Synthesis and Antimicrobial Activity of 8-Alkylberberine Derivatives with a Long Aliphatic Chain

• Abstract
• Materials and Methods
• References

Abstract

The compounds 8-ethyl- (2), 8-butyl- (3), 8-hexyl- (4), 8-octyl- (5), 8-decyl- (6) and 8-dodecylberberine chloride (7) were synthesized and tested for their antimicrobial activity in vitro to evaluate structure-activity relationships. Substitution of the alkyl groups at C-8 led to significant changes in the antimicrobial activity. All compounds were more potent against the tested microorganisms than berberine (1), especially against Gram-positive bacteria. The antimicrobial activity increased as the length of aliphatic chain was elongated and then decreased gradually when the alkyl chain exceeded eight carbon atoms. 8-Octylberberine (5) displayed the highest antimicrobial activity of all compounds. The toxicity of compounds 2 - 7 was stronger than that of 1. However, upon elongating the aliphatic chain, the toxicity decreased gradually.

Berberine (1) and its derivates are used for treating diarrhoea [1] and act towards inflammation [2]. Substituted derivatives at positions in the A, C or D-ring exhibit changes in their pharmacological effects [3], [4], [5]. Dioxymethylene replacement [3] at the C-2 and C-3 positions in the A-ring, as well as 8-alkyl- or 13-alkyl-substitution [5], [6] increase the antibacterial activity, but 13-hydroxy-substituted derivates show decreased antibacterial activity [7]. In the present study, 8-alkylberberine derivates with a long aliphatic chain (2 - 7) were synthesized and their antimicrobial activity and toxicity were tested in vitro to evaluate structure-activity relationships.

The data of UV, IR, TLC, ¹H-NMR and ¹³C-NMR measurements confirmed that the 8-alkyl-substituted berberine derivates 2 - 7 with long aliphatic chain had been synthesized successfully (see Supporting Information, Table 1S).

The antimicrobial activity of compounds 2 - 7 was higher than that of berberine (1) and displayed more potency against Gram positive bacteria than towards other microbes (Table [1]). The antimicrobial activity of compounds 2 - 5 increased as the aliphatic chain length increased and then gradually decreased (compounds 6 and 7) when the number of carbon atoms was higher than eight; 8-octylberberine (5) showed the strongest activity against the microbes tested.

The LD₅₀ values of berberine derivatives towards mice are listed in Table [2]. The toxicity of compounds 2 - 7 was stronger than that of 1. However, the longer the aliphatic chain of 2 - 7, the lower was their toxicity; compound 7 showed almost the same low toxicity as 1 (Table [2]).

A previous report showed that the antibacterial activity increased as the length of the aliphatic chain increased [7]. In the present study, we discovered that the antibacterial activity decreased when the aliphatic chain exceeded eight carbon atoms. While a long aliphatic chain is beneficial for inhibiting the growth of microbes [8], a long aliphatic chain favors the stability of the cell membrane leading to lower toxicity; thus a long chain is unfavorable to inhibit bacterial growth [12]. These two counteractions obviously made 8-octylberberine the strongest antimicrobial in the series of 2 - 7.

Materials and Methods

To efficiently obtain 8-alkyl-substituted berberine derivates with long carbon chains, THF was used as an optimal reaction solvent instead of ether. The synthesis was carried out according to previous methods [6]. To obtain pure products, the compounds with long chains needed to be recrystallized three times from MeOH at -20 °C. Melting points, UV, IR, ¹H-NMR and ¹³C-NMR and TLC were used to identify the structures of 2 - 7 (see Supporting Information, Table 1S). The mobile phase used in TLC was C₆H₆/EtOAc/i-PrOH/MeOH/NH₄OH, 6 : 3:1.5 : 1.5 : 0.5. TLC analysis confirmed purity of the recrystallized compounds.

Antimicrobial activities against Gram-positive (G⁺-) bacteria (B. subtilis, S. aureus, and methicillin resistant S. aureus), Gram-negative (G^--) bacteria (E. coli and S. dysenteriae) and a fungus (C. albicans) (see Supporting Information for suppliers) were evaluated by the minimum inhibitory concentration using the two-fold serial dilution test [9]. Compounds 1 - 7 were dissolved in H₂O containing 2 % DMF and 0.8 % Tween-80 and diluted into different concentrations from 1 to 1000 μg/mL with liquid medium. The mixture of berberine derivatives and the microbes (2 × 10⁸ cfu/mL) was incubated at 37 °C for 24 h for bacteria in broth medium, and at 25 °C for 48 h for the fungus in improved Sabouraud medium. Microbial growth was examined by measuring the absorbance at 655 nm with a spectrophotometer. The H₂O/DMF/Tween-80 was used as a blank and 1 as positive control. All experiments were run in triplicate.

The LD₅₀ was determined to evaluate the toxicity of compounds 1 - 7 according to Karber [10]. Healthy Kunming mice of both genders (20 ± 2 g, 7 weeks old, see Supporting Information for supplier) were randomly divided into 36 groups with 10 mice each. Animal care and toxicity test procedure were carried out according to [11]. The given dosages of compounds 1 - 7 were designed according to Table [3]. The normal control group was fed with the same volume of physiological saline. Mice were housed in stainless cages in a room with controlled temperature (23 ± 2 °C) and humidity (40 - 60 %) and a 12 h light/dark cycle. The protocol complied with the guidelines of Chongqing City Laboratory Animal Administration Committee of China for the care and use of laboratory animals. Animals were then kept under observation for 7 days to record toxic signs and total mortality.

References

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Prof. Dr. Li Xuegang

Chemistry Institute of Pharmaceutical Resources

School of Pharmaceutical Science

Southwest University

Chongqing 400715

People’s Republic of China

Phone: +86-23-6825-0728

Email: Xuegangli2000@yahoo.com.cn

References

• 1 Kupeli E, Kosar M, Yesilada E, Husnu K, Baser C. A comparative study on the anti-inflammatory, anti-nociceptive and antipyretic effects of isoquinoline alkaloids from the roots of Turkish Berberis species.  Life Sci. 2002;  72 645-57.
  CrossrefPubMedSearch in Google Scholar
• 2 Jiang J Y, Geng D S, Tursonjan T, Liu F. Anti-inflammatory effects and mechanism of berberine.  Chin Pharmacol Bull. 1998;  14 434-7.
  PubMedSearch in Google Scholar
• 3 Iwasa K, Nishiyama Y, Ichimaru M, Moriyasu M, Kim H -S, Wataya Y. et al . Structure-activity relationships of quaternary protoberberine alkaloids having an antimalarial activity.  Eur J Med Chem. 1999;  34 1077-83.
  CrossrefPubMedSearch in Google Scholar
• 4 Hong S W, Kim S H, Jeun J A, Lee S J, Kim S U, Kim J H. Antimicrobial activiy of 9-O-acyl- and 9-O-benzoyl-substituted berberines.  Planta Med. 2000;  66 361-3.
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Iwasa K, Kamigauchi M, Sugiura M, Nanba H. Antimicrobial activity of some 13-alkyl-substituted protoberberinium salts.  Planta Med. 1997;  63 196-8.
  PubMedSearch in Google Scholar
• 6 Iwasa K, Lee D U, Kang S I, Wiegrebe W. Antimicrobial activity of 8-alkyl- and 8-phenyl-substituted berberines and their 12-bromo derivatives.  J Nat Prod. l998;  61 1150-3.
  PubMedSearch in Google Scholar
• 7 Iwasa K, Kamigauchi M, Ueki M, Taniguchi M. Antibacterial activity and structure-activity relationships of berberine analogs.  Eur J Med Chem. 1996;  31 469-78.
  CrossrefPubMedSearch in Google Scholar
• 8 Ye X L, Li X G, Yuan L J, He H M. Effect of the surface activity on the antibacterial activity of octadecanoyl acetal sodium sulfite series.  Colloids Surf A Physicochem Eng Asp. 2005;  268 85-9.
  CrossrefPubMedSearch in Google Scholar
• 9 Ma X R. Drug microbial test handbook.  Beijing: People Health Publishing. House;  2001 50-3.
  PubMedSearch in Google Scholar
• 10 Zhou L G. Drug toxicology.  Beijing: People’s Medical Publishing. House;  2003 59-161.
  PubMedSearch in Google Scholar
• 11 GB 5193.3-2003. Acute toxicity test. Beijing; Standards Press of China 2005: 1-15.
  Search in Google Scholar
• 12 Ye X L, Li X G, Yuan L J, Ge L H, Zhang B S, Zhou S B. Interaction of houttuyfonate homologues with the cell membrane of Gram-positive and Gram-negative bacteria. Colloids Surf A Physicochem Eng Asp, in press.

Prof. Dr. Li Xuegang

Chemistry Institute of Pharmaceutical Resources

School of Pharmaceutical Science

Southwest University

Chongqing 400715

People’s Republic of China

Phone: +86-23-6825-0728

Email: Xuegangli2000@yahoo.com.cn

• Supplementary Material