Novel phospha-oxazepinoquinazolinyl derivatives of ibuprofen as nitric oxide synthase inhibitors: Synthesis and biological evaluation

2.1 Chemistry

As a continuation effort of our research group in chemical synthesis of quinazolin-4-one derivatives, novel phospha-oxazepinoquinazolinyl derivatives were produced and examined as NOS inhibitor (Algohary and Hassan, 2021). Our recently described benzoxazinone (1) was refluxed with NH₂OH.HCl, in dry pyridine to yield the 3-hydroxy derivative of quinazolin-4(3H)-one (2) through a ring-opening ring closure (RORC) process (Yu and Wu, 2003). Oxygen of benzoxazinone (1) that is swapped by a nitrogen-bearing hydroxyl group, is proposed for this reaction. The hydroxylamine nitrogen nucleophile attacks the benzoxazinone nucleus, followed by ring closure of the intermediate's carbonyl carbon with highly electrophilic sp2 hybridization, yielding the quinazolinone 2 (El-Hashash et al., 2016). The spectroscopic and analytical results of 3-hydroxyquinazolin-4-one 2 were used to determine its identity. Due to both (OH and C=O_qunazoline) groups, its IR spectra revealed significant intensity bands at v 3465 and 1665 cm⁻¹, respectively in addition to the D₂O exchangeable proton characteristic for its OH group which was found at δ 10.50 ppm in its ¹H NMR spectrum.

When hydroxyquinazolin-4-one 2 was oxidized with SeO₂ in aprotic solvent dioxane, the new 2-(3-hydroxy-6-nitro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-(4-isobutylphenyl)acetaldehyde (3) was synthesized using approaches similar to those recommended by (Abbas et al., 2017) (Scheme 1). Correct elemental studies and comprehensive examinations of spectrum data characterized the structural elucidation of 3. In the IR spectra of aldehyde 3, carbonyl peaks at v 1720 (C=O_formyl) and v 1670 (C=O_{quinazolinine}) cm⁻¹ were found. A doublet signal at δ 9.70 ppm was also detected in its ¹H NMR spectra, which is indicative of the aldehydic CH proton. The characteristic CHO carbon atom was identified at δ 190.05 ppm in compound 3 ¹³C NMR spectrum. Furthermore, its mass spectra showed a molecular ion peak at m/z 381 corresponding to its molecular formula (C₂₀H₁₉N₃O₅).

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Scheme 1 Synthesis of aldehyde 3.

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As further validation for compound 3, a homonuclear NOE experiment (NOESY 1D) was performed in addition to its standard analytical techniques. According to our knowledge, this method of determining the structure of quinazolinyl derivatives is not frequently used. Irradiation of the OH singlet proton of compound 3 with a high proton frequency (11.20 ppm) has a specific impact on only the aldehydic CH singlet proton, whereas irradiation of the aldehydic CH singlet proton with a proton frequency of (9.70 ppm) has a significant effect on the adjacent chiral proton in addition to both OH and the nearest neighbor aromatic proton. According to the results of that experiment, selenium oxide (SeO₂) only oxidized the chiral methyl group, leaving the other methyl groups unaffected by aldehydic transformation Fig. 2.

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Fig. 2 Structure elucidation of compound 3 using NOE effect.

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As a result of the investigation of new synthetic strategies based on multi-component methods for preparation of our quinazolinones and studying of its therapeutic properties, we establish here a lithium bromide-catalyzed condensation three-component of our new aromatic aldehyde 3, phosphorus reagents, and active cyanomethylenes namely: cyanoacetamide, and/or malononitrile for the synthesis of our desired hybrid phospho-oxazepino quinazolinyl derivatives (4, 7, 10–12). On the other hand, to compare the results we obtained here with the outcomes of our previously reported procedures (Ali et al., 2019; Ali and Hassan, 2017). The LiBr catalytic method was installed because of the oxophilicity of Li cation, as evidenced by previous researches. It was found that LiBr activates oxygen-containing electrophiles for nucleophilic attack with high yield anticipated products (Singh et al., 2011). To begin, a domino phospha-Michael reaction mixture involving each of aldehyde 3, cyanoacetamide, and phosphorous (phosphonic) acid was refluxed in the presence of catalytic LiBr (0.1 mmol) in H₂O (5 mL) to yield the oxazepinoquinazolinylphosphonic acid 4. The formation of compound 4 was established through its spectroscopic and physical data analysis. The formyl moiety vanished from compound 4′s IR spectra, which verified the existence of absorption at ν 3310–3100 and 1660 cm⁻¹ related to both NH₂ and C=O_amide groups, respectively. Its ¹H NMR spectrum clearly revealed the removal of the typical CHO proton and the existence of the characteristic two NH₂ groups. Furthermore, the C atoms of P–CH and C=O_amide were detected at δ 33.50 and 168.05 ppm, respectively, in its ¹³C NMR spectra. Furthermore, the phosphorus atom resonated at δ 21.50 ppm in the ¹³P NMR spectra of compound 4. In addition, the molecular ion peak [M] ⁺ at m/z 529 was discovered in the ESI mass spectrum of compound 4.

Under the suggested approach, we suppose that the synergy of LiBr and aqueous medium has a major influence on the reaction's product. The LiBr/H₂O system's success is assumed to be attributed to the lithium ion's ability to activate the aldehyde moeity, due to the generation of the intermediate Knoevenagel cyanoolefin product I and, eventually, the imino-oxazepinoquinazoline II. Following that, both imino and phosphorous (phosphonic) acid may also be activated in an unexpected manner by LiBr and H₂O, resulting in the phospha-Michael addition to provide the desired product 4 Fig. 3.

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Fig. 3 A proper mechanism for the synthesis of oxazepinoquinazolinylphosphonic acid 4.

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In absolute ethanol containing piperidine, adehyde 3 was allowed to react with cyanoacetamide according to typical preparation procedures (Ibrahim and Hassanin, 2018), yielding the anticipated product (3E)-5,11-dihydro-2-imino-5-(4-isobutylphenyl)-9-nitro-11-oxo-2H-[1,2]oxazepino[3,2-b]quinazoline-3-carboxamide (5). Compound 5′s ¹H NMR spectra revealed a doublet signal ascribed to the H-4 _oxazepino proton at δ 5.85, while the protons NH₂ and NH emerged as D₂O-exchanged signals at δ 9.20 and 8.70 ppm, respectively. The distinctive carbon atoms were found at δ 161.10(C=NH) and 163.00(C=O_amide) ppm in the ¹³C NMR spectra of compound 5. The molecular ion peak M⁺ at m/z 447 was visible in its mass spectrum, confirming the suggested structure.

Phosphorous acid was directed to react with compound 5 in dry dioxane in the presence of 4-toulenesulfonic acid as a catalyst to produce the intermediate A, that was generated through the phospha-Michael addition process (Zhao et al., 2009). Latter the transitional intermediate A was cyclized upon attack of an amino group nucleophile on a phosphorus atom, followed by the removal of a water molecule, yielding the predicted nonisolable intermediate B, which was then auto-oxidized to provide the end product 6 (Scheme 2). The occurrence of the addition reaction at this location was confirmed by the absence of the H-4_oxazepino of compound 6 in its ¹H NMR spectra. Furthermore, its ³¹P NMR spectrum was identical to that of the suggested structure, with chemical shifts at δ 21.50 ppm. The MS, which shown a formula ion peak at m/z 509 supported the formation of this product.

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Scheme 2 Synthesis of compounds 4, 5, 6.

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As is evident from our results that by using the LiBr-catalyzed one-pot multi-component approach, we were able to isolate the Michael addition high yielded non-closed-loop phosphorous compounds, in the shortest time and with the least reagents and solvents toxicity. That had never been able to be achieved easily in most of our previous research articles (Ali et al., 2019a,2019b; Ali and Hassan, 2017; Ali et al., 2018), allowing us to investigate the predicted biological effect of such open chained compounds.

In the same approach, compound 7 was synthesized using the same phospha-Michael reaction mixture as for compound 4 synthesis, however with replacement of cyanoacetamid by malononitrile. The NH₂ and CN functions were detected in the IR spectra of compound 7 at ν 3320, and 2195 cm⁻¹. Its ¹H NMR spectra indicated the characteristic NH₂ protons as a singlet at δ 8.69 ppm, as well as the distinctive double doublet for P-CH proton at δ 3.90 ppm. The CN carbon atom was identified at δ 110.0 ppm in its ¹³C NMR spectrum. Furthermore, the phosphorus atom resonated at δ 20.10 ppm in the ¹³P NMR spectrum of compound 7. Compound 7′s ESI mass spectrum shown a (M⁺) peak at m/z 511, which matches the suggested formula.

Condensation of aldehyde 3 with the malononitril, followed by cycloaddition of the hydroxyl group into the nitrile functional group, results in compound 8. In the ¹H NMR spectra of compound 8, the oxazepino H-5 proton was given a doublet at δ 2.70, whilst the protons NH were detected as D₂O-exchanged signals at δ 8.30 ppm. The carbon atom CN was found at δ 116.50 ppm in the compound 8 ¹³C NMR spectrum. The molecular ion peak M at m/z 429 was visible in its mass spectrum, confirming the hypothesized structure.

Instead of the anticipated compound 6, compound 8 was reacted with H₃PO₂ in the presence 4-toluenesulfonic acid as a catalyst to provide low yielded derivative 9 as an unexpected result under the same reaction conditions as compound 6 synthesis (Scheme 3). The reaction was initiated by an unanticipated nucleophilic assault of a phosphorus atom on the nitrile group, rather than the phospha-Michael addition mechanism, which bring about the formation of nonisolable intermediate D. The nonisolable intermediate E was formed by heterocyclization of the later intermediate by removing the water molecule·H₂O and H₃PO₂ in the reaction media aided the imino group hydrolysis into C=O group, resulting in the end product 9.

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Scheme 3 Synthesis of compounds 7, 8, 9.

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The amidic carbonyl group was detected at 1675 cm⁻¹ in the IR spectra of 9. In addition to the existence of C=O_amide at δ 166.10 ppm in its ¹³C NMR spectra, its ¹H NMR spectrum revealed a distinct signal at δ 3.25 (P-OH) ppm. Furthermore, at δ 25.50 ppm, its ³¹P NMR chemical shift was found, validating the suggested structure 9. Under electron impact, compound 9 recorded its molecular ion peak at m/z 494 which concurred with the future structure.

Because of the promising outcomes of the efficient and rapid LiBr catalyzed phospha-Michael addititon protocol, we decided to try to make additional potential open-loop phosphorous derivatives for biological testing and compare them to compounds made with the same phosphorous reagents using our standard methods detailed in our research papers (Assiri et al., 2018a, 2018b). Accordingly and in the same manner as compound 4 synthesis, phospha-Michael reaction mixture including each of aldehyde 3, cyanoacetamide, and some selective phosphrous reagents namely, tris(2-chloroethyl)phosphite, O,O-diethyldithiophosphoric acid, and P,P-dichloro(phenyl)phosphine in the presence of LiBr/H₂O under refluxing condition afforded the phospha–oxazepinoquinazolinyl derivatives 10, 11 and 12, respectively (Scheme 4). All ¹H-, ¹³C- and ³¹P NMR spectra proved all proposed structures (cf: ‘‘Experimental’’).

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Scheme 4 Synthesis of compounds 10, 11, 12.

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The reaction of component 5 with tris(2-chloroethyl)phosphite and boron trifloride followed the parameters of our non-catalytic synthesis. At 80–90 ⁰C, BF₃.Et₂O as a catalyst experienced Michael addition of a phosphorus atom at position C–5_oxazepin, resulting in the intermediate G. Adding droplets of water to the last intermediate, the 2-chloroethanol molecule was removed, resulting in the nonisolable oxazepinoquinazolinyl phosphonate H. With the elimination of the 2-chloroethanol molecule, this intermediate was cyclized by nucleophilic attack of the amino group at the phosphorus atom, yielding compound 13 via auto oxidation process with hydrogen molecule elimination (Scheme 5). One moiety of OCH₂CH₂Cl was detected as two triplets at δ 3.35 and 4.40 (J_HH = 6.6 Hz) ppm in the ¹H NMR spectrum of compound 13, and one amidic NH proton was detected as a wide signal at δ 9.70 ppm. The specific carbon atoms ClCH₂ and OCH₂ were discovered in spectrum of ¹³C NMR of compound 13 at δ 35.80 and 54.70 ppm, respectively. Additionally, the MS of compound 13 revealed molecular ion peaks at m/z 573 (M + 2, 15 %) and 571 (M⁺ , 10 %) at a ratio of 1:3, confirming the existence of one chlorine atom, this pair of molecular ion peaks, often described as doublets confirming the suggested structure (Pavia et al., 2001).

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Scheme 5 Synthesis of compounds 13, 14, 15.

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The novel thiazaphosphinine 14 was created by reacting compound 5 with O,O-diethyldithiophosphoric acid (generated in situ) in dry ethanol. This product's ESI mass spectrum revealed the expected peak (M) ⁺ at m/z 585. It also had significant absorption bands at ν 1710 (C=O_amide), ν 1070 (P–O–C), and ν 740 (P=S) cm⁻¹ in its IR spectra. The ethoxy group's distinctive triplet at δ 1.25 ppm and quartet at δ 3.80 ppm were found in 14′s ¹H NMR spectra. It also revealed a novel singlet corresponding to NH proton at δ 9.40 ppm. The structure of 14 was further corroborated by its ¹³C NMR spectra, which revealed the particular carbon atoms at δ 15.00 (CH₃) and δ 62.50 (CH₂). Furthermore, at δ 50.50 ppm, its ³¹P NMR chemical shift was detected. The reaction began with the addition of a sulfur-Michael group to the oxazepino CH=C to generate the nonisolable intermediate I, which was then cyclized by removing ethanol molecules, followed by the removal of hydrogen via an internal autoxidation mechanism (Scheme 5).

Compound 5 was allowed to react with P,P-dichloro(phenyl)phosphine and triethylamine in 1,4-dioxane as a catalyst to give the nonisolable intermediate J, after that Phospha-Michael added to the oxazepino CH=C to give the intermediate K, which was subsequently rearranged to give the product 15. The lack of the H-4_oxapino proton in the starting material 5 and the development of a new doublet at δ 5.10 (H-3a) ppm in the ¹H NMR spectrum of compound 15 confirmed the suggested formula. Due to its link through double bond with the phosphorus atom, its ¹³C NMR spectra recorded the typical carbon atom C-3a at δ 70.10 ppm. Furthermore, its ³¹P NMR spectrum exhibited a singlet at δ 30.10 ppm. Its MS presented the molecular ion peak at m/z 589, which assured its structure.

2.2 Inhibition properties of nNOS and iNOS

Using recombinant isoenzymes, the pharmacological activity of the newly synthesized derivatives (3–15) as nNOS and iNOS inhibitors, was tested in vitro (Bredt and Snyder, 1990). A pilot experiment was carried out utilizing 0.001 mol of each compound to choose the most potent structure Table 1.

In general, compounds having nitro substituents have a strong enzymatic N-Oxygenation action with high inhibitory values (Winkler and Hertweck, 2017). Non-closed-loop phosphorous compounds (4, 7, 10–12) are the best inhibitors of all other investigated products, exhibiting more than 60% inhibition, according to iNOS inhibition values (Table 1). Nonetheless, certain assumptions may be drawn from the experimental results. From a qualitative viewpoint, the effect of different substituents on iNOS inhibitory efficacy varies in closed-loop phosphorous compounds (6, 9, 13–15). In general, substituted molecules with sulphur and halogen ring closed atoms isoforms (13, 14 and 15) exhibited higher inhibitory activity than substituted molecules with free out closed hydroxyl group (6 and 9) which had similar inhibition effects. The oxyazepino free phosphrous compounds (5 and 8) showed considerable inhibition as compared to the beginning formylquinazolinone 3, with no significant inhibition values as predicted. Supporting our concept of increasing the inhibitory impact by employing a hybrid molecular structure with several affected species that we synthasized.

The inhibition values of nNOS by our newly created products at a concentration of 1 mM are also shown in Table 1. Of all the substances examined in this case, products (4, 7, 10–12) exhibited the highest nNOS inhibitory values. Compounds (13, 14 and 15) had stronger inhibition effects than compounds (6 and 9) with moderate nNOS inhibition values, and hence had a better inhibitory effect than compounds (5 and 8). The inhibitory impact of compound 3 without the phosphaoxazeoino character is weaker than that of any other derivatives.

Table 2 shows the IC₅₀ values for the most interesting phosphaoxazeoinoquinazolinyl substances (4, 7, 10–12). The IC₅₀ values for both 10 and 11 for iNOS are around 18 M, showing that it is a very potent inhibitor. With IC50 values of 27.02 and 18.30 M, respectively, the derivatives 10 and 11 were the most efficient against nNOS, indicating that 11 is the best inhibitor of all synthesized compounds.

Our newly synthesized compounds have significantly high potency against iNOS and nNOS in comparison with L-NAME. Fig. 4 shows that our newly synthesized phospha-oxazepinoquinazolinyl derivatives enhance NOS inhibition where each value is reported from the mean of three triplicate experiments using iNOS or nNOS enzymes.

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Fig. 4 nNOS and iNOS activity as a percentage of control by adding 1 mM novel synthesized derivatives (3–15).

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3.1 Instruments and reagent

Merck and Aldrich Chemical Company provided all of the essential chemicals. For TLC (Silica gel 60 F254, Merck Germany) were used, and UV light was applied to detect spots. Measuring of melting points were determined by a Veego REC-22038 A2 instrument and are uncorrected. Elementar Vario analyser was used for elemental analyses to performance. Bruker FTIR spectrophotometer was used to record the IR spectra (KBr). On a Bruker Spectrospin DPX 400 MHz and a Bruker Spectrospin DPX 75 MHz spectrometer, ¹H, ¹³C and ³¹P NMR were recorded using DMSO‑d₆ as a solvent and trimethylsilane (TMS) as an internal standard. The mass spectra were recorded using ESI-MS (AB-Sciex 2000, Applied Biosystem).

3.2 Synthesis

3.3 In vitro nNOS and iNOS inhibition activities

Sigma Aldrich provided all of the necessary chemicals. The Bredt and Snyder (Bredt and Snyder, 1990) technique for evaluating nNOS activity was used to track the conversion of L-[3H]-arginine to L-[3H]-citrulline. Here, we use 100 µl of total incubation volume, which contain 10 µl of a recombinant nNOS (specific activity 21.05 nmol/min/mg protein) added to a buffer solution with a concentration of 0.025 M TriseHCl, 0.001 M DTT, 0.004 mM of H4-biopterin, 0.1 mM CaCl2, 0.5 mM inosine, 0.01 mM of FAD, 0.5 mg/ml BSA, 10 µM L-arginine, and, 10 µg/mL calmodulin (only for nNOS) and 50 nM L-[³H]-arginine at pH 7.0 and 7.6 for nNOS and iNOS, respectively. In ethanol, 10 mL of 0.75 mM NADPH and 10 mL of each quinazolinyl derivative were added to 1 mM final concentration of (20 %). The tubes were incubated and vortexed for 30 min at 37 °C. The absence of NADPH was used to create control incubations. The process was ended by adding up 400 mL of cold 0.1 M HEPES, 0.175 mg/ml L-citrulline pH 5.5 and, 10 mM EGTA. The reaction mixture was poured into a 2 mL column package and washed with 1.2 mL water using Dowex-50 W ion-exchange resin (Na + form). liquid scintillation spectroscopy was utilized for determination of L-[3H]-citrulline. In this technique, L-[3H]-arginine was maintained at a rate of more than 98 percent. To determine particular enzyme activity, the control value, which was often less than 1% of the radioactivity administered, was removed. Picomoles of L-[3H]-citrulline produced per milligram of protein per minute were used to measure nNOS activity. The technique for detecting iNOS activity was very identical to that for determining nNOS activity, with the exception that instead of nNOS, an aliquot of 10 L recombinant iNOS was used, and the mixture was incubated without calmodulin.

3.4 Statistical analysis

The statistics data are labelled as a mean with SEM. The Newmane-Keuls multiple scale analysis was employed after the one-way analysis of variance. Statistical significance was defined as AP value of less than 0.05.