Synthesis of New Multitarget-Directed Ligands Containing Thienopyrimidine Nucleus for Inhibition of 15-Lipoxygenase, Cyclooxygenases, and Pro-Inflammatory Cytokines

Abstract

A new series of thieno[2,3-d]pyrimidine derivatives 4, 5, 6a-o, and 11 was designed and synthesized starting from cyclohexanone under Gewald condition with the aim to develop multitarget-directed ligands (MTDLs) having anti-inflammatory properites against both 15-LOX and COX-2 enzymes. Moreover, the potential of the compounds against the proinflammatory mediators NO, ROS, TNF-α, and IL-6 were tested in LPS-activated RAW 264.7 macrophages. Compound 6o showed the greatest 15-LOX inhibitory effect (IC₅₀ = 1.17 μM) that was superior to that of the reference nordihydroguaiaretic acid (NDGA, IC₅₀ = 1.28 μM); meanwhile, compounds 6h, 6g, 11, and 4 exhibited potent activities (IC₅₀ = 1.29–1.77 μM). The ester 4 (SI=137.37) and the phenyl-substituted acetohydrazide 11 (SI=132.26) showed the highest COX-2 selectivity, which was about 28 times more selective than the reference drug diclofenac (SI=4.73), however, it was lower than that of celecoxib (SI = 219.25). Interestingly, compound 6o, which showed the highest 15-LOX inhibitory activity and 5 times higher COX-2 selectivity than diclofenac, showed a greater poteny in reducing NO (IC₅₀ =7.77 μM) than both celecoxib (IC₅₀ =22.89 μM) and diclofenac (IC₅₀ =25.34), but comparable activity in inhibiting TNF-α (IC₅₀ = 11.27) to diclofenac (IC₅₀ = 10.45 μM). Similarly, compounds 11 and 6h were more potent in reducing TNF-α and IL6 levels than diclofenac, meanwhile, compound 4 reduced ROS, NO, IL6, and TNF-α levels with comparable potency to the reference drugs celecoxib and diclofenac. Furthermore, docking studies for our compounds within 15-LOX and COX-2 active sites revealed good agreement with the biological evaluations. The proposed compounds could be promising multi-targeted anti-inflammatory candidates to treat resistant inflammatory conditions.

Graphical Abstract

1. Introduction

Inflammation is defined as the body response to an external stimuli such as a pathogen or injury in order to destroy the invading pathogen or repair the damaged tissue [1, 2]. However, inflammation is strongly associated with a variety of diseases, such as Alzheimer’s disease [3, 4], asthma, atherosclerosis, multiple sclerosis, osteoarthritis, rheumatoid arthritis, diabetes mellitus, carcinoma in addition to bacterial and viral infections, that may cause chronic inflammation [5, 6].

Arachidonic acid (AA) represents a major precursor for many inflammatory mediators released during inflammation [7]. Moreover, lipoxygenases (LOXs) represent a class of non-heme iron-containing enzymes which cause production of various pro-inflammatory mediators from arachidonic acid, including leukotrienes, eoxins, and lipoxins [8, 9]. There are three isozymes of lipoxygenases, 5-LOX, 12-LOX, and 15-LOX, which are characterized according to the peroxidation site of arachidonic acid. All of these isozymes are associated with a number of physiological processes, such as inflammation, hyperproliferative and neurodegenerative disorders [8]. 15-Lipoxygenase (15-LOX) is involved in one of the main metabolic pathways that are responsible for the conversion of arachidonic acid into 15-hydroxyeicosatetraenoic acid (15-HETE) and other pro-inflammatory mediators. Recent studies have shown the direct contribution of 15-HETE in airway inflammation [10], induced dysfunction of the retina in diabetic retinopathy [11], and various types of cancer [12, 13]. Thus far, zileuton is the only approved LOX inhibitor, which acts by chelating the iron metal located in the active site of the LOX enzyme, however its pharmacokinetics are unfavorable, and it has been linked to several liver problems [14]. Additionally, there has been an increased interest in developing safe and effective anti-LOX therapies. Although several lipoxygenase inhibitors have been reported [15], it is challenging to synthesize potent small molecule inhibitors with favorable physicochemical properties.

In addition to LOX enzymes, cyclooxygenases, COX-1 and COX-2, are the other metabolic arm of the arachidonic acid pathway[16]. COX-1 and COX-2 convert arachidonic acid into prostaglandins, thromboxanes, and prostacyclins, which play a major role in inflammation [16]. However, cyclooxygenase inhibitors, e.g., NSAIDs, shunt the metabolism of arachidonic acid toward 15-LOX [17]. In fact, this can worsen the patient’s condition if the 15-LOX enzyme has a significant role in the pathogenesis of the disease, such as in asthma [18]. There have been reports suggesting that 15-LOX might be used as a target for inhibiting the synthesis of eoxines, a pro-inflammatory mediator, as well as a cancer promoter [19].

Accordingly, the use of multitarget-directed ligands (MTDLs) to inhibit both enzymes 15-LOX and COX is a promising strategy in drug therapy that could provide maximum efficiency and minimal side effects [20, 21].

Noticeable, several clinically used NSAIDs (Figure 1) possess thiophene rings [22, 23], such as tiaprofenic acid (Surgam^®, I) and suprofen (Profenal^®, II). Moreover, compound CBS-1108 (III) which has the thiophene pharmacophore is a selective COX-2 inhibitor [22].

Figure 1:

Some clinically used NSAIDs (I,II) and compound CBS-1108 (III)

Furthermore, the tetrahydrobenzothienopyrimidine derivative (IV,Figure 2) was reported to have an increased COX-2 activity with a high selectivity index [24]. On the other hand, other sulfur-bicyclic compound bearing 4-thiazolidinone moiety such as compound V has been reported to inhibit 15-LOX enzyme (IC₅₀ = 17.7 μM), and those with thienopyrimidine nucleus (VI, Figure 2) exhibited significant inhibition of COX-2 enzyme (IC₅₀ = 0.09 μM) [25, 26].

Figure 2:

Different structures for sulfur-containing compounds and new thienopyrimidine derivatives 6a-o,11

Based on the aforementioned data that several sulfur-nitogen heterocycles either bicyclic e.g. compound V and VI or tricyclic e.g. compound IV exhibited activity against 15-LOX or COX-2, we decided to synthesize new thienopyrimidine derivatives 6a-o,11 (Figure 2) with structure similarities to those compounds (IV-VI) hoping to act as multitarget-directed ligands (MTDLs) to not only inhibit 15-LOX and COX-2 enzymes (compound 6o, Figure 3) but also to suppress the production of other pro-inflammatory mediators, e.g., ROS, iNOS, IL-6 and TNF-α Moreover, in order to provide a clear insight into the degree of COXs selectivity of our newly synthesized thienopyrimidines, their in vitro COX-1/COX-2 inhibitory activities were determined in comparison to celecoxib (the highly selective COX-2 inhibitor), indomethacin (relatively COX-1 selective inhibitor) [27], and diclofenac sodium (preferential COX-2 inhibtor) [28] as reference drugs (Figure 3). In addition their 15-LOX inhibitory activities were determined using the reference drug nordihydroguaiaretic acid (NDGA) [29]. The multitarget-directed ligands (MTDLs) represents a promising strategy to treat special inflammatory conditions that are exacerbated by the solo inhibition of one target, e.g. asthma. Moreover, molecular docking study of the novel thienopyrimidine derivatives 6a-o,11 was performed to postulate their potential binding poses with 15-LOX and COX-2.

Figure 3.

Chemical structures of thienopyrimidine 6o and nordihydroguaiaretic acid (NDGA), celecoxib, indomethacin and diclofenac references drugs

2. Results and Discussion

2.1. Chemistry

The synthetic strategies adopted for the synthesis of the key intermediates and final compounds are illustrated in Schemes 1–3. Intermediate 1, ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate, was prepared under Gewald condition [30] through refluxing cyclohexanone with ethyl cyanoacetate in the presence of sulfur in ethanol for 1 h. Then, intermediate 1 was cyclized by formamide [31] to afford the thieno[2,3-d]pyrimidinone 2, which was chlorinated under the effect of POCl₃ to give 4-chloro-5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidine 3 [30, 32]. The reaction of ethyl glycinate hydrochloride with the chloro derivative 3 under basic condition (sodium acetate) afforded the ester derivative 4. Hydrazinolysis of ethyl ester 4 via refluxing the reactants in ethanol afforded the acid hydrazide 5 (Scheme 1), which is a precursor for target compounds 6a-o.

Scheme 1.

Synthetic pathway for preparing key intermediate 5; 2-((5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin-4-yl)amino) acetohydrazide.

Reagents and conditions:

(a) Ethylcyanaoacetate, S, EtOH, reflux, 1 h. (b) Formamide, reflux, 2 h. (c) POCl₃, 1 h. (d)Ethyl glycinate, sodium acetate, EtOH, reflux, 24 h. (e) Hydrazine hydrate, EtOH, reflux, 18 h.

Scheme 3.

Synthetic pathway for preparation of acetohydrazide 11.

Reagents and conditions:

(a) 2-Cyanoacetamide, sulfur, EtOH, reflux, 2.5 h. (b) Benzaldehyde, I₂, CH₃CN, stir, 2 h. (c) POCl₃, Na₂CO₃. (d) Ethyl glycinate, sodium acetate, EtOH, reflux, 24 h. (e) Hydrazine hydrate, EtOH, reflux, 18 h.

Condensation of the acid hydrazide 5 with different aromatic aldehydes in ethanolic solution containing catalytic amount of glacial acetic acid afforded the new hydrazone derivatives 6a-i, while its condensation with isatin in dioxane in the presence of drops of acetic acid gave the novel oxoindoline derivative 6j as shown in Scheme 2.

Scheme 2.

Synthetic pathway for preparing 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives 6a-o.

Reagents and conditions:

(a) Appropriate aldehyde, ethanol, acetic acid (drops), reflux, 4 h. (b) Isatin, dioxane, acetic acid (drops), reflux, 4 h. (c) Phenyl isocyanate, ethanol, reflux, 18h. (d) Ethyl chloroformate, butanol, reflux, 4 h. (e) Acetylacetone /benzoylacetone, TEA, ethanol, reflux, 24 h. (f) Maleic anhydride, ethanol, acetic acid (drops), reflux, 2 h.

The novel semicarbohydrazide 6k was obtained by heating a mixture of phenyl isocyanate and the acid hydrazide 5 in ethanolic solution at reflux for 18 h. Moreover, the oxadiazole 6l was synthesized by heating a mixture of the acid hydrazide 5 and ethyl chloroformate in butanol for 4 h.

The novel thienopyrimidine derivatives bearing pyrazoline ring, 6m and 6n, were prepared by stirring a mixture of the acid hydrazide 5 with acetylacetone or benzoylacetone in ethanol containing 10 drops of triethylamine (TEA) at reflux for 24 h. Condensation reaction of the acid hydrazide 5 and maleic anhydride via refluxing the reactants in ethanol containing drops of acetic acid for 2 h led to the formation of the acyclic derivative 6o rather than the cyclized maleimide 6p (Scheme 2).

Intermediate 7 was also prepared under Gewald [30] condition through heating cyclohexanone with 2-cyanoacetamide in the presence of sulfur in ethanol for 2.5 h as shown in Scheme 3. Compound 7 was cyclized with benzaldehyde in acetonitrile in the presence of iodine as catalyst to afford intermediate 8 [33]. The chloro derivative 9 was obtained by chlorination of intermediate 8 using POCl₃.

The reaction of the chlorinated product 9 with ethyl glycinate in the presence of sodium acetate as a base gave the ester 10, which was then converted into the final acid hydrazide 11 upon its reaction with hydrazine hydrate.

The chemical structures of the novel compounds 4, 5, 6a–o and 11 were determined by ¹H, ¹³C NMR, and HRMS.

IR spectrum of ester 4 showed characteristic absorption bands at 3334.7 and 1748.5 cm⁻¹ due to NH and C=O ester groups, respectively, while IR spectrum of the acid hydrazide 5 exhibited two new bands at 3321.5 and 3301.4 cm⁻¹ which are characteristic for NH₂ group in addition to the presence of two bands at 3357.5 and 1654.0 cm⁻¹ which are attributable to an amide NH and C=O groups. Moreover, ¹H NMR spectrum of the ester derivative 4 showed a very characteristic pattern for ethyl ester group; CH₃ resonated at δ 1.32 as a triplet and OCH₂ as a quartet at δ 4.29 ppm. Moreover, the two resonances at δ 4.33, as a doublet, and a broad singlet at δ 6.02 ppm were assigned to NCH₂ and NH protons, respectively. On the other hand, the acid hydrazide 5 ¹H NMR spectra lacks the ethyl ester group resonances, however, showed exchangeable protons at δ 5.02 and 9.33 atributed to NH₂ and NH of the hydrazide moiety.

The chemical structures of compounds 6a-j were established using different spectroscopic methods, whereas their ¹H NMR spectra showed the disappearance of NH₂ signal at δ 5.02 ppm of the starting hydrazide 5 in addition to the appearance of a deshielded signal at about δ 8.17–8.25 ppm, which, in turn, revealed the presence of azomethine protons.

It is noteworthy to mention that hydrazones can exist as a pair of diastereomers, Z and E around the C=N bond of the hydrazone moiety, and as cis and trans conformers originated from the rotation of the molecular fragment around the C(O)-N bond of the amide [34]. In ¹H NMR spectra of this series of compounds (6a-i) two sets of methylene doublet were observed, along with two sets of triplet of NH (CH₂NH), two sets of singlet of azomethine proton, and two sets of singlet of amide NH protons. These resonances were attributed to the protons of the cis and trans amide conformers and the E and Z isomers of hydrazone. However, oxoindoline derivative 6j showed only E/Z isomers. Moreover, the presence of double sets of resonances was seen in ¹³C NMR spectra as well, confirming the presence of different diastereomers/conformers. The structure of the novel semicarbohydrazide 6k was established using ¹H NMR, which showed the disappearance of NH₂ signal at δ 5.02 ppm of the starting hydrazide 5 in addition to the appearance of 5 signals for aromatic protons and two singlet signals at δ 9.75 and 9.31 ppm due to CONHNH and ArNHCO), respectively.

¹H NMR spectrum of compound 6l showed characteristic signals for a butoxy group which appeared as a triplet at δ 0.86 ppm for CH₃ group, two multiplets at δ 1.24–1.34 and 1.50–1.57 ppm for the two CH₂ groups and a triplet at δ 4.07 ppm for OCH₂ group. Moreover, ¹H NMR spectra of the pyrazolines 6m and 6n showed two exchangeable protons at δ 6.72 and 6.89, and the pyrazoline isolated methylene protons, which appeared along with the cyclohexane protons as a multiplet at δ 2.98– 2.78 ppm for 6m and appeared quartet at 3.10 ppm for 6n. Moreover, the two CH₃ protons on the pyrazoline ring in 6m resonated at δ 2.00 and δ 1.85–1.75 ppm, while the one CH₃ protonsin 6n resonated as a singlet at δ 2.08. Another characteristic tool that confirming the structure of 6m and 6n is mass spectrum, since the molecular ion peak was m/z = 359.51 for 6m and 421.42 for 6n. ¹H NMR for 6o showed two broad singlets at δ 13.30 and 10.58 ppm, due to OH and NH protons, respectively. Additionally, the two alkenyl protons of the maleic acid moiety appeared as a pair of doublets at δ 6.27 and 6.35 ppm. Furthermore, HRMS revealed the exact MW of 6o at m/z = 376.1095 [M+H], which confirmed the presence of the acyclic structure 6o rather than the cyclized one 6p. ¹H NMR spectrum of compound 11 showed three singlet signals at δ 4.39, 6.90 and 9.25 ppm, which showed no corss contours in the HSQC spectrum. These protons were assigned to the protons of NH₂, NHCH_2, and the amide NH, respectively. MS showed the molecular ion peak at m/z = 353.46 and the base peak at m/z = 294.28 atributed to N-methyl-2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine radical fragment.

2.2. Biological activity

2.2.1. In vitro 15-LOX inhibition assay

The inhibitory activities of all the synthesized thienopyrimidines 4, 5, 6a-o and 11 (Figure 4) against 15-LOX are shown in Table 1. The anti-15-LOX activity for the starting ester 4 (IC₅₀ = 1.77 μM) is nearly the same as its hydrazide derivative 5 (IC₅₀ = 1.67 μM). In our attempt to improve anti-15-LOX activity, the drug extension tactic was carried out via forming acyclic hydrazones (6a-j) and semicarbohydrazide (6k) derivatives to study the inhibitory effects of different substituents on 15-LOX. It was noted that the hydrazone bearing an electron-donating group, as seen in thienopyrimidine containing 4-methoxyphenyl moiety (6g, IC₅₀ = 1.30 μM), is more active than that bearing an electron-withdrawing group, as that containing 4-nitrophenyl group (6f, IC₅₀ = 3.12 μM), however, is comparable to that of the reference nordihydroguaiaretic acid (NDGA) (IC₅₀ = 1.28 μM). Among the hydrazones containing a halogen atom, the 4-chloro derivative 6a (IC₅₀ = 1.80 μM) is the most active one (Table 1).

Figure 4.

The effect of compounds 11 and 6o on the activities of COX-1 (A), COX-2 (B) and 15-LOX (C). Also, the effect of compound 11 on the production of ROS (D), the effect of compound 6o on the production of NO (H), and the effect of both 11 and 6o on TNF-α, IL-6 (G,K) in LPS-stimulated RAW264.7 macrophages. Measurements of ROS (D-F), NO (H-J), IL-6 (G), and TNF-α (K) were performed in RAW264.7 macrophages that were pretreated with different concentrations (12.5, 25, or 50 μM) of test compounds, celecoxib, or diclofenac for 2 h followed by LPS challenge (1 μg/mL) for an additional 20 h. The results are representative of three independent experiments. *p < 0.05 vs control, #p < 0.05 vs LPS. IL-6 (interleukin-6); NO (Nitric Oxide); ROS (Reactive Oxygen Species); TNF-α (Tumor necrosis factor-α).

Table 1.

In vitro 15-LOX, COX-1 and COX-2 enzyme inhibition assay of the thienopyrimidines 4, 5, 6a-o, 11 and reference drugs celecoxib, indomethacin, diclofenac, and NDGA

Compound	IC50 (μM)			aCOX-2 SI
	15-LOX	COX-1	COX-2
4	1.77±1.04	15.15±3.00	0.11±1.03	137.73
5	1.67±1.38	0.07±1.57	0.21±1.03	0.33
6a	1.80±0.71	4.80±1.34	0.66±1.74	7.28
6b	2.11±0.22	28.60±1.42	0.65±1.12	44.00
6c	3.23±1.00	0.01±1.16	0.07±1.39	0.14
6d	3.33±1.51	0.01±1.90	0.10±1.12	0.10
6e	2.02±0.37	>100	21.99±2.05	0.00
6f	3.12±1.57	14.17±2.79	0.53±1.40	26.74
6g	1.30±0.25	>100	2.98±1.05	>100
6h	1.29±0.54	7.03±1.82	0.92±1.27	7.64
6i	2.17±1.25	0.14±1.01	1.13±1.9	0.12
6j	2.70±1.22	>100	3.86±1.04	>100
6k	2.13±1.18	>100	7.68±1.79	>100
6l	2.43±0.86	0.03±1.46	4.78±1.06	0.006
6m	1.92±1.97	10.77±1.83	2.15±100	5.00
6n	2.29±0.95	37.05±3.09	0.96±1.48	38.59
6o	1.17±0.52	10.11±3.88	0.45±1.32	22.46
11	1.58±0.23	92.58±2.51	0.70±1.12	132.26
Celecoxib	-----	8.77±1.26	0.04±1.16	219.25
Indomethacin	-----	0.50±1.20	1.61±1.09	0.31
Diclofenac	-----	3.88±1.21	0.82±1.14	4.75
NDGA	1.28±0.26	-----	-----	-----

^aIC₅₀, μM: The half-maximal inhibitory concentration values;

^aCOX-2 selectivity index (SI) = IC₅₀ (COX-1)/IC₅₀ (COX-2).

It was also noted that the replacement of the oxygen atom of the furyl group of compound 6i (IC₅₀ = 2.17 μM) by a sulphur atom, as seen in compound 6h (IC₅₀ = 1.29 μM), led to a twice as active compound as the reference NDGA (IC₅₀ = 1.28 μM). Moreover, the cyclization of the side chain of thienopyrimidine in the form of oxadiazole 6l (IC₅₀ = 2.43 μM) or pyrazolines 6m (IC₅₀ = 1.92 μM) and 6n (IC₅₀ = 2.29 μM) led to a dramatic decrease in activity, while the acyclic derivative 6o (IC₅₀ = 1.17 μM) showed the highest activity in this series which was superior to that of the reference NDGA (IC₅₀ = 1.28 μM), as well. Comparing the unsubstituted acetohydrazide 5 (IC₅₀ = 1.67 μM) to the phenyl-substituted derivative 11 (IC₅₀ = 1.58 μM), it was found that the phenyl group at C-2 caused an increase in the activity. It can be concluded that the decreasing order of the activity towards 15-LOX is as follows, 6o > NDGA > 6h > 6g > 11.

2.2.2. In vitro COX-1 and COX-2 inhibitory assay

In vitro COX-1/COX-2 inhibition assays were performed for all the synthesized thienopyrimidines 4, 5, 6a-o and 11 (Figure 4) using celecoxib, indomethacin, and diclofenac sodium as reference drugs (Table 1). It was found that the ester 4 exhibited better COX-2 inhibitory activity (IC₅₀ = 0.11 μM) than its hydrazide 5 (IC₅₀ = 0.21 μM). It is reported that there is a difference in the size of the binding pockets between of COX-1 and COX-2 [35], where COX-1 has a 25% smaller substrate binding pocket than COX-2 [36]. The COX-2 binding site contains an extra side-pocket that could be filled by selective COX-2 NSAIDs. Regarding the halogen derivatives 6a-e, it was noted that the thienopyrimidine bearing large size substituents, as 3,4-dichlorophenyl 6c (IC₅₀ = 0.07 μM) and 4-bromophenyl 6d (IC₅₀ = 0.10 μM), showed a higher COX-2 inhibitory activity than those bearing smaller size substituents, like 4-fluorophenyl 6e (IC₅₀ = 21.99 μM). Their activities were comparable to that of the reference drug celecoxib (IC₅₀ = 0.04 μM), however. It was observed also that thienopyrimidine bearing an electron-withdrawing group as 4-nitrophenyl group (6f) exhibited a higher COX-2 inhibitory activity (IC₅₀ = 0.53 μM) than ones with electron-donating groups as 4-methoxyphenyl (6g) (IC₅₀ = 2.98 μM).

Thienopyrimidines bearing heterocyclic ring showed variable activities. Those with a thiophene ring (6h) and a phenyl-substituted pyrazoline (6n) showed higher COX-2 inhibitory activities, (IC₅₀ = 0.92 μM) and (IC₅₀ = 0.96 μM), respectively, than other derivatives bearing oxadiazole ring (6l) (IC₅₀ = 4.78 μM). It was also noted that pyrazoline bearing a bulky substituent, e.g., a phenyl group as in 6n (IC₅₀ = 0.96 μM) fits comfortably in the bigger COX-2 active site, and not the smaller COX-1 site. Therefore, its selectivity index (SI =38.59) is higher than that the methyl-substituted one (6m) (SI =5.00).

Moreover, among the thienopyrimidines, the ester derivative 4 and the C-2 phenyl-substituted pyrimidine ring 11 showed the greatest COX-2 selectivity index, SI=137.37 and 132.26, respectively, that was about 28 times more selective than the reference drug diclofenac (SI=4.73). However, their COX-2 selectivity was significantly lower than that of celecoxib (SI= 219.25) which could be an advantage to avoid the cardiovascular side effects reported for highly selective COX-2 inhibitors [37, 38].

2.2.3. Effects on NO and ROS production in LPS-activated RAW 264.7 macrophages

The novel thienopyrimidines 4, 5, 6a-o and 11 (Figure 4) were evaluated for their ability to inhibit nitric oxide (NO) and reactive oxygen species (ROS) production in lipopolysaccharides (LPS)-activated RAW 264.7 macrophage cells (Table 2).

Table 2.

Effects of thienopyrimidines 4, 5, 6a-o, 11 and reference drugs celecoxib and diclofenac on the suppression of ROS, NO, TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages cells

Compound	IC50(μM)
	ROS	NO	TNF-α	IL-6
4	7.07±1.39	4.72±1.30	26.03±1.38	6.00 ± 1.42
5	1.07±3.24	31.78±1.25	18.48±1.19	14.66±1.52
6a	>50	12.28±1.06	>100	12.60±1.11
6b	12.82±1.03	18.93±1.04	25.06±1.04	3.21±2.67
6c	>50	1.98±1.65	23.22±1.21	9.75±1.15
6d	15.68±1.25	0.22±3.16	11.87±1.5	8.23±1.25
6e	>50	9.92±1.15	45.41±1.21	3.36±1.19
6f	45.24±1.10	8.66±1.23	11.05±1.12	11.89±1.06
6g	36.47±1.87	13.95±1.03	16.48±1.22	24.60±1.16
6h	>50	36.87±1.06	7.90±1.06	5.30±1.22
6i	18.72±1.29	3.12±1.85	7.16±1.16	9.71±1.05
6j	18.55±1.18	15.86±1.06	9.67±1.14	5.20±1.23
6k	21.42±1.39	37.13±1.07	>100	14.75±1.04
6l	32.81±1.06	1.02±3.43	>100	7.04±1.25
6m	5.14±1.54	8.60±1.24	7.90±1.11	11.42±1.06
6n	5.31±1.32	11.69±1.09	14.94±1.04	7.29±1.10
6o	>50	7.77±2.07	11.27±1.07	33.77±1.14
11	39.85±2.20	>50	11.06±1.05	66.84±1.16
Celecoxib	16.52±1.12	22.89±1.06	4.14±1.12	3.12±1.10
Diclofenac	26.26±1.08	25.34±1.35	10.45±1.03	19.59±1.35

It was reported that the exposure of RAW 264.7 cells to the bacterial toxin LPS causes an increased expression of multiple inflammatory mediators including COX-2 [39], while inducing nitric oxide (NO) production by upregulating the inducible isoform of nitric oxide synthase [40]. In addition, LPS also triggers the generation of ROS and other inflammatory cytokines [41]. It was also reported that numerous compounds with antioxidant potential are effective as anti-inflammatory and anti-cancer drugs [41–43].

In this study, it was observed that thienopyrimidine carrying unsubstituted acetohydrazide (5) was the most potent derivative in reducing ROS levels as it showed IC₅₀ value of 1.07 μM, which was 15-fold more potent than celecoxib (IC₅₀=16.52 μM). Moreover, several thienopyrimidine derivatives such as the ester 4, hydrazones bearing either 3-chlorophenyl (6b) or 4-bromophenyl (6d) and also those carrying a pyrazoline ring (6m and 6n) showed superior potency (IC₅₀ = 5.14 – 15.68 μM) in reducing ROS levels than the reference drugs celecoxib and diclofenac (IC₅₀ = 16.52 and 26.26 μM, respectively). Moreover, thienopyrimidines 6i, 6j and 6k showed higher activities (IC₅₀ = 18.55 – 21.42 μM) in reducing ROS levels than the reference drug diclofenac (IC₅₀ = 26.26 μM). Unfortunately, some thienopyrimidines (6f, 6g and 6l) expressed inferior activities in reducing ROS levels, while others (6a, 6c, 6e, 6h and 6o) caused non-significant reduction of ROS levels (Table 2).

Regarding NO production, most of the tested compounds exhibited potent inhibitory activities when compared to celecoxib and diclofenac. Theinopyrimidine with 4-bromo benzyl acetohydrazide (6d) exhibited an exceptional potency (IC₅₀ = 0.22 μM) that was approximately 100 times more potent than celecoxib (IC₅₀ = 22.89 μM). Among the most potent thienopyrimidines (IC₅₀ = 0.22–4.72 μM), the preference of substitution was arranged as 6d (4-bromophenyl) > 6l (oxadiazole) > 6c (3,4-dichlorophenyl) > 6i (furan) > 4 (ester) (Table 2).

Notably, none of the tested theinopyrimidines were toxic to RAW 264.7 macrophages when tested by MTS cell viability assay at 50 μM.

2.2.4. Effects on TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages

The novel thienopyrimidines 4, 5, 6a-o and 11 (Figure 4) were evaluated for their ability to inhibit TNF-α and IL-6 production in LPS-activated RAW 264.7 macrophages (Table 2). The excessive production of these cytokines results in a severe systemic inflammatory response, tissue damage, and sepsis [44].

Regarding the inhibition of IL-6, our findings showed that the hydrazones carrying 3-chlorophenyl 6b (IC₅₀ = 3.21 μM) or 4-fluorophenyl 6e (IC₅₀ = 3.36 μM) are the most potent ones in this series of compounds and their potency are comparable to that of celecoxib (IC₅₀ = 3.12 μM), yet exceeding that of diclofenac (IC₅₀ = 19.59 μM ). Moreover, the thienopyrimidines 4, 5, 6a, 6c, 6d, 6f, 6h, 6i, 6j, 6k, 6l and 6n (IC₅₀ = 5.20–9.75 μM) were significantly more potent than the reference drug diclofenac (IC₅₀= 19.59 μM) (Table 2).

Concerning the inhibition of the production of the inflammatory mediator TNF-α, the thienopyrimidines 5, 6d, 6f-j, 6m-o and 11 (Figure 4) exhibited inhibitory activities that are comparable to or higher (IC₅₀= 7.16–18.48 μM) than that of the reference drug diclofenac (IC₅₀=10.45 μM). As shown in Table 2, some thienopyrimidines bearing heterocyclic ring systems such as thiophene (6h), furan (6i), methyl-substituted pyrazoline (6m) showed the greatest TNF-α inhibitory activities (IC₅₀ = 7.16–7.90 μM) which was more potent than diclofenac (IC₅₀= 10.45 μM). Unfortunately, compounds 6a, 6k and 6l did not show any significant inhibitory activities against the production of TNF-α.

2.3. Molecular docking and in silico study

2.3.1. Docking study

The novel thienopyrimidine derivatives 4, 6g, 6h, 6o and 11 which showed a high anti-inflammatory activity through their prominent action not only against the dual 15-LOX/COX-2 enzymes, but also through suppressing other inflammatory mediators, including NO, ROS, TNF-α and IL-6, were selected to study their molecular docking to gain insight into their potential binding modes with the active sites of 15-LOX and COX-2.

2D and 3D molecular modeling studies of compounds 4, 6g, 6h, 6o and 11 were carried out using Molecular Operating Environment (MOE) version 2019 software (Chemical Computing Group, Montreal, CA). The X-ray crystallographic complex structures of 15-lipoxygenase-2 enzyme (15-LOX-2) with ligand C8E4 (PDB entry 4NRE) and cyclooxygenase-2 enzyme (COX-2) with ligand SC-558 (PDB entry 1CX2) were downloaded from the Protein Data Bank website (http://www.rcsb.org). We used C8E4 and SC-558 as references where both were redocked for validation.

2.3.1.1. Docking into 15-LOX active site

The selected compounds 4, 6g, 6h, 6o and 11 filled the 15-LOX pocket completely, forming hydrogen/hydrophobic interactions with the surrounding amino acids, Ile 412, Ile 676, Gln 425, Leu 420, Phe 192, Tyr 185, Ala188, leu 419, Asp 602, His 378, and His 373 with favored docking scores ranging from −7.3987 to −5.7047 kcal/mol.

Notably, compound 6o showed the best docking score of −7.3987, which matchs with the in vitro 15-LOX binding assay, as it also showed the lowest IC₅₀ value (1.17 μM) against 15-LOX (Figure 5). Interestingly, compound 6o showed three hydrogen bond interactions with amino acids Leu419, Leu 415 through its nitrogen atoms of pyrimidine ring, Ala416 through its carbonyl group with docking score of −7.3987 kcal/mol (Figure 5).

Figure 5.

2D and 3D representations of the proposed binding interaction pattern of compound 6o (green) in the binding site of 15-LOX (PBD:4NRE); S= −7.3987, rmsd= 1.3631

It is noteworthy that compound 6h displayed hydrogen bond interaction between the amidic carbonyl group and Ala 416 and pi-pi interaction between thiophene ring of 6h and His 378 imidazole ring (face-to-face) (Figure 6). Furthermore, Compound 6g formed hydrogen bond interactions with Arg 429 at the entrance of the pocket [45]. Additionally, it formed hydrophobic interactions with Leu 420, which possibly explains its good inhibitory properties (Figure 7).

Figure 6.

2D and 3D representations of the proposed binding interaction pattern of compound 6h (yellow) in the binding site of 15-LOX (PBD:4NRE); S= −7.2516, rmsd= 1.74009 (H-bonding interaction between amidic carbonyl group and Ala 416, and pi-pi interaction between 6h thiophene ring and the imidazole ring of His 378 (face-to-face).

Figure 7.

2D and 3D representations of the proposed binding interaction pattern of compound 6g (cyan) in the binding site of 15-LOX (PBD:4NRE); S= −6.4023, rmsd= 1.4818 (H-bonding with Arg 429 and H-pi interaction with Leu 420)

Moreover, compound 11 was shown to form H-bonding with Asp 602 [46], H-pi interaction with Leu 420 (Figure 8). However, compound 4 showed the least docking score (S= −5.7047 kcal/mol) among of the selected compounds, yet fits to 15-LOX binding site (Figure 9).

Figure 8.

2D and 3D representations of the proposed binding interaction pattern of compound 11 (tan) in the binding site of 15-LOX (PBD:4NRE); S= −6.2359, rmsd= 1.7159 (H-bonding with Asp 602 and H-pi interaction with Leu 420)

Figure 9.

2D and 3D representations of the proposed binding interaction pattern of compound 4 (orange) in the binding site of 15-LOX (PBD:4NRE); S= −5.7047, rmsd= 1.8966 (H-bonding with Ala 606)

2.3.1.2. Docking into COX-2 active site

Based on their in vitro COX-2 inhibitory activity, the thienopyrimidine derivatives 4, 6o and 11, which completely filled the pocket, formed hydrogen/hydrophobic interactions with several amino acid residues surrounding it, Gln192, Tyr355, His 90 Ala 527, Val 523, Ser 530, Leu 352, Ala 516, Arg 120, Gly 354 and Arg 513. According to the docking results, the selected compounds scored well within COX-2 active site, −7.3338 to − 6.9835. As shown in Figure 10, compound 4 formed three hydrogen bond interactions with the amino acids Arg 513, Tyr 355 and Ser 530. A similar phenomenon was observed with compound 11, which formed three hydrogen bonds with Arg 513, Leu 352, and Ser 530, as well as hydrophobic interactions with Ala 527. (Figure 11). Both compounds 4 and 11 were able to form hydrogen bonds with Arg 513; COX-2 has been shown to be highly selectively inhibited by these interactions [47, 48]. Interestingly, these results are consistent with the enzyme-binding assay and the high selectivity index for compounds 4 and 11, SI =137.73 and 132.26, respectively (Table 1). Throughout COX-2’s active site, compound 6o formed five hydrogen bonds with key amino acids Leu 352, His 90, Arg513 and Gly354. A hydrophobic interations with Trp387 and Phe 518 also formed (Figure 12).

Figure 10.

2D and 3D representations of the proposed binding interaction pattern of compound 4 (orange) in the binding site of COX-2 (PBD: 1CX2), S = −7.3464, rmsd = 1.4376.

Figure 11.

2D and 3D representations of the proposed binding interaction pattern of compound 11 (tan) in the binding site of COX-2 (PBD: 1CX2), S = −6.9835, rmsd = 1.1837.

Figure 12.

2D and 3D representations of the proposed binding interaction pattern of compound 6o (green) in the binding site of COX-2 (PBD: 1CX2), S = −7.3338, rmsd = 0.9863.

3. Conclusion

It can be concluded that a new series of thieno[2,3-d]pyrimidine derivatives 4, 5, 6a-o and 11 were synthesized and their inhibitory activity against 15-LOX, COX-2, NO, ROS, TNF-α and IL-6 in RAW 264.7 macrophages were evaluated in vitro. Compound 6o was the most active 15-LOX inhibitor (IC₅₀ = 1.17 μM) and its activity was superior to that of the reference NDGA (IC₅₀ = 1.28 μM). Meanwhile, compounds 4, 6h, 6g and 11 exhibited high activities (IC₅₀ = 1.29–1.77 μM. Addressing COX-2 selectivity, the ester 4 (SI=137.37) and the phenyl substituted acetohydrazide 11 (SI=132.26) showed the greatest COX-2 selectivity that was about 28 times more selective than the reference drug diclofenac (SI=4.73), however, it was lower than that of celecoxib (SI= 219.25).

Compounds 6o showed high activities in reducing NO with IC₅₀ values of 7.77 μM (3-fold more potent than celecoxib, IC₅₀ =22.89 μM). Compound 11 and 6o caused a reduction of TNF-α level (IC₅₀ =11.06 and 11.27 μM) that was comparable to that of diclofenac, IC₅₀ = 10.45 μM. Moreover, compound 6h caused a reduction of IL6 and TNF-α levels by 4- and 1.3 fold more potent than diclofenac, respectively. Further, compound 4 has anti-inflammatory activity due to its dual functionality against 15-LOX and COX-2, as well as its suppression of other inflammatory mediators such as ROS (IC₅₀ = 21.34 μM), NO (IC₅₀ = 4.72 μM), IL6 (IC₅₀ = 6.0 μM) and TNF-α (IC₅₀ = 26.03μM). The results were further supported by molecular docking studies, which identify the key interactions needed for COX-2, and 15-LOX inhibitors to act successfully. Our study demonstrated that the thienopyrimidine nucleus is a promising scaffold in designing multi-target candidates for development potential anti-inflammatory agents.

4. Materials and Methods

4.1. Chemistry

Melting points were determined with a Gallenkamp (London, U.K.) melting point apparatus and are uncorrected. IR spectra (KBr, cm⁻¹) were recorded by JASCO FTIR-6300 spectrophotometer. ¹H NMR and ¹³C NMR spectra were obtained using Bruker Avance II 600 MHz and/or Bruker NEO 400 MHz spectrometers. All spectra were recorded in deuterated solvents (CDCl₃ or DMSO-d₆), and the chemical shift values were expressed in parts per million (ppm) relative to the internal standard, tetramethylsilane (TMS). Mass spectra were determined on Mass Spectrometer Double-Focusing Sector (GS-MS DFS/Thermo) and Shimadzu QP1000 EX (Shimadzu Corporation, Tokyo, Japan) with ionization energy 70 eV. HREIMS were measured using a UPLC–MS/MS (Q-TOF-ESI) (Waters Corp., USA) with an electrospray ionization (ESI) technique. Progress of the reaction was monitored on precoated thin layer chromatography plates with UV₂₅₄ indicator (Merck 60F₂₅₄). The plates were visualized by exposing them to a short (λ_max = 245 nm) wavelength UV light, and iodine vapors. Compounds 1–3 and 7–9 were synthesized according to the previously reported procedure [30–34].

Synthesis of ethyl 2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino) acetate (4)

4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine 3 (1 gm, 4.45 mmol) was added to a stirred suspension of fused sodium acetate and ethyl glycinate HCl in ethanol. The stirred reaction mixture was heated at reflux for 24 h. The reaction mixture was filtered while hot and allowed to cool to room temperature. The resulting precipitate was collected by filtration and washed successively with n-hexane (3 × 25 mL). The solid residue was dried under reduced pressure and crystallized from ethanol to give 0.5 g of yellow solid (38.6%); mp 193–195 °C; IR (KBr) ν_max 3334 (N–H), 2940 (C–H aliphatic), 1748 (C=O), 1572 cm⁻¹ (C-C aromatic);¹H NMR (600 MHz, CDCl₃) δ = 8.38 (s, 1H, NCHN), 6.02 (br s, 1H, NH), 4.33 (d, J = 4.8 Hz, 2H, CH₂), 4.29 (q, J = 7.1, 2H, CH_{2 ethyl}), 2.99–3.01 (m, 2H, CH₂, _cyclohexane), 2.80–2.82 (m, 2H, CH₂, _cyclohexane), 1.87–1.96 (m, 4H, 2CH₂, _cyclohexane), 1.32 (t, J = 7.1 Hz, 3H, CH_{3 ethyl}) ppm; ¹³C NMR (CDCl₃, 150 MHz,) δ = 170.9 (CH₂COO), 164.9 (SCN), 156.6 (NCNH), 152.5 (NCHN), 134.1 (SCCH₂), 125.8 (CCCH₂), 116.6 (NHCC), 61.9 (OCH₂CH₃), 43.2 (NHCH₂CO), 26.2, 25.6, 22.7 (4 CH_{2 cyclohexane}), 14.4 (OCH₂CH₃) ppm; EIMS m/z (rel. int. %) 291.33 [M⁺] (51), 218.18 (100), 262.36 (4), 190.30 (16), 174.29 (6); HRESIMS m/z 291.1037 [M⁺] (calcd for C₁₄H₁₇O₂N₃S, 291.1041).

Preparation of 2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)aceto hydrazide (5)

To a stirred solution of thieno[2,3-d] pyrimidine ester 4 (1 gm, 3.43 mmol), hydrazine hydrate (100%, 10.59 mmol) was added and the reaction mixture was refluxed for 18 h. The reaction mixture was cooled to room temperature. The produced solid was filtered and washed with ethanol (2 × 20 ml) and crystallized from ethanol to yield 0.6 g of white solid (57%); mp 232–234 °C; IR ν _max 3357 (N–H), 3321 and 3301 (H–N–H), 2937 (C–H aliphatic), 1654 (C=O), 1571 cm⁻¹ (C-C aromatic); ¹H NMR (600 MHz, DMSO-d₆) δ = 9.33 (br s, 1H, CONH), 8.25 (s, 1H, NCHN), 6.80 (t, J = 5.4 Hz, 1H, NHCH₂), 5.02 (br s, 2H, NH₂), 4.04 (d, J = 5.4 Hz, 2H, COCH₂NH), 2.94 (t, J = 5.4 Hz, 2H, CH_{2, cyclohexane}), 2.76 (br s, 2H, CH_{2 cyclohexane}), 1.83 (d, 4H, 2CH_{2 cyclohexane}) ppm; ¹³C NMR (150 MHz, DMSO-d6) δ = 168.5 (CONH), 164.4 (SCN), 156.5 (NCNH), 152.5 (NCHN), 131.4 (SCCH₂), 126.8 (CCCH₂), 115.8 (NHCC), 42.7 (NHCH₂CO), 25.5, 24.9, 22.1, 21.9 ppm (4 CH_{2 cyclohexane}). EIMS m/z (rel. int. %) 277.37 [M⁺] (22), 246.32 (93), 218.17 (100), 189.28 (17),174.29 (10), 97.01 (18), 82.97 (22), 68.98 (31); HRESIMS m/z 277.0993 [M⁺] (calcd for C₁₂H₁₅ON₅S, 277.0997).

General procedure for preparing compounds 6a-j

To a solution of thieno[2,3-d]pyrimidine acid hydrazide 5 (0.1g, 0.36 mmol) in 30 ml of an appropriate solvent (ethanol or dioxane) containing few drops of acetic acid (9 drops), the appropriate aldehyde or ketone (0.36 mmol) was added. The reaction mixture was heated at reflux for 4 h, then filtered while hot, and recrystallized from ethanol.

(E, Z)-N’-(4-chlorobenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetohydrazide (6a)

White solid, yield: 67%; mp 286–288 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 11.63, 11.52 (2 sets of s, 1H, CONH), 8.25, 8.22 (2 sets of s, 1H, NCH _{benzylidenimine}), 8.03 (s, 1H, NCHN), 7.73 (m, 2H, H-2, H-6), 7.52 (m, 2H, H-3, H-5), 6.94, 6.81 (2 sets of t, J = 5.4 Hz, 1H, NHCH₂), 4.62, 4.19 (2 sets of d, J = 5.6 Hz, 2H, NHCH₂), 2.99 (s, 2H, CH_{2 cyclohexane}), 2.80 (s, 2H, CH_{2 cyclohexane}), 1.85 (d, J = 4.0 Hz, 4H, 2 CH_{2 cyclohexane}) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ = 171.0 and 166.7 (2 CONH), 165.1 (NCNH), 157.1 (SCN), 153.1 (NCHN), 145.8 and 142.9 (2 ArCHN), 134.9 (SCCH₂), 133.6 (C-4), 132.3 (C-1), 129.4 (C-2, C-6), 129.0 (C-3, C-5), 126.9 (CH₂CC), 116.1 (NHCC), 43.9 and 42.5 (2 NHCH₂CO), 26.2, 25.4, 22.6 (4 CH_{2 cyclohexane}) ppm; EIMS m/z (rel. int., %) [M⁺] 399.15 (10%), 246.34 (71%), 218.08 (100%), 189.30 (19%), 174.30 (7%), 88.94 (5%), 72.94 (9%); HRESIMS m/z 399.0916, [M⁺] (calcd for C₁₉H₁₈ON₅ClS, 399.0921)

(E, Z)-N’-(3-chlorobenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl) amino)acetohydrazide (6b)

White solid, yield: 50%, mp =269–270 °C. ¹H NMR (400 MHz, DMSO-d₆) δ = 11.68, 11.59 (2s, 1H, CONH), 8.26, 8.23 (2s, 1H, NCH _{benzylidenimin}), 8.02 (s, 1H, NCHN), 7.78, 7.73 (2s, 1H, H2), 7.66 – 7.68 (m, 1H, H6), 7.48 (t, J = 5.4 Hz, 2H, H4, H5), 6.94, 6.82 (2t, J = 5.3 Hz, 1H, NHCH₂), 4.63, 4.20 (2d, J = 5.2, 2H, NHCH₂), 2.98 (s, 2H, CH_{2 cyclohexane}), 2.79 (s, 2H, CH_{2 cyclohexane}), 1.86 (d, J = 3.2 Hz, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 170.63 and 166.16 (2s, CONH), 164.54 and 164.48 (2s, NCNH), 156.69 and 156.56 (2s, SCN), 152.62 and 152.51 (2s, NCHN), 144.97, 142.11 (2s, ArCHN), 136.51 and 136.26 (2s, SCCH₂), 133.66 and 133.57 (2s, C1), 131.79 (s, C4), 130.73, 130.68 (2s, C5), 129.54 (s, C6), 126.30 (d, J = 9.5 Hz, CH₂CC), 126.60 and 126.03 (2s, C3), 125.61 and 125.56 (s, C2), 115.80 and 115.62 (2s, NHCC), 43.27, 42.17 (2s, NHCH2CO), 25.66 (s, CH2 cyclohexane), 24.86 (s, CH2 cyclohexane), 22.09 (s, CH2 cyclohexane), 21.92 (s, CH_{2 cyclohexane}) ppm. EIMS m/z (rel. int.) [M⁺] 399.12 (9%), 246.33 (45%), 218.08 (100%), 189.29 (19%), 174.28 (11%), 110.94 (8%), 88.94 (15%). HRESIMS m/z [M⁺] 399.0914, (calcd for C₁₉H₁₈ON₅ClS, 399.0915)

(E, Z)-N’-(3,4-dichlorobenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin −4-yl) amino)acetohydrazide (6c)

White solid, yield: 71%, m.p.= 289–291°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 11.79 (s, 1H, CONH), 8.58, 8.36 (2s, 1H, NCH _{benzylidenimin}), 8.26 (s, 1H, NCHN), 7.92, 7.99 (2d, J = 8.8 Hz, 1H, H5), 7.71 – 7.70 (m, 1H, H6), 7.53–7.49 (m, 1H, H4), 6.95, 6.81 (2t, J = 5.0 Hz, 1H, NHCH₂), 4.62, 4.20 (2d, J = 5.3 Hz, 2H, NHCH₂), 2.98 (s, 2H, CH_{2 cyclohexane}), 2.79 (s, 2H, CH_{2 cyclohexane}), 1.86 (s, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 170.65 and 166.26 (2s, CONH), 164.48 (s, NCNH), 156.63 (s, SCN), 152.62 and 152.58 (2s, NCHN), 141.40 (s, ArCHN), 138.70 (s, C2), 134.95 and 134.86 (2s, SCCH₂), 133.57 (s, C4), 131.78 (s, C5), 130.59 and 130.35 (2s, C3), 129.33 (s, C1), 127.98 (s, C6), 126.57 and 126.30 (2s, CH₂CC), 115.79 and 115.60 (2s, NHCC), 43.34 and 42.12 (2s, NHCH₂CO), 25.63 (s, CH_{2 cyclohexane}), 24.84 (s, CH_{2 cyclohexane}), 22.07 (s, CH_{2 cyclohexane}), 21.90 (s, CH_{2 cyclohexane}) ppm. EIMS m/z (rel. int.) [M⁺] 433.28 (13%), 246.33 (75%), 218.08 (100%), 189.29 (30%), 174.28 (12%), 123.04 (5%). HRESIMS m/z [M⁺] 433.0525, (calcd for C₁₉H₁₇ON₅Cl₂S, 433.0525)

(E, Z)-N’-(4-bromobenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl) amino)acetohydrazide (6d)

White solid, Yield: 74%, m.p. 286–288°C. ¹H NMR (600 MHz, DMSO-d₆) δ = 11.64 and 11.52 (2s, 1H, CONH), 8.25 and 8.21 (2s, 1H, 1H, NCH _{benzylidenimin}), 8.00 (s, 1H, NCHN), 7.69 – 7.63 (m, 4H, ArH), 6.93 and 6.81 (2t, J = 5.4, 5.5 Hz, 1H, NHCH₂), 4.61 and 4.18 (2d, J = 5.4 Hz, 2H, NHCH2), 2.98 (br s, CH2 cyclohexane), 2.79 (s, 2H, CH2 cyclohexane), 1.86 (m, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (151 MHz, DMSO-d₆) δ = 170.51 and 166.02 (2s, CONH), 164.52 and 164.47 (s, SCN), 156.67 and 156.55 (2s, HNCN), 152.62 and 152.51 (2s, NCHN), 145.40 and 142.52 (2s, ArCHN), 133.53 and 133.29 (2s, C3, C5), 131.82 and 131.77 (2s, C1), 131.72 (s, s, SCCH₂), 128.81 and 128.66 (2s, C2, C6), 126.60 and 126.33 (s, CH₂CC), 123.15 and 123.07 (2s, C4), 115.78 and 115.61 (2s, NHCC), 43.26 and 42.11 (2s, NHCH₂CO), 25.64 (s, CH_{2 cyclohexane}), 24.85 (s, CH₂ cyclohexane), 22.08 (s, CH2 cyclohexane), 21.91 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 443.27 (11%), 246.33 (94%), 218.18 (100%), 189.30 (25%), 174.29 (9%), 88.49 (8%), 76.93 (29%). HRESIMS m/z [M⁺] 443.0415, (calcd for C₁₉H₁₈ON₅BrS, 443.0410)

(E, Z)-N’-(4-fluorobenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetohydrazide (6e)

White solid, Yield: 80 %, mp 281–282 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 11.57 (s, 1H, CONH), 8.24, 8.26 (2S, 1H, NCH _{benzylidenimin}), 8.03 (s, 1H, NCHN), 7.79–7.72 (m, 2H, H2, H6), 7.32– 7.25 (m, 2H, H3, H5), 6.93, 6.81 (2t, J = 5.4 Hz, 1H, NHCH₂), 4.62, 4.19 (2d, J = 5.3 Hz, 2H, NHCH₂), 2.99 (s, 2H, CH_{2 cyclohexane}), 2.80 (s, 2H, CH_{2 cyclohexane}), 1.86 (d, J = 3.9 Hz, 4H, 2CH₂ cyclohexane) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 170.45 and 165.94 (2s, CONH), 164.52, 164.64 (s, SCN), 163.02, 162.97 (2d, J = 247.6, 247.6 Hz, C4), 156.68 and 156.57 (2s, HNCN), 152.63 and 152.52 (2s, NCHN), 145.55 and 142.59 (2s, ArCHN), 130.84, 130.64 (2d, J = 2.8, 3 Hz, C1), 128.96, 129.12 (2d, J = 8.5, 8.7 Hz, C2, C6), 126.60 (s, CH₂CC), 126.34 (s, SCCH₂), 115.88 (d, J = 22.1 Hz, C3), 115.83 (d, J = 21.9 Hz, C5), 115.62 (s, NHCC), 43.24 and 42.13 (2s, NHCH₂CO), 25.65 (s, CH_{2 cyclohexane}), 24.85 (s, CH_{2 cyclohexane}), 22.00 (d, J = 16.7 Hz, 2 CH_{2 cyclohexane}) ppm. EIMS m/z (rel. int.) [M⁺] 383.19 (3%), 246.15 (31%), 218.08 (100%), 189.20 (27%), 121.99 (13%), 107.91 (20%), 82.89 (12%). HRESIMS m/z [M⁺] 383.1218, (calcd for C₁₉H₁₈ON₅FS, 383.1211)

(E, Z)-N’-(4-nitrobenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl) amino)acetohydrazide (6f)

Yellow solid, yield: 56%, m.p. 280– 282°C. ¹H NMR (600 MHz, DMSO-d₆) δ = 11.78 and 11.68 (2s, 1H, CONH), 8.30– 8.27 (m, 3H, NCH _{benzylidenimin}, C3 and C5), 8.14 (s, 1H, NCHN), 7.98 (d, J = 8.6 Hz, 2H, C2 and C6), 6.89, 6.77 (2s, 1H, CH₂NH), 4.67 and 4.25 (2d, J = 5.3 Hz, 2H, NHCH₂), 3.01 (s, 2H, CH_{2 cyclohexane}), 2.81 (d, 2H, J = 5.4 Hz, 2H, CH_{2 cyclohexane}), 1.87–188 (m, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (151 MHz, DMSO-d₆) δ = 170.89 (s, CONH), 164.52 (s, C4), 156.69 (s, SCN), 152.60 and 152.51 (2s, NCHN), 147.71 (s, HNCN), 141.37 (s, ArCHN), 140.31 (s, C1), 131.82 (s, SCCH₂), 127.89 and 127.75 (2s, C2, C6), 126.35 (s, CH₂CC), 124.05 (d, J = 7.0 Hz, C3, C5), 115.64 (s, NHCC), 43.35 and 42.14 (2s, NHCH₂CO), 25.66 (s, CH_{2 cyclohexane}), 24.86 (s, CH2 cyclohexane), 22.09 (s, CH2 cyclohexane), 21.92 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 409.95 (13%), 246.32 (35%), 218.17 (100%), 189.29 (27%), 147.28 (13%), 135.16 (18%), 150.25 (48%), 88.94 (14%), 75.93 (8%). HRESIMS m/z [M⁺] 410.1155, (calcd for C₁₉H₁₈O₃N₆S, 410.1156)

(E, Z)-N’-(4-methoxybenzylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetohydrazide (6g)

White solid, Yield: 62%, m.p. 278–279°C. ¹H NMR (400 MHz, DMSO-d₆) δ =11.48 and 11.34 (2s, 1H, CONH), 8.27 and 8.18 (2S, 1H, NCH _{benzylidenimin}), 7.98 (s, 1H, CH _pyrimidine), 7.66– 7.61 (m, 2H, H2, H6), 7.01 (t, J = 7.9 Hz, 2H, H3, H5), 6.93 and 6.81 (2t, J = 5.5 Hz, 1H, NHCH₂), 4.60 and 4.17 (2d, J = 5.4 Hz, 2H, NHCH₂), 3.81 and 3.79 (2s, 3H, OCH₃), 2.99 (s, 2H, CH₂ cyclohexane), 2.79 (s, 2H, CH_{2 cyclohexane}), 1.86 (s, 4H, 2 CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 170.20 and 165.61 (2s, CONH), 164.46 (s, C4), 160.72 (d, J = 6.2 Hz, SCN), 156.70 (s, HNCN), 152.67 and 152.57 (2s, NCHN), 146.57 and 143.64 (2s, ArCHN), 131.78 (s, C1), 128.57 (s, C2), 128.38 (s, C6), 126.61 (s, SCCH₂), 126.36 (s, CH₂CC), 115.63 (s, NHCC), 114.32 (d, J = 5.4 Hz, C3, C5), 55.29 (s, OCH₃), 43.23, 42.16 (2s, NHCH₂CO), 25.66 (s, CH_{2 cyclohexane}), 24.87 (s, CH2 cyclohexane), 22.11 (s, CH2 cyclohexane), 21.94 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 395.21 (5%), 246.32 (100%), 218.16 (97%), 189.29 (25%), 150.25 (48%), 134.15 (23%), 76.93 (29%). HRESIMS m/z [M⁺] 395.1406, (calcd for C₂₀H₂₁O₂N₅S, 395.1410)

(E, Z) 2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-N’-(thiophen-2-yl methylene) acetohydrazide (6h)

Light black solid, Yield: 84%, m.p. 235–237°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 11.55 and 11.39 (2s, 1H, CONH), 8.47 and 8.20 (2s, 1H, NCH _{benzylidenimin}), 8.26 (s, 1H, CH _pyrimidine), 7.64 (t, J = 4.4 Hz, 1H, CH _thiophene), 7.43 (dd, J = 6.9, 3.2 Hz, 1H, CH _thiophene), 7.15– 7.12 (m, 1H, CH thiophene), 6.92 and 6.78 (2t, J = 5.3, 5.4 Hz, 1H, CH₂NH), 4.53 and 4.16 (2d, J = 5.3 Hz, 2H, CH₂NH), 2.99 (d, J = 5.1 Hz, 2H, CH_{2 cyclohexane}), 2.79 (s, 2H, CH_{2 cyclohexane}), 1.86 (d, J = 4.9 Hz, 4H, 2CH2 cyclohexane) ppm. ¹³C NMR (101 MHz, DMSO-d6) δ = 170.57 and 166.29 (2s, CONH), 165.02 and 164.45 (2s, NHCN), 157.16 and 157.03 (2s, SCN), 153.12 and 153.01 (2s, NCHN), 141.99 (s, C3), 138,86 (s, C5), 132.28 (s, C2), 131.25 (s, C4), 128.39 (s, ArCHN), 128.26 (s, SCCH₂) and 126.82 (2s, CH₂CC), 115.81 and 115.62 (2s, NHCC), 43.78 and 42.46 (2s, CH₂NH), 26.14 (s, CH2 cyclohexane), 25.35 (s, CH2 cyclohexane), 22.41 (s, CH2 cyclohexane), 21.52 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 371.37 (5%), 246.32 (96%), 218.18 (100%), 189.29 (37%), 147.28 (13%), 135.12 (8%), 95.92 (22%), 56.96 (37%). HRESIMS m/z [M⁺] 371.0874, (calcd for C₁₇H₁₇ON₅S₂, 371.0869)

(E, Z) N’-(furan-2-ylmethylene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetohydrazide (6i)

White solid, Yield: 82%, m.p. 231–233°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 11.55 and 11.39 (s, 1H, CONH), 8.26 (s, 1H, CH _pyrimidine), 8.15 and 7.92 (2s, 1H, NCH _{benzylidenimin}), 7.83 (d, J = 7.8 Hz, 1H, CH _furan), 6.90 (dd, J = 15.1, 3.3 Hz, 1H, CH _furan), 6.77 (t, J = 5.0 Hz, 1H, CH₂NH), 6.64–6.62 (m, 1H, CH _furan), 4.54 and 4.18 (2d, J = 5.4 Hz, 2H, CH₂NH), 2.98 (s, 2H, CH_{2 cyclohexane}), 2.79 (s, 2H, CH_{2 cyclohexane}), 1.86 (d, J = 4.3 Hz, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 170.27 and 165.88 (2s, CONH), 164.45 (s, NHCN), 156.60, 156.55 (2s, SCN), 152.61 and 152.49 (2s, NCHN), 149.28 and 149.01 (2s, C2), 145.03 (d, J = 5.0 Hz, C5), 136.60 and 133.85 (2s, ArCHN), 131.82 and 131.72 (2s, SCCH₂), 126.60 and 126.31 (2s, CH₂CC), 115.80 and 115.61 (2s, NHCC), 113.61 and 113.39 (2s, C3), 112.10 (s, C4), 43.22 and 42.12 (2s, CH₂NH), 25.61 (s, CH2 cyclohexane), 24.85 (s, CH2 cyclohexane), 22.08 (s, CH2 cyclohexane), 21.92 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 355.36 (10%), 246.15 (100%), 218.18 (94%), 189.20 (14%), 147.20 (8%), 95.89 (5%), 56.93 (5%). HRESIMS m/z [M⁺] 355.1092, (calcd for C₁₇H₁₇O₂N₅S, 355.1097).

N’-(2-oxoindolin-3-ylidene)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetohydrazide (6j)

Yellow solid, Yield: 75%, m.p. 281–283°C.¹H NMR (400 MHz, DMSO-d₆) δ = 13.47, 12.62 (s, 1H, CONH _isatin), 11.21 (s, 1H, NH), 8.29 (s, 1H, NCHN), 7.60 (d, J = 29.5 Hz, 1H, ArH), 7.53–6.89 (m, 4H, 3 ArH and NHCHC), 4.76 (s, 1H, NHCH₂), 4.30 (s, 1H, NHCH₂), 3.03 (d, J = 22.0 Hz, 2H, CH2 cyclohexane), 2.80 (s, 2H, CH2 cyclohexane), 1.87 (s, 4H, 2CH2 cyclohexane) ppm. 13C NMR (101 MHz, DMSO-d₆) δ = 164.56 (d, J = 13.3 Hz, CHCOH), 162.52 (s, CONH _isatin), 156.54 (s, NCNH), 152.49 (s, NCHN), 143.76 (s, SCN), 132.61 (s, NHCC _isatin), 131.91 (s, SCCH₂), 126.41 (s, C4 _isatin), 126.06 (s, C2 _isatin), 122.58 (s, CCCH₂), 121.59 (s, C3 _isatin), 119.69 (s, C5 _isatin), 115.71 (s, NHCC), 115.21 (s, COCC _isatin), 111.09 (s, CHCOH), 110.59 (s, CHCOH), 25.66 (s, CH₂ cyclohexane), 24.88 (s, CH2 cyclohexane), 22.11 (s, CH2 cyclohexane), 21.92 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 405.97 (4%), 246.15 (91%), 218.08 (100%), 188.18 (93%), 161.17 (35%), 117.98 (29%), 103.91 (61%), 76.90 (44%). HRESIMS m/z [M⁺] 406.1208, (calcd for C₁₂H₁₅ON₅S, 406.1206)

Synthesis of N-phenyl-2-(2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino) acetyl)hydrazinecarboxamide (6k)

Phenyl isocyanate (0.36 mmol) was added to a stirred solution of compound 5 (0.1g, 0.36 mmol) in ethanol. The reaction mixture was refluxed for 18 h, concentrated and the precipitated solid was crystallized from ethanol/dioxane (10:3). White solid, Yield: 55%, m.p. 164–166°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 10.27 (s, 1H, CH₂CONH), 9.75 (s, 1H, CONHNH), 9.31 (s, 1H, ArNHCO), 8.19 (s, 1H, NCHN), 7.45 – 7.32 (m, 4H, ArH), 7.20 (s, 1H, H4), 6.89 (t, J = 5.3 Hz, 1H, CH₂NH), 4.22 (d, J = 4.9 Hz, 2H, CH₂NH), 3.00 (s, 2H, CH_{2 cyclohexane}), 2.79 (s, 2H, CH₂ cyclohexane), 1.84 (s, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 180.87 (s, CONH), 169.12 (s, CONH), 164.48 (s, NHCN), 156.52 (s, SCN), 152.40 (s, NCHN), 138.83 (s, C1), 131.80 (s, SCCH₂), 128.19 (s, C3, C5), 126.74 (s, CH₂CC), 125.79 (s, C4), 125.35 (s, C2, C6), 115.99 (s, CH₂CC), 43.05 (s, NHCH₂), 25.60 (s, CH_{2 cyclohexane}), 24.88 (s, CH_{2 cyclohexane}), 22.13 (s, CH2 cyclohexane), 21.90 (s, CH2 cyclohexane) ppm. EIMS m/z (rel. int.) [M⁺] 394.19 (47%), 317.03 (34%), 245.31 (64%), 218.18 (100%), 204.25 (89%), 188.28 (66%), 135.12 (28%), 76.93 (50%). HRESIMS m/z [M⁺] 396.1366, (calcd for C₁₉H₂₀O₂N₆S, 396.1363).

Preparation of N-((5-butoxy-1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8-tetrahydrobenzo-[4,5]thieno[2,3-d]pyrimidin-4-amine (6l)

To a stirred solution of compound 5 (0.1g, 0.36 mmol) in butanol (10 ml), ethylchloroformate (0.27 mmol) was added. The reaction mixture was heated at reflux for 4 h, cooled and concentrated under rotary evaporated. The obtained solid was recrystallized from ethanol to give black solid, yield 45%, m.p. 120–124°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.24 (s, 1H, NCHN), 7.00 (t, J = 5.6 Hz, 1H, CH₂NH), 4.19 (d, J = 5.8 Hz, 2H, CH₂NH), 4.07 (t, J = 6.5 Hz, 2H, OCH₂), 2.94 (s, 2H, CH2 cyclohexane), 2.79 (s, 2H, CH2 cyclohexane), 1.85 (s, 4H, 2CH2 cyclohexane), 1.57–1.50 (m, 2H, OCH₂CH₂), 1.34–1.24 (m, 2H, CH₂CH₂CH₃), 0.86 (t, J = 7.4 Hz, 3H, CH₂CH₂CH₃) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ 170.19 (s, C2 _oxadiazole), 164.68 (s, C5 _oxadiazole), 156.39 (s, NHCN), 152.34 (s, NCHN), 131.85 (s, SCCH₂), 126.34 (s, CH₂CC), 115.57 (s, NHCC), 63.91 (s, OCH₂), 42.51 (s, CH₂NH), 30.14 (s, OCH₂CH₂), 25.63 (s, CH_{2 cyclohexane}), 24.84 (s, CH_{2 cyclohexane}), 22.08 (s, CH₂ cyclohexane), 21.85 (s, CH_{2 cyclohexane}), 18.45 (s, CH₂CH₃), 13.44 (s, CH₃) ppm. HRESIMS m/z [M⁺] 359.1405, (calcd for C₁₇H₂₁O₂N₅S, 359.1410).

Preparation of 1-(5-hydroxy-3,5-dimethyl-4,5-dihydro-1H-pyrazol-1-yl)-2-((5,6,7,8-tetrahydrbenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)ethanone (6m)

To a solution of acid hydrazide 5 (0.135gm, 0.49 mmol) and triethylamine (10 drops) in ethanol (30 mL), acetylacetone (0.05gm, 0.49 mmol) was added. The reaction mixture was stirred under reflux for 24 h and cooled to room temperature. The separated solid was filtered and crystallized from ethanol. Light red solid, yield 80%, m.p. 199–201°C. ¹H NMR (600 MHz, DMSO-d₆) δ = 8.24 (s, 1H, NCHN), 6.72 (s, 1H, OH, exchangeable), 6.43 (s, 1H, CH₂NH, exchangeable), 4.42 (d, J = 4.8 Hz, 2H, CH₂NH), 2.98– 2.78 (m, 6H, 2CH_{2 cyclohexane} & CH_{2 pyrazole}), 2.00 (s, 3H, CH₃ pyrazole), 1.85– 1.74 (m, 7H, 2CH_{2 cyclohexane} & CH_{3 pyrazole}) ppm. ¹³C NMR (151 MHz, CDCl₃-d₆) δ = 167.92 (s, CH₂CON), 156.70 (s, SCN), 156.64 (s, NCNH), 151.81 (s, NCHN), 134.40 (s, NCCH_{3 pyrazole}), 126.14 (s, SCCH₂), 116.74 (s, CCCH₂), 91.99 (s, COH _pyrazole), 51.66 (s, CH₂ pyrazole), 44.05 (s, NHCH₂CO), 27.15 (s, CH_{3 pyrazole}), 26.18 (s, CH_{2 cyclohexane}), 25.64 (s, CH₂ cyclohexane), 22.69 (d, J = 3.3 Hz, 2CH_{2 cyclohexane}), 16.32 (s, CH_{3 pyrazole}), 11.49 (s, CH_{3 pyrazole}) ppm. EIMS m/z (rel. int.) [M⁺] 359.51 (13%), 302.27 (4%), 245.34 (100%), 218.19 (97%), 188.29 (49%), 162.27 (5%), 97.00 (27%) ppm. HRESIMS m/z [M⁺] 359.1411, calcd for C₁₇H₂₁O₂N₅S, 359.1410).

Preparation of 1-(5-hydroxy-5-methyl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)ethenone (6n)

To a solution of acid hydrazide 5 (0.1g, 0.36 mmol) and benzoylactone (0.058 gm, 0.36 mmol) in ethanol (30 mL), few drops of acetic acid (10 drops) was added. The reaction mixture was stirred under reflux for 24 h and cooled to room temperature. The separated solid was filtered and crystallized from ethanol. White solid, yield= 55%, m.p.255–257°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 8.27 (s, 1H, NCHN), 7.38 (d, J = 7.0 Hz, 2H, H2, H6), 7.31 (t, J = 7.3 Hz, 2H, H3, H5), 7.23 (t, J = 6.7 Hz, 1H, H4), 6.89 (s, 1H, OH), 6.68 (s, 1H, NHCH₂), 4.52 (d, J = 4.5 Hz, 2H, NHCH₂), 3.10 (q, J = 18.9 Hz, 2H, CH_{2 pyrazole}), 2.90 (s, 2H, CH_{2 cyclohexane}), 2.75 (s, 2H, CH₂ cyclohexane), 2.08 (s, 3H, CH_{3 pyrazole}), 1.80 (s, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 165.54 (s, CH₂CON), 164.38 (s, NCNH), 156.43 (s, SCN), 155.02 (s, NCCH_{3 pyrazole}), 152.61 (s, NCHN), 143.60 (s, SCCH₂), 131.75 (s, C1), 127.85 (s, C4), 127.03 (s, C3, C5), 126.24 (s, C2, C6), 124.64 (s, CCCH₂), 115.55 (s, NHCC), 92.12 (s, ArCOH _pyrazole), 55.32 (s, CH_{2 pyrazole}), 43.51 (s, NHCH2CO), 25.56 (s, CH2 cyclohexane), 24.79 (s, CH2 cyclohexane), 22.03 (s, CH2 cyclohexane), 21.83 (s, CH_{2 cyclohexane}), 15.72 (s, CH_{3 pyrazole}) ppm. EIMS m/z (rel. int.) [M⁺] 421.42 (25%), 368.68 (8%), 302.26 (18%), 246.34 (54%), 218.19 (100%), 188.29 (25%), 158.30 (22%), 104.99 (20%), 76.94 (14%). HRESIMS m/z [M⁺] 421.1560, calcd for C₂₂H₂₃O₂N₅S, 421.1567).

Synthesis of (Z)-4-oxo-4-(2-(2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetyl)hydrazinyl)but-2-enoic acid (6o)

To a solution of acid hydrazide 5 (0.1gm, 0.36 mmol) in ethanol (30 mL) containing drops of acetic acid (9 drops), maleic anhydride (0.035 gm, 0.36 mmol) was added. The reaction mixture was heated at reflux for 2h and the formed solid was filtered while hot. The solid was crystallized from ehanol/dioxane (10:3) to give a white powder 0.08 gm (60%) mp 242–244 °C; ¹H NMR (400 MHz, DMSO-d₆) δ =13.30 (br s, 1H, OH), 10.58 (br s, 1H, NH), 10.27 (s, 1H, CONH), 8.25 (s, 1H, NCHN), 6.91 (t, J = 5.3 Hz, 1H, NHCH₂), 6.35 (d, J =12.1 Hz, 1H, CH=CH _maleic), 6.27 (d, J = 12.2 Hz, 1H, CH=CH _maleic), 4.16 (d, J = 5.5 Hz, 2H, NHCH₂), 2.98 (s, 2H, CH_{2 cyclohexane}), 2.78 (s, 2H, CH_{2 cyclohexane}), 1.84 (s, 4H, 2CH_{2 cyclohexane}) ppm. ¹³C NMR (101 MHz, DMSO-d₆) δ = 167.98 (s, CH₂CONH), 166.73 (s, COOH _maleimide), 164.52 (s, CONH _maleimide), 162.65 (s, NHCN), 156.51 (s, SCN), 152.50 (s, NCHN) 132.70 (s, CH=CH _maleimide), 131.63 (s, SCCH₂), 127.38 (s, CH=CH maleimide), 126.70 (s, CCCH₂), 115.81 (s, NHCC), 42.60 (s, NHCH₂CO), 25.58 (s, CH_{2 cyclohexane}), 24.89 (s, CH2 cyclohexane), 22.62 (s, CH2 cyclohexane), 22.14 (s, 2 CH2 cyclohexane). EIMS m/z (rel. int.) [M⁺] 356.90 (6%), 277 (14%), 246.9 (10%), 218 (100%), 189 (20%), 174 (17%), 135 (11%), 98 (13%), 54 (73%) ppm. HRESIMS m/z [M+H] 376.1095, (calcd for C₁₆H₁₇N₅O₄S, 376.1074)

Synthesis of ethyl 2-((2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetate (10)

4-chloro-2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine 9 (1 gm, 3.33 mmol) was added to a stirred suspension of fused sodium acetate and ethyl glycinate HCl (3.33 mmol) in ethanol. The stirred reaction mixture was heated at reflux for 24 h. The reaction mixture was filtered while hot and allowed to cool to room temperature. The resulting precipitate was collected by filtration and washed successively with n-hexane (3 × 25 mL). The solid was dried under reduced pressure and crystallized from ethanol to give yellow solid (35 %) mp 161–163 °C. ¹H NMR (400 MHz, DMSO-d₆) δ. 8.35 (m, 2H, ArH), 8.00 (s, 1H, NCHN), 7.61 – 7.39 (m, 3H, ArH), 7.19 (t, J = 5.5 Hz, 1H, CH₂NH), 4.23 (d, J = 5.5 Hz, 2H, CH₂NH), 4.13 (q, J = 7.1 Hz, 2H, OCH2CH3), 2.97 (s, 2H, CH2 cyclohexane), 2.80 (s, 2H, CH2 cyclohexane), 1.86 (s, 4H, 2CH2 cyclohexane), 1.18 (t, J = 5.5 Hz, OCH₂CH₃).

Preparation of 2-((2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetohydrazide (11)

To a stirred solution of ethyl 2-((2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)acetate (0.1 gm, 0.27 mmol), hydrazine hydrate (100%, 1.36 mmol) was added. The reaction mixture stirred at reflux for 24 h. The obtained solid was filtered while hot, washed with ethanol and dried to give a white solid 0.05 gm (35%) mp 183–185°C; ¹H NMR (400 MHz, DMSO-d₆) δ. 9.25 (s, 1H, NH), 8.36 (br s, 2H, H2, H6), 7.46 (br s, 3H, H3, H4, H5), 6.90 (s, 1H, NH), 4.39 (br s, 2H, NH₂), 4.17 (d, J = 4.5 Hz, 2H, NHCH₂CO), 3.01 (s, 2H, CH_{2 cyclohexane}), 2.80 (s, 2H, CH_{2 cyclohexane}), 1.86 (s, 4H, 2CH_{2 cyclohexane}). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.75 (s, CONH), 165.54 (s, NCN), 157.48 (s, NCNH), 156.40 (s, NCS), 137.74 (s, SCCH₂), 131.55 (s, C1), 129.89 (s, C4), 128.23 (s, C3), 127.60 (s, C2), 126.82 (s, CCCH₂), 114.45 (s, NHCC), 42.93 (s, NHCH2CO), 25.46 (s, CH2 cyclohexane), 24.95 (s, CH2 cyclohexane), 22.17 (s, CH2 cyclohexane), 21.91 (s, CH_{2 cyclohexane}). EIMS m/z (rel. int.) [M⁺] 353.46 (31), 321.98 (20), 294.28 (100), 265.35 (26), 135.10 (8), 103.98 (29), 76.93 (47). HRESIMS m/z [M⁺] 353.1307, calcd for C₁₂H₁₅ON₅S, 353.1305).

4.2. Biological activity

4.2.1. In vitro 15-LOX inhibition assay

The in vitro 15-LOX assay was performed for the synthesized compounds using soybean 15-LOX inhibitor screening assay (Cayman, Ann Arbor, MI) and nordihydroguaiaretic acid (NDGA) as reference and according to the manufacturer’s instructions.

4.2.2. In vitro COX-1 and COX-2 inhibitory assay

The in vitro COX-1/COX-2 inhibition assay was performed using ovine COX-1 and human recombinant COX-2 inhibitory assay kits (Cayman, Ann Arbor, MI) for all the synthesized thienopyrimidines 4, 5, 6a-o and 11, using celecoxib, indomethacin and diclofenac sodium as reference drugs. The half-maximal inhibitory concentration (IC₅₀, μM) was determined and used to calculate the COX-2 selectivity index (SI) using the following equation: SI= IC₅₀ (COX-1)/IC₅₀ (COX-2).

4.2.3. Effects on NO, ROS, and cytokines production in LPS-activated RAW 264.7 macrophages cells

The levels of the inflammatory mediators TNF-α, IL-6, and NO, as well as ROS were measured in LPS-induced RAW 264.7 macrophage cells purchased from ATCC (Manassas, VA) in the absence and presence of the different synthesized compounds as detailed in our previous studies [20, 49].

4.3. Molecular modeling

Material and methods

2D and 3D modeling studies of the thieno[2,3-d]pyrimidine derivatives 4, 6g, 6h, 6o, 11 were conducted using the Molecular Operating Environment (MOE), version MOE 2019.0102 (Chemical Computing Group, Montreal, CA). The X-ray crystal structure of COX-2 enzyme along with its ligand SC-558 (PDB code 1CX2) [50], as well as 15-LOX (PDB code 4NRE) [51] with substrate mimic were downloaded from the Protein Data Bank website (http://www.rcsb.org). Using MOE’s quick preparation tool, the protein structures were prepared after downloading. In 15-LOX, H₂O molecules, repeated chains, and unwanted surfactants except for a metal ion (Fe) were removed. In order to prepare each compound data, energy was minimized, hydrogen atoms were added, partial charges, and potential energies were calculated. The validation was achieved by redocking SC-558 within the binding sites of their respective enzymes in order to restore the original orientation. Following the completion of the experiment, the poses with the best ligand-enzyme interactions, scores, and root mean square deviation (RMSD) values were selected.

Highlights.

• Novel thieno[2,3-d]pyrimidine derivatives 4, 5, 6a-o, and 11 were designed and synthesized.

• The synthesized compounds were evaluated in vitro as COX-2 and 15-LOX inhibitors.

• Compound 6o was the most active 15- LOX inhibitor (IC₅₀ = 1.17 μM).

• Compound 4 (SI=137.37) and 11 (SI=132.26) showed the greatest COX-2 selectivity.

• Compounds 6o showed high activities in reducing NO production (3-fold more potent than celecoxib).

• Compound 11 and 6o caused a reduction of TNF-α level comparable to that of diclofenac.

• Compound 6h caused a reduction of IL6 and TNF-α levels more potent than diclofenac.