Compound with T-type calcium channel inhibitory activity, composition and application and screening method thereof

Abstract

The invention discloses a compound with T-type calcium channel inhibitory activity, a composition and application and a screening method thereof, and belongs to the technical field of medical chemistry and biological medicine. The compound takes a 6-isopentenyl isomer of parvacol as a parent nucleus, has excellent inhibitory activity and selectivity through structural optimization design and virtual screening, can improve DHT-induced hair follicle function inhibition or regulate scalp microenvironment calcium steady state, and has a good application prospect. The compound can be used for preparing Cav3 family T-type calcium channel inhibitors and medicines for preventing alopecia and / or promoting hair growth. The compound can be prepared into a solution, a gel, a cream or a liposome in vitro preparation. The compound disclosed by the invention not only has the effects of preventing alopecia, promoting hair growth and the like, but also reduces the risk of skin sensitization and reactivity, and improves the stability and process accessibility of an external preparation.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical chemistry and biomedicine, and in particular to compounds, compositions, and their applications and screening methods that have T-type calcium channel inhibitory activity. Background Technology

[0002] Hair loss disorders such as androgenetic alopecia (AGA) are closely related to the imbalance of the hair follicle microenvironment, typically manifested as decreased activity of dermal papilla cells (DPCs), upregulation of inflammatory factors, insufficient blood supply and nutritional support, and excessive activation of apoptotic signals. Dihydrotestosterone (DHT) induces antagonism of multiple downstream pathways in the hair follicle anagen phase through transcriptional regulation mediated by the androgen receptor (AR). In DHT-induced human dermal papilla cell models, commonly used functional and mechanistic evaluation indicators include: AR expression and activation levels; the secretory repressor Dickkopf-1 (DKK-1); β-catenin, a key protein in the Wnt / β-catenin pathway; transforming growth factor TGF-β2; and caspase-3, an apoptosis-executing protein. Abnormal changes in these indicators are closely related to premature transition of hair follicles from the anagen to catagen phase, suppressed dermal papilla cell proliferation, and increased apoptosis.

[0003] Voltage-gated T-type calcium channels (Cav3 family, including Cav3.1, Cav3.2, and Cav3.3) play a pivotal role in low-threshold depolarization and cellular rhythmic activities. In hair follicle-associated cells, abnormal calcium influx can disrupt intracellular calcium homeostasis, thereby affecting downstream calcium-dependent signaling networks such as CaM / CaMK and calcineurin-NFAT, manifested as follows:

[0004] (1) It has an adverse effect on the regulation of cell cycle and proliferation signals, and inhibits the response of dermal papilla cells to growth factors (such as IGF-1 and VEGF);

[0005] (2) The negative effect on the Wnt / β-catenin axis hinders the stability and nuclear translocation of β-catenin, and reduces the ability to maintain the growth phase;

[0006] (3) Through cross-linking between mitochondria and the apoptosis pathway, it promotes the activation of the caspase-3 cascade, inducing or aggravating apoptosis;

[0007] (4) It has a functional coupling with the DHT / AR axis. Abnormal calcium signals can amplify the inhibitory biological effects downstream of AR, including the upregulation of DKK-1 and TGF-β2.

[0008] Based on the above biological connections, selectively inhibiting the overactivation of the Cav3 family is expected to restore calcium homeostasis in dermal papilla cells, indirectly alleviate the activity of DHT-induced AR downstream hair regrowth inhibition biological pathways, increase the expression levels of Wnt / β-catenin signaling and growth factors, and reduce caspase-3-mediated apoptosis tendency, thereby generating comprehensive benefits in preventing hair loss and promoting hair growth.

[0009] In terms of compound design and screening, developing a compound that has both good membrane compatibility and transmembrane permeability, and also has the potential to form beneficial interactions with hydrophobic pockets, so that it can inhibit the activity of Cav3 family T-type calcium channels and achieve the effects of preventing hair loss and / or promoting hair growth, is one of the important research topics at present. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a compound with T-type calcium channel inhibitory activity, which can have both good membrane compatibility and transmembrane permeability, and at the same time have the potential to form beneficial interactions with hydrophobic pockets. It can inhibit Cav3 family T-type calcium channels and achieve the effects of preventing hair loss and / or promoting hair growth, thereby overcoming the shortcomings of existing compounds in preventing hair loss and / or promoting hair growth.

[0011] To address the aforementioned technical problems, this invention provides compounds (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), or (Ⅴ) with T-type calcium channel inhibitory activity:

[0012]

[0013] Wherein, R1 and R3 are independently selected from -H, -OH, -OCH3, -OEt, -OAc, -COOH, -NH2, -SH, -Cl, and -Br, respectively; R2 is selected from -H, -isopentenyl, -1-butenyl, -2-butenyl, or -cyclopentenyl; R4 is selected from any group in R1 or a phenyl group, and when selected from a phenyl group, ... The a, b, and c sites on the surface are substituted by any one of the groups in R1; X is S or O.

[0014] As an improvement of the present invention, R2 is selected from -isopentenyl or -cyclopentenyl, and R4 contains at least one donor or acceptor group capable of forming a hydrogen bond.

[0015] As an improvement to the present invention, the compound is selected from the following structures:

[0016] .

[0017] As a further improvement, the compound is selected from the following structures:

[0018] .

[0019] As a further improvement, the compound includes pharmaceutically acceptable salts, solutions, crystal forms, enantiomers, racemates, or mixtures thereof.

[0020] As a further improvement, the preparation method of the compounds is based on the parent nucleus of 6-isopentenylcoumarin isomer, chromone, naphthol, quinoline, benzothiophene or benzofuran, respectively, and introduces R4 at the 2 or 3 position of the respective parent nucleus through halogenation-cross coupling and / or Heck / Sonogashira reaction, and then introduces R1-R3 at the 5-7 position of the respective parent nucleus through selective halogenation and Suzuki coupling reaction.

[0021] Furthermore, the present invention also provides the use of the above-described compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments that inhibit Cav3.1, Cav3.2 or Cav3.3 T-type calcium channels.

[0022] This invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for preventing hair loss and / or promoting hair growth. Specifically, the compounds or pharmaceutically acceptable salts thereof are used to improve DHT-induced inhibition of hair follicle function or regulate calcium homeostasis in the scalp microenvironment.

[0023] As a further improvement of the present invention, the present invention also provides a pharmaceutical composition for preventing hair loss and / or promoting hair growth, said pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof.

[0024] As a further improvement, the pharmaceutical composition contains the above-mentioned compound or its pharmaceutically acceptable salt in a mass percentage of 0.001-10.0%.

[0025] As a further improvement, the pharmaceutical composition is a topical skin preparation, which is a solution, gel, cream, or liposome.

[0026] As a further improvement of the present invention, the present invention also provides a method for screening pharmaceutical compositions for preventing hair loss and / or promoting hair growth, the method comprising:

[0027] a) Establishment of the parent nucleus and construction of chemical space

[0028] Using coumarin, chromone, naphthol, benzofuran, benzothiophene, quinoline, and their heteroatom variants as the core ring system, substituents R1, R2, and R3 are introduced at positions 5, 6, and 7 of the core ring, and a side chain R4 is introduced at position 2 or 3. The type and site combination of substituents are controlled by programmed rules, resulting in a construction scale of approximately 10. 6 The standardized chemical space was established, and duplicates were eliminated, physicochemical boundaries were pre-screened, and warning groups were excluded, thus completing the construction of the compound library;

[0029] Wherein, R1 and R3 are independently selected from -H, -OH, -OCH3, -OEt, -OAc, -COOH, -NH2, -SH, -Cl, and -Br, respectively; R2 is selected from -H, -isopentenyl, -1-butenyl, -2-butenyl, or -cyclopentenyl; R4 is selected from any group in R1 or a phenyl group, and when selected from a phenyl group, ... The a, b, and c sites on the surface are replaced by any one of the groups in R1;

[0030] This step, without altering the core pharmacophore logic, expands the core from coumarin to at least three synthetic and engineerable aromatic (hetero)cyclic systems, such as chromones and naphthols, covering a broader conformational and electronic distribution space. Furthermore, the selection of substituents aims to form a dual-characteristic pharmacophore design framework of "hydrophobic fragment + polar anchor."

[0031] b) Target-guided parallel virtual screening

[0032] The GPU-based Uni-Dock engine establishes a unified receptor preparation and grid parameter system for the three isozymes Cav3.1, Cav3.2, and Cav3.3. It implements multi-batch parallel docking and multi-pose evaluation, using the comprehensive score of the three isozymes as the main criterion, supplemented by pose consistency and pocket key interaction integrity. It completes the dimensionality reduction screening from millions of compounds to tens of thousands of compounds in stages, and performs joint sorting of cross-core results to determine the priority set of compounds to enter experimental verification.

[0033] c) Drugability and Synthetizability Access

[0034] The physicochemical and drug-likeness verification of the priority compound set for experimental validation is carried out (including but not limited to molecular weight, cLogP, topological polar surface area, number of hydrogen bond donors and acceptors, number of rotatable bonds, and skin safety warnings) to form a parameter window that matches the topical drug delivery scenario; a synthesis feasibility score and raw material accessibility assessment are introduced, and combined with the overall steps, key coupling strategies, metal residue control and scale-up friendliness, a set of candidate compounds suitable for small-scale synthesis is screened out.

[0035] d) Small-scale synthesis and activity confirmation

[0036] For the candidate compounds in the set of candidate compounds that have entered the pilot synthesis, formulate a total synthesis route of no more than ten steps, complete the preparation and structural characterization of 20mg-level samples, and ensure that the purity and residue meet the requirements of external raw materials;

[0037] The candidate compounds that met the requirements for small-scale synthesis were subjected to whole-cell patch-clamp experiments to determine the inhibitory activity and selectivity of the Cav3 tri-isoenzyme, thereby forming electrophysiological evidence for "target confirmation" and confirming its IC50. 50 With selectivity, a set of candidate compounds suitable for entering the cell validation stage was further screened;

[0038] In a DHT-induced human dermal papilla cell model, the candidate compounds that have entered the cell validation stage were used to validate potential candidate compounds with comprehensive regulatory effects on growth-promoting and anti-apoptotic pathways, focusing on key indicators such as AR, DKK-1, β-catenin, TGF-β2, and VEGF.

[0039] e) External dosage form and conversion validation

[0040] Based on the physicochemical properties and skin compatibility of the obtained candidate compounds with development potential, we designed topical dosage forms such as solutions, gels, creams, or liposomes, completed accelerated and long-term stability studies, and clarified the manufacturability boundaries of the formulation parameters.

[0041] Through the above technical solutions, this invention obtains a group of novel Cav3 family calcium channel inhibitor candidate compounds with clear targets and potential for external application, and provides their preparation methods and pharmaceutical compositions, providing new technical solutions for preventing hair loss and promoting hair growth.

[0042] With this design, the present invention has at least the following advantages:

[0043] This invention constructs a diverse compound library by introducing substituents R1 / R2 / R3 at positions 5 / 6 / 7 of the coumarin core and variable side chains R4 at positions 2 or 3. Based on the coumarin core, the library can be expanded to include various aromatic / heteroaromatic ring systems such as chromones and naphthols. Using a Python-based automatic compound library generation tool and the Uni-Dock (GPU) molecular docking engine, parallel virtual screening of Cav3 family (Cav3.1 / Cav3.2 / Cav3.3) T-type calcium channel receptors is performed to preferentially select candidate inhibitors with low binding energy that simultaneously meet the requirements of druggability and synthetic feasibility. The preparation methods and their validation and utilization in topical applications such as hair loss prevention and hair growth promotion are also presented.

[0044] The compounds obtained by this invention form a dual-characteristic pharmacophore of "hydrophobic fragment + polar anchor", which optimizes the interaction with hydrophobic pockets and aromatic sites. For example, the polar anchoring unit containing hydrogen bond donor receptor enhances the binding with polar residues in the channel pocket. At the same time, by regulating the ligand conformational freedom, skin penetration and lipophilic-hydrophilic balance through flexible chain segments or ring systems, it can take into account both metabolic stability and skin compatibility, providing favorable conditions for the preparation of drugs for preventing hair loss and / or promoting hair growth.

[0045] The compounds obtained by this invention achieve a balance between lead properties, druggability, and syntheticability. By combining constraints on physicochemical dimensions such as hydrophobic volume, electronic parameters, topological polar surface area, and number of rotatable bonds, the risk of skin sensitization and reactivity is reduced, and the stability and processability of topical formulations are improved. Attached Figure Description

[0046] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] Figure 1 This is a schematic diagram of the general formula of the core parent nucleus of the compound of this invention and its derivatives.

[0048] Figure 2 This is a flowchart of the candidate molecule library construction and experimental verification based on the multi-dimensional screening strategy of this invention.

[0049] Figure 3a and 3b This is a summary diagram of the chemical structures of the Top 50 candidate molecules in the embodiments of this invention.

[0050] Figure 4 This is a comprehensive score and distribution diagram of the activity of candidate molecules based on patch-clamp experimental data in an embodiment of the present invention.

[0051] Figure 5 This is a Venn diagram showing the multi-target regulation of 20 preferred compounds in the DHT-hDPCs model in this embodiment of the invention.

[0052] Figure 6 These are detailed chemical structure diagrams of 20 final preferred compounds in the embodiments of the present invention.

[0053] Figure 7 This is a simulation diagram of the predicted binding of the representative compound NAP355002 of this invention to the target sites Cav3.1, Cav3.2 and Cav3.3. Detailed Implementation

[0054] Several typical embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be particularly noted that the embodiments shown in the drawings are merely illustrative examples of the present invention and are not intended to limit the scope of protection of the present invention. The present invention can be implemented through various methods, and the embodiments described herein are intended to fully illustrate the technical principles of the present invention and ensure that those skilled in the art can fully understand the technical boundaries of the present invention.

[0055] The overall goal of this invention is to construct a novel small molecule inhibitor discovery and validation pathway for Cav3 family T-type calcium channels. Using the 6-isopentenyl isomer of parsleyol as the parent core, and expanding from the coumarin core to aromatic (heterocyclic) systems such as chromones, naphthols, quinolines, benzothiophenes, and benzofurans, the invention aims to obtain candidate compounds and their topical compositions with clear target mechanisms and industrially scalable properties through an integrated algorithm-chemistry process for the exploration of applications in preventing hair loss and promoting hair growth.

[0056] Specific implementation examples are described below:

[0057] Example 1: Core Design and Structural Isomer Establishment

[0058] This embodiment first takes the natural product Apiole as the starting structure, considers its substitution mode on the aromatic ring and its influence on target binding ability, and designs a novel isomer molecule as the core of the entire compound library.

[0059] Parsleyol is characterized by an isopentenyl group at the 8-position of its benzene ring. Based on the analysis of the structure-activity relationship of the Cav3 protein family of calcium channels, it was found that hydrophobic substituents at specific positions on the aromatic ring have a significant effect on the localization of the binding pocket, while polar groups may interact specifically with hydrogen bond donors or acceptors at the target site. Therefore, this embodiment designs a method to move the isopentenyl group at the 8-position to the 6-position to enhance its integration with the protein's hydrophobic pocket.

[0060] By constructing this isomer's core structure, the electronic distribution and spatial configuration of the molecule can be significantly improved, providing a stable skeletal basis for the introduction of diverse substituents. In the new core, the 5th, 6th, and 7th positions of the original aromatic ring are renumbered and defined as three substitution sites, R1, R2, and R3. At the same time, a derivatization site R4 at position 3 is introduced, greatly expanding the possibilities for molecule modification in three-dimensional space.

[0061] This parent nucleus has the following notable characteristics:

[0062] (1) Novel structure: The isopentenyl group migrates from the traditional 8 position to the 6 position, which has not been systematically reported in the natural styrene skeleton;

[0063] (2) High synthetic feasibility: The construction of isomers can be achieved through existing organic methods such as regioselective CH activation and palladium-catalyzed cross-coupling;

[0064] (3) Sufficient modification space: The three-position substitution at the 5-7 position of the aromatic ring and the derivatization at the 3-position R4 provide more than 100 possible combinations, which can meet the needs of subsequent construction of a diverse compound library;

[0065] (4) Facilitates target binding: According to the pre-constructed docking model, the spatial orientation of the 6-position isopentenyl group is highly matched with the hydrophobic pocket of the Cav3 protein family channel, which has the potential for stronger binding force.

[0066] Based on this parent core, the following examples will elaborate on the systematic design of each substitution site (R1-R4) and the main ring system (coumarin and its alternative structures) to construct a complete compound library system.

[0067] Example 2: Substituent Design and Compound Library Construction

[0068] After establishing the core structure with the 6-isopentenylbenzene ring as its core, this embodiment further systematically designed and constructed a diverse library of substituent-derived compounds. The construction of this compound library was based on the following objectives:

[0069] (1) Structural diversity: providing the most diverse possible combinations of substitutions to cover a wide range of chemical spaces;

[0070] (2) Drug-likeness: Prioritize functional groups with known good efficacy.

[0071] (3) Synthetic accessibility: Ensure that there is a good synthetic route and the possibility of industrial scale-up in the later stage.

[0072] The specific design is as follows:

[0073] 2.1 Aromatic ring substitution design at R1, R2, and R3 positions

[0074] The 5th, 6th, and 7th positions (labeled R1, R2, and R3) of the parent aromatic ring are the main substitution sites. The R2 position has been introduced with an isopentenyl group, which can also be used to replace other hydrophobic groups, such as -H, -1-butenyl, -2-butenyl, or -cyclopentenyl.

[0075] In this embodiment, the following functional groups are introduced into R1 and R3 respectively:

[0076] (1) R1 and R3 can be independently: hydrogen (-H), hydroxyl (-OH), methoxy (-OMe), ethoxy (-OEt), acetoxy (-OAc), carboxyl (-COOH), amino (-NH2), mercapto (-SH), halogen (-Cl, -Br);

[0077] These groups can modulate the hydrophilicity / hydrophobicity, electronic effects, and potential hydrogen bonding ability of molecules.

[0078] (2) R2 position design: selected from -H, -isopentenyl, -1-butenyl, -2-butenyl or -cyclopentenyl; R2 is the middle position, and can also be used as a polar-hydrophobic synergistic regulation region.

[0079] 2.2 R4-position design: Aromatic ring 3-position derivatization

[0080] The R4 position is located at the "leader" end of the molecule, allowing for the introduction of a large aromatic substituent group. The design is as follows:

[0081] R4 can be: an R1 class group (small polar group) or a phenyl group, when selected from phenyl. The a, b, and c sites on the structure are replaced by any one of the groups in R1 to form a disubstituted aryl structure.

[0082] The substituents in this section are designed to improve compatibility and selective recognition with the external binding pocket regions of the Cav3 protein family.

[0083] 2.3 Diverse Replacement of the Main Skeleton

[0084] In addition to the modifications of the aforementioned groups, this embodiment also considers the substitution of two six-membered aromatic rings in the parent skeleton to construct "ring system diversity". Without destroying the main planar rigid structure, the coumarin core structure is substituted with the following: chromone, naphthol, quinoline, benzofuran, and benzothiophene.

[0085] These structures are spatially similar to coumarin, possessing a natural pharmacological basis and capable of enhancing target-specific binding or improving pharmacokinetic properties. (See attached image) Figure 1 As shown.

[0086] Appendix Figure 2 This paper details the complete technical route and logical flow for efficient screening from an initial molecular library and subsequent experimental validation. The flowchart systematically presents key steps such as virtual screening, activity testing, and validation, as well as their interrelationships. The specific details are described below.

[0087] 2.4 Compound Library Size and Encoding Strategy

[0088] Using combinatorial chemistry principles, a systematic arrangement and combination was performed, resulting in over 300 theoretical structures. For ease of management and screening, the following naming logic was adopted for structure coding:

[0089] C-[Parental Ring System]-[R1]-[R2]-[R3]-[R4]

[0090] For example, C-Coumarin-OH-Prenyl-OMe-2,6-diOMePh represents a molecule with a coumarin core, R1 being -OH, R2 being isopentenyl, R3 being -OMe, and R4 being 2,6-dimethoxyphenyl.

[0091] This coding system will provide a systematic basis for subsequent molecular docking and synthesis planning.

[0092] 2.4.1 Library grouping rules, parameters and output format

[0093] (1) Objective: To construct a standardized and traceable chemical space that supports a scale of millions of sites and can be precisely scaled according to site rules.

[0094] (2) Rules and parameters

[0095] a) Site rules: The availability and mutual exclusion of R1 / R2 / R3 are defined through configuration files (e.g., R3=NO2 is prohibited when R1=OH; R1=Cl is preferentially retained when R3=Br).

[0096] b) Substitution set: Provides three categories of lists for each site: "mandatory", "optional", and "prohibited", and allows setting sampling weights for substituents to balance the chemical spatial distribution.

[0097] c) R4 template library: chain length and branching degree are limited to C2–C 10 .

[0098] d) Physicochemical properties and drug-likeness screening indicators: molecular weight 250–550 Da, cLogP 2.0–5.0, TPSA 40–90 A², HBA≤10, HBD≤5, rotatable bonds≤10; PAINS, aggregates and highly reactive substructures are eliminated.

[0099] (3) Output

[0100] a) Fields: ID, parent nucleus, discrete descriptions of R1 / R2 / R3 / R4 and SMILES fragments, whole molecule SMILES, whether standardization was successful, physicochemical properties, prior warning markers, and syntheticity screening score (SCScore).

[0101] b) Fragmentation: Each batch of files contains 50k–100k molecules, with uniform naming and verification digests (hash and record count), which facilitates GPU parallelism and result recording.

[0102] c) Scale: Taking coumarin as an example, the complete enumeration scale is about 600,000; other parent kernels are generated and processed in parallel according to the same rules.

[0103] Example 3: Molecular docking and cross-core co-sequencing

[0104] To screen candidate compounds with potential inhibitory activity against the Cav3 protein family (members of the T-type calcium channel family) from the constructed compound library, this embodiment employs molecular docking technology to systematically screen all designed structures. The specific method is as follows:

[0105] The target protein Cav3 protein family channel (T-type calcium channel alpha-1H subunit) has become one of the important targets for screening voltage-gated ion channel drugs because of its key role in neurological diseases such as pain signal transduction and epileptic seizures.

[0106] Since there is currently no high-resolution crystal structure for this protein, this embodiment uses the AlphaFold predicted structure model as the initial structure and employs the following processing steps to optimize the target model.

[0107] In order to screen for candidate compounds that perform well in both the T-type calcium channel Cav3 family (3.1, 3.2, 3.3) and the L-type calcium channel Cav1.2, this embodiment uses the Uni-Dock (GPU) platform combined with an SDF molecular library built in Python for multi-target docking and subsequent screening.

[0108] 3.1 Molecular library preparation

[0109] The Python script automatically generates the 3D conformations of all derived compounds based on the parent nucleus formula and exports them as SDF files.

[0110] In Uni-Dock, perform the "Prepare Ligands" process on the SDF file, which includes preprocessing such as protonation, depolarized hydrogen transfer, and force field minimization.

[0111] 3.2 Target Model Preparation

[0112] A prediction / experimental structure for four targets (Cav3.1, Cav3.2, Cav3.3, Cav1.2) was constructed based on AlphaFold / PDB;

[0113] In Uni-Dock, water molecules and heteroatoms are removed, missing residues are repaired, and energy is minimized.

[0114] The conserved binding sites of the four targets were identified, and the same range of receptor binding sites was set for each model.

[0115] 3.3 Interface Setup and Execution

[0116] In the Uni-Dock “Dock Ligands” module, select the Ligand SDF and target models that were just preprocessed;

[0117] Using the CDOCKER or LibDock method, rigid receptor-flexible ligand docking is performed with a conservative pocket as the center.

[0118] Each ligand was attempted at least 10 times in each target model, and the docking score was taken as the highest LibDock Score (or the lowest CDOCKER Energy) as the evaluation metric.

[0119] Specific docking implementation methods: ① Engine and computing power: Uni-Dock (GPU) parallel processing, processing 50k–100k molecules per batch; ② Multiple pose settings: Initial screening generates 10 initial binding poses for each molecule, and secondary screening improves the exhaustiveness and enables more refined energy assessment; ③ Possession consistency: Selecting the top 40 poses with the best docking scores for each molecule in the initial screening, and calculating their conformational consistency. The proportion of poses with RMSD ≤ 2.0 Å is used as the key indicator of binding stability, and high consistency indicates more reliable prediction results.

[0120] 3.4 Multi-target scoring and screening

[0121] Docking parameters:

[0122] L0 initial screening: moderate exhaustiveness, n_poses=5–10; sorted by the comprehensive score of the three targets, retaining approximately 200,000.

[0123] L1 re-screening: Improve the exhaustiveness and energy assessment accuracy. The consistency criterion is n_poses = 20–40, and the proportion of conformations with RMSD ≤ 2.0 Å must be ≥ 50%. Recalculate at a higher accuracy, and retain 20,000 for each parent nucleus (denoted as the "top 20,000" for each nucleus).

[0124] Overall score

[0125] Primary criterion: The comprehensive score of the three isoenzymes is the primary ranking indicator. Example weights: Cav3.2 0.40, Cav3.1 0.35, Cav3.3 0.25. That is, Score = 0.40×Score_Cav3.2 + 0.35×Score_Cav3.1 + 0.25×Score_Cav3.3.

[0126] Auxiliary criteria: proportion of pose consistency, integrity of key interactions (hydrophobic stacking, π-π, H bond, salt bridge), and docking energy distribution quantile.

[0127] Output: docking energy, optimal pose coordinates, list of key interactions, pose consistency index, batch log and parameter seed, all written into the results table.

[0128] Joint ranking: The top 20,000 of each of the three dominant parent nuclei (coumarins, chromones, and naphthols) are merged to form a unified candidate set (approximately 60,000), and then screened and ranked again with consistent parameters to obtain the overall top 20,000.

[0129] 3.5 Docking Results

[0130] L0→L1: The coumarin core is reduced from 1,000,000 to 200,000 (L0), and then to 20,000 (L1).

[0131] Cross-core: Coumarin / Cryonosine / Naphthol three cores are entered into the merge list and jointly sorted to obtain the overall "top 20,000".

[0132] Representative molecule NAP355002: forms a π–π, hydrophobic stacking and polar-anchored hydrogen bond network in the Cav3.2 pocket; interactions are shown in [link to relevant documentation]. Figure 7 The posture consistency rate reached 85%.

[0133] Conclusion: This embodiment yielded a structurally sound and robust high-priority ensemble of compounds that will proceed to the next stage of druggability and synthetic accessibility filtration.

[0134] Example 4: Drugability / Skin Safety Filtration and Synthetic Accessibility Assessment

[0135] 4.1 Materials and Methods

[0136] Drugability / Safety (L3): Verify molecular weight, cLogP, TPSA, HBA / HBD, rotatable bonds, gastrointestinal absorption, blood-brain barrier penetration, etc.; eliminate high-risk fragments and highly reactive groups that may cause skin sensitization / phototoxicity; control halogen density.

[0137] Synthesibility (L4): SCScore ≤ 3.5 preferred; total steps ≤ 10; key coupling uses commercial catalytic systems; mild redox and scale-up friendly; starting materials are available.

[0138] Output list: A synthetic strategy card is generated, including route tags, key intermediates, bottlenecks, and alternatives. Approximately 5,000 candidate compounds are output for syntheticity review.

[0139] 4.2 Results and Discussion

[0140] This embodiment successfully established a crucial access bridge from computational screening to experimental synthesis. After rigorous druggability and skin safety filtering in the L3 stage, approximately 5,000 high-dosing-score molecules were selected from about 20,000 high-potential-score molecules. These high-quality candidates met the predefined physicochemical window (molecular weight: 250-550 Da, cLogP: 2.0-5.0, TPSA: 40-90 Ų) and had no known structures indicating skin sensitization, phototoxicity, or high reactivity. Statistical analysis showed that the average initial synthetic feasibility score (SCScore) of this set was 3.2, indicating promising synthetic prospects.

[0141] In the subsequent L4 synthetic accessibility review, we further selected 500 of the most promising candidate compounds from 5,000 molecules based on four principles: "total steps ≤ 10," "commercialization of key catalytic reactions," "high availability of raw materials," and "scale-up friendly process," forming a "priority synthesis cohort." This cohort not only represents the calculated activity advantages but also ensures its high feasibility in real-world chemical synthesis. This step significantly reduces the investment risk of subsequent experimental research and lays a solid material foundation for efficient and targeted lead compound optimization.

[0142] Example 5: Synthetic route design for candidate molecules

[0143] For the top 50 candidate molecules in terms of final activity score and with good synthetic feasibility, see attached... Figure 3a and 3b As shown in the figure. This embodiment, based on the substituent type and core structure characteristics, formulates a corresponding organic synthetic route. The overall synthetic strategy adheres to the principles of "modular assembly" and "highly selective reaction," ensuring mild reaction conditions, available starting materials, and easy expansion of structural diversity. The following are the general design principles and representative examples of the synthetic route.

[0144] 5.1 General Reaction Formula

[0145] The general synthetic formula begins with simple starting materials (such as hydroxyl compounds or haloalkanes) and progressively constructs intermediates through a series of reactions. The formula utilizes a variety of reagents and conditions, including bases (such as potassium carbonate, NaHCO3), catalysts (such as palladium catalysts, AlCl3), solvents (such as tetrahydrofuran, toluene, DMF, DCM, etc.), and temperature control. The intermediates are compounds with reactive sites that facilitate subsequent derivatization. Synthetic routes for 50 candidate compounds include:

[0146] (1) Compounds NAP82809, NAP82819, NAP82101, and NAP52890 share the following general reaction formula:

[0147]

[0148] (2) Compounds NAP35082, NAP35809, NAP35581, NAP35859, NAP35591, NAP35519, NAP35819, NAP35806, and NAP35119 share the following general reaction formula:

[0149]

[0150] (3) Compounds NAP42819, NAP42502, and NAP42136 share the following general reaction formula:

[0151]

[0152] (4) Compounds NAP355809, NAP355109, NAP355801, NAP355102, NAP355209, NAP355609, and NAP355002 share the following general reaction formula:

[0153]

[0154] (5) Compounds NAP354002, NAP354012, and NAP354119 share the following general reaction formula:

[0155]

[0156] (6) Compounds NAP83101 and NAP83102 share the following general reaction formula:

[0157]

[0158] (7) Compounds NAP362101 and NAP362102 share the following general reaction formula:

[0159]

[0160] (8) Compounds NAP34816 and NAP34012 share the following general reaction formula:

[0161]

[0162] (9) Compound NAP74112 shares the following general reaction formula:

[0163]

[0164] (10) Compounds NAP75102, NAP75002, and NAP75812 share the following general reaction formula:

[0165]

[0166] (11) Compounds CHR354101 and CHR74002 share the following general reaction formula:

[0167]

[0168] (12) Compounds CHR394111 and CHR362012 share the following general reaction formula:

[0169]

[0170] (13) Compounds CHR13082 and CHR13191 share the following general reaction formula:

[0171]

[0172] (14) Compounds CHR05090 and CHR05190 share the following general reaction formula:

[0173]

[0174] (15) Compounds CHR12191 and CHR12182 share the following general reaction formula:

[0175]

[0176] 5.1.2 Quality Standards

[0177] HPLC purity ≥95%; metal residue ≤5 ppm; ¹H / ¹³C NMR and HRMS spectral data are consistent with the structure.

[0178] 5.2 Results and Discussion

[0179] This embodiment systematically planned and implemented synthetic routes for the top 50 preferred candidate compounds, successfully translating computational design into physical molecules. The core of the synthetic strategy lies in "modular" and "convergent" synthesis, using key intermediates such as the precursor compound 6-isopentenylcoumarin as the core. Through efficient Knoevenagel condensation, regioselective halogenation (e.g., NBS), and various cross-coupling reactions (e.g., Suzuki, Heck, Sonogashira), diverse R4 side chains were efficiently introduced, and R1-R3 substituents were precisely modified. The synthetic routes for the Top 50 molecules are listed in Table 1 below.

[0180] Table 1 Top 50 Molecular Synthetic Pathways

[0181]

[0182]

[0183] Using compound NAP355002 as the target molecule, it was synthesized efficiently with an overall yield of 42% via the synthetic route shown in Appendix 1. The structure of the product was confirmed by ¹H NMR, ¹³C NMR and HRMS, and the HPLC purity was as high as 98.5%.

[0184] The synthetic route of compound NAP355002 will be described in detail below based on the corresponding reaction formula.

[0185] Step 1: Synthesis of intermediate 8 (Suzuki-Miyaura coupling)

[0186] Under nitrogen protection, intermediate 7, 2-iodophenol, and sodium bicarbonate obtained from the above reaction formula (4) were added to a dry 500 mL three-necked flask. After purging the system three times, a mixed solvent of toluene, ethanol, and water was added under a nitrogen stream. Subsequently, the catalyst Pd(dppf)Cl2 was added. The reaction mixture was heated to 80 °C and stirred at this temperature. The reaction was monitored by TLC until the starting material was almost completely eliminated, and the reaction took about 6-8 hours.

[0187] After the reaction was complete, the mixture was cooled to room temperature, diluted with 100 mL of water, and extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5:1, v / v) to give intermediate 8.

[0188] Step 2: Synthesis of Intermediate 9 (Nickel-catalyzed synthesis of silica-boronate esters)

[0189] Under nitrogen protection, intermediate 8, 4,4,5,5-tetramethyl-2-(triethylsilyl)-1,3,2-dioxaborhecyclopentane, potassium fluoride, and copper fluoride obtained in step one were added to a dry 100 mL Schlenk tube. The system was evacuated and purged with nitrogen, and this process was repeated three times. Toluene was then injected under a nitrogen flow. Subsequently, the ligand tributylphosphine and bis(1,5-cyclooctadiene)nickel (catalyst) were added sequentially. The reaction mixture was stirred at 60 °C and monitored by TLC or ¹¹B NMR until the reaction was complete, approximately 12 h.

[0190] After the reaction was complete, the mixture was cooled to room temperature, filtered through a diatomaceous earth filter, and washed with ethyl acetate. The filtrate was washed successively with saturated ammonium chloride solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10:1, v / v) to give intermediate 9 as a colorless oil.

[0191] Step 3: Synthesis of compound NAP355002 (halogenation of N-chlorosuccinimide)

[0192] Under nitrogen protection, intermediate 9 obtained in step two above was dissolved in acetonitrile. N-chlorosuccinimide was added to this solution in portions. The reaction mixture was heated to 80°C and stirred. The reaction was monitored by TLC or LC-MS until the starting material was completely converted, which took 2-4 hours.

[0193] After the reaction was complete, the mixture was cooled to 0°C, and excess NCS was quenched by adding saturated sodium thiosulfate solution. The mixture was extracted with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3:1, v / v) to give the final product compound NAP355002 as a white solid.

[0194] The synthetic pathways of the 50 target compounds shown in Table 1 above demonstrate that the compound library designed in this embodiment not only possesses rich structural diversity but also has clear, reliable, and scalable synthetic pathways, fully meeting the requirements for high-quality chemical entities for subsequent in-depth biological functional verification.

[0195] Example 6: Patch-clamp validation—Optimal compound and overall statistics

[0196] 6.1 Materials and Methods

[0197] System: HEK293 cell lines stably expressing human Cav3.1, Cav3.2, Cav3.3, and Cav1.2 channels were used. All experiments were performed at room temperature using a uniform voltage clamping protocol: the potential was maintained at -100 mV and stepped to -30 mV (to activate T-type calcium channels) to induce inward calcium currents.

[0198] Controls: A negative control (extracellular fluid containing 0.1% DMSO) was set up for each experiment to determine the baseline current, and a positive control (mibediil, a known T-type calcium channel inhibitor) was set up to verify the sensitivity of the experimental system.

[0199] Endpoint: IC of sample to Cav3 channel 50 In addition to studies on the reversibility of inhibition; the activity of L-type (Cav1.2) calcium channels was selectively evaluated by comparing compounds.

[0200] Statistics: All data points were derived from at least three (n≥3) independent cell experiments, and results are expressed as mean ± standard deviation. IC 50 The values ​​were obtained through nonlinear fitting of a four-parameter Logistic equation. The correlation between virtual screening scores and experimental activity was evaluated using the Spearman rank correlation coefficient.

[0201] 6.2 Results and Discussion

[0202] Patch-clamp experiments provided crucial electrophysiological evidence to verify the target inhibitory activity of the compounds in this embodiment. Through a systematic review of approximately 500 synthetic compounds, we obtained data on their inhibitory activity against three T-type calcium channels: Cav3.1, Cav3.2, and Cav3.3. Based on this data, the inventors ranked them according to a weighted scoring system: IC... 50 Weighted average = 0.40 × IC 50 _Cav3.2+0.35×IC 50 _Cav3.1+0.25×IC 50Cav3.3 comprehensively evaluates the inhibitory effects and selectivity of each compound on the three channels, and selects the top 200 molecules with the best comprehensive scores for further in-depth research. For example... Figure 4 As shown in the data distribution, for the top 200 preferred compound molecules screened in this embodiment, their bioactivity (in IC50) is as follows: 50 The weighted average (as a metric) exhibits a highly concentrated and favorable distribution trend. 50 The weighted average is a key indicator for evaluating the inhibitory efficacy of compounds; the lower the value, the stronger the biological activity of the compound. Detailed structural information for the aforementioned 200 compounds is listed in Table 2 below.

[0203] Table 2. Structural information of 200 preferred compounds screened based on patch-clamp experiments.

[0204]

[0205]

[0206]

[0207]

[0208] To systematically evaluate the reversibility of the inhibitory effect, the inventors selected the top 20 compounds by weighted score for in-depth analysis. As shown in Table 3 below, these compounds generally exhibit rapid onset and highly reversible inhibitory effects on the Cav3.2 channel. In the experiment, we recorded the calcium current under normal conditions, the current response after adding each compound, and the current recovery after elution. All compounds were assigned their corresponding IC50 values. 50 The addition of certain concentrations caused the calcium current to be generally suppressed to about 50% of the initial level after drug addition. After removing the compound and eluting thoroughly, the current could be significantly recovered, with the final recovery level reaching 75%-90% of the initial current.

[0209] Table 3. Recovery rate of Cav3.2 calcium current after compound elution

[0210]

[0211] The results in Table 3 consistently show that the selected compounds interact with the Cav3.2 channel in a non-covalent and reversible manner, exhibiting good reversible inhibitory properties, which provides an important safety basis for their subsequent development as therapeutic lead molecules.

[0212] Crucially, this study yielded satisfactory results in evaluating the selectivity of the compounds. As a control, we tested the inhibitory activity of these compounds against the L-type calcium channel subtype Cav1.2 in parallel. Selectivity test data showed that the vast majority of compounds exhibited inhibition rates of less than 50% against Cav1.2 channels at a high concentration of 10 µM. This significant difference strongly demonstrates that the compounds of this invention possess high selectivity for T-type calcium channels (especially Cav3.2) and effectively avoid cross-inhibition of L-type calcium channels.

[0213] Given the crucial role of the Cav1.2 channel in the cardiovascular system (such as myocardial contraction and electrophysiology), its inhibition is a major cause of cardiovascular side effects in many clinical calcium channel blockers. Therefore, the high selectivity exhibited by this series of compounds is a key feature that distinguishes them from traditional non-selective blockers and gives them superior safety potential.

[0214] Furthermore, statistical analysis of all test data shows that the comprehensive matching score of the virtual screening is consistent with the IC measured in the experiment. 50 A significant negative correlation was found between the values ​​(Spearman r = -0.73, p < 0.001). This strong correlation further validates the accuracy and effectiveness of the computational screening process used in this study, indicating that this screening method can efficiently enrich lead molecules with real activity from a large-scale virtual library, providing a reliable foundation for subsequent targeted drug development.

[0215] Example 7: Cellular Validation of DHT-hDPCs

[0216] 7.1 Materials and Methods

[0217] Model: DHT 100nM pretreatment for 24-48h to simulate the in vivo pathological environment of androgenetic alopecia; test compound 0.1–10μM intervention; solvent control and positive control were set up.

[0218] Detection indicators: The gene expression levels of AR, DKK-1, β-catenin, TGF-β2, and caspase-3 were detected using real-time quantitative PCR (qPCR) technology, and combined with ALP, IGF-1, VEGF, EdU, and Ca²⁺ imaging.

[0219] Statistics: All experiments were independently repeated at least 3 times (n≥3), and data are expressed as mean ± standard deviation; t-test or Mann-Whitney U was used for comparisons between two groups; ANOVA or Kruskal-Wallis was used for multiple groups, with correction for multiple comparisons.

[0220] 7.2 Results and Discussion

[0221] To evaluate the therapeutic potential of the lead compound for androgenetic alopecia, an in vitro pathological model was successfully constructed by stimulating hDPCs cells with DHT. Compared with the blank control group, the cells in the DHT model group exhibited typical molecular characteristics associated with androgenetic alopecia: the expression of androgen receptor (AR) was significantly upregulated (p < 0.001), indicating enhanced cell sensitivity to androgens; simultaneously, the gene expression of key hair follicle growth inhibitors DKK-1 and TGF-β2 was also significantly increased (p < 0.01), while the gene expression of β-catenin, a core signaling molecule promoting hair follicle growth and development, and the protein secretion level of the pro-angiogenic factor VEGF were significantly inhibited (p < 0.01). These changes collectively confirm that the DHT model successfully simulates the core pathological state of excessive activation of the AR signaling pathway, inhibition of the Wnt / β-catenin germinal pathway, and deterioration of the hair follicle growth microenvironment during in vivo hair follicle miniaturization.

[0222] Based on this model, we screened a library of synthetic compounds. The results showed that several candidate molecules could effectively reverse DHT-induced pathological phenotypes. To systematically screen potential compounds with multi-target synergistic regulatory effects, we conducted a comprehensive analysis of the expression regulatory effects of each compound on five key genes (AR, DKK-1, β-catenin, TGF-β2, and VEGF), and selected the top 50 compounds with the most significant expression regulation at each target for Venn diagram intersection analysis. The results are as follows: Figure 5 As shown.

[0223] From the appendix Figure 5 It was found that a total of 20 compounds were able to significantly regulate all five targets mentioned above. These compounds demonstrated the ability to reverse the DHT effect in multiple key pathological processes, including reducing AR overexpression, antagonizing DKK-1 and TGF-β2 upregulation, and restoring β-catenin and VEGF expression levels.

[0224] The screening results indicate that these 20 compounds ( Figure 6 The chemical structures of the 20 compounds shown above all possess the potential to mimic ideal therapeutic interventions through multi-target synergistic effects. They can inhibit pathogenic signals, actively activate hair follicle pathways, and improve hair follicle microcirculation, thus exhibiting comprehensive anti-hair loss activity at the cellular level. These candidate molecules provide an important foundation for the development of potential drugs to treat androgenetic alopecia, and their specific in vivo efficacy and mechanisms of action require further in-depth research.

[0225] The inventors selected NAP355002, a representative compound with the best regulatory effect on the expression of key AR genes, from the above 20 compounds for subsequent experiments and verification.

[0226] Example 8: Optimal Molecular Docking Conformation and Interaction Verification

[0227] 8.1 Materials and Methods

[0228] (1) Subjects: The lead compound NAP355002, which showed the best performance in cell experiments, was selected to perform molecular docking verification with the Cav3 family T-type calcium channels (PDB numbers: 6kzo (Cav3.1), 9ayg (Cav3.2), and 7wli (Cav3.3)).

[0229] (2) docking software and platform: Molecular docking was performed using the GPU-based Uni-Dock v2.1. The computing platform was an Intel Core i9-14900K, NVIDIA GeForce RTX 4090 (24GB VRAM), and 64GB RAM workstation (operating system and CUDA / driver version).

[0230] (3) Acceptor preparation: All three acceptors were pretreated in UCSF ChimeraX, where water of crystallization and heteroatoms were removed, hydrogen was added, and the hydrogen bond network was optimized. Then, the energy was minimized using the AMBER force field (convergence threshold of approximately 0.05 kcal·mol⁻¹·Å⁻¹). Based on the definition of the pocket as the hydrophobic core region surrounding the selective filtering region, the search box center coordinates were set as (192.812131, 182.559482, 189.254018), (145.149987, 140.971244, 149.035425), and (152.771016, 138.812260, 145.642250) for the three subtypes, respectively, with a uniform box size of 15×15×15Å.

[0231] (4) Ligand preparation: Using the lead compound NAP355002 as the ligand, the 3D conformation was generated from the input structure using Open Babel 3.0, hydrogen was added and geometry optimization was performed under the MMFF94 force field (using the default convergence settings), and finally exported as SDF format for subsequent docking.

[0232] (5) Docking parameters: A flexible ligand docking mode was adopted, generating 50 initial poses per molecule, with 1000 iterations and a pose clustering RMSD threshold of 2.0 Å. The scoring function incorporates van der Waals, electrostatic, hydrogen bonding, and desolvation terms.

[0233] (6) Interaction Analysis and Plotting: Interaction identification and visualization were performed in UCSF ChimeraX (v 1.10.1): the hbonds and contacts commands were used to detect and display ligand-receptor hydrogen bonds and hydrophobic / van der Waals contacts, respectively. Key interactions were labeled with distance in Å units; residue labels and numbers were kept consistent with the receptor structure. The three-dimensional pocket view was presented as a molecular surface and colored with the Coulombic potential. The pocket profile was displayed using clip slab, and a magnified view was generated in the ligand neighborhood for detailed presentation. The two-dimensional schematic (planar interaction labels of ligands and neighboring residues) was exported from ChimeraX's 2D labeling and ranging information, and then all images were exported as high-resolution PNGs. The results are attached. Figure 7 As shown.

[0234] 8.2 Results and Discussion

[0235] Molecular docking results Figure 7 The results show that NAP355002 exhibits extremely strong predicted affinity (Vina score, kcal / mol) for all three subtypes of the Cav3 family: Cav3.2 = -11.9, Cav3.1 = -11.2, and Cav3.3 = -10.8. Compared to Cav3.1 and Cav3.3, Cav3.2 shows a more pronounced preferred binding tendency. The optimal docking conformation is shown in the figure. The main interactions (hydrogen bonds, hydrophobic / van der Waals interactions, and possible aromatic ring stacking, etc.) are shown below. Key distances are labeled in Å, and residue numbering is consistent with the acceptor structure.

[0236] Based on the docking results of Cav3.2 and NAP355002, the optimal binding conformation is mainly stabilized by hydrophobic and aromatic interactions. The naphthol backbone forms significant "π-alkyl / π-σ" contacts with pocket residues PHE A:1441 / ILE A:1442 / LEU A:1494 (distances: PHE A:1441 5.15 Å, ILE A:1442 5.48 Å, LEU A:1494 2.93 Å, respectively). The aromatic ring also exhibits "π-S" interactions with sulfur-containing residues CYS A:1438 and MET A:1814, at distances of 4.28 Å and 5.79 Å, respectively, constituting lateral anchoring. The cyclopentenyl side chain forms multiple π-alkyl / alkyl contacts with ligands such as VAL A:1841, MET A:1495, and PHE A:1441 (distances of 4.06 Å, 4.51 Å, and 4.28 Å, respectively). Furthermore, ligands such as TRP A:1810, VAL A:1499, VAL A:1434, LEU A:1845, and PHE A:1498 on the periphery of the pocket exhibit van der Waals contacts with the ligands, collectively providing hydrophobic encapsulation. Overall, the interaction characteristics are predominantly hydrophobic stacking and π-alkyl / π-S anchoring, consistent with the slightly hydrophobic nature of the Cav3.2 pocket and supporting its superior docking score.

[0237] Postural consistency: Postural clustering analysis showed that the top 12 best conformations had high consistency (RMSD consistency of 85%) and all retained the above key interactions, indicating that the binding mode was highly stable.

[0238] These interactions reveal the mechanism of NAP355002's high inhibitory activity: the naphthol core acts as a hydrophobic anchoring unit embedded in the pocket, while the polar groups of the R4 side chain directionally bind to the polar residues of the channel via hydrogen bonds and salt bridges, thereby blocking calcium ion influx. This result is consistent with the nanomolar inhibitory activity (IC50) of NAP355002 against Cav3.2 observed in electrophysiological experiments. 50 = 400.2nM) and high selectivity (for Cav1.2 IC) 50 The results showed a high degree of consistency (>10μM), verifying the accuracy of the virtual screening.

[0239] Example 9: Formulation and Stability Study

[0240] 9.1 Materials and Methods

[0241] 9.1.1 Active Ingredients

[0242] The lead compound NAP355002 (purity ≥95%, verified by HPLC) was used as the API.

[0243] 9.1.2 Dosage Form Design and Formulation

[0244] Four topical formulations have been developed, with API concentrations ranging from 0.001 to 10.0 wt%.

[0245] (1) Solution: API is dissolved in ethanol / water (50:50, v / v) system, containing 1% propylene glycol as a penetration enhancer and 0.01% EDTA as a stabilizer.

[0246] (2) Gel agent: Carbomer 940 (1.0%) was used as the gel matrix, neutralized to pH 6.0 with triethanolamine, and API was uniformly dispersed in the aqueous phase.

[0247] (3) Cream: The oil phase (8% stearic acid, 3% glyceryl monostearate) and the aqueous phase (5% glycerol, 2% Tween 80) are emulsified at 70°C to form an O / W type cream, and API is added to the oil phase.

[0248] (4) Liposomes: Prepared using microfluidic technology (Nanowetech OBM III), the lipid composition was phosphatidylcholine:cholesterol:DSPE-PEG2000 (molar ratio 60:35:5), API was dissolved in the lipid phase (prepared with 40% ethanol aqueous solution to a final concentration of 2 mg / mL), and the aqueous phase was PBS (pH 7.4). Target particle size 80-180 nm, polydispersity index (PDI) <0.25, encapsulation efficiency >60%.

[0249] 9.1.3 Characterization Methods

[0250] (1) Particle size and PDI: Measured using a Malvern Zetasizer Nano ZS dynamic light scattering instrument.

[0251] Encapsulation efficiency: Free API was separated by ultracentrifugation (100,000 g, 30 min) and HPLC (C 18 Encapsulation efficiency was determined by column chromatography (acetonitrile-water gradient elution).

[0252] (2) Stability study:

[0253] Accelerated stability testing: Samples were taken at 0, 1, 2, and 3 months after being placed at 40℃ / 75% RH for 3 months.

[0254] Long-term stability: Samples were taken at 0, 3, 6, 9 and 12 months after being placed at 25℃ / 60% RH.

[0255] Detection indicators: API content (HPLC), related substances (degradation products), particle size / viscosity, appearance, pH value, and microbial limits (according to USP). <51> ).

[0256] 9.1.4 Solubility and Permeability

[0257] (1) Solubility: The equilibrium solubility of API in water, ethanol and propylene glycol was determined by the shake flask method (25℃).

[0258] (2) In vitro permeation: Franz diffusion cell (effective diffusion area 1.0 cm²) and fresh pig skin (thickness 0.4 mm) were used. The acceptor solution was PBS (containing 0.01% NaN3). The samples were collected for 24 hours and the cumulative permeation was analyzed by HPLC.

[0259] 9.2 Results and Discussion

[0260] 9.2.1 Dosage form characterization

[0261] (1) Liposomes: The average particle size was 125±5nm, the PDI was 0.18±0.02, the encapsulation efficiency was 72.5±3.2%, and the Zeta potential was -32.1mV, indicating that the system was stable.

[0262] (2) Other dosage forms: The solution is clear and transparent with a pH of 6.5; the gel is uniform and free of bubbles with a viscosity of 4500 cP; the cream has a fine texture and shows no separation in the centrifugation test.

[0263] 9.2.2 Solubility and Permeability

[0264] API has the highest solubility in ethanol (15.2 mg / mL) and only 0.12 mg / mL in water.

[0265] 24-hour cumulative penetration of liposome formulations (Q 24 The concentration of liposomes was 45.2 μg / cm², which is 2.5 times that of the solution (18.1 μg / cm²), indicating that liposomes significantly enhance skin penetration.

[0266] 9.2.3 Stability Results

[0267] (1) Accelerated stability: The API content of all dosage forms remained >95% within 3 months, and the growth of related substances was <1.0%. The liposome particle size increased slightly to 135nm, and the PDI remained <0.25; the viscosity of the cream decreased by <10%. There was no microbial growth or appearance change.

[0268] (2) Long-term stability: Within 12 months, the API content is >90%, the related substances are <2.0%, and the physical properties (particle size, viscosity, pH) of each dosage form meet the initial standards.

[0269] (3) Compatibility of excipients: API has good compatibility with commonly used excipients (such as propylene glycol, carbomer, phospholipids), and no new degradation peaks appear.

[0270] 9.2.4 Conclusion

[0271] All four developed topical dosage forms exhibit good stability, compatibility, and permeability, with the liposome formulation demonstrating superior performance in skin delivery. The formulation process is robust, key quality indicators are controllable, and the formulation meets the requirements for industrial production, providing a solid foundation for subsequent clinical translation.

[0272] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. Compounds (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), or (Ⅴ) possessing Cav3 T-type calcium channel inhibitory activity: in, R1 and R3 are independently selected from -H, -OH, -OCH3, -OEt, -OAc, -COOH, -NH2, -SH, -Cl, and -Br, respectively; R2 is selected from -H, -isopentenyl, -1-butenyl, -2-butenyl, or -cyclopentenyl. R4 is selected from any group in R1 or a phenyl group. When R4 is selected from a phenyl group, The a, b, and c sites on the surface are replaced by any one of the groups in R1; X is either S or O.

2. The compound according to claim 1, characterized in that, in, R2 is selected from -isopentenyl or -cyclopentenyl, and R4 contains at least one donor or acceptor group capable of forming a hydrogen bond.

3. The compound according to claim 1, characterized in that, The compound is selected from the following structures: 。 4. The compound according to claim 3, characterized in that, The compound is selected from the following structures: 。 5. The compound according to claim 1, characterized in that, The compound comprises a pharmaceutically acceptable salt, solute, crystal form, enantiomer, racemate, or mixture thereof of the compound of claim 1.

6. The compound according to claim 1, characterized in that, The preparation methods of the compounds are based on the parent nuclei of 6-isopentenylcoumarin isomer, chromone, naphthol, quinoline, benzothiophene or benzofuran, respectively, and introduce R4 at the 2 or 3 position of their respective parent nuclei through halogenation-cross coupling and / or Heck / Sonogashira reaction, and then introduce R1-R3 at the 5-7 position of their respective parent nuclei through selective halogenation and Suzuki coupling reaction.

7. Use of the compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting Cav3.1, Cav3.2 or Cav3.3 T-type calcium channels.

8. The use of the compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing hair loss and / or promoting hair growth.

9. The use of the compound of claim 8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing hair loss and / or promoting hair growth, characterized in that, The compound or a pharmaceutically acceptable salt thereof is used to improve DHT-induced inhibition of hair follicle function or regulate calcium homeostasis in the scalp microenvironment.

10. A pharmaceutical composition for preventing hair loss and / or promoting hair growth, characterized in that, The pharmaceutical composition comprises the compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition for preventing hair loss and / or promoting hair growth according to claim 10, characterized in that, The pharmaceutical composition comprises 0.001-10.0% by mass of any one of the compounds of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical composition for preventing hair loss and / or promoting hair growth according to claim 11, characterized in that, The pharmaceutical composition is a topical skin preparation, which is a solution, gel, cream, or liposome.

13. A method for screening pharmaceutical compositions for preventing hair loss and / or promoting hair growth, characterized in that, The method includes: a) Establishment of the parent nucleus and construction of chemical space Using coumarin, chromone, naphthol, benzofuran, benzothiophene, quinoline, and their heteroatom variants as the core ring system, substituents R1, R2, and R3 are introduced at positions 5, 6, and 7 of the core ring, and a side chain R4 is introduced at position 2 or 3. The type and site combination of substituents are controlled by programmed rules, resulting in a construction scale of approximately 10. 6 The standardized chemical space was established, and duplicates were eliminated, physicochemical boundaries were pre-screened, and warning groups were excluded, thus completing the construction of the compound library; Wherein, R1 and R3 are independently selected from -H, -OH, -OCH3, -OEt, -OAc, -COOH, -NH2, -SH, -Cl, and -Br, respectively; R2 is selected from -H, -isopentenyl, -1-butenyl, -2-butenyl, or -cyclopentenyl; R4 is selected from any group in R1 or a phenyl group, and when selected from a phenyl group, ... The a, b, and c sites on the surface are replaced by any one of the groups in R1; b) Target-guided parallel virtual screening The GPU-based Uni-Dock engine establishes a unified receptor preparation and grid parameter system for the three isozymes Cav3.1, Cav3.2, and Cav3.

3. It implements multi-batch parallel docking and multi-pose evaluation, using the comprehensive score of the three isozymes as the main criterion, supplemented by pose consistency and pocket key interaction integrity. It completes the dimensionality reduction screening from millions of compounds to tens of thousands of compounds in stages, and performs joint sorting of cross-core results to determine the priority set of compounds to enter experimental verification. c) Drugability and Synthetizability Access The physicochemical and pharmaceutical properties of the priority compound set that enters the experimental verification are reviewed to form a parameter window that matches the topical administration scenario; a synthesis feasibility score and raw material accessibility assessment are introduced, and a set of candidate compounds suitable for small-scale synthesis is screened by combining the overall steps, key coupling strategies, metal residue control and scale-up friendliness. d) Small-scale synthesis and activity confirmation For the candidate compounds in the set of candidate compounds that have entered the pilot synthesis, formulate a total synthesis route of no more than ten steps, complete the preparation and structural characterization of 20mg-level samples, and ensure that the purity and residue meet the requirements of external raw materials; The candidate compounds that met the requirements for small-scale synthesis were subjected to whole-cell patch-clamp experiments to determine the inhibitory activity and selectivity of the Cav3 tri-isoenzyme, confirming its IC50. 50 With selectivity, a set of candidate compounds suitable for entering the cell validation stage was further screened; In a DHT-induced human dermal papilla cell model, the candidate compounds that have entered the cell validation stage were used to validate the potential candidate compounds with comprehensive regulatory effects on growth-promoting and anti-apoptotic pathways in the candidate compound set, focusing on key indicators such as AR, DKK-1, β-catenin, TGF-β2, and VEGF. e) External dosage form and conversion validation Based on the physicochemical properties and skin compatibility of the obtained candidate compounds with development potential, a topical dosage form was designed, accelerated and long-term stability studies were completed, and the manufacturability boundaries of the formulation parameters were clarified.

Images