Design and Efficient Synthesis of New 4-Amino-Substituted 2-(4-Bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines of Anticancer Interest and Their In Silico Study

Abstract

Thienopyrimidines are an emerging class of fused pyrimidines due to their broad spectrum of pharmacological properties, including antimicrobial, anti-inflammatory, antimalarial, anticancer, etc. The anticancer activity of these compounds has been mechanistically proven via the inhibition of validated drug targets, such as EGFR, VEGFR-2, PI3K, and c-kit. In this research article, we designed and synthesized new 4-amino-substituted 2-(4-bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines to explore their anticancer potential. These heterocycles were designed based on pharmacophoric features of the core heterocycle, varying its C4 substitution with a variety of amines and considering cancer protein-ligand interactions with the aim to obtain potent lead molecules. The target compound-protein interaction complexes were analyzed, and lead compounds were identified based on their better binding affinity in molecular docking studies.

Key words

thienopyrimidine - benzo[4,5]thieno[2,3-d]pyrimidines - microwave irradiation - anticancer - synthesis - molecular docking - ADME

The thienopyrimidine scaffold stands out as a widely utilized structure in pharmaceutical development.[1] [2] Bearing structural and isoelectronic similarities to purine, thienopyridine-containing compounds are a compelling drug design element.[3,4] These compounds boast diverse pharmacological properties, including antimicrobial, antibacterial, antiviral, antiprotozoal, anti-inflammatory, and anticancer activities.[5–11] Thienopyrimidine-based drugs, each exhibiting diverse biological activity profiles, e.g. sufugolix (TAK-013), a GnRHR antagonist developed by Takeda Pharmaceutical, underwent clinical investigation for the treatment of prostate cancer and endometriosis.[12] However, the exploration of sufugolix ceased at phase II clinical trials, and it was replaced by relugolix, another thieno[2,3-d]pyrimidine derivative. The drug has successfully completed phase III trials, demonstrating efficacy in treating endometriosis and prostate carcinoma as a GnRHR antagonist.[13]

Similarly, DDP-225, a thienopyrimidine compound developed as a serotonin receptor (5-HT3) antagonist and noradrenaline reuptake inhibitor, progressed to phase II clinical trials for treating irritable bowel syndrome (IBS) and gastrointestinal tract (GIT) diseases.[14] [15] Pictilisib (GDC-0941), a derivative of thieno[3,2-d]pyrimidine, is currently undergoing phase II clinical trials for inhibiting phosphatidylinositol 3-kinase (PI3K) in advanced solid tumors.[16] Additionally, olmutinib inhibits epidermal growth factor receptor (EGFR) and is indicated for treating Non-small cell lung cancer.[17] [18] PRX-08066, identified as an inhibitor of fibroblasts, shows potential as an anticancer candidate.[19] PF-03758309, another thienopyrimidine-based drug targeting advanced solid tumors, is in phase I clinical trials.[20] SNS-314, a potent inhibitor of Aurora kinases A, B, and C, is undergoing phase I clinical trials for treating advanced solid tumors.[21] Additionally, GNE 490 and GNE-493, two thienopyrimidine derivatives designed from structural modification of GDC-0941, exhibit promising pharmacokinetic parameters and high potency/selectivity in inhibiting the PI3K pathway, undergoing phase II clinical trials for breast cancer treatment.[22] Apitolisib, another PI3K inhibitor, is also in phase II clinical trials (Figure [1]).[23]

Figure 1 Chemical structures of drugs containing thienopyrimidine moiety

From the perspective of anticancer drug design, thieno[2,3-d]pyrimidines have been extensively investigated as kinase and topoisomerase inhibitors. Studies on their structure-activity relationship (SAR), as documented in the literature, emphasize the importance of a 6/7-membered carbocyclic ring fused with a thiophene ring. Some of these compounds are summarized in Figure [2].[24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] Interestingly, it has been noted that 4-amino substituents, whether aryl/aryl alkyl or urea, play a crucial role in binding to cancer proteins. These findings, particularly regarding the 6-membered ring-fused thieno[2,3-d]pyridines and their medicinal properties, prompted us to develop a more efficient delivery system for new 4-amino-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines. These compounds feature a C2-substituted 4-bromobenzyl group designed to enhance their binding affinity towards cancer kinases and topoisomerases, thereby forming stable complexes that might lead to improved anticancer efficacy. Moreover, the C2-substitution strategy is anticipated to augment pharmacokinetic stability and bioavailability by minimizing susceptibility to metabolizing enzymes, including xanthine and aldehyde oxidases.[39] [40] [41]

Figure 2 Some reported compounds having carbocyclic ring-fused benzo[4,5]thieno[2,3-d]pyrimidine scaffold with their respective biological action

To obtain the designed leads structures, we were interested in the synthesis of new 4-amino-substituted 2-(4-bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines to explore their anticancer potential.

To introduce the p-bromobenzyl moiety in the target compounds, we first prepared compound 4 by reacting the intermediate 2-amino ester derivative 2 with 4-bromophenylacetonitrile (3) under acidic conditions. 2-Amino ester derivative 2 was prepared by condensing cyclohexanone (1) with active nitrile and elemental sulfur under typical Gewald­ reaction conditions.[42] Further, a chloro derivative 5 as the precursor of target compounds 7 was synthesized by treating compound 4 with POCl₃ for 3 h under reflux, as shown in Scheme [1].

Scheme 1 Synthetic route for the synthesis of compound 5 as the precursor of target compounds 7

Our next aim was to carry out S_NAr at 5 efficiently using various amines to generate the drug candidates in a short period of time for their biological screening.

To find out the optimum reaction conditions, we first set up a model reaction for the formation of 7a; we treated 5 (1 mmol, 1 equiv) with 6a (1.1 equiv) as a model substrate under various reaction conditions (Table [1]). The maximum yield (entry 17, 91%) of product 7a was obtained by carrying out the reaction in DMF for 5 min at 80 °C under microwave conditions (CEM discover), indicating the importance of aprotic polar solvent and microwave irradiations accelerating the rate of reaction in a short time duration.[43] [44] [45] Adding ice water to the reaction mixture yielded white precipitates 7a with sufficient purity.

Table 1 Optimization of the Reaction of 2-(4-Bromobenzyl)-4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine with 1-(Pyridin-4-yl)piperazine

Entry	Reaction conditions	Yield (%)a,b
1	neat	trace
2	PhMe, rt, 24 h	10
3	PhMe, 80 °C, 10 h	13
4	PhMe, 80 °C, MW, 15 min	20
5	hexane, rt, 10 h	11
6	hexane, 80 °C, 10 h	15
7	hexane, 80 °C, MW, 15 min	16
8	CHCl3, rt, 10 h	12
9	CHCl3, 80 °C, 10 h	16
10	CHCl3, 80 °C, MW, 13 min	21
11	DCM, rt, 10 h	18
12	DCM, 80 °C, 10 h	22
13	DCM, 80 °C, MW, 13 min	32
14	THF, rt, 10 h	40
15	THF, 80 °C, 10 h	45
16	DMF, rt, 10 h	65
17	DMF, 80 °C, MW, 5 min	91
18	DMSO, rt, 10 h	30
19	DMSO, 80 °C, 10 h	34
20	DMSO, 80 °C, MW, 10 min	70
21	MeOH, 80 °C, 10 h	67
22	MeOH, rt, 24 h	42
23	MeOH, 80 °C, MW, 9 min	74
24	H2O, rt, 10 h	30
25	H2O, 80 °C, 10 h	42
26	H2O, 80 °C, MW, 20 min	49

^a Isolated yield.

^b 1 mL of the solvent was used wherever mentioned.

Having optimized reaction conditions in hand, we synthesized a series of target compounds by treating a variety of amines (Table [2]) in good to excellent yields (76–91%). There was not much significant impact on the yields of the products (7b–7g) due to the electronic nature of the substituents, e.g., entries 1–6 (Table [2]).

Table 2 Synthesis of New 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines 7b–7s

Entry	R2NH or RNH2	Product	Time (min)	Isolated yield (%)
1			8	81
2			9	79
3			8	83
4			5	80
5			7	85
6			5	78
7			10	84
8			6	87
9			7	81
10			8	76
11			6	82
12			5	78
13			5	80
14			4	90
15			5	88
16			5	83
17			8	91
18			6	90

Interestingly, selective product formation occurred with the substrates having additional nucleophilic groups such as amine and alcohol (6o, 6q–6r). Moreover, no bis-adduct formation with ethanolamine (6o) or propane-1,3-diamine (6q) was observed. Fortunately, no de-Boc product formation was observed with Boc-protected piperazine 6s. Furthermore, a Boc-protected target compound 7s provides scope for linkerology for Proteolysis Targeting Chimeras (PROTACs) development, an important modality for protein degradation.[46] All the target compounds 7a–7s were fully characterized by mp, NMR, and HRMS (see Supporting Information, Figures S2–S68).

To evaluate the binding affinity of the newly designed 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines 7, all synthesized derivatives 7a–7s were docked against key anticancer­ targets, including EGFR, VEGFR-2, PI3K, and c-kit (see Supplementary Information, Table S1) using Schrödinger (version 2023-1) through the Glide module.

Our analysis indicated that 7o exhibited the most favorable binding among all docked compounds at the active sites of EGFR (PDB ID – 1M17), VEGFR-2 (PDB ID – 4ASE), c-kit (PDB ID – 4U0I), and PI3K-γ (PDB ID – 3DBS) as shown in Figure [3]A–3H. Remarkably, compound 7o demonstrated increased binding affinity, which was attributed to favorable interactions with critical amino acids at the EGFR active site, facilitated by its polar terminal group. The terminal hydroxy group of 7o formed a hydrogen bond with CYS773 within 1.96 Å, resulting in non-covalent targeting distinct from afatinib,[47] and additionally established hydrogen bonding with the THR766 amino acid residue. Conversely, compound 7q, with one extra carbon in the chain length, exhibited a conserved hydrogen bonding interaction between the terminal amino group and CYS773 within 2.03 Å. However, it displayed unfavorable interactions with LYS721 and lacked interaction with LEU764, leading to a lower docking score of –5.360 and binding affinity than 7o (dock score: –6.859). Likewise, compound 7r, featuring an aminoethylpiperazine group, failed to form a hydrogen bond with CYS773. Instead, the piperazine group formed hydrogen bonds with the ASP-831 amino acid residue, resulting in a modified binding orientation at the EGFR active site and consequently yielding a lower docking score of –5.505 compared to 7o (dock score: –6.859) (see Supporting Information, Figure S67). 7o exhibited significant interactions at the ATP binding site of EGFR, resembling those of erlotinib, with the bromine attached to the phenyl ring forming halogen bonds with LYS721 and LEU764 amino acid residues. Close proximity interactions were also observed with LEU768, MET769, GLY772, and THR830 (Figure [3]A and3 B).

Figure 3 Three-dimensional representation of: (A) binding pocket of 7o at EGFR (1M17), (B) displaying significant binding interactions of 7o at active site of EGFR, (C) binding pocket of 7o at VEGFR-2 (4ASE), (D) displaying important binding interactions of 7o at active site of VEGFR-2, (E) binding pocket of 7o at c-KIT (4U0I), (F) displaying meaningful binding interactions of 7o at the active site of c-KIT, (G) binding pocket of 7o at PI3K-γ (3DBS), and (H) displaying crucial binding interactions of 7o at the active site of PI3K-γ

Docking analysis of 7o at the ATP binding site of VEGFR-2 (Figure [3]C) revealed key interactions, including aromatic hydrogen bonding between one of the nitrogens of the thienopyrimidine ring and PHE1047, hydrogen bonding between the sulfur of the thienopyrimidine ring and CYS919, and hydrogen bonding between the cyclohexane ring and PHE918 amino acid residues. The bromine attached to the phenyl ring also formed halogen bonds with LYS868. Compared to vandetanib, 7o also exhibited hydrogen bonding with GLU885 and aromatic hydrogen bonding with VAL914 and ASP1046 (Figure [3]D).

In the case of c-kit, 7o demonstrated a best-fitting pose in the adenine pocket of the ATP-binding site (Figure [3]E), akin to ponatinib, with interactions such as hydrogen bonding between the hydroxy group and GLU640 amino acid residue, hydrogen bonding between the bromobenzene ring and THR670, ASP810, and proximity with CYS788 (Figure [3]F).

Moreover, docking studies of all the derivatives at the active site of PI3K (α, β, and γ) revealed 7o as the most favorable fit within the active site of PI3K-γ (Figure [3]G), closely resembling the pose of the standard compound GDC0941 (a co-crystallized ligand). Key interactions observed included hydrogen bonding between the OH group of ethanolamine linked to the pyrimidine of 7o and ASH841 and TYR867. Additionally, the bromo phenyl ring of 7o exhibited aromatic hydrogen bonding with ASP950 and ASP964 amino acid residues. Furthermore, the cyclohexyl ring of 7o demonstrated hydrogen bonding with GLU880 and MET953 amino acid residues (Figure [3]H).

To explore the druglike ability of compound 7o, ADME analysis via silico studies was performed. The parameters including partition coefficient (QlogPo/w) value 4.782 (acceptable range: –2.0 to 6.5), aqueous water solubility (QPlogS) value –5.715 (acceptable range: –6.5 to 0.5), cell permeability (QPPCaco) value 2452.753 (acceptable range: <25 poor, >500 great) and Lipinski violations value 0 (maximum is 4) were obtained and found to be in the acceptable range. Additionally, MMGBSA dG bind values of all the synthesized compounds 7a–7s with respect to EGFR are reported in Table S1.

Furthermore, we performed molecular dynamics (MD) simulations using the 7o complex with VEGFR-2 to explore protein-ligand interactions and determine the thermodynamic stability of the docked ligand at the VEGFR-2 active site. The simulations were conducted for 100 ns, and the interaction patterns between 7o and the amino acids were analyzed. During the MD simulations, stable trajectories were observed for 7o with VEGFR-2 for the initial 60 ns. This stability was evident from the temporal changes in potential energy and the root-mean-square deviation (RMSD) of the protein-ligand complex, as shown in Figure [4]C. A stable interaction range of 1 Å to 3 Å was observed during the first 60 ns of the simulation. However, fluctuations were noted in the protein-ligand complex trajectory between 60 and 80 ns, with a sudden increase in RMSD possibly due to conformational changes following initial protein-ligand interactions (Figure [4]C). Despite these fluctuations, no substantial structural alterations were observed, and the ligand conformation remained consistently oriented within the VEGFR-2 active site, where 7o interacts with LEU840, PHE918, CYS919, ASN923, PHE1047, and ARG1051 (Figure [4]A). Additionally, stable interactions of 7o with key amino acids, such as LEU840, ASN923, and PHE1047, with interaction fractions of ≥0.5, were visualized (Figure [4]B). The interaction with ASN923 remained almost 100% stable throughout the simulation time, as shown in Figure [4]D. Throughout the MD simulations, ligand 7o maintained most of the interactions observed in the docking studies.

Figure 4 Molecular dynamic simulation studies of 7o at the active site of VEGFR-2: (A) 2D MD simulation pose of 7o at the active site of VEGFR-2, (B) interactions (fraction) of 7o at the VEGFR-2 active site via MD simulation at 100 ns showing the type of protein-ligand contacts, (C) RMSD graph showing 7o interaction at the VEGFR-2 active site for 100 ns, and (D) a timeline representation of the interactions and contacts of 7o with VEGFR-2

Furthermore, we investigated the preliminary in vitro antiproliferative effect (MTT assay) of 7o at 10 μM concentration against a variety of cancer cell lines such as breast (MCF-7, T47D, and MDAMB-231), lung (A549), hepatocellular carcinoma (HCC) and head and neck carcinoma (FaDu). The results revealed that there was 50.22% growth inhibition of FaDu cells; however, there was not much decrease in the cell viability (<25%) of other cancer cells. These results highlight the role of the above mentioned targets (in particular EGFR and VEGFR) in cancer cell signaling especially in case of FaDu cells where their expression is around 90%. However, this further warrants the detailed in vitro biological investigations.

In conclusion, we have demonstrated an efficient, greener, fast, and high-yielding approach for the synthesis of designed 4-amino-substituted 2-(4-bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines under microwave conditions for the development of anticancer agents. The method allows for the generation of a compound library for structure-activity relationship (SAR) studies. Our in silico findings suggest that compounds with polar or electron-donating groups at the 4-amino-substituted thieno[2,3-d]pyrimidines may enhance binding affinity through hydrogen bonding compared to bulkier or electron-withdrawing groups for the inhibition of different anticancer targets, such as EGFR, VEGFR, c-kit, and PI3K. This insight underscores the importance of such substitutions in modulating the biological activity of the molecule, guiding further optimization for enhanced binding and therapeutic potential. Through QikProp, the ADME analysis revealed that the target compounds might have improved efficacy and bioavailability with no cytotoxicity toward normal cells. Furthermore, in vitro and in vivo anticancer evaluation of 7o is underway, and results will be published in due course.

Synthetic experiments were conducted using an oven-dried apparatus. Microwave-assisted reactions were performed using a CEM microwave synthesizer. HRMS were acquired on a quadrupole/TOF mass spectrometer equipped with an ESI source. Solvents were distilled using standard distillation procedures. NMR spectra (¹H at 600 MHz and ¹³C at 151 MHz) were recorded on a JEOL-NMR instrument. Chemical shifts for ¹H and ¹³C were referenced to the residual signals of the solvents [CDCl₃ δ 7.26 for ¹H and δ 77.16 for ¹³C, DMSO-d ₆ δ 2.50 for ¹H and δ 39.52 for ¹³C] at 25 °C. TLC was employed for reaction monitoring using 0.25 mm Merck silica gel plates (60 F₂₅₄); spots were visualized in EtOAc/petroleum ether (1:9). All reagents utilized in the preparation of the 4-amino substituted 2-(4-bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines were procured from Avra.

Ethyl 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2)

To a stirred solution of cyclohexanone (12 mL, 0.12 mmol), ethyl 2-cyanoacetate (20 mL, 0.12 mmol), and sulfur (3.8 g, 0.12 mmol) in EtOH (50 mL) was added Et₂NH (12 mL, 0.12 mmol) dropwise. The mixture was then refluxed at 60 °C for 10 h. After the completion of the reaction (TLC), excess EtOH was evaporated using a rotary evaporator, and ice-cold water was added to the dried crude solid. The resulting precipitates were thoroughly washed with excess water (3 × 20 mL), filtered, and air-dried to obtain product 2, which was further purified by column chromatography to give a yellow solid;[48] yield: 18.78 g (72%); mp 107–109 °C.

¹H NMR (400 MHz, DMSO-d ₆): δ = 7.17 (s, 2 H), 4.12–4.06 (dd, J = 7.28 Hz, 2 H), 2.46–2.33 (m, 4 H), 1.64–1.58 (m, 4 H), 1.19 (s, 3 H).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 165.62, 163.44, 131.78, 115.92, 103.13, 59.11, 27.04, 24.44, 23.36, 22.96, 14.83.

MS (EI): m/z = 225 [M]⁺.

2-(4-Bromobenzyl)-5,6,7,8-tetrhydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one (4)

To a mixture of 2 (5 g, 0.022 mmol) in 1,4-dioxane (20 mL), was added 4-bromophenylacetonitrile (6.4 g, 0.033 mmol). The mixture was stirred at rt for 24 h with continuous addition of 2.0 M ethereal HCl (2.0 mL) dropwise. After the completion of the reaction (TLC), excess 1,4-dioxane was evaporated using a rotary evaporator, and the crude mixture was neutralized by 1 M aq NaOH solution (50 mL). The resulting precipitates were thoroughly washed with excess water (3 × 20 mL), filtered, and air-dried to obtain a crude product that was further purified using column chromatography to afford 4 [48] as a white solid; yield: 6.25 g (75%); mp 176–178 °C.

¹H NMR (400 MHz, DMSO-d ₆): δ = 12.24 (s, 1 H), 7.48 (d, J = 8.28 Hz, 2 H), 7.24 (d, J = 8.28 Hz, 2 H), 3.851 (s, 2 H), 2.79 (t, J = 5.32 Hz, 2 H), 2.66 (t, J = 5.16 Hz, 2 H), 1.74–1.67 (m, 4 H).

MS (EI): m/z = 374 [M]⁺

2-(4-Bromobenzyl)-4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (5)

POCl₃ (3 mL, 3 equiv) was added to 4 (2 g, 0.022 mmol) with continuous stirring under ice-cold conditions. The mixture was further reflexed for 10 h. After the reaction was complete (TLC), the resulting mixture was poured over crushed ice with stirring to yield precipitates. The precipitates were washed thoroughly with excess of water, filtered, dried, and purified using column chromatography to obtain 5 as a white solid; yield: 1.47 g (70%); mp 171–173 °C.

¹H NMR (400 MHz, DMSO-d ₆): δ = 7.46 (d, J = 7.6 Hz, 2 H), 7.22 (d, J = 7.2 Hz, 2 H), 4.19 (s, 2 H), 2.79 (t, J = 5.4 Hz, 2 H), 2.66 (t, J = 5.1 Hz, 2 H), 1.78–1.68 (m, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 169.7, 162.8, 153.2, 138.7, 137.0, 131.6, 131.0, 127.0, 126.7, 120.7, 44.5, 26.3, 26.1, 22.6, 22.3.

HRMS (ESI-TOF): m/z [M + 2 + H]⁺ calcd for C₁₇H₁₄BrClN₂S: 394.9823; found: 394.9771.

2-(4-Bromobenzyl)-4-(4-(pyridin-4-yl)piperazin-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (7a); Typical Procedure

To a mixture of 5 (393.72 mg, 1 mmol) in DMF (1 mL), 1-(4-pyridyl)piperazine (179.54 mg, 1.1 mmol) was added. The mixture was heated under microwave irradiation (CEM, Discover) for 5 min. After the completion of the reaction (TLC), crushed ice was charged to the mixture and it was vigorously stirred resulting in precipitate formation. The precipitates were thoroughly washed with excess water (5 × 20 mL), filtered, and air-dried to obtain the crude product, which was further purified by crystallization (5% CH₂Cl₂/MeOH) to obtain 7a as a white solid; yield: 422.60 mg (91%); mp 250–252 °C.

¹H NMR (600 MHz, CDCl₃): δ = 8.33 (s, 2 H), 7.39 (d, J = 8.6 Hz, 2 H), 7.28 (s, 2 H), 6.72 (s, 2 H), 4.14 (d, J = 7.8 Hz, 2 H), 3.48 (d, J = 24.9 Hz, 8 H), 2.89 (d, J = 12.5 Hz, 2 H), 1.94 (s, 2 H), 1.80 (s, 2 H), 1.68 (s, 2 H).

¹³C NMR (151 MHz, CDCl₃): δ = 169.3, 162.0, 161.9, 155.2, 150.0, 138.0, 135.0, 131.4, 131.1, 126.7, 120.3, 119.3, 108.6, 50.0, 45.8, 44.9, 26.9, 25.9, 23.1, 22.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₆H₂₆BrN₅S: 520.1171; found: 520.1172.

N-Benzyl-2-(4-bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7b)

White solid; yield: 95 mg (81%); mp 230–232 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.34 (d, J = 1.6 Hz, 1 H), 7.33 (d, J = 1.6 Hz, 1 H), 7.31 (d, J = 1.6 Hz, 1 H), 7.30 (d, J = 3.9 Hz, 2 H), 7.28–7.27 (m, 2 H), 7.23 (s, 1 H), 7.21 (s, 1 H), 5.55 (s, 1 H), 4.74 (d, J = 6.2 Hz, 2 H), 4.04 (s, 2 H), 2.84 (t, J = 4.7 Hz, 2 H), 2.77 (t, J = 4.7 Hz, 2 H), 1.88–1.84 (m, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.3, 163.3, 157.3, 139.1, 138.3, 132.6, 131.2, 131.1, 128.8, 127.7, 127.4, 125.2, 120.0, 114.1, 45.2, 44.7, 26.4, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₄H₂₂BrN₃S: 464.0796; found: 464.0797.

2-(4-Bromobenzyl)-N-(2-chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7c)

White solid; yield: 100 mg (79%); mp 222–2224 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.36 (s, 4 H), 7.20 (s, 2 H), 7.13 (s, 1 H), 7.07 (s, 1 H), 5.95 (d, J = 59.2 Hz, 1 H), 4.77 (t, J = 108.6 Hz, 2 H), 4.05 (s, 1 H), 3.94 (s, 1 H), 2.89 (s, 2 H), 2.77 (s, 2 H), 1.90 (s, 2 H), 1.61 (s, 2 H).

¹³C NMR (151 MHz, CDCl₃): δ = 163.2, 157.1, 138.4, 136.3, 133.7, 132.7, 131.3, 131.2, 131.0, 129.7, 129.6, 129.4, 129.0, 128.8, 128.3, 127.2, 126.9, 125.3, 45.2, 44.7, 42.6, 26.3, 25.5, 22.7.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₄H₂₁BrClN₃S: 498.0406; found: 498.0407.

2-(4-Bromobenzyl)-N-(4-fluorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7d)

White solid; yield: 101 mg (83%); mp 235–237 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.35 (d, J = 8.6 Hz, 2 H), 7.23–7.20 (m, 4 H), 6.97 (t, J = 8.6 Hz, 2 H), 5.53 (t, J = 4.9 Hz, 1 H), 4.69 (d, J = 6.2 Hz, 2 H), 4.04 (s, 2 H), 2.83 (d, J = 6.2 Hz, 2 H), 2.78 (d, J = 5.4 Hz, 2 H), 1.87 (d, J = 4.7 Hz, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.4, 163.3, 163.0, 161.4, 157.1, 138.3, 134.9, 132.8, 131.2, 131.1, 129.5, 129.4, 125.1, 120.1, 115.6, 115.5, 114.1, 45.2, 44.0, 26.4, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₄H₂₁BrFN₃S: 482.0702; found: 482.0714.

2-(4-Bromobenzyl)-N-(2-fluorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7e)

White solid; yield: 98 mg (80%); mp 235–237 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.35 (d, J = 8.6 Hz, 2 H), 7.23 (d, J = 7.8 Hz, 1 H), 7.21 (d, J = 7.8 Hz, 2 H), 7.14 (t, J = 7.8 Hz, 1 H), 7.03 (t, J = 9.3 Hz, 1 H), 6.98 (t, J = 7.4 Hz, 1 H), 5.69 (t, J = 5.4 Hz, 1 H), 4.76 (d, J = 6.2 Hz, 2 H), 4.05 (s, 2 H), 2.86 (t, J = 5.4 Hz, 2 H), 2.76 (t, J = 5.8 Hz, 2 H), 1.90–1.85 (m, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.3, 163.2, 162.2, 160.6, 157.2, 138.4, 132.7, 131.3, 131.2, 130.6, 130.6, 129.1, 129.1, 125.2, 124.2, 124.2, 120.0, 115.4, 115.3, 114.2, 45.2, 38.8, 26.3, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₄H₂₁BrFN₃S: 482.0765; found: 482.0812.

2-(4-Bromobenzyl)-N-(3,4,5-trifluorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7f)

White solid; yield: 112 mg (85%); mp 215–217 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.34 (d, J = 8.6 Hz, 2 H), 7.16 (d, J = 8.6 Hz, 2 H), 6.86 (t, J = 7.4 Hz, 2 H), 5.61 (t, J = 5.4 Hz, 1 H), 4.65 (d, J = 6.2 Hz, 2 H), 4.02 (s, 2 H), 2.88 (d, J = 3.1 Hz, 2 H), 2.79 (t, J = 5.8 Hz, 2 H), 1.92–1.87 (m, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.6, 163.2, 156.9, 138.0, 135.8, 133.3, 131.3, 131.0, 125.0, 120.2, 114.1, 111.5, 111.5, 111.4, 111.4, 45.1, 43.6, 26.5, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₄H₁₉BrF₃N₃S: 518.0513; found: 518.0500.

2-(4-Bromobenzyl)-N-(4-methoxybenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7g)

White solid; yield: 98 mg (78%); mp 218–220 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.36 (t, J = 7.4 Hz, 2 H), 7.18 (d, J = 8.6 Hz, 2 H), 6.93–6.88 (m, 1 H), 6.88 (t, J = 4.3 Hz, 1 H), 6.83 (d, J = 8.6 Hz, 2 H), 5.49 (t, J = 5.1 Hz, 1 H), 4.65 (d, J = 5.4 Hz, 2 H), 4.05 (s, 2 H), 3.81 (s, 3 H), 2.81 (d, J = 5.4 Hz, 2 H), 2.77 (d, J = 4.7 Hz, 2 H), 1.85 (s, 2 H), 1.72–1.73 (2 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.2, 163.3, 161.1, 159.0, 157.2, 138.4, 132.5, 131.3, 131.2, 129.9, 129.3, 129.2, 125.3, 120.0, 114.1, 114.1, 114.0, 64.4, 55.4, 55.4, 45.2, 44.2, 26.4, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₅H₂₄BrN₃OS: 494.0902; found: 494.0895.

2-(4-Bromobenzyl)-4-(1H-imidazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (7h)

Yellow solid; yield: 91 mg (84%); mp 239–241 °C.

¹H NMR (400 MHz, DMSO-d ₆): δ = 8.09 (s, 1 H), 7.62 (s, 1 H), 7.09 (s, 1 H),7.43 (d, J = 8.28 Hz, 2 H), 7.26 (d, J = 7.8 Hz, 2 H), 4.25 (s, 2 H), 2.85 (t, J = 5.9 Hz, 2 H), 2.17 (t, J = 5.72 Hz, 2 H), 1.78–1.56 (m, 4 H).

¹³C NMR(100 MHz, DMSO-d ₆): δ = 171.14, 163.07, 150.08, 139.13, 138.71, 137.99, 131.88, 131.81, 129.41, 126.51, 122.98, 121.65, 120.23, 44.02, 25.92, 24.96, 22.52, 22.13.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₀H₁₇BrN₄S: 425.0430; found: 425.0440.

2-(4-Bromobenzyl)-N-(pyridin-2-ylmethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7i)

White solid; yield: 103 mg (87%); mp 244–246 °C.

¹H NMR (600 MHz, CDCl₃): δ = 8.56 (d, J = 3.9 Hz, 1 H), 7.61 (t, J = 6.6 Hz, 1 H), 7.36–7.34 (m, 2 H), 7.24 (d, J = 8.6 Hz, 2 H), 7.20 (t, J = 3.9 Hz, 2 H), 6.76 (s, 1 H), 4.80 (d, J = 4.7 Hz, 2 H), 4.06 (s, 2 H), 3.03 (t, J = 6.2 Hz, 2 H), 2.79 (t, J = 6.2 Hz, 2 H), 1.91 (dt, J = 33.0, 5.3 Hz, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.0, 163.3, 157.1, 157.1, 149.0, 138.5, 136.7, 132.3, 131.2, 131.1, 125.8, 122.3, 120.0, 114.5, 45.7, 45.2, 26.3, 25.5, 22.7.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₃H₂₁BrN₄S: 465.0749; found: 465.0748.

2-(4-Bromobenzyl)-N-cyclopropyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7j)

White solid; yield: 85 mg (81%); mp 211–213 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.38 (d, J = 7.8 Hz, 2 H), 7.35 (d, J = 8.6 Hz, 2 H), 5.40 (s, 1 H), 4.08 (s, 2 H), 2.90 (q, J = 3.4 Hz, 1 H), 2.79 (t, J = 5.8 Hz, 2 H), 2.76 (t, J = 5.1 Hz, 2 H), 1.86 (t, J = 4.3 Hz, 4 H), 0.85 (t, J = 6.2 Hz, 2 H), 0.51 (dd, J = 9.5, 6.0 Hz, 2 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.0, 163.3, 162.6, 158.4, 138.4, 132.6, 131.2, 131.1, 125.1, 120.0, 114.2, 45.2, 36.5, 31.5, 26.3, 25.4, 24.0, 22.6, 7.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₀H₂₀BrN₃S: 414.0640; found: 414.0662.

2-(4-Bromobenzyl)-4-(pyrrolidin-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (7k)

White solid; yield: 83 mg (76%); mp 216–218 °C.

¹H NMR (400 MHz, DMSO-d ₆): δ = 7.41 (d, J = 7.92 Hz, 2 H), 7.24 (d, J = 8.08 Hz, 2 H), 3.89 (s, 2 H), 3.54 (d, J = 5.8 Hz, 4 H), 2.73 (s, 4 H), 1.76–1.63 (m, 8 H).

¹³C NMR (100 MHz, DMSO-d ₆): δ = 168.08, 161.12, 158.51, 138.91, 131.80, 131.50, 130.94, 128.02, 119.77, 115.38, 51.07, 44.46, 29.24, 25.84, 25.55, 23.43, 22.82.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₁H₂₂BrN₃S: 428.0796; found: 428.0810.

2-(4-Bromobenzyl)-N,N-diethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7l)

White solid; yield: 90 mg (82%); mp 195–197 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.38 (d, J = 7.6 Hz, 2 H), 7.29 (d, J = 7.6 Hz, 2 H), 4.04 (s, 2 H), 3.58-3.53 (m, 2 H), 3.49 (s, 2 H), 2.87 (t, J = 5.5 Hz, 2 H), 2.77 (t, J = 5.4 Hz, 2 H), 1.91-1.86 (m, 4 H), 1.21-1.25 (m, 6 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.0, 163.3, 157.5, 138.5, 132.2, 131.175, 131.099, 125.3, 120.0, 114.0, 45.3, 35.8, 26.4, 25.4, 22.6, 14.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₁H₂₄BrN₃S: 402.0653; found: 402.0666.

2-(4-Bromobenzyl)-N-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7m)

White solid; yield: 77 mg (78%); mp 189–191 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.39–7.37 (m, 2 H), 7.30 (d, J = 8.6 Hz, 2 H), 5.21 (d, J = 3.9 Hz, 1 H), 4.05 (s, 2 H), 3.06 (d, J = 4.7 Hz, 3 H), 2.85 (t, J = 5.8 Hz, 2 H), 2.77 (t, J = 5.8 Hz, 2 H), 1.91–1.86 (m, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 165.8, 163.3, 158.1, 138.4, 132.3, 131.2, 131.1, 125.2, 120.1, 114.2, 45.2, 26.4, 25.4, 22.6, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₈H₁₈BrN₃S: 388.0483; found: 388.0482.

N-Allyl-2-(4-bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7n)

White solid; yield: 85 mg (80%); mp 190–192 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.37 (d, J = 8.6 Hz, 2 H), 7.29 (d, J = 7.8 Hz, 2 H), 5.18 (s, 1 H), 4.04 (s, 2 H), 3.55 (dt, J = 13.6, 6.2 Hz, 2 H), 2.86 (t, J = 5.8 Hz, 2 H), 2.77 (t, J = 5.1 Hz, 2 H), 1.91–1.85 (m, 4 H), 1.23 (t, J = 7.0 Hz, 3 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.0, 163.3, 157.5, 138.5, 132.2, 131.2, 131.1, 125.3, 120.0, 114.0, 45.3, 35.8, 26.4, 25.4, 22.7, 15.0.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₀H₂₀BrN₃S: 414.0640; found: 414.0640.

2-((2-(4-Bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (7o)

White solid; yield: 96 mg (90%); mp 201–203 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.39 (d, J = 8.6 Hz, 2 H), 7.26 (s, 2 H), 5.73 (d, J = 4.7 Hz, 1 H), 4.03 (s, 2 H), 3.81 (t, J = 4.7 Hz, 2 H), 3.68 (q, J = 4.9 Hz, 2 H), 2.88 (t, J = 5.8 Hz, 2 H), 2.79 (t, J = 5.8 Hz, 2 H), 1.92–1.87 (m, 4 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.3, 162.8, 158.0, 137.8, 133.2, 131.4, 131.0, 125.3, 120.3, 114.2, 63.7, 45.1, 44.7, 26.4, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₉H₂₀BrN₃OS: 418.0589; found: 418.0586.

2-(4-Bromobenzyl)-N-propyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7p)

White solid; yield: 94 mg (88%); mp 192–194 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.38–7.37 (m, 2 H), 7.28 (d, J = 8.6 Hz, 2 H), 5.25 (t, J = 5.1 Hz, 1 H), 4.03 (s, 2 H), 3.48 (q, J = 6.7 Hz, 2 H), 2.86 (t, J = 5.8 Hz, 2 H), 2.76 (t, J = 5.8 Hz, 2 H), 1.91–1.85 (m, 4 H), 1.61 (td, J = 14.6, 7.3 Hz, 2 H), 0.96 (t, J = 7.4 Hz, 3 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.0, 163.3, 157.6, 138.4, 132.2, 131.2, 125.2, 120.0, 114.0, 45.2, 42.7, 26.4, 25.4, 22.8, 22.7, 22.6, 11.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₀H₂₂BrN₃S: 416.0796; found: 416.0796.

N ¹-(2-(4-Bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)propane-1,3-diamine (7q)

White solid; yield: 91 mg (83%); mp 205–207 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.36 (d, J = 6.6 Hz, 2 H), 7.27 (d, J = 5.1 Hz, 2 H), 5.99 (s, 1 H), 4.03 (s, 2 H), 3.63 (s, 2 H), 2.80 (d, J = 63.1 Hz, 6 H), 1.85 (s, 4 H), 1.76 (s, 2 H), 1.25 (s, 2 H).

¹³C NMR (151 MHz, CDCl₃): δ = 166.0, 163.1, 157.6, 138.4, 132.1, 131.2, 131.1, 131.1, 125.6, 120.0, 114.1, 45.2, 39.1, 26.5, 25.5, 22.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₀H₂₃BrN₄S: 431.0905; found: 431.0912.

2-(4-Bromobenzyl)-N-(2-(piperazin-1-yl)ethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7r)

White solid; yield: 113 mg (91%); mp 221–223 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.38 (s, 2 H), 7.27 (s, 2 H), 4.07 (d, J = 43.6 Hz, 2 H), 3.40 (s, 2 H), 2.85 (s, 4 H), 2.56 (d, J = 42.0 Hz, 2 H), 2.07 (s, 4 H), 1.85 (d, J = 84.1 Hz, 2 H), 1.26 (s, 6 H), 0.86 (d, J = 22.6 Hz, 1 H).

¹³C NMR (151 MHz, CDCl₃): δ = 162.1, 161.8, 138.0, 138.0, 134.4, 131.3, 131.1, 127.0, 120.2, 119.1, 52.8, 50.8, 50.3, 44.8, 37.8, 29.8, 26.8, 25.8, 23.1, 22.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₃H₂₈BrN₅S: 486.1327; found: 486.1319.

tert-Butyl 4-(2-(4-Bromobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylate (7s)

White solid; yield: 125 mg (90%); mp 230–232 °C.

¹H NMR (600 MHz, CDCl₃): δ = 7.39 (d, J = 8.6 Hz, 2 H), 7.27 (d, J = 4.7 Hz, 2 H), 4.11 (s, 2 H), 3.56 (t, J = 4.7 Hz, 4 H), 3.32 (s, 4 H), 2.86 (d, J = 5.4 Hz, 4 H), 1.93–1.91 (m, 2 H), 1.77 (t, J = 5.4 Hz, 2 H), 1.49 (s, 9 H).

¹³C NMR (151 MHz, CDCl₃): δ = 169.2, 162.2, 161.8, 154.9, 138.0, 134.7, 131.3, 131.1, 126.8, 120.2, 119.2, 80.1, 44.9, 28.5, 26.8, 25.8, 23.1, 22.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₆H₃₁BrN₄O₂S: 487.0803; found: 487.0806.

Molecular Modeling Studies

The molecular docking was conducted utilizing the Maestro (version 13.5) molecular docking suite from Schrödinger (version 2023-1) through the Glide module. Ligands were initially drawn using ChemDraw Professional and then imported into Maestro in sdf format. Ligand preparation was carried out using the Ligprep tool to generate 3D structures. The OPLS4 force field was employed, and ionization states were set at pH 7.0 ± 2.0. Tautomers were generated for each neutralized or ionized molecule, with specified chiralities retaining up to 5 per ligand. The three-dimensional (3D) structures of the proteins EGFR, VEGFR-2, PI3K, and c-Kit were obtained from the RCSB Protein Data Bank and imported into Maestro. Protein preparation involved assigning bond orders, adding hydrogens, and including zero-order bonds to metal and disulfide bonds. Additionally, water molecules beyond 5.00 Å were removed. The active chain, where the co-crystallized ligand was bound, was selected for further refinement: optimization, removal of remaining water molecules, and restrained minimization. Following ligand and protein preparation, receptor grid generation was performed to define the active site, generating a receptor-grid file. Flexible ligand-protein docking was executed by docking the prepared ligand molecules into the active ATP binding site of the prepared protein, utilizing the receptor grid file. The extra precision mode was applied, and RMSD was computed to obtain the ligands' dock score and binding energy affinity.[49]

Molecular Dynamics Simulation Studies

To investigate the behavior and stability of compound 7o at the active site of VEGFR-2, a molecular dynamics (MD) simulation was conducted. The initial structure for the simulation was based on the docking complex of ligand 7o with VEGFR-2. Following the standard Desmond protocol, the system underwent a 100 ns simulation for both equilibration and production runs. The simulation system was solvated via the TIP3P water model and neutralized by adding 0.15 M Na⁺ and Cl^– ions, maintaining a water layer thickness of 10 Å. Before starting the MD simulations, the complex was subjected to energy minimization, with a maximum of 2000 steps. The temperature and pressure were maintained at 300 K and 1.01325 bar, respectively, using an isothermal-isobaric (NPT) ensemble. Coulomb interactions were calculated with a cut-off radius of 9 Å.

Furthermore, we also assessed the druglike properties and pharmacokinetic ADME properties of the compounds using the Qikprop module of the Schrödinger software.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

The authors acknowledge the Hon’ble Vice-Chancellor, Central University of Punjab for providing facilities to compile this work. Authors thank Dr. Santosh, NIPER Hyderabad for providing preliminary MTT assay results of the compound 7o. The detailed results will be published in due course.

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Publication History

Received: 05 April 2024

Accepted after revision: 16 July 2024

Accepted Manuscript online:
16 July 2024

Article published online:
13 August 2024