Combination of chiral linkers with thiophenecarboximidamide heads to improve the selectivity of inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2014.07.079

To develop potent and selective nNOS inhibitors, a new series of double-headed molecules with chiral linkers that derive from natural amino acid derivatives have been designed and synthesized. The new structures integrate a thiophenecarboximidamide head with two types of chiral linkers, presenting easy synthesis and good inhibitory properties. Inhibitor (S)-9b exhibits a potency of 14.7 nM against nNOS and is 1134 and 322-fold more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 9b and propionate A on the heme and tetrahydrobiopterin (H₄B) in nNOS, but not eNOS, contributes to its high selectivity. This work demonstrates the advantage of integrating known structures into structure optimization, and it should be possible to more readily develop compounds that incorporate bioavailability with these advanced features. Moreover, this integrative strategy is a general approach in new drug discovery.

Full text of DOI:10.1016/j.bmcl.2014.07.079

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4504–4510
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Combination of chiral linkers with thiophenecarboximidamide
heads to improve the selectivity of inhibitors of neuronal nitric oxide
synthase
Qing Jing a,b,c,d, Huiying Li e,f,g, Linda J. Roman h, Pavel Martásek h,i, Thomas L. Poulos e,f,g,
,
⇑
Richard B. Silverman a,b,c,d,
⇑
a Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
b Department of Molecular Biosciences, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
c Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
d Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
e Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
f Department of Pharmaceutical Chemistry, University of California, Irvine, CA 92697, USA
g Department of Chemistry, University of California, Irvine, CA 92697, USA
h Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78384-7760, USA
i Department of Pediatrics and Center for Applied Genomics, 1st School of Medicine, Charles University, Prague, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
To develop potent and selective nNOS inhibitors, a new series of double-headed molecules with chiral
linkers that derive from natural amino acid derivatives have been designed and synthesized. The new
structures integrate a thiophenecarboximidamide head with two types of chiral linkers, presenting easy
synthesis and good inhibitory properties. Inhibitor (S)-9b exhibits a potency of 14.7 nM against nNOS and
is 1134 and 322-fold more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show
that the additional binding between the aminomethyl moiety of 9b and propionate A on the heme and
tetrahydrobiopterin (H4B) in nNOS, but not eNOS, contributes to its high selectivity. This work demon-
strates the advantage of integrating known structures into structure optimization, and it should be pos-
sible to more readily develop compounds that incorporate bioavailability with these advanced features.
Moreover, this integrative strategy is a general approach in new drug discovery.
Received 9 July 2014
Revised 25 July 2014
Accepted 29 July 2014
Available online 12 August 2014
Keywords:
Nitric oxide synthase
Selective inhibition
Thiophenecarboximidamide
Heme
Ó 2014 Elsevier Ltd. All rights reserved.
Tetrahydrobiopterin
Protein crystallography
Nitric oxide (NO), an inorganic free radical, has diverse roles in
both normal and pathological processes, including the regulation
of blood pressure, neurotransmission, and macrophage defense
systems.1 NO is synthesized from the enzymatic catalysis of
these conditions must be selective for nNOS since inhibition of
eNOS and iNOS could result in unwanted effects, such as hyperten-
sion, atherogenesis, and interference with normal immune system
functions.9 Therefore, the development of selective nNOS inhibi-
tors is of critical interest and derives both from a therapeutic per-
spective and from their use as specific pharmacological tools.10
Early NOS inhibitors were structural analogs of the natural sub-
L-arginine to L-citrulline by three isoforms of nitric oxide synthase
(NOS): neuronal NOS (nNOS), endothelial NOS (eNOS), and induc-
ible NOS (iNOS). Although NO plays numerous physiological roles
beneficial to the host organism, the overproduction of NO has been
implicated in numerous disease states.2 In particular, excess NO
produced by nNOS has been shown to play an important role in
the development of several disorders including stroke,3 pain
(migraine, chronic tension-type headache, visceral, and neuro-
pathic),4 and neurodegenerative disorders5 (Parkinson’s,6 Alzhei-
mer’s,7 Huntington’s diseases8). However, any inhibitors to treat
strate
L
-arginine, which lack isoform selectivity.11,12 Some later
L
-arginine mimetic NOS inhibitors are potent and selective.13,14
Although many recent reports disclose the diversity of NOS inhib-
itors,15 very few provided integrative profiles because of either low
selectivity or poor bioavailability. Therefore, investigations into the
synthesis and chemistry of novel isoform-selective NOS inhibitors
and improvement of structure optimization and bioavailability still
remain as ongoing challenges.
Our research group has developed two lead compounds, 216 and
317 (Fig. 1), which feature easy synthesis and potent and selective
nNOS inhibitory properties. To develop an integrative strategy and
⇑
Corresponding authors. Tel.: +1 949 824 7020 (T.L.P.), +1 847 491 5653 (R.B.S.).
E-mail addresses: poulos@uci.edu (T.L. Poulos), Agman@chem.northwestern.edu
(R.B. Silverman).
http://dx.doi.org/10.1016/j.bmcl.2014.07.079
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Q. Jing et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4504–4510
4505
H2N
(R)
(Z)
H
N
(Z)
O
O
HN
S
(S)
O
O
N
N
N
NH2
H2N
N
Cl
(R)
H2N
NH2
NH
1 (ARR17477)
N
H
2
3
Figure 1. Structures of lead compounds 1, 2 and 3.
diversify the scope of NOS inhibitors, an early nNOS-selective
inhibitor with good bioavailability properties (1)18 was combined
with our two natural chiral linkers in 2 and 3 to explore new
opportunities for structure optimization. These new compounds
are expected to incorporate the features of easy accessibility, excel-
lent inhibitory properties, and improved bioavailability.
The synthesis of 9 began with a commercially available amino
compound, which was protected as the dimethylpyrrole (4) fol-
lowed by a one-step ether synthesis, connecting two 3-nitrobenzyl
heads simultaneously (compound 5, Scheme 1). The nitro groups in
5 were then reduced to amines with hydrazine in the presence of
Raney-Ni to afford 6. Coupling with 7 in ethanol19 led to double
substituted 8. After removal of the protecting group under micro-
wave conditions, 9 was successfully obtained.
the fluorobenzyl group to the prolinyl linker via an ether synthesis
was achieved prior to the Mitsunobu reaction to install the nitro-
phenyl group. The remaining synthetic steps are the same as those
employed for 21 with similar yields.
All of the inhibitors were assayed against the three different
isoforms of NOS, including rat nNOS, bovine eNOS, and murine
macrophage iNOS using L-arginine as a substrate (Table 1). The
(S)-enantiomer of 9 (9b) was found to be four times more potent
(14.7 nM) than the (R)-isomer with 5-fold enhancement of eNOS
selectivity (1134-fold) and 2.5-fold enhancement of iNOS selectiv-
ity (322-fold). Of the four isomers of 14 with a pyrrolidine linker,
compound 14a is the most potent (13.2 nM; although the other
three range from 21.4 to 28.1 nM), but 14d is the most selective
(658-fold for n/e and 98-fold for n/i). Again, the (S)-stereochemistry
at the amino group is favored. Two analogs of 25, with two differ-
ent bis-substituted phenyl linkages, show similar binding behav-
iors. Replacing one of the phenyl thiophenecarboximidamide
moieties in 25a with a fluorobenzene does not lead to any
improvement, as shown by 21 and 32.
Four isomers of 14 followed a similar synthetic route as 9
except for starting compounds 10a–d, which were previously
reported.17 Compounds 14 were obtained in good yields after
removal of the Boc protecting group in 13 under acidic conditions
(Scheme 2).
The synthesis of 21 is described in Scheme 3. Starting from 15, a
Mitsunobu reaction was used to give 16, followed by reduction of
the methyl carboxylic ester to alcohol 17 with LiBH4. Coupling of
the fluorobenzyl group to the prolinyl linker moiety afforded 18.
After reduction of the nitro group to the amine, coupling with 7,
and removal of the Boc protecting group, 21 was obtained in good
yield.
The synthetic route to 25a,b uses intermediate 17 from the syn-
thesis of 21 (Scheme 4). A second Mitsunobu reaction connected the
second nitrophenyl head (either 3- or 4-substituted) to the prolinyl
linker moiety, providing 22. Upon reduction of the nitro groups, the
resulting 23 was allowed to react with amidothiol ether 7 to gener-
ate the double-headed thiophene scaffold (24). Compound 25 was
obtained after removal of the Boc protecting group.
The only change between the two double headed inhibitors, 9a
and 9b, is the chirality at the position of the aminomethyl moiety,
but their potencies and selectivities show significant differences.
To reveal the structural basis for these observations we have deter-
mined the crystal structures of both nNOS and eNOS in complex
with 9a and 9b. As shown in Figure 2 for the nNOS structures, both
9a and 9b use the carboximidamide moiety to H-bond with the
Glu592 side chain, and the rest of the heme distal side is occupied
by the thiophene and phenyl rings. However, the orientations of 9a
and 9b are flipped 180° to one another. For 9a, it is the phenyl thio-
phenecarboximidamide moiety that is two atoms away from the
chiral center, whereas for 9b it is the one that is three atoms away
from the chiral center that binds to the NOS active site near
Glu592. The phenyl thiophenecarboximidamide moiety is impor-
tant in NOS active site recognition and thus is the fundamental
source of the inhibitory potency. Because of different binding
Compound 32 has a similar structure as 21 (Scheme 5), but with
transposition of the two head groups. Starting from 26, coupling of
N
N
H2N
N
b
a
c
O
O
O
O
HO
OH
HO
OH
O2N
NO2
H2N
NH2
(R)-4a
(S)-4b
(R)-5a
(R)-6a
(S)-6b
(S)-5b
H2N
N
d
e
O
O
O
O
S
S
HN
NH
HN
HN
NH
NH
HN
NH
HI
S
NH
S
S
S
7
(R)-8a
(S)-8b
(R)-9a
(S)-9b
Scheme 1. Synthesis of 9a,b. Reagents and conditions: (a) p-TsOH, toluene, 2,5-hexanedione, reflux, 77–79%; (b) 3-nitrobenzyl bromide, NaH, NaI, DMF, 0 °C, 75–78%;
(c) Raney-Ni, hydrazine hydrate, CH3OH, 98%; (d) compound 7, EtOH, 75–83%; (e) HCl in EtOH, microwave, 120 °C, 61–63%.

4506
Q. Jing et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4504–4510
BocN
BocN
BocN
a
b
O
NH2
O
NO2
HO
O
O
H2N
O2N
OH
(S, R)-10a
(R, R)-10b
(R, S)-10c
(S, S)-10d
(S, R)-11a
(S, R)-12a
(R, R)-11b
(R, S)-11c
(S, S)-11d
(R, R)-12b
(R, S)-12c
(S, S)-12d
NH
NH
HN
BocN
H
N
H
N
d
O
O
c
S
S
S
S
O
N
O
N
H
H
NH
NH
(S, R)-13a
(R, R)-13b
(R, S)-13c
(S, S)-13d
(S, R)-14a
(R, R)-14b
(R, S)-14c
(S, S)-14d
Scheme 2. Synthesis of 14a–d. Reagents and conditions: (a) 3-nitrobenzyl bromide, NaH, NaI, DMF, 0 °C, 57–85%; (b) Raney-Ni, hydrazine hydrate, CH3OH, 97–99%;
(c) compound 7, EtOH, 66–87%; (d) 1.25 M HCl in CH3OH, 89–96%.
BocN
BocN
BocN
BocN
a
b
c
O
O
HO
O
O
NO2
OH
O
NO2
F
O
NO2
O
O
(S, S)-15
(S, R)-16
(S, R)-17
(S, R)-18
d
NH
HN
NH
BocN
BocN
f
e
O
S
O
O
F
S
O
N
H
F
F
O
O
N
H
NH2
(S, R)-21
(S, R)-19
(S, R)-20
Scheme 3. Synthesis of 21. Reagents and conditions: (a) PPh3, DIAD, 3-nitrophenol, THF, 83%; (b) LiBH4, THF, 98%; (c) 3-fluorobenzylbromide, NaH, NaI, DMF, 0 °C, 91%;
(d) Raney-Ni, hydrazine hydrate, CH3OH, 97%; (e) compound 7, EtOH, 77%; (f) 1.25 M HCl in CH3OH, 85%.
BocN
BocN
BocN
a
b
O
O
HO
O
NO2
O
NH2
O
NO2
O2N
H2N
(S, R)-17
(S, R)-22a: 3-NO2
(S, R)-22b: 4-NO2
(S, R)-23a: 3-NO2
(S, R)-23b: 4-NO2
NH
NH
BocN
HN
c
d
O
O
S
S
H
N
H
N
O
N
O
N
H
H
S
S
NH
NH
(S, R)-25a: 3-NO2
(S, R)-25b: 4-NO2
(S, R)-24a: 3-NO2
(S, R)-24b: 4-NO2
Scheme 4. Synthesis of 25. Reagents and conditions: (a) PPh3, DIAD, 3-nitrophenol, THF, 72–82%; (b) Raney-Ni, hydrazine hydrate, CH3OH, 98%; (c) 7, EtOH, 74–77%;
(d) 1.25 M HCl in CH3OH, 86–94%.
orientations, the aminomethyl group at the chiral center exhibits
two distinct interactions with the protein: the one in 9b is sand-
wiched in between the H4B and heme propionate A, making
H-bonds with both and replacing a water molecule normally
bound at this position (Fig. 2B); however, the one in 9a points away
from the water site and thus the water remains (Fig. 2A). These
extra H-bonds with 9b are likely the main source of the 4-fold better
potency of 9b compared to 9a. The second phenyl thiophenecarb-
oximidamide moiety makes non-bonded contacts with Leu337 and
Trp306 of chain B, a little tighter in the case of 9b than in 9a, but
this portion of the inhibitors is partially disordered in both struc-
tures, and hence it is difficult to make a comparison with high
certainty.
site. The aminomethyl groups in both inhibitors, regardless the chi-
rality, make a H-bond with the water molecule bound in between
the H4B and heme propionate A rather than replacing the water. Its
capabilities of replacing the water and making direct H-bonds with
both the H4B and heme seems to be the underlying reasons for 9b,
compared to 9a, being both a more potent nNOS inhibitor and
more selective for nNOS over eNOS. Moreover, how the second
phenyl thiophenecarboximidamide moiety that extends out of
the active site in both of the inhibitor-bound structures depends
on the protein environment in each isoform. In nNOS, the second
thiophene ring points toward a pocket defined by Trp306 (chain
B), Ser602, and Arg603 (Fig. 2), while in eNOS, the second phenyl
thiophenecarboximidamide is rather disordered but is pointing
toward the opening of the active site access channel near
Leu107, with some differences in exact positions between 9a and
9b (Fig. 3). The aforementioned pocket in nNOS, where the second
thiophene ring points, is surrounded in eNOS by Trp76 (chain B),
His373, and Arg374 (Fig. 3). The bulkier His373 (compared to
The structures of eNOS with 9a and 9b bound, shown in Figure 3,
also provide an explanation for the isoform selectivity of 9b. Differ-
ent from the case of the nNOS structures, the same phenyl thio-
phenecarboximidamide moiety, with 3-atoms from the chiral
center, in both 9a and 9b anchors the inhibitor to the eNOS active

Q. Jing et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4504–4510
4507
BocN
b
BocN
BocN
BocN
c
a
HO
F
O2N
O
O
O
O
F
F
F
O
O
O
OH
O
O
(R, S)-27
(R, S)-26
(R, S)-28
(R, S)-29
d
BocN
BocN
HN
H
N
H
N
f
e
H2N
O
O
F
F
S
S
O
O
O
NH
NH
(R, S)-31
(R, S)-32
(R, S)-30
Scheme 5. Synthesis of 32. Reagents and conditions: (a) 3-fluorobenzylbromide, NaH, NaI, DMF, 0 °C, 68%; (b) LiBH4, THF, 98%; (c) PPh3, DIAD, 3-nitrophenol, THF, 76%;
(d) Raney-Ni, hydrazine hydrate, CH3OH, 98%; (e) compound 7, EtOH, 71%; (f) 1.25 M HCl in CH3OH, 93%.
Table 1
Kia values of inhibitors for rat nNOS, bovine eNOS and murine iNOS
Compounds
Ki [
lM]
Selectivityb
nNOS
eNOS
iNOS
e/n
210
1134
190
130
151
658
86
i/n
9a
9b
0.0590
0.0147
0.0132
0.0281
0.0214
0.0221
0.0684
0.0282
0.0270
0.101
12.44
16.68
2.47
3.63
3.17
1.45
5.89
3.34
1.15
1.86
8.11
4.73
1.11
1.99
1.23
2.11
6.17
2.42
4.40
9.28
137
322
85
71
58
96
90
86
163
92
14a
14b
14c
14d
21
25a
25b
32
119
142
18
a
The IC50 values were measured for three different isoforms of NOS including rat nNOS, bovine eNOS, and murine macrophage iNOS using
standard deviation of 10%. The corresponding Ki values were calculated from the IC50 values using the equation Ki = IC50/(1 + [S]/Km) with known Km values (nNOS, 1.3
iNOS, 8.3 M; eNOS, 1.7 M).
L
-arginine as a substrate with a
lM;
l
l
b
The Ki values are used to calculate the selectivity.
Ser602 in nNOS) side chain makes this pocket too shallow to allow
the thiophene to fit optimally. One additional, potentially impor-
tant, difference between the two isoforms is another amino acid
variant that is flanking this pocket, Asp309 in nNOS versus Gly79
in eNOS. The exposed N atom of the carboximidamide group of
9b is less than 6.0 Å from Asp309 in the nNOS dimer (Fig. 2B). This
could provide more favorable electrostatic interactions in nNOS
than in eNOS when the second thiophenecarboximidamide moiety
fits in the pocket. Optimization of these interactions in nNOS
requires 9b to adopt a compact conformation, which favors close
interactions between the aminomethyl group and heme propio-
nate. In sharp contrast, the lack of these additional interactions
with the carboximidamide group in eNOS enables the tail end of
9b to adopt an extended conformation and clearly binds less
tightly, given the poor quality of the electron density in this region.
This extended tail conformation of 9b in eNOS could also be the
reason why the aminomethyl group at the chiral center is pulled
away from the heme propionate and H4B, leaving a structural
water untouched (Fig. 3B).
Using chiral proline analogs (10a–d) as the linker, we synthe-
sized inhibitors 14a–d. These four compounds show very similar
binding behaviors, with 14a being the most potent (13.2 nM) but
14d the most selective (658-fold for nNOS over eNOS). We deter-
mined the structures of only 14d bound to both nNOS and eNOS
(Fig. 4). Again, 14d relies on the phenyl thiophenecarboximida-
mide moiety, the one with a 3-atom linker to the center pyrrolidine
ring, to anchor itself to the NOS active site. The longer linker length
pushes the pyrrolidine ring out so that in nNOS the ring
nitrogen sits in between the two heme propionates at about
4.0 Å without making H-bonds (Fig. 4A). The second phenyl

4508
Q. Jing et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4504–4510
Figure 2. View of the active site of nNOS complexed with inhibitor 9a (A) (PDB: 4UPM) and 9b (B) (PDB: 4UPN), showing also the omit Fo–Fc electron density for the bound
inhibitor at the 2.5 contour level. Note that a different phenyl thiophenecarboximidamide moiety in each case is used to anchor the inhibitor to the active site. Key
r
hydrogen bonds are depicted with dashed lines. Structural figures were prepared with PyMOL (www.pymol.org).
Figure 3. View of the active site of eNOS complexed with inhibitor 9a (A) (PDB: 4UPQ) and 9b (B) (PDB: 4UPR), showing also the omit Fo–Fc electron density for the bound
inhibitor at the 2.5
r contour level. Note that the same phenyl thiophenecarboximidamide moiety in both cases is used for active site recognition. Key hydrogen bonds are
depicted with dashed lines.
Figure 4. View of the active site of nNOS (A) (PDB: 4UPO) and eNOS (B) (PDB: 4UPS) complexed with inhibitor 14d, showing also the omit Fo–Fc electron density for the
bound inhibitor at the 2.5
r contour level. Key hydrogen bonds are depicted with dashed lines.

Q. Jing et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4504–4510
4509
Figure 5. View of the active site of nNOS (A) (PDB: 4UPP) and eNOS (B) (PDB: 4UPT) coordinated with inhibitor 25b, showing also the omit Fo–Fc electron density for the
bound inhibitor at the 2.5 contour level. Key hydrogen bonds are depicted with dashed lines.
r
thiophenecarboximidamide moiety of 14d in both nNOS and eNOS
is poorly defined with weak density. The disordering is more
severe in eNOS than in nNOS. However, the residual density is clear
enough to indicate that the thiophene ring is heading out of the
active site access channel and near Pro708 in nNOS (Pro479 in
eNOS). That means that even in nNOS 14d is too lengthy, compared
to 9a–b, to fit into the pocket next to Ser602 and Arg603.
To gain information on how 14a binds to nNOS, we simply
placed a model of 14a in the structure of nNOS-14d according to
the binding mode observed for 14d. Considering the structural
similarity between these two analogs, it is not surprising to see
that the first phenyl thiophenecarboximidamide moiety including
the pyrrolidine ring nitrogen of 14a can mimic the exact position
seen for 14d (Fig. S1). Differences only start from the different chi-
rality at the pyrrolidine ring. Even so, the second thiophene ring
can still end up with the same position seen for 14d. Apparently,
in the cases of 14a–d, the different stereochemistry does not
impact the inhibitor binding mode large enough to significantly
change the potency or isoform selectivity.
The linker length in 14a–d is too long to allow pyrrolidine inter-
actions with the heme propionates, and is flexible enough to com-
pensate for any differences generated at the chiral centers.
Therefore, we furtherdeveloped inhibitors25a,b toincludethesame
chiral pyrrolidine at the center as in 14a but with one less methylene
in the linker than their parent compound, in the hope that a shorter
structure might fit the substrate pocket of NOS better. We also tried
both meta- (25a) and para-substitution (25b) at the phenyl ring to
adjust the inhibitor binding conformation. The structures of 25b
bound to nNOS and eNOS (Fig. 5) show that the para-substituted
phenyl thiophenecarboximidamide moiety with a 2-atom linker to
the pyrrolidine is not only able to anchor the inhibitor to the NOS
active site in both cases but also allows the pyrrolidine nitrogen to
make direct H-bonds (ꢀ3.0 Å) with both heme propionates in nNOS
(Fig. 5A). To our surprise, the additional H-bonds in 25b do not trans-
late to better binding affinity of 25b to nNOS compared to 14a–d
(Table 1). The same H-bonds in eNOS cannot be established
(Fig. 5B), most likely because the second thiophene head is pointing
in a different direction in eNOS from that seen in nNOS. Similar to
what was seen in nNOS-9a the second thiophene of 25b fits into
the pocket defined by Trp306 (chain B), Ser602, and Arg603
(Fig. 5A). In contrast, the second thiophene of 25b in eNOS is more
disordered than that in the nNOS-25b structure, and the residual
density at the lower contour level guides the tail toward the opening
of the active site access channel (Fig. 5B). This tail orientation pulls
the pyrrolidine ring away from the heme in eNOS.
By superimposing the meta-substituted ring of 25a and 25b
(Fig. S2), we found that 25a can still orient the pyrrolidine nitrogen
in the same position seen for that of 25b, thus making H-bonds
with both heme propionates. The second thiophene head can also
fall into the same pocket, although the exact position of the second
meta-substituted phenyl ring is different in the two cases (Fig. S2).
Overall, the model provides a good explanation as to why the two
compounds show similar binding affinity to nNOS (Table 1), even
though the conformation around the phenyl ring is different.
To further investigate the structure-activity relationship of the
inhibitors, we designed and synthesized 21 and 32, which inte-
grate features from the structure of 1, but with the linker from
25a. The transposed position of the thiophenecarboximidamide
and fluorobenzyl heads was aimed to optimize the structure; how-
ever, neither provided improved results compared with 25a. On
the basis of what we learned from the binding of 14d (Fig. 4A)
and 25b (Fig. 5A) to nNOS, we expected that if 21 were bound to
the nNOS active site via its phenyl thiophenecarboximidamide
moiety, the short one-atom ether linker might not be able to bring
the pyrrolidine nitrogen to an optimal position to make good
H-bonds with heme as observed for 25b, which could lead to poorer
binding. As for 32, with its phenyl thiophenecarboximidamide
anchored to the nNOS active site, the 2-atom ether linker should
be able to position the pyrrolidine to interact with the heme propi-
onates. However, the fluorobenzyl group attached to a short linker
might not make significant van der Waals contacts with the pro-
tein to establish a binding better than that with 25a.
In conclusion, we designed and synthesized a new series of dou-
ble-headed inhibitors with two types of chiral linkers derived from
amino acids. By combining a phenyl thiophenecarboximidamide
head with our previously-developed skeleton, inhibitor 9b
emerges as being both a potent nNOS inhibitor, Ki = 14.7 nM and
exhibiting dual selectivities of 1134-fold (e/n) and 322-fold (i/n).
Compared to 1, the redesigned structures contain two ether bonds,
which allows for easy synthesis and structure-ready-to-optimize
features. Crystallography shows that additional binding of the ami-
nomethyl moiety of 9b to both the propionate A on heme and H4B,
replacing a structural water molecule there, explains the high
selectivity. A similar feature, in which a secondary amine is bound
to the same site leading to good potency and isoform selectivity,
was also observed with another series of NOS inhibitors developed
in our lab.20 Without synthesizing a large number of small mole-
cules, we were able to discover new, potent and selective inhibi-
tors, easily accessed, which confirm the efficiency of utilizing an
integrative strategy.

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Q. Jing et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4504–4510
8. Norris, P. J.; Waldvogel, H. J.; Faull, R. L. M.; Love, D. R.; Emson, P. C.
Acknowledgments
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The authors are grateful for financial support from the National
Institutes of Health (GM049725 to R.B.S. and GM057353 to T.L.P.).
We thank Dr. Bettie Sue Siler Masters (NIH grant GM52419, with
whose laboratory P.M. and L.J.R. are affiliated). B.S.S.M. also
acknowledges the Welch Foundation for a Robert A. Welch Distin-
guished Professorship in Chemistry (AQ0012). P.M. is supported by
grants 0021620849 from MSMT of the Czech Republic. We also
thank the beamline staff at SSRL and ALS for their assistance during
the remote X-ray diffraction data collections.
14. Ji, H.; Gomez-Vidal, J. A.; Martásek, P.; Roman, L. J.; Silverman, R. B. J. Med.
Chem. 2006, 49, 6254.
15. (a) Ramnauth, J.; Speed, J.; Maddaford, S. P.; Dove, P.; Annedi, S. C.; Renton, P.;
Rakhit, S.; Andrews, J.; Silverman, S.; Mladenova, G.; Zinghini, S.; Nair, S.;
Catalano, C.; Lee, D. K. H.; Felice, M. D.; Porreca, F. J. Med. Chem. 2011, 54, 5562;
(b) Annedi, S. C.; Ramnauth, J.; Maddaford, S. P.; Renton, P.; Rakhit, S.;
Mladenova, G.; Dove, P.; Silverman, S.; Andrews, J. S.; Felice, M. D.; Porreca, F. J.
Med. Chem. 2012, 55, 943; (c) Liang, G.; Neuenschwander, K.; Chen, X.; Wei, L.;
Munson, R.; Francisco, G.; Scotese, A.; Shutske, G.; Black, M.; Sarhan, S.; Jiang,
J.; Morize, I.; Vaz, R. J. Med. Chem. Commun. 2011, 2, 201; (d) Silverman, R. B.
Acc. Chem. Res. 2009, 42, 439.
16. Jing, Q.; Li, H.; Chreifi, G.; Roman, L. J.; Martásek, P.; Poulos, T. L.; Silverman, R.
B. Bioorg. Med. Chem. Lett. 2013, 23, 5674.
17. Jing, Q.; Li, H.; Roman, L. J.; Martásek, P.; Poulos, T. L.; Silverman, R. B. ACS
MedChemLett 2014, 5, 56.
Supplementary data
Supplementary data (detailed synthetic procedures and full
characterization (1H NMR, 13C NMR) of compounds 9, 14, 21, 25,
and 32; crystal preparation, X-ray diffraction data collection and
structure refinement procedures and statistics) associated with
this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2014.07.079.
18. (a) Zhang, Z. G.; Reif, D.; Macdonald, J.; Tang, W. X.; Kamp, D. K.; Gentile, R. J.;
Shakespeare, W. C.; Murray, R. J.; Chopp, M. J. Cereb. Blood Flow Metab. 1996, 16,
599; (b) Salerno, L.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.; Siracusa, M. A. Curr.
Pharm. Des. 2002, 8, 177.
19. The synthesis of 7 followed the reported method Collins, J. L.; Shearer, B. G.;
Oplinger, J. A.; Lee, S.; Garvey, E. P.; Salter, M.; Duffy, C.; Burnette, T. C.; Furfine,
E. S. J. Med. Chem. 1998, 41, 2858.
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