Enantioselective Effect of Chiral Small Molecules in SARS-CoV-2 Vaccine-Induced Immune Response.

Although numerous chiral small molecules have been discovered and synthesized, the investigation on their enantioselective immunological effects remains limited. In this study, we designed and synthesized a pair of small molecule enantiomers (R/S-ResP) by covalently bonding two immunostimulators (resiquimod/Res) onto a planar chiral framework (paracyclophane/P). Notably, we found that S-ResP exhibits a 4.05-fold higher affinity for toll-like receptor 7 (TLR7) than R-ResP, thereby more effectively enhancing the functions of dendritic cells and macrophages in cytokine secretion and antigen internalization. Furthermore, we observed that S-ResP significantly enhances RBD antigen-induced cross-neutralization against various SARS-CoV-2 strains compared to R-ResP. These findings demonstrate the enantioselective effects of small molecules on regulating vaccine-induced immune responses and emphasize the significance of chirality in designing small molecular adjuvants.

Additive-controlled asymmetric iodocyclization enables enantioselective access to both α- and ß-nucleosides.

ß-Nucleosides and their analogs are dominant clinically-used antiviral and antitumor drugs. α-Nucleosides, the anomers of ß-nucleosides, exist in nature and have significant potential as drugs or drug carriers. Currently, the most widely used methods for synthesizing ß- and α-nucleosides are via N-glycosylation and pentose aminooxazoline, respectively. However, the stereoselectivities of both methods highly depend on the assisting group at the C2' position. Herein, we report an additive-controlled stereodivergent iodocyclization method for the selective synthesis of α- or ß-nucleosides. The stereoselectivity at the anomeric carbon is controlled by the additive (NaI for ß-nucleosides; PPh3S for α-nucleosides). A series of ß- and α-nucleosides are prepared in high yields (up to 95%) and stereoselectivities (ß:α up to 66:1, α:ß up to 70:1). Notably, the introduced iodine at the C2' position of the nucleoside is readily functionalized, leading to multiple structurally diverse nucleoside analogs, including stavudine, an FDA-approved anti-HIV agent, and molnupiravir, an FDA-approved anti-SARS-CoV-2 agent.

Molecular modifications, biological activities, and applications of chitosan and derivatives: A recent update.

Polysaccharides arouse great interest due to their structure and unique properties, such as biocompatibility, biodegradability, and absence of toxicity. Polysaccharides from marine sources are particularly useful due to the wide variety of applications and biological activities. Chitosan, a deacetylated derivative of chitin, is an example of an interesting bioactive marine-derived polysaccharide. Moreover, a wide variety of chemical modifications and conjugation of chitosan with other bioactive molecules are responsible for improvements in physicochemical properties and biological activities, expanding the range of applications. An overview of the synthetic approaches for preparing chitosan, chitosan derivatives, and conjugates is described and discussed. A recent update of the biological activities and applications in different research fields, mainly focused on the last 5 years, is presented, highlighting current trends.

Design, Synthesis, and Application of Chiral Bicyclic Imidazole Catalysts.

ConspectusAsymmetric organocatalysis has been considered to be an efficient and reliable strategy for the stereoselective preparation of optically active chemicals. In particular, chiral tertiary amines as Lewis base organocatalysts bearing core structures including quinuclidine, dimethylaminopyridine (DMAP), N-methylimidazole (NMI), amidine, etc. have provided new and powerful tools for various chemical transformations. However, due to the limitations in structural complexity, synthetic difficulty, low catalytic efficiency, and high cost, the industrial application of such catalysts is still far from being widely adopted. Therefore, the development of new chiral tertiary amine catalysts with higher activity and selectivity is greatly desired.In order to address the contradiction between activity and selectivity caused by the ortho group, a bicyclic imidazole structure bearing a relatively large bond angle ∠θ was designed as the skeleton of our new catalysts. 6,7-Dihydro-5H-pyrrolo[1,2-a]imidazole (abbreviated as DPI) and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (abbreviated as TIP) are two of the utilized skeletons. In addition to obtaining satisfactory catalytic activity, excellent enantioselectivity would also be expected because the stereocontrol R group is neither far nor close to the catalytic active site (sp2-N atom) and is adjustable. Based on this skeleton, a family of chiral bicyclic imidazole catalysts were easily prepared and successfully applied in several enantioselective reactions for the synthesis of a variety of valuable chiral compounds.6,7-Dihydro-5H-pyrrolo[1,2-a]imidazole (abbreviated as DPI) is the predominantly utilized skeleton. First, HO-DPI, the key intermediate of the designed chiral bicyclic imidazole catalysts, could be efficiently synthesized from imidazole and acrolein, then separated by kinetic resolution or optical resolution. Second, Alkoxy-DPI, the alkyloxy-substituted chiral bicyclic imidazole catalysts, were synthesized by a one-step alkylation from HO-DPI. This type of catalyst has been successfully applied in asymmetric Steglich rearrangement (C-acylation rearrangement of O-acylated azlactones), asymmetric phosphorylation of lactams, and a sequential four-step acylation reaction. Third, Acyloxy-DPI, the acyloxy-substituted chiral bicyclic imidazole catalysts, were synthesized with a one-step acetylative kinetic resolution from racemic HO-DPI or acylation from enantiopure HO-DPI. The catalyst AcO-DPI has been successfully applied in enantioselective Black rearrangement and in direct enantioselective C-acylation of 3-substituted benzofuran-2(3H)-ones and 2-oxindoles. Fourth, Alkyl-DPI was synthesized via a two-step reaction from racemic HO-DPI and separated easily by resolution. The catalyst Cy-DPI has been successfully applied in dynamic kinetic resolution of 3-hydroxyphthalides through enantioselective O-acylation. Cy-PDPI was synthesized through a Cu-catalyzed amidation from Cy-DPI and successfully applied in the kinetic resolution of secondary alcohols with good to excellent enantioselectivities. Finally, the carbamate type chiral bicyclic imidazole catalysts, Carbamate-DPI, were readily synthesized from HO-DPI, and the catalyst Ad-DPI bearing a bulky adamantyl group was successfully applied in the synthesis of the anti-COVID-19 drug remdesivir via asymmetric phosphorylation. Alongside our initial work, this Account also introduces four elegant studies by other groups concerning asymmetric phosphorylation utilizing chiral bicyclic imidazole catalysts.In summary, this Account focuses on the chiral bicyclic imidazole catalysts developed in our group and provides an overview on their design, synthesis, and application that will serve as inspiration for the exploration of new organocatalysts and related reactions.

Rh(I)-Catalyzed Allenic Pauson-Khand Reaction to Access the Thapsigargin Core: Influence of Furan and Allenyl Chloroacetate Groups on Enantioselectivity.

Thapsigargin (Tg) is a potent SERCA pump inhibitor with the potential to treat cancer and COVID-19. We have extended the scope of the asymmetric allenic Pauson-Khand reaction to furan-tethered allene-ynes, a stereoconvergent transformation affording the 5,7,5-ring system of Tg in good yields and high enantioselectivity. Computational studies of the oxidative cyclization step show that the furan and chloroacetate groups contribute to this high selectivity.

Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies.

The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe-Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro.

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

Direct and Catalytic C-Glycosylation of Arenes: Expeditious Synthesis of the Remdesivir Nucleoside.

Since early 2020, scientists have strived to find an effective solution to fight SARS-CoV-2, in particular by developing reliable vaccines that inhibit the spread of the disease and repurposing drugs for combatting its effects on the human body. The antiviral prodrug Remdesivir is still the most widely used therapeutic during the early stages of the infection. However, the current synthetic routes rely on the use of protecting groups, air-sensitive reagents, and cryogenic conditions, thus impeding a cost-efficient supply to patients. We have, therefore, focused on the development of a straightforward, direct addition of (hetero)arenes to unprotected sugars. Here we report a silylium-catalyzed and completely stereoselective C-glycosylation that initially yields the open-chain polyols, which can be selectively cyclized to provide either the kinetic α-furanose or the thermodynamically favored ß-anomer. The method significantly expedites the synthesis of Remdesivir precursor GS-441524 after a subsequent Mn-catalyzed C-H oxidation and deoxycyanation.

Synthesis and evaluation of enantiomers of hydroxychloroquine against SARS-CoV-2 in vitro.

Since the end of 2019, the outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has evolved into a global pandemic. There is an urgent need for effective and low-toxic antiviral drugs to remedy Remdesivir's limitation. Hydroxychloroquine, a broad spectrum anti-viral drug, showed inhibitory activity against SARS-CoV-2 in some studies. Thus, we adopted a drug repurposing strategy, and further investigated hydroxychloroquine. We obtained different configurations of hydroxychloroquine side chains by using chiral resolution technique, and successfully furnished R-/S-hydroxychloroquine sulfate through chemical synthesis. The R configuration of hydroxychloroquine was found to exhibit higher antiviral activity (EC50 = 3.05 µM) and lower toxicity in vivo. Therefore, R-HCQ is a promising lead compound against SARS-CoV-2. Our research provides new strategy for the subsequent research on small molecule inhibitors against SARS-CoV-2.

Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method.

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).

Clarifying the Use of Benzylidene Protecting Group for D-(+)-Ribono-1,4-Lactone, an Essential Building Block in the Synthesis of C-Nucleosides.

In the last two years, nucleosides analogues, a class of well-established bioactive compounds, have been the subject of renewed interest from the scientific community thanks to their antiviral activity. The COVID-19 global pandemic, indeed, spread light on the antiviral drug Remdesivir, an adenine C-nucleoside analogue. This new attention of the medical community on Remdesivir prompts the medicinal chemists to investigate once again C-nucleosides. One of the essential building blocks to synthetize these compounds is the D-(+)-ribono-1,4-lactone, but some mechanistic aspects linked to the use of different carbohydrate protecting groups remain unclear. Here, we present our investigations on the use of benzylidene as a ribonolactone protecting group useful in the synthesis of C-purine nucleosides analogues. A detailed 1D and 2D NMR structural study of the obtained compounds under different reaction conditions is presented. In addition, a molecular modeling study at the B3LYP/6-31G* level of theory with the SM8 solvation model for CHCl3 and DMSO to support the obtained results is used. This study allows for clarifying mechanistic aspects as the side reactions and structural rearrangements liked to the use of the benzylidene protecting group.

(-)-6-epi-Artemisinin, a Natural Stereoisomer of (+)-Artemisinin in the Opposite Enantiomeric Series, from the Endemic Madagascar Plant Saldinia proboscidea, an Atypical Source.

Chemical and biological investigation of the Madagascar endemic plant Saldinia proboscidea led to the isolation of an isomer of artemisinin, (-)-6-epi-artemisinin (2). Its structure was elucidated using a combination of NMR and mass spectrometry. The absolute configuration was established by chemical syntheses of compound 2 as well as a new stereoisomer (3). The comparable bioactivities of artemisinin (1) and its isomer (-)-6-epi-artemisinin (2) revealed that this change in configuration was not critical to their biological properties. Bioactivity was assessed using an apoptosis induction assay, a SARS-CoV-2 inhibitor assay, and a haematin polymerization inhibitory activity (HPIA) assay. This is the first report of an artemisinin-related compound from a genus not belonging to Artemisia and it is the first isolation of an artemisinin-related natural product that is the opposite enantiomeric series relative to artemisinin from Artemisia annua.

Overestimation of 3α- over 3ß-25-Hydroxyvitamin D3 Levels in Serum: A Mechanistic Rationale for the Different Mass Spectral Properties of the Vitamin D Epimers.

The metabolism of vitamin D3 includes a parallel C-3 epimerization pathway-in addition to the standard metabolic processes for vitamin D3-reversing the stereochemical configuration of the -OH group at carbon-3 (ß→α). While the biological function of the 3α epimer has not been elucidated yet, the additional species cannot be neglected in the analytical determination of vitamin D3, as it has the potential to introduce analytical errors if not properly accounted for. Recently, some inconsistent mass spectral behavior was seen for the 25-hydroxyvitamin D3 (25(OH)D3) epimers during quantification using electrospray LC-MS/MS. The present work extends that of Flynn et al. ( Ann. Clin. Biochem. 2014, 51, 352-559) and van den Ouweland et al. ( J. Chromatogr. B 2014, 967, 195-202), who reported larger electrospray ionization response factors for the 3α epimer of 25(OH)D3 in human serum samples as compared to the regular 3ß variant. The present work was concerned with the mechanistic reasons for these differences. We used a combination of electrospray ionization, atmospheric pressure chemical ionization, and density functional theory calculations to uncover structural dissimilarities between the epimers. A plausible mechanism is described based on intramolecular hydrogen bonding in the gas phase, which creates a small difference of proton affinities between the epimers. More importantly, this mechanism allows the explanation of the different ionization efficiencies of the epimers based on kinetic control of the ionization process, where ionization initially takes place at the hydroxyl group with subsequent proton transfer to a basic carbon atom. The barrier for this transfer differs between the epimers and is in direct competition with H2O elimination from the protonated hydroxyl group. The "hidden" site of high gas phase basicity was revealed through computational calculations and appears to be inaccessible via direct protonation.

Effects of a Peripherally Restricted Hybrid Inhibitor of CB1 Receptors and iNOS on Alcohol Drinking Behavior and Alcohol-Induced Endotoxemia.

Alcohol consumption is associated with gut dysbiosis, increased intestinal permeability, endotoxemia, and a cascade that leads to persistent systemic inflammation, alcoholic liver disease, and other ailments. Craving for alcohol and its consequences depends, among other things, on the endocannabinoid system. We have analyzed the relative role of central vs. peripheral cannabinoid CB1 receptors (CB1R) using a "two-bottle" as well as a "drinking in the dark" paradigm in mice. The globally acting CB1R antagonist rimonabant and the non-brain penetrant CB1R antagonist JD5037 inhibited voluntary alcohol intake upon systemic but not upon intracerebroventricular administration in doses that elicited anxiogenic-like behavior and blocked CB1R-induced hypothermia and catalepsy. The peripherally restricted hybrid CB1R antagonist/iNOS inhibitor S-MRI-1867 was also effective in reducing alcohol consumption after oral gavage, while its R enantiomer (CB1R inactive/iNOS inhibitor) was not. The two MRI-1867 enantiomers were equally effective in inhibiting an alcohol-induced increase in portal blood endotoxin concentration that was caused by increased gut permeability. We conclude that (i) activation of peripheral CB1R plays a dominant role in promoting alcohol intake and (ii) the iNOS inhibitory function of MRI-1867 helps in mitigating the alcohol-induced increase in endotoxemia.

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections.

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3-H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC50 = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC50 = 12 µM by visual CPE score; EC50 = 8.3 µM by TMS score; MCC > 100 µM, CC50 = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC50 in the range 14.5-97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at ß-lactamase, thus protecting the antibiotic from undesirable biotransformation.

Developments in Treatment Methodologies Using Dendrimers for Infectious Diseases.

Dendrimers comprise a specific group of macromolecules, which combine structural properties of both single molecules and long expanded polymers. The three-dimensional form of dendrimers and the extensive possibilities for use of additional substrates for their construction creates a multivalent potential and a wide possibility for medical, diagnostic and environmental purposes. Depending on their composition and structure, dendrimers have been of interest in many fields of science, ranging from chemistry, biotechnology to biochemical applications. These compounds have found wide application from the production of catalysts for their use as antibacterial, antifungal and antiviral agents. Of particular interest are peptide dendrimers as a medium for transport of therapeutic substances: synthetic vaccines against parasites, bacteria and viruses, contrast agents used in MRI, antibodies and genetic material. This review focuses on the description of the current classes of dendrimers, the methodology for their synthesis and briefly drawbacks of their properties and their use as potential therapies against infectious diseases.

Concise Synthesis of Both Enantiomers of Pilocarpine.

Furan-2-carboxylic acid was used as a starting material for the synthesis of dehydro-homopilopic acid. Esterification, hydrogenation and enzymatic hydrolysis followed by the reduction of Weinreb amides and a single-step attachment of a 1-methyl-imidazole residue allowed for the concise synthesis of both enantiomers of pilocarpine.

Dynamic Profiling of Binding and Allosteric Propensities of the SARS-CoV-2 Spike Protein with Different Classes of Antibodies: Mutational and Perturbation-Based Scanning Reveals the Allosteric Duality of Functionally Adaptable Hotspots.

The functional adaptability and conformational plasticity of SARS-CoV-2 spike proteins allow for the efficient modulation of complex phenotypic responses to the host receptor and antibodies. In this study, we combined atomistic simulations with mutational and perturbation-based scanning approaches to examine binding mechanisms of the SARS-CoV-2 spike proteins with three different classes of antibodies. The ensemble-based profiling of binding and allosteric propensities of the SARS-CoV-2 spike protein residues showed that these proteins can work as functionally adaptable and allosterically regulated machines. Conformational dynamics analysis revealed that binding-induced modulation of soft modes can elicit the unique protein response to different classes of antibodies. Mutational scanning heatmaps and sensitivity analysis revealed the binding energy hotspots for different classes of antibodies that are consistent with the experimental deep mutagenesis, showing that differences in the binding affinity caused by global circulating variants in spike positions K417, E484, and N501 are relatively moderate and may not fully account for the observed antibody resistance effects. Through functional dynamics analysis and perturbation-response scanning of the SARS-CoV-2 spike protein residues in the unbound form and antibody-bound forms, we examine how antibody binding can modulate allosteric propensities of spike protein residues and determine allosteric hotspots that control signal transmission and global conformational changes. These results show that residues K417, E484, and N501 targeted by circulating mutations correspond to a group of versatile allosteric centers in which small perturbations can modulate collective motions, alter the global allosteric response, and elicit binding resistance. We suggest that the SARS-CoV-2 S protein may exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter the response to antibody binding without compromising the activity of the spike protein.

Fragment-based in silico design of SARS-CoV-2 main protease inhibitors.

3CLpro is essential for SARS-CoV-2 replication and infection; its inhibition using small molecules is a potential therapeutic strategy. In this study, a comprehensive crystallography-guided fragment-based drug discovery approach was employed to design new inhibitors for SARS-CoV-2 3CLpro. All small molecules co-crystallized with SARS-CoV-2 3CLpro with structures deposited in the Protein Data Bank were used as inputs. Fragments sitting in the binding pocket (87) were grouped into eight geographical types. They were interactively coupled using various synthetically reasonable linkers to generate larger molecules with divalent binding modes taking advantage of two different fragments' interactions. In total, 1,251 compounds were proposed, and 7,158 stereoisomers were screened using Glide (standard precision and extra precision), AutoDock Vina, and Prime MMGBSA. The top 22 hits having conformations approaching the linear combination of their constituent fragments were selected for MD simulation on Desmond. MD simulation suggested 15 of these did adopt conformations very close to their constituent pieces with far higher binding affinity than either constituent domain alone. These structures could provide a starting point for the further design of SARS-CoV-2 3CLpro inhibitors with improved binding, and structures are provided.

Development of a chiral HPLC method for the separation and quantification of hydroxychloroquine enantiomers.

Hydroxychloroquine (2-[[4-[(7-Chloroquinolin-4-yl) amino]pentyl](ethyl) amino]-ethanol, HCQ), an effective anti-malarial drug, has been tested in the clinics for potential treatment of severe coronavirus disease 2019 (COVID-19). Despite the controversy around the clinical benefits of HCQ, the existence of a chiral center in the molecule to possess two optical isomers suggests that there might be an enantiomeric difference on the treatment of COVID-19. Due to their poor resolution and the inability of quantification by previously reported methods for the analysis of HCQ enantiomers, it is necessary to develop an analytical method to achieve baseline separation for quantitative and accurate determination of the enantiomeric purity in order to compare the efficacy and toxicity profiles of different enantiomers. In this study, we developed and validated an accurate and reproducible normal phase chiral high-performance liquid chromatography (HPLC) method for the analysis of two enantiomers of HCQ, and the method was further evaluated with biological samples. With this newly developed method, the relative standard deviations of all analytes were lower than 5%, and the limits of quantification were 0.27 µg/ml, 0.34 µg/ml and 0.20 µg/ml for racemate, R- and S-enantiomer, respectively. The present method provides an essential analytical tool for preclinical and clinical evaluation of HCQ enantiomers for potential treatment of COVID-19.