Synthesis of uniflorol B, a chromanone metabolite of Calea uniflora, and investigation of novel analogues as anti-leishmanial agents

Abstract

In this work, we report the first synthesis of the anti-leishmanial natural product uniflorol B. Uniflorol B is a chromanone metabolite first isolated from the Brazilian medicinal plant Calea uniflora Less. and possesses anti-leishmanial activity. We prepared uniflorol B, the E isomer of the natural product, through an 8-step approach featuring Kabbe condensation, regioselective ketone reduction and Morita–Baylis–Hillman elaboration of the side chain. We extended the methodology to four novel analogues and tested all compounds for their activity against various species of Leishmania. The most potent activity was seen with compound 22, with an IC₅₀ of 64.8 µM against L. braziliensis.

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Introduction

Chromanones are one of the key divisions of the broader benzopyran class and are defined by the presence of a keto functionality. They occur widely in nature and have found many applications in medicinal chemistry. They have been described as privileged structures and their pharmacological properties and potential have been widely reviewed.^1,2 Diverse examples have been reported, including chroman-4-one derivatives found to act as selective inhibitors of sirtuin-2 (SIRT2) associated with age-related neurodegenerative disease,³ CDK6 (cyclin-dependent kinase 6) inhibitors for the treatment of chemotherapy-induced myelosuppression,⁴ or NO-donor derivatives that act as vasodilators in the search for potential antihypertensive agents (Fig. 1).⁵

Fig. 1 Examples of biologically active chromanone-containing molecules and structures of uniflorols A (Z isomer) and B (E isomer).

A subset of the chromanones is those with a 2,2-dimethyl substitution.

These compounds may be prepared through various routes,⁶ which include the reaction of phenols with 3,3-dimethylacrylic acid or its derivatives (tandem esterification/Fries rearrangement/intramolecular oxa-Michael addition sequence),⁷ or Claisen–Schmidt condensation of o-hydroxy-acetophenones with acetone (Kabbe reaction),^8,9 the latter approach being perhaps the most convenient and practical method (Scheme 1). We were particularly interested in 6-substituted chromanones, notably the metabolites of various Calea species, typified by the diastereomeric uniflorols A and B (Fig. 1).¹⁰ These compounds possess a 2,2-dimethylchroman-4-one skeleton with a 2-(hydroxymethyl)but-2-enoate side chain esterified to a hydroxyethyl group at position 6. Both the natural uniflorols⁴ and various derivatives^11,12 have demonstrated interesting anti-parasitic activity and prompted our exploration of a route to the natural product uniflorol B.

Scheme 1 Synthetic approaches to 2,2-dimethylchromanones.

Results and discussion

Our retrosynthetic analysis for the total synthesis of uniflorol B (1) is shown in Scheme 2.

Scheme 2 Retrosynthetic analysis of uniflorol B.

It was envisioned that uniflorol B (1) could be elaborated from acrylate 3 via the Morita–Baylis–Hillman reaction (MBHR) followed by isomerization of allyl alcohol 2. The acrylate 3 would be obtained from alcohol 4 by reaction with acryloyl chloride. The alcohol 4 in turn could be prepared from chromanone 5 by regioselective reduction of the acetyl moiety. The chromanone core could be elaborated through Fries rearrangement of a suitable phenylacetate to furnish 6, followed by cyclisation, Claisen–Schmidt condensation of o-hydroxyacetophenone 6 with acetone (Kabbe reaction), as previously reported.¹¹ Scheme 3 depicts the synthetic route to the chromanone core of uniflorol B.

Scheme 3 Access to the acetylated chromanone core from p-hydroxyacetophenone.

Commercial p-hydroxyacetophenone 7 was acetylated to 8 in excellent yield and subjected to Fries rearrangement in an aluminium chloride melt. Kabbe condensation in the ionic liquid [bmim]Br afforded chromanone 5 in 67% yield. This reaction was also performed in ethanol in place of the ionic liquid, which resulted in the obtention of 5 in 61% yield.

As the acetyl electron-withdrawing group disfavoured the Fries rearrangement and cyclization, we also investigated the use of p-ethylphenol as the starting material (Scheme 4).

Scheme 4 Access to the acetylated chromanone core from p-ethylphenol.

Commercial p-ethylphenol 9 was acetylated as previously described to 10 in excellent yield and subjected to Fries rearrangement in triflic acid (conditions easier to manage than the aluminium chloride melt on a multigram scale) to afford 11 in good yield. It is also interesting to mention that this intermediate (11) is commercially available and cheap. Kabbe condensation in refluxing ethanol afforded chromanone 12 in 79% yield. Finally, oxidation of intermediate 11 in its benzylic position was accomplished using an excess of potassium persulfate and a catalytic amount of Cu(II) salt to furnish 5 in 81% yield. Attempts using a stoichiometric amount of oxidizing agent were made in order to obtain alcohol 4. However, this resulted in the obtention of a mixture of the starting material, alcohol 4 and ketone 5, respectively, in a ratio of 32 : 32 : 36.

With 5 in hand, the regioselective reduction of methylketone was then investigated (Scheme 5).

Scheme 5 Regioselective reduction to alcohol 4.

Enantioselective bioreduction of the prochiral methylketone 5 was already reported, and therefore, both R and S enantiomers are accessible depending on the selected biocatalyst.^13–15 However, this transformation is slow, inefficient on a multigram scale, and the yield is low (29%). As the absolute configuration of the natural product uniflorol has not yet been determined, we turned our attention towards more conventional reduction procedures of ketones. In this way, we showed that methylketone 5 could be reduced using sodium borohydride to a separable mixture of the desired alcohol 4 and its regioisomer 13 and, furthermore, via hydrogenation over Pd/C to furnish 4 in an improved yield (68%), along with side product 12.

Next, with alcohol 4 in hand, we focused on the synthesis of the side chain via a Morita–Baylis–Hillman approach from the corresponding acrylate (Scheme 6).

Scheme 6 Synthetic approach to the side chain of uniflorol B 1.

Thus, esterification using acryloyl chloride in the presence of triethylamine and a catalytic amount of DMAP quantitatively furnished intermediate 3, which was subjected to the Morita–Baylis–Hillman reaction with acetaldehyde to give allylic alcohol 2 in moderate yield (40%). Finally, we completed the synthesis of the natural product uniflorol 1 by brominative allylic transposition of 2 using N-bromosuccinimide and Me₂S, affording 14 regioselectively and stereoselectively as the (Z)-isomer as a result of an S_N2′ substitution of a bromide ion.¹⁶ Subsequent hydrolysis of 14 under buffered conditions (NaH₂PO₄/Na₂HPO₄) to prevent any ester hydrolysis gave uniflorol B 1 (as a mixture with less than 5% of its diastereomer uniflorol A).¹⁷ Its ¹H and ¹³C NMR data are consistent with those reported for the natural uniflorol B in ref. 10.

Furthermore, in attempts to decrease the number of required steps, esterification of 4 with crotonyl chloride was attempted, in order to avoid isomerization during the last step. Unfortunately, intermediate 15, resulting from isomerization of the double bond, was obtained as the major product instead of the desired intermediate 16. Such deconjugation of α,β-unsaturated acid chlorides in the presence of triethylamine and benzyl alcohol to afford β,γ-unsaturated benzylic esters and proceeding via the formation of an intermediate ketene was already reported.¹⁸ Using the strategy developed to afford 2, some analogues were also synthesised from acrylate intermediate 3 (Scheme 7). Thus, upon the MBH reaction using various aldehydes, analogues 17–20 were obtained in low to moderate yields.

Scheme 7 Synthetic approach to analogues 17–20 derived from the same synthetic route.

As this reaction proceeds with difficulty, a late-stage esterification strategy was also envisioned. The Baylis–Hillman carboxylic acid adducts are easily accessible on the gram scale. However, their esterification with chromanol under Steglich conditions is inefficient. In fact, such a reaction is scarcely mentioned in the literature. Esterification of Baylis–Hillman carboxylic acid adducts is performed from halogenated partners via an S_N2 mechanism. The few experiments carried out on spiranic analogues have resulted in the formation of the elimination product rather than the esterification product.

Finally, some analogues were also synthesized from alcohols 4 and 13 (Scheme 8).

Scheme 8 Synthetic approach to analogues 21, 22, 26, and 27 derived from alcohol intermediates 4, 13 and 25.

Thus, esterification under catalysed coupling conditions afforded esters 21 and 22. In an analogous manner, compounds 26 and 27 were obtained from commercially available 2′-hydroxyacetophenone 23 through Kabbe condensation in the presence of pyrrolidine and acetone, followed by reduction to 25 prior to esterification to the final analogues 26 and 27.

Some of these compounds were evaluated for anti-leishmanial activity against promastigote cultures of Leishmania amazonensis, L. braziliensis and L. infantum, with activity expressed as inhibition of 50% growth (IC₅₀) (Table 1).^19,20

Table 1 Anti-leishmanial activity of the synthesised compounds

Compound	MW (g mol^−1)	Anti-leishmanial activity IC50 (µM)
		Leishmania amazonensis	Leishmania braziliensis	Leishmania infantum
MW: molecular weight.
1	318.4	—	—	—
2	318.4	286.7	122.7	352.1
21	302.4	—	—	—
22	364.4	536.4	64.8	149.5
26	258.4	137.6	135.4	121.1
27	246.3	454.3	320.6	338.4
AMB	924.1	0.23	0.33	0.19

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Amphotericin B (AMB) was used as positive control. Among the compounds tested, the uniflorol B analogue 26 showed the greatest activity against L. amazonensis, with an IC₅₀ of 137.6 μM, while 22 showed the greatest activity against L. braziliensis with an IC₅₀ of 64.8 μM and 26 showed the greatest activity against L. infantum (IC₅₀ 121.1 μM).

Conclusions

In conclusion, the first total synthesis of uniflorol B was completed in 8 steps from the commercially available 4′-hydroxyacetophenone 7 in 6.4% overall yield and 7 steps from the commercially available 4′-ethyl-2′-hydroxyacetophenone 11 in 8.0% overall yield. The synthetic method involved esterification of a secondary benzylic alcohol with acryloyl chloride followed by a Morita–Baylis–Hillman reaction and alcohol transposition to achieve the target molecule. This synthetic strategy could be applied to the synthesis of further analogues to better understand structure–activity relationships.

Author contributions

Luana Budny Niero: investigation (lead) and writing – review and editing (equal). Daniela Pagliara Lage: biological evaluation (lead). Eduardo Antonio Ferraz Coelho: biological evaluation (lead). Ricardo Andrez Machado-de-Avila: biological evaluation (lead). James W. Barlow: conceptualization (supporting), methodology (supporting), writing – original draft (lead), and writing – review and editing (equal). Patricia de Aguiar Amaral: conceptualization (supporting), supervision (lead), methodology (supporting), and writing – review and editing (equal). Nicolas Gouault: conceptualization (lead), methodology (lead), and writing – review and editing (equal).

Conflicts of interest

There are no conflicts to declare.

Data availability

Supplementary information (SI): experimental details and characterisation data (including NMR spectra). See DOI: https://doi.org/10.1039/d6ob00708b.

Acknowledgements

The authors thank the University of Rennes, Rennes Métropole, Région Bretagne, Centre National de la Recherche Scientifique (CNRS), for financial support. The CRMPO of the Institut des Sciences Chimiques de Rennes for mass measurement is gratefully acknowledged. Part of this work was performed using the PRISM core facility (Biogenouest©, UMS Biosit, Université de Rennes 35000 RENNES, FRANCE).

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