Aryl Itaconic Acids from Aryl Aldehydes and (Triphenylphosphoranylidene)succinic Anhydride via a One-Pot Ring-Opening–Wittig Olefination–Hydrolysis Reaction ^†

Abstract

(Triphenylphosphoranylidene)succinic anhydride, which is prepared from triphenylphosphine and maleic anhydride and is itself not reactive towards aryl aldehydes, is ring-opened with methanol to methyl (triphenylphosphoranylidene)succinate. In one pot, the newly formed phosphorane is reacted with aryl aldehydes to methyl aryl itaconates, which are subsequently hydrolyzed with aqueous sodium hydroxide to aryl itaconic acids. The biological activity of 12 aryl itaconic acids thus prepared against four gram-positive and four gram-negative bacterial strains has been studied.

1. Introduction

Itaconic acid (1) (Figure 1) is a fatty acid that is produced by macrophages and monocytes in various organisms under stress response, as well as by certain myeloid-derived suppressor cells [1]. It is a side product of the Krebs cycle. Thus, some cells, upon experiencing stress, suppress the tricarboxylic acid cycle, and this is when the metabolite cis-aconitate starts accumulating. The enzyme aconitate decarboxylase (ACOD1) metabolizes the excess cis-aconitate to itaconate. Itaconic acid and some of its ester derivatives are known as immunoregulators, limiting inflammation. Also, the compounds have an effect on bacterial and viral infections.

Both E- and Z-isomers of phenyl itaconic acid (2a) (Figure 1) have been isolated as metabolites from bacterial strains such as Azoarcus tolulyticus [2]. Also, E-phenyl itaconic acid has been found to be a constituent of Artemisia argyi (Levl and Vant) [3]. Aryl itaconic acids are usually prepared via a Stobbe-type reaction of aryl aldehydes with dialkyl succinate [4,5] with subsequent ester hydrolysis. The products of the Stobbe-type reaction are often Z-configurated, but mixtures of E- and Z-configurated compounds are also known. In the following study, a Wittig route to E-aryl itaconic acids was pursued. The biological activities of the E-aryl itaconic acids were assayed, initially against four gram-positive and four gram-negative bacterial strains.

2. Materials and Methods

2.1. General—Chemical Preparation

Infrared spectroscopy was carried out on a Perkin Elmer Spectrum Two FT-IR spectrometer. Samples were measured as KBr pellets. ¹H and ¹³C NMR spectra were recorded with a Varian 400 NMR (¹H at 395.7 MHz, ¹³C at 100.5 MHz) spectrometer in DMSO-d⁶ and CDCl₃ as solvents. The chemical shifts (δ) were reported in ppm and were referenced to the residual protonated solvent (e.g., δ = 2.49 ppm for DMSO-d⁶). Coupling constants (J) are given in Hz. Proton multiplicity was assigned using the following abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Additionally, the abbreviation for broad (br) was used. Mass spectra were measured on a Shimadzu LCMS-8045 with an electrospray ionization (ESI) ion source, using a mobile phase of acetonitrile and 0.1% formic acid buffer in a 70/30 ratio, with a flow rate of 0.5 mL/min under isocratic elution, where a C18 column (150 × 2 mm) was used. Column chromatography was carried out on recycled [6] silica gel (60 Å, 230–400 mesh, Sigma-Aldrich). Analytical thin layer chromatography (TLC) was carried out on silica on TLC Alu foils from Fluka (with fluorescent indicator at λ = 254 nm).

Aryl aldehydes, alkyl iodides (CH₃I and C₂H₅I), triphenyl phosphine, maleic anhydride, conc. hydrochloric acid, methanol, ethanol, acetone, sodium methoxide, sodium hydroxide, and potassium hydroxide were acquired commercially, and used without further purification. First, 2-methoxybenzaldehyde (from salicylaldehyde), 3-ethoxy-4-methoxybenzalde-hyde (from 3-hydroxy-4-methoxybenzaldehyde), 2,4-dimethoxybenzaldehyde (from 2,4-dihydroxybenzaldehyde), and 4-ethoxy-3-methoxybenzaldehyde (from 4-hydroxy-3-methoxybenzaldehyde [vanillin]) were prepared via alkylation of the respective phenols (KOH, CH₃I or C₂H₅I, DMSO), analogous to a known procedure [7].

Triphenylphosphoranylidene)succinic anhydride (5) was obtained from the reaction of triphenylphosphine (3) and freshly sublimated maleic anhydride (4) in acetone as the solvent, according to a known procedure (Scheme 1) [8].

2.2. Experimental—Chemical Synthesis

Procedure: (E)-2-(2,5-dimethoxyphenyl)methylenebutane-1,4-doic acid (2b). Phosphorane (5) (2.50 g, 6.94 mmol) was stirred in MeOH (20 mL) at rt for 10 h. Thereafter, the red–orange solution was cooled to 0 °C, and solid sodium methoxide (NaOMe, 390 mg, 7.22 mmol) and immediately thereafter 2,5-dimethoxybenzaldehyde (9b, 1.13 g, 6.80 mmol) were added. The resulting solution was stirred at rt for 1.5 h, thereafter it was stirred under reflux for 6 h. The solution was re-cooled to rt, and a solution of aq. NaOH (2.0 g in 30 mL H₂O) was added. The resulting mixture was stirred for 3 h at 65 °C. Thereafter, the mixture was cooled to rt and extracted with CH₂Cl₂ (3 × 20 mL). The basic aq. phase was cooled to 0 °C and acidified with half conc. HCl. The crystallization of the product sets in with the acidified phase cooled to 0 °C. Crystallization can continue for an extended amount of time. The last two purification steps should be carried out at speed, otherwise crystallization can occur in the organic phase, as well as in the interphase. In products where crystallization does not occur readily, the acidified organic phase can be extracted with CH₂Cl₂. Then, the organic phase is dried over MgSO₄ and concentrated in vacuo. 2b (760 mg, 42%) is obtained as a colorless solid, mp. 175–176 °C [Lit. 173–175 °C [9]]; IR (KBr, ν_max) 3450–2550 (OH), 2921, 2851, 1702, 1680, 1504, 1233, 1221, 1048, 1019 cm⁻¹. ¹H NMR (400 MHz, DMSO-d⁶) δ 12.46 (br, 2H), 7.71 (s, 1H), 6.97 (d, 1H, ³J = 8.8 Hz), 6.93 (dd, 1H, ³J = 8.8 Hz, ⁴J = 2.8 Hz), 6.80 (d, 1H, ⁴J = 2.8 Hz), 3.71 (s, 3H, OCH₃), 3.65 (s, 3H), 3.25 (s, 2H, CH₂) ppm. ¹³C NMR (100 MHz, DMSO-d⁶) δ 172.8 (CO₂H), 168.7 (CO₂H), 153.0, 151.7, 136.3, 127.7, 124.4, 115.6, 115.1, 112.5, 56.2 (OCH₃), 55.7 (OCH₃), 34.1 (CH₂) ppm. MS (m/z, ESI) 265 [M-H]⁺.

2.3. Antibacterial Assay—Methodology

All the bacterial cultures were obtained from the microbial collection center, IMTECH Chandigarh, India. Active cultures of the four gram-positive bacteria Staphylococcus aureus MTCC3160, Staphylococcus lentus MTCC 2292, Bacillus cereus MTCC6629, Bacillus subtilis MTCC1305, and the four gram-negative bacteria Pseudomonas aeruginosa MTCC1748, Pseudomonas putida MTCC 2492, Escherichia coli MTCC 1554, and Klebsiella pneumoniae MTCC 3040 were used. Streptomycin (50 µg/mL) was used as a standard.

2.4. Antibacterial Assay—Experimental

A single bacterial colony of a pure culture was transferred to a 150 mL conical flask containing 50 mL nutrient broth media and incubated at 37 °C for 8–12 h. All the samples were dissolved in 1 mL DMSO and made into aliquots of different concentrations for minimum inhibitory concentration (MIC) assay. The antibacterial assay was carried out by performing the swab streak method. For this, nutrient agar media were prepared and sterilized at 121 °C with 15 lbs pressure for 15 min. The sterile media were poured into petri dishes and allowed to solidify. A sterile cotton swab was taken, and the culture was uniformly spread onto the nutrient agar surface. Active bacterial cultures were taken and 100 µL of culture were added onto the agar surface. After the plates were solidified, wells were made using sterile well borer, and samples were loaded, 100 µL each, into the wells, respectively. Plates were incubated at 37 °C for 18–24 h in a bacterial incubator. The bacterial plates were observed after the incubation period.

3. Results and Discussion

3.1. Wittig Olefination Preparation of Aryl Itaconic Acids

(Triphenylphosphoranylidene)succinic anhydride (5) can prepared easily via the reaction of triphenylphosphine (PPh₃, 3) with maleic anhydride (4) (Scheme 1) [8]. 5 undergoes the Wittig olefination reaction only with the most reactive ketones and aldehydes, such as chloral [8]. It does, however, ring-open in the presence of alcohols, to provide 7, such as 7a (Scheme 2). Adding a base such as sodium methoxide to 7a creates phosphorane 8a. 8a undergoes a Wittig reaction when aryl aldehydes (9) are present, to give monoalkyl aryl itaconates as E-isomers, which can be subjected directly to base-catalyzed hydrolysis by the addition of either aq. NaOH or aq. KOH, providing (E)-aryl itaconic acids (2) in one pot from maleic anhydride (4) (Scheme 3). Here, it needs to be said that 7 is prone to a thermal fragmentation reaction to acrylates, which has been used in the synthesis of alkyl acrylates from maleic anhydride in the presence of triphenylphosphine (3) and an alcohol (6) [10] (Scheme 4), and in the synthesis of Michael adducts of such acrylates with aryl thiols 11, such as in the synthesis of aryl 3-thioarylthiopropionate 12 (Scheme 5) [11]. It is because of this that the addition of sodium methoxide to 7a in the featured reaction (Scheme 3) has to be performed carefully at 0 °C. Thereafter, the reaction is stirred at rt and only at the end is the temperature raised to 65 °C for 6 h. Hydrolysis of the formed methyl aryl itaconates is carried out in situ, with the addition of an aq. solution of NaOH or KOH. For the purification of the product, the reaction mixture is diluted with water, and all side products, including triphenylphosphine oxide, are extracted with CH₂Cl₂. Thereafter, the aq. phase is carefully acidified, where in most instances, the aryl itaconic acids will crystallize slowly. Where this does not happen, the aqueous phase is extracted with CH₂Cl₂ and the organic phase is dried over MgSO₄ and subsequently evaporated, to give the solid aryl itaconic acids, which can be washed with a small amount of diethyl ether. The aryl itaconic acids thus obtained with their respective yields are shown in Figure 2.

3.2. Antibacterial Activity of the Synthesized Compounds

The impact of 12 synthesized aryl itaconic acids on four gram-positive bacterial pathogen S. aureus, S. lentus, B. cereus, and B. subtilis was studied using the swab streak method. After the incubation period, the bacterial plates were observed. 2-(3-Ethoxy-4-methoxyphenyl)methylenebutane-1,4-dioic acid (E-2g) and 2-(2-bromophenyl)methylenebutane-1,4-dioic acid (E-2j) showed activity against all four gram-positive bacterial pathogens, while 2-(2,5-dimethoxyphenyl)methylenebutane-1,4-dioic acid (E-2b) and 2-(2,3-dimethoxyphenyl)methylenebutane-1,4-dioic acid (E-2e) showed activity against only two pathogen strains: S. aureus and S. lentus. The results are summarized in

Table 1. Antibacterial activity of compounds 2a–2L against gram positive pathogens.

Scheme	Sample Name	Gram Positive Bacterial Pathogens
		S. aureus	S. lentus	B. cereus	B. subtilis
1	(E)-2a	No activity	No activity	No activity	No activity
2	(E)-2b	10 mm	10 mm	No activity	No activity
3	(E)-2c	No activity	No activity	No activity	No activity
4	(E)-2d	No activity	No activity	No activity	No activity
5	(E)-2e	10 mm	12 mm	No activity	No activity
6	(E)-2f	No activity	No activity	No activity	No activity
7	(E)-2g	10 mm	10 mm	08 mm	08 mm
8	(E)-2h	No activity	No activity	No activity	No activity
9	(E)-2i	08 mm	08 mm	08 mm	10 mm
10	(E)-2j	No activity	No activity	No activity	No activity
11	(E)-2k	No activity	No activity	No activity	No activity
12	(E)-2L	No activity	No activity	No activity	No activity
13	Standard	14 mm	14 mm	14 mm	16 mm

and Figure 3.

The activity study for gram negative bacterial pathogens showed a similar pattern to the gram positive pathogens but varies for compound (E)-2L. 2-(2-Ethoxyphenyl)methylenebutane-1,4-dioic acid [(E)-2L] showed activity against E. coli and K. pneumoniae. The results are summarized in

Table 2. Antibacterial activity of compounds 2a-2L against gram negative pathogens.

S.no	Sample Name	Gram Negative Bacterial Pathogens
		P. aeruginosa	P. putida	E. coli	K. pneumoniae
1	(E)-2a	No activity	No activity	No activity	No activity
2	(E)-2b	12 mm	10 mm	No activity	No activity
3	(E)-2c	No activity	No activity	No activity	No activity
4	(E)-2d	No activity	No activity	No activity	No activity
5	(E)-2e	12 mm	14 mm	No activity	No activity
6	(E)-2f	No activity	No activity	No activity	No activity
7	(E)-2g	10 mm	10 mm	08 mm	10 mm
8	(E)-2h	No activity	No activity	No activity	No activity
9	(E)-2i	10 mm	12 mm	10 mm	10 mm
10	(E)-2j	No activity	No activity	No activity	No activity
11	(E)-2k	No activity	No activity	No activity	No activity
12	(E)-2L	No activity	No activity	10 mm	10 mm
13	Standard	14 mm	14 mm	14 mm	16 mm

and Figure 4.

3.3. Minimum Inhibitory Concentration (MIC) Assay of Compounds Against Gram Positive and Gram-Negative Bacteria

The samples tested for antibacterial activity were assayed for their minimum inhibitory concentrations (MIC) by loading 25 µL, 50 µL, 75 µL, 100 µL of samples diluted with DMSO to a final volume of 100 µL in each well of the plate. In this assay, 10 mg/mL samples were used, and appropriate dilutions were made. The MIC assay concentration for each compound against gram positive bacteria are summarized in

Table 3. Minimum inhibitory concentration (MIC) assay of compounds against four gram-positive bacterial pathogens.

S.no	Sample ID	MINIMUM INHIBITORY CONCENTRATION—MIC (mm)				MIC of Sample (µL)
		S. aureus
		25 µL	50 µL	75 µL	100 µL
1	(E)-2b	-	-	08 mm	10 mm	75 µL
2	(E)-2e	-	-	12 mm	14 mm	75 µL
3	(E)-2g	-	-	10 mm	14 mm	75 µL
4	(E)-2i	-	-	08 mm	10 mm	75 µL
		S. lentus
1	(E)-2b	-	08 mm	10 mm	12 mm	50 µL
2	(E)-2e	-	-	08 mm	12 mm	75 µL
3	(E)-2g	-	-	08 mm	12 mm	75 µL
4	(E)-2i	-	08 mm	10 mm	12 mm	50 µL
		B. cereus
1	(E)-2g	-	-	-	10 mm	100 µL
2	(E)-2i	-	-	-	10 mm	100 µL
		B. subtilis
1	(E)-2g	-	-	-	10 mm	100 µL
2	(E)-2i	-	-	-	08 mm	100 µL

and Figure 5 and for gram—negative bacterial pathogens in

Table 4. Minimum inhibitory concentration (MIC) assay of compounds against four-gram negative bacterial pathogens.

S.no	Sample ID	MINIMUM INHIBITORY CONCENTRATION—MIC (mm)				MIC of Sample (µL)
		P. aeruginosa
		25 µL	50 µL	75 µL	100 µL
1	(E)-2b	-	-	08 mm	10 mm	75 µL
2	(E)-2e	-	-	08 mm	10 mm	75 µL
3	(E)-2g	-	-	08 mm	10 mm	75 µL
4	(E)-2i	-	-	08 mm	10 mm	75 µL
		P. putida
1	(E)-2b	-	-	08 mm	12 mm	75 µL
2	(E)-2e	-	-	10 mm	12 mm	75 µL
3	(E)-2g	-	-	08 mm	12 mm	75 µL
4	(E)-2i	-	-	08 mm	12 mm	75 µL
		E. coli
1	(E)-2g	-	-	08 mm	10 mm	75 µL
2	(E)-2i	-	-	08 mm	12 mm	75 µL
3	(E)-2L	-	-	08 mm	12 mm	75 µL
		K. pneumoniae
1	(E)-2g	-	-	08 mm	10 mm	75 µL
2	(E)-2i	-	-	10 mm	12 mm	75 µL
3	(E)-2L	-	-	08 mm	12 mm	75 µL

and Figure 6.

4. Conclusions

Twelve aryl itaconic acids (2) were synthesized via a one-pot ring-opening–Wittig olefination–hydrolysis reaction. Among them, 2-(3-ethoxy-4-methoxyphenyl) methylenebutane-1,4-dioic acid (E-2g) and 2-(2-bromophenyl) methylenebutane-1,4-dioic acid (E-2j) demonstrated activity against gram-positive bacteria, including S. aureus, S. lentus, B. cereus, and B. subtilis. Furthermore, 2-(2,5-dimethoxyphenyl) methylenebutane-1,4-dioic acid (E-2b) and 2-(2,3-dimethoxyphenyl) methylenebutane-1,4-dioic acid (E-2e) exhibited activity against S. aureus and S. lentus only. Notably, these compounds also showed activity against gram-negative bacteria, although the activity varied for (E)-2L. Specifically, 2-(2-ethoxyphenyl) methylenebutane-1,4-dioic acid [(E)-2L] was effective against E. coli and K. pneumoniae. The minimum inhibitory concentration (MIC) assay was also conducted to evaluate the potency of these compounds.