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New Classification Framework and Structure-Activity- Relationship (SAR) of Tetracycline-Structure-Based Drugs

Domenico Fuoco, Pharm.D., Ph.D BioTech Consultant; Member of Italian National Order of Chemists and Italian Chemical Society in Rome, Italy

By studying the literature about Tetracyclines (TCs), it becomes clearly evident that TCs are very dynamic molecules. In some cases, their structure-activity-relationship (SAR) are known, especially against bacteria, while against other targets, they are virtually unknown. In other diverse yields of research, such as neurology, oncology and virology the utility and activity of the tetracyclines are being discovered and are also emerging as new technological fronts. The first aim of this paper is classify the compounds already used in therapy and prepare the schematic structure in which include the next generation of TCs. The aim of this work is introduce a new framework for the classification of old and new TCs, using a medicinal chemistry approach to the structure of that drugs. A fully documented Structure-Activity-Relationship (SAR) is presented with the analysis data of antibacterial and nonantibacterial (antifungal, antiviral and anticancer) tetracyclines. Lipophilicity of functional groups and conformations interchangeably are determining rules in biological activities of TCs.

Upper peripheral modification region Introduction

The number of articles published on tetracycline drugs reached the threshold of 50,000 papers since 1948. Over the last 10 years, technological fields are emerging in bacteriology and cellular physiology of eukaryotic cells. However, Lower peripheral non modifiable region chemical mechanisms of tetracyclines are not completely understood as for their action in human cells and to this day, no (Q)SAR model Figure 1. Structure-Activity-Relationship (SAR) of Tetracyclines (TCs). Shaded: contact region with 30S is validated without doubts. Tetracyclines were rRNA. In blue polygonal same Anthracycline region. first discovered by Dr. Benjamin Dugger of Lederle Laboratories in the mid 1940s as the fermentation product of an unusual golden- colored soil bacterium aptly named Corresponding author Tel/FAX Streptomyces aureofacians 1. Tetracyclines (TCs) (001)514.913.1983. [email protected] are a class of antibiotics able to inhibit protein synthesis in gram positive and gram negative Parkinson and others neurodegeneration bacteria by preventing the attachment of diseases; (IV) antiviral and anticancer 15-17 ; (V) aminoacyl-tRNA to the ribosomal acceptor (A) Tet repressor controlled gene switch 18 ; site .2 . This mechanism has been confirmed by X- ray cristallography 3. TCs bind specifically to the Antibacterial use bacterial ribosome and not with specifically I. Currently, as a consequence of their large use, eukaryotic ribosomes. TCs belong to a notable bacteria has developed TC resistance (efflux class of biologically active and commercially pump type) as opposed to the oldest compounds. valuable compounds. This fact may be simply Medicinal chemists with the intention to illustrated by mentioning the most important optimize structure and improve the antibacterial clinical application of TCs, their employment as power have successfully introduced an alkaline broad antimicrobial-spectrum antibiotics for 4 group on C-9 of minocycline skeleton, starting human and veterinary use . While discovered as a compound from total synthesis: Tigecycline and initially developed as antibiotics, they also (a patent of Pfizer and Wyeth, available in hold promise as non-antibiotic compounds for therapy from 2005). Searching for new future study and use. Tetracyclines, as dynamics molecules, it is not only important to study the entities, possess unique chemical and biological binding of drugs specifically to bacterial characteristics that may explain their ability to ribosome, but also to understand how the interact with so many different cellular targets, 5 skeleton of tetracycline can act like chelator and receptors and cellular properties . The discovery 19 ionophore . Moreover, the next generation of of new uses of tetracyclines and their novel antibacterial tetracyclines, is in progress, highly biological properties against both prokaryotes specific for bacterial species and with new and eukaryotes is currently under investigation groups and new rings on the classical skeleton 20. by numerous scientists throughout the world. Mechanism of action of TCs is divide in TCs as drugs show only few side effects: One is “typical”, if they act as bacteriostatic, or chelation of calcium and subsequent “atypical” if they act as batericidic. Typical TCs intercalation in bones and teeth and the other is bind specifically the bacterial ribosomal an action like photosensitizing drugs, given their subunits. All of them that don’t have ribosomal phototoxic action on keratinocytes and as primary target are consider atypical. fibroblasts. The mechanism of phototoxicity in 6 Moreover, these atypical mechanism of action vitro and in vivo is not yet entirely clear. are very toxic both for prokaryotic and Pharmacological activities eukaryotic (even for mammalian cells). Until now all TCs used in therapy are broad-spectrum The therapeutic uses are as follows: antibacterial against microbial agents, but researchers are and non-antibacterial. In the literature, these developing a platform to introduce in therapy uses fall into five main categories, namely: (I) only novel TCs with a narrow-spectrum for newer and more potent tetracyclines use in infection diseases. anitibacterial resistance 7, (II) the nonantibacterial uses of tetracyclines targeted toward: a) inflammation 8 and arthritis 9-12 ; (III) in neurology: a) in tissue destructive diseases acting like antifibrilogenics 13 ; b) inhibiting caspase-1 and caspase-3 expression in Hungtington disease 14 ; c) ischemia; d) Scheme 1 . Wide-spectrum of activities of tetracyclines (TCs) as antibiotic drugs. TCs are subdivided in: antibacterial (with typical and atypical mechanism of action), antifungal and antineoplastic. During the last 60 years were introduce some chemical modification around the four condensaded rings to obtain more therapeutic selectivity. Anthracyclines, Chemical Modified Tetracyclines, Glycilcycline and Aminomethylcycline are considered members of the same family of chemicals structurally correlated to Tetracycline. Non-antibacterial use cells and subsequently prevented virus-induced apoptosis 32 . II . Both laboratory and clinical studies have investigated the anti-Inflammatory properties of V. The tetracycline-controllable expression tetracyclines, acting as inhibitor of proliferation system offers a number of advantages: strict of lymphocytic9, suppression of neutrophilic on/off regulation, high inducibility, short 10 11 migration , inhibition of phospholipase A 2 and response times, specificity, no interference with accelerated degradation of nitric oxide the cellular pathway, bioavailability of a non- synthetase 12 . toxic inducer, and dose dependence. The tet-off system 33 , which uses the tetracycline-responsive III . In recent times, starting from the end of the transcriptional activator (tTA), and the tet-on 21 90s , TCs have showed to be anti-caking of β- system 34 , which uses the reverse tetracycline- amyloid protein and therefore useful in the responsive transcriptional activator (rtTA), treatment of neurodegenerative diseases like provide respectively negative and positive 22-23 Alzheimer’s and the Prion Diseases . In control of transgene expression. particular, Minocycline reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic windows24. Also, Minocycline inhibits caspase-3 expression and Classification of Tetracyclines delays mortality in a transgenic mouse model of 25 Historically, Tetracyclines are divided into Huntington Disease . Researchers focused on First generation if they are obtained by the mechanisms of intracellular pathway biosynthesis: Tetracycline; Chlortetecycline; communication and genetic control leading to Oxytetracycline; Demeclocycline; Second the attenuation of microglia activation 26 and 27 generation if they are derivatives of semi- protection of Schwann cells . synthesis: Doxycycline; Lymecycline; IV . In the same way that tetracyclines act as pro- Meclocycline; Methacycline; Minocycline; apoptotic in neuronal cells, they act in peripheral Rolitetracycline; And Third generation if they metastasis of generalized tumor. Experimental are obtained from total synthesis: Tigecycline. data using various carcinoma cell lines and However, some researchers consider Tigecycline animal carcinogenesis models showed that a part from Tetracyclines drugs as a new family doxycycline, minocycline and chemical of antibacterial called Glycylcycline. The modified tetracyclines (CMTs) inhibit tumor present paper introduces a new schematic point growth by inhibiting matrix metalloproteinase of view about the denomination and the (MMPs) and by having a direct effect on cell classification of Tetracycline-Structure-Based proliferation 16-17 . The first use of tetracyclines drugs. In the years to come, new TCs, that now in viral infections was reported by Lemaitre in are advanced in the clinical trials (Phase III of 1990 30 . Minocycline and doxycycline imparted Pharmaceutical Trials Protocol), will be in which case, protection against human available in therapy. TCs obtained via total immunodeficiency virus (HIV). In 2005, Zink 31 synthesis, such as Tigecycline, are considered documented the first anti-inflammatory and members of the Third generation if they show neuroprotective activity of an antibiotic against widespectrum activities (both gram + and gram -). a highly pathogenic virus infection. Minocycline Aminomethylcycline derivatives are considered is also significantly active versus West Nile at the same way of Glycylcycline. All new virus replication in cultured human neuronal compounds with highly bacterial species specific activities (narrow-spectrum) are considered destruction TCs appear to inhibit collagenase members of the Fourth generation (scheme 1). activity by a mechanism unrelated to the drug’s In the last five years, thousands of medicinal antibacterial efficacy. In fact, all CMTs have chemists around the world have synthesized, modified with the removal of the tested and patented more than 300 tetracyclines dimethylamino group from the C4 position on similar compounds in particular in the USA and the A ring. To better classify the new in Japan. Harvad University made available compounds it is very important to understand pentacycline antibacterials, a structural what are the chemical properties that make it modification of Doxycycline with five rings and possible for tetracycline-structure-based drugs to from other laboratories azatetracycline and act as a “chameleonic” entity. As discussed in alkylaminotetracycline antibacterials. All the previous paragraph, TCs can be considered these compounds are the “logical’’ results of as wide-spectrum antibiotics versus bacteria, modification around the four rings of fungi, virus and cancer cells. In this view TCs Tetracyclines that historically started with the can be considerate an optimus example of multi master work of Golub and McNamara 35 , when target drugs and the first well documented in twenty years ago, the first eight literatureMoreover, Doxorubicine and all the compoundscalled Chemical Modified other Anthracyclines are structurally correlated Tetracyclines (CMTs) were introduce in to Tetracyclines and it is appropriate to classify literature. In 1983, Golub and McNamara 35 both of them in the same scheme because of introduced a new concept concerning the their chemical similarities, chemical physics therapeutic usefulness of tetracyclines. They properties and mechanisms of action as anti- proposed two main ideas. First, tetracyclines, but cancer drugs not other antibiotics, can inhibit the activity of collagenase (a specific collagenolytic metallo- Strucutre Activities Relationship (SAR) neutral protease produced by host tissues which Figure 1 shows the TCs rigid skeleton with the has repeatedly been implicated in periodontal numeration of the four rings, groups and the destruction. Second, this newly discovered upper and lower sides of the molecule such as property of the drugs could provide a novel they are common called. Amino group in C4 approach to the treatment of diseases, such as position if pivotal for the antibacterial activities periodontal diseases, but also including certain (scheme 2). All TCs that act as inhibitor of medical disorders (e.g., non-infected corneal protein synthesis in bacterial ulcers), which involve excessive collagen

Table 1. Experimental data of TCs and their pharmacokinetics values .

Compounds LogP LogS % Enteric % Serum protein Renal clearance Half-Life absorption binding (ml/min) (hours) Oxytetracycline -1.3 -3.14 58 30 90 10 Tetracycline -0.3 -3.12 80 60 65 9 Doxycycline -0.2 -2.87 93 85 16 15 Demeclocycline 0.2 -2.52 66 75 31 13 Chlorotetracycline - - 30 55 35 6 Meclocycline ------Minocycline 0.5 - 95 90 10 20 Rolitetracycline ------Tigecycline 0.8 - - 90 - 32 Doxorububicine -0.5 - - 70 - 55 need the amino group in position C4 and keto- the specific tautomers that in aqueous solution enolic tautomers in position C1 and C3 of A are more stable than others. It is important to ring. Modification of amide in C2 is possible but analyze all the possible tautomers of this with loss of potency. Groups R1, R2 and R3 are molecule in their different degrees of modifiable to give more selectivity to the protonation and conformations to understand the biological target in antifungal TCs, but not for role of tautomerism in the chemical behavior of the antibacterial activity. D ring is the most TC. Simas optimized the structures of all 64 flexible to change. All modifications of group tautomers and calculated their heats of

R4, R5 and R6 are allowed to give highly formation(∆Hf°). There are different tautomers bacterial specificity. Hydroxyl group in C10, in equilibrium in each degree of protonation of C11 and C12 positions, with the respective TC. They have similar stabilities and they are tautomeric equilibrium are indispensable for present in considerable amount in the medium. recognition and bonding in ribosomal subunit. Antifungal and antitumor TCs act in a different way from antibacterial TCs. Once passed into the eukaryotic cells, TCs change the electronic balance equilibrium sequestering divalent ions (e.g., Ca 2+ ) much more then monovalent ions (e.g., K +). Hydroxyl group are a source of radical oxygen species (ROS) that damage irreversible the macromolecules such as DNA, A RNA and proteins. All these effect are consider as fundamentals in cellular death for oxidative stress. In the case of antitumor Anthracyclines and Metastat act both blocking the enzymatic transcriptional complex formation on DNA and then inducing apoptotic events. TCs have different acid groups in its structure and the possibility to adopt different conformations. The different proton-donating group of this molecule B offer several possibilities of metal ion substitution. The complexation with metal ions increases the stability of the various TC derivatives. In some cases metal ion complexation reduces the availability of TC in the blood plasma or eliminates its biological activity (Table 1). It is known that TC forms complexes in different position with calcium and magnesium ions that are available in the blood plasma. In 1999, Simas 36 brings out a C computational and experimental study to Figure 2. 3D geometrical shape of Tetracycline (A), evaluate the weight of the various chemical Tigelcycline (B) and SF2575 (C). TCs show different tautomeric behavior of tetracyclines in solution. conformation and biological activities due their functional The degree of protonation of TC depends upon groups on the same rigid molecular skeleton. Conclusions 5 Nelson, M.L. Chemical and Biological Dynamics In this work is introduce a new framework for of Tetracycline. Advanced Dental Research (1998). the classification of old and novel TCs and their 6 Carlotti B, Fuoco D, Elisei F. Fast and ultrafast Structure Activity Relationship. TCs show in spectroscopic investigation of tetracycline derivatives this paper were chosen as the best in class from in organic and aqueous media. Phys Chem Chem a data set of 1325 isomeric and structural Phys. 2010 Dec 28;12(48):15580-91. correlated tetracyclines. The new classification proposed is based on a medicinal chemistry 7 Chopra, Ian,; Roberts, Marilyn; Tetracycline antibiotics: mode of action, application, molecular approach due to the complexity of the novel biology and epidemiology of bacterial resistance. TCs drugs, e.g.: Chemical Modified Microbiol Mol Biol Rev (2001), 65 (2), 232-260. Tetracycline (CMT), Anthracycline drugs 8 D'Agostino, P; Ferlazzo V; Milano S; La Rosa M; (Doxorubicine) and Structurally Correlated Di Bella G; E; Anti-inflammatory effects of Tetracycline (Glycylcycline and chemically modified tetracyclines by the inhibition of Aminomethylcycline). The role of lipophilicity nitric oxide and interleukin-12 synthesis in J774 cell of TCs as the main factor to explain the line, International Immunopharmacology (2001), 1, 1765-1776 biological potency and dynamics of this old family of drugs. It is important to understand the 9 Thong YH, Ferrante A. Inhibition of mitogen- conformational flexibility and the affinity of induced human lymphocyte proliferative responses to tetracycline analogues. Clin Exp Immunol. 1979;35. TCs of metals ions due the several functional group present into the molecular skeleton to 10 Martin RR, Warr GA, Couch RB, Yeager H, obtain a significant SAR study. With more than Knight V. Effects of tetracyclines on leukotaxis. J 60 years under scientific investigation TCs are Infect Dis. 1974;129:110–116 consider a master example of pleitropic family 11 Pruzanski W, , Laliberte F, Stefanski E, Vadas P. of compounds with promising therapeutic Inhibition of enzymatic activity of phospholipase A2 properties. Due to the wide use in therapy and by minocycline and doxycycline. Biochem Pharmacol. 1992;P44:1165–1170 the low toxicity TCs can be consider the first Multi-Target Drugs fully and well discussed in 12 Amin AR, Patel RN, Thakker GD, Lowenstein CJ, the history of pharmacology. Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by References doxycycline and chemically modified tetracyclines. FEBS Lett. 1997;410:259–264

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