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Antibacterial, Antitubercular and Antiviral Activity Evaluations of Some Arylidenehydrazide Derivatives Bearing Imidazo[2,1-b]thiazole Moiety/Imidazo[2,1-b]tiyazol Cekirdegi Tasiyan Bazi Arilidenhidrazit Turevlerinin Antibakteriyel, Antituberkuler ve Antiviral Aktivite Tayinleri.

INTRODUCTION

Infectious diseases caused by bacteria have increased dramatically in recent years. Despite many significant advances in antibacterial therapy, the widespread use and misuse of antibiotics have led to the emergence of bacterial resistance to antibiotics, which is a serious threat to public health. On the other hand, tuberculosis (TB), still remains the leading cause of worldwide death among infectious diseases. (1,2) In 2014, there were an estimated 9.6 million new TB cases: 5.4 million among men, 3.2 million among women and 1.0 million among children. (3) Additionally, viral infections caused by the rapid emergence of antiviral drug resistant strains have become a serious threat globally. (4) Many diseases are actually caused by the different members of DNA- and RNA-containing viruses. Among DNA-containing viruses, the herpes group of viruses, particularly herpes simplex virus-1 (HSV-1) primarily causes encephalitis, stomatitis, ocular infections and HSV-2 primarily causes genital lesions, skin eruptions or cytomegalovirus is related with severe morbidity and mortality in patients at risk for disease because of immune system disabilities and varicella-zoster virus is the ethiological agent of chickenpox and shingles. (5,6) Influenza (INF) viruses, parainfluenza-3 virus, alphaviruses (e.g. sindbis virus), respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) are examples of enveloped single-stranded RNA-containing viruses. VSV causes an economically important disease in horses and cattle. (7) Both RSV and parainfluenza-3 virus are an important cause of respiratory tract infections. (8,9)

Among the heterocyclic rings containing bridgehead nitrogen atom, imidazo[2,1-b]thiazoles derivatives are especially attractive because of their different biological activities such us antibacterial, (10) antituberculosis, (11) antiviral, (12) anticancer, (13) anti-inflammatory (14) and diuretic (15) activities. On the other hand, arylidenehydrazide moiety are also associated with various biological properties including antibacterial, (16) antitubercular, (17) antiviral, (18) anticancer, (19) antiinflammatory and analgesic (20) activities.

In continuation of our previous studies on the biological properties of imidazo[2,1-b]thiazole derivatives, (21-27) in this study, we reported the antibacterial, antitubercular and antiviral activity evaluation of some arylidenehydrazide derivatives bearing imidazo[2,1-b]thiazole moiety.

MATERIALS AND METHODS

Chemistry

All chemicals were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA) chemical companies. Using a Buchi B-540 melting point apparatus (Flawil, Switzerland) with open capillaries, melting points were determined and are uncorrected. Elemental analyses were performed on a Thermo Finnigan Flash EA 1112 elemental analyser. Infrared spectra were recorded (in KBr) using a Perkin Elmer Spectrum 100 fourier transform infrared (FTIR) spectrometer and Shimadzu IRAffinity-1 FTIR spectrophotometer. [.sup.1]H and [.sup.13]C-nuclear magnetic resonance spectra were obtained on Varian UNITY INOVA 500 MHz spectrometer using dimetil sulfoxide-[d.sub.6] as an internal standard. All chemical shifts were reported as d (ppm) values and spin-spin couplings (J) were exposed in Hz. MS (ESI-) were determined on a Finnigan LCQ Advantage Max mass spectrometer.

General synthesis of [N.sup.2]-arylidene-(6-(4-chlorophenyl) imidazo[2,1-b]thiazol-3-yl)acetic acid hydrazides (3a-3j) (28) A solution of 0.005 mol compound 2 and 0.005 moL of an appropriate aromatic aldehyde in 100 mL ethanol was heated under reflux for 5 h. The precipitate obtained was purified either by recrystallization from ethanol or by washing with hot ethanol.

Biological activity

Antibacterial activity

Minimum inhibitory concentrations (MICs) were determined by the microbroth dilution method using the National Committee for Clinical Laboratory Standards recommendations. (29) Mueller-Hinton broth (Oxoid, Hemakim, Turkey) was used as the test medium. An inoculum of approximately 5x[10.sup.5] CFU [cm.sup.-3] was delivered per well. Serial twofold dilutions of the test compounds (128-0.25 [micro]g/mL) and extra dilutions (256-0.25 [micro]g/mL) for antibiotic standards were prepared. Plates were incubated for 16-20 h at 35[degrees]C in an ambient air incubator. The lowest concentration of the test compounds inhibiting visible growth was taken as the MIC value.

Antitubercular activity

In vitro evaluation of antitubercular activity

Primary screening was conducted at 6.25 mg/mL against Mycobacterium tuberculosis [H.sub.37]Rv in BACTEC 12B medium using a broth microdilution assay the Microplate Alamar Blue Assay (MABA). (30) Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. (31) Compounds effecting <90% inhibition in the primary screen were not generally evaluated further. Compounds demonstrating at least 90% inhibition in the primary screen were re-tested at lower concentrations against M. tuberculosis [H.sub.37]Rv in order to determine the actual MIC using MABA. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to the controls. Concurrently with the determination of MICs, compounds were tested for cytotoxicity (I[C.sub.50]) in VERO cells at concentrations [pounds sterling]6.25 mg/mL or 10 times the MIC for M. tuberculosis [H.sub.37]Rv (solubility in media permitting). After 72 h exposure, viability was assessed on the basis of cellular conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide into a formazan product using the Promega CellTiter 96 Non-radioactive Cell Proliferation Assay. Compounds for which the selectivity index (I[C.sub.50]: MIC ratio) SI>10 were assumed to possess in vitro activity confirmed in the BACTEC 460 at 6.25 mg/mL.

Microplate alamar blue susceptibility assay

Antimicrobial susceptibility testing was performed in black, clear-bottomed, 96-well microplates (black view plates; Packard Instrument, Meriden, Connecticut, USA) in order to minimize background fluorescence. Outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. Initial drug dilutions were prepared in either dimethyl sulfoxide or distilled deionized water, and subsequent twofold dilutions were performed in 0.1 mL of 7H9GC (no Tween 80) in the microplates. BACTEC 12B-passaged inocula were initially diluted 1:2 in 7H9GC, and 0.1 mL was added to wells. Subsequent determination of bacterial titers yield 1X[10.sup.6] CFU/mL in plate wells for [H.sub.37]Rv. Frozen inocula were initially diluted 1:20 in BACTEC 12B medium followed by a 1:50 dilution in 7H9GC. Addition of 1/10 mL to wells resulted in a final bacterial titers of 2.0X[10.sup.5] CFU/mL for [H.sub.37]Rv. Wells containing drug only were used to detect autofluorescence of compounds. Addition control wells consisted of bacteria only (B) and medium only (M). Plates were incubated at 37[degrees]C. Starting at day 4 of incubation, 20 mL of 10x Alamar Blue solution (Alamar Biosciences/Accumed, Westlake, Ohio, USA) and 12.5 mL of 20% Tween 80 were added to one B well an done M well, and plates were reincubated 37[degrees]C. Wells were observed at 12 and 24 h for a color change from blue to pink and for a reading of [greater than or equal to]50.000 fluorescence units (FU). Fluorescence was measured in a Cytofluor II microplate fluorometer (Perseptive Biosystems, Framingham, Massachusetts, USA) in bottom-reading mode with excitation at 530 nm and emission at 590 nm. If the B wells became pink by 24 h, reagent was added to the entire plate. If the well remained blue or [pounds sterling]50.000 FU was measured, additional M and B wells were tested daily until a color change occurred, at which time reagents were added to all remaining wells. Plates were then incubated at 37[degrees]C, and results were recorded at 24 h post-reagent addition. Visual MICs were defined as the lowest concentration of drug that had prevented a color change. For fluorometric MICs, a background subtraction was performed on all wells with a mean of triplicate M wells. Percent inhibition was defined as 1-(test well FU/mean FU triplicate B wells) X 100. The lowest drug concentration effecting an inhibition of [greater than or equal to]90% was considered the MIC.

BACTEC radiometric method of susceptibility testing

Inocula for susceptibility testing were either from a positive BACTEC isolation vial with a growth index (GI) of 500 or more, or a suspension of organisms isolated earlier on a conventional medium. The culture was well mixed with a syringe and 0.1 mL of a positive BACTEC culture was added to each of the vials containing the test compounds (6.25 mg/mL). The standard vials contained rifampicin (0.25 mg/mL). A control vial was inoculated with a 1:100 dilution of the culture. Each vial was tested immediately on a BACTEC instrument to provide C[O.sub.2] in the headspace. The vials were incubated at 37[degrees]C and tested daily with a BACTEC instrument. When the GI in the control read at least 30, the increase in GI ([DELTA]GI) from the previous day in the control was compared with that in the drug vial. The following formula was used to interpret the results:

[DELTA]GI control > [DELTA]GI drug = susceptible

[DELTA]GI control < [DELTA]GI drug = resistant

If a clear susceptibility pattern (the difference of [DELTA]GI of control and the drug bottle) was not seen at the time the control GI was 30 the vials were read for 1 or 2 additional days to establish a definite pattern of [DELTA]GI differences.

Antiviral activity

The compounds (3a-j) were evaluated for activity against diverse RNA- and DNA-viruses, using the following cell-based assays (32): (a) Madin-Darby Canine Kidney (MDCK) cells infected with INF A/H1N1 subtype (A/Ned/378/05), INF A/H3N2 subtype (A/HK/7/87) or INF B (B/Ned/537/05); (b) Crandell-Rees Feline Kidney (CRFK) cells infected with feline corona virus (FCoV) or feline herpes virus (FHV); (c) African green monkey kidney Vero cells infected with parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie B4 virus or Punta toro virus; (d) human embryonic lung (HEL) fibroblast cells infected with HSV-1 or -2, an acyclovir-resistant HSV-1 strain, vaccinia virus, VSV; (e) human cervixcarcinoma Henrietta Lacks (HeLa) cells infected with VSV, coxsackie B4 virus or RSV.

To perform the antiviral assays, the virus was added to subconfluent cell cultures in 96-well plates, and at the same time, the test compounds were added at serial dilutions. Appropriate reference compounds were included, i.e. the virus entry inhibitor dextran sulfate 5000, the broad antiviral agent ribavirin, the antiherpetic drug ganciclovir, and the HIV inhibitor azidothymidine. After 3-6 days incubation at 37[degrees]C (or 35[degrees]C in the case of INF virus), the cultures were examined by microscopy to score the compounds' inhibitory effect on virus-induced cytopathic effect or their cytotoxicity. For some viruses, antiviral and cytotoxic activities were confirmed by the colorimetric 3-(4,5-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay.

RESULTS AND DISCUSSION

The key intermediate 2 was prepared from ethyl (6-(4-chlorophenyl)imidazo[2,1-b]thiazol-3-yl)acetate hydrobromide (1) and hydrazine hydrate following the literature method. (33) The synthetic route of the compounds is outlined in Scheme 1. Condensation of 2 with appropriate aromatic aldehyde afforded the corresponding [N.sup.2]-arylidene-(6-(4-chlorophenyl) imidazo[2,1-b]thiazol-3-yl)acetic acid hydrazides (3a-j). (28)

Compounds 3a-j were evaluated for in vitro antibacterial activity against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 using the microbroth dilution method (29). As can be seen in Table 1, 3i (2,4-dichlorobenzylidene derivative) showed the highest activity against S. aureus ATCC 29213 and E. coli ATCC 25922 (MIC: 2 [micro]g/mL, 64 [micro]g/mL, respectively).

Compounds 3a-j were evaluated against M. tuberculosis [H.sub.37]Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the MABA. The primary antituberculosis screening was performed in accordance with the protocol of the Tuberculosis Antimicrobial Acquisition and Coordinating Facility Southern Research Institute (30). Rifampin was used as the control drug in the tests. Compounds demonstrating a percent inhibition of bacterial growth of greater than or equal to 90% in the primary screen were retested against M. tuberculosis [H.sub.37]Rv, to determine the actual MIC in the MABA. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of 90%, relative to controls. This value was determined from the dose-response curve as the I[C.sub.90] using a curve fitting program. Any I[C.sub.90] value of [less than or equal to]10 [micro]g/mL was considered "Active" for antitubercular activity. Compounds active in the initial screen were tested for I[C.sub.50] in Vero cells. Cytotoxicity was determined from the dose-response curve as the I[C.sub.50] using a curve fitting program. Concurrent with the determination of MICs, compounds were tested for cytotoxicity in Vero cells at concentrations 10x the MIC for M. tuberculosis [H.sub.37]Rv. Most of the tested compounds showed weakly antitubercular activity and cytotoxicities of the compounds were found to be very high (Table 2).

The compounds (3a-j) were also evaluated against INF A/H1N1 subtype (A/Ned/378/05), INF A/H3N2 subtype (A/HK/7/87), INF B (B/Ned/537/05) in MDCK, FCoV, FHV in CRFK, parainfluenza-3 virus, reovirus-1, sindbis virus, coxsackie B4 virus, punta toro virus in Vero, HSV-1 (KOS), HSV-2 (G), HSV-1 TK KOS ACV, vaccinia virus, VSV, in HEL and VSV, coxsackie B4 virus and RSV in HeLa cell cultures. As can be seen in Table 3, the most active compound was R=2,4-dichlorophenyl substituted 3i. It inhibited FCoV with E[C.sub.50] of 7.5 [micro]M. R=4-hydroxyphenyl substituted derivative 3c, inhibited HSV-1 (KOS), HSV-2 (G), HSV-1 TK KOS ACV, vaccinia virus and VSV with E[C.sub.50] of 9, 27, 32, 16 and 32 [micro]M, respectively. R=3-methoxy-4-hydroxyphenyl substituted 3g showed E[C.sub.50] values of 20 and 14 [micro]M for HSV-1 (KOS) and v virus, respectively (Table 4). However, tested compounds (3a-j) didn't show any inhibition against INF A/H1N1 subtype (A/Ned/378/05), INF A/H3N2 subtype (A/HK/7/87), INF B (B/Ned/537/05), parainfluenza-3 virus, reovirus-1, sindbis virus, coxsackie B4 virus, punta toro virus, VSV, coxsackie B4 virus and RSV strains (i.e. minimal antivirally effective concentration [greater than or equal to]5-fold lower than minimal cytotoxic concentration) (Table 5).

CONCLUSION

In this work, a series of arylidenehydrazide derivatives bearing imidazo[2,1-b]thiazole moiety was evaluated for antibacterial, antitubercular and antiviral activities. The results showed that some compounds exhibited antibacterial, antimycobacterial and antiviral activities with different percentage of inhibition. Therefore, we have identified a novel series of imidazo[2,1-b] thiazoles, which may develop into the potential class of antibacterial, anti-tubercular and antiviral agents.

ACKNOWLEDGEMENTS

We thank Prof. Lieve Naesens from the Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium for evalution of antiviral activity. We thank Dr. Joseph A. Maddry from the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), National Institute of Allergy and Infectious Diseases Southern Research Institute, Alabama, USA, for the evaluation of anti-TB activity. The present work was supported by Istanbul University Scientific Research Projects (Project No: 49399).

Conflict of Interest: No conflict of interest was declared by the authors.

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Nuray ULUSOY GUZELDEMIRCI (1*), Berin KARAMAN (1), Omer KUCUKBASMACI (2)

(1) Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey

(2) Istanbul University, Cerrahpasa Faculty of Medicine, Department of Microbiology, Istanbul, Turkey

(*) Correspondence: E-mail: nulusoy@istanbul.edu.tr, Phone: +90 532 574 92 63

ORCID ID: orcid.org/0000-0002-4495-4282

Received: 16.08.2016, Accepted: 23.10.2016

DOI: 10.4274/tjps.25743
Table 1. Antibacterial activity of compounds 3a-j (MIC mg/mL)

Comp.
/(*) microorg.  R

3a              C6H5
3b              [C.sub.6][H.sub.4](OH)(2-)
3c              [C.sub.6][H.sub.4](OH)(4-)
3d              [C.sub.6][H.sub.4](OCH3)(4-)
3e              [C.sub.6][H.sub.4]([NO.sub.2])(4-)
3f              [C.sub.6][H.sub.4](N(C[H.sub.3])[.sub.2])(4-)
3g              [C.sub.6][H.sub.3](OC[H.sub.3])(OH)(3,4-)
3h              [C.sub.6]H(OC[H.sub.3])[.sub.2](2,5-)
3i              [C.sub.6]H([Cl.sub.2])(2,4-)
3j              5-nitro-2-furyl
Amikacin        -

Comp.
/(*) microorg.   A       B       C

3a               128    >128     128
3b              >128    >128    >128
3c               128     128    >128
3d                64     128     128
3e              >128    >128    >128
3f               128    >128     128
3g               128    >128    >128
3h              >128    >128     128
3i                32    >128      64
3j               128     128     128
Amikacin           1       1       2

MIC: Minimum inhibitory concentrations, (*) A: Staphylococcus aureus
ATCC 29213, B: Pseudomonas aeruginosa ATCC 27853, C: Escherichia coli
ATCC 25922

Table 2. Antimycobacterial activity screening results of 3a-j
(MIC mg/mL)

Compound    Assay  I[C.sub.50] (mg/mL)  I[C.sub.90] (mg/mL)

3a          n.t.   n.t.                 n.t.
3b          MABA   >100                 >100
3c          MABA     22.710               33.060
3d          MABA     69.170             >100
3e          MABA   >100                 >100
3f          MABA   >100                 >100
3g          MABA     20.670               36.860
3h          MABA     44.720             >100
3i          MABA   >100                 >100
3j          MABA      6.16                14.390
Rifampicin                                 0.125

Compound    Activity

3a          n.t.
3b          Inactive
3c          Weakly active
3d          Weakly active
3e          Inactive
3f          Weakly active
3g          Weakly active
3h          Weakly active
3i          Inactive
3j          Weakly active
Rifampicin

MIC: Minimum inhibitory concentrations, MABA: Microplate Alamar Blue
Assay, n.t.: not tested

Table 3. Anti-feline corona virus and anti-feline herpes virus
activity and cytotoxicity of the compounds 3a-j in Crandell-Rees
Feline Kidney cell cultures

Compound           C[C.sub.50] (a)   E[C.sub.50] (b) ([micro]M)
                   ([micro]M)        FCoV            FHV

3a                 >100            >100              >100
3b                   50.6           >20               >20
3c                   20.7           >20               >20
3d                 >100            >100              >100
3e                    4.4            >4                >4
3f                   50.8           >20               >20
3g                   24. 5          >20               >20
3h                 >100            >100              >100
3i                 >100               7. 5           54.8
3j                    9. 7           >4                >4
HHA ([micro]g/mL)  >100               5.3             8.8
UDA ([micro]g/mL)  >100              17. 7           12.9
Ganciclovir
([micro]M)         >100            >100               3.6

FCoV: Feline corona virus, FHV: Feline herpes virus, HHA: Hippeastrum
hybrid agglutinin, UDA: Urtica dioica agglutinin, MTS:
3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)
-2-(4-sulfophenyl)-2H-Tetrazolium, (a) 50% cytotoxic concentration, as
determined by measuring the cell viability with the colorimetric,
formazan-based MTS assay, (b) 50% effective concentration, or
concentration producing 50% inhibition of virus-induced, cytopathic
effect, as determined by measuring e cell viability with the
colorimetricformazan-based MTS assay

Table 4. Antiviral activity and cytotoxicity of the compounds 3a-j in
human embryonic lung cell cultures

                                            E[C.sup.50] (b) ([micro]M)
Compound     MC[C.sub.a]                    Herpes simplex virus-1
             ([micro]M)                     (KOS)

3a                                    >100  >100
3b                                    >100  >100
3c           [greater than or equal to]100     9
3d                                     100   >20
3e                                    >100  >100
3f                                    >100  >100
3g                                     500    20
3h                                     100   >20
3i                                     100   >20
3j                                    >100  >100
Brivudin                              >250     0.05
Ribavirin                             >250     2
Cidofovir                             >250     0.7
Ganciclovir                           >100     0.03

Compound     Herpes simplex  Herpes simplex virus-1  Vaccinia virus
             virus-2 (G)     TK KOS AC[V.sup.r]

3a           >100            >100                    >100
3b           >100            >100                    >100
3c             27              32                      16
3d            >20             >20                     >20
3e           >100            >100                    >100
3f           >100            >100                    >100
3g           >100            >100                      14
3h            >20             >20                     >20
3i            >20             >20                     >20
3j           >100            >100                    >100
Brivudin      199              10                      10
Ribavirin       2               2                      10
Cidofovir       1.1             3.5                  >250
Ganciclovir     0.03            0.1                  >100

Compound     Vesicular stomatitis
             virus

3a           >100
3b           >100
3c             32
3d            >20
3e           >100
3f           >100
3g           >100
3h            >20
3i            >20
3j           >100
Brivudin     >250
Ribavirin    >250
Cidofovir    >250
Ganciclovir  >100

(a) Required to cause a microscopically detectable alteration of
normal cell morphology, (b) Required to reduce virus-induced
cytopathogenicity by 50%

Table 5. Antiviral activity and cytotoxicity of the compounds 3a-j in
Vero cell cultures

Compound              MCC (a)
                      ([micro]M)

3a                                            >100
3b                                             100
3c                                              20
3d                                            >100
3e                                              20
3f                                            >100
3g                                              40
3h                                             100
3i                    [greater than or equal to]20
3j                                             100
DS-5000 ([micro]M)                            >100
(S)-DHPA ([micro]M)                           >250
Ribavirin ([micro]M)                          >250

Compound                                     E[C.sub.50] (b) ([micro]M)
                      Parainfluenza-3 virus  Reovirus-1  Sindbis virus

3a                    >100                   >100        >100
3b                     >20                    >20         >20
3c                      >4                     >4          >4
3d                    >100                   >100        >100
3e                      >4                     >4          >4
3f                    >100                   >100        >100
3g                      >8                     >8          >8
3h                     >20                    >20         >20
3i                     >20                    >20         >20
3j                     >20                    >20         >20
DS-5000 ([micro]M)    >100                   >100          15
(S)-DHPA ([micro]M)   >250                   >250        >250
Ribavirin ([micro]M)    29                    146        >250

Compound
                      Coxsackie virus B4  Punta Toro virus

3a                    >100                >100
3b                     >20                 >20
3c                      >4                  >4
3d                    >100                >100
3e                      >4                  >4
3f                    >100                >100
3g                      >8                  >8
3h                     >20                 >20
3i                     >20                 >20
3j                     >20                 >20
DS-5000 ([micro]M)    >100                  20
(S)-DHPA ([micro]M)   >250                >250
Ribavirin ([micro]M)  >250                 112

(a) Required to cause a microscopically detectable alteration of
normal cell morphology, (b) Required to reduce virus-induced
cytopathogenicity by 50%
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Title Annotation:ORIGINAL ARTICLE
Author:Guzeldemirci, Nuray Ulusoy; Karaman, Berin; Kucukbasmaci, Omer
Publication:Turkish Journal of Pharmaceutical Sciences
Article Type:Report
Date:Aug 1, 2017
Words:4427
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