Neuroprotective Effect of N-Cyclohexylethyl-[A/G]-[D/E]-X-V Peptides on Ischemic Stroke by Blocking nNOS-CAPON Interaction

Peptides on Ischemic Stroke by Blocking nNOS −CAPON Interaction

Research Article

Neuroprotective Eﬀect of N‑Cyclohexylethyl-[A/G]-[D/E]-X‑V

†

Yajuan Qin,* Lingling Feng, Xin Fan, Liping Zheng, Yu Zhang, Lei Chang, and Tingyou Li*

Cite This: ACS Chem. Neurosci. 2021, 12, 244 −255

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sı Supporting Information

ABSTRACT: The protein−protein interaction between neuronal nitric oxide

syntheses (nNOS) and the carboxy-terminal PDZ ligand of nNOS (CAPON) is a

potential target for the treatment of ischemic stroke. Our previous study had

identiﬁed ZLc-002 as a promising lead compound for inhibiting nNOS−CAPON

coupling. To ﬁnd better neuroprotective agents disrupting the ischemia-induced

nNOS−CAPON interaction, a series of N-cyclohexylethyl-[A/G]-[D/E]-X-V

peptides based on the carboxy-terminal tetrapeptide of CAPON was designed,

synthesized, and evaluated in this study. Herein, we reported an aﬃnity-based

ﬂuorescence polarization (FP) method using 5-carboxyﬂuorescein (5-FAM)

labeled CAPON (496−506) peptide as the probe for high-throughput screening

of the small-molecule inhibitors of the PDZ domain of nNOS. N-Cyclohexylethyl-ADAV displayed the most potent aﬃnity for the

nNOS PDZ domain in the FP and isothermal titration calorimetry (ITC) (ΔH = −1670 ± 151.0 cal/mol) assays. To improve

bioavailability, lipophilicity, and membrane permeability, the Asp methylation was employed to get N-cyclohexylethyl-AD(OMe)AV,

which possesses good blood−brain barrier (BBB) permeability in vitro parallel artiﬁcial membrane permeability assay (PAMPA)-

BBB (Pe = 6.07 cm/s) and in vivo assays. In addition, N-cyclohexylethyl-AD(OMe)AV (10 mg/kg body weight, i.v., immediately

after reperfusion) substantially reduced infarct size in rats, which was measured 24 h after reperfusion and subjected to 120 min of

middle cerebral artery occlusion (MCAO).

KEYWORDS: nNOS−CAPON interaction blockers, anti-ischemic stroke, FP, ITC, MCAO

. INTRODUCTION

vascular coagulation (DIC). Therefore, it is critical to explore

a new, eﬀective, safe, and extended therapeutic time window

drug to treat the ischemic stroke.

Ischemic stroke is a major public health problem that leads to

high rates of death and long-term disability in adults. At the

present, research on stroke targets mainly includes calcium

antagonists, glutamate release inhibitors, γ-amino butyric acid

Reportedly, a stroke enhances the nNOS−CAPON

interaction in the acute phase. Studies have shown that

inhibiting the coupling of nNOS−CAPON has a neuro-

protective eﬀect. In 2013, Courtney et al. reported that a cell-

permeable peptide TAT-GESV, which competes for nNOS

PDZ domain interacting with CAPON, can inhibit excitatory

toxicity signals and damage in cultured neurons and animal

(

GABA) receptor agonists, free radical scavengers, MMP-9

inhibitors, N-methyl-D-aspartate receptor (NMDAR) antago-

nists, NOS inhibitors, nNOS-PSD95 PPI, neuronal nitric

oxide syntheses (nNOS)−carboxy-terminal PDZ ligand of

nNOS (CAPON) interaction blockers, etc. But unfortu-

nately, no other new eﬀective drug for ischemic stroke has

been approved by the Food and Drug Administration (FDA),

currently, since the recombinant tissue plasminogen activator

7,8

models of cerebral ischemia. The interaction between the

nNOS-PDZ domain and CAPON mediates the recruitment of

and requirement for CAPON in p38MAPK-dependent

excitotoxic neuronal death. In 2019, we published that a

stroke increased nNOS−CAPON association in the peri-

infarct cortex in the delayed period. In our previous work, we

reported a potent blocker ZLc-002(N-(2-carbomethoxyace-

tyl)-D-valine methyl ester) of nNOS−CAPON binding in

(

tPA) was licensed in 1996. However, only 2−5% stroke

patients received rtPAs treatment due to its short therapeutic

time window. Edaravone, a free radical scavenger, was

approved by the Japanese Ministry of Health in 2001 for the

treatment of acute ischemic stroke (AIS). It was reported that

combination therapy with edaravone and endovascular

reperfusion therapy was signiﬁcantly associated with greater

functional independence at hospital discharge, lower in-

hospital mortality, and reduced intracranial hemorrhage in

AIS patients. However, the potential adverse eﬀects of

edaravone were reported in some cases, such as acute renal

failure (ARF), fulminant hepatitis, and disseminated intra-

Received: November 18, 2020

Accepted: December 10, 2020

Published: December 23, 2020

2020 American Chemical Society

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Research Article

Table 1. List of Designed and Synthesized Compounds

cultured hippocampal neurons from ICR mice in Nature

concentration of the protein and probe, incubation time,

dimethyl sulfoxide (DMSO) tolerance and stability, and so on,

and demonstrated the feasibility of applying this method for

the high-throughput screening of inhibitors binding to the

nNOS PDZ domain. The aﬃnities of compounds for nNOS

PDZ domain were preliminary and secondary screened by FP

assay. Subsequently, ITC was employed to analyze the binding

activities of nNOS PDZ and two compounds. The results of

ITC correlated well with the data of FP experiment. To

improve the bioavailability, lipophilicity, and membrane

permeability, the Asp methylation was employed to get Che-

AD(OMe)AV, which possesses good blood−brain barrier

Medicine. ZLc-002 improved motor function in transient

middle cerebral artery occlusion (tMCAO) mice. nNOS−

CAPON interaction could serve as a novel pharmacological

target for functional restoration after a stroke, and it could

serve as a new therapeutic target in the delayed phase for

stroke recovery.

CAPON is a natural ligand of nNOS and is able to bind to

the pocket constituted by the αB helix and βB fold of the

nNOS PDZ domain through its carboxy-terminal tetrapeptide

EIAV. The binding site is a shallow and long groove

containing the binding pocket of the conserved sequence

(

BBB) permeability in vitro parallel artiﬁcial membrane

GLGF (Gly21, Leu 22, Gly23, Phe24). The N-cyclo-

permeability assay (PAMPA)-BBB and in vivo assays. In

addition, Che-AD(OMe)AV (10 mg/kg body weight, i.v.,

immediately after reperfusion) substantially reduced the infarct

size in rats, which was measured 24 h after reperfusion

subjected to 120 min of MCAO.

hexylethyl group (-Che) could increase the binding potency

19,20

with PDZ domains, plasma stability, and cell permeation.

On the basis of the carboxy-terminal tetrapeptide of CAPON

and our previous research, we designed a series of N-

cyclohexylethyl-tetrapeptides (Che-tetrapeptide) to uncouple

nNOS and CAPON by competitively binding to the GLGF

regional pocket of nNOS PDZ domain.

Here, we have established a method for the high-throughput

screening assay based on ﬂuorescence polarization (FP) for

testing the binding aﬃnities of compounds to nNOS PDZ

domain. 5-Carboxyﬂuorescein (5-FAM) labeled CAPON

. RESULTS AND DISCUSSION

.1. Design and Synthesis. PDZ domains typically

interacted with the carboxyl terminus of their binding

protein. It was generally acknowledged that the ﬁrst (P )

2−

and the third (P ) amino acids of the binding ligand

(

496−506) terminalpeptide was synthesized and employed

(

counting from the C-terminal amino acid) were considered

22,23

as the probe (5-FAM-KV-14). GST-nNOS PDZ was ex-

pressed, puriﬁed, and identiﬁed as the protein. Isothermal

titration calorimetry (ITC) was used to ﬁt the binding process

of 5-FAM-KV-14 and GST-nNOS₁₋₁₃₃protein to verify that

the binding was a typical single-point binding. In addition, we

optimized the experimental conditions, such as the optimal

quite important for aﬃnity with the PDZ domain.

the ﬁrst position (P ) was critical for aﬃnity for nNOS PDZ

and CAPON. Because if the Val (P ) on the carboxyl terminal

Val in

of CAPON was substituted to other amino acids, even a close

relative Ala, the binding activities with nNOS were almost

completely disappeared. The carboxyl at the side chain of the

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Scheme 1. Synthesis of Compound 6

Reagents and conditions: (a) 2-nitrobenzenesulfonyl (Nos) chloride, Et N, THF/H O, 0 °C-rt, 2 h; (b) 2-cyclohexylethan-1-ol, DIAD, Ph P;

DMF, 0 °C −rt, overnight; (c) NaOH, MeOH, rt, overnight; (d) sodium thiophenolate, DMF, rt, overnight; (e) Et N, (Boc) O, 1,4-dioxane, rt.

Figure 1. (A) Determination of the optimal concentration of the free probe for ﬂuorescence polarization assay by varying the concentration of 5-

FAM-KV-14 (0.01 nM−5 μM). (B) Optimization of the binding concentration for GST-nNOS using a 50 nM probe and increasing the

−133

concentrations of GST-nNOS

protein (from 1 nM to 20 μM). At the concentration of 1 μM, the binding of 5-FAM-KV-14 and GST-

−133

nNOS₁₋₁₃₃was saturated. (C) Stability of binding experiments over a 24 h period. Binding experiments were performed using a 50 nM probe and

increasing the concentrations of GST-nNOS₁₋₁₃₃protein (from 1 nM to 20 μM). (D) Eﬀect of DMSO on the binding experiments (0−12%).

P²amino acid could form a hydrogen-bond interaction with

−

carboxy-terminal tetrapeptide of the CAPON and our previous

research, we designed and synthesized a series of N-

cyclohexylethyl-[G/A]-[D/E]-X-V peptides (Table 1) to

uncouple nNOS and CAPON by competitively binding to

the GLGF regional pocket of the nNOS PDZ domain.

The synthetic route for the preparation of compounds N-

Boc-N-Che-Val-OH (6) is shown in Scheme 1. Compound 2

was synthesized by the treatment of H-Ala-OMe with 2-

nitrobenzenesulfonyl chloride in the presence of triethylamine

in dry DCM. Then, intermediate 2 was used in the subsequent

Mitsunobu reaction with 2-cyclohexylethan-1-ol mediated by

the phenolic hydroxyl of Tyr-71 on the nNOS PDZ domain.

So Asp and Glu, with carboxyl side chains, were selected in the

2−

position in this study. The results of peptides library

screening for the nNOS PDZ domain showed that the

occurrence rate of acidic and basic amino acids in the P

1−

site was extremely low. However, the total occurrence rate of

lipophilic amino acids, such as Ala, Leu, Ile, Phe, Pro, and Trp,

was 76%, indicating that a lipophilic amino acid is favorable at

this position. Considering the optimal sequence “-G-[D/E]-

x-v” screened by a peptide library, steric hindrance, and

polarity of the tetrapeptide, Gly and Ala with small side chains

were selected at the P³location. The N-cyclohexylethyl group

could increase the binding potency with PDZ domains, plasma

stability, and cell permeation. Therefore, on the basis of the

−

diisopropyl azadicarboxylate (DIAD) and Ph P to generate

compound 3. Intermediate 3 was hydrolyzed by NaOH to get

compound 4, which was deprotected by sodium thiophenolate

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to obtain compound 5. Then, compound 5 was protected by

the above result and saving time, the incubation time for the

binding experiment was chosen as 1 h.

(

Boc) O to aﬀord the desired intermediate 6.

Peptides (Table 1) were synthesized by manually applying

We also explored the inﬂuence of dimethyl sulfoxide

(DMSO) on the FP assay, which was a commonly used

solvent for dissolving some insoluble compounds in the high-

throughput screening. The data showed that the binding

aﬃnity of 5-FAM-KV-14 was unchanged and the FP assay

performed well in the presence of up to 12% DMSO (Figure

the standard Fmoc methodology by 2-chlorotrityl chloride

resin and Fmoc-protected amino acids, as we reported

before. The synthesized peptides were puriﬁed by semi-

preparative reversed-phase high-performance liquid chroma-

tography (RP-HPLC). The identiﬁcation and purity of the

ﬁnal compounds were veriﬁed using high-resolution mass

spectrometry (HRMS), nuclear magnetic resonance (NMR),

and analytical HPLC (see the Supporting Information).

D).

To identify the reliability and quality of the FP assay for the

high-throughput screening, the assay performance parameters

Z′ factor and signal-to-noise (S/N) ratio were determined.

The FP assay resulted in an excellent S/N (140.9) ratio and Z′

factor value (0.96). Therefore, it was conﬁrmed that the FP

assay conditions were adequate for high-throughput screening

^o^f^b^l^o^c^k^e^r^s^f^o^r^G^S^T^-ⁿ^N^O^S1−133 with 5-FAM-KV-14 as the

probe.

.2. Biological Activity. 2.2.1. Development and

Optimization FP-Based Competitive Assay. To test the

binding aﬃnities between the designed peptides and the nNOS

PDZ domain, FP assay was performed. THe GST fusion

protein of the N-terminal 1−133 of nNOS PDZ domain was

utilized as the FP assay protein. GST-tagged nNOS

was

1−133

In conclusion, a FP-based assay to evaluate the binding

aﬃnity of small-molecule blockers that target the nNOS PDZ

domain was developed and veriﬁed. The assays using 5-FAM-

KV-14 as the probe and GST-nNOS₁₋₁₃₃as the protein were

stable with up to 12% DMSO and 1−24 h incubation time and

had a Z′ factor value of 0.96, making it suitable for high-

throughput screening.

prepared from E. coli 21(DE3) strain as described in the

experimental section. The C-terminal amino acid (496−506)

sequence of CAPON is LGDGLDDEIAV. We designed a

ﬂuorescence probe 5-FAM labeled CAPON (496−506)

peptide, 5-FAM-KV-14 (5-FAM-KSGLGDGLDDEIAV),

which can be used for FP-based assay. The peptide was

synthesized by Fmoc-solid phase peptide synthesis with 2-

chlorotrityl chloride resin. 5-Fam was coupled to CAPON

terminal 11 peptide by a tripeptide spacer (KSG) with HATU

2.2.2. Binding Aﬃnity Evaluated with FP Assay. To

evaluate the binding aﬃnities between our designed peptides

and the nNOS PDZ, the FP assay was performed as described.

9,21

reagent.

After removing the protective group, 5-FAM

All IC₅₀and K values reported in the present study were

labeled peptide was cleaved from the resin by treatment with

TFA, precipitated with ice ether, ﬁltered, dried, and puriﬁed by

preparative HPLC. The identiﬁcation and purity of 5-FAM-

KV-14 was veriﬁed using MS and analytical HPLC (Figure

S1 ).

Initially, to determine of the optimal concentration of the

free probe (5-FAM-KV-14) for the FP assay, the experiment

was performed by varying 18 concentrations of the free probe

from 0.1 to 1000 nM. As shown in Figure 1A, with the 5-FAM-

KV-14 concentration increasing, the FP value decreased

rapidly and ﬁnally reached a stable level. We chose a low

concentration (50 nM) of 5-FAM-KV-14, which could yield a

stable FP value and reasonable ﬂuorescent signal.

obtained from at least three independent experiments. The

preliminary and secondary screening results are summarized in

Table 1 and Figures 2 and 3. Compounds Che-ADWV, Che-

ADAV, Che-ADIV, Che-GDLV, and Che-GDWV showed

preferable inhibitory rates (>86%, Table 1) with a concen-

tration of 1 mM. Compounds Che-AEAV, Che-AEWV, Che-

GDAV, Che-GDPV, and Che-GDFV also displayed moderate

potencies (>65%). However, Che-AEFV and Che-AEIV

<13%) showed poor competitive binding activities compared

To determine the optimal concentration of GST-tagged

nNOS₁₋₁₃₃, we evaluated 18 diﬀerent concentrations of the

GST-tagged nNOS₁₋₁₃₃, which varied from 1 nM to 20 μM

using a ﬁxed probe concentration of 50 nM. As presented in

Figure 1B, with the protein concentration increasing, the FP

values showed an s-curve change, which conforms to the

general rule of intermolecular interaction. The concentration

corresponding to 80% of the maximum FP value was the

optimal protein concentration. So, we chose 1 μM as the

optimal protein concentration. When the protein concen-

tration was 1 μM, the range of the FP value changed the most

signiﬁcantly. This concentration point made the assay sensitive,

and the consumption of protein was also relatively economical.

To evaluate the eﬀect of incubation time, the binding

experiments were performed using 50 nM 5-FAM-KV-14 with

increasing concentrations of GST-nNOS₁₋₁₃₃protein (from 1

nM to 20 μM). The plates were measured at 1, 2, 4, 8, and 24

h, and the results are shown in Figure 1C. After incubation for

Figure 2. Preliminary FP screening results of designed compounds

and reference compounds ZLc-002-1 (a nNOS−CAPON interaction

blocker). The assay was performed with 50 nM 5-FAM-KV-14, 1 μM

GST-nNOS₁₋₁₃₃protein, 1 h incubation time, and 5% DMSO. The

concentration of the tested compounds was 1 mM.

, 2, 4, 8, and 24 h, the FP values remained basically

unchanged and stable. The change of the K values was very

slight (1 h K = 0.30 μM, 2 h K = 0.35 μM, 4 h K = 0.31 μM,

h K = 0.31 μM, and 24 h K = 0.31 μM). Therefore, given

d d

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Research Article

experiment, also indicating that FP could be used as a

screening method for nNOS−CAPON binding blockers.

2.2.4. Molecule Docking Study. In order to explore the

binding mode between Che-ADAV and nNOS PDZ, we

performed a semiﬂexible molecular docking between Che-

ADAV and the nNOS PDZ domain (PDB code: 1B8Q) using

Discovery Studio 3.5. The interaction energy was −61.73 kcal/

mol. As shown in Figure 5, Che-ADAV was tightly bound into

the groove between the αB helix and βB folding of the nNOS

PDZ domain. In the Che-ADAV−nNOS PDZ domain

complex, there were hydrogen-bonding interactions between

the Che-ADAV and GLGF loop (Gly21-Leu22-Gly23-Phe24)

of the nNOS PDZ domain. The -COOH of Val (Che-ADAV)

formed a H-bond with Gly23; the hydrogen-bond distance was

.03 Å. The Asp (Che-ADAV) made double H-bonds with

Tyr71 and Arg79, respectively, and the H-bond distances were

.07 and 2.04 Å, respectively. In addition, the Ala (Che-

ADAV) interacted with Val26 using a H-bond.

.2.5. Structure Optimization of Che-ADAV and in Vitro

PAMPA-BBB Assay of Che-ADAV and Che-AD(OMe)AV. To

improve bioavailability, lipophilicity, and membrane perme-

ability, the Asp methylation of Che-ADAV was employed to

get Che-AD(OMe)AV. Compounds Che-AD(OMe)AV and

Che-ADAV were assayed in the FP binding experiment

(Figure 2). Che-ADAV displayed more potent binding aﬃnity

with the nNOS PDZ domain than did Che-AD(OMe)AV. As

shown in Figure 4D and Table 2, these two compounds were

also compared by the ITC assay to further conﬁrm the binding

aﬃnity with nNOS PDZ domain. Che-AD(OMe)AV (ΔH =

Figure 3. Secondary FP screening results of Che-ADIV, Che-ADAV,

Che-GDWV, and Che-GDLV. The assay was performed with 50 nM

5-FAM-KV-14, 1 μM GST-nNOS₁₋₁₃₃protein, 1 h incubation time,

and 5% DMSO.

with 5-FAM-KV-14 to the GST-nNOS₁₋₁₃₃. Those compounds

with high aﬃnities were picked out for the secondary

screening. Furthermore, comparing the aﬃnities of compounds

Che-AEAV (inhibition ratio = 65.87%) and Che-ADAV

−

967.7 ± 312.4 cal/mol) exhibited a weaker aﬃnity to GST-

nNOS compared with Che-ADAV (ΔH = −1670 ± 151.0

(

inhibition ratio = 96.38%, K = 27.37 μM), we observed

1−133

2−

cal/mol). The data indicated that the conversion of the Asp

carboxylate to an ester greatly reduced the binding aﬃnity

because the salt bridge/electrostatic interaction with Arg79

would be eliminated and H-bond interactions would be

reduced. To verify and compare the BBB permeability of

compounds Che-ADAV and Che-AD(OMe)AV, we carried

out the parallel artiﬁcial membrane permeability assay of BBB

(PAMPA-BBB) (Figure 6). The result showed that compound

Che-AD(OMe)AV (Pe = 6.07 cm/s) displayed a much higher

permeability value than Che-ADAV (Pe = 2.65 cm/s), which

suggested that compound Che-AD(OMe)AV could more

easily penetrate into the BBB than Che-ADAV.

that the Asp improved the aﬃnity more than Glu in the P

position. A similar eﬀect was observed when comparing Che-

AEIV (inhibition ratio = 12.79%) and Che-ADIV (onhibition

ratio = 87.44%) or Che-AEWV (inhibition ratio = 78.45%)

and Che-ADWV (inhibition ratio = 96.06%, K = 73.15 μM).

The -COOH in the side chain of Asp could probably more

easily form H-bond interactions with the phenolic hydroxyl of

Tyr71 or guanidino of Arg79 of the nNOS PDZ domain. In

−

the P position, the substitution of Ala with Gly did not aﬀect

the binding potency signiﬁcantly, as in Che-ADAV (inhibition

ratio = 96.38%, K = 27.37 μM) and Che-GDAV (inhibition

ratio = 82.99%) or Che-ADWV (inhibition ratio = 86.61%, K_i

73.15 μM) and Che-GDWV (inhibition ratio = 94.84%, K =

2.2.6. In Vivo Brain Pharmacokinetic Study. To evaluate

the permeability across the BBB in vivo of compound Che-

AD(OMe)AV and whether it was converted to Che-ADAV or

not, we measured the concentrations of Che-AD(OMe)AV

and Che-ADAV in brain tissue at 1 min, 5 min, 30 min, 45

min, 1 h, and 2 h after intraperitoneal injection (20 mg/kg). As

shown in Table 3, the precursor ion of Che-AD(OMe)AV was

499.3 and the product ion was 154.3. The precursor ion of

Che-ADAV was 485.3 and the product ion was 154.3.

Fluoxetine was used as an internal reference (the precursor

ion of ﬂuoxetine was 310.2 and the product ion was 148.1. In

vivo brain pharmacokinetic studies indicated that Che-

AD(OMe)AV displayed a good brain uptake (136.7 ng/g at

45 min). Che-AD(OMe)AV was acting as a prodrug and was

converted to the active carboxylate form Che-ADAV. The

concentration of Che-ADAV reached the high pot in brain

tissue at about 45 min after administration (Figure 7). Detailed

data can be found in the Supporting Information.

5.74 μM), demonstrating that lipophilic amino acids in the

−

position had a lower eﬀect on the overall activity of the

−

compounds. At the P position, it accommodated several

amino acids, such as Trp, Ala, Ile, Pro, and Leu, however, Phe

was hardly bound into the nNOS PDZ domain as shown in

Che-GDFV and Che-AEFV. Although further investigation on

the SAR was needed, these ﬁndings could provide more

experience for the further optimization of nNOS−CAPON

interaction blockers.

.2.3. ITC Assay. Isothermal titration calorimetry (ITC) was

used to ﬁt the binding process of 5-FAM-KV-14 and GST-

nNOS₁₋₁₃₃protein to verify that the binding was a typical

single-point binding. (Figure 4A). The titration results of Che-

AEAV and Che-ADAV to GST-nNOS₁₋₁₃₃are shown in

Figure 4. The thermodynamic parameters of the binding of the

designed peptides to the nNOS PDZ domain obtained by ITC

are summarized in Table 2. Che-ADAV exhibited a high

aﬃnity to GST-nNOS₁₋₁₃₃(ΔH = −1670 ± 151.0 cal/mol).

The results of the ITC screening correlated well with the FP

2.2.7. Che-AD(OMe)AV Attenuated Nerve Injury of MCAO

Rats. Having conﬁrmed the eﬀective blood−brain barrier

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Figure 4. ITC data for binding of (A) 5-FAM-KV-14, (B) Che-AEAV, (C) Che-ADAV, and (D) Che-AD(OMe)AV GST-nNOS₁₋₁₃₃. The upper

panel of the graph shows the heat ﬂow of each injection; the lower panel indicates the integration of each peak (kcal/mol) as a function of the

molar ratio of compounds to GST-nNOS₁₋₁₃₃after subtracting the heat of dilution. The thermodynamic parameters (K, ΔH, and ΔS) are indicated

in the picture.

Table 2. Thermodynamic Parameters of the Binding of the Designed Peptides to the nNOS PDZ Domain Obtained by ITC

compounds

K (M⁻¹)

ΔH (cal/mol)

ΔS (cal/mol/deg)

-FAM-KV-14

Che-AEAV

Che-ADAV

Che-AD(OMe)AV

1.11

6.56 × 10 ± 1.92 × 10

−4503 ± 1662

+13.26 ± 9.314

−1670 ± 151.0

−967.7 ± 312.4

6.94

36.6

17.0

18.0

9.56 × 10⁷

1.02 × 10

1.72

8.81 × 10 ± 1.22 × 10⁴

3.89

4.48 × 10 ± 2.43 × 10

permeability of compound Che-AD(OMe)AV, we next moved

on to investigate its neuroprotective ability in vivo by a middle

cerebyal artery occlution (MCAO) model that was widely used

for the ischemic stroke study. Infarct size was measured

following sacriﬁce at 24 h by staining of 1 mm thick brain slices

with 2,3,5-triphenylterazolium hydrochloride (TTC). We used

edaravone as the positive drug for its superior neuroprotective

ability described in previous reports. As shown in Figure 8, a

white region of the brain sections (23.9, vehicle) indicated an

infarction that was induced by MCAO. Treatment with 10

mg/kg compound Che-AD(OMe)AV signiﬁcantly reduced the

ischemia area to 4.8% (p < 0.001 vs vehicle). As a comparison,

ACS Chem. Neurosci. 2021, 12, 244−255

(20 mg/kg) for 1 min, 5 min, 30 min, 45 min, 1 h, and 2 h.

Research Article

Figure 7. Concentrations of Che-AD(OMe)AV and Che-ADAV:

time column chart of mice brain tissue after intraperitoneal injection

Figure 5. Interaction of Che-ADAV with the nNOS PDZ domain.

Figure 6. Ratio of penetrability in vitro of compounds in the PAMPA-

BBB assay was carried out and calculated by HPLC after an

incubation of 18 h. Carbamazepine (high brain penetration) was

selected as the positive control, and atenolol (low brain penetration)

was selected as the negative control. Data were shown as mean ±

SEM of three independent experiments.

Figure 8. Representative of triphenyltetrazolium chloride stained

brain slices.

the edaravone group reduced the ischemia area to 9.2% (p <

.01 vs vehicle). The compound Che-AD(OMe)AV decreased

the ischemia area by 4.4% compared with the edaravone group

Figure 9). These results suggested that the compound Che-

Table 3. Mass Parameters for the Detection of Che-

AD(OMe)AV and Its Metabolite Che-ADAV

(

AD(OMe)AV could be a potential neuroprotective compound

against cerebral ischemia damage.

precursor

ion

product

ion

collision

energy

compounds

fragmentor

Che-AD(OMe)AV

Che-ADAV

Fluoxetine

499.3

485.3

310.2

154.3

148.1

145

105

. CONCLUSION

In summary, we designed and synthesized a series of Che-[A/

G]-[D/E]-X-V peptides based on the carboxy-terminal

tetrapeptide of CAPON that is capable of blocking nNOS−

CAPON coupling. Herein, we reported an aﬃnity-based FP

ACS Chem. Neurosci. 2021, 12, 244−255

Research Article

.1.3. Synthesis of N-(2-Cyclohexylethyl)-N-((2-nitrophenyl)-

sulfonyl) Alanine Methyl Ester (3). To a cold mixture of compound

(15 g, 52 mmol), triphenylphosphine (20.46 g, 78 mmol 1.5 equiv),

and 2-cyclohexylethan-1-ol (7.25 mL, 52 mmol, 1 equiv) in 500 mL of

dry THF and under a N atmosphere was slowly added diisopropyl

azodiformate (DIAD, 15.76 g, 78 mmol, 1.5 equiv), and the mixture

was stirred overnight at room temperature. The solvent was

evaporated, and the residue was extracted with EtOAc (200 mL ×

). The EtOAc layers were combined, washed with brine, dried with

Na SO , and concentrated. The crude product was chromatographed

on silica to obtain 15.1 g of the expected compound 3. Yield: 73%. H

NMR (400 MHz, CDCl ): δ 8.16−7.97 (m, 1H), 7.72 (m, 2H),

.65−7.55 (m, 1H), 4.75 (q, J = 7.3 Hz, 1H), 3.59 (s, 3H), 3.53−3.04

(m, 2H), 1.68−0.82 (m, 16H).

.1.4. Synthesis of N-(2-Cyclohexylethyl)-N-((2-nitrophenyl)-

sulfonyl) Alanine (4). Compound 3 (15 g, 37.6 mmol) and 1 N

NaOH (45 mL, 45 mmol) were dissolved in MeOH (50 mL). The

mixture was reacted overnight at room temperature. After the

reaction, the residue was dissolved in water, acidiﬁed to pH 1−2 with

N HCl, and extracted with EtOAc (150 mL × 3). The organic

phases were combined, washed with a saturated aqueous solution of

NaCl, and dried and evaporated to dryness to aﬀord a yellow oil. The

yellow oil was puriﬁed by silica gel column chromatography (CH Cl /

Figure 9. Eﬀects of Che-AD(OMe)AV and positive compound

edaravone on cerebral ischemia in rats subjected to 120 min MCAO.

Infarct size was measured at 24 h after reperfusion. (n = 16−18, data

are presented as mean ± SEM, **P < 0.01, ***P < 0.001, vs vehicle-

treated MCAO group, one-way ANOVA, followed by post hoc Scheﬀe

test).

MeOH = 20:1) to give intermediate compound 4 at 13.5 g. Yield:

(

3%. H NMR (400 MHz, CDCl ): δ 9.75 (s, 1H), 8.18−7.98 (m,

H), 7.71 (m, 2H), 7.64 (m, 1H), 4.78 (q, J = 7.3 Hz, 1H), 3.58−3.00

m, 2H), 1.99−0.79 (m, 16H). MS (ESI, m/z): 407.1 [M + Na] .

.1.5. Synthesis of N-(tert-Butoxycarbonyl)-N-(2-cyclohexylethyl)

Alanine (6). To a solution of compound 4 (13 g, 34 mmol) in dry

DMF (50 mL) was added sodium thiophenolate (9 g, 68 mmol), and

then, the mixture was stirred overnight at room temperature. The

mixture was concentrated under reduced pressure. Water was added

to dissolve the crude mixture. The solution was acidiﬁed to pH 4 with

2 N HCl and extracted with ether three times. The aqueous layers

were collected, pH adjusted to 5−6, and freeze-dried to achieve a

pale-yellow solid, which could be used in the next reaction without

method using 5-FAM-KSGLGDGLDDEIAV as a probe for

high-throughput screening of the binding aﬃnities of our

designed peptides with the PDZ domain of nNOS. The most

potent peptide (Che-ADAV) binds to nNOS PDZ with a K_i

value of 27.37 μM in the FP assay. The ITC was employed to

conﬁrm the binding activity between Che-ADAV to GST-

nNOS₁₋₁₃₃. Che-ADAV exhibited a high aﬃnity to GST-

nNOS₁₋₁₃₃(ΔH = −1670 ± 151.0 cal/mol). The results of

ITC screening correlated well with the FP experiment. To

improve bioavailability, lipophilicity, and membrane perme-

ability, the Asp methylation was employed to get Che-

AD(OMe)AV, which possesses good BBB permeability in vitro

PAMPA-BBB (Pe = 6.07 cm/s) and in vivo assays. In addition,

Che-AD(OMe)AV (10 mg/kg body weight, i.v., after

reperfusion) substantially reduced the infarct size in rats

subjected to 120 min of MCAO.

further puriﬁcation. MS (ESI, m/z): 222.1 [M + Na] .

To compound 5 and triethylamine (19 mL, 136 mmol) in 1,4-

dioxane was added (Boc) O (22.3 g, 102 mmol) at room

temperature. After stirring overnight, the residue was concentrated,

and then taken up in water as the solvent and pH adjusted to 3−4 by

0% citric acid. The mixture was extracted with ethyl acetate three

times. The organic phase was dried, evaporated to dryness, and

puriﬁed by silica gel column chromatography (CH Cl /MeOH =

20:1) to get compound 6 at 5.1 g. Yield: 50%. H NMR (400 MHz,

CDCl ): δ 11.31 (s, 1H), 4.42−3.99 (m, 1H), 3.36−3.06 (m, 2H),

1.81−0.84 (m, 26H). MS (ESI, m/z): 322.2 [M + Na] .

.1.6. General Procedure for the Fmoc Solid-Phase Synthesis.

Peptides were synthesized by manual applying the standard Fmoc

methodology. 2-Chlorotrityl chloride resin (100−200 mesh, 0.3−0.8

mmol/g, 1% DVB) and Fmoc-protected amino acids (Fmoc-Val-OH,

Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH,

Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp-

. MATERIALS AND METHODS

4.1. Chemistry. 4.1.1. RP-HPLC was Performed on a Waters

Delta 600 Liquid Chromatograph Using the Following Solvent

System. Solvent A, 0.05% TFA in water; solvent B, 0.05% TFA in

CH CN. Semipreparative RP-HPLC was performed on a Sunﬁre Prep

C18 column (20 mm × 250 mm) with a linear gradient of 5−50% of

solvent B over 40 min at a ﬂow rate of 10 mL/min. Analytical RP-

HPLC was performed on a Sunﬁre Prep C18 column (4.6 mm × 250

mm) with a linear gradient of 5−50% of solvent B over 40 min at a

ﬂow rate of 1.2 mL/min. H and C NMR spectra were recorded on

a JEOL 400 MHz spectrometer at 25 °C.

(

tBu)-OH, and Fmoc-Gly-OH) were purchased from GL Biochem

Ltd. (Shanghai, China). The coupling reagents HBTU, HOBt, and

DIPEA were also purchased from GL Biochem Ltd. (Shanghai,

China). Dichloromethane (DCM), N,N-dimethylformamide (DMF),

tetrahydrofuran (THF), piperidine, and other solvents were redistilled

and stored with molecular sieves. All reagents and chemicals were

ACS grade or better and were used without further puriﬁcation. The

growing peptide chain was added to the swelled 2-chlorotrityl chloride

resin using the general amino acid cycle as follows: (1) the addition of

Fmoc-amino acid (4 equiv), HBTU (4 equiv), DIPEA (4 equiv), and

HOBt (4 equiv) in dry DMF, mixing and shaking for 4 h at 25 °C; (2)

monitoring the completion of the reaction with the ninhydrin test;

(3) ﬁltering and washing with DMF (15 × 3 mL) to totally take oﬀ

the unreacted Fmoc amino acid; (4) deprotecting the Fmoc group

with 20% (v/v) piperidine in DMF (10 min) and Fmoc deprotection

one more time (30 min); (5) monitoring the completion of the

reaction with the ninhydrin test; (6) washing with DMF (10 mL × 3).

.1.2. Synthesis of N-(2-Nitrobenzenesulfonyl)-L-alanine Methyl

Ester (2). To a solution of L-alanine methyl ester (6 g, 58.2 mmol, 1

equiv) in DCM (100 mL) was added 2-nitrobenzenesulfonyl chloride

(12.9 g, 58.2 mmol, 1 equiv) and triethylamine (16 mL, 116.4 mmol,

equiv) at 0 °C. The mixture was stirred at room temperature for 2 h

and then washed with water. The organic layer was separated and

dried with Na SO and concentrated. The brown solid was puriﬁed by

column chromatography to give desired compound 2 at 15 g. Yield:

9%. H NMR (400 MHz, DMSO-d ): δ 8.73 (s, 1H), 8.04−7.92 (m,

H), 7.89−7.82 (m, 2H), 4.06 (q, J = 7.1 Hz, 1H), 3.46 (s, 3H), 1.28

(

d, J = 7.2 Hz, 3H). MS (ESI, m/z): 311.0 [M + Na] .

ACS Chem. Neurosci. 2021, 12, 244−255

ACS Chemical Neuroscience

Research Article

4.14−4.02 (m, 1H), 3.71 (d, J = 6.7 Hz, 1H), 2.73−2.61 (m, 3H),

2.66 (d, J = 4.5 Hz, 1H), 2.52−2.37 (m, 2H), 2.03−1.87 (m, 1H),

1.63−1.50 (m, 6H), 1.48−1.35 (m, 3H), 1.31 (d, J = 6.7 Hz, 4H),

1.17−0.98 (m, 3H), 0.91 (s, 2H), 0.87 (d, J = 2.5 Hz, 3H), 0.84 (d, J

= 4.9 Hz, 6H), 0.77 (d, J = 7.4 Hz, 3H). HRMS (ESI): m/z calcd for

C H N O (M + H) , 527.3445; found, 527.3446.

4.1.6.8. N-Cyclohexylethyl-Gly-Asp-Ala-Val-OH (Che-GDAV).

Yield 248.6 mg (26.42%). HPLC: t = 16.50 min. H NMR (300

Prep C18 column (20 mm × 250 mm). After semipreparation, the

product peak was collected and freeze-dried to obtain the desired

peptide.

MHz, DMSO-d ): δ 12.59 (s, 2H), 8.73−8.61 (m, 1H), 8.10 (d, J =

7.0 Hz, 1H), 7.88−7.72 (m, 1H), 4.69−4.53 (m, 1H), 4.43−4.26 (m,

1H), 4.15−4.04 (m, 1H), 3.70 (s, 2H), 2.98−2.81 (m, 2H), 2.69−

2.67 (m, 1H), 2.61−2.50 (m, 2H), 2.04−1.89 (m, 1H), 1.63−1.41

(m, 5H), 1.45−1.23 (m, 2H), 1.19 (t, J = 6.3 Hz, 4H), 1.00−0.93(m,

3H), 0.95 (s, 2H), 0.85 (s, 6H). HRMS (ESI): m/z calcd for

.1.6.1. N-Cyclohexylethyl-Ala-Glu-Ala-Val-OH (Che-AEAV). Yield

55.5 mg (15.61%). HPLC: t = 13.45 min. H NMR (300 MHz,

DMSO-d ): δ 12.30 (s, 2H), 8.64 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 7.1

Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 4.34 (t, J = 7.0 Hz, 2H), 4.10 (dd, J

C H N O (M + H) , 471.2819; found, 471.2837.

8.5, 5.7 Hz, 1H), 3.89 (s, 1H), 2.78 (s, 3H), 2.24 (t, J = 8.0 Hz,

4.1.6.9. N-Cyclohexylethyl-Gly-Asp-Pro-Val-OH (Che-GDPV).

H), 2.01−1.89 (m, 1H), 1.87 (d, J = 5.2 Hz, 1H), 1.83−1.68 (m,

Yield 176.3 mg (17.78%). HPLC: t = 14.17 min. H NMR (300

H), 1.61−1.47 (m, 5H), 1.42 (t, J = 7.6 Hz, 2H), 1.33 (d, J = 6.8 Hz,

MHz, DMSO-d ): δ 12.51 (s, 2H), 8.90 (d, J = 7.7 Hz, 1H), 7.81 (d, J

H), 1.18 (d, J = 7.0 Hz, 6H), 0.84 (d, J = 6.8 Hz, 9H). HRMS (ESI):

= 8.5 Hz, 1H), 4.87 (d, J = 5.4 Hz, 1H), 4.40 (d, J = 6.3 Hz, 1H),

4.12−4.03 (m, 1H), 3.69 (s, 2H), 3.65−3.54 (m, 2H), 2.90 (s, 2H),

2.72−2.66 (m, 1H), 2.50 (s, 2H), 2.47−2.38 (m, 2H), 2.04−1.97 (m,

1H), 1.89 (s, 2H), 1.54−1.31 (m, 7H), 1.30−1.08 (m, 4H), 0.92 (d, J

= 7.0 Hz, 2H), 0.86 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calcd for

m/z calcd for C H N O (M + H) , 499.3132; found, 499.3142.

.1.6.2. N-Cyclohexylethyl-Ala-Glu-Trp-Val-OH (Che-AEWV).

Yield 76.3 mg (12.45%). HPLC: t_R= 14.15 min. H NMR (300

MHz, DMSO-d ): δ 13.04−11.60 (m, 2H), 10.75 (s, 1H), 8.58 (d, J =

.5 Hz, 1H), 8.08 (t, J = 8.5 Hz, 2H), 7.82−6.38 (m, 5H), 4.65 (d, J =

.9 Hz, 1H), 4.33 (d, J = 5.4 Hz, 1H), 4.21−4.06 (m, 1H), 3.81 (d, J

C H N O (M + H) , 497.2975; found, 497.2987.

4.1.6.10. N-Cyclohexylethyl-Gly-Asp-Leu-Val-OH (Che-GDLV).

7.0 Hz, 1H), 3.09 (d, J = 10.0 Hz, 2H), 2.97−2.63 (m, 3H), 2.15−

Yield 318 mg (31.06%). HPLC: t_R= 13.90 min. H NMR (300

.05 (m, 3H), 2.06 (s, 1H), 1.87 (s, 1H), 1.73 (d, J = 7.9 Hz, 1H),

.60 (d, J = 9.5 Hz, 5H), 1.55−1.32 (m, 3H), 1.23−1.12 (m, 4H),

.13 (s, 3H), 0.87−0.71 (m, 6H). HRMS (ESI): m/z calcd for

MHz, DMSO-d ): δ 8.75 (d, J = 7.9 Hz, 1H), 8.09 (d, J = 8.1 Hz,

1H), 7.80 (d, J = 8.4 Hz, 1H), 4.64−4.58 (m, 1H), 4.34−4.27 (m,

1H), 4.13−4.00 (m, 1H), 3.77−3.56 (m, 2H), 2.90 (s, 2H), 2.75−

2.58 (m, 1H), 2.50 (d, J = 9.5 Hz, 2H), 2.02−1.87 (m, 1H), 1.64 (s,

2H), 1.61 (s, 4H), 1.46−1.38 (m, 4H), 1.28−1.04 (m, 4H), 0.89 (d, J

= 9.2 Hz, 2H), 0.87−0.78 (m, 12H). HRMS (ESI): m/z calcd for

C H N O (M + H) , 614.3554; found, 614.3556.

.1.6.3. N-Cyclohexylethyl-Ala-Asp-Trp-Val-OH (Che-ADWV).

Yield 38.4 mg (6.41%). HPLC: t_R= 14.35 min. H NMR (300

MHz, DMSO-d ): δ 10.75 (s, 2H), 8.66 (s, 1H), 7.96 (s, 2H), 7.55

C H N O (M + H) , 513.3288; found, 513.3304.

25 44

(

d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.13−6.77 (m, 4H),

4.1.6.11. N-Cyclohexylethyl-Gly-Asp-Phe-Val-OH (Che-GDFV).

.58 (s, 2H), 4.12 (s, 1H), 3.68 (s, 1H), 3.08 (s, 2H), 2.93 (s, 2H),

Yield 151.5 mg (13.74%). HPLC: t = 14.15 min. H NMR (300

.67−2.35 (m, 3H), 2.01 (s, 1H), 1.58 (s, 5H), 1.38 (d, J = 7.5 Hz,

MHz, DMSO-d ): δ 12.62 (s, 2H), 8.69−8.43 (m, 1H), 8.22−7.93

H), 1.23 (d, J = 6.8 Hz, 5H), 1.12−0.99 (m, 3H), 0.84 (d, J = 4.1

(m, 2H), 7.22−6.89 (m, 5H), 4.72−4.41 (m, 2H), 4.13 (dd, J = 8.2,

6.0 Hz, 1H), 3.62−3.54 (m, 2H), 3.43 (s, 2H), 3.08−2.95 (m, 1H),

2.67−2.59 (m, 2H), 2.50−2.36 (m, 2H), 2.05−1.78 (m, 1H), 1.53−

1.44 (m, 7H), 1.31−1.00 (m, 4H), 0.94−0.74 (m, 8H). HRMS (ESI):

Hz, 6H). HRMS (ESI): m/z calcd for C H N O (M + H) ,

00.3397; found, 600.3393.

.1.6.4. N-Cyclohexylethyl-Ala-Glu-Phe-Val-OH (Che-AEFV). Yield

97.8 mg (17.03%). HPLC: t_R= 10.17 min. H NMR (300 MHz,

m/z calcd for C H N O (M + H) , 547.3132; found, 547.3251.

DMSO-d ): δ 8.56 (d, J = 8.0 Hz, 1H), 8.12−8.00 (m, 2H), 7.26−

4.1.6.12. N-Cyclohexylethyl-Gly-Asp-Trp-Val-OH (Che-GDWV).

7.07 (m, 5H), 4.62 (s, 1H), 4.29 (d, J = 5.2 Hz, 1H), 4.12 (d, J = 8.4

Yield 189.1 mg (16.16%). HPLC: t = 14.10 min. H NMR (300

Hz, 1H), 3.80 (d, J = 7.2 Hz, 1H), 2.99−2.90 (m, 2H), 2.85−2.65 (m,

MHz, DMSO-d ): δ 10.78 (d, J = 8.8 Hz, 1H), 8.67−8.57 (m, 1H),

H), 2.16 (t, J = 8.0 Hz, 2H), 2.03−1.89 (m, 2H), 1.83 (s, 1H),

8.16−7.94 (m, 2H), 7.57 (d, J = 6.4 Hz, 1H), 7.30 (d, J = 7.9 Hz,

1H), 7.13−6.91 (m, 3H), 4.69−4.52 (m, 2H), 4.12 (d, J = 8.8 Hz,

1H), 3.70−3.53 (m, 2H), 3.12−3.01 (m, 2H), 2.98−2.80 (m, 3H),

2.76−2.50 (m, 2H), 2.04−1.66 (m, 1H), 1.56−1.44 (m, 6H), 1.42 (s,

1H), 1.27−1.07 (m, 4H), 0.87 (d, J = 6.4 Hz, 8H). HRMS (ESI): m/z

.77−1.44 (m, 7H), 1.42−1.17 (m, 6H), 1.13 (s, 3H), 0.89−0.80 (m,

H). HRMS (ESI): m/z calcd for C H N O (M + H) , 575.3445;

found, 575.3450.

.1.6.5. N-Cyclohexylethyl-Ala-Asp-Ala-Val (Che-ADAV). Yield

3.2 mg (6.86%). HPLC: t_R= 11.18 min. H NMR (300 MHz,

calcd for C H N O (M + H) , 586.3241; found, 586.3250.

DMSO-d ): δ 12.47 (s, 2H), 9.09−8.73 (m, 1H), 7.99 (d, J = 6.9 Hz,

4.1.6.13. N-Cyclohexylethyl-Ala-Asp-OMe-Ala-Val (Che-AD-

H), 7.90 (s, 1H), 4.63 (s, 1H), 4.44−4.27 (m, 1H), 4.22−4.03 (m,

H), 3.81 (s, 1H), 2.80 (s, 2H), 2.73−2.50 (m, 2H), 2.52 (d, J = 9.6

(OMe)AV−OH). Yield 88 mg (16.7%). HPLC: t = 19.85 min. H

NMR (400 MHz, DMSO-d ): δ 8.88 (d, J = 8.0 Hz, 1H), 8.14 (d, J =

Hz, 1H), 2.10−1.94 (m, 1H), 1.63−1.42 (m, 5H), 1.44 (d, J = 6.7 Hz,

8.9 Hz, 1H), 7.89 (d, J = 9.6 Hz, 1H), 4.68−4.63 (m, 1H), 4.34−4.27

(m, 1H), 4.11−4.07 (m, 1H), 3.81−3.76 (m, 1H), 3.54 (s, 3H),

2.87−2.72 (m, 3H), 2.55−2.42 (m, 2H), 2.05−1.97 (m, 1H), 1.61−

1.54 (m, 5H), 1.44−1.38 (m, 2H), 1.29 (d, J = 6.9 Hz, 3H), 1.24−

1.05 (m, 7H), 0.83 (d, J = 6.7 Hz, 8H). HRMS (ESI): m/z calcd for

H), 1.36 (d, J = 6.8 Hz, 3H), 1.19 (d, J = 7.0 Hz, 6H), 0.87 (s, 3H),

.85 (s, 6H). HRMS (ESI): m/z calcd for C H N O (M + H) ,

85.2975; found, 485.3009.

.1.6.6. N-Cyclohexylethyl-Ala-Glu-Ile-Val-OH (Che-AEIV). Yield

23.5 mg (4.35%). HPLC: t_R= 12.95 min. H NMR (300 MHz,

C H N O (M + H) , 499.3132; found, 499.3137.

DMSO-d ): δ 8.54 (s, 1H), 7.92 (d, J = 8.5 Hz, 1H), 7.85 (s, 1H),

4.2. Biological Evaluation. 4.2.1. Expression, Puriﬁcation and

Identiﬁcation of GST-nNOS PDZ. E. coli 21(DE3) containing

pGEX4T-1-GST-nNOS₁₋₁₃₃was inoculated into 5 mL of LB medium

(1:1000) with 50 μg/mL ampicillin and cultured overnight in a shaker

at 37 °C at 180 r/min. It was transferred to incubate in 300 mL of LB

medium (1:100) with 50 μg/mL ampicillin for 2.5 h. The protein

expression was induced by the addition of lactose to a ﬁnal

concentration of 2.5 g/mL for 3 h at 37 °C. Cells were collected

by spinning at 3000g for 30 min at 4 °C and then resuspended in 10

mL of cell lysis buﬀer (50 mM Tris-HCl, pH 8.0, 60 μg/mL lysozyme,

0.5% Triton X-100, 10 U/mL DNA enzyme, 1 mM PMSF) per 1 g of

.40 (s, 1H), 4.33−4.21 (m, 1H), 4.14−4.05 (m, 1H), 3.73 (s, 1H),

.75−2.27(m, 3H), 2.20 (d, J = 7.5 Hz, 3H), 2.13−1.96 (m, 2H), 1.88

(

s, 1H), 1.74 (s, 2H), 1.63−1.51 (m, 5H), 1.50−1.36 (m, 4H), 1.35−

.10 (m, 7H), 0.87 (d, J = 2.7 Hz, 3H), 0.83 (d, J = 7.3 Hz, 6H), 0.78

(

d, J = 7.4 Hz, 3H). HRMS (ESI): m/z calcd for C H N O (M +

H) , 541.3601; found, 541.3601.

.1.6.7. N-Cyclohexylethyl-Ala-Asp-Ile-Val-OH (Che-ADIV). Yield

7.5 mg (12.82%). HPLC: t_R= 10.90 min. H NMR (300 MHz,

DMSO-d ): δ 8.75 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.71

(d, J = 9.3 Hz, 1H), 4.65 (d, J = 4.8 Hz, 1H), 4.33−4.21 (m, 1H),

ACS Chem. Neurosci. 2021, 12, 244−255

ACS Chemical Neuroscience

The Supporting Information is available free of charge at

Research Article

cell. The cell lysate was spun down at 15 000g for 10 min at 4 °C, and

the supernatant was ﬁltered with a 0.22 μm ﬁlter. Puriﬁcation was

performed by a GST aﬃnity chromatography column, and elution was

done by PBS buﬀer (KH PO 2 mM, Na HPO 8 mM, NaCl 136 mM,

dodecane; subsequently, 150 μL of PBS was added. The acceptor well

was placed onto the donor well and incubated at the room

temperature for 18 h. The permeability (Pe) was measured using

HPLC. After the removal of the acceptor well, the donor/acceptor

solutions and standard samples were measured using an Agilent 1200

Inﬁnity Series LC at 235 nm.

KCl 2.6 mM) with reduced glutathione (0.154 g/50 mL PBS) to get

GST-nNOS₁₋₁₃₃

The GST-nNOS₁₋₁₃₃protein was identiﬁed by a commassie blue

staining method. The puriﬁed and concentrated protein was subjected

to SDS-PAGE, dyed with coomassie blue staining solution overnight,

and then eluted with the decolorization solution and imaged in white

light.

4.2.6. Analysis of Che-AD(OMe)AV and Che-ADAV Concen-

6,33

trations in Brain.

For pharmacokinetic studies, C57BL/6J mice

(20−25 g) were housed individually and fasted overnight before use.

Water was allowed ad libitum. The concentrations of Che-

AD(OMe)AV and Che-ADAV of brain tissue at 1 min, 5 min, 30

min, 45 min, 1 h, and 2 h after intraperitoneal injection (compound

AD(OMe)AV, 20 mg/kg,) were measured by HPLC with tandem

mass spectrometry (LC−MS). The mice were sacriﬁced by cervical

dislocation, and the brain tissue was rapidly removed at the

aforementioned time, rinsed with cold normal saline, and weighed.

Then, the whole brain was quickly homogenized on ice using a glass

homogenizer and ultrasounded for 10 min. The ice-cold mixture (300

μL, methanol/acetonitrile = 1:1) along with 10 μL of the internal

standard solution (ﬂuoxetine) mixture were added to 100 μL of

homogenate to precipitate the proteins. The mixture was centrifuged

at 10 000g at 4 °C for 10 min, and 20 μL of the supernatant was

injected for LC−MS analysis. The analysis was performed on an

Agilent 1200 series LC system using a Sunﬁre Prep C18 column (4.6

mm × 250 mm). The mobile phase consisted of 0.1% formic acid in

water (A) and acetonitrile (B) (70:30, v/v) with a ﬂow rate of 0.25

mL/min. The detection was carried out with electron spray ionization

(ESI) operating at positive ion and multiple reactions monitoring

(MRM) mode. Mass parameters including the precursor ion, product

ion, fragmentor voltage, and collision energy are shown in Table 3.

4.2.7. Surgical Preparation and Infarct Volume Measure-

7,28

.2.2. Fluorescence Polarization Assay.

Fluorescence polar-

ization experiments were conducted in 96-well, black, ﬂat bottom

plates (Corning). The polarization values in milli polarization units

(

mP) were measured by a plate reader (BMG, LABTECH,

POLARstar Omega, Oﬀenburg, Germany). In each well, the ﬁnal

volume was 125 μL of the PBS buﬀer (KH PO 2 mM, Na HPO 8

mM, NaCl 136 mM, KCl 2.6 mM). To determinate the optimal

concentration of the free probe (5-FAM-KV-14) for ﬂuorescence

polarization assay, the polarization values were tested with increasing

concentrations of 5-FAM-KV-14 (from 0.01 nM to 5 μM). To

determine the optimal protein concentration, the GST-nNOS₁₋₁₃₃

concentration was varied from 1 nM to 20 μM at a ﬁxed

concentration of 5-FAM-KV-14 (50 nM). To assay the inﬂuence of

DMSO, increasing concentrations of 1−8% DMSO were added to the

well containing 50 nM 5-FAM-KV-14 and 1 μM GST-nNOS

. To

1−133

test the stability, the polarization values were measured at various time

points over 24 h.

Z′ factor and signal-noise ratio (S/N) statistical experiments were

performed to determine the assay quality and suitability in a group (5-

FAM-KV-14 bound to GST-nNOS₁₋₁₃₃and another group (5-FAM-

KV-14 alone) in 30 replicates, and experiments were repeated two

more times on diﬀerent days. The Z′ factor was calculated using the

following equation:

1,34

ment..

Focal cerebral ischemia was performed in male SD rats

(

Model Animal Research Center of Nanjing University, China)

through intraluminal middle cerebral artery occlusion (MCAO) as

reported in a method previously. Male SD rats (280−320 g) were

randomly divided into four groups (n = 15 per group): (1) sham, (2)

vehicle (I/R), (3) I/R + Che-AD(OMe)AV, (4) I/R+edaravone.

Under chloral hydrate anesthesia (350 mg/kg, i.p.), a 4/0 surgical

nylon monoﬁlament with rounded tip was introduced into the left

internal carotid artery through the external carotid stump and

advanced 20−21 mm past the carotid bifurcation until a slight

resistance was felt. At this point, the intraluminal ﬁlament blocked the

origin of the middle cerebral artery and occluded all sources of blood

ﬂow from the internal carotid artery, anterior cerebral artery, and

posterior cerebral artery. Throughout the procedure, the rat body

temperature was maintained at 37 ± 0.5 °C. The ﬁlament was left in

place for 120 min and then withdrawn for reperfusion. Saline (Sham),

saline (vehicle I/R), Che-AD(OMe)AV (10 mg/kg), and edaravone

(6 mg/kg) were injected through the tail vein immediately after

reperfusion. The infarct volume measurement was performed 24 h

after the MCAO as described previously. Brains were removed rapidly

and frozen at −20 °C for 5 min. Coronal slices were made at 1−2 mm

from the frontal tips, and sections were immersed in 2% 2,3,5-

tripenyltetrazolium chloride (TTC; Sigma-Aldrich) at 37 °C for 30 h.

The infarct volume was expressed as a percentage of the area of the

coronal section in the infracted hemisphere. The infarct areas (white)

in the individual sections were measured and analyzed using Image

software.

Z′ = 1 − (3σ_b_i_n_d_i_n_g+ 3σ_f_r_e_e)/|μ_b_i_n_d_i_n_g− μ_f_r_e_e|;

S/N = (μ_b_i_n_d_i_n_g− μ_f_r_e_e)/σ_f_r_e_e

where σ is the standard deviation, μ is the mean, binding is the FP

value of bound 5-FAM- 5-FAM-KV-14 with GST-nNOS₁₋₁₃₃, and free

is the FP value of free 5-FAM-KV-14.

Competitive binding experiments were performed by adding

increasing concentrations of compounds to a ﬁxed concentration of

-FAM-KV-14 (50 nM) and GST-nNOS₁₋₁₃₃domain (1 μM) at 37

C for 1 h. All graphs were prepared by GraphPad 7.0 (GraphPad

Software, San Diego, CA). All values reported are the average of at

least three individual experiments.

9,30

.2.3. Isothermal Titration Calorimetry (ITC).

All ITC

experiments were performed at 25 °C using an ITC200 micro-

calorimeter (MicroCal Inc., Northampton, MA) with a 200 μL cell

capacity and a 40 μL syringe volume. Protein solution (200 μL) was

added to the sample cell. Injections of 2 μL of ligand (peptides)

solution were added at an interval of 2 min into the GST-nNOS₁₋₁₃₃

solution. A reference titration of ligand into buﬀer was used to correct

for the heat of dilution. Finally, all ITC data was analyzed using the

Origin 7 software provided by MicroCal.

.2.4. Molecular Docking Study. The X-ray crystallographic

.2.8. Statistical Analysis. Data are presented as mean ± SEM.

structures of nNOS PDZ (PDB code: 1B8Q) were obtained from

the PDB (http://www.rcsb.org/pdb/). Discovery Studio (vision 3.5,

Accelrys) was used to perform molecular docking. Che-ADAV bound

into the three-dimensional structure of nNOS PDZ was carried out

following the CDOCKER protocol.

The data were analyzed by one-way ANOVA followed by post hoc

Scheﬀe test. Statistical signiﬁcance was established at a p value <0.05.

ASSOCIATED CONTENT

sı Supporting Information

■

1,32

.2.5. In Vitro PAMPA-BBB Assay.

Carbamazepine and

atenolol were elected as the positive and negative controls,

respectively. The stock solution of the compounds (5 mg/mL) was

prepared in DMSO. The stock solution was diluted to 50 μg/mL with

PBS (1% DMSO) to prepare a test solution. The test solution was

added to every donor well (300 μL per well), and then, the acceptor

well was soaked with 5 μL of porcine polar brain lipid (20 mg/mL) in

https://pubs.acs.org/doi/10.1021/acschemneuro.0c00739.

Figures of MS, H NMR, and HRMS spectra, reversed-

phase HPLC chromatograms, standard curve regression

ACS Chem. Neurosci. 2021, 12, 244−255

ACS Chemical Neuroscience

Huguenard, J. R., Bliss, T. M., and Steinberg, G. K. (2016) Enhanced

Research Article

plots, and chromatograms and table of analytical HPLC

data (PDF )

(3) Hiu, T., Farzampour, Z., Paz, J. T., Wang, E. H., Badgely, C.,

Olson, A., Micheva, K. D., Wang, G., Lemmens, R., Tran, K. V.,

Nishiyama, Y., Liang, X., Hamilton, S. A., O’Rourke, N., Smith, S. J.,

phasic GABA inhibition during the repair phase of stroke: A novel

therapeutic target. Brain 139 (2), 468−480.

AUTHOR INFORMATION

Corresponding Authors

Yajuan Qin − School of Pharmacy, Nanjing Medical

University, Nanjing 211166, China; Phone: +86-25-8686-

486; Email: yjqin@njmu.edu.cn

Tingyou Li − School of Pharmacy, Nanjing Medical

University, Nanjing 211166, China; orcid.org/0000-

■

(4) Nakase, T., Yoshioka, S., and Suzuki, A. (2011) Free radical

scavenger, edaravone, reduces the lesion size of lacunar infarction in

human brain ischemic stroke. BMC Neurol. 11 (1), 39.

(5) Cai, H., Ma, Y., Jiang, L., Mu, Z., Jiang, Z., Chen, X., Wang, Y.,

Yang, G. Y., and Zhang, Z. (2017) Hypoxia response element-

regulated MMP-9 promotes neurological recovery via glial scar

degradation and angiogenesis in delayed stroke. Mol. Ther. 25 (6),

002-4216-2206 ; Phone: +86-25-8686-8486;

Email: l_tingyou@njmu.edu.cn

6) Hertelendy, P., Toldi, J., Fulop, F., and Vecsei, L. (2019)

448−1459.

Authors

Ischemic stroke and kynurenines: Medicinal chemistry aspects. Curr.

Med. Chem. 25 (42), 5945−5957.

Lingling Feng − School of Pharmacy, Nanjing Medical

University, Nanjing 211166, China

Xin Fan − School of Pharmacy, Nanjing Medical University,

Nanjing 211166, China

Liping Zheng − School of Pharmacy, Nanjing Medical

University, Nanjing 211166, China

Yu Zhang − School of Pharmacy, Nanjing Medical University,

Nanjing 211166, China

(7) Nash, K. M., Schiefer, I. T., and Shah, Z. A. (2018) Development

of a reactive oxygen species-sensitive nitric oxide synthase inhibitor

for the treatment of ischemic stroke. Free Radical Biol. Med. 115, 395−

04.

(8) Bauer, V., and Sotnikova, R. (2010) Nitric oxide –the

endothelium-derived relaxing factor and its role in endothelial

functions. Gen. Physiol. Biophys. 29 (4), 319−340.

Lei Chang − School of Pharmacy, Nanjing Medical University,

(9) Zhou, L., Li, F., Xu, H. B., Luo, C. X., Wu, H. Y., Zhu, M. M., Lu,

W., Ji, X., Zhou, Q. G., and Zhu, D. Y. (2010) Treatment of cerebral

ischemia by disrupting ischemia-induced interaction of nNOS with

PSD-95. Nat. Med. 16 (12), 1439−43.

Nanjing 211166, China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschemneuro.0c00739

(10) Li, L. L., Ginet, V., Liu, X., Vergun, O., Tuittila, M., Mathieu,

M., Bonny, C., Puyal, J., Truttmann, A. C., and Courtney, M. J.

(2013) The nNOS-p38MAPK pathway is mediated by NOS1AP

during neuronal death. J. Neurosci. 33 (19), 8185−201.

Author Contributions

Y.Q. and L.F. contributed equally to this work. Y.Q. and T.L.

†

designed the compounds and the whole study. Y.Q., L.F., and

Y.Z. performed the FP assay, analyzed the data, and drafted the

manuscript. L.F., X.F., and L.Z. performed the ITC, in vitro

PAMPA-BBB assay, and in vivo pharmacokinetic study. Y.Q.

performed the molecular docking study. Y.Q., L.F., and C.L.

investigated the neuroprotective ability of the potential

compound by MCAO model. Y.Q., L.F., and T.L. drafted

the main text of the manuscript. All authors have given their

ﬁnal approval for the manuscript.

(11) Wardlaw, J. M., Murray, V., Berge, E., and del Zoppo, G. J.

(

2014) Thrombolysis for acute ischaemic stroke. Cochrane Database

Syst. Rev. 2014 (7), CD000213.

12) Zahuranec, D. B., and Majersik, J. J. (2012) Percentage of acute

Otomo, Y. (2019) Clinical Effects of Early Edaravone Use in Acute

stroke patients eligible for endovascular treatment. Neurology 79 (13),

S22−5.

(13) Enomoto, M., Endo, A., Yatsushige, H., Fushimi, K., and

Ischemic Stroke Patients Treated by Endovascular Reperfusion

Therapy. Stroke 50 (3), 652−658.

(14) Abe, M., Kaizu, K., and Matsumoto, K. (2007) A case report of

acute renal failure and fulminant hepatitis associated with edaravone

administration in a cerebral infarction patient. Ther. Apheresis Dial. 11

Notes

The authors declare no competing ﬁnancial interest.

(

3), 235−40.

ACKNOWLEDGMENTS

■

(

15) Ni, H. Y., Song, Y. X., Lin, Y. H., Cao, B., Wang, D. L., Zhang,

This work was supported by the National Natural Science

Foundation of China (No. 81803349), Natural Science

Foundation of Jiangsu Province (CN) (No. BK20171048).

Y., Dong, J., Liang, H. Y., Xu, K., Li, T. Y., Chang, L., Wu, H. Y., Luo,

C. X., and Zhu, D. Y. (2019) Dissociating nNOS (neuronal NO

synthase)-CAPON (carboxy-terminal postsynaptic density-95/discs

large/zona occludens-1 ligand of nNOS) interaction promotes

functional recovery after stroke via enhanced structural neuro-

plasticity. Stroke 50 (3), 728−737.

ABBREVIATIONS LIST

■

nNOS, neuronal nitric oxide syntheses; CAPON (NOSA1AP),

carboxy-terminal PDZ ligand of nNOS; FP, ﬂuorescence

polarization; ITC, isothermal titration calorimetry; PPI,

protein−protein interaction; PDZ, PSD-95, discs large, zonula

occludens-1; LC−MS, liquid chromatography−mass spectrom-

etry; DCM, dichloromethane; PBS, phosphate-buﬀer saline

(16) Zhu, L. J., Li, T. Y., Luo, C. X., Jiang, N., Chang, L., Lin, Y. H.,

Zhou, H. H., Chen, C., Zhang, Y., Lu, W., Gao, L. Y., Ma, Y., Zhou, Q.

G., Hu, Q., Hu, X. L., Zhang, J., Wu, H. Y., and Zhu, D. Y. (2014)

CAPON-nNOS coupling can serve as a target for developing new

anxiolytics. Nat. Med. 20 (9), 1050−4.

(17) Jaffrey, S. R., Snowman, A. M., Eliasson, M. J., Cohen, N. A.,

and Snyder, S. H. (1998) CAPON: a protein associated with neuronal

nitric oxide synthase that regulates its interactions with PSD95.

Neuron 20 (1), 115−24.

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