Introduction

Benzopyran compounds are broadly characterized by the fusion of a benzene ring to a pyran (oxine) ring. Depending on the saturation and oxidation level within the core structure, benzopyrans may be further classified as derivatives of chroman, 2H-chromene, 4H-chromene, 3-chromanone, 4-chromanone, chromone or 2,4-chromandione [1]. The relative significance of such frameworks was illustrated by a recent review examining the frequency of ring systems in natural products and synthetic compounds, which showed that 4H-chromen-4-one, 4-chromanone and chroman held ranks 3, 14, and 15 respectively [2]. Diverse biological activities have been documented for various benzopyrans, notably chromones, including anti-inflammatory, anti-platelet, anti-cancer, anti-microbial, and anti-obesity effects, and as potential drugs for various neurodegenerative conditions [3, 4]. Chromones have as a result been described as privileged structures [5].

In Nature, benzopyran derivatives are found in various assemblies. The benzopyran unit can be found in diverse metabolites, including coumarins, catechins, and precocenes, and also as base structures for various types of polyphenols, flavonoids, and anthocyanins [1]. Natural products containing the benzopyran skeleton are widely distributed throughout the plant kingdom, but are particularly common in certain families, including the Asteraceae, a family that contains many valuable medicinal species. An interesting group of the Asteraceae is the genus Calea, from which several benzopyran derivatives have been isolated [6]. Species of Calea containing these compounds include Calea serrata [7], C. hispida [8], C. oxylepis [9], C. rotundifolia [10], C. peckii [11], C. clausseniana [12] and C. uniflora [13]. The latter species has a history of ethnomedicinal use in Brazil, primarily as a topical anti-inflammatory [14], and has been the subject of some investigation. Benzopyran derivatives isolated from C. uniflora (Fig. 1) include 2,2-dimethyl-6-(1-hydroxyethyl)-chroman-4-one 1 [13], uniflorol-A 2 [13], uniflorol-B 3 [13], orobol 4 [15], noreugenin 5 [15], quercetin 3-O-glucopyranosyl 6 [15], quercetin-3-O-β-galactopyranoside 7 [16] and 3´,4´,7-trihydroxyflavone-7-O-β-glucopyranoside 8 [16].

Fig. 1
figure 1

Benzopyran derivatives identified in C. uniflora

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Pharmacologic rationale for the application of C. uniflora extracts in inflammatory conditions was evaluated using the carrageenan induced pleurisy model in mice. Leaf alcoholic extracts given i.p. at a dose of 50 mg/kg showed statistically relevant anti-inflammatory activity and reduced cytokine levels [17]. Certain isolated metabolites of C. uniflora were also investigated using the same experimental model, and among these, chromen-4-one 5 (5 mg/kg i.p.) reduced TNF-α, IFN-γ and IL-6 levels, and also phosphorylation of p65 NF-κb and p38 MAPK. These results support the anti-inflammatory activity of both the plant and of isolated compounds. As a group, chromen-4-ones have been reviewed as a source of anti-inflammatory agents, with mechanisms of action encompassing inhibition of enzymes such as cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), and of production of mediators such as interleukin-5 (IL-5), and nitric oxide (NO) [18].

In addition to anti-inflammatory activity, certain Calea metabolites have also been evaluated for anti-parasitic activity, and while compounds 2 and 3 had leishmanicidal [13] activity, compound 4 was trypanocidal [15]. In other work, we have described the anti-leishmanial activity of compounds based on 2 and 3, both chromanones [19] and thiochromanones [20]. We noted that among such compounds, activity was improved with a lipophilic chain at C6, in the form of an amine or amide. Continuing our interest in similar molecular architectures, we designed and synthesized novel benzopyrans inspired by metabolites 1-3 of C. uniflora. Four different lineages were designed, with modifications at C2, C4 and C6 (Fig. 2), and were based on 6-(1-hydroxyethyl)-2,2-dimethylchroman-4-one (series 1), 1-(2,2-dimethylchroman-6-yl)ethanol (series 2), 6-(1-hydroxyethyl)chroman-4-one (series 3) and 6-(aminomethyl)-2,2-dimethylchroman-4-one (series 4). The four classes of compounds were envisaged via esterification and amidation reactions of these parent compounds.

Fig. 2
figure 2

Benzopyran target structures based on metabolites of C. uniflora

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Results and Discussion

Chemistry

Synthetic approaches to chromanone skeletons have been reviewed; common approaches include Michael addition protocols, aldol condensations, the heating of o-(trimethylsilyl)phenyltriflates with acrylic acids, the electrophilic cyclization of ethyl 3-phenoxypropanoate derivatives or the acylation of resorcinols [21]. We chose to prepare compounds of series 1 by the synthetic route presented in Scheme 1 [19], starting with acetylation of commercial 4-hydroxyacetophenone 9 with acetic anhydride to afford compound 10. Migration of the acetyl group via Fries rearrangement yielded 11, albeit in low yield. Cyclization to obtain 6-acetyl-2,2-dimethylchroman-4-one 12 was achieved by Kabbe condensation with acetone [22], yielding the desired compound in 67% yield. Interestingly, metabolite 12, which occurs as a metabolite of several plant species, has recently been shown to exhibit anti-obesity effects [23]. Regioselective reduction of diketone 12 was proposed using vegetable biocatalysis using Daucus carota, which showed efficiency in the selective reduction of diketone compounds in previous studies [24]. Using similar conditions, compound 1 was obtained at a yield of 29%. Finally, esterification of compound 1 with different acids was performed using dicyclohexylcarbodiimide (DCC) and (4-dimethylamino) pyridine (DMAP) catalysis, to obtain compounds 13a-d, in yields between 11–64%.

Scheme 1
scheme 1

Series 1, reagents and conditions: (a) Ac2O, Et3N, DCM, rt, 3 h; (b) AlCl3, 160 °C, 3 h; (c) acetone, morpholine, [bbim]Br, 100 °C, 8 h; (d) Daucus carota, DMF, water, rt, 7 days; (e) appropriate acid, DMAP, DCC, DCM, rt, 20 h

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Compounds in series 2 were formed in 3 steps, as shown in Scheme 2, starting with cyclization of 9 using 2-methylbut-3-en-2-ol in formic acid [25], to prepare 14 in 20% yield. Quantitative reduction of the carbonyl group using sodium borohydride in methanol afforded secondary alcohol 15. Finally, esterification or amidation of 15 afforded 16a-g. Esterifications were performed analogously to 13a-d, obtaining compounds 16a-e in 7–37% yields. Amidations of 15 were performed using FeCl3, nitromethane and either acetamide or acrylamide [26] to afford 16f-g in yields of 24 and 53% respectively.

Scheme 2
scheme 2

Series 2, reagents and conditions: (a) 2-methylbut-3-en-2-ol, HCOOH, reflux, 10 days; (b) MeOH, NaBH4, rt, 2 h; (c) 16a-e: appropriate acid, DMAP, DCC, DCM, rt, 20 h; 16f-g: appropriate amide, FeCl3, nitromethane, overnight, rt

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Compounds 21a-d (Series 3) were prepared as described in Scheme 3. Compound 17 was synthesized by alkylation of 9 with 3-chloro-1-propanol, yielding 41% of the desired compound. Permanganate oxidation of compound 17 gave 18, followed by cyclization with polyphosphoric acid to give 19 in 44% yield. Diketone 19, when subject to reduction analogously to 12, instead of solely affording the exocyclic alcohol, instead resulted in a predominance of chromanol 20 in 79% yield. Esterification of compound 20 as for compounds 1 and 15 gave 21a-d in 7–34% yields.

Scheme 3
scheme 3

Series 3, reagents and conditions: (a) 3-chloro-1-propanol, K2CO3, KI, DMF, 100 °C, 18 h; (b) KMnO4, acetone, rt, 18 h; (c) PPA, 100 °C, 5 h; (d) Daucus carota, DMF, water, rt, 3 days; (e) appropriate acid, DMAP, DCC, DCM, rt, 20 h

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Compounds of series 4 were obtained from commercial p-cyanophenol 22 as previously described [19]. Briefly, as shown in Scheme 4, Fries rearrangement of acetylated 22 was followed by Kabbe cyclisation to obtain a nitrile-substituted chromanone, which was reduced using nickel(II)/NaBH4 to afford amine 23. EDC coupling of 23 with different acids afforded amides 24a-i in 21–61% yields. In certain cases, the required unsaturated acids were commercially available. In the case of the C5-C8 acids, these required preparation via Wittig reaction of benzaldehyde 27 with an ylide generated from an appropriate phosphonium salt 26. In the case of the C5 and C6 synthons, the required (carboxy-alkyl)triphenylphosphonium bromides were commercially available; however, the C7 and C8 analogues required preparation through the reaction of the appropriate ω-bromo carboxylic acid 25 with triphenylphosphine under prolonged reflux.

Scheme 4
scheme 4

Series 4, reagents and conditions: (a) appropriate acid, EDC, Et3N, DCM, 0 °C to rt, 24 h; (b) PPh3, toluene, 24–48 h, Δ, N2; (c) 1 M LHMDS in THF, −78 °C to rt, 24 h

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Antileishmanial activity

Selected compounds were screened for their leishmanicidal ability as previously described [27], by examining their activity on L. infantum axenic amastigotes, and the results are expressed in Table 1 as IC50 values. In parallel, evaluation of the cytotoxicity of test compounds was performed by MTT assay using the J774A.1 macrophage cell line. Results are presented as CC50 values.

Table 1 Activity of chromanones against L. infantum amastigotes and evaluation of cytotoxicity
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Screening of the tested compounds on the growth of L. infantum axenic amastigotes revealed IC50 values between 7.29 and >260 μM, revealing a broad range of activity. Of these, 12 compounds demonstrated activity values of <50 μM and can be considered active. Within the four categories of compounds tested, some common features were observed. Ten of the 12 active compounds contained either a phenylalkenyl motif, such as cinnamyl, styryl, or a more lipophilic extension. Of these, compounds 13c and 24c were less active (IC50 38.18 and 42.09 μM respectively), suggesting a styryl motif or longer at position 6 on the chromanone skeleton is optimal for activity. A comparison of compound 13d (IC50 12.10 μM) with compound 16d (IC50 7.29 μM) suggests that the keto functionality at position 4 in the parent chromanones is not essential. Likewise, amide analogues retained activity, and a comparison of 13d (IC50 12.10 μM) versus 24e (IC50 11.64 μM) suggests that a benzylamido linkage at position 6 is as acceptable as a benzylic ester. Extension of the amide chain from 24d to 24i revealed optimum activity with phenylbutenamide 24e (IC50 11.64 μM), with extension beyond phenylpentenamide 24f showing higher cytotoxicity. Finally, the activity of 2,2-demethylated compounds 21b and 21d (IC50 28.1 μM and 7.82 μM respectively) suggests the 2,2-dimethyl group is not essential. Particularly, the activity of 21d, with a styryl substituent at C4 rather than C6, implies that there is flexibility in the positioning of this feature. Two of the most potent compounds, 16d and 21d, had selectivity indices of greater than 10, demonstrating considerable selectivity to amastigotes over macrophages. Considering these screening observations, alongside already published work [19], allows us to suggest a general structure for activity among this class of chromanones (Fig. 3):

Fig. 3
figure 3

Structure activity map of anti-leishmanial uniflorol analogues

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Our results align with observations of Harel et al. [28], who demonstrated impressive anti-L. donovani activity of chromanamines with a phenylalkyl residue at the N-atom of a 7-methoxy-substituted chromane. In that work, an increased number of methylene moieties between the N-atom and the phenyl ring lead to increased activity, with a phenyl-butylamine the most potent compound (IC50 0.57 μM). Phenylalkenylamide structural motifs are also present in other compounds with activity against various forms of Leishmania, including diverse natural products and their synthetic derivatives. Ferreira et al. evaluated a group of synthetic amides based on piperine against various forms of L. amazonensis [29]. Both piperine and a simplified phenylamide were shown to display toxicity towards L. amazonensis, via modification of parasite mitochondrial structure and function. In other work, the amide piperlongumine had IC50 values of 7.9 and 3.3 μM against promastigote forms of L. infantum and L. amazonensis, respectively, and 0.4 μM against the intracellular amastigote form of L. amazonensis [30]. Our results add to the growing body of knowledge regarding the structure-activity relationships of chromanones and allied compounds as potential anti-leishmanial agents, with further work warranted to optimise their physicochemical and pharmacotoxicological properties. This may include examination of the consequences of compound exposure on the cytoskeleton, on mitochondrial integrity (via assessment of mitochondrial membrane potential), and on lipid metabolism.

Conclusions

Among 24 novel uniflorol derivatives, 12 were active against the amastigote form of L. infantum. Initial observations on SARs suggested that within the prepared compounds, those bearing a phenylalkenyl motif, such as cinnamyl, styryl or a more lipophilic extension, and amide analogues retained activity. Compound 16d was the most promising compound from those described, with good potency and selectivity, and warrants description as a promising hit for Leishmania. It may be synthesized in four steps from commercially available precursors, displays promising activity (IC50 7.29 μM) and low cytotoxicity (SI > 10.99).

Experimental

All required chemicals, solvents, and reagents were purchased from Sigma-Aldrich or Merck and were of reagent grade. All synthesized compounds were purified by column chromatography (silica gel 60; gradient elution with ethyl acetate (EtOAc)/petroleum ether (PE) /dichloromethane (DCM)). Merck Silica Gel 60 F254 plates were used for analytical TLC. Melting points were estimated using a Gehaka PF 1000 melting point apparatus. Compounds were fully characterized by 1H NMR, 13C NMR and 19F NMR if appropriate, using either a Bruker Avance 300 or 400 spectrometer. Splitting patterns are described as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q) and multiplet (m). Chemical shifts are expressed as δ (ppm) and coupling constants (J) in Hertz (Hz). Mass spectral analyses were recorded using a LCT Premiere XE (ESI-TOF MS) instrument or a Bruker micrOTOF-QIII instrument. All calculated exact mono isotopic mass distributions were calibrated against internal reference standards. IR data was obtained with a Thermo scientific microlet I s10 instrument model 1510 or a Shimadzu IR Prestige-21 spectrophotometer.

Synthesis and characterization of series 1 compounds

4-acetylphenyl acetate (10)

The compound was prepared and characterized as previously described in the literature [31]. Yield: 98%.

1,1-(4-hydroxy-1,3-phenylene)diethanone (11)

The compound was prepared and characterized as previously described in the literature [32]. Yield: 17%.

6-acetyl-2,2-dimethylchroman-4-one (12)

Compound 11 (2.5 g, 14.2 mmol), acetone (2.3 mL), and morpholine (0.7 mL) were added to the ionic liquid [bbim]Br (2 g) and stirred at 95–100 °C for 8 hours [22]. The reaction mixture was extracted with ethyl acetate (3 × 30 mL) and the organic layers dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by column chromatography (Petroleum ether (PE): ethyl acetate (EtOAc)). Characterization agreed with previously reported values [33]. Yield: 67%.

6-(1-hydroxyethyl)-2,2-dimethylchroman-4-one (1)

Compound 12 (200 mg, 0.9 mmol), dissolved in dimethylformamide (1.5 mL) was added to water (500 mL) containing D. carota slices (150 g). The reaction mixture was stirred vigorously for 7 days at room temperature. The reaction was then extracted with ethyl acetate (6 × 50 mL), and the organic layers dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by column chromatography (PE: EtOAc). Yellow oil, yield: 29%, αD (20 °C) −18.3 (c = 0.95, CH2Cl2); IR: 3393, 1724, 1679, 1616, 1488, 1372, 1252, 1203, 1128, 930, 835 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.37 (6H, s, (CH3)2), 1.41 (3H, d, J = 6.53 Hz, CH3CH), 2.63 (2H, s, CH2CO), 4.79 (1H, q, J = 6.53 Hz, CH3CH), 6.85 (1H, d, J = 8.53 Hz, Ar-H), 7.47 (dd, J = 8.53, 2.26 Hz, Ar-H), 7.74 (1H, d, J = 2.26 Hz, Ar-H); 13C NMR (100 MHz, CDCl3) δ = 24.9 (CH3), 26.6 (2CH3), 48.8 (CH2), 69.6 (COH), 79.3 (C2), 118.6 (Ar-CH), 119.6 (Ar-C), 123.3 (Ar-CH), 133.8 (Ar-C), 138.1 (Ar-CH), 159.4 (Ar-C), 192.8 (CO). Characterization agreed with previously reported values [13].

1-(2,2-dimethyl-4-oxochroman-6-yl)ethyl but-2-enoate (13a)

Compound 1 (20 mg, 0.09 mmol), but-2-enoic acid (15 mg, 0.18 mmol), 4-dimethylaminopyridine (5 mg, 0.045 mmol) and N,N′-dicyclohexylcarbodiimide (37 mg, 18 mmol) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 20 h. The reaction mixture was filtered, washed with 1 M NaOH and extracted with dichloromethane (3 ×20 mL). The organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE: EtOAc). Colourless oil, yield: 15%. IR: 1716, 1690, 1618, 1489, 1436, 1372, 1255, 1176, 1062, 967, 833 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.45 (6H, s, (CH3)2), 1.54 (3H, d, J = 6.6 Hz, CH3), 1.87 (3H, dd, J = 6.9, 1.4 Hz, CH3C=C), 2.71 (2H, s, CH2), 5.84-5.91 (2H, m, CH and CH=C), 6.90 (1H, d, J = 8.6 Hz, Ar-H8), 6.99 (1H, m, CH=C), 7.48 (1H, dd, J = 8.6, 2.2 Hz, Ar-H7), 7.87 (1H, d, J = 2.1 Hz, Ar-H5); 13C NMR (100 MHz, CDCl3) δ = 18.0 (CH3), 22.0 (CH3), 26.6 (CH3), 26.7 (CH3), 48.8 (CH2), 71.2, 79.3, 118.5 (Ar-CH), 119.9 (Ar-C), 122.8 (CH), 124.0 (Ar-CH), 134.4 (Ar-C), 134.5 (Ar-CH), 145.0 (CH), 159.5 (Ar-C), 165.8 (COO), 192.5 (CO). HRMS (M + Na)+ 311.1257, C17H20O4Na requires 311.1259.

1-(2,2-dimethyl-4-oxochroman-6-yl)ethyl 3-methylbut-2-enoate (13b)

50 mg (0.22 mmol) of 1 was reacted as for 13a with trans-3-methylbut-2-enoic acid (27 mg, 0.45 mmol) to afford 13b. Colourless oil, yield: 11%. IR: 1695, 1653, 1619, 1263, 705 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.38 (6H, s, (CH3)2, 1.48 (3H, d, J = 6.8 Hz, CH3CH), 1.73 and 1.76 (6H, (CH3)2C=C), 2.64 (2H, s, CH2CO), 5.81 (2H, m, CH and CH=C), 6.83 (1H, d, J = 8.5 Hz, Ar-H), 7.41 (1H, dd, J = 8.5, 2.3 Hz, Ar-H), 7.79 (1H, d, J = 2.3 Hz, Ar-H); 13C NMR (100 MHz, CDCl3) δ = 20.3 (CH3), 26.7 (CH3)2), 27.5 (CH3), 48.8 (CH2), 64.6 (CH2), 79.4 (C2), 115.7 (C=CH), 118.7 (C = CH), 119.9 (quat. C), 126.4 (C=CH), 128.9 (quat. C), 136.3 (C=CH), 157.6 (quat. C), 159.8 (quat. C), 166.3 (COO), 192.3 (CO).

1-(2,2-dimethyl-4-oxochroman-6-yl)ethyl cinnamate (13c)

50 mg (0.22 mmol) of 1 was reacted as for 13a with cinnamic acid (67 mg, 0.45 mmol) to afford 13c. Colourless oil, yield: 61%. IR: 1710, 1692, 1618, 1490, 1255, 1168 cm−1. 1H NMR (400 MHz, CDCl3): δ = 1.46 (6H, d, J = 2.2 Hz, CH3CCH3), 1.60 (3H, d, J = 6.6 Hz, CH3CH), 2.72 (2H, s, CH2), 5.98 (1H, q, J = 6.4 Hz, CHCH3), 6.46 (1H, d, J = 15.9 Hz, CH=CH), 6.92 (1H, d, J = 8.6 Hz, ArH), 7.38 (3H, m, 3 x ArH), 7.52 (3H, m, 3 x ArH) 7.69 (1H, d, J = 16.1 Hz, CH=CH), 7.92 (1H, d, J = 2.2 Hz, ArH); 13C NMR (100 MHz, CDCl3) δ = 22.0, 26.6, 26.7, 48.8, 71.7, 79.0, 118.3, 118.6, 124.1, 128.1 (2C), 128.9 (2C), 130.3, 134.2, 134.4, 134.5, 145.0, 158.5, 166.2, 192.4 (one quat. C obscured). HRMS (M+Na)+ 373.1411, C22H22O4Na requires 373.1416.

1-(2,2-dimethyl-4-oxochroman-6-yl)ethyl (E)-4-phenylbut-3-enoate (13d)

60 mg (0.3 mmol) of 1 was reacted as for 13a with 4-phenylbut-3-enoic acid (50 mg, 0.6 mmol) to afford 13d. Colourless oil, yield: 64%. IR: 1734, 1700, 1616, 1257, 750 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.45 (6H, d, J = 3.2 Hz, CH3CCH3), 1.54 (3H, d, J = 6.6 Hz, CH3CH), 2.71 (2H, s, CH2), 3.26 (2H, d, J = 7 Hz, CH2C = C), 5.87 (1H, q, J = 6.5 Hz, CHCH3), 6.30 (1H, dt, J = 7 Hz, CH2CH=CH), 6.48 (1H, d, J = 16 Hz, CH2CH=CH), 6.90 (1H, d, J = 8.6 Hz, H8), 7.20–7.36 (6H, m, 5 x ArH), 7.47 (1H, dd, J = 8.6, 2.1 Hz, H7), 7.87 (1H, d, J = 2.1 Hz, H5); 13C NMR (100 MHz, CDCl3) δ = 21.9, 26.6, 26.7, 38.6, 48.8, 72.0, 79.4, 118.6, 119.9, 121.6, 124.1, 126.3 (2C), 127.6, 128.5 (2C), 133.5, 134.0, 134.5, 136.8, 159.6, 170.9, 192.4. HRMS (M+Na)+ 387.1563, C23H24O4Na requires 387.1567.

Synthesis and characterization of series 2 compounds

1-(2,2-dimethylchroman-6-yl)ethanone (14)

Compound 9 (2.50 g, 18.4 mmol) was dissolved in formic acid (75 mL) and 2-methylbut-3-en-2-ol (1.58 g, 18.3 mmol) was added slowly. The mixture was heated to reflux for 8 h. It was then cooled, ice cold H2O (200 mL) was added and the mixture was extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were washed with saturated NaHCO3 and then dried (Na2SO4), concentrated in vacuo and the residue purified by flash chromatography (PE: EtOAc). Colourless oil, yield: 20%. Characterization agreed with previously reported values [34].

1-(2,2-dimethylchroman-6-yl)ethanol (15)

To compound 14 (1 g, 4.9 mmol) dissolved in methanol (15 mL) was slowly added sodium borohydride (0.25 g, 6.6 mmol). The mixture was stirred at room temperature for 2 hours, and then extracted with dichloromethane (3 × 30 mL). The organic layers were dried over anhydrous sodium sulfate concentrated under reduced pressure and purified by column chromatography (PE: EtOAc). Yellow oil, yield: 100%. IR: 3354, 1616, 1587, 1495, 1367, 1252, 1121, 1070, 820, 607 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.25 (6H, s, (CH3)2), 1.39 (3H, d, J = 6.27 Hz, CH3), 1.72 (2H, t, CH2), 2.69 (2H, t, CH2), 4.72 (1H, q, CH), 6.69 (1H, d, J = 8 Hz, Ar-H), 7.01 (2H, m, 2 x Ar-H); 9.03 (OH); 13C NMR (100 MHz, CDCl3) δ = 22.6 (CH2), 24.9 (CH3), 26.9 (CH3), 26.9 (CH3), 32.8 (CH2), 70.1 (CHOH), 74.3 (C2), 117.2 (Ar-CH), 120.8 (Ar-C), 124.6 (Ar-CH), 126.6 (Ar-C), 136.9 (Ar-CH), 153.4 (Ar-C).

1-(2,2-dimethylchroman-6-yl)ethyl but-2-enoate (16a)

Compound 15 (100 mg, 0.5 mmol) and but-2-enoic acid (80 mg, 1 mmol), 4-dimethylaminopyridine (3 mg, 0.025 mmol) and N,N′-dicyclohexylcarbodiimide (200 mg, 1 mmol)) was dissolved in DCM (5 mL) and stirred at room temperature for 20 h. The reaction mixture was filtered, washed with 1 M NaOH and extracted with DCM. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure and purified by column chromatography (PE: EtOAc). Yellow oil, yield: 37%. IR: 1713, 1497, 1151, 1122, 1099, 1059, 967, 820 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.24 (6H, s, (CH3)2), 1.46 (3H, d, J = 6.53 Hz, CH3CH), 1.71 (2H t, J = 6.78 Hz, CH2), 1.79 (3H, dd, J = 6.9, 1.6 Hz, CH3C=C), 2.68 (2H, t, J = 6.78 Hz, CH2), 5.80 (2H, m, CH=CH and CH3CH), 6.68 (d, J = 8.28 Hz, Ar-H), 6.89 (1H, m, CH = CH), 6.99 (br. s, Ar-H), 7.03 (1H, dd, J = 8.53, 2.26 Hz, Ar-H); 13C NMR (100 MHz, CDCl3) δ = 18.0 (CH3), 22.0 (CH2), 22.5 (CH3), 26.9 (2CH3), 32.7 (CH2), 71.9 (CH), 74.3 (C), 117.2 (Ar-CH), 120.6 (CH), 123.1 (Ar-C), 125.3 (Ar-CH), 127.7 (Ar-CH), 132.7 (C), 144.5 (CH), 153.7 (Ar-C), 165.9 (CO). HRMS (M + Na)+ 297.1462, C17H22O3Na requires 297.1467.

1-(2,2-dimethylchroman-6-yl)ethyl 2-methylbut-2-enoate (16b)

Compound 15 (200 mg, 1 mmol) was reacted as for 16a with trans-2-methyl-but-2-enoic acid (100 mg, 2 mmol) to afford 16b. Yellow oil, yield: 7%. IR: 1714, 1497, 1224, 1142, 1059, 947, 819 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.25 (6H, s, (CH3)2), 1.46 (3H, d, J = 6.53 Hz, CH3), 1.70–1.80 (8H, m, 2 x CH3 and CH2), 2.69 (2H, t, CH2), 5.80 (1H, q, CH), 6.68 (1H, d, J = 8.53 Hz, Ar-H), 6.81 (1H, m, CH=CH), 6.98–7.04 (2H, m, 2x CH=CH); 13C NMR (100 MHz, CDCl3) δ = 12.1 (CH3), 14.3 (CH3), 22.2 (CH3), 22.5 (CH2), 26.9 (2CH3), 32.8 (CH2), 72.0 (CH), 74.3 (C2), 117.2 (CH=CH), 120.6 (quat. C), 125.2 (CH=CH), 127.5 (CH=CH), 129.0 (quat. C), 133.1 (quat. C), 136.9 (CH=CH), 153.6 (Ar-C), 167.4 (CO). HRMS (M+) 288.1723, C18H24O3 requires 288.1725.

1-(2,2-dimethylchroman-6-yl)ethyl 3-methylbut-2-enoate (16c)

Compound 15 (200 mg, 1 mmol) was reacted as for 16a with trans-3-methyl-but-2-enoic acid (100 mg, 2 mmol) to afford 16c. Yellow oil, yield: 7%. IR: 1705, 1527, 1304, 1121, 1059, 819, 733 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.24 (6H, s, (CH3)2), 1.44 (3H, d, J = 6.53 Hz, CH3CH), 1.71 (2H, t, CH2), 1.79 (3H, s, CH3), 2.08 (3H, s, CH3), 2.68 (2H, t, CH2), 5.62 (1H, d, J = 1.00 Hz, CH), 5.77 (1H, q, CH3CH), 6.67 (1H, d, J = 8.28 Hz, Ar-H), 6.99 (1H, s, Ar-H), 7.01 (dd, Ar-H); 13C NMR (100 MHz, CDCl3) δ = 19.2 (CH3). 21.1 (CH3), 21.5 (CH2), 25.9 (CH3), 26.3 (2CH3), 31.7 (CH2) 70.1 (CH), 73.2 (C2), 115.5 (CH), 116.2 (CH), 119.6 (Ar-C), 124.3 (Ar-CH), 126.5, (CH), 132.0 (Ar-C), 152.6 (quat. C), 155.4 (quat. C), 164.9 (CO). HRMS (M + Na)+ 311.1614, C18H24O3Na requires 311.1618.

1-(2,2-dimethylchroman-6-yl)ethyl 4-phenylbut-3-enoate (16d)

Compound 15 (200 mg, 1 mmol) was reacted as for 16a with 4-phenylbut-3-enoic acid (32 mg, 2 mmol) to afford 16d. Yellow oil, yield: 17%. IR: 1728, 1496, 1294, 1209, 1121, 1057, 819, 691 cm−1. 1H NMR (CDCl3, 400 MHz) δ = 1.24 (6H, s, C(CH3)2), 1.46 (3H, d, J = 6.53 Hz, CH3CH), 1.70 (2H, t, J = 6.78 Hz, CH2), 2.67 (2H, td, J = 6.65 Hz, CH2), 3.17 (2H, d, J = 6.3 Hz, CH2C=C), 5.78 (1H, q, J = 6.53 Hz, CH3CH), 6.23 (1H, m, CH2CH=CH), 6.39 (1H, d, J = 19 Hz, CH2CH=CH), 6.67 (1H, d, J = 8.3 Hz, H8), 6.99 (1H, br. s, H5), 7.01 (1H, dd, J = 8.3, 2.3 Hz, H7), 7.12–7.29 (5H, m, ArCH2´-6´); 13C NMR (100 MHz, CDCl3) δ = 20.9, 21.4, 25.9, 31.7, 37.7, 71.6, 73.3, 116.2, 119.6, 120.9, 124.3, 125.2 (2 x ArCH), 126.4, 126.7, 127.5 (2 x ArCH), 131.3, 132.3, 135.9, 152.8, 169.9. HRMS (M + Na)+ 373.1774, C23H26O3Na requires 373.1780.

1-(2,2-dimethylchroman-6-yl)ethyl 4,4,4-trifluoro-3-methylbut-2-enoate (16e)

Compound 15 (100 mg, 0.5 mmol) was reacted as for 16a with 4,4,4-trifluoro-3-methylbut-2-enoic acid (150 mg, 1 mmol) to afford 16e. Colourless oil, yield: 24%. IR: 1722, 1498, 1293, 1197, 1122, 1096, 1059, 947, 892 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.25 (6H, s, C(CH3)2), 1.49 (3H, d, J = 6.53 Hz, CH3CH), 1.72 (3H, t, CH2), 2.15 (3H, d, J = 1.51 Hz, CH3C=C), 2.69 (2H, t, CH2), 5.83 (1H, q, CH3CH) 6.25 (1H, d, CH = C), 6.69 (d, J = 8.28 Hz, Ar-H), 7.01 (2H, m, 2Ar-H); 13C NMR (100 MHz, CDCl3) δ = 11.3 (CH3), 20.8 (CH3), 21.5 (CH2), 25.9 (2CH3), 31.7 (CH2), 72.1 (CH), 73.4 (C2), 116.3 (CH=C), 119.8 (Ar-CH), 120.9 (Ar-C), 124.4 (Ar-CH), 126.7 (Ar-CH), 130.9 (Ar-C), 140.6 (quat. C), 153.0 (quat. C), 163.2 (CO); 19F NMR (376.5 MHz, CDCl3): δ = −71.3 (CF3). HRMS (M+) 342.1435, C18H21O3F3 requires 342.1443.

N-(1-(2,2-dimethylchroman-6-yl)ethyl)acetamide (16f)

Compound 15 (50 mg, 0.25 mmol), acetamide (16 mg, 0.25 mmol), and FeCl3 (20 mg, 0.12 mg) were dissolved in nitromethane (3 mL) and stirred overnight at room temperature, after which time the reaction was concentrated under reduced pressure and purified by column chromatography (PE: EtOAc). Colourless oil, yield: 34%. IR: 3282, 1738, 1644, 1494, 1456, 1367, 1257, 1121, 946, 818 cm−1. 1H NMR (400 MHz, CDCl3): δ = 1.25 (6H, s, (CH3)2), 1.39 (3H, d, J = 6.78 Hz, CH3CH), 1.71 (2H, t, CH2), 1.89 (3H, s, CH3CO), 2.68 (2H, t, CH2), 4.96 (1H, m, CH3CH), 5.60 (1H, br. s, NH), 6.67 (d, J = 8.3 Hz, Ar-H), 6.94 (2H, m, 2Ar-H); 13C NMR (100 MHz, CDCl3): δ = 21.5 (CH3), 22.5 (CH2), 25.9 (CH3), 28.7 (2CH3), 31.7 (CH2), 47.3 (CH) 73.3 (C2), 116.4 (Ar-CH), 119.9 (Ar-C), 124.0 (Ar-CH), 126.6 (Ar-CH), 133.1 (Ar-C), 152.3 (Ar-C), 168.0 (CO). HRMS (M + Na)+ 270.1469, C15H21NO2Na requires 270.1465.

N-(1-(2,2-dimethylchroman-6-yl)ethyl)acrylamide (16g)

Compound 15 (120 mg, 0.58 mg) was reacted as for 16f with acrylamide (40 mg, 0.58) to afford 16g. Colourless oil, yield: 53%. IR: 3269, 1653, 1624, 1494, 1233, 1208, 1121, 946, 728 cm−1. 1H NMR (400 MHz, CDCl3): δ = 1.24 (6H, s, (CH3)2), 1.41 (3H, d, J = 6.78 Hz, CH3CH); 1.70 (2H, t, CH2), 2.66 (2H, t, CH2), 5.05 (1H, m, CH3CH), 5.52 (1H, dd, H of CH2), 6.01 (2H, H of CH2 and NH), 6.19 (1H, C=CH), 6.65 (1H, d, J = 8.28 Hz, Ar-H), 6.95 (2H, m, 2Ar-H); 13C NMR (100 MHz, CDCl3): δ = 21.6 (CH3), 22.5 (CH2), 26.9 (2CH3) 32.7 (CH2), 48.3 (CH), 74.2 (C2), 117.3 (Ar-CH), 120.8 (Ar-C), 125.1 (Ar-CH), 126.3 (CH2), 127.7 (Ar-CH), 131.1 (CH), 134.1 (Ar-C), 153.3 (Ar-C), 164.6 (CO). HRMS (M + Na)+ 282.1477, C16H21NO2Na requires 282.1465.

Synthesis and characterization of series 3 compounds

1-(4-(3-hydroxypropoxy)phenyl)ethanone (17)

4-hydroxyacetophenone (10 g, 73.44 mmol), 3-chloro-1-propanol (7.3 mL), K2CO3 (13 g, 94 mmol), and KI (120 mg, 0.7 mmol) were dissolved in DMF (100 mL), and stirred at 100 °C for 18 h, after which time water was added, and the reaction extracted with diethyl ether (3 × 100 mL). The organic layers were washed sequentially with 1 M NaOH and saturated NaCl solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure and purified by column chromatography (PE: EtOAc). Characterization agreed with previously reported values [35].

3-(4-acetylphenoxy)propanoic acid (18)

Compound 17 (3.3 g, 17 mmol) and KMnO4 (13.7 g, 86.8 mmol) were dissolved in acetone (150 mL) and stirred at room temperature for 18 h. Sodium thiosulfate saturated solution was added, followed by acidification with 1 M HCl. The mixture was then extracted with DCM (3 × 100 mL), washed with saturated NaCl solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. White solid, yield: 100%; mp 142 °C; IR (KBr) 3489, 2921, 2349, 2306, 1727, 1660, 1559, 1421, 1362, 1268, 1173, 1034, 960, 833, 671 cm−1. 1H NMR (270 MHz, CDCl3): δ = 2.56 (3H, s, CH3), 2.89 (2H, t, CH2), 4.32 (2H, t, CH2), 6.95 (2H, d, 2Ar-H), 7.94 (2H, d, 2Ar-H); 13C NMR (67.5 MHz, CDCl3): δ = 26.5 (CH3), 34.1 (CH2), 63.4 (CH2), 114.4 (2Ar-CH), 130.8 (2Ar-CH), 162.4 (Ar-C), 175.1 (COOH), 197.1 (CO).

6-acetylchroman-4-one (19)

Compound 18 (1 g, 4.8 mmol) and PPA (20 g) were stirred at 100 °C for 5 h, after which time was added water, and the reaction stirred for 5 minutes. It was extracted with DCM, washed with saturated NaCl solution dried over anhydrous sodium sulfate, concentrated under reduced pressure and purified by column chromatography (DCM: EtOAc). White solid, yield: 44%; mp 115 °C; IR (KBr): 2998, 2919, 1686, 1604, 1491, 1424, 1357, 1247, 1129, 1027, 837, 754 cm−1. 1H NMR (270 MHz, CDCl3): δ = 2.60 (3H, s, CH3). 2.87 (2H, t, CH2), 4.62 (2H, t, CH2), 7.04 (1H, d, Ar-H), 8.14 (1H, dd, Ar-H), 8.48 (1H, d, Ar-H); 13C NMR (67.5 MHz, CDCl3): δ = 26.4 (CH3), 37.4 (CH2), 67.3 (CH2), 118.7 (Ar-CH), 120.4 (Ar-C), 128.7 (Ar-CH), 130.9 (Ar-C), 135.3 (Ar-CH), 165.2 (Ar-C), 190.9 (CO), 196.3 (CO).

6-(1-hydroxyethyl)chroman-4-one (20)

Compound 19 (100 mg, 0.53 mmol) dissolved in DMF (1.5 mL) was added to water (500 mL) and D. carota slices (40 g). The reaction mixture was vigorously stirred at room temperature for 3 days. The reaction was then extracted with ethyl acetate (6 × 100 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM: EtOAc). Yellow oil, yield: 79%; αD (20 °C) −3.62 (c = 1.28, CH2Cl2); IR (KBr): 3517, 2232, 1708, 1681, 1607, 1498, 1357, 1250, 1135, 1050, 831, 733 cm−1. 1H NMR (270 MHz, CDCl3): δ = 2.10 (2H, m, CH2). 2.57 (3H, s, CH3), 4.37 (2H, m, CH2), 4.88 (1H, s, CH), 6.90 (1H, d, Ar-H), 7.82 (1H, d, Ar-H), 8.01 (1H, d, Ar-H); 13C NMR (67.5 MHz, CDCl3): δ = 26.3, 30.5, 37.4, 62.6, 63.1, 117.2 (Ar-CH), 124.2 (Ar-C), 130.1 (Ar-C), 130.3 (Ar-CH), 130.8 (Ar-CH), 158.9 (Ar-C), 190.9 (CO), 196.8 (CO). HRMS (M + Na)+ 215.0677, C11H12O3Na requires 215.0679.

6-acetylchroman-4-yl (E)-but-2-enoate (21a)

Compound 20 (100 mg, 0.5 mmol), but-2-enoic acid (90 mg, 1 mmol), 4-dimethylaminopyridine (31 mg, 0.25 mmol) and N,N′-dicyclohexylcarbodiimide (214 mg, 1 mmol) were dissolved in DCM (5 mL) and stirred at room temperature for 20 h. The reaction mixture was filtered, washed with 1 M NaOH, and extracted with DCM. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE: EtOAc) to afford 21a. Colourless oil, yield: 7%; IR: 1712, 1675, 1247, 1169, 1049, 833 cm−1. 1H NMR (400 MHz, CDCl3): δ = 1.89 (3H, dd, J = 7.0, 1.6 Hz, CH = CHCH3), 2.13–2.30 (2H, m, CHCH2), 2.54 (3H, s, COCH3), 4.29–4.41 (2H, m, OCH2), 5.87 (1H, dd, J = 15.4, 1.7 Hz, CH=CH), 6.01 (1H, t, J = 3.7 Hz, CH), 6.91 (1H, d, J = 8.6 Hz, Ar-H8), 7.03 (1H, m, CH=CH), 7.87 (1H, dd, J = 8.8, 2.2 Hz, Ar-H7), 7.94 (1H, d, J = 2.2 Hz, Ar-H5); 13C NMR (100 MHz, CDCl3): δ = 18.0, 26.3, 28.1, 62.7, 64.6, 117.4, 120.1, 122.5, 130.2, 130.5, 132.1, 145.8, 159.4, 165.7, 196.6. HRMS (M + Na)+ 283.0948, C15H16O4Na requires 283.0941.

6-acetylchroman-4-yl (Z)-2-methylbut-2-enoate (21b)

Compound 20 (80 mg, 0.40 mmol) was reacted as for 21a with trans-2-methyl-but-2-enoic acid (83 mg, 0.8 mmol) to afford 21b. Colourless oil, yield: 17%; IR (KBr): 3050, 2921, 1671, 1605, 1494, 1435, 1250, 1134, 1057, 975, 828, 739 cm−1. 1H NMR (400 MHz, CDCl3): δ = 1.79 and 1.85 (6H, m, 2 x CH3). 2.17 (2H, m, CH2), 2.54 (3H, s, CH3), 4.37 (2H, m, CH2), 6.03 (1H, m, CH), 6.90 (2H, m, CH and Ar-H), 7.87 (1H, d, Ar-H), 7.95 (1H, d, Ar-H); 13C NMR (100 MHz, CDCl3): δ = 12.1, 14.4, 26.3, 28.1, 62.8, 64.7, 117.3 (Ar-CH), 120.2 (Ar-C), 128.5 (Ar-C), 130.2 (Ar-C), 130.5 (Ar-CH), 132.1 (Ar-CH), 138.2 (Ar-CH), 159.4 (Ar-C), 167.3 (CO), 196.7 (CO). HRMS (M + Na)+ 297.1099, C16H18O4Na requires 297.1097.

6-acetylchroman-4-yl 3-methylbut-2-enoate (21c)

Compound 20 (80 mg, 0.40 mmol) was reacted as for 21a with trans-3-methyl-but-2-enoic acid (83 mg, 0.8 mmol) to afford 21c. Colourless oil, yield: 20%. IR: 3055, 2925, 2223, 1713, 1677, 1607, 1501, 1358, 1253, 1140, 1076, 1018, 832, 735, 703 cm−1. 1H NMR (400 MHz, CDCl3): δ = 1.90 (3H, d, CH3). 2.11–2.29 (2H, m, CH2), 2.21 (3H, s, CH3), 2.54 (3H, s, COCH3), 4.26–4.20 (2H, m, OCH2), 5.69 (1H, t, CH), 5.98 (1H, 5, J = 3.5 Hz, CH), 6.90 (1H, d, J = 8.8 Hz, Ar-H8), 7.86 (1H, dd, J = 8.6, 2.2 Hz, Ar-H7), 7.95 (1H, d, J = 2.2 Hz, Ar-H5); 13C NMR (100 MHz, CDCl3): δ = 20.4, 26.3, 27.5, 28.1, 62.8, 63.8, 115.8, 117.3, 120.4, 130.2, 130.4, 132.0, 158.2, 159.4, 165.8, 196.7. HRMS (M + Na)+ 297.1102, C16H18O4Na requires 297.1097.

1-(4-oxochroman-6-yl)ethyl 4-phenylbut-3-enoate (21d)

Compound 20 (60 mg, 0.3 mmol), was reacted as for 21a with 4-phenylbut-3-enoic acid (50 mg, 0.6 mmol) to afford 21d. Colourless oil, yield: 34%; IR: 1729, 1672, 1606, 1358, 1248, 1139, 1046 cm−1. 1H NMR (400 MHz, CDCl3): δ = 2.13–2.29 (2H, m, CH2), 2.52 (3H, s, CH3), 3.30 (2H, d, J = 7.1 Hz, CH2), 4.28–4.41 (2H, m, CH2), 5.99 (1H, t, J = 3.5 Hz, CH), 6.30 (1H, dt, J = 15.9, 7.1 Hz, CH=CH), 6.51 (1H, d, J = 15.9 Hz, CH = CH), 6.91 (1H, d, J = 8.8 Hz, Ar-H8), 7.22–7.37 (5H, m, 5 x Ar-H),), 7.88 (1H, dd, J = 8.7, 1.8 Hz, Ar-H7), 7.94 (1H, d, J ~ 2 Hz, Ar-H5); 13C NMR (100Mz, CDCl3): δ = 26.3 (CH3), 27.9 (CH2), 38.6 (CH2C=C), 62.6 (OCH2), 65.3 (OCH), 117.4 (CH), 120.0 (ArC), 121.2 (CH), 126.3 (2CH), 127.7 (CH), 128.6 (2CH), 130.2 (ArC), 130.6 (CH), 132.1 (CH), 133.9 (CH), 136.7 (ArC), 159.3 (ArC), 171.0 (OC = O), 196.6 (C=O). HRMS (M + Na)+ 359.1253, C21H20O4Na requires 359.1254.

Synthesis and characterization of series 4 compounds

(E)-N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)but-2-enamide (24a)

To a solution of trans-2-butenoic acid (0.084 g, 0.98 mmol) in dichloromethane (15 mL) at 0oC under a N2 atmosphere was added EDC (0.28 g, 1.48 mmol) and triethylamine (0.18 mL, 1.30 mmol). After 30 minutes, 23 (200 mg, 0.98 mmol) was added to this solution. The reaction was allowed to reach room temperature and stirred for 24 hours. The residual solvent was removed in vacuo, the residue extracted with saturated bicarbonate/DCM, and the crude organic residue purified by flash column chromatography to afford amide (24a). Colourless oil, yield: 28%; IR (neat): 3267, 1690, 1615, 1574, 1485, 1191, 973, 924, 834 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.37 (6H, s, C(CH3)2), 1.78 (3H, dd, J = 6.9, 1.0 Hz, CH3CH=CH), 2.63 (2H, s, CH2C = O), 4.37 (2H, d, J = 6 Hz, NHCH2), 5.75 (1H, dd, J = 15.2, 1.6 Hz, CH=CH), 5.85 (1H, br., NH), 6.77–6.86 (2H, m, H8 and CH=CHCH3), 7.37 (1H, dd, J = 8.5, 2.3 Hz, H7), 7.65 (1H, d, J = 2.1 Hz, H5); 13CNMR (100Mz, CDCl3) δ = 17.8 (CH3C=C), 26.6 ((CH3)2), 42.6 (NHCH2), 48.8 (CH2C = O), 79.3 (C2), 118.9 (CH), 119.8 (ArC), 124.7 (CH), 125.2 (CH), 130.9 (ArC), 136.1 (CH), 140.6 (CH), 159.4 (ArC), 165.9 (NHC=O), 192.5 (C4). HRMS (M + Na)+ 296.1269, C16H19NO3Na requires 296.1263.

(E)-N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-2-methylbut-2-enamide (24b)

100 mg (0.49 mmol) of 23 was reacted as for 24a with trans-2,3-dimethylacrylic acid (49 mg, 0.49 mmol) to afford 24b. Colourless oil, yield: 61%; IR (neat): 3305, 1689, 1616, 1530, 1352, 1296, 1254, 1188, 1129, 683 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.37 (6H, s, C(CH3)2), 1.67 (3H, d, J = 6.8 Hz, CH3CH = C), 1.77 (3H, br. s, CH3C=CH), 2.62 (2H, s, CH2C=O), 4.35 (2H, d, J = 5.8 Hz, NHCH2), 6.18 (1H, br., NH), 6.40 (1H, q, J = 6.8 Hz, CH3CH=C), 6.81 (1H, d, J = 8.5 Hz, H8), 7.37 (1H, dd, J = 8.5, 2.5 Hz, H7), 7.65 (1H, dd, J = 2, 0.5 Hz, H5); 13CNMR (100Mz, CDCl3) δ = 12.4 (CH3C=C), 14.0 (CH3C = C), 26.6 ((CH3)2), 42.9 (NHCH2), 48.7 (CH2C=O), 79.3 (C2), 118.9 (CH), 119.8 (ArC), 125.2 (CH), 131.1 (ArC), 131.3 (CH), 131.4 (ArC), 136.1 (CH), 159.3 (ArC), 169.3 (NHC = O), 192.5 (C4). HRMS (M-H)+ 288.1446, C17H20NO3 requires 288.1443.

N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-3-(thiophen-2-yl)acrylamide (24c: E/Z mix)

200 mg (0.98 mmol) of 23 was reacted as for 24a with 3-(2-thienyl)acrylic acid (0.15 g, 0.97 mmol) to afford amide 24c. Off-white solid, yield: 60%; IR (neat): 3271, 1693, 1649, 1612, 1555, 1487, 1231, 1208, 828 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.37 (6H, s, C(CH3)2), 2.63 (2H, s, CH2C = O), 4.43 (2H, d, J = 5.9 Hz, NHCH2), 6.02 (1H, br., NH), 6.16 (1H, d, J = 15.3 Hz, CH=CH), 6.83 (1H, d, J = 8.6 Hz, H8), 6.95 (1H, m, CH), 7.13 (1H, d, J = 3.3 Hz, CH), 7.23 (1H, d, J = 5 Hz, CH), 7.40 (1H, dd, J = 8.6, 2.1 Hz, H7), 7.70 (2H, m, CH and H5); 13CNMR (100Mz, CDCl3) δ = 26.6 ((CH3)2), 42.9 (NHCH2), 48.8 (CH2C=O), 79.4 (C2), 119.0 (CH), 119.2 (CH), 119.9 (ArC), 125.3 (CH), 127.4 (CH), 128.0 (CH), 130.5 (CH), 130.7 (CH), 136.2 (CH), 134.3 (CH), 139.9 (ArC), 159.4 (ArC), 165.6 (NHC=O), 192.5 (C4). HRMS (M + H)+ 342.1149, C19H20NO3S requires 342.1164.

N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)cinnamamide (24d)

100 mg (0.49 mmol) of 23 was reacted as for 24a with trans-cinnamic acid (72 mg, 0.49 mmol) to afford amide 24d. White solid, yield: 21%; IR (neat): 3258, 2924, 1691, 1655, 1617, 1552, 1486, 1385, 1221 cm−1. 1H NMR (CDCl3, 400 MHz) δ = 1.44 (6H, s, C(CH3)2), 2.69 (2H, s, CH2C=O), 4.51 (2H, d, J = 5.9 Hz, NHCH2), 6.18 (1H, br., NH), 6.43 (1H, d, J = 15.7 Hz, COCH=CH), 6.90 (1H, d, J = 8.6 Hz, H8), 7.33–7.37 (3H, m, 3 x ArH), 7.46–7.50 (3H, m, 3 x ArH), 7.66 (1H, d, J = 15.7 Hz, CH=CHAr), 7.77 (1H, d, J = 2.2 Hz, H5); 13C NMR δ = 26.6 ((CH3)2), 42.9 (NHCH2), 48.8 (CH2C=O), 79.4 (C2), 119.0 (CH), 119.9 (ArC), 120.3 (CH), 125.3 (CH), 127.8 (2xCH), 128.8 (2xCH), 129.8(CH), 130.8 (ArC), 134.7 (ArC), 136.2 (CH), 141.6 (CH), 159.4 (ArC), 165.9 (NHC=O), 192.5 (C4). HRMS (M-H)+ 334.1434, C21H20NO3 requires 334.1443.

(E)-N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-4-phenylbut-3-enamide (24e)

100 mg (0.49 mmol) of 23 was reacted as for 24a with 4-phenylbut-3-enoic acid (79 mg, 0.49 mmol) to afford amide 24e. Colourless oil, yield: 36%; IR (neat): 3286, 1644, 1487, 1300, 1257, 1189, 966, 693 cm−1. 1H NMR (400 MHz, CDCl3) δ = 1.37 (6H, s, C(CH3)2), 2.63 (2H, s, CH2C=O), 3.14 (2H, dd, J = 7.3, 1.5 Hz, CH2C=C), 4.33 (2H, d, J = 5.8 Hz, NHCH2), 5.90 (1H, br., NH), 6.24 (1H, m, CH2CH=CH), 6.47 (1H, d, J = 16 Hz, CH2CH = CH), 6.82 (1H, d, J = 8.5 Hz, H8), 7.16–7.32 (5H, m, ArCH2´-6´), 7.35 (1H, dd, J = 8.5, 2.3 Hz, H7), 7.65 (1H, d, J = 2.3 Hz, H5); 13CNMR (100Mz, CDCl3) δ = 26.6 ((CH3)2), 40.8 CH2C = C), 42.9 (NHCH2), 48.8 (CH2C = O), 79.4 (C2), 119.0 (C8), 119.9 (ArC), 122.1 (ArCH), 125.3 (C5), 126.4 (2 x ArCH), 127.9 (ArC), 128.6 (2 x ArCH), 130.6 (ArC), 135.0 (ArCH), 136.0 (C7), 136.5 (ArC), 159.4 (ArC), 170.6 (NHC=O), 192.4 (C4). HRMS (M-H)+ 348.1612, C22H22NO3 requires 348.1600.

General procedure for the synthesis of phosphonium salts (26)

A mixture of ω-bromocarboxylic acid (1 equiv.) and triphenylphosphine (1 equiv.) in 20 mL of toluene was refluxed for 24–48 h under nitrogen. The mixture was allowed to cool at room temperature and concentrated in vacuo. The residue was crystallized from various solvents to give the corresponding phosphonium salt in yields between 46–96%.

General procedure for the synthesis of unsaturated carboxylic acids (28)

To a stirred suspension of phosphonium salt 26 (1 equiv) in dry THF (10 mL) was slowly added lithium bis(trimethylsilyl)amide (1.0 M in THF). The solution was stirred for 30 min, and then cooled to −78 °C. A solution of benzaldehyde 27 (1 equiv.) was then added dropwise. The reaction was allowed to warm to room temperature overnight. Water and ether were added. The water layer was separated and acidified with 1 M aqueous hydrochloric acid to pH 1, then extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was chromatographed on silica gel (hexane:ethyl acetate) to afford the acid products as oils.

5-phenylpent-4-enoic acid (28f) ~2:1 mix of E/Z.

Commercial (3-carboxypropyl)triphenylphosphonium bromide (1 g, 2.33 mmol) was reacted using the general procedure to obtain 28f. Colourless waxy semi-solid, yield: 75%. 1H NMR (CDCl3, 400 MHz) δ = 2.49–2.71 (4H, m, (CH2)2), 5.64 & 6.22 (1H, 2 x m, C=CH), 6.45 & 6. 49 (1H, 2 x d, C=CH), 7.19–7.38 (5H, m, 5 x ArH); 13C NMR δ = 27.9, 33.8, 126.1, 127.2, 128.0, 128.5, 128.7, 130.1, 131.2, 137.2, 179.1 (signals of dominant E isomer).

6-phenylhex-5-enoic acid (28g) (2.5:1 mix of E/Z)

Commercial (4-carboxybutyl)triphenylphosphonium bromide (1 g, 2.26 mmol) was reacted using the general procedure to obtain 28g. Colourless oil, yield: 74%. 1H NMR (CDCl3, 400 MHz) δ = 1.83 (2H, m, (CH2), 2.25–2.43 (4H, m, (CH2)2), 5.63 & 6.18 (1H, 2 x dt, C=CH), 6.41 & 6.47 (1H, 2 x d, C = CH), 7.18–7.35 (5H, m, 5 x ArH); 13C NMR δ = 24.2, 32.3, 33.4, 126.0, 127.1, 128.2, 128.5, 128.7, 129.3, 131.0, 137.5, 180.1 (signals of dominant E isomer).

7-phenylhept-6-enoic acid (28h) 1.3:1 mix of E/Z.

26h (1.0 g, 2.19 mmol) was reacted using the general procedure to obtain 28h. Colourless oil, yield: 58%. 1H NMR (CDCl3, 400 MHz) δ = 1.53 (2H, m, (CH2), 1.70 (2H, m, (CH2), 2.22–2.43 (4H, m, (CH2)2), 5.64 & 6.21 (1H, 2 x dt, C=CH), 6.39 & 6.43 (1H, 2 x d, C = CH), 7.17–7.35 (5H, m, 5 x ArH); 13C NMR δ = 24.2 & 24.3, 28.2 & 28.7, 29.3 & 32.6, 33.9 & 33.9, 125.9, 126.5, 126.9, 128.1, 128.5, 128.5, 128.7, 129.3, 130.2, 130.2, 130.2, 130.2, 132.2, 133.8, 137.6 & 137.7, 172.0 & 179.9.

8-phenyloct-7-enoic acid (28i) 1:1.7 mix of E/Z.

26i (1.7 g, 3.61 mmol) was reacted using the general procedure to obtain 28i. Orange-brown oil, yield: 7%. 1H NMR (CDCl3, 400 MHz) δ = 1.33–1.53 (4H, m, (CH2)2), 1.64 (2H, m, (CH2), 2.17–2.38 (4H, m, (CH2)2), 5.64 & 6.21 (1H, 2 x dt, C=CH), 6.38 & 6.41 (1H, 2 x d, C=CH), 7.17–7.35 (5H, m, 5 x ArH); 13C NMR δ = 24.6, 28.4, 28.8, 29.6, 32.8, 125.9, 126.5, 128.2, 128.5, 128.8, 129.0, 132.8, 137.7, 179.8 (signals of dominant Z isomer).

(E)-N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-5-phenylpent-4-enamide (24f)

100 mg (0.49 mmol) of 23 was reacted as for 24a with 28f (86 mg, 0.49 mmol) to afford amide 24f. Colourless oil, yield: 48%; IR (neat): 3297, 2925, 1689, 1637, 1618, 1488, 1372, 1253, 1208, 1191, 964 cm−1. 1H NMR (CDCl3, 400 MHz) δ = 1.44 (6H, s, C(CH3)2), 2.38 (2H, br. t, CH2), 2.58 (2H, m, CH2), 2.69 (2H, s, CH2C=O), 4.39 (2H, d, J = 5.9 Hz, NHCH2), 5.84 (1H, br., NH), 6.20 (1H, 2 x t, J = 6.9 Hz, CH2CH=CH), 6.43 (1H, d, J = 16 Hz, CH2CH=CH), 6.82 (1H, d, J = 8.6 Hz, H8), 7.18–7.33 (5H, m, ArCH2´-6´), 7.39 (1H, dd, J = 8.5, 2.4 Hz, H7), 7.72 (1H, d, J = 2.4 Hz, H5); 13C NMR δ = 26.6 ((CH3)2), 29.0 (CH2), 36.4 (CH2), 42.7 (NHCH2), 48.8 (CH2C=O), 79.4 (C2), 119.0 (CH), 119.8 (ArC), 125.3 (CH), 126.1 (2 x CH), 127.2 (CH), 128.5 (2 x CH), 128.6 (CH), 130.8 (ArC), 131.2 (CH), 136.1 (CH), 137.3 (ArC), 159.4 (ArC), 172.1 (NHC=O), 192.5 (C4). HRMS (M-H)+ 362.1772, C23H24NO3 requires 362.1756.

N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-6-phenylhex-5-enamide, 3:1 E/Z (24g)

110 mg (0.54 mmol) of 23 was reacted as for 24a with 28g (102 mg, 0.54 mmol) to afford amide 24g. Semi-solid gum, yield: 30%; IR (neat): 3292, 2929, 1687, 1644, 1617, 1487, 1256, 1188 cm−1. 1H NMR (CDCl3, 400 MHz) δ = 1.44 (6H, s, C(CH3)2), 1.85 (2H, m, CH2), 2.18–2.41 (4H, m, (CH2)2), 2.70 (2H, s, CH2C=O), 4.27 & 4.38 (2H, 2 x d, J = 5.9 Hz, NHCH2), 5.66 & 5.80 (1H, 2 x br., NH), 5.62 & 6.18 (1H, 2 x m, CH2CH = CH), 6.37 & 6.46 (1H, 2 x d, J = 15.9 & 11.7 Hz, CH2CH=CH), 6.88 (1H, 2 x d, J = 8.6 Hz, H8), 7.17–7.33 (5H, m, ArCH2´-6´), 7.36 & 7.41 (1H, 2 x dd, J = 8.6, 2.2 Hz, H7), 7.68 & 7.72 (1H, 2 x d, J = 2.1 Hz, H5); 13C NMR δ = 25.1 & 25.8 (CH2), 26.6 ((CH3)2), 27.8 & 32.4 (CH2), 35.9 & 35.9 (CH2), 42.6 & 42.7 (NHCH2), 48.8 (CH2C=O), 79.4 & 79.4 (C2), 118.9 & 119.0 (CH), 119.8 & 119.9 (ArC), 125.2 & 125.3 (CH), 126.0 (2 x CH), 126.7 & 127.0 (CH), 128.5 (2 x CH), 128.2 & 128.7 (CH), 129.7 & 130.8 (CH), 129.8 & 130.9 (ArC), 136.1 & 136.1 (CH), 137.4 & 137.5 (ArC), 159.4 & 159.4 (ArC), 172.6 & 172.7 (NHC=O), 192.5 (C4). HRMS (M-H)+ 376.1906, C24H26NO3 requires 376.1913.

N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-7-phenylhept-6-enamide, 1.5:1 E/Z (24h)

65 mg (0.32 mmol) of 23 was reacted as for 24a with 28h (65 mg, 0.32 mmol) to afford amide 24h. Pale oil, yield: 27%; IR (neat): 3284, 2925, 1686, 1644, 1616, 1538, 1487, 1436, 1255, 1189, 983 cm−1. 1H NMR (CDCl3, 400 MHz) δ = 1.45 & 1.45 (6H, 2 x s, C(CH3)2), 1.51 (2H, m, CH2), 1.69 (2H, m, CH2), 2.15–2.38 (4H, m, (CH2)2), 2.70 & 2.71 (2H, 2 x s, CH2C = O), 4.36 & 4.39 (2H, 2 x d, J = 5.7 Hz, NHCH2), 5.65 & 5.73 (1H, 2 x br., NH), 5.64 & 6.20 (1H, 2 x m, CH2CH = CH), 6.37 & 6.42 (1H, 2 x d, J ~ 16 & 11.6 Hz, CH2CH = CH), 6.90 (1H, 2 x d, J = 8.6 Hz, H8), 7.17–7.34 (5H, m, ArCH2´-6´), 7.42 (1H, 2 x dd, J = 8.4, 2.4 Hz, H7), 7.72 (1H, m, H5); 13C NMR δ = 25.2 & 25.3 (CH2), 26.6 ((CH3)2), 28.1 & 32.8 (CH2), 29.0 & 29.4 (CH2), 36.5 & 36.6 (CH2), 42.7 & 42.7 (NHCH2), 48.8 (CH2C=O), 79.4 (C2), 119.0 (CH), 120.0 (ArC), 125.3 (CH), 126.0 (2 x CH), 126.5 & 126.9 (CH), 128.5 (2 x CH), 128.2 & 128.7 (CH), 129.2 & 130.2 (ArC), 130.4 & 130.9 (CH), 136.1 (CH), 137.6 & 137.7 (ArC), 159.4 (ArC), 172.8 & 172.8 (NHC=O), 192.5 (C4). HRMS (M+Na)+ 414.2040, C25H29NO3Na requires 414.2045.

N-((2,2-dimethyl-4-oxochroman-6-yl)methyl)-8-phenyloct-7-enamide, 1:1 E/Z (24i)

51 mg (0.25 mmol) of 23 was reacted as for 24a with 28i (50 mg, 0.23 mmol) to afford amide 24i. Semi-solid gum, yield: 22%; IR (neat): 2927, 1689, 1644, 1617, 1539, 1488, 1434, 1258, 11882 cm−1. 1H NMR (CDCl3, 400 MHz) δ = 1.32–1.53 (4H, m, CH2, signal overlap), 1.45 (6H, s, C(CH3)2), 1.61–1.73 (2H, m, CH2), 2.17–2.38 (4H, m, (CH2)2), 2.71 (2H, s, CH2C=O), 4.38 (2H, m, NHCH2), 5.70 (1H, br., NH), 5.64 & 6.20 (1H, 2 x m, CH2CH=CH), 6.39 (1H, m, CH2CH=CH), 6.90 (1H, d, J = 8.6 Hz, H8), 7.17–7.35 (5H, m, ArCH2´-6´), 7.42 (1H, dd, H7), 7.72 (1H, d, H5); 13C NMR δ = 25.5 (CH2), 26.6 ((CH3)2), 28.4 (CH2), 26.9(CH2), 29.6(CH2), 36.7 (CH2), 42.7 (NHCH2), 48.8 (CH2C=O), 79.3 (C2), 118.9 (ArCH), 119.8 (ArC), 125.2 (ArCH), 125.9 (ArCH), 126.5 (ArCH), 128.1 (ArCH), 128.5 (ArCH), 128.7 (ArCH), 129.9 (CH=CH), 130.8 (CH=CH), 132.8 (ArC), 136.0 (ArCH), 137.7 (ArC), 159.4 (ArC), 172.9 (NHC=O), 192.5 (C4). HRMS (M+Na)+ 428.2197, C26H31NO3Na requires 428.2202.

Biologic activity

Antileishmanial activity on L. infantum axenic amastigotes

L. infantum promastigotes (MHOM/MA/67/ITMAP-263, CNR Leishmania, Montpellier, France, expressing luciferase activity) were cultivated in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS), 2 mM L-glutamine and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) and harvested in the logarithmic phase of growth by centrifugation at 900 g for 10 min. The supernatant was carefully removed and replaced by the same volume of RPMI 1640 complete medium at pH 5.4, and then incubated for 24 h at 24 °C. The acidified promastigotes were then incubated for 24 h at 37 °C in a ventilated flask to transform promastigotes into axenic amastigotes. The effects of the tested compounds on the growth of L. infantum axenic amastigotes were assessed as follows. L. infantum amastigotes were incubated at a density of 2 × 106 parasites/mL in sterile 96-well plates with various concentrations of compounds dissolved in DMSO (final con-centration less than 0.5% v/v), in duplicate. Appropriate controls, DMSO and amphotericin, were added to each set of experiments. After a 48 h incubation period at 37 °C, each plate-well was then microscopically-examined to detect any precipitate formation. To estimate the luciferase activity of axenic amastigotes, 80 μL of each well were transferred to white 96-well plates, Steady Glow® reagent (Promega) was added according to the manufacturer’s instructions, and plates were incubated for 2 min. The luminescence was measured using a Microbeta Luminescence Counter (PerkinElmer). The inhibitory concentration 50% (IC50) was defined as the concentration of drug required to inhibit by 50% the metabolic activity of L. infantum amastigotes compared to control. IC50 values were calculated by non-linear regression analysis on dose response curves, using TableCurve 2D V5 software. IC50 values represent the mean of three independent experiments.

Cytotoxicity evaluation on J774A.1 cells

Evaluation of the cytotoxicity of test compounds was performed by MTT assay using the J774A.1 cell line (mouse macrophage cell line, Sigma-Aldrich). Briefly, cells (5 × 104 cells/mL) in 100 μL of complete medium, [DMEM High glucose supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin)] were seeded into each well of 96-well plates and incubated at 37 °C in a humidified 5% CO2 with 95% air atmosphere. After 24 h incubation, 100 µL of medium with various product concentrations and appropriate controls were added and the plates were incubated for 72 h at 37 °C. Each plate-well was then examined under the microscope to detect possible precipitate formation before the medium was aspirated from the wells. 100 µL of MTT solution (0.5 mg/mL in RPMI) was then added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with a microplate spectrophotometer. DMSO was used as blank and doxorubicin (Sigma Aldrich) as positive control. CC50 values were calculated by non-linear regression analysis on dose–response curves, using TableCurve 2D V5 software.