Design, synthesis and biological evaluation of brain-targeted thiamine disulfide prodrugs of ampakine compound LCX001

Article abstract of DOI:10.3390/molecules21040488

: Ampakine compounds have been shown to reverse opiate-induced respiratory depression by activation of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors. However, their pharmacological exploitations are hindered by low blood-brain barrier (BBB) permeability and limited brain distribution. Here, we explored whether thiamine disulfide prodrugs with the ability of "lock-in" can be used to solve these problems. A series of thiamine disulfide prodrugs 7a-7f of ampakine compound LCX001 was synthesized and evaluated. The trials in vitro showed that prodrugs 7e, 7d, 7f possessed a certain stability in plasma and quickly decomposed in brain homogenate by the disulfide reductase. In vivo, prodrug 7e decreased the peripheral distribution of LCX001 and significantly increased brain distribution of LCX001 after i.v. administration. This compound showed 2.23- and 3.29-fold greater increases in the AUC0-t and MRT0-t of LCX001 in brain, respectively, than did LCX001 itself. A preliminary pharmacodynamic study indicated that the required molar dose of prodrug 7e was only one eighth that of LCX001 required to achieve the same effect in mice. These findings provide an important reference to evaluate the clinical outlook of ampakine compounds.

Full text of DOI:10.3390/molecules21040488

molecules
Article
Design, Synthesis and Biological Evaluation of
Brain-Targeted Thiamine Disulfide Prodrugs of
Ampakine Compound LCX001
Dian Xiao, Fan-Hua Meng, Wei Dai, Zheng Yong, Jin-Qiu Liu, Xin-Bo Zhou * and Song Li
Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and Toxicology,
Beijing 100850, China; be_xiaodian@163.com (D.X.); mengfh331@126.com (F.-H.M); twotou3@126.com (W.D.);
yongzhabc@126.com (Z.Y.); fri_autumn@163.com (J.-Q.L.); lis@bmi.ac.cn (S.L.)
* Correspondence: hapwave@163.com; Tel.: +86-10-6693-1634
Academic Editor: Jean Jacques Vanden Eynde
Received: 26 February 2016; Accepted: 8 April 2016; Published: 14 April 2016
Abstract: Ampakine compounds have been shown to reverse opiate-induced respiratory depression
by activation of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors.
However, their pharmacological exploitations are hindered by low blood-brain barrier (BBB)
permeability and limited brain distribution. Here, we explored whether thiamine disulfide prodrugs
with the ability of “lock-in” can be used to solve these problems. A series of thiamine disulfide
prodrugs 7a
–7f of ampakine compound LCX001 was synthesized and evaluated. The trials in vitro
showed that prodrugs 7e
,
7d 7f possessed a certain stability in plasma and quickly decomposed in
,
brain homogenate by the disulfide reductase. In vivo, prodrug 7e decreased the peripheral distribution
of LCX001 and significantly increased brain distribution of LCX001 after i.v. administration. This
compound showed 2.23- and 3.29-fold greater increases in the AUC0-t and MRT0-t of LCX001 in
brain, respectively, than did LCX001 itself. A preliminary pharmacodynamic study indicated that
the required molar dose of prodrug 7e was only one eighth that of LCX001 required to achieve the
same effect in mice. These findings provide an important reference to evaluate the clinical outlook of
ampakine compounds.
Keywords: opiate; respiratory depression; ampakines; thiamine disulfide prodrugs; brain distribution
1. Introduction
The use of opioid analgesics is still the most effective way to manage moderate to severe pain in
the clinic [1]. Nevertheless, opiate-induced respiratory depression is a life-threatening condition and a
leading cause of death that may arise not only from overdoses, but also during routine procedures
supervised by clinicians, including surgical anaesthesia, post-operative analgesia, and as a result of
routine out-patient management of pain from cancer, accidents, or other illnesses [
and related opioid antagonists are currently used to counter respiratory depression in an emergency
setting [ ]. However, those agents reverse analgesia, so selective antagonism of the respiratory
2–5]. Naloxone
6
depressive effects of opioids without decreasing their analgesic effects would be a major step toward
improving opioid safety.
Recent studies demonstrated that AMPA receptor was a critical component for the generation of
respiratory rhythm within the pre-Botzinger complex [7–10], and was becoming an ideal target for
reversing respiratory depression. Ampakine compounds, developed by Cortex Pharmaceuticals, such
as CX717, CX1739 and CX1942 etc., had been proved to modulate respiratory drive and rhythmogenesis
positively by activation of AMPA receptor [11
–18]. Most advanced ampakine compounds are CX717
and CX1739 [19], both of which are currently in Phase II clinical trials. Studies demonstrated that a
Molecules 2016, 21, 488; doi:10.3390/molecules21040488
www.mdpi.com/journal/molecules

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single oral dose of 1500 mg of CX717 had positive effects on respiratory depression induced by pain
relieving opiates, without affecting the pain relieving effects [14]. The reversal degree of the basal
respiratory rate was similar to that obtained with the opioid antagonist, naloxone. Similar studies in
respiratory depression with CX1739 at a high dose of 900 mg have also been planned [20].
Structurally these compounds belong to the benzamide derivative type
specific structures remained unknown. According to Cortex’s patents [21 22], we found that compound
(Figure 1, LCX001) was one of the most active compounds, with the best electrophysiological effect
1 (Figure 1), but their
,
2
giving a 21% increase in amplitude of the field excitatory postsynaptic potential (EPSP) in the rat
dentate gyrus and producing a 100% inhibition of damphetamine- stimulated locomotion in the
behavioral testing [21], so we infered that LCX001 was probably an analogue of the clinical compounds
or even one of them. To date, no further developments for these compounds were reported for this
indication. We assumed that besides poor absorption the low blood-brain barrier permeability (tPSA
of LCX001: 74.49) and limited brain distribution might hinder the development of the compounds. As
a result, high-dose use of drugs and non-targeting distribution could cause serious safety problems.
In 2007, Food and Drug Administration (FDA) even rejected an investigational new drug (IND)
application for CX717 as a treatment for attention-deficit hyperactivity disorder (ADHD) because of its
toxicity after chronic dosing at very high dose levels in animals.
Figure 1. Structure of benzamide derivatives, LCX001, thiamine disulfide, DOPA prodrug, naproxen
prodrug, ibuprofen prodrug and the designed prodrugs of LCX001.
The thiamine disulfide system (TDS), has been widely applied as a classic brain-targeted prodrug
strategy to a variety of drugs such as DOPA, naproxen, ibuprofen (Figure 1) etc. [23–25]. In this
approach, the increased lipophilic nature of the prodrugs could facilitate passive diffusion across the
BBB; subsequently, TDS was reduced and ring-closed to be a thiazolium by disulfide reductase once
across the BBB, and then the prodrugs with this system were “locked” in the brain where they can
provide sustained release of the active drug via hydrolysis (Figure 2) [23].
In this paper, we introduced the lipophilic TDS with “lock-in” ability to mediate the penetration
of LCX001 and designed some brain-targeted prodrugs 7a–7f (Figure 1), which were expected to
have synergism and attenuation effects and also provide an important reference value to evaluate the
clinical outlook of ampakine compounds. These designed compounds were sequentially synthesized.
The in vitro stabilities of these prodrugs were discussed. Furthermore, this account also included the
biodistribution and pharmacodynamic study of these prodrugs in mice after i.v. administration.

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Figure 2. Sequential metabolism and brain “lock-in” pathways of brain-targeted thiamine disulfide
prodrugs of LCX001.
2. Results and Discussion
2.1. Chemistry
The synthetic routes for the prodrugs 7a–7f are shown in Scheme 1. As shown, N,4-dimethyl-5-
(2-(hydroxy)ethyl)thiazolium iodide (
4-methylthiazole ( ). Halohydrocarbons 10a
refluxed for 7 h to afford intermediates 11a
by the reaction of N,4-dimethyl-5-(2-(hydroxy)ethyl) thiazolium iodide (
in the presence of NaOH at room temperature. Thiamine disulfide derivatives 12a
with glutaric anhydride to give the requisite intermediate 13a 13f. These compounds were conjugated
with LCX001 in the presence of EDCI, HOBT, DMAP and triethylamine to afford the prodrugs 7a 7f
The structures of all the synthesized prodrugs 7a 7f have been confirmed by NMR, and MS, and all
9
) can be obtained through methylation of 5-(2-hydroxyethyl)-
10f and sodium thiosulfate pentahydrate were mixed and
11f. Thiamine disulfide derivatives 12a 12f were obtained
) with intermediates 11a 11f
12f further react
8
–
–
–
9
–
–
–
–
.
–
the corresponding data can be found in the Experimental Section.
Scheme 1. General synthetic route for synthesis of compounds 7a–7f. Reagents and conditions:
(i) iodomethane, 50 ˝C, 2 h, 96.2%; (ii) Na2S2O3 5H2O , ethanol/H2O, 110 ˝C, 7 h; (iii) NaOH, H2O,
¨
rt, 1 h, two steps, 13.3%–70.5%; (iv) glutaric anhydride, DMAP, DCM, 50 ˝C, 5 h; (v) LCX001, DMAP,
HOBT, EDCI, TEA, DCM, 50 ˝C, 5 h, 38.3%–57.5%.

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2.2. Metabolism Studies of the Prodrugs
2.2.1. Metabolic Stability
To determine the plasma stability and brain stability of these prodrugs, we assayed the
pharmacokinetics of the prepared compounds 7a 7f in mice plasma extract and brain homogenate at
–
˝
37 C. The pseudo-first order rate constants of disappearance (Ke) of the prodrugs and their half lives
(t1/2) which were calculated by linear regression of Ln of peak area against time in minutes are listed
in Table 1.
Table 1. Metabolic stability of compounds 7a
–
7f in mice plasma extract and brain homogenate at 37 ˝C.
Kinetic Constants
Compound
Biological Matrix
R
Ke (min´1) (ˆ10´2
)
t1/2 (min)
Plasma
Brain
31.1
19.9
2.23
3.48
7a
7b
7c
Plasma
Brain
7.06
6.82
9.82
10.17
Plasma
Brain
6.77
4.05
10.24
17.10
Plasma
Brain
4.50
6.34
15.41
10.93
7d
7e
Plasma
Brain
4.34
3.69
15.97
18.78
15.47
11.99
Plasma
Brain
4.48
5.78
7f
In plasma, compounds 7a–7f decomposed at a high rate. This instability is due to the existence
of ester and disulfide linkages in 7a
–
7f. The stability of these prodrugs had a descending trend of
7d (t1/2 = 15.41 min) > 7c (t1/2 = 10.24 min) > 7b (t1/2 = 9.82 min) > 7a (t1/2 = 2.23 min), indicating
that the longer the carbon chain, the better the plasma stability. Larger steric hinderance produced by
longer carbon chain hindered the reduction of the disulfide bond. The half-lives of 7d and 7e were
similar, suggesting that substitution in the side chain had little influence on the plasma stability. We
tried to further increase the plasma stability of the compounds by introducing the benzyl group, but
no effect was obvious. In spite of a relatively high metabolic rate, it is reasonable to give enough time
(t1/2 = 15.41 min–15.97 min) for the compounds 7d, 7e, 7f to be distributed and reach the brain before
complete decomposition.
In brain homogenate, all the compounds decomposed swiftly (t1/2 = 3.48 min–18.78 min) probably
due to the abundance of disulfide reductase in brain. They underwent the expected reduction and
cyclization of the thiamine disulfide component to the corresponding thiazolium in brain homogenate.
These compounds could be reduced to strongly polar products with high rates, and thus, contributed
to the “lock-in” effect in the brain before they were effluxed to the periphery.
2.2.2. Release of the Parent Drug by Prodrugs in Brain Homogenate
To study the release of LCX001 by the prodrugs in brain, the peak in the LC-MS/MS corresponding
to the parent compound was also analyzed. A certain increase in peak area over time was observed,
indicating that the prodrugs were indeed converted to the parent drug by esterases in mice brain
homogenate. Although the deliverance of LCX001 seemed not fast, the concentration increased steadily
as time went by, which might indicate sustained release of LCX001 by the prodrugs in the brain. The

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release rates of different produgs were similar, which indicated that the release process of parent drug
was unlikely to be seriously affected by the formation of a thiazolium cation. Peak integrals were
normalized by dividing by the area for the peak of each prodrug itself at 5 min. The ratios are shown
in Figure 3.
Figure 3. Deliverence of LCX001 by the prodrugs in brain homogenate.
Several conclusions could be drawn from the above results: first, the half-lives of the prodrugs
were mainly influenced by the length of the carbon chains linked to the disulfide functional group.
Second, disulfide reductase produced reduction and ring closure of compounds 7a–7f predominated
over their clearance in brain with high efficiency, since the decomposition of the prodrugs consisted
of reduction and hydrolysis, and the hydrolysis occurs at a relatively low rate. Finally, the release
process of the parent drug was unlikely to be seriously affected by the formation of a thiazolium ion.
As the above in vitro studies demonstrated that prodrugs 7e, 7d, 7f possess preliminary and favorable
physicochemical properties, we selected compound 7e as a model compound to proceed with an
in vivo study.
2.3. In Vivo Studies
2.3.1. Pharmacokinetics in Plasma of the Prodrug 7e
To understand the in vivo behavior of the prodrug, we assessed the plasma pharmacokinetics of
LCX001 and prodrug 7e in mice (Figure 4).
The compounds were injected through the caudal vein of the mice with a single dose equivalent
to 1 mg/kg body weight of LCX001, and then blood was collected to analyze the concentration of
LCX001 at different intervals by an LC-MS/MS method. The pharmacokinetic parameters of LCX001
in blood are reported in Table 2.
Compared with the concentration curve of LCX001 after administration of underivatized LCX001,
prodrug 7e exhibited significantly lower concentrations at different time intervals. Free LCX001 of
prodrug 7e and LCX001 presented with an area under the concentration-time profile (AUC0-t) ratio
of 0.40 and the mean residence times (MRT) of LCX001 in plasma after i.v. administration of 7e was
0.77 times that of LCX001, indicating that 7e could increase brain distribution because of its increased
lipophilicity and be cleared more quickly than LCX001 due to the strongly polar thiazolium cation
produced by reduction of the corresponding thiamine disulfides.

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Figure 4. Concentration curve of LCX001 in plasma versus time after administration of LCX001 and
prodrug 7e. Error bars show the value of SD (n = 3).
Table 2. Pharmacokinetic parameters of LCX001 in plasma after administration of LCX001 and
prodrug 7e (n = 3).
Compound
Cmax (ng/mL)
AUC0-t (h¨ ng/mL)
Tmax (h)
t1/2 (h)
MRT0-t (h)
LCX001
7e
833.67 ˘ 79.85
951.66 ˘ 115.52
0.08
0.08
0.53 ˘ 0.17
0.48 ˘ 0.20
1.08 ˘ 0.04
590.36 ˘ 99.90 *
384.16 ˘ 39.21 *
0.83 ˘ 0.03 *
* p < 0.05 with respect to naked LCX001.
2.3.2. Brain Distribution of Prodrug 7e
To further evaluate the possibility of prodrug 7e being transported across the BBB, the brain
distribution of prodrug 7e and LCX001 after injection were detected at fixed time intervals. The
concentrations of LCX001 in brain versus time curves are displayed in Figure 5. The curves show that
prodrug 7e exhibited much higher distribution in brain. The pharmacokinetic parameters of LCX001
in brain are reported in Table 3. The data showed that Cmax of LCX001 in brain after i.v. administration
of prodrug 7e was not significantly improved, but the AUC0-t and MRT0-t of LCX001 was significantly
higher than that after the injection of the parent compound LCX001. The AUC0-t for prodrug 7e was
enhanced to 2.23 times that of LCX001. The MRT0-t was increased to 3.29 times that of LCX001. The
curves displayed that LCX001 could be quickly metabolized or effluxed while prodrug 7e showed a
certain stability. This agreed well with the fact that the “lock-in” effect and sustained release effect of
the prodrug 7e could effectively delay the clearance of LCX001.
2.4. Pharmacodynamic Study of Prodrug 7e
To investigate the effect of 7e on opiate-induced respiratory depression, we proceeded with a
preliminary pharmacodynamic study in mice. The potent opiate receptor agonist 030418 (Ki = 0.91 nM
for µ-opioid receptor) [26] was administered to induce fatal apneas. Varying doses of prodrug 7e
were administered 10 min before 030418 administration. The effect of prodrug 7e and LCX001 on
opiate-induced respiratory depression was evaluted by the mortality of mice. The results are reported
in Table 4. The data suggested that prodrug 7e reduced the death rate in a dose-dependent manner.
Moreover, 3 mg/kg prodrug 7e gave the same effect as 10 mg/kg LCX001 on reducing mortality, which
indicated that the required molar dose of 7e was only one eighth of that of LCX001 to achieve the same
effect. Further pharmacodynamic studies are currently underway.

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Figure 5. Concentration curve of LCX001 in brain versus time after administration of LCX001 and
prodrug 7e. Error bars show the value of SD (n = 3).
Table 3. Pharmacokinetic parameters of LCX001 in brain after administration of LCX001 and prodrug
7e (n = 3).
Compound
Cmax (ng/g)
AUC0-t (h¨ ng/g)
Tmax (h)
t1/2 (h)
MRT0-t (h)
LCX001
7e
609.40 ˘ 73.66
454.53 ˘ 74.69
504.32 ˘ 204.71
0.08
0.17
0.59 ˘ 0.08
0.70 ˘ 0.22
1125.49 ˘ 336.57 *
2.50 ˘ 0.42 *
2.30 ˘ 0.73 *
* p < 0.05 with respect to naked LCX001.
Table 4. Mortality of opiate poisoned mice after administration of LCX001 and prodrug 7e.
Dose
(mg/kg)
Death
Number (n)
Experiment
Treatment
Test Material
n
ROUTE
Death Rate
RD
RD
RD
RD
RD
RD
RD
030418, 15 mg/kg, sc
030418, 15 mg/kg, sc
030418, 15 mg/kg, sc
030418, 15 mg/kg, sc
030418, 15 mg/kg, sc
030418, 15 mg/kg, sc
030418, 15 mg/kg, sc
Saline
LCX001
7e
7e
7e
7e
7e
10
10
10
10
20
10
10
iv
iv
iv
iv
iv
iv
iv
8
2
0
0
4
3
5
80%
20%
0%
10
30
10
3
1
0.5
0%
20%
30%
50%
RD—Respiratory depression; sc—subcutaneous injection, iv—intravenous injection.
3. Experimental Section
3.1. General Information
TLC was performed using precoated silica gel plates (Yantai dexin Bio-Technology Co., Ltd.,
Yantai, China). Column chromatography was performed using silica gel (200–300 mesh; Yantai
Chemical Industry Research Institute, Yantai, China). Melting points were measured on a RY-1
apparatus (Tianda Tianfa Technology Co., Ltd., Tianjin, China). NMR spectra were recorded on a
JNM-ECA-400 400 MHz spectrometer (JEOL Ltd., Tokyo, Japan) using CDCl3, DMSO-d6 and D2O as
solvent. Chemical shifts are expressed in
δ (ppm), with tetramethylsilane (TMS) functioning as the
internal reference, coupling constants (J) were expressed in Hz. Mass spectroscopy (MS) was carried
out on an API 3000 triple-quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada) equipped

Molecules 2016, 21, 488
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with a Turbo IonSpray electrospray ionization (ESI) source was used for mass analysis and detection.
Analyst 1.4 software (AB Sciex) was used for data acquisition.
3.2. Liquid Chromatographic Conditions
An Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) consisting of a vacuum
degasser, a quaternary pump, and an autosampler was used for HPLC solvent and sample delivery.
Chromatographic separation was achieved using a Poroshell 120 C18 column (50 mm
ˆ
2.1 mm, 2.7 µm,
Agilent) and a C18 guard column (4 3.0 mm, 5 m; Phenomenex, Torrance, CA, USA). The column
ˆ
µ
˝
temperature was maintained at 25 C. Gradient elution was performed using purified water as solvent
A and acetonitrile as solvent B. Two elution gradient programs were used. The program applied for
in vitro samples was as follows: 0.0–0.5 min 10% B to 80% B; 0.5–1.5 min 80% B; 1.5–1.6 min 80% B to
10% B; 1.6–6.0 min 10% B. And the program applied for in vivo samples was as follows: 0.0–0.5 min
10% B to 80% B; 0.5–1.3 min 80% B; 1.3–1.5 min 80% B to 10% B; 1.5–7.0 min 10% B. The flow rate was
set at 0.3 mL/min. The injection volume was 10 µL.
3.3. Mass Spectrometric Conditions
The mass spectrometer was operated in the positive-ion mode. Ultrapure nitrogen was used as
a nebulizer, curtain, and collision-activated dissociation (CAD) gas at 8, 10, and 6 instrument units,
respectively. The optimized Turbo IonSpray voltage and temperature were set to 4500 V and 450 ˝C,
respectively. Quantification was performed with multiple reaction monitoring. Glipizide was chosen
as internal standard. The transitions and the optimized collision energies (CE) for each compound
were listed in Table 5. Each transition was monitored with a 150-ms dwell-time.
Table 5. Mass spectrometric conditions for the compounds.
[M + H]+
Precursor ion
Product ion
CE (V)
LCX001
Glipizide
276.2
446.0
607.5
621.5
635.5
649.5
649.5
669.5
178.2
321.0
258.2
258.2
258.2
258.2
258.2
258.2
20
20
25
26
27
29
29
29
7a
7b
7c
7d
7e
7f
3.4. Stability in Plasma Extract and Brain Homogenate
Blood was drawn from mice though the orbital sinus and was collected in a heparinized tube
paved with heparin sodium. Samples were centrifuged at 10,000 rpm for 16 min to separate plasma
which was diluted with four volumes of water. The brain was removed and homogenized. Samples
were then placed on ice and used immediately. Ten
compound was added to 50 L of plasma or brain homogenate and gently vortexed. Samples were
incubated at 37 C and aliquots were removed after 0, 5, 15, 30, 60 and 90 min, respectively. One
hundred L acetonitrile was added to each and vortexed. Samples were centrifuged for 10 min to
µL acetonitrile solution (10 µg/mL) of the
µ
˝
µ
remove proteins and the supernatants were analyzed by LC-MS/MS method as described.
3.5. Release of the Parent Drug by Prodrugs in Brain Homogenate
The prodrugs were treated in brain homogenate according to the procedure in Section 3.4 except
that the intervals were changed to 0, 5, 15, 30 and 60 min. The peak of LCX001 was analyzed by
LC-MS/MS described in Sections 3.2 and 3.3. Peak integrals were normalized by dividing by the area
for the peak of each prodrug itself at 5 min.

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3.6. Biodistribution Studies in Vivo
3.6.1. Test Animals
Adult Kunming mice weighing 20–22 g were obtained from the animal center of academy of
military medical sciences. The animals were left for two days to acclimatize to animal room conditions
and were maintained on standard pellet diet and water ad libitum. Food was withdrawn on the day
before the experiment, but free access to water was allowed. Since the experiments could be completed
within 24 h, there was no significant change in the mice body weight during the experiment. All
animals received human care, and the study protocols complied with the guidelines of the animal
center of academy of military medical sciences. Throughout the experiments, the animals were handled
according to the international ethical guidelines for the care of laboratory animals.
3.6.2. Biodistribution Studies of the Prodrug and LCX001
Mice were randomly divided into two groups, 27 in each group, for different sampling times and
housed in one cage. Each animal was injected with 7e or LCX001 in 20% w/v hydroxypropyl-β-cyclodextrin
and saline (0.45%) solution through the tail vein at a single dose equivalent to 1 mg/kg body weight of
LCX001. At appropriate time intervals (5, 10, 30, 45, 60, 90, 120, 240 and 480 min), the animals were
sacrificed and 1 mL blood samples withdrawn from the orbital sinus were collected in heparinized
tubes. Fifty
and stored at
µL plasma was immediately separated by centrifugation, diluted with 200 µL methanol
´
20 ˝C until assay. Meanwhile, the brain was removed, weigh˝ed, homogenized and
diluted with methanol to 1:5 (g/mL). The homogenates were also stored at
´20 C until assay. Internal
standard (200 ng/mL) was added to the samples. The samples were vortexed for 3 min and then
centrifuged at 9500 rpm for 15 min. Fifty L supernatants were withdrawn, then 50 L of water
L samples were analyzed by LC-MS/MS method
µ
µ
was added to each aliquot and vortexed. Ten
as described.
µ
3.7. Statistical Analysis
The Cmax, AUC0-t, Tmax, MRT0-t and t1/2 were calculated by WinNonlin software (version 5.2.1,
Pharsight, Mountain View, CA, USA). Statistical evaluation was performed using analysis of variance
followed by t-test. A value of p < 0.05 was considered significant.
3.8. Pharmacodynamic Study of Prodrug 7e
3.8.1. Test Animals
A total of 80 male Kunming mice weighing 20–22 g were used in this study. Animals were fed
ad libitum with a commercial rodent feed and had free access to drinking water. All animals received
human care, and the study protocols complied with the guidelines of the animal center of academy of
military medical sciences. Throughout the experiments, the animals were handled according to the
international ethical guidelines for the care of laboratory animals.
3.8.2. Model of Opiate-Induced Respiratory Depression
Respiratory depression in conscious mice was induced by hypodermic (15 mg/kg) administration
of 030418. Ten min prior to 030418 administration, animals were randomly assigned to the experimental
groups and received an intravenous injection of either saline solution (control), LCX001 (10 mg/kg),
prodrug 7e (30 mg/kg, 10 mg/kg, 3 mg/kg, 1 mg/kg or 0.5 mg/kg) (Table 4).
3.8.3. Assessment of the Prodrug 7e Effect
During the experiment, the poisoning time, poisoning symptoms and death time were recorded.
The death rate (death number/sample number) was used to evaluate the effect of 7e on opiate-induced
respiratory depression.

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3.9. Synthesis
N,4-Dimethyl-5-(2-(hydroxy)ethyl)thiazolium iodide (
9). 5-(2-Hydroxyethyl)-4-methylthiazole (100.0 g,
698.2 mmol) and methyl iodide (100.0 mL, 1500.0 mmol) were mixed and refluxed for 2 h. After
evaporation of excess methyl iodide, to the residual brown syrup was added ether (100 mL) and stirred
˝
9 as a pale-yellow solid (191.4 g, 96.2%). Mp: 82–84 C.
for 30 min, the precipitate was filtered to yield
1H-NMR (DMSO-d6, ppm): 2.43 (s, 3H), 3.03 (t, 2H, J = 5.5 Hz), 3.63 (t, 2H, J = 5.6 Hz), 4.09 (s, 3H), 9.96
(s, 1H); MS m/z [M]+ calculated for C7H12NS+: 158.1; found: 158.1.
General procedure for synthesis of S-substituted sodium thiosulfates 11a–11f. A solution of sodium thiosulfate
pentahydrate (24.8 g, 100.0 mmol) in water (60 mL) was added to the solution of alkyl bromide
˝
(100 mmol) in ethanol (30 mL) under stirring. The reation mixture was heated to 110 C and maintained
for 7 h. After evaporation of the solvent, the residual white solid was dried in a vacuum desiccator to
yield S-substituted sodium thiosulfates 11a–11f.
General procedure for synthesis of compounds 12a
quaternary ammonium salt (14.3 g, 50.0 mmol) and sodium hydroxide (4.0 g, 100.0 mmol) was
added S-substituted sodium thiosulfates 11a 11f (120.0 mmol). The reaction mixture was stirred
–12f. Under argon protection, to a 30 mL aqueous of
9
–
for 1 h at room temperature. The resultant oily substance was extracted with ethyl acetate (450 mL)
and the organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure.
Purification of the crude product by column chromatography with dichloromethane-methanol (100:1)
as eluent gave a yellow oil.
N-(3-(Ethyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide (12a, Figure S1). 40.9% yield as a
1
yellow oil. H-NMR (CDCl3, ppm): 1.19 (t, 3H, J = 7.3 Hz), 1.96, 1.86 (2s, 3H), 2.64 (q, 2H, J = 7.3 Hz),
2.70 (t, 2H, J = 6.7 Hz), 2.98, 2.83 (2s, 3H), 3.55–3.43 (m, 2H), 7.96, 7.84 (2s, 1H). MS m/z [M + H]+
calculated for C9H17NO2S2: 236.1; found: 236.2.
N-(3-(Propyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide (12b, Figure S2). 64.8% yield as a
1
yellow oil. H-NMR (CDCl3, ppm): 0.97 (t, 3H, J = 7.3 Hz), 1.69–1.63 (m, 2H), 2.01, 1.96 (2s, 3H), 2.47
(br, 1H), 2.59 (t, 2H, J = 7.6 Hz), 2.87 (t, 2H, J = 6.7 Hz), 3.06, 2.95 (2s, 3H), 3.81 (t, 3H, J = 6.4 Hz), 7.96,
7.92 (2s, 1H); MS m/z [M + H]+ calculated for C10H19NO2S2: 250.1; found: 250.1.
N-(3-(Butyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide (12c, Figure S3). 70.5% yield as a
1
yellow oil. H-NMR (CDCl3, ppm): 0.91(t, 3H, J = 7.3 Hz), 1.43–1.33 (m, 2H), 1.65–1.57 (m, 2H), 2.01,
1.96 (2s, 3H), 2.51 (br, 1H), 2.62 (t, 2H, J = 7.3 Hz), 2.87 (t, 2H, J = 6.7 Hz), 3.06, 2.95 (2s, 3H), 3.81 (t, 2H,
J = 6.8 Hz), 7.96, 7.92 (2s, 1H). MS m/z [M + H]+ calculated for C11H21NO2S2: 264.1; found: 264.2.
N-(3-(Amyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide (12d, Figure S4). 52.0% yield as a
1
yellow oil. H-NMR (CDCl3, ppm): 0.90 (t, 3H, J = 7.3 Hz), 1.36–1.29 (m, 4H), 1.65–1.60 (m, 2H), 2.01,
1.97 (2s, 3H), 2.49 (br, 1H), 2.61 (t, 2H, J = 7.3 Hz), 2.87 (t, 2H, J = 6.7 Hz), 3.06, 2.95 (2s, 3H), 3.80 (t, 2H,
J = 6.8 Hz), 7.96, 7.92 (2s, 1H). MS m/z [M + H]+ calculated for C12H23NO2S2: 278.1; found: 278.0.
N-(3-Isoamyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide (12e, Figure S5). 76.8% yield as a
1
yellow oil. H-NMR (CDCl3, ppm): 1.19 (t, 3H, J = 7.3 Hz), 1.53–1.47 (m, 2H), 1.68–1.61 (m, 1H), 2.01,
1.96 (2s, 3H), 2.49 (br, 1H), 2.62 (t, 2H, J = 7.6 Hz), 2.88 (t, 2H, J = 6.8 Hz), 3.06, 2.95 (2s, 3H), 3.80(t, 3H,
J = 6.4 Hz), 7.96, 7.92(2s, 3H). MS m/z [M + H]+ calculated for C12H23NO2S2: 278.1; found: 278.0.
N-(3-(Benzyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide (12f, Figure S6). 44.1% yield as a
1
yellow oil. H-NMR (d6-DMSO, ppm): 1.91, 1.85 (2s, 3H), 2.62 (t, 2H, J = 6.7 Hz), 2.82, 2.81 (2s, 3H),
3.52–3.47 (m, 2H), 3.92 (s, 2H), 4.74–4.70 (m, 1H), 7.35–7.25 (m, 5H), 7.96, 7.81 (2s, 1H). MS m/z [M + H]+
calculated for C14H19NO2S2: 298.1; found: 298.2.
General procedure for synthesis of 4-(N-methylformamido)pent-3-en-1-yl)oxy)-5-oxopentanoic acids 13a
–13f.
A stirred solution of intermediate 12a 12f (10.0 mmol) in dry dichloromethane (50 mL) was treated
–

Molecules 2016, 21, 488
11 of 14
with DMAP (0.1 g, 0.8 mmol) and glutaric anhydride (3.4 g, 30.0 mmol). The mixture was refluxed
for 5 h. The solvent was evaporated under vacuum and purification of the crude product by column
chromatography with dichloromethane-methanol (100:1) as eluent gave 13a–13f as yellow syrup.
General procedure for synthesis of compounds 7a–7f. The intermediate 13a–13f (4.7 mmol), LCX001
(1.0 g, 3.6 mmol), triethylamine (0.5 mL, 3.6 mmol) and HOBT (486 mg, 3.6 mmol) were dissolved in
dry dichloromethane (50 mL) in a round-bottomed flask under string for 10 min. After addition of
DMAP (44 mg, 0.36 mmol) and EDCI (1.1 g, 5.4 mmol), the mixture was refluxed for 5 h. The resulting
mixture was concentrated by a rotary evaporator to afford yellow oleosus residue that was purified
by column chromatography on silica gel (ethyl acetate/petroleum ether, (1:1)) to provide 7a–7f as
white solids.
S-3-(Ethyldisulfanyl)-4-(N-methylformamido)pent-3-en-1-yl((1R,4R)-4-(N-methylbenzo[c][1,2,5]oxadiazole-5-
˝
carboxamido)cyclohexyl) glutarate (7a, Figure S7). 43.2% yield as a white solid. Mp: 78.8–79.5 C;
1H-NMR (CDCl3, ppm): 1.27 (t, 3H, J = 7.3 Hz), 1.99, 1.97 (2s, 3H), 1.94–1.62 (m, 8H), 2.12–1.99 (m, 2H)
,
2.44–2.35 (m, 4H), 2.63 (q, 2H, J = 7.3 Hz), 3.04–2.87 (m, 8H), 4.70–4.59, 3.51 (m, 1H), 4.24 (t, 2H,
J = 6.7 Hz), 7.41 (d, 1H, J = 8.1 Hz); 13C-NMR (CDCl3, ppm): 172.6, 172.2, 168.6, 162.3, 161.2, 148.4,
139.7, 136.5, 130.6, 117.6, 114.5, 113.6, 71.8, 62.0, 51.9, 33.3, 33.0, 31.8, 30.3, 29.6, 29.6, 29.0, 29.0, 28.2, 26.9,
19.8, 18.4, 14.0. HRMS (ESI+) m/z [M + H]+ calculated for C28H38N4O7S2: 607.2182; found: 607.2255.
S-3-(Propyldisulfanyl)-4-(N-methylformamido)pent-3-en-1-yl((1R,4R)-4-(N-methylbenzo[c][1,2,5]oxadiazole-5-
˝
carboxamido)cyclohexyl) glutarate (7b, Figure S8). 40.7% yield as a white solid. Mp: 78.0–79.3 C;
1H-NMR (CDCl3, ppm): 0.96 (t, 3H, J = 7.3 Hz), 1.94–1.23 (m, 10H), 2.00, 1.97 (2s, 3H), 2.13–2.11
(m, 2H), 2.42–2.26 (m, 4H), 2.58 (t, 2H, J = 6.2 Hz), 3.04–2.88 (m, 8H), 4.70–4.56, 3.50 (m, 1H), 4.25 (t, 2H,
J = 5.6 Hz), 7.42 (d, 1H, J = 8.9 Hz), 7.98–7.83 (m, 3H); 13C-NMR (CDCl3, ppm): 172.7, 172.3, 168.8,
162.3, 161.2, 148.4, 139.7, 136.6, 130.7, 117.5, 114.6, 113.5, 71.8, 62.1, 51.9, 41.1, 33.3, 33.1, 30.4, 29.6, 29.6,
29.0, 29.0, 28.2, 26.9, 21.9, 19.9, 18.5, 12.9. HRMS (ESI+) m/z [M + H]+ calculated for C29H40N4O7S2:
621.2338; found: 621.2411.
S-3-(Butyldisulfanyl)-4-(N-methylformamido)pent-3-en-1-yl((1R,4R)-4-(N-methylbenzo[c][1,2,5]oxadiazole-5-
˝
carboxamido)cyclohexyl) glutarate (7c, Figure S9). 38.3% yield as a white solid. Mp: 67.6–68.4 C;
1H-NMR (CDCl3, ppm): 0.91 (t, 3H, J = 7.6 Hz), 2.00–1.25 (m, 12H), 2.03, 2.00 (2s, 3H), 2.15–2.09
(m, 2H), 2.40–2.26 (m, 4H), 2.61 (t, 2H, J = 7.3 Hz), 3.03–2.87 (m, 8H), 4.72–4.53, 3.51 (m, 1H), 4.25
(t, 2H, J = 7.0 Hz), 7.42 (d, 1H, J = 8.7 Hz), 7.94–7.83 (m, 3H); 13C-NMR (CDCl3, ppm): 172.7, 172.3,
168.6, 162.4, 161.2, 148.3, 139.8 136.6, 130.7, 117.5, 114.6, 113.7, 71.6, 62.1, 52.0, 39.0, 33.4, 33.3, 31,7,
30.4, 29.6, 29.6, 29.0, 29.0, 28.3, 26.9, 21.5, 20.0, 18.5, 13.6. HRMS (ESI+) m/z [M + H]+ calculated for
C30H42N4O7S2: 635.2495; found: 635.2568.
S-3-(Amyldisulfanyl)-4-(N-methylformamido)pent-3-en-1-yl((1R,4R)-4-(N-methylbenzo[c][1,2,5]oxadiazole-5-
˝
carboxamido)cyclohexyl) glutarate (7d, Figure S10). 45.5% yield as a white solid. Mp: 69.3–70.3 C;
1H-NMR (CDCl3, ppm): 0.89 (t, 3H, J = 6.8 Hz), 1.94–1.28 (m, 14H), 2.00, 1.97 (2s, 3H), 2.14–2.09
(m, 2H), 2.41–2.27 (m, 4H), 2.60 (t, 2H, J = 5.8 Hz), 3.04–2.88 (m, 8H), 4.71–4.59, 3.50 (m, 1H), 4.24 (t, 2H,
J = 6.8 Hz), 7.42 (d, 1H, J = 8.4 Hz), 7.94–7.84 (m, 3H); 13C-NMR (CDCl3, ppm): 172.7, 172.2, 168.8,
162.4, 161.2, 148.5, 139.8, 136.6, 130.4, 117.4, 114.6, 113.7, 71.9, 62.1, 51.9, 39.3, 33.4, 33.1, 31.9, 30.5,
30.2, 29.6, 29.6, 29.0, 29.0, 28.3, 26.9, 22.2, 19.2, 18.5, 13.9. HRMS (ESI+) m/z [M + H]+ calculated for
C31H44N4O7S2: 649.2651; found: 649.2724.
S-3-(Isoamyldisulfanyl)-4-(N-methylformamido)pent-3-en-1-yl((1R,4R)-4-(N-methylbenzo[c][1,2,5]oxadiazole-5-
˝
carboxamido)cyclohexyl) glutarate (7e, Figure S11). 45.0% yield as a white solid. Mp: 74.0–75.4 C;
1H-NMR (CDCl3, ppm): 0.89 (d, 6H, J = 6.5 Hz), 1.97–1.47 (m, 11H), 2.00,1.97 (2s, 3H), 2.15–2.09
(m, 2H), 2.41–2.26 (m, 4H), 2.62 (t, 2H, J = 6.7 Hz), 3.04–2.88 (m, 8H), 4.71–4.57, 3.51 (m, 2H), 4.24
(d, 2H, J = 6.4 Hz), 7.42 (d, 1H, J = 8.1 Hz), 9.97–7.83 (m, 3H); 13C-NMR (CDCl3, ppm): 172.7, 172.3,
168.6, 162.4, 161.3, 148.5, 139.7, 136.5, 130.8, 117.4, 114.6, 113.6, 71.8, 62.1, 51.9, 37.6, 37.4, 33.4, 33.1, 31.9,

Molecules 2016, 21, 488
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30.2, 29.6, 29.6, 29.0, 29.0, 28.3, 28.3, 26.9, 22.2, 20.0, 18.5. HRMS (ESI+) m/z [M + H]+ calculated for
C31H44N4O7S2: 649.2651; found: 649.2723.
S-3-(Benzyldisulfanyl)-4-(N-methylformamido)pent-3-en-1-yl((1R,4R)-4-(N-methylbenzo[c][1,2,5]oxadiazole-5-
˝
carboxamido)cyclohexyl) glutarate (7f, Figure S12). 57.5% yield as a white solid. Mp: 72.9–73.8 C;
1H-NMR (CDCl3, ppm): 1.91–1.25 (m, 8H), 1.94, 1.91 (2s, 3H), 2.10–2.03 (m, 2H), 2.41–2.25 (m, 4H),
3.00–2.75 (m, 8H), 4.69–4.59, 3.50 (m, 1H), 3.86 (s, 2H), 4.15 (t, 2H, J = 7.1 Hz), 7.42–7.28 (m, 5H),
7.97–7.83 (m, 3H). 13C-NMR (CDCl3, ppm): 172.6, 172.3, 168.8, 162.4, 161.3, 148.4, 139.7, 136.1, 135.8,
130.4, 129.2, 129.2, 128.5, 128.5, 127.7, 117.7, 114.6, 113.6, 71.8, 62.0, 51.9, 44.2, 33.3, 33.3, 31.9, 30.4, 29.5,
29.5, 28.7, 28.7, 26.9, 19.9, 18.4. HRMS (ESI+) m/z [M + H]+ calculated for C33H40N4O7S2: 669.2338;
found: 669.2411.
4. Conclusions
In this paper, a series of brain-targeted prodrugs of LCX001 was synthesized and evaluated.
The in vitro trials showed that prodrugs 7e
propitious to get sufficient time to be delivered across the BBB. Moreover, prodrugs 7e
,
7d
,
7f possessed a certain stability in plasma which was
7d 7f were
,
,
quickly decomposed in brain homogenate by the disulfide reductase, indicating the “lock-in” effect of
these compounds after they entered the central nervous system (CNS). In vivo, prodrug 7e decreased
the peripheral distribution of LCX001 and significantly increased brain distribution of LCX001 after
i.v. administration. As a result, a lower dosage of prodrug 7e could give the same effect as the parent
drug LCX001 on reversing opiate-induced respiratory depression, which was helpful in promoting the
therapeutic efficacy and safety. More significantly, this prodrug also provides an important reference
value to evaluate the clinical outlook of ampakine compounds.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/4/488/s1.
Acknowledgments: Funding for this study was provided by the Integrated Drug Discovery Technology Platform
(2012ZX09301003-001-006) of National Science and Technology Major Projects for “Major New Drugs Innovation
and Development” in China.
Author Contributions: X.-B.Z., Z.Y. and S.L. conceived the project; D.X. and X.-B.Z. designed the experiments
and executed the chemical synthesis; F.-H.M. and J.-Q.L. carried out the pharmacokinetics experiments; W.D. and
Z.Y. performed the pharmacodynamic experiments; D.X. wrote the paper. All authors discussed the results and
commented on the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
AMPA
BBB
Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
Blood-brain barrier
EPSP
FDA
IND
Excitatory postsynaptic potential
Food and Drug Administration
Investigational new drug
ADHD
TDS
Attention-deficit hyperactivity disorder
Thiamine disulfide system
EDCI
HOBT
DMAP
TEA
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
N-Hydroxybenzotriazole
4-Dimethylaminopyridine
Triethylamine

Molecules 2016, 21, 488
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DCM
rt
Dichloromethane
Room temperature
MRT
RD
sc
iv
NMR
LC
Mean residence times
Respiratory depression
Subcutaneous injection
Intravenous injection
Nuclear Magnetic Resonance
Liquid chromatography
Mass spectrometry
MS
ESI
CAD
CE
Electrospray ionization
Collision-activated dissociation
Collision energies
USA
TMS
United States of America
Tetramethylsilane
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).