Technical Field

The present invention relates to novel IL-1 antagonists which are synthesized as small molecules for treatment of different diseases. Most particularly, the present invention provides novel 5- fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3-(4-phenylthiosemicarbazone] derivatives as well as methods for manufacturing these compounds and novel use ofthe same as IL-1 antagonists.

Background of the Invention

Interleukin 1 (IL-1], a potent pro-inflammatory cytokine, is considered to be an important therapeutic target since it plays a critical role in the pathogenesis of several rare and common inflammatory diseases, especially of autoimmune nature. It is crucial to develop a molecule that can control the effects of IL-1 in the treatment of autoinflammatory diseases such as Behcet's disease as well as other chronic diseases, e.g. atherosclerotic vascular disease, diabetes, gout and Alzheimer's. IL-1 is one of the most potent pro-inflammatory cytokines, and to date, with the exception of protein-based IL-1 receptor (IL-1R] antagonist anakinra and anti-IL-Ib neutralization antibodies, rilonasept and canakinumab, no synthetic -small- chemical molecule has been developed that acts as an IL-1 antagonist

The development of a molecule capable of controlling the effects of IL-1 in the treatment of the diseases where IL-l’s principal role is of prime importance (Dinarello, CA, Simon , A. and Van der Meer, JWM 2012, Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nature Reviews Drug Discovery, 11 (8), 633-652; Schett, G., Dayer ; JM and Manger, B. 2016, Interleukin-1 function and role in rheumatic disease Nature Reviews Rheumatology, 12 (1), 14-24 ).

The amino acid Lysll2 through which IL-1 binds to IL-1R was determined by in silico methods, and 2-indolinone derivatives were screened using the "docking" approach. l//-indole-2,3-dione 3-thiosemicarbazone derivatives carrying the 3-sulfamoylphenyl residue with the highest affinity binding to the Lysll2 region and new 2-indolinone derivatives were identified, and their free binding energies were calculated (Eraslan Elma, P. 2017, lH-indole-2,3-dione 3-thiosemicarbazone derivatives carrying 3-sulfamoylphenyl moiety: Synthesis, structure determination, molecular modeling and biological activity evaluation. Istanbul University, Institute of Health Sciences, Department of Pharmaceutical Chemistry, Ph.D. Thesis, Istanbul, Turkey; Ermut, G., Steady, N., Cetin, Rel, Topcul, M. et Birteksoz, S. 2013, Synthesis and chemotherapeutic activities of 5-chloro-lH- indole-2,3-dione 3-thiosemicarbazones. Marmara Pharmaceutical Journal, 17 (2), 147-154; Guzel, 0., Karali, N. and Salman , A. 2008, Synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy- lH-indole-2,3-dione 3-thiosemicarbazone derivatives Bioorganic & Medicinal Chemistry, 16 (19), 8976-8987; Karali, N. and Gursoy, A. 1994, Synthesis and anticonvulsant activity of some new thiosemicarbazone and 4-thiazoIidone derivatives bearing an isatin moiety, Farmaco (Societa Chimica Italiana: 1989), 49 (12), 819-822; , Karali, N.L., Gursoy, A., Terzioglu, N., Ozkirimli, S., Ozer, H. and Ekinci, A.C. 1998, Synthesis and preliminary CNS depressant activity evaluation of new 3-[(3- substituted-5-methyl-4-thiazolidinon-2-ylidene)hydrazono]-lH -2-indolinones and 3-[(2-thioxo-3- substituted-4,5-imidazolidinedione-l-yl)imino]-lH-2-indolino nes. Archiv der Pharmazie, 331 (7-8), 254-258; Karali, N. L, Terzioglu, N. and Gursoy, A. 2002, Synthesis and primary evaluation of new 5- bromo-3-substituted-hydrazono-lH-2-indolinones. Archiv der Pharmazie, 335 (8), 374-380; Karali, N., Gursoy, A., Kandemirli, F., Shvets , N., Source, F.B., Ozbey, S. et al. 2007, Synthesis and structure- antituberculosis activity relationship of lH-indole-2,3-dione derivatives. Bioorganic & Medicinal Chemistry, 15 (17), 5888-5904; Karali, N., Akdemir, A., Goktas, F., Eraslan Elma, P., Angeli, A., Kizilirmak, M. et al. 2017a, Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-associated alpha carbonic anhydrases. Bioorganic & Medicinal Chemistry, 25 (14), 3714-3718; Karali, N., Goktas, F., Cihan-Ustundag, G., Eraslan-Elma, P. 2017b, N-[(aminosulfonyl)phenyl]-2-(l,2- dihydro-2-oxo-3H-indoIe)-3-yIidene)hydrazine-carbothio amide derivatives for treating cancer and immunological disorders. PCT Publication No: WO 2017/099695 Al; Karali, N., Gokta$, F. and Eraslan-Elma, P. 2015, lH-indole-2,3-dione 3-thiosemicarbazone derivatives carrying new sulfamoyl residue and their synthesis methods. Turkish Patent Application No, TR 2015/16135; Terzioglu, N., Karali, N., Gursoy A., Pannecouque C, Leysen P., Paeshuyse J. et al. 2006, Synthesis and primary antiviral activity evaluation of3-hydrazono-5-nitro-2-indoIinone derivatives. ARKIVOC, 1 , 109-118).

The newly synthesized and already registered 2-indolinone derivatives that bind to the Lysll2 region with the highest affinity were determined, and their free binding energies were calculated. In vitro tests have confirmed that most of the 2-indolinone derivatives have significant anti-IL-1 activity provided that the thiosemicarbazone structure is maintained. Thiazolidone, as well as semicarbazone derivatives, synthesized from this structure, are inactive. In vitro tests have confirmed that most of the 2-indolinone derivatives have significant IL-IR inhibitory activity provided that the thiosemicarbazone structure is maintained. Methyl or ethyl substitution in the lst-position of indole ring increased the activity whereas in most N-Mannich bases the activity was decreased. ( Gatfar ; U., 2015 Identification and characterization of specific caspase-1 and IL-1 inhibitors. KoV University Graduate School of Health Sciences, M.Sc. Thesis , Istanbul, Turkey; Karali, N., Gul, A., Lack, N., Guzel-Akdemir ; 0. and Erman, B. 2016, Original lH-indole-2,3-dione 3- thiosemicarbazone derivative IL-1 antagonists Patent Application No: TR 2016/18981). 1-methyl- 5-(trifluoromethoxy] substituted compounds in l//-indole-2,3-dione 3-(4-thiosemicarbazone] derivatives showed the highest in vitro IL-IR antagonist activity below the micromolar range in screening tests.

Among the previously synthesized l//-indole-2,3-dione 3-(4-thiosemicarbazone] compounds im prior art, l-methyl-5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(4-methylphenyl] thiosemi- carbazone] (D21), displayed the highest in vitro IL-IR blocking activity [TR 2016/18981):

In the 4th-position of the thiosemicarbazone moiety, the substitutions of methyl, ethyl and allyl groups with n-butyl, cyclohexyl, and benzyl reduced the activity. The most effective derivatives were phenyl and substituted phenyl derivatives which contain methyl, fluor, and trifluoromethoxy groups at 5th-position in indole ring.

Despite of the foregoing state of the art, there is an unmet need in the relevant field to provide alternative 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3-(4-phenylthiosemicarbazone] derivatives which may have sufficient or higher level of affinity to IL-IR and TLR2, sufficient or better level of antipyretic and antiinflamatory activity, as well as prominence for treatment of diseases including Behcet's disease, atherosclerosis, diabetes, gout or Alzheimer's disease.

Brief Description of the Figures

Figure 1 shows interaction sites between IL-Ib and IL-1RI.

Figure 2 shows complex structures of IL-1RI crystal structure with IL-Ib, anakinra and compound

D21.

Figure 3 shows amino acids at which the compound D21 interacts at the best binding position.

Figure 4 shows the molecular structure of (Z] 5-fluoro-l-methyl-l//-indole-2,3-dione 3-[4-(4- methylthiophenyl]thiosemicarbazone] (4E8-2]

Figure 5 shows hydrogen bonds of the compound designated as 4E8-2 according to the present invention. Figure 6 shows hydrogen bonds of the compound designated as 5-fluoro-l-methyl-l//-indole-2,3- dione 3-[4-(3-fluorophenyl]thiosemicarbazone] (3E3-2) according to the present invention.

Figure 7 shows layers in the a-axis of compound 3E3-2.

Figure 8 shows intramolecular hydrogen bonds of (Z]l-methyl-5-(trifluoromethoxy]-l//-indole- 2,3-dione 3-[4-(4-methoxyphenyl]thiosemicarbazone] (4D7-2]

Figure 9 shows a view of compound 4D7-2 along the a-axis (A], b-axis (B] and c-axis (C] of the hydrogen bonds and crystal sequence.

Figure 10 shows location of compound 4E5-4 on the electrostatic potential surface area map in the active zone.

Figure 11 shows interactions of compound 4E5-4 in the active site of the receptor.

Figure 12 shows placement of the compound 4D8-2 ligand on the electrostatic potential surface area map in the active site.

Figure 13 shows interactions of compound 4D8-2 ligand in the active site.

Figure 14 shows effect of compound 4E5-4 on LPS-induced body temperature increase in Balb-c mice.

Figure 15 shows effect of compound 4E5-4 on LPS-induced body temperature increase in Balb-c mice.

Figure 16 shows histopathological examination of the liver tissues obtained from Balb-c mice for the compound 4E5-4 (Hematoxylin-Eosin staining].

Figure 17 shows antipyretic effect of compound 4D8-2 on LPS-induced body temperature increase in Lewis rats.

Figure 18 shows antipyretic effect of compound 4D8-2 on LPS-induced body temperature increase in Balb-c mice.

Figure 19 shows histopathological examination of the kidney (left] and liver (right] tissues obtained from Lewis rats for the compound 4D8-2 (Hematoxylin-Eosin staining].

Figure 20 shows the effects of compound 4D8-2 on blood pressure in Balb-c mice (* P=0,0047 # P=0,0044] Detailed Description of the Invention

The present invention provides 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3-(4- phenylthiosemicarbazone] derivatives as well as methods for manufacturing these compounds and novel use of the same as IL-1 antagonists. In other aspects, the invention pertains to these compounds for use in treatment of autoinflammatory diseases such as Behcet's disease as well as other chronic diseases, e.g. atherosclerotic vascular disease, diabetes, gout and Alzheimer's disease.

In screening tests, it was found that new derivatives had no negative effect on cell viability, but they also showed an antagonistic effect on TLR2 receptors below the micromolar range. The previously synthesized l-methyl-5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(4-methyl- phenyl]thiosemicarbazone] (D21) compound has been used for comparison. A series of compounds with a higher affinity to IL-1R than D21, bearing the 5-fluoro/(trifluoromethoxy]-l//- indole-2,3-dione 3 -[4- (substituted phenyl]thiosemicarbazone] structure, were modelled and their free binding energies to IL-IRs were computed, as well as to that of the TLR2 and second messengers such as MyD88 and IRAK4.

Novel 2-indolinone derivatives showed significant targeted and non-toxic effects in both in silico screenings and in vitro tests. Based on the results of this study, the lead compounds, l-benzyl-5- fluoro-l//-indole-2,3-dione 3-[4-(4-bromophenyl]thiosemicarbazone] (4E5-4) and l-methyl-5- (trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(4-trifluoromethoxyphenyl]thiosemicarbazone] (4D8-2) were selected as the exemplary and preferred synthetic IL-IR inhibitors.

4E5-4 4D8-2

IL-1 mediated antipyretic effect, and the acute toxic effects of these agents have been examined in animal models (Sevinpli, Z.$. 2018, Synthesis and investigation of biological effects of 1 -substituted 5-fluoro-lH-2-indolinone derivatives. Health Sciences Institute, Department of Pharmaceutical Chemistry, Ph.D. Thesis , Istanbul, Soylu, O. 2019, Design, synthesis and investigation of activities of interleukin-1 inhibitor drug candidate new 5-(trifluoromethoxy)-lH-indole-2,3-dione 3-(4- phenylthiosemicarbazone j derivatives , Istanbul University, Institute of Health Sciences, Department of Pharmaceutical Chemistry, Ph.D. Thesis, Istanbul).

Therefore, in a more particular aspect, the present invention provides 5- fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3 -[4- (substituted phenylthiosemicarbazone] derivatives of general formula (IV):

Formula IV wherein Ri, R2 and R3 are selected approprietely as follows, with the proviso that resulting compound is one of those recited in the List-1 below:

Riis H, 2-CHs, 3-CHs, 4-CH _¾ 2-C2H5, 3-C2H5, 4-C2H5, 2-CF _¾ 3-CF _¾ 4-CF _¾ 4-/-C3H7 4-t-C ₄ H _9, 2-OCH3, 3-OCH _¾ 4-0CH _¾ 2-SCH _¾ 3-SCH _¾ 4-SCH _¾ 2-OCF _¾ 3-OCF _¾ 4-0CF _¾ 3-SCF _¾ 4-SCF _¾ 2-F, 3-F, 4-F, 2 -Cl, 3 -Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-1, 3-1, 4-1, 2-CN, 3-CN, 4-CN, 2-NH ₂ , 3-NH ₂ , 4-NH2, 2-NO2, 3-NO2, 4-NO2, 4-N(CH ₃ ] ₂ , 4-CONH2, 4-COOH, 4-SO3H, 2-OH, 3-OH, 4-OH, 4- N ₂ , 4-C ₆ H ₅ , 2,3-diCH ₃ , 2,4-diCH ₃ , 2,5-diCH ₃ , 2,6-diCH ₃ , 3,5-diCF ₃ , 2,4-diOCH ₃ , 2,5-diOCH ₃ , 3,4-diOCH ₃ , 3,5-diOCH ₃ , 2-CH ₃ -3-Cl, 2-CH ₃ -5-Cl, 4-CH ₃ -3-CF ₃ , 2-OCH ₃ -5-CH ₃ , 2-F-5-CF ₃ , 4- F-3-CF3, 2-Cl-5-CF ₃ , 4-Cl-3-CF ₃ , 5-Cl-2-OCH ₃ , 2,4-diF, 2,5-diF, 2,6-diF, 3,5-diF, 2,3-diCl, 2,4- diCl, 2,5-diCl, 2,6-diCl, 3,4-diCl, 3,5-diCl, 4-Br-2-F, 2,4,6-triCH ₃ , 3,4,5-triOCH ₃ , 2,3,4-triF, 2,4,5-triF, 2,4,6-triF, 2,4,5-triCl, 2,4,6-triCl or pentaF,

R2 is F or OCF ₃ ,

R ₃ is one of the groups listed below; H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , CH(CH ₃ ) ₂ , CH ₂ CH=CH ₂ , CH ₂ CH(CH ₃ ] ₂ , CH ₂ COOH, CH(CH ₃ )COOH, CH ₂ COONa, CH(CH ₃ )COONa, CH ₂ COOC ₂ H _5, CH(CH ₃ ]C00C ₂ H5, CH ₂ C00CH ₂ 0C0CH3, CH(CH ₃ ]C00CH ₂ C0CH3, CH ₂ COOCH(CH ₃ ]OCOCH _3, CH(CH3]C00CH(CH ₃ ]0C0CH3, CH2C00CH20C0C(CH ₃ ]3, CH(CH ₃ ]COOCH ₂ OCOC(CH ₃ ]3, CH2C00CH(CH ₃ ]0C0C(CH3]3, CH(CH3]C00CH(CH3]0C0C(CH ₃ ]3, CH2COOCH2OCOOCH3, CH(CH ₃ ]COOCH ₂ OCOOCH ₃ , CH ₂ C00CH(CH ₃ ]0C00CH3, CH(CH3]C00CH(CH ₃ ]0C00CH3, CH ₂ C00CH ₂ 0C00C ₂ H ₅ , CH(CH ₃ ]C00CH ₂ 0C00C ₂ H ₅ , CH ₂ C00CH(CH ₃ ]0C00C ₂ H5, CH(CH ₃ ]COOCH(CH ₃ ]OCOOC ₂ H ₅ ,

List 1- Compounds according to the invention

In more particular embodiments, the compounds with Formula IV according to the present invention are selected from the list below:

In another aspect, the present invention provides methods for synthesizing above compounds. The 4- (substituted phenyl]thiosemicarbazide derivative (I] is obtained from the reaction of a substituted aniline derivative (A] with thiophosgene and hydrazine hydrate. Second, by reaction of 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (II] with R ₃ X, 1-substituted 5- fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (III] is synthesized. Third, by reaction of I with II or III in ethanolic medium, 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3 -[4- (substituted phenyl]thiosemicarbazone] derivatives (IV] are synthesized (Scheme 1] It is noted that adding a catalytic amount of sulfuric acid into the medium significantly reduced the reaction times and increased the yield in the synthesis of the derivatives.

Scheme 1 Therefore, the present invention provides a process for producing compound of Formula IV selected from the List-1 above comprising the steps of: reacting compound of Formula II or III, with a compound of Formula I,

I and obtaining the compound of Formula IV, wherein; Ri, R ₂ and R ₃ are as defined above such that the resulting compound of Formula IV is one of those recited in the List-1.

In another aspect, the present invention provides a compound having Formula III which is a useful intermediate for the production of the compunds of Formula IV; wherein; R ₂ and R ₃ are selected approprietely as follows, with the proviso that resulting compound is one of those recited in the List-2 below:

R ₂ is; F or OCF ₃ ,

R3 is one of the groups listed below; H, CH ₃ , C ₂ H5, C ₃ H7, CH(CH ₃ ] ₂ , CH ₂ CF CH ₂ , CFhCHfCHs^, CH ₂ COOH, CH(CH ₃ ]C00H, CFLCOONa, CH(CH ₃ ]COONa, CHzCOOCzHs.CHfCHsJCOOCzHs _,

CH ₂ C00CH ₂ 0C0CH _3, CH(CH ₃ ]C00CH ₂ C0CH ₃ , CH ₂ COOCH(CH ₃ ]OCOCH _3, CH(CH ₃ ]C00CH(CH ₃ ]0C0CH _3, CH2C00CH20C0C(CH ₃ ] _3, CH(CH ₃ ]C00CH ₂ 0C0C(CH ₃ ] ₃ , CH2C00CH(CH ₃ ]0C0C(CH ₃ ] _3, CH(CH ₃ ]C00CH(CH ₃ ]0C0C(CH ₃ ] _3, CH2COOCH2OCOOCH ₃ , CH(CH ₃ ]COOCH ₂ OCOOCH ₃ , CH ₂ C00CH(CH ₃ ]0C00CH _3, CH(CH ₃ ]C00CH(CH ₃ ]0C00CH _3, CH ₂ COOCH ₂ OCOOC ₂ H5, CH(CH ₃ ]C00CH ₂ 0C00C ₂ H5, CH ₂ C00CH(CH ₃ ]0C00C ₂ H5,

CH(CH ₃ ]COOCH(CH ₃ ]OCOOC ₂ H ₅ ,

List-2: Intermediates according to the present invention In another aspect, the present invention provides a process for producing the above compounds with Formula III as provided in List-2 comprising: reacting compound of Formula II, with a compound of R3X, and obtaining the compound of Formula III, wherein;

R2 and R3 are as defined above, and X is a halogen, such that resulting compound with Formula III is one of those provided in List-2.

Still in a further aspect, the present invention provides the compounds of Formula IV for use as an IL-1 antagonist. These compounds have been shown to be useful as antipyretic and antiinflamatory agent. More particularly, these compounds are provided for use in treatment of Behcet's disease, atherosclerosis, diabetes, gout or Alzheimer's disease.

Additionally the present invention provides pharmaceutical compositions comprising a compound having general Formula IV as identified above along with a pharmaceutically acceptable excipient

X-ray single crystal crystallography studies showed that the molecular and isomeric structures of the compounds with Formula IV were in Z-configuration. Molecular modeling studies of IL-IR were performed, and physicochemical parameters were calculated.

The inventors have performed molecular biology experiments on cell lines tailored to express either IL-1, or TLR2 receptors were stimulated using IL-1 and lipopolysaccharide (LPS], respectively. The reporter system consisted of the measurement of the secreted embryonic alkaline phosphatase (SEAP] The activity and selectivity of the compound IV were determined. Tetrazolium proliferation assay was used for cell viability. Among the new compounds that were identified as relatively selective and non-toxic IL-IR antagonists in in vitro experiments, 1-benzyl- 5-fluoro-l//-indole-2,3-dione 3-[4-(4-bromophenyl]thiosemicarbazone] (4E5-4) and 1-methyl- 5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(4-trifluoromethoxyphenyl]thiosemi- carbazone] (4D8-2) were selected in order to be used in animal experiments. In vivo experiments assessing the antipyretic action showed that new lead compounds reversed LPS-induced increase in body temperature. Histopathological examination revealed no damage to kidney and liver tissues.

The following compounds were used in the Examples for comparison:

Example 1 - Synthesis and Purification Methods

Synthesis of N- (substituted phenyl) isothiocyanate derivatives (B)

A substituted aniline (A] (60 mmol] is dissolved in 100 mL of water containing 25 mL of concentrated HC1. After addition of the thiophosgene (60 mmol, 6.79 g], the mixture is stirred at room temperature until the red color of the thiophosgene disappears. The white crystalline product formed is filtered off and washed with water.

Synthesis of N- (substituted phenyl)thiosemicarbazide derivatives (I) A solution of N- (substituted phenyl]isothiocyanate (B] (50 mmol] in 20 mL of ethanol is added dropwise to a solution of hydrazine hydrate cooled in ice water (50 mmol] in 20 ml of ethanol with continuous stirring. The product immediately precipitated is filtered and crystallized from ethanol. General Synthesis of 1-Substituted 5-fluoro/(trifluoromethoxy)-lH-indoIe-2,3-dione derivatives (III)

1.17 g of 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (II] (5 mmol] and 0.97 g of anhydrous K ₂ CO ₃ (7 mmol] in 10 ml of DMF were continuously mixed at room temperature for 1 hour. R ₂ halide (15 mmol] and 0.17 gof KI (1 mmol] were added to the mixture as a catalyst and heated to 50-60 °C under reverse continuous stirring until the reaction is complete. The mixture is first evaporated to dryness under reduced pressure, then the solid product obtained was washed with water to remove excess K ₂ CO ₃ and KI, and purified by crystallization with ethanol to obtain the crude product.

General Synthesis of 5-Fluoro/(trifluoromethoxy)-lH-indole-2,3-dione 3 -[4- (substituted phenyl)thiosemicarbazone] derivatives I (IV)

4- (Substituted phenyl]thiosemicarbazide (I] (2.5 mmol] was added in 10 ml of ethanol solution containing either 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (II] or 1-substituted 5- fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (III] (2.5 mmol]. The mixture was heated in a back cooler until the reaction is complete under surveillance by thin layer chromatography. The precipitated crude product was filtered off, washed with ethanol or crystallized.

General Synthesis of 5-Fluoro/(trifluoromethoxy)-lH-indole-2,3-dione 3 -[4- (substituted phenyl)thiosemicarbazone] derivatives II (IV)

10 ml of 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (II] or 1-substituted 5- fluoro/(trifluoromethoxy]-l//-indole-2,3-dione (III] (2.5 mmol] is added to the solution in absolute ethanol from 4- (substituted phenyl]thiosemicarbazide (I] (2.5 mmol] and ethanol as a catalyst in trace amounts of acid (concentrated sulphuric acid, glacial acetic acid, concentrated hydrochloric acid or para-toluenesulfonic acid]. The mixture is heated in back cooler, under surveillance by thin-layer chromatography until the reaction is complete. The precipitated crude product is filtered off, washed with ethanol or crystallized.

Example 2 - Molecular Modeling Study for Interleukin- 1 Receptor Type 1 flL-lRll Structure

Preparation of Protein Structure

The crystal structure of IL-1R1 for determination of inhibitors in computer medium was obtained from Protein Data Bank with the code HTB.pdb (2,5 A] This crystal structure includes IL-Ib and IL-1R1 (Vigers, GP, Anderson , Lf Caffes , P. and Brandhuber, B] (1997). Crystal structure of type 1 interleukin-1 receptor complexed with interleukin- 1b. Nature 386 (6621), 190-194). In order not to affect the ligand-protein binding relationships in the Maestro® software, ligand IL-Ib protein structure was deleted with water molecules, the Protein Preparation Wizard application completed incomplete hydrogen atoms and amino acid fragments, energy structure was minimized, and protein structure was recorded as mae file.

Preparation of Ligands

Molecules were drawn by 2D Sketcher application in Maestro®, hydrogen atoms were added and energy minimization at pH 7 according to 0PLS3e method was created with LigPrep application, and three-dimensional ligand structures were created and saved as mae file. QikProp application was used to calculate the pharmacokinetic parameters of molecules and to examine their compliance with Lipinski rules.

Two active sites A and B were detected in the binding of IL-Ib to IL-1RI. The amino acids His30, Glnl5, Gln32, Argil and Thrl47 in the active site A and Ile56, Lys93, Glul05, and Lys92 in the active site B are essential for ligand-receptor interactions (Figure 1] (Vigers , GP, Anderson , Lf Caffes, P. and Brandhuber, B] (1997). Nature, 386 (6621), 190-194; Heidary, DK, Roy, M., Daumy, GO, Cong, Y, and Jennings, PA (2005). Coupling between separate docking sites in Interleukin-1 b, Journal of Molecular Biology; 353 (5), 1187-1198).

The results of in vitro tests with novel 2-indolinone derivatives correlated with the free binding energy levels to IL-lRin insilico docking experiments. The binding site on IL-1R1 was determined by the computer model using the most effective lead compound D21 with the structure 1-methyl- 5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(4-methylphenyl] thiosemi- carbazone] as a template. This region is similar to the regions to which IL-Ib and IL-1 receptor antagonist anakinra bind, as shown in Figure 2, and this supports the importance of Lysll2 amino acid in binding (Figure 3]

On the protein structure prepared in Maestro, a grid was created in the center of Lysll2 amino acid. The prepared ligands were docking in Glide application by SP (Standard Precision] and XP (Extra Precision] scanning methods. Each ligand was screened 10 times to find high-score binding poses. Five poses showing the highest score for each ligand were recorded for analysis.

Exair le 3 - Interleukin- 1 Inhibition Assays

Cellular Experiments

Secreted Embryonic Alkaline Phosphatase (SEAP) Assay Rationale: For in vitro activity measurements, HEK BLUE™ interleukin- 1b (IL-Ib] cells and HEK BLUE™ hTLR2 cells with binary cell culture in DMEM (Dulbecco's Modified Eagle's Medium] (10% fetal bovine serum (FBS] + 1% penicillin/streptomycin] culture series were formed. In these assays, where selectivity other than activity was also assessed, new compounds should inhibit IL- 1R while not inhibiting TLR2. In the presence of novel compounds, the amount of alkaline phosphatase secreted by IL-Ib, and LPS was measured in IL-lR-expressing cell lines, and TLR2- expressing cell lines, respectively. By comparing the IC50 values obtained with IL-Ib and LPS, the extent to which the compounds selectively block IL-1 receptors was evaluated.

Procedure: For this experiment, 1 L of a 2 M diethanolamine solution was prepared as a colorimetric solution, 1 mM MgCU and 0.5 mM ZnCU were added, 20 mg of this solution was added with 20 mg p-nitrophenyl phosphate. On the other hand, 15% CHAPS (detergent] was prepared, and the final concentration of 0.05% DPBS (Dulbecco’s Phosphate-Buffered Saline] was mixed with the culture medium, and 20 pL CHAPS was added to the plates. 40 pL of the cell suspension prepared 24 hours in advance, 200 pL of the colorimetric solution was added and allowed to incubate for 1 hour. After incubation, the absorbance in each well was measured at 405 nm. The results from novel compound cells were normalized according to the positive and viability controls, and the concentration-response curves were plotted.

MTS Cell Viability Test

Rationale: In the mitochondria of living cells, yellow MTS (3-(4,5-dimethylthiazol-2-yl]-5-(3- carboxymethoxyphenyl]-2-(4-sulfophenyl]-2H-tetrazolium bromide] is reduced to purple formazan. Since this reduction occurs only when mitochondrial reductase enzymes are active, the conversion is directly related to viable cell count and metabolic activity. This experiment aims to determine the mortality rate in drug-treated cells. The absorbance of the colored formazan product was determined by measuring at 490 nm.

Procedure: The stock solutions of the compounds were prepared by dissolving in DMSO to 10 mM. Each compound was prepared and tested with serial dilution at 12 different concentrations starting at 20 pM. For dilutions, preheated DMEM culture medium was used. DMEM containing 1% DMSO was prepared and added to the wells to prepare HEK BLUE cell lines. The cell suspension was prepared with 3xl0 ⁴ cells in each well, hIL-Ib protein for 150 HI HEK BLUETM IL-1 b cells and LPS agonist compound for HEK BLUETM TLR2 cells were added and incubated for 24 hours. After 24 hours, the test plates were used for the MTS assay after 40 pi were transferred for use in SEAP. 15 pL of MTS reagent was added to each well, and after 3 hours of incubation, the absorbance in each well was measured at 490 nm. Results from cells which contain novel compounds were normalized according to positive and viability controls, and concentration- response curves were plotted.

Example 4 - In vivo Experiments

Determination of the antipyretic effect

In the control and treatment of fever induced by lipopolysaccharide (LPS, Escherichia coli 0111: B4, Sigma], an IL-1 mediated response, Lewis breed rats and Balb-c breed mice were used for the leading compounds selected (4D8-2 and 4E5-4] Doses were determined by IC50 values obtained in in vitro experiments by allometric scaling.

Determination of the Least Effective Dose: In the experiment carried out in Lewis rats, since 4D8- 2 has been administered for the first time to living animals, groups of 1-3 animals were formed for each dose to be tested in the four-chamber Latin square order to determine the lowest effective dose. Injections of LPS (220 pg/kg, subcutaneous] and compound or vehicle were simultaneously administered. The compound was administered intraperitoneally (i.p.] by reducing one log unit starting from the highest dose (150 pg/kg] with a detected effect Body temperature was measured by infrared thermometers over rectal and abdominal skin every 20 minutes for five hours. Rectal and skin temperature measurements were correlated with each other.

Antipyretic effect measurement procedure: In the experiment, 16 rats weighing 150-250 g were divided into control and treatment groups. Infrared and rectal thermometers measured the body temperature of rats under isoflurane anesthesia, and the results were recorded as baseline values. Measurements were made by pushing the rectal thermometer up to 4 cm from the rectum. Since the skin temperature of the animal was measured by infrared method, measurements were performed by shaving 2x2 cm area in the abdominal region, and the results were recorded. After the basal measurement, 220 pg/kg LPS was injected subcutaneously, and the animals were taken to their cages and left to their natural shelter conditions for 3 hours to induce fever.

At the end of 3 hours, the lowest (1,5 pg/kg] of the 3 different doses detected in the dose determination experiments was injected intraperitoneally under isoflurane anesthesia. After 4D8-2 or 4E5-4 injections, fever measurements were performed with rectal and infrared thermometers at 20-minute intervals. Sixty minutes after the first injection, the second injection was made to reach 15 pg/kg. Likewise, a measurement was made in every 20 minutes, and at the end of the second hour, the compound dose was increased cumulatively to 150 pg/kg. After the last injection, the test was terminated at the end of 1 hour with 3 measurements at 20-minute intervals, and the animals were euthanized following intracardiac blood collection under anesthesia. Heart, lung, spleen, kidney, and liver tissues were collected for histopathological examination. The collected tissue samples were divided into different groups and stored in -80 ^Q C and 10% neutral formalin solutions for further analysis.

In the experiments with Balb-c mice, 10 mice weighing 25-30 g were divided into control and treatment groups. A rectal thermometer measured fever, and the results were recorded as the baseline. After the basal measurement, 220 pg/kg LPS was injected subcutaneously into the animals, and the animals were taken to their cages for 3 hours to induce fever and left to their natural shelter conditions. At the end of 3 hours, the lowest (2 pg/kg] of the 3 different doses derived from the IC ₅₀ values injected intraperitoneally into the animals. Fever measurements were made with a rectal thermometer at 30-minute intervals after 4D8-2 injection. 90 minutes after the first compound injection, the second dose was injected cumulatively to 20 pg/kg.

Similarly, measurements were taken at 30-minute intervals, and the compound dose was increased to 200 pg/kg at the end of the second 1.5 hours. The test was terminated at the end of 1.5 hours after the last dose with 3 measurements at 30-minute intervals, and animals were euthanized following intracardiac blood collection under anesthesia. Blood was centrifuged and stored in -80 °C for further analysis.

Balb-c mice weighing 25-30 g for 4E5-4 were used in the LPS-induced fever experiments as a second species. The doses used were derived from IC50 values obtained in in vitro experiments by allometric scaling. The experiments for the lowest effective dose detection, as stated above (4D8- 2 experiments] were not repeated for this molecule. Instead, the dose was estimated comparing the EC50 values of both molecules. This allowed the completion of the experiments with fewer animals.

IR Findings

Thioamide N-H stretching bands in the solid phase (KBr] IR spectra of 5- fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3 -[4- (substituted phenyl]thiosemicarbazone] compounds (IV] were observed in single, two or three separate bands. The most crucial finding showing the formation of thiosemicarbazone structure by condensation was that the ketone C=0 stretching band which is observed in the spectra of compounds II and III is not observed in the spectra of the compounds IV, but also the presence of the C=0 stretching (amide I] band which defines the lactam group. The presence of C=N stretching, aromatic C=C stretching, N-H bending combination bands, and C=S stretching band proved the presence of thioamide group; also the absence of S-H stretching band in the range of 2500-2600 cm ¹ wavelength altogether supported the solid-state thion form of compounds IV.

Single Crystal X-ray Diffraction Findings

The crystal and molecular structure of 4E8-2 was determined by single-crystal X-ray crystallography study (Figure 4] In the compound which is found to be in the Z form, the two intramolecular N — H ··· N and N — H ··· 0 hydrogen bonds are formed between thioamide N4-H (N4-H) and thioamide, N1 (N2), and thioamide N2-H (N3-H) and indole lactam oxygen (01), respectively (Figure 5). The hydrogen bonds CH ··· S which are found between the layers of molecules parallel to the plane are formed between indole C6-H (C3-H) and thioamide sulfur atom (Si) and between the methyl group (C9) protons in the l ^st -position of the indole ring and the thiomethyl group sulfur atom (S2) in the phenyl ring. So hydrogen bonds between these molecules stabilize the compound ( Atioglu , Z., Sevinfli, Z.§., Karah , N., Akkurt, M. and Ersanli, CC 2017a, 2- (5- FIuoro-l-methyI-2-oxoindoIein-3-yIidene)-N-[4-(methyIsuIfany I) phenyl] hydrazine-l-carbothio amide, IUCrData, 2 (5), xl 70671 ).

Molecular and isomeric structure of 5-fluoro-l-methyl-l//-indole-2,3-dione 3 -[4- (3 -fluorophenyl) thiosemicarbazone] (3E3-2), and inter and intramolecular hydrogen bonds were verified using single-crystal X-ray diffraction method (Figure 6). The compound was in the Z-form. Three intramolecular hydrogen bonds N — H ··· N, N — H ··· 0 and C — H ···. S bonds were formed between thioamide N4 (N4) proton and thioamide Ni (N2) proton, thioamide N2 (N3) proton and indole lactam oxygen (Oi), and phenyl C2 (C12) proton and thioamide sulfur (Si), respectively (Figure. 7). It was found that the hydrogen bonds CH ··· F between the molecular layers parallel to the plane stabilize the compound, these intermolecular hydrogen bonds were formed between indole C7-H (C2-H) and fluor (F2) at the 3rd- position of the phenyl ring ( Atioglu , Z., Sevincli, Z.S., Karali, N., Akkurt, M. and Ersanli, CC 2017b, ( 2Z)-2-(5-FIuoro-l-methyI-2-oxoindoIein-3-yIidene)-N - (3) -fluorophenyl) hydrazine-l-carbothio amide, IUCrData, 2 (6), xl70900).

Single-crystal X-ray diffraction method determined that l-methyl-5-(trifluoromethoxy)-lH- indole-2,3-dione 3-[4-(4-methoxyphenyl)thiosemicarbazone] (4D7-2) has Z-isomeric structure. Three intra-molecular hydrogen bonds, namely N — H ··· N, N — H ··· 0 and C — H ··· S, intermolecular hydrogen bonds C — H ··· 0, p-p interactions and weak CO ·· · p (ring) relationships contribute to the stabilization of the crystal structure. Intra-molecular N — H ··· N, N — H ··· 0 and C — H ··· S bonds with thionamide N4 proton (N4-H) and Ni (N2), thionamide N2 proton (N3-H) and lactam oxygen (Oi), and phenyl C2 proton (C13-H) and sulfur (Si), respectively p-p interactions were observed between the indole ring and the 4-methoxyphenyl ring (Figures 8 and 9). ¹ H-NMR Findings

Thioamide N2 proton signal attached to azomethine group and thioamide N4 proton signal to which the phenyl group is attached were observed as singlet in ^! H-NMR spectra of DMSO-c/ of 1- (non) substituted 5-fluoro/(trifluoromethoxy)-l//-indole-2,3-dione 3 -[4- (substituted phenyl)- thiosemicarbazone] derivatives (IV). The absence of the thiol peak in the spectrum proves that thiosemicarbazone derivatives are present in thion form. Intra- and/or intermolecular hydrogen bonds and electron densities decrease the thioamide N2 and N4 proton signals, thus yielding high ppm values. Interaction constant values of indole proton signals were in line with the literature, confirming present findings. Resonance values of aromatic protons were determined by considering ¹ H- ¹³ C relationships and substituent effects in HSQC-2D and HMBC-2D spectra of the compound.

In the ^! H-NMR spectra of the compounds taken in DMSO-c/ medium, the presence of singlet and single peaks of thioamide N2 and N4 proton signals, as well as a single signal of the phenyl protons, and singlet and single peaks of the methyl, ethyl, methoxy and thiomethyl groups in the phenyl ring confirmed that the compounds were eluded only in Z-isomer form. By illuminating their crystal structures, X-ray single-crystal diffraction method supports that the synthesized 4E8-2, 3E3-2 and 4D7-2 compounds are indeed in Z-isomer form.

Z izomer E izomer

¹³ C-NMR Findings

Selected prototype compounds were subjected to ¹³ C-NMR (Decoupled-DMSO-c/ _, 100 MHz) analysis for the verification of the ³ H-NMR findings of the synthesized compounds. The indole ring and thiosemicarbazide residue carbon signals were determined in the spectra of the compounds, and the confirmation by the interaction constants of the interruption of the indole and phenyl signals by ⁱ³ C- ¹⁹ F interaction supported this data. Indole carbon signals in the compounds IV were monitored following the literature, starting from the top domain, respectively C7, C4, C3 _¾ C ₆ , C3, C7 _¾ C5, and C2. In the compounds II and III, the signals of indole C3 ketone carbonyl carbon were not observed in thiosemicarbazone structure, the signals defining the azomethine (C=N) group and the signals of indole C2 lactam carbonyl were observed. HSQC-2D (Heteronuclear Single Quantum Coherence] was used to identify the carbon signals of some compounds carrying hydrogen and showing ¹³ 0- ^! H relationship at a single bond distance and to support ^! ff-NMR, ¹³ C-NMR (Decoupled] and DEPT findings. HMBC-2D (Heteronuclear Multiple Bond Correlation] analyses were performed to identify protonated carbon signals and to give accuracy to ^! ff-NMR, ¹³ C-NMR (Decoupled], DEPT and HSQC-2D findings.

LCMS Findings

In the mass spectra of some compounds obtained by positive ionization technique, [M+H] ⁺ peaks were observed as the base peak in all of the compounds and confirmed the molecular weights of the compounds. [M+H] ⁺ +2 peaks were determined in the spectra of both the 3-chlorophenyl and 4-chlorophenyl radical-bearing compounds at a relative abundance ratio of about 3: 1 (100: 37] resulting from isotopes 35 and 37 of the chlorine atom. In the spectrum of the compound carrying the 4-bromophenyl residue, [M+H] ⁺ +2 peak defined the relative abundance ratio of about 1:1 (100:94] resulting from the isotopes 79 and 81 of the bromine atom.

Molecular Modeling Findings

The molecular modeling of 4E5-4 and 4D8-2, selected as the leading compounds, was carried out with the components of the Schrodinger-Small-Molecule Drug Discovery Suite (v. 2018-3, Schrodinger LLC, New York, 2019]

For 4E5-4, XP G-Score: -2.320 AG (kcal/mol]: -2.310, IC ₅₀ (M]: 0.023 (Figures 10 and 11].

In the ligand 4D8-2, the IC50 value was calculated to be 20.31 mM with free binding energy of 2,400 kcal/mol (Figures 12 and 13].

Simulation of Pharmacokinetic Parameters

Some 1- (non] substituted 5-fluoro/(trifluoromethoxy]-l//-indole-2,3-dione 3 -[4- (substituted phenyl]thiosemicarbazone] compounds (IV] were evaluated for Lipinski rules. In the molecular docking calculations, the number of hydrogen bond donors was found to be 1, and the number of hydrogen bond acceptor number was between 5.5-6.25. These results show that all compounds comply with Lipinski rules in terms of hydrogen bond donor and acceptor groups. As far as the molecular weights of the compounds are concerned, all of the R3 methyl and ethyl substituted derivatives, as well as some R3 benzyl substituted compounds, have molecular weights of less than 500 Daltons and meet the criteria. In the D series, l-methyl-5-(trifluoromethoxy]-l//-indole-2,3- dione 3-[4-(3-methylphenyl]thiosemicarbazone] (3D2-2], l-methyl-5-(trifluoromethoxy]-l//- indole-2,3-dione 3-[4-(3-methoxyphenyl]thiosemicarbazone] (3D7-2], l-methyl-5-(trifluoro- methoxy]-l//-indole-2,3-dione 3-[4-(4-methoxyphenyl]thiosemicarbazone] (4D7-2], 1-methyl- 5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(3-fluorophenyl]thiosemicarbazone] (3D10-2], l-methyl-5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(3-chlorophenyl]thiosemicarbazone] (3D11-2), l-ethyl-5-(trifluoromethoxy)-l//-indole-2,3-dione 3-(4-phenyl)thiosemi- carbazone] (4D1-3], l-ethyl-5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(3-methoxyphenyl] thiosemicarbazone] (3D7-3] and l-ethyl-5-(trifluoromethoxy]-l//-indole-2,3-dione 3-[4-(4- methoxyphenyl]thiosemicarbazone] (4D7-3] have log P values in the range of 4.533-4.940 and meet the criterion. All of the methyl and ethyl substituted derivatives (except l-ethyl-5-fluoro- l//-indole-2,3-dione 3-[4-(4-trifluoromethoxyphenyl)thiosemicarbazone (4E10-3]] were found to have log P values in the range of 3.58-4.924 and meet the criteria. When examined in terms of the number of non-compliant Lipinski rules the compounds with code names 3D2-2, 3D7-2, 4D7-2, 3D10-2, 4D1-3, 3D7-3 and 4D7-3, and all of R3 methyl and ethyl substituted derivatives of E-coded compounds (except 4E10-3] were found all compliant despite the fact that some had log P values above 5, i.e., one R05 rule violation. In this classification system, "one violation" is the maximum number allowed to be compliant with R05 rules. Therefore, all described compounds have sufficient properties to become drug candidates. Since most of the benzyl derivatives of the D-coded compounds had log P values above 5 and molecular weights above 500, they were considered non-compliant with the Lipinski rules (Tables 1 and 2).

Table 1. Some pharmacological values of certain 1-substituted 5-fluoro-lfi-indole-2,3-dione 3-[4-(substituted phenyl)thiosemicarbazone derivatives and their compliance with Lipinski rules

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3

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H Table 2. Some pharmacological values of certain 1-substituted S-(trifluoromethoxy)-l//-indole-2,3-dione 3-[4-(substituted phenyl) thioseinicarbazone derivatives and their compliance with Lipinski rules

Table 2 (continued)

Water solubility (log S), blood-brain barrier partition coefficient (log BB), central nervous system activity (CNS), skin permeability (log Kp), human serum albumin binding rate (log Khsa) and human oral absorption values were calculated in silico. In terms of water solubility, it was observed that only 3D7-2 and 4D7-2 met this criterion where log S values are preferred to be in the range of (-6.5) -0.5. This value was -6.469 and -6.474 for 3D7-2 and 4D7-2 compounds, respectively. In the E coded compounds, most of the methyl and ethyl substituted derivatives in R3 (except 4E9-2, 4E10-2, 4E9-3 and 4E10-3) were found to have a log S value in the range of (- 5.165) -(- 6.465) and were considered to be of the appropriate solubility levels (Tables 1 and 2).

LogBB describes the blood-brain barrier partition coefficient and SSS the possible activity of the compounds on the central nervous system. For a compound to cross the central nervous system, the value of LogBB is preferred to be in the range of (-3) -1.2 and SSS in the range of (-2) -2. In the coded compounds, l-methyl-5-fluoro-l//-indole-2,3-dione 3-[4-(4-trifluoromethylphenyl)- thiosemi carbazone] (4E9-2), l-methyl-5-fluoro-l//-indole-2,3-dione 3-[4-(4-trifluoromethoxy- phenyl) thiosemicarbazone] (4E10-2), l-ethyl-5-fluoro-l//-indole-2,3-dione 3-[4-(4-trifluoro- methylphenyl)thiosemicarbazone] (4E9-3), l-ethyl-5-fluoro-l//-indole-2,3-dione 3-[4-(4- trifluoromethoxyphenyl)thiosemicarbazone] (4E10-3) and l-benzyl-5-fluoro-l//-indole-2,3- dione 3-[4-(4-trifluoromethylphenyl) thiosemicarbazone] (4E9-4), CNS values were found to be one, and zero in all the others. All LogBB values were in the range (-3) -1.2 (Table 1).

The logKp values of the compounds which define the skin permeability and preferred to be in the range (-8) - (-1) were determined as (-1.78) - (-0.53). The logKhsa value, which defines the binding rate of the compounds to human serum albumin and preferred to be in the range of (-1.5) -1.5, was found in the range of 0.164-1.194. In humans, the percentage of oral absorption for agents with high and poor oral efficacy were defined as >80, and <25, respectively. Calculating this value as 100 for all compounds is important to show that the compounds will have high oral absorption (Table 3).

Table 3. Regulation of docking results obtained through SP and XP scanning methods according to XP GScore and some other parameters of some of 1-substituted 5- (trifluoromethoxy) - lf/-indole-2, 3 -dione 3 - [4- (substituted phenyl) thiosemicarbazone derivatives

AG: Free binding energy Ki: Dissociation constant BEI: Binding coefficient index SEI: Surface binding coefficient index LLE: Ligand lipophility coefficient

Example 6 - Biological Activity Findings

In vitro Test Findings l-(Non] substituted 5-fluoro/trifluoromethoxy-l//-indole-2,3-dione 3 -[4- (substituted phenyl] thiosemicarbazone] derivatives (IV] were tested for their anti-inflammatory activity in HEK BLUE cell lines that specifically express IL-Ib and hTLR2 receptors and their selectivity for IL-1R was assessed. The amount of secreted alkaline phosphatase (SEAP] upon the stimulation by either IL- 1b or LPS was measured on respective IL-1R or TLR2 expressing cell lines. Most of the new compounds were found to exhibit non-toxic IL-1 antagonist activity below the microM level in vitro. By comparing the IC50 values obtained with IL-1 b and LPS, the extent to which the compound selectively blocks IL-1 receptors was evaluated. Cell viability was assessed by MTS tetrazolium proliferation assay (Tables 4 and 5]

Table 4. IL-1R, TLR2 activities and selectivities of certain 1-substituted 5-fluoro-lf/-indole- 2,3-dione 3 -[4- (substituted phenyl)thiosemicarbazone derivatives

Table 5. IL-1R, TLR2 activities and selectivities of certain 1-substituted 5- (trifluoromethoxy) - lf/-indole-2, 3 -dione 3 - [4- (substituted phenyl) thiosemicarbazone derivatives

Example 7 - In vivo Experimental Findings

Antipyretic Effect Test Findings

Balb-c mice weighing 25-30 g were used to prevent the fever induced by subcutaneous lipopolysaccharide (LPS, Escherichia coli 0111: B4, Sigma] injection, an IL-1 mediated effect The doses used were determined by IC50 values obtained from in vitro experiments by allometric scaling. For the least effective dose determination, experiments in four-cell Latin square scheme were performed, and then the dose was escalated and used at the maximum antipyretic effect range.

Mice were divided into two groups. Body temperatures were measured with a rectal thermometer and the results were recorded as the baseline (n= 5] Following basal measurement, 220 pg/kg LPS was injected subcutaneously into both groups to induce fever. The mice were allowed to rest for 3 hours under normal housing conditions to attain a steady level of body temperature, then 3 separate doses (1.5, 3 and 4.5 mg/kg in 0.3 cc] of 4E5-4 were administered to one group intraperitonelly in mineral oil solution. The responses to a given dose were monitored for 90 minutes with measurements in half-hour intervals. The control group has received an injection of mineral oil used as the vehicle (Figure 15] Basal body temperature increased approximately 2.5 °C in both groups within 3 hours following LPS injection. With increasing doses of 4E5-4, the increase was reversible (Figure 15). In mice injected with vehicle, the fever continued steadily until the experiment was terminated. The difference was significantly lower for the 4.5 mg/kg dose (Figure 16). The primary mediator of LPS-induced body temperature increase is the cytokine IL-1. With these results, a physiological effect was determined for the 4E5-4 in the expected antipyretic direction.

Histopathological examinations of liver tissues obtained 24 hours later in Balb-c mice treated with 4E5-4 (4.5 pg/kg) showed no micro-damage to the tissues (Figure 16).

The antipyretic effect of 4D8-2 was assessed in Lewis rats and Balb-c mice using the same experimental design as in 4E5-4 (see above). In the experiment carried out with Lewis rats, 16 rats were divided into control and treatment groups. Body temperature was measured by infrared and rectal thermometers and the results were recorded as baseline values. After basal measurement, 220 pg/kg LPS was injected subcutaneously into the animals, and 3 doses (1.5, 15 and 150 pg / kg) were administered to the animals i.p. Body temperature measurements were made before and after compound injection (Table 6).

Table 6. Effect of 4D8-2 on increase of body temperature induced by LPS in Lewis Rats Basal body temperature increased approximately 2.5 °C in both groups within 3 hours following the LPS injection. The increase was reversible with 4D8-2 administration in a dose-dependent manner (Figure 17). In the control group, body temperature remained high without any significant change until the end of the experiment The difference in two groups was significantly different for the dose of 150 pg / kg. The main mediator of LPS-induced body temperature increase is the cytokine IL-1. For the first time, a physiological effect was demonstrated for the lead molecule synthesized (Figure 17].

In the experiment with Balb-c mice, 10 mice were divided into control and treatment groups, the fever of the mice was measured by a rectal thermometer, and the results were recorded as the baseline. Following basal measurement, 220 gg/kg LPS was injected subcutaneously, and the mice were monitored for 3 hours to have a steady body temperature rise. Fever measurements were made before and after 4D8-2 injection (Table 7).

Table 7. Effect of 4D8-2 on increase of body temperature induced by LPS in Balb-c Rats

LPS 4 4D8-2 (n-5) LPS (n— 5) Mean ± SD (°C) Mean ± SD (°C)

LPS injection 36, 18+0, 15 36,20+0,16 8-2 38,86+0,40 38,82+0,22 4-20 min. 38,82±0,22 38,80+0,26 4-40 min. 38,84+0,32 39,00+0,34 8-2 38,88*0,54 39,08+0,68 +20 min. 38,54+0,62 38,96+0,63 +40 min. 38,40+0,51 38,98+0,50

200 gg/kg 4D8-2 38, 18+0,44 39,04+0,21

200 gg/kg 4D8-2 +20 min. 37,30+ 1 ,20 38,98+0,29

200 gg/kg 4D8-2 +40min. 36,98+0,56 39,20+0,22

200 gg/kg 4D8-2 +60min. 36,60+0,53 39,14+0,24

Basal body temperature increased by approximately 2.5 °C in both groups within 3 hours following the LPS injection. The increase was reversible with 4D8-2 given at increasing doses. In LPS mice not treated with 4D8-2, body temperature increased with small jumps until the experiment was terminated. The difference between the drug and vehicle groups was statistically significant for 200 gg/kg dose. The main cytokine which mediates the LPS-induced body temperature increase is the IL-1. With these results, a physiological effect was determined in the expected direction (antipyretic] for one of the candidate molecules synthesized for the first time (Figure 18]. The determination of the antipyretic activity of the 4D8-2 in both mice validated the results obtained from Lewis rats.

Histopathological examinations of kidney and liver tissues obtained 24 hours after 4D8-2 treatment (150 gg/kg] in Lewis rats showed no harm to the tissues atthe micro-level (Figure 19]. Effects of4E5-4 and 4D8-2 on Blood Pressure

Acute effects of the candidate molecule 4E5-4 on blood pressure were assessed by the non- invasive tail-cuff method in Balb-c mice. The aim is to determine the possible effects of candidate molecules on blood pressure at doses that previously produced antipyretic effects. Experiments were planned in quadratic Latin square design to investigate the effects of lipopolysaccharide (LPS]-induced fever and the candidate molecule on blood pressure. The four experimental groups were (1] LPS-treated with 4E5-4 (or 4D8-2], (2] LPS-naive with 4E5-4 (or 4D8-2], (3] LPS- treated with the vehicle, (4] vehicle-treated only (Table 8). The doses administered were those obtained in previous tests for the antipyretic effect and known to be antipyretic. Experiments were performed with n = 1 block repetition (4 replicates, n = 4]

Table 8: Experimental groups*

* Same design was used in experiments with 4D8-2

Blood pressure was measured with a high throughput noninvasive "tail-cuff” blood pressure system (C0DA-HT8, Kent Scientific Corp., MA, USA] For this purpose, Balb-c mice of both sexes weighing 25-30 g, previously acclimated for blood pressure measurement, were assayed in blocks of four. For each animal, the restrainer- time was limited to a maximum of 10 min to ensure the consistency of the measurements. Only the mean of the "accepted” values in 15 automatic consecutive measurements by the built-in mathematical algorithm was taken into account at every measurement point. If the standard deviation obtained in sequential measurements is greater than 30 mmHg, the measurement is considered invalid (Wang, Y., Thatcher, SE, & Cassis, LA (2017] Biol, 1614, 69-73.]. Blood pressure (BP] was expressed as mean blood pressure ± standard deviation. Mean blood pressure was calculated using the formula: 2 /3 Diastolic BP + 1/3 Systolic BP.

Two doses of 4E5-4 (4.5 and 6 mg/kg] and two doses of 4D8-2 (0.2 and 0,4 mg/kg] known to be antipyretic, were injected intraperitoneally and blood pressure measurements were started at the 30 ^th minute of drug administration. Since the peak antipyretic action was obtained in the nineteenth minute, the data obtained at this point were used in statistical analyses. Animals that receive LPS injections were more vulnerable and risk to not tolerate the measurements due to the possibility of worsening of their general condition, in particular. Therefore, extreme care was taken to limit the number of measurements, in such that six measurements were taken from each animal at 30-minute intervals. Ninety minutes after the low dose (4.5 mg/kg and 0.2 mg/kg for 4E5-4 and 4D8-2, respectively] drug administration, the dose was increased cumulatively to the high dose (i.e., 6 mg/kg and 0,4 mg/kg], and the experimental protocol resumed. Body temperatures were measured by a rectal thermometer at the beginning and at the end of the experiment, and recorded.

All data acquired on the computer were combined in an Excel table, then exported to Prism v. 8 (GraphPad Software Inc.] for subsequent analysis. Two-way analysis of variance (ANOVA], followed by t-test for comparisons between the two groups were used. P values <0.05 were considered significantly different Data obtained in 4E5-4 experiments were summarized in Table 9.

Table 9: Mean arterial pressure (mmHg)

* P <0.05 compared with G3 in the low dose group (Welch’s t-test]

As shown in Table 9, there is some decrease in blood pressure values in all groups over time. However, this decrease was not significant in the vehicle (5.2 mmHg] and only 4E5-4 (4.8 mmHg) groups (P> 0.05, two-way AN OVA] In itself, this data suggests that the effect of 4E5-4 on blood pressure is not significant. However, blood pressure was significantly lower in the LPS group over time. This is predictable since the hypotensive effects of LPS in endotoxic shock were well described ( Hallemeesch , MM, Janssen , BJ, de Jonge, WJ, Soeters, PB, Lamers, WH, & Deutz, NE (2003). NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice. Am J Physiol Endocrinol Metab, 285 (4), E871-875.). In the group where LPS injection was combined with high dose (6 mg/kg] 4E5-4, the blood pressure drop was lower than in the group receiving only LPS. However, this decrease was not significant Body temperature measurements confirmed the antipyretic effect obtained in previous experiments.

Data obtained from 4D8-2 experiments were summarized in Figure 20. The results are inline with the other lead compound. 4D8-2 did not cause a significant change in blood pressure like the other candidate molecule 4E5- 4. However, in the LPS-treated groups, it prevented the steady drop in blood pressure caused by LPS (Figure 20] This may infer that 2-indolinone derivatives have the potential to reverse endotoxin-induced hypotension ( Hallemeesch , MM, Janssen , BJ, de Jonge, WJ, Soeters, PB, Lamers, WH, & Deutz, NE 2003, NO production by cNOS and iNOS reflects blood pressure changes in LPS- challenged mice Am J Physiol Endocrinol Metab, 285 (4), E871-875 ).