A Comprehensive Review on Chemical Synthesis and Chemotherapeutic Potential of 3-Heteroaryl Fluoroquinolone Hybrids

Fluoroquinolones have been studied for more than half a century. Since the 1960s, four generations of these synthetic antibiotics have been created and successfully introduced into clinical practice. However, they are still of interest for medicinal chemistry due to the wide possibilities for chemical modification, with subsequent useful changes in the pharmacokinetics and pharmacodynamics of the initial molecules. This review summarizes the chemical and pharmacological results of fluoroquinolones hybridization by introducing different heterocyclic moieties into position 3 of the core system. It analyses the synthetic procedures and approaches to the formation of heterocycles from the fluoroquinolone carboxyl group and reveals the most convenient ways for such procedures. Further, the results of biological activity investigations for the obtained hybrid pharmacophore systems are presented. The latter revealed numerous promising molecules that can be further studied to overcome the problem of resistance to antibiotics, to find novel anticancer agents and more.


Introduction
The development of synthetic antibiotics of the quinolone group started in the 1960s. Up to the 2020s, four generations of well-known and highly efficient fluoroquinolones (FQs) have been developed [1]. However, medicinal chemistry is still concerned with investigations in this area. What are the reasons for such interest and attention?
First of all, FQs possess a wide range of antibacterial activity [2][3][4]. They are prescribed for the treatment of different diseases caused by Gram-negative [5] and Gram-positive microorganisms [6], for a tuberculosis cure [7,8] in the case of rare hospital strains [5,9]. This makes it possible to use them in the therapy of bacterial pneumonia related to COVID-19, as long as the pandemic is still in progress and the special treatment has not yet been created. Moreover, new derivatives reveal antiviral [10], antifungal [11], anticancer [12,13], antiinflammatory [14] and hypoglycemic [15] activity. That is why fluoroquinolone molecules are promising objects in the search for new biologically active compounds. In addition, the therapeutic use of FQs and other antibiotics is associated with the appearance of more dangerous and resistant strains of bacteria [16,17].
Secondly, these molecules are attractive from a chemists' point of view due to the structure that can be modified in different ways ( Figure 1). The general fluoroquinolone structure represents a bicyclic system with a substituent R 1 at the N-1 position, a substituent R 2 (often nitrogen-based heterocyclic moiety) at C-7, a benzene ring with or without R 3 substituents, a carboxyl group at the 3 position that can be modified in R 4 , an oxo group at structure that can be modified in different ways ( Figure 1). The general fluoroquinolone structure represents a bicyclic system with a substituent R1 at the N-1 position, a substituent R2 (often nitrogen-based heterocyclic moiety) at C-7, a benzene ring with or without R3 substituents, a carboxyl group at the 3 position that can be modified in R4, an oxo group at the 4 position and a fluorine atom at the 6 position [18]. Each position can be modified to change the pharmacodynamics and pharmacokinetics of the molecule, which opens broad horizons for both chemical and biological investigations [19][20][21][22]. Thirdly, we must admit that there is no way as yet to create a medicine without side effects, and fluoroquinolones are not an exception in this case. Therefore, it is necessary to improve the efficacy and safety of these medicines by synthesizing new analogues.
All these reasons together allow scientists to consider fluoroquinolones as a promising group of antibiotics, the structure of which can be modified to create novel, more active and safe medicines, with antibacterial properties and other valuable and needed types of biological activity.
Regarding the core systems that are subjected to different types of structural modifications, the first place is taken by the representatives of the second FQ generation, namely, ciprofloxacin (CF), norfloxacin (NF) and ofloxacin (OF) (Figure 2). Levofloxacin (LF) from the 3rd generation is also popular among medicinal chemists. As the second significant groups, 2nd-generation pefloxacin (PF) and 4th-generation enrofloxacin (EF) are presented. A few papers describe novel potent derivatives of 2nd-generation fleroxacin (FL), lomefloxacin (LoF), rufloxacin (RF) and 4th-generation gatifloxacin (GF) and moxifloxacin (MF).
For the 2nd-generation molecules, the most common way is to perform modifications for searching novel antimicrobials. It was backed by previous investigations and has already provided us with two more generations of FQs. All of them are effective and widely used against both Gram-negative and Gram-positive strains of bacteria, with increasing activity spectrum from generation to generation, as stated above, including multidrug-resistant bacteria. For instance, ciprofloxacin is one of the most often prescribed antibiotics in the world. Still, it is possible to improve the effectiveness safety profile as well as activity spectrum, which will be discussed further. As for other initial systems, they are often used for the expansion of the pharmacological profile, as they already have quite impressive antibacterial properties. Therefore, they are often investigated, for example, as anticancer agents.
In the current review, the results of investigations on the structural modification of FQs by introducing heterocyclic moieties into position 3 of the core system are analyzed and summarized, the approaches to the formation of heterocycles from the fluoroquinolone carboxyl group are shown, and the results of the studies of the biological activity of the obtained hybrid pharmacophore systems are presented. Thirdly, we must admit that there is no way as yet to create a medicine without side effects, and fluoroquinolones are not an exception in this case. Therefore, it is necessary to improve the efficacy and safety of these medicines by synthesizing new analogues.
All these reasons together allow scientists to consider fluoroquinolones as a promising group of antibiotics, the structure of which can be modified to create novel, more active and safe medicines, with antibacterial properties and other valuable and needed types of biological activity.
Regarding the core systems that are subjected to different types of structural modifications, the first place is taken by the representatives of the second FQ generation, namely, ciprofloxacin (CF), norfloxacin (NF) and ofloxacin (OF) (Figure 2). Levofloxacin (LF) from the 3rd generation is also popular among medicinal chemists. As the second significant groups, 2nd-generation pefloxacin (PF) and 4th-generation enrofloxacin (EF) are presented. A few papers describe novel potent derivatives of 2nd-generation fleroxacin (FL), lomefloxacin (LoF), rufloxacin (RF) and 4th-generation gatifloxacin (GF) and moxifloxacin (MF).
For the 2nd-generation molecules, the most common way is to perform modifications for searching novel antimicrobials. It was backed by previous investigations and has already provided us with two more generations of FQs. All of them are effective and widely used against both Gram-negative and Gram-positive strains of bacteria, with increasing activity spectrum from generation to generation, as stated above, including multidrugresistant bacteria. For instance, ciprofloxacin is one of the most often prescribed antibiotics in the world. Still, it is possible to improve the effectiveness safety profile as well as activity spectrum, which will be discussed further. As for other initial systems, they are often used for the expansion of the pharmacological profile, as they already have quite impressive antibacterial properties. Therefore, they are often investigated, for example, as anticancer agents.
In the current review, the results of investigations on the structural modification of FQs by introducing heterocyclic moieties into position 3 of the core system are analyzed and summarized, the approaches to the formation of heterocycles from the fluoroquinolone carboxyl group are shown, and the results of the studies of the biological activity of the obtained hybrid pharmacophore systems are presented.
The C-3 carboxylic acid group in a fluoroquinolone core can be easily modified, especially with different heterocycles via substitution and cyclization reactions that can enhance or change the basic activity or contribute to the positive effects on pharmacokinetics of the initial molecule.
However, this position did not gain enough attention, as C-7 modifications of FQ have been the most popular in recent years. Still, there are valuable investigations in this area, and their summary is presented in Figure 3 and Table 1. The text in this section provides both original and generalized reaction schemes. Details on synthetic procedures from the literature data are provided in the Supplementary Materials.  pecially with different heterocycles via substitution and cyclization reactions that can enhance or change the basic activity or contribute to the positive effects on pharmacokinetics of the initial molecule. However, this position did not gain enough attention, as C-7 modifications of FQ have been the most popular in recent years. Still, there are valuable investigations in this area, and their summary is presented in Figure 3 and Table 1. The text in this section provides both original and generalized reaction schemes. Details on synthetic procedures from the literature data are provided in the Supplementary Materials.  To prepare the review, we used the chemical and bibliographic information SciFinder database (https://scifinder-n.cas.org/ (accessed on 13 December 2022)) and PubMed database (https://pubmed.ncbi.nlm.nih.gov/, accessed on 13 December 2022). Marvin was used for drawing, displaying and characterizing chemical structures, substructures and reactions, Marvin 23.1.0, 2023, ChemAxon (https://chemaxon.com/, accessed on 1 February 2023). DataWarrior was used for drawing diagrams, OSIRIS Da-taWarrior 5.5.0, 2021 (https://openmolecules.org/datawarrior/, accessed on 1 February 2023). To prepare the review, we used the chemical and bibliographic information SciFinder database (https://scifinder-n.cas.org/ (accessed on 13 December 2022)) and PubMed database (https://pubmed.ncbi.nlm.nih.gov/, accessed on 13 December 2022). Marvin was used for drawing, displaying and characterizing chemical structures, substructures and reactions, Marvin 23.1.0, 2023, ChemAxon (https://chemaxon.com/, accessed on 1 February 2023). DataWarrior was used for drawing diagrams, OSIRIS DataWarrior 5.5.0, 2021 (https://openmolecules.org/datawarrior/, accessed on 1 February 2023).

Modification of FQs with Five-Membered Heterocycles
The most pronounced contribution in the investigated area was made by the scientists who worked with the substitution of the C-3 carboxylic acid group with subsequent cyclization, obtaining a wide range of 3-heteroaryl hybrids of initial FQs.
On the one hand, there is evidence of the importance of carboxylic acid group presence for the manifestation of the antimicrobial activity of fluoroquinolones. On the other hand, small heterocyclic moieties (five-membered heterocycles) have already proven themselves to be an effective modification tool for the piperazine ring of such well-known antibiotics as norfloxacin and ciprofloxacin [23,24].
They can be introduced into C-3 via the formation of different functional derivatives of carboxylic acids that are presented in Scheme 1. For instance, alkylation offers an opportunity to further obtain hydrazine derivatives . The carboxylic group can be easily turned into an amide group with the subsequent formation of nitriles [56]. Among other common derivatives are acid halides [57,58] and thiosemicarbazides [41,59]. All of them can be prepared easily and used for further cyclization. In addition, several works Antibiotics 2023, 12, 625 5 of 36 describe 3-acetyl derivatives obtained directly via the Gould-Jacobs reaction [60][61][62] and then converted into bromoacetyl derivatives; the formation of amines [63] from nitro derivatives is also an interesting case.
proven themselves to be an effective modification tool for the piperazine ring of such well-known antibiotics as norfloxacin and ciprofloxacin [23,24].
They can be introduced into C-3 via the formation of different functional derivatives of carboxylic acids that are presented in Scheme 1. For instance, alkylation offers an opportunity to further obtain hydrazine derivatives . The carboxylic group can be easily turned into an amide group with the subsequent formation of nitriles [56]. Among other common derivatives are acid halides [57,58] and thiosemicarbazides [41,59]. All of them can be prepared easily and used for further cyclization. In addition, several works describe 3-acetyl derivatives obtained directly via the Gould-Jacobs reaction [60][61][62] and then converted into bromoacetyl derivatives; the formation of amines [63] from nitro derivatives is also an interesting case. Scheme 1. General approaches to the formation of functional derivatives of FQs .
The basic structure of these derivatives is similar, differing only by one heteroatom in the ring. As a result, similar approaches are usually utilized for their synthesis, and the possibilities of converting one heterocycle into another are also used. Scheme 1. General approaches to the formation of functional derivatives of FQs .
The basic structure of these derivatives is similar, differing only by one heteroatom in the ring. As a result, similar approaches are usually utilized for their synthesis, and the possibilities of converting one heterocycle into another are also used.
A variation of derivatives 1.1 are mercapto/thio substituted compounds 1.2. A popular synthesis method for them consists of the formation of oxadiazolthiones via treatment of synthesized hydrazides with carbon disulfide (Scheme 4). The corresponding amino derivatives are obtained by the modification of the carboxyl group of fluoroquinolones by the action of semicarbazide in the presence of a cyclizing agent [25]. Scheme 4 also summarizes methods for the introduction of 1,2,4-triazole fragments into FQ molecules. One of them is the conversion of oxadiazoles, as mentioned above. For example, the reaction of compounds 1.3 with hydrazine hydrate yielded FQ containing a 3-thio-4-amino-1,2,4-triazole fragment in the 3 position (CT 1.3B) [25,49,50,53,54]. narily obtained hydrazides with aliphatic or aromatic acids in similar condition [29,35,37,52]. The resulting oxadiazole hybrids are often transformed into other derivatives. When interacting with various amino compounds, the oxadiazole fragment is modified into 1,2,4-triazole fragment. An example of such a modification is presented in Scheme 3 [64] Target products CT 1.3 are synthesized by refluxing the obtained oxadiazole hybrid with 2,3,4,5-tetrafluorobenzohydrazide in dry pyridine. Scheme 3. Conversion of oxadiazole hybrids into 1,2,4-triazoles [64].
A variation of derivatives 1.1 are mercapto/thio substituted compounds 1.2. A pop ular synthesis method for them consists of the formation of oxadiazolthiones via treat ment of synthesized hydrazides with carbon disulfide (Scheme 4). The corresponding amino derivatives are obtained by the modification of the carboxyl group of fluoroquin olones by the action of semicarbazide in the presence of a cyclizing agent [25]. Scheme 4 also summarizes methods for the introduction of 1,2,4-triazole fragment into FQ molecules. One of them is the conversion of oxadiazoles, as mentioned above. Fo example, the reaction of compounds 1.3 with hydrazine hydrate yielded FQ containing 3-thio-4-amino-1,2,4-triazole fragment in the 3 position (CT 1.3B) [25,49,50,53,54]. Scheme 3. Conversion of oxadiazole hybrids into 1,2,4-triazoles [64].
A variation of derivatives 1.1 are mercapto/thio substituted compounds 1.2. A popular synthesis method for them consists of the formation of oxadiazolthiones via treatment of synthesized hydrazides with carbon disulfide (Scheme 4). The corresponding amino derivatives are obtained by the modification of the carboxyl group of fluoroquinolones by the action of semicarbazide in the presence of a cyclizing agent [25]. example, the reaction of compounds 1.3 with hydrazine hydrate yielded FQ containing a 3-thio-4-amino-1,2,4-triazole fragment in the 3 position (CT 1.3B) [25,49,50,53,54].
For example, Lihumis H.S. et al. [25] described a modification of the carboxyl group of ofloxacin using all the hybrids shown in the scheme.
In the literature data, there are various options for implementing the last reaction fo hybrids of FQ with s-triazoles and oxadiazoles. For example, the use of methyl ketones in the Mannich reaction allowed scientists [46] to synthesize new compounds based on ofloxacin (Scheme 6). Scheme 6. Synthesis of new ofloxacin derivatives [46]. Scheme 6. Synthesis of new ofloxacin derivatives [46]. Schiff bases synthesized from 3-mercaptotriazole-modified levofloxacin were introduced by the authors [71] into the Mannich reaction with formaldehyde and phenols (Scheme 7). It has been proven by spectral methods that the reaction conditions proposed by them favor the reaction on the aryl ring with the formation of polymeric products.
Antibiotics 2023, 12, x FOR PEER REVIEW 9 of 3 Schiff bases synthesized from 3-mercaptotriazole-modified levofloxacin were in troduced by the authors [71] into the Mannich reaction with formaldehyde and phenol (Scheme 7). It has been proven by spectral methods that the reaction conditions propose by them favor the reaction on the aryl ring with the formation of polymeric products. The literature data also describe the preparation of the corresponding hydrazid from the product of alkylation of mercaptooxadiazole with chloroacetic ester and it transformation into a series of arylhydrazones (Scheme 8). For such transformations, th authors used ofloxacin [27,60] and pefloxacin [36]. Furthermore, to study the effect on antitumor activity, the synthesis of bioisosteri 3-hydrazinotriazole derivatives obtained as a result of sequential modification of th carboxyl group of ofloxacin was carried out [60]. The compounds were also used i interactions with arylaldehydes (Scheme 9). The literature data also describe the preparation of the corresponding hydrazide from the product of alkylation of mercaptooxadiazole with chloroacetic ester and its transformation into a series of arylhydrazones (Scheme 8). For such transformations, the authors used ofloxacin [27,60] and pefloxacin [36].
Antibiotics 2023, 12, x FOR PEER REVIEW 9 of Another direction of ofloxacin modification was the introduction of th 3-mercapto-s-triazole moiety into its molecule (via the reaction with potassium thiocyanate and subsequent cyclization under the action of alkali) and further alkylatio with bromoacetophenones. The resulting alkyl derivatives were converted to th corresponding thiosemicarbazones (Scheme 10) [41]. The previously synthesized N-butyl analog of FQs with a morpholine fragment a position 7 and a GHQ168 carboxyl group modified into benzylamide was used by scien tists [59] for further chemical modification (Scheme 11). Based on the starting acid, th corresponding heterocyclic derivatives were synthesized-1,3,4-thiadiazole an 1,2,4-triazole; the modification of the 1,2,4-triazole derivative by maleimides is also pre sented in this article. 2-Aminothiazole has been identified by scientists as one of the key pharmacophore in the structure of antibacterial medicines [61,62,83]. A plausible method for the con struction of a thiazole ring takes place via Hantzsch thiazole synthesis; the thiazole de rivative was formed by condensation of initial α-haloketone and thiourea (Scheme 12 [61,62,83]. The previously synthesized N-butyl analog of FQs with a morpholine fragment at position 7 and a GHQ168 carboxyl group modified into benzylamide was used by scientists [59] for further chemical modification (Scheme 11). Based on the starting acid, the corresponding heterocyclic derivatives were synthesized-1,3,4-thiadiazole and 1,2,4-triazole; the modification of the 1,2,4-triazole derivative by maleimides is also presented in this article. Another direction of ofloxacin modification was the introduction of th 3-mercapto-s-triazole moiety into its molecule (via the reaction with potassium thiocyanate and subsequent cyclization under the action of alkali) and further alkylation with bromoacetophenones. The resulting alkyl derivatives were converted to th corresponding thiosemicarbazones (Scheme 10) [41]. The previously synthesized N-butyl analog of FQs with a morpholine fragment a position 7 and a GHQ168 carboxyl group modified into benzylamide was used by scien tists [59] for further chemical modification (Scheme 11). Based on the starting acid, th corresponding heterocyclic derivatives were synthesized-1,3,4-thiadiazole and 1,2,4-triazole; the modification of the 1,2,4-triazole derivative by maleimides is also pre sented in this article. 2-Aminothiazole has been identified by scientists as one of the key pharmacophore in the structure of antibacterial medicines [61,62,83]. A plausible method for the con struction of a thiazole ring takes place via Hantzsch thiazole synthesis; the thiazole de rivative was formed by condensation of initial α-haloketone and thiourea (Scheme 12 [61,62,83]. 2-Aminothiazole has been identified by scientists as one of the key pharmacophores in the structure of antibacterial medicines [61,62,83]. A plausible method for the construction of a thiazole ring takes place via Hantzsch thiazole synthesis; the thiazole derivative was formed by condensation of initial α-haloketone and thiourea (Scheme 12) [61,62,83]. 2-Aminothiazole has been identified by scientists as one of the key pharmacophores in the structure of antibacterial medicines [61,62,83]. A plausible method for the construction of a thiazole ring takes place via Hantzsch thiazole synthesis; the thiazole derivative was formed by condensation of initial α-haloketone and thiourea (Scheme 12) [61,62,83]. Interestingly, an attempt to introduce a piperazine or a similar fragment into position 7 in order to obtain a norfloxacin analogue resulted in a mixture of amination products. The authors [62] synthesized 2-aminothiazole hybrids with each of the resulting products after their separation.
A few articles describe the incorporation of a tetrazole ring in a fluoroquinolone molecule [79,90]. Namely, the conversion of 3-amino derivatives into tetrazoles is possible (Scheme 13) [63]. Interestingly, an attempt to introduce a piperazine or a similar fragment into position 7 in order to obtain a norfloxacin analogue resulted in a mixture of amination products. The authors [62] synthesized 2-aminothiazole hybrids with each of th resulting products after their separation.
A few articles describe the incorporation of a tetrazole ring in a fluoroquinolon molecule [79,90]. Namely, the conversion of 3-amino derivatives into tetrazoles is possi ble (Scheme 13) [63]. The authors [63] used Chitosan-supported Cu nanoparticles to modify the carboxy group of 1-alkyl-6,7-dihalo-4-oxo-quinoline-3-carboxylic acids into a nitro group. Afte nitration, the piperidine fragment was successfully introduced into the molecule. T restore the nitro group to the amino group, tin chloride was used. The amino derivativ reacted with sodium azide and triethylorthoformate, as a result of which tetrazol hybrids of fluoroquinolones were obtained.
Another approach for the formation of a tetrazole ring based on the carboxyl group in the 3d position of tricyclic fluoroquinolones was used in the study [56]. Preliminarily synthesized primary amides were converted into nitriles by the action of POCl3. Th cyclization of the latter to tetrazoles was carried out by the action of hydroxylamine o sodium azide/zinc chloride (Scheme 14). The authors [63] used Chitosan-supported Cu nanoparticles to modify the carboxyl group of 1-alkyl-6,7-dihalo-4-oxo-quinoline-3-carboxylic acids into a nitro group. After nitration, the piperidine fragment was successfully introduced into the molecule. To restore the nitro group to the amino group, tin chloride was used. The amino derivative reacted with sodium azide and triethylorthoformate, as a result of which tetrazole hybrids of fluoroquinolones were obtained.
Another approach for the formation of a tetrazole ring based on the carboxyl group in the 3d position of tricyclic fluoroquinolones was used in the study [56]. Preliminarily synthesized primary amides were converted into nitriles by the action of POCl 3 . The cyclization of the latter to tetrazoles was carried out by the action of hydroxylamine or sodium azide/zinc chloride (Scheme 14). A similar approach-the action of azide on the corresponding nitrile-was used by the authors of [57] to introduce the tetrazole fragment (Scheme 15). In the presented study, the authors synthesized various bioisostere hybrids of FQs for subsequent comparison of their pharmacological activity. Derivatives of 1,3,4-oxadiazole obtained via the interaction of the acid chloride of the initial acid with diphenylacetic acid hydrazide or isomeric derivatives of 1,2,4-oxadiazole were synthesized using amidoxime. Synthesis of 1,3-oxazole hybrids was carried out using two methods, with acid chloride or nitrile as initial compounds.

Scheme 14. Tetrazole ring introduction via nitrile formation [56].
A similar approach-the action of azide on the corresponding nitrile-was used by the authors of [57] to introduce the tetrazole fragment (Scheme 15). In the presented study, the authors synthesized various bioisostere hybrids of FQs for subsequent comparison of their pharmacological activity. Derivatives of 1,3,4-oxadiazole obtained via the interaction of the acid chloride of the initial acid with diphenylacetic acid hydrazide or isomeric derivatives of 1,2,4-oxadiazole were synthesized using amidoxime. Synthesis of 1,3-oxazole hybrids was carried out using two methods, with acid chloride or nitrile as initial compounds.

Modification of the C-3 Carboxylic Acid Group with Fused Heterocycles
Among the published investigations, there are those related to fused heterocyles as C-3 substituents in the initial FQs. They utilize similar synthetic approaches, mainly, the formation of a triazole ring with its subsequent substitution and cyclocondensation or the reacton with o-aminophenol and its analogues.
For example, 12 novel C-3 thiazolotriazole derivatives were synthesized from levofloxacin by the authors of [48,88]

Modification of the C-3 Carboxylic Acid Group with Fused Heterocycles
Among the published investigations, there are those related to fused heterocyles as C-3 substituents in the initial FQs. They utilize similar synthetic approaches, mainly, the formation of a triazole ring with its subsequent substitution and cyclocondensation or the reacton with o-aminophenol and its analogues.
For example, 12 novel C-3 thiazolotriazole derivatives were synthesized from levofloxacin by the authors of [48,88]

Modification of the C-3 Carboxylic Acid Group with Fused Heterocycles
Among the published investigations, there are those related to fused heterocyles a C-3 substituents in the initial FQs. They utilize similar synthetic approaches, mainly, th formation of a triazole ring with its subsequent substitution and cyclocondensation or th reacton with o-aminophenol and its analogues.
For example, 12 novel C-3 thiazolotriazole derivatives were synthesized from levofloxacin by the authors of [48,88] according to Scheme 16.
A few more articles were devoted to benzimidazoles, benzoxazoles and benzothia zoles as fused derivatives of fluoroquinolones. First and foremost, a thorough molecula modelling study [93] revealed the promising biologically active molecules among thes compounds.
A few more articles were devoted to benzimidazoles, benzoxazoles and benzothiazoles as fused derivatives of fluoroquinolones. First and foremost, a thorough molecular modelling study [93] revealed the promising biologically active molecules among these compounds.
A series of novel ciprofloxacin and levofloxacin derivatives were synthesized via condensation following the Mannich reaction (Scheme 20) [94].
A few more articles were devoted to benzimidazoles, benzoxazoles and benzothia zoles as fused derivatives of fluoroquinolones. First and foremost, a thorough molecular modelling study [93] revealed the promising biologically active molecules among these compounds.
A series of novel ciprofloxacin and levofloxacin derivatives were synthesized via condensation following the Mannich reaction (Scheme 20) [94]. Similar cyclization of a quinolone derivative with o-phenylenediamin 2-aminophenol or 2-aminobenzenethiol in the presence of polyphosphoric acid was re ported by the authors of [95,96].
The introduction of the quinazoline fragment was carried out as a result of the in teraction of norfloxacin and ciprofloxacin acid chlorides with 5-chloroanthranilic aci (Scheme 23). The resulting benzoxazine reacted wit 4-(6-methylbenzo[d]thiazol-2-yl)aniline with the formation of the corresponding produc [58]. Similar cyclization of a quinolone derivative with o-phenylenediamine, 2-aminophenol or 2-aminobenzenethiol in the presence of polyphosphoric acid was reported by the authors of [95,96].
The introduction of the quinazoline fragment was carried out as a result of the interaction of norfloxacin and ciprofloxacin acid chlorides with 5-chloroanthranilic acid (Scheme 23). The resulting benzoxazine reacted with 4-(6-methylbenzo[d]thiazol-2-yl)aniline with the formation of the corresponding product [58].
The introduction of the quinazoline fragment was carried out as a result of the interaction of norfloxacin and ciprofloxacin acid chlorides with 5-chloroanthranilic acid (Scheme 23). The resulting benzoxazine reacted with 4-(6-methylbenzo[d]thiazol-2-yl)aniline with the formation of the corresponding product [58].  At last, esters of 1-

Bis-Fluoroquinolones and FQs with Two Heterocyclic Moieties as a Special Case in the Investigated Area
An appealing idea in the synthesis of bis-fluoroquinolones linked via the heterocyclic moiety in the 3d position appeared to be common among scientific publications related to the investigated topic. Such novel molecules were mostly studied as antitumor agents and revealed promising results.
First of all, synthesis of different symmetrical and non-symmetrical bis-fluoroquinolones via the five-membered heterocycle as a link was described. For instance, the carboxylic acid groups of two molecules of fluoroquinolones were successfully replaced with an 1,3,4-oxadiazole ring, and a series of the C3/C3 bis-fluoroquinolones as their HCl salts were obtained (Scheme 25) [52].

Investigated Area
An appealing idea in the synthesis of bis-fluoroquinolones linked via the heteroc clic moiety in the 3d position appeared to be common among scientific publications r lated to the investigated topic. Such novel molecules were mostly studied as antitum agents and revealed promising results.
First of all, synthesis of different symmetrical and non-symmetric bis-fluoroquinolones via the five-membered heterocycle as a link was described. For i stance, the carboxylic acid groups of two molecules of fluoroquinolones were succes fully replaced with an 1,3,4-oxadiazole ring, and a series of the C3/C bis-fluoroquinolones as their HCl salts were obtained (Scheme 25) [52].
Another study described the reaction of norfloxacin with hydrazine hydrate wi the formation of a hydrazide intermediate, followed by cyclization with the second no floxacin molecule in the presence of POCl3 to target bis-fluoroquinolone in a modera yield [97].

Investigated Area
An appealing idea in the synthesis of bis-fluoroquinolones linked via the heterocyclic moiety in the 3d position appeared to be common among scientific publications related to the investigated topic. Such novel molecules were mostly studied as antitumor agents and revealed promising results.
First of all, synthesis of different symmetrical and non-symmetrical bis-fluoroquinolones via the five-membered heterocycle as a link was described. For instance, the carboxylic acid groups of two molecules of fluoroquinolones were successfully replaced with an 1,3,4-oxadiazole ring, and a series of the C3/C3 bis-fluoroquinolones as their HCl salts were obtained (Scheme 25) [52].
Another study described the reaction of norfloxacin with hydrazine hydrate with the formation of a hydrazide intermediate, followed by cyclization with the second norfloxacin molecule in the presence of POCl3 to target bis-fluoroquinolone in a moderate yield [97].
In addition, there are bis-fluoroquinolones created via the fused heterocyclic link, as ref. [53] describes, taking, as an example, the [1,2,4]-triazolo [3,4-b] [1,3,4]-thiadiazole core. In this study, initial cipro-and levofloxacin molecules were substituted with hydrazine hydrate and then were subjected to acyclo-condensation with carbon disulfide in the presence of an excess alkali-ethanol solution (Scheme 30). Obtained oxadiazolethiols were converted into the amino s-triazolethiols using hydrazine hydrate. The following condensation into the target compounds was successful only in the presence of POCl3.

Biological Activity of the 3-Heteroaryl FQ Hybrids
Investigations of the biological activity of the FQ hybrids led to bifurcation and revealed two new potentialities, namely, creation of new potent antimicrobials and novel anticancer medicines. These two groups appear to be the most prominent for now. Still, there are a few more cases of other activity types that are also promising.

Novel FQ Hybrids as Antimicrobials and Antiviral Medicines
Among the core FQ molecules that served for the development and research of new potent antimicrobials, the first place is taken by norfloxacin. The carboxylic group of the initial compound was modified mainly with five-membered heterocyles with further investigation of antibacterial, antifungal and antiviral activities.
For example, a series of 1,3,4-oxadiazoles containing FQ derivatives was synthesized and screened for antibacterial and antimycobacterial properties in ref. [31] (Figure 5). The disk diffusion method revealed potent antibacterial activities against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae. In addition, the obtained norfloxacin derivatives showed antimycobacterial activity against Mycobacterium smegmatis H37Rv with minimal inhibitory concentrations

Biological Activity of the 3-Heteroaryl FQ Hybrids
Investigations of the biological activity of the FQ hybrids led to bifurcation and revealed two new potentialities, namely, creation of new potent antimicrobials and novel anticancer medicines. These two groups appear to be the most prominent for now. Still, there are a few more cases of other activity types that are also promising.

Novel FQ Hybrids as Antimicrobials and Antiviral Medicines
Among the core FQ molecules that served for the development and research of new potent antimicrobials, the first place is taken by norfloxacin. The carboxylic group of the initial compound was modified mainly with five-membered heterocyles with further investigation of antibacterial, antifungal and antiviral activities.
For example, a series of 1,3,4-oxadiazoles containing FQ derivatives was synthesized and screened for antibacterial and antimycobacterial properties in ref. [31] (Figure 5). The disk diffusion method revealed potent antibacterial activities against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae. In addition, the obtained norfloxacin derivatives showed antimycobacterial activity against Mycobacterium smegmatis H37Rv with minimal inhibitory concentrations (MICs) of 22.35, 16.20 and 20.28 µg/mL. The authors also studied absorption, distribution, metabolism and excretion (ADME) properties and proved the promising pharmacokinetic properties and drug-likeness for the obtained compounds.
It is worth noting that most of the investigations in this area are based on in silico studies of drug-likeness and binding properties. Thus, the authors of [26] described molecular docking investigations (AutoDock Tools-1.5.6) against the receptor GlcN-6P (2VF5) that revealed good binding affinities for synthesized norfloxacin derivatives. In this work, the core molecule was modified with 1,3,4-oxadiazole, thiazolidin-4-one, 1,3,4oxadiazoline, 1,2,4-triazole and 1,3,4-thiadiazole rings. The antimicrobial assessment via disk diffusion and serial dilution methods showed higher activity than for norfloxacin against S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, K. pneumoniae, Pseudomonas aeruginosa, E. coli and Proteus mirabilis.  It is worth noting that most of the investigations in this area are based on in silico studies of drug-likeness and binding properties. Thus, the authors of [26] described molecular docking investigations (AutoDock Tools-1.5.6) against the receptor GlcN-6P (2VF5) that revealed good binding affinities for synthesized norfloxacin derivatives. In this work, the core molecule was modified with 1,3,4-oxadiazole, thiazolidin-4-one, 1,3,4-oxadiazoline, 1,2,4-triazole and 1,3,4-thiadiazole rings. The antimicrobial assessment via disk diffusion and serial dilution methods showed higher activity than for norfloxacin against S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus luteus, K. pneumoniae, Pseudomonas aeruginosa, E. coli and Proteus mirabilis.
1,2,4-Triazole and 1,3,4-oxadiazole norfloxacin hybrids with suitable druglike, antibacterial and antifungal properties were successfully obtained by researches [30]. Especially pronounced was the activity against S. pneumoniae, with minimum inhibitory concentrations of 0.89 and 0.96 mg/mL and minimal bactericidal concentrations of 2.95 and 2.80 mg/mL. Combined with simple synthetic approaches, such results are promising for further investigations.
Interestingly, similar oxadiazole norfloxacin and ciprofloxacin derivatives showed good antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) strains, combined with promising antifungal activity against fungi (Saccharomyces cerevisiae) in comparison with reference drugs ciprofloxacin and fluconazole in the study [29].
A few interesting works were devoted to an exploration of the antimicrobial potential of aminothiazolyl hybrids of norfloxacin. It was proved that the 2-aminothiazole fragment at the 3-position of the quinolone core plays an important role in exerting antibacterial activity. For instance, in this case, the antibacterial activity investigation revealed higher values in comparison with the reference drugs against methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus 25923, with MIC values of 0.009 and 0.017 mM [60]. Figure 6 shows the detailed activity and concentrations of the synthesized compounds. 1,2,4-Triazole and 1,3,4-oxadiazole norfloxacin hybrids with suitable druglike, antibacterial and antifungal properties were successfully obtained by researches [30]. Especially pronounced was the activity against S. pneumoniae, with minimum inhibitory concentrations of 0.89 and 0.96 mg/mL and minimal bactericidal concentrations of 2.95 and 2.80 mg/mL. Combined with simple synthetic approaches, such results are promising for further investigations.
Interestingly, similar oxadiazole norfloxacin and ciprofloxacin derivatives showed good antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) strains, combined with promising antifungal activity against fungi (Saccharomyces cerevisiae) in comparison with reference drugs ciprofloxacin and fluconazole in the study [29].
A few interesting works were devoted to an exploration of the antimicrobial potential of aminothiazolyl hybrids of norfloxacin. It was proved that the 2-aminothiazole fragment at the 3-position of the quinolone core plays an important role in exerting antibacterial activity. For instance, in this case, the antibacterial activity investigation revealed higher values in comparison with the reference drugs against methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus 25923, with MIC values of 0.009 and 0.017 mM [60]. Figure 6 shows the detailed activity and concentrations of the synthesized compounds.
Another series of aminothiazolyl norfloxacin analogs was synthesized by the authors of [71] and was screened for antimicrobial properties. Most of the compounds synthesized were superior to reference drug inhibitory efficiencies against K. pneumoniae and Candida albicans, with MIC values of 0.005 and 0.010 mM. Furthermore, these compounds revealed better antibacterial activity against S. aureus ATCC 29213 and methicillin-resistant strains (Figure 7).
In addition, investigations of the inhibitory activity against the DNA gyrase from E. coli showed that aminothiazolyl norfloxacin analogs have good inhibitory potency for DNA gyrase (IC 50 1 4 16.7 mM), which was more effective than the reference drug norfloxacin (IC 50 1 4 18.6 mM). Altogether, the authors concluded that the replacement of the carboxyl group with the weak basic 2-aminothiazole moiety provides a similar antibacterial mechanism to norfloxacin by targeting DNA gyrase. Another series of aminothiazolyl norfloxacin analogs was synthesized by the authors of [71] and was screened for antimicrobial properties. Most of the compounds synthesized were superior to reference drug inhibitory efficiencies against K. pneumoniae and Candida albicans, with MIC values of 0.005 and 0.010 mM. Furthermore, these compounds revealed better antibacterial activity against S. aureus ATCC 29213 and methicillin-resistant strains (Figure 7).
In addition, investigations of the inhibitory activity against the DNA gyrase from E. coli showed that aminothiazolyl norfloxacin analogs have good inhibitory potency for DNA gyrase (IC50 ¼ 16.7 mM), which was more effective than the reference drug norfloxacin (IC50 ¼ 18.6 mM). Altogether, the authors concluded that the replacement of the carboxyl group with the weak basic 2-aminothiazole moiety provides a similar antibacterial mechanism to norfloxacin by targeting DNA gyrase.
They additionally proved their hypothesis via docking studies with the topoisomerase IV-DNA complex and gyrase-DNA complex. Among the found interactions, the sulfur atom in the 2-aminothiazole moiety participated in the non-covalent coordination with ARG-136 residue via hydrogen bond formation that is favorable for stabilizing the compound-enzyme-DNA supramolecular complex, which further accounted for the good inhibitory efficiency against the tested strains. They additionally proved their hypothesis via docking studies with the topoisomerase IV-DNA complex and gyrase-DNA complex. Among the found interactions, the sulfur atom in the 2-aminothiazole moiety participated in the non-covalent coordination with ARG-136 residue via hydrogen bond formation that is favorable for stabilizing the compoundenzyme-DNA supramolecular complex, which further accounted for the good inhibitory efficiency against the tested strains.
A tertrazolyl moiety appeared to be one more promising bioisostere that was introduced in the C-3 of norfloxacin and ciprofloxacin [63]. The authors conducted docking studies using a Molegro Virtual Docker (MVD) to prove their idea. The tested compounds showed a very similar binding mode with DNA gyrase compared to the co-crystallized ciprofloxacin. The tetrazole formed three hydrogen bonds with Ser1084 instead of one, which is formed by ciprofloxacin. The bond lengths of the three hydrogen bonds were 1.9, 2.1 and 3.1Å. The nitrogen of the piperazine formed a hydrogen bond with base pair DNA backbone DT4. The MolDock score (kcal mol −1 ) and the Rerank Score were −123.54 and −74.67, respectively. Overall, the tested compound revealed a similar manner to ciprofloxacin, with additional hydrogen bonds related to the 3-tetrazole scaffold, supporting the molecular design. A tertrazolyl moiety appeared to be one more promising bioisostere that was introduced in the C-3 of norfloxacin and ciprofloxacin [63]. The authors conducted docking studies using a Molegro Virtual Docker (MVD) to prove their idea. The tested compounds showed a very similar binding mode with DNA gyrase compared to the co-crystallized ciprofloxacin. The tetrazole formed three hydrogen bonds with Ser1084 instead of one, which is formed by ciprofloxacin. The bond lengths of the three hydrogen bonds were 1.9, 2.1 and 3.1Å. The nitrogen of the piperazine formed a hydrogen bond with base pair DNA backbone DT4. The MolDock score (kcal mol −1 ) and the Rerank Score were −123.54 and −74.67, respectively. Overall, the tested compound revealed a similar manner to ciprofloxacin, with additional hydrogen bonds related to the 3-tetrazole scaffold, supporting the molecular design.
In addition, this modification led to optimization of the solubility profile of the initial molecules. As for the antibacterial activity, the inhibition zones for S. aureus and MRSA were from 12.5 to 25 mM. Several derivatives revealed activity at 12.5 and 25 mM, respectively, against Salmonella typhi, while reference drugs were active at 100 mM. Moreover, high activity against Vibrio cholerae and E. coli was observed.
A few more investigations described modifications of ciprofloxacin. Thus, ref.
[32] evidenced the synthesis and evaluation of antibacterial activity of ciprofloxacin C3 hybrids with isatins, phthalimides and oxadiazoles. In vitro antibacterial evaluation was made using disk diffusion and serial dilution methods, and antitubercular activity was measured using the Lowenstein-Jensen (LJ) method. All the obtained compounds were highly effective against E. coli, K. pneumonia (Gram-negative) and S. aureus (Gram-positive) at concentrations of 75 and 100 µg/mL. In addition, they possessed antitubercular activity against normal, multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis. Therefore, they are more potent antimicrobial agents than ciprofloxacin. Similar research was conducted by scientists [37] who successfully obtained 1,3,4oxadiazole hybrids of ciprofloxacin and tested them against the standard group of Gram-positive and Gram-negative microorganisms. Here, again, promising activity was observed for both groups that exceeded the reference drug ciprofloxacin.
More pronounced activity against Gram-negative strains is described in ref. [65] for new C3 triazole ciprofloxacin derivatives. Antibacterial activity against Gram-positive strains, in this case, remained at the ciprofloxacin level. In addition, molecular docking studies using topoisomerase (3ILW) protein revealed correlation between the antibacterial activity and binding free energy of the molecules. The tested compound showed high affinity with low energy of −6.2 kcal/mol with the employed protein (for ciprofloxacin, it was −6.7 kcal/mol).
Among other core FQs that were utilized, we should mention levofloxacin triazole-3thiol, oxazole, oxadiazole and thiadiazol derivatives [25]. They were tested against E. coli and S. aureus and, in the disk diffusion method, exceeded the initial compound.
Furthermore, a series of new ofloxacin analogs was synthesized by modifying it by triazoles [46]. In the first stage of the research, in silico docking studies using Autodock vina 4.0 program were performed. Almost all the compounds used for docking showed a best-fit Root Mean Square Difference (RMSD) value of 0.000 with topoisomerase II (3ILW), and good inhibition, with an affinity range between −7.4 and −6.4 kcal/mol. The obtained data were verified via in vitro antimicrobial screening, where the obtained compounds showed promising activity against S. aureus, S. epidermidis and Bacillus subtilis (MIC 0.125 µg/mL).
Several patents on FQs hybridized via five-membered heterocyles at C3 were obtained by Chinese scientists. They claim antibacterial (against Gram-positive and Gram-negative strains), antifungal and DNA intercalating properties in the obtained thiazole [85] and aminothiazole derivatives [84,86]. In addition, they describe a simple and affordable preparation technique based on available raw materials. Another patent [119,120] describes the preparation of novel hybrids with rhodamine as promising antibacterial agents.
Among fused hybrids, the authors of [94] describe novel benzimidazole-quinolinone derivatives of ciprofloxacin and levofloxacin. All the synthesized compounds revealed promising antifungal activity when compared with the reference drug griseofulvin.
A series of new triazolothiadiazole derivatives of ciprofloxacin was obtained and tested against S. aureus and E. coli [47]. They showed stronger inhibitory activity against E. coli than that of S. aureus compared to ciprofloxacin. Therefore, the fused heterocyclebased substituted FQs are valuable for further investigations.
Lastly, a novel class of FQ oxadiazole derivatives that inhibit NS5B polymerase, a key enzyme of the Hepacivirus viral life cycle, is described in ref. [57] that may be the first step toward exploration of this dimension of FQs.
Altogether, the published studies definitely prove the logic and efficiency of these investigations. Even if they are now underestimated, their continuation gives a perfect chance to obtain fruitful results, namely, new potent antimicrobials to combat the problem of resistance to antibiotics.

Novel FQ Hybrids as Promising Antitumor Agents
Surprisingly, literature data revealed that many research groups searched for antitumor agents among new FQ hybrids. This approach is based on the concept of bioisosters that gained popularity in medicinal chemistry in recent years. Namely, the bioisosteric replacement of the carboxylic group with different heterocyclic moieties and synthesis of bis-fluoroquinolones linked via a heterocycle at C3 are two main strategies that are widely presented.
Another valuable point is the variety of cancer types against which the compounds synthesized were tested in the above-mentioned investigations.
Thus, new 1,3,4-thiadiazole derivatives of ciprofloxacin were synthesized and investigated via thorough in silico and in vitro studies (Figure 8). Theoretical and experimental DNA binding research revealed good correlation with human hepatocellular carcinoma (Huh-7) cell line activity. IC 50 values from Huh-7 cell line studies (25.75 µM) revealed synthesized compounds as potent anticancer agents, promising for further investigations [79]. synthesized were tested in the above-mentioned investigations.
Thus, new 1,3,4-thiadiazole derivatives of ciprofloxacin were synthesized and in vestigated via thorough in silico and in vitro studies (Figure 8). Theoretical and experi mental DNA binding research revealed good correlation with human hepatocellular carcinoma (Huh-7) cell line activity. IC50 values from Huh-7 cell line studies (25.75 μM revealed synthesized compounds as potent anticancer agents, promising for further in vestigations [79]. Other isosteres of the C-3 carboxylic group for the pefloxacin 1,3,4-oxadiazole-thione ring and oxadiazole thione Mannich bases were suggested by the authors of [31]. The obtained compounds were tested in vitro against a liver cancer (Hep-3B) cell line and according to the results, all the title compounds showed more significant potency than parent compounds. In addition, derivatives of aliphatic amines appeared to be more ac tive than the derivatives of aromatic amines. Furthermore, similar derivatives were re ported in patent [36], and it was shown that molecules with an electron-withdrawing group attached on the aryl ring had more potency than compounds with an elec tron-donating group.
The authors of [39] patented similar oxadiazole norfloxacin derivatives that were screened in vitro against the same liver cancer (Hep-3B) cell line. Evaluation was per formed via an MTT assay. The results revealed higher cytotoxicity compared to norflox acin for fifteen title compounds. Correspondent quaternary ammonium salts exhibited promising anticancer activity with IC50 values below 25.0 μmol/L.
Another search for agents against the human hepatoma (Hep-3B) cancer cell line and human pancreatic (Capan-1) cell line was made based on comparative molecular field analysis techniques [40]. Three-dimensional quantitative structure-activity relationship (3D-QSAR) investigations on the antitumor activity of s-triazole sulfide-ketone deriva Other isosteres of the C-3 carboxylic group for the pefloxacin 1,3,4-oxadiazole-thione ring and oxadiazole thione Mannich bases were suggested by the authors of [31]. The obtained compounds were tested in vitro against a liver cancer (Hep-3B) cell line and, according to the results, all the title compounds showed more significant potency than parent compounds. In addition, derivatives of aliphatic amines appeared to be more active than the derivatives of aromatic amines. Furthermore, similar derivatives were reported in patent [36], and it was shown that molecules with an electron-withdrawing group attached on the aryl ring had more potency than compounds with an electron-donating group.
The authors of [39] patented similar oxadiazole norfloxacin derivatives that were screened in vitro against the same liver cancer (Hep-3B) cell line. Evaluation was performed via an MTT assay. The results revealed higher cytotoxicity compared to norfloxacin for fifteen title compounds. Correspondent quaternary ammonium salts exhibited promising anticancer activity with IC 50 values below 25.0 µmol/L.
Another search for agents against the human hepatoma (Hep-3B) cancer cell line and human pancreatic (Capan-1) cell line was made based on comparative molecular field analysis techniques [40]. Three-dimensional quantitative structure-activity relationship (3D-QSAR) investigations on the antitumor activity of s-triazole sulfide-ketone derivatives of ciprofloxacin and levofloxacin gave the possibility to design four novel molecules with promising anti-tumor activity and to plan further in vitro research.
Research related to bladder tumors is also patented [73] and describes similar triazole derivatives for which an IC50 value of 0.6μM against a bladder tumor was detected.
Research related to bladder tumors is also patented [73] and describes similar triazole derivatives for which an IC 50 value of 0.6µM against a bladder tumor was detected.
A simple synthetic approach led to the development of novel citrate-triazole-oxadiazole norfloxacin hybrids that exhibited remarkable anticancer activity against cervical cancer (HeLa) cell lines with an IC 50 value 11.3 ± 0.41, comparable to the standard drug [30]. The compounds also revealed suitable druglike properties and are expected to present a good bioavailability profile.
Similar bis-fluoroquinolone oxadiazole carbazide N-methyl-ciprofloxacin derivatives were synthesized, tested against human A549, Capan-I and A375 cell lines; they showed promising activity and were patented [107,108]. In addition, they reduced toxic side effects on normal cells.
Many published papers are devoted to the search for potential antileukemic agents among 3-heteroaryl FQ hybrids. For instance, a series of [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazine and pyrazolo [5,1-c] [1,2,4]triazole derivatives of norfloxacin, ciprofloxacin and levofloxacin was successfully obtained [91]. Their in vitro antitumor activity was tested against murine leukemia (L1210) and Chinese hamster ovary (CHO) cell lines via the standard MTT assay. The results showed poor inhibitory activity for parent fluoroquinolones (IC 50 > 150 mmol/L), while isolated fused compounds had a potential activity with an IC 50 value within 10.0 mmol/L. Several in silico studies prove this idea. Thus, for the therapy of T-cell lymphoma, a molecular electronegativity distance vector of levofloxacin-thiadiazole histone deacetylase (HDAC) inhibitor was measured [76] and the compounds were described as promising.
Quantitative structure-activity relationship (QSAR) and molecular docking study of levofloxacin and thiadiazole as antitumor agents with HDAC1, HDAC2 and HDAC6 also revealed that the main factors affecting their biological activity are hydrogen bonding and hydrophobic interactions [78]. From the docking results, it can be seen that the active part of the molecule formed a hydrogen bond with the active part of the macromolecule, while the hydrophobic part of the small molecule had a hydrophobic interaction with non-polar amino acid residues in the active part of the macromolecule.
For instance, novel oxadiazole [35] and oxadiazolone thione Mannich base derivatives of enrofloxacin [34] were synthesized and studied as promising antitumor agents. Their antiproliferative activity against hepatocellular carcinoma (SMMC-7721), murine leukemia (L1210) and human leukemia (HL60) cell lines was evaluated via an MTT method. The results revealed improved activity compared to enrofloxacin.
Novel C-3 s-triazole-oxadiazole sulfide Mannich base pefloxacin derivatives were also screened against SMMC-7721, L1210 and HL60 cell lines and were evaluated by an MTT assay. The investigation revealed that the sulfides and their corresponding Mannich base compounds are more potent inhibitors than the starting compounds, especially against SMMC-7721 [42,43].
New s-triazole ofloxacine derivatives with functionalized side chains of Schiff bases and Schiff-Mannich bases are described in ref. [45]. Their in vitro antitumor activity against L1210, Chinese hamster ovary (CHO) and HL60 cell lines was evaluated in the MTT assay. Higher inhibitory activity was detected for compounds possessing a free phenol group. Other novel s-triazole-thiosemicarbazone ofloxacine derivatives were also evaluated by the MTT assay and revealed more significant antiproliferative activity than parent ofloxacin. Thiosemicarbazones, especially those containing a nitro group or fluorine atom, showed activity comparable to doxorubicin. Therefore, an azole ring modified with a functional side chain is a favorable bioisosteric replacement of the C-3 carboxylic group for an improvement in antitumor activity [41].
Aminothiadiazole ciprofloxacin derivatives and their Schiff bases were screened against SMMC-7721, HL60 and L1210 cell lines via MTT assay [77]. They showed potential cytotoxicity with IC 50 values that reached micro-molar concentration and were patented.
Another patent was obtained for the invention of floxacin diazole derivatives that exhibited IC 50 values of approximately 2, 5 and 2.5 µM against CHO, HL60 and L1210 cell lines, respectively [67].
In addition, there are papers devoted to the hybridization of ofloxacin at C-3 with an s-triazole ring [60], oxadiazole-5-sulfanylacetylhydrazone moiety [27], triazole-oxadiazole methylsulfide [44] and oxadiazole ring [37]. All of them state higher antitumor activity than for the parent ofloxacin in the MTT assay. Similar derivatives patented by the authors of [71] gave a CHO IC 50 value of 1.3 µM. A patent for bis-oxadiazolyl methylsulfides derived from ofloxacin describes in vitro antitumor activity evaluation against three cancer cell lines by the MTT method. The compounds synthesized showed potential anticancer activity (IC 50 < 25µmol/L). The activity of the quaternary ammonium salts was higher than that of the corresponding free bases [40].
Further, there are many inventions on bis-fluoroquinolones as antitumor agents that were patented. Namely, levofloxacin-containing bis-fluoroquinolone oxadiazole carbamide derivative is described as useful in the treatment of cancer [98]. A novel bis-fluoroquinolone thiadiazole urea-series fleroxacin derivative was developed by the authors of [109] to increase the antitumor activity and selectivity of fluoroquinolones and reduce the toxic side effects on normal cells. Novel thiadiazole urea rufloxacin derivatives exhibited IC 50 values in a range of 0.46 µM to 2.36 µM [110]. A series of bis-fluoroquinolone thiadiazole urea N-Me lomefloxacin derivatives is described in patent [112], similar bis-fluoroquinolone thiadiazole urea-based N-Me moxifloxacin derivatives designed as promising antitumor agents were patented [113] and the authors continued this project with thiadiazole ureabased pefloxacin [114], ofloxacin [115], levofloxacin [116] and gatifloxacin [117] derivatives. Furthermore, N-Me moxifloxacin-containing bis-fluoroquinolone oxadiazole urea derivatives for cancer treatment are proposed in the patent [100], N-Me gatifloxacin-containing bis-fluoroquinolone oxadiazole urea derivative in [101], oxadiazole urea fleroxacin derivatives with IC 50 values from 0.14 µM to 1.36 µM in [104] and oxadiazole urea pefloxacin bis-derivatives in [106].
At last, there are two unusual papers that are unique according to their research strategy. Thus, a novel series of topoisomerase I (Top I) inhibitors was designed via condensation of FQs with o-phenylenediamine, o-aminophenol or o-aminobenzenethiol in polyphosphoric acid (PPA). The most potent compound 1-ethyl-3-(6-nitrobenzoxazol-2-yl)-6,8-difluoro-7-(3-methylpiperazin-1-yl)-4(1H)-quinolone revealed a significant inhibitory effect on Top I, leading to Top I-mediated cleavage and influencing Top I expression at the cellular level. Moreover, it induced cell death via apoptosis and accelerated DNA strand breaks without significant alteration in cell cycle populations. The in vivo evaluation on the growth of HT-29 tumor xenografts in nude mice showed its therapeutic potential for further development [95].
The authors of [93] searched for P-glycoprotein ABCB1 inhibitors among fused FQ derivatives. The ABCB1 is involved in multidrug resistance of tumor cells by preventing intracellular accumulation of cytotoxic drugs. In addition, its overexpression limits drug oral bioavailability. To find new potent ABCB1 inhibitors, a 3D pharmacophore model was created based on known inhibitors. The inhibitory activities of the best hits were evaluated by several biological assays, such as rhodamine 123 accumulation assay, chemosensitization assay and multidrug resistance 1-Madin-Darby canine kidney cell/Madin-Darby canine kidney cell permeability assay. The most promising compounds were identified and taken for further development.
Altogether, we can conclude that the scope of antitumor activity of FQ derivatives is quite broad and promising.

Other Types of Biological Activity
Apart from antimicrobial and anticancer potency of FQs, we should emphasize the possibility to broaden the horizons of their utilization as biologically active molecules. This idea was proved by several noteworthy investigations presented below.
First of all, in line with the problem of combating infectious diseases, the authors of [59] searched for potent molecules to cure protozoal infections. It was already known that N-benzylamide derivative of norfloxacin is promising so they synthesized thiosemicarbazide of 1-butyl-6-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid and its heterocyclic derivatives-1,3,4-thiadiazole and 1,2,4-triazole. Furthermore, modification of the 1,2,4-triazole ring with maleimides was also described. For the obtained compounds, antitrypanosomal activity was also typical but lower than for N-benzylamide derivative. Still, the C3 substitution plays a key role in this type of activity.
Triazole derivatives of norfloxacin and their Schiff bases were successfully obtained and described in the paper [82]. The Schiff bases ( Figure 10) that were screened for analgesic and anti-inflammatory activity on the carrageenan-induced rat paw edema model revealed encouraging results and exceeded the reference drug ibuprofen. At the same time, unsubstituted triazoles revealed antibacterial and antifungal activity. Therefore, further investigations in this area may result in compounds with a versatile pharmacological profile.
were identified and taken for further development.
Altogether, we can conclude that the scope of antitumor activity of FQ derivatives is quite broad and promising.

Other Types of Biological Activity
Apart from antimicrobial and anticancer potency of FQs, we should emphasize the possibility to broaden the horizons of their utilization as biologically active molecules. This idea was proved by several noteworthy investigations presented below.
First of all, in line with the problem of combating infectious diseases, the authors of [59] searched for potent molecules to cure protozoal infections. It was already known that N-benzylamide derivative of norfloxacin is promising so they synthesized thiosemicarbazide of 1-butyl-6-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid and its heterocyclic derivatives-1,3,4-thiadiazole and 1,2,4-triazole. Furthermore, modification of the 1,2,4-triazole ring with maleimides was also described. For the obtained compounds, antitrypanosomal activity was also typical but lower than for N-benzylamide derivative. Still, the C3 substitution plays a key role in this type of activity.
Triazole derivatives of norfloxacin and their Schiff bases were successfully obtained and described in the paper [82]. The Schiff bases ( Figure 10) that were screened for analgesic and anti-inflammatory activity on the carrageenan-induced rat paw edema model revealed encouraging results and exceeded the reference drug ibuprofen. At the same time, unsubstituted triazoles revealed antibacterial and antifungal activity. Therefore, further investigations in this area may result in compounds with a versatile pharmacological profile. Figure 10. Triazole derivatives of norfloxacin as analgesic and anti-inflammatory agents [82]. The synthesized levofloxacin triazole-3-thiol, oxazole, oxadiazole and thiadiazol derivatives revealed antioxidant activity equivalent to ascorbic acid (IC 50 = 31.95 g/mL) in the investigation [25].
A series of novel benzimidazole-quinolinone derivatives of ciprofloxacin and levofloxacin was screened for in vitro antidiabetic activity by α-glucosidase inhibitory action and appeared to be promising at a 200 µg/mL concentration compared to acarbose [94].
Novel 5-amino-1,3,4-thiazidazole hybrids of norfloxacin and levofloxacin were synthesized, characterized and assessed for their acetyl cholinesterase enzyme (AChE) inhibitory activity [81]. The obtained derivatives showed promising results, especially levofloxacin derivative (IC 50 18.1 ± 0.9 nM), which substantially exceeded the reference drug neostigmine (IC 50 2186.5 ± 98.0 nM). In addition, the authors evaluated the ADMET parameters, and values of the hybrids showed appropriate correlation with the binding energy values (Kcal/mol). Combined with high drug-likeness scores and the results of molecular docking studies, this research can be a promising background for finding a treatment for Alzheimer's disease.
Altogether, we can assume that the scope of the probable biological activity of FQ hybrids is wide. That makes this area of investigation even more versatile and attractive for medicinal chemists. Table 1 summarizes all the data on the chemotypes that were utilized for the structural modification of FQs as well as the activities studied for the synthesized compounds. Table 1. The general scope of the chemical synthesis and chemotherapeutic potential of 3-heteroaryl fluoroquinolone hybrids.

Chemotype
Scheme FQ * Activities Studied ** molecular docking studies, this research can be a promising background for finding a treatment for Alzheimer's disease. Altogether, we can assume that the scope of the probable biological activity of FQ hybrids is wide. That makes this area of investigation even more versatile and attractive for medicinal chemists. Table 1 summarizes all the data on the chemotypes that were utilized for the structural modification of FQs as well as the activities studied for the synthesized compounds.

Conclusions
Fluoroquinolones, nowadays, belong to the medicinal sector. This is true both for clinical practice and for scientific investigations. While all four generations are widely used for treatment of different infections, the issue of resistance of microorganisms becomes demanding as well as their pharmacokinetics improving.
Therefore, medicinal chemists all over the world continue investigations on FQs structural modifications. As the current research revealed, there are promising studies in the area of 3-heteroaryl hybrids. The latter can be synthesized via different convinient methods with the formation of new derivatives with five-membered and fused heterocycles or creation of bis-fluoroquinolones with variable linking moieties. These novel compounds revealed not only good antimicrobial properties compared to the parent molecules but were also widely investigated as anticancer agents with promising activity.
Among the described novel molecules, there are potent antibacterial agents that possess activity against clinical and resistant strains. A big part of these investigations is supported by molecular docking studies that reveal all the nuances of interactions with proteins and binding modes. As for the anticancer activity, we observe a wide variety of cancer cell lines against which the compounds were tested and showed promising results. Moreover, there are studies devoted to potent 3-heteroaryl FQ hybrids as antiprotozoal, analgesic and anti-inflammatory, antioxidant, antidiabetic agents and even acetyl cholinesterase enzyme inhibitors. Furthermore, the big scope of patents obtained serves as proof of interest in this area and prospects for continuation of these studies.
Altogether, we can conclude that this scientific direction is gaining momentum and further investigations on chemical and pharmacological potential of 3-heteroaryl hybrids of fluoroquinolones will be of a great value.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics12030625/s1/. File S1: Synthesis methods of 3-Heteroaryl Fluoroquinolone Hybrid Chemotypes. Ref. [122] is cited in Supplementary Materials. Data Availability Statement: All data generated or analyzed during this study are included in this article.