A New Coumarin from Murraya paniculata

• Abstract
• Materials and Methods
• References

Abstract

A new natural product, 2′-O-ethylmurrangatin (1) was isolated along with two previously known compounds murranganone (2) and paniculatin (3) from the leaves of Murraya paniculata. The structure of compound 1 was elucidated with the help of spectroscopic studies and by chemical reactions. Compounds 2 and 3 have been found to be cholinesterase inhibitors.

Murraya paniculata (L.) Jack (syn. M. exotica) [1] or Orange Jasmine (Rutaceae) occurs widely in Southeast Asia, Southern China and Malay Peninsula. The leaves are used for the treatment of diarrhea and dysentery. The leaves possess antibiotic activity against Micrococcus pyogenes var. aureus and Escherichia coli [1], [2]. Previous phytochemical studies on this plant have resulted in the isolation of a number of coumarins and carbazole alkaloids [3] [4] [5] [6]. We now report here the isolation and structure elucidation of a new natural product 1 along with the two known compounds 2 and 3.[*]

2′-O-Ethylmurrangatin (1) was obtained as a colorless powder. The HREIMS displayed the M⁺ at m/z 304.1334 corresponding to the molecular formula C₁₇H₂₀O₅ (Calc. 304.1311). The mass fragmentation pattern [m/z (rel. int. %) 233 (83), 205 (100), 175 (9) and 162 (7)] was in agreement with the proposed structure 1. The UV absorptions at 204, 258 and 320 nm indicated the presence of a coumarin nucleus [7]. The IR bands at 1496, 1562 and 1605 (aromatic C=C), 1728 (α,β-unsaturated lactone) [8] and a broad peak at 3500 (OH) cm^-1. The ¹H-NMR spectrum displayed characteristic signals for a methoxy group δ = 3.91 (s), a O-CH₂-CH₃ system δ = 3.43 (2H, m) and δ = 1.12 (3H, t, J = 7.0 Hz), exomethylene group δ = 4.62 (1H, m) and 4.67 (1H, m), olefinic protons of coumarin nucleus δ = 6.23 and 7.59 (1H, d each, J _3,4 = 9.5 Hz), and two aromatic ortho C-5 and C-6 protons δ = 6.84 and 7.36 (1H, d each, J _5,6 = 8.5 Hz) and the two vicinal protons were apparent from signals at δ = 4.88 and δ 5.12 (1H, d each, J _{1′ , 2′} = 8.5 Hz). H-1′ (δ = 5.12) indicated the presence of a geminal secondary hydroxy group. The position of the secondary OH was further confirmed by acetylation. In the ¹H-NMR of 2′-O-ethylmurrangatin acetate (1a), the H-1′ appeared at δ = 6.06 (1H, d, J _{1′ , 2′} = 8.5 Hz). The difference of ∼ 1 ppm in chemical shifts of H-1′ in 1 and 1a makes it rather clear that OH is present at C-1′ [9]. The ¹³C-NMR spectra showed the resonances of all seventeen carbons. The DEPT spectra revealed the presence of six methine, two methylene and three methyl carbons, while the difference between the BB and DEPT spectra indicated the presence of six quaternary carbons in the molecule. The downfield signals at δ = 160.3 and 161.5 were assigned to the C-2 lactone and quaternary C-7, respectively. The direct ¹H/¹³C chemical shift correlations for protonated carbons were deduced from the HMQC spectrum. From HMBC studies, the H-2′ showed ² J correlation with C-3′ and ³ J correlation with C-4′ (see Fig. [1]). The stereochemistry at C-1′ was found to be “S” by Horeau’s method [10]. In this method, compound 1 was reacted with racemic 2-phenylbutanoic anhydride, and (R)-2-phenylbutanoic acid was recovered (after hydrolysis of the left-over anhydride) unreacted, which indicated that only the “S” form of the racemic 2-phenylbutanoic anhydride was consumed in the ester formation. The stereochemistry at the adjacent C-2′ was concluded to be “R” on the basis of spectral comparison and molecular model [9]. These spectroscopic studies led to the structure 1 for this new coumarin.

Compound 2 (10 mg, 2 × 10^-5 %) was isolated as white amorphous powder with m.p. 103 °C and [α]²⁵ _D: 105 (c = 0.06, CHCl₃). The HREIMS displayed the molecular ion at m/z 276.2046 (C₁₅H₁₆O₅). The UV absorptions at 247, 257 and 320 nm. The IR absorption bands at 1612 (C=C), 1733 (lactonic carbonyl) and 3502 (OH) cm^-1. The ¹H-NMR spectrum displayed the characteristic signal for a methine proton at δ = 5.90 (1H, s, H-1′) indicating the presence of a secondary hydroxy group. The spectral data (¹H-NMR, MS, UV, IR) of 2 matched well with the previously reported literature values [11]. This compound was earlier isolated from M. paniculata.

Compound 3 (C₂₀H₂₄O₆) (15 mg, 3 × 10^-2 %) was isolated as white amorphous powder with m.p. 120 °C and [α]²⁵ _D: 120 (c = 0.1, CHCl₃). The UV absorptions at 247, 256 and 320 nm. The IR bands appeared at 1608 (C=C), 1728 (lactonic carbonyl) cm^-1. The compound 3 was identified as paniculatin by comparing its spectral data (¹H-NMR, MS, UV, IR) with the previously reported literature values [12]. This compound was also earlier isolated from the same plant.

Some coumarins are known to inhibit acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) [13]. However, no 7-methoxycoumarin is yet reported to have this activity. The compounds 1 - 3 were therefore subjected to cholinesterase inhibition screening. Inhibition of AchE is considered to be a promising approach for the treatment of Alzheimer’s disease (AD) and for possible therapeutic applications in the treatment fo Parkinson's disease, aging and myasthenia gravis [14], [15]. The role of BuchE in the normal, aging and diseased brain is still unknown. Recently, it has been found that the BuchE is existing in significantly higher quantities in Alzheimer’s palques than in plaques of age related non-demented brains. Moreover, the inclusion of cymserine which is a very potent selective BuchE inhibitor in the clinical trials for AD treatment do prove that BuchE inhibition could be an important tool for the treatment of AD and related dementias. Coumarins 1 - 3 isolated from M. paniculata were tested for cholinesterase inhibition activity. The concentration of compounds 2 and 3 that inhibited the enzymes activity by 50 % (IC₅₀) is shown in Table [1]. Eserine [(-)-physostigmine] was used as a positive control. Compound 3 has been found to be more potent against AchE, while compound 2 was more potent against BuchE. Both compounds did not show any significant selectivity towards any of the two enzymes. These coumarins were found to be moderate inhibitors of cholinesterase.

Materials and Methods

The melting point was determined on a Buchi 510 apparatus. The ¹H-NMR spectra were recorded in CDCl₃ at 400 and 500 MHz on Bruker AM-400 and AMX-500 NMR spectrometers with TMS as internal standard. ¹³C-NMR was recorded in CDCl₃ at 125 MHz on a Bruker AMX-500 NMR spectrometer. Mass spectra were recorded on a Varian MAT 312 double focussing mass spectrometer connected to DEC PDP 11/34 computer system. The IR spectra were recorded on a Jasco IRA-1 IR spectrophotometer. The UV spectra were recorded on a Shimadzu UV 240 instrument. The optical rotations were measured on JASCO DIP-360 digital polarimeter. Purity of the samples was checked by TLC on pre-coated silica gel GF-254 preparative plates (20 × 20 cm, 0.25 mm thick).

The fresh leaves of M. paniculata (L.) Jack (50 kg) were collected from suburban areas of Karachi (Pakistan) in June, 1992. The plant was identified by the Mr. Tahir Ali, Plant Taxonomist, Department of Botony, University of Karachi. A voucher specimen (KUH # 63485) has been deposited in the herbarium of the department of Botony, University of Karachi. The air-dried leaves of M. paniculata (19 kg) were extracted with methanol. The methanolic extract (2 liters) was concentrated to give a residue (500 g), which was diluted with H₂O and extracted first with chloroform and then with ethyl acetate by adjusting the pH 3 - 8 by HCl and ammonium hydroxide. All these extracts were subjected to column chromatography on silica gel. The CHCl₃ fraction (12 g) extracted at pH 3 was loaded on a silica gel (250 g) and column was eluted with about 6 liters of CHCl₃ : hexane to CHCl₃ : Me₂CO mixtures (from 5 : 5 to 5 : 5, respectively). Three parts were thus obtained. Part A (CHCl₃ : hexane, 9 : 1, 200 mL) containing mainly compound 1, Part B (CHCl₃ : Me₂CO, 8 : 2, 300 mL) containing mainly 2 and Part C (pure CHCl₃, 250 mL) containing mainly 3. Rechromatography of Part A by TLC (silica gel) yielded compound 1 (50 mg, CHCl₃ : CH₃OH, 9.5 : 0.5). Part B was purified by column chromatography (silica gel 20 g) to yield compound 2 (10 mg, CHCl₃ : CH₃OH, 9.8 : 0.2). Part C was also purified on column chromatography (silica gel 15 g) to obtain compound 3 (15 mg, CHCl₃ : hexane, 9.5 : 0.5).

2′-O-Ethylmurrangatin (1): White amorphous powder (50 mg, 1 × 10^-4 %); m.p. 128 °C; [α]²⁵ _D: 130 (c = 0.1, CHCl₃); UV (MeOH): λ_max = 204 nm (log ε = 4.31); IR (CHCl₃): ν_max = 1496, 1562, 1605 (C=C), 1728 (coumarinic carbonyl), 3500 cm^-1 (OH); ¹H-NMR (CD₃Cl, 400 MHz): δ = 1.12 (3H, t, J = 7.0 Hz, CH₃), 1.68 (3H, s, CH₃), 3.43 (2H, m, OCH₂CH₃), 3.91 (3H, s, OCH₃), 4.62 (1H, m, H-4′a), 4.67 (1H, m, H-4′b), 4.88 (1H, d, J _{2′ , 1′} = 8.5 Hz, H-2′), 5.12 (1H, d, J _{1′ , 2′} = 8.5 Hz, H-1′), 6.23 (1H, d, J _3,4 = 9.5 Hz, H-3), 6.84 (1H, d, J _6,5 = 8.5 Hz, H-6), 7.36 (1H, d, J _5,6 = 8.5 Hz, H-5), 7.59 (1H, d, J _4,3 = 9.5 Hz, H-4); ¹³C-NMR (CDCl₃, 125 MHz): δ = 161.5 (C-7), 160.3 (C-2), 154.0 (C-9), 143.6 (C-4), 143.5 (C-3′), 128.8 (C-5), 114.7 (C-8), 114.0 (C-4′), 113.5 (C-3), 113.0 (C-10), 108.0 (C-6), 76.4 (C-2′), 75.9 (C-1′), 65.2 (OCH₂), 56.2 (OCH₃), 17.4 (CH₃), 15.3 (CH₃); EI MS m/z (rel. int. %), 233 (83), 205 (100), 175 (9), 162 (7): HREI MS m/z 304.1334 (C₁₇H₂₀O₅, calc. 304.1311).

2′-O-Ethylmurrangatin acetate (1a): White amorphous powder; ¹H-NMR (CD₃Cl, 400 MHz): δ = 1.14 (3H, t, J = 7.0 Hz, CH₃), 1.55 (3H, s, CH₃), 3.43 (2H, m, OCH₂CH₃). 3.91 (3H, s, OCH₃), 4.64 (1H, m, H-4′a), 4.89 (1H, m, H-4′b), 5.35 (1H, d, J _{2′ , 1′} = 8.5 Hz, H-2′), 6.06 (1H, d, J _{1′ , 2′} = 8.5 Hz, H-1′), 6.23 (1H, d, J _3,4 = 9.5 Hz, H-3), 6.84 (1H, d, J _6,5 = 8.5 Hz, H-6), 7.36 (1H, d, J _5,6 = 8.5 Hz, H-5), 7.59 (1H, d, J _4,3 = 9.5 Hz, H-4); EI MS: m/z (rel. int. %) = 233 (88), 205 (100), 175 (37), 162 (25), 57 (27).

In Horeau's method, a mixture of 2′-O-ethylmurrangatin (1) (5 mg), C₅H₅N (0.5 mL) and racemic 2-phenylbutanoic anhydride (0.1 mL) was kept at room temperature for overnight with continuous stirring. Usual work-up afforded 2-phenylbutanoic acid (after hydrolysis of the left-over anhydride) which was found to have a negative optical rotation, thereby establishing the “S” configuration at C-1′ in the compound 1.

Electric eel AchE, horse BuchE, acetylthiocholine iodide, butyrylthiocholine chloride, 5,5′-thiobis-2-nitrobenzoic acid (DTNB) and eserine were purchased from Sigma (St. Louis, MO). Buffers and other chemicals were of analytical grade. Cholinesterase inhibition was determined spectrophotometrically by using the modified Ellman method [16].

References

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• 2 Perry L M, Metzger J. Medicinal Plants of East and Southeast Asia. The MIT Press Cambridge; 1980: 367
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• 3 Talapatra S K, Dutta L N, Talapatra B. Structure of murralongin, a novel monomeric coumarin from Murraya elongata: Stereochemistry and preferred conformation of its unique side chain. Tetrahedron Letters 1973: 5005-8
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• 4 Ramstad E, Lin W C, Lin T J, Koo W Y. Coumurrayin, a new coumarin from Murraya paniculata (L.) Jack. Tetrahedron Letters 1968: 811-3
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• 5 Dreyer D L. Chemotaxonomy of the Rutaceae. IV. Constituents of Murraya paniculata (Linn.) Jack.  The Journal of Organic Chemistry. 1968;  33 3574-6
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• 8 Barik B R, Kundu A B. A cinnamic acid derivative and a coumarin from Murraya exotica .  Phytochemistry. 1987;  26 3319-21
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• 11 Ito C, Furukawa H. Constituents of Murraya exotica L. Structure elucidation of new coumarins.  Chemical & Pharmaceutical Bulletin. 1987;  35 4277-85
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• 12 Steck W. Paniculatin, a new coumarin from Murraya paniculata (L.) Jack.  Canadian Journal of Chemistry. 1972;  50 443-5
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• 13 Simeon-Rudolf V, Kovarik Z, Radic Z, Reiner E. Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4′-bipyridine and by a coumarin derivative.  Chemico-Biological Interaction. 1999;  119 - 120 119-28
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• 14 Quinn D M. Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states.  Chemical Reviews. 1987;  87 955-79
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• 15 Nochi S, Asakawa N, Sato T. Kinetic study on the inhibition of acetylcholinesterase by 1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine hydrochloride (E2020).  Biological & Pharmaceutical Bulletin. 1995;  18 1145-7
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• 16 Ellman G L, Courtney K D, Andres V, Featherstone R M. A new and rapid colorimetric determination of acetylcholinesterase activity.  Biochemical Pharmacology. 1961;  7 88-95
  CrossrefPubMedSearch in Google Scholar

M. Iqbal Choudhary

International Center for Chemical Sciences

H.E.J. Research Institute of Chemistry

University of Karachi

Karachi-75270

Pakistan

Email: hejric@digicom.net.pk, zainraa@digicom.net.pk

Fax: (92-21) 924-3190, 924-3191

References

• 1 Sastri B N. The Wealth of India. Vol. 6. Council of Scientific and Industrial Research New Delhi; 1962: 446-8
  Search in Google Scholar
• 2 Perry L M, Metzger J. Medicinal Plants of East and Southeast Asia. The MIT Press Cambridge; 1980: 367
  Search in Google Scholar
• 3 Talapatra S K, Dutta L N, Talapatra B. Structure of murralongin, a novel monomeric coumarin from Murraya elongata: Stereochemistry and preferred conformation of its unique side chain. Tetrahedron Letters 1973: 5005-8
  Search in Google Scholar
• 4 Ramstad E, Lin W C, Lin T J, Koo W Y. Coumurrayin, a new coumarin from Murraya paniculata (L.) Jack. Tetrahedron Letters 1968: 811-3
  Search in Google Scholar
• 5 Dreyer D L. Chemotaxonomy of the Rutaceae. IV. Constituents of Murraya paniculata (Linn.) Jack.  The Journal of Organic Chemistry. 1968;  33 3574-6
  PubMedSearch in Google Scholar
• 6  Atta-ur-Rahman, Choudhary M I, Shabbir M, Sultani S Z, Jabbar A. Cinnamates and coumarins from the leaves of Murraya paniculata .  Phytochemistry. 1997;  44 683-5
  CrossrefPubMedSearch in Google Scholar
• 7 Murray R DH, Mendez J, Brown S A. The Natural Coumarins. John Wiley & Sons Ltd Bristol; 1982: 27
  Search in Google Scholar
• 8 Barik B R, Kundu A B. A cinnamic acid derivative and a coumarin from Murraya exotica .  Phytochemistry. 1987;  26 3319-21
  CrossrefPubMedSearch in Google Scholar
• 9 Das S, Baruah R H, Sharma R P, Barua J N, Kulanthaival P, Herz W. 7-Methoxycoumarins from Micromelum minutum .  Phytochemistry. 1984;  23 2317-21
  CrossrefPubMedSearch in Google Scholar
• 10 Eliel E L, Wilen S H, Mander L N. Stereochemistry of Organic Compounds. John Wiley & Sons, Inc. New York; 1994: 140-2
  Search in Google Scholar
• 11 Ito C, Furukawa H. Constituents of Murraya exotica L. Structure elucidation of new coumarins.  Chemical & Pharmaceutical Bulletin. 1987;  35 4277-85
  PubMedSearch in Google Scholar
• 12 Steck W. Paniculatin, a new coumarin from Murraya paniculata (L.) Jack.  Canadian Journal of Chemistry. 1972;  50 443-5
  PubMedSearch in Google Scholar
• 13 Simeon-Rudolf V, Kovarik Z, Radic Z, Reiner E. Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4′-bipyridine and by a coumarin derivative.  Chemico-Biological Interaction. 1999;  119 - 120 119-28
  PubMedSearch in Google Scholar
• 14 Quinn D M. Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states.  Chemical Reviews. 1987;  87 955-79
  CrossrefPubMedSearch in Google Scholar
• 15 Nochi S, Asakawa N, Sato T. Kinetic study on the inhibition of acetylcholinesterase by 1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine hydrochloride (E2020).  Biological & Pharmaceutical Bulletin. 1995;  18 1145-7
  PubMedSearch in Google Scholar
• 16 Ellman G L, Courtney K D, Andres V, Featherstone R M. A new and rapid colorimetric determination of acetylcholinesterase activity.  Biochemical Pharmacology. 1961;  7 88-95
  CrossrefPubMedSearch in Google Scholar

M. Iqbal Choudhary

International Center for Chemical Sciences

H.E.J. Research Institute of Chemistry

University of Karachi

Karachi-75270

Pakistan

Email: hejric@digicom.net.pk, zainraa@digicom.net.pk

Fax: (92-21) 924-3190, 924-3191