Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure-activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes.
Selective inhibition of pan class="Gene">neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure-activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes.
pan class="Chemical">Nitric oxide (NO) is an important biological
second messenger in humans, which plays a critical role in cell and
neuronal signaling, blood pressure regulation, and the immune response.[1] NO is produced from oxidation of l-arginine
(l-Arg) in the presence of NADPH by a class of heme-dependent
enzymes, nitric oxide synthases (NOS).[2] Mammals have three dominant isoforms of NOS: constitutively expressed
neuronal NOS (nNOS), present throughout the nervous system and skeletal
muscles, endothelial NOS (eNOS), also a constitutive enzyme located
in the endothelium and functioning in regulation of blood pressure
and blood flow, and inducible NOS (iNOS), which is associated with
the immune response.
In the brain, low nanomolar concentpan class="Species">rations
of NO produced by nNOS are neuroprotective, and downstream NO, along
with cyclic guanosine 5′-monophosphate (cGMP) in the protein
kinase G (PKG) signaling pathway, plays an important role in neurotransmission
and other metabolic processes.[3] However,
overexpression and overactivation of nNOS following neuronal damage
causes NO levels to jump several orders of magnitude,[4] which is neurotoxic. Such NO-mediated neurotoxicity leads
to protein degradation, misfolding, and aggregation through tyrosine-nitration,[5]S-nitrosylation,[6] and oxidative stress damage through formation of reactive
oxygen species (ROS) and reactive nitrogen species (RNS).[7] This neurotoxicity has been implicated in several
neurodegenerative disorders that include Alzheimer’s, Parkinson’s,
and Huntington’s diseases and amyotrophic lateral sclerosis
(ALS).[8] Furthermore, progressive neuronal
damage and loss of neural tissue associated with NO overproduction
are seen in cerebral palsy, stroke, ischemic brain damage, and migraine
headaches.[9] Therefore, with the increasing
human and economic costs associated with neurodegenerative diseases,
and the lack of existent treatments, there is an urgent need for the
development of new therapeutics that would prevent, cure, or attenuate
neurodegeneration. With high levels of NO implicated in these neurodegenerative
conditions, and target validation linking nNOS to these pathological
conditions,[10] the development of nNOS inhibitors
is an important therapeutic approach for neuroprotection.[11]
All NOS isoforms are active only as homodimeric
enzymes, where each monomer contains an N-terminal pan class="Chemical">oxygenase domain
and a C-terminal reductase domain.[12] The
reductase domain contains the n>n class="Chemical">FMN, FAD, and NADPH binding-sites,[13] while the oxygenase domain binds the cofactor
(6R)-5,6,7,8-tetrahydrobiopterin (H4B)
and the substrate l-Arg at the heme catalytic site. Upon
dimerization and activation of NOS by Ca2+-mediated calmodulin
binding,[14] electrons flow from NADPH to
FAD and FMN in the reductase domain to the heme in the oxygenase domain,
where conversion of l-Arg to l-citrulline and NO
takes place in the presence of oxygen.[15] Therefore, quite predictably, the major approach in the development
of NOS inhibitors involves the utilization of arginine mimetics as
competitive nNOS inhibitors.[11,16] However, in addition
to potent inhibition of nNOS, there are many challenges associated
in designing nNOS inhibitors. First, selectivity of nNOS inhibitors
over eNOS and iNOS (NOS isoforms share nearly identical active site)
is essential to minimize undesired side effects.[17] Second, as arginine isosteres, these inhibitors are highly
polar; therefore, they suffer from poor bioavailabilty. Hence, the
design of potent nNOS inhibitors with improved pharmacokinetic properties
that address high isoform selectivity, blood–brain barrier
permeability, and minimal off-target efficacy is crucial.
In
this respect, continuing efforts from our labopan class="Species">ratories, guided by
structure-based drug design and fragment hopping, have resulted in
a series of highly potent and selective small molecule nNOS inhibitors
based on pyrrolidinomethyl-2-aminopyridine scaffolds (Figure 1, compounds 1 and 2).[18] Compounds 1 and 2 showed
high potency and excellent selectivity over iNOS and eNOS, while 2, when administered intravenously to a pregnant rabbit dam
with induced uterine hypoxia, showed a complete reversal of hypoxia–ischemia
induced death in the newborn kits.[19] However,
further development of these compounds stalled because of their inability
to cross the blood–brain barrier. This was presumably the result
of the hydrophilic nature of the molecule (too many basic amines and
hydrogen-bond donors, high polar surface area, and a large number
of rotatable bonds).[20] Modifications of
these pyrrolidinomethyl-2-aminopyridine scaffolds, such as reducing
the number of polar charges and basicity by alkylation,[21] fluorination,[22] and
intramolecular hydrogen bonding,[23] met
with either diminished potency or selectivity or without notable improvement
in cellular permeability. Furthermore, synthesis of these pyrrolidinomethyl-2-aminopyridine
scaffolds involved more than 12 steps, difficult chiral resolutions,
and diastereomer separations. Later modifications of simplified double-headed
2-aminopyridine scaffolds displayed good potency and selectivity;[24] however, they still suffered from poor permeability
in a Caco-2 assay, which is used to estimate intestinal cellular permeability
and also reflects potential brain permeation.[25]
Figure 1
Representative
nNOS and iNOS inhibitors and nNOS inhibitor design strategy.
Representative
pan class="Gene">nNOS and iNOS inhibitors and nNOS inhibitor design strategy.
Therefore, as bioavailability
was a major challenge in these pan class="Gene">nNOS inhibitors, one avenue we explored
to improve the cellular permeability of the NOS inhibitors, while
maintaining a good potency and selectivity, was replacement of the
more basic arginine isosteres with fewer basic groups that engage
in heme-coordination in the active site of NOS to arrest l-arginine turnover (for example, the pKa of conjugate acid of 2-aminopyridine is 7.1). Compared to many inhibitors
designed as arginine mimetics, only a few reports exist that explore
the heme-coordination of NOS.[11] However,
imidazole (pKa of its conjugate acid is
6.9) is known to weakly bind to nNOS (IC50 200 μM),
while the less-donating 1-phenylimidazole shows nearly 10-fold improved
potency (IC50 25 μM).[26] Furthermore, 2-(1-imidazolyl)pyrimidine scaffolds, such as 3, have shown very good potency as iNOS dimerization inhibitors.[27] In addition, 3 demonstrated good
cellular permeability and in vivo efficacy against iNOS in rats.[27b]
Toward this end, we designed simplified
compounds 4 and 5 with improved predicted
physicochemical properties by hybridizing selective molecular fragments
from inhibitors 1 and 3. The key hydrophobic
tail of 1 was incorpopan class="Species">rated into structures 4 and 5 based on previous precedence, where contacts
between 1 and residues lining a hydrophobic pocket adjacent
to the substrate access channel[28] were
implicated in improving isoform selectivity.[18,29] On the basis of inhibitory assay results, docking studies, and crystallographic
analysis, further modifications on 5, such as changing
the linker length between the two aromatic heads, the number of secondary
amines, and utilizing various substitutions on the aromatic rings,
were employed to maximize rat and humannNOS potency, improve isoform
selectivity, enable cellular permeability, and minimize off-target
effects. Figure 2 summarizes the different
nNOS inhibitors designed and studied in this series. Through these
structure–activity optimizations, highly potent and selective
inhibitors of nNOS were discovered. These compounds showed improved
cellular permeability, high selectivity against a panel of central
nervous system (CNS) receptors, and attenuated CYP inhibition.
Figure 2
nNOS inhibitors
synthesized in this study.
pan class="Gene">nNOS inhibitors
synthesized in this study.
Chemistry
Synthesis of the pan class="Chemical">2,4-disubstituted pyrimidine
scaffolds in this study was designed to utilize common intermediates
and proceed in relatively few steps. Thus, the synthesis of the 2,4-disubstituted
pyrimidine cores in 4 and 5 was obtained
by following a modified procedure reported by Davey et al.[27b] Alkylation of N-Boc piperazine
(21) by 3-fluorophenethyl bromide gave 22, which underwent a sequential one-pot substitution on 4-chloro-2-methanesulfonylpyrimidine by itself and imidazole, respectively, to provide target
compound 4 (Scheme 1). Similar
substitutions on 4-chloro-2-methanesulfonyl pyrimidine by 26 and imidazole, respectively, gave the primary framework as a Boc-protected
precursor of 5, which was subsequently Boc-deprotected
with trifluoroacetic acid and obtained as a trihydrochloride salt
upon precipitation from methanolic HCl. Compound 25 was
synthesized following a reported procedure,[30] from where a Mitsunobu reaction with diphenylphosphoryl azide gave
the corresponding azide, which was subsequently reduced to give the
primary amine 26.
Scheme 1
Synthesis of 4 and 5
Reagents and conditions: (a) (i)
NaH, THF, 0 °C, 1 h, then 3-fluorophenethyl bromide, TBAI, 60
°C, 48 h, 56%, (ii) TFA, CH2Cl2, 0 °C
→ RT, 3 h, 85%; (b) 4-chloro-2-methanesulfonyl pyrimidine,
K2CO3, MeCN, 40 °C, 19 h, then imidazole,
65 °C, 30 h, 82%; (c) 3-fluorophenyl acetaldehyde, NaBH(OAc)3, RT, 14 h, 91%; (d) (i) H2, Pd/C, MeOH, 12 h,
86%, (ii) Boc2O, CH2Cl2, 3 h, 76%;
(e) (i) DIAD, PPh3, DPPA, THF, 12 h, (ii) PPh3, THF/H2O, 41%; (f) (i) same as (b), 60%, (ii) TFA, CH2Cl2, 2 h, (iii) HCl in MeOH, 10 min, 83%.
Synthesis of 4 and 5
Reagents and conditions: (a) (i)
pan class="Chemical">NaH, THF, 0 °C, 1 h, then 3-fluorophenethyl bromide, TBAI, 60
°C, 48 h, 56%, (ii) TFA, CH2Cl2, 0 °C
→ RT, 3 h, 85%; (b) 4-chloro-2-methanesulfonyl pyrimidine,
K2CO3, MeCN, 40 °C, 19 h, then imidazole,
65 °C, 30 h, 82%; (c) 3-fluorophenyl acetaldehyde, NaBH(OAc)3, RT, 14 h, 91%; (d) (i) H2, Pd/C, MeOH, 12 h,
86%, (ii) Boc2O, CH2Cl2, 3 h, 76%;
(e) (i) DIAD, PPh3, DPPA, THF, 12 h, (ii) PPh3, THF/H2O, 41%; (f) (i) same as (b), 60%, (ii) TFA, CH2Cl2, 2 h, (iii) HCl in MeOH, 10 min, 83%.
In 6 and 7, where the secondary
pan class="Chemical">amine was benzylic to the pyrimidine, a reductive amination to form
the C–N bond with an aromatic aldehyde was conceived as a favorable
disconnection (Scheme 2). Therefore, 2-methylthio-4-pyrimidine
carboxaldehyde (30) was first obtained by condensation
between pyruvaldehydedimethylacetal (27) and thiourea
(28) and then acetal deprotection.[31] This intermediate underwent efficient reductive amination
with 3-fluorophenylethylamine and 3-fluorophenylpropylamine to form
secondary amines 33 and 34, respectively.
Then Boc protection of the secondary amine and oxidation of the thioether
group to methanesulfonyl by mCPBA enabled the successive displacement
by imidazole to get the main structural frameworks of 6 and 7. Finally, deprotection of the Boc group under
acidic conditions and subsequent precipitation from methanolic HCl
gave 6 and 7 as dihydrochloride salts.
Scheme 2
Synthesis of 6 and 7
Reagents
and conditions: (a) (i) N,N-dimethylformamide
dimethyl acetal, 110 °C, 8 h, (ii) NaOMe, MeOH, RT, 16 h, (iii)
MeI, 60 °C, 12 h, 87%; (b) HCl, 85 °C, 6 h, 58%; (c) NaBH(OAc)3, cat. AcOH, 3 Å sieves, CH2Cl2, 14 h, 85–89%; (d) (i) Boc2O, THF, 12 h, 97–99%,
(ii) mCPBA, CH2Cl2, 12 h, 75–89%; (e)
imidazole, K2CO3, MeCN, 60 °C, 4 h, 84–93%;
(f) (i) TFA, CH2Cl2, 3 h, (ii) HCl in MeOH,
10 min, 79–93%.
Synthesis of 6 and 7
Reagents
and conditions: (a) (i) N,N-dipan class="Chemical">methylformamide
dimethyl acetal, 110 °C, 8 h, (ii) NaOMe, MeOH, RT, 16 h, (iii)
MeI, 60 °C, 12 h, 87%; (b) HCl, 85 °C, 6 h, 58%; (c) NaBH(OAc)3, cat. AcOH, 3 Å sieves, CH2Cl2, 14 h, 85–89%; (d) (i) Boc2O, THF, 12 h, 97–99%,
(ii) mCPBA, CH2Cl2, 12 h, 75–89%; (e)
imidazole, K2CO3, MeCN, 60 °C, 4 h, 84–93%;
(f) (i) TFA, CH2Cl2, 3 h, (ii) HCl in MeOH,
10 min, 79–93%.
pan class="Chemical">2,4-Disubstituted pyrimidine
scaffolds possessing two methylene units between the secondary amine
and the pyrimidine ring (8–14) were
synthesized via a Michael addition on the electron-deficient vinyl
pyrimidine ring (Scheme 3). Therefore, the
2-methylthio-4-vinyl pyrimidine (40) was synthesized
by a Stille coupling between commercially available 4-chloro-2-methylthio
pyrimidine (39) and tributylvinyltin in the presence
of tetrakistriphenylphosphine Pd(0).[32] A
Michael addition between 40 and the particular primary
amines gave the corresponding homobenzylic amines (41–47). The primary amines (31–32, S3–S12 (see Supporting Information), 62) were
synthesized either from commercially available bromides or carboxylic
acids in 2–3 steps, as elaborated in the Supporting Information. Note that in the case of the primary
amines containing a cyclopropyl group, the cis and trans isomers were completely separable by silica gel column
chromatography (see Supporting Information for further details), and the trans isomer was
carried forward for the Michael addition reaction.
Scheme 3
General Scheme for
Synthesis of 8–14
Reagents and conditions: (a) tributylvinyl tin, Pd(PPh3)4, DCE, 70 °C, 48 h, 92%; (b) R-NH2,
cat. AcOH, EtOH, 8–48 h, 62–97%; (c) (i) Boc2O, THF, 3 h, 80–95%, (ii) mCPBA, CH2Cl2, 3 h, 65–91% (note in case of 46, oxone was
used instead of mCPBA in a 1:1 THF/H2O mixture for 4 h);
(d) imidazole, K2CO3, MeCN, 65 °C, 5–10
h, 76–92%; (e) (i) TFA, CH2Cl2, 3 h,
(ii) HCl in MeOH, 10 min, 80–99%.
General Scheme for
Synthesis of 8–14
Reagents and conditions: (a) pan class="Chemical">tributylvinyl tin, Pd(PPh3)4, DCE, 70 °C, 48 h, 92%; (b) R-NH2,
cat. AcOH, EtOH, 8–48 h, 62–97%; (c) (i) Boc2O, THF, 3 h, 80–95%, (ii) mCPBA, CH2Cl2, 3 h, 65–91% (note in case of 46, oxone was
used instead of mCPBA in a 1:1 THF/H2O mixture for 4 h);
(d) imidazole, K2CO3, MeCN, 65 °C, 5–10
h, 76–92%; (e) (i) TFA, CH2Cl2, 3 h,
(ii) HCl in MeOH, 10 min, 80–99%.
Following
a similar route as illustpan class="Species">rated for the synthesis of 6 and 7, final compounds 8–14 were obtained (Scheme 3). However,
in the case of 46, after Boc protection, mCPBA oxidation
(regardless of conditions) always led to undesired oxidation of the
pyridine ring to the N-oxide. Finally, oxidation
of 46 in a 1:1 mixture of THF and H2O by oxone
at room temperature for 4 h gave desired sulfone 53.[33] Similar imidazole substitution, Boc deprotection,
and acidification gave final compound 13 as a pure trihydrochloride
salt.
When sepapan class="Species">ration of the two enantiomers of 12 became crucial, the trans-isomer of 2-(3-fluorophenyl)-1-cyclopropyl
amine (62) was subjected to DCC-mediated amidation with
(S)-(+)-α-methoxyphenylacetic acid (Scheme 4).[34] The diastereomers, 63a and 63b, were obtained pure by silica gel
column chromatographic separation. Finally, hydrolysis of the auxiliary
under refluxing ethanolicHCl gave the enantioenriched amines 62a and 62b in >95% enantiopurity. These amines
were then independently converted to the final enantiomerically enriched
isomers (R,R)-12 and
(S,S)-12 by following
the exact same route as used to synthesize 12 from 62 and 2-methylthio-4-vinylpyrimidine (40).
Scheme 4
Synthesis of (R,R)-12 and (S,S)-12
Reagents and conditions: (a) (i)
(S)-(+)-α-methoxyphenylacetic acid, DCC, CH2Cl2, −20 °C to RT, 12 h, (ii) chiral
resolution of diastereomers on silica gel column; (b) HCl in EtOH,
reflux, 12 h.
Synthesis of (R,R)-12 and (S,S)-12
Reagents and conditions: (a) (i)
(S)-(+)-α-pan class="Chemical">methoxyphenylacetic acid, DCC, CH2Cl2, −20 °C to RT, 12 h, (ii) chiral
resolution of diastereomers on silica gel column; (b) HCl in EtOH,
reflux, 12 h.
When the pan class="Chemical">2,4-disubstituted pyrimidine
was replaced by a 3,5-disubstituted pyridine ring, as in 15, synthesis was initiated from commercially available 5-bromo nicotinic
acid (64) by reduction to alcohol 65 (Scheme 5). Homologation by one methylene unit was obtained
by conversion of the alcohol to a nitrile (66) and then
to its aldehyde by DIBAL. As the intermediate aldehyde was photosensitive
and unstable, it was synthesized in the dark, and the crude reaction,
after work up, was immediately subjected to reductive amination with
3-fluorophenethyl amine (31) to provide 67 in a 41% yield (over two steps). Boc protection of the secondary
amine, followed by a Cu-catalyzed amination of the 3-bromopyridine
group, gave 68,[35] which was
Boc deprotected and treated with methanolic HCl to give final compound 15 as a trihydrochloride salt.
Scheme 5
Synthesis of 15
Reagents and conditions: (a) (i)
isobutyl chloroformate, Et3N, THF, RT, 1 h, (ii) NaBH4, H2O, 12 h, 43%; (b) (i) SOCl2, CH2Cl2, 2 h, (ii) KCN, DMF, RT, 12 h, 70%; (c) (i)
DIBAL, CH2Cl2, −78 °C, 1 h, (ii)
3-fluorophenethylamine, cat. AcOH, MgSO4, NaBH(OAc)3, 12 h, 41%; (d) (i) Boc2O, THF, 12 h, 92%, (ii)
CuBr, 8-acetyl-5,6,7,8-tetrahydroquinoline, imidazole, Cs2CO3, DMSO, 100 °C, 12 h, 67%; (e) (i) TFA, CH2Cl2, 3 h, (ii) HCl in MeOH, 10 min, 88%.
Synthesis of 15
Reagents and conditions: (a) (i)
pan class="Chemical">isobutyl chloroformate, Et3N, THF, RT, 1 h, (ii) NaBH4, H2O, 12 h, 43%; (b) (i) SOCl2, CH2Cl2, 2 h, (ii) KCN, DMF, RT, 12 h, 70%; (c) (i)
DIBAL, CH2Cl2, −78 °C, 1 h, (ii)
3-fluorophenethylamine, cat. AcOH, MgSO4, NaBH(OAc)3, 12 h, 41%; (d) (i) Boc2O, THF, 12 h, 92%, (ii)
CuBr, 8-acetyl-5,6,7,8-tetrahydroquinoline, imidazole, Cs2CO3, DMSO, 100 °C, 12 h, 67%; (e) (i) TFA, CH2Cl2, 3 h, (ii) HCl in MeOH, 10 min, 88%.
The different aromatic substitutions at the 2-position
of the pan class="Chemical">pyrimidine in 16–20 were synthesized
from advanced intermediate 49 by substituting the sulfone
with the different methyl imidazoles or triazoles (Scheme 6). Thereafter, a similar TFA-mediated Boc deprotection
and salt formation gave the compounds 16–20. However, when 4-methylimidazole was used, both 4- and
5-methyl-2-imidazolyl pyrimidines 72 and 73 were obtained, which were inseparable by chromatographic conditions.
Therefore, a subsequent Boc deprotection of the mixture of 72 and 73, followed by a chromatographic separation, provided
the free bases of 19 and 20. These were
subsequently treated with methanolic HCl to obtain pure dihydrochloride
salts 19 and 20.
Scheme 6
Synthesis of 16–20
Reagents
and conditions: (a) imidazole/triazole, K2CO3, MeCN, 65 °C, 5–24 h, 82–88%; (b) (i) TFA, CH2Cl2, 1 h, (ii) HCl in MeOH, 10 min, 87–99%.
Synthesis of 16–20
Reagents
and conditions: (a) pan class="Chemical">imidazole/triazole, K2CO3, MeCN, 65 °C, 5–24 h, 82–88%; (b) (i) TFA, CH2Cl2, 1 h, (ii) HCl in MeOH, 10 min, 87–99%.
Results and Discussion
Table 1 summarizes the binding affinity of 4–20 in the in vitro enzyme inhibitory assays
against several isoforms of NOS. An oxyhemoglobin NO capture assay
was used to determine the IC50 pan class="Chemical">value of inhibitors against
the purified n>n class="Species">rat and humannNOS, murine macrophage iNOS, and bovineeNOS.[36] The apparent Ki values of the inhibitors were determined from IC50 and substrate Km values using
the Cheng–Prusoff equation, and the corresponding isoform selectivities
as ratios of their respective Ki values.
Table 1
Determination of Ki Values
of Inhibitors 4–20a
Ki (μM)
selectivity
compd
rnNOS
eNOS
iNOS
hnNOS
n/e
n/i
r/hnNOS
4
4.7
NT
NT
NT
5
0.368
40.0
6.4
NT
109
17
6
8.7
NT
NT
NT
7
2.7
90.0
10.5
NT
33
4
8
0.138
4.0
1.1
0.758
30
8
5.5
9
0.019
4.95
0.77
0.193
260
41
10.1
10
0.032
8.1
0.91
0.125
253
28
3.9
11
0.056
4.0
1.9
0.359
71
34
6.4
12
0.040
14.5
2.9
0.358
363
73
8.9
13
0.054
10.9
1.8
0.125
202
33
2.3
14
0.183
10.5
3.4
0.138
57
19
0.75
15
5.5
NT
NT
NT
16
60.0
NT
NT
NT
17
27.0
NT
NT
NT
18
11.5
NT
NT
NT
19
81.0
NT
NT
NT
20
0.060
15.4
24.0
0.303
257
400
5.0
Compounds 4–20 were assayed in vitro against four purified NOS isoforms:
rat nNOS (rnNOS), bovine eNOS (eNOS), murine iNOS (iNOS), and human
nNOS (hnNOS) using known literature methods. Ki values are determined using the Cheng–Prusoff equation
directly from IC50 values (see Experimental
Section for details). IC50 values are the average
of at least two replicates with nine data points; all experimental
standard error values are less than 15%, and all correlation coefficients
are >0.9. Selectivity values are the ratios of respective Ki values. NT = not tested.
Compounds 4–20 were assayed in vitro against four purified NOS isoforms:
pan class="Species">rat nNOS (rnNOS), bovineeNOS (eNOS), murineiNOS (iNOS), and humannNOS (hnNOS) using known literature methods. Ki values are determined using the Cheng–Prusoff equation
directly from IC50 values (see Experimental
Section for details). IC50 values are the average
of at least two replicates with nine data points; all experimental
standard error values are less than 15%, and all correlation coefficients
are >0.9. Selectivity values are the ratios of respective Ki values. NT = not tested.
Between the two initial inhibitors
designed, 4 and 5, compound 4 only weakly inhibited pan class="Gene">nNOS (Ki ∼
5 μM) while 5 displayed a stronger inhibition of
n>n class="Gene">nNOS (Ki 0.368 μM). This nearly
10-fold difference in potency can be attributed to the key structural
difference in the linker between the pyrimidine and 3-fluorophenyl
ring in 4 and 5. While a piperazine ring
may be sterically favorable in a more open active site, in case of 3 binding to an iNOS monomer (and hence preventing dimerization),[27a] the same may cause unfavorable steric clashes
with the peptide backbone in the nNOS dimer. Furthermore, the open
and flexible linker in 5 can also engage in favorable
interactions via the secondary amines, thus orienting the hydrophobic
end of the molecule properly. To gain more insight into the structural
basis for potencies and selectivity, we determined the crystal structure
of 5 bound to nNOS and eNOS. Indeed, the crystal structure
of 5 bound to nNOS (Figure 3A)
shows that while the 2-imidazolyl pyrimidine head in the molecule
ligates to the heme Fe,[27a] the void left
between the imidazole ring and Glu592 is filled with a water molecule.
The secondary amine next to the pyrimidine ring is engaged in a salt
bridge with heme propionate A, while the other secondary amine makes
a hydrogen bond with a water bridging in between H4B and
the heme. This also orients the rest of the linker toward the hydrophobic
pocket lined by Tyr706, Met336, Leu337, and Trp306 (from the other
monomer) in ratnNOS.[18] The aromatic ring
of 5 engages in quite a few van der Waals interactions
with these four residues at distances from 3.5 to 4.0 Å, except
for the closer distance (3.3 Å) from the fluorine atom of 5 to the carbonyl oxygen of Met336. This also represents the
first structure of nNOS with a type II imidazole-based heme-bound
inhibitor. The crystal structure of 5 bound to eNOS (Figure 3B) reveals two major differences compared to its
binding conformation seen in nNOS. First, similar to nNOS, the tail
fluorophenyl ring can still make van der Waals contacts with Tyr477,
Leu107, and Trp76 (the other monomer), but the fluorine atom is no
longer inserted into the pocket as in the nNOS case. Instead, the
aromatic ring simply caps the pocket at its edge with the F atom pointing
sideways. The reason for this binding orientation is very likely an
amino acid variation, Val106 (eNOS) vs Met336 (nNOS), which is also
part of this hydrophobic pocket. The more degrees of freedom of Met336
in nNOS can more readily adapt to inhibitor binding than Val106 in
eNOS. As a result, the CG1 atom of Val106 in eNOS would clash with
the aromatic ring of 5 if it were in its position seen
in nNOS. Consequently, in eNOS the secondary amine in the linker of 5 makes a hydrogen bond with heme propionate D, rather than
with a water molecule as in nNOS. The Val/Met variation is, therefore,
the structural basis for the observed 100-fold selectivity for 5 (Table 1).
Figure 3
Active site structure
of 5 bound to rat nNOS ((A) PDB 4D3B) and bovine eNOS
((B) PDB 4D33). Key hydrogen bonds are shown by dashed lines, and distances are
in Å. The omit Fo–Fc map for the ligand is contoured at 2.5 σ.
The heme pyrrole rings are labeled in order to identify the propionate
positions. All structural figures were prepared with PyMol (www.pymol.org).
Active site structure
of 5 bound to pan class="Species">rat nNOS ((A) PDB 4D3B) and bovineeNOS
((B) PDB 4D33). Key hydrogen bonds are shown by dashed lines, and distances are
in Å. The omit Fo–Fc map for the ligand is contoured at 2.5 σ.
The heme pyrrole rings are labeled in order to identify the propionate
positions. All structural figures were prepared with PyMol (www.pymol.org).
Because the part of inhibitor 5 positioned directly over the pan class="Chemical">heme appeared optimal, we sought
to improve the enzyme–inhibitor interactions beyond the active
site (compounds 6–9), utilizing different
linker lengths between the pyrimidine and 3-fluorophenyl rings. The
rationale behind the design of the shorter 4-atom-linker, as in 6, was to avoid the twisting of methylene units on top of
each other, as seen in the conformation of 5, but still
enable the aromatic end to reach the hydrophobic pocket. Also, we
reasoned that moving the secondary amine away from the pyrimidine
by one or two methylene units might enable it to hydrogen bond with
both the heme propionates and improve the potency of the molecule.
With this amine moving up along the linker, the other secondary amine
in 5 becomes obsolete and thus can be removed to reduce
the basicity of the inhibitors.
From epan class="Chemical">valuation of the binding
affinity of these inhibitors against nNOS (Table 1), we found two crucial factors that contribute to their potency
and selectivity: the position of the secondary amine and the length
of the linker. Moving the secondary amine by one more methylene unit
from the pyrimidine ring (8 and 9) dramatically
decreased the Ki values relative to 6 and 7 (8.7 and 2.7 μM, respectively).
The structure of 7 bound to nNOS (Figure 4A) reveals that the secondary amine can no longer make a hydrogen
bond with either of the heme propionates, thus suggesting that this
interaction is critical for imparting potency to these pharmacophores.[28,37] This polar interaction was regained in 8 and 9, where the secondary amine is one carbon farther away from
the pyrimidine than in 6 and 7. The structure
of 8 bound to nNOS (Figure 4B)
indicates that this position of the amine enables it to engage in
dual salt bridges with both the heme propionates, with the secondary
amine positioned equidistant between the two. However, the five-atom
linker of 8 is not long enough to bring the fluorophenyl
ring into tighter van der Waals contacts with the hydrophobic pocket.
This, in fact was attained with the six-atom linker between the fluorophenyl
and the pyrimidine to provide maximum potency. Thus, 9 is one of the most potent nNOS inhibitors in this series of 2-imidazolylpyrimidine
scaffolds (Ki = 0.019 μM). Indeed,
the structure of nNOS–9 (Figure 5A) demonstrates the structural basis for this good potency.
Like 8, the secondary amine of 9 can establish
dual salt bridges with both heme propionates. In addition, owing to
the longer linker of 9, the fluorine atom of the aromatic
ring can be inserted into the hydrophobic pocket, as in the case of 5. Extensive van der Waals contacts provided by the inhibitor’s
aromatic ring should be responsible for the 7-fold higher potency
when comparing 9 with 8 (Figure 5A vs Figure 4B). Furthermore, 9 displayed 260-fold selectivity over eNOS, with an improved
41-fold selectivity over iNOS. The differences in interactions with
residues lining the hydrophobic pocket also contribute to isoform
selectivity. As shown in Figure 5B, even though 9 in eNOS can retain the dual salt bridges from its secondary
amine in the linker, its fluorophenyl ring cannot insert into the
hydrophobic pocket as it does in nNOS. This is the result of an amino
acid variation (Val106 in eNOS vs Met336 in nNOS), as postulated for 5. The lack of these van der Waals contacts reflects in the
poorer potency of 9 to eNOS, which is supported by the
fact that both 8 and 9 share similar potency
to eNOS (see Figure S1 in the Supporting Information for structure of 8 bound to eNOS).
Figure 4
Active site structures
of 7 ((A) PDB 4V3V) and 8 ((B) PDB 4V3W) bound to rat nNOS.
Key hydrogen bonds are shown by dashed lines and distances are in
Å. The omit Fo–Fc map for the ligand is contoured at 2.5 σ.
Figure 5
Active site structures of 9 bound
to rat nNOS ((A) PDB 4V3X) and bovine eNOS ((B) PDB 4D35). Key hydrogen bonds are shown by dashed lines and
distances are in Å. The omit Fo–Fc map for the ligand is contoured at 2.5 σ.
Active site structures
of 7 ((A) PDB 4V3V) and 8 ((B) PDB 4V3W) bound to pan class="Species">rat nNOS.
Key hydrogen bonds are shown by dashed lines and distances are in
Å. The omit Fo–Fc map for the ligand is contoured at 2.5 σ.
Active site structures of 9 bound
to pan class="Species">rat nNOS ((A) PDB 4V3X) and bovineeNOS ((B) PDB 4D35). Key hydrogen bonds are shown by dashed lines and
distances are in Å. The omit Fo–Fc map for the ligand is contoured at 2.5 σ.
Encouraged by the good potency
and selectivity of 9, we sought to replace the fluorophenyl
with a chlorophenyl ring at the hydrophobic end of 10. Prior studies have shown that pan class="Chemical">halogen replacements could result
in enhanced inhibitory potency and selectivity from contacts between
the inhibitor and residues in the hydrophobic pocket.[28,30] However, in our case, this change results in slightly diminished
inhibitor potency in nNOS (32 nM for 10 vs 19 nM for 9). The structure of nNOS–10 (Figure S2 in Supporting Information) shows that
because of the bulkiness of the chlorine atom, the aromatic ring actually
retreats from the hydrophobic pocket, leading to less extensive van
der Waals contacts with the protein compared to 9. As
expected, the chlorophenyl ring of 10 in the eNOS–10 structure (Figure S3 in Supporting
Information) is entirely outside of the pocket, similar to
the situation in eNOS–9 (Figure 5B), and therefore 10 still maintains good n/e
selectivity (253-fold).
While 9 showed a high level
of selectivity against pan class="Gene">eNOS, we looked deeper into further improving
its iNOS selectivity. The three NOS isoforms have a 50–60%
overall sequence identity and highly conserved, almost identical active
site sequences.[17] Therefore, specificity
among these NOS isoforms have relied on key differences in residues
and their conformational flexibility along the substrate access channel
that connects the active site to the hydrophobic pocket (vide supra)
lying at the farthest end of this channel. Previous designs of nNOS
inhibitors have shown that contacts between the inhibitor and residues
of this pocket could be in part responsible for imparting isoform
selectivity.[18,28] In murineiNOS, for example,
a polar Asn115 replaces Leu337 of rat NOS in this pocket, which would
strongly disfavor hydrophobic interactions. In addition, superimposition
of the nNOS–9 and iNOS crystal structures reveals
subtle differences in spatial orientations of residues along the substrate
access channel. We reasoned that conformational restrictions and an
increase of steric bulk in 9, by introduction of cyclopropane
rings along the methylene linker, might impose steric clashes and
thereby lower binding affinity to iNOS. Furthermore, installing a
cyclopropane ring as in 11 increases the metabolic stability
of a more labile benzylic methylene group in 9, while
the electron-withdrawing nature of the ring can reduce the basicity
of the secondary amine as in 12, thereby increasing the
probability of blood–brain barrier permeation. Although both 11 and 12 displayed a slightly lower binding
affinity for nNOS, they displayed a weaker inhibitory effect on iNOS
than parent compound 9. In fact, 12 demonstrated
an improved 73-fold selectivity over iNOS, more than the previous
lead, and the highest n/e selectivity (363-fold) obtained so far in
this series of scaffolds.
To gain more concrete evidence, we
determined the crystal structures of both 11 and 12 in pan class="Gene">nNOS (parts A and B of Figure 6, respectively) and eNOS (Supporting Information,
Figures S4 and S5, respectively). Because of the rigidity of
the linker’s cyclopropyl ring, the fluorophenyl ring of both
inhibitors in nNOS retreats from the deeper position seen for 9 (Figure 5A) but is oriented similarly
to 10 (Supporting Information, Figure
S2). Again, less extensive van der Waals contacts for 11 and 12 relative to those by 9 are the reason for the slightly weaker potency of these two inhibitors
(Table 1). Interestingly, although racemic
samples of 11 and 12 were used for the crystal
preparations, the electron density indicated that only one enantiomer
preferably binds in each case: (R,R)-11 in nNOS–11 and (R,R)-12 in nNOS–12. Similarly, the same enantiomer also dominates the binding of 11 or 12 to eNOS (Supporting
Information, Figures S4 and S5, respectively); however, in
both cases, the fluorophenyl ring caps at the edge of the hydrophobic
pocket. Therefore, the less favorable enzyme–inhibitor contacts
are the origin of poorer binding affinity for 11 and 12 toward eNOS. Owing to a different location of the cyclopropane
in the linker, the kink caused by the cyclopropyl ring in 12 pulls the fluorophenyl ring away from the protein more severely
than that observed in 11. This explains the even poorer
affinity of 12 to eNOS, which results in better n/e selectivity
(Table 1).
Figure 6
Active site structures of 11 ((A) PDB 4V3Z) and 12 ((B) PDB 4D2Y) bound to nNOS. There is preferential
binding of one enantiomer: (R,R)-11 and (R,R)-12 for 11 and 12, respectively. Key hydrogen
bonds are shown by dashed lines, and distances are in Å. The
omit Fo–Fc map for the ligand is contoured at 2.5 σ.
Active site structures of 11 ((A) PDB 4V3Z) and 12 ((B) PDB 4D2Y) bound to pan class="Gene">nNOS. There is preferential
binding of one enantiomer: (R,R)-11 and (R,R)-12 for 11 and 12, respectively. Key hydrogen
bonds are shown by dashed lines, and distances are in Å. The
omit Fo–Fc map for the ligand is contoured at 2.5 σ.
The observation that one enantiomer of 12 dominates the binding prompted us to synthesize and test the two
enantiomers of 12 sepapan class="Species">rately to identify the more potent
enantiomer in the pair and determine the effect of chirality on the
binding of the inhibitor to the NOS active site. Table 2 summarizes the binding affinity to NOSs by the two enantiomers
of 12. While the “ring up” (R,R)-12 shows a much tighter binding
to nNOS, comparable to the affinity of 9, this particular
enantiomer also preferably binds to eNOS and iNOS. However, the very
low Ki for nNOS (18 nM) compared to that
for eNOS and iNOS makes (R,R)-12 the best inhibitor in this series with 573- and 119-fold
selectivities against eNOS and iNOS, respectively.
Table 2
Determination of Ki Values of Inhibitors
(R,R)-12 and (S,S)-12a
Ki (μM)
selectivity
compd
rnNOS
eNOS
iNOS
hnNOS
n/e
n/i
r/hnNOS
(R,R)-12
0.018
10.32
2.14
0.137
573
119
7.6
(S,S)-12
0.150
40.0
3.0
0.873
267
20
5.8
See Table 1 and Experimental Section for details. Ki values were determined using the Cheng–Prusoff
equation directly from IC50 values. IC50 values
are the average of at least two replicates with nine data points;
all experimental standard error values are less than 10%, and all
correlation coefficients are >0.95. Selectivity values are the
ratios of respective Ki values.
See Table 1 and Experimental Section for details. Ki pan class="Chemical">values were determined using the Cheng–Pruspan class="Chemical">off
equation directly from IC50 values. IC50 values
are the average of at least two replicates with nine data points;
all experimental standard error values are less than 10%, and all
correlation coefficients are >0.95. Selectivity values are the
ratios of respective Ki values.
These results are also in agreement
with the structural studies. The pan class="Gene">nNOS structure with (R,R)-12 bound confirms that this enantiomer
is indeed the one that dominates the binding to nNOS when racemic 12 was used for crystal preparation (Figure 6B). It is surprising that the structure of (S,S)-12 enantiomer bound to nNOS (Figure S6 in Supporting Information) also shows
a binding mode that is not that much different from that of (R,R)-12; both the positions
of the secondary amine and the fluorine of the aromatic ring more
or less overlap between the two enantiomers, even though different
chiralities at the cyclopropane lead to differences in the linker
conformation and phenyl ring orientation (Figure
S6 in Supporting Information). Therefore, the differences in
linker conformation and position of the cyclopropane ring may result
in the 8-fold variation in the nNOS affinities between these two enantiomers
(Table 2).
Alongside determining the
potencies of these inhibitors in the three lower animal isoforms of
NOS, we assayed these compounds with the pan class="Species">human nNOS enzyme to see
if the potency and selectivity ratios are also reflected similarly
in the human isoform. Prior work on isosteric arginine mimetics developed
from our lab, such as the highly potent and selective inhibitor 1, showed a humannNOS potency of 0.070 μM, with a 5-fold
selectivity difference between the human and rat isoform. This is
one of the most potent humannNOS inhibitors developed in our lab
to date. When compounds 8–12 were
assayed against humannNOS, we were pleased to see that inhibitors 9–12 displayed good potency in humannNOS,
and a similar 4–10-fold selectivity difference between the
human and rat isoform of nNOS, as seen with previously reported inhibitors
from our lab. The humannNOS exhibits an exact sequence identity with
the rat isoform, except for the hydrophobic pocket, where a hydrophilic
His342 replaces the Leu337 present in ratnNOS. Thus, the pocket in
humannNOS is smaller and more hydrophilic on one side. Therefore,
the modifications that we made in our chemical structures to improve
nNOS potency, although generally well tolerated in humannNOS, can
be modified to improve their humannNOS potency and lower the rat/human
selectivity. For this target, we designed and tested compounds 13–14, with smaller and polar aromatic
ends that may interact effectively with His342 residue in the pocket
in humannNOS. Indeed, we found that 13 is a potent inhibitor
of humannNOS (0.125 μM) and ratnNOS (0.054 μM), where
the selectivity between the two has dropped down to 2.3-fold (Table 1). The structure of the humannNOS–13 complex (Figure 7A) shows, as expected,
that the nitrogen of the pyridine ring indeed makes a hydrogen bond
with the side chain of His342. Although the bond distances vary from
2.8 to 3.2 Å in the four independent copies of 13, all are well ordered in structure. In contrast, the polar pyridine
ring of 13 does not behave as well in the structure of
ratnNOS–13 (Figure 7B),
where the ring shows the sign of disordering with weak electron density.
The more hydrophobic pocket in ratnNOS prefers a phenyl ring over
the pyridine ring for interactions.
Figure 7
Active site structures of 13 bound to human nNOS ((A) PDB 4V3U) or rat nNOS ((B) PDB 4D30). Key hydrogen bonds
are shown by dashed lines and distances are in Å. The omit Fo – Fc map
for the ligand is contoured at 2.5 σ.
Active site structures of 13 bound to pan class="Species">human nNOS ((A) PDB 4V3U) or ratnNOS ((B) PDB 4D30). Key hydrogen bonds
are shown by dashed lines and distances are in Å. The omit Fo – Fc map
for the ligand is contoured at 2.5 σ.
Compound 10 (with a pan class="Chemical">3-chlorophenyl tail) also
displayed potent humannNOS inhibition similar to 13.
With a 3-chloro substitution being bigger than a 3-fluoro, it is difficult
to explain why 10 is a more potent humannNOS inhibitor
than 9. With the known behavior of 10 in
ratnNOS (Figure S2 in Supporting Information), the bulkier chlorophenyl ring in humannNOS is likely pulled out
a bit from the depth of the hydrophobic pocket compared to the position
of a fluorophenyl ring in 9. This movement may allow
the 3-chlorophenyl ring to fit better in the pocket, which might contribute
to the slightly better affinity of 10 than 9 to humannNOS. On the other hand, it is easier to explain the preference
for more polar aromatic heads being tolerated better in humannNOS
with 14, where a polar 3-cyanophenyl with a shorter 4-atom
linker between the arene and the pyrimidine head, demonstrated a 138
nM binding affinity for humannNOS, while a 183 nM binding affinity
for ratnNOS. Thus, this is the first case of a potent humannNOS
inhibitor from our lab, where the selectivity is reversed in preference
toward its human isoform potency over the rat isoform. With a shorter
linker, the cyanophenyl ring of 14 locates right outside
the hydrophobic pocket in ratnNOS (Figure S7
in Supporting Information). The cyano group is in nonbonded
contact with the Leu337 side chain. However, in humannNOS, the larger
side chain of His342 is at the right distance for a hydrogen bond,
as shown in Figure S8 in Supporting Information, which makes 14 a better inhibitor for humannNOS than
ratnNOS. Therefore, these inhibitors clearly demonstrate that we
can utilize the same pocket in the substrate access channel for both
potency and selectivity determining factors in designing the NOS inhibitors.
Finally, with compounds 15–20,
we investigated variations in the pan class="Chemical">2-imidazolylpyrimidine part of the
scaffold that may result in improving potency or minimizing n>n class="Chemical">off-target
affinity. To this end, preliminary docking studies indicated that
a 3,5-disubstituted pyridine could replace the 2,4-disubstituted pyrimidine
in 8 and engage in a salt bridge formation with the Glu592
in the active site of nNOS, the same residue that forms crucial hydrogen
bonds with the guanidine group of l-arginine or other arginine
mimetics.[38] However, when 15 was assayed against nNOS, it showed a poor 5.5 μM inhibition
against nNOS. Therefore, further variations on the imidazole ring
fragment were made based on 9, keeping the pyrimidine
ring intact. We were interested from the literature examples of using
of heme-nitrogenous ligands as inhibitors of aromatase enzymes for
the treatment of breast cancer, and inhibitors of lanosterol demethylase
enzymes as antifungal drugs.[39] Among the
triazoles and methyl-substituted imidazoles incorporated as 2-pyrimidyl
substituents in compounds 16–20,
both the 1,2,4- and 1,2,3-triazole-substituted scaffolds were poor
inhibitors of nNOS, presumably because of a major depletion of the
heme-Fe binding affinity by the electron-deficient triazoles. Among
the substituted imidazoles, only (5-methyl)-2-imidazolylpyrimidine
(20) retained very high potency against nNOS, and along
with high selectivity against eNOS, it demonstrated a remarkably high
400-fold selectivity against iNOS as well, in sharp contrast to all
the imidazole-substituted inhibitors mentioned previously. This presumably
arises from the residues lining the active site over the heme-porphyrin,
which makes a smaller and less flexible pocket in iNOS compared to
nNOS.[28] This pocket is surrounded by Pro565,
Val567, and Phe584 from two β-strands in ratnNOS (Figure 8A), but by Pro344, Val346, and Phe363 in murineiNOS. While residues facing the heme are conserved, the residue that
stabilizes the second β-strand is Ser585 in nNOS and Asn364
in iNOS. Asn364 is involved in more hydrogen bonds than Ser585, which
should help rigidify this part of the structure; however, the Asn364
polypeptide chain in iNOS also shrinks the active site pocket to accommodate
its larger side chain compared to Ser585 in nNOS. The crystal structure
of 20 bound to nNOS (Figure 8A)
shows that the methyl group sticks in to this pocket just described,
and the imidazole ring is bent away from the pyrimidine ring plane
at a larger angle than usual because of steric hindrance between the
pyrimidine ring and the methyl group. Therefore, the methyl group
might create some clashes in the active site pocket of iNOS, thereby
making it a very poor iNOS inhibitor. On the other hand, the 257-fold
selectivity of 20 against eNOS stems from different structural
features at the other end of inhibitor. Similar to parent compound 9, the fluorophenyl ring of 20 in eNOS (Figure 8B) cannot insert into the hydrophobic pocket on
the far end of the substrate access channel. Thus, lack of good van
der Waals contacts in the pocket in eNOS compared to those encountered
by 20 in nNOS leads to poorer affinity for eNOS.
Figure 8
Active site
structures of 20 bound to rat nNOS ((A) PDB 4D32) and bovine eNOS
((B) PDB 4D3A). Key hydrogen bonds are shown by dashed lines and distances are
in Å. The omit Fo – Fc map for ligand is contoured at 2.5 σ.
Active site
structures of 20 bound to pan class="Species">rat nNOS ((A) PDB 4D32) and bovineeNOS
((B) PDB 4D3A). Key hydrogen bonds are shown by dashed lines and distances are
in Å. The omit Fo – Fc map for ligand is contoured at 2.5 σ.
Encouraged by the high pan class="Gene">nNOS affinity
and good isoform selectivities of several compounds in this series,
as one of the lead nNOS inhibitors, 9 was subjected to
a Caco-2 monolayer permeability assay (Table 3). The Caco-2 cell line is a human intestinal epithelial line used
to approximate the compound’s permeabililty in the gastrointestinal
tract as well as the blood–brain barrier.[25] Compound 9 showed good cellular permeability,
thus reflecting a better potential for oral bioavailability. In addition,
the efflux ratio of 9 was below 2, which indicates that
it is likely not a favorable substrate for P-gp or any active transport
system that might shuttle it out of the cells.
Table 3
Caco-2 Permeability Assay for 9a
apparent
permeability (Papp, 10–6 cm s–1)b
recovery
compd
mean A→B
mean B→A
efflux ratioc
A→B (%)
B→A (%)
9
17.8
32.4
1.8
73
83
ranitidined
0.18
1.6
8.9
warfarine
53.4
13.2
0.2
talinololf
0.11
11.0
100.0
All assays
were performed over 2 h at 10 μM concentration.
Papp: apparent
permeability rate coefficient.
Efflux ratio: Papp (B→A)/Papp (A→B).
Low permeability control.
High permeability control.
High efflux control.
All assays
were performed over 2 h at 10 μM concentpan class="Species">ration.
pan class="Chemical">Papp: apparent
permeability pan class="Species">rate coefficient.
Efflux n class="Species">ratio: Papp (B→A)/Papp (A→B).
Low permeability control.High permeability control.High efflux control.We also sought to determine the pan class="Chemical">off-target effects
of 9 against a panel of 50 CNS receptors, which included
G-protein coupled receptors such as the serotonin, adrenergic, dopamine,
and histamine receptors, as well as muscarinic and σ receptors.[40] Compound 9 showed significant inhibition
(>75%) at a high 10 μM concentration in a primary binding
assay only at the following targets: humanserotonin 5-HT1A and 5-HT2A
(83 and 88%), adrenergic α-2C (95%), σ-1 and -2 receptors
(94 and 88%), and dopamine D3 receptor (87%). Therefore, a consecutive
secondary binding assay on these targets were evaluated, which revealed
a binding affinity (Ki) of ∼0.2
μM only for α-2C and σ receptors, while the rest
were 0.5 μM or higher. So, even a 0.2 μM off-target affinity
holds 10-fold selectivity (relative to the nNOS Ki), which nonetheless is about 7% of the total receptors
assayed. Thus, overall 9 displays a good safety profile,
which is very promising for further development of this class of scaffolds.
Finally, the presence of a pan class="Chemical">heme-coordinating group increases the
likelihood of these compound’s ability to inhibit cytochromes
P450 (CYPs), the xenobiotic-metabolizing enzymes in humans. Therefore, 9 was evaluated against five major human liver microsomal
P450s and, at 10 μM concentration, was found to decrease the
activity of CYP2C19, CYP2D6, and CYP3A4 by more than 70%. Because
CYP3A4 is the major liver and intestinal P450 that metabolizes the
majority of drugs,[41] we determined IC50 values for 9, (R,R)-12, 13, and 20 using an
in vitro CYP3A4 activity assay and 7-benzyloxy-4-trifluoromethylcoumarin
as a substrate (Table 4). Compound 9 displayed moderate inhibitory potency for CYP3A4 (IC50 of 2.5 μM) and maintained >130-fold selectivity toward
nNOS. When hydrophobicity in the molecules is increased through the
cyclopropane ring insertion or methyl substitution of the imidazole
in (R,R)-12 and 20, respectively, the CYP3A4 inhibitory potency is substantially
increased (IC50 <1 μM). This is understandable
given the bigger and more hydrophobic active site of CYP3A4 that preferably
binds large, nonpolar molecules. The more polar and potent humannNOS
inhibitor 13 acts as a weak CYP3A4 inactivator (IC50 of 70 μM), which suggests that modulation of hydrophobicity
and bulkiness of compounds containing the heme-coordinating group
can attenuate CYP inhibition, even with the presence of heme-ligating
groups like imidazole.
Table 4
Comparison of nNOS
and CYP3A4 Binding Affinity of Selected Compoundsa
compd
nNOS Ki (μM)
CYP3A4 IC50 (μM)
selectivity nNOS/CYP3A4
9
0.019
2.5 ± 0.5
132
(R,R)-12
0.018
0.3 ± 0.02
17
13
0.054
70 ± 5
1296
20
0.060
0.9 ± 0.08
15
See Table 1 and Experimental Section for details. Ki values of nNOS are determined
using the Cheng–Prusoff equation directly from IC50 values. IC50 values are the average of at least two replicates
with nine data points; all experimental standard error values are
less than 10%, and all correlation coefficients are >0.95.
See Table 1 and Experimental Section for details. Ki pan class="Chemical">values of nNOS are determined
using the Cheng–Prusoff equation directly from IC50 values. IC50 values are the average of at least two replicates
with nine data points; all experimental standard error values are
less than 10%, and all correlation coefficients are >0.95.
Conclusions
We have designed and
synthesized a new series of pan class="Chemical">2,4-disubstituted pyrimidine scaffolds
by exploiting the much less-explored heme coordinating ability of
inhibitors in the active site of NOS. This design was based on the
rationale that 2-imidazolylpyrimidines might bind and inhibit nNOS
with groups that are less polar and less basic than the 2-aminopyridines
and therefore more bioavailable. We also speculated that by modulating
other parts of the inhibitor molecule, we would be able to incorporate
selectivity into the inhibitor against other NOS isozymes and heme-containing
CYP enzymes. Indeed, we were able to obtain 2,4-disusbtituted pyrimidines
that are highly potent inhibitors of nNOS, as shown by its low nanomolar
binding affinity to both rat and humannNOS, and >200-fold and
>100-fold selectivity over eNOS and iNOS, respectively. Crystal
structures of the compounds bound to both rat and humannNOS indicate
heme-Fe coordination by the 2-imidazolyl fragment, and the noncoordinating
aryl rings are stabilized in a hydrophobic pocket at the far end of
the substrate access channel. Access to this pocket is important for
gaining inhibitory potency in both rat and humannNOS isoforms (via
extensive van der Waals contacts and polar interactions) and isoform
selectivity (owing to the sequence diversity in the pocket among NOS
isoforms). Very promising compounds that came from this study were 9, (R,R)-12, and 13, which showed good Caco-2 permeability, minimal
off-target binding efficacy, and good selectivity. Even with the presence
of imidazole rings, CYP3A4 inhibition could be attenuated by modifications
to other parts of the molecule. Therefore, these results exhibit high
potential among these compounds to be orally bioavailable and brain
permeable. Hence, further developments of these compounds are currently
in progress.
Experimental Section
General
Methods
Anhydrous solvents were purified by passage through
a solvent column composed of activated pan class="Chemical">alumina and a supported n>n class="Chemical">copper
redox catalyst. All remaining solvents and reagents were purchased
from commercial vendors and used without further purification. Moisture
or oxygen-sensitive reactions were performed under an atmosphere of
dry N2 or argon. Analytical thin-layer chromatography was
performed on Silicycle precoated silica gel 60 Å F254 plates.
An Agilent 971-FP flash purification system with various SiliaSep
(Silicycle, 40–63 μm, 60 Å) prepacked silica gel
cartridges was used for flash column chromatography. 1H
NMR and 13C NMR spectra were recorded at 500 and 126 MHz
respectively on a Bruker Avance-III instrument. Low-resolution ESIMS
was performed on a Thermo Finnigan LCQ or Bruker Amazon SL mass spectrometer
consisting of an electrospray ionization (ESI) source. High-resolution
mass spectral data were obtained at the Integrated Molecular Structure
Education and Research Facility (Northwestern University) on an Agilent
6210A TOF mass spectrometer in positive ion mode using electrospray
ionization, with an Agilent G1312A HPLC pump and an Agilent G1367B
autoinjector. The purity of the compounds was evaluated on a Beckman
Gold reverse phase analytical HPLC system using a Phenonemex Gemini
C-18 (4.6 mm × 250 mm, 5 μm) reverse phase column with
UV absorbance and evaporative light scattering detection. Purities
of all final compounds that were subjected to enzymatic assays were
found to be >95%. Preparative HPLC was performed at the Northwestern
University Center for Molecular Innovation and Drug Discovery ChemCore
lab, using an Agilent 1200 series HPLC and Agilent 6120 quadrupole
mass spectrometer (API-MS mode), and a Phenomenex Gemini-NX 5 μm
C18 column (150 mm × 21.2 mm). Chiral HPLC to determine enantiopurity
of precursors of racemic and enantiomers of 11 was performed
on an Agilent 1260 Series HPLC using a 0.46 cm × 25 cm Chiralpak
AD-H column, with hexanes and 2-propanol (isocratic 10% 2-propanol
in hexanes) as the mobile phases, and the flow rate of 0.5 mL/min
with UV detection. 4-Chloro-2-methanesulfonyl pyrimidine,[27b]24, 25, 26,[30]40,[32]65,[42] and 66(43) were synthesized following
previously reported procedures. Syntheses of the remaining primary
amines are detailed in Supporting Information.
Compound 26 (0.146 g, 0.516
mmol) was diluted with pan class="Chemical">MeCN (3 mL), followed by addition of 4-chloro-2-methanesulfonylpyrimidine (0.1 g, 0.516 mmol) and K2CO3 (0.143
g, 1.033 mmol) and heated at 40 °C for 19 h. Imidazole (0.176
g, 2.583 mmol) was added to the resulting mixture, and heating continued
at 65 °C for another 30 h. The mixture was cooled, diluted in
CH2Cl2 (30 mL), washed with H2O (2
× 20 mL), and the organic layer dried over anhydrous sodium sulfate.
The solution was concentrated, and the resulting crude oil was purified
by flash chromatography (EtOAc/MeOH) to yield the Boc-protected precursor
of 5 as a clear sticky oil (0.132 g, 60%). This intermediate
Boc-protected carbamate was dissolved in CH2Cl2 (3 mL), cooled to 0 °C, and trifluoroacetic acid was added
(1.5 mL) to it. The resulting solution was stirred at room temperature
for 2 h, when it was diluted with more CH2Cl2 (15 mL), and treated slowly with saturated K2CO3 (10 mL). The layers were separated, and the aqueous layer was extracted
with CH2Cl2 (2 × 10 mL). The combined organic
layers were dried with anhydrous sodium sulfate, concentrated, and
chromatographed with EtOAc/MeOH to give an oily residue. MethanolicHCl (∼2 M, 2 mL) was added to the residue, stirred for 10 min,
concentrated to 1 mL, and treated with excess Et2O, when
a white crystalline precipitate formed. The solid was collected by
filtration and dried to obtain 5 as a white crystalline
solid (0.11 g, 83%); mp = 208–210 °C. 1H NMR
(500 MHz; DMSO-d6): δ 10.32 (s,
1 H), 9.49 (s, 2 H), 8.66 (t, J = 6.0 Hz, 1 H), 8.52
(t, J = 1.8 Hz, 1 H), 8.22 (d, J = 6.0 Hz, 1 H), 7.89 (t, J = 1.7 Hz, 1 H), 7.39
(td, J = 7.9, 6.3 Hz, 1 H), 7.19–7.07 (m,
3 H), 6.69 (d, J = 6.0 Hz, 1 H), 3.90–3.86
(m, 2 H), 3.23 (dq, J = 11.4, 6.3 Hz, 2 H), 3.18–3.14
(m, 2 H), 3.06 (dd, J = 9.7, 6.5 Hz, 2 H), 1.30 (dd, J = 15.1, 6.8 Hz, 2 H). 13C NMR (126 MHz; DMSO-d6): δ (164.16, 162.23, d, J = 243.9 Hz, 1 C), 164.1, 155.8, 153.1, (141.09, 141.00, d, J = 11.3 Hz, 1 C), 136.2, (131.52, 131.46, d, J = 7.6 Hz, 1 C), (125.79, 125.78, d, J = 1.3 Hz,
1 C), 122.1, 119.7, (116.48, 116.31, d, J = 21.4
Hz, 1 C), (114.69, 114.48, J = 26.4 Hz, 1 C), 107.7,
48.4, 46.7, 37.2, 32.0. HRMS (ESI): calcd for C17H20FN6 [M + H]+, 327.1728; found, 327.1731.
General pan class="Chemical">method A for trifluoroacetic
acid mediated Boc-group deprotection: Compound 37 (0.257
g, 0.647 mmol) was diluted in CH2Cl2 (5.5 mL)
and cooled to 0 °C. Trifluoroacetic acid (2.75 mL) was added
to the resulting solution and stirred at room temperature for 3 h.
The reaction mixture was diluted with more CH2Cl2 (25 mL) and treated with saturated K2CO3 (15
mL). The layers were separated, and the aqueous layer was extracted
again with CH2Cl2 (2 × 15 mL). The organics
together were dried over sodium sulfate and concentrated. The resulting
oily residue was purified by flash column chromatography (CH2Cl2/MeOH) to give the free base of 6 as a
yellow oil. The oil was treated with methanolic HCl (∼2 M,
2 mL) for 10 min, when a white precipitate started forming. This suspension
was concentrated to ∼0.5 mL and treated with excess Et2O and sonicated. The white solid was filtered, washed twice
with Et2O, and dried to give a white amorphous solid of 6 as a dihydrochloride salt (0.223 g, 93%); mp = 220–222
°C. 1H NMR (500 MHz, DMSO-d6): δ 10.07 (s, 2 H), 10.02 (s, 1 H) 9.05 (d, J = 5.1 Hz, 1 H), 8.57 (s, 1 H), 7.78 (d, J = 5.0
Hz, 2 H), 7.44–7.37 (m, 1 H), 7.21–7.09 (m, 3 H), 4.61
(t, J = 5.2 Hz, 2 H), 4.01 (br s, 1 H), 3.39–3.28
(m, 2 H), 3.24–3.15 (m, 2 H). 13C NMR (126 MHz;
DMSO-d6): δ 164.8, (164.17, 162.23,
d, J = 244.4 Hz, 1 C), 161.2, 153.4, (141.05, 140.99, J = 7.6 Hz, 1 C), 136.9, (131.51, 131.44, d, J = 8.8 Hz, 1 C), (125.80, 125.78, d, J = 2.5 Hz,
1 C), 125.1, 120.7, 119.3, (116.48, 116.31, d, J =
21.4 Hz, 1 C), (114.68, 114.45, d, J = 29 Hz, 1 C),
49.7, 48.2, 31.9. HRMS (ESI): calcd for C16H17FN5 [M + H]+, 298.1463; found, 298.1462.
Compounds 19 and 3-(3-fluorophenyl)-N-2-[2-(5-pan class="Chemical">methyl-1H-imidazol-1-yl)pyrimidin-4-yl]ethylpropan-1-amine
dihydrochloride (20) were obtained in an inseparable
15:1 ratio (by 1H NMR) from the reaction of 15:1 mixture
of compounds 72 and 73 (0.668 g, 1.52 mmol)
following general method A, as a white amorphous solid of the corresponding
dihydrochloride salts (0.62 g, 99% total). This mixture was separated
by preparative HPLC on an Agilent 1200 series instrument using a Phenomenex
Gemini-NX 5 μm C18 column (150 mm × 21.2 mm), using a gradient
of 1–15% MeCN in H2O with 0.1% formic acid, to isolate 19 and 20 separately in >95% purity. Following
this, 19 and 20 were converted to their
corresponding dihydrochloride salts by similar precipitation with
methanolic HCl and excess Et2O.
To a suspension of pan class="Chemical">NaH (0.107 g, 2.686
mmol) in THF (4 mL) cooled to 0 °C, a solution of 21 (0.5 g, 2.686 mmol) in THF (2.5 mL) was added dropwise. The reaction
was stirred at room temperature for 1 h followed by the addition of
2-(3-fluorophenyl)ethyl bromide (0.66 g, 3.242 mmol) in THF (2 mL)
and a pinch of tetrabutylammonium iodide (∼0.05 g, 0.13 mmol).
The resulting solution was heated at 60 °C for 48 h, when it
was cooled to room temperature and diluted with ethyl acetate (20
mL) and water (20 mL). The layers were separated, and the aqueous
layer was washed with EtOAc (2 × 10 mL). The combined organic
layers were washed with brine (20 mL), dried over sodium sulfate,
and concentrated. The residue was purified by flash column chromatography
(hexanes/EtOAc), and the resulting oil (0.464 g, 1.504 mmol, 56%)
was diluted with CH2Cl2 (12 mL). This solution
was cooled to 0 °C and treated with trifluoroacetic acid (6 mL).
The reaction was stirred for 3 h at room temperature, after which
it was concentrated. The residue was diluted with EtOAc (20 mL) and
treated with saturated K2CO3 (15 mL). The organic
layer was extracted, and the aqueous layer was re-extracted with more
EtOAc (2 × 10 mL). The combined organic layers were dried and
concentrated to yield a colorless oil (0.266 g, 85%), clean by NMR. 1H NMR (500 MHz, CDCl3): δ 7.22 (q, J = 7.2 Hz, 1H), 6.96 (d, J = 7.7 Hz, 1H),
6.92–6.85 (m, 2H), 3.98 (s, 1H), 2.96 (t, J = 5.0 Hz, 4H), 2.78 (dd, J = 11.2, 8.3 Hz, 2H),
2.58 (dd, J = 11.0, 8.5 Hz, 2H), 2.53 (s, 4H). 13C NMR (126 MHz, CDCl3): δ (163.75, 161.80,
d, J = 245.7 Hz, 1 C), (142.71, 142.65, d, J = 7.56 Hz, 1 C), (129.75, 129.69, d, J = 7.56 Hz, 1 C), (124.29, 124.27, d, J = 2.52 Hz,
1 C), (115.55, 115.38, d, J = 21.42 Hz, 1 C), (112.98,
112.81, d, J = 21.42 Hz, 1 C), 60.4, 53.5, 45.5,
33.0.
To a solution of 41 (0.356 g, 1.222 mmol) in pan class="Chemical">THF (10
mL), a solution of di-tert-butyl dicarbonate (0.293
g, 1.344 mmol) in THF (7 mL) was added. The resulting solution was
stirred at room temperature for 3 h and, thereafter, it was diluted
with EtOAc/H2O (30 mL, 1:1). The layers were separated,
and the aqueous layer extracted with EtOAc (2 × 10 mL). Combined
organic layers were dried, concentrated, and purified by column chromatography
(hexanes/EtOAc). The resulting oil (0.39 g, 0.996 mmol, 82%) was dissolved
in CH2Cl2 (2 mL) and added to a solution of m-chloroperbenzoic acid (0.636 g, 3.685 mmol) in CH2Cl2 (3 mL) at 0 °C. The resulting solution
was stirred at room temperature for 3 h. At that point, the reaction
was filtered, and the white residue was washed with cold CH2Cl2 (∼5 mL). The collected filtrate was washed
with 10% aqueous K2CO3 (5 mL), the organic layer
was dried, concentrated, and purified by flash column chromatography
(hexanes/EtOAc) to give a clear viscous oil (0.337 g, 80%). 1H NMR (500 MHz, CDCl3): δ 8.76 (d, J = 4.1 Hz, 1 H), (7.43 (s), 7.31 (s), 7:3, 1 H), 7.23 (q, J = 6.5 Hz, 1 H), 6.97–6.84 (m, 3 H), (3.61–3.53
(m), 3.49–3.44 (m), 7:3, 2 H), 3.37 (t, J =
7.1 Hz, 2 H), 3.34 (s, 3 H), (3.18–3.10 (m), 3.08–2.98
(m), 7:3, 2 H), ((2.86–2.80 (m), 2.78–2.72 (m), 3:7,
2 H), 1.38 (s, 9 H). 13C NMR (126 MHz, CDCl3): δ (170.93, 170.82, 1 C), (165.98, 165.76, 1 C), (163.79,
161.84, d, J = 245.7 Hz, 1 C), (158.01, 157.88, 1
C), (155.22, 154.87, 1 C), 141.38, (130.00, 129.94, d, J = 7.56 Hz), 124.54, 123.56, (115.75, 115.59, d, J = 20.16 Hz, 1 C), (113.37, 113.21, d, J = 20.16
Hz, 1 C), 80.05, (49.37, 49.18, 1 C), (46.80, 46.14, 1 C), 39.09,
(36.91, 36.29, 1 C), (34.90, 34.19, 1 C), 28.25. MS (ESI) m/z [M + Na]+: calcd, 446.15;
found, 446.06.
Compounds 49–52 were synthesized from compounds 42–45 following the same procedures used to synthesize 48.
Compound 46 (0.443
g, 1.536 mmol) was dissolved in pan class="Chemical">THF (15 mL), and a solution of di-tert-butyl dicarbonate (0.369 g, 1.689 mmol) in THF (6 mL)
was added. The resulting solution was stirred at room temperature
for 3 h, and thereafter, it was diluted with EtOAc/H2O
(40 mL, 1:1). The layers were separated, and the aqueous layer was
extracted with EtOAc (2 × 15 mL). Combined organic layers were
dried, concentrated, and purified by column chromatography (hexanes/EtOAc).
The resulting oil (0.567 g, 1.459 mmol, 95%) was dissolved in THF
(12 mL). Water (12 mL) and oxone (1.345 g, 2.188 mmol) were sequentially
added, and the reaction was stirred at room temperature for 4 h. At
that point, the reaction was diluted with EtOAc/H2O (30
mL, 1:1) and the layers separated. The aqueous layer was basified
to pH 10 with 6 N NaOH, saturated by addition of solid K2CO3, and extracted with EtOAc (3 × 15 mL). Combined
organic fractions were dried over sodium sulfate, and concentrated.
The resulting light-yellow oil (0.527 g, 86%) was clean by NMR and
was used in the next step without further purification. 1H NMR (500 MHz, CDCl3): δ 8.74 (d, J = 3.9 Hz, 1 H), 8.42 (s, 2 H), 7.48 (d, J = 6.9
Hz, 1 H), (7.42 (s), 7.34 (s), 3:2, 1 H), 7.20 (dd, J = 7.3, 4.9 Hz, 1 H), 3.66–3.54 (m, 2 H), 3.31 (s, 3 H), 3.24–3.03
(m, 4 H), 2.56 (t, J = 7.8 Hz, 2 H), 1.80 (p, J = 7 Hz, 2 H), 1.36 (s, 9 H). 13C NMR (126 MHz,
CDCl3): δ 170.87, 165.81, 157.85, 155.27, 149.53,
147.30, 136.70, 135.74, 123.47, 123.37, 79.93, (47.30, 46.94, 1 C),
46.01, 39.05, (36.87, 36.33, 1 C), 30.11, (29.84, 29.58, 1 C), 28.25.
MS (ESI) m/z [M + Na]+: calcd, 443.16; found, 443.03.
Chiral Resolution
of 2-(3-Fluorobenzyl)cyclopropan-1-amine (62)
pan class="Chemical">2-(3-Fluorobenzyl)cyclopropan-1-amine (62; 0.851 g,
5.15 mmol) was diluted in CH2Cl2 (20 mL) and
cooled to −20 °C. (S)-(+)-α-Methoxylphenylacetic
acid (1.0 g, 6.18 mmol) was added to the resulting solution, followed
by the addition of dicyclohexylcarbodiimide (1.275 g, 6.18 mmol).
The reaction mixture was gradually warmed to room temperature and
stirred overnight. The resulting suspension was filtered, and the
white precipitate was washed with cold CH2Cl2 (15 mL). The combined filtrate was concentrated and chromatographed
in silica gel using a gradient of hexanes/EtOAc to obtain the two
diastereomers (R,R,S)-63a and (S,S,S)-63b.
To a solution of
pan class="Chemical">(R,R,S)-63a (0.775 g, 2.472 mmol) in ethanol (12 mL), 12 N HCl (12 mL) was added,
and the reaction was heated to reflux for 12 h. The resulting solution
was cooled to room temperature, concentrated, and treated with Et2O/6 N HCl (30 mL, 1:1). The layers were separated, and the
aqueous layer was washed with Et2O (10 mL), basified to
pH 10–12 with 6 N NaOH, and extracted with CH2Cl2 (4 × 15 mL). The combined CH2Cl2 extracts were dried over sodium sulfate and concentrated to obtain
(R,R)-62a (0.347 g,
85%), which was pure by NMR and used directly in the next step without
further purification.
pan class="Chemical">(S,S)-62b was obtained
from (S,S,S)-63b (0.756 g, 2.42 mmol) in 88% yield (0.352 g) following
the same procedure used to synthesize pan class="Chemical">(R,R)-62a.
Compounds pan class="Chemical">(R,R)-12 and (S,S)-12 were synthesized from 62a and 62b, respectively, following the same procedures
used to synthesize racemic 12 from 2-methylthio-4-vinylpyrimidine
(40) and 62. Enantiopurities of (R,R)-59 (enantiomeric ratio:
97:3) and (S,S)-59 (enantiomeric
ratio: 96:4) were determined by chiral HPLC using a Chiralpak AD-H
column using an isocratic gradient of 10% 2-propanol/hexanes at flow
rate 0.5 mL/min.
Compound 66 (0.55 g, 2.78 mmol) was dissolved in pan class="Chemical">CH2Cl2 (50 mL) and cooled to −78 °C. DIBAL in THF (25
wt % in toluene; 2.8 mL, 2.37 g, 4.17 mmol) was added dropwise, and
the reaction continued at −78 °C for 1 h. At this point,
sodium sulfate decahydrate was added to the reaction mixture, which
was gradually warmed to room temperature over 1 h. The resulting suspension
was filtered through Celite and washed with CH2Cl2 (20 mL), and the combined organic layers were dried and concentrated.
To this crude 2-(5-bromopyridin-3-yl)acetaldehyde, CHCl3 (50 mL) was added, followed by the addition of anhydrous MgSO4 (∼3.0 g). 3-Fluorophenethylamine (31;
0.36 mL, 0.387 g, 2.78 mmol) and acetic acid (60 μL) were sequentially
added and stirred at room temperature for 1 h. The reaction mixture
was cooled to 0 °C, and sodium triacetoxyborohydride (0.71 g,
3.336 mmol) was added in one portion. The mixture was allowed to warm
to room temperature and stirred overnight. The reaction was filtered,
and the filtrate was washed with saturated aqueous NaHCO3 (20 mL), and the aqueous layer was extracted with CHCl3 (2 × 10 mL). The organic phase was washed with brine (20 mL),
dried over sodium sulfate, concentrated, and purified by flash column
chromatography with CH2Cl2/MeOH to obtain 67 (0.37 g, 41%) as a pale-yellow oil. 1H NMR (500
MHz, CDCl3): δ 8.51 (d, J = 2.1
Hz, 1 H), 8.34 (d, J = 1.6 Hz, 1 H), 7.64 (t, J = 1.8 Hz, 1 H), 7.25–7.20 (m, 1 H), 6.93 (d, J = 7.5 Hz, 1 H), 6.91–6.84 (m, 2 H), 2.88 (td, J = 7.1, 4.0 Hz, 4 H), 2.76 (dt, J = 14.5,
7.1 Hz, 4 H), 1.31 (s, 1 H). 13C NMR (126 MHz, CDCl3): δ (163.81, 161.86, d, J = 245.7
Hz, 1 C), 148.7, 148.2, (142.27, 142.21, d, J = 7.56
Hz, 1 C), 138.6, 137.2, (129.89, 129.83, d, J = 7.56
Hz, 1 C), (124.26, 124.24, d, J = 2.52 Hz, 1 C),
120.6, (115.47, 115.31, d, J = 20.16 Hz, 1 C), (113.20,
113.04, d, J = 20.16 Hz, 1 C), 50.5, 50.2, 36.0,
33.1. MS (ESI) m/z [1:1; (M + H])+]: calcd, 323.0, 325.0; found, 322.77, 324.76.
Compound 67 (0.37
g, 1.145 mmol) was dissolved in pan class="Chemical">THF (10 mL), and a solution of di-tert-butyl dicarbonate (0.275 g, 1.26 mmol) in THF (6 mL)
was added. The resulting solution was stirred overnight, and then
it was diluted with EtOAc/H2O (30 mL, 1:1). The layers
were separated and the aqueous layer extracted with EtOAc (2 ×
10 mL). The combined organic layers were dried, concentrated, and
purified by column chromatography (hexanes/EtOAc). The resulting oil
(0.445 g, 1.051 mmol, 92%) was added to a vial along with CuBr (7.5
mg, 0.052 mmol) and Cs2CO3 (0.685 g, 2.102 mmol).
The reaction vial was evacuated and backfilled with argon; anhydrous
DMSO (previously purged for 5 min with argon), 8-acetyl-5,6,7,8-tetrahydroquinoline
(17 μL, 0.018 g, 0.105 mmol), and imidazole (0.107 g, 1.576
mmol) were sequentially added, the reaction sealed, and heated at
100 °C for 12 h. It was cooled to room temperature, treated with
EtOAc/H2O (30 mL, 1:1), and the layers separated. The aqueous
layer was extracted with EtOAc (2 × 10 mL), and the combined
EtOAc extracts were washed with brine (20 mL), dried, and concentrated.
The residue was purified by flash column chromatography using CH2Cl2/MeOH to obtain 68 (0.289 g, 67%)
as a cream-colored oil. 1H NMR (500 MHz, CDCl3): δ 8.59 (s, 1 H), 8.43 (s, 1 H), 8.01 (s, 1 H), 7.59 (s,
1 H), 7.42 (s, 2 H), 7.24 (q, J = 6.5 Hz, 1 H), 7.02–6.79
(m, 3 H), 3.42–3.33 (m, 4 H), 2.96–2.69 (m, 4 H), 1.38
(s, 9 H). 13C NMR (126 MHz, CDCl3): δ
(163.81, 161.86, d, J = 245.7 Hz, 1 C), 155.17, (154.88,
154.85, 1 C), 149.15, 141.51, (140.86, 140.71, 1 C), 135.86, 133.89,
(130.02, 129.97, d, J = 6.3 Hz, 1 C), 129.13, (124.49,
124.47, d, J = 2.52 Hz, 1 C), (115.74, 115.57, d, J = 21.42 Hz, 1 C), (113.44, 113.28, d, J = 20.16 Hz, 1 C), 79.98, (49.33, 49.04, 1 C), 48.19, (34.95, 34.27,
1 C), (32.01, 31.88, 1 C), 28.27. MS (ESI) m/z [M + H]+: calcd, 411.21; found, 411.33.
Compounds 69–73 were synthesized
from 49 following the same procedure used to synthesize 56.
All isozymes of NOS, pan class="Species">rat and humannNOS,
murine macrophage iNOS, and bovineeNOS, were recombinant enzymes,
overexpressed in Escherichia coli and
purified following previously reported procedures.[44] The enzyme inhibition was determined by measuring the production
of nitric oxide from l-arginine using the hemoglobin capture
assay in the presence of different concentrations of inhibitors.[36] The assay was performed at 37 °C in 100
mM HEPES buffer with 10% glycerol (pH 7.4) in the presence of 10 μM l-arginine and tetrahydrobiopterin, 100 μM NADPH, 0.83
mM CaCl2, ∼320 units/mL of calmodulin, and 3 μM
human oxyhemoglobin. For iNOS, CaCl2, and calmodulin were
substituted by HEPES buffer. All assays were performed in 96-well
plates using a Synergy H1 hybrid multimode microplate reader with
automated dispensing of NOS enzyme and hemoglobin after 30 s (maximum
delay), which initiated the assay. The initial rates of NO production
were determined by monitoring the formation of methemoglobin (NO mediated
conversion of oxyhemoglobin to methemoglobin) by monitoring the absorbance
at 401 nm. The entire kinetic readout was performed for 5 min with
measurements at every 22 s interval. Each compound was assayed at
least in duplicate, and nine concentrations (100 μM to 10 nM
for nNOS; 500 μM to 50 nM for iNOS and eNOS) were used to construct
dose–response curves with slopes from initial
readouts. IC50 values were calculated by nonlinear regression
using GraphPad Prism (standard error values reported are from the
Log IC50 calculations), and apparent Ki values were determined using the Cheng–Prusoff
equation [Ki = IC50/(1 + [S]/Km)] with the following Km values for l-arginine: 1.3 (ratnNOS), 1.6 (humannNOS), 8.2 (murine macrophage iNOS), and 1.7 μM (bovineeNOS).
The selectivity of an inhibitor was defined as the ratio of their
respective Ki values.
Inhibitor Complex
Crystal Preparation
The prepapan class="Species">rations of ratnNOS, bovineeNOS, and humannNOSheme domains used for crystallographic studies
were carried out by the procedures described previously.[45] The heme domain samples of nNOS (at 9 mg/mL
containing 20 mM histidine), bovineeNOS (10 mg/mL containing 2 mM
imidazole), and humannNOS (13 mg/mL) were used for the sitting drop
vapor diffusion crystallization setup under conditions reported.[45] A new orthorhombic crystal form of humannNOS
was obtained when the pH was raised to 6.2 from 5.0 and the protein
concentration dropped to 10 mg/mL. The well solution compositions
were only slightly shifted from what were reported: 9–11% PEG3350,
40 mM citric acid, 60 mM Bis-Tris-propane, 10% glycerol, and 5 mM
TCEP. From the sitting drop setup, plate-like crystals grew to full
size at 4 °C in 3–4 days without seeding. Fresh crystals
were first passed stepwise through cryoprotectant solutions and then
soaked with 10 mM inhibitor for 4–6 h at 4 °C before being
flash cooled with liquid nitrogen.
X-ray Diffraction Data
Collection, Data Processing, and Structural Refinement
The
cryogenic (100 K) X-ray diffraction data were collected remotely at
the Stanford Synchrotron Radiation Light Source (SSRL) or Advanced
Light Source (ALS) through the data collection control software pan class="Gene">Blu-Ice[46] and a crystal-mounting robot. When a Q315r CCD
detector was used, 90–100° of data were typically collected
with 0.5° per frame. If a Pilatus pixel array detector was used,
140–160° of fine-sliced data were collected with 0.2°
per frame. Raw CCD data frames were indexed, integrated, and scaled
using HKL2000[47] or MOSFLM,[48] but the pixel array data were processed with XDS[49] and scaled with Scala (Aimless).[50] For ratnNOS or bovineeNOS structures, the
binding of inhibitors was detected by the initial difference Fourier
maps calculated with REFMAC.[51] For humannNOS structures, molecular replacement was performed with PHASER[52] to provide the initial electron density. One
homodimer in the known humannNOS structure (4D1N) was used as the
search model. The new humannNOS structure closely resembles that
of ratnNOS to have only one homodimer in the asymmetric unit. The
inhibitor molecules were then modeled in COOT[53] and refined using REFMAC or PHENIX.[54] Water molecules were added in REFMAC or PHENIX and checked manually
in COOT. The TLS[55] protocol was implemented
in the final stage of refinements with each subunit as one TLS group.
The omit Fo – Fc density maps were calculated by removing inhibitor coordinates
from the input PDB file before running one more round of TLS refinement
in REFMAC or in PHENIX (simulated annealling protocol with a 2000
K initial temperature). The resulting map coefficients DELFWT and
SIGDELWT were used to generate maps that are displayed in figures.
The refined structures were validated with the validation service
in the RCSB Protein Data Bank. The crystallographic data collection
and structure refinement statistics are summarized in Table S1 of the Supporting Information, with
the PDB accession codes included.
Caco-2 Permeability Assay
Caco-2 permeability assays were performed by pan class="Gene">Cyprotex (Watertown,
MA) using the Caco-2 epithelial monolayers. Caco-2 cells, grown in
tissue culture flasks, were trypsinized, suspended in media, and plated
in 96-well plates to be grown for 3 weeks; the proper formation of
monolayer was determined by fluorescent measurement of transport of
an impermeable dye, Lucifer yellow. All assays were performed with
compounds at a concentration of 10 μM for 2 h. For apical to
basolateral (A→B) permeability, compounds were added on the
apical side (A) and permeation determined on the basolateral side
(B), where the receiving buffer was removed for analysis by LC/MS/MS
using an Agilent 6410 mass spectrometer (ESI, MRM mode) coupled with
an Agilent 1200 HPLC. The buffers used were 100 μM Lucifer yellow
in transport buffer (1.98 g/L glucose in 10 mM HEPES, 1× Hank’s
Balanced Salt Solution, pH 6.5) (apical side) and transport buffer,
pH 7.4 (basolateral side). The apparent permeability (Papp) is expressed using the following equation: Papp = (dQ/dt)/C0A, where dQ/dt is the rate of permeation, C0 is initial concentration, and A is the monolayer area. For bidirectional permeability, the efflux
ratio was defined as Papp(B→A)/Papp(A→B); high efflux ratio (>3) indicates
that a compound is a potential substrate for P-gp or other active
transport systems.
CNS Receptors Screening Assay
Screening
of compound 9 for pan class="Chemical">off-target receptor activity was performed
at the NIMH Psychoactive Drug Screening Program at UNC Chapel Hill.
In the primary radioligand binding assays, the compound was tested
at a single concentration (10 μM) in quadruplicate in 96-well
plates. For receptors with which a compound displayed more than 50%
inhibition at 10 μM concentration, the compound was subjected
to secondary radioligand binding assays to determine equilibrium binding
affinity at specific targets. In the secondary binding assays, compound 9 was tested at 11 concentrations (10–0.1 μM)
and in triplicate. Both primary and secondary radioligand binding
assays were carried out in a final volume of 125 μL per well
in the appropriate binding buffer, and the radioligand concentration
was at a concentration close to the Kd. In a typical assay, 25 μL of radioligand was added to each
well of a 96-well plate, followed by addition of 25 μL binding
buffer with or without compound. The reaction started upon addition
of 75 μL of fresh membrane protein (typically 25–50 μg
per well), and the reaction was incubated in the dark at room temperature
for 90 min. The reaction was stopped by vacuum filtration onto cold
0.3% polyethylenimine (PEI)-soaked 96-well filter mats using a 96-well
Filtermate harvester, followed by three washes with cold wash buffers.
Scintillation cocktail was then melted onto the microwave-dried filters
on a hot plate, and radioactivity was counted in a Microbeta counter.
For primary binding assay analysis, nonspecific binding in the presence
of 10 μM of an appropriate reference compound was set as 100%
inhibition; total binding in the absence of test compound or reference
compound was set as 0% inhibition. The radioactivity in the presence
of test compound was calculated with the equation: % inhibition =
(sample cpm – nonspecific cpm)/(total cpm – nonspecific
cpm) × 100, where the radioactivity was measured in counts per
minute (cpm/well). For secondary binding results, counts (cpm/well)
were pooled and fitted to a three-parameter logistic function for
competition binding in Prism to determine IC50 values: Y = bottom + (top – bottom)/1 + 1050, where Y is the
total binding in the presence of a corresponding concentration of
compound (X, in this case, concentration of 9), and top and bottom are the total and nonspecific binding
in the absence and presence of 10 μM reference compound. Ki is determined from the corresponding IC50 value using the Cheng–Prusoff equation.
CYP Inhibition
Assay
The pan class="Gene">CYP inhibition assay of 9 was performed
against the five major liver microsomal CYP enzymes: CYP1A2, CY2C9,
CYP2C19, CYP2D6, and CYP3A4 (by Sai Life Sciences). In the assay,
a 25 μL aliquot of microsomes diluted in Kphos buffer (0.4 mg/mL)
was added to individual wells of the reaction plate. Fluvoxamine,
sulfaphenazole, quinidine, ticlopidine, and ketoconazole (positive
control inhibitors for CYP1A2, CYP2C9, CYP2D6, CYP2C19, and CYP3A4,
respectively), diluted in buffer (25 μL), were added separately
to the respective wells. Compound 9, diluted in DMSO
to a concentration of 10 μM, was directly spiked into microsomal
mix (2×), and 50 μL was aliquoted into individual wells.
An aliquot of 25 μL of phenacetin, diclofenac, bufuralol, S-mephenytoin, and midazolam (4×) for CYP1A2, CYP2C9,
CYP2D6, CYP2C19, and CYP3A4, respectively, was added separately to
wells and incubated for 5 min at 37 °C. The reactions were initiated
using 25 μL of NADPH (4×) and further incubated for 5 min
for CYP3A4 (Midazolam), 10 min for CYP1A2, CYP2C9, and CYP2D6, and
20 min for CYP2C19. All reactions were terminated using 100 μL
of ice-cold acetonitrile containing internal standard (imipramine
and glipizide at 1 μM). The plates were centrifuged at 4000
rpm for 15 min, 100 μL aliquots were subjected to LC-M/MS on
a Shimadzu API 4000 system (MRM mode), and the metabolites were detected.
The respective peak area ratios (PA) of metabolites and internal standard
was used to determine % inhibition, where % activity = (PA ratios
in the presence of compound/PA ratios in DMSO control) × 100,
and % inhibition = 100 – % activity.
CYP3A4 Inhibition Assay
The inhibitory potency of 9, pan class="Chemical">(R,R)-12, 13, and 20 on the 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) debenzylase
activity of humanCYP3A4 was evaluated fluorimetrically in a reconstituted
system with cytochrome P450 reductase (CPR). The reaction was carried
out at room temperature in 100 mM phosphate buffer, pH 7.4, containing
catalase and superoxide dismutase (2 U/mL each). A mixture of 1 μM
CYP3A4 and 1 μM CPR was preincubated for 1 h at room temperature
and diluted by 20-fold before measurements. BFC (50 μM) and
various concentrations of inhibitors were added 2 min prior to initiation
of the reaction with 100 μM NADPH. Formation of 7-hydroxy-4-trifluoromethylcoumarin
(λex = 430 nm; λem = 500 nm) was
followed in a Hitachi F100 fluorimeter. IC50 values were
derived from the [% activity] vs [inhibitor] plots.
Authors: H Ischiropoulos; L Zhu; J Chen; M Tsai; J C Martin; C D Smith; J S Beckman Journal: Arch Biochem Biophys Date: 1992-11-01 Impact factor: 4.013
Authors: Haitao Ji; Benjamin Z Stanton; Jotaro Igarashi; Huiying Li; Pavel Martásek; Linda J Roman; Thomas L Poulos; Richard B Silverman Journal: J Am Chem Soc Date: 2008-03-06 Impact factor: 15.419
Authors: Anthony V Pensa; Maris A Cinelli; Huiying Li; Georges Chreifi; Paramita Mukherjee; Linda J Roman; Pavel Martásek; Thomas L Poulos; Richard B Silverman Journal: J Med Chem Date: 2017-08-04 Impact factor: 7.446
Authors: Maris A Cinelli; Huiying Li; Anthony V Pensa; Soosung Kang; Linda J Roman; Pavel Martásek; Thomas L Poulos; Richard B Silverman Journal: J Med Chem Date: 2015-10-27 Impact factor: 7.446
Authors: Heng-Yen Wang; Yajuan Qin; Huiying Li; Linda J Roman; Pavel Martásek; Thomas L Poulos; Richard B Silverman Journal: J Med Chem Date: 2016-04-20 Impact factor: 7.446
Authors: Soosung Kang; Huiying Li; Wei Tang; Pavel Martásek; Linda J Roman; Thomas L Poulos; Richard B Silverman Journal: J Med Chem Date: 2015-07-10 Impact factor: 7.446
Authors: Ha T Do; Heng-Yen Wang; Huiying Li; Georges Chreifi; Thomas L Poulos; Richard B Silverman Journal: J Med Chem Date: 2017-11-01 Impact factor: 7.446
Authors: Dhananjayan Vasu; Huiying Li; Christine D Hardy; Thomas L Poulos; Richard B Silverman Journal: Bioorg Med Chem Date: 2022-06-11 Impact factor: 3.461
Authors: Maris A Cinelli; Cory T Reidl; Huiying Li; Georges Chreifi; Thomas L Poulos; Richard B Silverman Journal: J Med Chem Date: 2020-04-17 Impact factor: 7.446
Figure 1
Figure 2
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Scheme 6
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8