WO1996007409A1

WO1996007409A1 - Alkaloids and their antiviral agents

Info

Publication number: WO1996007409A1
Application number: PCT/GB1995/002091
Authority: WO
Inventors: Peter John Houghton; Tibebe Zewdie Woldermarian; Naheed Mahmood
Original assignee: King's College London
Priority date: 1994-09-07
Filing date: 1995-09-05
Publication date: 1996-03-14
Also published as: GB9417969D0; AU3395295A

Abstract

The present invention relates to chromone alkaloids isolated from the root, stem and root-bark of Schumanniophyton magnificum and Schumanniophyton problematicum, to analogues thereof and to therapeutic use thereof in the treatment of viral infection.

Description

ALKALOIDS AND THEIR ANTIVIRAL AGENTS

The present invention relates to chromone alkaloids isolated from the root, stem and root -bark of Schumanniophyton magnificum and S. problematicum, which are trees found in West Central Africa. The present invention also relates to analogues of the alkaloids and to therapeutic uses of the alkaloids and their analogues. In particular, the invention relates to use of the alkaloids and their analogues in the prophylaxis and treatment of infection by human immunodeficiency virus (HIV), which is believed to be the aetiological agent in human acquired immunodeficiency syndrome (AIDS), and herpes simplex virus (HSV).

Chromone alkaloids were first isolated from S. problematicum by Schlittler et al. (Tetrahedron Letts., 2911-2914 (1978)), who reported three alkaloids, schumanniophytine (1) and two unnamed piperidin-2-ones (2a) and (2b):

Further chromone alkaloids were isolated from S. magnificum

Harms. (Rubiaceae) by Okogun et al. (Planta Medica, Journal of Medicinal Plant Research, 49, 95-98, (1983)), who reported two alkaloids, schumannificine and N-methyl schumannificine, and the acetyl derivatives thereof which were prepared in the course of the isolation of the parent alkaloids.

Houghton et al . (Planta Medica, Journal of Medicinal Plant Research, 23-27, (1985); Ibid., 262-264 (1987); Ibid., 264- 266 (1987); Ibid., 239-242 (1988); Phytochemical Analysis, 4, 9-13, (1993)) isolated a number of additional alkaloids including anhydroschumannificine (3), N-methyl anhydroschumannificine (3a), isoschumanniophytine (4) and rohitukine (5) and corrected the original structural formulae assignments of schumannificine 6a and N-methyl- schumannificine 6b. Structure 6a is now believed the correct structure of schumannificine. The compound is generally isolated as a mixture of 7'-isomers, although the isomers may be separated by HPLC The stereochemistry at the 3' and 4' positions has not been determined.

Rohitukine (5) has also been isolated from other plants belonging to the family Meliaceae. Rohitukine (5) and a number of derivatives thereof are reported (United States Patents 4,603,137 and 4,900,727 and Australian Patent Application AU-A-43841/89) to exhibit anti-inflammatory, analgesic, immuno-suppressive and anti-tumour activity.

HIV is believed to be the aetiological agent in AIDS. (Barre-Sinoussi et al . , Science, 220, 868-870, (1983); Gallo et al . , Science, 224, 500-503, (1984)) and there are numerous reports of chemical compounds, such as AZT (zidovudine), possessing HIV- inhibitory activity. Such compounds, however, often exhibit problems with toxicity and other undesirable side effects in individual patients. There remains, therefore, the need for alternative and improved compounds for use in the prophylaxis and treatment of HIV infection. It has now been found that Schumanniophyton alkaloids and derivatives thereof are effective viral inhibitors whilst exhibiting low toxicity.

According to a first aspect of the present invention there is provided use of a compound of a formula selected from the group comprising:-

wherein

R¹, R², R⁴, R⁵, R⁶, R⁷ and R⁸ may be the same or different and are selected from the group comprising hydrogen, hydroxy and substituted alkyl, alkoxy, alkoyloxy, aryl, aryloxy and aryloyloxy groups;

R³ is selected from the group comprising hydrogen, carbohydrates and oligosaccharides, and substituted or unsubstituted alkyl, alkoyl, aryl and aryloyl groups;

R⁹ is an alkyl group;

X is selected from -CH₂- and - C (O) - ;

Y is selected from -CHR¹⁰- and - C (O) - ;

Z is selected from N and O;

n is selected from 0, 1 and 2;

R¹⁰ is selected from the group comprising hydrogen, hydroxy, carbohydrates and oligosaccharides, and substituted or unsubstituted alkyl, alkoxy, alkoyloxy, aryl, aryloxy and aryloyloxy groups;

and pharmaceutically acceptable derivatives thereof, in the manufacture of a medicament for use in the treatment or prophylaxis of viral infection.

Preferably, the viral infection comprises HIV or HSV infection.

Pharmaceutically acceptable derivative means any pharmaceutically acceptable salt or addition compound or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) the parent compound or an anti-virally active metabolite or residue thereof.

Pharmaceutically acceptable salts include, for example, the hydrochloride, hydrobromide, sulphate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate. Pharmaceutically acceptable addition compounds include, for example, quaternary amines and esters of the compounds.

Reference to an alkyl group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical. Where cyclic, the alkyl group is preferably C₃ to C₁₂, more preferably C₅ to C₁₀, more preferably C₅ to C₇. Where acyclic, the alkyl group is preferably C₁ to C₁₀, more preferably C₁ to C₆, more preferably methyl. Reference to an aryl group means an aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more, preferably one, heteratom, such as pyridyl, pyrrolyl, furanyl and thiophenyl. Preferably, the aryl group comprises phenyl.

The alkyl and aryl groups may be substituted or unsubstituted, preferably unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. Substituents may include halogen atoms; oxygen containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyl, alkoyloxy; nitrogen containing groups such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulphur containing groups such as thiol, alkylthiol, sulphonyl and sulphoxide; heterocyclic groups containing one or more, preferably one, heteroatom, such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, morpholinyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl and carbolinyl; and aryl groups such as phenyl and substituted phenyl. Alkyl includes substituted and unsubstituted benzyl.

Reference to alkoxy means alkyl-0-. Reference to alkoyloxy means alkyl -C(O)-O-. Reference to aryloxy means aryl-O-. Reference to aryloyloxy means aryl -C(O)-O-.

Carbohydrates and oligosaccharides preferably comprise carbohydrates and oligosaccharides that improve the bioavailability of the compound, such as mono- up to penta- saccharides comprising, for example, glucose, glucuronic acid or rhamnose or their derivatives.

Preferably, R¹ is methyl or substituted or unsubstituted phenyl. More preferably, R¹ is methyl or unsubstituted phenyl, preferably methyl.

Preferably, R² is hydroxy or alkoyloxy. More preferably, R² is hydroxy.

Preferably, R³ is hydrogen or alkyl. More preferably, R³ is hydrogen.

Preferably, R⁴ is hydrogen.

Preferably, R⁵ is hydroxy or alkoyloxy. More preferably, R⁵ is hydroxy.

Preferably, R⁶ is hydroxy or alkoyloxy. More preferably, R⁶ is hydroxy. Preferably, R⁷ is hydroxy or alkoyloxy.

Preferably, R⁸ is hydrogen. Preferably, R¹⁰ is hydroxy or alkoyloxy. More preferably, R¹⁰ is hydroxy.

With regard to compounds of formula I and III, x is preferably -C(O)-. With regard to compounds of formula IV, X is preferably -CH₂-.

Preferably, Y is -CHR¹⁰-

Preferably, Z is O.

Preferably, n is 1

Preferably, R⁴, R⁵ and R⁸ are hydrogen and Y is -CHR¹⁰-. Preferably, the compound is selected from the group comprising formulae I, II, III and IV, more preferably I and III, more preferably I.

Preferably, the compound is selected from the group comprising

Schumanniophytine

Isoschumanniophytine

N-methylschumanniophytine

Rohitukine

N-methylschumannificine

N-methylanhydroschumannificine

N-dimethylschumannificine

7'-(4-bromobenzoyl) N-methylschumannificine

Schumannificine

Anhydroschumannificine

N-demethyl-3'-acetyl-rohitukine

N,7'-diacetylschumannificine

N,7',5-triacetylschumannificine 7'-(4-bromobenzoyl)-schumannificine

1',5-di(4-bromobenzoyl)-schumannificine

7'-methoxyschumannificine

7',5-dimethoxyschumannificine

It will be appreciated that the compounds of the present invention exist in various diastereomeric and enantiomeric forms as a result of asymmetric centres in the compounds. The present invention incudes different diastereomers and enantiomers in isolation from each other, as well as mixtures.

The compounds of the present invention may be synthesised by conventional synthetic organic chemistry or may be prepared by isolation of the natural product from the root, stem or root-bark of S. magnificum or S. problematicum (Flora of West Tropical Africa, 2nd edition, (1963), ed. N. Hepper, volume II, pages 104-105 and 116, J. Hutchinson and J.M. Dalziel, pub. Crown Agents.) followed, where appropriate, by derivatisation using conventional synthetic organic chemistry.

For example, compounds of formula II may be prepared from compounds of formula I (where X = -C(O)-) by treatment with BF₃ and R⁹-O-R⁹; compounds in which Y = -CH₂- may be prepared from the corresponding compounds in which Y = -CHOH- by tosylation and reduction; compounds in which Z = N can be prepared from the corresponding compounds in which Z = O by treatment with ammonia; compounds in which X = - CH₂- may be prepared from the corresponding compounds in which X = -C(O) by reduction with LiAlH₄; compounds in which Y = -C(O)- may be prepared from the corresponding compounds in which Y = -CHOH- by oxidation with Jones' reagent.

Whilst it is believed that the structural formulae assigned to the natural schumanniophyton alkaloids identified above are correct, and that the alkaloids have been purified to homogeneity, it will be understood that the present invention extends to the natural product isolates described herein irrespective of the assigned formulae, to any co- isolates thereof and to derivatives thereof.

Accordingly, a further aspect of the present invention provides use of a schumanniophyton alkaloid or derivative thereof in the manufacture of a medicament for the treatment or prophylaxis of viral infection. A schumanniophyton alkaloid comprises an alkaloid isolatable from S . magnifi cum or S . problema ti cum . Derivatives thereof comprise alkaloids bearing alkyl, alkoxy, alkoyloxy, aryl, aryloxy and aryloyloxy substituents as defined hereinbefore.

According to a further aspect of the present invention there is provided a compound of a formula selected from the group comprising

wherein R¹-R⁵, X, Y, Z and n are as defined above,

with the proviso that when R¹ is methyl, R³ is hydrogen or methyl, R⁴ is hydrogen, R⁵ is hydrogen, X is -C(O)-, Y is

CHR ¹⁰-, R¹⁰ is OH or OAc, Z is 0 and n is 1, then R² is not the same as R¹⁰;

wherein R¹-R⁵, R⁹, Y, Z and n are as defined above;

wherein R¹-R⁵, X, Z and n are as defined above,

with the proviso that when R¹ is methyl, R³ is hydrogen cr methyl, R⁴ is hydrogen, X is -C(O)-, Z is 0 and n is 1, then R² is not OH or OAc;

wherein R¹-R⁷, X, Z and n are as defined above,

with the proviso that either or both X is -C(O)- and/or Z is

N;

wherein R¹, R² , R⁴ , R⁵ and Z are as defined above,

with the proviso that when R¹ is methyl, R⁴ and R⁵ are hydrogen and Z is 0, then R² is not OH or OAc;

wherein R¹, R², R⁴, R⁸ and Z are as defined above,

with the proviso that when R¹ is methyl, R⁴ and R⁸ are hydrogen and Z is 0 then R² is not OH or OAc.

According to a further aspect of the present invention, there is provided a pharmaceutical composition for use in the treatment or prophylaxis of viral infection comprising a compound as hereinbefore defined in combination with a pharmaceutically acceptable excipient. The compounds of the invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal and topical administration.

For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension. Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil. For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl- pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

According to a further aspect of the present invention there is provided a process for the production of a pharmaceutical composition comprising the step of combining a compound as hereinbefore defined with a pharmaceutically acceptable excipient. According to a further aspect of the invention there is provided a method of treatment or prophylaxis of viral infection comprising administering to a patient in need of such treatment or prophylaxis an effective dose of a compound as hereinbefore defined.

The invention will now be described with reference to the following examples. It will be appreciated that what follows is by way of example only and that modifications to detail may be made whilst still falling within the scope of the invention.

EXPERIMENTAL

A. EXTRACTION AND ISOLATION OF ALKALOIDS FROM

SCHUMANNIOPHYTON MAGNIFICUM

All solvents and reagents used were of AnalaR grade. Dried S . magnificum stem- and root-bark was obtained from Southeast Nigeria. Samples of dried stem- and root-bark of S . problema ticum were obtained from Tiassle, Ivory Coast. Fresh S. magnificum stem and root material was collected from a forest reserve near Calabar and flown to London within 48 h where it was stored at -70°C. Reference vouchers are stored in the herbarium of the Chelsea Department of Pharmacy, King's College London.

500 g of powdered dried rootbark were extracted with hot methanol for 12 hours using a Soxhlet apparatus. The extract was concentrated to a thick syrup and mixed with 2L chloroform-water 1:1. The two layers were separated using a separating funnel. Chloroform layer

The chloroform layer was washed with 2 x 150mL water and then taken to dryness. 2.5g of the residue was adsorbed on to silica gel for Flash chromatography and fractionated on a silica column 1.5 x 25 cm. The following eluting solvents were used and 5ml aliquots collected.

The fractions were screened for content using TLC (silica gel GF₂₅₄) chloroform:butanone 4:1 (system A), chloroform:methanol 12:1 (system B) and chloroform:methanol 6:1 (syscem C).

Developed plates were examined under UV (254nm and 365nm) light before spraying and in daylight after spraying with Dragendorff's reagent. Plates were then oversprayed with aqueous Fe(III)Cl₃ and the colours of zones noted.

Like fractions were bulked and taken to dryness. Compounds were extracted in the pure form by preparative TLC using systems A, B or C or butanone:methanol 12:1 (System D) and elution using methanol.

The compounds are eluted in the following order. (Details of the R_f values, colour reactions and spectroscopic characteristics are given below).

Noreugenin (non-alkaloid)

N-methylanhydroschumannificine (3b)

Isoschumanniophytine (4)

Schumanniophytine (1)

N-methylschumannificine (6b)

Anhydroschumannifine (3a)

Schumannificine (6a)

Rohitukine (5)

Water Layer Alkaloids were precipitated from the water layer with Dragendorff's reagent. The suspension was filtered and the residue dissolved in acetone :methanol :water 6:2:1. This solution was passed through an ion exchange column 15 x 1.5cm IRA 400 Cl- . The eluate was concentrated and the residue separated by droplet counter-current chromatography butan-1-ol:methanol:water 5:1:5 using descending mode. Fractions (10mL) were collected and monitored on TLC (Silica gel/acetone:methanol:13.5M ammonia 4:1:1 - System E; detection as above for chloroform layer).

Similar fractions were bulked, concentrated and individual compounds extracted by prep TLC (silica gel GF₂₅₄/ethyl acetate:propan-2-ol:ammonia 65:35:10 - System F). The two alkaloids isolated were (in order of elution):

N-methylschumanniophytine (7)

Rohitukine (5) (Details of the R_f values, colour reactions and spectroscopic characteristics are given below). Example 1

N-METHYLANHYDROSCHUMANNIFICINE (3b) TLC behaviour

Colour:

UV 254nm Quenches

UV 365nm No colour

After Dragendorff's Brown

After FeCl₃ Dark Brown

R_f values

System A 0.28

System B 0.86

System D 0.45

Crystallisation Could not obtain crystals

UV spectrum (Methanol; maxima nm (log ε))

224 (4.20), 246 (4.10), 256 (4.10), 310 (2.30)

IR spectrum (liquid paraffin)

1660 1620 1592 ¹H NMR spectrum in CDCl₃ (δ ppm from TMS)

12.73 1H s (5-OH), 7.72 1H s (7'-H), 6.46 1H s (6-H), 6.12 1H s (3-H), 3.85 1H dd (4'-H), 3.6-3.0 4H m (5',6'-CH₂), 3.14 3H s (N-CH₃), 2.41 3 s (2-CH₃)

Mass spectrum

313 (25, M⁺), 298(20), 192 (100) Example 2 SCHUMANNIOPHYTINE (1) TLC behaviour

Colour

UV 254nm Pale yellow

UV 365nm Lemon yellow

After Dragendorff's Orange

After FeCl₃ Dark orange

R_f values

System A 0.09

System B 0.70

System D 0.06 Crystallisation

No crystals obtained

UV spectrum (Methanol; maxima nm (log ε))

225 (4.4) , 237 (4.41) , 251 (4.46) , 256 (4.45) , 292 (4.07) , 318 (4.11)

IR spectrum (liquid paraffin)

3200-2400, 3070, 1750, 1660, 1620, 1580

¹H NMR spectrum in CDCl₃ (δ ppm from TMS) 13.47 1H bs (5-OH), , 9.58 1H s (2'-H), 8.97 1H d (6'-H), 8.48 1H d (5'-H), 6.82 1H s (6-H), 6.34 1H s (3-H), 2.66 3H s (2-CH₃) Mass spectrum

295 (100, M⁺), 267, 255 (18), 227

Example 3

ISOSCHUMANNIOPHYTINE (4)

TLC behaviour Colour:

UV 254nm Pale yellow

UV 365nm Golden yellow

After Dragendorff's Brown

After FeCl₃ Dark Brown

R_f values

System A 0.09

System B 0.72

System D 0.04

Crystallisation

No crystals could be obtained

UV spectrum (Methanol; maxima nm (log ε))

226 (4.11), 245 (3.96), 260 (3.96), 272 (3.56), 314 (2.98)

IR spectrum (liquid paraffin)

1750, 1660, 1620, 1590 ¹NMR spectrum in CDCl₃ (δ ppm from TMS)

15.6 1H s (5-OH), 9.52 1H S (2'-H), 8.93 2H s (5',6'-H), 6.94 1H s ( 6 -H), 6.24 1H s (3-H), 2.50 3H S (2-CH₃) Mass spectrum

295 (100 M⁺) , 267 (12), 255 (15) Example 4 N-METHYLSCHUMANNIFICINE (6b)

TLC behaviour Colour:

UV 254nm Quenches

UV 365nm No colour

After Dragendorff's Brown

After FeCl₃ Dark Brown

R_f values System A 0.10

System B 0.72

System D 0.40

Crystallisation

Cream crystals from methanol MPt218-220°C

UV spectrum (Methanol; maxima nm (log ε)) 220 (4.12), 225 (4.12), 253 (3.89), 260 (3.90), 277 (3.89), 290 (3.90), 310 (3.95), 320 (3.96)

IR spectrum (liquid paraffin) 3300, 1670, 1630, 1575 ¹H NMR spectrum in CDCl₃ ( δ ppm from TMS)

12.6 1H bs (5-OH), 6.87 1H bs (7' -OH), 6.35 1H s (6-H), 6.09 1H s (3-H), 5.60 1H d (7'-H), 3.7 1H m (4'-H), 3.3-3.1 3H m (5' -CH₂,3' -H), 3.05 3H s (N-CH₃), 2.65 1H m (6'-H), 2.39 3H s (2-CH₃), 2.20 1H m (6' -H)

Mass spectrum 331 (46, M⁺) , 313 (24), 205 (43), 192 (100)

Example 5

ANHYDROSCHUMANNIFICINE (3a)

TLC behaviour

Colour: UV 254nm Quenches

UV 365nm No colour

After Dragendorff's Blue

After FeCl₃ Blue-black R_f values

System A 0.18

System B 0.79

System D 0.47

Crystallisation

No crystals could be obtained UV spectrum (Methanol: maxima nm (log ε)) 224 (4.08), 253 (4.32), 310 (2.40) IR spectrum (liquid paraffin)

1670, 1640, 1620

¹H NMR spectrum in CDCl₃ (δ ppm from TMS)

12.7 1H s (5-OH), 7.77 1H dd (7'-H), 7.2 1H bs (NH) , 6.27 1H s (6-H), 6.20 1H s (3-H), 4.00 1H dd (4'-H), 2.45 3H s (2- CH₃), 3.6-1.5 4 Hm (5', 6'-CH₂)

Mass spectrum

299 (14, M⁺), 192 (100) Example 6

SCHUMANNIFICINE (6a)

TLC behaviour

Colour:

UV 254nm Quenches

UV 365nm No colour

After Dragendorff's Pale orange

After FeCl₃ Blue-black

R_f values System A 0.05

System B 0.37

System D 0.29

Crystallisation

Crystals from methanol MPt 244-246°C UV spectrum (Methanol; maxima nm (log ε))

220 (4.13), 255 (4.13), 253 (3.86), 260 (3.88), 280 (3.88), 290 (3.85), 290 (3.85), 310 (3.95), 320 (3.96), 333 (3.97)

IR spectrum (liquid paraffin)

1650, 1620, 1165, 1090 ¹H NMR spectrum in CDCl₃ ( δ ppm from TMS)

12.6 1H bs (5-OH), 7.17 1H bs (N-H), 6.34 1H s (6-H), 6.11

1H s (3-H), 5.76 1H d (7'-H), 2.40 3H s (2-CH₃)

Mass spectrum

317 (21, M⁺), 299 (15), 287 (17), 192 (100) Example 7 ROHITUKINE (5)

TLC behaviour Colour :

UV 254nm Quenches

UV 365nm No colour

After Dragendorff's Pale orange

After FeCl₃ Blue-black

R_f values

System A 0.00

System E 0.95

System F 0.38 Crystallisation

Yellow crystals from absolute ethanol UV spectrum (Methanol; maxima nm (log ε))

208 (4.37), 228 sh (4.12), 250 sh (3.97), 262 (4.10), 330 (3.68) IR spectrum (liquid paraffin)

3400, 1660, 1612, 1560

¹H NMR spectrum in CDCl₃ (δ ppm from TMS)

6.79 1H s (6-H), 6.17 1H s (3-H), 4.44 1H d (3' -H), 3.63 1H dt (4' -H), 3.16-2.36 5H m (2' , 5, 6'-H) , 2.27 3H s (2-CH₃) , 2.21 3H s (N-CH₃), 1.57 1H m (6'-H) Mass spectrum

305 (M⁺)

Example 8

N-METHYLSCHUMANNIOPHYTINE (7)

TLC behaviour Colour:

Daylight Bright yellow

UV 254nm Yellow

UV 365nm Bright yellow

After Dragendorff's Orange

After FeCl₃ Dark orange R_f values

System A 0.00

System E 0.75

System F 0.10

Crystallisation

Yellow crystals from absolute ethanol

UV spectrum (Methanol; maxima nm (log ε))

204 (4.69), 225 (4.4), 236 (4.39), 282 sh (4.07), 355 (4.12) IR spectrum (liquid paraffin)

3200, 1750, 1660, 1620, 1585

¹H NMR spectrum in CDCl₃ (δ ppm from TMS)

8.99 1H s (2' -K), 8.69 1H d (5' -H), 8.10 1H d (6' -H), 6.32 1H s (6-H), 6.10 1H s (3-H), 4.39 3H s (N-CH₃), 2.26 3H s (2-CH₃)

Mass spectrum

310 (5, M⁺) , 295 (20), 243 (76), 228 (56), 200 (32), 196 (20)

B. SYNTHESIS OF CHROMONE ALKALOID DERIVATIVES

Example 9

4-BROMOBENZOYL DERIVATIVES

60mg of the alkaloid was dissolved in 1mL pyridine. 60mg dimethylaminopyridine (DMAP) and 120 mg 4-bromo benzoyl chloride (4BrBzCl) were dissolved in 1mL dichloromethane and were added to the alkaloid solution. The solution was made up to 6mL with dichloromethane and kept at 25º for 72 hours.

The reaction mixture was poured into 20mL 1M NaHCO₃ and extracted with 3 x 10mL CHCl₃. The chloroform was washed with water and evaporated off under reduced pressure. The residue was examiend on TLC and the products isolated by prep TLC (silica gel/chloroform:methanol 12:1). The mass spectrum and ¹H NMR spectrum of each product was obtained.

Schumannificine when treated in this way gave two products Example 9a

Di (4-bromobenzoyl) schumannificine (6f) R_f value 0.73

Mass spectrum

684 (15) M⁺, 301 (100).

¹H NMR spectrum (CDCl₃) δ ppm from TMS

8.04 (2H d J=8.6 3", 5"-H). 7.85 (2H, d J=8.6 Hz 3'5'-H), 7.85 (2H d J=8.6 2", 6"-H), 7.60 (2H, d J=8.6Hz 2', 6'-H), 7.17 (1H. d J=3.7Hz 7' -H), 6.76 ( 1H s 6-H), 6.01 (1H s 3-H) , 4.82 (1H bs N-H) 3.89 (1H m 4'-H), 3.35 (1H m 6' -H), 3.24 (1H m 3'-H), 3.15 (1H m 6'-H), 2.80 (1H m 5'-H), 2.43 (3H s

2-CH₃), 2.25 (1H m 5' -H).

Example 9b Mono (4-bromobenzoyl) schumannificine (6c) R_f value 0.64

Mass spectrum

5?0 (40) M⁺, 301 (100).

¹H NMR spectrum (CDCl₃) δ ppm from TMS

12.7 (1H s 5-OH), 7.81 (2H, d J=8.6 Hz 3'5'-H), 7.54 (2H, d J=8.6Hz 2',6'-H), 7.14 (1H. d J=3.7Hz 7'-H), 6.38 (1H s 6- H), 6.12 (1H s 3-H), 4.82 (1H bs N-H) 3.82 (1H m 4' -H), 3.32 (1H m 6'-H), 3.18 (1H m 3'-H), 3.07 (1H m 6'-H), 2.80 (1H m 5'-H), 2.43 (3H s 2-CH₃), 2.19 (1H m 5'-H).

Example 9c

N-methylschumannificine gave one product

Mono (4-bromobenzoyl) N-methylschumannificine (6e) R_f value

0.69 Mass spectrum

514 ( 40 ) M⁺ , 331 ( 100 ) . ¹H NMR spectrum (CDCl₃) δ ppm from TMS

12.64 (1H s 5-OH), 7.84 (2H d J=8.6 Hz 3'5'-H), 7.54 (2H, d J=8.6Hz 2',6'-H), 7.17 (1H. d J=3.7Hz 7'-H), 6.38 (1H s 6- H), 6.12 (1H s 3-H), 3.81 (1H m 4'-H), 3.24-3.08 (3H m 3'-H, 6-CH₂), 2.92 (3H s N-CH₃), 2.81 (1H m 5'-H), 2.43 (3H s 2- CH₃), 2.22 (1H m 5' -H).

Example 10 ACETYLATION

Cold acetylation

60mg alkaloid is mixed with 1 ml pyridine and 2 ml acetic anhydride and kept for 72 hours at 25º. The solvents were evaporated under reduced pressure and the residue acidified with 1M HCl (20mL) and shaken with 3 x 10mL chloroform. The chloroform was evaporated to small volume and the products purified by prep TLC using silica gel/chlcroform:methanol 12:1. The mass spectrum and ¹H NMR spectrum of each product was obtained.

Schumannificine treated in this way gave two products Example 10a

N,5,7'-triacetylschumannificine (6g) R_f 0.67

Mass spectrum

443 (M⁺ 20) 317 (100) ¹H NMR spectrum (CDCl₃) δ ppm from TMS

2.09 (3H s 7' -acetate) 2.41 (3H s 7' -acetate) 2.41 (3H s 2- CH₃) 2.60 (3H s NAc) 6.12 (1H s 3-H) 6.62 (1H s 3-H) 6.95 (1H d 7'-H) 12.60 (3H s 5-OAc)

Example 10b

N,7'-diacetylschumannificine (6f) R_f 0.60

Mass spectrum

401 (M⁺, 24), 359 (21), 341 (14), 317 (100)

1H NMR spectrum (CDCl₃) δ ppm from TMS

2.09 (s 3H 7' -acetate), 2.40 (s 3H 2-CH₃), 2.60 (s 3H N-Ac), 6.12 (s 1H 3-H), 6.62 (s 1H 3-H), 6.96 (d 1H 7'-H), 12.60 (3H s 5-OH) N-methylschumannificine treated in this way gave two products

Example 10c

5,7'-diacetyl N-methylschumannificine (6h) R_f 0.75 Mass spectrum

415 (M⁺, 24), 373 (18), 331 (100), 313 (22) ¹H NMR spectrum (CDCl₃ δ ppm from TMS)

2.08 (s 3H 7' -acetate), 2.36 (s 3H 5-acetate), 2.40 (s 3H 2- CH₃), 2.91 (s 3H N-CH₃), 6.04 (s 1H 3-H), 6.60 (s 1H 6-H), 6.97 (d 1H J = 4.0 7' -H) Example 10d

7'-acetyl N-methylschumannificine (6j) R_f 0.66

Mass spectrum

363 (M⁺ 30) 331 (100)

¹H NMR spectrum (CDCl₃) δ ppm from TMS

2.08 (s 3H 7' -OAc), 2.40 (s 3H 2-CH₃), 2.91 (s 3H N-CH₃), 6.97 (1H d J=4 7'-H), 6.60 (1H s 6-H), 6.04 (1H s 3-H), 12.61 (1H s 5 -OH) Hot Acetylation

60mg alkaloid is mixed with 1 ml pyridine and 2 ml acetic anhydride and refluxed on a water bath for 3 hours. The solvents were evaporated under reduced pressure and the residue acidified with 1M HCl (20mL) and shaken with 3 x 10mL chloroform. The chloroform was evaporated to small volume and the products purified by prep TLC using silica gel/chloroform:methanol 12:1. The mass spectrum and *H NMR spectrum of each product was obtained.

Schumannificine treated in this way gave two dehydrated acetylated products

Example 10e

N,5,-diacetylanhydroschumannificine (3d) R_f 0.69

Mass spectrum 383 (28) M⁺ , 323 (45), 301 (51), 263 (100). ¹H NMR spectrum (CDCl₃) δ ppm from TMS

7.57 (1H d J=1.4 7'-H), 6.66 (1H S 6-H), 6.06 (1H s 3-H), 4.05 (1H dd J=8.2 J=2.2 6'-H), 3.95 (1H d J=3 . 8 4 ' -H), 3.68 (1H m 6'-H), 2.88 (1H m 5'-H), 2.62 (1H s N-OAc), 2.42 (3H s 2-CH₃), 2.36 (3H s 5-OAc), 1.94 (1H m 5'-H)

Example 10f N-acetylanhydroschumannificine (3c) R_f 0.64

Mass spectrum

341 (60) M⁺, 239 (100)

1H NMR spectrum (CDCl₃) δ ppm from TMS

12.71 (1H s 5-OH), 7.57 (1H d J=1.4 7'-H), 6.43 (1H S 6-H),

6.15 (1H s 3-H), 4.04 ( 1H dd J=8.2 J=2.2 6'-H), 3.87 (1H d

J=3.8 4'-H), 3.65 (1H m 6'-H), 2.89 (1H m 5' -H), 2.62 (1H s N-OAc), 2.42 (3H s 2-CH₃), 1.90 (1H m 5'-H).

Example 11

METHOXYLATION

30mg alkaloid was dissolved in 1ml 0.2m trimethylanilium hydroxide in methanol and refluxed for 1 hr. The solution was evaporated to dryαess and acidified with 1M HCl and extracted with 3 x 15 mL chloroform.

The products were isolated using prep TLC (silica gel/chloroform: methanol 12:1) and the mass spectrum and ¹H NMR spectrum of each product was obtained. Schumannificine gave two products Example 11a

5,7'-dimethoxyschumannificine (6j) R_f 0.62

Mass spectrum

345 (100) M⁺

¹H NMR spectrum (CDCl₃) δ ppm from TMS

7.16 (1H bs NH), 6.38 (1H s 6-H), 6.09 (1H s 3-H), 5.57 ( 1H J= 4.0 7'-H), 3.81 (1H m 4'-H), 3.78 (3H s 5-CH₃), 3.76 (3H s 7'-OCH₃), 3.24-3.08 (3H m 3'-H, 6-CH₂), 2.81 (1H m 5'-H), 2.41 (3H s 2-CH₃), 2.22 (1H m 5'-H)

Example lib

7'-methoxyschumannificine (6k) R_f 0.46 Mass spectrum 331 (100) M⁺

¹H NMR spectrum (CDCl₃) δ ppm from TMS

12.68 (1H s 5-OH), 7.16 (1H bs NH), 6.38 (1H s 6-H), 6.09 (1H s 3-H), 5.57 (1H J=4.0 7' -H), 3.81 (1H m 4'-H), 3.76 (3H s 7' -OCH₃), 3.24-3.08 (3H m 3'-H, 6-CH₂), 2.81 (1H m 5' -H), 2.41 (3H s 2-CH₃), 2.22 (1H m 5'-H)

Example 12

FORMATION OF QUATERNARY AMINE FROM N-METHYLSCHUMANNIFICINE 20 mg N-methylschumannificine was refluxed with methyl iodide (5mL) for 30 min. The mixture was evaporated and the residue dissolved in 1 mL methanol. The product was purified by prep TLC (silica gel/ethyl acetate :propan-2- ol:ammonia 65:35:10) and the mass spectrum and ¹H NMR spectrum obtained.

N-dimethylschumannificine (61)

Mass spectrum

346 (100) M⁺

¹H NMR spectrum (C₅D₅N) δ ppm from TMS

12.61 (1H s 5-OH 6.78 (1H bs 7'-OH), 6.33 (1H s 6-H), 6.11 (1H s 3-H), 5.58 (1H d J=4.0 7'-H), 4.35 (6 H s N-CH₃), 3.71 (1H m 4'-H), 3.31-3.10 (3H m 6 ' - CH₂ , 3'-H), 2.65 (1H m 5- CH), 2.39 (3H s 2-CH₃), 2.21 (1H m 5-CH).

Example 13

FORMATION OF IMIDATES (Paquette, Kalihana, Hansen and Philips (1971) J. Am. Chem. Soc . 93 152.)

Boron trifluoride/ether reagent is dissolved in dry CH₂Cl₂ to form 0.27M solution. The alkaloid is added to the mixture which is then kept under nitrogen at 0º. 0.3M epichlorhydrin in CH₂Cl₂ is added dropwise and the mixture stirred continuously for 7 hours at 0°. Cold 5% aqueous K₂CO₃ is then added to the mixture and after careful shaking the organic layer is removed, washed with water and the products isolated.

Example 14

REMOVAL OF HEMIACETAL OH BY TOSYLATION

The alkaloid is dissolved in dry benzene. p-Toluene sulphonic acid and dry CaCl₂ are added and the mixture refluxed for 3 hours.

The benzene is washed with water and the alkaloid products are recovered from the benzene layer.

An alternative method

The alkaloid is dissolved in dry CH₂Cl₂ and equimolar p- toluene sulphonylhydrazine added. The mixture is refluxed at 100° for 30 min and after cooling, sodium borohydride is added. The reaction mixture is washed with water and the products recovered from the organic layer. Exampl e 15

REPLACEMENT OF O BY N IN THE CHROMONE RING

The alkaloid is dissolved in water:ethanol 1:1 and 10M NaOH added to make the mixture pH 12. 13.5M ammonia is then added to the mixture which is then refluxed at 100° for 30 min.

The pH of the reaction mixture is adjusted to 7 with dilute acid and CH₂Cl₂ is added to extract the products.

Example 16

REDUCTION OF THE PIPERIDINE RING (Weintraub, Oles and Kalish (1968) J. Org. Chem. 33 1679)

Boron trifluoride/ether reagent is dissolved in dry CH₂Cl₂ to form 0.27M solution. The alkaloid is added to the mixture which is then kept under nitrogen at 0°. 0.3M epichlorhydrin in Ch₂Cl₂ is added dropwise and the mixture stirred continuously for 7 hours at 0°. LiBH₄ is then added to the mixture which is then stirred at 0° for 3 hours.

Cold dilute acid is then added to the mixture and the alkaloid is recovered from the organic layer or by basification of the acid layer and extraction with CH₂Cl₂.

Example 17

OXIDATION OF THE HEMIACETAL TO A LACTONE (Bowden, Helibron, Jones and Weeden (1946) J. Chem. Soc. 39)

The alkaloid is dissolved in acetone and the solution maintained at 15°. Equimolar chromic acid solution is added dropwise with stirring. When all the chromic acid has been added sufficient CH₂Cl₂ is added to the mixture to form two layers. The products are recovered from the organic layer after washing. C. BIOLOGICAL TESTING

The anti-HIV activity and toxicity of compounds were assessed in C8166 cells infected with HIV-1_III-B. Cells were grown in RMPI 1640 with 10% fetal calf serum. Forty- thousand cells per microtitre plate well were mixed with 5- fold dilutions of compound prior to addition of 10 CCID₅₀ (50% cell culture infectious dose) units of virus and incubated for 5-7 days. Formation of syncytia was examined from 2 days post-infection. Gp120 antigen produced at 5-7 days was measured by ELISA, using the lectin GNA (from Galanthus nivalis) to capture the glycoprotein and human anti-HIV serum for detection, as described by Mahmood and Hay, (J. Immunol. Methods, 151, 9-13, (1992)) . Cell viability of virus-infected and uninfected control cells was measured by the MTT-Formazan method as described by Pauweis e t al . (J. Virol. Meth., 20, 309-321 (1988)).

Results are presented in Table I.

The antiviral activity against herpes simplex type I (HSV-1, strain 17-1) was determined by measuring viral antigen produced in injected Vero or human lung embryonic cells MRC5 as described by Mahmood et al (Antiviral Chem. Cher. ther . 4, 235-240. (1993)). Five fold dilutions of compounds were added to duplicate cells just before adding virus at a multiplicity of infection of 0.01 plaque-forming units per cell. The cells were incubated 16-18h at 37° and then fixed with 3% for 1-2h. Antigen

was detected by ELISA using rabbit anti HSV-I antibodies obtained from Dakopatts, Denmark. The cytotoxicity was assessed using the MTT-Formazan assay on growing Vero and human lung embryonic cells as described by Pauweis et al (J. Virol. Meth. 20, 309-321, (1983)).

Results are presented in Tables II and III.

Claims

1. Use of a compound of a formula selected from the group comprising:-

wherein

R¹, R², R⁴ , R⁵, R⁶ , R⁷ and R⁸ may be the same of different and are selected from the group comprising hydrogen, hydroxy and substituted alkyl, alkoxy, alkoyloxy, aryl, aryloxy and aryloyloxy groups;

R³ is selected from the group comprising hydrogen, carbohydrates and oligosaccharides, and substituted or unsubstituted alkyl, alkoyi, aryl and aryloyl groups;

R⁹ is an alkyl group;

X is selected from -CH₂- and -C(O)-;

Y is selected from -CHR¹⁰- and -C(O)-;

Z is selected from N and O;

n is selected from 0, 1 and 2;

R¹⁰ is selected from the group comprising hydrogen, hydroxy, carbohydrates and oligosaccharides, and substituted or unsubstituted alkyl, alkoxy, alkocloxy, aryl, aryloxy and aryloyloxy groups;

and pharmaceutically acceptable derivatives thereof, in the manufacture of a medicament for use in the treatment cr prophylaxis of viral infection.

2. Use of a compound according to claim 1 wherein

R⁴, R⁵ and R⁸ are hydrogen; and

Y is -CH(OH) -

3. Use of a compound according to claim 1 or 2 wherein

Z = O.

4. Use of a compound according to any preceding claim wherein the compound is of formula I or III.

5. Use of a compound according to any one of claims 1 to 3 wherein the compound is of formula V or VI.

6. Use of a compound according to any preceding claim wherein the viral infection comprises HIV infection.

7. Use of a compound according to any one of claims 1 to 5 wherein the viral infection comprises HSV infection.

8. A pharmaceutical composition for use in the treatment or prophylaxi of viral infection comprising a compound as defined in any one of claims 1 to 5 in combination with a pharmaceutically acceptable excipient.

9. A method of treatment or prophylaxis of a viral infection comprising administering to a patient in need of such treatment or prophylaxis an effective dose of a compound as defined in any one of claims 1 to 5.

10. A compound of a formula selected from the group comprising

wherein R¹-R⁵, X, Y, Z and n are as defined above,

CHR¹⁰-, R¹⁰ is OH or OAc, Z is 0 and n is 1, then R² is not the same as R ¹⁰;

wherein R¹-R⁵, R⁹, Y, Z and n are as defined above;