CN1048489C - Novel anti-virus compound methyl brevifolin carboxylic ester and method for prepn. of same - Google Patents

Abstract

The present invention relates to a novel antiviral compound (brevifolincarboxylic acid methyl ester). Underleaf pearl whole grass is used as raw materials, a crude product is prepared by the gradient elution of chloroform, methanol and water through pulverization, degreasing, methanol extraction and silica gel column low pressure chromatography, and a pure brervifolincaboxylic acid methyl ester product is obtained after the recrystallization of methanol and chloroform. Experiments prove that the compound has obvious antiviral activity. The present invention lays chemical and biological foundations for developing novel antiviral medicine and can be directly used for developing novel antiviral medicine.

Description

Novel anti-virus compound Methy Brevifolin-carboxlate and preparation method thereof

The present invention relates to a kind of antiviral activity compound Methy Brevifolin-carboxlate and preparation method thereof.

Hepatitis B is one of main transmissible disease of harm humans health.Domestic have 10% population to carry hepatitis B surface antigen approximately.Though the medicine of treatment hepatitis B and the research of Hepatitis B virus vaccine thereof get along with at present, still do not have good specifics so far.In order to study the medicine of treatment hepatitis B, very big manpower, financial resources have all been dropped in countries in the world.Doctor Bu Lunbai (Dr.B.Blumberg) of U.S. Philadelphia DKFZ adopts the plant amedica scalping, finds the Common Leafflower Herb crude extract, and the activity that suppresses hepatitis B surface antigen reaches 82%, but all belongs to crude extract.

The object of the present invention is to provide a kind of new compound with antiviral activity, and the method for from Common Leafflower Herb, extracting this new compound.

This antiviral activity compound that the present invention separates in Common Leafflower Herb, purifying obtains is Methy Brevifolin-carboxlate (Methy Brevifolin-carboxlate), and its structural formula is:

This compound can be used as the preparation new antiviral drug.

With modern spectral methods such as chemical reaction, ultraviolet, infrared, nucleus magnetic resonance and mass spectrums the All new compounds of extracting is identified.In structure is identified, also adopted two dimensional NMR and ¹H- ¹H COSY, ¹³C- ¹HCOSY, long-range ¹³C- ¹New technology, novel methods such as H and DEPT, INEPT, qualification result is as follows:

Methy Brevifolin-carboxlate is little yellow needle .m.p.200 ℃ (decomposing methyl-sulphoxide).High resolution mass spectrum provides molecular formula C ₁₄H ₁₀O ₀(M306.0376, calculated value 306.0376).The ferric chloride reaction positive, the potassium hydroxide reacting positive.Displaing yellow fluorescence on the polyamide layer.

UV (λ _Max ^MEOHNm, log ε): 218.5 (4.57), 279.0 (4.95), 349.0 (4.62), 369.0 (4.62) with ethyl brevifolincarboxylate (ethyl brevifolincarboxylate) basically identical of bibliographical information.

IR (V _Max ^KBrCm ^-1): 3387,3140 (OH), 1720,1698 (C=o), 1634 (C=C), 1598,1498 (aromatic ring frame vibrations).

Comparison this product and short leaf wood phenol acetate ¹H-NMR, and its fragrant hydrogen signal δ 7.29 (1H, S), phenolic hydroxyl group signal δ 10.20 (3H, b) and the ABX system signal that is presented, δ 2.46 (1H, dd, j=2HZ), 2.98 (1H, dd, j=8HZ, 19HZ), 4.42 (1H, dd, j=8HZ is 2HZ) all close with ethyl brevifolincarboxylate.Different is that ethyl brevifolincarboxylate presents OCH ₂CH ₃A ₂X ₃The signal of system, and this product replaces OCH ₃δ 3.62 (3H, S) hydrogen signal.

Comparison this product and ethyl brevifolincarboxylate ¹³C-NMR (seeing Table-1).Ethyl brevifolincarboxylate has OCH ₂CH ₃In two carbon signals, and this product replaces OCH ₃Carbon signal.Other carbon signal is identical.

Shown that by above deduction this product is leaf bush phenolic acid methyl esters (Methyl brevifolin carboxlate), is a new compound, its structural formula is:

¹H- ¹H COSY, ¹³C- ¹H COSY, long-range ¹³C- ¹H COSY has further verified the structure of Methy Brevifolin-carboxlate.And in view of the above to Methy Brevifolin-carboxlate ¹³C-NMR belongs to.Thereby by relatively to ethyl brevifolincarboxylate ¹³C-NMR has carried out belonging to (seeing Table-1) again.

The cleavage of mass spectrum rule of Methy Brevifolin-carboxlate of the present invention is seen accompanying drawing 1.

The high resolution fragment composition of Methy Brevifolin-carboxlate of the present invention sees Table-2.

Methy Brevifolin-carboxlate of the present invention ¹H- ¹H COSY spectrum is seen accompanying drawing 2; ¹³C- ¹The HCOSY spectrum is seen accompanying drawing 3; Long-range ¹³C- ¹H COSY spectrum is seen accompanying drawing 4; The DEPT spectrum is seen accompanying drawing 5.

Table-1 Methy Brevifolin-carboxlate ¹³The C-NMR data, ¹H- ¹³COSY, ¹H- ¹³The carbon spectrum of C long-range coupling and ethyl brevifolincarboxylate.

Carbon atom	Chemical shift			Relevant spectrum	Long-range coupling
						Ethyl brevifolincarboxylate		Methy Brevifolin-carboxlate
	A	B
			1 2 3 3a 5 5a 6 7 8 9 9a 9b 1′ 1″ 1			41.0 41.0 36.8 36.8 193.0 193.0 149.7 145.4 160.2 160.2 113.1 115.1 108.2 108.2 143.7/145.9 140.3 138.5 149.7 145.9/143.7 143.7 115.1 113.1 140.3 138.5 172.1 172.1 60.7 60.7 13.9 13.9			40.7264(d) 37.0874(l) 193.144(s) 145.951(s) 160.240(s) 115.106(s) 108.227(d) 140.448(s) 149.775(s) 143.709(s) 113.121(s) 138.525(s) 172.632(S) 52.1807(q)	δ 4.42(H-1) δ 2.46(H-2B) δ 2.98(H-2A) δ 7.29(H-6) δ 3.62(H-1″)	H-1 H-6 H-6 H-6 H-6 H-6 H-1 H-1，H-1″

A: the document ownership of ethyl brevifolincarboxylate C-NMR

B: this paper is to the document ownership of ethyl brevifolincarboxylate C-NMR

The high resolution mass spectrum fragment of table-2 Methy Brevifolin-carboxlates is formed

Fragment	Heavy		Ownership
				Measured value	Calculated value
	C14H1008 C13H607 C12H606 C11H605 C10H704 C9H603 C8H602 C8H50 C7H50	306.0376 306.0376 274.0125 274.0113 246.0167 246.0165 218.9436 218.0215 191.0345 191.0345 162.0304 162.0317 134.0366 134.0368 117.0324 117.0324 105.0343 105.0346				M + M +-CH3OH M +-CH3OH-CO M +-CH3OH-CO-H M +-CH3OH-3CO+H M +-CH3OH-4CO M +-CH3OH-5CO M +-CH3OH-5CO-OH M +-CH3OH-6CO-H

Further measured the antiviral activity of new compound Methy Brevifolin-carboxlate.

By the method for measuring the medicine antiviral activity usually, measure medicine pair cell toxicity (TO with the human cervical carcinoma Hela cell ₅₀), reproduce and examine the antivirus action of medicine the simple herpes virus HSV-1 of people STAKER strain and HSV-2 SAR strain, measure ED ₅₀, and calculate its protection index (PI).The positive control medicine adopts virazole, and (ribavirin, virazole), the result is shown in table 3,4,5,6,7.

Table 3, the simple herpes virus Hsv-1 of people, Hsv-2 cause Hela cytopathy record (CPE)

Virus infection amount 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6
	HSV-1 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++++ SHV-2 ++ ++ ++ ++ ++ ± ± ±-
Annotate :-there is not an obvious cytopathy ++ cytopathy＜50% ± occur suspicious cells pathology +++cytopathy＜75%+cytopathy＜25% ++ ++ cytopathy＞80%

HSV-1, HSV-2 are to Hela cell-lethal effect for table 4 people simple cell virus

And TCID ₅₀(mtt assay)

The virus infection amount	10 -1 10 -2 10 -3 10 -4 10 -5 10 -6	TCID 50
			Cell mortality (%)
	HSV-1 HSV-2			100.0 96.0 80.0 65.0 25.0 18.0 96.2 76.0 20.0 0 0 0	4.5×10 3.80×10

Table 5 different concns medicine is to the kill rate (%) and the TD of Hela cell ₅₀

Drug level (μ g)	7.88 1.95 4.8×10 -11.2×10 -13.0×10 -27.5×10 -31.8×10 -34.6×10 -4	TD 50
			Cell mortality (%)
	Methy Brevifolin-carboxlate			97.0 80.0 34.0 14.0 11.0 5.0 --- ---	5.67×10
Drug level (μ g)		1.25 1.25×10 -11.25×10 -21.25×10 -31.25×10 -41.25×10 -5	TD 50
	Cell mortality (%)
		Virazole		83.4 44.0 24.0 20.0 0 0	9.92×10

The anti-HSV-1 virus function of table 6 Methy Brevifolin-carboxlate (ED ₅₀)

And protection index (PI)

Medicine name	TD 50	ED 50	PI
				The Methy Brevifolin-carboxlate virazole	0.567 0.0992	0.00375 0.000625	151.2 158.0

The anti-HSV-2 virus function of table 7 Methy Brevifolin-carboxlate (ED ₅₀) and

Protection index (PI)

Medicine name	TD 50	ED 50	PI
				The Methy Brevifolin-carboxlate virazole	0.567 0.0992	0.0018 0.000125	302.40 793.60

Table 3-7 result show, Methy Brevifolin-carboxlate has sure and significant antiviral activity to human herpes simplex vicus HSV-1, HSV-2 strain.

Methy Brevifolin-carboxlate of the present invention is the new compound that extraction from Common Leafflower Herb, separation, purifying obtain.Common Leafflower Herb is an Euphorbiaceae phyllanthus emblica platymiscium, and the used Common Leafflower Herb of the present invention all picks up from mountain area, Wuyi, Fujian, extracts this new compound and mainly comprises following step:

1, gets dry Common Leafflower Herb herb and be crushed to suitable fineness;

2, use petroleum ether degreasing earlier, and isolate degreasing fluid;

3, the isolated dregs of a decoction fully extract with methyl alcohol;

4, concentrate methanol extract liquid with underpressure distillation, obtain a kind of methyl alcohol medicinal extract;

5, add the water suspendible, medicinal extract also distributes with chloroform and propyl carbinol successively;

6, isolate propyl carbinol and distribute phase, concentrate again;

7, be that eluent carries out gradient elution with the silica gel low pressure column chromatography and with chloroform-methanol-water, collect elutriant respectively, 250 milliliters every part, obtain the Methy Brevifolin-carboxlate crude product at the 36th part;

8, secondary purification by silica gel column chromatography;

9, heavily tie product with methyl alcohol-chloroform, obtain pure Methy Brevifolin-carboxlate.

Below accompanying drawing is done an explanation:

Accompanying drawing 1 is the cleavage of mass spectrum figure of Methy Brevifolin-carboxlate of the present invention;

Accompanying drawing 2 is a Methy Brevifolin-carboxlate of the present invention ¹H- ¹The HCOSY spectrogram.

Accompanying drawing 3 is a Methy Brevifolin-carboxlate of the present invention ¹³C- ¹The HCOSY spectrogram.

Accompanying drawing 4 is long-range for Methy Brevifolin-carboxlate of the present invention ¹³C- ¹The HCOSY spectrogram.

Accompanying drawing 5 is the DEPT spectrogram of Methy Brevifolin-carboxlate of the present invention.

With a concrete instance the present invention extracts Methy Brevifolin-carboxlate in Common Leafflower Herb method is further described again below.Get dry Common Leafflower Herb herb, break into meal, insert in the extractor of the rope third constellations, add petroleum ether degreasing earlier after, dregs of a decoction airing, fully extract with methyl alcohol again.Tell methanol extract liquid, concentrate methanol extract liquid, obtain methyl alcohol medicinal extract with underpressure distillation.This medicinal extract is added the suitable quantity of water suspendible, distribute with chloroform and propyl carbinol successively, after propyl carbinol taken out concentrating under reduced pressure, with silica gel and diatomite spice, the silica gel low pressure column chromatography is made gradient elution with chloroform-methanol-water, collects elutriant respectively, 250 milliliters every part, obtain the Methy Brevifolin-carboxlate crude product at the 36th part.It is secondarily purified that this raw product passes through silicagel column again, with methyl alcohol-chloroform recrystallization, promptly obtains pure Methy Brevifolin-carboxlate.

This Methy Brevifolin-carboxlate of the present invention to extract in the Common Leafflower Herb, has significant antiviral activity through this new compound of experiment confirm, thereby, and can be directly used in the exploitation new antiviral drug for this new new antiviral drug of exploitation has been established chemistry and biological basis.

Claims (2)

1, a kind of antiviral activity compound that extracts from Common Leafflower Herb is characterized in that said antiviral activity compound is a Methy Brevifolin-carboxlate, and its structural formula is: This compound can be used as the preparation new antiviral drug.

2,, it is characterized in that comprising following extraction step according to the extracting method of the described antiviral activity compound of claim 1:

2-1, pulverizing exsiccant Common Leafflower Herb herb;

2-2, use petroleum ether degreasing, separate degreasing fluid;

2-3, leaching slag fully extract with methyl alcohol;

2-4, underpressure distillation concentrate methanol extract liquid, get methyl alcohol medicinal extract;

2-5, medicinal extract add the water suspendible;

2-6, distribute with chloroform and propyl carbinol successively;

2-7, isolate the n-butanol extraction liquid phase, and concentrate;

2-8, with the silica gel low pressure column chromatography;

2-9, with chloroform-methanol-water gradient elution, collect elutriant respectively, 250 milliliters every part, rise at the 36th part, obtain the Methy Brevifolin-carboxlate crude product;

2-10, secondary purification by silica gel column chromatography;

2-11, with methyl alcohol-chloroform recrystallization, obtain pure Methy Brevifolin-carboxlate.

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