Cytotoxic Coumarins from Mammea harmandii

• Abstract
• Materials and Methods
• References

Abstract

Two new naturally occurring coumarins, isomesuol (1) and mammearin A (2), together with nine known Mammea coumarins 3 - 11 were isolated from the EtOAc extract of the leaves and twigs of Mammea harmandii. Coumarins 1, 3 and 4 showed cytotoxicity against a panel of mammalian cancer cell lines. Their structures were determined by spectroscopic methods. The assignments of ¹³C-NMR signals of isomesuol (1), which was isolated for the first time as a natural product, have been revised.

Mammea harmandii (Pierre) Kosterm (Guttiferae) is an evergreen tree found in Vietnam, Laos and Thailand [1]. As part of our continuing search for anticancer agents, the investigation of the cytotoxic EtOAc extract from the leaves and twigs of this unexplored species has led to the isolation of two new naturally occurring Mammea coumarins, isomesuol (mammea A/BD) (1) [2], [3], [4] and mammearin A (2), together with the known mammea A/BA (3) [4], [5], [6], [7] mammea A/BB (4) [4], [5], [6], [7], [8], [9], mammea A/AA cyclo D (mammeigin) (5) [7], [10], [11], [12], mammea A/AB cyclo D (6) [10], [11], [12], mammea A/AD cyclo D (7) [11], [12], mammea B/AC cyclo D (8) [12], [13], mammea B/AD cyclo D (9) [12], mammea E/BC cyclo D (10) [14] and mammea E/BD cyclo D (11) [14]. We now report the details of the isolation and structure elucidation of these coumarins, as well as the cytotoxic activities of the active compounds 1, 3 and 4.

The structure of compound 1 was identified as 5,7-dihydroxy-6-(3-methylbut-2-enyl)-8-(2-methylpropanoyl)- 4-phenylbenzopyran-2(2H)-one, which has been synthetically known as isomesuol [2], [3]. Its IR and UV data (see Materials and Methods) as well as the ¹H-NMR data are in agreement with those reported [4]. However, the reported ¹³C-NMR data [3] are different from our assignments. Based on the information obtained from the COSY-45, HMQC, HMBC spectra, the ¹H and ¹³C signals were assigned (Table [1]). The HMBC information confirmed the connectivities in 1 (Fig. [1]). Moreover, a series of NOE experiments was performed to distinguish the signals of 4″-CH₃ and 5″-CH₃. Enhancements of 2.6 % and 2.4 % were obtained at the 2″-H signal when the signals of 1″-H and 5″-CH₃ were irradiated, respectively. Irradiation of the 2″-H signal gave rise to an enhancement of the 5″-CH₃ signal (6.0 %). The 1″-H signal was enhanced by 1.0 % on irradiating the 4″-H signal, while irradiation of the 1″-H signal enhanced the 4″-H signal (2.6 %).

Compound 2 was isolated as a white powder. Its HR-MS shows (M + 1)⁺ at m/z = 371.1874, corresponding to C₂₂H₂₇O₅. The molecular ion at m/z = 370 (M^+·), the fragment ion at m/z = 313 (M - C₄H₉)⁺, as well as the base peak at m/z = 257 (313 - C₄H₈)⁺ in its EI-MS suggested the presence of C₅-acyl and 3-methylbut-2-enyl units. The IR spectrum displayed the bands at 3423, 1729 and 1659 cm^-1 of the hydroxy, the conjugated δ-lactone and the chelated aryl keto groups, respectively. Compound 2 showed UV maxima at λ = 222, 291 and 330 nm which are consistent with those observed for 1, but shifted to 279 and 399 nm upon addition of base [4], [7]. Comparable to compound 1, the ¹H-NMR spectrum of 2 (Table [1]) indicated the presence of singlet of 3-H in the coumarin skeleton at δ = 5.80. Compound 2 was analyzed as a furocoumarin with an ethyl group substituted at C-2 of the furobenzopyran-4(2H)-one skeleton by the presence of a triplet of -O-C-H at δ = 5.56 (J = 5.9 Hz, 2-H), two multiplets of methylene protons (2H, 1′-H) at δ = 1.89 and 1.99, along with a triplet of the 2′-methyl group at δ = 1.02 (J = 7.3 Hz), supported by comparison with the data of the synthetic compounds with a similar skeleton [15]. The presence of a very low-field singlet of 7-OH group at δ = 15.10 indicated the chelation to the 1″′-carbonyl of the 6-acyl substituent. This acyl substituent was identified as a 2-methylbutanoyl group by the signals at δ = 3.72 (m, 2″′-H), 1.40 (m, 3″′a-H), 1.82 (m, 3″′b-H), 0.94 (t, J = 7.6 Hz, 4″′-H) and 1.17 (d, J = 7.1 Hz, 5″′-H). Two sharp singlets of 4″-CH₃ and 5″-CH₃ at δ = 1.69 and 1.62, together with a two protons doublet of 1″-H at δ = 3.23 (J = 7.4 Hz) and a triplet of 2″-H at δ = 5.20 (J = 7.4 Hz) were the signals of the 3-methylbut-2-enyl group substituted at C-8. By analyses of the vicinal couplings and the cross peaks observed in its COSY-45 spectrum (see Materials and Methods), the complete assignments of ¹H signals were made. The ¹³C-NMR spectrum of 2 was analyzed to consist of five methyl carbons, three methylene carbons, four methine carbons, eight quaternary carbons and two carbonyl carbons (Table [1]). The chemical shift assignments and the connectivities in structure 2 were obtained from the information of HMQC and HMBC experiments (Fig. [1]).

Compounds 3 - 11 were identified by analyses of their ¹H- and ¹³C-NMR spectra including 2D-experiments. The reported physical properties (Materials and Methods) and the spectroscopic data of these compounds, except for 9, agreed well with the literature values, where available. Although compound 9 is known, its physical and spectroscopic data have not been reported. The literature [12] mentioned only GC-MS studies, thus our data are also included (Materials and Methods). It should be noted that structures of compounds 10 and 11 isolated from Mammea siamensis were published after this work had been completed [14].

Compounds 1 - 11 were screened for cytotoxicity against a panel of cultured mammalian cancer cell lines according to an established protocol [16], (Table [2]). ED₅₀ values of ≤ 5 μg/mL are considered as active. Compounds 2 and 5 - 11 show cytotoxicity > 5 μg/mL.

Materials and Methods

Melting points are uncorrected. IR and UV spectra were recorded on a Spectrum GX-FTIR system (Perkin Elmer) and JASCO V-530 spectrophotometers, respectively. NMR spectra were recorded in CDCl₃ on Bruker DPX 300 and Bruker AV 500 spectrometers, using TMS as an internal standard. HR-MS was recorded on a Finnigan MAT 95 instrument at the University of Illinois at Chicago, USA. Silica gel 60 (Merck, 70 - 230 mesh) and silica gel plates (Merck, Kieselgel 60 F₂₅₄, 0.5 mm) were used for CC and preparative TLC, respectively.

The leaves and twigs of Mammea harmandii were collected in March 1996 from Doi Tung, Chaingmai, Thailand. The plant was identified by one of us (TS) and a voucher specimen (BKF 118 397) has been deposited at the Forest Herbarium, Royal Forestry Department, Bangkok.

Air-dried finely powdered leaves and twigs (1.85 kg) of Mammea harmandii were percolated with MeOH (5 × 7 L) at room temperature. Evaporation of the solvent yielded a crude MeOH extract (316.5 g), which was further partitioned between a water layer (500 mL) and an EtOAc layer (13 × 500 mL). After removal of solvents, the EtOAc extract (147.3 g) and H₂O extract (142.6 g) were obtained. The EtOAc extract (146.8 g) was separated by CC over silica gel (1.5 kg), gradient eluting with hexane (4 L), 2 %, 4 %, 6 %, 8 %, 10 %, 15 %, 20 %, 30 %, 40 %, 60 %, 80 % acetone-hexane and acetone (3 L for each solvent mixture), followed by 5 % , 10 %, 30 %, 50 % MeOH-acetone and MeOH (3 L for each solvent mixture). The collected fractions (500 mL each) were combined on the basis of their TLC characteristics and after removal of solvents gave 12 separated fractions (A1-A12). Fraction A2 (7.5 g, eluted with 4 % acetone-hexane) was further separated by CC (silica gel, EtOAc-hexane gradient followed by MeOH-EtOAc gradient) to give fractions B1-B9. Fraction B4 (3.0 g, eluted with 4 - 5 % EtOAc-hexane) was further purified by CC (silica gel, EtOAc-hexane gradient followed by MeOH-EtOAc gradient), to afford fractions C1 - C9. Separation of fraction C3 (400 mg, eluted with 2 % EtOAc-hexane) by preparative TLC (cyclohexane:EtOAc:Et₃N, 30 : 1:3) gave four separated bands. The three less polar bands after preparative TLC (silica gel, 20 % EtOAc-hexane) gave pure 5 {10.8 mg, yellow needles, m. p. 140 - 142 °C (CH₂Cl₂-hexane); lit. [7]}, 8 {29.1 mg, yellow needles, m. p. 111 - 113 °C (CH₂Cl₂-hexanes); lit. [13]} and 9 (12.4 mg), respectively. The more polar band provided pure 6 {18.7 mg, t_R: 13.2 min, yellow needles, m. p. 96 - 97 °C (CH₂Cl₂-hexanes), [α]_D ³⁰: + 16.0^° (c 0.1, CHCl₃); lit. [11]} and 7 {15 mg, t_R: 11.1 min, yellow needles, m. p. 147 - 148 °C (CH₂Cl₂-hexane); lit. [11]} after separation by HPLC (Partisil 10 ODS-3 M20, 9.5 mm × 50 cm, 4.5 μL/min, MeOH:H₂O, 9 : 1, UV detection at 280 nm). Fraction A3 (2.4 g, eluted with 6 % acetone-hexane) was further purified as described for fraction A2 to give additional quantities of pure 5 (20.4 mg), 6 (27.0 mg), 7 (19.6 mg) and 9 (4.2 mg). Fraction A4 (10.1 g, eluted with 8 % acetone-hexane) was separated by CC (silica gel, EtOAc-hexane gradient followed by MeOH-EtOAc gradient) yielded fractions D1 - D11. Upon addition of hexane to fraction D2 (865 mg, eluted with 2 % EtOAc-hexane), the obtained white precipitate (402 mg) was rechromatographed by preparative TLC (20 % EtOAc-hexane) to afford pure 4 {16.8 mg, pale brown powder, m. p. 120 - 121 °C (CH₂Cl₂-hexane), [α]_D ²⁸: -14.0° (c 0.1 %, CHCl₃); lit. [7]}. Similarly, the precipitate (1.0 g) obtained from fraction D3 (2.0 g, eluted with 4 % EtOAc-hexane) was separated by preparative TLC (20 % EtOAc-hexane) to give pure 1 (40 mg) and 3 {13.4 mg, colorless semisolid; lit. [4]}. Further separation of fraction D4 (3.0 g, eluted with 5 % EtOAc-hexane) was not successful by CC or preparative TLC. The yellow powdered residue (56.1 mg) was further separated by HPLC, using the conditions mentioned above to afford pure 10 {11.0 mg, t_R: 14.1 min, pale yellow semisolid, [α]_D ²⁹: -50.0° (c 0.1, CHCl₃); lit. [14]} and 11 {(14.0 mg, t_R: 12.2 min, pale yellow semisolid, [α]_D ²⁹: -52.0° (c 0.1, CHCl₃); lit. [14]}. Fraction A5 (16.8 g, eluted with 10 % acetone-hexane) was further purified by CC (silica gel, EtOAc-hexane gradient followed by MeOH-EtOAc gradient) to give fractions E1-E10. Fraction E5 (4.0 g, eluted with 5 % EtOAc-hexane) was further separated by CC (EtOAc-hexane gradient). One isolated fraction (40 mg, eluted with 5 % EtOAc-hexane) was rechromatographed by preparative TLC (50 % CH₂Cl₂-hexane) to provide pure 2 (11.3 mg).

Isomesuol (mammea A/BD) (1): White powder, m. p. 167 - 169 ° C (CH₂Cl₂-hexane), lit.: white needles, m. p. 169 - 171 ° C (hexane-CHCl₃) [4]; FT-IR (KBr): ν_max = 3482, 1735, 1620, 1594, 1440, 1393, 1232, 1183, 1132, 883, 856 cm^-1; UV (EtOH): λ_max (log ε) = 229 (4.95), 299 (4.75), 335 (4.95) nm; λ_max (EtOH + 0.1N KOH) (log ε) = 232 (4.96), 259 (4.77), 336 (5.01), 388 nm (infl); ¹H-NMR (500 MHz) and ¹³C-NMR (125 MHz) in CDCl₃: see Table [1]; HR-MS: found m/z = 392.1628 [M]^+· (calcd. for C₂₄H₂₄O₅ : 392.1624); EI-MS: m/z (rel. int.) = 392 [M]^+· (30), 349 (65), 293 (100).

Mammearin A (2): White powder, m. p. 110 - 111 °C (CH₂Cl₂-hexanes); [α]_D ²⁹: + 14.0° (c 0.1, CHCl₃); FT-IR (KBr): ν_max = 3423, 1730, 1659, 1635, 1601, 1463, 1399, 1231, 1190, 1133, 1063, 875 cm^-1; UV (EtOH): λ_max (log ε) = 222 (4.83), 291 (4.79), 330 (4.58) nm; λ_max (EtOH + 0.1N KOH) (log ε) = 279 (4.65), 399 (4.45) nm; ¹H-NMR (300 MHz) and ¹³C-NMR (75 MHz) in CDCl₃: see Table [1]; COSY correlations [δ_H/δ_H (H/H)]: 0.94/1.40 (4″′-H/3″′a-H), 0.94/1.82 (4″′-H/3″′b-H), 1.02/1.89 (2′-H/1′a-H) 1.02/1.99 (2′-H/1′b-H), 1.17/3.72 (5″′-H/2″′-H), 1.40/1.82 (3″′a-H/3″′b-H), 1.40/3.72 (3″′a-H/2″′-H), 1.82/3.72 (3″′b-H/2″′-H), 1.62/5.20 (5″-H/2″-H), 1.69/5.20 (4″-H/2″-H), 1.89/1.99 (1′a-H/1′b-H), 1.89/5.56 (1′a-H/2-H), 1.99/5.56 (1′b-H/2-H), 3.23/5.20 (1″-H/2″-H); HR-MS: found m/z = 371.1874 [M + H]⁺ (calcd. for C₂₂H₂₇O_{5 :} 371.1859); EI-MS: m/z (rel. int.) = 370 [M]^+· (59), 313 (44), 257 (100).

Mammea B/AD cyclo D (9) [12]: Yellow needles, m. p. 112 - 114 °C (CH₂Cl₂-hexane); FT-IR (KBr): ν_max = 3434, 1736, 1609, 1586, 1458, 1419, 1378, 1264, 1192, 1146, 1114 cm^-1; UV (EtOH): λ_max (log ε) = 227 (4.53), 287 (4.75) nm; ¹H-NMR (300 MHz, CDCl₃): δ = 0.93 (t, J = 7.4 Hz, 3′-H), 1.14 (d, J = 6.7 Hz, 3″-H, 4″-H), 1.46 (s, 5″′-H, 6″′-H), 1.56 (m, 2′-H), 2.80 (septet, J = 6.7 Hz, 2″-H), 2.86 (t, J = 7.4 Hz, 1′-H), 5.51 (d, J = 10.0 Hz, 3″′-H), 5.88 (s, 3-H), 6.76 (d, J = 10.0 Hz, 4″′-H), 15.20 (s, 5-OH); ¹³C-NMR (75 MHz, CDCl₃): δ = 14.0 (C-3′), 19.4 (C-3″, C-4″), 22.7 (C-2′), 28.1 (C-5″′, 6″′), 38.5 (C-1′), 39.9 (C-2″), 79.7 (C-2″′), 101.4 (C-8), 103.3 (C-4a), 106.4 (C-6), 110.3 (C-3), 115.7 (C-4″′), 126.1 (C-3″′), 155.1 (C-8a), 157.3 (C-7), 159.5 (C-4), 160.1 (C-2), 165.4 (C-5), 211.9 (C-1″); HRMS: found m/z = 356.1625 [M]^+· (calcd. for C₂₁H₂₄O_{5 :} 356.1624); EI-MS: m/z (rel. int.) = 356 [M]^+· (43), 341(100), 313 (30).

References

• 1 Kostermans A JGH. A Monograph of the Asiatic and Pacific Species of Mammea L. Lembaga Pusat Pen-jelidikan Kehutanan Bogor, Indonesia; 1961: pp 22-3
  Search in Google Scholar
• 2 Chakraborty D P, Das B C. The structure of mesuol. Tetrahedron Lett 1966: 5727-30
  Search in Google Scholar
• 3 Chakraborty D P, Chatterjee D, Guha S. On mesuol and isomesuol. A regiospecific synthesis of isomesuagin.  J Indian Chem Soc. 1985;  LXII 993-8
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• 4 Crombie L, Jones R CF, Palmer C J. Synthesis of the Mammea coumarins. Part I. The coumarins of the Mammea A, B and C series.  J Chem Soc Perkin Trans. 1987;  I 317-31
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• 7 Crombie L, Games D E. Extractives of Mammea americana L. Part II. The 4-phenylcoumarins. Isolation and structure of Mammea A/AA, A/A cyclo D, A/BA, A/AB and A/BB. J Chem Soc (C) 1967: 2553-9
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• 8 Crombie L, Games D E. Isolation and structure of Mammea A/BA, A/AB and A/BB: a group of 4-arylcoumarin extractives of Mammea africana L. Tetrahedron Lett 1966: 145-9
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• 9 Morel C, Guilet D, Oger J M, Seraphin D, Sevenet T, Wiart C, Hadi A HA, Richomme P, Bruneton J. 6-Acylcoumarins from Mesua racemosa .  Phytochemistry. 1999;  50 1243-7
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• 10 Carpenter I, McGarry E J, Scheinmann F. Extractives from Guttiferae. Part XXI. The isolation and structure of nine coumarins from the bark of Mammea africana G. Don. J Chem Soc (C) 1971: 3783-9
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• 11 Prachyawarakorn V, Mahidol C, Ruchirawat S. NMR study of seven cumarins from Mammea siamensis .  Pharmaceutical Biology. 2000;  38 58-62
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• 12 Games D E. Identification of 4-phenyl and 4-alkylcoumarins in Mammea americana L., Mammea americana G. Don and Calophyllum inophyllum by gas chromatography-mass spectrometry. Tetrahedron Lett 1972: 3187-90
  Search in Google Scholar
• 13 Kaweetripob W, Mahidol C, Ruchirawat S. Chemical investigation of Mammea siamensis .  Pharmaceutical Biology. 2000;  38 55-7
  PubMedSearch in Google Scholar
• 14 Mahidol C, Kaweetripob W, Prawat H, Ruchirawat S. Mammea coumarins from the flowers of Mammea siamensis .  J Nat Prod. 2002;  65 757-60
  CrossrefPubMedSearch in Google Scholar
• 15 Crombie L, Jones R CF, Palmer C J. Synthesis of Mammea coumarins. Part 2. Experiments in the Mammea E series and synthesis of Mammea E/AC. J Chem Soc Perkin Trans 1 1987;: 333-43
  Search in Google Scholar
• 16 Likhitwitayawuid K, Angerhofer C K, Cordell G A, Pezzuto J M, Ruangrungsi N. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta .  J Nat Prod. 1993;  56 30-8
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Prof. Vichai Reutrakul
Dr. Patoomratana Tuchinda

Department of Chemistry

Faculty of Science

Mahidol University

Bangkok 10400

Thailand

Phone: +66-2-2015152

Fax: +66-2-6445126

Email: scvrt@mahidol.ac.th

Email: scptc@mahidol.ac.th

References

• 1 Kostermans A JGH. A Monograph of the Asiatic and Pacific Species of Mammea L. Lembaga Pusat Pen-jelidikan Kehutanan Bogor, Indonesia; 1961: pp 22-3
  Search in Google Scholar
• 2 Chakraborty D P, Das B C. The structure of mesuol. Tetrahedron Lett 1966: 5727-30
  Search in Google Scholar
• 3 Chakraborty D P, Chatterjee D, Guha S. On mesuol and isomesuol. A regiospecific synthesis of isomesuagin.  J Indian Chem Soc. 1985;  LXII 993-8
  PubMedSearch in Google Scholar
• 4 Crombie L, Jones R CF, Palmer C J. Synthesis of the Mammea coumarins. Part I. The coumarins of the Mammea A, B and C series.  J Chem Soc Perkin Trans. 1987;  I 317-31
  PubMedSearch in Google Scholar
• 5 Crombie L, Games D E, Haskins N J. Extractives of Mammea americana L. Part V. The insecticidal compounds.  J Chem Soc Perkin Trans. 1972;  I 2255-60
  PubMedSearch in Google Scholar
• 6 Crombie L, Jones R CF, Palmer C J. Synthesis of mammeins and surangin A.  Tetrahedron Lett. 1985;  26 2929-32
  CrossrefPubMedSearch in Google Scholar
• 7 Crombie L, Games D E. Extractives of Mammea americana L. Part II. The 4-phenylcoumarins. Isolation and structure of Mammea A/AA, A/A cyclo D, A/BA, A/AB and A/BB. J Chem Soc (C) 1967: 2553-9
  Search in Google Scholar
• 8 Crombie L, Games D E. Isolation and structure of Mammea A/BA, A/AB and A/BB: a group of 4-arylcoumarin extractives of Mammea africana L. Tetrahedron Lett 1966: 145-9
  Search in Google Scholar
• 9 Morel C, Guilet D, Oger J M, Seraphin D, Sevenet T, Wiart C, Hadi A HA, Richomme P, Bruneton J. 6-Acylcoumarins from Mesua racemosa .  Phytochemistry. 1999;  50 1243-7
  CrossrefPubMedSearch in Google Scholar
• 10 Carpenter I, McGarry E J, Scheinmann F. Extractives from Guttiferae. Part XXI. The isolation and structure of nine coumarins from the bark of Mammea africana G. Don. J Chem Soc (C) 1971: 3783-9
  Search in Google Scholar
• 11 Prachyawarakorn V, Mahidol C, Ruchirawat S. NMR study of seven cumarins from Mammea siamensis .  Pharmaceutical Biology. 2000;  38 58-62
  PubMedSearch in Google Scholar
• 12 Games D E. Identification of 4-phenyl and 4-alkylcoumarins in Mammea americana L., Mammea americana G. Don and Calophyllum inophyllum by gas chromatography-mass spectrometry. Tetrahedron Lett 1972: 3187-90
  Search in Google Scholar
• 13 Kaweetripob W, Mahidol C, Ruchirawat S. Chemical investigation of Mammea siamensis .  Pharmaceutical Biology. 2000;  38 55-7
  PubMedSearch in Google Scholar
• 14 Mahidol C, Kaweetripob W, Prawat H, Ruchirawat S. Mammea coumarins from the flowers of Mammea siamensis .  J Nat Prod. 2002;  65 757-60
  CrossrefPubMedSearch in Google Scholar
• 15 Crombie L, Jones R CF, Palmer C J. Synthesis of Mammea coumarins. Part 2. Experiments in the Mammea E series and synthesis of Mammea E/AC. J Chem Soc Perkin Trans 1 1987;: 333-43
  Search in Google Scholar
• 16 Likhitwitayawuid K, Angerhofer C K, Cordell G A, Pezzuto J M, Ruangrungsi N. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta .  J Nat Prod. 1993;  56 30-8
  PubMedSearch in Google Scholar

Prof. Vichai Reutrakul
Dr. Patoomratana Tuchinda

Department of Chemistry

Faculty of Science

Mahidol University

Bangkok 10400

Thailand

Phone: +66-2-2015152

Fax: +66-2-6445126

Email: scvrt@mahidol.ac.th

Email: scptc@mahidol.ac.th