You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction {#sec1-molecules-21-00488}
===============

The use of opioid analgesics is still the most effective way to manage moderate to severe pain in the clinic \[[@B1-molecules-21-00488]\]. 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 \[[@B2-molecules-21-00488],[@B3-molecules-21-00488],[@B4-molecules-21-00488],[@B5-molecules-21-00488]\]. Naloxone and related opioid antagonists are currently used to counter respiratory depression in an emergency setting \[[@B6-molecules-21-00488]\]. However, those agents reverse analgesia, so selective antagonism of the respiratory 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 \[[@B7-molecules-21-00488],[@B8-molecules-21-00488],[@B9-molecules-21-00488],[@B10-molecules-21-00488]\], 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 \[[@B11-molecules-21-00488],[@B12-molecules-21-00488],[@B13-molecules-21-00488],[@B14-molecules-21-00488],[@B15-molecules-21-00488],[@B16-molecules-21-00488],[@B17-molecules-21-00488],[@B18-molecules-21-00488]\]. Most advanced ampakine compounds are CX717 and CX1739 \[[@B19-molecules-21-00488]\], both of which are currently in Phase II clinical trials. Studies demonstrated that a 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 \[[@B14-molecules-21-00488]\]. 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 \[[@B20-molecules-21-00488]\].

Structurally these compounds belong to the benzamide derivative type **1** ([Figure 1](#molecules-21-00488-f001){ref-type="fig"}), but their specific structures remained unknown. According to Cortex's patents \[[@B21-molecules-21-00488],[@B22-molecules-21-00488]\], we found that compound **2** ([Figure 1](#molecules-21-00488-f001){ref-type="fig"}, LCX001) was one of the most active compounds, with the best electrophysiological effect 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 \[[@B21-molecules-21-00488]\], 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.

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](#molecules-21-00488-f001){ref-type="fig"}) *etc.* \[[@B23-molecules-21-00488],[@B24-molecules-21-00488],[@B25-molecules-21-00488]\]. 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](#molecules-21-00488-f002){ref-type="fig"}) \[[@B23-molecules-21-00488]\].

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](#molecules-21-00488-f001){ref-type="fig"}), 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.

2. Results and Discussion {#sec2-molecules-21-00488}
=========================

2.1. Chemistry {#sec2dot1-molecules-21-00488}
--------------

The synthetic routes for the prodrugs **7a**--**7f** are shown in [Scheme 1](#molecules-21-00488-sch001){ref-type="scheme"}. As shown, *N*,4-dimethyl-5-(2-(hydroxy)ethyl)thiazolium iodide (**9**) can be obtained through methylation of 5-(2-hydroxyethyl)-4-methylthiazole (**8**). Halohydrocarbons **10a**--**10f** and sodium thiosulfate pentahydrate were mixed and refluxed for 7 h to afford intermediates **11a**--**11f**. Thiamine disulfide derivatives **12a**--**12f** were obtained by the reaction of *N*,4-dimethyl-5-(2-(hydroxy)ethyl) thiazolium iodide (**9**) with intermediates **11a**--**11f** in the presence of NaOH at room temperature. Thiamine disulfide derivatives **12a**--**12f** further react 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 the corresponding data can be found in the [Experimental Section](#sec3-molecules-21-00488){ref-type="sec"}.

2.2. Metabolism Studies of the Prodrugs {#sec2dot2-molecules-21-00488}
---------------------------------------

### 2.2.1. Metabolic Stability {#sec2dot2dot1-molecules-21-00488}

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 (*K*~e~) of the prodrugs and their half lives (*t*~1/2~) which were calculated by linear regression of Ln of peak area against time in minutes are listed in [Table 1](#molecules-21-00488-t001){ref-type="table"}.

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** (*t*~1/2~ = 15.41 min) \> **7c** (*t*~1/2~ = 10.24 min) \> **7b** (*t*~1/2~ = 9.82 min) \> **7a** (*t*~1/2~ = 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 (*t*~1/2~ = 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 (*t*~1/2~ = 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 {#sec2dot2dot2-molecules-21-00488}

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 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](#molecules-21-00488-f003){ref-type="fig"}.

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 {#sec2dot3-molecules-21-00488}
--------------------

### 2.3.1. Pharmacokinetics in Plasma of the Prodrug **7e** {#sec2dot3dot1-molecules-21-00488}

To understand the *in vivo* behavior of the prodrug, we assessed the plasma pharmacokinetics of LCX001 and prodrug **7e** in mice ([Figure 4](#molecules-21-00488-f004){ref-type="fig"}).

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](#molecules-21-00488-t002){ref-type="table"}.

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 (AUC~0-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.

### 2.3.2. Brain Distribution of Prodrug **7e** {#sec2dot3dot2-molecules-21-00488}

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](#molecules-21-00488-f005){ref-type="fig"}. The curves show that prodrug **7e** exhibited much higher distribution in brain. The pharmacokinetic parameters of LCX001 in brain are reported in [Table 3](#molecules-21-00488-t003){ref-type="table"}. The data showed that *C*~max~ of LCX001 in brain after i.v. administration of prodrug **7e** was not significantly improved, but the AUC~0-t~ and MRT~0-t~ of LCX001 was significantly higher than that after the injection of the parent compound LCX001. The AUC~0-t~ for prodrug **7e** was enhanced to 2.23 times that of LCX001. The MRT~0-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*** {#sec2dot4-molecules-21-00488}
----------------------------------------------

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 (K~i~ = 0.91 nM for μ-opioid receptor) \[[@B26-molecules-21-00488]\] 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](#molecules-21-00488-t004){ref-type="table"}. 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.

3. Experimental Section {#sec3-molecules-21-00488}
=======================

3.1. General Information {#sec3dot1-molecules-21-00488}
------------------------

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 CDCl~3~, DMSO-d~6~ and D~2~O 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 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 {#sec3dot2-molecules-21-00488}
--------------------------------------

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 C~18~ column (50 mm × 2.1 mm, 2.7 μm, Agilent) and a C~18~ guard column (4 × 3.0 mm, 5 μm; Phenomenex, Torrance, CA, USA). The column 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 {#sec3dot3-molecules-21-00488}
----------------------------------

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](#molecules-21-00488-t005){ref-type="table"}. Each transition was monitored with a 150-ms dwell-time.

3.4. Stability in Plasma Extract and Brain Homogenate {#sec3dot4-molecules-21-00488}
-----------------------------------------------------

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 μL acetonitrile solution (10 μg/mL) of the compound was added to 50 μL 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 μL acetonitrile was added to each and vortexed. Samples were centrifuged for 10 min to 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 {#sec3dot5-molecules-21-00488}
---------------------------------------------------------------

The prodrugs were treated in brain homogenate according to the procedure in [Section 3.4](#sec3dot4-molecules-21-00488){ref-type="sec"} 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 [Section 3.2](#sec3dot2-molecules-21-00488){ref-type="sec"} and [Section 3.3](#sec3dot3-molecules-21-00488){ref-type="sec"}. Peak integrals were normalized by dividing by the area for the peak of each prodrug itself at 5 min.

3.6. Biodistribution Studies in Vivo {#sec3dot6-molecules-21-00488}
------------------------------------

### 3.6.1. Test Animals {#sec3dot6dot1-molecules-21-00488}

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 {#sec3dot6dot2-molecules-21-00488}

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 μL plasma was immediately separated by centrifugation, diluted with 200 μL methanol and stored at −20 °C until assay. Meanwhile, the brain was removed, weighed, 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 μL supernatants were withdrawn, then 50 μL of water was added to each aliquot and vortexed. Ten μL samples were analyzed by LC-MS/MS method as described.

3.7. Statistical Analysis {#sec3dot7-molecules-21-00488}
-------------------------

The *C*~max~, AUC~0-t~, *T*~max~, MRT~0-t~ and *t*~1/2~ 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*** {#sec3dot8-molecules-21-00488}
----------------------------------------------

### 3.8.1. Test Animals {#sec3dot8dot1-molecules-21-00488}

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 {#sec3dot8dot2-molecules-21-00488}

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](#molecules-21-00488-t004){ref-type="table"}).

### 3.8.3. Assessment of the Prodrug **7e** Effect {#sec3dot8dot3-molecules-21-00488}

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.

3.9. Synthesis {#sec3dot9-molecules-21-00488}
--------------

*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 for 30 min, the precipitate was filtered to yield **9** as a pale-yellow solid (191.4 g, 96.2%). Mp: 82--84 °C. ^1^H-NMR (DMSO-*d*~6~, 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 C~7~H~12~NS^+^: 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**--**12f**. Under argon protection, to a 30 mL aqueous of quaternary ammonium salt **9** (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 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 Na~2~SO~4~ 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](#app1-molecules-21-00488){ref-type="app"}). 40.9% yield as a yellow oil. ^1^H-NMR (CDCl~3~, 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 C~9~H~17~NO~2~S~2~: 236.1; found: 236.2.

*N-(3-(Propyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide* (**12b**, [Figure S2](#app1-molecules-21-00488){ref-type="app"}). 64.8% yield as a yellow oil. ^1^H-NMR (CDCl~3~, 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 C~10~H~19~NO~2~S~2~: 250.1; found: 250.1.

*N-(3-(Butyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide* (**12c**, [Figure S3](#app1-molecules-21-00488){ref-type="app"}). 70.5% yield as a yellow oil. ^1^H-NMR (CDCl~3~, 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 C~11~H~21~NO~2~S~2~: 264.1; found: 264.2.

*N-(3-(Amyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide* (**12d**, [Figure S4](#app1-molecules-21-00488){ref-type="app"}). 52.0% yield as a yellow oil. ^1^H-NMR (CDCl~3~, 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 C~12~H~23~NO~2~S~2~: 278.1; found: 278.0.

*N-(3-Isoamyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide* (**12e**, [Figure S5](#app1-molecules-21-00488){ref-type="app"}). 76.8% yield as a yellow oil. ^1^H-NMR (CDCl~3~, 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 C~12~H~23~NO~2~S~2~: 278.1; found: 278.0.

*N-(3-(Benzyldisulfanyl)-5-hydroxypent-2-en-2-yl)-N-methylformamide* (**12f**, [Figure S6](#app1-molecules-21-00488){ref-type="app"}). 44.1% yield as a yellow oil. ^1^H-NMR (*d*~6~-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 C~14~H~19~NO~2~S~2~: 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 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](#app1-molecules-21-00488){ref-type="app"}). 43.2% yield as a white solid. Mp: 78.8--79.5 °C; ^1^H-NMR (CDCl~3~, 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); ^13^C-NMR (CDCl~3~, 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 C~28~H~38~N~4~O~7~S~2~: 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](#app1-molecules-21-00488){ref-type="app"}). 40.7% yield as a white solid. Mp: 78.0--79.3 °C; ^1^H-NMR (CDCl~3~, 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); ^13^C-NMR (CDCl~3~, 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 C~29~H~40~N~4~O~7~S~2~: 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](#app1-molecules-21-00488){ref-type="app"}). 38.3% yield as a white solid. Mp: 67.6--68.4 °C; ^1^H-NMR (CDCl~3~, 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); ^13^C-NMR (CDCl~3~, 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 C~30~H~42~N~4~O~7~S~2~: 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](#app1-molecules-21-00488){ref-type="app"}). 45.5% yield as a white solid. Mp: 69.3--70.3 °C; ^1^H-NMR (CDCl~3~, 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); ^13^C-NMR (CDCl~3~, 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 C~31~H~44~N~4~O~7~S~2~: 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](#app1-molecules-21-00488){ref-type="app"}). 45.0% yield as a white solid. Mp: 74.0--75.4 °C; ^1^H-NMR (CDCl~3~, 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); ^13^C-NMR (CDCl~3~, 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, 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 C~31~H~44~N~4~O~7~S~2~: 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](#app1-molecules-21-00488){ref-type="app"}). 57.5% yield as a white solid. Mp: 72.9--73.8 °C; ^1^H-NMR (CDCl~3~, 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). ^13^C-NMR (CDCl~3~, 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 C~33~H~40~N~4~O~7~S~2~: 669.2338; found: 669.2411.

4. Conclusions {#sec4-molecules-21-00488}
==============

In this paper, a series of brain-targeted prodrugs of LCX001 was synthesized and evaluated. The *in vitro* trials showed that prodrugs **7e**, **7d**, **7f** possessed a certain stability in plasma which was propitious to get sufficient time to be delivered across the BBB. Moreover, prodrugs **7e**, **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.

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.

**Sample Availability**: Samples of the compounds **7**--**13** are available from the authors.

###### 

Click here for additional data file.

Supplementary materials can be accessed at: <http://www.mdpi.com/1420-3049/21/4/488/s1>.

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.

The authors declare no conflict of interest.

The following abbreviations are used in this manuscript: AMPAAmino-3-hydroxy-5-methyl-4-isoxazolepropionic acidBBBBlood-brain barrierEPSPExcitatory postsynaptic potentialFDAFood and Drug AdministrationINDInvestigational new drugADHDAttention-deficit hyperactivity disorderTDSThiamine disulfide systemEDCI1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlorideHOBT*N*-HydroxybenzotriazoleDMAP4-DimethylaminopyridineTEATriethylamineDCMDichloromethanertRoom temperatureMRTMean residence timesRDRespiratory depressionscSubcutaneous injectionivIntravenous injectionNMRNuclear Magnetic ResonanceLCLiquid chromatographyMSMass spectrometryESIElectrospray ionizationCADCollision-activated dissociationCECollision energiesUSAUnited States of AmericaTMSTetramethylsilane

Figures, Scheme and Tables
==========================

![Structure of benzamide derivatives, LCX001, thiamine disulfide, DOPA prodrug, naproxen prodrug, ibuprofen prodrug and the designed prodrugs of LCX001.](molecules-21-00488-g001){#molecules-21-00488-f001}

![Sequential metabolism and brain "lock-in" pathways of brain-targeted thiamine disulfide prodrugs of LCX001.](molecules-21-00488-g002){#molecules-21-00488-f002}

![General synthetic route for synthesis of compounds **7a**--**7f**. *Reagents and conditions*: (i) iodomethane, 50 °C, 2 h, 96.2%; (ii) Na~2~S~2~O~3~·5H~2~O , ethanol/H~2~O, 110 °C, 7 h; (iii) NaOH, H~2~O, 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%.](molecules-21-00488-sch001){#molecules-21-00488-sch001}

![Deliverence of LCX001 by the prodrugs in brain homogenate.](molecules-21-00488-g003){#molecules-21-00488-f003}

![Concentration curve of LCX001 in plasma *versus* time after administration of LCX001 and prodrug **7e**. Error bars show the value of SD (*n* = 3).](molecules-21-00488-g004){#molecules-21-00488-f004}

![Concentration curve of LCX001 in brain *versus* time after administration of LCX001 and prodrug **7e**. Error bars show the value of SD (*n* = 3).](molecules-21-00488-g005){#molecules-21-00488-f005}

molecules-21-00488-t001_Table 1

###### 

Metabolic stability of compounds **7a**--**7f** in mice plasma extract and brain homogenate at 37 °C.

![](molecules-21-00488-i001.jpg)

  Compound   R                                  Biological Matrix   Kinetic Constants   
  ---------- ---------------------------------- ------------------- ------------------- -------
  **7a**     ![](molecules-21-00488-i002.jpg)   Plasma              31.1                2.23
  Brain      19.9                               3.48                                    
  **7b**     ![](molecules-21-00488-i003.jpg)   Plasma              7.06                9.82
  Brain      6.82                               10.17                                   
  **7c**     ![](molecules-21-00488-i004.jpg)   Plasma              6.77                10.24
  Brain      4.05                               17.10                                   
  **7d**     ![](molecules-21-00488-i005.jpg)   Plasma              4.50                15.41
  Brain      6.34                               10.93                                   
  **7e**     ![](molecules-21-00488-i006.jpg)   Plasma              4.34                15.97
  Brain      3.69                               18.78                                   
  **7f**     ![](molecules-21-00488-i007.jpg)   Plasma              4.48                15.47
  Brain      5.78                               11.99                                   

molecules-21-00488-t002_Table 2

###### 

Pharmacokinetic parameters of LCX001 in plasma after administration of LCX001 and prodrug **7e** (*n* = 3).

  Compound   *C*~max~ (ng/mL)    AUC~0-t~ (h·ng/mL)   *T*~max~ (h)   *t*~1/2~ (h)   MRT~0-t~ (h)
  ---------- ------------------- -------------------- -------------- -------------- ----------------
  LCX001     833.67 ± 79.85      951.66 ± 115.52      0.08           0.53 ± 0.17    1.08 ± 0.04
  **7e**     590.36 ± 99.90 \*   384.16 ± 39.21 \*    0.08           0.48 ± 0.20    0.83 ± 0.03 \*

\* *p* \< 0.05 with respect to naked LCX001.

molecules-21-00488-t003_Table 3

###### 

Pharmacokinetic parameters of LCX001 in brain after administration of LCX001 and prodrug **7e** (*n* = 3).

  Compound   *C*~max~ (ng/g)   AUC~0-t~ (h·ng/g)     *T*~max~ (h)   *t*~1/2~ (h)     MRT~0-t~ (h)
  ---------- ----------------- --------------------- -------------- ---------------- ----------------
  LCX001     609.40 ± 73.66    504.32 ± 204.71       0.08           0.59 ± 0.08      0.70 ± 0.22
  **7e**     454.53 ± 74.69    1125.49 ± 336.57 \*   0.17           2.50 ± 0.42 \*   2.30 ± 0.73 \*

\* *p* \< 0.05 with respect to naked LCX001.

molecules-21-00488-t004_Table 4

###### 

Mortality of opiate poisoned mice after administration of LCX001 and prodrug **7e**.

  Experiment   Treatment              Test Material   *n*   ROUTE   Dose (mg/kg)   Death Number (*n*)   Death Rate
  ------------ ---------------------- --------------- ----- ------- -------------- -------------------- ------------
  RD           030418, 15 mg/kg, sc   Saline          10    iv                     8                    80%
  RD           030418, 15 mg/kg, sc   LCX001          10    iv      10             2                    20%
  RD           030418, 15 mg/kg, sc   **7e**          10    iv      30             0                    0%
  RD           030418, 15 mg/kg, sc   **7e**          10    iv      10             0                    0%
  RD           030418, 15 mg/kg, sc   **7e**          20    iv      3              4                    20%
  RD           030418, 15 mg/kg, sc   **7e**          10    iv      1              3                    30%
  RD           030418, 15 mg/kg, sc   **7e**          10    iv      0.5            5                    50%

RD---Respiratory depression; sc---subcutaneous injection, iv---intravenous injection.

molecules-21-00488-t005_Table 5

###### 

Mass spectrometric conditions for the compounds.

  \[M + H\]^+^   Precursor ion   Product ion   CE (V)
  -------------- --------------- ------------- --------
  LCX001         276.2           178.2         20
  Glipizide      446.0           321.0         20
  **7a**         607.5           258.2         25
  **7b**         621.5           258.2         26
  **7c**         635.5           258.2         27
  **7d**         649.5           258.2         29
  **7e**         649.5           258.2         29
  **7f**         669.5           258.2         29