Synthesis and structure−activity relationship of 8-substituted protoberberine derivatives as a novel class of antitubercular agents

Background The emergence of multi-drug resistant tuberculosis (MDR-TB) has heightened the need for new chemical classes and innovative strategies to tackle TB infections. It is urgent to discover new classes of molecules without cross-resistance with currently used antimycobacterial drugs. Results Eighteen new 8-substituted protoberberine derivatives were synthesized and evaluated for their anti-mycobacterial activities against Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv. Among them, compound 7g was the most effective antitubercular agent with minimum inhibitory concentration (MIC) of 0.5 μg/mL. Moreover, it also afforded a potent antitubercular effect against clinically isolated MDR strains of M. tuberculosis with MICs ranging from 0.25 to 1.0 μg/mL, suggesting a novel mode of action. Conclusions The structure−activity relationship (SAR) analysis revealed that introduction of a substituent at the 8-position in pseudoprotoberberine, especially an n-decyl, could significantly enhance the anti-TB activity. We consider 8-n-decylberberines to be a novel family of anti-tubercular agents with an advantage of inhibiting MDR strains of M. tuberculosis.


Background
Currently, one third of the world's population is infected with Mycobacterium tuberculosis (M. tuberculosis) [1]. It is anticipated that there will be about 8.9−9.9 million new and relapse tuberculosis (TB) cases this year, more than in any other year in history [2,3]. The limited effectiveness and long-term treatment lead to poor patient compliance, which often causes multi-drug resistant (MDR) and extensively-drug-resistant (XDR). The emergence of new cases, the increased incidence of MDR strains of M. tuberculosis, the adverse effects of first-line anti-TB drugs isoniazid (INH) and rifampin (RIF) [4,5], and the increased incidence of TB associated with HIV infections [6][7][8] have led to renewed research interest in discovering novel anti-TB drugs. Especially, the emergence of MDR-TB and of the virtually untreatable MDR-TB has heightened the need for new chemical class and innovative strategies to tackle TB infections. However, truly novel antitubercular drugs other than repurposed drugs have not been developed since the 1970s [9,10]. Though new anti-TB drug Bedaquiline was just approved by FDA last year [11], there is still an urgent need to discover new classes of molecules without crossresistance with currently used antimycobacterial drugs.
We have identified 13-substituted protoberberine derivatives to be a novel family of anti-TB agents [12] with poor solubility. The primary structure−activity relationship (SAR) indicated that the berberine ring (BBR, 1, Figure 1) might be beneficial for keeping good antitubercular activity. In our ongoing efforts to discovering new anti-TB agents, we turned our SAR analysis on the substituents at the 8-position of BBR derivatives, by which nitrogen ion at the 7-position might be blocked by the 8-substituents with bigger volume, thereby enhancing the solubility of this kind of compounds. Based on this strategy, several 8-substituted protoberberine derivatives (6a-e) were designed, semi-synthesized and evaluated for their antimycobacterial activity against M. tuberculosis strain H 37 Rv. Furthermore, by replacing 1 with pseudoberberine (2, Figure 1) or palmatine (3, Figure 1) core, two natural products extracted from Chinese herb Huanglian, by which a group of new 8-substituted pseudoberberine (7a-h) or palmatine (8a-e) derivatives was generated for testing. Herein, eighteen 8-substituted protoberberine derivatives ( Figure 2) were designed and synthesized, and their anti-mycobacterial effects were evaluated afterwards.

Results and discussion
Chemistry Eighteen target compounds were synthesized with commercially available 1, 3 or 2 (synthesized in our laboratory) [13,14] as the starting material as described in Scheme 1. The Grignard reagents were first prepared with Mg turnings and the corresponding alkyl and aryl iodide in absolute ether under N 2 protection. The key intermediates dihydroberberine, dihydropseudoberberine or dihydropalmatine (4) were obtained via nucleophilic substitution of newly synthesized Grignard reagents with 1, 2 or 3 under N 2 protection, respectively [15,16]. Then, the intermediate 4 was oxidized using bromine as a oxidizing agent in HOAc at refluxing temperature to yield the 8-substituted berberine bromate 5, which was converted into the corresponding chloride 6-8 with AgCl in MeOH at room temperature. Finally, the desired products in series 6, 7 and 8 were purified by flash column chromatography using methanol/dichloromethane as the gradient eluent with overall yields of 72%-81%.  Biological activity and SAR analysis of 8-substituted protoberberine derivatives for anti-mycobacterial activity Our SAR strategy was first focused on the modifications of the substituents at the 8-position in BBR. According to our previous SAR results, several lipophilic groups including p-methoxyphenyl (6a), m-methoxyphenyl (6b), p-methylphenyl (6c), 1-naphthyl (6d) and n-decyl (6e) were introduced into the 8-position aiming to improve the cLogP value (Table 1), thereby enhancing the antimycobacterial activity. Among these analogues, compound 6e possessing an n-decyl afforded the hightest antibacterial activity with a MIC of 2.0 μg/mL against M. tuberculosis. The results supported that the increased cLogP value might be helpful for enhancing the anti-TB activity of this kind of compounds. In order to further explore the influence of the BBR core, a variety of lipophilic substituents were attached to the 8-position of 2, by which the 8 new 8-substituted pseudoberberine classes (7a−h) were generated for testing. The results showed ( Table 1) that the majority of them (7b−h) exhibited potential anti-mycobacterial activities with MICs ranging from 0.5 μg/mL to 2.0 μg/mL. It seems that the 10,11-dimethoxy on the ring D would be beneficial for their binding affinity to the target molecular. Compound 7g bearing an n-decyl afforded the best anti-TB activity with a MIC of 0.5 μg/mL. Similarly, the lipophilic side-chains were also introduced to the same position in 3, and then 5 new 8-substituted palmatine analogues (8a−e) were made. As expected, compound 8e with an n-decyl at position 8 had the most potent activity with an MIC of 1.0 μg/mL. It was deduced that an n-decyl at the 8-position would improve the anti-mycobacterial activity as regarding to this kind of compounds. The pesudoberberine ring might be beneficial for the antimycobacterial activity, and thus the representative compounds in 7 series were chosen for further investigation.
Anti-resistance TB effect of 7g and 7f As compound 7g bearing an n-alkyl at position 8 possessed an excellent activity against drug-susceptible M. tuberculosis strain H 37 Rv, it was selected to test the anti-TB activity against MDR strains. In this experiment, M. tuberculosis strains 87, 192, 262 and 266 isolated from the patient infected with tuberculosis in China, were resistant to both RIF and INH. RIF and INH showed a decreased activity against the drugresistant stains partially or completely with MIC ranges between 2 and > 32 μg/mL (Table 2), while compound 7g afforded a potential effect against MDR strains with comparable MIC ranges of 0.25−1 μg/mL. In addition, compound 7f possessing an aromatic moiety at the 8position in 7 series afforded a moderate cLogP value (cLogP = 1.60) and then was chosen to evaluate for the drug-resistant strains as well. As described in Table 2, 7f showed an equivalent potency against the drugsusceptible strain H 37 Rv and multidrug-resistance isolates of M. tuberculosis strains 257, 373, 559 and 164 with a MIC range between 2 and 4 μg/mL as well. The results indicated that 7g and 7f were effective for drug- susceptible M. tuberculosis as well as MDR strains isolated from TB patients in China, suggesting a mode of action different from currently used anti-TB drugs.

Cytotoxicity of 7g and 7f in Vero and MRC-5 cells
Both of compounds 7g and 7f were further tested their cytotoxicity in African green monkey kidney (Vero) and human lung fibroblast (MRC-5) cells with MTT assay. Cytotoxicity activity was expressed with CC 50 value, and the selectivity index (SI), as an important therapeutic indication, was calculated as the ratio of CC 50 to MIC value. Anti-TB effect of compounds 7g and 7f was evaluated by combining their MIC with SI values. As described in Table 3, compound 7g showed a moderate SI value of 10.3 and 17.6 in Vero and MRC-5 cells, respectively.

Experimental Instruments
Melting point (m.p.) was uncorrected and recorded on a Mettler Toledo MP90 melting point apparatus. 1    General procedure to obtain final 8-substituted protoberberine derivatives

H NMR
Grignard reagents were prepared via Magnesium turnings (3.8 g) with the corresponding alkyl and aryl iodides (0.13 mol) in absolute ether (100 mL) at 0°C. The synthesized Grignard reagents were added to the suspension of dry 1, 2 or 3 (0.03 mol) in absolute ether (100 mL) dropwise under N 2 protection at 0°C [15,16]. After refluxing for 2 h, saturated NH 4 Cl solution (200 mL) was added to quench the reaction. The aqueous phase was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with saturated brine (100 mL) and dried (Na 2 SO 4 ). The mixture was concentrated in vacuo to give 4. Then, bromine solution (1N/ HOAc, 30 mL) was added to oxidize compound 4 in HOAc at refluxing temperature to generate the 8-substituted BBR derivatives bromate 5, which was treated with excessive AgCl in MeOH at room temperature thus converted into the corresponding chloride form. Finally, the desired products in series 6, 7 and 8 were purified by flash column chromatography using methanol/ dichloromethane as the gradient eluent with overall yields of 72%-81%.