ZA200805010B - Compound and composition for treating sexual dysfunction - Google Patents

Description

DE

BACKGROUND OF THE INVENTION -

The invention relates to a novel compound having erection stimulation activity, to compositions containing this compound and to the use of the compound and the compositions containing it for the treatment of impotence.

The compound may be used for stimulating penile erection in a human or animal, and in particular may be used for the treatment of impotence. Impotence can be defined as a lack of power, in the male, to copulate and may involve an inability to achieve penile erection or ejaculation, or both. More specifically, erectile impotence or dysfunction may be defined as an inability to obtain or sustain an erection adequate for intercourse.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a process for preparing an extract of a plant of the genus Securidaca, especially S. longependunculata, the extract comprising an erection stimulant agent, the process including the steps of treating collected plant material with a solvent to extract a fraction having erection stimulating activity, separating the extraction solution from the rest of the plant material, removing the solvent from the extraction solution and recovering the extract.

According to a further embodiment of the invention, there is also provided a plant extract made of plants of the genus Securidaca, especially S. longependunculata, and having erection stimulating activity.

The extract may be prepared from plant material such as the stem bark, root bark, leaves and seeds of plants of the genus Securidaca. In particular, the extract may be made from the root bark.

The extract may include an active compound (1) having the structural formula:

OH 0 OH 7 8a 84 6 4b 43

HO 5 0 4 OH

OMe (1

The compound of formula (I), according to S.I. nomenclature, may be referred to as 1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone.

According to a further embodiment of the invention, there is provided a composition having erection stimulating activity, the composition comprising an extract as described above. The composition may further include a pharmaceutically acceptable excipient, diluent or carrier.

According to a further embodiment of the invention, there is provided a compound of formula (1) and/or salts, derivatives, acids or analogues thereof which have erection stimulating activity.

According to a further embodiment of the invention, there is provided a process for synthetically producing a compound of formula (I) and/or a salt, derivative, acid or analogue thereof which has erection stimulating activity.

According to a further embodiment of the invention, there is provided a composition having erection stimulating activity, the composition comprising a compound of formula (I) or a salt, derivative, acid or analogue thereof. The composition may further include a pharmaceutically acceptable excipient, diluent or carrier.

According to a further embodiment of the invention, there is provided a method for stimulating an erection in a human or animal, the method including the step of administering to the human or animal an effective dosage of a compound of formula (I) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof or a composition comprising the compound of formula (I) or a salt, derivative, acid or analogue thereof as active ingredient.

According to a further embodiment of the invention, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof in a method of manufacturing a medicament for use in stimulating an erection in a human or animal.

The compound or composition is envisaged primarily for the curative or prophylactic treatment of erectile dysfunction or sexual dysfunction in a human or animal male, but may also be used for the treatment of female sexual dysfunction including orgasmic dysfunction.

The compound or composition may be administered orally or parenterally, e.g. sublingually or buccally. For veterinary use, the compound or composition may be administered as a suitably acceptable formulation in accordance normal veterinary practice.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a proton spectrum for a compound of the formula (1);

Figure 2 shows HMQC spectra for the compound of formula (I);

Figure 3 shows a UV spectrum in MeOH for the compound of formula (1);

Figure 4 shows a UV spectrum with AICI; for the compound of formula (1);

Figure 5 shows a UV spectrum in NaOAc for the compound of formula (1);

Figure 6 shows a HMBC spectrum for the compound of formula (1);

Figure 7 shows a UV spectrum in MeOH for a compound of the formula (ll);

Figure 8 shows a UV spectrum with AICI; for the compound of formula (ll);

Figure 9 shows UV spectra with NaOAc for the compound of formula (ll);

Figure 10 shows a proton spectrum for the compound of formula (li);

Figure 11 shows a "*C spectrum for the compound of formula (11);

Figure 12 shows a HMBC spectrum for the compound of formula (li); and

Figure 13 shows a MBQC spectrum for the compound of formula (II).

DETAILED DESCRIPTION OF THE INVENTION

A novel xanthone isolated from Securidaca longependunculata is described herein.

The xanthone, named 1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone, has the following

OH 0 OH 7 8a 8H 6 4 4a

HO 5 © 4 OH

OMe structure: 0

A second novel xanthone isolated from S. longependunculata, is also described herein.

This xanthone, named 1,6,8-trihydroxy-2,3,4,7-tetramethoxyxanthone, has the following structure:

OH 0 OH

MeO, . 8 | , OMe 8a 8b 6 4b 4a 3

HO 0 OMe s 4

OMe

The erection stimulation ability of the compound of formula (I), similar to sildenafil citrate (marketed commercially as Viagra™ and Revatio™) is also described herein.

Xanthones are secondary metabolites known to occur in a few higher plant families, fungi and lichen (Peres & Nagem, 1997). There is a growing interest in xanthones because of their high taxonomic value within the families (Cardona et al, 1990) and their pharmacological properties. Their pharmacological properties include in vitro cytotoxicity, in vivo antitumour activity, antifungal activity, antibacterial activity, anti- inflammatory properties and immunomodulatory activity (Sordat-Diserens et al., 1992).

Example

Plant material

Root bark of S. longependunculata (also referred to as the violet tree or krinkhout) was collected in Limpopo, South Africa. Voucher specimen (NCR. 16) was deposited and identified at the H.G.W.J. Schweickerdt Herbarium (PRU), University of Pretoria.

Extraction, purification and isolation of chemical constituents ® Dry root bark (634 g) of S. longependunculata was extracted in acetone by homogenizing the plant material using a fire protected blender and leaving it in the dark at room temperature for 8 days. The extraction process was repeated twice. The extract was filtered and concentrated to dryness under reduced pressure at 37 °C yielding 11g. The total extract was subjected to column chromatography using silica gel 60 (Merck, 7724, 500 g), and eluded with the following solvent ratios of n-

hexane:ethyl acetate 0%, 20%, 40%, 60%, 80%, 100%, thereafter 10% methanol:ethyl acetate, and finally 100% methanol. 46 fractions were collected, which were reduced to 7 subfractions based on the TLC results. The TLC plates were developed using a chloroform:methanol mixture (8:2). The results showed that fractions 2 to 6 were the most active fractions.

Fraction 3 was subjected to column chromatography again, using a silica gel column.

The column was eluded with n-hexane:ethyl acetate (8:2) and 6 sub-fractions were obtained. Further purification of fraction 4 was achieved with about 15 g of Sephadex

LH 20. The column was eluded with methanol (100%) and 18 fractions were obtained.

Fractions 2-14 (46mg) appeared to be pure. The same procedure was followed for fractions 2 (8mg), 3 (23mg) and finally 6 (21mg), which yielded four major pure compounds, which were further analyzed by different physicochemical methods to determine their chemical structures.

Two of the compounds isolated from the S. longependunculata root bark were found to be 2-hydroxy-1,7-dimethoxyxanthone (compound A) and 1, 4-dihydroxy-7- methoxyxanthone (compound B). Both of these compounds are known and have previously been isolated from S. longependunculata (Geleffi et al., (1990); Rakuamba et al., (2005)).

The remaining two compounds, compounds C and D, were found to be novel and have not previously been described.

Compound C: 1,3,6,8-tetrahydroxy-2.5-dimethoxyxanthone

Compound C was isolated as yellow crystals, mp 152-5°C. The molecular formula of

C15H1,0s was deduced from MS (M+, 310.0), 1H (fig 1),"*C and HMQC (fig 2) spectra.

UV spectral data showed absorption in MeOH (fig 3) at 237;,, 258, 330 nm. A strong bathochromic shift observed with AICI; (fig 4), which was not affected by the addition of

HCI, indicated the presence of peri-hydroxyl group(s) at 1 and/or 8. A bathochromic shift in NaOAc (fig 5) spectra (20 nm) indicated the presence of acidic hydroxyl(s) at 3 and/or 6 position(s).

NMR spectra of compound C showed two proton singlets at d 6.25, 6.45 attached to carbons d; 93.1, 98.3 ppm respectively, two three proton singlets at d 3.84, 3.88 attached to carbons at d, 61.54, 60.73, suggesting two methoxyl groups O- disubstituted. The two signals at 12.1, 11.80 indicated the presence of two peri- hydroxyls.

This data suggested that compound C is a hexa oxygenated xanthone, with two positions occupied by free hydroxyls at C-1 and C-8, the other two being methoxylated and the last two positions being occupied by two free hydroxyl groups.

HMBC (fig 6) spectra of compound C showed correlations between the methoxyl proton at d 3.84 and the C-127.5, the other methoxyl group d 3.88 with C-130.5. The chemical shift of both carbons suggested the positions 2, 4, 5 or 7 for the methoxyl groups. The bathochromic shift of compound C in NaOAc and the '*C values of the hydroxyls’ attached carbons (d. 157.7, 158.3) suggested that both the 3 and 6 positions are occupied by free hydroxyls. 'C data of compound C is in good agreement with 1,3-dihydroxy-2-methoxyxanthones (Ortega et al., 1981), which suggests a part of the structure of compound C.

The 6,8-dihydroxy-5-methoxyxanthone substitution pattern of ring B was confirmed by the facts that firstly, the NMR data of compound C showed no symmetry, and secondly, the *C NMR spectra showed aromatic carbons oxygen-substituted at d. 130.5 (attached to one methoxy group), 158.8, 152.7, which suggest the presence of 5, 7, 8- trisubstituted ring B. The methoxyl carbon at d, 60.73 suggested the O-disubstitution, which require -OH substitution at C-6. The structure was finally confirmed by the correlation observed in HMBC spectra, and compound C was determined to be of formula (1):

' * ad \] 50 |! 0 - Be 2008 /

OH 0 OH 8 1 2 OMe 7 8a 8 6 4b 4a

HO 5 © 1 OH

OMe 0)

Compound D: 1,6,8-trihydroxy-2,3,4,7-tetramethoxyxanthone

Compound D was isolated as pale yellow crystals, mp 201-4°C, having a molecular formula Cy7HsOg deduced from MS. The UV spectra of compound D showed (in

MeOH) (fig 7) absorptions at 235, 265, 282, and 245. A strong bathochromic shift was observed by addition of AICI; (fig 8). The addition of NaOAc (fig 9) also gave a small bathochromic shift. "H-NMR (fig 10) spectra showed one singlet aromatic proton at d 6.82, four methoxyl signals at d 4.14, 3.99, 3.97, 3.92, and two peri-hydroxyl signals at 11.38, 11.88.

The "H- and C-NMR (fig 11) data indicated a hepta oxygenated xanthone with only one unsubstituted position. From the seven oxygenated positions, three are free hydroxyl groups, two of them located at C-1 and C-8 as indicated from the "H-NMR spectra and the strong bathochromic shift in AICI; spectral UV data.

The UV spectra gave a bathochromic (10 nm) shift by addition of NaOAc, which indicated the presence of an acidic hydroxyl at position 3 or 6. The remaining four oxygenated positions should be methoxylated. "°C data showed chemical shifts at d. 61.8, 61.6, 61.2 and 61.1, which indicated four O-disubstituted methoxyls. Ring A is therefore completely substituted with three methoxyl groups at positions 2, 3 and 4.

According to the '*C data, the fourth methoxyl group should be located at a position between the two hydroxy! groups in ring B.

As indicated from the previous data, it was deduced that compound D is 1,6,8- trinydroxy-2,3,4,7-tetramethoxyxanthone. The aromatic proton d 6.82 showed correlations with C-6, C-7, C-4b and C-8a in HMBC spectra. Other data of HMBC (fig 12) and HMQC (fig 13) are only compatible with the structure of formula (li):

OH [0] OH

MeO, ; 8 1 , OMe 8a 8b 6 4 4a 3

HO 0 OMe 4

OMe (I)

Table 1: 'H and *C-NMR chemical shifts for compounds A-D (d, ppm, in CDCI;)

Compound A Compound B Compound C Compound D

H, mult, J H, mult, J H, mult, J H, mult, J 3 | 123.4 | 7.36,d, 9.01 119.3 [7.15,4d,8.79 157.7 154.0 5 113.9 | 7.20, d, 9.31 119.8% | 7.42, d, 9.06 130.5 | | 989 [636s 124.6 7,30, dd, 125.8 7.22, dd, 158.3 159.8 9,31, 3.11 9.05, 2.97 8 | 105.6 | 7.66, d, 3.11 108.6" | 745, d, 2.97 152.7 145.6 Co] (OW | #08 [Ss | wz [9ws | ois sms | 68 [99s

I EE I I cE RL KE

EE ET IE EO

I EE I A A I I EC

Note: **, "= interchangeable signals.

Erectile dysfunction activity of xanthones isolated from S. longependunculata

A bioassay was performed as described by Levin et al. (1997) with some minor changes. Strips (12 mm long and 1-2 mm thick) of rabbit corpus carvernosal smooth muscle were dissected and mounted in an organ-bath chamber containing Krebs-PSS solution with the following composition: NaCl = 7.01g/l, KCI = 0.34g/l, KH,PO, = 0.1g/,

NaHCO; = 1.99g/l, CaCl, = 0.2g/l, MgSO, = 0.3g/l and glucose = 1.8g/l. One end of the muscle was tied to the inside bottom of the perfusion bath and the other end to a thin wire connected to a Harvard isotonic force transducer for isotonic tension measurements. Changes in isotonic tension were recorded on a computerized calibrating program. The corpus cavernosum muscle was perfused with 2mil Krebs-

PSS buffered saline and oxygenated with 95% O, and 5% CO, for 5 min to ascertain a stable baseline recording. This was followed by perfusion with 2ml CaCl, in Krebs-PSS (17.8 mg/ml) for muscle contraction. Baseline tension was set at the point of maximal contraction following the addition of CaCl, into the experimental bath. The compounds fo be analyzed were added after a stable contraction baseline. The final compound concentration in the perfusion bath was 1.8 x 10° mg/ml. The same procedure (and concentration) was repeated for the positive control, Viagra™ (Pfizer). In these experiments, the stimulation frequency used for rabbit strips was 9 Hz. The results are shown in Table 2.

Table 2: Averages of the relaxation % of the pre-contracted rabbit corpus cavernosum smooth muscle at 1.8 x 10° mg/ml

Compound Average % relaxation

CT] ee

Known Compound A 63 (5.8)

Known Compound B 0 (0)

New Compound C 97 (5.7)

New Compound D 30.5(4.8)

Viagra™ 100 (0)

The results show that compounds A and C have significant smooth muscle relaxation properties and that the erection stimulating action of compound C (having formula (1)) is comparable to that of Viagra™.

Further studies will be conducted on the erection stimulating activity of compounds of the formula (I) or salts, acids, analogues and derivatives thereof, and on methods of isolating or synthesizing these compounds.

References

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: ¢ =~ 1

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Claims (15)

LE Sa CLAIMS:

1. A compound of formula (!) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof OH 0 OH 8 U5 OMe 7 8a 8b 6 4b 4a Ho" 5 © 4 OH OMe (1 [1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone].

2. A compound according to claim 1, for treating erectile dysfunction and/or stimulating erection in a male mammal.

3. A compound according to claim 1, for treating sexual dysfunction in a female mammal.

4. A composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof and a suitable diluent, excipient or carrier OH 0 OH 8 U5 OMe 7 8a 8b 6 4 4a HO 5 © 7 OH OMe

(0.

5. A composition according to claim 4, for treating erectile dysfunction and/or stimulating erection in a male mammal.

RE

6. A composition according to claim 4, for treating sexual dysfunction in a female mammal.

7. A process for obtaining an extract of a plant of the genus Securidaca, the process including the steps of: (i) treating collected plant material with a solvent to extract a fraction including a compound of formula (I) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof; OH 0 OH 7 8a 8H 6 4b 4a HO 5 0 4 OH OMe 0) (ii) separating the fraction from the rest of the plant material; (iii) removing the solvent from the fraction; and (iv) recovering the extract.

8. A process according to claim 7, wherein the plant is S. longependunculata.

9. A plant extract from plant material of the genus Securidaca, which includes a compound of formula (I) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof OH 0 OH 8 Ul, OMe 7 8a 8H 6 4p 4a HO 5 © 3 OH OMe

(M.

‘ ’

10. A plant extract according to claim 9, wherein the plant material is from S. longependunculata.

11. A plant extract according to either of claims 9 or 10, for treating erectile dysfunction and/or stimulating erection in a male mammal.

12. A plant extract according to either of claims 9 or 10, for treating sexual dysfunction in a female mammal.

13. The use of a compound of formula (I) or a pharmaceutically acceptable salt, derivative, acid or analogue thereof in a method of manufacturing a medicament for use in a method of treating sexual dysfunction in a mammal and/or stimulating erection in a male mammal OH 0 OH 8 Ul, OMe 7 8a 8H 6 4b 4a HO 5 © 1 OH OMe

(Mn.

14. The use according to claim 13, wherein the sexual dysfunction is in a female mammal.

15. The use according to claim 13, wherein the sexual dysfunction is erectile dysfunction in a male mammal. DATED THIS 9" DAY OF JUNE 2008 = SRPEOR & FISHER APPLICANT'S PATENT ATTORNEYS