Treatment of 1-(4-(pyrrolidin-1-ylsulfonyl)phenyl)ethanone (1) with bromine in a mixture of dioxane/diethy- lether afforded the 2-bromo-1-(4-(pyrrolidin-1-ylsulfonyl)phenyl)ethanone (2) in a good yield, (Scheme 1). The IR spectrum of compound 2 showed strong absorption band at ν = 1707 cm−1 assignable to ketonic carbonyl group. Other important bands revealed at ν = 1336 and 1161 cm−1 characterized for sulfonyl group. The 1H NMR spectrum showed two triplet signals at δ = 1.99 and 3.27 ppm corresponding to pyrrolidine protons, and a singlet signal at δ = 4.56 ppm due to active methylene of bromoacetyl moiety. Other important signal appeared at δ = 7.84 and 8.22 ppm due to aromatic protons. Furthermore, the 13C NMR spectrum of compound 2 displayed two important signals at δ = 32.7 and 193.5 ppm corresponding to the active methylene of bromoacetyl and ketonic carbonyl carbons, respectively. The mass spectrum of compound 2 revealed molecular ion peaks at m/z = 331 and 333 reflecting the isotopes of bromine. Condensation of 1 with thiosemicarbazide gave the corresponding thiosemicarbazone derivative 3, which when reacted with phenacyl bromide derivative 2 afforded the corresponding thiazole derivative 4, which exhibited singlet signal in 1H NMR due to CH-thiazole at δ = 7.28 ppm and (D2O exchangeable) signal at δ = 8.87 ppm due to NH proton (Scheme 1).
2-Aminothiazole derivative 5, was synthesized by two different methods, either starting with an acetophenone derivative 1 (Method 1) or phenacyl bromide derivative 2 (Method 2). Method 1, which involves the reaction of 1 and thiourea in the presence of equivalent amount of iodine. The yield was low, furthermore, iodine had to be recycled because of it is the pollution problems. In order to overcome these drawbacks, the second method was employed. When phenacyl bromide derivative 2, was reacted with thiourea in ethanol (Method 2), the yield could be raised to 90% and the reaction time was decreased (Scheme 2). The IR spectrum of 5 showed, two bi-forked characteristic absorption bands at ν = 3384 and 3334 cm−1 assignable to amino group. Its 1H NMR spectrum revealed a single signal of one proton appeared in aromatic region at δ = 6.99 ppm, corresponding to CH-thiazole, and (D2O exchangeable) singlet at δ = 7.45 ppm, corresponding to amino protons. The 13C NMR spectrum of compound 5 revealed nine carbon types; for thirteen carbon atoms; the most important signals appeared at δ = 105.2 and 170.3 ppm corresponding to thiazole-C5 and thiazole-C2, respectively. The mass spectrum of 5 showed a molecular ion peak at m/z = 309. The investigation was extended to include the behavior of 2-aminothiazole derivative 5 towards some electrophiles. Thus, treatment of 5 with ethyl cyanoacetate gave acyclic cyanoacetamide derivative 6, rather than the expected cyclic product of thiazolo[3,2-a]pyrimidine derivative 7. The obtained product was established based on elemental analysis and spectral data. Thus, IR spectrum of 6 revealed absorption bands at ν = 3273, 2220 and 1696 cm−1 due to NH, cyano and carbonyl groups, respectively. 1H NMR spectrum showed singlet signal at δ = 4.20 ppm due to active methylene protons, and (D2O exchangeable) signal at δ = 9.15 ppm due to NH proton. On other hand, condensation of 5 with 4-flourobenzaldehyde in boiling ethanol gave the corresponding 4-fluorobenzylidene derivative 8 (Scheme 2).
Interaction of phenacyl bromide derivative 2 with thiosemicarbazide afforded 2-hydrazinyl thiazole derivative 10, instead of 2-aminothiadiazine derivative 9, the appearance of NH absorption band at ν = 3269 cm−1, in IR spectrum, and at δ = 10.35 ppm, in 1H NMR spectrum, supported the structure 10 and ruled out the other possible structure 9. Cyclocondensation of 2 with 4-fluorobenzylidenethiosemicarbazide gave thiazole derivative 11, an equivocal support for structure 11 was achieved via its synthesis through condensation of 2-hydrazinyl thiazole derivative 10 with 4-fluorobenzaldehyde in refluxing ethanol (Scheme 3).
Scheme 1. Synthesis of compounds 1-4.
Cyclocondensation of 2 with thioacetamide derivatives namely thioacetamide and phenylthiourea gave thiazole derivatives 12a,b, respectively (Scheme 4). Thiazole derivatives 12a,b were established on the basis of elemental analysis and spectral data. Thus, IR spectrum of 12a lacked the absorption band of carbonyl function and revealed absorption bands at ν = 1339, 1161 cm−1 due to sulfonyl group. However, 1H NMR spectrum of 12b
Scheme 2. Synthesis of compounds 5,6,8.
Scheme 3. Synthesis of compounds 10,11.
Scheme 4. Synthesis of compounds 12a,b-17.
showed singlet signal at δ = 8.97 ppm due to NH proton, which discharged with D2O. Also, compound 2 reacted with potassium cyanide in refluxing ethanol to afford acyclic product identified as 3-oxo-3-(4-(pyrrolidin-1- ylsulfonyl)phenyl)propanenitrile (13) which confirmed by elemental analysis and spectral data. Thus, IR spectrum revealed an absorption band at ν = 2218 cm−1 corresponding to cyano group. 1H NMR spectrum showed a singlet signal at δ = 3.67 ppm corresponding to active methylene protons. Additionally, interaction of phenacyl bromide derivative 2 with 2-aminopyridine and 3-amino-1,2,4-triazole afforded fused imidazo derivatives 14, 15, respectively. IR spectrum of 14 lacked the absorption band of carbonyl function of bromoacetyl moiety and its mass spectrum was compatible with molecular formula C17H17N3O2S (M+: 327). 1H NMR spectrum of 15 showed two triplets signals at δ = 1.92 and 3.77 ppm corresponding to pyrrolidine protons. Besides, singlet signals at δ = 7.51 and 8.81 ppm due to CH-6 and CH-3, respectively, of imidazotriazole ring, in addition to, D2O exchangeable signal at δ = 12.01 ppm due to NH proton. Aligned with the aim of synthesis of different substituted pyrrolidine benzenesulfonamide, compound 2 was reacted either with 4-chloroaniline or ammonium diethylcarbamodithioate to afford compounds 16 and 17, respectively (Scheme 4).
Treatment of a solution of ethyl acetoacetate in DMF with phenyl isothiocyanate in the presence of potassium hydroxide, at room temperature followed by the addition of an equimolar amount of phenacyl bromide derivative 2 afforded only one isolable product (TLC) for which three proposed structures 18, 19 or 20 seemed possible (Scheme 5). Structures 18 and 19 were ruled out on the basis of 1H NMR spectrum of the isolated product. Thus, 1H NMR spectrum of 20 showed singlet signal at δ = 6.73 ppm due to CH-thiazole. On the other hand, when potassium salt of malononitrile was treated with 2 furnished only one isolable product (TLC) for which two proposed structures 21 or 22 seemed possible (Scheme 5). Structure 21 was ruled out based on IR, 1H NMR
Scheme 5. Synthesis of compounds 20,22.
and mass spectral data. Thus, IR spectrum of 22 showed no absorption bands for NH, NH2 or C=O groups, 1H NMR spectrum showed singlet signal at δ = 7.21 ppm due to CH-thiazole, and the mass spectrum was compatible with the molecular formula C22H18N4O2S2 (M+; 434).
The goal was extended to include the behavior of 2 towards heterocyclic amines for building different fused heterocyclic rings. Thus, treatment of 2 with 2-aminothiazole and 2-aminobenzothiazole in refluxing ethanol yielded imidazo[2,1-b]thiazole 23 and benzo[d]imidazo[2,1-b]thiazole 24, respectively (Scheme 6). Efforts to
Scheme 6. Synthesis of compounds 23-25,27.
cyclize 2 with 2-mercapto-4,6-dimethylnicotinonitrile [
Treatment of phenacyl bromide derivative 2 with o-phenylenediamine in refluxing ethanol afforded a crystalline product identified as 2-(4-(pyrrolidin-1-ylsulfonyl)phenyl)quinoxaline(30) in an excellent yield (Scheme 7). A plausible mechanism may involve the condensation of one of phenylenediamine amino groups with the carbonyl group of bromoacetyl moiety, while the second amino group replaced bromine atom via nucleophilic substitution. The expected product is the dihydroquinoxalinyl derivative 29, however, the spectral data of the isolated product established that the dihydroquinoxalinyl derivative 29 was oxidized under the reaction conditions
Scheme 7. Synthesis of compounds 30,31,33.
to give quinoxaline derivative 30. The IR spectrum of 30 showed no absorption band for NH group. The 1H NMR spectrum showed a singlet, of one proton, at δ = 8.99 ppm due to quinoxaline-H3. The presence of nine aromatic carbon types for fourteen aromatic carbon atoms on 13C NMR spectrum of the isolated product between δ = 121.0 and 160.2 ppm, in addition to two aliphatic carbon types for four carbon atoms of pyrrolidine moiety at δ = 20.9 and 62.7 ppm, confirmed structure 30. Cyclocondensation of phenacyl bromide derivative 2 with o-aminothiophenol afforded benzo[b][1,4]thiazine derivative 31. Similarly treatment of 2 with 1H-benzo [d]imidazole-2-thiol afforded benzo[4,5]imidazo[2,1-b]thiazole derivative 33 via cyclization of acyclic intermediate 32 by dehydration under the reaction conditions (Scheme7).
Interaction of phenacyl bromide derivative 2 with 3-methyl-5-oxo-4,5-dihydro-1H-pyrazole-1-carbothioamide [
Thymidylate synthase and dihydrofolate reductase are among the main targets involved in anticancer and antimicrobial activity [
The active site revealed that hydrogen bond interactions beside hydrophobic interactions were considered responsible for the observed affinity as it acts as a hydrogen bond donor to the backbone Ile 5 and Ile 94 residues and the side chain Asp 27 residue. It also acts as a hydrogen bond accepter to Arg 52 and Arg 57 residues. This beside many hydrophobic interactions with various amino acid residues: ILe 5, Ala 6, Ala7, Asp 27, Leu 28, Phe
Scheme 8. Synthesis of compounds 34,35.
31, Lys 32, Ser 49, Ile 50, Arg 52, Leu 54, Arg 57, Ile 94, Tyr 100 and Thr 113, as shown in (
MOE docking studies of the inhibitors were performed using dihydrofolate reductase co-crystallized with methotrexate (PDB ID: 4DFR) as a template.
1) Docking of compound 1 into DHFR
The active site revealed the presence of one hydrogen bond interaction as one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the amino acid residue Ser 59 (2.80 Å) with a strength of 72.5%. In addition to, hydrophobic interactions involving carbon atom of carbonyl function, C2,3,5,6 of phenyl ring, C2,3,4 of pyrrolidine ring and oxygen atom of SO2 moiety with the following amino acid residues: Val 8, Ala 9, Asp 21, Glu 30, Phe 31, Phe 34, Thr 56 and Ser 59, as shown in (
2) Docking of compound 2 into DHFR
The active site revealed that several molecular interactions were considered responsible for the observed affinity, as the one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the side chain residues; Thr 56 and Ser 59 (3.63 Å and 3.16 Å, respectively) with a strength of 2.4% and 10.9%, respectively. Besides to, hydrophobic interactions involving the bromine atom, oxygen atom of carbonyl function and other carbons as well the second oxygen atom of SO2 moiety and the following amino acid residues: Ile 16, Leu 22, Phe 31, Ile 60, Pro 61, Val 115 and Tyr 121, as shown in (
3) Docking of compound 4 into DHFR
The active site revealed the presence of hydrogen bond interaction between hydrogen atom of NH function as it acted as a hydrogen bond donor with the side chain residue Asp 21 (2.16 Å) with a strength of 23%. Moreover, one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor with the amino acid residue Thr 56 (2.83 Å) with a strength of 29.7%. Besides to, arene-arene cation interaction between the phenyl ring of benzothiazole moiety and the amino acid residue Phe 31. In addition to, hydrophobic interactions among other atoms of the compound with the following amino acid residues: Ala 9, Ile 16, Gly 17, Asp 21, Leu 22, Gln 35, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Lys 68, Arg 70, Val 115 and Tyr 121, as shown in (
4) Docking of compound 5 into DHFR
The active site illustrated the presence of several interactions of the one oxygen atom of SO2 moiety with different amino acid residues as it acted as a hydrogen bond acceptor with the side chain residues; Thr 56 and Ser 59 (3.57 Å and 3.02 Å, respectively) with a strength of 2.4% and 21.4%, respectively. This beside hydrophobic interaction among the amino function, sulfur atom and C4 of thiazole moiety, C2,6 of benzene ring, oxygen atoms of SO2 moiety and pyrrolidine C2, C3, C5 and the following amino acid residues: Ile 16, Leu 22, Phe 31, Phe 34, Ile 60, Pro 61, Leu 67, Val 115 and Tyr 121, as shown in (
5) Docking of compound 8 into DHFR
The active site revealed the presence of hydrogen bond interaction between the one oxygen atom of SO2 moiety as it acted as a hydrogen bond acceptor with the side chain residues; Thr 56 and Ser 59 (3.35 Å and 3.23 Å, respectively) with a strength of 4.1% and 7.4%, respectively. In addition to, hydrophobic interactions involving the other atoms of the compound with the following amino acid residues: Ile 16, Asp 21, Leu 22, Phe 31, Gln 35, Ile 60, Pro 61, Asn 64, Leu 67, Lys 68, Val 115 and Tyr 121, as shown in (
6) Docking of compound 11 into DHFR
The active site revealed the presence of several molecular interactions in which the one oxygen atom of SO2 moiety acted as a hydrogen bond acceptor for with the amino acid residue Thr 56 (3.57 Å) with a strength of 2.6%. In addition to, hydrophobic interactions involving other atoms of the compound with the following amino acid residues: Ala 9, Ile 16, Asp 21, Leu 22, Phe 31, Phe 34, Gln 35, Ser 59, Pro 61, Asn 64, Arg 70, Val 115 and Tyr 121, as shown in (
7) Docking of compound 13 into DHFR
The active site revealed the presence of hydrogen bond interactions between one oxygen atom of SO2 moiety and the cyano group as they acted as a hydrogen bond acceptor with the side chain residues; Thr 56 and Thr 136 (3.41 Å and 3.25 Å, respectively) with a strength of 1.6% and 13%, respectively. There is also hydrophobic interactions involving the pyrrolidine C2, C3, C4 as well as the oxygen atoms of SO2 moiety with the following amino acid residues: Ile 7, Val 8, Ala 9, Ile 16, Leu 22, Glu 30, Phe 34, Ser 59, Ile 60, Val 115 and Tyr 121, as shown in (
8) Docking of compound 20 into DHFR
The active site revealed the presence of several molecular interactions, including two hydrogen bonds. In which both oxygen atoms of SO2 moiety acted as a hydrogen acceptor via two hydrogen bonds with the amino acid residue Asn 64 (2.80 Å and 3.18 Å, respectively) with a strength of 39.1% and 7.2%, respectively). Besides to hydrophobic interactions involving the cyano function as well as the other atoms of the compound with the following amino acid residues: Val 8, Ile 16, Asp 21, Leu 22, Phe 31, Phe 34, Thr 56, Ser 59, Ile 60, Pro 61, Asn 64, Val 115 and Tyr 121, as shown in
9) Docking of compound 24 into DHFR
The active site revealed the presence of arene-arene cation interaction between the phenyl ring of benzo[d]imidazothiazole with the amino acid residue Phe 31. In addition to, hydrophobic interactions involving other atoms of the compound with many amino acid residues: Val 8, Ala 9, Ile 16, Leu 22, Phe 31, Thr 56, Ser 59, Ile 60, Pro 61 and Val 115, as shown in (
10) Docking of compound 33 into DHFR
The active site revealed only hydrophobic interactions concerning C2,3,4 of pyrrolidine ring and C2,7,8 of benzo [d]imidazothiazole with the following amino acid residues: Ile 16, Leu 22, Phe 31, Phe 34, Ser 59, Ile 60, Pro 61, Asn 64, Val 115 and Tyr 121, as shown in (
Docking was performed for the compounds 1, 2, 4, 5, 8, 11, 13, 20, 24 and 33 on the dihydrofolate reductase in a trial to predict their mode of action as anticancer drugs. The compounds show several interactions with dihydrofolate reductase enzyme. Particularly noteworthy are the compounds 4, 8, 11, 20, 24 and 33, which suggest that they might exert their action through inhibition of the DHFR enzyme (
The newly synthesized compounds were evaluated for their in-vitro cytotoxicity against human liver hepatocel-
| Comp. No. | Score | E-conf | E-place | E-score 1 | E-score 2 | E-refine |
|---|---|---|---|---|---|---|
| 1 | −16.87 | −05.76 | −61.37 | −08.38 | −16.87 | −13.19 |
| 2 | −16.30 | −00.84 | −57.58 | −08.81 | −16.30 | −15.73 |
| 4 | −28.18 | 08.55 | −105.27 | −09.29 | −28.18 | −07.14 |
| 5 | −16.91 | −61.16 | −65.08 | −09.14 | −16.91 | −13.89 |
| 8 | −23.18 | −02.87 | −93.77 | −11.43 | −23.18 | −15.38 |
| 11 | −22.42 | 17.10 | −78.04 | −10.31 | −22.42 | −18.02 |
| 13 | −16.91 | −24.24 | −61.74 | −08.16 | −16.91 | −08.57 |
| 20 | −27.21 | 24.44 | −100.42 | −09.65 | −27.21 | −03.40 |
| 24 | −21.15 | 26.14 | −99.85 | −09.28 | −21.15 | −12.28 |
| 33 | −21.24 | 18.53 | −65.44 | −09.05 | −21.24 | −07.83 |
Score: for all scoring functions, lower scores indicate more poses that are favorable. The unit for all scoring functions is kcal/mol. E-conf: the energy of the conformer. If there is a refinement stage, this is the energy calculated at the end of the refinement. E-place: Score from the placement stage (Placement. A collection of poses is generated from the pool of ligand conformations using one of the placement methods). E-score 1: Score from the first rescoring stage. E-score 2: Score from the second rescoring stage. E-refine: Score from the refinement stage (Refinement: Energy minimization of the system is carried out using the conventional molecular mechanics setup).
lular carcinoma cell line (HepG2). Some of the tested compounds were more potent compared with methotrexate as the reference drug. From the obtained results in
Chart 1. Comparison of the Selective index (SI) for the synthesized compounds against human liver hepatocellular carcinoma cell line (HepG2).
Chart 2. Comparison of the docking Score energy of the selective newly synthesized compounds.
22 > 12a > 33 > 35 > 27 > 24 > MTX > 20 > 3 > 1 > 13 > 17 > 16 > 34 > 23 > 14 > 25 > 2 > 6 > 5 > 30 > 15 > 10 > 31 > 12b. These preliminary results of biological screening of the tested compounds could offer an encouraging framework in this field that may lead to the discovery of potent anticancer agent.