A General Review of Tetracyclines Antibiotics
Chang Liu Instructor: Prof. Jasson Sello Department of Chemistry, Brown University Providence 02906, RI Tetracyclines are the first broad-spectrum antibiotic to be applied to clinic use. Nevertheless, the increasing incidence of bacterial resistance of tetracyclines led to a series of studies on the development of semisynthetic tetracyclines to circumvent the resistant organisms. In order to better design the structures of tetracycline derivatives, research on the action mode of tetracyclines, mechanisms of resistance, biosynthesis and total synthesis of tetracyclines were also performed.

Keywords: Tetracyclines, Structure-activity Relationship, Mode of Action, Mechanisms of Resistance, Biosynthesis, Total Synthesis

1. Introduction Tetracyclines are a group of polyketide broad-spectrum antibiotics that has activity against a variety of gram-positive and gram-negative bacteria, mycoplasmas, chlamydiae and peotozoan parasites [1]. The discovery of tetracycline was in the 1940s. At that time, the problems related to the production of Pennicillin has been solved and pharmaceutical industry

and academic institutes started to concentrate their energy on the development of new antibiotics. In 1948, the first member of tetracycline family—chlorotetracyclin, or Aureomycin was discovered as an isolate of Streptomyces Aureofaciens in an antibiotic screening program functioned in Lederle Labs [2]. In 1950, oxytetracycline or Terramycin—second member of tetracycline was found as a metabolite of Streptomyces rimosus by Finlay et al. [3] and the chemical structure of oxytetracycline was defined by Woodward, which was a hallmark in tetracycline research. Since then, tetracyclines has been intensively used in both human therapy and animal feed use [4]. They have been extensively applied to human therapy for treatment of bacterial respiratory and urogenital diseases, as well as periodontal, Lyme, and reckettsial diseases. What’s more, tetracycline is also used as an important animal growth promoter in food industry, and in some countries the amount of tetracycline used for food industry has surpassed that for human. The widely application of tetracycline results in a dramatic increase of bacterial species that contain tetracycline-resistant genes and a serious decrease of tetracycline effectiveness over time [4]. Currently, 40 different tetracycline resistant genes has been discovered [5]. Therefore, in the period of 1970-1980, enormous effort has been made to modify the structure of tetracyclines in order to improve their antibacterial activity and overcome the bacterial resistance [6]. These semisynthetic approaches contributed to a second

generation of tetracyclines, including rolitetracycline, methacycline, doxycycline and minocycline, etc. The most recently synthesized compound is the glycylcycline [7], which could overcome the two most common mechanisms of resistance, efflux and ribosomal protection. And in 2005, one of the glycylcyclines—tigecycline has been approved by FDA and is now commercially available. In this review, we will cover a wide range of topics concerning with tetracyclines, including description of key structure features, mode of action, mechanisms of resistance, biosynthesis and chemical synthesis.

(a)

(b)

(c)

(d)

(e)

(f)

(g)
Figure 1.1 structure of tetracycline and its derivatives (a) tetracycline (b) chlorotetracycline (c) oxytetracycline (d) rolitetracycline (e) methacycline (f) doxycycline (g) minocycline

2. Structure-Activity Relationship By the analysis of tetracycline structure, we can conclude that the dimethylamino group at 4 position is basic, while the hydroxyl group at 3,5,6,10,12,12a is weakly acidic. Therefore, the whole molecule is amphoteric.

Figure 2.1 structure-activity relationship

The inviolate structures that relate to the antibacterial activities are the linear fused tetracycle, the substituents at position 1,2,3,4, the enol system of 11,12 and 12a, and the α stereochemical configurations of the hydroxyl group at 12a and the hydrogen at 4a [8]. The α configuration of the dimethylamino group at 4 is exceedingly important. It has been found that the β-epimers（4-Epitetracycline）accounts for about 5% of the activity of normal tetracyclines in vitro [9]. What’s more, the 4-Epitetracycline is 2~3 times more toxic to eukaryotes than normal tetracycline. The empimerization is reported to occur in various conditions with a PH ranging from 2 to 6. Since the hydroxyl group at position 5 of oxytetracycline can form a hydrogen bond with the dimethylamino group, the oxytetracycline can better retain its α

configuration of the dimethylamino group and thus be more therapeuticly efficient [10].

Figure 2.2 structure of oxytetracycline and the hydrogen bonding between hydroxyl group at 5 and dimethylamino group at 4

The hydroxyl group at 6 makes the molecule unstable to both acids and bases [6]. It goes on to anti-elimination reactions under acidic conditions to give its orange dehydration product—anhydrotetracycline. Under basic conditions, it goes on to ring open reaction to form a lactone isomer of tetracycline. The hydroxyl group at 6 can be deprotonated to oxygen anion and the enol at 12 can turn to keto form. Then the oxygen anion can conduct intramolecular nucleuphilic attack towards the keto at 12, break the ring, and form a lactone [6].

Figure 2.3 structural transformation of tetracycline under acidic condition and basic condition

Therefore, enormous efforts have been made to modify the structure of tetracycline at position 6. Dehydroxy product like Doxycycline (6-Deoxy-5-hydroxytetracycline) and has Minocycline the best

(7-Dimethylamino-6-demethyl-6-deoxytetracycline)

antibiotic activity [6]. They are much more stable and lipophilic than the original tetracycline and can be take up more efficiently. And they are the first long-term tetracyclines first applied to clinic use and can be taken only one time a day [11]. Demethylation at position 6 does not affect its activity seriously, which gets to Demecycline. And Sancycline (6-deoxy-6-Demethyltetracycline) is the simplest tetracycline that retains its anti-bacterial activity [12]. It is

regarded as the pharmacophore of tetracyclines. If the carbon at position 6 is substituted by sulfur, we get 6-thiatetracycline. Nevertheless, it possesses activity against

gram-positive bacteria but not against gram-negative bacteria [13]. This implies that it exhibits a different structure-activity relationship from the major tetracyclines. Further study shows that the tetracycline could be trapped in the hydrophobic environment of the cytoplasmic membrane and disrupt the bacterial membrane function [13].

Figure 2.4 structure of 6-thiotetracycline

Position

7

and

9

are

free

for

substitution

to

take

place.

Electro-withdrawing groups like chloro and nitro group could amplify its activity against both gram-positive and gram-negative bacteria [6]. Nevertheless, the electro-donating groups would diminish its activity. The substitution of an N-alkylglycylamido group at position 9 gives the most up-to-date tetracycline antibiotic glycylcyclines [14]. One of them are tigecycline, which has a 9-t-butyl-glycylamido side chain and could overcome resistance of both efflux and ribosome protection mechanisms

.
Figure 2.5 structure of tigecycline

The keto-enol system at position 11 and 12 is also crucial to the antibacterial activity of tetracycline. The result of the theoretical calculation indicates that the increase of electron density at these positions could result in the increase of activity [6]. This is consistent with the mode of action of tetracyclines. The keto-enol system could function as chelation site and bind to metal ions on the 30S RNA of bacterial ribosome and form chelation compounds [1].

3. Mode of Action The mode of action of typical tetracyclines like tetracycline, chlorotetracycline, minocycline and doxycycline is that it binds to the ribosomal RNA to inhibit the binding of Aminoacyl-tRNA to the ribosomal A-Site in a reversible way [6]. To reach their targets in bacteria they need to cross two membranes for gram-positive bacterial (outer and cytoplasmic membranes) or the cytoplasmic membrane for gram negative bacterials [15, 16]. Tetracyclines get through the bacterial cell wall by passive diffusion through hydrophilic pores in the outer cell membrane and trough the inner cytoplasmic membrane by both passive diffusion and

energy-dependent uptake. Thus，to discuss the factors that influence the action of tetracycline we need to consider both its uptake by cells and the mechanism of binding to ribosomes. Study shows that tetracycline binds to the S7 protein on the 30S subunit with high-affinity binding, while low-affinity binding is observed in both subunits [17]. Also, it is reported that binding of tetracycline to the ribosome weakens the ribosome-tRNA interaction [18], which may be the cause of its inhibition of protein synthesis. It is possible that the position S7 on the 30S subunit overlap with the aminoacyl-tRNA binding site. From what is stated above, there is no convincible evidence that binding of tetracycline to the ribosome is directly related to its antibiotic activity. We cannot make the conclusion until direct evidence has been found which correlates the ribosome-tetracycline interaction to its binding to the ribosomes. The partially energy-dependent uptake of tetracycline through the cell membranes has been explained by the following model [16]: Tetracycline could chelate a M2+ ion and pass the cell outer membrane through the porins OmpF and OmpC. Then, the cationic [M-tc]+ reaches the periplasm motivated by the Donnan potential. Possibly, the [M-tc]+ complex will dissociate and forms uncharged tetracycline. And the neutral tetracycline is slightly lipophilic and could do passive diffusion through the inner membrane. Due to the high concentration of M2+ and

the internal pH in the cytoplasm, the tetracycline may be converted to an ionic compound again.

Figure 3.1 uptake of tetracycline by outer and inner membrane of bacterial cells. Tetracycline, protons, metal cations and metal-tetracycline complex are depicted as tc, H+, M2+ and [M-tc]+ respectively

4. Mechanisms for Resistance There are major two mechanisms that are responsible for the resistance of tetracyclines: (1) to protect the ribosome from interacting with tetracycline (2) energy-dependent efflux of tetracycline [6]. Study shows that the ribosome protection proteins Tet M, Tet O, Tet S, Tet B, Tet Q and Otr A has similar amino acids sequences as the elongation factors Tu and G, which suggests that the ribosome protection proteins may function as tetracycline-resistant elongation factors, or by

hindering the binding of tetracycline to the ribosome [19]. On the other hand, the genes which determine the efflux proteins are identified as tetA, tetB, tetC, tetD, tetE, tetG, tetH, tetK, tetL and otrB. All these genes code for energy-dependent proteins which could pump tetracyclines through the cell membrane. The export of tetracycline decreases the concentration of tetracycline within the cell and thus protects the ribosomes from interacting with tetracyclines [20].

5. Biosynthesis One of the hallmarks for elucidating the biosynthesis pathway of tetracycline was the sequencing of the gene clusters associated with chlorotetracycline and oxytetracycline [21]. As there are a lot of similarities between the two clusters and the enzymes which are essential in the pathways also function in similar ways, here we concentrate our discussion only on the oxytetracycline biosynthesis [22].

Figure 5.1 gene clusters associated with chlorotetracycline and oxytetracycline biosynthesis

As a member of polyketide family, the polyketide synthase of tetracycline contains ketosynthase domain (OxyA), chain length factor (OxyB) and

acyl-carrier protein (OxyC) and amidotransferase (OxyD). The synthase utilizes the malonamyl-CoA as the starting substrate and then elongate the polyketide chain with 9 malonyl-CoAs to form the amidated polyketide backbone of the tetracycline. Then the reduction of the ketone at position 9 is catalyzed by ketoreductase OxyJ. The next steps are cyclization of the backbone to form the linear fused tetracycle structure catalyzed by cyclases OxyK and OxyN. And then, the C-methyltransferase OxyF catalyzed the methylation at position 6. What follow are the hydroxylation at 4 and 12a catalyzed by OxyL, the amidation at position 4 catalyzed by OxyQ and the N,N-dimethylation catalyzed by OxyT. The final steps are two hydroxylations and a dehydration. The OxyS catalyzes the hydroxylation at position 6. Then the hydroxylation at position 5 and the final reduction step of the ketone at position 12 are catalyzed by unknown proteins.

Figure 5.2 oxytetracycline biosynthesis pathway

6. Chemical Synthesis The first total synthesis of a fully active tetracycline was that of 6-demethyl-6-deoxytetracycline by Woodward and Conover (25 steps, ~0.002 yield) [23]. Then several pathways for synthesis of tetracycline derivatives came out for 5-oxytetracycline,

12a-deoxy-5a,6-anhydrotetracycline and tetracycline [6]. But the yield was quite limited due to the complexity of the pathways. The most efficient synthesis of tetracycline derivatives that has been ever achieved is the synthesis of 12a-deoxytetracycline (16 steps, 18~25% yield) [24]. However the antibiotic activity of the 12a-deoxy product diminishes seriously due to the absent of hydroxyl group at the key position. The most recent progress of the chemical synthesis of tetracyclines is the work published in Science in 2005, by Charest etc., for a convergent enantioselective route to synthesis 6-deoxytetracycline [26]. The reaction route is briefly summarized below. The central idea of this synthesis is to couple a D-ring precursor with an AB precursor and construct the C-ring diastereoselectively, which could be utilized to synthesize a diverse range of 6-deoxytetracyclines. The synthesis started from the microbial dihydroxylation of benzoic acid by a mutant strain of Alcaligenes eutrophus. The diol product then went through epoxidation to the epoxide compound. Then esterification of the compound is carried out by trimethylsilyldiazomethane. Then the

tert-butyldimethylsilyl triflate was used to conduct the bis-silylation and the epoxide isomerization and formed the epoxy ester. Next, an organolithium reagent prepared by

benzyloxy-5-dimethylaminomethylisoxazole and n-butyllithium was added to the epoxy ester and form a ketone. The next step is the closure of the A ring achieved by heating the ketone product with lithium triflate. The author believes that this ring closure proceeds through a rearrangement mechanism which is similar to the Sommelet-Hauser rearrangement. Then trifluoroacetic acid was added to selectively remove the protection group. Then the hydroxyl group at position 11a was replaced by thiophenyl group, and a chiral oxidant was added to conduct the sulfoxidation, follows by a Mislow-Evans rearrangement to gibe the allylic alcohol. The next step is the protection of the allylic alcohol by benzyl chloroformate, follows by the hydrolysis of the silyl ether protection group, the oxidation of the allylic alcohol and the protection of the remaining carbinol with tert-butyldimethylsilyl triflate. Finally, we achieve the enone product which is the AB precursor of the tetracycline. The AB precursor and the D-ring precursor is then fused by a Michael reaction and Dieckmann reaction sequence.

Figure 6.1 a convergent enantioselective route to synthesis 6-deoxytetracycline

7. Conclusion The emergence of resistance genes of tetracycline is the major challenge for its clinic usage. Thus the examination of newly developed derivatives which could circumvent the bacterial resistance is crucial, eg. the glycylcyclines and the dactylocyclines. The approval of tigicycline by FDA proves the potential of the tetracyclines to retain its status as a front-line antibiotic. However, the resistance of new tetracyclines could probably occur quickly after its widely application. We need to develop new classes of drugs with unique antibacterial mechanisms, or we should understand the mechanisms of resistance better to help us design new drugs that could be hardly resisted by bacteria.

References
[1] Chopra, I.; Roberts, M. Microbiology and Molecular Biology Reviews , 232 (2001). [2] Duggar, B.M. Ann. NY Acad. Sci. 51, 177 (1948). [3] Finlay, A.C. Science 111, 85 (1950). [4] Levy, S.B. The Antibiotic Paradox: How Miracle Drugs are Destroying the Miracle. Plenum Press, New York. (1992). [5] Roberts, M.C. FEMS Microbiol. Lett. 245, 195 (2005). [6] Hlavka, J.J.; Boothe J.H. Handbook of experimental pharmacology 78, 179 (1985). [7] Agwuh, K.N.; MacGowan, A. J. Antimicrob. Chemother. 58, 256 (2006). [8] Bahrami, F.; Morris, D.L. Pourgholami, M.H., Mini-Reviews in Medicinal Chemistry 12, 44 (2012). [9] Doerschuk, A.P.; Bitler, B.A. J. Am. Chem. Soc. 77, 4687 (1955). [10] Hussar, D.A.; Niebergall, P.J.; Sugita, E.T. Doluisio J.T. J. Pharm. Pharmacol. 20, 539 (1968). [11] McMurry, L.M., et al. Antimicrob. Agents Chemother. 22, 791 (1982). [12] Mitscher, L.A. The Chemistry of the Tetracycline Antibiotics. Marcel Dekker, Inc., New York, N.Y. (1978). [13] Chopra, I. Antimicrob. Chemother. 38, 637 (1994). [14] George, A.P. J. Antimicrobial Chemotherapy 56, 470 (2005). [15] Argast, M.; Beck, C.F. Antimicrob. Agents Chemother. 141, 260 (1985). [16] Nikaido, H.; Thanassi, D.G. Antimicrob. Agents Chemother. 37, 1393 (1993). [17] Rudolf, O.; Norbert, P.; Andrea, B. Nucleic Acids Research 25, 1219 (1997). [18] Epe, B.; Wooley, P.; Hornig, H. FEBS Lett. 213, 443 (1987). [19] Sanchez-Pescador, R.; Brown, J.T.; Roberts, M.; Urdea, M.S. Nucleic Acids Res. 16, 1218 (1988). [20] Roberts, M. FEMS Microbiol. Rev. 19, 1 (1996). [21] Fierro, F.; Martin, J.F. Microbial Secondary Metabolites: Biosynthesis, Genetics and Regulation. Research Signpost, Lucknow, 141 (2002). [22] Lauren, B.P.; Tang, Y. Metabolic Engineering 11, 69 (2009). [23] Korst, J.J. et al. J. Am. Chem. Soc. 90, 439 (1968). [24] Stork. G.; Clair, J.J.L.; Spargo, P.; Nargund, R.P.; Totah, N. J. Am. Chem. Soc. 118, 5304 (1996). [26] Charest, M.G. et al. Science 308, 15 (2005).