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. 2023 Feb 8;8(7):6234–6243. doi: 10.1021/acsomega.2c05390

Bio-Oriented Synthesis of Novel Polyhydroquinoline Derivatives as α-Glucosidase Inhibitor for Management of Diabetes

Faiz Talab †, Saeed Ullah ‡, Aftab Alam †, Sobia Ahsan Halim ‡, Najeeb Ur Rehman ‡, Zainab §, Mumtaz Ali †,*, Abdul Latif †, Ajmal Khan ‡,*, Ahmed Al-Harrasi ‡,*, Manzoor Ahmad †,*
PMCID: PMC9948207  PMID: 36844517

Abstract

graphic file with name ao2c05390_0007.jpg

Polyhydroquinoline derivatives (1-15) were synthesized through an unsymmetrical Hantzsch reaction in excellent yields by treating 3,5-dibromo-4-hydroxybenzaldehyde, dimedone, ammonium acetate, and ethyl acetoacetate in ethanol solvent. The structures of the synthesized compounds (1-15) were deduced through different spectroscopic techniques such as 1H NMR, 13C NMR, and HR–ESI–MS. The synthesized products were tested for their α-glucosidase inhibitory activity where compounds 11 (IC50= 0.56 ± 0.01 μM), 10 (IC50= 0.94 ± 0.01 μM), 4 (IC50= 1.47 ± 0.01 μM), 2 (IC50= 2.20 ± 0.03 μM), 6 (IC50= 2.20 ± 0.03 μM), 12 (IC50= 2.22 ± 0.07 μM), 7 (IC50= 2.76 ± 0.04 μM), 9 (IC50= 2.78 ± 0.03 μM), and 3 (IC50= 2.88 ± 0.05 μM) exhibited high potential for the inhibition of α-glucosidase, while the rest of the compounds (8, 5, 14, 15, and 13) showed significant α-glucosidase inhibitory potential with IC50 values of 3.13 ± 0.10, 3.34 ± 0.06, 4.27 ± 0.13, 6.34 ± 0.15, and 21.37 ± 0.61 μM, respectively. Among the synthesized series, two compounds, i.e., 11 and 10, showed potent α-glucosidase inhibitory potential higher than the standard. All the compounds were compared with standard drug “acarbose” (IC50 = 873.34 ± 1.67 μM). An in silico method was used to predict their mode of binding within the active site of enzyme to understand their mechanism of inhibition. Our in silico observation complements with the experimental results.

1. Introduction

Compounds containing at least one ring member other than carbon are termed as heterocyclic compounds. Over the past two decades, studies of heterocyclic compounds show promising results in the field of pharmaceuticals, biochemistry, and agriculture chemistry.1 In this context, 3, 4, 5, 6, and 7 membered heterocyclic compounds are known up-to-date, and their biological characteristics had been evaluated; the chemical world is also familiar with fused heterocyclic compounds.2 Nitrogen-containing heterocycles have gained a great consideration owing to their certain role in medicinal chemistry, as they are extensively studied in biological processes comprising investigations on anti-diabetic, anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor agents.3 Quinolines are well-known heterocyclic compounds containing a benzene ring attached with pyridine at two adjacent carbon atoms.4 Several alkaloids are members of the quinolone family like quinine, while chloroquine, mefloquine, and amodiaquine (Figure 1) are synthetic quinolines and act as anti-malarial drugs.5

Figure 1.

Figure 1

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Typical anti-malarial drugs.

Polyhydroquinolines are asymmetrical compounds (Hantzsch products) achieved through several approaches, and one of most common approach is by a four-component coupling reaction.6,7 Usually, preparation of polyhydroquinoline requires a four-component one-pot reaction between various substituted aromatic or aliphatic aldehydes, ethylaceto acetate, dimadone, and ammonium acetate that leads to the synthesis of PHQs in the presence of different catalysts such as palladium chloride, l-proline, glycine, nicotinic acid, etc.8 Polyhydroquionlines (PHQ) have a wide range of therapeutic use, such as calcium channel blockers, anti-malarial, anti-diabetic, and anti-cancer agents.9 Apart from this, polyhydroquinoline derivatives are reported for anti-bacterial, anti-asthmatic, anti-inflammatory, and anti-tumor properties.10

The major purpose of organic synthesis and medicinal chemistry is to prepare compounds with high bioactivity. In past the few decades, heterocyclic compounds gained a unique position because of their therapeutic benefits in pharmaceutical chemistry.11,12 Most of the bicyclic compounds and their derivatives bear a wide range of medicinal applications.13 Among heterocycles, the derivatives of PHQ are more crucial in the nitrogen-containing heterocycles due to their promising pharmacological/biological properties. Moreover, these heterocycles have been reported as vasodilator,14 antiatherosclerosis,15 anti-tumor,16 anti-hyperglycemic,17 anti-proliferative,18,19 and antioxidant agents.20,21

α-Glucosidase is found in the brush border surface of the small intestine, which is the key catabolic enzyme of carbohydrates. It breaks down carbohydrates into glucose through the mechanism of hydrolysis, as the human intestine can only absorb glucose in the form of a monomer into the bloodstream.22 The digestion of carbohydrates and the processing of glycoproteins basically depend on the enzymatic action of α-glucosidase. Therefore, type 2 diabetes are directly linked to hyperactivity of α-glucosidase and can be cured by α-glucosidase inhibitors.23 It has been deemed that the synthetic inhibitors of α-glucosidase, including acarbose, miglitol, and voglibose, have attracted widespread attention in the field of medicinal chemistry and have limitations due their side effect. Hence, identification of new candidates has become the immense need for the management of type 2 diabetes.24,25

These α-glucosidase inhibitors slow down the digestion of polysaccharides and thus control the blood glucose level in diabetic patient.26 In contrast to other hypoglycemic agents that regulate certain biochemical processes, these inhibitors act locally in the human intestine.27 However, the clinical uses of these are associated with side effects such as abdominal discomfort, diarrhea, and flatulence.28−30 Therefore, synthesis of new α-glucosidase inhibitors with no or minimal side effects is still a reasonable need.

The objective of this study was to synthesize new polyhydroquinoline derivatives and to evaluate their α-glucosidase inhibitory activity to develop a strategy for the treatment of type 2 diabetes.

2. Results and Discussion

2.1. Chemistry

In nonstop efforts, to create biologically/pharmacologically active and potent glucosidase inhibitors, 15 derivatives (1–15) with a polyhydroquinoline nucleus were synthesized and purified. Initially, polyhydroquinoline was synthesized from the reaction between dimedone, ethyl acetoacetate, 3,5-dibromo-4-hydroxybenzaldehyde, and ammonium acetate in the presence of absolute ethanol. The desired PHQ was dissolved in DMF containing potassium carbonate (K2CO3) and stirred for 40 min, and various aliphatic alkyl halides were added to the reaction mixture and refluxed for 4–5 h (Scheme 1). The completion of the reaction of the synthesized products was checked with the help of thin layer chromatography (TLC) using a 40% solvent system of n-hexane and ethyl acetate (4:6). To get the desired products, all the compounds were recrystallized with ethanol or methanol in good to excellent yield. The compounds were characterized with the help of various spectroscopic techniques (HR–ESI–MS, 1H NMR, and 13C NMR) and finally evaluated for their α-glucosidase inhibitory potential.

Scheme 1. Synthesis of Polyhydroquinoline Derivatives (1–15).

Scheme 1

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2.2. α-Glucosidase Inhibitory Activity and Structure–Activity Relationship

To explore the anti-diabetic potential of quinoline carboxylate derivatives, compounds 2–15 were proceeded to evaluate their inhibitory effect against the main carbohydrates’ digestive enzyme “α-glucosidase” because of its prime importance in diabetes. In this series, all the compounds showed significant inhibitory activities when compared with the standard drug, acarbose (IC50 = 873.34 ± 1.67 μM) (Table 1). The synthesized derivatives of hexahydroquinoline-3-carboxylate indicated that different substituent (R) groups have very strong binding affinity on the pocket of α-glucosidase enzyme, which is clearly demonstrated by the higher potent holding back effect of all derivatives against α-glucosidase. A careful analysis of the structure–activity relationship (SAR) is required to determine the impact of the substituted aliphatic chains (R-group) on the suppression of the α-glucosidase catalytic activity.

Table 1. Inhibition Studies of Polyhydroquinoline Derivatives (2–15)a.

2.2.

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a

SEM = standard error mean, N/A = not active.

To explain our findings, we grouped the target molecules into a hypothetical R-group, which have different substituted alkyl chains at the similar position. For instance, compound 2 with a pentane moiety displayed several folds of potent inhibitory capability against α-glucosidase with IC50 = 2.20 ± 0.03 μM, as compared to acarbose, a marketed drug (IC50 = 873.34 ± 1.67 μM). Similarly, compound 3, which contain a hexane group, exhibited 800 times more potent inhibitory potential against α-glucosidase with an IC50 value of 2.88 ± 0.05 μM. The anti-diabetic potential of compound 4 with the addition of heptane further enhanced the anti-diabetic potency of 4 (IC50 = 1.47 ± 0.01 μM), as compared to compounds 2 and 3. While in compound 5, substitution of the octane group was responsible to slightly decline the anti-diabetic potential (IC50 = 3.34 ± 0.06 μM), as compared to compounds 2–4. Interestingly, compounds 6–9, with different alkyl substituents including C3H3, C3H5, C12H25, and C14H29, respectively, demonstrated close highly potent anti-diabetic capability with IC50 values of 2.20 ± 0.03, 2.76 ± 0.04, 3.13 ± 0.10, and 2.78 ± 0.03 μM, respectively (Figure 2), which might be due to the similar suppressive effect of the attached groups. In contrast, in compound 10, the increase in the length of the alkyl chain (C16H33) interestingly enhanced the α-glucosidase inhibitory activity of this compound (IC50 = 0.94 ± 0.01 μM) and made it the second most potent compound in this series. The enhanced α-glucosidase inhibitory activity with the increase in strength of the R-group was also observed in compound 11 with an octadecane chain (IC50 = 0.56 ± 0.01 μM) and made it the most potent agent in the series as compared to all other α-glucosidase inhibitors reported in the current study. Compound 12, substituted with butane (attached at the second position) showed strong inhibition of α-glucosidase with an IC50 value of 2.22 ± 0.07 μM, as compared to acarbose.

Figure 2.

Figure 2

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Structure–activity relationship of the most potent compounds.

On the other hand, compound 13, with bromo butane substituent (C3H6Br) showed reduced α-glucosidase potential (IC50 = 21.37 ± 0.61 μM), as compared to other compounds in the series. The decline in the α-glucosidase repressive potential might be due to low access of this molecule in the catalytic pocket of enzyme. Unlike 13, the addition of bromo propane group in compound 14 slightly decreased the α-glucosidase inhibitory potential of 14 (IC50 value = 4.27 ± 0.13 μM). However, in compound 15, the reduced strength of the R-substituent (ethyl group) is responsible to slightly weaken the inhibitory activity of this compound (IC50 = 6.34 ± 0.15 μM). It reflects that the longer carbon chain aids the binding of ligands with the enzyme active site or ligand binding pocket. This hypothesis is further confirmed by molecular docking analysis.

The SAR studies explore the key role of the basic skeleton and the R-groups attached in the α-glucosidase inhibition because the strength of the R-group showed slight variations in the inhibitory capability neither drastic variation.

2.3. Docking Studies

All the compounds showed high activity against the α-glucosidase enzyme; therefore, their mode of interaction within the active site was predicted through molecular docking. Acarbose, which is a standard drug of the α-glucosidase, was docked in the active site of the α-glucosidase. The docked orientation of acarbose reflects that the drug mediates hydrogen bonds (H-bonds) with the side chains of Glu277, Asp215, and Asp352, which are residues of the catalytic triad. In addition, the side chains of Asp69, Asp242, and Arg442 also participate in ligand binding through H-bonds. The binding mode of acarbose is given in Figure S1 (Supporting Information). This indicates that binding with any residue of the catalytic triad can enhance the inhibitory activity of molecules. Compounds 11, 10, and 4 were identified as the most active inhibitors with IC50 in range of 0.56 to 1.47 μM. The mode of binding of the most active molecule, 11, reflects that its quinoline amino group donated a H-bond to the side chain of one of the catalytic residues, Asp352, whereas one of its bromine atoms mediated a halogen bond with the side chain of Thr310. In addition, its long alkyl chain is fixed in between several residues including Asp307, Pro312, His280, Ser240, Tyr158, Phe178, Tyr72, Val216, Tyr316, Gln279, and Arg315. Due to these specific interactions, compound 11 exhibited the lowest docking score, i.e., −11.09 kcal/mol. The docked orientation of the second most active hit, compound 10, was similar to the binding mode of 11; however, the quinoline moiety of 10 was tilted toward Phe303, Thr306, Asp307, Arg315, Tyr347, and Gln353. Instead of interacting with the residues of catalytic triad, the amino group of 10 mediated a H-bond with the side chain of Thr307 at 2.30Å, while its alkyl chain adapted the same position as the alkyl chain of compound 11. The docking score of 10 (−10.81 kcal/mol) also reflect its good stability in the active site of the enzyme. The docked conformation of compound 4 was found a bit different than the docked orientation of 10 and 11. The hydroquinoline carboxylate moiety of 4 was tilted 120° from the hydroquinoline moiety of compounds 10 and 11. Because of this conformational difference, the carboxylate of compound 4 formed a H-bond with the side chain of Arg442, whereas its Br atoms formed halogen bonds with Glu277 and Thr306. Meanwhile, the alkyl chain of 4 was fixed in between Phe303, Tyr347, and Asn350. Compound 2 also took a similar conformation like compound 4, and Br atoms of 2 mediated halogen bonding with Glu277 and Asp307; however, its quinoline amino group formed a H-bond with the side chain of Gln279 at 1.93Å and its alkyl chain of fitted between Phe301, Tyr347, Asp352, and Asn350. The docking scores of compounds 4 (−9.37 kcal/mol) and 2 (−7.82 kcal/mol) also reflect that these are good inhibitors; however, their docking scores are less than the docking scores of compounds 10 and 11, which is due to the conformational variation. Similarly, the binding mode of compound 6 was like the docked orientation of compound 2. The quinoline amino of 6 donated a H-bond to the side chain of Gln279 at 2.03Å, whereas it is one of the Br atoms that formed a halogen bond with Asp307, while the butyne moiety did not form any interaction due to its small size; instead, the dibromo-substituted phenyl ring of this compound formed hydrophobic (π–π) interaction with the side chain of Phe303. The docking score of compound 6 (−7.89 kcal/mol) was comparable to the docking score of 2.

The conformational sampling of compound 12 indicates that the compound has taken an entirely different conformation than the other compounds. Unlike other compounds, the hydroquinoline carboxylate moiety of 12 was oriented toward Glu411, Tyr316, Phe314, Arg315, Gln353, and Tyr158. The amino nitrogen and carboxyl group of this molecule formed H-bonds with the side chain of Glu411 and Gln353, respectively. While the bromo-substituted phenyl ring was bent toward the catalytic residues, however, it did not make any interaction. A similar orientation was observed for compound 7, and its amino nitrogen was also H-bonded with the side chain of Glu411. This conformational variation could be a reason of the slightly lower activities of those compounds than compounds 10 and 11. The binding modes of compounds 9 and 3 are completely identical, and their quinoline nitrogen interacts with the side chain of Asp307 through the H-bond, while their alkyl chain was tilted toward the core of the active site (Asp69, Arg213, Asp215, Glu277, His351, Asp352, and Arg442).

Compounds 8, 5, 14, and 15 showed the inhibitory activities with IC50 values in the range of 3.34 to 3.34 μM. The binding mode of compound 8 reflect that its quinoline nitrogen formed a H-bond only with the side chain of Glu277 (2.14Å), whereas several residues including Tyr158, Ser240, Ser311, Asp242, Pro312, Thr310, and His280 surrounds the alkyl chain of this compound. The docked orientation of compound 5 was similar with the binding mode of compound 4; however, the carboxylate of 4 mediated a H-bond with the side chain of Arg442; this H-bond is not seen in compound 5, which may be the reason of lesser inhibitory activity of 5 as compared to compound 4. Whereas, like compound 4, one of the Br atoms of compound 5 also formed a halogen bond with the side chain of Thr306. The binding conformations of compounds 14 and 15 are more similar with the docked conformation of compound 8. Like compound 8, the quinoline nitrogen of 14 and 15 also formed only one H-bond with the side chain of Glu277; however, the only difference between these molecules is their alkyl chain. Due to the small structure, the propoxy and ethoxy groups of 14 and 15, respectively, did not fit well into the groove (comprising of Tyr158, Ser240, Ser311, Asp242, Pro312, Thr310, and His280) where long alkyl chain of 8 was fitted. This may be the reason of the lower activity of compounds 14 and 15. The binding mode of compound 13 was found similar with the docked conformation of compound 4. Likewise, the hydroquinoline-substituted carboxylate of 13 also formed a H-bond with the side chain of Arg442, whereas Br atoms of this compound did not form any interaction.

With the docking analysis, we can conclude that the higher number of alkyl groups in the compounds have a great chance of rotation in the active site; therefore, the compounds with big alkyl chains can enter deep inside the active site and subsequently block the access of substrate in the active site. This is also confirmed with the docking scores of the compounds, which complement well with our experimental results. The results of docking are given in Table 2. The binding modes of all the compounds are shown in Figure 3.

Table 2. Docking Scores and Binding Interactions of Compoundsa.

    interactions
compounds score (kcal/mol) ligand atom receptor atom bond distance (Å)
11 –11.09 N1 OD2-ASP352 HBD 2.45
    Br49 OG1-THR310 halogen 2.66
10 –10.81 N1 OG1-THR306 HBD 2.10
4 –9.37 Br57 OG1-THR306 halogen 2.25
    Br58 OE2-GLU277 halogen 2.84
    O18 NH1-ARG442 HBA 2.93
2 –7.82 N1 OE1-GLN279 HBD 2.80
    Br58 OD1-ASP307 halogen 2.41
    Br59 OE2-GLU277 halogen 2.34
6 –7.89 N1 OE1-GLN279 HBD 2.74
    Br46 OD1-ASP307 halogen 2.55
    6-ring 6-ring-PHE303 π–π 2.90
12 –7.76 N1 OE2-GLU411 HBD 3.02
    O18 NE2-GLN353 HBA 2.19
7 –7.61 N1 OE2-GLU411 HBD 2.50
9 –7.59 N1 OD1-ASP307 HBD 2.41
3 –7.44 N1 OD1-ASP307 HBD 2.56
8 –7.32 N1 OE2-GLU277 HBD 2.32
5 –7.24 Br58 OG1-THR306 halogen 2.34
    6-ring 6-ring-PHE303 π–π 3.03
14 –6.89 N1 OE2-GLU277 HBD 2.72
15 –5.92 N1 OE2-GLU277 HBD 2.84
13 –3.50 O18 NH1-ARG442 HBA 2.90
    6-ring 6-ring-PHE303 π–π 3.14
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a

HBA = hydrogen bond acceptor, HBD = hydrogen bond donor.

Figure 3.

Figure 3

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(A) The docked conformations of all the compounds (depicted in magenta ball and stick model) are shown in the active site of enzyme (presented in yellow surface). (B). The binding interactions of compounds (presented in magenta ball and stick model) are shown with the active site residues (yellow stick model). H-bonds are shown in black dotted lines.

3. Conclusions

In conclusion, a series of polyhydroquinoline was synthesized and structurally characterized through different spectroscopic techniques. Finally, the synthesized compounds were scrutinized for their α-glucosidase inhibitory activity. All the compounds showed significant activity when compared to standard drug acarbose. Compounds 11 (IC50= 0.56 ± 0.01 μM), 10 (IC50= 0.94 ± 0.01 μM), 4 (IC50= 1.47 ± 0.01 μM), 2 (IC50= 2.20 ± 0.03 μM), 6 (IC50= 2.20 ± 0.03 μM), 12 (IC50= 2.22 ± 0.07 μM), 7 (IC50= 2.76 ± 0.04 μM), 9 (IC50= 2.78 ± 0.03 μM), and 3 (IC50= 2.88 ± 0.05 μM) showed highly potent inhibition of α-glucosidase, while compounds 8, 5, 14, 15, and 13 reflected significant inhibitory potential against α-glucosidase with IC50 in range of 3.13 to 21.37 μM. In this study, compounds 10 and 11 are retrieved as the strongest inhibitors of α-glucosidase enzyme. In addition, in silico binding analysis predict that the long alkyl chains in the compounds are favorable to fit well in the active site and, consequently, block the active site for substrate entry and thus exhibit higher inhibition of the enzyme.

4. Experimental Section

4.1. Materials and Methods

All the regents used in this work were of synthetic grade obtained from Merck, BDH, and Sigma-Aldrich (USA). Merck Silica gel 60 F254 aluminum cards were used to performed thin layer chromatography (TLC) on using solvent system n-hexane/ethyl acetate. Optical rotation was measured on an ATAGO, POLAX-2L polarimeter. A Stuart apparatus was used to check the melting points of the synthesized compounds. Modern high-resolution electrospray ionization mass spectrometry (HR–ESI–MS) (Agilent 6530 LC Q-TOF, USA/EU, made in Singapore) was used to determine the masses of the synthesized compounds.

The NMR spectra were recorded on a BRUKER Avance 600 MHz spectrometer (BRUKER, Zürich, Switzerland) using solvent peaks and internal reference (CDCl3, δH: 7.26; δC: 77.2–76.8). Abbreviations used in the discussion of NMR spectra are s: singlet, d: doublet, t: triplet, m: multiplet, J: coupling constant (in Hz), and δ for chemical shifts in parts per million (ppm).

The main features of the final products (2–16) in their 1H NMR spectra included protons of the methyl group −CH3) at δ 0.96–2.35, aromatic protons appearing at 7.37, methyne proton (−CH) appearing at 4.92, and −NH proton at 5.96 ppm. The HR-ESIMS (positive) of the synthesized products showed (M + H)+ molecular ion peaks and various fragment peaks of the compounds. The spectra confirmed the structures and masses of the synthesized compounds and details of spectral interpretation are given in experimental section. After confirmation of structures of the compounds through 1H & 13C NMR and HR-ESIMS and melting point determinations; they were evaluated for anti-microbial activity.

4.2. Synthesis of Ethyl-4-(3,5-dibromo-4-hydroxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (1)

In a 100 mL round bottom (RB) flask, a mixture of 3,5-dibromo-4-hydroxy benzaldehyde (7.1448 mmole, 2 g), dimedone (7.1448 mmole, 1 g), ethyl acetoacetate (7.1448 mmole, 0.929 g), and ammonium acetate (14.28 mmole, 1.1 g) was refluxed for 6–8 h. The product formation was checked through thin layer chromatography (TLC) in a solvent system containing n-hexane and ethyl acetate (6:4). After completion, the reaction mixture was cooled to room temperature and poured into ice cold distilled water; precipitates were formed, filtered, and washed with distilled water, dried under air, weighed, and collected for further reactions.

4.3. General Procedure for the Synthesis of Ethers of Polyhydroquinoline (PHQ) (2–15)

Fourteen (2-15) derivatives of polyhydroquinoline were synthesized from ethyl 4-(3,5-dibromo-4-hydroxyphenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate 1 using the standard protocol. Compound 1 was dissolved in DMF containing equimolar K2CO3 and stirred for 40 min. After stirring, various aliphatic alkyl halides (Table 2) were added to the reaction mixture and refluxed for 4–5 h. The reaction progress was confirmed using thin layer chromatography having n-hexane and ethyl acetate (6:4). After completion of the reaction, the mixture was cooled to room temperature and poured onto crushed ice. Precipitates were formed, filtered, washed with distilled water, dried under air, weighed, and collected. In some cases, precipitates were not formed, and the reaction mixture was extracted with ethyl acetate to obtain pure product. [α]D25 =

4.4. Spectral Data of the Synthesized Compounds (1–15)

4.4.1. Ethyl-4-(3,5-dibromo-4-hydroxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (1)

White solid, yield: 87%; m.p. 156–157 °C; [α]D25 – 0.35 (C = 10 mg, MeOH) 1H NMR: (600 MHz, CDCl3; δ in ppm): 9.59 (s, 1H, −OH), 7.37 (s, 2H, Ar–H, H-2′, H-6′), 6.12 (s, 1H, H-1), 4.92 (s, 1H, H-4), 3.91 (m, 2H, −O–CH2–CH3), 2.33 (s, 3H, CH3), 2.20 (m, 4H, H-6, H-8), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.05 (s, 3H, CH3), 0.95 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3 δ (ppm): 195.4 (C-5), 167.0 (C=O), 151.4 (C-2), 139.2 (C-1′), 145.3 (C-10), 144.0 (C-4′), 134.2 (C-2′, C-6′), 112.2 (C-9), 111.2 (C-3′, C-5′), 105.2 (C-3), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.0 (C-8), 36.0 (C-4), 32.8 (C-7), 29.3 (CH3), 27.3 (CH3), 19.5 (CH3), 14.2 (−O–CH2–CH3). HR-EIMS (ESI+): found [M + H]+: 514.0523; C21H23Br2NO4.

4.4.2. Ethyl-4-(3,5-dibromo-4-(pentyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (2)

Off white solid, yield: 80%; m.p. 161–162 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 6.19 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.00 (m, 2H, H-2″), 1.80 (m, 2H, H-3″), 1.45 (m, 2H, H-4″), 3.91 (m, 2H, −O–CH2–CH3), 2.33 (s, 3H, CH3), 2.31–2.14 (m, 4H, H-6, H-8), 1.35 (m, 2H, H-5″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.05 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.90 (t, J = 7.2 Hz, 3H, H-6″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.4 (C-5), 167.0 (C=O), 151.4 (C-2), 148.5 (C-4′), 145.3 (C-10), 144.0 (C-1′), 132.2 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.2 (C-9), 105.2 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.0 (C-8), 36.0 (C-4), 32.8 (C-7), 29.7 (C-3″), 29.3 (CH3), 28.0 (C-4″), 27.3 (CH3), 22.5 (C-5″), 19.5 (CH3), 14.2 (−O–CH2–CH3), 14.0 (C-6″). HR-EIMS (ESI+): found [M + H]+: 583.0586; C26H33Br2NO4.

4.4.3. Ethyl-4-(3,5-dibromo-4-(hexyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3)

White solid, yield: 85%; m.p. 156–157 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 6.00 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.12–4.00 (m, 2H, H-2″), 3.93 (m, 2H, −O–CH2–CH3), 2.34 (s, 3H, CH3), 2.32–2.15 (m, 4H, H-6, H-8), 1.82 (m, 2H, H-3″), 1.62 (br. s, 2H, H-4″), 1.49 (m, 4H, H-5″, H-6″), 1.48 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.05 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.94 (t, J = 7.2 Hz, 3H, H-7″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.3 (C-5), 166.9 (C=O), 151.4 (C-2), 148.3 (C-4′), 145.4 (C-10), 143.9 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.3 (C-9), 105.3 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.1 (C-8), 36.0 (C-4), 32.8 (C-7), 31.7 (C-3″), 30.0 (C-4″), 29.3 (CH3), 27.3 (CH3), 25.5 (C-5″), 22.6 (C-6″), 19.6 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-7″). HR-EIMS (ESI+): found [M + H]+: 596.0842; C27H35Br2NO4.

4.4.4. Ethyl-4-(3,5-dibromo-4-(heptyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (4)

White solid, yield: 85%; m.p. 151–152 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 5.98 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.03 (m, 2H, H-2″), 4.02 (m, 2H, −O–CH2–CH3), 2.35 (s, 3H, CH3), 2.32–2.15 (m, 4H, H-6, H-8), 1.80 (m, 2H, H-3″), 1.47 (m, 2H, H-4″), 1.34 (m, 2H, H-5″), 1.28 (m, 4H, H-6″, H-7″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.86 (t, J = 7.2 Hz, 3H, H-8″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.3 (C-5), 166.9 (C=O), 151.4 (C-2), 148.3 (C-4′), 145.2 (C-10), 143.9 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.3 (C-3), 105.3 (C-9), 73.4 (C-2″), 60.0 −O–CH2–CH3), 50.6 (C-6), 41.1 (C-8), 36.0 (C-4), 32.8 (C-7), 31.8 (C-3″), 30.0 (C-4″), 29.3 (CH3), 27.3 (CH3), 29.1 (C-5″), 25.8 (C-6″), 22.6 (C-7″), 19.6 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-8″). HR-EIMS (ESI+): found [M + H]+: 610.0938; C28H37Br2NO4.

4.4.5. Ethyl-4-(3,5-dibromo-4-(octyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (5)

Off white solid, yield: 90%; m.p. 145–146 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 5.98 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.10–4.00 (m, 2H, H-2″), 3.89 (m, 2H, −O–CH2–CH3), 2.34 (s, 3H, CH3), 2.31–2.14 (m, 4H, H-6, H-8), 1.79 (m, 2H, H-3″), 1.46 (m, 2H, H-4″), 1.30 (m, 8H, H-5″, H-6″, H-7″, H-8″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.05 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.85 (t, J = 6.6 Hz, 3H, H-9″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.5 (C-5), 167.0 (C=O), 151.4 (C-2), 148.8 (C-4′), 145.2 (C-10), 144.1 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.1 (C-9), 105.2 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 40.9 (C-8), 36.0 (C-4), 32.8 (C-7), 31.8 (C-3″), 30.0 (C-4″), 29.4 (C-5″), 29.3 (C-6″), 29.2 (CH3), 27.2 (CH3), 25.9 (C-7″), 22.6 (C-8″), 19.4 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-9″). HR-EIMS (ESI+): found [M + H]+: 624.0661; C29H39Br2NO4.

4.4.6. Ethyl-4-(3,5-dibromo-4-(prop-2-yn-1-yloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (6)

White solid, yield: 78%; m.p. 162–163 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.39 (s, 2H, Ar–H, H-2′, H-6′), 5.92 (s, 1H, H-1), 4.94 (s, 1H, H-4), 4.63 (m, 2H, −O–CH2–CH3), 2.51 (br. s, 1H, H-3″), 2.35 (s, 3H, CH3), 2.33–2.15 (s, 4H, H-6, H-8), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3 δ (ppm): 195.3 (C-5), 166.8 (C=O), 150.1 (C-4′), 148.3 (C-2), 146.2 (C-10), 144.0 (C-1′), 132.4 (C-2′, C-6′), 117.7 (C-3′, C-5′), 111.2 (C-9), 105.2 (C-3), 78.2 (C-2″), 60.4 (−O–CH2–CH3), 50.6 (C-6), 48.5 (C-3″), 41.1 (C-8), 36.2 (C-4), 32.8 (C-7), 29.3 (CH3), 27.3 (CH3), 19.6 (CH3), 14.2 (−O–CH2–CH3). HR-EIMS (ESI+): found [M + H]+: 550.0111; C24H23Br2NO4.

4.4.7. Ethyl-4-(4-(allyloxy)-3,5-dibromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (7)

white solid, Yield: 88%; m.p. 165–166 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.39 (s, 2H, Ar–H, H-2′, H-6′), 6.12 (s, 1H, H-1), 6.07 (m, 1H, H-3″), 5.42 (d, J = 16.8 Hz, 1H, H-4″), 5.25 (d, J = 11.4 Hz, 1H, H-4″), 4.93 (s, 1H, H-4), 4.44 (m, 2H, −O–CH2–CH3), 4.12–4.09 (m, 2H, H-2″), 2.34 (s, 3H, CH3), 2.32–2.15 (m, 4H, H-6, H-8), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3δ (ppm): 195.4 (C-5), 166.9 (C=O), 151.0 (C-2), 148.4 (C-4′), 145.6 (C-10), 144.0 (C-1′), 133.2 (C-3″), 132.3 (C-2′, C-6′), 118.3 (C-4″), 117.7 (C-3′, C-5′), 111.2 (C-9), 105.2 (C-5), 73.9 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.1 (C-8), 36.0 (C-4), 32.8 (C-7), 29.3 (CH3), 27.3 (CH3), 19.5 (CH3), 14.2 (−O–CH2–CH3). HR-EIMS (ESI+): found [M + H]+: 552.0309; C24H27Br2NO4.

4.4.8. Ethyl-4-(3,5-dibromo-4-(dodecyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahyd roquinoline-3-carboxylate (8)

White solid, yield: 70%; m.p. 163–164 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 5.96 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.03 (m, 2H, H-2″), 4.01 (m, 2H, −O–CH2–CH3), 2.35 (s, 3H, CH3), 2.32–2.15 (m, 4H, H-6, H-8), 1.79 (m, 2H, H-3″), 1.46 (m, 2H, H-4″), 1.33 (m, 2H, H-5″), 1.27 (m, 14H, H-6″-H-11″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.85 (t, J = 7.2 Hz, 3H, H-13″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.3 (C-5), 166.9 (C=O), 151.4 (C-2), 148.2 (C-4′), 145.2 (C-10), 143.9 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.3 (C-9), 105.3 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.1 (C-8), 36.2 (C-4), 32.8 (C-7), 31.9 (C-11″), 30.0 (C-3″), 29.7 (C-5″), 29.6 (C-6″, C-7″), 29.5 (C-8″, C-9″), 29.3 (CH3, C-10), 27.3 (CH3), 25.9 (C-4″), 22.7 (C-12″), 19.6 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-13″). HR-EIMS (ESI+): found [M + H]+: 680.1796; C33H47Br2NO4.

4.4.9. Ethyl-4-(3,5-dibromo-4-(tetradecyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexah ydroquinoline-3-carboxylate (9)

White solid, yield: 75%; m.p. 154–155 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 5.78 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.01 (m, 2H, H-2″), 3.91 (m, 2H, −O–CH2–CH3), 2.36 (s, 3H, CH3), 2.33–2.15 (m, 4H, H-6, H-8), 1.80 (m, 2H, H-3″), 1.46 (m, 2H, H-4″), 1.33 (m, 2H, H-5″), 1.23 (m, 18H, H-6″-H-14″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.07 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.85 (t, J = 7.2 Hz, 3H, H-15″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.3 (C-5), 166.9 (C=O), 151.4 (C-2), 148.2 (C-4′), 145.2 (C-10), 143.9 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.3 (C-9), 105.3 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.2 (C-8), 36.0 (C-4), 32.8 (C-7), 31.9 (C-13″), 30.0 (C-3″), 29.7 (C-5″, C-6″), 29.6 (C-7″, C-8″, C-9″), 29.5 (C-10″, C-11″), 29.4 (C-12″), 29.2 (CH3), 27.3 (CH3), 25.9 (C-4″), 22.7 (C-14″), 19.5 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-15″). HR-EIMS (ESI+): found [M + H]+: 708.2084; C35H51Br2NO4.

4.4.10. Ethyl-4-(3,5-dibromo-4-(hexadecyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (10)

White solid, yield: 77%; m.p. 155–156 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.24 (s, 2H, Ar–H, H-2′, H-6′), 5.70 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.01 (m, 2H, H-2″), 3.90 (m, 2H, −O–CH2–CH3), 2.36 (s, 3H, CH3), 2.33–2.15 (m, 4H, H-6, H-8), 1.80 (m, 2H, H-3″), 1.46 (m, 2H, H-4″), 1.33 (m, 2H, H-5″), 1.23 (m, 22H, H-6″-H-16″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.07 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.85 (t, J = 7.2 Hz, 3H, H-17″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.2 (C-5), 166.9 (C=O), 151.5 (C-2), 147.9 (C-4′), 145.2 (C-10), 143.7 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.4 (C-9), 105.4 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.2 (C-8), 36.0 (C-4), 32.8 (C-7), 31.9 (C-15″), 30.0 (C-3″), 29.7 (C-5″, C-6″), 29.6 (C-7″-C-12″), 29.5 (C-13″), 29.4 (C-14″), 29.3 (CH3), 27.3 (CH3), 25.9 (C-4″), 22.7 (C-16″), 19.6 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-17″). HR-EIMS (ESI+): found [M + H]+: 736.2431; C37H55Br2NO4.

4.4.11. Ethyl-4-(3,5-dibromo-4-(octadecyloxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (11)

White solid, yield: 90%; m.p. 136–137 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 5.85 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.01 (m, 2H, H-2″), 3.91 (m, 2H, −O–CH2–CH3), 2.35 (s, 3H, CH3), 2.32–2.15 (m, 4H, H-6, H-8), 1.81 (m, 2H, H-3″), 1.46 (m, 2H, H-4″), 1.23 (m, 28H, H-5″-H-18″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.85 (t, J = 7.2 Hz, 3H, H-19″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.3 (C-5), 166.9 (C=O), 151.4 (C-2), 148.1 (C-4′), 145.2 (C-10), 143.8 (C-1′), 132.3 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.4 (C-9), 105.3 (C-3), 73.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.1 (C-8), 38.7 (C-17″), 36.0 (C-4), 32.8 (C-7), 30.0 (C-3″), 29.7 (C-5″, C-6″), 29.6 (C-7″-C-14″), 29.4 (CH3), 29.3 (C-16″), 27.3 (CH3), 25.9 (C-4″, C-15″), 22.7 (C-18″), 19.6 (CH3), 14.2 (−O–CH2–CH3), 14.1 (C-19″). HR-EIMS (ESI+): found [M + H]+: 764.2623; C39H59Br2NO4.

4.4.12. Ethyl-4-(3,5-dibromo-4-(sec-butoxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (12)

Brownish yellow solid, yield: 85%; m.p. 164–165 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.38 (s, 2H, Ar–H, H-2′, H-6′), 6.24 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.45–4.01 (m, 1H, H-2″), 4.13–4.02 (m, 2H, −O–CH2–CH3), 2.34 (s, 3H, CH3), 2.30–2.14 (m, 4H, H-6, H-8), 1.82–1.76 (m, 1H, H-3″), 1.69–1.62 (m, 1H, H-3″), 1.23 (d, J = 6.0 Hz, 3H, H-5″), 1.20 (m, 3H, −O–CH2–CH3), 1.05 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.93 (t, J = 7.2 Hz, 3H, H-4″). 13C NMR (150 MHz, CDCl3δ (ppm): 195.4 (C-5), 167.0 (C=O), 150.2 (C-2), 148.5 (C-4′), 144.6 (C-10), 144.1 (C-1′), 132.5 (C-2′, C-6′), 118.2 (C-3′, C-5′), 111.2 (C-9), 105.2 (C-3), 81.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.0 (C-8), 35.9 (C-4), 32.8 (C-7), 29.5 (C-3″), 29.3 (CH3), 27.2 (CH3), 19.5 (H-5″), 19.1 (CH3), 14.2 (−O–CH2–CH3), 9.9 (C-4″). HR-EIMS (ESI+): found [M + H]+: 568.0471; C25H31Br2NO4.

4.4.13. Ethyl-4-(3,5-dibromo-4-(3-bromopropoxy)phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (13)

Off white solid, yield: 82%; m.p. 197–198 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.38 (s, 2H, Ar–H, H-2′, H-6′), 6.10 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.10–4.01 (m, 4H, H-2″, −O–CH2–CH3), 3.67 (t, J = 6.6 Hz, 2H, H-4″), 2.36 (s, 3H, CH3), 2.34–2.14 (m, 6H, H-8, H-3″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3δ (ppm): 195.2 (C-5), 166.8 (C=O), 150.6 (C-2), 148.2 (C-4′), 145.6 (C-10), 143.9 (C-1′), 132.2 (C-2′, C-6′), 117.3 (C-3′, C-5′), 111.0 (C-9), 105.0 (C-3), 70.4 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.7 (C-8), 36.1 (C-4), 33.5 (C-4″), 32.6 (C-7), 30.1 (C-3″), 29.3 (CH3), 27.3 (CH3), 19.5 (CH3), 14.2 (−O–CH2–CH3). HR-EIMS (ESI+): found [M + H]+: 631.9469;C24H28Br3NO4.

4.4.14. Ethyl-4-(3,5-dibromo-4-propoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (14)

Yellowish solid, yield: 75%; m.p. 200–202 °C; [α]D25 – 1.15 (C = 10 mg, MeOH)1H NMR: (600 MHz, CDCl3; δ in ppm): 7.37 (s, 2H, Ar–H, H-2′, H-6′), 6.12 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.11–4.01 (m, 2H, H-2″), 3.89 (m, 2H, −O–CH2–CH3), 2.34 (s, 3H, CH3), 2.32–2.14 (m, 4H, H-6, H-8), 1.83 (m, 2H, H-3″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 1.03 (t, J = 7.2 Hz, 3H, H-4″), 0.96 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3δ (ppm): 195.4 (C-5), 166.9 (C=O), 151.4 (C-2), 148.5 (C-4′), 145.3 (C-10), 143.9 (C-1′), 132.2 (C-2′, C-6′), 117.6 (C-3′, C-5′), 111.2 (C-9), 105.3 (C-3), 74.9 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.0 (C-8), 36.0 (C-4), 32.8 (C-7), 29.3 (CH3), 27.3 (CH3), 23.3 (C-3″), 19.5 (CH3), 14.2 (−O–CH2–CH3), 10.5 (C-4″). HR-EIMS (ESI+): found [M + H]+: 554.0359;C24H29Br2NO4.

4.4.15. Ethyl-4-(3,5-dibromo-4-ethoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydro quinoline-3-carboxylate (15)

White solid, yield: 80%; m.p. 184–185 °C; 1H NMR: (600 MHz, CDCl3; δ in ppm): 7.38 (s, 2H, Ar–H, H-2′, H-6′), 6.09 (s, 1H, H-1), 4.92 (s, 1H, H-4), 4.12–4.00 (m, 4H, H-2″, −O–CH2–CH3), 2.34 (s, 3H, CH3), 2.32–2.15 (m, 4H, H-6, H-8), 1.41 (t, J = 6.6 Hz, 3H, H-3″), 1.20 (t, J = 7.2 Hz, 3H, −O–CH2–CH3), 1.06 (s, 3H, CH3), 0.96 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3δ (ppm): 195.4 (C-5), 166.9 (C=O), 151.4 (C-2), 148.4 (C-4′), 145.3 (C-10), 143.9 (C-1′), 132.2 (C-2′, C-6′), 117.2 (C-3′, C-5′), 111.2 (C-9), 105.3 (C-3), 69.3 (C-2″), 60.0 (−O–CH2–CH3), 50.6 (C-6), 41.1 (C-8), 36.0 (C-4), 32.8 (C-7), 29.3 (CH3), 27.3 (CH3), 19.5 (CH3), 15.5 (−O–CH2–CH3), 14.2 (C-3″). HR-EIMS (ESI+): found [M + H]+: 540.0184; C23H27Br2NO4.

4.5. In Vitro α-Glucosidase Inhibition Assay

The α-glucosidase (E.C.3.2.1.20) enzyme inhibition assay was developed by using 0.1M phosphate buffer (pH 6.8) at 37 °C.31,32 The enzyme (0.2 μ/mL) was incubated in phosphate buffered saline with different concentrations of tested compounds at 37 °C for 15 min. The substrate (0.7 mM, p-nitrophenyl-α-d-glucopyranoside) was then added, and the variation in absorbance at 400 nm was observed for 30 min using a spectrophotometer (xMark microplate spectrophotometer, BIO-RAD). Test compounds were substituted with DMSO-d6 (7.5% final) in the control. Acarbose was used as the standard inhibitor. The % inhibition was calculated by using the following formula:

4.5.

4.6. Molecular docking

Initially, the X-ray crystal structure of Saccharomyces cerevisiae isomaltase in complex with its competitive inhibitor maltose (α-d-glucopyranose) was taken from the RCSB Protein databank with PDB code: 3A4A, and resolution: 1.60 Å. In silico experiment was performed on Molecular Operating Environment (MOE version 2020.0901). Previously,33−35 we scrutinized the docking efficiency of MOE through redocking of maltose in its binding site where MOE proved good performance. In this work, QuickPrep module of MOE was used to prepare the enzyme file before docking. QuickPrep adds missing hydrogen atoms on each residue and calculates partial charges via a predefined force field. We used here an Amber10: EHT force field. The structures of compounds were imported from ChemDraw into MOE database in mol format. In the MOE database, the structures of all compounds were minimized with an RMS gradient of 0.1 RMS kcal/mol/Å, and their partial charges were calculated. For docking, the Triangle Matcher docking algorithm and London dG scoring function were used.

Acknowledgments

The authors are thankful to the Higher Education Research Endowment Fund for financial support, under research project (HED-041). One of the authors is also thankful to The Oman Research Council (TRC) through the Research Grant Program (BFP/RGP/CBS/21/002).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c05390.

  • Binding mode of acarbose (Figure S1); representative numbered structure of the series (Figure S2); mass, 1H NMR and 13C NMR spectra of compounds 2–15 (Figures S3–S16) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao2c05390_si_001.pdf (2.8MB, pdf)

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Supplementary Materials

ao2c05390_si_001.pdf (2.8MB, pdf)

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