Synthesis, Characterization and In Vitro Antibacterial Derivatives of Doxycycline

Authors’ Contributions 1 Conception & Study Design. 2 Data Collection. 3,6 Data Analysis. 4,5,8 Drafting. 7 Critical Review. Article info. Received: January 17, 2019 Accepted: November 13, 2019 Funding Source: Nil Conflict of Interest: Nil Cite this article: Khan MR, Khan MA, Ahmad K, Hamad A, Sajid-ur-Rehman M, Asif HM, Younus M, Kamal Y. Synthesis, Characterization and In Vitro Antibacterial Derivatives of Doxycycline. RADS J Pharm Pharm Sci. 2019; 7(4): 215-226. *Address of Correspondence Author: ansari.khalil@gmail.com Objective: Nowadays antibacterial drugs resistance is major problem in the world. To overcome this problem, some novel derivates of doxycycline were synthesized by single step condensation reaction with ten different types of aromatic and aliphatic aldehydes and ketones in ethanol as solvent and acetic acid as catalyst. In these reactions, deprotonation of primary amine occurs that results in formation of imine. Methods: All the derivatives physically characterized and confirmed by analytical techniques i.e. FTIR and 1H NMR and 13C NMR. The derived compounds have exhibited significantly more active against both gram positive as well as gram negative bacterial strains as compared to parent drug. Results: Derived Schiff bases RDC2, RDC4 and RDC10 showed zone of inhibition against Bacillus subtilis as compared to doxycycline and derived Schiff bases RDC1, RDC2, RDC5, RDC6, RDC7 and RDC8 showed more zone of inhibition against Micrococcus luteus as compared to doxycycline. Yield (75%), m.p. 180-185°C, Mol. Wt. 1063, Elemental Analysis: (Calculated) for C58H54N4O16: iC, 65.53; H, 5.12; N, 5.27; (Found): C, 65.49; H, 5.18; N, 5.37; FTIR (ν, cm‐1): 3066 (=C-H), 1665(C=N), 1585, 1490(C=C, phenyl), 3650(OH), 1692 (C=O); 1H NMR (DMSO−d6, δ, ppm), 6.02-6.03 d, 6.91-6.92 d, (=CH-); 6.88-6.89 t (=CH), 2.89-2.90 d, (-CH); 1.07-1.08 q, (-CH3), 3.383.39 d; 3.17-3.18 d, (CH), 1.47-1.48 t (CH), 1.48 s, (CH3), 6.96-6.97d, 7.5777.588 d, (-CH=) 15.21 s (OH); 4.62 s (OH); 1.46-1.47 s (OH); 13C NMR (DMSO−d6, δ, ppm). Conclusion: Doxycycline is among broad-spectrum tetracycline. The Schiff bases derived from doxycycline show significantly highly active against gram negative bacteria as compared to doxycycline. In future further study on these derived compounds will help in market new derivative of doxycycline, which will have more broad-spectrum activity than doxycycline.


I N T R O D U C T I O N
Hugo Schiff in 1864 first time made Schiff base. Schiff bases have functional group containing carbon nitrogen double bond (R1R2C=NR3). Here R3 may be alkyl or aryl group but not hydrogen atom, R1 and R2 is organic side chain [1].
In 1864 Hugo Schiff made first Schiff base by reaction of aniline with benzaldehyde, acetaldehyde, valeraldehyde and cinnamaldehyde [2]. He opened the new door of synthetic chemistry by synthesis of imine [3]. First beta lactam is produced by reaction of Schiff base with diphenyl ketenes [4].
Aryl substituent containing Schiff bases are more stable then alkyl substituent [5]. Aldehydes react rapidly with imine than ketones because aldehydes have less stearic hindrance than ketones. The second carbon attached with carbonyl group make ketones less electrophilic than aldehydes [6]. The condensation products of aromatic amines and aldehydes and ketones are known as azomethines imines or anils. The aldehydes and ketone derived Schiff bases are known as aldimines and ketamine, respectively. The C=N of imines have wide biological and chemical importance. The C=N have excellent chelating properties the presence of hydroxyl and thiol group with azomethine group may form penta or hexa ring structure with metal ions. Schiff bases may be bidentate, tridentate, tetradentate, and polydentate. Aliphatic aldehydes derived Schiff bases less stable than aromatic aldehydes because conjugation make them more stable.
Schiff bases have interesting biological properties because of electron donating and electron accepting groups [7]. There is wide range of biological activity of Schiff bases i.e. antimicrobial activity against bacteria protozoa fungal and viral infection [8] also have anticancer activity [9] anti-inflammatory and analgesic activity [10] and anticonvulsant activity [11] antianthelmintic activity [12]. The carbon nitrogen double bond is mainly responsible for chelating activity of Schiff bases [13]. Schiff bases have important chemical properties these are precursor four different reactions organometallic reactions carbon nitrogen double bond have capability of incorporation of metals [14], in coordination chemistry Schiff bases have unique application due to its chelating property [15]. These metal complexes have antifungal antibacterial anti-cancer activities [16,17].

Doxycycline
Pfizer chemists team led by Robert Blackwood first time synthesized doxycycline from methacycline and it was approved 1967 by FDA and marketed as vibramycin [18]. It is one of the drug in WHO essential drugs list most effective and safe medicine among antibiotics for human body system [19]. The mechanism of action of doxycycline "it inhibits microorganism macromolecule synthesis by binding to the 30S microorganism organelle and preventing access of aminoacyl soluble RNA to the acceptor site on the mRNA-ribosome advanced. These medications enter gram-negative microorganism by passive diffusion through the hydrophilic channels fashioned by the porin proteins of the outer plasma membrane associated by transport via an energydependent system that pumps all doxycycline across the living substance membrane. Entry of those medication into gram-positive microorganism needs metabolic energy, however isn't in addition understood" [20].
It is use in acute pelvic inflammatory disease with combination of cefoxitin [21] in ocular rosacea [22].

List of Chemicals
All the chemicals used were analytical grade and obtained from the following sources (Table 1)

Preparation of Schiff Bases
In 250 ml round bottom flask equimolar solution of available aldehydes and ketones and doxycycline in 30 ml of ethanol added in flask. Few drops of acetic acid also added as catalyst. The reaction mixture refluxed for 4 hours, cool at room temperature and filter. The solvent was evaporated by rotary evaporator the solid residue collected and dried at room temperature. Recrystallize with ethanol. Characterized by using different physio-chemical procedure.

Instrumentation and Spectral Determination
All the physical properties of prepared Schiff base drugs like solubility and melting points were determined.
Solubility of all the derived ligands were determined by using Ela E 30H, D78224 Singe /Htw sonicator.
Galen Kemp apparatus used to determine the melting point of Schiff base drugs.
FTIR spectra of Schiff bases obtained from Brooker TENSOR 27 FTIR spectrophotometer. 1 H NMR and 13 C NMR spectra of synthesized were obtained by Bruker 400-spectrophotometer.
Antibacterial activity of all synthesized ligand was performed by using agar well diffusion method. Autoclave was used for sterilization of equipment and media used for activity.

Solubility
Solubility of all derived Schiff bases is determined one by one in different solvents like water, ethanol, DMSO, methanol and acetone using Ela E 30H, D-78224 Singe /Htw sonicator.

Melting Point
Melting point of all synthesized Schiff bases were determined one by one sample by adding small amount of powder of synthesized Schiff base in capillary tube and tube heated on Galen Kemp melting point apparatus.

Infrared Spectrum
Infrared spectrum of Schiff bases was determined by Bruker TENSOR-27 FTIR spectrophotometer using KBR pellets in the range of 4000-400cm -1 at speed of 16 times per sample.

NMR Spectrum
1 H NMR and 13 C NMR spectra of synthesized were obtained by Bruker 400-spectrophotometer.

General Scheme
General scheme for synthesis of Schiff bases of doxycycline

Antibacterial Activity
Antibacterial action of all the prepared Schiff base ligands were checked by agar well diffusion method against the following strains (Table 2a, 2b)   Alphabets ABC indicates concentration of novel Schiff bases 5 mg/ml, 2.5 mg/ml and 1 mg/ml respectively against sevens trains. Table 3. Standard drug against bacterial strains. Antimicrobial activity of standard drug doxycycline against seven strains are mentioned in Table 3.

D I S C U S S I O N Gram +ve and Gram -ve Bacteria Compared with Standard Drug Doxycycline
The Schiff bases synthesized by simple one-step reaction. All the ligands synthesized by simple condensation reaction, characterized and confirmed by using different techniques of spectroscopy i.e. FTIR and NMR.
Antibacterial activity of all the derived Schiff bases ligands performed against both gram +ve and gramve bacteria and compared with standard drug doxycycline from which all of these Schiff base ligands derived.

C O N C L U S I O N
Antibacterial resistance against antibiotics is major problem in all over the world. We have developed resistance against many broad-spectrum antibiotics.
To overcome this resistance against we need new antibiotics discovery or derived new compounds from pre-existing antibiotics. These derived compounds show activity against resistant bacteria. Doxycycline is among broad-spectrum tetracycline. The Schiff bases derived from doxycycline show significantly highly active against gram negative bacteria as compared to doxycycline. In future further study on these derived compounds will help in market new derivative of doxycycline, which will have more broadspectrum activity than doxycycline.