Synthesis and antimalarial and antituberculosis activities of a series of natural and unnatural 4-methoxy-6-styryl-pyran-2-ones, dihydro analogues and photodimers

Abstract

Previous studies have identified the 3,6-dialkyl-4-hydroxy-pyran-2-one marine microbial metabolites pseudopyronines A and B to be modest growth inhibitors of Mycobacterium tuberculosis and a range of tropical diseases including Plasmodium falciparum and Leishmania donovani. In an effort to expand the structure-activity relationship of this compound class towards. infectious diseases, a library of natural product and natural product-like 4-methoxy-6-styryl-pyran-2-ones and a subset of catalytically reduced examples were synthesised. In addition, the photochemical reactivity of several of the 4-methoxy-6-styryl-pyran-2-ones were investigated yielding head-to-head and head-to-tail cyclobutane dimers as well as examples of asymmetric aniba-dimer A-type dimers. All compounds were evaluated for cytotoxicity and activity against M. tuberculosis, P. falciparum, L. donovani, Trypanosoma brucei rhodesiense and T. cruzi. Of the styryl-pyranones, natural product 3 and non-natural styrene and naphthalene substituted examples 13, 18, 21, 22 and 23 exhibited antimalarial activity (IC₅₀ < 10 μM) with selectivity indices (SI) > 10. Δ⁷ Dihydro analogues were typically less active or lacked selectivity. Head-to-head and head-to-tail photodimers 5 and 34 exhibited moderate IC₅₀s of 2.3 to 17 μM towards several of the parasitic organisms, while the aniba-dimer-type asymmetric dimers 31 and 33 were identified as being moderately active towards P. falciparum (IC₅₀ 1.5 and 1.7 μM) with good selectivity (SI ∼ 80). The 4-tert-butyl aniba-dimer A analogue 33 also exhibited activity towards L. donovani (IC₅₀ 4.5 μM), suggesting further elaboration of this latter scaffold could lead to the identification of new leads for the dual treatment of malaria and leishmaniasis.

1. Introduction

4-Methoxy-6-styryl-2H-pyran-2-ones are polyketide synthase-derived natural products commonly reported from fungi and primitive angiosperms.^1,2 The most well known representatives of this class of compounds are the kavapyrones being neuroactive and potentially hepatotoxic metabolites of Kava (Piper methysticum).³ The related dihydrostyryl- and styryl-pyrones 1, 2 and 3, isolated from a number of sources, including Alpinia speciosa⁴ and Polygala sabulosa,⁵ exhibit plant growth inhibiting properties⁴ as well as anxiolytic⁶ and antinociceptive⁷ (visceral pain) effects in mice. There are however few reports of the evaluation of this family of natural products for activity towards infectious diseases such as trypanosomiasis, leishmaniasis, malaria or tuberculosis.^8-10 Pyrone 3 displays weak activity against American Trypanosoma cruzi blood trypomastigotes,⁹ while 2 and 4, isolated from Piper sanctum, were growth inhibitors of Mycobacterium tuberculosis H₃₇Rv with MICs of 32 μg/mL and 4 μg/mL respectively.¹⁰

Styrylpyrones such as 4 are also known to undergo photochemically-induced dimerization, to form symmetrical head-to-head (5)^11,12 or head-to-tail (6)^13,14 cyclobutane dimers or unsymmetrical dimers (7).^15,16 Although examples of each dimer class have been reported as natural products, it is still unclear whether they are true natural products or artifacts generated during the process of isolation and purification. Head-to-tail and asymmetric dimers related to 6 and 7, isolated from extracts of the Colombian medicinal plant Achyrocline bogotensis, were recently reported to modulate cytokine production by chemically stimulated human peripheral blood mononuclear cells.¹⁷

During our recent study of the structures and anti-infective bioactivities of the marine microbial pyrones pseudopyronines A (8) and B (9), the latter was found to be a modest growth inhibitor of Mycobacterium tuberculosis (MIC 2.7 μM), Trypanosoma brucei rhodesiense (IC₅₀ 42 μM), Leishmania donovani (IC₅₀ 4.8 μM) and Plasmodium falciparum (K1 strain, chloroquine resistant) (IC₅₀ 48 μM) while the methylether analogue of pseudopyronine A 10 was similarly active towards L. donovani, less active towards M tuberculosis (MIC 45 μM) but more potent towards both T. brucei rhodesiense (IC₅₀ 14 μM) and P. falciparum (IC₅₀ 11 μM).¹⁸ In continuation of our interest in this class of natural products, we have synthesized a library of natural and unnatural 4-methoxy-6-styryl-2H-pyran-2-ones, and a selection of Δ⁷ dihydro and photochemically dimerized analogues, and evaluated their abilities to act as growth inhibitors of the neglected disease target organisms M. tuberculosis, L. donovani, T. brucei rhodesiense, T. cruzi and P. falciparum.

2. Chemistry

Target styrylpyrones were chosen to explore the effects of steric bulk, lipophilicity and extended conjugation on biological activity. To this end, the pyrones were prepared by aldol condensation of 4-methoxy-6-methyl-2H-pyran-2-one 11¹⁹ and a variety of aryl aldehydes (Scheme 1, Table 1).²⁰ Thus a solution of pyrone 11 and the appropriate aldehyde in anhydrous MeOH was added to a suspension of magnesium methoxide in anhydrous methanol and the resulting mixture heated at reflux for 6 hours. Upon cooling, the solvent was removed in vacuo and the crude product purified by either recrystallization or silica gel column chromatography.

Scheme 1.

Reagents and conditions: (a) RCHO, Mg(OMe)₂, MeOH

Table 1. 6-Styryl-pyran-2H-ones and their biological activities.

entry	No.	structure	M. tb.a	T.b.rhod.b	T. cruzic	L.don.d	P. falc.e	L6f	SI Pf.g
1	3		>350	31	170	270	3.0	160	54
2	4		>440	120	64	190	13	160	12
3	12		>370	290	>330	270	12	180	15
4	13		>370	38	240	49	6.0	190	32
5	14		>350	44	26	20	5.3	24	4
6	15		>370	47	180	230	5.6	16	3
7	16		>310	8.8	43	30	4.9	13	3
8	17		>270	34	40	25	6.3	64	10
9	18		>390	150	>350	220	6.7	190	29
10	19		170	290	>300	>300	7.2	26	4
11	20		>460	76	>410	240	17	140	8
12	21		>360	18	34	47	4.6	110	24
13	22		>360	2.7	>320	>320	1.3	28	22
14	23		>260	119	>240	>240	8.6	>240	>28

^aMycobacterium tuberculosis H₃₇Rv grown in GAST medium, MIC (μM). Highest test concentration was 100 μg/mL;

^bTrypanosoma brucei rhodesiense. IC₅₀ (μM);

^cTrypanosoma cruzi. IC₅₀ (μM);

^dLeishmania donovani. IC₅₀ (μM);

^ePlasmodium falciparum. IC₅₀ (μM);

^fL6 rat skeletal myoblast cell line. IC₅₀ (μM);

^gSelectivity index for Plasmodium falciparum against L6. All data is the mean value from duplicate assays.

The synthesis and characterisation of target pyrones 3,^5,21,22 4,^4,23 12,⁴ 13,⁴ 15,^22,24 and 18²⁰ have been previously reported and in all cases the spectroscopic and spectrometric data observed in the current study agreed with the previously reported values. The remaining new compounds were fully characterized by MS and NMR, and the data observed was consistent with the structures (see Experimental). Diffusion of hexane into a solution of 3 in acetone and slow evaporation of a methanolic solution of 14 yielded crystals of a suitable quality for X-ray analysis. The crystal structures of 3 and 14 and details of the crystallographic studies are given in the Supplementary data. Noteworthy in both cases is that these particular pyrones pack in the crystal unit cell in head-to-tail fashion, with alignment of Δ⁵ and Δ⁷ at intermolecular distances of 3.6-3.7 Å and 3.5-3.9 Å for 3 and 14, respectively (see Supplementary data). Previous studies have concluded that 3.6-4.1 Å is a typical intermolecular distance for cinnamic acid analogues to achieve [2 + 2] cycloaddition.²⁵ The alignment and intermolecular distances have a direct bearing on the observed solid state photochemical reactivities of these pyrones to yield aniba-dimer-type products (see later).

Catalytic hydrogenation²⁰ of pyrones 3, 4, 14, 15, 17, 21, 22, and 23 with 10% Pd/C under a hydrogen atmosphere yielded exclusively the Δ⁷ dihydro analogues 25⁵, 1²⁶, 24, 2⁵, 26, 27, 28, and 29, respectively, in yields of 49-96% (Table 2). The new compounds prepared were fully characterized by MS and NMR, and the data observed was consistent with the structures (see Experimental). Slow evaporation of a H₂O-MeOH solution of 2 gave crystals suitable for X-ray analysis, the structure and details of which are given in Supplementary data. Reduction of Δ⁷ led to loss of overall planarity of the molecule, compared to styryl pyrones 3 and 14.

Table 2. Alkyl-pyran-2H-ones and their biological activities.

entry	No.	R/structure	M. tb.a	T.b.rhod.b	T. cruzic	L.don.d	P. falc.e	L6f	SI Pfg
1	1		>430	280	240	190	>22	290	-
2	2		>360	>330	>330	>330	>18	150	-
3	24		170	78	55	24	11	95	9
4	25		>340	200	>310	270	>17	>310	-
5	26		130	65	36	15	7.2	50	7
6	27		270	85	76	55	>18	110	-
7	28		180	72	65	42	16	91	6
8	29		>260	200	>240	>240	3.6	50	14

^aMycobacterium tuberculosis H₃₇Rv grown in GAST medium, MIC (μM). Highest test concentration was 100 μg/mL;

^bTrypanosoma brucei rhodesiense. IC₅₀ (μM);

^cTrypanosoma cruzi. IC₅₀ (μM);

^dLeishmania donovani. IC₅₀ (μM);

^ePlasmodium falciparum. IC₅₀ (μM);

^fL6 rat skeletal myoblast cell line. IC₅₀ (μM);

^gSelectivity index for Plasmodium falciparum against L6. All data is the mean value from duplicate assays.

As noted earlier, the solid state photodimerization of 4-methoxy-6-styryl-2H-pyran-2-one 4 has been extensively studied and is known to yield three distinct dimer products, 5 - 7.^11-16 The three established photocyclisation products arise from [2+2] cycloadditions of styryl-pyrones in either head-to-head (5) or head-to-tail (6) orientations or cycloaddition between Δ⁵ of one molecule with Δ⁷ of a second molecule (7). Preliminary studies of the propensity of styryl pyrones 3, 4, 12 – 23 to undergo photochemical reactions identified 3, 4, 14, 15, 17 and 22 as being reactive and yielding of stable products. Samples of these pyrones were each suspended in water (∼1 mL) with vigorous stirring and exposed to a 300 W sunlamp from a distance of 15 cm for 5 hrs. Under these conditions pyrone 4 afforded dimers 5^11,12 and 7^14-16 in isolatable quantities. Trace quantities of dimer 6^13,14 in the crude product mixture were detected by ¹H NMR spectroscopy but could not be purified from the other major products of the reaction. The ¹H NMR data observed for 5 agreed with those reported for the cis-trans-cis head-to-head dimer previously obtained as a natural product from the leaves of Aniba gardneri (though the structure was originally assigned as 6-cis-styryl-4-methoxy-2-pyrone)¹⁵ or as a synthetic photodimerization product.^11,12 The ¹H NMR data observed for 7 also agreed with those previously reported for aniba-dimer-A.¹⁴ Photodimerization of pyrone 3 gave the previously reported natural products 30 and 31,²² while pyrones 14, 15, 17 and 22 each gave a single product of 33 (asymmetric), 32 (head-to-head), 35 (asymmetric), and 34 (head-to-tail) respectively (Table 3). The structures and relative configuration of asymmetric dimers 33 and 35 were readily established by interpretation of 2D NMR data and comparison with NMR chemical shifts observed for aniba-dimer A (7). Dimer 32 was judged to be of head-to-head orientation by comparison of NMR chemical shifts with that of 5, and by ESI-MS-MS analysis, which identified a major fragment of the pseudomolecular parent ion to be m/z 277.0690 ([M+H]⁺, calcd for C₁₄H₁₃O₆ 277.0707), which is only possible for a head-to-head dimer. A similar ion was observed in ESI-MS-MS analysis of 5. The cis-trans-cis relative configuration of 32 was assumed to match that of 5, which has been rigorously established.¹² In contrast, 2-naphthalene dimer 34 was determined to be of head-to-tail orientation, based upon ESI-MS-MS analysis which only detected a fragment at m/z 279.1005 ([M+H]⁺, calcd for C₁₈H₁₅O₃ 279.1021). Similar retro-[2+2] fragmentation was observed for dimers 6 and 30 (see Experimental).

Table 3. Dimeric pyranones and their biological activities.

entry	No.	R	M. tb.a	T.b.rhod.b	T. cruzic	L.don.d	P. falc.e	L6f	SI Pfg
1	30	3,4-dimethoxyphenyl	>170	6.2	38	75	3.1	22	7
2	34	2-naphthyl	>180	4.9	90	10	2.3	95	42
3	5	phenyl	>220	15	32	17	3.8	51	13
4	32	benzo[3,4]dioxomethylene	>180	ndh	nd	nd	nd	nd	nd
5	7	phenyl	>220	13	28	27	7.1	>200	>28
6	31	3,4-dimethoxyphenyl	>170	61	130	79	1.5	120	80
7	33	4-tert-butyl-phenyl	>180	21	71	4.5	1.7	130	76
8	35	3-(4-tert-butyl-phenoxy)-phenyl	>130	>120	>120	66	4.4	>120	>27

^aMycobacterium tuberculosis H₃₇Rv grown in GAST medium, MIC (μM). Highest test concentration was 100 μg/mL;

^bTrypanosoma brucei rhodesiense. IC₅₀ (μM);

^cTrypanosoma cruzi. IC₅₀ (μM);

^dLeishmania donovani. IC₅₀ (μM);

^ePlasmodium falciparum. IC₅₀ (μM);

^fL6 rat skeletal myoblast cell line. IC₅₀ (μM);

^gSelectivity index for Plasmodium falciparum against L6. All data is the mean value from duplicate assays.

3. Biological results and discussion

3.1. Overview

The resultant library of 6-styryl-pyran-2-ones, dihydro analogues and photodimers were evaluated for anti-tuberculosis, anti-parasitic and cytotoxic activities. Anti-tuberculosis testing was carried out against the Mycobacterium tuberculosis H₃₇Rv strain grown in GAST medium. The reported MIC was determined as the minimal concentration of test compound leading to 100% growth inhibition. To investigate the potential of the compounds to act as drug leads against human African trypanosomiasis (HAT), American trypanosomiasis, leishmaniasis or malaria, their abilities to inhibit the growth of Trypanosoma brucei rhodesiense (strain STIB 900, trypomastigote stage), T. cruzi (strain Tulahuen C2C4, amastigote stage), Leishmania donovani (strain MHOM-ET-67/L82, amastigote/axenic stage) and Plasmodium falciparum (strain K1, IEF stage) were evaluated. In order to establish selectivity indices, the cytotoxicity of compounds towards the rat skeletal myoblast cell line L6 were also determined. Summaries of these assay results are presented in Tables 1, 2 and 3, with compounds grouped into the three different classes of 6-styryl-pyran-2-ones (Table 1), their dihydro analogues (Table 2) and photodimers (Table 3).

3.2. Anti-tuberculosis activity

Overall, none of the library compounds exhibited any appreciable level of anti-tuberculosis activity. This finding was somewhat surprising given the reported activities of the styryl-pyranone 4 and dihydro analogue 2 natural products towards M. tuberculosis H₃₇Rv.¹⁰ When compared to the moderately antimycobacterial activities observed for 3,6-dialkyl-pyranones 8 - 10 and other 6-alkyl substituted pyranones¹⁸ the current results indicate that the presence of aryl-alkenyl/alkyl substitution at C-6 is detrimental to activity.

3.3 Anti-parasitic activity

On the whole, the library of 6-styryl-pyran-2-ones exhibited moderate activity towards Plasmodium falciparum, with IC₅₀s varying between 1.3 to 17 μM (Table 1). Of particular note was the potency of antimalarial activity coupled with lack of cytotoxicity towards the L6 cell line of dimethoxy 3 (entry 1), para-nitro 13 (entry 4), aryl-diene 18 (entry 9), both naphthalene analogues 21 and 22 (entries 12 and 13) and dipyrone 23 (entry 14). In all cases, these pyrones exhibited antimalarial activity with IC₅₀ < 10 μM, and with a selectivity index greater than 10, identifying them as potentially useful starting points for further structure-activity optimisation studies. Only two examples of styryl-pyran-2-ones, 16 and 22, achieved activity towards Trypanosoma brucei rhodesiense with IC₅₀ < 10 μM. In the case of the former compound, broad ranging anti-parasitic activity was observed suggesting a general mechanism related to toxicity. Collectively, the styryl-pyran-2-ones exhibited only modest to poor activity towards T. cruzi and L. donovani. There was no apparent correlation of specific structural features with biological activity in this series of compounds.

The Δ⁷ dihydro analogues 1, 2, and 24-29 were typically less potent and lacked selectivity against the parasitic targets (Table 2) compared to their Δ⁷ counterparts. Three examples were identified as being more active growth inhibitors of P. falciparum (24, 26, 29; entries 3, 5 and 8) with IC₅₀s of 11, 7.2 and 3.6 μM respectively, however they lacked selectivity (SI 9, 7 and 14 respectively) versus the L6 cell line, limiting their therapeutic potential.

Symmetrical head-to-tail dimers 30 and 34 (Table 3, entries 1 and 2) and head-to-head dimer 5 (Table 3, entry 3) were found to be moderate growth inhibitors of two organisms, P. falciparum and T. brucei rhodesiense. In the cases of 5 and 34, limited cytotoxicity towards the L6 cell line identifies both classes of dimers as being useful starting points for further development. In contrast, the asymmetric aniba-dimer A-type compounds 7, 31, 33 and 35 exhibited antimalarial activity (IC₅₀ 1.5 to 7.1 μM) with moderate selectivity versus the L6 cell line. Of special note is the identification of tert-butyl-phenyl dimer 33 as a dual organism growth inhibitor exhibiting anti-leishmanial (IC₅₀ 4.5 μM) and anti-malarial (IC₅₀ 1.7 μM) activities. Although limited in scope, the results presented in Table 3 suggest that enhanced lipophilicity analogues of asymmetric aniba-dimer A-type compounds have the potential to act as novel scaffolds for the development of anti malarial or dual organism anti-malarial and anti-leishmanial drugs.

4. Conclusions

A library of natural product and natural product-like 4-methoxy-6-styryl-pyran-2-ones, selected Δ⁷ dihydro analogues and photodimers have been synthesized and several candidates identified as potential anti-malarial or dual organism anti-malarial / anti-T. brucei rhodesiense or anti-malarial / anti-leishmanial agents. Overall the styryl-pyranones were moderate growth inhibitors of P. falciparum but in many cases lacked selectivity. Similar conclusions were drawn for the Δ⁷ dihydro analogues. Cyclobutane head-to-tail and head-to-head photodimers were found to represent a new dual organism P. falciparum / T. brucei rhodesiense inhibiting scaffold while asymmetric dimers related to aniba-dimer A inhibited P. falciparum and in one example, also inhibited L. donovani. Further exploration of structure-activity relationships of these latter photodimers is somewhat constrained by the method of their synthesis – the crystal packing of styryl-pyranone subunits dictates the nature of the resultant dimeric product. We are currently exploring how the judicious choice of functionalization of the benzenoid ring of 4-methoxy-6-styryl-pyran-2-ones can be used to direct crystal packing and to facilitate the preparation of new leads for the treatment of neglected diseases.

5. Experimental

5.1. Chemistry: general methods

Mass spectra were recorded on either a VG-7070 or a a Bruker micrOTOF Q II mass spectrometer. Infrared spectra were run as dry films on sodium chloride or ATR crystal and acquired with a Perkin Elmer Spectrum One Fourier Transform infrared spectrometer with a Universal ATR Sampling Accessory. Melting points were obtained on an Electrothermal melting point apparatus and are uncorrected. ¹H NMR (300.13 or 400.13 MHz) and ¹³C NMR (75.47 or 100.62 MHz) spectra were run on a Bruker Avance 300 MHz or a Bruker DRX 400 MHz spectrometer. Chemical shifts are expressed in parts per million (ppm) relative to TMS in ¹H NMR and to deuterated solvent in ¹³C NMR (CDCl₃: ¹³C 77.0 ppm). Complete assignment of ¹H and ¹³C NMR resonances was based on interpretation of standard 2D NMR data. Pressurized (flash) column chromatography was performed on Kieselgel 60 0.063 - 0.200 mesh (Merck) silica gel. Analytical thin layer chromatography (TLC) was carried out on 0.2 mm thick plates of Kieselgel F₂₅₄ (Merck). Reactions were heated by immersion in oil or by use of DrySyn™ MULTI reaction block kit while the temperature was taken from a thermometer touching the bottom of the pyrex bath. Microanalyses were carried out by the Campbell Laboratory, University of Otago, Dunedin, New Zealand. 4-Methoxy-6-methyl-2-pyrone (11) was prepared by a literature method.¹⁹ The ¹H NMR data observed for pyrones 3 ⁵, 4 ²⁶, 12 ⁴, 13 ⁴, 15 ²⁴, 18 ²⁰, dihydro styryl pyrones 1 ²⁶ and 2 ⁵, and dimers 5, ^11,12 7 ¹⁴, 30 ²², and 31 ²² agreed with those previously reported in the literature.

5.2. General procedure for the preparation of 6-substituted 4-methoxy-2H-pyran-2-ones

To a suspension of magnesium methoxide, freshly prepared by gently heating Mg (104 mg) in anhydrous MeOH (10 mL), were added 4-methoxy-6-methyl-2-pyrone (11)¹⁹ (200 mg, 1.4 mmol) and aldehyde (1.7 mmol) under nitrogen.²⁰ The reaction mixture was heated at reflux for 6 hrs, allowed to cool and dried in vacuo. The solid was then suspended in acetic acid (3.3 M, 20 mL) and extracted with dichloromethane (4 × 50 mL), and the combined organic layers washed with water (2 × 50 mL) and dried in vacuo. Purification of the resultant pyrone was achieved either by trituration with diethylether followed by recrystallization from MeOH or by silica gel column chromatography eluting with 0-1% MeOH in dichloromethane.

5.2.1. 6-[2-(3,4-Dimethoxyphenyl)ethenyl]-4-methoxy-2H-pyran-2-one (3)

From pyrone (201 mg, 1.4 mmol) and 3,4-dimethoxybenzaldehyde (285 mg, 1.7 mmol). Repeated recrystallization from MeOH gave the product as a yellow crystalline solid (115 mg, 28%). Mp 162-163 °C (lit.²² 160-162 °C); R_f = 0.42 (3% MeOH:CH₂Cl₂); IR (ATR) 1700, 1550, 1408, 1252, 1153 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.42 (1H, d, J = 15.8 Hz, H-8), 7.05 (1H, dd, J = 8.3, 1.9 Hz, H-14), 7.00 (1H, d, J = 1.9 Hz, H-10), 6.84 (1H, d, J = 8.3 Hz, H-13), 6.43 (1H, d, J = 15.8 Hz, H-7), 5.88 (1H, d, J = 2.2 Hz, H-5), 5.44 (1H, d, J = 2.2 Hz, H-3), 3.90 (3H, s, OMe-11), 3.88 (3H, s, OMe-12), 3.79 (3H, s, OMe-4);¹³C NMR (CDCl₃, 75 MHz) δ 171.2 (C-4), 164.1 (C-2), 158.9 (C-6), 150.4 (C-12), 149.2 (C-11), 135.6 (C-8), 128.2 (C-9), 121.6 (C-14), 116.5 (C-7), 111.2 (C-13), 109.3 (C-10), 100.5 (C-5), 88.4 (C-3), 55.9 (2×OMe), 55.8 (OMe); (+)-ESIMS m/z 289 [M+H]⁺, (+)-HRESIMS m/z 289.1070 (calcd for C₁₆H₁₇O₅ 289.1071).

Crystallographic data (excluding structure factors) for the structure of compound 3 have been deposited with the Cambridge Crystallographic Data Centre as Supplementary Publication No. CCDC 831983. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

Crystal data

C₁₆H₁₆O₅, M 288.30, T 93(2) K, Triclinic, P-1, a = 8.6990(7) Å, b = 9.3889(8) Å, c = 10.0485(8) Å, α = 89.624(7)°, β= 68.010(6)°, γ = 67.643(6)°, V = 694.90(10) ų, Z = 2, D_c = 1.378 Mg/m³, μ = 0.103 mm⁻¹, F(000) = 304, crystal size 0.18 × 0.16 × 0.10 mm³, Θ_max = 28.17°, index ranges −11<=h<=11, −12<=k<=12, −13<=l<=13, reflections collected = 8711, independent reflections = 3359 [R(int) = 0.0688], data / restraints / parameters 3359 / 0 / 193. All non hydrogen atoms were identified after isotropic refinement of the initial solution. Full matrix least-squares refinement on F² was carried out to give R indices [I>2σ(I)], R₁ = 0.0730, wR₂ = 0.2232 and GOF = 1.084.

5.2.2. 4-Methoxy-6-styryl-2H-pyran-2-one (4)

From pyrone (200 mg, 1.4 mmol) and benzaldehyde (180 μL, 1.8 mmol). Recrystallization from MeOH gave the product as a white microcrystalline solid (153.1 mg, 47%). Mp 137-138 °C (lit.²³ 134-136 °C); R_f = 0.78 (3% MeOH:CH₂Cl₂); IR (smear) v_max 3076, 1719, 1635, 1607, 1555, 1446, 1406, 1256, 1152, 1001, 954, 831, 748, 685 cm^{−1; 13}C NMR (CDCl₃, 100 MHz) δ 171.1 (C-4), 164.0 (C-2), 158.6 (C-6), 135.8 (C-8), 135.2 (C-9), 129.4 (C-12), 128.9 (C-11), 127.4 (C-10), 118.6 (C-7), 101.3 (C-5), 88.9 (C-3), 55.9 (OMe); EIMS m/z 228 [M]⁺; HREIMS m/z 228.0788 (calcd for C₁₄H₁₂O₃ 228.0786); Anal.Calcd for C₁₄H₁₂O₃: C, 73.67; H, 5.30. Found: C, 73.63; H, 5.44. ¹H NMR data agreed with literature.²³

5.2.3. 4-Methoxy-6-(3-nitrostyryl)-2H-pyran-2-one (12)

From pyrone (202 mg, 1.4 mmol) and 3-nitrobenzaldehyde (259 mg, 1.7 mmol). Recrystallization from MeOH gave the product as a yellow amorphous solid (115 mg, 29%). Mp 235-236 °C; R_f = 0.76 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3078, 1726, 1526, 1347, 1257 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (1H, t, J = 1.8 Hz, H-10), 8.17 (1H, ddd, J = 8.1, 2.2, 1.0 Hz, H-12), 7.75 (1H, br d, J = 7.7 Hz, H-14), 7.55 (1H, t, J = 8.0 Hz, H-13), 7.52 (1H, d, J = 16.0 Hz, H-8), 6.70 (1H, d, J = 16.0 Hz, H-7), 6.02 (1H, d, J = 2.2 Hz, H-5), 5.53 (1H, d, J = 2.2 Hz, H-3), 3.84 (3H, s, OMe); ¹³C NMR (CDCl₃, 75 MHz) δ 170.7 (C-4), 163.5 (C-2), 157.4 (C-6), 148.8 (C-11), 137.0 (C-9), 133.6 (C-14), 132.9 (C-8), 130.0 (C-13), 123.6 (C-12), 121.5 (C-7), 121.1 (C-10), 102.9 (C-5), 89.7 (C-3), 56.1 (OMe); EIMS m/z 273 [M]⁺; HREIMS m/z 273.0637 (calcd for C₁₄H₁₁NO₅ 273.0637). ¹H NMR data agreed with literature.⁴

5.2.4. 4-Methoxy-6-(4-nitrostyryl)-2H-pyran-2-one (13)

From pyrone (199 mg, 1.4 mmol) and 4-nitrobenzaldehyde (258 mg, 1.7 mmol). Recrystallization from MeOH gave the product as an orange solid (21 mg, 5%). Mp 213-214 °C (lit⁴ 211.5-214 °C); R_f = 0.77 (3% MeOH:CH₂Cl₂); IR (smear) v_max 3081, 1694, 1610, 1591, 1553, 1513, 1448, 1337, 1254, 1154, 955, 810 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.23 (2H, d, J = 8.8 Hz, H-11), 7.62 (2H, d, J = 8.8 Hz, H-10), 7.51 (1H, d, J = 16.0 Hz, H-8), 6.70 (1H, d, J = 16.0 Hz, H-7), 6.03 (1H, d, J = 2.1 Hz, H-5), 5.54 (1H, d, J = 2.1 Hz, H-3), 3.84 (3H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 170.6 (C-4), 163.4 (C-2), 157.3 (C-6), 147.8 (C-12), 141.4 (C-9), 132.9 (C-8), 127.9 (C-10), 124.2 (C-11), 122.6 (C-7), 103.3 (C-5), 89.9 (C-3), 55.1 (OMe); EIMS m/z 273 [M]⁺; HREIMS m/z 273.06323 (calcd for C₁₄H₁₁NO₅ 273.06372). ¹H NMR data agreed with literature.⁴

5.2.5. 4-Methoxy-6-(4-tert-butyl-styryl)-2H-pyran-2-one (14)

From pyrone (201 mg, 1.4 mmol) and 4-tert-butylbenzaldehyde (290 μL, 1.7 mmol). Repeated recrystallization from MeOH gave the product as a white microcrystalline solid (104 mg, 25%). Mp 146-147 °C; R_f = 0.77 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2960, 1713, 1638, 1552, cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.48 (1H, d, J = 16.0 Hz, H-8), 7.43 (2H, m, H-10), 7.39 (2H, m, H-11), 6.54 (1H, d, J = 16.0 Hz, H-7), 5.92 (1H, d, J = 2.2 Hz, H-5), 5.47 (1H, d, J = 2.2 Hz, H-3), 3.81 (3H, s, OMe), 1.32 (9H, s, H-14); ¹³C NMR (CDCl₃, 100 MHz) δ 171.1 (C-4), 164.1 (C-2), 158.9 (C-6), 152.9 (C-12), 135.7 (C-8), 132.4 (C-9), 127.3 (C-10), 125.9 (C-11), 117.8 (C-7), 100.9 (C-5), 88.6 (C-3), 55.9 (OMe), 34.8 (C-13), 31.2 (C-14); EIMS m/z 284 [M]⁺; HREIMS m/z 284.1412 (calcd for C₁₈H₂₀O₃ 284.1412); Anal. Calcd for C₁₈H₂₀O₃: C, 76.03; H, 7.09. Found: C, 75.87; H, 7.35.

Crystallographic data (excluding structure factors) for the structure of compound 14 in this paper have been deposited with the Cambridge Crystallographic Data Centre as Supplementary Publication No. CCDC 831984. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

Crystal data

C₁₈H₂₀O₃, M 284.34, T 93(2) K, Triclinic, P2₁/n, a = 14.6633(5) Å, b = 7.1228(3) Å, c = 15.6152(6) Å, α= 90°, β= 113.468(3)°, γ = 90°, V = 1496.01(10) ų, Z= 4, D_c = 1.262 Mg/m³, μ = 0.085 mm⁻¹, F(000) = 608, crystal size 0.44 × 0.12 × 0.12 mm³, Θ_max = 27.99°, index ranges −19<=h<=19, −9<=k<=9, −20<=l<=20, reflections collected = 18895, independent reflections = 3588 [R(int) = 0.0656], data / restraints / parameters 3588 / 0 / 202. All non hydrogen atoms were identified after isotropic refinement of the initial solution. Full matrix least-squares refinement on F² was carried out to give R indices [I>2σ(I)], R₁ = 0.0465, wR₂ = 0.1007 and GOF = 1.013.

5.2.6. 6-[2-(1,3-Benzodioxo-5-yl)ethenyl]-4-methoxy-2H-pyran-2-one (15)

From pyrone (201 mg, 1.4 mmol) and piperanal (260 mg, 1.7 mmol). Recrystallization from MeOH gave the product as a yellow crystalline solid (155 mg, 40%). Mp 231-232 °C (lit.²⁴ 234-235°C); R_f = 0.43 (3% MeOH:CH₂Cl₂); ¹³C NMR (CDCl₃, 100 MHz) δ 171.1 (C-4), 164.0 (C-2), 158.8 (C-6), 148.9 (C-12), 148.3 (C-11), 135.5 (C-8), 129.7 (C-9), 123.5 (C-14), 116.8 (C-7), 108.6 (C-10), 105.9 (C-13), 101.4 (C-15), 100.7 (C-5), 88.5 (C-3), 55.9 (OMe); EIMS m/z 272 [M]⁺; HREIMS m/z 272.0686 (calcd for C₁₅H₁₂O₅ 272.0685); Anal. Calcd for C₁₄H₁₂O₃: C, 66.17; H, 4.44. Found: C, 66.12; H, 4.53. IR and ¹H NMR data agreed with literature.²⁴

5.2.7. 4-Methoxy-6-(3-phenoxy-styryl)-2H-pyran-2-one (16)

From pyrone (200 mg, 1.4 mmol) and 3-phenoxybenzaldehyde (295 μL, 1.7 mmol). Purification by repeated silica gel column chromatography eluting with 1% MeOH in dichloromethane gave the product as a yellow oil (104 mg, 23%). R_f = 0.58 (2% MeOH:CH₂Cl₂); IR (ATR)v_max 3078, 1721, 1641, 1607, 1554, 1484, 1233 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.42 (1H, d, J = 16.0 Hz, H-8), 7.35 (2H, m, H-18), 7.32 (1H, m, H-13), 7.21 (1H, m, H-14), 7.13 (1H, m, H-19), 7.11 (1H, m, H-10), 7.01 (2H, m, H-17), 6.97 (1H, m, H-12), 6.51 (1H, d, J = 16.0 Hz, H-7), 5.92 (1H, d, J = 2.0 Hz, H-5), 5.48 (1H, d, J = 2.0 Hz, H-3), 3.81 (3H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 163.8 (C-2), 158.3 (C-6), 157.9 (C-11), 156.7 (C-16), 137.0 (C-9), 135.1 (C-8), 130.2 (C-13), 129.9 (C-18), 123.6 (C-19), 122.5 (C-14), 119.7 (C-12), 119.3 (C-7), 119.1 (C-17), 116.9 (C-10), 101.6 (C-5), 89.0 (C-3), 55.9 (OMe); EIMS m/z 320 [M]⁺; HREIMS m/z 320.1051 (calcd for C₂₀H₁₆O₄ 320.1049).

5.2.8. 4-Methoxy-6-(3-(4-tert-butyl-phenoxy)-styryl)-2H-pyran-2-one (17)

From pyrone (202 mg, 1.4 mmol) and 3-(4-tert-butyl-phenoxy)benzaldehyde (434 mg, 1.7 mmol). Repeated recrystallization from MeOH gave the product as a white microcrystalline solid (121 mg, 22%). Mp 138-139 °C; R_f = 0.64 (2% MeOH:CH₂Cl₂); IR (ATR) v_max 2959, 1717, 1638, 1565, 1505, 1442, 1242 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.42 (1H, d, J = 16.0 Hz, H-8), 7.36 (2H, m, H-18), 7.31 (1H, m, H-13), 7.20 (1H, m, H-14), 7.10 (1H, br m, H-10), 6.97 (1H, m, H-12), 6.95 (2H, m, H-17), 6.52 (1H, d, J = 16.0 Hz, H-7), 5.92 (1H, d, J = 2.2 Hz, H-5), 5.48 (1H, d, J = 2.2 Hz, H-3), 3.81 (3H, s, OMe), 1.33 (9H, s, H-21); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 163.9 (C-2), 158.3 (C-6), 158.2 (C-11), 154.1 (C-16), 146.6 (C-19), 136.9 (C-9), 135.2 (C-8), 130.1 (C-13), 126.7 (C-18), 122.0 (C-14), 119.4 (C-12), 119.2 (C-7), 118.7 (C-17), 116.8 (C-10), 101.6 (C-5), 89.0 (C-3), 55.9 (OMe), 34.3 (C-20), 31.5 (C-21); EIMS m/z 376 [M]⁺; HREIMS m/z 376.1674 (calcd for C₂₄H₂₄O₄ 376.1675); Anal. Calcd for C₂₄H₂₄O₄: C, 76.57; H, 6.43. Found: C, 76.69; H, 6.58.

5.2.9. 4-Methoxy-6-(4-phenyl-1,3-butadienyl)-2H-pyran-2-one (18)

From pyrone (201 mg, 1.4 mmol) and cinnamaldehyde (450 μL, 3.6 mmol). Recrystallization from MeOH gave the product as a yellow solid (23 mg, 6%). Mp 189-190 °C (lit.²⁰ 188.5-190 °C); R_f = 0.81 (3% MeOH:CH₂Cl₂); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 164.0 (C-2), 158.7 (C-6), 138.3 (C-10), 136.4 (C-11), 136.3 (C-8), 128.8 (C-13), 128.6 (C-14), 127.1 (C-9), 127.0 (C-12), 122.0 (C-7), 100.9 (C-5), 88.7 (C-3), 55.9 (OMe); EIMS m/z 254 [M]⁺; HREIMS m/z 254.0936 (calcd for C₁₆H₁₄O₃ 254.0943); Anal. Calcd for C₁₄H₁₂O₃: C, 75.57; H, 5.55. Found: C, 75.28; H, 5.68. IR and ¹H NMR data agreed with literature.²⁰

5.2.10. 4-Methoxy-6-(4-(4-dimethylaminophenyl)-1,3-butadienyl)-2H-pyran-2-one (19)

From pyrone (200 mg, 1.4 mmol) and 4-dimethylaminocinnamaldehyde (298 mg, 1.7 mmol) and heating with magnesium methoxide in MeOH under nitrogen for 22 hrs. Recrystallization from MeOH gave the product as a red solid (21 mg, 5%). Mp 239-240 °C; R_f = 0.86 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 1720, 1595, 1541, 1523, 1258 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.33 (2H, m, H-12), 7.28 (1H, m, H-8), 6.75 (1H, m, H-10), 6.70 (1H, m, H-9), 6.65 (2H, m, H-13), 6.02 (1H, d, J = 15.2 Hz, H-7), 5.79 (1H, d, J = 2.0 Hz, H-5), 5.41 (1H, d, J = 2.0 Hz, H-3), 3.78 (3H, s, OMe), 2.98 (6H, s, H-16); ¹³C NMR (CDCl₃, 100 MHz) δ 171.2 (C-4), 164.3 (C-2), 159.4 (C-6), 150.7 (C-14), 139.1 (C-10), 137.4 (C-8), 128.4 (C-12), 124.6 (C-11), 122.7 (C-9), 119.2 (C-7), 112.1 (C-13), 99.7 (C-5), 87.9 (C-3), 55.8 (OMe), 40.2 (C-16); EIMS m/z 297 [M]⁺; HREIMS m/z 297.1365 (calcd for C₁₈H₁₉NO₃ 297.1365).

5.2.11. 4-Methoxy-6-[2-(2-furyl)ethenyl]-2H-pyran-2-one (20)

From pyrone (200 mg, 1.4 mmol) and furfuraldehyde (145 μL, 1.8 mmol). Purification by recrystallization from MeOH gave the product as a yellow solid (66 mg, 21%). Mp 174-175 °C; R_f = 0.46 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3091, 2917, 1697, 1634, 1543, 1449, 1145, 943 cm⁻¹; ¹H NMR (CDCl₃, 400 mHz) δ 7.43 (1H, d, J = 1.5 Hz, H-12), 7.23 (1H, d, J = 15.6 Hz, H-8), 6.49 (1H, m, H-10), 6.47 (1H, m, H-7), 6.44 (1H, m, H-11), 5.89 (1H, d, J = 2.2 Hz, H-5), 5.45 (1H, d, J = 2.2 Hz, H-3), 3.81 (3H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 163.9 (C-2), 158.4 (C-6), 151.6 (C-9), 144.0 (C-12), 122.6 (C-8), 116.5 (C-7), 113.3 (C-10), 112.3 (C-11), 101.2 (C-5), 88.7 (C-3), 55.9 (OMe); EIMS m/z 218 [M]⁺; HREIMS m/z 218.0575 (calcd for C₁₂H₁₀O₄ 218.0579); Anal. Calcd for C₁₂H₁₀O₄: C, 66.05; H, 4.62. Found: C, 66.34; H, 4.85.

5.2.12. 4-Methoxy-6-[2-(1-naphthyl)ethenyl]-2H-pyran-2-one (21)

From pyrone (200 mg, 1.4 mmol) and 1-napthaldehyde (233 μL, 1.7 mmol). Purification by repeated recrystallization from MeOH gave the product as a yellow solid (160 mg, 40%). Mp 120-121 °C; R_f = 0.83 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3079, 1724, 1636, 1556, 1411 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.29 (1H, d, J = 15.7 Hz, H-8), 8.23 (1H, d, J = 8.2 Hz, H-16), 7.85 (2H, m, H-12, 13), 7.72 (1H, d, J = 7.2 Hz, H-10), 7.58 (1H, m, H-15), 7.52 (1H, m, H-14), 7.47 (1H, m, H-11), 6.66 (1H, d, J = 15.7 Hz, H-7), 5.99 (1H, d, J = 2.2 Hz, H-5), 5.53 (1H, d, J = 2.2 Hz, H-3), 3.85 (3H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 164.0 (C-2), 158.6 (C-6), 133.7 (C-12a), 132.7 (C-9), 132.6 (C-8), 131.4 (C-16a), 129.8 (C-12), 128.6 (C-13), 126.7 (C-15), 126.2 (C-14), 125.4 (C-11), 124.1 (C-10), 123.6 (C-16), 121.3 (C-7), 101.6 (C-5), 89.0 (C-3), 55.9 (OMe); EIMS m/z 278 [M]⁺; HREIMS m/z 278.0942 (calcd for C₁₈H₁₄O₃ 278.0943).

5.2.13. 4-Methoxy-6-[2-(2-naphthyl)ethenyl]-2H-pyran-2-one (22)

From pyrone (201 mg, 1.4 mmol) and 2-napthaldehyde (269 mg, 1.7 mmol). Purification by repeated recrystallization from MeOH gave the product as a yellow crystalline solid (138 mg, 35%). Mp 201-202 °C; R_f = 0.56 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3081, 1709, 1638, 1549, 1448, 1408, 1251 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.87 (1H, d, J = 1.0 Hz, H-16), 7.82 (3H, m, H-11, H-12, H-15), 7.64 (1H, d, J = 15.9 Hz, H-8), 7.63 (1H, m, H-10), 7.48 (2H, m, H-14 and H-13), 6.68 (1H, d, J = 15.9 Hz, H-7), 5.96 (1H, d, J = 2.2 Hz, H-5), 5.49 (1H, d, J = 2.2 Hz, H-3), 3.81 (3H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 164.0 (C-2), 158.7 (C-6), 135.8 (C-8), 133.8 (C-11a), 133.4 (C-15a), 132.7 (C-9), 128.9 (C-16), 128.6 (C-11), 128.4 (C-15), 127.7 (C-12), 126.9 (C-13), 126.6 (C-14), 123.2 (C-10), 118.8 (C-7), 101.3 (C-5), 88.8 (C-3), 55.9 (OMe); EIMS m/z 278 [M]⁺; HREIMS m/z 278.0945 (calcd for C₁₈H₁₄O₃ 278.0943).

5.2.14. 6,6′-(1E,1′E)-2,2′-(1,4-phenylene)bis(ethene-2,1-diyl)bis(4-methoxy-2H-pyran-2-one) (23)

From pyrone (200 mg, 1.4 mmol) and terephthaldicarboxaldehyde (95 mg, 0.7 mmol). Purification by recrystallization from MeOH gave the product as a yellow solid (72.4 mg, 27%). Mp 330 °C (decomp.); R_f = 0.15 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3078, 1726, 1640, 1556, 1409 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.50 (4H, s, H-10), 7.48 (2H, d, J = 16.0 Hz, H-8), 6.60 (2H, d, J = 16.0 Hz, H-7), 5.96 (2H, d, J = 2.0 Hz, H-5), 5.50 (2H, d, J = 2.0 Hz, H-3), 3.83 (6H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 163.9 (C-2), 158.4 (C-6), 136.3 (C-9), 134.8 (C-8), 128.0 (C-10), 119.3 (C-7), 101.8 (C-5), 89.1 (C-3), 56.0 (OMe); FABMS m/z 379 [M+H]⁺; HRFABMS m/z 379.1188 (calcd for C₂₂H₁₉O₆ 379.1182).

5.3. General procedure for catalytic hydrogenation of pyrones

Pyrone was dissolved in MeOH/CHCl₃ (5 mL, 1:1) and stirred under a hydrogen atmosphere (balloon) for 2 h in the presence of 10% Pd/C. The suspension was filtered and the solvent evaporated in vacuo. Purification by silica gel column chromatography eluting with MeOH (0-1%) in dichloromethane gave the product.

5.3.1. 7,8-Dihydro-4-methoxy-6-styryl-2H-pyran-2-one (1)

From pyrone 4 (24.9 mg, 0.11 mmol) and Pd/C (10%, 6.8 mg) to give product 1 as a white solid (16.9 mg, 67%). Mp 99-100 °C (lit.²⁶ 96 - 97 °C); R_f = 0.59 (3% MeOH:CH₂Cl₂); EIMS m/z 230 [M]⁺; HREIMS m/z 230.0939 (calcd for C₁₄H₁₄O₃ 230.0943); Anal. Calcd for C₁₄H₁₄O₃: C, 73.03; H, 6.13. Found: C, 73.30; H, 6.11. ¹H and ¹³C NMR data agreed with the literature.²⁶

5.3.2. 6-[2-(1,3-Benzodioxo-5-yl)ethyl]-4-methoxy-2H-pyran-2-one (2)

From pyrone 15 (17.9 mg, 0.066 mmol) and Pd/C (10%, 4.8 mg) to give product 2 as white crystals (13.0 mg, 72%). Mp 143-144 °C (lit.⁵ 138-140 °C); R_f = 0.39 (3% MeOH:CH₂Cl₂); IR (smear) v_max 3108, 2927, 1705, 1645, 1567, 1239 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.71 (1H, d, J = 7.8 Hz, H-13), 6.64 (1H, d, J = 1.6 Hz, H-10), 6.60 (1H, dd, J = 7.8, 1.6 Hz, H-14), 5.91 (2H, s, H₂-15), 5.70 (1H, d, J = 2.3 Hz, H-5), 5.40 (1H, d, J = 2.1 Hz, H-3), 3.77 (3H, s, OMe), 2.88 (2H, t, J = 7.6 Hz, H₂-8), 2.68 (2H, t, J = 7.6 Hz, H₂-7); ¹³C NMR (CDCl₃, 100 MHz) δ 171.1 (C-4), 164.9 (C-2), 164.2 (C-6), 147.7 (C-11), 146.1 (C-12), 133.6 (C-9), 121.2 (C-14), 108.7 (C-10), 108.3 (C-13), 100.9 (C-15), 100.3 (C-5), 87.7 (C-3), 55.8 (OMe), 35.7 (C-7), 32.6 (C-8); EIMS m/z 274 [M]⁺; HREIMS m/z 274.0841 (calcd for C₁₅H₁₄O₅ 274.0841); Anal. Calcd for C₁₅H₁₄O₅: C, 65.69; H, 5.15. Found: C, 65.76; H, 5. 23.

Crystallographic data (excluding structure factors) for the structure of compound 2 have been deposited with the Cambridge Crystallographic Data Centre as Supplementary Publication No. CCDC 831985. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

Crystal data

C₁₅H₁₄O₅, M 274.26, T 98(2) K, Triclinic, P-1, a = 8.0036(2) Å, b = 8.3760(3) Å, c = 10.5599(3) Å, α= 90.8060(10)°, β= 105.8980(10)°, γ = 110.4210(10)°. V = 633.31(3) ų, Z = 2, D_c = 1.438 Mg/m³, μ = 0.109 mm⁻¹, F(000) = 288, crystal size 0.43 × 0.29 × 0.20 mm³, Θ_max = 27.98°, index ranges −10<=h<=10, −11<=k<=10, −13<=l<=13, reflections collected = 15272, independent reflections = 3024 [R(int) = 0.0243], data / restraints / parameters 3024 / 0 / 182. All non hydrogen atoms were identified after isotropic refinement of the initial solution. Full matrix least-squares refinement on F² was carried out to give R indices [I>2σ(I)], R₁ = 0.0342, wR₂ = 0.0944 and GOF = 1.033.

5.3.3. 4-Methoxy-6-[2-(4-tert-butylphenyl)ethyl]-2H-pyran-2-one (24)

From pyrone 14 (19.3 mg, 0.068 mmol) and Pd/C (10%, 5.2 mg) to give product 24 as a white solid (17.4 mg, 90%). Mp 77-79 °C; R_f = 0.42 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2959, 1713, 1567, 1451, 1411, 1248 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.31 (2H, d, J = 8.3 Hz, H-11/13), 7.11 (2H, d, J = 8.3 Hz, H-10/14), 5.75 (1H, d, J = 2.1 Hz, H-5), 5.41 (1H, d, J = 2.1 Hz, H-3), 3.77 (3H, s, OMe), 2.92 (2H, m, H₂-8), 2.73 (2H, m, H₂-7), 1.30 (9H, s, H-16); ¹³C NMR (CDCl₃, 100 MHz) δ 171.2 (C-4), 165.0 (C-2), 164.6 (C-6), 149.3 (C-12), 136.8 (C-9), 127.9 (C-10/14), 125.5 (C-11/13), 100.1 (C-5), 87.7 (C-3), 55.8 (OMe), 35.4 (C-7), 34.4 (C-15), 32.3 (C-8), 31.3 (C-16); (+)-ESIMS m/z 287 [M+H]⁺, (+)-HRESIMS m/z 287.1639 (calcd for C₁₈H₂₃O₃ 287.1642).

5.3.4. 6-[2-(3,4-Dimethoxyphenyl)ethyl]-4-methoxy-2H-pyran-2-one (25)

From pyrone 3 (20.8 mg, 0.072 mmol) and Pd/C (10%, 3.9 mg) to give 25 as a white solid (20.1 mg, 96%). Mp 71-73 °C; (lit.²⁷ 74 – 75 ^°C); R_f = 0.18 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 1709, 1651, 1569, 1518, 1453, 1241, 1136 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.77 (1H, d, J = 8.1 Hz, H-13), 6.69 (1H, dd, J = 8.1, 1.9 Hz, H-14), 6.66 (1H, d, J = 1.9 Hz, H-10), 5.69 (1H, d, J = 2.2 Hz, H-5), 5.40 (1H, d, J = 2.2 Hz, H-3), 3.84 (3H, s, OMe-11*), 3.83 (3H, s, OMe-12*), 3.76 (3H, s, OMe-4), 2.91 (2H, m, H₂-8), 2.71 (2H, m, H₂-7); ¹³C NMR (CDCl₃, 100 MHz) δ 171.2 (C-4), 165.1 (C-2), 164.3 (C-6), 148.8 (C-11), 147.5 (C-12), 132.4 (C-9), 120.1 (C-14), 111.6 (C-10), 111.3 (C-13), 100.4 (C-5), 87.6 (C-3), 55.8 (OMe), 55.7 (OMe), 35.7 (C-7), 32.4 (C-8); (+)-ESIMS m/z 313 [M+Na]⁺, (+)-HRESIMS m/z 313.1056 (calcd for C₁₆H₁₈NaO₅ 313.1046).

5.3.5. 4-Methoxy-6-[2-(3-(4-tert-butylphenoxy)phenyl)ethyl]-2H-pyran-2-one (26)

From pyrone 17 (21.2 mg, 0.056 mmol) and Pd/C (10% 3.8 mg) to give 26 as a colorless oil (19.6 mg, 92%). R_f = 0.49 (3% MeOHCH₂Cl₂); IR (smear) v_max 2960, 1721, 1568, 1247 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.33 (2H, m, H-18/20), 7.22 (1H, t, J = 7.8 Hz, H-13), 6.90 (2H, m, H-17/21), 6.87 (1H, m, H-14), 6.83 (1H, m, H-12), 6.81 (1H, m, H-10), 5.71 (1H, d, J = 2.1 Hz, H-5), 5.40 (1H, d, J = 2.1 Hz, H-3), 3.77 (3H, s, OMe), 2.93 (2H, m, H₂-8), 2.72 (2H, m, H₂-7), 1.32 (9H, s, H-23);¹³C NMR (CDCl₃, 75 MHz) δ 171.1 (C-4), 164.8 (C-2), 164.2 (C-6), 157.8 (C-11), 154.5 (C-16), 146.2 (C-19), 141.7 (C-9), 129.8 (C-13), 126.5 (C-18,20), 122.9 (C-14), 118.4 (C-17/21), 118.3 (C-10), 116.6 (C-12), 100.3 (C-5), 87.7 (C-3), 55.8 (OMe), 35.2 (C-7), 34.3 (C-22), 32.7 (C-8), 31.5 (C-23); (+)-ESIMS m/z 401 [M+Na]⁺, (+)-HRESIMS m/z 401.1725 (calcd for C₂₄H₂₆NaO₄ 401.1723).

5.3.6. 4-Methoxy-6-[2-(1-naphthyl)ethyl]-2H-pyran-2-one (27)

From pyrone 21 (21.1 mg, 0.076 mmol) to give 27 as a white solid (16.0 mg, 75%). Mp 75-77 °C; R_f = 0.40 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3078, 1714, 1647, 1563, 1465, 1257, 1138 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.02 (1H, d, J = 8.4 Hz, H-16), 7.86 (1H, d, J = 7.8 Hz, H-13), 7.73 (1H, d, J = 8.2 Hz, H-12), 7.50 (2H, m, H-15/H-14), 7.38 (1H, t, J = 7.6 Hz, H-11), 7.30 (1H, d, J = 7.6 Hz, H-10), 5.71 (1H, d, J = 2.2 Hz, H-5), 5.42 (1H, d, J = 2.2 Hz, H-3), 3.76 (3H, s, OMe), 3.42 (2H, m, H₂-8), 2.86 (2H, m, H₂-7); ¹³C NMR (CDCl₃, 100 MHz) δ 171.1 (C-4), 164.9 (C-2), 164.4 (C-6), 135.9 (C-9), 133.9 (C-12a), 131.5 (C-16a), 128.9 (C-13), 127.3 (C-12), 126.2 (C-10/15), 125.6 (C-11*), 125.5 (C-14*), 123.2 (C-16), 100.3 (C-5), 87.8 (C-3), 55.8 (OMe), 34.8 (C-7), 30.2 (C-8); (+)-ESIMS m/z 281 [M+H]⁺, (+)-HRESIMS m/z 281.1174 (calcd for C₁₈H₁₇O₃ 281.1172).

5.3.7. 4-Methoxy-6-[2-(2-naphthyl)ethyl]-2H-pyran-2-one (28)

From pyrone 22 (19.4 mg, 0.070 mmol) to give 28 as a white solid (16.3 mg, 84%). Mp 72-74 °C; R_f = 0.42 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3082, 1722, 1649, 1564, 1412, 1250, 1142 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.77 (3H, m, H-11,12,15), 7.61 (1H, br s, H-16), 7.43 (2H, m, H-13,14), 7.29 (1H, dd, J = 8.4, 1.6 Hz, H-10), 5.71 (1H, d, J = 2.1 Hz, H-5), 5.40 (1H, d, J = 2.1 Hz, H-3), 3.74 (3H, s, OMe), 3.13 (2H, m, H₂-8), 2.83 (2H, m, H₂-7); ¹³C NMR (CDCl₃, 100 MHz) δ 171.2 (C-4), 165.1 (C-2), 164.2 (C-6), 137.2 (C-9), 133.5 (C-15a), 132.1 (C-11a), 128.2 (C-11*), 127.6 (C-12*), 127.5 (C-15*), 126.7 (C-10), 126.6 (C-16), 126.1 (C-13*), 125.5 (C-14*), 100.4 (C-5), 87.7 (C-3), 55.8 (OMe), 35.3 (C-7), 32.9 (C-8); (+)-ESIMS m/z 281 [M+H]⁺, (+)-HRESIMS m/z 281.1172 (calcd for C₁₈H₁₇O₃ 281.1172).

5.3.8. 6,6′-(2,2′-(1,4-Phenylene)bis(ethane-2,1-diyl))bis(4-methoxy-2H-pyran-2-one) (29)

From pyrone 23 (19.7 mg, 0.052 mmol) and Pd/C (10%, 4.9 mg) to give 29 as a white solid (9.8 mg, 49%). Mp 196-198 °C; R_f = 0.06 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 1710, 1651, 1566, 1450, 1413, 1250, 1143 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.08 (4H, s, H-10), 5.70 (2H, d, J = 2.2 Hz, H-5), 5.39 (2H, d, J = 2.2 Hz, H-3), 3.77 (6H, s, OMe), 2.92 (4H, m, H₂-8), 2.70 (4H, m, H₂-7); ¹³C NMR (CDCl₃, 100 MHz) δ 171.1 (C-4), 164.8 (C-2), 164.3 (C-6), 138.0 (C-9), 128.5 (C-10), 100.2 (C-5), 87.7 (C-3), 55.8 (OMe), 35.4 (C-7), 32.4 (C-8); (+)-ESIMS m/z 405 [M + Na]⁺, (+)-HRESIMS m/z 405.1316 (calcd for C₂₂H₂₂NaO₆ 405.1309).

5.4. General procedure for photodimerization of 6-styryl-pyran-2-ones

6-Styryl-pyran-2-one was suspended, with stirring, in water and exposed to a sun lamp (300W, 15 cm from vial) for three 5 hr periods at which time the resulting crude product was purified by repeated column chromatography eluting with 0-5% MeOH in dichloromethane.

5.4.1. 6,6′-(3,4-Diphenylcyclobutane-1,2-diyl)bis(4-methoxy-2H-pyran-2-one) (5) and Aniba-dimer-A (7)

From pyrone 4 (43.0 mg, 0.19 mmol) to give the head-to-tail dimer 5 ^11,12 (8.0 mg, 19%) and aniba-dimer A^14-16 7 (4.2 mg, 10%), both as amorphous colorless solids.

Data for 5

Mp 82-84 °C (lit.¹¹ 103-105 °C; lit.¹² 170-172 ^°C); R_f = 0.24 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2918, 1714, 1646, 1563, 1454, 1409, 1239 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.12 (4H, m, H-11,13), 7.06 (2H, m, H-12), 6.95 (4H, m, H-10,14), 5.99 (2H, d, J = 2.3 Hz, H-5), 5.36 (2H, d, J = 2.3 Hz, H-3), 4.50 (2H, m, H-8), 4.11 (2H, m, H-7), 3.75 (6H, s, OMe); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 164.3 (C-2), 162.4 (C-6), 137.9 (C-9), 128.2 (C-11,13), 127.7 (C-10,14), 126.7 (C-12), 101.5 (C-5), 88.0 (C-3), 55.9 (OMe), 44.9 (C-8), 44.0 (C-7); (+)-ESIMS m/z 457 [M+H]⁺, (+)-HRESIMS m/z 457.1650 (calcd for C₂₈H₂₅O₆ 457.1646); (+)-HRESIMSMS (parent m/z 457) fragment m/z 277.0701 (calcd for C₁₄H₁₃O₆ 277.0707), 229.0853 (calcd for C₁₄H₁₃O₃ 229.0859).

Data for 7

Mp 178-180 °C (lit.¹⁵ 178-179 °C; lit.¹⁴ 185-188 oC); R_f = 0.20 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3078, 1704, 1647, 1623, 1567, 1244 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.41 (2H, m, H-10′,14′), 7.29 (8H, m, H-10,11,12,13,14,11′,12′,13′), 6.94 (1H, d, J = 15.9 Hz, H-8′), 6.59 (1H, d, J = 15.9 Hz, H-7), 5.91 (1H, d, J = 2.2 Hz, H-5), 5.34 (1H, d, J = 2.2 Hz, H-3), 5.28 (1H, s, H-3′), 4.35 (1H, t, J = 10.3 Hz, H-8), 4.16 (1H, d, J = 11.1 Hz, H-7), 3.71 (3H, s, OMe-4), 3.59 (1H, d, J = 10.3 Hz, H-5′), 3.27 (3H, s, OMe-4′); ¹³C NMR (CDCl₃, 100 MHz) δ 170.5 (C-4), 169.9 (C-4′), 164.6 (C-2*), 163.9 (C-2′*), 158.6 (C-6), 135.9 (C-9′), 135.6 (C-9), 131.5 (C-8′), 128.7, 128.5, 128.3, 127.8 (C-11,12,13,11′,12′,13′), 127.5 (C-10/14), 126.9 (C-10′/14′), 124.4 (C-7), 102.7 (C-5), 91.8 (C-3′), 88.7 (C-3), 79.4 (C-6′), 55.9 (OMe-4), 55.4 (OMe-4′), 54.5 (C-7), 45.7 (C-5′), 39.2 (C-8); (+)-ESIMS m/z 479 [M+Na]⁺, (+)-HRESIMS m/z 479.1453 (calcd for C₂₈H₂₄NaO₆ 479.1465).

5.4.2. 6,6′-(2,4-Bis(3,4-dimethoxyphenyl)cyclobutane-1,3-diyl)bis(4-methoxy-2H-pyran-2-one) (30) and 11,12,11′,12′-Tetramethoxy-aniba-dimer-A (31)

From pyrone 3 (19.9 mg, 0.069 mmol) to give the head-to-tail dimer 30 as an amorphous colorless solid (4.1 mg, 14%) and asymmetric dimer 31 as an amorphous yellow solid (6.9 mg, 35%).

Data for 30

Mp 86-88 °C (lit.²² 112-114 °C); R_f = 0.09 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2918, 2849, 1706, 1641, 1563, 1515, 1454, 1409, 1249, 1142, 1023 cm^{−1; 13}C NMR (CDCl₃, 100 MHz)δ 170.6 (C-4), 164.0 (C-2), 162.9 (C-6), 148.9 (C-11), 148.2 (C-12), 129.8 (C-9), 119.4 (C-14), 111.04 (C-10*), 110.96 (C-13*), 101.3 (C-5), 87.8 (C-3), 56.0 (OMe-11), 55.82 (OMe-12), 55.76 (OMe-4), 45.5 (C-7), 43.5 (C-8); (+)-ESIMS m/z 577 [M+H]⁺, (+)-HRESIMS m/z 577.2073 (calcd for C₃₂H33O₁₀ 577.2068); (+)-HRESIMSMS (parent m/z 577) fragment m/z 289.1055 (calcd for C₁₆H₁₇O₅ 289.1071). ¹H and ¹³C NMR chemical shifts agreed with the literature, however the previously reported assignments of C-2 and C-4 should be reversed.²²

Data for 31

Mp 99-100 °C (lit.²² 195-197 °C); R_f = 0.05 (3% MeOH:CH₂Cl₂); (+)-ESIMS m/z 599 [M+Na]⁺, (+)-HRESIMS m/z 599.1881 (calcd for C3₂H₃₂NaO₁₀ 599.1888). ¹H and ¹³C NMR data were in agreement with literature values, however the previously reported assignments of C-2 and C-4 should be reversed.²²

5.4.3. 6,6′-(3,4-Di(benzo[d][1,3]dioxo-5-yl)cyclobutane-1,2-diyl)bis(4-methoxy-2H-pyran-2-one) (32)

From pyrone 15 (19.3 mg, 0.071 mmol) to give 32 as an amorphous colorless solid (2.0 mg, 10%) and unreacted starting material 15 (5.6 mg, 29%). Mp 101-103 °C; R_f = 0.14 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2905, 1706, 1646, 1561, 1490, 1445, 1408, 1237, 1034 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.62 (2H, d, J = 8.0 Hz, H-13), 6.46 (2H, dd, J = 8.0, 1.7 Hz, H-14), 6.43 (2H, d, J = 1.7 Hz, H-10), 5.96 (2H, d, J = 2.1 Hz, H-5), 5.86 (4H, br s, H₂-15), 5.35 (2H, d, J = 2.1 Hz, H-3), 4.35 (2H, m, H-8), 3.96 (2H, m, H-7), 3.75 (6H, s, OMe-4); ¹³C NMR (CDCl₃, 100 MHz) δ 171.0 (C-4), 164.2 (C-2), 162.2 (C-6), 147.7 (C-11), 146.3 (C-12), 131.8 (C-9), 120.8 (C-14), 108.1 (C-10), 101.4 (C-5), 100.9 (C-15), 88.1 (C-3), 55.9 (OMe-4), 44.8 (C-8), 44.4 (C-7); (+)-ESIMS m/z 545 [M+H]⁺, (+)-HRESIMS m/z 545.1435 (calcd for C₃₀H₂₅O₁₀ 557.1959), (+)-HRESIMSMS (parent m/z 545) fragment m/z 277.0690 (calcd for C₁₄H₁₃O₆ 277.0707).

5.4.4. 12,12′-Di-tert-butyl-aniba-dimer-A (33)

From pyrone 14 (20.6 mg, 0.036 mmol) to give 33 as an amorphous colorless solid (9.5 mg, 46%). Mp 130-131 °C; R_f = 0.26 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2960, 1713, 1626, 1567, 1455, 1390, 1244 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.35 (4H, s, H-10′,11′,13′,14′), 7.34 (2H, d, J = 9.9 Hz, H-11,13), 7.16 (2H, d, J = 8.3 Hz, H-10,14), 6.91 (1H, d, J = 15.9 Hz, H-8′), 6.55 (1H, d, J = 15.9 Hz, H-7), 5.89 (1H, d, J = 2.1 Hz, H-5), 5.33 (1H, d, J = 2.1 Hz, H-3), 5.27 (1H, s, H-3′), 4.33 (1H, t, J = 10.4 Hz, H-8), 4.14 (1H, d, J = 10.4 Hz, H-7), 3.70 (3H, s, OMe-4), 3.53 (1H, d, J = 10.4 Hz, H-5′), 3.24 (3H, s, OMe-4′), 1.30 (9H, s), 1.29 (9H, s, H-16,16′); ¹³C NMR (CDCl₃, 100 MHz) δ 170.5 (C-4), 170.1 (C-4′), 164.8, 164.0 (C-2,2′), 158.8 (C-6), 151.4 (C-12′), 150.9 (C-12), 133.1 (C-9′), 132.6 (C-9), 131.1 (C-8′), 127.2 (C-10,14), 126.6 (C-10′,14′), 125.7 (C-11′,13′), 125.3 (C-11,13), 123.6 (C-7′), 102.6 (C-5), 91.7 (C-3′), 88.7 (C-3), 79.5 (C-6′), 55.8 (OMe-4), 55.2 (OMe-4′), 54.5 (C-7), 45.8 (C-5′), 38.8 (C-8), 34.6, 34.5 (C-15,15′), 31.3, 31.2 (C-16,16′); (+)-ESIMS m/z 591 [M + Na]⁺, (+)-HRESIMS m/z 591.2711 (calcd for C₃₆H₄₀NaO₆ 591.2717).

5.4.5. 6,6′-(2,4-Di(naphthalen-2-yl)cyclobutane-1,3-diyl)bis(4-methoxy-2H-pyran-2-one) (34)

From pyrone 22 (21.3 mg, 0.077 mmol) to give 34 as an amorphous colorless solid (4.1 mg, 19%). Mp 130-131 °C; R_f = 0.20 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 3055, 2946, 1714, 1644, 1564, 1454, 1410, 1246, 1146 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.80 (8H, m, ArH), 7.45 (6H, m, ArH), 5.77 (2H, d, J = 2.2 Hz, H-5), 5.13 (2H, d, J = 2.2 Hz, H-3), 4.67 (2H, m, H-8), 4.48 (2H, m, H-7), 3.60 (6H, s, OMe-4); ¹³C NMR (CDCl₃, 100 MHz) δ 170.4 (C-4), 163.8 (C-2), 162.6 (C-6), 134.9 (C-9), 133.3, 132.5 (C-11a,15a), 128.3, 127.9, 127.6, 126.2, 126.1, 125.9, 125.8 (C-10,11,12,13,14,15,16), 101.5 (C-5), 87.9 (C-3), 55.6 (OMe-4), 45.3 (C-7), 44.0 (C-8); (+)-ESIMS m/z 557 [M+H]⁺, (+)-HRESIMS m/z 557.1963 (calcd for C₃₆H₂₉O₆ 557.1959); (+)-HRESIMSMS (parent m/z 557) fragment m/z 279.1005 (calcd for C₁₈H₁₅O₃ 279.1021).

5.4.6. 11,11′ Di (4-tert-butyl-phenoxy) aniba-dimer-A (35)

From pyrone 17 (19.9 mg, 0.026 mmol) to give 35 as an amorphous colorless solid (9.7 mg, 49%). Mp 103-104 °C; R_f = 0.36 (3% MeOH:CH₂Cl₂); IR (ATR) v_max 2960, 1713, 1626, 1568, 1507, 1444, 1240 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 7.34 (4H, m, H-18,20,18′,20′), 7.26 (2H, m, H-13,13′), 7.17 (1H, d, J = 7.9 Hz, H-14′), 6.98 (1H, br s, H-10′), 6.90 (8H, m, H-10,12,14,17,21, 12′,17′,21′), 6.85 (1H, m, H-8′), 6.53 (1H, d, J = 15.9 Hz, H-7), 5.89 (1H, d, J = 2.1 Hz, H-5), 5.35 (1H, d, J = 2.1 Hz, H-3), 5.26 (1H, s, H-3′), 4.28 (1H, t, J = 10.2 Hz, H-8), 4.09 (1H, d, J = 10.2 Hz, H-7), 3.72 (3H, s, OMe-4), 3.55 (1H, d, J = 10.2 Hz, H-5′), 3.34 (3H, s, OMe-4′), 1.33 (9H, s, H-23,23′), 1.32 (9H, s, H-23,23′); ¹³C NMR (CDCl₃, 100 MHz) δ 170.4 (C-4), 169.7 (C-4′), 164.3, 163.7 (C-2,2′), 158.4 (C-6), 158.0, 157.8 (C-11,11′), 154.3, 154.3 (C-16,16′), 146.5, 146.3 (C-19,19′), 137.6 (C-9,9′), 131.2 (C-8′), 130.0, 129.6 (C-13,13′), 126.65, 126.60 (C-18,20,18′,20′), 124.8 (C-7′), 121.8 (C-14), 120.8 (C-14′), 118.6, 118.4, 118.2, 117.9, 117.8 (C-10,12,17,21,12′,17′,21′), 117.3 (C-10′), 102.6 (C-5), 91.8 (C-3′), 88.8 (C-3), 79.2 (C-6′), 55.9 (OMe-4), 55.5 (OMe-4′), 54.4 (C-7), 45.6 (C-5′), 38.9 (C-8), 34.3 (C-22,22′), 31.5 (C-23,23′); (+)-ESIMS m/z 753 [M + H]⁺, (+)-HRESIMS m/z 753.3402 (calcd for C₄₈H₄₉O₈ 753.3422).

5.5. Biological assays

5.2.1. Anti-tuberculosis Activity

Procedures for the determination of MIC against M. tuberculosis H₃₇Rv in 7H9 broth have been reported elsewhere.²⁸ In the present study, the protocol was modified slightly by utilizing 1:1000 diluted cultures rather than 1:100 and by growth of M tuberculosis H₃₇Rv in GAST medium²⁹ which does not contain bovine serum albumin. Positive control was isoniazid which gave MIC 0.22 μM (0.03μg/mL).

5.2.2. Anti-protozoal Activity

The in vitro activities against the protozoan parasites T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum and cytotoxicity assessment against L6 cells were determined as reported elsewhere.³⁰ The following strains, parasite forms and positive controls were used: T.b. rhodesiense, STIB900, trypomastigote forms, melarsoprol, IC₅₀ of 0.01 μM (4 ng/mL); T. cruzi, Tulahuen C2C4, amastigote forms in L6 rat myoblasts, benznidazole, IC₅₀ of 1.4 μM (0.352 μg/mL); L.donovani, MHOM/ET/67/L82, axenic amastigote forms, miltefosine, IC₅₀ of 0.5 μM (0.213 μg/mL); P. falciparum, K1 (chloroquine and pyrimethamine resistant), erythrocytic stages, chloroquine, IC₅₀ of 0.20 μM (0.065 μg/mL) and L6 cells, rat skeletal myoblasts, podophyllotoxin, IC₅₀ of 0.01 μM (0.004 μg/mL).