ALTINTOP MEHLIKA DILEK (TR)
KUCUKOGLU KAAN (TR)
FAYDALI NAGIHAN (TR)
BUL DILEK (TR)
SEVER BELGIN (TR)
ATATURK UNIV BILIMSEL ARASTIRMA PROJELERI BIRIMI (TR)
| CLAIMS The compound of formula (I) or pharmaceutically acceptable salt and/or solvate thereof, characterized by: Formula (I) Ri: CH3, C2H5, R2: CH3, C2H5, Ar: 4-CH3-C6H4, 4-OCH3-C6H4, 4-Cl-C6H4, 4-Br-C6H4, 4-F-C6H4, 4-CF3-C6H4, 4-NO2- C6H4, 4-(3-Thienyl)-C6H4. The compound according to Claim 1, characterized in that it is selected from the following group: • N,N-Dimethyl-l-(3-(p-tolyl)-l,2,4-oxadiazol-5-yl)methanamine • N-Methyl-N-((3-/2-tolyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine • l-(3-(4-Methoxyphenyl)-l,2,4-oxadiazol-5-yl)-N,N-dimethylmethanamine • N-((3-(4-Methoxyphenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-methylethanamine • N-((3-(4-Chlorophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-methylethanamine • 1 -(3 -(4-Bromophenyl)- 1 ,2,4-oxadiazol-5-yl)-N,N-dimethylmethanamine • N-((3-(4-Bromophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-methylethanamine • N-((3-(4-Bromophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-ethylethanamine • l-(3-(4-Fluorophenyl)-l,2,4-oxadiazol-5-yl)-N,N-dimethylmethanamine • N-((3-(4-Fluorophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-methylethanamine • N-Ethyl-N-((3-(4-fluorophenyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine • N,N-Dimethyl-l-(3-(4-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)methanamine • N-Methyl-N-((3-(4-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine • N-Ethyl-N-((3-(4-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine • N-Methyl-N-((3-(4-nitrophenyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine • N,N-Dimethyl-l-(3-(4-(3-thienyl)phenyl)-l,2,4-oxadiazol-5-yl)methanamine • N-Methyl-N-((3-(4-(3-thienyl)phenyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine • N-Ethyl-N-((3-(4-(3-thienyl)phenyl)-l,2,4-oxadiazol-5-yl)methyl)ethanamine A pharmaceutical composition, characterized in that it comprises a compound according to any one of the preceding claims. A pharmaceutical composition according to Claim 3, characterized in that it is used in the preparation of medications for the treatment of diseases such as cancer, glaucoma, obesity, and epilepsy. A compound according to Claim 2, characterized in that it is used as a carbonic anhydrase I and/or II enzyme inhibitor. |
I AND II ENZYMES
Technical Field
The invention relates to the synthesis of 3,5-disubstituted-l,2,4-oxadiazole derived compounds, molecular docking studies and high inhibitory activities on carbonic anhydrase enzymes I and II (CA I and CA II).
State of the Art (Background)
Carbonic anhydrase (CA) enzymes are a metalloenzyme superfamily found in many organisms. They carry a tightly bound Zn 2+ ion that can interact with water and histidine molecules in their active regions. These enzymes have the function of catalyzing the conversion between CO2 and HCOs". CA enzymes have 16 isoforms, some of which are cytosolic (CA I, CA II, CA III, CA VII, and CA XIII), while others (CA IV, CA IX, CA XII, CA XIV, and CA XV) are membrane bound. CA enzymes play critical roles in important cellular processes such as electrolyte secretion, calcification, osteoporosis respiration and pH homeostasis, and in biosynthetic processes such as ureagenesis, gluconeogenesis, and lipogenesis. Abnormal CA levels or activities occur in many pathological conditions. Therefore, selective inhibition of CA isoforms is a frequently used approach in the design of new therapeutic compounds that can be used in the treatment of different diseases. Some CA inhibitors are used in the clinic to treat glaucoma, cancer, obesity, and epilepsy.
CA isozymes are targets for many diseases, and drugs that inhibit these isozymes are used in the clinic to treat some diseases. In addition to this, studies on the design, synthesis and biological activities of compounds that inhibit CA isozymes are still ongoing in the world. Since CA isozymes are targets for many diseases, there is a need to design new compounds that have a strong inhibitory effect against these enzymes.
Brief Description and Objects of the Invention The invention is intended to investigate the activities on CA enzyme activity by synthesizing a group of new compounds that have been substituted from their 3rd and 5th positions and have an alkyl amine structure, considering the studies involving 1,2,4-oxadiazole ring-bearing compounds in recent years.
Another object of the invention is to use the compounds of the invention, which have a high level of CA I and CA II isoenzyme inhibitor, as a drug candidate in the treatment of diseases such as glaucoma, cancer, obesity, epilepsy.
In this context, substituted benzonitriles and hydroxylamine hydrochloride compounds were reacted in an environment containing water, ethanol, and sodium carbonate to obtain substituted benzamide oxides. The obtained substituted benzamide oxides were first mixed with chloroacetyl chloride in acetone at room temperature and then heated under toluene- recycling refrigerant to obtain 3-substitutedphenyl-5-chloromethyl-l,2,4-oxadiazole compounds. In the last stage, alkyl amines and these oxadiazole compounds were reacted in potassium carbonate medium and acetonitrile and the resulting compounds were synthesized. The properties of the resulting compounds to inhibit CA I and CA II isoenzymes were investigated.
Of the synthesized resulting compounds OAld-OA18d, all but OAld have stronger inhibitory effect on CA I isoenzyme compared to the reference compound acetazolamide (AAZ). In addition, the OAlOd compound showed stronger inhibitory activity on the CA I isoenzyme than the other reference compound sulfanilamide (SSA). All of the OAld-OA18d compounds have stronger inhibitory activity on the CA II isoenzyme than the reference compound AAZ.
Definitions of Figures Describing the Invention
Figure 1. Docking exposures of OAld-OA18d compounds along with acetazolamide (AAZ) and sulfanilamide (SSA) at the substrate binding site of CA I isoenzyme.
Figure 2. Docking interactions of OAlOd and OA17d compounds with AAZ and SSA at the substrate binding site of CA I isoenzyme Figure 3. Docking exposures of OAld-OA18d compounds along with acetazolamide (AAZ) and sulfanilamide (SSA) at the substrate binding site of CA II isoenzyme.
Figure 4. Docking interactions of OA3d, OA7d, OAlOd, and OA17d compounds with AAZ and SSA at the substrate binding site of CA II isoenzyme
Detailed Description of the Invention
The invention relates to 18 novel 3,5-disubstituted-l,2,4-oxadiazole derivative compounds and their synthesis, molecular docking studies, and high inhibitory activities on CA I and CA II enzymes.
The present invention is a compound of Formula (I) or pharmaceutically acceptable salt and/or solvate thereof,
Formula (I)
Ri: CH 3 , C2H5,
R 2 : CH 3 , C2H5,
Ar: 4-CH 3 -C 6 H 4 , 4-OCH 3 -C 6 H 4 , 4-Cl-C 6 H 4 , 4-Br-C 6 H 4 , 4-F-C 6 H 4 , 4-CF 3 -C 6 H 4 , 4-NO 2 -C 6 H 4 , 4-(3-Thienyl)-C 6 H 4 .
The invention relates to a compound of formula (I) or pharmaceutical composition comprising pharmaceutically acceptable salt and/or solvate thereof; it may be used in the preparation of medications for the treatment of diseases such as cancer, glaucoma, obesity, and epilepsy.
SYNTHESIS OF OAld-OA18d DERIVATIVE COMPOUNDS The detailed synthesis of the compounds synthesized within the scope of the present invention is presented in Diagram 1, and the codes of the synthesized compounds and the substituents they carry are presented in Table 1.
Diagram 1: Detailed synthesis diagram of the OAld-OA18d compounds of the invention.
The method of synthesis of the compounds of the invention shown above is described in detail below:
General Synthesis of N'-Hydroxybenzimidamide Derivatives (la-8a)
Hydroxylamine hydrochloride (8.4 g, 24.2 mmol) was dissolved in distilled water (10 mL). Sodium carbonate (8.2 g, 15.51 mmol) was dissolved in distilled water (10 mL) and added to hydroxylamine hydrochloride solution. The solution of the benzonitrile derivative (9.69 mmol) was prepared by dissolving in ethanol (20 mL) and added to hydroxylamine hydrochloride solution dropwise. The reaction mixture was controlled by thin layer chromatography (TLC) and heated under the refrigerant, which was refluxed for 3-5 hours. After cooling the reaction mixture to room temperature, the solvent was removed using a vacuum. Distilled water (10 mL) was added to the residue and extracted with di chloromethane (3x10 mL) dried over sodium sulfate. n-Hexane (20-40 mL) was added to the residue obtained, filtered and dried at room temperature. The purity of compounds la-8a was controlled by TLC, and compounds obtained without further purification were used in the synthesis of (2-chloroacethoxy) benzimidamide derivatives (lb-8b).
General Synthesis of (2-Chloroacethoxy) Benzimidamide Derivatives (lb-8b)
A'-Hydroxybenzimidamide derivative (la-8a) (4.7 mmol) and chloroacetyl chloride (4.7 mmol) were dissolved separately in acetone (10 mL). Then, chloroacetyl chloride solution was added dropwise into the A'-hydroxybenzimidamide derivative solution. The mixture was stirred at room temperature for 3-5 hours and the course of the reaction was monitored with TLC. When the reaction was over, the acetone was condensed and the residue washed with sodium bicarbonate solution (100 mL) and water, filtered and dried. The purity of compounds lb-8b was controlled by TLC and compounds obtained without using any other purification method were used in the synthesis of 5-chloromethyl-3-substitutedphenyl-l,2,4-oxadiazole (lc-8c).
General Synthesis of 5-Chloromethyl-3-substituophenyl-l ,2,4-oxadiazole Derivatives (lc-8c)
Compounds lb-8b were dissolved in toluene (30 mL) and controlled with TLC and heated under a refrigerant for 3-5-hour reflux. Toluene was removed from the medium using a vacuum and diethyl ether (60-80 mL) was added to the residue. After solidification of the residue, diethyl ether was removed. The resulting residue was filtered and dried at room temperature. The purity of compounds lc-8c was controlled by TLC and the compounds obtained without using another purification method were used in the synthesis of the resulting compounds OAld-OA18d.
General Synthesis of 5-Substituted aminophenyl- 3 -substituted phenyl-l,2,4-oxadiazole Derivatives (OAld-OA18d)
Compounds lc-8c (0.25 mmol) were dissolved in acetonitrile (10 mL) and potassium carbonate (1 mmol) was added. Suitable amine (1 mmol) was dissolved in acetonitrile (10 mL) and added dropwise to the mixture. When diethylamine or dimethylamine was used as amine, the mixture was heated under refrigerant, which turned back for 3-5 hours. On the other hand, when A-ethylmethylamine was used as amine, the mixture was stirred at room temperature for 2-3 days. The reactions were controlled by TLC and terminated and acetonitrile was removed from the medium by vacuum. Dichloromethane and water were added to the residue and extracted (3x10 mL), the obtained organic phase was dried over sodium sulfate. After removal of dichloromethane from the medium by vacuum, ethanol (20 mL) and activated carbon were added to the residue and heated under the refluxing refrigerant for 5 minutes. Then, the mixture was filtered while hot. After the solvent was condensed, the final compounds were purified by preparative chromatography using the n-hexane: ethyl acetate (3:2) solvent system. Table 1. Synthesized 3,5-disubstituted-l,2,4-oxadiazole derived compounds
Compound Ar Ri R2
OAld 4-CH3-C6H4- CH 3 CH 3
OAld 4-CH 3 -C 6 H 4 - CH 3 C2H5
OA3d 4-OCH 3 -C 6 H 4 - CH 3 CH 3
OA4d 4-OCH 3 -C 6 H 4 - CH 3 C2H5
OA5d 4-Cl-C 6 H 4 - CH 3 C2H5
OA6d 4-Br-C 6 H 4 - CH 3 CH 3
OA7d 4-Br-C 6 H 4 - CH 3 C2H5
OA8d 4-Br-C 6 H 4 - C2H5 C2H5
OA9d 4-F-C 6 H 4 - CH 3 CH 3
OAlOd 4-F-C 6 H 4 - CH 3 C2H5
OAlld 4-F-C 6 H 4 - C2H5 C2H5
OAlld 4-CF 3 -C 6 H 4 - CH 3 CH 3
OA13d 4-CF 3 -C 6 H 4 - CH 3 C2H5
OA14d 4-CF 3 -C 6 H 4 - C2H5 C2H5
OA15d 4-NO 2 -C 6 H 4 - CH 3 C2H5
OA16d 4-(3-Thienyl)-C 6 H 4 - CH 3 CH 3
OA17d 4-(3-Thienyl)-C 6 H 4 - CH 3 C2H5
OA18d 4-(3-Thienyl)-C 6 H 4 - C2H5 C2H5
N,N-Dimethyl-l-(3-(p-tolyl)-l,2,4-oxadiazole-5-yl)methana mine (OAld)
Colorless oily liquid
Yield: 50%
’H NMR (400 MHz, CDCh, 6): 2.41 (s, 3H, CH 3 ), 2.42 (s, 6H, 2 x N-CH 3 ), 3.85 (s, 2H, -CH 2 - ), 7.28 (d, J = 7.9 Hz, 2H, Ar-H), 7.99 (d, J = 7.9 Hz, 2H, Ar-H).
13 C NMR (100 MHz, DMSO-de, 6): 177.56, 167.87, 141.97, 130.27, 127.43, 123.86, 53.54, 45.01, 21.54.
MS (APCI) m/z: 218.2 (M+H + ).
N-Methyl-N-((3-(p-tolyl)-l,2,4-oxadiazol-5-yl)methyl)etha namine (OA2d)
Colorless oily liquid
Yield: 45%
X H NMR (400 MHz, DMSO-de, 6): 1.16 (t, J = 7.2 Hz, 3H, CH 3 ), 2.41 (s, 3H, CH 3 ), 2.43 (s, 3H, N-CH 3 ), 2.62 (q, J = 7.2 Hz, 2H, N-CH2-), 3.95 (s, 2H, -CH2-), 7.28 (d, J = 8.1 Hz, 2H, Ar-H), 7.98 (d, J = 8.1 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 176.08, 168.34, 141.61, 129.56, 127.44, 123.80, 51.25, 50.91, 41.77, 21.57, 12.27.
MS (APCI) m/z: 232.3 (M+H + ). l-(3-(4-Methoxyphenyl)-l,2,4-oxadiazol-5-yl)-N,N-dimethylmet hanamine (OA3d)
Colorless oily liquid
Yield: 45%
’H NMR (400 MHz, CDCh, 6): 2.42 (s, 6H, 2 x N-CH 3 ), 3.84 (s, 2H, -CH 2 -), 3.86 (s, 3H, O- CH 3 ), 6.98 (d, J = 8.6 Hz, 2H, Ar-H), 8.05 (d, J = 8.6, 2H, Ar-H).
13 C NMR (100 MHz, DMSO-de, 6): 177.39, 167.60, 162.18, 129.18, 118.91, 115.14, 55.87, 53.54, 45.02.
MS (APCI) m/z: 234.2 (M+H + ).
N-((3-(4-Methoxyphenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-me thylethanamine (OA4d)
Colorless oily liquid
Yield: 45%
’H NMR (400 MHz, DMSO, 5): 1.15 (t, J = 7.2 Hz, 3H, CH 3 ), 2.42 (s, 3H, N-CH 3 ), 2.61 (q, J = 7.2 Hz, 2H, N-CH2-), 3.86 (s, 3H, O-CH 3 ), 3.94 (s, 2H, -CH 2 -), 7.45 (dd, J = 8.7 Hz, J o = 2.0 Hz, 2H, Ar-H), 8.04 (dd, J = 8.7 Hz, J o = 2.0 Hz, 2H, Ar-H).
13 C NMR (100 MHz, DMSO-de, 6): 177.63, 167.58, 162.17, 129.18, 118.93, 115.14, 55.88, 51.33, 50.58, 41.78, 12.80.
MS (APCI) m/z: 248.2 (M+H + ).
N-((3-(4-Chlorophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-met hylethanamine (OA5d)
Colorless oily liquid
Yield: 45%
’H NMR (400 MHz, DMSO, 5): 1.15 (t, J = 7.2 Hz, 3H, CH 3 ), 2.42 (s, 3H, N-CH 3 ), 2.61 (q, J = 7.2 Hz, 2H, N-CH2-), 3.94 (s, 2H, -CH 2 -), 7.45 (dd, J = 8.7 Hz, J o = 2.0 Hz, 2H, Ar-H), 8.04 (dd, J = 8.7 Hz, J o = 2.0 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 176.03, 167.64, 137.53, 129.23, 128.84, 125.03, 50.93, 41.67, 12.10.
MS (APCI) m/z: 252.2 (M+H + ). l-(3-(4-Bromophenyl)-l,2,4-oxadiazol-5-yl)-N,N-dimethylmetha namine (OA6d)
Colorless oily liquid
Yield: 45%
’H NMR (400 MHz, CDCh, 8): 2.42 (s, 6H, 2 x N-CH 3 ), 3.85 (s, 2H, -CH 2 -), 7.61 (dd, J = 8.6 Hz, Jo = 1.9 Hz, 2H, Ar-H), 7.98 (dd, J = 8.6, Jo = 1.9 Hz, 2H, Ar-H).
13 C NMR (100 MHz, DMSO-de, 8): 177.73, 167.27, 132.85, 129.46, 125.80, 125.65, 53.36, 44.92.
MS (APCI) m/z: 282.2 (M+H + ).
N-((3-(4-Bromophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-meth ylethanamine (OA7d)
Colorless oily liquid
Yield: 40%
X H NMR (400 MHz, DMSO, 8): 1.02 (t, J = 7.1 Hz, 3H, CH 3 ), 2.26 (s, 3H, N-CH 3 ), 2.46- 2.50 (m, 2H, N-CH2-), 3.97 (s, 2H, -CH2-), 7.76 (d, J = 8.5 Hz, 2H, Ar-H), 7.93 (d, J = 8.5 Hz, 2H, Ar-H).
13 C NMR (100 MHz, DMSO-de, 6): 178.26, 167.23, 133.13, 132.87, 129.47, 125.85, 125.62, 51.31, 50.55, 41.76, 12.81.
MS (APCI) m/z: 296.2 (M+H + ).
N-((3-(4-Bromophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-ethy lethanamine (OA8d)
Colorless oily liquid
Yield: 55%
X H NMR (400 MHz, CDCh, 8): 1.16 (t, J = 7.2 Hz, 6H, 2 x N-CH 3 ), 2.71 (q, J = 7.2 Hz, 4H, 2 x N-CH2-), 4.07 (s, 2H, -CH2-), 7.62 (dd, J = 8.6 Hz, Jo = 1.8 Hz, 2H, Ar-H), 8.97 (dd, J = 8.6 Hz, Jo = 1.8 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 174.92, 163.00, 132.52, 129.04, 126.75, 125.14, 49.03, 38.16, 31.25, 14.13, 9.97.
MS (APCI) m/z: 310.2 (M+H + ). l-(3-(4-Fluorophenyl)-l,2,4-oxadiazol-5-yl)-N,N-dimethylmeth anamine (OA9d)
Colorless oily liquid
Yield: 48%
’H NMR (400 MHz, CDCh, 6): 2.42 (s, 6H, 2 x N-CH 3 ), 3.85 (s, 2H, -CH 2 -), 7.13-7.78 (m, 2H, Ar-H), 8.12-8.08 (m, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 6): 167.60, 165.89, 163.39, 129.74, 129.66, 122.84, 116.18, 115.96, 53.68, 45.02.
MS (APCI) m/z: 222.2 (M+H + ).
N-((3-(4-Fluorophenyl)-l,2,4-oxadiazol-5-yl)methyl)-N-met hylethanamine (OAlOd)
Colorless oily liquid
Yield: 52%
’H NMR (400 MHz, DMSO, 8): 2.15 (t, J = 7.1 Hz, 3H, CH 3 ), 2.42 (s, 3H, N-CH 3 ), 2.61 (q, J = 7.1 Hz, 2H, N-CH2-), 3.94 (s, 2H, -CH 2 -), 7.14-7.19 (m, 2H, Ar-H), 8.08-8.13 (m, 2H, Ar- H).
13 C NMR (100 MHz, DMSO-de, 6): 167.10, 165.65, 163.18, 130.09, 130.00, 123.23, 117.02, 116.80, 51.29, 50.56, 41.75, 12.78.
MS (APCI) m/z: 236.2 (M+H + ).
N-Ethyl-N-((3-(4-fluorophenyl)-l,2,4-oxadiazol-5-yl)methy l)ethanamine (OAlld)
Colorless oily liquid
Yield: 45%
’H NMR (400 MHz, CDCh, 5): 1.13 (t, J = 7.1 Hz, 6H, 2 x N-CH 3 ), 2.67 (q, 7 = 7.1 Hz, 4H, 2 x N-CH 2 -), 4.01 (s, 2H, -CH 2 -), 7.13-7.18 (m, 2H, Ar-H), 8.07-8.12 (m, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 6): 167.50, 165.86, 163.36, 129.73, 129.65, 122.93, 116.16, 115.94, 47.74, 46.96, 12.01.
MS (APCI) m/z: 250.3 (M+H + ).
N,N-Dimethyl-l-(3-(4-(trifluoromethyl)phenyl)-l,2,4-oxadi azol-5-yl)methanamine
(OA12d)
Colorless oily liquid
Yield: 30%
X H NMR (400 MHz, CDCh, 6): 2.44 (s, 6H, 2 x N-CH 3 ), 3.88 (s, 2H, -CH 2 -), 7.75 (d, J = 8.7 Hz, 2H, Ar-H), 8.24 (d, J = 8.7 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 6): 176.53, 167.45, 133.21, 132.89, 129.94, 127.90, 125.90, 125.09, 53.62, 45.00.
MS (APCI) m/z: 272.3 (M+H + ).
N-Methyl-N-((3-(4-(trifluoromethyl)phenyl)-l,2,4-oxadiazo l-5-yl)methyl)ethanamine
(OA13d)
Colorless oily liquid
Yield: 40%
’H NMR (400 MHz, DMSO, 8): 1.17 (t, J = 7.2 Hz, 3H, CH 3 ), 2.43 (s, 3H, N-CH 3 ), 2.62 (q, J = 7.2 Hz, 2H, N-CH2-), 3.99 (s, 2H, -CH 2 -), 7.75 (d, J = 7.6 Hz, 2H, Ar-H), 8.24 (d, J = 7.8 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 177.34, 165.27, 144.86, 141.56, 129.72, 127.96, 126.03, 125.99, 50.87, 29.71, 22.70, 14.13.
MS (APCI) m/z: 286.3 (M+H + ).
N-Ethyl-N-((3-(4-(trifluoromethyl)phenyl)-l,2,4-oxadiazol -5-yl)methyl)ethanamine
(OA14d)
Colorless oily liquid
Yield: 50%
X H NMR (400 MHz, CDCh, 8): 1.17 (t, J = 7.0 Hz, 6H, 2 x N-CH 3 ), 2.72 (q, J = 7.0 Hz, 4H, 2 x N-CH2-), 4.08 (s, 2H, -CH 2 -), 7.74 (d, J = 8.2 Hz, 2H, Ar-H), 8.23 (d, J = 8.5 Hz, 2H, Ar- H).
13 C NMR (100 MHz, CDCh, 8): 177.03, 167.48, 132.97, 129.84, 127.92, 125.93, 125.07, 122.36, 59.53, 48.00, 38.15, 29.70, 11.65.
MS (APCI) m/z: 300.3 (M+H + ).
N-Methyl-N-((3-(4-nitrophenyl)-l,2,4-oxadiazol-5-yl)methy l)ethanamine (OA15d)
Colorless oily liquid
Yield: %40
X H NMR (400 MHz, DMSO, 8): 1.16 (t, J = 7.1 Hz, 3H, CH 3 ), 2.43 (s, 3H, N-CH 3 ), 2.62 (q, J = 7.1 Hz, 2H, N-CH2-), 3.98 (s, 2H, -CH 2 -), 8.30 (d, J = 9.1 Hz, 2H, Ar-H), 8.35 (d, J = 9.3 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 166.86, 162.82, 149.52, 133.47, 128.52, 124.12, 51.23, 50.98, 41.80, 12.23.
MS (APCI) m/z: 263.2 (M+H + ).
N,N-Dimethyl-l-(3-(4-(thiophen-3-yl)phenyl)-l,2,4-oxadiaz ol-5-yl)methanamine
(OA16d)
Colorless oily liquid
Yield: %40
’H NMR (400 MHz, CDCh, 8): 2.44 (s, 6H, 2 x N-CH 3 ), 3.87 (s, 2H, -CH 2 -), 7.41-7.46 (m, 2H, thienyl), 7.55-7.57 (m, 1H, thienyl), 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 8.42 (d, J = 8.0 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 176.12, 168.16, 141.35, 138.56, 128.06, 126.74, 126.64, 126.16, 125.15, 121.43, 53.74, 45.05.
MS (APCI) m/z: 286.3 (M+H + ). N-Methyl-N-((3-(4-(thiophen-3-yl)phenyl)-l,2,4-oxadiazol-5-y l)methyl)ethanamine
(OA17d)
Colorless oily liquid
Yield: 50%
’H NMR (400 MHz, CDCh, 6): 1.16 (td, J = 7.2 Hz, Jo = 2.13 Hz, 3H, CH 3 ), 2.42 (s, 3H, N- CH 3 ), 2.61 (q, J = 7.1 Hz, Jo = 2.0 Hz, 2H, N-CH2-), 3.95 (s, 2H, -CH2), 7.40-7.46 (m, 2H, thienyl), 7.55-7.57 (m, 1H, thienyl), 7.70 (d, J = 8.0 Hz, 2H, Ar-H), 8.42 (d, J = 8.0 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 8): 176.24, 168.11, 141.36, 138.54, 128.06, 126.73, 126.64, 126.16, 125.19, 121.42, 51.25, 50.94, 41.79, 12.28.
MS (APCI) m/z: 300.3 (M+H + ).
N-Ethyl-N-((3-(4-(thiophen-3-yl)phenyl)-l,2,4-oxadiazol-5 -yl)methyl)ethanamine
(OA18d)
Colorless oily liquid oily liquid
Yield: 45% X H NMR (400 MHz, CDCh, 8): 1.15 (t, J = 7.2 Hz, 6H, 2 x N-CH 3 ), 2.69 (q, J = 7.2 Hz, 4H, 2 x N-CH2-), 4.04 (s, 2H, -CH 2 -), 7.41-7.46 (m, 2H, thienyl), 7.55-7.57 (m, 1H, thienyl), 7.71 (d, J = 8.2 Hz, Jo = 1.7 Hz, 2H, Ar-H), 8.26 (d, J = 8.1 Hz, Jo = 1.7 Hz, 2H, Ar-H).
13 C NMR (100 MHz, CDCh, 6): 176.89, 168.01, 141.38, 138.46, 128.05, 126.71, 126.63, 126.16, 125.32, 121.39, 47.70, 47.24, 12.17.
MS (APCI) m/z: 314.3 (M+H + ).
Molecular Docking Studies
Molecular docking studies were conducted in the active sites of CA I (PDB code: 1AZM) and CA II (PDB code: 3DC3) isoenzymes for resulting compounds (OAld-OA18d). AAZ and SSA were selected as reference compounds in accordance with in vitro enzyme studies. In general, the 1,2,4-oxadiazole ring system also plays a very important role in the high affinity to the active site of the CA I isoenzyme by making 7t-7t interactions with the amino acid Phe91. In addition, aryl groups (except for the 4-(3-thienyl)phenyl group) connected to the 3rd position of the 1,2,4-oxadiazole ring system formed 7t-7t interactions with the His94 amino acid. The 4-methoxyphenyl substituted compound OA4d methoxyphenyl group formed a metal coordination and salt bridge with Zn261 through the 4-nitrophenyl substituted compound OA15d nitrophenyl group. As seen in OA3d and OA16d compounds, alkylamino groups in the 5th position of the 1,2,4-oxadiazole ring formed hydrogen bonds with the amino acid Alai 32. Despite the strong affinity of compound OA18d, it demonstrated no interference in the active site of CA I isoenzyme. OAd compounds did not have significant interactions with Thrl99 and Zn261, as seen with AAZ and SSA (Figures 1 and 2).
1,2, 4- Alkylamino groups in the 5th position of the oxadiazole ring played an important role in the activity by forming hydrogen bonds with Gln92 amino acid, salt bridge with Glu69 and it- cation interactions with Phel31 amino acid in the active region of CA II isoenzyme. In addition, the aryl groups in the 3rd position of the 1,2,4-oxadiazole ring formed 7t-7t interactions with the amino acid His94. 4-Methoxyphenyl-bearing OA3d and OA4d compounds have hydrogen bonded with the amino acid Thrl99 through methoxy substituents. The OA15d compound, which is a 4-nitrophenyl derivative, formed a hydrogen bond with Thrl99 and participated in the formation of salt bridge and metal bond with Zn262. As seen in AAZ and SSA, significant interactions with the amino acids Thrl99 and Zn262 did not usually occur in OAd compounds (Figures 3 and 4). The docking scores of the compounds are used to compare the docking data of different compounds, and the glide emodel scores are usually used to compare the docking data of different conformation of the same compounds. Table 2 shows the docking scores, glide gscore and glide emodel results calculated for CA I and CA II isoenzymes of the compounds.
While the docking scores of the compounds for CA I are determined to be between -3.213 and -4.487 kcal/mol, the docking scores for CA II are determined to be between -3.645 and - 4.461. The docking scores of the compounds are generally in agreement with the biological data. The OAlOd compound with the highest docking score for both enzymes showed high inhibitory potential and high binding capacity to the active regions of CA I and CA II. The docking scores of AAZ and SSA were found to be higher than those of OAld-OA18d compounds and were -6.414 kcal/mol and -7.184 kcal/mol for CA I and -6.282 kcal/mol and - 7.046 kcal/mol for CA II, respectively (Table 2). Table 2. Results of docking scores (kcal/mol), glide gscores (kcal/mol) and glide emodel (kcal/mol) for CA I (PDB code: 1AZM) and CA II (PDB code: 3DC3) isozymes of all compounds
1AZM 3DC3
Compound Docking Glide Glide Docking Glide Glide criteria gscore emodel criteria gscore emodel
OAld -3.213 -3.215 -29.156 -3.645 -3.647 -44.076
OA2d -3.459 -3.461 -26.723 -3.734 -3.736 -47.405
OA3d -3.600 -3.602 -26.012 -4.201 -4.203 -37.428
OA4d -3.411 -3.412 -28.091 -3.971 -3.973 -40.331
OA5d -3.538 -3.540 -24.821 -3.879 -3.880 -43.298
OA6d -3.804 -3.805 -25.306 -3.966 -3.968 -38.982
OA7d -3.750 -3.751 -29.360 -4.337 -4.338 -41.476
OA8d -3.918 -3.919 -33.761 -4.391 -4.392 -43.011
OA9d -3.843 -3.845 -29.178 -3.912 -3.914 -40.898
OAlOd -4.487 -4.488 -30.429 -4.461 -4.463 -43.264
OAlld -3.471 -3.473 -28.168 -3.739 -3.741 -43.810
OA12d -4.136 -4.136 -26.637 -3.887 -3.888 -38.145
OA13d -4.049 -4.051 -27.764 -3.995 -3.997 -38.079
OA14d -4.017 -4.018 -28.589 -3.844 -3.845 -42.721
OA15d -3.414 -3.416 -26.221 -3.964 -3.965 -42.214
OA16d -3.775 -3.777 -29.296 -4.141 -4.143 -41.246
OA17d -4.251 -4.253 -28.641 -4.472 -4.474 -45.184 OA18d -3.667 -3.669 -23.644 -4.063 -4.065 -40.860
AAZ -6.414 -6.865 -62.378 -6.282 -8.069 -78.846
SSA -7.184 -7.656 -53.035 -7.046 -7.518 -56.774
In silico Pharmacokinetic Studies
Pharmacokinetic properties of OAld-OA18d compounds and Jorgensen's 3 rules and 5 Lipinski's 5 rules were investigated using the QikProp module (Schrodinger Release 2016-2: QikProp, Schrodinger, LLC, New York, NY, 2016). Based on in silico data, it was found that OAld-OA18d compounds have the appropriate octanol/water partition coefficient and can cross the blood-brain barrier. It was also found that all synthesized compounds showed significant serum albumin binding within the specified range (-1.5-1.5). All tested 10 compounds except compound OA15d had high levels of oral absorption and were in agreement with Jorgensen's 3 rules and Lipinski's 5 rules. As a result, it can be concluded that these compounds may be drug candidates with high oral bioavailability (Table 3).
Table 3. Estimated ADME properties of OAld-OA18d compounds
Oral
QPlogBB* QPlogPo/w* QPlogS* QPlogKhsa* absorption % Lipinski's 5 Jorgensen's
Compound (-3 - 1.2) (-2.0 - 6.5) (-6.5 - 0.5) (-1.5 - 1.5) (>80% high, Rules ** 3 Rules **
<25% low)
OAld 0.26 1.21 -1.07 -0.445 83.84 0 0
OA2d 0.27 1.51 -1.65 -0.322 87.33 0 0
OA3d 0.26 1.14 -0.88 -0.645 84.57 0 0
OA4d 0.51 1.55 -1.37 -0.522 87.54 0 0
OA5d 0.45 1.95 -1.99 -0.368 89.87 0 0
OA6d 0.51 1.60 -1.69 -0.466 87.29 0 0
OA7d 0.46 2.08 -2.11 -0.342 90.64 0 0
OA8d 0.44 2.36 -2.12 -0.266 92.81 0 0
OA9d 0.44 1.28 -1.09 -0.573 85.44 0 0
OAlOd 0.40 1.60 -1.61 -0.450 87.82 0 0
OAlld 0.38 2.01 -1.61 -0.373 90.79 0 0
OA12d 0.60 2.09 -2.27 -0.339 90.16 0 0
OA13d 0.55 2.49 -2.70 -0.215 93.05 0 0
OA14d 0.53 2.77 -2.70 -0.139 95.23 0 0
OA15d 0.26 0.74 -1.26 -0.595 66.26 0 0
OA16d 0.42 2.68 -3.02 -0.033 93.61 0 0 OA17d 0.38 3.08 -3.44 0.167 96.50 0
OA18d 0.36 3.36 -3.45 0.091 100.00 0
* QPlogBB: Estimated blood/brain partition coefficient, QPlogPo/w: Estimated octanol/water partition coefficient, QPlogS: Estimated water solubility, QPlogKhsa: Estimated binding to human serum albumin.
* * Lipinski's 5 Rule: The number of violations of Lipinski's 5 rule. These rules are: Molecular weight of the molecule < 500, QPlogPo/w < 5, Hydrogen bond donor atom count < 5, Hydrogen bond acceptor atom count < 10. Compounds that meet these rules are considered drug-like molecules. Jorgensen's 3 Rules: The number of violations of Jorgensen's 3 Rules. These rules are: QPlogS > -5.7, Estimated apparent Caco-2 cell permeability (QPPCaco) > 22 nm/s, # Primary metabolites < 7. Compounds that violate these rules less (preferably not at all) are considered to be more appropriate for oral use.
CA ENZYME INHIBITOR ACTIVITY RESULTS OF OAld-OA18d-DERIVED COMPOUNDS
Data on the inhibitory effects of the invention OAld-OA18d coded compounds on CA I and CA II enzymes in vitro are presented in Table 4. Acetazolamide (AAZ) and Sulfanilamide (SSA) were used as reference compounds in this study. All compounds tested with the invention, except for OAld, have been found to have a significant inhibitory effect on the CA I enzyme compared to the reference compound AAZ (ICso value 6.523) (ICso values 0.68- 17.38 pM). The compound OAlOd showed higher inhibitory activity with an ICso value of 0.68 pM than the other reference compound, SSA (ICso value 0.923 pM). Furthermore, the compound OA17d showed a comparably high CA I inhibition activity with an ICso value of 0.96 pM. Trifluorom ethyl substituted compounds OA12d, OA13d, OA14d and thienyl ring system-bearing compound OA18d showed better inhibitory activity against CA I than other compounds in this series with ICso values of 1.89, 1.92, 2.03 and 1.81 pM, respectively. Similarly, the inhibitory effects of the tested compounds on CA II isoenzyme are remarkable (ICso values 0.40-7.45 pM). OA7d and OA17d compounds are the compounds with the strongest inhibitory effect on CA II isoenzyme compared to the reference compound AAZ (ICso= 7.85 pM) and these compounds have the same IC50 value (0.40 pM). Furthermore, the compounds methoxy-substituted OA3d and fluoro- substituted OAlOd showed a significant inhibitory activity against CA II with values of 0.71 and 0.65 pM IC50, respectively. It is a remarkable finding that OA16d-OA18d compounds carrying the thiophene ring system exhibit significant CA II inhibitory activity in the range of 0.40-1.49 pM, unlike other derivatives. The inhibitory activity of OAd series compounds on CA II isozyme was found to be lower than that of the other reference compound, SSA (IC50 value 0.256 pM).
Compounds OA2d, OA12d, and OA14d were found to show more pronounced inhibitory activity on CA I than on CA II; however, other compounds tested showed stronger inhibitory activity on CA II than on CA I except for OAlOd. Fluoro-substituted compound OAlOd was found to have a very close inhibitory effect on CA I and CA II isozymes with IC50 values of 0.68 pM and 0.65 pM, respectively.
Table 4. Inhibitory effects of OAld-OA18d coded compounds on CA I and CA II enzymes
CA I inhibition CA II inhibition
CA I/
Compound IC50 IC50 r 2 Ki (uM) r 2 Ki (uM) CA II
OiM) (pM)
OAld 17.38 0.9935 15.84 7.45 0.9921 5.71 2.33
OA2d 6.03 0.9959 5.57 6.52 0.9907 5.00 0.92
OA3d 4.09 0.9928 3.71 0.71 0.9927 0.54 5.76
OA4d 5.58 0.9903 4.97 2.85 0.9989 2.19 1.96
OA5d 4.44 0.9954 4.12 3.65 0.9921 2.80 1.22
OA6d 3.53 0.9951 3.26 2.6 0.9935 1.99 1.36
OA7d 4.33 0.9985 3.70 0.40 0.9976 0.31 10.83
OA8d 3.36 0.9955 2.97 1.18 0.9963 0.90 2.85
OA9d 2.73 0.9961 1.84 1.95 0.9987 1.495 1.4
OAlOd 0.68 0.9964 0.36 0.65 0.9960 0.498 1.05
OAlld 5.19 0.9940 4.64 4.05 0.9988 3.11 1.28
OA12d 1.89 0.9943 1.74 3.35 0.9984 2.57 0.56
OA13d 1.92 0.9958 1.76 1.77 0.9952 0.77 1.084
OA14d 2.03 0.9979 1.84 2.56 0.9970 1.84 0.79
OA15d 5.89 0.9974 4.64 2.41 0.9928 1.53 2.44
OA16d 2.38 0.9907 2.19 1.49 0.9980 0.77 1.59
OA17d 0.96 0.9931 0.87 0.40 0.9901 0.31 2.40
OA18d 1.81 0.9968 0.91 1.14 0.9935 0.77 1.59
AAZ 6.523 0.9915 60.85 7.85 0.9906 5.37 8.31
SSA 0.923 0.9965 0.83 0.256 0.9856 0.19 3.61