Alkaloids of Dicranostigma franchetianum (Papaveraceae) and Berberine Derivatives as a New Class of Antimycobacterial Agents

Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis. The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has created a need for new antiTB agents with new chemical scaffolds to combat the disease. Thus, the key question is: how to search for new antiTB and where to look for them? One of the possibilities is to search among natural products (NPs). In order to search for new antiTB drugs, the detailed phytochemical study of the whole Dicranostigma franchetianum plant was performed isolating wide spectrum of isoquinoline alkaloids (IAs). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. Alkaloids were screened against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. avium, M. kansasii, and M. smegmatis). Alkaloids 3 and 5 showed moderate antimycobacterial activity against all tested strains (MICs 15.625–31.25 µg/mL). Furthermore, ten semisynthetic berberine (16a–16k) derivatives were developed and tested for antimycobacterial activity. In general, the derivatization of berberine was connected with a significant increase in antimycobacterial activity against all tested strains (MICs 0.39–7.81 μg/mL). Two derivatives (16e, 16k) were identified as compounds with micromolar MICs against M. tuberculosis H37Ra (MIC 2.96 and 2.78 µM). All compounds were also evaluated for their in vitro hepatotoxicity on a hepatocellular carcinoma cell line (HepG2), exerting lower cytotoxicity profile than their MIC values, thereby potentially reaching an effective concentration without revealing toxic side effects.


Introduction
Dicranostigma Hooker f. & Thomson is a small genus of the Papaveraceae family with eight accepted species native to the Himalayas and western China [1,2]. Plants of this genus are also known as eastern horned poppies. Although resembling the true horned poppies of Glaucium, they have stigmas with two lobes and fruit with only traces of "horns". These plants have been used for a long time in folk medicine to treat various diseases in derivatives of NPs (i.e., sampangine, cleistopholine, berberine, and galanthamine) have been studied for their antimycobacterial potential [24][25][26][27].
The interesting biological activities of the IAs and the absence of current phytochemical study of D. franchetianum encouraged us to examine this species. As a part of our ongoing research on IAs and their semisynthetic derivatives as potential drugs, this work reports on the isolation of alkaloids from the whole plant of D. franchetianum, preparation of berberine derivatives, and their in vitro antimycobacterial activity against Mtb H37Ra, and four other mycobacterial strains (M. aurum, M. avium, M. kansasii, and M. smegmatis).

General Experimental Procedures
All solvents were treated using standard techniques before use. All reagents and catalysts were purchased from Sigma Aldrich, Czech Republic, and used without purification. NMR spectra were recorded in either CDCl 3 or CD 3 OD on a VNMR S500 (Varian, Palo Alto, CA, USA) spectrometer operating at 500 MHz for proton nuclei and 125.7 MHz for carbon nuclei at ambient temperature. ESI-HRMS were obtained with a Waters Synapt G2-Si hybrid mass analyzer of a quadrupole-time-of-flight (Q-TOF) type, coupled to a Waters Acquity I-Class UHPLC system (Waters, Millford, MA, USA). The EI-MS were obtained on an Agilent 7890A GC 5975 inert MSD operating in EI mode at 70 eV (Agilent Technologies, Santa Clara, CA, USA). A DB-5 column (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, USA) was used with a temperature program: 100-180 • C at 15 • C/min, 1 min hold at 180 • C, and 180-300 • C at 5 • C/min and 5 min hold at 300 • C; and detection range m/z 40-600. The injector temperature was 280 • C. The flow rate of carrier gas (helium) was 0.8 mL/min. A split ratio of 1:15 was used. Fractionation of fractions II and V was performed with a BÜCHI Sepacore flash system ×10 equipped with a BÜCHI control unit C-620, BÜCHI fraction collector C-660, UV photometer C-640, and BÜCHI pump modules C-605 (BÜCHI, Flawil, Switzerland). TLC was carried out on Merck precoated silica gel 60 F254 and Merck precoated silica gel 60 RP-18 F254 plates. Compounds on the plate were observed under UV light (254 and 366 nm) and visualized by spraying with Dragendorff's reagent.

Plant Material
The plant material used from Dicranostigma franchetianum came from a monoculture introduced to the Garden of Medicinal Plants at the Faculty of Pharmacy, Charles University in Hr. Králové in 2014 (seeds from the Centre of Medicinal Plants of Masaryk University in Brno). Botanical identification was performed by Prof. L. Opletal. A voucher specimen is deposited in the Herbarium of the Faculty of Pharmacy in Hradec Králové under number: CUFPH-16130/AL-540.

Extraction and Isolation of Alkaloids
Finely cut and dried aerial parts of D. franchetianum (11.87 kg) were minced and sequentially extracted with 95% EtOH (500 g of material boiled with 3 L of EtOH) for 30 min. The combined extracts were evaporated to the consistency of thin syrup, to which 6 L of distilled water at 80 • C was added, and the pH adjusted to 1-1.5 by the addition of 2% H 2 SO 4 . The suspension was filtered through Celite 545. The filtrate was alkalized by 10% Na 2 CO 3 (pH 9-10) and extracted with CHCl 3 (3 × 5 L). The organic layer was evaporated to give 120 g of dark brown syrup. This alkaloid summary extract was again dissolved in 2% H 2 SO 4 (3 L), defatted with Et 2 O (3 × 2 L), and alkalized to pH 9-10 with 10% Na 2 CO 3 . The water layer was extracted with EtOAc (4 × 2 L) to give 47 g of concentrated alkaloidal extract in the form of brown syrup.
Other fractions and subfractions have not been used to isolate alkaloids due to either the low amount or complex mixture of compounds.

Preparation of Berberine Derivatives
The same procedure as described previously has been used to afford the corresponding ethers. NMR and mass spectra of the synthesized derivatives (16a-16k) were in agreement with a previous report [28].
The mycobacterial strains were cultured on Middlebrook 7H9 agar and suspensions were prepared in Middlebrook 7H9 broth. The final density was adjusted to 1.0 on the McFarland scale and diluted in the ratio of either 1:20 (for rapidly growing mycobacteria) or 1:10 (for slow-growing mycobacteria) with broth.
The tested compounds were dissolved in DMSO (Sigma-Aldrich, Steinheim, Germany), then Middlebrook broth was added to obtain a concentration of 2000 µg/mL. The standards used for activity determination were INH, RIF, and CPX (Sigma-Aldrich, Steinheim, Germany).

Cytotoxicity Assay
Human hepatocellular carcinoma HepG2 cells (ATCC HB-8065; passage 20-25), purchased from Health Protection Agency Culture Collections (ECACC, Salisbury, UK), were cultured in Minimum Essential Eagle Medium supplemented with 10% fetal bovine serum and 1% L-glutamine solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C in a humidified atmosphere containing 5% CO 2 . For passage, the cells were treated with trypsin/EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C and then harvested. For cytotoxicity evaluation, the cells treated with the test substances were used, while untreated HepG2 cells served as the control group; blank was included, as well. The cells were seeded in a 96-well plate at a density of 50,000 cells per well and incubated for 24 h. The following day, the cells were treated with each of the test compounds dissolved in DMSO (the highest DMSO concentration used was 0.5% v/v). The test substances were prepared at different concentrations in triplicates according to their solubility and incubated for 24 h in a humidified atmosphere containing 5% CO 2 at 37 • C. After incubation, a solution of thiazolyl blue tetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 without phenol red (BioTech, Praha, Czech Republic) was added and incubated for 30 min in a humidified atmosphere containing 5% CO 2 at 37 • C. Then, the formazan crystals formed were dissolved in DMSO and the absorbance of the samples was recorded at 570 nm (BioTek, Synergy Neo2 Multi-Mode Reader NEO2SMALPHAB). IC 50 values were calculated by nonlinear regression from a semi-logarithmic plot of incubation concentration versus percentage of absorbance relative to untreated controls using GraphPad Prism software (version 9; GraphPad Software, Inc., La Jolla, CA, USA). The obtained results of the experiments are presented as the concentration that reduces the viability of cells from the maximal viability to 50% (IC 50 ).
Considering the anti-TB potency of alkaloids containing an isoquinoline heterocycle in their structure [43], the compounds isolated in sufficient amounts have been screened for their antimycobacterial potential against five Mycobacterium or Mycolicibacterium strains ( Table 1). Most of the tested alkaloids showed either weak or no antimycobacterial potency (MIC ≥ 125 µg/mL, Table 1). Alkaloids 3 and 5, which contain a benzophenanthridine skeleton in their structure, displayed moderate activity against all the tested mycobacterial strains (MICs 15.625-31.25 µg/mL, Table 1). However, alkaloid 3 was recognized as an isolation artifact only. According to the isolation conditions, chelerythrine was unintentionally introduced to ethanol in an acidic environment at the very beginning of the phytochemical work, where the iminium electrophile was readily trapped by a nucleophile. Thus, this nucleophilic addition furnished a hemiaminal ether 3. Compound 5 is a dimer of two molecules of dihydrochelerythrine connected at position C-6 by an ether bridge and has been isolated previously from Zanthoxylum paracanthum and Z. monophyllum [44,45]. Dostál et al. have previously reported chelerythrine-like compounds in a thorough description of the dimerization. We assume that the action of the basic aqueous conditions of the phytochemical process yielded this dimer 5 [46]. Due to the induced dimerization, chelerythrine has never appeared in our final steps of isolation. Nevertheless, compound 5 exhibited strong activity against Aspergillus fumigatus (IC 50 = 0.9 µM) and methicillin-resistant Staphylococcus aureus (IC 50 = 1 µM) [45]. The improved antimycobacterial activity of 3 and 5, compared to dihydrochelerythrine itself (2), can be an inspiration for the preparation of more potent antimycobacterial compounds derived from the benzophenanthridine skeleton.
In previous studies, berberine (16) exerted potent antimycobacterial activity against M. intracellulare with a MIC value of 1.56 µg/mL [47], and moderate activity against Mycolicibacterium smegmatis (MIC 25 µg/mL) [48]. On the contrary, the study of Gentry et al.  (Table 1). Semisynthetic derivatives of 16 are reported as compounds with strong antimycobacterial potential, e.g., 2,3,9-triethoxy-3,9-dibenzyloxy-10-methoxy-13-n-octylprotoberberine chloride displaying activity against drug-susceptible Mtb strain H37Rv with a MIC value of 0.125 µg/mL. Moreover, this berberine derivative showed an intriguing effect against RIF-and INH-resistant Mtb, which implies a new mechanism of action [27]. Because berberine (16) was isolated in quantity allowing preparation of its semisynthetic derivatives, we continued our study with the design and synthesis of C-9 substituted berberine analogues to inspect their antimycobacterial activity.

Berberine Derivatives (16a-16k) and Their Antimycobacterial Activity
Berberine is currently one of the most studied NPs, with a wide range of biological activities, including antimicrobial, hepatoprotective, antihyperlipidemic, anticancer, antidiabetic, anti-inflammatory, and antiarrhythmic effects [49][50][51][52][53]. It is available globally, mainly as a root extract, providing antimicrobial and antidiabetic effects. Recent studies have shown that berberine can enhance the inhibitory efficiency of antibiotics against clinically multi-drug resistant isolates of Staphylococcus aureus [54]. Besides, berberine administration to mice with pulmonary tuberculosis resulted in decreased lung pathology with no additive or synergistic effects on bacterial burden [55]. Berberine has low permeability through biological membranes and poor bioavailability, limiting its application and further development. Structural modification of NPs is a powerful method to improve their pharmacological properties. For this reason, structural modifications of berberine (16) that increase its lipophilicity, mostly at the C-9 position, have been pursued to address these drawbacks [28,56]. Based on our previous report on the antimycobacterial activity of semisynthetic derivatives of Amaryllidaceae alkaloids, which showed promising activity against three Mycobacterium strains, we selected berberine (16) for the preparation of a small library of its semisynthetic derivatives, which have been screened for antimycobacterial activity against five Mycobacterium strains (Table 1). Given the necessity of long-term administration of anti-TB drugs, we have also determined their potential hepatotoxicity in vitro using a hepatocellular HepG2 cell line.
istration to mice with pulmonary tuberculosis resulted in decreased lung pathology with no additive or synergistic effects on bacterial burden [55]. Berberine has low permeability through biological membranes and poor bioavailability, limiting its application and further development. Structural modification of NPs is a powerful method to improve their pharmacological properties.
For this reason, structural modifications of berberine (16) that increase its lipophilicity, mostly at the C-9 position, have been pursued to address these drawbacks [28,56]. Based on our previous report on the antimycobacterial activity of semisynthetic derivatives of Amaryllidaceae alkaloids, which showed promising activity against three Mycobacterium strains, we selected berberine (16) for the preparation of a small library of its semisynthetic derivatives, which have been screened for antimycobacterial activity against five Mycobacterium strains (Table 1). Given the necessity of long-term administration of anti-TB drugs, we have also determined their potential hepatotoxicity in vitro using a hepatocellular HepG2 cell line.

Figure 2.
Berberine derivatives from our library investigated as antitubercular agents within this study [28].
In all cases, replacing the methoxy group at position C-9 by differently substituted benzyl appendages was associated with a significant increase in antimycobacterial activity against all studied Mycobacterium strains (MIC 0.39-7.81 µg/mL, Table 1). For all the tested compounds, Mycolicibacterium smegmatis was the most sensitive strain. Among the methyl-substituted benzylberberine derivatives (16b-16d), a para position of the methyl group on the aromatic ring yielded the most active compound. Bromine substitution within the benzyl moiety resulted in better activity against all strains, generating the most active compound within the study 9-O-(2-brombenzyl)berberine (16e) (MIC 0.390-3.125 µg/mL). For this halogen substituent, the ortho position at the aromatic ring was connected with the highest activity. 3,4-Dichloro-substituted analogue 16k showed the same activity Figure 2. Berberine derivatives from our library investigated as antitubercular agents within this study [28].
In all cases, replacing the methoxy group at position C-9 by differently substituted benzyl appendages was associated with a significant increase in antimycobacterial activity against all studied Mycobacterium strains (MIC 0.39-7.81 µg/mL, Table 1). For all the tested compounds, Mycolicibacterium smegmatis was the most sensitive strain. Among the methyl-substituted benzylberberine derivatives (16b-16d), a para position of the methyl group on the aromatic ring yielded the most active compound. Bromine substitution within the benzyl moiety resulted in better activity against all strains, generating the most active compound within the study 9-O-(2-brombenzyl)berberine (16e) (MIC 0.390-3.125 µg/mL). For this halogen substituent, the ortho position at the aromatic ring was connected with the highest activity. 3,4-Dichloro-substituted analogue 16k showed the same activity against Mtb as 16e, and either the same or slightly lower potency against the other tested strains ( Figure 2, Table 1).

Lipophilicity versus Activity
Lipophilicity is one of the most important physicochemical properties of a compound that is crucially related to cell transmembrane transport. The mycobacterial cell wall is rich in mycolic acids, a distinctive feature of the mycobacterial cell wall, which efficiently prevents the penetration of drugs. Drugs with higher lipophilicity may exert better activity against Mtb [57], and thus we calculated ClogP values using the ChemBioDraw Ultra program (ver 18.1; PerkinElmer, Waltham, MA, USA). The ClogP value is correlated directly to the molecular hydrophobicity, presuming the diffusion through the biological membranes. All synthesized derivatives showed a higher ClogP value (0.99-2.30) in comparison with parent berberine (−0.77).

Cytotoxicity of Berberine Derivatives
Compounds with MIC values lower than 20 µM for Mtb H37Ra were also evaluated for in vitro cytotoxicity on hepatocellular carcinoma cells (HepG2) using an MTT assay. The HepG2 cell line serves as an in vitro model for hepatotoxicity for early drug screening. Moreover, the hepatocellular model was chosen since anti-TB drugs are known to carry the risk of hepatotoxicity, mostly ascribed to their long-term administration [58].
Evaluation of cytotoxicity allows the calculation of selectivity indexes (SI) as the ratio of IC 50 , HepG2 to MIC of Mtb H37Ra (Table 1; Table S1). In general, values of SI higher than 10 indicate more acceptable toxicity (analogous to the therapeutic index). The active compounds were screened at an initial concentration of 50 µM, and the IC 50 values were subsequently determined. The effective cytotoxic concentrations of all tested compounds are listed in Table 1.
Among berberine derivatives, compound 16h exhibited the highest cytotoxicity with an IC 50 value of 9.44 ± 0.29 µM. The most active derivatives in terms of the highest antimycobacterial potency, i.e., 16e and 16k, showed cytotoxicity, with IC 50 values of 11.61 ± 0.29 µM, and 12.66 ± 2.51 µM, respectively, reaching SI values of 3.92 for 16e, and 4.55 for 16k. These values indicate a potential risk of hepatotoxicity. Thus, further research to find a balance between antimycobacterial efficacy and hepatotoxicity will be necessary to optimize the berberine scaffold.

Conclusions
In conclusion, the phytochemical investigation of the alkaloidal extract of the whole plant of Dicranostigma franchetianum allowed the isolation of 16 known isoquinoline alkaloids of different structural types. Compounds isolated in sufficient amounts were evaluated for their in vitro antimycobacterial activity against Mtb H37Ra and four other mycobacterial strains of clinical importance or usually used in antimycobacterial screening (Mycolicibacterium aurum, Mycobacterium avium, Mycobacterium kansasii, and Mycolicibacterium smegmatis). 6-Ethoxydihydrochelerythrine and bis-[6-(5,6-dihydrochelerythrinyl)]ether, both containing a benzophenanthridine nucleus in their structure, demonstrated moderate activity against all tested strains. The strong antimycobacterial potential of berberine, reported in previous studies, was not confirmed.
On the other hand, its semisynthetic derivatives demonstrated significant activity increment against all tested mycobacterial strains. Unfortunately, the most active berberine derivatives showed a relatively high cytotoxicity profile, as determined on HepG2 cell lines, indicating a potential hepatotoxicity risk. Therefore, further research on the berberine scaffold will focus on developing novel agents retaining high antimycobacterial profile while also reducing the cytotoxicity of the final entity. Further research warrants testing the top-ranked drug candidates in a combinatorial regimen by checkerboard studies to determine their synergistic properties with known antitubercular drugs. In vivo toxicity and in vivo efficacy will be determined using Galleria mellonella animal model. As promising antimycobacterial compounds, this pilot study presents prospective C-9 substituted berberine derivatives. Nevertheless, structure optimization needs further development and endeavor.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.