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Title:
COMPOUND
Document Type and Number:
WIPO Patent Application WO/2014/173957
Kind Code:
A2
Abstract:
A compound of formula (I) wherein R is a C9-23 polyunsaturated alkenyl group; X is SO3R3, SO2R3, PO3R3, PO2R3, COCHal3, or COOR3; R1 and R2 are each independently H, a C1-6 a iky I group optionally substituted with OH, C6-10 aryl group optionally substituted with OH, C7-12 arylalkyl group optionally substituted with OH, COC1-6 alkyl group optionally substituted with OH, COC6-10 aryl group optionally substituted with OH, COC7-12 arylalkyl group optionally substituted with OH, or COCHaI3 group; hal is halide, preferably F; each R3 is independently H, C1-6 alkyl, C6-10 aryl, or C7-12arylalkyl; or a salt thereof.

Inventors:
SIMIC NEBOJŠA (NO)
JOHANSEN BERIT (NO)
IGIC ALEKSANDAR (RS)
Application Number:
PCT/EP2014/058248
Publication Date:
October 30, 2014
Filing Date:
April 23, 2014
Export Citation:
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Assignee:
SIMIC NEBOJŠA (NO)
JOHANSEN BERIT (NO)
IGIC ALEKSANDAR (RS)
International Classes:
C07H15/04; A61K31/216; A61K31/351; A61K31/7032; A61K31/7034; A61P29/00; C07C69/734; C07D407/04; C07H15/18; C07H17/07
Domestic Patent References:
WO2011039365A12011-04-07
Other References:
SYED BUKHARI ET AL: "Anti-inflammatory and antioxidant activities of Sclerochloa dura (Poaceae)", JOURNAL OF THE SERBIAN CHEMICAL SOCIETY, no. 00, 21 January 2014 (2014-01-21), pages 3-3, XP055120426, ISSN: 0352-5139, DOI: 10.2298/JSC131202003B
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 28 May 2009 (2009-05-28), HUA, JIN MEI ET AL: "Inhibitory activity of ethanol extracts of Ailanthus altissima and luteolin-7-glucoside on phospholipase A2 activity", XP002725059, retrieved from STN Database accession no. 150:489348
Attorney, Agent or Firm:
CAMPBELL, Neil (St Bride's House10 Salisbury Square, London EC4Y 8JD, GB)
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Claims:
Claims

1. A compound of formula (I)

wherein R is a C9-23 polyunsaturated alkenyl group;

X is SO3R3, S02R3, PO3R3, PO2R3, COC Ha!,, or COOR3;

Ri and R? arc each independently II , a C ! -6 a iky I group optionally

substituted with OH, C6-10 aryl group optionally substituted with OH, C7-12 arylalkyl group optionally substituted with OH, COC I -6 alkyl group optionally substituted with OH, COC6-10 aryl group optionally substituted with OH, COC7-12 arylalkyl group optionally substituted with OH, or COCHal3 group;

hal is halide, preferably F;

each R3 is independently H, Cl-6 alkyl, C6-10 aryl, or C7-12arylalkyl; or a salt thereof.

2. A compound as claimed in any preceding claim being a compound of formula (V)

where X. R . and R1-R3 are as hereinbefore defined;

or a salt thereof.

3. A compound as claimed in any preceding claim being a compound of formula (VI)

where X and R, are as hereinbefore defi ned;

or a salt thereof.

4. A compound as claimed in any preceding claim wherein X is SO3H

5. A compound as claimed in any preceding claim wherein -COR is linoleic acid derivative.

6. A compound as claimed in any preceding claim wherein the hexose is dcoxyglucose.

7. A compound as claimed in any preceding claim wherein the hexose is

6-deoxyglucopyransoyl.

8. The compound:

or a salt thereof.

9. A pharmaceut ical composit ion comprising a compound of formula (I) as hereinbefore defined and at least one carrier or cxcipient. 10. A compound of formula (I) for use as a medicament.

1 1 . A compound of formula (I) for use in the treatment of in flammat ion, e.g. via the inhibit ion of cPLA2.

12. A compound of formula (II)

(II)

wherein

each EL, is H, OH , OR3, Cl -6alkyi, C6- 10 aryl, C7- 12 arylal kyi or the group:

wherein each R3 is as hereinbefore defined;

p is 0-3;

n is 1 to 3;

w is 0 to 2;

R9 is Ci-6 alkyl;

R5 is a monosaccharide linked to the phenyl ring via a C-C bond or l inked via a C-0 bond at the 1 -position of the saccharide, and

R6 is H, OH , CI -6 alky! or OR3;

or a salt thereof;

for use in the treatment of inflammation.

13. A compound of formula (III)

wherein R7 is H or CI -6 alkyl; or salt thereof for use in the treatment of inflammat ion.

A compound of formula (IV)

wherein each Rg is H or a Cl-6-alkyl;

Rio is H or CI -6 alky!;

q is 0 to 3, such as 1 or 2;

each R3 is as hereinbefore defined with at least one OR3 group being OH; a salt thereof; for use in the treatment of inflammation.

15. A compound of formula (I) to (IV) as claimed in claim 1 to 14 for use as an inhibitor of cPLA2.

16. A compound of formula (I) to (IV) as claimed in claim 1 to 14 for use in treatment of a chronic inflammatory disease such as acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, and vasculitis.

17. A compound of formula (I) to (IV) as claimed in claim 1 to 14 for use in treatment of hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and cancer.

18. Use of a compound of form la (I) to (IV) as claimed in claim 1 to 14 in the manufacture of a medicament for the treatment of in flammat ion.

19. A method of treating inflammation comprising administering to a patient in need thereof, such as a human, a therapeutical ly effect ive amount of a compound of formula (I) to (IV) as claimed in claim 1 to 14.

Description:
Compound

This invention relates to a class of new compounds which have been found to exhibit ant i-inflammatory properties. The invention also relates to a series of known compounds which have been surprisingly found to possess anti- inflammatory properties by affecting the activ ity PL .42 enzyme in the early stage of the inflammat ion cascade.

I n particular, the invent ion relates to a new compound is lated fr m

Sclerochloa dura (L.)P. Beauv and analogues thereof which is an anti- inflammatory agent. The inv ent ion also relates to a pharmaceutical composit ion comprising the compounds of the invention as well as the uses thereof in the treatment of inflammation and conditions associated with inflammation.

Background of invention it is general ly known that plants produce many chemical compounds during their life time such as alkaloids, flavonoids, proteins, amino acids, bioactive peptides, sugars, bio pesticides and others. Plants have always been a centre of attention in the history of traditional medicine, because they produce vital and complex molecules, which can be used either directly or in modified form to cure a number of diseases.

For example, berberine is well known for its anti-inflammatory activity. It is present in a number of plants such as Hydrastis canadenis, Berberis vulgaris, Barberis aristata and Rhizoma coptidis. Similarly, quercetin belongs to tlavonoid class of compounds and is also anti- inflammatory in nature. It can be isolated from many plants, for instance Helminthostachys zeylanica. Some other classes of compounds showing ant i-in flammatory activ ity are terpenoids, phenols, polyphenols and some sulphur contai ning compounds.

The present inventors have developed a class of new ant i inflammatory drugs with an improved mode of act ion compared to exist ing drugs. I n particular, the compounds of the inv ent ion are inhibitors of cPLA2. Most non-steroidal ant i- inflammatory drugs (NSAIDs) act on specific enzymes late i n the in flammat ion reaction pathway (e.g. COX-inhibitors ). The problem with acting at such a late stage in the process is that it causes an imbalanced inflammation reduction due to over- activation of parallel pathways (Figure 1). Such an imbalance can cause severe side- effects such as heart attack, stroke and liver failure. The use of existing NSAIDs, therefore, has been notoriously linked w ith unwanted and serious side-effects.

For example, the NSAID Rofecoxib (sold under the name Vioxx, Merck) was shown to give serious cardiovascular problems in patients having been prescribed the drug in the period 1999-2004 (between 88 000 and 139 000 heart attacks in US only, 30 to 40 % of which were probably fatal ). The drug was withdrawn from the market in 2004. These deaths could have been prevented if the drug had targeted enzymes earlier in the inflammation process.

I n 201 1 , a British study reported that another NSAID, Diclofenac increases the probability of experiencing cardiovascular problems with 40%. Voltaren was in 2010 prescribed to 500 000 patients in Norway alone. The drug is still on the market worldw ide. Other NSAIDs that have been withdraw n from the market due to serious side effects include Valdecoxib (Bextra, Pfizer, 2005), Lumiraeoxib (Prexige, Novartis, 2007 ), Etorico ib (Arcoxia, Merck,) and Parecoxib (Dynastat, Pfizer).

The long term use of most of the NSAIDs on the market today ( e.g. COX inhibitors) are related to quite severe side-effects. Therefore, there is a need for NSAIDs with an improv ed mode of act ion.

The compounds of the presence invention affect ( reduce ) the activ ity of PLA2 enzyme although a complete mechanism and exact point of action has yet to be elucidated.

The core inv ention is the use of the compounds of the inv ention to interrupt the anti-inflammatory cascade by inhibiting PL A . Moreover, the inv ent ion relates to the characterisation of a new anti-inflammatory compound from a biological source and with that knowledge the design of a new class o f a n t i - i n 11 a m m a t o ry agents.

The preferred compound of the present invention derives from Sclerochloa dura which has prev iously been ident ified as hav ing medicinal uses. This plant has a traditional use in smal l communities in South-East Serbia for treatment of menstrual disorders characterised by excessive bleeding and pai n. Oral intake of a boiled water extract of the plant allegedly results in pain relief and significantly reduces bleeding.

The present inventors have identified a series of chemical compounds present in S. dura, which have anti-inflammatory properties and based on this knowledge designed the claimed subject matter to achieve a new class of anti ¬ inflammatory drugs with an improved mode of act ion and fewer side effects.

Grade extracts and subsequent fractions were tested on human synovial sarcoma cell line SW982 (stimulated with interleukin- 1 β) to determine the inhibition of arachidonic acid (AA) release, which corresponds to the activation level of phosph l ipase A.> (PLA2) enzymes. The results prove that the crude extract and subsequent methanoiic fraction of S. dura inhibits IL-1 induced release of AA in a time- and dose-dependent manner. Thus, they contain active ingredient which can inhibit the activity of the P LA > cascade.

The PL A.> enzymes hydro Iyze fatty acids such as A A from the sn-2 ester bond of membrane glycerophospho l ipids. These enzymes are sorted into five main categories, namely cytosolic PLA >s (CPLA2), secretory PLA?s (SPLA2), C ' - independent PLAis (1PLA2), PAF acetyl hydrolases ( PAF-AH ). and lysosomal PLA2S. Apart fr m their role in normal physiological functions, the di fferent isotypes are implicated in a variety of diseases and thus there is currently great interest in the development of inhibitors that target specific PL A ? subtypes. The Ω6 fatty acid (AA) is the substrate for eicosanoids such as prostaglandins and leukotrienes, which are potent lipid mediators of inflammation. As cPLAi group IVa (cPLA.>-I Va ) is the only P LA > with a preference for AA in the sn-2 position, it is a central enzyme of eicosanoid production and thus considered to be a main

contributor in inflammatory processes.

Most chronic diseases, such as atherosclerosis, rheumatoid arthritis, diabetes and chronic respiratory diseases are characterized by a considerable in flammatory component. These arc the leading cause of global death ( WHO, 201 1 ). i nflammat ion is an acute response to harmful stimuli that normally abates after the challenge is cleared. In chronic inflammatory diseases, the response is not attenuated and the inflammation in the tissue is not completely resolved. Instead, a prolonged signaling ensures a persistent inflammatory env ironment. Pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interlcukin 1 β ( I L- 1 β), are abundant and activate cPLAi-I Va, which results in the subsequent release of AA and generation of eicosanoids, creating a sel f-perpetuating process. Several studies using knockout mice or cPLA >-I Va inhibitors have highlighted the potential for targeting this enzyme in order to quench the inflammation and ameliorate disease. The S. dura derived compounds that inhibit AA release exert an overall anti- inflammatory action.

The present inventors hav e isolated and elucidated compounds from

Sclerochloa dura. The compounds expressed ant i-inflammatory activity by showing the inhibition of cPLA? in an in vitro AA release assay. The invention further relates to the use of the information gleaned to devise a new group of anti- inflammatory agents as well as the possible organic synthesis of these molecules.

Summary of Invention

Thus, viewed from one aspect the invention provides a compound of formula

wherein R is a C9-23 polyunsaturated alkenyl group;

X is SO3R3, SO2R3, PO3R3, PO2R3, COCHai 3 , or COOR 3 ;

R] and R ? are each independently H, a CI -6 alkyi group optionally subst ituted with OH , C6- 10 aryl group optionally subst ituted with OH , C7- 12 arylalkyl group optional ly subst ituted with OH , COC 1 -6 alkyl group optionally siibst ituted with OH, COC6- 10 aryl group optionally substituted with OH, COC7- 12 arylalkyl. group optionally substituted with OH , or COO Iah group;

hal is halide, preferably F;

each R 3 is independently H . Cl -6 alkyl, C6- 10 aryl, r C7- 12arylalkyi; or a salt thereof.

For the avoidance of doubt each substituent in formula (I) must bind a separate carbon atom in the six membered ring. It is not therefore possible for two substituents to bind the same carbon atom. The substituents can otherwise attach in any order.

Preferably the compound of formula (I) is a compound of formula (V)

where X, R, and R1-R 3 are as hereinbefore defined;

or a salt thereof.

Most preferably, it is a compound of formula (VI)

(VI) where X and R, arc as hereinbefore defined;

or a salt thereof.

Viewed from another aspect the invention provides a compound of formula

(II)

wherein

each R.4 is H, OH, OR 3 , Cl-6alkyl, C6-10 aryl, C7- 12 aryialkyl or the group:

wherein each R ¾ is as hereinbefore defined;

p is 0-3;

n is 1 to 3;

w is 0 to 2;

R5 is a monosaccharide linked to the phenyl ring via a C-C bond or linked

C-0 bond at the 1 -position of the saccharide, and

R 6 is H, OH, CI -6 alkyl or OR 3 ;

or a salt thereof:

for use in the treatment of inflammation.

Viewed from another aspect the invent ion provides a compound of formula

wherein R 7 is H or CI -6 alkyl; or salt thereof for use in the treatment f inflammation.

Viewed fr m another aspect the invention prov ides a compound of formula

(IV)

wherein each Rs is H or a C I -6-aikyl;

Rio is H or CI -6 alkyl;

q is 0 to 3, such as 1 or 2;

each R 3 is as hereinbefore defined with at least one OR 3 group being OH; or a salt thereof; for use in the treatment of inflammation.

Viewed from another aspect the invention provides a pharmaceutical composition comprising a compound of formula (I) as hereinbefore defined and at least one carrier or excipient.

Viewed from another aspect the invention prov ides a compound of formula (I) for use as a medicament. Viewed from another aspect the invention provides a compound of formula (I) for use in the treatment of inflammation.

Viewed from another aspect the invention provides a compound of formula (I) to (IV) for use as an inhibitor of cPLA>.

Viewed from another aspect the invent ion provides a compound of formula (I) to (IV) for use in the treatment of a clironic inflammatory disease such as acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, and vasculitis.

V iewed fr m another aspect the invention provides a compound of formula (I) to (IV) for use in the treatment of hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and cancer.

Viewed from another aspect the invention provides the use of a compound of formula (I) to (IV) as hereinbefore defined in the manufacture of a medicament for the treatment of inflammation.

Viewed from another aspect the invent ion provides a method of treating inflammation comprising administering to a patient in need thereof, such as a human, a therapeutically effective amount of a compound of formula (I) to (IV) as hereinbefore defined.

Detailed Description of Invention

This invention relates to a group of compounds which possess antiinflammatory properties. The compounds are defined by formula (I) to (IV).

Compounds of formula (I) are new. Compounds of formula (II) to (IV) are new for use in the treatment of inflammatory conditions, e.g. v ia the inhibit ion of cPLA 2 .

Definitions I n compounds of formula (I), for the avoidance of doubt each subst ituent must bind a separate carbon atom. It is not therefore possible for two subst ituents to bi nd the same carbon atom on the ring.

The group "aryialkyl" comprises an alky! port ion and an aryl portion. I t may l ink to another group either via the aryl portion or a Iky I portion. Thus, this term covers the groups tolyl and benzyl for example.

i n compounds of formula (III), the monosaccharide is linked to the phenyl ring d irect ly either via a C-C l ink from a saccharide carbon ring atom to a phenyl ring atom. It can also be linked via a C-0 link at the 1 -posit ion of the saccharide direct ly to a carbon atom of the phenyl ring.

I n general is preferably H or Ci-6-alkyl, such as 0 1-4 a Iky I, especially methyl, especially H .

Compounds (I)

I n a preferred embodiment, the invent ion relates to compounds of formula

with substituents definitions as hereinbefore defined. As will be noted below, the specific compound (X), can be isolated from Sclerochloa dura (L.)P. Beauv but the invention covers analogues of this isolated compound as well.

Whilst the polyunsaturated long chain hydrocarbon subst ituent in the isolated molecule of formula (X) as defined herein is based on iinolenic acid (C I 8, 3 double bonds), it will be appreciated that other polyunsaturated long chain side groups would also be suitable.

In preferred R groups of the invention therefore, there are at least 3, such as 3 to 6 double bonds, preferably 3 to 5 double bonds. Such double bonds are preferably not conjugated.

The last double bond (i.e. furthest from the 6 membered sugar ring) is preferably 3 carbons from the end of the chain, i.e. the side chain is preferably based on an omega-3 type structure.

The total number of carbons present in group R may range from 1 1 to 23, preferably 13 to 21 , such as 15 to 19. Preferably, there are an even number of carbon atoms in the side chain R when you include the CO carbon atom in the calculat ion. The use of a lino leic side chain is preferred (when including the CO carbon atom - i.e. 18 carbon atoms).

The sugar ring is preferably in the alpha configurat ion at the anomeric carbon. It should also be in the pyranose configuration. Ideally, the hexose is based on a 6-deoxyglucose mo iety and ideal ly it is a D-glucopyranosyl ring, it will be appreciated that the glucose moiety is 6-deoxy in that the group at the 6-position is not OH but a group X as defined herein.

I t is preferred i the -OCOR group lies equatorial ly on the ring. Ideal ly that group is opposite the O atom (i.e. on the para position to that O atom).

It is preferred if the C¾X group lies equatorial ly on the ring. Ideally, the ring is based on glucose and all substituents other than the anomeric carbon are equatorial.

The X group is preferably SO3R3, S0 2 R 3 , COCHai 3 , or COOR 3 . More preferably it is SO 3 R 3 , especial ly SO 3 H. The R ¾ group in the definition of X is preferably H or Me.

It is preferred if at least one of Ri and R ? is H. Ideally both are H . If not IT, it is preferred if R] or R > is a C 1 -6 alkyl. such as a C 1 -4 alkyl especial ly methyl group.

The group OR 3 in the compounds of formula (I) is preferably OH. If not OH , OR 3 is preferably OC l-6 alkyl such as OMe.

The glycero l based subst ituent group ideal ly sits axially on the ring.

Preferably the compound of formula (I) is a compound of formula (V)

such as a compound of formula (VI)

where X, R, and R i -R < arc as hereinbefore defined; or a salt thereof. More preferably, it is a compound of formula (VII)

wherein R, and R r R < are as hereinbefore defined; or a salt thereof, e.g. where SO3R3 is S0 3 H.

Most preferably, it is a compound of formula (VIII)

where X and R, arc as hereinbefore defined;

or a salt thereof.

Ideally, the compound of the invention is one of formula (IX)

where R is as hereinbefore defined.

Most especially, the compound of formula (I) is a compound of formula (X) as herein described or a salt thereof:

Compounds (II)

In a second embodiment, the invention provides a compound of formula (II) (II)

wherein

each R.( is H , -6alkyl, C6- 10 aryl, C7- 12 arylal kyl or the group:

wherein each R ¾ is as hereinbefore defined;

p is 0-3;

n is 1 to 3;

w is 0 to 2;

R 9 is Ci-6 aikyl;

Rj is a monosaccharide linked to the phenyl ring via a C-C bond or l inked v ia a C-0 bond at the 1 -position of the saccharide, and

Re is H, OH , CI -6 a Iky I or OR 3 ;

or a salt thereof;

for use in the treatment o inflammat ion.

It is preferred if the saccharide employed in compounds of formula (II) as subst it uent R> is glucose or deoxy glucose. It may also be a 1 -deoxy

monosaccharide such as l-deoxyglucose. In particular, the saccharide may be bound to the phenyl of the bicyciic ring via the anomeric oxygen of the sugar or via a direct C-C link, preferably at the 1 -position of the ring.

A further preferred compound f formula (II) is therefore of formula (XI) or

(XII)

It is preferred if the sugar is bound at the position adjacent the OH group (ortho) or meta to the OH group.

The subscript n is preferably 1 or 3. The R.i group may independently be OH or OC' i /.a lkyl such as OMe. The group R may also preferably represent:

In this group each R is preferably H or CI -6 alkyl and p can be 1 or 2. The

R3 groups outside the ring arc ideally H . Ideally the group is

5

(XV) Compounds (III)

The invention also relates to a compound of formula

wherein R < is H or C I -6 alky I; or salt thereof for use in the treatment of inflammation.

It is preferred if at least two R ? groups are I I, preferably at least 3 R7 groups are H . Preferably, one of the R? groups on the Ph ring is CI -6 alkyl, ideally methyl.

Ideally, the compound is:

(XVI)

Compounds IV

The invention also prov ides a compound of formula (IV)

wherein each R N is H or a (Ί -6-alkyl;

Rio is H or CI -6 alky I;

q is 0 to 3, such as 1 or 2;

each is as hereinbefore defined with at least one OR3 group being OH ; or a salt thereof; for use in the treatment of inflammation.

Preferred Rg groups arc C 1 -4 a Iky I groups, such as methyl. Rio is preferably ethyl or especially methyl.

It is preferred if at least two OR 3 groups in formula (IV) are OH, such as at least three groups OR s are OH . Ideally, all OR 3 groups are OH in formula (IV).

The hexose ring is again preferably based on glucose.

In a preferred embodiment, the compound is of formula (XVII)

Synthesis As we noted below compounds of formu la (I) to (IV) can be isolated from natural sources. I n order to make analogues of these compounds as described in the general formulae above, it may be easiest to start with the isolated compounds and effect transformations from those compounds using well known chemistry.

Conversion of OH to Oalkyl groups is trivial and so on.

I t is also possible however to synthesize the compounds of the invention, and in particular those of formula (I). The compounds of formula (I) can be made synthetically using known protocols, in general, any synthesis of a compound of formula (X) and related molecules of formula (I) may start from the saccharide, e.g. glucose. I ntroduction of the SO d l group at C-6 may be accomplished via conversion of the OH group to a good leaving group ( Br, CI, tosylate, mesylate, trifiate); reaction with thioacetate followed by oxidation.

Introduction of the glycerol substituent at C- l might involve an acetonide of glycerol.

The polyunsaturated fatty acid chain required is a readily available product which can be coupled to the sugar ring using know n chemistry with protection if required. Nucleophi!ic attack of the 3 -position hydroxyl group onto to an acid chloride modified fatty acid chain allows the side chain to be introduced.

Esterfieation of the 3-position-OH might occur via a mixed anhydride and there is also a set of methods which can be used on the linolenic acid directly. Possibly enzymatic react ions could have some benefit in offeri ng h igh

regioselectivity, mild conditions and easy clean up.

Protocols discussed in the following papers can be employed:

Kv rno, Lisbet, Werdcr, Moritz, Hauser, Helmut, and Carreira, Erick M. Synthesis and in Vitro Evaluation of Inhibitors of Intestinal Cholesterol Absorption. J.Med.Chem. 48[ 19], 6035-6053. 2005;

Kv rno, Lisbet, W order, Moritz, Hauser, Helmut, and Carreira, Erick M. Carbohydrate Sulfonyl Chlorides for Simple, Conven ient Access to

Glycoconjugates. Org. Lett. 7[6], 1 145- 1 148. 2005;

Yamazaki, Takayuki, Aoki, Satoko, Ohta, Keisuke, Hyuma, Sh inj i,

Sakaguch i, Kengo, and Sugawara, Fumio. Synthesis of an immunosuppressant SQAG9 and determination of the binding peptide by T7 phage display.

Bioorg.Med.Chem.Lett. 14[ 16], 4343-4346. 2004;

Liptak, Andras, Bal la. Edit, Janossy, Lorant. Sajtos, Fcrcnc, and Szi lagyi, Laszlo. The first synthesis of secondary sugar sulfonic acids by nucleophilic displacement reactions. Tetrahedron Lett. 45 [4], 839-842. 2004;

Sahara, H iroeki, Hanashima, Shinya, Yamazaki, Takayuki, Takahashi, Shunya, Sugawara, Fumio, Ohtani, Seij i, ishikawa, Masako, M izush ina, Yoshiyuki, Ohta, Keisuke, Shimozawa, Kumiko, Gasa, Shinsei, Jimbow, Kouichi, Sakaguchi, Kengo, Sato, Noriyuki, and Takahashi, obuaki. Ant i-tumor effect of chemical ly synthesized sulfolipids based on sea urchin's natural sulfonoquinovosylmono- acylglycerols. Jpn. J . Cancer Res. 93 [ 1], 85-92. 2002;

Sugawara, Fumio, Sakaguchi, Kengo, Yamazaki, Takayuki, M urata, H iroshi, Ohta, Keisuke. and Masaki, Kazuyoshi. Novel 6-deoxy-6- carbonyhhioglucopyranosyl acetimidates, novel intermediates for production thereof and processes for production of both. (Toyo Suisan Kaisha, Ltd. Japan. 2001 - JP 1607[2001064699], 60. 2001 0907. WO. 2-3 -2001 ;

Hanashima, S., M izushina, Y., Yamazaki, T., Ohta, K., Takahashi, S., Sahara. 11., Sakaguchi, K.. and Sugawara, F. Synthesis of

sulfoquinovosylacylglycerols, inhibitors of Eukaryotic DNA Polymerase I± and P. Bioorg. Mcd.Chcm. 9[2], 367-376. 2001 ; and

Hanashima, Shinya, M izushina, Yoshiyuki, Yamazaki, Takayuki, Ohta, Keisuke. Takahashi, Shunya. Kosh ino, H iroyuki, Sahara, H iroeki, Sakaguch i, Kengo, and Sugawara, Fumio. Structural determination of

sulfoquinovosyldiacylgiycerol by chiral syntheses. Tetrahedron Lett. 41 [22], 4403- 4407. 2000;

The specific compound of formula (X) can, however be iso lated from Sclerochloa dura (Linneaus) P Beauvois, also known as common hard grass. The experimental section contains a detailed description of the isolation procedure. In particular, a compound of formula (X) can be isolated from the aerial parts of the plant. Compounds of formula (II) to (IV) are also isoiable from this plant. Tlius viewed from another aspect the invention provides a process for the isolation of a compound of formula (X) comprising extracting said compound from Sclerochloa dur. Applications

The compounds of the invention have surprisingly been found to hav e potential as anti-inflammatory agents. They have been found to be potent inhibitors of PL A.> in particular cPLA.> The compounds of the invention are therefore of use as possible therapies for chronic inflammatory condition that can generally be associated with PL.A> inhibition.

it is a particular feature of the inv ention that the compounds of the inv ention are able to reduce inflammation via inhibit ion of cPLA2 as opposed to some enzyme later in the inflammation reaction pathway.

Conditions of interest therefore include acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensit iv ities, inflammatory bowel disease , pelv ic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, and vasculitis.

The compounds of the invention may also have utility in the treatment of hay fev er, periodontitis, atherosclerosis, rheumatoid arthritis, and cancer.

The animal being treated will typically be a human although these compounds could also be used by a vet field to treat domest ic, farm or even wild animals.

As used herein, the expression "pharmaceutically acceptable salts" refers to deriv atives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Any pharmaceutically acceptable salt can be used in the invention such as a

rifluoroacetatc. sulphate, phosphate, acetate, fumarate, maleate, citrate, pyruvate, succinate, oxalate, methanesulphonate, p-toluenesulphonate, bisulphate, benzcncsulphonate, ethanesulphonatc, ma!onate. xinafoate, ascorbatc, oleatc, nicotinate, saccharinatc, adipate, formate, glycolatc, L-lactate, D-lactatc, aspartate, malate, L-tartrate, D-tartrate, stcaratc, 2-furoate, 3-furoate, napadisylate

( naphthalene- 1 ,5-disu!f nate or naphthalene- 1 -( sulfonic acid)-5-suifonate), edisylate (ethane- 1 ,2-d isulfonate or ethane- 1 -( sulfonic acid )-2-sulfonatc ), isethionate (2- hydroxyethylsulfonate), 2-mcsitylenesulphonate, 2-naphthalenesulphonate, D- mandelate or L-mandelate.

Of course the salt may well be a base salt such as ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.

By treating or treatment is meant at least one of:

(i) . prevent ing or delaying the appearance of cl inical symptoms of the disease developing in a mammal;

(ii) . inhibit ing the disease i.e. arresting, reducing or delaying the development of the disease or a relapse thereof or at least one cl inical or subcl inical symptom thereof, or

(iii) . rel ieving or attenuating one or more of the cl inical or subcl inical symptoms of the disease.

The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician. I n general a skilled man can appreciate when "treatment" occurs. I t is particularly preferred i f the compounds of the invention are used therapeutically, i.e. to treat a condit ion which has manifested rather than prophylactical ly. it may be that the compounds of the inv ention are more effect ive when used therapeutical ly than prophylact ically.

The compounds of the inv ent ion can be used on any animal subject, in particular a mammal and more particularly to a human or an an imal serving as a model for a disease ( e.g., mouse, monkey, etc.).

I n order to treat a disease an effect ive amount of the active agent needs to be adm inistered to a patient. A "therapeutically effect ive amount" means the amount of a compound that, when adm inistered to an animal for treating a state, disorder or condit ion, is sufficient to effect such treatment. The "therapeutically effect ive amount" will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated and will be ultimately at the discretion of the attendant doctor.

It may be that to treat an inflammatory condition according to the invent ion that the compound of formula (I) has to be reapplied at certain intervals. Suitable dosage regimes can be prescribed by a physician.

While it is possible that, for use in the methods of the inv ention, a compound may be administered as the bulk substance, it is preferable to present the active ingredient in a pharmaceutical formulation, for example, wherein the agent is in admixture with a pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

The term "carrier" refers to a diluent, excipient, and/or v ehicle with which an active compound is administered. The pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers arc well known in the art.. The pharmaceutical compositions may also comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s) and so on. The compositions can also contain other active components, e.g. other drugs for the treatment of inflammation.

It will be appreciated that pharmaceutical compositions for use in accordance with the present invention may be in the form of oral, parenteral, transdermal, sublingual, topical, implant, nasal, or enterally administered (or other mucosally administered) suspensions, capsules or tablets, which may be formulated in conv entional manner using one or more pharmaceutically acceptable carriers r excipients. The compositions of the invent ion could also be formulated as nanoparticie formulat ions.

For some inflammatory conditions, the compositions of the invent ion will preferably be applied topically. They may be in the form of ointments, gels, syrups, creams and so on. Any conventional topical formulation vehicle may be used.

The pharmaceutical compositions of the invention may contain from 0.01 to 99% weight of the activ e compound.

A therapeutically effect ive amount of the compound of the present invention can be determined by methods known in the art. The therapeutically effective quant it ies will depend on the age and on the general physiological condit ion of the patient, the route of administration and the pharmaceutical formulation used. The therapeutic doses will generally be between about 10 and 2000 mg/day and preferably between about 30 and 1500 mg/day. Other ranges may be used,

includi ng, for example, 50-500 mg/day, 50-300 mg day, 100-200 mg/day.

Administration may be once a day. twice a day, or more often, and may be decreased during a maintenance phase of the disease or disorder, e.g. once every second or third day instead o every day or twice a day. The dose and the

adm inistration frequency will depend on the cl inical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skil led in the art.

The compounds of the invention may be used to treat inflammation in combinat ion with other known pharmaceut icals f r said purpose and this forms a further aspect of the invention. Other useful pharmaceuticals include

corticosteroids and anti-histamines.

The invent ion is described further below wit h reference to the following non- lim it ing examples and figures.

Figure 1 : The inflammation process showing point of act ion f r both the compounds of the invent ion and most NSA lDs. Inhibit ion of one of the three parallel pathw ays late in the process gives an over-expression of the two other pathw ays, thus an unbalanced inhibit ion.

Figure 2: describes the IC5 0 v alue of compound 1. Examples

For extraction, VWR ultrasound cleaner. BTJCHI rotavapor R-200. BLICH I rotavapor Rl l and iso mant le of KEBO-GRAVE isopadisomantle were used. MPLC system used was Kronlab GmbH with data system Prepcon 4.47. Stationary phase used in MPLC was polygoprep 60-50 RP- 18 ( Macherey & Nagel ) and mobi le phase used was distilled water, methanol and isopropanol. For preparative and semi preparative 11 PLC, the system used was SEPBOX (Fa. SEP I A tec ), the mobile phase components were water, methanol and acetonitrile. Detection had been made by ELSD ( Sedex 75) and UV (Merck, 250nm) detectors. For preparative HPLC Kromasil C18 250x50 mm, 10 μιη was used as separation phase. For semi preparative HPLC Kromasil CI 8 250x16 mm, 10 um and Nucleodur C18 Isis, 100x10; 5 iim were used as separation phases. The human synovial sarcoma cell line SW982 was purchased from ATCC (UK). Recombinant human I L- 1 [3 was purchased from Roche (UK); phosphate-buffered saline solution (PBS) was from Oxo id (UK); Dulbecco's Modified Eagle Medium ( DM EM ), foetal bov ine serum (FBS), fatty acid-free bov ine serum albumin (fBSA), gentamicin and L-glutamine were from Sigma-Aldrich (USA). Labelled (5,6,8,9,1 1, 12, 14, 15- 3 H)-AA (specific activity 180-240 Ci/mmol) and liquid scintillation cocktail OptiPhase Supermix were from NEN Perk in Elmer (USA). The ' ll- MR, 13 C-NMR, HMBC, COSY, NOESY. HSQC and HSQC-TOCSY experiments for structure elucidat ion of the isolated compounds were run on a Bruker A VANCE 11 instrument, operating at a proton frequency of 600.1 M Hz, with a 5-mm triple-resonance cryo probe equipped with a z-gradient.

Sclerochloa dura (Linnaeus) P. Beauvois, known also as common hardgrass, belongs to family Poaceae Barnhart, which involves more than 700 genera and almost 50,000 species. Sclerochloa dura is an annual plant. It has:

flat leaves and procumbent or erect steam up to 16 cm;

inflorescence 1 -4 cm, rigid, more or less second;

pedicels stout, up to 1 mm;

spikeiets 6- 10 mm, narrowly oblong, crowded;

glumes somew hat asymmetrical, glabrous, oblong ovate, with wide hyaline margins;

lower glume 2-3 mm, obtuse to emarginated, with (1) 3 (5) veins;

upper glume 3-5 mm, rounded to emarginated, with (5) 7-9 veins;

lowest lemma 4.8-6 mm, the upper much shorter, glabrous, rounded to emarginated, sometimes very shortly mucronatc;

anthers 0.8- 1 .5; and

lowest rhachilia segments 2-2.5 mm, the upper shorter. 2n=14. Tlie plant is a common inhabitant of areas with heavy traffic, e.g. along dirty roads, on play yards and walking pathways. It is idely spread in the moderate climate zone on almost all the cont inents on North Hemisphere and introduced in

Australia as well.

S. dura was collected in the city of Nis, Serbia, near Gabrov acka reka creek.

The plant was air dried in the shade for ten days. The dry plant material was packed in plastic bags and kept in a coolish and dark place until extraction.

Extraction

Aerial plant parts were crushed into smaller pieces. Clean plant material weighing 1.4 kg was exhaustively extracted by soaking in methanol for 24 hours at room temperature twice. The extraction was aided by 90 minute of sonication. The obtained extracts were filtered ( Blue ribbon filter paper from Schleicher and Schuell) and combined. The volume was reduced on rotavapour and the concentrated solution transferred into separation funnels. Extraction with n-hexane (three times) was used for detailing and the separated methanoiic phase dried on rotav apour. The dry methanoiic extract yielded 1 70g (12.14%) and was used for further work.

Bioassay guided isolation

Isolation was done by using a multistage fractionation approach by liquid chromatography. The crude extract was subjected to pre fractionation by MPLC using a double inject ion, 85 g per injection. The method used for MPLC is given in Table 1. The obtained pre- fractions were tested on human synovial sarcoma ceil line SW982 to determine any effect on PLA2 activ ity by measuring A A release. Three p re-fractions found very active were subsequently fractionated by 3 respective runs on preparative HPLC into 259 fractions. The method used for preparative HPLC is giv en in Table 2. All the obtained fractions were tested for inhibit ion of PLA2 activity by measuring AA release. Based on the similarity of chromatograms and bio-activity, some of the fractions of pre- tract ion F were pooled into groups, e.g. F40-F44 as one group and F70-F74 as the other. These groups were subjected to semi-preparat ive HPLC runs. The method used for semi preparat ive runs is given in Table 3. Based on TLC, fractions G50-G51 of pre-fraction G were pooled together and subjected to semi-preparative HPLC run. The method used for semi-preparative run is given in Table 3.

TABLE 1 Method 1. Used for MPLC

MPLC system Kronlab GmbH

Data System Prepcon 4.47

Stationary Phase Polygoprep 60-50 RP- 18 (Macherey & Nagel )

Mobi le Phase A: distilled Water

B: Methanol

C: Isopropanol

Injected amounts 2 x 85 gram

Grad ient Time (min) Flow rate (ml/min) % A % B % C

0.0 100 100 0 0

5.0 100 100 0 0

5. 1 130 100 0 0

10.0 130 100 0 0

10.1 100 100 0 0

18.0 100 100 0 0

I ndividual gradient

5 1 .0 100 30 70 0

61.0 100 10 90 0

61.1 1 50 0 100 0

66.0 1 50 0 100 0

66.1 30 0 0 100

70.0 30 0 0 100

70. 1 75 0 0 100

74.0 75 0 0 100 ine fractions were collected starting at 10 min, 8 min col lection t ime each. Table 2 Method 2. Used for preparative H PLC

HPLC SEPBOX (Fa. SEPIAtec) system

Separation Kromasil CI 8 250x50 mm,

phase

Amounts 2.3638 g "E", 0.7078 g "F" and 1.0822 g "G"

injected

Flow rate 109 ml/min

Detection ELSD ( Sede.x 75), UV ( Merck, 250 nm)

M bi le phase A : water

B: Methanol, Aeetonitrile 1 : 1

Gradients Time (min) % A % B

Fraction E 00.0 85 15

57.7 58 42

58.0 0 100

63.0 0 100

Fract ion F 00.0 77 23

57.7 43 57

58.0 0 100

63.0 0 100

Fraction G 00.0 45 55

57.7 9 91

58.0 0 100

63.0 0 100

A total of 80 fractions from pre-fraction E, 90 fractions from pre- fraction F and 89 fractions from pre-fraction G were obtained. All fractionations were time based: 18 to 20 second per fraction

Table 3 Method 3. Used for semi preparative HPLC

H PLC system SEP BOX (Fa. SEPIAtec)

Separation Kromasil C18 250x16 mm, 10 μ ηι (Group G50-G5 1 ); all other plia.sc separations: Nucleodur CI 8 Isis, 100x 10; 5 μιη

Amounts 34.3 mg "Group F40-F44 " , 39.6 mg "Group F70-F74 " and 45.0 injected mg "Group G50-G5 1 "

Flow rate 15 ml m in (Group G50-G5 1 ); al l other separations: 5 m l/min Detection ELSD (Sedex 75), UV (Merck, 250

Mobile phase A: water

B: Methanol/ Acetonitrile 1 : 1

Gradients Time (min) % A % B

Group F40-F44 00.0 100 0

57.7 94 6

58.0 0 100

63.0 0 100

Group F70-F74 00.0 94 6

57.7 86 14

58.0 0 100

63.0 0 100

Group G50-G5 1 00.0 37 63

25.0 20 80

25. 1 0 100

30.0 0 100

Five sub fract ions from group F40-F44, 7 sub fractions from group F-70-74 and 2 sub fractions from group G50-G51 were obtained. All sub fract ionat ions were time based: 18 to 20 second per sub fraction

Cell culture and treatment of SW982 cells

The human synovial sarcoma cell line SW982 is used as a model system to determine cPLA > activity by measuring AA release. SW982 cells are widely used as a model for rheumatoid synovial inflammation and express c LA.>-i Va and cytokines after inflammatory st imulat ion by e.g., IL- Ι β. Cells were sub-cultured biweekly by routi ne trypsin detachment and kept in a sub-confluent state. The cel ls were cultured in DM EM supplemented 10% FBS, 0. 1 mg m l gentamicin and 0.3 mg m l, L-glutamine in a humidified 10% CO.) atmosphere at 37°C. For AA release experiments, 5* 10 s cells/well were seeded in a 48-weii/plate format. At two days post-con fluency, the cel ls were label led with Tl-AA in serum-free DM EM ov ernight and processed at day three post-confluence to ensure differentiation and synchronization of the cells. The experiments were performed in serum-free DM EM in triplicates of wells. Untreated cells without inducing agents or plant extract were included for unstimulated control; distilled water was added to these for vehicle control. Following treatments, cel ls were routinely observ ed by microscopy to ensure unaltered cell morphology, integrity and viability.

A A release assay

The AA release assay determines the amount of AA released from ceils under inflammatory conditions. The enzyme cPLA > is the only arachidonyl-specific PLA ) enzyme and thus the A A release mainly corresponds to the activity of cP LA.> that cleaves off AA in the sn-2 position of phospholipids. In the presence of inhibiting compound(s), the AA release is reduced, which is taken as evidence that the compound(s) target some level in the arachidonyl cascade, such as the cPLA ? enzyme.

At two days post -con fluency, SW982 cells were serum-starved and labelled overnight with Tl-AA ( 0.4 μθ/πιΐ). Prior to the addition of S. dura fractions, the cel ls were washed with PBS containing fBSA (2 mg/ml) to remove un incorporated radioactivity. Cells were pre-treated for 1 h with various dilutions ef fractions of the plant extract, followed by addit ion of 1 I .- 1 β (10 ng ml. ) to induce an in flammatory state. Following 24 h of IL- 1 β stimulation, the supematants were cleared of detached cel ls by centrifugat ion (13000 rpm, 10 min ). Adherent cells were dissolved in 1M NaOH in order to measure the amount of labelled AA incorporated into the cells. The activity in the supematants and cellular fractions was assessed by liquid scint i l lat ion count ing in LS 6500 u It i- Purpose Scint i llat ion Counter ( Beckman Coulter, Inc USA). The results are given as released Tl-AA in the supematants relative to total Tl-AA incorporated into the cells. Inhibition was calculated as AA release in inhibited relative to stimulated cells, normalized to control, subtracted by 100.

By means o MPLC, the crude extract was separated into 9 pre fractions (A- I), which were tested for inhibitory effects in AA-release assay. Out of these, E, F and G were found to exert the highest inhibitory effect, as shown in Table 4.

The results of inhibition activity of the three most active pre-fractions E, F and G are shown in the Table 4. Table 4. Comparison of act ivity of fractions E, F and G. Experiments were done triplicates. Inhibition and IC5 0 values are based on two independent experiments. IC50 could not be calculated for fraction F as inhibition did not go below 50 % in any concentration tested.

Concentration % Inhibition (mean

Fraction ICso

(mg/ml) ± S.D.)

E 0.10 63.1 ± 18.4 0.10

F 0.10 68.3 ± 4.8

G 0.08 63.5 ± 25.5 0.60

By the bioact ivity guided study, from pre fraction F and G a total of six compounds with pronounced ant i- inflammatory activity were identified. Compound 1

Compound 1 was obtained by semi-preparative HPLC run of group G50-G5 1 (Table 3). The analysis o f spectral data revealed that compound 1 is a novel compound. HRMS pointed out to a m lecular formula C27H46O11S ( ESI -) with a molecular mass 578.2751 ( theoretical va lue 578.2761). The analysis of 1 D and 2 D NMR spectra with homo- and hetero- nuclear direct or long range correlations allowed structure elucidation and a complete 1 H and 13 C assignment, as listed in Table 5. The carbons 1 -6, belonging to glucose moiety, had shift values for 13 C ranging from 6 54.4 to 100.3 ppm and for protons from δ 2.92 to 4.77 ppm. The shift values of both I3 C and protons at C-6 indicated a direct attachment to an SO3H group, which was also confirmed by MS analysis. The 18 C-atoms with shift values ranging from δ 14.8 to 1 76.2 ppm w ith attached protons from δ 0.98 to 5.37 ppm belong to linolenoyl chain of the molecule. An H M BC correlat ion peak of H-3 (at δ 3.65 ppm) of glucose and carbon C- l "(6 176.2 ppm) of the linolenoyl chain revealed the attachment position. Glycerol moiety exhibited shift values for carbons ranging fr m δ 64.4 to 72.7 ppm and protons from δ 3.36 to 4.05 ppm. The correlat ion peaks between carbon C- 1 of glucose and protons ΙΤ,.η- of glycerol in H M BC unambiguously revealed connect ivity between glucose and glycerol. Both the m lecular formula and NM R data ind icated that 1 was a deriv at iv e of glycerol with the chemical name l -0-(3-0-linolenoyl-6-deoxy-6-suifo-a-D-glucopyranosyl)- glycerol. Compound 2

Compound 2 was identified as isovitexin. It was isolated as the main compound in fraction F-07 obtained by preparative HPLC run of pre fraction F (Table 2). HRMS (ESI +) was 432.1055 which corresponded to molecular formula C21H20O10, ( theoretical value 432.1056). The compound was identified by a scries of 1 D and 2D NMR experiments. The signal assignments for carbons and protons can be seen in Table 5. The NMR data were in agreement with those already published (Leong et a!., 2010).

Compound 3

Compound 3 was isolated as the main compound in fract ion F-51. This fraction was obtained by preparative H PLC run of pre fraction F (Table 2). Compound 3 was ident ified as tricin 4 -O-(erythro- -guaiacyiglyceryl) ether 7-Ο-β- glucopyranoside. H RMS (ESI +) found was 688.2003 ( theoretical value 688.2003) and it corresponded to the molecular formula C33H36O16. The NMR data of the compound (Table 5) fits the published ones (Bouaziz et al., 2002).

Compound 4

Semi preparative H PLC run of group F-40-F44 (Table 3) yielded a fraction containing 4 as the main compound. H RMS (ESI +) for 4 was 372.2147 compared to the theoretical value of 372.2148 and matches the molecular formula C19H32O7. Compound 4 was identified as byzantionoside B. The NMR data (Table 5) are in agreement with those published earlier ( Matsunami et al., 2010 ).

Compound 5

The semi preparative HPLC run f group F-70-F74 (Table 3) gave 5 as the main compound in the resulted sub fract ions. I t was identified as 1-O-feruloyl glycerol. H RMS (ESI +) was found to be 268.0952 (corresponds to molecular formula with theoretical value 268.0947. . The NMR resonances of l H and 13 C for 5 are in agreement with those reported prev iously (Table 5) (Luo et al., 2012). Com pound 6

Compound 6 was the main compound in fraction F-32, obtained by preparative HPLC run o pre fraction F (Table 2). It was identified as trie in 7- glucoside. FIRMS (ESI +) 492.1268 for C23H24O12, found 492.1263. The NMR data of 6 (Table 5) fits to those reported earlier (Avvaad et al., 2008).

The structures of compounds 1 to 6 are shown below:

4 = formula (XVII)

5 = formula (XVI)

6 = formula (XIII)

The structures of identified compounds from methanolic extract of S. dura. Compound 1 : 1 - O-(3-0-linolenoyl-6-deoxy-6-sulfo-a-D-glucopyranosyl)-glycer ol; Compound 2: Isovitexin; Compound 3: Tricin 4 -0-(erythro-|3-guaiacylglyceryl) ether 7-0-P-glucopyranoside; Compound 4: Byzantionoside B; Compound 5: 1-O-feruloyl glycerol; Compound 6: Tricin 7-glucoside. Table 5. Assignment of ! H and 13 C resonances of compounds 1-6.

18" 14.8 0.98, t - - - -

7.5 Hz

V" - - - - 133.4 -

2'" - - - - 111.5 7.00, m

3'" - - - - 148.6 -

4'" - - - - 146.8 -

5'" - - - - 115.6 6.73, m

6'" - - - - 120.5 6.81, m

0

Me

3", - - 57.0 3.92 5"

3'" 56.5 3.83

6" - - - - 104.9 7.30

OMe

3' 56.3 3.90

3", 5" 56.2 3.84

' " ' Assignments marked with the same letter are interchangeble

' Tentative assignments

Comparison of bioactivity of the identified compounds from S. dura

The respect ive fractions containing each of these compounds were again tested for bioact iv ity. confirming inhibitory effects on PLA 2 enzyme act ivity. IC5 0 values of inhibition were estimated to be 0. 1 1 mg/ml both for compound 1 and 3 (Table 6). The identified compounds from S. dura inhibited IL- 1 induced release of A A in SW982 cells in this order of decreasing efficiency:

Compound 5 > Compound 4 > Compound I and 3 > Compound 6 > Compound 2.

Table 6. Comparison of act ivity of compounds 1 - 6 based on experiments in triplicates. Inhibition and IC50 values are based on at least three independent experiments, unless otherwise mentioned.

* Based on one experiment ** Based on two independent experiments

The graph showing IC50 value of compound 1 is given in Figure 1.

The m le percentage of compound 1 in the act ive fract ion was est imated to be 60%, based on integral ratio in Ή- M R spectrum. Therefore, the IC5 0 v alue of 1 from three independent experiments corresponds to 1 14. 1 ± 31.1 μΜ. The obtained IC50 value shows that 1 has the potential to be used as a lead compound for the dev elopment o f a n t i- i n flam matory drugs. Furthermore, the spectroscopic study f 1 shows unambiguous presence of an -SO3H group. The presence of sulfonyl group in 1 might be important for its bioact ivity.