Synthesis and anti-leishmanial activities of uniflorol
analogues

Paula da Silva Cardoso, Luana Budny Niero, Tiago Elias Allievi Frizon, Silvia Dalbo, Anne-Cécile Le Lamer, Nicolas Gouault, Patrícia de Aguiar Amaral, James W. Barlow

► To cite this version:

Paula da Silva Cardoso, Luana Budny Niero, Tiago Elias Allievi Frizon, Silvia Dalbo, Anne-Cécile Le Lamer, et al.. Synthesis and anti-leishmanial activities of uniflorol analogues. Medicinal Chemistry Research, 2024, 10.1007/s00044-024-03275-3 . hal-04690529

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# Synthesis and anti-leishmanial activities of uniflorol analogues

Paula da Silva Cardoso<fnref n="1" />,<fnref n="2" /> · Luana Budny Niero<fnref n="1" /> · Tiago Elias Allievi Frizon<fnref n="3" /> · Silvia DalBó<fnref n="1" /> · Anne Cécile Le Lamer<fnref n="4" /> · Nicolas Gouault<fnref n="5" /> · Patrícia de Aguiar Amaral<fnref n="1" /> · James W. Barlow ID<fnref n="2" />

Received: 14 May 2024 / Accepted: 29 June 2024 / Published online: 8 July 2024
© The Author(s) 2024

## Abstract

Chromanones are a subset of the benzopyran family, and display diverse biological activities, both as natural products and synthetic derivatives. Among these, we selected the natural product uniflorol, a 4-chromanone with an α,β-unsaturated ketone side chain, as a lead compound due to its reported anti-leishmanial properties. We designed and synthesised four series of novel compounds, varying the substitution patterns around the benzopyran core, and evaluated the compounds for anti-leishmanial activity against amastigotes of *L. infantum*. We prepared and characterised 24 novel compounds; upon screening, 12 compounds demonstrated activity values of <50 µM, with the most potent compound, **16d**, having an IC₅₀ of 7.29 µM. Activity was favoured in compounds bearing a phenylalkenyl motif, such as cinnamyl, styryl or a more lipophilic extension, and amide analogues retained activity. Uniflorol analogues display promise as novel architectures towards the development of potential anti-leishmanial agents.

<figure><img src="image_3.png" /></figure>

**Keywords** Chroman-4-one · Chroman · *Calea uniflora* · uniflorol · anti-leishmanial.

## 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

<figure><img src="image_4.png" /></figure>

<footnote>James W. Barlow
jambarlow@rcsi.com</footnote>

<footnote n="1">¹ Laboratory of Medicinal Plants (LaPlaM/PPGCA), Universidade do Extremo Sul Catarinense (UNESC), Avenida Universitária 1105, Bairro Universitário, 88806-000 Criciúma, SC, Brazil</footnote>

<footnote n="2">² Department of Chemistry, RCSI University of Medicine and Health Sciences, 123 St. Stephen's Green, Dublin 2, Ireland</footnote>

<footnote n="3">³ Laboratory of Advanced Materials (LMA/PPGES), Federal University of Santa Catarina, Araranguá, SC, Brazil</footnote>

<footnote n="4">⁴ Faculté des Sciences Pharmaceutiques, Université Toulouse III - Paul Sabatier, 35 Chemin des Maraîchers, 31062 Toulouse, Cedex 09, France</footnote>

<footnote n="5">⁵ Chimie Organique et Interfaces (CORINT), UMR 6226 Sciences Chimiques, Université de Rennes1, Rennes, France</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 1** Benzopyran derivatives identified in *C. uniflora*</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2** Benzopyran target structures based on metabolites of *C. uniflora*</figcaption></figure>

Series

<table><thead><tr><th></th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>R<sub>1</sub> = CH<sub>3</sub></td><td>R<sub>1</sub> = CH<sub>3</sub></td><td>R<sub>1</sub> = H</td><td>R<sub>1</sub> = CH<sub>3</sub></td><td>R<sub>1</sub> = CH<sub>3</sub></td></tr><tr><td>R<sub>2</sub> = CH<sub>3</sub></td><td>R<sub>2</sub> = CH<sub>3</sub></td><td>R<sub>2</sub> = H</td><td>R<sub>2</sub> = CH<sub>3</sub></td><td>R<sub>2</sub> = CH<sub>3</sub></td></tr><tr><td>R<sub>3</sub> = O</td><td>R<sub>3</sub> = H<sub>2</sub></td><td>R<sub>3</sub> = OH</td><td>R<sub>3</sub> = O</td><td>R<sub>3</sub> = O</td></tr><tr><td>R<sub>4</sub> = OH</td><td>R<sub>4</sub> = OH</td><td>R<sub>4</sub> = O</td><td>R<sub>4</sub> = NH<sub>2</sub></td><td>R<sub>4</sub> = NH<sub>2</sub></td></tr><tr><td>R<sub>5</sub> = CH<sub>3</sub></td><td>R<sub>5</sub> = CH<sub>3</sub></td><td>R<sub>5</sub> = CH<sub>3</sub></td><td>R<sub>5</sub> = H</td><td>R<sub>5</sub> = H</td></tr></tbody></table>

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].

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.
**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

<figure><img src="image_7.png" /></figure>

1

e

<figure><img src="image_8.png" /></figure>

13

$$R = \mathrm{CH}_{3}\mathrm{CH}=\mathrm{CHCO}\quad \mathbf{13a}$$

$$R = (\mathrm{CH}_{3})_{2}\mathrm{C}=\mathrm{CHCO}\quad \mathbf{13b}$$

$$R = \mathrm{C}_{6}\mathrm{H}_{5}\mathrm{CH}=\mathrm{CHCO}\quad \mathbf{13c}$$

$$R = \mathrm{C}_{6}\mathrm{H}_{5}\mathrm{CH}=\mathrm{CHCH}_{2}\mathrm{CO}\quad \mathbf{13d}$$

<figure><img src="image_9.png" /></figure>

9

14

15

c
7-53%

<figure><img src="image_10.png" /></figure>

16a-g

$$R = \mathrm{CH}_{3}\mathrm{CH}=\mathrm{CHCO}_{2}\quad \mathbf{16a}$$

$$R = \mathrm{CF}_{3}\mathrm{C}(\mathrm{CH}_{3})=\mathrm{CHCO}_{2}\quad \mathbf{16e}$$

$$R = \mathrm{CH}_{3}\mathrm{CH}=\mathrm{C}(\mathrm{CH}_{3})\mathrm{CO}_{2}\quad \mathbf{16b}$$

$$R = \mathrm{CH}_{3}\mathrm{CONH}\quad \mathbf{16f}$$

$$R = (\mathrm{CH}_{3})_{2}\mathrm{C}=\mathrm{CHCO}_{2}\quad \mathbf{16c}$$

$$R = \mathrm{CH}_{2}=\mathrm{CHCONH}\quad \mathbf{16g}$$

$$R = \mathrm{C}_{6}\mathrm{H}_{5}\mathrm{CH}=\mathrm{CHCH}_{2}\mathrm{CO}_{2}\quad \mathbf{16d}$$

**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

# Results and Discussion

## Chemistry

Synthetic approaches to chromanone skeletons have been reviewed; common approaches include Michael addition protocols, aldol condensations, the heating of *o*--(tri-methylsilyl)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%.

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.

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.

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
<figure><img src="image_11.png" /><figcaption>**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</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**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</figcaption></figure>

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.

## 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.

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):

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

<table><caption>**Table 1** Activity of chromanones against *L. infantum* amastigotes and evaluation of cytotoxicity</caption><thead><tr><th>Compound</th><th><i>L. infantum</i> IC<sub>50</sub> (µM)<sup>a</sup></th><th>J774A.1 CC<sub>50</sub> (µM)<sup>a</sup></th><th>SI<sup>b</sup></th></tr></thead><tbody><tr><td><b>1</b></td><td>&gt;227.00</td><td>&gt;227.00</td><td>ND<sup>c</sup></td></tr><tr><td><b>13a</b></td><td>101.44</td><td>116.63</td><td>1.15</td></tr><tr><td><b>13c</b></td><td>38.18 (3.48)</td><td>75.20 (1.87)</td><td>1.97</td></tr><tr><td><b>13d</b></td><td>12.10 (2.00)</td><td>53.20 (4.96)</td><td>4.40</td></tr><tr><td><b>16a</b></td><td>55.51</td><td>&gt;182.24</td><td>ND</td></tr><tr><td><b>16b</b></td><td>&gt;173.37</td><td>&gt;173.37</td><td>ND</td></tr><tr><td><b>16c</b></td><td>78.71</td><td>162.25</td><td>2.06</td></tr><tr><td><b>16d</b></td><td>7.29 (1.13)</td><td>80.09 (5.65)</td><td>10.99</td></tr><tr><td><b>16e</b></td><td>70.54</td><td>&gt;146.04</td><td>ND</td></tr><tr><td><b>16f</b></td><td>&gt;202.15</td><td>&gt;202.15</td><td>ND</td></tr><tr><td><b>16g</b></td><td>34.71 (2.20)</td><td>85.71 (9.22)</td><td>2.47</td></tr><tr><td><b>20</b></td><td>&gt;260.13</td><td>&gt;260.13</td><td>ND</td></tr><tr><td><b>21b</b></td><td>28.1 (1.12)</td><td>57.79 (0.38)</td><td>2.06</td></tr><tr><td><b>21c</b></td><td>63.1</td><td>92.34</td><td>1.46</td></tr><tr><td><b>21d</b></td><td>7.82 (0.78)</td><td>80.47 (6.58)</td><td>10.29</td></tr><tr><td><b>24a</b></td><td>&gt;182.93</td><td>95.75</td><td>ND</td></tr><tr><td><b>24b</b></td><td>&gt;174.00</td><td>101.57</td><td>ND</td></tr><tr><td><b>24c</b></td><td>42.09</td><td>41.27</td><td>0.98</td></tr><tr><td><b>24d</b></td><td>63.15 (5.40)</td><td>68.07 (3.06)</td><td>1.08</td></tr><tr><td><b>24e</b></td><td>11.64 (2.85)</td><td>60.30 (11.74)</td><td>5.18</td></tr><tr><td><b>24f</b></td><td>27.9 (2.88)</td><td>104.74 (10.46)</td><td>3.75</td></tr><tr><td><b>24g</b></td><td>20.63 (2.87)</td><td>9.46 (1.97)</td><td>0.46</td></tr><tr><td><b>24h</b></td><td>30.78 (1.19)</td><td>44.8 (5.01)</td><td>1.46</td></tr><tr><td><b>24i</b></td><td>&gt;12.33 (ND)</td><td>&gt;12.33 (ND)</td><td>ND</td></tr><tr><td colspan="4">Amphotericin B 0.072 (0.011)</td></tr><tr><td colspan="4">Doxorubicin 0.047 (0.004)</td></tr></tbody></table>

<figure><img src="image_13.png" /><figcaption>**Fig. 3** Structure activity map of anti-leishmanial uniflorol analogues</figcaption></figure>

phenyl-butylamine the most potent compound (IC<sub>50</sub> 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 IC<sub>50</sub> 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 (IC<sub>50</sub> 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.

<footnote><sup>a</sup>Results are reported as the mean value ± standard deviation of the half-maximum concentration in µM. <sup>b</sup>SI (Selectivity Index) = CC<sub>50</sub>/IC<sub>50</sub>. <sup>c</sup>Non-determinable as IC<sub>50</sub> and/or CC<sub>50</sub> > 50µg/mL (max. concentration tested).</footnote>
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, CH₃CH), 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 (100 MHz, 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-enoamide (24a)
To a solution of trans-2-butoenoic acid (0.084 g, 0.98 mmol) in dichloromethane (15 mL) at 0°C 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 (NHCC=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 (NHCC=O), 192.5 (C4). HRMS (M-H)⁺ 288.1446, C₁₇H₂₀NO₃ requires 288.1443.

*N*-(2,2-dimethyl-4-oxochroman-6-yl)methyl)-3-(thio-
phen-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 (NHCC=O), 192.5 (C4). HRMS (M + H)⁺ 342.1149, C₁₉H₂₀NO₃S requires 342.1164.

*N*-(2,2-dimethyl-4-oxochroman-6-yl)methyl)cinnama-
mide (**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 (NHCC=O), 192.5 (C4). HRMS (M-H)⁺ 334.1434, C₂₁H₂₀NO₃ requires 334.1433.

*(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
(NHCC=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 tri-
phenylphosphine (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, 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 (NHCC=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 (NHCC=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, ArCH<sup>2-6</sup>), 7.42 (1H, 2 x dd, *J* = 8.4, 2.4 Hz, *H*7), 7.72 (1H, m, *H*5); <sup>13</sup>C NMR *δ* = 25.2 & 25.3 (CH<sub>2</sub>), 26.6 ((CH<sub>3</sub>)<sub>2</sub>), 28.1 & 32.8 (CH<sub>2</sub>), 29.0 & 29.4 (CH<sub>2</sub>), 36.5 & 36.6 (CH<sub>2</sub>), 42.7 & 42.7 (NHCH<sub>2</sub>), 48.8 (CH<sub>2</sub>C=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)<sup>+</sup> 414.2040, C<sub>25</sub>H<sub>29</sub>NO<sub>3</sub>Na requires 414.2045.

*N*--((2,2-dimethyl-4-oxochroman-6-yl)methyl)-8-phenyl-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, *H*8), 7.17–7.35 (5H, m, ArCH<sup>2-6</sup>), 7.42 (1H, dd, *H*7), 7.72 (1H, d, *H*5); <sup>13</sup>C 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)<sup>+</sup> 428.2197, C<sub>26</sub>H<sub>31</sub>NO<sub>3</sub>Na 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.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1007/s00044-024-03275-3.

**Acknowledgements** This research was funded by CAPES-CSF-PVE-S (Programa Pesquisador Visitante Especial) Processo: 88881.064993/2014-01.

**FundingInformation** Open Access funding provided by the IReL Consortium.

### Compliance with ethical standards

**Conflict of interest** The authors declare no competing interests.
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