, 2Department of Pharmaceutical Sciences, and Department of Chemistry,
University of California, Irvine, California 92697-3900, United States
#Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
fDepartment of Pediatrics and Center for Applied Genomics, 1st School of Medicine, Charles University, Prague, Czech Republic
S Supporting Information
b
ABSTRACT: We report novel neuronal nitric oxide synthase
(nNOS) inhibitors based on a symmetric double-headed ami-
nopyridine scaffold. The inhibitors were designed from crystal
structures of leads 1 and 2 (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) and synthesized using a highly efficient route. The
best inhibitor, 3j, showed low nanomolar inhibitory potency
and modest isoform selectivity. It also exhibited enhanced membrane permeability. Inhibitor 3j binds to both the substrate site and
the pterin site in nNOS but only to the substrate site in eNOS. These compounds provide a basis for further development of novel,
potent, isoform selective, and bioavailable inhibitors for nNOS.
’ INTRODUCTION
Our research group has recently identified a number of chiral
pyrrolidine-based inhibitors (1 and 2, Figure 1) that possess
excellent potency and remarkable isoform selectivity for nNOS
over eNOS and iNOS.17,18 However, results from rodent in vivo
studies indicated that these inhibitors have poor ability to penetrate
the blood-brain barrier (BBB),19 which limits their further appli-
cations as drug candidates for neurodegenerative diseases. We
reasoned that the two high pKa nitrogen atoms on 1 and 2 (the
pyrrolidine nitrogen and the secondary amine in the middle of the
alkyl chain) gave them multiple positive charges at physiological
pH, which impaired the molecules to diffuse passively into the brain.
Moreover, the complex structures of these inhibitors and low
overall yield of the syntheses presented additional obstacles to
optimizing the pharmacokinetic properties of the inhibitors.17,19-22
Crystal structures of nNOS with both lead compounds 1 and 2
have been solved.18 As shown in Figure 1B, the aminopyridine
moiety of 1 binds to the heme, leaving the m-fluorophenyl tail
Neurodegenerative disorders, such as Alzheimer’s, Parkin-
son’s, and Huntington’s disease, have emerged as a rapidly
growing age-related health problem in the world. Despite years
of efforts invested by scholars in the pharmaceutical and aca-
demic community, to date, neurodegeneration caused by various
neurodegenerative diseases remains essentially untreatable.
Thus, the development of clinically useful therapeutic agents
for the affected population has become an urgent need.
Biological studies have demonstrated that overexpression of the
neuronal isoform of nitric oxide synthase (nNOS),1-4 and the
subsequent overproduction of the small molecule neurotransmitter
nitric oxide (NO), is implicated in various neurological diseases,
including Parkinson’s,5 Alzheimer’s,6 and Huntington’s diseases,7 as
well as neuronal damage due to stroke.8 Accordingly, inhibition of
the abnormal activity of nNOS has provided a promising target for
the development of new therapeutic agents for neurodegenera-
tion.9-12 In the past two decades, significant research has been
devoted to the design and synthesis of potent inhibitors of
nNOS.13-16 Inhibition of nNOS activity has also focused on
isoform selectivity over endothelial NOS (eNOS), the NOS iso-
form that regulates blood pressure and inducible NOS (iNOS), the
NOS isoform that plays a critical role in immune responses.16,17
extending into a peripheral pocket (Tyr706, Leu337, and Met336
)
at the edge of the active site.18 This was the predicted binding
mode. The aminopyridine of 1 was designed to replace the
Received: August 17, 2010
Published: March 16, 2011
r
2011 American Chemical Society
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Figure 1. (A) Chemical structures and inhibitory activities of 1 and 2. (B, C) View of the active site of nNOS complexed with inhibitor 1 (gray) (B) and
2 (gray) (C), showing also heme (pink), tetrahydrobiopterin (H4B, yellow), and key residues (cyan) in the active site.
guanidinium group of the substrate, L-arginine, which ion-pairs
with Glu592 or its homologue in all NOS isoforms. The pyrroli-
dine nitrogen of 1 replaces the interaction between the R-amino
group of L-arginine, which extends up to and interacts with
Asp597. Inhibitor 1 therefore mimics many of the interactions
between active site groups and L-arginine. The main reason that 1
is selective for nNOS is that the residue in eNOS that corresponds
to Asp597 in nNOS is an Asn, and thus, there is greater electro-
Figure 2. General structure of inhibitor 3.
static stabilization between the positive charge on the pyrrolidine
N atom of the inhibitor and the electronegative environment
treated with NaH, and the resulting anion was allowed to react
created by both Asp597 and Glu592 in nNOS. Interestingly,
with bis-bromide 5a to give 6a in good yields. Next, the two Boc-
however, inhibitor 2 binds to the active site of nNOS employing
protecting groups of 6a were removed with TFA to generate 3a
a totally different binding mode, the “flipped” binding mode
in high yields.
(Figure 1C); the aminopyridine binds in the peripheral pocket
The synthesis of 3b-d began with 2,5-dimethylpyrrole-pro-
(Tyr706, Leu337, and Met336) and makes hydrogen bonds with a
tected 2-amino-4,6-dimethylpyridine (7,23 Scheme 2). The uti-
heme propionate, while the m-fluorophenyl moiety sits above the
lization of 2,5-dimethylpyrrole-protecting group has found its
heme.18 The fact that the aminopyridine moiety can bind to two
application in synthesizing NOS inhibitors.23,24 Compound 7
different binding sites in the active site of nNOS by virtue of the
was treated with n-butyllithium (n-BuLi) at -78 °C, followed by
molecule flipping over provides an intriguing strategy for design-
the addition of 5a or 5b to produce 6b-d. Then the 2,5-
ing inhibitors for nNOS. We hypothesized that effective inhibi-
dimethylpyrrole protecting groups were removed with hydro-
tion of nNOS could be achieved by double-headed amino-
xylamine hydrochloride (NH2OH HCl) at 100 °C to generate
3
pyridine derivatives (3, Figure 2) with an optimizable linker.
Compared to the “parent compounds” (1 and 2), inhibitor 3
possesses attractive advantages. First, the highly polar amino
groups in the pyrrolidine core and lipophilic tail of the lead
compounds are replaced by a hydrophobic linker. The significant
increase in lipophilicity of 3 (Table S1 in Supporting In-
formation), as a consequence, could potentially improve the
BBB permeability of the inhibitors, providing a strategy to bypass
the difficulty in modifying the chemical structure of compounds 1
and 2. Moreover, the simplicity of the chemical structure of 3
could significantly increase the efficiency of the synthesis, which is
essential for future preclinical and clinical studies.
3b-d in excellent yields.
To synthesize inhibitors 3e-h, compound 7 was treated
with n-BuLi at -78 °C, followed by the addition of 5c or 5d to
give protected inhibitors 6e-h (Scheme 3). The 2,5-dimethyl-
pyrrole protecting groups of 6e-h were removed with NH2-
OH HCl at 100 °C to generate 3e-h in excellent yields. The
3
synthesis of 3i is similar to that for 3e-h except 7 was treated
with lithium diisopropylamide (LDA) at 0 °C following a reported
procedure.24
The syntheses of inhibitors 3j-n, with 3,5-bis-substituted
pyridine or 2,6-bis-substituted pyridine or 3,5-bis-substituted
aniline as the linker, began with 7 (Scheme 4). Compound 7
was treated with n-BuLi at -78 °C, and the resulting anion was
allowed to react with 5e, 5f, or 5g to give 6j-n.25 The 2,5-
dimethylpyrrole protecting groups of 6j-m were removed with
’ CHEMISTRY
Inhibitor 3a was synthesized using a two-step procedure
(Scheme 1). N-Boc-2-amino-4,6-dimethylpyridine (4) was
NH2OH HCl at 100 °C to generate 3j-n in very good yields.
3
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Scheme 1. Synthesis of 3aa
a Reagents and conditions: (a) (i) NaH, 0 °C, 15 min, (ii) 5a, 6 h, 81%; (b) TFA/CH2Cl2 (1:1), room temp, 8 h, 90%.
Scheme 2. Synthesis of 3b-da
a Reagents and conditions: (a) (i) n-BuLi, -78 to 0 °C, 30 min, (ii) 5a or 5b; (b) NH2OH HCl, EtOH/H2O (2:1), 100 °C, 20 h, 90-92%.
3
Scheme 3. Synthesis of 3e-ha
a Reagents and conditions: (a) (i) n-BuLi, -78 to 0 °C, 30 min, (ii) 5c or 5d; (b) NH2OH HCl, EtOH/H2O (2:1), 100 °C, 20 h, 85-90%; (c) (i) LDA,
3
0 °C, 30 min, (ii) 5d, 65%.
’ RESULTS AND DISCUSSION
NO capture assay,26 as summarized in Table 1. The compounds
with a naphthalene linker, 3a-d, were tested against nNOS
because the length of these compounds is similar to those of the
The inhibition constants (Ki) of these compounds were
determined for all three NOS isoforms using the oxyhemoglobin
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Scheme 4. Synthesis of 3j-na
a Reagents and conditions: (a) (i) n-BuLi, -78 to 0 °C, 30 min, (ii) 5e, 5f, or 5g; (b) NH2OH HCl, EtOH/H2O (2:1), 100 °C, 20 h, 80-88%; (c)
3
NH2OH HCl, EtOH/H2O (2:1), 100 °C, 36 h, 85%.
3
parent aminopyridine-pyrrolidine inhibitors (Figure 1, 1 and 2).
Compound 3a, an internal amidine dimer, showed poor binding,
whereas the compounds with the external amidine functionality,
3b and 3c, exhibited submicromolar binding affinity to nNOS
and modest selectivity against eNOS and iNOS. However, the
asymmetric dimer, 3d, was a poor inhibitor. Compounds 3e-i,
having a shorter 1,3- or 1,4-bis-substituted benzene linker
between the two aminopyridine head groups, showed improved
potency by 5- to 10-fold. Again, the orientation of the amidine
functionality was important. Compound 3i binds rather poorly to
nNOS when the methyl group is next to the ring nitrogen in both
aminopyridine groups. To further improve inhibitor potency and
selectivity, we sought to modify the 1,3-bis-substituted benzene
by additional heteroatoms. Compounds 3j-n were synthesized
with a pyridine linker, and 3n was synthesized with an aniline
linker. The best inhibitor in this series, 3j, showed better potency
(25 nM) than its phenyl analogue 3g (49 nM), with a 107-fold
selectivity for nNOS over eNOS.
We have also determined the crystal structures of several of
these inhibitors complexed to both nNOS and eNOS;27 how-
ever, we confine the present discussion to the most potent
inhibitor, 3j. Compound 3j binds as designed with one amino-
pyridine interacting with Glu592 and the second extending out of
the active site, where it interacts with the heme propionate.
However, in nNOS a second 3j molecule (3jB) also binds
(Figure 3). NOS is a homodimer, and the H4B cofactor binds
at the dimer interface, where it interacts with both subunits A and
B of the dimer. One aminopyridine of 3jB displaces the H4B
cofactor. This places the bridging pyridine of 3jB in position to
coordinate a new Zn2þ ion along with Asp600, a chloride ion, and
His692(B) of subunit B at the dimer interface. For this to occur,
Arg596 must move from its position where it normally interacts
with H4B. The new position of Arg596 enables its interactions
with Asp597 and Glu592. In order for His692(B) to coordinate to
the Zn2þ ion, there must be a slight tightening of the dimer
interface and His692 (B) must adopt a new rotomer conforma-
tion. None of this occurs in eNOS, and only one 3j molecule
binds in the active site as initially predicted. On the basis of an
extensive mutagenesis analysis,27 we concluded that the en-
hanced stability of the dimer in eNOS compared to nNOS
prevents the motions required to coordinate the Zn2þ ion in
eNOS, thereby preventing the binding of 3jB.
Binding of only one 3j in eNOS compared to the binding of
two molecules of 3j and displacement of H4B in nNOS may
account for the 107-fold selectivity of this inhibitor for nNOS
over eNOS. However, this modest isoform selectivity implies
that the inhibitory potency is likely dominated by the binding of
the first 3j that occupies the substrate binding site, which occurs
in both nNOS and eNOS. The binding of a second 3jB in nNOS
and displacement of H4B enhance the potency to nNOS about
100-fold over eNOS. Despite the modest selectivity, the identi-
fication of a second binding pocket in nNOS that requires
substantial structural changes that do not take place in eNOS
provides a new window of opportunity for the design of novel
nNOS-selective inhibitors. Inhibitor 3j can serve as a NOS active
site recognizing building block for developing other inhibitors
that can further explore the subtle differences in the active site of
nNOS and eNOS, which the good isoform selectivity of the
parental inhibitors, 1 and 2, was based on.18
The activities of the best compounds, 3g, 3h, 3j, and 3k, were
further tested in a cell-based assay, in direct comparison with lead
compound 2.28 Inhibitors 3g and 3h, as well as lead 2, showed
almost no activity in the cell-based assay, suggesting that these
compounds are blocked by the cellular membrane (Figure 4).
Inhibitors 3j and 3k, however, showed good membrane penetra-
tion ability. The best compound, 3j, has an IC50 value in the
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Table 1. Ki a Values and Selectivity of 3a-n
inhibitor
nNOS (nM)
eNOS (nM)
iNOS (nM)
n/e b
n/i b
3a
3b
3c
3d
3e
3f
>1000
241
102
>1000
97
NDc
NDc
2270
2050
NDc
2410
NDc
682
NDc
7.22
14.9
NDc
24.6
NDc
28.8
111
NDc
107
156
95
NDc
9.42
20.1
NDc
24.8
NDc
13.9
71.8
NDc
58
1740
1520
NDc
2390
NDc
167
49
3g
3h
3i
1410
4200
NDc
38
2730
NDc
1450
1910
4750
12600
3400
>1000
25
3j
2680
16100
9400
20000
4950
3k
3l
103
99
19
48
3m
195
102
64
3n
85
58
40
a The Ki values were calculated based on the directly measured IC50 values, which represent at least duplicate measurements with standard deviations of
(10%. b The ratio of Ki(eNOS or iNOS) to Ki(nNOS). c Not determined.
Figure 4. Inhibition of nNOS in 293T cells overexpressing nNOS
(293T/nNOS cells). 293T/nNOS cells were treated with A23187 (5
μM) and various concentrations of inhibitors 2 and 3g-k. After 8 h, the
amount of nitrite in culture medium was quantified with the Griess
reagent. Each data point is an average of two independent experiments.
Figure 3. Crystal structure of nNOS complexed with 3j.27 Major
cell-based assay of 5 μM, which is significantly better than that of
hydrogen bonds and the ligation bonds of zinc ion are depicted with
lead compound 2, which, we believe, is due to the result of its
increased lipophilicity (Table S1). Compared to its in vitro
dashed lines. The native H4B binding site that is occupied by 3jB is
labeled.
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activity, the potency of 3j in the cell-based assay is weaker, which
may be the result of its affinity for one of the active efflux transporters
(e.g., p-glycoprotein)29-32 or may just represent the Kd for mem-
brane transport and not Ki nNOS inhibition. These results indicate
that double-headed scaffold 3 is a promising candidate for future
development of bioavailable nNOS inhibitors.
extracted with Et2O (2 ꢀ 25 mL), and the combined organic layers were
dried over Na2SO4. The solvent was removed by rotary evaporation, and
the resulting yellow oil was purified by flash chromatography (5-10%
MeOH in CH2Cl2) to yield inhibitors 6a-m.
Di-tert-butyl Naphthalene-2,6-diylbis(methylene)bis(4,6-
dimethylpyridin-2-ylcarbamate) (6a). To a solution of 4 (444
mg, 2 mmol) in dimethylformamide (DMF, 10 mL) at 0 °C was added
NaH (60% in mineral oil, 88 mg, 2.2 mmol). After 15 min, bis-bromide
5a (314 mg, 1 mmol) was added slowly. The reaction mixture was
allowed to stir at room temperature for 6 h and then concentrated. The
crude product was purified by flash column chromatography (EtOAc/
hexanes, 1:9 to 1:4) to yield 6a (960 mg, 81%) as a white solid: 1H NMR
(500 MHz, CDCl3) δ 1.41 (s, 18H), 2.29 (s, 6H), 2.45 (s, 6H), 5.33 (s,
4H), 6.72 (s, 2H), 7.28 (s, 2H), 7.42-7.44 (d, J = 8.5, 2H), 7.68-7.70
(m, 4H); 13C NMR (125 MHz, CDCl3) δ 14.4, 21.3, 24.4, 28.5, 31.9,
50.5, 81.2, 117.7, 120.6, 126.1, 126.3, 127.8, 132.6, 137.1, 148.7, 154.0,
154.7, 156.5; LC-TOF (M þ Hþ) calcd for C36H45N4O4 597.344 08,
found 597.344 92.
2,6-Bis(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)naphthalene (6b). Compound 6b was synthesized
starting with 7 and 5a using general method A (60%): 1H NMR (500
MHz, CDCl3) δ 2.18 (s, 12H), 2.39 (s, 6H), 3.20-3.40 (m, 8H), 5.94 (s,
4H), 6.90 (s, 2H), 6.97 (s, 2H), 7.34-7.36 (d, J = 8.5 Hz, 2H), 7.61 (s,
2H), 7.69-7.71 (d, J = 8.5 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ
13.5, 21.3, 36.2, 39.9, 107.0, 120.4, 122.9, 126.6, 127.6, 127.7, 128.8,
132.5, 138.6, 149.7, 151.9, 161.2; LC-TOF (M þ Hþ) calcd for
C38H41N4 553.333 12, found 553.333 82.
’ SUMMARY
Double-headed aminopyridine inhibitors (3a-m) were de-
signed based on the unexpected binding mode of chiral pyrro-
lidine leads 1 and 218 and synthesized as a novel class of inhibitors
for nNOS. Inhibitor 3j, the best inhibitor discovered from the
series, exhibits high potency and modest selectivity for nNOS. It
showed improved membrane permeability based on a cell-based
assay. The simplicity of the chemical structures of 3a-n makes
the synthesis of the new class of inhibitors much more efficient
than the lead compounds (1 and 2). The crystal structure of the
nNOS-3j complex also revealed an unexpected second binding
site unique to nNOS and illustrated the importance of the
substrate and H4B binding sites in inhibitor development. This
new binding site also provides the basis for the design of novel
and isoform-selective inhibitors.
’ EXPERIMENTAL SECTION
All experiments were conducted under anhydrous conditions in an
atmosphere of argon using flame-dried apparatus and employing
standard techniques in handling air-sensitive materials. All solvents were
distilled and stored under an argon or nitrogen atmosphere before use.
All reagents were used as received. Aqueous solutions of sodium
bicarbonate, sodium chloride (brine), and ammonium chloride were
saturated. Analytical thin layer chromatography was visualized by
ultraviolet light. Flash column chromatography was carried out under
a positive pressure of nitrogen. 1H NMR spectra were recorded on 500
MHz spectrometers. Data are presented as follows: chemical shift (in
ppm on the δ scale relative to δ = 0.00 ppm for the protons in
tetramethylsilane (TMS)), integration, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling
constant (J/Hz). Coupling constants were taken directly from the
spectra and are uncorrected. 13C NMR spectra were recorded at 125
MHz, and all chemical shift values are reported in ppm on the δ scale
with an internal reference of δ 77.0 or 49.0 for CDCl3 or MeOD,
respectively. High-resolution mass spectra were measured by liquid
chromatography/time-of-flight mass spectrometry (LC-TOF). The
purity of the tested compounds was determined by elemental analysis
and was >95%.
General Method A: Synthesis of 2,5-Dimethylpyrrole-Pro-
tected Inhibitors 6b-m. To a solution of 7 (500 mg, 2.5 mmol) in
THF (10 mL) at -78 °C was added n-BuLi (1.6 M solution in hexanes,
1.64 mL, 2.63 mmol) dropwise. The resulting dark red solution after the
addition was transferred to an ice bath. After 30 min, a solution of
bromide 5a, 5b, 5c,20 or 5d20 (1 M in THF) was added dropwise until
the dark red color disappeared. The reaction mixture was allowed to stir
at 0 °C for an additional 10 min and then quenched with H2O (20 μL).
The solvent was removed by rotary evaporation, and the resulting yellow
oil was purified by flash chromatography (EtOAc/hexanes) to yield 2,5-
dimethylpyrrole-protected inhibitors 6b-m.
2,7-Bis(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)naphthalene (6c). Compound 6c was synthesized
starting with 7 and 5b using general method A (55%): 1H NMR (500
MHz, CDCl3) δ 2.18 (s, 12H), 2.39 (s, 6H), 3.15-3.25 (m, 8H), 5.94 (s,
4H), 6.90 (s, 2H), 6.98 (s, 2H), 7.32-7.34 (d, J = 8.5 Hz, 2H), 7.57 (s,
2H), 7.75-7.76 (d, J = 8.5 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ
13.5, 21.3, 36.3, 39.9, 107.0, 120.4, 122.9, 126.4, 126.9, 127.9, 128.8,
130.9, 134.1, 139.4, 149.7, 151.9, 161.2; LC-TOF (M þ Hþ) calcd for
C38H41N4 553.333 12, found 553.333 79.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-(2-(7-(2-(6-(2,5-dimeth-
yl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)naphthalen-
2-yl)ethyl)-6-methylpyridine (6d). Compound 6d was synthe-
sized starting with 7 and 5b using general method A (35%): 1H NMR
(500 MHz, CDCl3) δ 2.02 (s, 6H), 2.16 (s, 6H), 2.36 (s, 3H), 2.57 (s,
3H), 3.00-3.15 (m, 4H), 3.15-3.30 (m, 4H), 5.86 (s, 2H), 5.93 (s,
2H), 6.79 (s, 1H), 6.88 (s, 1H), 6.96 (s, 1H), 7.03 (s, 1H), 7.25-7.27 (d,
J = 10 Hz, 1H), 7.31-7.33 (d, J = 10 Hz, 1H), 7.47 (s, 1H), 7.54 (s, 1H),
7.73-7.74 (d, J = 9.0 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 13.2,
13.5, 21.2, 24.5, 36.2, 36.98, 37.02, 39.9, 106.8, 107.0, 119.5, 120.4, 122.5,
122.9, 126.4, 126.5, 126.6, 127.2, 127.9, 128.1, 128.6, 128.7, 131.0, 134.0,
138.2, 139.6, 149.8, 151.7, 151.9, 153.1, 158.5, 161.1; LC-TOF (M þ
Hþ) calcd for C38H41N4 553.333 12, found 553.332 96.
1,4-Bis(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)benzene (6e). Compound 6e was synthesized start-
ing with 7 and 5c using general method A (65%): 1H NMR (500 MHz,
CDCl3) δ 2.21 (s, 12H), 2.44 (s, 6H), 3.12-3.15 (m, 8H), 5.97 (s, 4H),
6.94 (s, 2H), 7.01 (s, 2H), 7.18 (s, 4H); 13C NMR (125 MHz, CDCl3) δ
13.6, 21.3, 35.8, 40.1, 107.0, 120.3, 122.9, 128.8, 139.4, 149.7, 151.9, 161.4;
LC-TOF (M þ Hþ) calcd for C34H39N4 503.317 47, found 503.318 28.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-(4-(2-(6-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)phenethyl)-6-
methylpyridine (6f). Compound 6f was synthesized starting with 7
and 5c using general method A (35%): 1H NMR (500 MHz, CDCl3) δ
2.12 (s, 6H), 2.19 (s, 6H), 2.43 (s, 3H), 2.61 (s, 3H), 2.99 (s, 4H), 3.05-
3.20 (s, 4H), 5.91 (s, 2H), 5.95 (s, 2H), 6.83 (s, 1H), 6.92 (s, 1H), 6.99
(s, 1H), 7.04 (s, 1H), 7.10-7.12 (d, J = 8.0 Hz, 2H), 7.16-7.18 (d, J =
7.5, 2H); 13C NMR (125 MHz, CDCl3) δ 13.4, 13.5, 21.3, 24.5, 35.7,
General Method B: Synthesis of Inhibitors 3a-m. To a
solution of 6a-m (0.5 mmol) in EtOH (10 mL) was added hydro-
xylamine hydrochloride (NH2OH HCl, 340 mg, 5 mmol) followed by
3
H2O (5 mL). The reaction mixture was heated at 100 °C for 20 h. After
cooling to room temperature, the reaction mixture was partitioned
between Et2O (50 mL) and 2 N NaOH (25 mL). The aqueous layer was
2044
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Journal of Medicinal Chemistry
ARTICLE
36.4, 37.2, 40.1, 106.9, 107.0, 119.5, 120.3, 122.5, 122.8, 128.67, 128.70,
128.73, 128.9, 138.3, 139.8, 149.7, 151.7, 151.9, 153.3, 158.5, 161.3; LC-
TOF (M þ Hþ) calcd for C34H39N4 503.317 47, found 503.318 45.
1,3-Bis(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)benzene (6g). Compound 6g was synthesized start-
ing with 7 and 5d using general method A (60%): 1H NMR (500 MHz,
CDCl3) δ 2.19 (s, 12H), 2.42 (s, 6H), 3.00-3.20 (m, 8H), 5.94 (s, 4H),
6.91 (s, 2H), 6.97 (s, 2H), 7.06-7.08 (m, 3H), 7.21-7.23 (m, 1H); 13C
NMR (125 MHz, CDCl3) δ 13.5, 21.7, 36.2, 40.1, 107.0, 120.3, 122.9,
126.4, 128.68, 128.74, 129.0, 141.9, 149.7, 151.9, 161.3; LC-TOF (M þ
Hþ) calcd for C34H39N4 503.317 47, found 503.318 14.
MHz, CDCl3) δ 2.08 (s, 6H), 2.13 (s, 6H), 2.38 (s, 3H), 2.56 (s, 3H),
2.94 (s, 4H), 3.06 (s, 4H), 5.88 (s, 2H), 5.91 (s, 2H), 6.80 (s, 1H), 6.89
(s, 1H), 6.92 (s, 1H), 6.97 (s, 1H), 7.31 (s, 1H), 8.25 (s, 1H), 8.29 (s,
1H); 13C NMR (125 MHz, CDCl3) δ 13.4, 13.5, 21.2, 24.5, 32.8, 33.8,
36.7, 39.4, 107.0, 119.2, 120.6, 122.3, 122.9, 128.60, 128.66,
128.69, 135.6, 136.2, 136.8, 149.88, 149.93, 151.9, 152.0, 152.3, 158.8,
160.3; LC-TOF (M þ Hþ) calcd for C33H38N5 504.312 72, found
504.312 36.
2,6-Bis(bromomethyl)pyridine (5f). To pyridine-2,6-diyldi-
methanol (2 g, 14 mmol) was added 60% HBr (15 mL) slowly. The
mixture was heated at 125 °C for 6 h and then cooled to room
temperature. The resulting residue was dissolved in H2O (50 mL) to
give a yellow solution. To this solution was added saturated NaHCO3 to
pH 8. The resulting aqueous solution was extracted with CH2Cl2 (4 ꢀ
50 mL), and the combined organic layers were dried over Na2SO4. The
solvent was removed by rotary evaporation, and the resulting material
was purified by flash column chromatography (EtOAc/hexanes, 1:9 to
1:4) to yield 2,6-bis(bromomethyl)pyridine (5f, 3.5 g, 96%) as a white
solid: 1H NMR (500 MHz, CDCl3) δ 4.53 (s, 4H), 7.36-7.38 (d, J = 8.0
Hz, 2H), 7.68-7.71 (dd, J = 7.5, 8.0 Hz, 1H); 13C NMR (125 MHz,
CDCl3) δ 33.8, 123.1, 138.4, 157.0; LC-TOF (M þ Hþ) calcd for
C7H8Br2N 263.902 35, found 263.901 93.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-(3-(2-(6-(2,5-dimeth-
yl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)phenethyl)-6-
methylpyridine (6h). Compound 6h was synthesized starting with 7
and 5d using general method A (35%): 1H NMR (500 MHz, CDCl3) δ
2.12 (s, 6H), 2.19 (s, 6H), 2.41 (s, 3H), 2.60 (s, 3H), 2.97 (s, 4H), 3.05-
3.20 (m, 4H), 5.91 (s, 2H), 5.94 (s, 2H), 6.83 (s, 1H), 6.91 (s, 1H), 6.97
(s, 1H), 7.02 (s, 1H), 7.05-7.15 (m, 3H), 7.21-7.25 (m, 1H); 13C
NMR (125 MHz, CDCl3) δ 13.4, 13.5, 21.2, 24.5, 36.1, 36.8, 37.2, 40.1,
106.9, 107.0, 119.4, 120.3, 122.5, 122.8, 126.3, 126.7, 128.65, 128.71,
128.8, 128.9, 140.8, 142.1, 149.8, 151.7, 151.9, 153.2, 158.5, 161.2; LC-
TOF (M þ Hþ) calcd for C34H39N4 503.317 47, found 503.318 40.
1,3-Bis(2-(2-(2,5-dimethyl-1H-pyrrol-1-yl)-6-methylpyri-
din-4-yl)ethyl)benzene (6i). To a solution of 7 (500 mg, 2.5 mmol)
in THF (10 mL) at 0 °C was added lithium diisopropylamide (2.0 M
solution in THF, 1.32 mL, 2.63 mmol) dropwise. The resulting dark red
solution after the addition was allowed to warm to room temperature.
After 30 min, a solution of bromide 5d (1 M in THF) was added
dropwise until the dark red color disappeared. The reaction mixture was
allowed to stir at 0 °C for an additional 10 min and then quenched with
H2O (20 μL). The solvent was removed by rotary evaporation, and the
resulting yellow oil was purified by flash chromatography (EtOAc/
hexanes, 1:9) to yield 2,5-dimethylpyrrole-protected inhibitors 6i (405
mg, 65%): 1H NMR (500 MHz, CDCl3) δ 2.09 (s, 12H), 2.56 (s, 6H),
2.94 (s, 6H), 5.88 (s, 3H), 6.81 (s, 1H), 6.95-7.05 (m, 4H), 7.20-7.23
(m, 1H); 13C NMR (125 MHz, CDCl3) δ 13.4, 24.5, 36.7, 37.1, 106.9,
119.3, 122.4, 126.6, 128.6, 128.8, 141.0, 151.8, 153.1, 158.5; LC-TOF
(M þ Hþ) calcd for C34H39N4 503.317 47, found 503.318 01.
2,6-Bis(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)pyridine (6l). Compound 6l was synthesized starting
1
with 7 and 5f using general method A (58%): H NMR (500 MHz,
CDCl3) δ 2.08 (s, 12H), 2.37 (s, 6H), 3.25 (s, 8H), 5.91 (s, 4H), 6.85-
6.87 (d, J = 9.5 Hz, 2H), 6.93-6.95 (d, J = 7.5 Hz, 2H), 7.00 (s, 2H),
7.42-7.46 (dd, J = 7.5, 8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ
13.5, 21.2, 37.9, 38.1, 106.9, 120.2, 120.5, 122.9, 128.7, 136.7, 149.6,
151.8, 160.8, 161.3; LC-TOF (M þ Hþ) calcd for C33H38N5
504.312 72, found 504.312 62.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-(2-(6-(2-(6-(2,5-dimeth-
yl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-2-
yl)ethyl)-6-methylpyridine (6m). Compound 6m was synthesized
1
starting with 7 and 6f using general method A (37%): H NMR (500
MHz, CDCl3) δ 2.07 (s, 6H), 2.11 (s, 6H), 2.37 (s, 3H), 2.55 (s, 3H),
3.10-3.12 (t, J = 3.0 Hz, 4H), 3.21-3.23 (t, J = 3.0 Hz, 4H), 5.87 (s,
2H), 5.90 (s, 2H), 6.80-7.10 (m, 6H), 7.40-7.50 (m, 1H); 13C NMR
(125 MHz, CDCl3) δ 13.3, 13.5, 21.2, 24.4, 34.98, 35.041, 37.9, 38.08,
38.15, 38.8, 106.9, 119.3, 120.3, 120.5, 120.8, 122.4, 122.8, 122.9, 128.6,
128.7, 136.9, 149.6, 151.8, 153.2, 153.3, 158.4, 159.7, 160.8, 161.1, 161.3;
LC-TOF (M þ Hþ) calcd for C33H38N5 504.312 72, found 504.312 26.
16,60-(2,20-(5-(2,5-Dimethyl-1H-pyrrol-1-yl)-1,3phenyle-
ne)bis(ethane-2,1-diyl))bis(2-(2,5-dimethyl-1H-pyrrol-1-
yl)-4-methylpyridine) (6n). Compound 6n was synthesized starting
3,5-Bis(bromomethyl)pyridine (5e). To pyridine-3,5-diyldi-
methanol (2 g, 14 mmol) was added 60% HBr (15 mL) slowly. The
mixture was heated at 125 °C for 6 h and then cooled to room
temperature. The resulting residue was dissolved in H2O (50 mL) to
give a yellow solution. To this solution was added saturated NaHCO3 to
pH 8. The resulting aqueous solution was extracted with CH2Cl2 (4 ꢀ
50 mL), and the combined organic layers were dried over Na2SO4. The
solvent was removed by rotary evaporation, and the resulting material
was purified by flash column chromatography (EtOAc/hexanes, 1:4) to
yield 3,5-bis(bromomethyl)pyridine (5e, 3.5 g, 95%) as a white solid: 1H
NMR (500 MHz, CDCl3) δ 4.45 (s, 4H), 7.74-7.75 (t, J = 2.0 Hz, 1H),
8.53-8.54 (d, J = 2.0 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 29.3,
134.0, 137.3, 149.8; LC-TOF (M þ Hþ) calcd for C7H8Br2N
263.902 35, found 263.901 82.
1
with 7 and 5g using general method A (55%): H NMR (500 MHz,
CDCl3) δ 1.94 (s, 6H), 2.11 (s, 12H), 2.35 (s, 6H), 3.07 (s, 8H), 5.85 (s,
2H), 5.88 (s, 4H), 6.81-6.82 (d, J = 1.5 Hz, 2H), 6.85 (s, 2H), 6.87 (s,
2H), 7.06 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 13.0, 13.3, 21.0, 35.3,
39.4, 105.5, 106.6, 106.7, 120.1, 122.6, 125.7, 128.0, 128.4, 128.5, 138.9,
142.5, 149.4, 151.7, 160.5; LC-TOF (M þ Hþ) calcd for C40H46N5
596.375 32, found 596.375 19.
N,N0-(Naphthalene-2,6-diylbis(methylene))bis(4,6-dimet-
hylpyridin-2-amine) (3a). To a solution of 6a (590 mg, 1.0 mmol) in
CH2Cl2 (8 mL) was added trifluoroacetic acid (TFA, 8 mL). The
reaction mixture was allowed to stir at room temperature for 8 h and
then was concentrated. The crude product was purified by flash column
chromatography (2-5% MeOH in CH2Cl2) to yield 3a (355 mg, 90%)
as a white solid: 1H NMR (500 MHz, CD3OD/CDCl3) δ 2.28 (s, 6H),
2.48 (s, 6H), 4.60 (s, 4H), 6.46 (s, 4H), 7.45-7.47 (d, J = 8.5, 2H), 7.78
(s, 2H), 7.84-7.86 (d, J = 8.5, 2H); 13C NMR (125 MHz, CD3OD/
CDCl3) δ 18.6, 22.0, 45.9, 106.8, 114.5, 125.6, 129.0, 133.1, 134.3, 147.6,
153.8, 158.0; LC-TOF (M þ Hþ) calcd for C26H29N4 397.239 22,
found 397.239 99.
3,5-Bis(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)pyridine (6j). Compound 6j was synthesized starting
with 7 and 5e using general method A (55%): H NMR (500 MHz,
1
CDCl3) δ 2.13 (s, 12H), 2.38 (s, 6H), 3.05 (s, 8H), 5.90 (s, 4H), 6.88 (s,
2H), 6.91 (s, 2H), 7.34 (s, 1H), 8.21-8.22 (d, J = 2.5 Hz, 2H); 13C
NMR (125 MHz, CDCl3) δ 13.5, 21.2, 32.8, 39.4, 107.0, 120.5, 122.9,
128.6, 136.2, 136.5, 147.9, 149.8, 152.0, 160.3; LC-TOF (M þ Hþ)
calcd for C33H38N5 504.312 72, found 504.312 65.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-(2-(5-(2-(6-(2,5-dimeth-
yl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-3yl)
ethyl)-6-methylpyridine (6k). Compound 6k was synthesized
starting with 7 and 5e using general method A (30%): 1H NMR (500
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Journal of Medicinal Chemistry
ARTICLE
6,60-(2,20-(Naphthalene-2,6-diyl)bis(ethane-2,1-diyl))bis(4-
methylpyridin-2-amine) (3b). Inhibitor 3b was synthesized using
general method B (90%): 1H NMR (500 MHz, CDCl3) δ 2.19 (s, 6H),
2.94-2.98 (dd, J = 7.5, 10.5 Hz, 4H), 3.12-3.16 (dd, J = 6.0, 9.0 Hz,
4H), 4.52 (br s, 4H), 6.20 (s, 2H), 6.37 (s, 2H), 7.35-7.36 (d, J = 8.5 Hz,
2H), 7.64 (s, 2H), 7.70-7.72 (d, J = 8.5 Hz, 2H); 13C NMR (125 MHz,
CDCl3) δ 21.2, 36.4, 40.0, 106.9, 115.7, 126.4, 127.6, 132.4, 139.0, 149.6,
158.5, 159.6; LC-TOF (M þ Hþ) calcd for C26H29N4 397.239 22,
found 397.239 54.
2H), 4.47 (br s, 4H), 6.13 (s, 1H), 6.19 (s, 1H), 6.35 (s, 1H), 6.38 (s,
1H), 6.99-7.01 (d, J = 7.5 Hz, 1H), 7.04 (s, 1H), 7.06-7.08 (d, J = 7.5
Hz, 1H), 7.19-7.22 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 21.2,
24.2, 36.3, 36.8, 37.4, 40.2, 105.6, 106.8, 114.2, 114.6, 126.1, 126.5, 128.6,
128.9, 141.5, 142.3, 149.5, 153.3, 156.6, 158.3, 158.5, 159.7; LC-TOF
(M þ Hþ) calcd for C22H27N4 347.223 57, found 347.224 17. Anal.
Calcd for C22H26N4 0.5CH3OH: C, 74.55; H, 7.79; N, 15.46. Found:
3
C, 73.45; H, 7.48; N, 14.97.
4,40-(2,20-(1,3-Phenylene)bis(ethane-2,1-diyl))bis(6-met-
hylpyridin-2-amine) (3i). Inhibitor 3i was synthesized using general
method B (85%): 1H NMR (500 MHz, CDCl3) δ 2.33 (s, 6H), 2.46 (br
s, 4H), 2.69-2.72 (m, 4H), 2.81-2.84 (m, 4H), 6.10 (s, 2H), 6.35 (s,
2H), 6.91 (s, 2H), 6.99-7.00 (d, J = 8.0 Hz, 2H), 7.17-7.20 (m, 1H);
13C NMR (125 MHz, CDCl3) δ 23.8, 36.6, 37.4, 105.9, 114.1, 126.4,
128.7, 128.8, 141.5, 153.5, 156.1, 158.2; LC-TOF (M þ Hþ) calcd for
C22H27N4 347.223 57, found 347.223 89. Anal. Calcd for
6,60-(2,20-(Naphthalene-2,7-diyl)bis(ethane-2,1-diyl))bis(4-
methylpyridin-2-amine) (3c). Inhibitor 3c was synthesized using
general method B (90%): 1H NMR (500 MHz, CDCl3) δ 2.18 (s, 6H),
2.95-2.99 (m, 4H), 2.13-2.17 (m, 4H), 4.55 (br s, 4H), 6.18 (s, 2H),
6.38 (s, 2H), 7.32-7.33 (d, J = 8.5 Hz, 2H), 7.60 (s, 2H), 7.72-7.74 (d, J
= 8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 21.2, 36.5, 40.0, 106.8,
114.6, 126.3, 126.9, 127.8, 130.8, 134.1, 139.8, 149.5, 158.6, 159.7; LC-
TOF (M þ Hþ) calcd for C26H29N4 397.239 22, found 397.239 46.
C22H26N4 0.5CH3OH: C, 74.55; H, 7.79; N, 15.46. Found: C, 73.49;
3
Anal. Calcd for C26H28N4 0.5H2O: C, 77.15; H, 7.33; N, 13.58. Found:
H, 7.36; N, 14.79.
3
6,60-(2,20-(Pyridine-3,5-diyl)bis(ethane-2,1-diyl))bis(4m-
ethylpyridin-2-amine) (3j). Inhibitor 3j was synthesized using
C, 77.53; H, 7.14; N, 11.72
4-(2-(7-(2-(6-Amino-4-methylpyridin-2-yl)ethyl)naphtha-
len-2-yl)ethyl)-6-methylpyridin-2-amine (3d). Inhibitor 3d was
synthesized using general method B (92%): 1H NMR (500 MHz,
CDCl3) δ 2.19 (s, 3H), 2.37 (s, 3H), 2.82-2.84 (m, 2H), 2.85-2.99
(m, 2H), 3.00-3.03 (m, 2H), 3.14-3.17 (m, 2H), 4.48 (br s, 4H), 6.16
(s, 1H), 6.19 (s, 1H), 6.38 (s, 1H), 6.43 (s, 1H), 7.25-7.27 (d, J = 8.5
Hz, 1H), 7.33-7.35 (d, J = 8.5 Hz, 1H), 7.55 (s, 1H), 7.61 (s, 1H),
7.73-7.75 (d, J = 8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 21.2,
24.3, 36.4, 36.9, 37.3, 40.0, 105.6, 106.8, 114.2, 114.6, 126.29, 126.32,
126.7, 127.1, 127.8, 127.9, 130.9, 134.0, 139.0, 140.0, 149.4, 153.1, 156.8,
158.4, 158.6, 159.7; LC-TOF (M þ Hþ) calcd for C26H29N4
397.239 22, found 397.239 70.
1
general method B (86%): H NMR (500 MHz, CDCl3) δ 2.16 (s,
6H), 2.80-2.83 (m, 4H), 2.93-2.96 (m, 4H), 4.47 (br s, 4H), 6.17 (s,
2H), 6.27 (s, 2H), 7.31 (s, 1H), 8.26-8.27 (d, J = 1.5 Hz, 2H); 13C NMR
(125 MHz, CDCl3) δ 21.2, 33.1, 39.6, 106.9, 114.7, 136.3, 136.8, 147.79,
147.82, 149.5, 158.6, 158.9; LC-TOF (M þ Hþ) calcd for C21H26N5
348.218 82, found 348.218 90. Anal. Calcd for C21H25N5 0.5CH3OH: C,
3
71.04; H, 7.49; N, 19.27. Found: C, 70.28; H, 7.33; N, 19.11.
4-(2-(5-(2-(6-Amino-4-methylpyridin-2-yl)ethyl)pyridin-
3-yl)ethyl)-6-methylpyridin-2-amine (3k). Inhibitor 3k was
synthesized using general method B (80%): 1H NMR (500 MHz,
CDCl3) δ 2.18 (s, 3H), 2.36 (s, 3H), 2.69-2.73 (m, 2H), 2.82-2.85
(m, 4H), 2.94-2.98 (m, 2H), 4.46 (br s, 2H), 4.53 (br s, 2H), 6.09 (s,
1H), 6.19 (s, 1H), 6.31 (s, 1H), 6.35 (s, 1H), 7.27 (s, 1H), 8.26-8.29
(m, 2H); 13C NMR (125 MHz, CDCl3) δ 21.2, 24.2, 33.0, 33.1, 33.7,
37.0, 39.5, 105.6, 107.0, 114.1, 114.7, 136.1, 136.3, 136.8, 136.9, 147.7,
147.8, 148.1, 149.5, 149.6, 152.4, 156.8, 158.3, 158.5, 158.7, 158.8; LC-
TOF (M þ Hþ) calcd for C21H26N5 348.218 82, found 348.218 78.
6,60-(2,20-(1,4-Phenylene)bis(ethane-2,1-diyl))bis(4-met-
hylpyridin-2-amine) (3e). Inhibitor 3e was synthesized using gen-
1
eral method B (85%): H NMR (500 MHz, CDCl3) δ 2.20 (s, 6H),
2.85-2.88 (m, 4H), 2.94-2.98 (m, 4H), 4.39 (s, 4H), 6.19 (s, 2H), 6.36
(s, 2H), 7.16 (s, 4H); 13C NMR (125 MHz, CDCl3) δ 21.2, 35.9, 40.2,
106.7, 114.7, 128.6, 139.7, 149.4, 158.4, 160.0; LC-TOF (M þ Hþ)
calcd for C22H27N4 347.223 57, found 347.223 99. Anal. Calcd for
Anal. Calcd for C21H25N5 0.5CH3OH: C, 71.04; H, 7.49; N, 19.27.
3
C22H26N4 0.5CH3OH: C, 74.55; H, 7.79; N, 15.46. Found: C, 73.68;
Found: C, 70.11; H, 7.30; N, 19.10.
3
6,60-(2,20-(Pyridine-2,6-diyl)bis(ethane-2,1-diyl))bis(4me-
thylpyridin-2-amine) (3l). Inhibitor 3l was synthesized using gen-
H, 7.27; N, 15.05.
4-(4-(2-(6-Amino-4-methylpyridin-2-yl)ethyl)phenethyl)-
6-methylpyridin-2-amine (3f). Inhibitor 3f was synthesized using
general method B (88%): 1H NMR (500 MHz, CDCl3) δ 2.20 (s, 3H),
2.37 (s, 3H), 2.73-2.76 (m, 2H), 2.83-2.88 (m, 4H), 2.95-2.98 (m,
2H), 4.42 (s, 2H), 4.45 (s, 2H), 6.13 (s, 1H), 6.19 (s, 1H), 6.37 (s, 1H),
6.40 (s, 1H), 7.09-7.19 (d, J = 8.0 Hz, 2H), 7.15-7.17 (d, J = 8.0 Hz,
2H); 13C NMR (125 MHz, CDCl3) δ 21.2, 24.2, 35.9, 36.4, 37.5, 40.2,
105.6, 106.7, 114.3, 114.6, 128.6, 128.7, 138.9, 140.0, 149.5, 153.2, 156.7,
158.3, 158.4, 159.8; LC-TOF (M þ Hþ) calcd for C22H27N4
1
eral method B (80%): H NMR (500 MHz, CDCl3) δ 2.14 (s, 6H),
2.97-3.01 (m, 4H), 3.13-3.16 (m, 4H), 4.68 (br s, 4H), 6.15 (s, 2H),
6.34 (s, 2H), 6.93-6.95 (d, J = 7.5 Hz, 2H), 7.41-7.44 (dd, J = 7.5, 7.5
Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 21.2, 38.2, 38.5, 106.7, 114.5,
120.3, 136.6, 149.3, 158.6, 159.7, 161.1; LC-TOF (M þ Hþ) calcd for
C21H26N5 348.218 82, found 348.218 71. Anal. Calcd for C21H25-
N5 0.5CH3OH: C, 71.04; H, 7.49; N, 19.27. Found: C, 69.80; H,
3
7.23; N, 19.01.
347.223 57, found 347.224 10. Anal. Calcd for C22H26N4 0.5CH3OH:
4-(2-(6-(2-(6-Amino-4-methylpyridin-2-yl)ethyl)pyridin-
2-yl)ethyl)-6-methylpyridin-2-amine (3m). Inhibitor 3m was
synthesized using general method B (88%): 1H NMR (500 MHz,
CDCl3) δ 2.17 (s, 3H), 2.34 (s, 3H), 2.89-2.92 (m, 2H), 2.99-3.06
(m, 4H), 3.13-3.17 (m, 2H), 4.40 (br s, 2H), 4.43 (br s, 2H), 6.10-6.18
(m, 2H), 6.37-6.40 (m, 2H), 6.87-6.90 (dd, J = 8.0, 8.0 Hz, 1H),
6.95-6.98 (dd, J = 8.0, 8.0 Hz, 1H), 7.42-7.46 (m, 1H); 13C NMR (125
MHz, CD3OD) δ 19.9, 22.2, 35.3, 37.5, 37.6, 37.9, 105.7, 106.8, 112.9,
113.4, 120.8, 120.9, 121.0, 137.46, 137.55, 150.0, 153.3, 155.3, 158.0,
159.3, 159.5, 160.3, 160.4, 160.8; LC-TOF (M þ Hþ) calcd for
C21H26N5 348.21882, found 348.21880. Anal. Calcd for C21H25-
3
C, 74.55; H, 7.79; N, 15.46. Found: C, 73.86; H, 7.37; N, 14.93.
6,60-(2,20-(1,3-Phenylene)bis(ethane-2,1-diyl))bis(4-met-
hylpyridin-2-amine) (3g). Inhibitor 3g was synthesized using gen-
1
eral method B (85%): H NMR (500 MHz, CDCl3) δ 2.20 (s, 6H),
2.84-2.87 (m, 4H), 2.94-2.98 (m, 4H), 4.44 (br s, 4H), 6.19 (s, 2H),
6.35 (s, 2H), 7.06-7.11 (s, 3H), 7.19-7.22 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 21.2, 36.4, 40.2, 106.7, 114.6, 126.2, 128.5, 129.0, 142.2,
149.4, 158.5, 159.9; LC-TOF (M þ Hþ) calcd for C22H27N4
347.223 57, found 347.224 02. Anal. Calcd for C22H26N4 0.5CH3OH:
3
C, 74.55; H, 7.79; N, 15.46. Found: C, 73.59; H, 7.62; N, 15.09.
4-(3-(2-(6-Amino-4-methylpyridin-2-yl)ethyl)phenethyl)-
6-methylpyridin-2-amine (3h). Inhibitor 3h was synthesized using
general method B (90%): 1H NMR (500 MHz, CDCl3) δ 2.19 (s, 3H),
2.36 (s, 3H), 2.71-2.75 (m, 2H), 2.82-2.87 (m, 4H), 2.95-2.98 (m,
N5 0.5CH3OH: C, 71.04; H, 7.49; N, 19.27. Found: C, 70.77; H,
3
7.33; N, 19.21.
6,60-(2,20-(5-Amino-1,3-phenylene)bis(ethane-2,1diyl))-
bis(4-methylpyridin-2-amine) (3n). Inhibitor 3n was synthesized
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using general method B (85%): 1H NMR (500 MHz, CDCl3) δ 2.20 (s,
6H), 2.83-2.84 (t, J = 2.5 Hz, 8H), 3.49 (s, 2H), 4.49 (br s, 4H), 6.19 (s,
2H), 6.35 (s, 2H), 6.43 (s, 2H), 6.52 (s, 1H); 13C NMR (125 MHz,
CDCl3) δ 21.3, 36.3, 39.9, 106.8, 113.2, 114.6, 119.5, 143.3, 146.6, 149.7,
158.2, 159.5; LC-TOF-MS (M þ Hþ) calcd for C22H28N5 362.234 47,
n-BuLi, n-butyllithium; NH2OH HCl, hydroxylamine hydro-
3
chloride;LDA, lithium diisopropylamide; TMS, tetramethylsilane;
LC-TOF, liquid chromatography/time-of-flight;DMF, dimethyl-
formamide; TFA, trifluoroacetic acid;DTT, dithiothreitol
found 362.233 99. Anal. Calcd for C22H27N5 0.5CH3OH: C, 71.58; H,
3
’ REFERENCES
(1) Marletta, M. A. Nitric oxide synthase structure and mechanism. J.
Biol. Chem. 1993, 268, 12231–12234.
(2) Marletta, M. A. Nitric oxide synthase: aspects concerning
structure and catalysis. Cell 1994, 78, 927.
(3) Hall, A. V.; Antoniou, H.; Wang, Y.; Cheung, A. H.; Arbus, A. M.;
Olson, S. L.; Lu, W. C.; Kau, C. L.; Marsden, P. A. Structural organization
of the human neuronal nitric oxide synthase gene (NOS1). J. Biol. Chem.
1994, 269, 33082–33090.
(4) Wang, Y.; Newton, D. C.; Marsden, P. A. Neuronal NOS: gene
structure, mRNA diversity, and functional relevance. Crit. Rev. Neurobiol.
1999, 13, 21–43.
(5) Zhang, L.; Dawson, V. L.; Dawson, T. M. Role of nitric oxide in
Parkinson’s disease. Pharmacol. Ther. 2006, 109, 33–41.
(6) 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.
(7) Norris, P. J.; Waldvogel, H. J.; Faull, R. L. M.; Love, D. R.;
Emson, P. C. Decreased neuronal nitric oxide synthase messenger RNA
and somatostatin messenger RNA in the striatum of Huntington’s
disease. Neuroscience 1996, 72, 1037–1047.
7.74; N, 18.55. Found: C, 69.21; H, 7.33; N, 17.46.
NOS Inhibition Assays. IC50 values for inhibitors 3a-n were
measured for three different isoforms of NOS including rat nNOS,
bovine eNOS, and murine macrophage iNOS using L-arginine as a
substrate. The three enzyme isoforms were recombinant enzymes
overexpressed (in E. coli) and isolated as reported.18 These enzymes
are known to have very high sequence identity. The formation of nitric
oxide was measured using a hemoglobin capture assay as described
previously. All NOS isozymes were assayed at room temperature in a
100 mM hepes buffer (pH 7.4) containing 10 μM L-arginine, 1.6 mM
CaCl2, 11.6 μg/mL calmodulin, 100 μM dithiothreitol (DTT), 100 μM
NADPH, 6.5 μM H4B, and 3.0 μM oxyhemoglobin (for iNOS assays, no
additional Ca2þ and calmodulin). The assay was initiated by the addition
of enzyme, and the initial rates of the enzymatic reactions were
determined on a UV-vis spectrometer by monitoring the formation
of NO-hemoglobin complex at 401 nm from 0 to 60 s after mixing. The
corresponding Ki values of inhibitors were calculated from the IC50
values using eq 1 with known Km values (human nNOS, 1.6 μM; rat
nNOS,1.3 μM; iNOS, 8.3 μM; eNOS, 1.7 μM).
(8) Sims, N. R.; Anderson, M. F. Mitochondrial contributions to
tissue damage in stroke. Neurochem. Int. 2002, 40, 511–526.
(9) Hobbs, A. J.; Higgs, A.; Moncada, S. Inhibition of nitric oxide
synthase as a potential therapeutic target. Annu. Rev. Pharmacol. 1999,
39, 191–220.
(10) Southan, G. J.; Szabo, C. Selective pharmacological inhibition
of distinct nitric oxide synthase isoforms. Biochem. Pharmacol. 1996,
51, 383–394.
(11) Babu, B. R.; Griffith, O. W. Design of isoform-selective in-
hibitors of nitric oxide synthase. Curr. Opin. Chem. Biol. 1998,
2, 491–500.
(12) Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Nitric oxide
synthase: structure, function and inhibition. Biochem. J. 2001,
357, 593–615.
(13) Salerno, L.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.; Siracusa,
M. A. Progress in the development of selective nitric oxide synthase
(NOS) inhibitors. Curr. Pharm. Des. 2002, 8, 177–200.
(14) Alcaraz, M. J.; Guillꢀen, M. I. The nitric oxide related therapeutic
phenomenon: a challenging task. Curr. Pharm. Des. 2002, 8, 215–231.
(15) Vallance, P.; Leiper, J. Blocking NO synthesis: how, where and
why? Nat. Rev. Drug Discovery 2002, 1, 939–950.
(16) Ji, H.; Erdal, E. P.; Litzinger, E. A.; Seo, J.; Zhu, Y.; Xue, F.; Fang,
J.; Huang, J.; Silverman, R. B. Frontiers in Medicinal Chemistry; Reitz,
A. B., Choudhary, M. I., Atta-ur-Rahman, Eds.; Bentham Science
Publishers: Oak Park, IL, 2009; Vol. 5, pp 842-882.
(17) Silverman, R. B. Design of selective neuronal nitric oxide
synthase inhibitors for the prevention and treatment of neurodegenera-
tive diseases. Acc. Chem. Res. 2009, 42, 439–451.
(18) 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.
(19) Lawton, G. R.; Ranaivo, H. R.; Wing, L. K.; Ji, H.; Xue, F.;
Martesek, P.; Roman, L. J.; Watterson, D. M.; Silverman, R. B. Analogues
of 2-aminopyridine-based selective inhibitors of neuronal nitric oxide
synthase with increased bioavailability. Bioorg. Med. Chem. 2009,
17, 2371–2380.
Ki ¼ IC50=ð1 þ ½Sꢁ=KmÞ
ð1Þ
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis of 5g, Table S1, and
NMR spectra of new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
b
’ AUTHOR INFORMATION
Corresponding Author
*For T.L.P.: phone, 949-824-7020; fax, 949-824-3280; e-mail,
poulos@
uci.edu. For R.B.S.: phone, 847-491-5653; fax, 847-491-7713;
e-mail, Agman@chem.northwestern.edu.
Present Addresses
bDepartment of Chemistry, University of Louisiana at Lafayette,
Lafayette, Louisiana 70504.
3Department of Chemistry, Lanzhou University, Lanzhou,
China.
’ ACKNOWLEDGMENT
The authors are grateful to the National Institutes of Health
for financial support to R.B.S. (Grant GM49725), T.L.P (Grant
GM57353), and Dr. Bettie Sue S. Masters (Grant GM52419,
with whose laboratory P.M. and L.J.R. are affiliated). Dr. Bettie
Sue S. Masters is the Robert A. Welch Distinguished Professor in
Chemistry (AQ0012). P.M. is supported by Grants 0021620806
and 1M0520 from MSMT of the Czech Republic.
’ ABBREVIATIONS USED
(20) Xue, F.; Fang, J.; Lewis, W. W.; Martasek, P.; Roman, L. J.;
Silverman, R. B. Potent and selective neuronal nitric oxide synthase
inhibitors with improved cellular permeability. Bioorg. Med. Chem. Lett.
2010, 20, 554–557.
NO, nitric oxide; eNOS, endothelial nitric oxide synthase; nNOS,
neuronal nitric oxide synthase;iNOS, inducible nitric oxide
synthase; BBB, blood-brain barrier; H4B, tetrahydrobiopterin;
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Journal of Medicinal Chemistry
ARTICLE
(21) Xue, F.; Huang, J.; H., Li; Ji, H.; Fang, J.; Poulos, T, L.;
Silverman, R. B. Structure-based design, synthesis, and biological
evaluation of lipophilic-tailed monocationic inhibitors of neuronal nitric
oxide synthase. Bioorg. Med. Chem. 2010, 18, 6526–6537.
(22) Xue, F.; Li, H.; Fang, J.; Poulos, T. L.; Silverman, R. B.
Peripheral but crucial: the 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) Xue, F.; Gu, W.; Silverman, R. B. A concise route to the chiral
pyrrolidine core of selective inhibitors of neuronal nitric oxide synthase.
Org. Lett. 2009, 11, 5194–5197.
(24) Hagmann, W. K.; Caldwell, C. G.; Chen, P.; Durette, P. L.;
Esser, C. K.; Lanza, T. J.; Kopka, I. E.; Guthikonda, R.; Shah, S. K.;
MacCoss, M.; Chabin, R. M.; Fletcher, D.; Grant, S. K.; Green, B. G.;
Humes, J. L.; Kelly, T. M.; Luell, S.; Meurer, R.; Moore, V.; Pacholok,
S. G.; Pavia, T.; Williams, H. R.; Wong, K. K. Substituted 2-aminopyr-
idines as inhibitors of nitric oxide synthases. Bioorg. Med. Chem. Lett.
2000, 10, 1975–1978.
(25) Momenteau, M.; Mispelter, J.; Loock, B.; Lhoste, J. M. Both-
faces hindered porphyrins. Part 2. Synthesis and characterization of
internally five-co-ordinated iron(II) basket-handle porphyrins derived
from 5,10,15,20-tetrakis(o-hydroxyphenyl)porphyrin. J. Chem. Soc., Per-
kin Trans. 1 1985, 61–70.
(26) Hevel, J. M.; Marletta, M. A. Nitric-oxide synthase assays.
Methods Enzymol. 1994, 233, 250–258.
(27) Delker, S. L.; Xue, F.; Li, H.; Jamal, J.; Silverman, R. B.; Poulos,
T. L. Role of zinc in isoform-selective inhibitor binding to neuronal nitric
oxide synthase. Biochemistry 2010, 49, 10803–10810.
(28) Fang, J.; Silverman, R. B. A cellular model for screening
neuronal nitric oxide synthase inhibitors. Anal. Biochem. 2009,
390, 74–78.
(29) Linnet, K.; Ejsing, T. B. A review on the impact of P-glycopro-
tein on the penetration of drugs into the brain. Focus on psychotropic
drugs. Eur. Neuropsychopharmacol. 2008, 18, 157–169.
(30) Juliano, R. L; Ling, V. A. A surface glycoprotein modulating
drug permeability in Chinese hamster ovary cell mutants. Biochim.
Biophys. Acta 1976, 455, 152–162.
(31) Schinkel, A. H.; Mayer, U.; Wagenaar, E.; Mol, C. A.; van
Deemter, L.; Smit, J. J.; van der Valk, M. A.; Voordouw, A. C.; Spits, H.;
van Tellingen, O.; Zijlmans, J. M.; Fibbe, W. E.; Borst, P. Normal
viability and altered pharmacokinetics in mice lacking mdr1-type (drug-
transporting) P-glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 4028–4033.
(32) Schinkel, A. H. P-Glycoprotein, a gatekeeper in the blood-brain
barrier. Adv. Drug Delivery Rev. 1999, 36, 179–194.
2048
dx.doi.org/10.1021/jm101071n |J. Med. Chem. 2011, 54, 2039–2048