Scientific Patents:


01. GR/2012/0100266 – 21.05.2012 (Pub) – 201201605 – 21.05.2012
Kossida S, Vlachakis D, Tsiliki G, Pavlopoulou A.

3D PHARMACOPHORE FOR THE DESIGN OF HUMAN POLYADENOSINE RIBONUCLEASE(PARN) INHIBITORS

This invention is about a highly specific, custom-made three dimensional Pharmacophore model for the catalytic site of the human poly-adenosine ribonuclease (PARN).

Description:

The present invention relates to means and methods for the design of highly specific anti-PARN agent using our 3D pharmacophore model. Using a library of previously published modified nucleoside analog substrates acting in the proximity of the scissile bond of PARN, we conducted structure – activity relationship (SAR) analysis, structural characterization and complex (receptor-ligand) based molecular dynamics simulations that led to the design of a PARN-specific pharmacophore model. Our full pharmacophore model consists of five pharmacophoric annotation points, which include two hydrogen donating PAPs, one hydrophobic PAP, one aromatic PAP and a hydrogen accepting PAP, in the vicinity of human poly-Adenosine ribonuclease catalytic site.

02. WO/2009/125191 – 15.10.2009 (Pub) – PCT/GB2009/000936 – 09.04.2009
Brancale A, Vlachakis D, Berry C, Neyts J.

HCV ANTIVIRAL DRUG DESIGN, BENZENE DERIVATIVES

Compounds, which according to the invention can display potent inhibition against Hepatitis C Virus (HCV) helicase and potent anti-viral activity against HCV.

Description:

BENZENE DERIVATIVES

The present invention relates to compounds suitable for use in the treatment of a viral infection, particularly a HCV infection.

Hepatitis C is a liver infection caused by a flavivirus (HCV) that affects over 170 million people worldwide. In most cases the infection becomes chronic and 20% of the carriers develop cirrhosis. Currently the infection is treated with interferon, but this therapy does not completely eliminate the virus and more efficacious treatments are needed.

According to a first aspect of the present invention there is provided a compound having the formula:


wherein:

R1, R2, R3, R4 and R5, are each independently selected from the group consisting of hydrogen, Cl, Br, F, I, NH2, CN, COOH, COONH2, OCH3, CH3, NO2 and SH; Y either does not exist or is selected from the group consisting of CH2 and CO;

X is selected from the group consisting of CH2, CH2CH2, CH=CH cis or trans,

C≡C, CO, NH and O;

Z is selected from the group consisting of O, S and NH;

Q either does not exist or is selected from the group consisting of cycloalkyl (C3 to C20), heterocyclic having 3 to 20 ring members including one or more atoms selected independently from O, N and S, aryl (C6 to C20), naphthyl, heteroaryl having 4 to 20 ring members including one or more atoms selected independently from O, N and S;

T is selected from the group consisting of CH=CHCOCH3, CH-CHCOOH, CH=CHCONH2, CH=CHCN, CH=CHNO2, SH, SSCH2CH2OH, SSCH2CH(NH2)COOH, CH(NH2)CH2SH, CH(NH2)CH2CH2SH, CH2F, CH2Cl,

CH2Br and CH2I; and pharmacologically acceptable salts, prodrugs, including esters thereof, especially methyl, ethyl, isopropyl, tbutyl and benzyl, solvates and hydrates thereof.

The present invention provides compounds that are believed to be HCV helicase inhibitors and thus useful in the treatment of HCV infections.

Although we do not wish to be bound by any theory, the compounds of the present invention are believed to target a specific cysteine (Cys 431) on the enzyme surface that could react with an inhibitor, forming a covalent complex. This specific cysteine is close to arginine 393, which is a key residue involved in the binding of the RNA.

Other than possibly T, and possibly Q, no part of the compounds of the present invention exhibits a chiral centre. Preferably, Q, if present, does not include any chiral centre. Preferably, the compound contains no chiral centres.

A compound of the present invention can thus provide a single structure, which can be readily prepared, with a relatively controlled configuration between an active part of a substituent selected from R1, R2, R3, R4 and R5 and an active part of the moiety T. Suitably, the spacing which can be exhibited between the active part of a substituent Ri, R2, R3, R4 or R5 and the active part of moiety T lies in the range of from 10 nm to 14 nm. More suitably, this spacing lies in the range of from 11 nm to 13 nm.

A compound with such a spacing between these recited active parts of the molecule is believed to be particularly suitable for binding with Cys 431 on HCV helicase and thus acting as a helicase inhibitor.

Where each OfR1, R2, R3, R4 and R5 is hydrogen, the above defined spacing is measured

Where one or more of Rj, R2, R3, R4 and R5 is other than hydrogen, which is preferred, the above defined spacing is measured form the active part of the substituent or substituents other than hydrogen. Preferably one, two or three substituents are other than hydrogen.

More preferably, only one Of R1, R2, R3, R4 and R5 is other than hydrogen. Where only one substituent is other than hydrogen, it is preferably R1 or R2, more preferably it is Rj. Preferably the any one substituent other than hydrogen is COOH.

Where two substituents are other than hydrogen, they are preferably the same and they are preferably R2 and R5.

Where three substituents are other than hydrogen, they are preferably the same and they are preferably R5, Ri and R2, with the above defined spacing being measured from Rj.

Preferably Q is an aryl, naphthyl or heteroaryl moiety, so as to provide further rigidity to the configuration of the molecule of the above formula and/or further control of the above defined spacing.

Preferably, R2, R3, R4 and R5 are hydrogen and R] is COOH or a prodrug thereof such as an ester thereof such as the methyl, ethyl, isopropyl, tbutyl and benzyl ester, or Rj, R3, R4 and R5 are hydrogen and R2 is COOH, or a prodrug thereof such as an ester thereof such as the methyl, ethyl, isopropyl, Λutyl and benzyl ester thereof. Methyl esters are especially preferred. In use, the ester is hydrolysed to the active acid moiety. In the acid moiety, O is the active part with respect to which the above defined spacing is measured.

Where T is CH=CHCOCH2, CH=CHCOOH, CH=CHCONH2, CH=CHCN or CH=CHNO2, the active part of the moiety T with respect to which the above defined spacing is measured is the C atom of T which is attached either to Q or to the C atom bearing Z. In use to treat, for example, HCV, the cysteine (Cys 431) reacts with this C atom of the C=C double bond in T.

Where T is SSCH2CH2OH or SSCH2CH(NH2)COOH, the active part of the moiety T with respect to which the above defined spacing is measured is the S atom of T which is attached either to Q or to the C atom bearing Z. In use to treat, for example, HCV, T cleaves at the S-S bond.

Where T is SH or CH(NH2)CH2SH or CH(NH2)CH2CH2SH, the active part of the moiety T with respect to which the above defined spacing is measured is the S atom. In use to treat, for example, HCV, the cysteine (Cys 431) reacts this S atom of T.

Where T is CH2F, CH2Cl, CH2Br or CH2I, the active part of the moiety with respect o which the above defined spacing is measured is the C atom. In use to treat, for example, HCV, T cleaves at the C-halogen bond.

Preferably T is selected from the group consisting of CH=CHCOCH33 SH and SSCH2CH(NH2)COOH and CH(NH2)CH2SH.

Where Q is heterocyclic or heteroaryl, it preferably contains from 1 to 6 hetero atoms in total of O, N and S. Preferably where Q is heterocyclic or heteroaryl, it contains a maximum of 12 ring members, more preferably a maximum of 7 ring members. Even more preferably, where Q is heterocyclic or heteroaryl, it is a five membered ring and contains one hetero atom, most preferably N.

Preferably Q is selected from the following moieties, where the substitution pattern refers to the relationship in the respective bonds between Q and T and between Q and the

C atom bearing Z: a phenyl with a 1,2 or 1,3 or 1,4 substitution pattern; a pyridine ring with a 2,3 or 2,4 or 2,5 or 2,6 or 3,4 or 3,5 or 3,6 substitution pattern; a pyrimidine ring with a 2,4 or 2,5 or 4,5 or 4,6 substitution pattern; a pyrazine ring with a 2,3 or 2,5 or 2,6 substitution pattern; a pyrrole or a thiophene or a furan with a 1,3 or 2,4 or 2,5 or 3,4 substitution pattern, preferably a 2,5 substitution pattern, even more preferably a pyrrole with a 2,5 substitution pattern; a pyrazole with a 1,3 or 1,4 or 2,4 substitution pattern; a imidazole with a 1 ,4 or 2,4 or 2,5 substitution pattern; a oxazole with a 2,4 or 2,5 substitution pattern; a thiazole with a 2,4 or 2,5 substitution pattern; a isoxazole with a 3,5 substitution pattern; and a isothiazole with a 3,5 substitution pattern.

Where Q does not exist, T is bonded directly to the C atom bearing Z.

Preferably X is NH.

Preferably Y is CH2.

Where Y does not exist, X is bonded directly to the C6 aryl group bearing R1, R2, R3, R4 and R5.

Preferably Z is O.

Preferred compounds of the present invention have, in combination, X as NH, Y as CH2 and Z as O.

Preferred compounds of the present invention include compounds where T is present as any of CH=CHCOCH3, SH, SSCH2CH(NH2)COOH and CH(NH2)CH2SH, preferably as CH=CHCOCH3 or SH, in combination with R1 present as COOH or COOMe, preferably as COOH, and each of R2, R3, R4 and R5 present as H; further particularly preferred compounds include compounds with such T and R1, R2, R3, R4 and R5 moieties where, further, Q is present as an aryl, naphthyl or heteroaryl moiety, more preferably where Q is present as any of the specific moieties recited above, especially pyrrole with a 2,5 substitution pattern or phenyl with a 1,4 substitution pattern; and further particularly preferred compounds include compounds with such T, Ri, R2, R3, R4, R5 and Q moieties where, further, X is NH, Y is CH2 and Z is O.

Any chiral centre present in the compound, in for example T, can be present as pure S or pure R.

Where a chiral centre is present in the compound it can be present as a mixture of S and R. The compound can be a racemic mixture. Where a chiral centre is present in the compound according to the above formula, it is to be understood that the term "compound", as used herein, includes, unless otherwise stated, a mixture of the S and R chiral forms of that compound.

Suitably, R1 is not Cl. Suitably, none of Ri, R2, R3 R4 and R5 is CL Suitably, the compound of the present invention is none of N-(2,4-dichlorobenzyl)chloroacetamide, N-(4-chlorobenzyl)chloroacetamide, N-(3 -chlorobenzytychloroacetamide, N-(2,3 -dicholorobenzyl)chloroacetamide and N-(2,5-dichlorobenzyl)chloroacetamide.

Suitably, Q is not 4-oxo-4,7-dihydrofuro[2,3-b]pyridine. Suitably, Q is none of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine, 7-oxo-4,7-dihydrothieno[3,2-b]pyridine, 4-thioxo-4,7-dihydro-thieno[2,3-b]pyridine and 4-hydroxyquinoline.

According to a further aspect of the present invention there is provided a pharmaceutical formulation comprising a compound of the present invention, together with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any conventional carrier appropriate for the format in which it is desired to deliver the present compound to a subject in need of the compound. Suitably, the present compound can be administered parenterally, transdermally, mucosally, nasally, buccally, sublingually, topically or orally.

If desired, the present compound can be used in the treatment of a viral infection such as HCV in combination with a therapeutically effective amount of a second antiviral agent.

According to a further aspect of the present invention there is provided a compound of the present invention for use as a medicament for treating or preventing a viral infection. Suitably, the viral infection is HCV.

According to a further aspect of the present invention there is provided a use of a compound of the present invention for the manufacture of a medicament for treating or preventing a viral infection. Suitably, the viral infection is HCV.

According to a further aspect of the present invention there is provided a method for treating or preventing a viral infection, the method including the step of administering a compound of the present invention in a pharmaceutically effective amount to a subject suffering from or susceptible to a viral infection. Preferably, the viral infection is an HCV infection.

According to a further aspect of the present invention there is provided a method of treating an HCV infection in a subject by administering to said subject an effective amount of a compound of the present invention such that the HCV infection is treated.

Where a compound of the present invention is employed to treat a subject, the compound is suitably administered in a pharmaceutically effective amount. Such an amount is suitably in the range of from 10 mg to 1000 mg per day, more preferably from 50 to 500 mg per day, even more preferably, about 100 mg per day. The compound can be administered either in a single daily dose or in a number of doses throughout the day, e.g. between 2 to 5, suitably about 3 doses per day.

Preferably the compounds of the present invention are used to treat or to prevent a viral infection, such as an HCV infection, in homo sapiens.

The compounds of the present invention can be prepared by techniques known in the art.

The potential absence of any chiral centres in the molecule between the active part of a substitutent selected from R1, R2, R3, R4 and R5 and the active part of the moiety T, as defined above, means that the compound can be readily prepared with a single structural configuration with a suitable potential spacing in the range of from 10 nm to 14 ran, preferably from 11 nm to 13 nm, between the active part of moiety T and the active part of a substituent Rj, R2, R3, R4 or R5.

Embodiments of the present invention will now be described by way of example only.

Compounds set out in Table 1 below were prepared.

TABLE 1


Starting materials

(EVMethyl 5-(3-oxobut-l-envI)-lH-pyrrole-2-carboxylate and (E)-methyl 4-(3-oxobut-l-enyl)-lH-pyrroIe-2-carboxylate



To a stirring mixture of either of the methyl formyl-lH-pyrrole-2-carboxylate derivatives (0.01 mol), acetone (3.7 mL, 0.05 mol), water (50 mL) and amine (30 mol %) were added. The reaction was stirrred overnight at room temperature and on completion as monitored by TLC was extracted with (3x50 mL) dichloromethane. The organic layer was washed with water (2x 50 mL) and dried over anhydrous Na2SO4 and evaporated to obtain crude product. Column chromatography of the crude on silica gel using mixture of DCM/MeOH (99:1) as eluent gave pure products.(134)


Methyl 5-(3-oxobut-l-enyl)-lH-pyrroIe-2-carboxyIate;

1H NMR (DMSO-d6): δ 10.06 (br. s, IH), 7.37 (d, J = 16.3 Hz, IH), 6.85 (d, J = 3.95 Hz, IH), 6.58 (d, J 16.25 Hz, IH), 6.51 (d, J = 3.9 Hz, IH), 3.86 (s, 3H), 2.27 (s, 3H). 13C NMR (DMSO-d6) : δ 27.53 (CH3). δ 52.13 (CH3). δ 125.66, 132.69, 161.62, 197.76. (4C5 quaternary C). δ 114.47, 116.85, 125.05, 131.92. (4C, CH).

Methyl 4-(3-oxobut-l-enyl)-lH-pyrroIe-2-carboxylate 1H NMR (DMSO-d6): δ 9.56 (br. s, IH), 7.37 (d, J = 16.1 Hz, IH), 7.14 (m, IH), 7.04 (s, IH), 6.43 (d, J

16.05 Hz, IH), 3.81 (s, 3H), 2.25 (s, 3H).

13C NMR (DMSO-dό) : δ 27.27 (CH3). δ 52.82 (CH3). δ 122.20, 124.51, 161.22, 198.39. (4C, quaternary C). δ 112.99, 125.02, 136.51, 185.53. (4C, CH).

(E)-5-(3-oxobut-l-enyl)-lH-pyrrole-2-carboxylic acid and (E)-methyl 4-(3-oxobut-1 enyl)-lH-pyrrole-2-carboxylic acid



A mixture of the corresponding Methyl pyrrole-2-carboxylate ester (0.01 mol), KOH IN solution (9 mL), MeOH (30 niL) and THF (30 mL) was refluxed 15 hrs. The resulting solution was cooled to room temperature, water was added and pH was adjusted to pH 2 using IN HCl solution. This was extracted with ethyl acetate (3x 5OmL). the organic layer was washed with brine solution and dried over dry Na2SO4 then evaporated under vacuum. The crude product was purified on silica gel using increasing proportions of MeOH in DCM.



5-(3-oxobut-l-enyl)-lH-pyrroIe-2-carboxyIic acid;

1H NMR (DMSO-d6): δ 12.62 (br. s, IH), 12.21 (br. s, IH), 7.43 (d, J = 16.35 Hz, IH), 6.81 (d, J = 16.35 Hz, IH), 6.78 (m, IH), 6.69 (dd, J = 2.35, 3.75 Hz, IH), 2.25 (s, 3H). 13C NMR (DMSO-d6) : δ 27.28 (CH3). δ 126.56, 132.57, 161.47, 197.46. (4C, quaternary C). δ 113.67, 116.02, 124.92, 132.37. (4C, CH).

4-(3-oxobut-l-envI)-lH-pyrroIe-2-carboxyIic acid

1H NMR (PMSO-d6): δ 12.73 (br. s, IH), 12.11 (br. s, IH), 7.51 (d, J = 16.2 Hz, IH), 7.44 (dd, J = 1.6, 3.1 Hz, IH), 7.1 (m, IH), 6.49 (d, J = 16.2 Hz, IH), 2.24 (s, 3H). 13C NMR (DMSO-d6) : δ 26.86 (CH3). δ 120.94, 124.96, 161.56, 197.55. (4C, quaternary C). δ 112.82, 123.80, 126.69, 137.65. (4C, CH). Compounds 1 and 2 Compound 1 (E)-methyl 4-((5-(3-oxobut-l-enyI)-lH-pyrroIe-2-carboxamido) methyl) benzoate

Compound 2 (E)-methyl 4-((4-(3-oxobut-l-envI)-lH-pyrroIe-2-carboxamido)methyl)benzoate


To a solution of the corresponding oxobutenyl pyrrole-2-carboxylic acid derivative (0.179 gm, 0.001 mol) in dry dichloromethane (5 mL), 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (0.383 gm, 0.002 mol) and dimethylaminopyridine (DMAP) (0.244 gm, 0.002mol) were added. Methyl 4-(aminomethyl)benzoate ( 0.165 gm, 0.001 mol) in dry THF (5mL) was stirred for 3 min under N2 atmosphere then was added to the above mentioned DCM mixture. The resulting mixture was stirred at RT for 48 hr under N2. The resulting mixture was evaporated under reduced pressure. The crude product was chromatographed using ethyl acetate as eluent.


Methyl 4-((5-(3-oxobut-l-enyl)-lH-pyrrole-2-carboxamido)methyl) benzoate (compound 1): 1H NMR (DMSO-d6): δ 12.09 (br. s, IH), 8.90 (t, J = 6.05 Hz, IH), 7.94 (d, J = 8.3 Hz, 2H), 7.44 (m, 3H)5 6.93 (dd, J = 2.05, 3.7 Hz, IH), 6.75 (d, J = 16.3 Hz, IH), 6.69 (dd, J - 2.2, 3.6 Hz, IH), 4.54 (d, J = 5.95 Hz, 2H), 3.84 (s, 3H), 2.24 (s, 3H).

13C NMR (DMSO-d6):

6 27.17 (CH3).

5 41.78 (CH2). 5 52.05 (CH3).

5 128.08, 129.79, 131.14, 145.36, 160.08, 166.08, 197.45 (7C, quaternary C). δ 111.99, 113.76, 123.88, 127.34, 129.26, 132.79 (6C, CH).

Methyl 4-((4-(3-oxobut-l-enyl)-lH-pyrroIe-2-carboxamϊdo)methyI) benzoate (compound 2);

1H NMR (DMSO-d6): δ 12.00 (br. s, IH), 8.83 (t, J = 6 Hz, IH), 7.98 (d, 8.3 Hz, 2H), 7.58 (d, J - 16.1 Hz, IH),

7.49 (d, J = 8.35 Hz, 2H), 7.42 (dd, J = 1.6, 2.7 Hz, IH), 7.22 (br. s, IH), 6.37 (d, J =

16.1 Hz, IH), 4.54 (d, J = 6.05 Hz, 2H), 3.89 (s, 3H), 2.29 (s, 3H).

13C NMR (DMSO-d6): δ 26.74 (CH3).

5 41.76 (CH2). δ 52.0 (CH3). δ 120.47, 128.02, 128.12, 145.38, 160.26, 166.08, 197.39 (7C, quaternary C). δ 108.03, 123.16, 125.60, 127.27, 129.14, 138.19 (6C, CH).

Compound 3 (E)-4-((4-(3-oxobut-l-enyI)-lH-pyrrole-2-carboxamido)methyl) benzoic acid


A mixture of (E)-methyl 4-((4-(3-oxobut-l-enyl)-lH-pyrrole-2-carboxamido) methyl)benzoate ester (0.326 gm, 0.001 mol), KOH IN solution (5 mL), MeOH (30 mL) and THF (30 mL) was stirred overnight. The resulting solution was evaporated under reduced pressure, water was added and pH was adjusted to pH 2 using IN HCl solution.

This was extracted with ethyl acetate (3x 50 mL). the organic layer was washed with brine solution and dried over dry Na2SO4 then evaporated under vacuum. The crude product was purified on silica gel using increasing proportions of MeOH in DCM (0-3 %) to yield 0.3 gm (96.2 %) of compound 3.

1H NMR (DMSO-d6): δ 12.78 (br. s, IH), 11.95 (br. s, IH), 8.77 (t, J = 6.05 Hz, IH), 7.91 (d, J = 8.25 Hz, 2H), 7.55 (d, J = 16.15 Hz, IH), 7.43 (d, J = 8.3 Hz, 2H), 7.37 (dd, J = 1.4, 2.95 Hz, IH), 7.18 (t, J = 1.85 Hz, IH), 6.34 (d, J - 16.1 Hz, IH), 4.52 (d, J = 6 Hz, 2H), 2.25 (s, 3H).

13C NMR (DMSO-d6): δ 26.75 (CH3). δ 41.78 (CH2). δ 120.45, 128.05, 129.27, 144.85, 160.24, 167.14, 197.40 (7C, quaternary C). δ 108.01, 123.15, 125.59, 127.15, 129.29, 138.20 (6C, CH).

Compounds 4 and 5 4-((4-Iodobenzamido)methvDbenzoic acid and Methyl 4-((4-Iodobenzamido)methvl)benzoate

R= H, Me

A mixture of 4-iodobenzoyl chloride (2.65 gm, 0.01 mol) and the corresponding 4-aminomethyl benzoic acid derivative (0.01 mol) and triethylamine (1.4 mL) in dichloromethane (50 mL) was refluxed for 6 hr. The resulting reaction mixture was evaporated under vacuum, water (50 mL) was added where the crude product was precipitated out, fltered and dried. Purification of the crude product was carried out by column chromatography using silica gel and (DCM: EtOAc, 70:30) as eluent in case of the free carboxylic acid derivative, or (EtOAc:hexane, 60:40) in case of the ester counterpart.


4-((4-Iodobenzanudo)methyr)benzoic acid:

1H NMR (DMSO-d6): δ 12.90 (br. s, IH), 9.22 (t, J = 5.95 Hz, IH), 7.91 (d, J = 8.25 Hz, 2H), 7.87 (d, J = 8.55 Hz, 2H), 7.7 (d, J = 8.55 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 4.54 (d, J = 5.95 Hz, 2H).

13C NMR (DMSO-d6): δ 42.47 (CH2). δ 98.94, 129.24, 133.55, 144.61, 165.62, 167.12. (6C, quaternary C). δ 127.06, 129.29, 129.36, 130.26, 137.21, 137.55. (6C, aromatic CH).

Methyl 4-((4-iodobenzamido)methyl)benzoate:

1H NMR (DMSO-d6): δ 7.92 (d, J = 8.35 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.48 (br. s, IH), 4.6 (d, J = 5.9 Hz, 2H), 3.84 (s, 3H).

13C NMR (DMSO-d6):

5 43.78 (CH2). 5 52.15 (CH2). δ 98.71, 129.55, 133.51, 143.16, 166.68, 166.74. (6C, quaternary C). δ 127.63, 128.58, 130.10, 137.71, 137.89, 138.40. (6C, aromatic CH)

Compound 5 (E)-4-((4-(3-Oxobut-l-enyl)benzamido)inethyl)benzoic acid

Compound 4 (E)-methyl 4-((4-(3-oxobut-l-enyl)benzamido)methyl)benzoate


R= H, Me tetrabutyl amm acetate Pd acetate K2CO3/ KCl


To a stirred solution of the appropriate iodo derivative [viz; 4-((4-iodo benzamido)methyl)benzoic acid or methyl 4-((4-iodobenzamido)methyl) benzoate] (0.003 mol) in DMF (5 mL) were added methyl vinyl ketone (0.63 gm , 0.009 mol), tetrabutyl ammonium acetate (1.81 gm, 0.006 mol), potassium carbonate (0.93 gm, 0.0045 mol), potassium chloride (0.22 gm, 0.003 mol) and palladium II acetate (0.02 gm, 0.00009 mol). The mixture was stirred at 90 °C for 9 hr. After the mixture was cooled, 2N HCl was slowly added and the reaction mixture was stirred at room temperature for 10 min. then, the mixture was diluted with ether and washed with water. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo under reduced pressure. The residue was purified by column chromatography using (MeOH in EtOAc in increasing proportions up to 1%) as eluent in case of the free carboxylic acid derivative and (EtOAc:DCM 30:70) as eluent in case of the ester derivative.


4-((4-(3-Oxobut-l-enyI) benzamido)methvDbenzoic acid (compound 5):

1H NMR (PMSO-d6): δ 9.19 (t, J = 5.95 Hz, IH), 7.95 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.25 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 16.4 Hz, IH), 7.44 (d, J = 8.25 Hz, 2H), 6.91 (d, J = 16.4 Hz,

IH), 4.56 (d, J = 5.95 Hz, 2H), 2.36 (s, 3H).

13C NMR (DMSO-dό): δ 27.43 (CH3). δ 42.49 (CH2). δ 128.46, 130.32, 135.21, 137.20, 166.5, 189.01, 198.31. (1C, quaternary C). δ 127.17, 127.81, 128.29, 128.65, 129.37, 141.91. (6C, aromatic CH).

methyl 4-((4-(3-oxobut-l-enyl) benzamido)methvDbenzoate (compound 4):

1H NMR (DMSO-d6): δ 9.21 (t, J - 5.9 Hz, IH), 7.95 (m, 3H), 7.82 (d, J = 8.35 Hz, 2H), 7.66 (d, J = 16.35 Hz, IH), 7.46 (d, J = 8.3 Hz, IH), 6.90 (d, J = 16.4 Hz, IH), 4.56 (d, J = 5.95 Hz, 2H), 3.84 (s, 3H), 2.36 (s, 3H). 13C NMR (DMSO-d6): 5 27.42 (CH3). δ 42.47 (CH2). δ 52.03 (CH3). δ 128.38, 135.31, 137.22, 145.14, 165.74, 166.11, 198.21. (7C, quaternary C). δ 127.35, 127.81, 128.13, 128.30, 128.64, 129.09, 129.23, 129.74, 141.73, 141.91. (1OC, CH).

Compounds 6 and7 Compound 6 Methyl-4-((4-mercaptobenzamido)methyl) benzoate


F.W .= 154.18634 F.W.= 306.3568

The 4-mercaptobenzoic acid (0.53 g; 3.4 mmol) and Iodine (0.44g; 1.7 mmol) were dissolved in 10 ml of ethanol. Triethylamine (1.5ml; 10.2 mmol) was added and the solution was stirred overnight (16h).

The excess of iodine was removed with the addiction of a 10% solution of sodium thiosulfate . The cloudy solution was concentrated and combined with HCl 0,01 M till the pH became acid.

The pallid yellow precipitate was collected, dried under vacuum and washed with petroleum ether.

This step did not need further purification.

YIELD: 80%

1H NMR (DMSO) δ: 13,04 (bs, 2H, COOH); 7,94 (d, 4H, J = 8.2 Hz, H2); 7,65 (d, 4H, J = 8.2 Hz, H3);


I)SOCl2

2)DMA, TEA, DMAP, methyl(4-aminomethyl)benzoate


F.W. = 600.70452

0.345 g of dithiobis benzoic acid (1.126 mmol) was refluxed at 80°C in excess of thionyl chloride (3 ml) for 4 h under nitrogen atmosphere. When the reaction was finished (checked by TLC) the solvent was evaporated, 10ml of toluene were added and the solvent was evaporated again. The resulted oil was combined with the methyl(4-aminomethyl)benzoate ( 0.453g; 2.25 mol) in 15 ml of N5N' Dimethylacetamide and mixed with DMAP (3.5 mg) and Et3N ( 0,35 ml). The reaction proceeded overnight under nitrogen atmosphere. Quenching with water (~50 ml) produced an off-white precipitate. The flask cooled in ice bath for 2 h and the solid formed was filtered under vacuum and washed with hot methanol, a saturated solution of NaHCO3, hot water and finally dried under vacuum. The residue was recrystallised with MeOH. Melting Point: 219-220°C TLC: Chloroform:MeOH (9:1)

YIELD: 50%

1R NMR (DMSO) δ: 9,19 (t, 2H, J=6.00 Hz, NH); 7,97 (d, 4H, J = 8.2 Hz, H2'); 7,96 (d, 2H, J = 8.5 Hz, H2); 7,70 (d, 4H, J=8.5 Hz, H3); 7,49 (d, 4H, J = 8.2 Hz, H3'); 4,59 (d, 4H, J=6.00 Hz, CH2); 3,89(s, 6H, OCH3);


F.W.= 301.3602 (compound 6)

The compound obtained from the previous reaction (0,200 g; 0,33 mmol) was dissolved in 10 ml of DMF and DTT (62 mg; 0.4 mmol) was added. The mixture was stirred at room temperature under nitrogen atmosphere overnight.

The solvent was evaporated and the yellow solid was purified by silica flash column chromatography Chloroform/MeOH (99:1) YIELD:70% Compound 6

1H NMR (CDCh) δ: 7,91 (d, 2H3 J=8.2 Hz, H2.); 7,58 (d, 2H, J = 8.3 Hz, H2); 7,29 (d,

2H, J = 8.2 Hz, H3-); 7,19 (d, 2H, J=8.3 Hz, H3); 6,57 (t, IH, J = 5.3 Hz, NH); 4,58 (d,

2H, J=5.3 Hz, CH2);

3,82(s, 3H, OCH3 ); 3,50 (s, IH, SH)

13C NMR (CDCh) δ: 166.79, 166.77, 143.45, 136.58, 131.04, 130.04, 129.42, 128.58,

127.74, 127.58, 52.13, 43.68

Compound 7 10 4-((4-mercaptobenzaniido)-methyl) benzoic acid


F.W.: 301,36 (compound 6) F.W.: 287,33 (compound 7)

15 Methyl-4-((4mercaptobenzamido)-methyl) benzoate (compound 6) (71mg,; 0.235 mmol) was hydrolized with LiOH IM (0,517 ml; 0,517 mmol) in a mixture of water (0,1 ml) and THF (3 ml) .When the reaction was finished (monitorated by TLC) the THF was evaporated and the residual water solution was adjusted at pH 2 with HCL IM. The resulting white precipitate was filtered and washed with water to give the desiderate

20 compound, which did not required further purification. TLC: Chloroform/MeOH ( 98:2) YIELD:77%

Compound 7

25 1H NMR (DMSO) δ: 12,91 (BS, IH, COOH); 9,08 (t, IH, J=5.7 Hz, NH); 7,96 (d, 2H,

J=8.3 Hz, H2'); 7,83 (d, 2H, J = 8.3 Hz, H2); 7,47 (d, 2H, J = 8.3 Hz, H3.); 7,44 (d, 2H,

J=8.3 Hz, H3); 5,79 (s, IH, SH) 4,58 (d, 2H, J=5.7 Hz, CH2);

13C NMR (DMSO) δ: 167.13, 165.50, 144.63, 139.01, 133.18, 129.35, 129.26, 128.35,

127.13, 126.38, 42.44 30

Experimental data Compounds 1 to 9 were assessed for their anti HCV activity using the following assay.

A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated and the other one DIG-labelled, at their respective 5' termini. This DNA duplex was immobilised, via the biotin molecule, on the surface of a neutravidin-coated 96 well plate. Helicase will initiate its unwinding activity upon activation with ATP, leading eventually in the release of the DIG labelled oligonucleotides, which translates in signal (luminescence) reduction with respect to control wells. This signal can be produced and quantified with the aid of a chemiluminescence antibody.

Other assay methods for this type of enzyme may analyse ATPase activity but assessment of the effects on helicase unwinding activity has been identified as the best way to evaluate inhibitors of this class of enzyme (1). Helicase activity assays rely on the ability of the enzyme to displace one strand (release strand) of DNA or RNA from another (template) strand. Many reports use displacement of radio-labelled release strands, detected either after gel electrophoresis (eg (2)), thin layer chromatography (eg (3)) or scintillation counting (eg (4)). The latter method may be amenable to high-throughput screening but carries the practical disadvantages of the use of radioactive materials. Further methods have incorporated DIG labelled release strands to allow detection by ELISA (5). We propose a new combined method that uses chemiluminescent antibody detection of residual release strand to give a robust helicase assay, that does not employ radiolabeled compounds and gives a stable read-out that is highly suited to high-throughput screening.

As proof of concept, the HCV helicase was expressed and isolated using recombinant protein techniques and then used in our assay. We found this assay to be highly reproducible since only slight variation was observed when a total of 96 helicase reactions (including controls) were performed on one plate. Therefore, our suggested rapid helicase assay is fast, convenient and highly reproducible while obviating the need to employ radiochemicals. These criteria make it suitable for high throughput screening of potential helicase inhibitors.

METHOD: MATERIALS: Oligonucleotide 1:

5 '-biotin-GCTGACCCTGCTCCCAATCGTAATCTATAGTGTCACCTA-3 ' Oligonucleotide 2:

5 '-DIG-CGATTGGG AGCAGGGTC AGC-3 ' Purified helicase Neutravidin - (Pierce Protein Research Products)

CSPD - luminescence kit (Pierce Protein Research Products) 96 well plates

EQUIPMENT: Incubator (Bioline shacking floor incubator)

Luminometer (luci II, Anthos Labtec) PCR block

PROCEDURE:

1) Setup the annealing reaction

Oligonucleotides 1 :1 Molar ratio

HEPES 2 mM

NaCl 0.05 M

EDTA 0.1 mM

SDS 0.01% (w/v)

Prepare for annealing by heating the oligonucleotide mix at 1000C for 5 min. Incubate at 65°C for 30 min. Incubate at 220C for 4 h to allow gradual annealing. Store the annealed NS3 helicase substrate at -200C

2) Neutravidin Coating of the 96-weIl plates

Prepare a stock solution of neutravidin at a final concentration of 1 mg/ml in phosphate buffered saline (PBS) Each of the 96 wells was coated overnight at 4°C with 100 μl/well of a 5 μg/ml neutravidin solution in a 0.5 M sodium carbonate buffer pH 9.3. Plates were then washed three times with lOOμl/well of PBS and air-dried at room temperature.

3) Blocking with BSA

Wells were subsequently blocked upon addition of 100 μl of a 0.1% (w/v) BSA solution followed by an incubation at 22°C for 2 h. Plates were then washed three times with 200 μl/well of PBS, air-dried at room temperature and stored at 4°C with desiccant.

4) Substrate Application in the 96-well plate

For standard assays, mix 75 μl of 1 M phosphate buffer (PBS) pH 7.0, containing 1 M NaCl with 2.5 ng of the partially annealed DNA duplex for each well. Then incubate at 22°C for 4 h. Finally, wash each well twice with 200 μl PBS and once with 200 μl of 50 mM Tris HCl pH 7.5, 50 mM NaCl. All solutions were pre-warmed to 370C.

5) Helicase Reaction

Helicase reactions were initiated upon addition of 90 μl of a reaction mix consisting of 11 nM of purified full-length HCV NS3 protein, 25 mM 4-morpholine-propanesulphonic acid (MOPS) pH 7.0, 5 mM ATP, 2 mM DTT, 3 mM MnCl2, and 100 μg/ml of BSA to the wells in which 2.5 ng of DNA substrate was previously applied. For negative controls, the reaction mix contained no ATP. Reactions were carried out for 60 min at 37°C. Wells were then washed twice with 200 μl of 150 mM NaCl and dried at room temperature for 15 min.

6) Activity determination - chemiluminescence preparation

The wells of the multi-well plate were washed for 5 min with detection washing buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5, 0.3% Tween20). Then each well was filled up with Blocking Solution (10% BSA (w/v) 0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for 30 min followed by a 30 min incubation in 20 μl Antibody solution (anti-Dig, Roche, 1:10,000 solution of the antibody -75 mU/ml- in Blocking solution). The wells were washed twice with 100 μl of detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5). Then 20 μl of detection buffer was applied for equilibration. 1 μl of chemiluminescence substrate (CSPD - 0.25 mM) working solution was applied to each well and the plates were incubated for 5 min at 170C. The wells were drained and incubated at 370C for 30 min, in order to dry the wells completely. The luminescence continues for almost 48 hours with a constant-intensity phase lasting for the first 24 hours. Remaining DIG label in each well of the 96 well-plate was counted for 10 min against controls (one with no protein and one with no ATP) in a luminescence multi-well plate reader.

CRITICAL STEPS:

Step 1: Choice of oligonucleotides. The oligonucleotides used here are those described by Hicham Alaoui-Ismaili et at. (4), modified by DIG labelling of the release strand. Other sequences are possible but a 3 '-single stranded region in the substrate appears to be required to initiate strand displacement (6).

Step 3: During the blocking step it is important to ensure that all potential binding sites are occupied, to prevent direct binding of the detection antibody to the well in step 6. The wells should be filled with blocking solution to achieve full coating of the plate. Step 4: Pre-warming the solutions allows reactions to proceed at their optimum temperatures and avoids rate changes due to temperature equilibration.

Step 6: Reaction wells should be completely filled up with Blocking Solution in order to ensure that the whole well has been blocked, thus preventing non-specific binding of any components of the detection system.

References:

1. Borowski, P., Niebuhr, A., Schmitz, H., Hosmane, R. S., Bretner, M., Siwecka, M. A. & Kulikowski, T. (2002) Acta Biochim Pol 49, 597-614.

2. Borowski, P., Niebuhr, A., Mueller, O., Bretner, M., Felczak, K., Kulikowski, T. & Schmitz, H. (2001) J Virol 75, 3220-3229.

3. Bartelma, G. & Padmanabhan, R. (2002) Virology 299, 122-132.

4. Hicham Alaoui-Ismaili, M., Gervais, C, Brunette, S., Gouin, G., Hamel, M., Rando, R. F. & Bedard, J. (2000) Antiviral Res. 46, 181-193.

5. Hsu, C. C, Hwang, L. H., Huang, Y. W., Chi, W. K., Chu, Y. D. & Chen, D. S. (1998) Biochem Biophys Res Commun 253, 594-599.

6. Tai, C. L., Chi, W. K., Chen, D. S. & Hwang, L. H. (1996) J Virol 70, 8477- 8484.

The compounds 1-9 were biologically evaluated using the above enzymatic assay against the HCV helicase. The measured IC50 is reported in TABLE 2.

TABLE 2

Cpd. IC50

1 26O nM

2 777 mM

3 956 μM

4 >1M

5 1O mM

6 H mM

7 365 nM

8 5 mM

9 8 mM

As can be seen from the data reported in Table 2 compounds 1 and 7 showed exceptionally high potency in the helicase assay with respect to HCV.
 
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