Design, Synthesis and Biological Activity of Novel Methoxy- and Hydroxy-Substituted N-Benzimidazole-Derived Carboxamides.

This work presents the design, synthesis and biological activity of novel N-substituted benzimidazole carboxamides bearing either a variable number of methoxy and/or hydroxy groups. The targeted carboxamides were designed to investigate the influence of the number of methoxy and/or hydroxy groups, the type of substituent placed on the N atom of the benzimidazole core and the type of substituent placed on the benzimidazole core on biological activity. The most promising derivatives with pronounced antiproliferative activity proved to be N-methyl-substituted derivatives with hydroxyl and methoxy groups at the phenyl ring and cyano groups on the benzimidazole nuclei with selective activity against the MCF-7 cell line (IC50 = 3.1 µM). In addition, the cyano-substituted derivatives 10 and 11 showed strong antiproliferative activity against the tested cells (IC50 = 1.2-5.3 µM). Several tested compounds showed significantly improved antioxidative activity in all three methods compared to standard BHT. In addition, the antioxidative activity of 9, 10, 32 and 36 in the cells generally confirmed their antioxidant ability demonstrated in vitro. However, their antiproliferative activity was not related to their ability to inhibit oxidative stress nor to their ability to induce it. Compound 8 with two hydroxy and one methoxy group on the phenyl ring showed the strongest antibacterial activity against the Gram-positive strain E. faecalis (MIC = 8 µM).

Novel anthraquinone amide derivatives as potential glyoxalase-I inhibitors.

This study aimed to identify novel Glyoxalase-I (Glo-I) inhibitors with potential anticancer properties, focusing on anthraquinone amide-based derivatives. We synthesized a series of these derivatives and conducted in silico docking studies to predict their binding interactions with Glo-I. In vitro assessments were performed to evaluate the anti-Glo-I activity of the synthesized compounds. A comprehensive structure-activity relationship (SAR) analysis identified key features responsible for specific binding affinities of anthraquinone amide-based derivatives to Glo-I. Additionally, a 100 ns molecular dynamics simulation assessed the stability of the most potent compound compared to a co-crystallized ligand. Compound MQ3 demonstrated a remarkable inhibitory effect against Glo-I, with an IC50 concentration of 1.45 µM. The inhibitory potency of MQ3 may be attributed to the catechol ring, amide functional group, and anthraquinone moiety, collectively contributing to a strong binding affinity with Glo-I. Anthraquinone amide-based derivatives exhibit substantial potential as Glo-I inhibitors with prospective anticancer activity. The exceptional inhibitory efficacy of compound MQ3 indicates its potential as an effective anticancer agent. These findings underscore the significance of anthraquinone amide-based derivatives as a novel class of compounds for cancer therapy, supporting further research and advancements in targeting the Glo-I enzyme to combat cancer.

Design of Mixed Medicinal Plants, Rich in Polyphenols, Vitamins B, and Palmitoylethanolamide-Based Supplement to Help Reduce Nerve Pain: A Preclinical Study.

Neuropathy affects 7-10% of the general population and is caused by a lesion or disease of the somatosensory system. The limitations of current therapies highlight the necessity of a new innovative approach to treating neuropathic pain (NP) based on the close correlation between oxidative stress, inflammatory process, and antioxidant action. The advantageous outcomes of a novel combination composed of Hop extract, Propolis, Ginkgo Biloba, Vitamin B, and palmitoylethanolamide (PEA) used as a treatment was evaluated in this study. To assess the absorption and biodistribution of the combination, its bioavailability was first examined in a 3D intestinal barrier model that replicated intestinal absorption. Further, a 3D nerve tissue model was developed to study the biological impacts of the combination during the essential pathways involved in NP. Our findings show that the combination could cross the intestinal barrier and reach the peripheral nervous system, where it modulates the oxidative stress, inflammation levels, and myelination mechanism (increased NRG, MPZ, ERB, and p75 levels) under Schwann cells damaging. This study proves the effectiveness of Ginkgo Biloba, Propolis, Hop extract, Vitamin B, and PEA in avoiding nerve damage and suggests a potential alternative nutraceutical treatment for NP and neuropathies.

Effect of Phenol and Alkylamide Interaction on α-Glucosidase Inhibition and Cellular Antioxidant Activity during In Vitro Digestion: Using Szechuan Pepper (Zanthoxylum genus) as a Model.

Although recent evidence indicated significant phenol and alkylamide interaction in aqueous solutions, the gastrointestinal digestion influence of the combination remains unclear. This study aims to investigate phenol and alkylamide interaction during in vitro digestion, focusing on bioaccessibility and bioactivity, including α-glucosidase inhibition and cellular antioxidant activity. Additionally, the structural mechanism of phenol and alkylamide interaction during in vitro digestion was explored. The results indicated that the presence of phenols and alkylamides significantly increased or decreased their respective bioaccessibility, depending on the Zanthoxylum varieties. Furthermore, although antagonistic phenol/alkylamide interaction was evident during α-glucosidase inhibition, cellular oxidative stress alleviation, and antioxidant gene transcription upregulation, this effect weakened gradually as digestion progressed. Glycoside bond cleavage and the methylation of phenols as well as alkylamide isomerization and addition were observed during digestion, modifying the hydrogen bonding sites and interaction behavior. This study provided insights into the phenol/alkylamide interaction in the gastrointestinal tract.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

TMV-CP based rational design and discovery of α-Amide phosphate derivatives as anti plant viral agents.

The tobacco mosaic virus coat protein (TMV-CP) is indispensable for the virus's replication, movement and transmission, as well as for the host plant's immune system to recognize it. It constitutes the outermost layer of the virus particle, and serves as an essential component of the virus structure. TMV-CP is essential for initiating and extending viral assembly, playing a crucial role in the self-assembly process of Tobacco Mosaic Virus (TMV). This research employed TMV-CP as a primary target for virtual screening, from which a library of 43,417 compounds was sourced and SH-05 was chosen as the lead compound. Consequently, a series of α-amide phosphate derivatives were designed and synthesized, exhibiting remarkable anti-TMV efficacy. The synthesized compounds were found to be beneficial in treating TMV, with compound 3g displaying a slightly better curative effect than Ningnanmycin (NNM) (EC50 = 304.54 µg/mL) at an EC50 of 291.9 µg/mL. Additionally, 3g exhibited comparable inactivation activity (EC50 = 63.2 µg/mL) to NNM (EC50 = 67.5 µg/mL) and similar protective activity (EC50 = 228.9 µg/mL) to NNM (EC50 = 219.7 µg/mL). Microscale thermal analysis revealed that the binding of 3g (Kd = 4.5 ± 1.9 µM) to TMV-CP showed the same level with NNM (Kd = 5.5 ± 2.6 µM). Results from transmission electron microscopy indicated that 3g could disrupt the structure of TMV virus particles. The toxicity prediction indicated that 3g was low toxicity. Molecular docking showed that 3g interacted with TMV-CP through hydrogen bond, attractive charge interaction and π-Cation interaction. This research provided a novel α-amide phosphate structure target TMV-CP, which may help the discovery of new anti-TMV agents in the future.

Isolation and Culture of Primary Human Mammary Epithelial Cells.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Structure-Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents.

Aminopyrazoles represent interesting structures in medicinal chemistry, and several derivatives showed biological activity in different therapeutic areas. Previously reported 5-aminopyrazolyl acylhydrazones and amides showed relevant antioxidant and anti-inflammatory activities. To further extend the structure-activity relationships in this class of derivatives, a novel series of pyrazolyl acylhydrazones and amides was designed and prepared through a divergent approach. The novel compounds shared the phenylamino pyrazole nucleus that was differently decorated at positions 1, 3, and 4. The antiproliferative, antiaggregating, and antioxidant properties of the obtained derivatives 10-22 were evaluated in in vitro assays. Derivative 11a showed relevant antitumor properties against selected tumor cell lines (namely, HeLa, MCF7, SKOV3, and SKMEL28) with micromolar IC50 values. In the platelet assay, selected pyrazoles showed higher antioxidant and ROS formation inhibition activity than the reference drugs acetylsalicylic acid and N-acetylcysteine. Furthermore, in vitro radical scavenging screening confirmed the good antioxidant properties of acylhydrazone molecules. Overall, the collected data allowed us to extend the structure-activity relationships of the previously reported compounds and confirmed the pharmaceutical attractiveness of this class of aminopyrazole derivatives.

Discovery of novel amide derivatives as potent quorum sensing inhibitors of Pseudomonas aeruginosa.

With the increasing reports of antibiotic resistance in this species, Pseudomonas aeruginosa is a common human pathogen with important implications for public health. Bacterial quorum sensing (QS) systems are potentially broad and versatile targets for developing new antimicrobial compounds. While previous reports have demonstrated that certain amide compounds can inhibit bacterial growth, there are few reports on the specific inhibitory effects of these compounds on bacterial quorum sensing systems. In this study, thirty-one amide derivatives were synthesized. The results of the biological activity assessment indicated that A9 and B6 could significantly inhibit the expression of lasB, rhlA, and pqsA, effectively reducing several virulence factors regulated by the QS systems of PAO1. Additionally, compound A9 attenuated the pathogenicity of PAO1 to Galleria mellonella larvae. Meanwhile, RT-qPCR, SPR, and molecular docking studies were conducted to explore the mechanism of these compounds, which suggests that compound A9 inhibited the QS systems by binding with LasR and PqsR, especially PqsR. In conclusion, amide derivatives A9 and B6 exhibit promising potential for further development as novel QS inhibitors in P. aeruginosa.

Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Insights into the structure-activity relationship of the anticancer compound ZJ-101: A role played by the amide moiety.

ZJ-101, a structurally simplified analog of marine natural product superstolide A, was previously designed and synthesized in our laboratory. In the present study four new analogs of ZJ-101 were designed and synthesized to investigate the structure-activity relationship of the acetamide moiety of the molecule. The biological evaluation showed that the amide moiety is important for the molecule's anticancer activity. Replacing the amide with other functional groups such as a sulfonamide group, a carbamate group, and a urea group resulted in the decrease in anticancer activity.

Synthesis and biological evaluation of Halogen-Substituted novel α-Ketoamides as potential protein aggregation modulators in Alzheimer's disease.

The escalating prevalence of Alzheimer's disease (AD) has prompted extensive research into potential therapeutic interventions, with a specific focus on molecular targets such as amyloid beta (Aß) and tau protein aggregation. In this study, a series of α-ketoamide derivatives was synthesized from ß,γ-unsaturated α-keto thioesters, achieving high purity and good yield. Thioflavin T based Aß aggregation assay identified four promising compounds (BD19, BD23, BD24, and BD27) that demonstrated significant inhibitory effects on Aß aggregation. BD23, selected for its better solubility (0.045 ± 0.0012 mg/ml), was further subjected to in vitro Parallel Artificial Membrane Permeability Assay to determine the Blood-Brain-Barrier permeability and emerged as BBB permeable with permeability rate (Pe) of 10.66 ± 8.11 × 10-6 cm/s. In addition to its Aß inhibitory properties, BD23 exhibited significant inhibition of heparin-induced tau aggregation and demonstrated non-toxicity in SHSY5Y cell lines. Subsequent in vivo assays were conducted, administering compound BD23 to an Aß induced mouse model of AD at various doses (1, 2, & 5 mg/kg). The results revealed a noteworthy enhancement in cognitive functions, particularly when BD23 was administered at a dosage of 5 mg/kg, comparable to the effects observed with the standard dose of Donepezil (DNP). In silico investigations, including molecular docking, molecular dynamics simulations, and Density Functional Theory calculations provided insights into BD23's interactions with the targets and electronic properties. These analyses contribute to the understanding of the therapeutic potential of the lead compounds BD23 which further pave the way for further exploration of its therapeutic potential in the context of AD.

Rho kinase inhibitor Y-27632 downregulates IL-1ß expression in mice with experimental autoimmune myocarditis.

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1ß expression in the myocardial tissue of EAM mice. Notably, IL-1ß expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1ß expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings.

Protein mimetic 2D FAST rescues alpha synuclein aggregation mediated early and post disease Parkinson's phenotypes.

Abberent protein-protein interactions potentiate many diseases and one example is the toxic, self-assembly of α-Synuclein in the dopaminergic neurons of patients with Parkinson's disease; therefore, a potential therapeutic strategy is the small molecule modulation of α-Synuclein aggregation. In this work, we develop an Oligopyridylamide based 2-dimensional Fragment-Assisted Structure-based Technique to identify antagonists of α-Synuclein aggregation. The technique utilizes a fragment-based screening of an extensive array of non-proteinogenic side chains in Oligopyridylamides, leading to the identification of NS132 as an antagonist of the multiple facets of α-Synuclein aggregation. We further identify a more cell permeable analog (NS163) without sacrificing activity. Oligopyridylamides rescue α-Synuclein aggregation mediated Parkinson's disease phenotypes in dopaminergic neurons in early and post disease Caenorhabditis elegans models. We forsee tremendous potential in our technique to identify lead therapeutics for Parkinson's disease and other diseases as it is expandable to other oligoamide scaffolds and a larger array of side chains.

Design, synthesis, insilco study and biological evaluation of new isatin-sulfonamide derivatives by using mono amide linker as possible as histone deacetylase inhibitors.

OBJECTIVE: Aim: To evaluate the cytotoxic activity of newly synthesized a series of novel HDAC inhibitors comprising sulfonamide as zinc binding group and Isatin derivatives as cap group joined by mono amide linker as required to act as HDAC inhibitors. PATIENTS AND METHODS: Materials and Methods: The utilization of sulfonamide as zinc binding group joined by N-alkylation reaction with ethyl-bromo hexanoate as linker group that joined by amide reaction with Isatin derivatives as cap groups which known to possess antitumor activity in the designed of new histone deacetylase inhibitors and using the docking and MTT assay to evaluate the compounds. RESULTS: Results: Four compounds have been synthesized and characterized successfully by ART-FTIR, NMR and ESI-Ms. the compounds were synthesized and characterized by successfully by ART-FTIR, NMR and ESI- Ms. Assessed for their cytotoxic activity against human colon adenocarcinoma MCF-7 (IC50, I=105.15, II=60.00, III=54.11, IV=56.57, vorinostat=28.41) and hepatoblastoma HepG2 (IC50, I=63.91, II=135.18, III=118.85, IV=51.46, vorinostat=37.50). Most of them exhibited potent HDAC inhibitory activity and significant cytotoxicity. CONCLUSION: Conclusions: The synthesized compounds (I, II, III and IV) showed cytotoxicity toward MCF-7 and HepG2 cancer cell lines and their docking analysis provided a preliminary indication that they are viable [HDAC6] candidates.

Synthesis, Pharmacokinetic Profile, Anticancer Activity and Toxicity of the New Amides of Betulonic Acid-In Silico and In Vitro Study.

Betulonic acid (B(O)A) is a pentacyclic lupane-type triterpenoid that widely exists in plants. There are scientific reports indicating anticancer activity of B(O)A, as well as the amides and esters of this triterpenoid. In the first step of the study, the synthesis of novel amide derivatives of B(O)A containing an acetylenic moiety was developed. Subsequently, the medium-soluble compounds (EB171 and EB173) and the parent compound, i.e., B(O)A, were investigated for potential cytotoxic activity against breast cancer (MCF-7 and MDA-MB-231) and melanoma (C32, COLO 829 and A375) cell lines, as well as normal human fibroblasts. Screening analysis using the WST-1 test was applied. Moreover, the lipophilicity and ADME parameters of the obtained derivatives were determined using experimental and in silico methods. The toxicity assay using zebrafish embryos and larvae was also performed. The study showed that the compound EB171 exhibited a significant cytotoxic effect on cancer cell lines: MCF-7, A-375 and COLO 829, while it did not affect the survival of normal cells. Moreover, studies on embryos and larvae showed no toxicity of EB171 in an animal model. Compared to EB171, the compound EB173 had a weaker effect on all tested cancer cell lines and produced less desirable effects against normal cells. The results of the WST-1 assay obtained for B(O)A revealed its strong cytotoxic activity on the examined cancer cell lines, but also on normal cells. In conclusion, this article describes new derivatives of betulonic acid-from synthesis to biological properties. The results allowed to indicate a promising direction for the functionalization of B(O)A to obtain derivatives with selective anticancer activity and low toxicity.

Discovery of Novel Anti-Resistance AR Antagonists Guided by Funnel Metadynamics Simulation.

Androgen receptor (AR) antagonists are widely used for the treatment of prostate cancer (PCa), but their therapeutic efficacy is usually compromised by the rapid emergence of drug resistance. However, the lack of the detailed interaction between AR and its antagonists poses a major obstacle to the design of novel AR antagonists. Here, funnel metadynamics is employed to elucidate the inherent regulation mechanisms of three AR antagonists (hydroxyflutamide, enzalutamide, and darolutamide) on AR. For the first time it is observed that the binding of antagonists significantly disturbed the C-terminus of AR helix-11, thereby disrupting the specific internal hydrophobic contacts of AR-LBD and correspondingly the communication between AR ligand binding pocket (AR-LBP), activation function 2 (AF2), and binding function 3 (BF3). The subsequent bioassays verified the necessity of the hydrophobic contacts for AR function. Furthermore, it is found that darolutamide, a newly approved AR antagonist capable of fighting almost all reported drug resistant AR mutants, can induce antagonistic binding structure. Subsequently, docking-based virtual screening toward the dominant binding conformation of AR for darolutamide is conducted, and three novel AR antagonists with favorable binding affinity and strong capability to combat drug resistance are identified by in vitro bioassays. This work provides a novel rational strategy for the development of anti-resistant AR antagonists.

Chemical Constituents from the Heartwood of Solanum Verbascifolium L. And Their Anti-Inflammatory Activities Combined Network Pharmacology.

Phytochemical studies on 95 % ethanol extract of the heartwood of Solanum verbascifolium L. resulted in the isolation of one new amide derivative (1), and 21 known phenylpropanoids compounds. The structures were characterized by spectral analysis and high-resolution mass spectrometric analysis. The anti-inflammatory activity of amide compounds 1-4 and 6-9 by investigating their impact on the release of nitric oxide (NO) in MH-S cells. Our findings unveiled significant inhibitory effects on NO secretion. Compound 1 exhibited robust dose-dependent suppression, with pronounced inhibition observed at both 20 µM (P<0.01) and 40 µM (P<0.01). Furthermore, compound 9 demonstrated noteworthy inhibitory effects at 40 µM (P<0.01). Similarly, compounds 3 and 4 displayed substantial inhibition of NO secretion at the same concentration, although the significance level was slightly lower (P<0.05). It is expected that there is a substantial association between the anti-inflammatory activities of amides and their targets, specifically PTGS2, by combining network pharmacology and molecular docking techniques. This discovery emphasizes amides' potential as an interesting subject for additional study in the realm of anti-inflammatory medications.

Synthesis and in†vitro Anti-Inflammatory Activity of Novel Dendrobine Amide/Sulfonamide Derivatives.

A traditional Chinese medicine ingredient, dendrobine, has been demonstrated to have anti-inflammatory properties. However, due to its poor anti-inflammatory properties, its clinical use is limited. Consequently, we have designed and synthesized 32 new amide/sulfonamide dendrobine derivatives and screened their anti-inflammatory activities in vitro. Experiments showed that nitric oxide (NO) generation in lipopolysaccharide (LPS)-induced RAW264.7 cells was strongly reduced by derivative 14, with an IC50 of 2.96 µM. Western blot research revealed that 14 decreased the concentration-dependent expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS). Molecular docking was used to predict the binding of the inflammation-associated proteins COX-2 and INOS to compound 14.

Amide-to-chloroalkene substitution for overcoming intramolecular acyl transfer challenges in hexapeptidic neuromedin U receptor 2 agonists.

CPN-116 is a peptidic agonist that activates human neuromedin U receptor type 2 (NMUR2) but suffers from chemical instability due to inherent backbone isomerization on the Dap residue. To address this, a Leu-Dap-type (Z)-chloroalkene dipeptide isostere was synthesized diastereoselectively as a surrogate of the Leu-Dap peptide bond to develop a (Z)-chloroalkene analogue of CPN-116. The synthesized CPN-116 analogue is stable in 1.0 M phosphate buffer (pH 7.4) without backbone isomerization and can activate NMUR2 with similar potency to CPN-116 at nM concentrations (EC50 = 1.0 nM).