WO2020202239A1

WO2020202239A1 - Glycosylated 3-substituted fluoroquinolone derivatives, preparation methods thereof, and their use in the treatment of antimicrobial infections

Info

Publication number: WO2020202239A1
Application number: PCT/JO2019/050011
Authority: WO
Inventors: Ghadeer SUAIFAN; Mayadah SHEHADEH; Aya MOHAMMED
Original assignee: The University Of Jordan
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2020-10-08

Abstract

The present disclosure relates to 3-substituted fluoroquinolone derivatives, and more particularly to glycosylated 3-substitutred fluoroquinolone derivatives, methods of preparation thereof, and uses thereof for treating microbial infections.

Description

GLYCOSYLATED 3 -SUBSTITUTED FLUOROQUINOLONE

DERIVATIVES, PREPARATION METHODS THEREOF, AND THEIR USE

IN THE TREATMENT OF ANTIMICROBIAL INFECTIONS

TECHNICAL FIELD

[01] The present disclosure relates to 3-substituted fluoroquinolone derivatives, and more particularly to glycosylated 3-substitutred fluoroquinolone derivatives, methods of preparation thereof, and uses thereof for treating microbial infections.

BACKGROUND

[02] Since the discovery of nalidixic acid, the prototype of quinolones, extensive research has been conducted around the quinolone basic scaffold to develop novel synthetic analoges of value as reserve agents with improved antimicrobial activity. For instance, the fluoro-derived quinolones as norfloxacin and ciprofloxacin were developed, and are considered to be clinically effective against a wide range of Gram positive bacteria including Streptococci , Enterococci and Staphylococci and/or Gram negative bacteria. Hence, the fluoroquinolones are used in the treatment of various infections including urinary tract, respiratory tract, gastrointestinal, skin and bone infections through the inhibition of microorganism topoisomerase II. This widespread consumption of fluoroquinolones including ciprofloxacin led to the development of bacterial resistance. This continued emergence of bacterial resistance and the prevalnce of new virulence pathogens incites the discovery and development of novel effective fluoroquinolone analogues.

[03] Several attempts have been reported in the prior arts to develop quinolone derivatives.

For instannce the US patent publication number 20050107310 discloses the synthesis of carboxylic glycuronides, glycosamines and glycosides of quinolones among other antibiotics. However, these agents were reported to possess fair activity against staphylococci and poor activity to streptococci but exhibiting good activity against P. aeruginosa.

[04] Other attempts included 3-substituted carboxylic acids, 3-substituted carboxamides and sugar moiety incorporation at the piperazine ring position 7 [05] The international publication number 03079980 discloses carboxylic acid glycuronides, glycosamides and glycosides of quinolones and analogs thereof to treat conditions and diseases such as bacterial infections.

[06] In the present disclosure, the amidation of 1,3-fluoroquinolones such as ciprofloxacin, norfloxacin and moxifloxacin with glucosamine as a protecting moiety is surprisingly found to enhance bacterial drug-uptake, hence, improving activity and lowering possible cytotoxicity. Therefore, the aim of this invention is to provide novel 3 -glucosamine- fluoroquinolone derivatives with good antimicrobial activity against various Gram positive and Gram negative bacteria in addition to pathogenic fungi along with methods for preparing these novel derivatives.

SUMMARY

[07] Aspects of the present disclosure provides a compound of the Formula (I), or a salt thereof:

Formula (I) wherein Ri may be selected from a group consisting of hydrogen; alkyl; dialkylamino; substituted or unsubstituted cycloalkyl; alkenyl; unsubstituted or substituted cycloalkenyl; alkynyl; aryl; or unsubstituted or substituted heteroaryl;

R_2, R₄ may be independently selected from a group consisting of hydrogen; amino; halogen; sulfur; alkyl; dialkylamino; unsubstituted or substituted alkenyl; unsubstituted or substituted cycloalkyl; alkynyl; alkoxy preferably methoxy; aryloxy; alkanoyl; unsubstituted or substituted heteroaryloxy; hydroxyl; cyano; or unsubstituted or substituted heteroaryl

R₃ may include

R₅, R._6, R₇, R₈ may be selected from a group consisting of free hydroxy groups; fully acylated hydroxy groups; or partially acylated hydroxy groups having the group R₉-(C=O)-;

R₉ may be selected from a group consisting of alkyl; substituted or unsubstituted aryl; or arylalkyl.

R₁₀ may be selected from a group consisting of hydrogen; amino; halogen; sulfur; alkyl; dialkylamino; unsubstituted or substituted alkenyl; unsubstituted or substituted cycloalkyl; alkynyl; alkoxy preferably methoxy; aryloxy; alkanoyl; unsubstituted or substituted heteroaryloxy; hydroxyl; cyano; or unsubstituted or substituted heteroaryl.

[08] In some aspects, the acyl groups may include acetyl.

[09] In other aspects, R₉ may include phenyl; nitrophenyl; halophenyl; alkyl substituted phenyl; alkoxy; phenyl; or benzyl.

[010] Other aspects of the present disclosure provide a method of preparing the compound of the Formula (I), the method may include the steps of:

Dissolving ciprofloxacin hydrochloride in water followed by an addition of aqueous NaHCO₃ to form a first precipitate, then filtering the first precipitate washing it with cold water and drying it under reduced pressure to produce neutral ciprofloxacin;

Adding AC₂O to solution of the produced free ciprofloxacin in glacial acetic acid to form a first mixture, warming up the first mixture and allowing it to crystallize forming a first set of crystals, followed by filtering, washing, and drying the first set of precipitate;

Adding AC₂O drop-wise to a solution of norfloxacin in glacial acetic acid to form a second mixture, warming up the second mixture and allowing it to crystallize forming a second set of crystals, followed by filtering, washing, and drying the second set of precipitate;

Dissolving moxifloxacin hydrochloride in CH₂C₁₂, followed by an addition of AC₂O along triethylamine to form a third mixture, warming and stirring the third mixture, followed by reducing it under pressure and adding MeOH to form a fourth mixture, and evaporating and adding Et₂O to the fourth mixture to provide a third set of precipitate;

Adding ethyl chloroformate drop-wise to a solution of triethylamine and first set of precipitate in CH₂C₁₂ while stirring till an intermediate is formed, followed by an addition of 1,3, 4, 6- Tetra-0 -acetyl- b-D-glucosamine in drop-wise to form a fifth mixture while stirring, then extracting the fifth mixture followed by drying an organic layer and triturating a fourth precipitate;

Adding ethyl chloroformate drop-wise to a solution of triethylamine and second set of precipitate in CH₂C₁₂ while stirring till an intermediate is formed, then adding 1,3,4, 6-tetra- O-acetyl - b-D-gl ucosam i ne and CH₂C₁₂ drop-wise while stirring to form a sixth mixture, followed by extracting the sixth mixture followed by drying an organic layer and triturating a fifth precipitate;

Adding ethyl chloroformate to a solution of the third set of precipitate in CH₂C₁₂ while stirring till an intermediate is formed, then a solution of 1 , 3 ,4, 6-tetra-O-acetyl -b-D-glucosam i ne in CH₂C₁₂ to form a seventh mixture while stirring, followed by extracting the seventh mixture and drying an organic layer and triturating a sixth precipitate;

Adding NaOMe portion-wise to a solution of second white solid precipitate to form an eighth mixture till a product is formed, followed by neutralizing, filtering, evaporating and re dissolving the eighth mixture in water, followed by freeze drying to provide a seventh precipitate;

Adding NaOMe portion-wise to a solution of second white solid precipitate in MeOH to form a ninth mixture, neutralizing, filtering, and evaporating and re-dissolving the ninth mixture in water, followed by freeze drying to provide an eighth precipitate; and

Adding NaOMe portion-wise to a solution of the first residue in MeOH to form a tenth mixture, followed by neutralizing the tenth mixture and evaporating a filtrate to form a fifth residue, then recrystallizing the ninth precipitate.

[Oil] Yet other aspects of the present disclosure provide a pharmaceutical composition including a compound of the Formula (I) and/or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier/excipient. [012] The pharmaceutical composition may be used in treating a microbial infection.

[013] In some aspects, the microbial infection is caused by one or more of Escherichia coli 0157:H7, Salmonella entericae ATCC 13312, Listeria monogenesis ATCC 19115 Pseudomonas aeruginosa ATCC 9627, Escherichia coli NCTC 11954, Escherichia coli ATCC 8739, Escherichia coli isolate, Methicillin resistant staphylococcus aureus ATCC 33591, and Methicillin sensitive staphylococcus aureus ATCC 9253, or a combination thereof.

[014] In other aspects, the microbial infection is caused by one or more of Candida albicans ATCC 10231, Aspergillus flavus ATCC 9643, Fusarium solani ATCC 36031, and Stachybotrys chartarum, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[015] The disclosure will now be described with reference to the accompanying drawings, which illustrate embodiemnts of the present disclosure, and in which:

[016] FIG. 1 illustrates a flowchart of a method of preparing 1,3,4,6-O-acetylated-Z)- glucosamine in accordance with embodiments of the present disclosure.

[017] FIG. 2A illustrates a flowchart of a method of preparing a first group of glycosylated 3-substituted fluoroquinolone derivatives in accordance with embodiments of the present disclosure.

[018] FIG. 2B illustrates a flowchart of a method of preparing a second group of glycosylated 3-substituted fluoroquinolone derivatives in accordance with embodiments of the present disclosure.

[019] FIG. 2C illustrates a flowchart of a method of preparing a third group of glycosylated 3-substituted fluoroquinolone derivatives in accordance with embodiments of the present disclosure. DETAILED DESCRIPTION

[020] It is an object of the present disclosure to provide glycosylated 3-substituted fluoroquinolone derivatives according to Formula (I), or a slat thereof:

Formula (I) wherein Ri may include hydrogen; alkyl; dialkylamino; substituted or unsubstituted cycloalkyl; alkenyl; unsubstituted or substituted cycloalkenyl; alkynyl; aryl; or unsubstituted or substituted heteroaryl;

R₂, R₄ may independently include hydrogen; amino; halogen; sulfur; alkyl; dialkylamino; unsubstituted or substituted alkenyl; unsubstituted or substituted cycloalkyl; alkynyl; alkoxy preferably methoxy; aryloxy; alkanoyl; unsubstituted or substituted heteroaryloxy; hydroxyl; cyano; or unsubstituted or substituted heteroaryl;

R₃ may independently include:

or

R₅, R.₆, R₇, R₈ may include free hydroxy groups; fully acylated hydroxy groups; or partially acylated hydroxy groups having the group R₉-(C=0)-; wherein R₉ may include alkyl; substituted or unsubstituted aryl; or arylalkyl. and R₁₀ is selected from a group consisting of hydrogen; amino; halogen; sulfur; alkyl; dialkylamino; unsubstituted or substituted alkenyl; unsubstituted or substituted cycloalkyl; alkynyl; alkoxy preferably methoxy; aryloxy; alkanoyl; unsubstituted or substituted heteroaryl oxy; hydroxyl; cyano; or unsubstituted or substituted heteroaryl. [021] In embodiments of the present disclosure, the acyl groups may include acetyl.

[022] In some embodiments, R₉ may include phenyl; nitrophenyl; halophenyl; alkyl substituted phenyl; alkoxy; phenyl; or benzyl.

[023] Embodiments of the present disclosure further provide a pharmaceutical composition including a compound of general formula (I) and/or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier/excipient.

[024] The term“pharmaceutical composition”, as used herein, is intended to include a pharmaceutical active compound of general formula (I) and/or a pharmaceutically acceptable salt thereof.

[025] The pharmaceutical composition can be, for example, in a liquid form, e.g. a solution, syrup, emulsion and suspension, or in a solid form, e.g. a capsule, caplet, tablet, pill, powder and suppository. Granules, semi-solid forms and gel caps are also considered. In case that the pharmaceutical composition is a liquid or a powder, dosage unit optionally is to be measured, e.g. in the dosage unit of a teaspoon.

[026] The pharmaceutical composition of this disclosure can be formulated for oral administration in solid or liquid form, for parenteral injection or for rectal administration. The pharmaceutical composition can be administered to humans and other mammals orally, sublingually, rectally, parenterally, intracisternally, intraurethrally, intraperitoneally, topically (as powder, ointment or drop), as buccal or as an oral or nasal spray. The term "parenterally", as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, subcutaneous, intra-articular injection and infusion.

[027] Pharmaceutical compositions of this disclosure for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. [028] The term“pharmaceutical acceptable carrier/excipient”, as used herein, means a non toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as com starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; binding agents such as hypromellose; disintegrating agents such as crosscarmellose; water; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil; cottonseed oil; safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgement of the formulator.

[029] All components of the pharmaceutical composition have to be pharmaceutically acceptable. The term "pharmaceutically acceptable" means at least non-toxic. The therapeutically active component should preferably be present in the above-mentioned pharmaceutical composition, the concentration of about 0.1 to 99.5% by weight, preferably of about 0.5 to 95% by weight of the total mixture.

[030] The above-mentioned pharmaceutical composition can further contain other pharmaceutical active compounds in addition to the compound of general formula (I) according to the disclosure.

[031] In embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of microbial infections.

[032] In accordance with embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of infections caused by Gram positive and Gram negative bacteria.

[033] In accordance with embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of infections caused by E. coir, S. entericae; P. aeruginosa and L. monocytogenes bacteria. In accordance with embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of fungal infections.

[034] In accordance with embodiments of the present disclosure, the pharmaceutical composition is for use in the treatment of infections caused by C. albicans ; S. chartarum; and P. chrysogenum fungi.

[035] The disclosure is now further illustrated on the basis of Examples and a detailed description from which further features and advantages may be taken. It is to be noted that the following explanations are presented for the purpose of illustrating and description only; they are not intended to be exhaustive or to limit the disclosure to the precise form disclosed.

Example 1

Preparation of 1,3,4,6-O-acetylated-D-glucosamine

Preparation scheme

[036] l,3,4,6-O-acetylated-D-glucosamine was prepared in accordance with the following scheme:

Wherein (i) represents aqueous NaOH and 4-methoxybenzaldehyde; (ii) represents Py and AC₂O; (iii) represents acetone and aqueous 5M HC1.

(iv) represents CH₂C₁₂ and aqueous lM NaHCO₃.

[037] Reference is now being made to FIG. 1, which illustrates a flowchart of a method of preparing 1 ,3, 4, 6-O-acetyl ated-D-glucosamine in accordance with embodiments of the present disclosure. For the preparation of compound (1), 4-methoxybenzaldehyde (having a volume of about 12 mL, and a number of moles of about 93 mmol) was added drop-wise to a solution of D-(+)-glucosamine hydrochloride (having a mass of about 20 g, and a number of moles of about 93 mmol) in aqueous 1 M NaOH (of volume about 96 mL). The mixture was stirred at room temperature until crystallization commenced and left standing overnight (process block 1-1). The crystallized mixture was filtered, washed with cold water (of about 100 mL) followed by EtOH and Et₂0 solution (according to a ratio of about 1 : 1, and a volume of 100 mL), and dried (process block 1-2). For the preparation of compound (2), compound 1 (having a mass of about 15 g, and a number of moles of about 51 mmol) was added portion -wise to a cold solution of pyridine (of about 83 mL volume) and AC₂O (of about 45 mL volume). The mixture was stirred at a temperature of about 0 °C for about one hour and at room temperature for two days, or until reaction completion as indicated by TLC (n-Hexane: EtOAc 4:6, R_f = 0.70). Cold water (about 300 mL volume) was then added to initiate precipitation. The precipitate was filtered, washed with cold water (of about 185 mL volume) and dried (process block 1 -3). For the preparation of compound (3), compound 2 (having a mass of about 16 g, and a number of moles of about 35 mmol) was added to an aqueous solution of aqueous 5 M HC1 (of about 8 mL volume) and warm acetone (of about 150 mL volume), which led to the formation of a precipitate. The mixture was then cooled, and Et₂0 (of about 150 mL volume) was added and left stirring at room temperature for about two hours. The precipitate was filtered, washed with Et₂0 (of about 50 mL volume) and dried (process block 1-4). For the preparation of compound (4), compound 3 (having a mass of about 6 g, and a number of moles of about 16 mmol) was dissolved in CH₂C₁₂ (of about 85 mL volume). Then, about 85 mL of aqueous 1 M NaHCO₃ solution was added and mixed for 30 about minutes. After that, the mixture was extracted, dried over anhydrous Na₂SO₄ and evaporated (process block 1-5).

Example 2

Preparation of glycosylated 3-substituted fluoroquinolone derivatives

Preparation scheme [038] Compounds of the general formula (I) can be prepared by the following scheme:

wherein (i) represents aqueous 5% NaHCO₃;

(ii) represents AC₂O and glacial acetic acid;

(iii) represents AC₂O and glacial acetic acid;

(iv) AC₂O, triethylamine and CH₂C₁₂;

(v) CICOOC₂H₅, triethylamine and CH₂C₁₂;

(vi) MeOH, NaOMe and amberlite resin.

[039] Reference till the end of this example will now be made to FIGs 2A-2C, which illustrate flowcharts of a method of preparing glycosylated 3 -substituted fluoroquinolone derivatives in accordance with embodiments of the present disclosure. Referring now to FIG. 2A, for the preparation of compound (5), about 8 g of ciprofloxacin hydrochloride having a number of moles of about 21.7 mmol was dissolved in about 267 mL of water followed by an addition of about 300 mL of excess aqueous 5% NaHCO₃ forming a first precipitate. The first precipitate was filtered, washed with cold water and dried to produce neutral ciprofloxacin (process block 2-1). [040] For the preparation of compound (6), about 0.5 mL of AC₂O was added to about 1.5 g and 4.5 mmol solution of the produced neutral ciprofloxacin in about 12 mL of glacial acetic acid. The mixture was warmed up to less than about 35°C for about 1 hour and left for a few minutes to crystallize at room temperature. Then, the crystals were filtered, washed with Et20 and dried to afford the white solid compound (6) (process block 2-2).

[041] For the preparation of compound (7), about 1.8 mL of AC₂O having number of moles of 19.5 mmol was added drop-wise to about 4 g and 13 mmol solution of norfloxacin in about 150 ml of glacial acetic acid. The mixture was warmed up to less than about 35°C for about 1 hour and left for a few minutes to crystallize at room temperature. Then, the crystals were filtered, washed with Et₂0 and dried to afford the white solid compound (7) (process block 2-3).

[042] For the preparation of compound (8), about 4.9 g, 11 mmol moxifloxacin hydrochloride was dissolved in about 30 mL CH₂C₁₂, followed by an addition of about 1.6 ml and 16.5 mmol AC₂O along with about 3.08 ml and 22 mmol triethylamine. The mixture was slightly warmed and stirring was continued until TLC (Thin Layer Chromatography) indicated the end of the reaction. The solvent was reduced under pressure and MeOH was added, the reaction was stirred overnight. This was followed by evaporation and addition of Et₂0 to a compound (8) as a white solid precipitate (process block 2-4).

[043] For the preparation of compound (9), about 0.5 mL and 5.4 mmol of Ethyl chloroformate was added drop-wise to a solution of about 0.8 mL and 5.4 mmol triethylamine and about 2.0 g and 5.4 mmol of compound (6) in about 150 mL cold CH₂C₁₂ at a temperature of about -20 °C while stirring (process block 2-5). The stirred solution was monitored by TLC until intermediate formation. After that, about 1.88 g and 5.4 mmol solution of compound (4) in about 30 mL cold CH₂C₁₂ having a temperature of about -20 °C was added drop-wise, then the reaction mixture was stirred at a temperature of about - 20 °C for about 5 hours and then stirred at room temperature for about 2 days (process block 2-6). The stirred mixture was extracted with about 180 mL water, about 180 mL aqueous 1M HC1, and about 180 mL aqueous 1M NaHCO₃. Then, organic layer was dried over anhydrous Na₂SO₄, and the concentrated gummy residue was triturated with Et20 to afford solid material and purified using silica gel column chromatography to afford compound (9) (process block 2-7). [044] Referring now to FIG. 2B, for the preparation of compound (10), about 0.2 mL and 2.1 mmol of ethyl chloroformate was added drop-wise to a solution of about 0.3 mL and 2.1 mmol triethylamine and about 0.5 g and 1.39 mmol of compound (6) in about 250 mL of cold CH₂C₁₂ having a temperature of about -20 °C while stirring. The stirred mixture was monitored by TLC until intermediate formation (process block 2-8). After that, about 0.7 g and 2.0 mmol of compound (4) and about 30 mL of cold CH₂C₁₂ having a temperature of about -20 °C was added drop-wise, stirred at -20 °C for 5 hours and then left stirring at room temperature for about 2 days. The reaction mixture was extracted with about 300 mL water about 300 mL of aqueous 1M HC1, about 300 mL of aqueous 1M NaHCO₃ (process block 2-9). Finally, the organic layer was dried over anhydrous Na₂SO₄, the concentrated gummy residue was triturated with Et20 to afford solid material and purified using silica gel column chromatography (process block 2-10).

[045] For the preparation of compound (11), about 0.3 mL and 2.7 mmol of ethyl chloroformate was added to about 0.3 mL and 2.1 mmol of a solution containing about 1.0 g and 2.3 mmol of compound (7) in about 40 ml CH₂C₁₂ having a temperature of about -20 °C while stirring (process block 2-11). The stirred solution was monitored by TLC (CHCl₃: MeOH) until intermediate formation. Following, about 945 mg and 3.45 mmol of a solution of compound (4) in about 30 ml of CH₂C₁₂ having a temperature of about -20 °C was added drop-wise to the stirred solution, the reaction mixture was stirred at a temperature of about -20 °C for about 5 hours and then left stirring at room temperature for about 2 days (process block 2-12). Then, the solution was extracted with about 75 mL of water, about 75 mL of aqueous 1M HC1, about 75 mL of aqueous lM NaHCO₃ (process block 2-13). The organic layer was dried over anhydrous Na₂SO₄, the concentrated gummy residue was triturated with Et20 to afford solid material and purified using silica gel column chromatography (process block 2-14).

[046] Reference is now being made to FIG. 2C, for the preparation of compound (12), about 0.13 g and 2.1 mmol of NaOMe was added portion-wise to a solution containing about 0.3 g and 0.35 mmol of compound 8 in about 30 mL MeOH (process block 2-15). The reaction was continued for about 2 hours, until TLC indicated the formation of product. The solution was neutralized using amberlite resin, filtered, evaporated and redissolved in water. The solution was freeze dried to obtain compound (12) (process block 2-16). [047] For the preparation of compound (13), about 0.2 g and 2.58 mmol of NaOMe was added portion-wise to a solution containing about 0.4 g and 0.46 mmol of compound (9) in about 50 mL MeOH. After about 2 hours, TLC indicated the formation of product (process block 2-17). Then, the reaction solution was neutralized using amberlite resin, filtered, evaporated and redissolved in water. The solution was freeze dried to obtain a pale yellow solid compound (13) (process block 2-18).

[048] For preparing compound (14), about 0.21 g and 3.9 mmol NaOMe was added portion- wise to a solution containing about 0.5 g and 0.65 mmol of compound (10) in about 30 mL MeOH. After about 2 hours, TLC indicated the formation of product (process block 2-19). The solution was neutralized using amberlite resin. The filtrate was evaporated and yellow powder was obtained. The solid was recrystallized from MeOH/Et₂0 to afford a solid powder compound (14) (process block 2-20).

Example 3

Structure Elucidation Data

[049] Compounds (1) to (14) may have chemical characterization as follows:

2-Deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose (1) m.p: 164-168 °C, IRv_max: 1650 cm^-1 (N=C), 3200 cm^-1 (O-H); d_H 2.80 (1H, t, J = 8.4 Hz, CH), 3.13-3.18 (1H, m, CH), 3.22-3.25 (1H, m, CH), 3.41-3.43 (1H, m, CH), 3.45-3.52 (1H, m, CH), 3.73(1H, d, J= 5.4, 10.4 Hz, CH), 3.81 (3H, m, OCH₃), 4.53 (1H, t, J= 5.7 Hz, OH), 4.70 (1H, t, J= 7.2 Hz, OH), 4.80 (1H, d, J= 5.5 Hz, OH), 4.91 (1H, d, J= 5.2 Hz, OH), 6.50 (1H, d, J= 5.5 Hz, CH), 6.99 (2H, d, J = 8.5 Hz, Ar-H), 7.70 (2 H, d, J = 8.6 Hz, Ar-H), 8.12 (1H, s, N=CH).5_H2.80 (lH, t, J= 8.4 Hz, CH), 3.13-3.18 (1H, m, CH), 3.22-3.25 (1H, m, CH), 3.41- 3.43 (1H, m, CH), 3.45-3.52 (1H, m, CH), 3.73(1H, d, J= 5.4, 10.4 Hz, CH), 3.81 (3H, m, OCH3), 4.53 (1H, t, J= 5.7 Hz, OH), 4.70 (1H, t, J= 7.2 Hz, OH), 4.80 (1H, d, J= 5.5 Hz, OH), 4.91 (1H, d, J= 5.2 Hz, OH), 6.50 (1H, d, J= 5.5 Hz, CH), 6.99 (2H, d , J = 8.5 Hz, Ar-H), 7.70 (2 H, d, J = 8.6 Hz, Ar-H), 8.12 (1H, s, N=CH). l,3,4,6-Tetra-O-acetyl-2-deoxy-2-[p-methoxybenzylidene(amino)] b-D-glucopyranose

(2) m.p: 180-182 °C),IR v_max 1750cm^-1 (C=0), 1650cm^-1 (C=N); ¾-NMR (DMSO-de. 500 MHz) d_H 1.82 (3H, s, CH₃CO), 1.99 (6H, s, 2 x CH₃CO), 2.48 (3H, s, CH₃CO), 3.45 (1H, m, H-2), 3.77 (3H, s, CH₃O), 3.95-4.04 (1H, m, H-5), 4.20-4.40 (2H, m, H-6a, H-6b), 4.97 (1H, t, J= 9.7 Hz, H-4), 5.45 (1H, t, J= 9.7 Hz, H-3), 6.07 (1H, d, J= 8.2 Hz, H-l), 6.99 (2H, d , J = 8.6 Hz, Ar-H), 7.66 (2 H, d, J = 8.6 Hz, Ar-H), 8.29 (1H, s, N=CH).

1.3.4.6-Tetra-0-Acetyl-b-D-glucosamine hydrochloride (3) m.p: 235 °C, IR v_max: 2800 cm^-1 (NH₃CI), 1750cm^-1 (C=0) ; ¾-NMR (DMSO-de. 500 MHz) d_H 2.00 (3H, s, CH₃CO), 2.04 (6H, s, 2 x CH₃CO), 2.12 (3H, s, CH₃CO), 3.55 (2H, t, J= 9.5Hz, H-2), 3.99-4.05 (2H, m, H-5, H-6), 4.20 (1H, dd, J=4.1, 12.4 Hz, H-6), 4.95 (1H, t, J= 9.6 Hz, H-4), 5.37 (1H, t, J= 9.7Hz, H-3), 5.94 (1H, d, J= 8.6 Hz, H-l), 8.87 (3H, s, NH₃CI).

1.3.4.6-Tetra-O-acetyl- b-D-glucosamine (4) m.p: 138 °C, ¾-NMR (DMSO-de. 500 MHz) d_H 1.65 (2H, s, NH₂), 2.00 (9H, s, 3 x CH₃CO), 2.12 (3H, s, CH₃CO), 2.76 (1H, dd, J= 8.8, 9.7 Hz, H-2), 3.96-3.98 (2H, m, H-5, H-6), 4.17 (1H, dd, J= 5.1, 13.0 Hz, H-6), 4.82 (1H, t, J= 9.6 Hz, H-4), 5.06 (1H, t, J= 9.6 Hz, H-3), 5.55 (1H, d, J= 8.5 Hz, H-l). l-Cyclopropyl-6-fluoro-4-oxo-7-piperazin-l-yl-quinoline-3-carboxylic acid (5) m.p: 253-255 °C, ¹H-NMR (DMSO-de. 500 MHz) d_H 1.14 (2H, bs, NCHCH_{2 )}. 1.28 (2H, q, J = 6.2 Hz, NCHCH₂). 2.87 (4H, m, 2 x NCH₂). 3.20 (4H, m, 2 x NCH₂). 3.78 (1H, m, NCHCH₂ ). 7.49 (1H, d , J= 7.4 Hz, H-8), 7.85 (1H, d , J= 13.4 Hz, H-5), 8.62 (1H, s, H-2).

7-(4-(acetyl)-piperazin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-l,4-dihydroquinoline-3- carboxylic acid (6) m.p: 253-256 °C; (R_f= 0.41); IR_vmax 1716.12 cm^-1 (C=0); ¹H-NMR (DMSO-de. 500 MHz) d_H l .15 (2H, bs, NCHCH₂). 1.27 (2H, q , J= 6.2 Hz, NCHCH₂). 2.02 (3H, s CH₃CO), 3.32 (4H, m, 2 x NCH₂). 3.63 (4H, m, 2 x NCH₂). 3.77 (1H, m, NCHCH₂). 7.53 (1H, d , J= 7.4 Hz, H- 8), 7.89 (1H, d, J= 13.2 Hz, H-5), 8.61 (1H, s, H-2), 15.02 (1H, s, COOH1.

7-(4-(acetyl)-piperazin)-l-ethyl-6-fluoro-4-oxo-l,4-dihydro-quinoline-3-carboxylic acid

(7) m.p: 295 °C; (R_f= 0.43); IRv_max 1722 (C=0); ¹H-NMR (DMSO-de. 500 MHz) d_H 1.37 (3H, t, J= 6.9 Hz, CH₂CH₃), 2.02 (3H,s, C¾CO), 3.35 (4H, m, 2 x NCH₂). 3.61 (4H, m, 2 x NCH₂). 4.54 (2H, q, J= 6.9 Hz, CH₂CH3), 7.16 (1H, s, H-8), 7.90 (1H, d, J= 12.0 Hz, H-5), 8.92 (1H, s, NCH=C), 15.27 (1H, s, COOH).

7-(l-acetylhexahydro-lH-pyrrolo[3,4-b]pyridin-6(2H)-yl)-l-cyclopropyl-6-fluoro-8- methoxy-4-oxo-l,4-dihydroquinoline-3-carboxylic acid (8)

(R_f= 0.65); IR_vmax 1746.86 cm^-1 (C=0); ¾-NMR (DMSO-d₆, 500 MHz) d_H 0.77, 1.083 and l .25 (4H, m, NCHCH₂), 1.39 (2H, m, CH₂), 1.83 (4H, m, CH₂), 2.11 (3H, s, CH3CO), 2.22 (1H, m, CH), 3.22-4.06 (9H, m, CH₂, CH, OCH₃) 7.73 (1H, d, J =13.8 Hz, H-5), 8.72 (1H, s, NCH=C), 14.96 (1H, bs, COOH).

7-(4-(acetyl)-piperazin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-N-(l,3,4,6-tetra-0-acetyl-2- deoxy-D-glucopyranose-2-yl)-l,4-dihydroquinoline-3-carboxamide (9)

7-(4-(acetyl)-piperazin-l-yl)-l-ethyl-6-fluoro-4-oxo-N-(l,3,4,6-tetra-0-acetyl-2-deoxy-D- glucopyranose-2-yl)-l,4-dihydroquinoline-3-carboxamide (10)

7-(l-acetylhexahydro-lH-pyrrolo[3,4-b]-pyridin-6(2H)-yl)-8-methoxy-4-oxo-N-(l, 3,4,6- tetra-0-acetyl-2-deoxy-D-glucopyranose-2-yl)-l-cyclopropyl-6-fluoro-l,4- dihydroquinoline-3-carboxamide (11)

7-(4-(acetyl)-piperazin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-N-(2-deoxy-D- glucopyranose-2-yl)- l,4-dihydroquinoline-3-carboxamide (12)

7-(4-(acetyl)-piperazin-l-yl)-l-ethyl-6-fluoro-4-oxo-N-(2-deoxy-D-glucopyranose-2-yl)- l,4-dihydroquinoline-3-carboxamide (13)

7-(l-acetylhexahydro-lH-pyrrolo[3,4-b]-pyridin-6(2H)-yl)-8-methoxy-4-oxo-N-(2- deoxy-D-glucopyranose-2-yl)-l-cyclopropyl-6-fluoro-l,4-dihydroquinoline-3- carboxamide (14)

Example 4

In vitro antimicrobial activity and antifungal testing

[050] Antimicrobial activities of the synthesized compounds (9) to (14) were investigated in vitro against food-borne Gram negative bacteria ((Escherichia coli 0157:H7, Salmonella entericae, ATCC 13312 and Listeria monogenesis ATCC 19115); Gram negative bacteria (Pseudomonas aeruginosa ATCC 9627; bscherichia coli NCTC 11954; Escherichia coli ATCC 8739 and a resistant clinical Escherichia coli isolate (Resistant to nalidixic acid, ciprofloxacin HC1 and norfloxacin, acquired from Biolab (Amman-Jordan))); Gram positive bacteria included (Methicillin resistant staphylococcus aureus ATCC 33591 and Methicillin sensitive staphylococcus aureus , ATCC 9253); non spore forming fungi (Candida albicans , ATCC 10231) and spore forming fungi (Aspergillus flavus ATCC 9643; Fusarium solani ATCC 36031; Stachybotrys chartarum, was provided by Professor Naresh Magan (Cramfield University, U.K.); and Penicillium chrysogenum ATCC 10106). All media were prepared under sterilized conditions in accordance to manufacturer instructions. Bacterial broth cultures stocks were cultivated in the appropriate medium at about 37 °C for about 24 hours prior to testing. 0.5 McFarland standards were used to visually approximate the concentration of cells in a suspension.

[051] Fungi conidia was attained from fungi stock previously cultivated on malt extract agar at about 30°C for 7 days. The fungi conidia were transferred into about 3 mL normal saline with a cotton swap previously immerged in Tween 20. The conidial suspension was vigorously vortexed for about 20 seconds to prevent spore clumping and then left standing at room temperature for about 15 minutes. Then, the upper layer was transferred into a sterile falcon tube and adjusted to 0.5 McFarland at 530 nm to yield 4.38 X 10⁶ sporangiospore suspension (A. flavus , 5xl0⁶; F. solani , 4xl0⁶; S. chartarum , 7x10⁶; P. chrysogenum , 10x10⁶). Mean spores count from three trials was determined using hemocytometer. The final working suspension was obtained after diluting the stock suspension by about 1 :50 malt extract broth.

[052] Minimal inhibitory concentration (“MIC”) (mg/mL) of the synthesized compounds (9) to (14) against representative bacterial strains and Candida albicans were determined using micro-broth dilution method. MIC is defined as the lowest concentration of the tested compound showing no microbial viability. Compounds (9) to (14) were first dissolved in dimethyl sulfoxide (“DMSO”) to form a stock solution of about 2 mg/mL. Then, a volume of about 100 mL was transferred into each well of 96-well plate having about 100 mL of the adequate broth, followed by two-fold serial dilution in subsequent wells. After that, a volume of about 10 mL of the microbial suspension (10⁶ strength concentration) was added to each well and incubated at a temperature of about 37°C for about 24 hours and the wells were completed to a volume of about 200 mL with adequate broth. Experiments were conducted in triplicates. Following, absorbance of light scattering at about 625 nm was measured using Epoch spectrophotometer. Ciprofloxacin HC1, Norfloxacin and Moxifloxacin HC1 were used as positive control and proper sterility and negative control DMSO samples were prepared and evaluated. Table 1 shows compounds (9) to (14) antimicrobial activity.

Table (1)

wherein

* refers to Penicillinase without Extended Spectrum b -Lactamase;

* * refers to food-poisoning bacterial strain;

* * * refers to clinical isolate of Escherichia coli resistant to quinolones and fluoroquinolones; * * * * refers to Methicillin resistant Staphylococcus aureus ;

ND means not detected;

MC means Microorganism characteristics;

G +ve means Gram positive;

G -ve means Gram negative; and

Comp means compound.

[053] The data in table (1) reveals that the 7-(4-(acetyl)-piperazin-l-yl)-l-cyclopropyl-6- fluoro-4-oxo-N-( 1 ,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose-2-yl)- l ,4- dihydroquinoline-3-carboxamide (compound 9) exhibited potential activity against Salmonella entericae, ATCC 13312 and Pseudomonas aeruginosa , ATCC 962719115 (MIC 0.0078 and 0.0078 mg/mL, respectively) whereas it exhibited comparable antibacterial activity to its parent drug against coli clinical isolate (MIC 0.1875 mg/mL, for both compounds). The potential improvement in activity against Gram negative bacteria could be attributed to the increase in the compound’s lipophilicity which might enhance bacterial uptake via the non-porin pathways.

[054] Norfloxacin derivatives (i.e. compounds (10) and (13) showed moderate activity against E. coli 0157:H7 (MIC 0.0313 mg/mL, each). Both compounds also exhibited potent activity against E. coli clinical isolate (MIC 0.0918 and 0.0469 mg/mL, respectively), comparable to norfloxacin (0.0918 mg/mL) and against Salmonella entericae, ATCC 13312 (MIC 0.0078 mg/mL, each). Similarly to the compound (9), compounds (10) and (13) exhibited no potential activity against Gram positive bacteria.

[055] MIC protocol for the spore-forming fungi was similar to bacterial protocol, however, MICs were recorded after about 48 hours of incubation at a temperature of about 30°C and absorbance was measured at about 405 nm. Compounds stock solutions had a 4 mg/mL concentration. Fluconazole were used as positive control; DMSO was used as negative control along with proper sterility and growth control. Experiments were also conducted in triplicates. Table (2) shows the antifungal activity of compounds (9) to (14).

Table (2)

[056] As depicted from Table 2, compound (9) exhibited potential antifungal activity against the spore forming P. chrysogenum which has been reported as an allergenic, toxic and pathogenic fungi (MIC <0.0039 mg/mL) more potent than the antifungal fluconazole (MIC 0.0625 mg/mL).

[057] Similarly, as depicted from table 2, compounds (10) and (13) exhibited promising antifungal activity against P. chrysogenum in comparison to fluconazole (MIC 0.0313, 0.0625 and 0.0625 mg/mL, respectively). Moreover, moxifloxacin target derivatives exhibited less antibacterial activity/no activity with respect to their positive control. But, compound (14) which is the diacetyl-glycosylated derivative exhibited strong antifungal activity in comparison to fluconazole against S. chartarum (MIC 0.0010 and 0.0313 mg/mL, respectively) andP. chrysogenum (MIC 0.0010 and 0.0625 mg/mL, respectively).

[058] Compound (10) was found to be a potent inhibitor against C. albicans in comparison to fluconazole (MIC 0.0039 and 0.1250 mg/mL, respectively). Both compounds (10 and 13) showed comparable activity against Stachybotry chartarum with regard to fluconazole (0.0313, 0.0625 and 0.0313 mg/mL, respectively).

[059] While embodiments of the present disclosure have been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various additions, omissions, and modifications can be made without departing from the spirit and scope thereof.

[060] In describing and claiming the present invention, the following terminology will be used.

[061] The singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise.

[062] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a defacto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[063] Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub range is explicitly recited. For example, a numerical range of approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly recited limits of 1 to approximately 4.5, but also to include individual numerals such as 2, 3, 4, and sub ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as“less than approximately 4.5,” which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.

[064] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

[065] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[066] As used herein, the term“about”, when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[067] As used herein, the term“and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase“A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and sub-combinations of A, B, C, and D.

Claims

CLAIMS What is claimed is:

1. A compound of the Formula (I), or a salt thereof:

Formula (I) wherein R₁ is selected from a group consisting of hydrogen; alkyl; dialkylamino; substituted or unsubstituted cycloalkyl; alkenyl; unsubstituted or substituted cycloalkenyl; alkynyl; aryl; or unsubstituted or substituted heteroaryl;

R_2, R₄ are independently selected from a group consisting of hydrogen; amino; halogen; sulfur; alkyl; dialkylamino; unsubstituted or substituted alkenyl; unsubstituted or substituted cycloalkyl; alkynyl; alkoxy preferably methoxy; aryloxy; alkanoyl; unsubstituted or substituted heteroaryl oxy; hydroxyl; cyano; or unsubstituted or substituted heteroaryl

R₃ is

R5, R6, R7, R₈ are selected from a group consisting of free hydroxy groups; fully acylated hydroxy groups; or partially acylated hydroxy groups having the group R₉-(C=O)-;

R₉ is selected from a group consisting of alkyl; substituted or unsubstituted aryl; or arylalkyl. R₁₀ is selected from a group consisting of hydrogen; amino; halogen; sulfur; alkyl; dialkylamino; unsubstituted or substituted alkenyl; unsubstituted or substituted cycloalkyl; alkynyl; alkoxy preferably methoxy; aryloxy; alkanoyl; unsubstituted or substituted heteroaryloxy; hydroxyl; cyano; or unsubstituted or substituted heteroaryl

2. The compound of claim 1, wherein the acyl groups comprise acetyl.

3. The compound of claim 1, wherein R₉ comprises phenyl; nitrophenyl; halophenyl; alkyl substituted phenyl; alkoxy; phenyl; or benzyl.

4. A method of preparing the compound of claim 1, the method comprises the steps of:

Dissolving moxifloxacin hydrochloride in CH₂C₁₂, followed by an addition of AC₂O along triethylamine to form a third mixture, warming and stirring the third mixture, followed by reducing it under pressure and adding MeOH to form a fourth mixture, and evaporating and adding Et20 to the fourth mixture to provide a third set of precipitate;

Adding ethyl chloroformate drop- wise to a solution of triethylamine and first set of precipitate in CH₂C₁₂ while stirring till an intermediate is formed, followed by an addition of 1, 3,4,6- Tetra-O-acetyl- b-D-glucosamine in drop-wise to form a fifth mixture while stirring, then extracting the fifth mixture followed by drying an organic layer and triturating a fourth precipitate; Adding ethyl chloroformate drop-wise to a solution of triethylamine and second set of precipitate in CH₂C₁₂ while stirring till an intermediate is formed, then adding 1,3,4, 6-Tetra- O-acetyl-b-D-glucosamine and CH₂C₁₂ drop-wise while stirring to form a sixth mixture, followed by extracting the sixth mixture followed by drying an organic layer and triturating a fifth precipitate;

Adding ethyl chloroformate to a solution of the third set of precipitate in CH₂C₁₂ while stirring till an intermediate is formed, then a solution of l,3,4,6-Tetra-0-acetyl-b-D-glucosamine in CH₂C₁₂ to form a seventh mixture while stirring, followed by extracting the seventh mixture and drying an organic layer and triturating a sixth precipitate;

Adding NaOMe portion-wise to a solution of second white solid precipitate to form an eighth mixture till a product is formed, followed by neutralizing, filtering, evaporating and re- dissolving the eighth mixture in water, followed by freeze drying to provide a seventh precipitate;

Adding NaOMe portion- wise to a solution of the first residue in MeOH to form a tenth mixture, followed by neutralizing the tenth mixture and evaporating a filtrate to form a fifth residue, then recrystallizing the ninth precipitate.

5. A pharmaceutical composition comprising a compound of claim 1 and/or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier/excipient.

6. The pharmaceutical composition of claim 5, for use in treating a microbial infection.

7. The pharmaceutical composition of claim 6, wherein the microbial infection is caused by one or more of the following: Escherichia coli 0157:H7, Salmonella entericae ATCC 13312, Listeria monogenesis ATCC 19115 Pseudomonas aeruginosa ATCC 9627, Escherichia coli NCTC 11954, Escherichia coli ATCC 8739, Escherichia coli isolate, Methicillin resistant staphylococcus aureus ATCC 33591, and Methicillin sensitive staphylococcus aureus, ATCC 9253, or a combination thereof.

8. The pharmaceutical composition of claim 6, wherein the microbial infection is caused by one or more of of the following Candida albicans ATCC 10231 , Aspergillus flavus ATCC 9643, Fusarium solani ATCC 36031, Stachybotrys chartarum, and Penicillium chrysogenum ATCC 10106, or a combination thereof.