Synthesis and Antibacterial Study of Novel Harmine Derivatives and Tetrahydro-β-Carboline Derivatives In Vitro

Dairy mastitis is a disease of dairy cattle caused by a variety of pathogenic microorganisms which has biought huge economic losses aused huge economic losses to the world. In this paper, Harmine derivatives and tetrahydro-β-carboline derivatives synthesized by the splice method are shown to have a good inhibitory effect on the pathogenic bacteria of dairy mastitis. The results of a bacteriostatic test on pathogenic bacteria of dairy cow mastitis (S. dysgalactiae, S. pyogenes, B. subtilis and P. vulgaris) showed that compound 7l had the best bacteriostatic effect on Streptococcus dysgalactiae, with a mic value of 43.7 μ g/mL. When the concentration of 7l was 1 × MIC and 2 × MIC, it had a significant inhibitory effect on Streptococcus dysgalactiae, and there was almost no growth of Streptococcus dysgalactiae at 4 × MIC. The binding properties of target compound 7l to amine oxidase [flavin-containing] A protein were simulated by the molecular docking technique. The ligand 7l achieved strong binding with the receptor through three hydrogen bonds. The hydrogen bonds were amino acid residues thr-52, arg-51 and ser-24, which are the main force for the compound to bind to active sites.


Introduction
Harmel is a plant of the genus Pederma of Tribulus teristaceae, growing in arid areas [1]. Harmine is the main active ingredient of Pederma teristaceae [2]; The structure is shown in Figure 1. It has antibacterial properties and protects the seeds from bacterial invasion. Harmine has abundant natural distribution and extensive pharmacological activities, i.e., anti-tumor, anti-inflammatory, antioxidant and other effects. Additionally, its excellent antibacterial effect has attracted extensive attention from researchers [3]. Structural modification of harmine is expected to improve its antibacterial activity. Cinnamic acid is isolated from cinnamon or benzoin resin and has the characteristics of low molecular weight and low price. Harmine and cinnamic acid are both plant extracts with certain bacteriostatic effects. Compounds 7l and 7h were obtained by concatenating the cinnamate moiety at the harmine N9 site. Compounds 11b, 11f and 11g are obtained by linking different benzene ring groups at N2 site of tetrahydro-β-carboline through ester bonds. The structure of tetrahydro-β-carboline derivative is similar to that of the Harmine derivative. The spatial structure of the compound was obtained by single crystal diffraction [4,5]. This is a common chemical modification method to connect two compounds to produce new compounds. In this paper, target compound 7l is shown to have significantly improved antibacterial performance, and notably, enhanced inhibition of Streptococcus agalactiae. In

Minimum Inhibitory Concentration Test
A microplate dilution method was used to study the inhibitory effect of compounds 7l, 7h, 11b, 11f and 11g on Gram-positive bacteria (S. dysgalactiae, S. pyogenes, B. subtilis) and gram-negative bacteria (P. vulgaris). Harmine and cinnamic acid are used as control drugs. And the results were shown in Table 1. Compounds 7l, 7h, 11b, 11f and 11g had a good inhibitory effect on both Gram-positive and Gram-negative bacteria, and 7l had the best inhibitory effect on S. dysgalactiae.

Minimum Inhibitory Concentration Test
A microplate dilution method was used to study the inhibitory effect of compounds 7l, 7h, 11b, 11f and 11g on Gram-positive bacteria (S. dysgalactiae, S. pyogenes, B. subtilis) and gram-negative bacteria (P. vulgaris). Harmine and cinnamic acid are used as control drugs. And the results were shown in Table 1. Compounds 7l, 7h, 11b, 11f and 11g had a good inhibitory effect on both Gram-positive and Gram-negative bacteria, and 7l had the best inhibitory effect on S. dysgalactiae.

Results of Bactericidal Time-Kill Kinetics
The results of bactericidal time-kill kinetics were shown in Figure 2. Accord to the effect of Harmine and 7l on Streptococcus dysgalactiae, the growth inhibition of 1 × MIC in the Harmine group was shown. The growth inhibition was significant in the 2 × MIC group, and no growth was found in the 4 × MIC group at 10 h. In the 7l group, 1 × MIC was significantly inhibited, and 2 × MIC and 4 × MIC did not grow at 12 h and 6 h, respectively. Therefore, compound 7l has a stronger inhibitory effect on Streptococcus dysgalactiae than Harmine.

Results of Bactericidal Time-Kill Kinetics
The results of bactericidal time-kill kinetics were shown in Figure 2. Accord to the effect of Harmine and 7l on Streptococcus dysgalactiae, the growth inhibition of 1 × MIC in the Harmine group was shown. The growth inhibition was significant in the 2 × MIC group, and no growth was found in the 4 × MIC group at 10 h. In the 7l group, 1 × MIC was significantly inhibited, and 2 × MIC and 4 × MIC did not grow at 12 h and 6 h, respectively. Therefore, compound 7l has a stronger inhibitory effect on Streptococcus dysgalactiae than Harmine.

Synthesis and Characterization
The synthesis of 7l and its intermediates is described in Scheme 1. After Pictet-Spengler reaction, 6-methoxytryptamine forms β -carboline framework under acidic conditions, and is dehydrogenated under Pd/C catalysis to obtain Harmine. After Perkin reaction, substituted benzaldehyde and substituted acetic acid produce substituted cinnamic acid. The Harmine N9 site reacts with dibromoalkane in nucleophilic substitution. The nucleophilic substitution reaction between the product and substituted cinnamic acid is the key of the whole process.

Synthesis and Characterization
The synthesis of 7l and its intermediates is described in Scheme 1. After Pictet-Spengler reaction, 6-methoxytryptamine forms β-carboline framework under acidic conditions, and is dehydrogenated under Pd/C catalysis to obtain Harmine. After Perkin reaction, substituted benzaldehyde and substituted acetic acid produce substituted cinnamic acid. The Harmine N9 site reacts with dibromoalkane in nucleophilic substitution. The nucleophilic substitution reaction between the product and substituted cinnamic acid is the key of the whole process.
Tetrahydro-β-carboline derivatives and their intermediates are synthesized in Scheme 2. The formation of the amide bond generally occurs via the condensation reagent method; however, as the condensation reagent method progresses too quickly, this experiment used the acyl halide method, which is relatively mild. In this setting, commonly used acylation reagents are SOCl 2 and COCl 2 . First, the corresponding carboxylic acid reacts with its acylation reagent to produce the corresponding acyl chloride; it then reacts with the amino group to form an amide bond while the acyl halide method synthesizes the amide bond. The purpose of this is to improve the controllability of the whole reaction process. Tetrahydro-β-carboline derivatives and their intermediates are synthesized in Scheme 2. The formation of the amide bond generally occurs via the condensation reagent method; however, as the condensation reagent method progresses too quickly, this experiment used the acyl halide method, which is relatively mild. In this setting, commonly used acylation reagents are SOCl2 and COCl2. First, the corresponding carboxylic acid reacts with its acylation reagent to produce the corresponding acyl chloride; it then reacts with the amino group to form an amide bond while the acyl halide method synthesizes the amide bond. The purpose of this is to improve the controllability of the whole reaction process. Scheme 2. Synthesis of tetrahydro -β-carboline derivatives 11a-11i.

Crystal Structure Description of the Compound
wRref(F2) = 0.1067. The crystal temperature remains 296(2) K. Detailed crystallographic data and structural refinement parameters are shown in Table 2. Draw spatial structure diagram ( Figure 3) and packing diagram ( Figure 4) according to compound geometric parameters of 7l.

Crystal Structure Description of the Compound
Crystal data of 7l compound were obtained by X-ray single crystal diffraction. The molecular formula of the compound is C28H29N3O5. Detailed: monoclinic P 21/c (no.14) a = 8.7050 (11)    The crystals of 7l is a colorless, transparent and shiny block particles. The crystal size is 0.3 mm × 0.25 mm × 0.25 mm. The data were calculated by the least square method.
The cinnamic acid benzene part of target compound 7l is parallel to the harmine benzene part of another 7l molecule, and the distance between them is 3.6687 Å, which is conducive to a stable structure between molecules. According to the crystal structure analysis of the target compound, the plane where C1-C11 and C23-C28 are located is an immutable region, and C14-C22 is a single chain with a long distance which can be torsional and folded. This allows target compound 7l to enter the active pocket and generate hydrogen bonds and other secondary bonds with the receptor. The crystals of 7l is a colorless, transparent and shiny block particles. The crystal size is 0.3 mm × 0.25 mm × 0.25 mm. The data were calculated by the least square method. The molecule is not coplanar, according to the torsion angles C21-C22-C23-C28(-165.49°) and C21-C22-C23-C24(17.8°). The angle between Cinnamic acid nuclear plane C23-C28 and Harmine nuclear plane C1-C11 is 68.075(33), The C20-O1 bond (1.3252 Å) is shorter than an ordinary C19-O1 bond (1.4517 Å) but longer than a C20-O2 bond (1.1979 Å) due to p-π conjugation effect. The sum of the angles of O2-C20-O1(123.15°), O2-C20-C21(123.79°) and O1-C20-C21(113.06°) is 360°, indicating the sp 2 hybridization state of the C(22) atom.
The cinnamic acid benzene part of target compound 7l is parallel to the harmine benzene part of another 7l molecule, and the distance between them is 3.6687 Å, which is conducive to a stable structure between molecules. According to the crystal structure analysis of the target compound, the plane where C1-C11 and C23-C28 are located is an immutable region, and C14-C22 is a single chain with a long distance which can be torsional and folded. This allows target compound 7l to enter the active pocket and generate hydrogen bonds and other secondary bonds with the receptor.

Molecular Docking Results
The protein receptor PDBID: 2Y5Z is matched and docked with ligands 7l and 7h through AutoDock Vina software. The results showed that 7l was bound to amino acid residues Thr-52, Arg-51 and Ser-24 of Amine oxidase [flavin-containing] A protein by hydrogen bonds, and the bond lengths were 3.4 Å, 3.4 Å and 3.4 Å respectively [6]. Additionally, 7h binds to the Gly-22 amino acid residue of Amine oxidase [flavin-containing] A receptor and produces a 2.3 Å long hydrogen bond at Gly-22. Which is the main force for molecules to enter the active site. The results are shown in Figure 5. The 7l crystal structure was compared with the ligand structure obtained by docking. The C19-O1-C20 angle of the ligand structure is larger than that of 7l. The included angle of the target compound C19-O1-C20 is 117.00 (11). The plane of C1-C11 and C23-C28 is an immutable region, and the structure has not changed. The single chain region of C14-C22 is twisted and folded, so that the oxygen atom atO2 position forms a hydrogen bond with Thr-52. After molecular docking, the molecular energy is −9.6 kcal/mol. The ligand structure of 7h is compared with the crystal structure. The Angle of crystal structure C12-O5-C13 is 115.747 • , that of C1-C6 and C18-C28 is 14.2 • , that of the ligand structure C12-O5-C13 is 109.8 • , and the dihedral Angle of C1-C6 and C18-C28 is 165.5 • . The regional structure of the indole ring and benzene ring did not change, and the molecular energy after docking was −7.9 kcal/mol. According to the data recorded in the PDB database, a combination of Amine oxidase [flavin-containing] A receptor and Harmine does not produce a hydrogen bond. Target compounds 7l and 7h have good binding characteristics with Amine oxidase [flavin-containing] A, and their strength is greater than that of Harmine, Detailed crystal data are shown in Tables S1-S30 of the supplementary document.
The protein receptor PDBID:2Y5Z was combined with ligands 11b, 11f and 11g in a flexible docking manner. Ligands have different binding properties at receptor binding sites. The binding diagram is preferentially drawn with the conformation with the lowest binding energy, as shown in Figure 6. Notably, 11b has no hydrogen bond when entering the active site, and the molecular energy after docking is −8.1 kcal/mol. Additionally, 11F binds to the active site of Amine oxidase [flavin containing] A protein by two hydrogen bonds, producing hydrogen bonds with lengths of 2.0 Å and 2.5 Å at the amino acid residues of Gln-215 and Tyr-444, respectively. The molecular energy after binding is −8.7kcal/mol. The N2-C13-C19 angle of the ligand structure of 11f is the same as that of the crystal structure, which is 119.9 • . The dihedral angle between the C1-C7 plane and C14-C19 plane of the crystal structure is 40.3 • , and the dihedral angle between the C1-C7 plane and C14-C19 plane of the ligand structure is smaller than that of crystal structure, which facilitates the formation of the hydrogen bond between 11f and Gln-215. and benzene ring did not change, and the molecular energy after docking was -7.9 kcal/mol. According to the data recorded in the PDB database, a combination of Amine oxidase [flavin-containing] A receptor and Harmine does not produce a hydrogen bond. Target compounds 7l and 7h have good binding characteristics with Amine oxidase [flavincontaining] A, and their strength is greater than that of Harmine, Detailed crystal data are shown in Tables S1-S30 of the supplementary document. The protein receptor PDBID:2Y5Z was combined with ligands 11b, 11f and 11g in a flexible docking manner. Ligands have different binding properties at receptor binding sites. The binding diagram is preferentially drawn with the conformation with the lowest binding energy, as shown in Figure 6. Notably, 11b has no hydrogen bond when entering the active site, and the molecular energy after docking is -8.1 kcal/mol. Additionally, 11F binds to the active site of Amine oxidase [flavin containing] A protein by two hydrogen bonds, producing hydrogen bonds with lengths of 2.0 Å and 2.5 Å at the amino acid residues of Gln-215 and Tyr-444, respectively. The molecular energy after binding is -8.7kcal/mol. The N2-C13-C19 angle of the ligand structure of 11f is the same as that of the crystal structure, which is 119.9°. The dihedral angle between the C1-C7 plane and C14-C19 plane of the crystal structure is 40.3°, and the dihedral angle between the C1-C7 plane and C14-C19 plane of the ligand structure is smaller than that of crystal structure, which facilitates the formation of the hydrogen bond between 11f and Gln-215. The binding site of 11g entering the active pocket is similar to that of 11f. It is also connected in the form of a hydrogen bond with Tyr-444 amino acid residue. The hydrogen bond length is 3.5 Å and the molecular energy after binding is −7.7kcal/mol. The N2-C11-C12 angle of the ligand structure of 11g is the same as that of the crystal structure of 11g: 120.0°. The C1-C8 region of the crystal structure and ligand structure is the same as that of the C12-C17 region, and there is no change. The torsion angle of N2-C11-C12-C13 and the dihedral angle between the C1-C6 plane and C12-C17 plane of the ligand structure are larger than those of the crystal structure. The torsion angle of N2-C11-C12-C13 is 59.7°, and the dihedral angle formed by the C1-C6 plane and C12-C17 plane is 35.7°. Notably, 7l, 7h, 11b, 11f and 11g all have high binding ability to Amine oxidase [flavincontaining] A protein. In the flexible docking process, the conformation of the ligand compound is automatically adjusted according to the active site, while that of the crystal structure is different. A comparison of the two conformations showed that the indole ring and benzene ring hardly changed. Because C-C and C-N bonds can be twisted, the single chain region between indole ring and benzene ring is an important factor for conformational change, and is conducive to the reduction of steric hindrance of ligand binding to the receptor, as well as to the compound binding to the receptor. The binding of 7l produces three hydrogen bonds, the binding of 7h produces one, the binding of 11b produces no hydrogen bonds, the binding of 11f produces two hydrogen bonds, and the binding of 11g produces one hydrogen bond. The greater the number of hydrogen bonds, the stronger the binding ability at the active site, and the more advantages compared with competitive binding ligands. The molecular energies of 7l, 7h, 11b, 11f and 11g after docking with the receptor were -9.6 kcal/mol, -7.9 kcal/mol, -8.1 kcal/mol, -8.7 kcal/mol and - The binding site of 11g entering the active pocket is similar to that of 11f. It is also connected in the form of a hydrogen bond with Tyr-444 amino acid residue. The hydrogen bond length is 3.5 Å and the molecular energy after binding is −7.7kcal/mol. The N2-C11-C12 angle of the ligand structure of 11g is the same as that of the crystal structure of 11g: 120.0 • . The C1-C8 region of the crystal structure and ligand structure is the same as that of the C12-C17 region, and there is no change. The torsion angle of N2-C11-C12-C13 and the dihedral angle between the C1-C6 plane and C12-C17 plane of the ligand structure are larger than those of the crystal structure. The torsion angle of N2-C11-C12-C13 is 59.7 • , and the dihedral angle formed by the C1-C6 plane and C12-C17 plane is 35.7 • .
Notably, 7l, 7h, 11b, 11f and 11g all have high binding ability to Amine oxidase [flavincontaining] A protein. In the flexible docking process, the conformation of the ligand compound is automatically adjusted according to the active site, while that of the crystal structure is different. A comparison of the two conformations showed that the indole ring and benzene ring hardly changed. Because C-C and C-N bonds can be twisted, the single chain region between indole ring and benzene ring is an important factor for conformational change, and is conducive to the reduction of steric hindrance of ligand binding to the receptor, as well as to the compound binding to the receptor. The binding of 7l produces three hydrogen bonds, the binding of 7h produces one, the binding of 11b produces no hydrogen bonds, the binding of 11f produces two hydrogen bonds, and the binding of 11g produces one hydrogen bond. The greater the number of hydrogen bonds, the stronger the binding ability at the active site, and the more advantages compared with competitive binding ligands. The molecular energies of 7l, 7h, 11b, 11f and 11g after docking with the receptor were −9.6 kcal/mol, −7.9 kcal/mol, −8.1 kcal/mol, −8.7 kcal/mol and −7.7 kcal/mol respectively. Among them, 7l had the lowest molecular energy after binding with the receptor, indicating that its structure is more stable.

Discussion
Target compounds 7l and 7h were obtained by connecting the cinnamic acid moiety to the Harmine N9 site, while compounds 11b, 11f and 11g were obtained by linking the benzene ring-containing group to tetrahydro-β-Carboline. They were characterized by 1 H NMR, 13 C NMR and ESI-MS. The crystal structure of compound 7l was determined. By simulating the binding process between ligand molecule and receptor, compounds 7l, 7h, 11f and 11g were shown to be able to form strong hydrogen bonds with receptor Amine oxidase [flavin-containing] A. Target compound 7l connects to the receptor Amine oxidase [flavin-containing] A protein as a target molecule. Compared with harmine, target compound 7l can bind to the receptor Amine oxidase [flavin-containing] A protein more effectively through hydrogen bonding. Cow mastitis is a disease caused by a variety of pathogenic bacteria. Compound 7l, synthesized in the present research, has good inhibitory activity against its pathogenic bacteria. In vitro, the antibacterial activity of compound 7l was better than that of Harmine. Compound 7l had good bactericidal activity against Streptococcus lactis with a minimum inhibitory concentration (MIC) of 43.7 µg/mL. This study is of great significance for the treatment of dairy cow mastitis, and is of guiding significance for the transformation of Harmine and tetrahydro-β-Caroline. The antibacterial activity of the compound is worthy of further study.

Instruments and Reagents
Positive control Harmine and cinnamic acid were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). All chemical reagents were purchased from Energy Reagent Co., Ltd. (Beijing, China). Or Shanghai Huangdi Chemical Co., Ltd. (Shanghai, China). Unless otherwise stated, all commercial reagents were used immediately upon receipt. HRMS was obtained with an ESI-Brucker APEX II49e mass spectrometer (Bruker, Billerica, MA, USA). The 1 H NMR and 13 C NMR spectra were recorded with a JNM-ECS (JEOL Co., Ltd., Tokyo, Japan) and Bruke AM-400 spectrometer (Bruker, Marietta, GA, USA), respectively. The solvents used were deuterated reagents (DMSO-d6 and CDCl 3 ). Data are presented as follows: chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet, br = broad), coupling constant J (Hz) and integration. To monitor the reaction, thin layer chromatography (TLC) was performed on a silica gel plate (Qingdao Ocean Chemical Co., Ltd., Shandong, China) and observed with an ULTRAVIOLET lamp. The product is further purified by column chromatography on silica gel (200-300 mesh, Gansu Yihua Chemical Glass Instrument Co., Ltd., Lanzhou, China) and eluted by air pressure in an appropriate solvent mixture. Crystals of the target compounds were obtained by solvent evaporation at 25 • C and their crystal structures were determined using a Supernova single crystal diffractometer.

MIC Testing
In this study, the microplate dilution method was used to screen the antibacterial activity of 7l in vitro [7]. The investigation included three Gram-positive strains, Streptococcus dysgalactiae ATCC 35666, Bacillus subtilis CMCC(B) 63501 and Streptococcus pyogenes ATCC 19615, and one Gram-negative strain, Proteusbacillus vulgaris CMCC(B) 49027. Harmine and cinnamic acid were used as control drugs.

Synthesis and Crystallization
The 6-methoxtryptamine was dissolved in 40 mL HCl solution (0.1N) and stirred at room temperature until clarified. Next, 5 mL of 35% acetaldehyde solution (20 mmol) was slowly added to the reaction solution. The reaction was carried out at 40 • C for 6 h, the reaction liquid was cooled to room temperature and the pH was adjusted to 10. After extraction with dichloromethane, the organic phase was combined, washed with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered, decompressed and suspended by steaming, and purified by column chromatography to prepare the compound for use [10].