Design, Synthesis, Antifungal Activity, and Molecular Docking of Streptochlorin Derivatives Containing the Nitrile Group

Based on the structures of natural products streptochlorin and pimprinine derived from marine or soil microorganisms, a series of streptochlorin derivatives containing the nitrile group were designed and synthesized through acylation and oxidative annulation. Evaluation for antifungal activity showed that compound 3a could be regarded as the most promising candidate—it demonstrated over 85% growth inhibition against Botrytis cinerea, Gibberella zeae, and Colletotrichum lagenarium, as well as a broad antifungal spectrum in primary screening at the concentration of 50 μg/mL. The SAR study revealed that non-substituent or alkyl substituent at the 2-position of oxazole ring were favorable for antifungal activity, while aryl and monosubstituted aryl were detrimental to activity. Molecular docking models indicated that 3a formed hydrogen bonds and hydrophobic interactions with Leucyl-tRNA Synthetase, offering a perspective for the possible mechanism of action for antifungal activity of the target compounds.


Introduction
Natural products are well known as one of the most important sources for lead discovery in medicinal and agricultural chemistry, because their novel scaffolds can afford an opportunity to discover novel candidates with different modes of action from the existing agents. Streptochlorin is a marine natural product with the structure of 4-chloro-5-(3-indolyl)oxazole; it has been reported to display a range of biological activity [1][2][3][4]. Pimprinine is an indole alkaloid produced by many species of Streptomyces, first isolated from the filtrates of Streptomyces pimprina cultures in 1963 [5,6]; it is a monoamine oxidase (MAO) inhibitor. Both of these natural products belong to the class of naturally occurring 5-(3 -indolyl)oxazoles, and compounds of this family, including Pimprinethine; Pimprinaphine; WS-30581 A and B; Labradorins 1 and 2; Almazole A, B, and C; and Martefragin A, exhibit a wide range of potent biological activities [7] (Figure 1), such as anti-angiogenesis [3], antibiotic [8], anticancer [1], anti-cell proliferation [9], antioxidant [10], and antiviral activity [11,12]. Bioassay conducted at Syngenta showed that streptochlorin and pimprinine are also promising antifungal substances demonstrating good bioactivity against many phytopathogens [13][14][15][16]; for example, streptochlorin displayed excellent In classical medicinal chemistry, the nitrile group was commonly considered as bioisosteres of carbonyl, hydroxyl, and carboxyl groups, as well as halogen atoms [17]. As nitrile-containing drugs account for 2.4% of the 2327 approved small-molecule drugs according to the DrugBank database by 2018 [18], the presence of the nitrile group in the structure of compounds is a very common feature of drug molecules [19,20], such as Enzalutamide, a hormone treatment that blocks testosterone from reaching prostate cancer cells [21], Escitalopram, a medication used in the management and treatment of major depressive disorder and generalized anxiety disorder [22]; Tofacitinib, as an oral JAK3 inhibitor to treat adults with moderately to severely active rheumatoid arthritis [23]; Verapamil, a medication for treating hypertension, angina, and certain heart rhythm disorders [24]; Rilpivirine, a non-nucleoside reverse transcriptase inhibitor that inhibits the replication of HIV-1 [25]; and Vildagliptin, an orally administered dipeptidyl peptidase-4 (DPP-4) inhibitor for treating diabetes [26]. Meanwhile, Cyazofamid is a novel fungicide exhibiting specific activity against diseases caused by Oomycetes [27]; Azoxystrobin is a broad-spectrum β-methoxyacrylate fungicide that was first introduced in 1998, which inhibits mitochondrial respiration by binding to the Qo site of the cytochrome bc1 complex [28,29]; and Phenamacril is a Fusarium-specific fungicide used for Fusarium head blight management [30,31]. (Figure 2). In classical medicinal chemistry, the nitrile group was commonly considered as bioisosteres of carbonyl, hydroxyl, and carboxyl groups, as well as halogen atoms [17]. As nitrile-containing drugs account for 2.4% of the 2327 approved small-molecule drugs according to the DrugBank database by 2018 [18], the presence of the nitrile group in the structure of compounds is a very common feature of drug molecules [19,20], such as Enzalutamide, a hormone treatment that blocks testosterone from reaching prostate cancer cells [21], Escitalopram, a medication used in the management and treatment of major depressive disorder and generalized anxiety disorder [22]; Tofacitinib, as an oral JAK3 inhibitor to treat adults with moderately to severely active rheumatoid arthritis [23]; Verapamil, a medication for treating hypertension, angina, and certain heart rhythm disorders [24]; Rilpivirine, a non-nucleoside reverse transcriptase inhibitor that inhibits the replication of HIV-1 [25]; and Vildagliptin, an orally administered dipeptidyl peptidase-4 (DPP-4) inhibitor for treating diabetes [26]. Meanwhile, Cyazofamid is a novel fungicide exhibiting specific activity against diseases caused by Oomycetes [27]; Azoxystrobin is a broad-spectrum β-methoxyacrylate fungicide that was first introduced in 1998, which inhibits mitochondrial respiration by binding to the Qo site of the cytochrome bc 1 complex [28,29]; and Phenamacril is a Fusariumspecific fungicide used for Fusarium head blight management [30,31]. (Figure 2).  Introducing the nitrile group into the molecules is an effective protocol for structural optimization ( Figure 3). For example, the nitrile-containing structure exhibited a 277-fold improvement in potency over the non-substituted structure as selective inhibitors of cFMS. For casein kinase 2 (CK2) inhibitor, the nitrile-containing structure improved binding affinity more than 90-fold compared with the non-substituted structure, and the nitrile group was engaged in hydrogen bond interactions with the conserved water molecules in a cocrystal structure (PDB Code: 5H8B) [17]. Introducing the nitrile group into the molecules is an effective protocol for structural optimization ( Figure 3). For example, the nitrile-containing structure exhibited a 277-fold improvement in potency over the non-substituted structure as selective inhibitors of cFMS. For casein kinase 2 (CK2) inhibitor, the nitrile-containing structure improved binding affinity more than 90-fold compared with the non-substituted structure, and the nitrile group was engaged in hydrogen bond interactions with the conserved water molecules in a cocrystal structure (PDB Code: 5H8B) [17].
In this study, based on the parent structures of streptochlorin and pimprinine (Figure 4), we designed and synthesized a series of streptochlorin derivatives containing the nitrile group, and carried out the evaluation for antifungal activity, aiming at the discovery of natural product derivatives with improved antifungal activity. Furthermore, the structure-activity relationships (SARs) around these compounds and the molecular docking of the most active compound with potential target enzyme were further performed.  In this study, based on the parent structures of streptochlorin and pimprinine (Figu 4), we designed and synthesized a series of streptochlorin derivatives containing the trile group, and carried out the evaluation for antifungal activity, aiming at the discove of natural product derivatives with improved antifungal activity. Furthermore, the stru ture-activity relationships (SARs) around these compounds and the molecular docking the most active compound with potential target enzyme were further performed.

Synthetic Chemistry
The series of streptochlorin derivatives containing the nitrile group were synthesiz as shown in Scheme 1, using the reported methods [32,33]. The synthesis started w cheap and readily available indole (1). After the acylation of indole, 3-cyanoacetylind (2) was obtained. Then, the target compounds 3 were synthesized by the oxidative ann lation of 3-cyanoacetylindole. With DMF as solvent and TBHP as oxidant, 3-cyanoa tylindole reacted with methylene amine under the catalysis of iodine to give compoun  In this study, based on the parent structures of streptochlorin and pimprinine ( Figure  4), we designed and synthesized a series of streptochlorin derivatives containing the nitrile group, and carried out the evaluation for antifungal activity, aiming at the discovery of natural product derivatives with improved antifungal activity. Furthermore, the structure-activity relationships (SARs) around these compounds and the molecular docking of the most active compound with potential target enzyme were further performed.

Synthetic Chemistry
The series of streptochlorin derivatives containing the nitrile group were synthesized as shown in Scheme 1, using the reported methods [32,33]. The synthesis started with cheap and readily available indole (1). After the acylation of indole, 3-cyanoacetylindole (2) was obtained. Then, the target compounds 3 were synthesized by the oxidative annulation of 3-cyanoacetylindole. With DMF as solvent and TBHP as oxidant, 3-cyanoacetylindole reacted with methylene amine under the catalysis of iodine to give compounds 3. The structures and yields of 20 target compounds are shown in Figure 5. Copies of the NMR spectra and HR-MS (ESI) spectra can be found in the Supplementary Materials.

Synthetic Chemistry
The series of streptochlorin derivatives containing the nitrile group were synthesized as shown in Scheme 1, using the reported methods [32,33]. The synthesis started with cheap and readily available indole (1). After the acylation of indole, 3-cyanoacetylindole (2) was obtained. Then, the target compounds 3 were synthesized by the oxidative annulation of 3-cyanoacetylindole. With DMF as solvent and TBHP as oxidant, 3-cyanoacetylindole reacted with methylene amine under the catalysis of iodine to give compounds 3. The structures and yields of 20 target compounds are shown in Figure 5

Antifungal Activity and Structure-Activity Relationships (SARs)
The antifungal activity of the target compounds and positive controls was evaluated against six common phytopathogenic fungi at the concentration of 50 μg/mL, including Botrytis cinerea (BOT), Alternaria solani (ALS), Gibberella zeae (GIB), Rhizoctorzia solani (RHI), Colletotrichum lagenarium (COL), and Alternaria Leaf Spot (ALL). The screening results are presented in Table 1.

Antifungal Activity and Structure-Activity Relationships (SARs)
The antifungal activity of the target compounds and positive controls was evaluated against six common phytopathogenic fungi at the concentration of 50 μg/mL, including Botrytis cinerea (BOT), Alternaria solani (ALS), Gibberella zeae (GIB), Rhizoctorzia solani (RHI), Colletotrichum lagenarium (COL), and Alternaria Leaf Spot (ALL). The screening results are presented in Table 1.

Antifungal Activity and Structure-Activity Relationships (SARs)
The antifungal activity of the target compounds and positive controls was evaluated against six common phytopathogenic fungi at the concentration of 50 µg/mL, including Botrytis cinerea (BOT), Alternaria solani (ALS), Gibberella zeae (GIB), Rhizoctorzia solani (RHI), Colletotrichum lagenarium (COL), and Alternaria Leaf Spot (ALL). The screening results are presented in Table 1.  As compounds 3a, 3b, 3g, and 3h exhibited relatively good antifungal activity in primary screening; EC50 values of them and commercial fungicides Boscalid and Carbendazim were further determined ( Table 2). The most active compound 3a was compared with Osthole, Boscalid, and Flutriafol in the radar chart shown in Figure 6, and its antifungal activity against four kinds of fungi was more active than at least one of the positive controls. As compounds 3a, 3b, 3g, and 3h exhibited relatively good antifungal activity in primary screening; EC 50 values of them and commercial fungicides Boscalid and Carbendazim were further determined ( Table 2). The most active compound 3a was compared with Osthole, Boscalid, and Flutriafol in the radar chart shown in Figure 6, and its antifungal activity against four kinds of fungi was more active than at least one of the positive controls.
Although the antifungal activity of most of streptochlorin derivatives containing the nitrile group was relatively poor, making it difficult to find clear structure-activity relationships, some preliminary conclusions could still be drawn.
Firstly, it is worth noting that the target compounds lack antifungal activity potency, though compounds 3a and 3g showed a more than 50% antifungal effect against at least three kinds of fungi. 3a could be regarded as the most promising candidate, as it demonstrated over 85% growth inhibition against Botrytis cinerea, Gibberella zeae, and Colletotrichum lagenarium, as well as a broad antifungal spectrum.
Secondly, this series of streptochlorin derivatives showed relatively stronger antifungal activity against Rhizoctorzia solani than the other five phytopathogenic fungi. This was highlighted by compounds 3b, 3g, and 3h, which were equivalent to or even more active than Osthole.  Although the antifungal activity of most of streptochlorin derivatives containing th nitrile group was relatively poor, making it difficult to find clear structure-activity rela tionships, some preliminary conclusions could still be drawn.
Firstly, it is worth noting that the target compounds lack antifungal activity potency though compounds 3a and 3g showed a more than 50% antifungal effect against at leas three kinds of fungi. 3a could be regarded as the most promising candidate, as it demon strated over 85% growth inhibition against Botrytis cinerea, Gibberella zeae, and Colleto trichum lagenarium, as well as a broad antifungal spectrum.
Secondly, this series of streptochlorin derivatives showed relatively stronger antifun gal activity against Rhizoctorzia solani than the other five phytopathogenic fungi. This wa Thirdly, the antifungal activity data indicated that non-substituent or alkyl substituent at the 2-position of oxazole ring were favorable for antifungal activity, while aryl and monosubstituted aryl were detrimental to activity, though compound 3h also demonstrated 67.5% growth inhibition against Rhizoctorzia solani. This might be due to the presence of methylene on the benzyl group.

Molecular Docking
Although streptochlorin and pimprinine exhibited widely potent biological activities, the mechanism of action for the antifungal activity is still unclear. In our previous studies [16,34], molecular docking was performed on streptochlorin, which indicated that streptochlorin binds with tLeuRS in a similar mode to AN2690, and provided some ideas for the possible mechanism of action for antifungal activity of synthesized target compounds.  Figure 7) was selected and analyzed according to the minimum value of the docking energy.
ties, the mechanism of action for the antifungal activity is still unclear. In our previous studies [16,34], molecular docking was performed on streptochlorin, which indicated that streptochlorin binds with tLeuRS in a similar mode to AN2690, and provided some ideas for the possible mechanism of action for antifungal activity of synthesized target compounds.
Molecular docking of the most active compound 3a with receptor protein tLeuRS (PDB Code: 2V0C) was performed using Autodock 4.2. The protein was downloaded in high resolution solved at 1.85 Å from RCSB Protein Data Bank (https://www.rcsb.org/, accessed on 29 October 2022). After the molecular docking, the best binding mode of 3a (cyan in Figure 7) was selected and analyzed according to the minimum value of the docking energy. The simulated binding models indicated that compound 3a formed hydrogen bonds and hydrophobic interactions with the amino acid residues. The nitrile group of 3a formed a hydrogen bond with residue Met338, the indole N-H bond formed hydrogen bonds with Thr247 and Thr252, and the oxazole ring formed a weak hydrogen bond with Arg346. The indole ring formed hydrophobic interactions with Arg249, Thr252, Val340, His343, and Asp344 (Figure 7).

Chemicals
All commercially available chemicals were purchased from Nanjing Crystal Chemical Co. Ltd. (Nanjing, China) or Alfa Aesar (Beijing, China) and were analytically pure. The specification of silica gel for column chromatography was 200-300 mesh. All target The simulated binding models indicated that compound 3a formed hydrogen bonds and hydrophobic interactions with the amino acid residues. The nitrile group of 3a formed a hydrogen bond with residue Met338, the indole N-H bond formed hydrogen bonds with Thr247 and Thr252, and the oxazole ring formed a weak hydrogen bond with Arg346. The indole ring formed hydrophobic interactions with Arg249, Thr252, Val340, His343, and Asp344 (Figure 7).

Chemicals
All commercially available chemicals were purchased from Nanjing Crystal Chemical Co., Ltd. (Nanjing, China) or Alfa Aesar (Beijing, China) and were analytically pure. The specification of silica gel for column chromatography was 200-300 mesh. All target compounds were characterized by melting point, 1 H NMR, 13  Furthermore, 3-cyanoacetylindole (2) and the target compounds (3) were synthesized using the reported methods [32,33]. All of the reaction yields were not optimized.

Preparation of 3-(1H-indol-3-yl)-3-oxopropanenitrile (2)
Cyanoacetic acid (3.40 g, 40 mmol) was dissolved in Ac 2 O (76 mL) with stirring and heating to 50 • C. Indole (4.69 g, 40 mmol) was then added and the solution was heated to 85 • C. The reaction was monitored by TLC and, after the reaction was complete, the mixture was cooled in ice water. The solid was collected under suction and washed with MeOH to obtain pure compound 2.

Biological Assays
Antifungal activity testing was carried out using the mycelia growth-inhibitory rate method. The six common phytopathogenic fungi selected were Botrytis cinerea, Alternaria solani, Gibberella zeae, Rhizoctonia solani, Colletotrichum lagenarium, and Alternaria leaf spot, which were provided by the Laboratory of Plant Disease Control, Nanjing Agricultural University. The experimental procedure of the antifungal activity was performed according to the paper from the Department of Plant Pathology, Nanjing Agricultural University [35]. The compounds and three positive controls, Osthole, Boscalid, and Flutriafol, were tested at 50 µg/mL in the primary screening. The strains were activated in Potato Dextrose Agar Medium (PDA) at 25 • C for 2-15 days to afford new mycelia; the edge of the mycelia was punched before the antifungal activity assay. The screening results are listed in Table 1.

Molecular Docking Strategy
First, removing the water molecules in the protein was performed using PyMol 2.5.4 (Schrödinger, New York, NY, USA). Drawing and energy minimization of ligand molecules were completed in Chemdraw (Version 14.0, CambridgeSoft, Cambridge, MA, USA) and Chem3D (Version 14.0, CambridgeSoft, Cambridge, MA, USA). Then, the preparation of the protein and ligand was performed using Autodock 4.2 (The Scripps Research Institute, La Jolla, CA, USA). For protein, we added the hydrogen atoms, calculated the charge, and added the atom type (Assign AD4type). As for ligand, we checked the charge, "detect Root", and "Choose Torsions". Finally, we ran docking after setting the Grid (cen-ter_x = 53.489, center_y = −26.319, center_z = 33.004, size_x = size_y = size_z = 22.5 Å) and docking parameters, and the number of runs was 50. The best binding mode was analyzed in PyMol.

Conclusions
Based on the natural product structures of streptochlorin and pimprinine derived from marine or soil microorganisms, 20 kinds of streptochlorin derivatives containing the nitrile group were effectively synthesized from indole, through acylation and oxidative annulation. The antifungal activity of the target compounds against six common phytopathogenic fungi was evaluated at 50 µg/mL. Evaluation of antifungal activity showed that compound 3a could be regarded as the most promising candidate-it demonstrated over 85% growth inhibition against Botrytis cinerea, Gibberella zeae, and Colletotrichum lagenarium, as well as a broad antifungal spectrum in the primary screening at a concentration of 50 µg/mL, though the target compounds lack antifungal activity potency as a whole. The SAR study revealed that non-substituent or alkyl substituent at the 2-position of oxazole ring were favorable for antifungal activity, while aryl and monosubstituted aryl were detrimental to activity. Molecular docking models indicated that 3a formed hydrogen bonds and hydrophobic interactions with Leucyl-tRNA Synthetase, offering a perspective for the possible mechanism of action for antifungal activity of the target compounds. Further structural optimization is well under way.