AU626983B2 - N6-substituted 9-methyladenines - Google Patents

Description

n OPI DATE 05/09/90 PCT AOJP DATE 11/10/90 APPLN. ID 49414 PCT NUMBER PCT/US90/00210 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/09178 A61K 31/52, C07D 473/34 Al C07D 473/18 (43) International Publication Date: 23 August 1990 (23.08.90) (21) International Application Number: PCT/US90/00210 (81) Designated States: AT, AT (European patent), AU, BB, BE (European patent), BF (OAPI patent), BG, BJ (OAPI (22) International Filing Date: 16 January 1990 (16.01.90) patent), BR, CA, CF (OAPI patent), CG (OAPI patent), CH (European patent), CM (OAPI patent), DE (European patent), DK (European patent), ES (European pa- Priority data: tent), FI, FR (European patent), GA (OAPI patent), GB 304,346 31 January 1989 (31.01.89) US (European patent), HU, IT (European patent), JP, KP, KR, LK, LU (European patent), MC, MG, ML (OAPI 1i-. 1 patent), MR (OAPI patent), MW, NL (European pa- (71) Applicant: WHI-TBY-RESEARGH-I-NGORPORATED tent), NO, RO, SD, SE (European patent), SN (OAPI [US/US]; 1001 Health Sciences Road West, Irvine, CA patent), SU, TD (OAPI patent), TG (OAPI patent).

92715 (US).

(72) Inventors: OLSSON, Ray, A. 5114 W. Cleveland Street, Published Tampa, FL 33609 MARCUS, Ted 3781 Magno- With international search report.

lia, Irvine, CA 92714 Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of (74) Agent: BARAN, Robert, Whitby Research, Inc., 1001 amendments.

Health Scir.es Road West, Irvine, CA 92715 (US).

/2.1 626983 (54)Title: N 6 -SUBSTITUTED 9-METHYLADENINES: A NEW CLASS OF ADENOSINE RECEPTOR ANTAGON-

ISTS

(57) Abstract A serieq of N 6 -substituted adenines are disclosed to be antagonists of A 2 -adenosine receptor-mediated stimulation of adenylate cyclase in A 2 -adenosine receptors and antagonists of A 1 -adenosine receptor-mediated inhibition of adenylate cyclase. These compounds are useful in reversal of adenosine-mediated lipolysis, reversal of aenosine-mediated deleterious cardiovascular effects (conduction defects, hypotension), reversal of adenosine-mediated vascular actions in kidney, bronchodilation, antiarrhythmic action, reversal of adeno-mediated relaxation of smooth muscle, anti-narcoleptic action, CNS stimulation, and blockade of adenosine mediated inhibition of neurotransmitter release.

WO 90/09178 PCT/US90/00210 -1-

N

6 -SUBSTITUTED 9-METHYLADENINES: A NEW CLASS OF ADENOSINE RECEPTOR ANTAGONISTS SUMMARY OF THE INVENTION Novel compounds and a method of using them to antagonize adenosine receptors are provided wherein the compounds are represented by the general formula:

I

RR

wherein R 2 is selected from the group consisting of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13, preferably 6 to carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to 10, carbon atoms, and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen; R 1 may be hydrogen or R 2 and R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl-substituted amine, cycloalkyl-substituted amine, alkyl radicals having from 1 to 10 carbon atoms, and cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms. R 4 is selected from the group consisting of benzyl, phenyl, and WO 90/09178 PCT/US90/00210 -2alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for example ethers and alcohols. R 5 is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with alkyl, cycloalkyl, or phenyl.

BACKGROUND OF THE INVENTION This application is a continuation-in-part of U.S. patent application Serial No. 042,383, filed April 23, 1987 entitled "N 6 -Substituted 9-Methyladenines: A New Class of Adenosine Receptor Antagonists," which is incorporated herein by reference in its entirety.

Adenosine receptors have been divided into two subtypes, based on adenylate cyclase activity: A 1 (Ri) receptors mediate inhibition and A 2 (Ra) receptors mediate stimulation of adenylate cyclase activity. Some N 6 -substituted adenosine analogs, like N 6 -u-phenyl isopropyl adenosine (R-PIA) have very high affinity for A 1 adenosine receptors, but at

A

2 receptors 5'-N-ethylcarboxamido-adenosine (NECA) is more potent than N 6 -substituted analogs. Alkylxanthines, such as caffeine and theophylline, are the best known antagonists at adenosine receptors.

Adenine was generally believed to have no effect on adenosine receptor-controlled systems. However, it was found that at low concentrations adenine displays specific competitive antagonism of adenosine-induced cyclic Amp accumulation in a human fibroblast cell line. Methylation of adenine at the 9-position increases potency about 4-fold i u:~ L. WO 90/09178 PCT/US90/00210 -3in this assay. At higher concentration, both compounds show non-specific inhibitory activity.

DETAILED DESCRIPTION OF THE INVENTION The compounds of this invention are represented by the general formula: RNR2

R

5 N

N

R

4 wherein R 2 is selected from the group consisting of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13, preferably 6 to carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to' 10, carbon atoms, and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen; R 1 may be hydrogen or R 2 and R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, alkyl radicals having 1 to 10 carbon atoms, cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms, thio, sulfonate, sulfonamide, sulfon, sulfoxamide, phenyl, alkyl-substitued amine, and cycloalkyl substituted amine. R 4 is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for instance ethers and alcohols. R 5 is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein WO 90/09178 Pcr/Us90/ooz10o -4said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with phenyl and alkyl and cycloalkyl groups comprising 1 to 6 carbon atoms.

The preferred compounds are those wherein R, is hydrogen; wherin 2 is endo-2-Norbornyl or cyclopentyl; wherein R 3 is bromine, chlorine, amino, hydrogen, thio, cyclopentyl or cyclopentylamine; wherein R 4 is methyl, ethyl, 2-hydroxyethyl, phenyl, or 2-hydroxyethoxy methyl; and wherein R 5 is hydrogen, hydroxy or chlorine.

The following is a list of compounds useful in the practice of the present invention. This list is intended to be illustrative and the scope of the invention is not limited to compounds named therein:

N

6 -Cyclobutyl-9-Methyl Adenine (MA)

N

6 -Cyclopentyl-9

-MA

N

6 -Methylcyclopentyl-9 -MA(

N

6 -Cyclohexyl-9-MA

N

6 -Methyl-9-MA

N

6 -3-Pentyl-9-MA

N

6 -Phenyl-9-MA

N

6 -2 -Fluorophenyl-9-MA

N

6 -Benzyl-9-MA

N

6 -2-Phenethyl-9-MA

N

6 5-Trimethoxyphenyl) ethyl-9-MA

N

6 (3-Pyridylethyl) -9-MA

N

6 (3-Thienylethyl) -9-MA

N

6 -R-1-Phenyl-2-propyl-9-MA

N

6 1-Phenyl- 2-propyl-9-MA

N

6 (tndo-2-Norborny1) -9-MA

N

6 (2-Thienyl) -2-butyl-9-MA WO090/09178 PCIP/US90/00210

N

6 (exo-2-Norbornyl) -9-MA

N

6 2-diphenylethyl-9-MA

N

6 -2 -phenylethyl-9-MA

N

6 (2-chlorophenyl) ethyl-9-MA

N

6 1-indanyl-9-MA

N

6 -2-ami aoethyl-9-14%

N

6 (N,1',-Dimethylam-Lnoethyl) -9-MA

N

6 -R-1-phenyl-1-ethyl-9-MA

N

6 -S-1-phenyl-l'-ethyl-9-MA

N

6 -2-th~ienyIl=9-MA N6-0(-chloro-2-methyl phenyl) -9-MA,

N

6 2- (3-ethylindole) -9-MA

N

6 (l--methyl1- 2-pheny 1ethyl) -9-MA

N

6 (l-methyl-2-phenoxyethyl) -9-MA

N

6 -1-carboxy-1-butyl-9-MA

N

6 (endo-2-norbornyl) -2-chloro-9-MA

N

6 (endo-2-norbornyl) -8-cyclopentyl-9-MA

N

6 (endo-2-norbornyl) -8-hydroxy-9-MA

N

6 (endo-2-norbornyl) -8-brono-9-MA

N

6 (endo-2-norbornyl) -8-amino-9-MAI

N

6 (endo-2-norbornyl) -8-carboxy-9-MA

N

6 -cyclopentyl-8-cyclopentyl-9-MA

N

6 (endo-2-norbornyl) (2-hydroxyethoxy) methyl] adenine

N

6 (endo-2-nk-rbornyl) -8-thio-9-MA N 6 (endo-2-norbornyl) -8-chloro-9-MA

N

6 -(endo-2-norbornyl) -8-sulfonate-9-MA sodium salt

N

6 (endo-2 -norbornyl) -2 -hydroxy-9-MA

N

6 (endo-2-norbornyl) -8-cyclopentylamine-9-MA

N

6 (endo-2-norbornyl) -8-propy).amine-9-MA

N

6 -(endo-2-norborpyl)-9-phenyl adenine

N

6 -cyclopentyl-2-~Chloro-9 -MA

N

6 -phenyl-2 -chloro-9-MA

N

6 -cyclopentyl-9-phenyl adenine

N

6 -R-1-phenyl-2-propyl-9-phenyl adeninej WO 90/09178 WO 9009178PCF/US90/00210 -6-

N

6 -S-1-phenyl-2-propyl-9-phenyl adenine

N

6 (3-chloro-endo-2-florbornyl) ]-9-MA

N

6 -phenyl-9-phenyl adenine 2 -ethoxy-9 -MA 2 -propoxy- 9-MA 2 -butoxy-9 -MA 2-isopropoxy-9-MA 2- (2-butoxy) -9-MA 2-(2-methy. propoxy) 9-MA 2 -pentoxy- 9-MA 2-(2-phenylethoxy) 9-MA 2 -phenyl amino- 9-MA 9 -hydroxyethyl1adenine

N

6 -cyclopentyl-9-benzyl adenine

N

6 -cyclohexyl-9 -ethyl adenine The preparation o~f 9-methyl adenines is well known.

See R. K. Robins, K. J. Dille, and B. E. Christensen, J.

Org. Chem., 19, 930 (1954) R. K. Robins and H. H. Lin, J.

An. Chem. Soc., 79, 490 (1957; and J. A. Montgomery and Carroll Temple, Jr., J. Am. Chem. Soc., 79, 5238 (1957).

Preparation of N 6 -Cyclopentyl-9-Methyl Adenine To prepare N 6 -'cyclopentyl-9-methyl Adenine the following additional steps were taken. A mixture of 6chloro-9-methyl Adenine (0.82g), cyclopentylamine (0.52 ml) triethylamine (0.53 ml) and ethanol (60 mil), was ref luxed for 24 hours. The solution was concentrated in vacuo to a yellow syrup. The syrup was passed through a C-18 column to give 0.78g or 74% yield of with 108-109 0 C. 1

HNMR-

(He 2 SO-d6): 61-2(m,9 3.7(S,Cfl 3 7.6(d,NI); 8.l(S,lH); 8.2(S,lH).

Preparation of N 6 -3-Peftyl-9-Methyladenine WO 90/09178 PCT/US90/00210 -7- A mixture of 6-chloro-9-methyladenine 3-pentylamine (1.3 ml), triethylamine (1.3ml) and ethanol ml), was refluxed for 24 hours. The solution was concentrated and passed through a C-18 column to give a white solid having m.p. 107-109 0

C.

Preparation of N 6 -(2-Aminonorbornyl)-9-methyl Adenine A mixture of 1.5g 6-chloro-9-methyl Adenine, 1.75 g endo- 2-aminonor-bornane, 2.9 ml triethylamine and 60 ml ethanol was refluxed overnight. The solution was then concentrated in vacuo and the remainder was passed through C-18 prepchromatography to give 1.6g (75% yield) m.p. 130-131 0

C.

1 HNMR(Me 2 SO-d6): 61-2.6(m,10 3.8(S, CH 3 4.1(m,lH); 7.2(S,NH); 7.4(S,1H); 7.6(S,1H).

Preparation of N 6 -(endo-2-Norbornyl)-8-Bromo-9-MA To a stirred suspension of N 6 -(endo-2-norbornyl)-9-MA (6g, 24.66 mmoles) in 150 ml of 1M sodium acetate buffer (pH 3.9) was added a solution of bromine (3.0 ml) in 300 ml of 1M sodium acetate buffer (pH The mixture was stirred overnight and the resulting precipitate was filtered and washed with water. To the residue was added silica gel and the suspension was evaporated to a powder. The powder was added to a silica gel column (150g, packed with petroleum ether). The purine was eluted with 10% to 25% ethylacetate in petroleum ether. Evaporation of the appropriate fractions gave 6.7g, 84% yield of N 6 -(endo-2-Norbornyl)-8- Bromo-9-MA.

Preparation of N 6 -(endo-2-Norbornyl)-8-Azido-9-MA To a solution of N 6 -(endo-2-Norbornyl)-9-Bromo-9-MA (0.72g, 2.23 mmoles) in DMF was added sodium azide (0.91g, 13.98 mmoles). The mixture was heated at 70-80 0 C overnight.

The crude was dissolved in water, extracted with ethyl L I WO 90/09178 WO 9009178PCF/US90/00210 -8acetate, and then dried over magnesium sulf ate and the organic phase was evaporated in vacuo to give 0. 62g, 98% yield.

Preparation of N 6 (endo-2-Norbornyl) -8-Aniino-9-MA The crude product, N 6 (endo-2-Norbornyl) -8-Azido-9-MA 1.75 mmole) was dissolved in ethanol. The solution, in presence of 10% palladium charcoal was shaken with H 2 at 35 atm overnight. The suspension was filtered and evaporated to a small volume, and then poured through a C-18 column (HPLC) to give 0.36g 80% yield of N 6 -(endo-2norbornyl) -8-Amino-9-MA.

Preparation of N 6 (endo-2-Norbornyl) -8-Oxo-9-MA To a mixture of N 6 (endo-2-Norbornyl)-9-Bromo-9-MA (0.15g, 0.62 mmole) in 12 ml acetic acid was added sodium acetate and 1.2 ml acetic anhydride. The mixture was allowed to ref lux overnight. The mixture was then evaporated under vacuc and purified on a chromatotron using CHCl 3 stepping to. 2% ethanol, and finally to 4% ethanol on 2 mm plate giving 90 mg, 75% yield of N 6 -(endo-2-Norbornyl)-8-oxo-9-MA.

Preparation of N 6 (endo-2-Norbornyl) -8-Cyclopentylamine-9-MA~ To a solution of N 6 -(endo-2-Norbornyl)-8-Bromo-9-.MA 1.55 mmols) in 20 ml ethanol was added 20rn.1 of cyclopentylamine; the reaction mixture was ref luxed overnight. The mixture was then evaporated under vacuo and passed through a C-18 column (HPLC) to~give 0.32g, 77% yield of N 6 (endo-2-Norbornyl) -8-Cyclopentylamine-9-Mij.

Preparation of N 6 (endo-2-Norbornyl) 8-Bromo-2-Chloro-9-MA

N

6 (endo-2-Norbornyl)2-Chloropurine was first prepared as follows: A mixture of 2,6-dichioropurine (5.0g, 26.45 umoles) endo-2-aminobornane hydrochloride (5.0g, 33.86 WO 90/09178 pcr/US90/00210 -9mmoles) and triethyl amine (10 ml) in absolute ethanol was refluxed for 48 hours. The solution was then cooled to room temperature and evaporated in vacuo to a white solid. The white solid was washed with water and dried to yield 84% yield of N 6 -(endo-2-Norbornyl)2-Chloropurine used as is with no further purification for next step.

A mixture of N 6 -(endo-2-Norbornyl)-2-chloropurine 18.96 mmoles), triethyl ammonium hydroxide (18.9 ml), and methyl iodide (1.41 ml, 22.68 mmoles) in dichloromethane was heated to 35 0 C for 24 hours. The solution was then evaporated in vacuo and the syrup was crystallized in methanol to give 4.0g, 76% yield of N 6 -(endo-2-Norbornyl)2chloro-9-MA.

To a stirred solution of N 6 -(endo-2-Norbornyl)-2-chloro- 9-MA (4.3g, 14.4 mmoles) in acetate buffer (1 molar acetic acid and 1 M sodium acetate mixture, 45:1 ratio respectively; pH 3.9) was added dropwise Bromine (3.12g, 19.56 mmoles) dissolved in the acetate buffer. The reaction mixture was stirred for 72 hours; the mixture was then filtered and the solid material collected was eluted from ethyl acetate/petroleum ether on silica gel column to yield 4.9g, 85% of N 6 -(endo-2-Norbornyl)8-Bromo-2-Chloro-9-MA.

Preparation of N 6 -(endo-2-Norbornyl)-8-Cyclopentyl-9-MA To a vigorously stirred solution of 2g (12.2 mmoles) of 4-methylamino-5-amino-6-chloropyrimidine in CHCl 3 was added dropwise over a period of 20 minutes cyclopentane carbonyl chloride (1.6g, 12.2 mmoles). The mixture was stirred overnight and then evaporated in vacuo to a yellow syrup.

The syrup was then dissolved in methanol and purified through a C-18 column (HPLC) to give 2.2g, 71% yield of 4 WO 90/09178 PC/US90/00210 4-methylamino-6-chloro-5-cyclopentylamido-pyrimidine 2.2g, 8.6 mmoles) was refluxed i POCl 3 for approximately 2 hours. The solution was concentrated in vacuo to a syrup.

The syrup was added dropwise to ice. The aqueous mixture was then extracted with chloroform. The organic layer was evaporated and the syrup was passed through a C-18 column (HPLC) giving 1.25g, 63% yield of 8-cyclopentyl-6-chloro-9methyladenine.

A mixture of 8-cyclopentyl-6-chloro-9-methyladenine (0.48g 2.0 mmoles) and endo-2-aminonorbornane hydrochloride 3.4 mmoles) in absolute ethanol was refluxed for 48 hours. The mixture was then evapo-sted in vacuo and purified through a C-18 column (HPLC) to give 0.45g, 71% yield of N 6 -(endo-2-Norbornyl) -8-cyclopentyl-9-MA.

Preparation of N 6 -(endo-2-Norbornyl)-8-Chloro-9-MA A mixture of N 6 -(endo-2-Norbornyl) -8-bromo-9-M,, (1.25g, 3.7 mmoles) and POC1 3 was refluxed for 1 hour. Then the phosphorous oxychloride was removed in vacuo and the yellow solid was passed through a C-18 column (HPLC) to give 0.96g, 84% yield of N 6 -(endo-2-Norbornyl)-8-chloro-9-MA.

Preparation of N 6 -(endo-2-Norbornyl)-9-[(2-hydroxyethoxy) methyl)purine.

To a solution of 6-chloropurine (6g, 38.8 mmoles) in DMF was added sodium hydride 60% (0.93g) over 1.5 hour period.

(2-acetoxyethoxy)methyl bromide was then added at room temperature; the reaction mixture was allowed to stir for 2 hours under N 2 atmosphere. H 2 0 was added and the product was extracted with ethyl acetate. The organic phase was dried over MgSO4, filtered, and evaporated in vacuo to give a light yellow solid 7.1g, 68% yield of 9 -[(2-Acetoxy-

L

WO 90/09178 PCT/US90/00210 -11- -llethoxy)methyl]-6-chloro-purine. The crude was used without further purification.

To a solution of 9-[(2-acetoxyethoxy)methyl]-6-chloropurine (5.1g, 18.8 mmoles) in ethanol and triethylamine was added endo-2-aminonorbornane hydrochloride (4.0g, 27.1 mmoles). The mixture was refluxed in vacuo and the residue was purified by HPLC to give 4.70g, 77% yield of N 6 -(endo-2- Norbornyl)-9-[(2-acetoxyethoxy)methyl]purine.

A solution of N 6 -(endo-2-Norbornyl)-9-[(2-acetoxyethoxy) methyl]purine (3.75g, 10.8 mmoles) in methanol was saturated with NH 3 gas under N 2 The mixture was stirred overnight, then evaporated in vacuo to give 2.03g, 62% yield of

N

6 -(endo-2-Norbornyl)-9-[(2-hydroxyethoxy)methyl]'purine.

The invention is further illustrated by the following examples which are illustrative of various aspects of the invention. These examples are not intended as limiting the scope of the invention as defined by the appended claims.

PHARMACOLOGIC TESTING A series of N 6 -substituted 9-methyladenines were assayed as adenosine antagonists in A 1 and A 2 test systems (Ukena, et al, FEBS Lett. 215(2), 203-208, 1987). For activity at

A

1 receptors, compounds were tested as inhibitors of the binding of N 6 3 H]-Phenylisopropyladensine in rat brain membranes and for their ability to prevent R-PIA-induced inhibition of adenylate cyclase in rat fat cell membranes.

For activity at A 2 receptors, compounds were tested as antagonists of NECA-stimulated adenylate cyclase in membranes of human platelets and rat PC12 cells.

WO 90/09178 PCffUS90/00210 -12- It is known that A 1 receptors influence inhibition of adenylate cyclase in fat, brain and heart cells; whereas

A

2 receptors stimulate adenylate cyclase in endothelial and smooth muscle cells. (See John W. Daly, et al., "Structure- Activity Relationship for N 6 -Substituted Adenosines at a Brain Al-Adenosine Receptor With A Comparison to an A 2 Adenosine Receptor Regulating Coronary Blood Flow," Biochemical Pharmacology, Vol. 35. No. 15, pp. 2467-2471 (1986)).

The results summarized below in Table I show that N 6 substitution can markedly increase the potency of 9methyladenine at adenosine receptors. The lower apparent affinity values (KB, Ki) identify the most potent compounds.

The most pronounced effect is seen at Al receptors. For example, N 6 -Cyclopentyl-9-methyladenine is at least 100-fold more potent than 9-methyladenine at A 1 receptors. At A 2 receptors, this compound is 5-fold more potent than 9methyladenine in the human platelet assay. Thus, this data demonstrates the activity of a novel series of adenosine antagonists.

T ABL E 1 A 2 E ffe c ts A 1 E ffe ct s KBI(sM) vs NECA Stimulation KBOLM) Kf($%M) (Adenytate Cyclase) vs3AIHBTONV H t (Adenylate Cyctase) (Binding)

(C)

1.Aeie760 570 >1000 >100 2. 9-Methytadenine (9-MA) 24 24 112 106 N -substftuted 9-methyladenines 3. N 6 -Cyctobutyt-0-MA 5.5 23 0.89 1.2 4. N 6 -Cyctopentyt-9-MA 4.9 25 1.3 0.54

N

6 -Methytcytopentyt-9-MA 45 56 9.0 6. N 6 -Cyctohexyt-9-MA 7.4 21 0.65 0.94 7. N 6 .Methyl-9-MA 150 130 220 >100 a. N 6 3-Penty-9-MA 1 1 53 7. 6 3 .3 9. N 6 -Phenyt-9-MA 21 107 10 1 0. N 6 _2-Fluorophenyl -9 MA 1 1 29 17 8 11. N 6 2-Ienzyt-9-MA 57 100 49 17 12. N 6 2-Phenethyl-9-MA 170 120 >300 >1,00 13. N 6 -2-(3,4,5-Trimethoxyphenylethyt) ,9-MA 23 40 122 >100 14. N 6 -2-(3-PyrfdyLethyt)-9-MA 92 117 96 41

N

6 _2-(3-ThlenytethyL)-9-MA 14 25 24 16. N 6 -R-1-PhenyL-2-propyl-9-MA 13 25 7.2 17. N 6 _S I-Phenyt-2-propyl-9-MA 23 74 23 1 0 Human Platelet Membranes Rat PC12 Membranes Rat Fat Cell Membranes Rat Brain Membranes WO 90/09178 PCT/US90/00210 -14- FURTHER FUNCTIONAL ASSAYS To test the selectivity of the compounds of the invention, in vitro assays were conducted utilizing model tissues that are thought to contain homogenous populations of either the A 1 or A 2 adenosine receptors. Four examples were characterized by their ability to antagonize competitively the action of adenosine agonists in eliciting two responses: the reduction in force of contraction of guinea pig atrium (A 1 and the decrease in the contractile tone of the guinea pig taenia caecum (A 2 The left atria from male guinea pigs were isolated, suspended between two punctate electrodes, and placed in a ml organ bath that contained Krebs-Hertsileit solution that was continuously gassed with 95% 02 5% CO 2 and maintained at 310C. The resting tension was one gram. The atria were stimulated electrically at 1 Hz, 1 msec duration pulses at supramaximal voltage. The force of contraction was recorded isometrically.

Taenia from the guinea pig caecum were cut into lengths of 1.5-2 cm. The tissues were suspended in a 20 ml organ bath containing de Jalon's solution that was gassed with 95% 02 5% CO 2 and maintained at 31 0 C. The resting tension was 1.5 g. The contractile response was measured isotonically. Tissues were contracted with 10 M furmethide and allowed sufficient time to reach a stable contraction before addition of adenosine agonists.

The ability of the compounds to antagonize the effects of agonists was analyzed using modified Schild plots.

12 WO90/09178 PCT/US90/00210 Although there was some sensitization of the tissue, i.e. addition of the agonist -produced a larger response in the presence of high concentrations of the subject compounds, N 6 -3-Pentyl-9-MA, N 6 -Cyclopentyl-9-MA and N 6 -(endo- 2-Norbornyl)-9-MA did not competitively antagonize the effects of adenosine agonists in relaxing the taenia caecum.

Sensitization is also observed when using high concentrations of 8-phenyltheophylline a non-selective adenosine receptor antagonist. 8-PT did antagonize the effects of agonists at low concentrations. The lack of competitive antagonism by the other compounds suggests that the latter compounds do not interact appreciably with A 2 adenosine receptors and are, thus, selective for A 1 adenosine receptors.

However, N 6 -3-Pentyl-9-MA, N 6 -Cyclopentyl-9-MA, N 6 (endo-2-Norbornyl)-9-MA and N 6 -4-(2-thienyl)-3-butyl)-9-MA all were found to be competitive antagonists at adenosine receptors in the atria. N 6 -3-Pentyl-9-MA and N 6 thienyl)-2-butyl-9-MA also produced increases in basal force of contraction in the atria. Affinity constants (pKB) for the present compounds determined using known methods are summarized in Table 2 below: Table 2 Drug

PKB

N

6 -3-Pentyl-9-MA 5.4 0.14

N

6 -Cyclopentyl-9-MA 6.17 0.11

N

6 -(endo-2-Norbornyl)-9-MA 6.28 0.09

N

6 -1-(2-Thienyl)-2-butyl)-9-MA 5.36 0.1 These results show that the above examples display selectivity towards the A 1 adenosine receptor, with N 6 (Qndo-2-Norbornyl)-9-MA being the most potent antagonist.

WO 90/09178 PCT/US90/00210 -16- IN VIVO ASSAY In vitro selectivity of the present antagonists was confirmed by in vivo tests on rat heart rate and blood pressure, the former associated with A 1 receptors and the latter associated with A 2 receptors.

Rats were anesthetized with urethan and blood pressure was monitored via a carotid cannula. Drug injections were made intravenously through a jugular cannula.

Blood pressure, EGC, and heart rate were recorded on a Grass polygraph.

Adenosine produced a dose dependent decrease in blood pressure and heart rate, with a concommitant increase in the P-R interval of the ECG. Administration of N 6 -(endo Norbornyl)-9-methyladenine attenuated the effects of subsequently idministered adenosine on all parameters measured. At ,gh doses, adenosine causes heart block; this effect was also substantially reduced by the agonist. Due to the short duration of action and direct route of administration of adenosine, it is often difficult to determine whether adenosine decreased blood pressure by causing peripheral vasodilation or by reducing cardiac output. To overcome these problems, NECA adenosine), which is longer-acting and selective for A 2 adenosine receptors, was used as an adenosine receptor agonist. Prior administration of N-0861 attenuated the effects of NECA on the heart while minimally affecting the NECA-induced decrease in blood pressure. These results show that N 6 -endo-2-Norbornyl)-9-methyladenine is a cardioselective adenosine receptor antagonist in vivo and support the data above showing selectively of the N-6 substituted 9-methyladenines of the invention as A 1 adenosine receptor antagonists.

Ii WO 90/09178 PCT/US90/00210 -17- FURTHER RECEPTOR AFFINITY ASSAYS Further tests to discover the affinities of test compounds at A 2 receptors were conducted. 3 H]-N-ethylcarboxamido adenosine 3 H-J-NECA) was used as the radioligand, bovine caudate was the source of membranes, and the assay buffer was 50 mM Tris; 10 mM MgC12, pH 7.4.

To provide bovine caudate nuclei, bovine brains were obtained fresh from a local slaughterhouse. The caudate nuclei were dissected out and homogenized in Buffer A (50 mm Tris; 1 mm Na 2 -EDTA; 5 mm KC1; 1 mm MgC12; 2 mm CaC12; pH 7.4) using a Brinkman Polytron. The homogenate was centrifuged at 40,000 x g for 20 minutes and washed once. The pellet was resuspended in 3uffer A, incubated at 37 0 C for minutes, then centrifuged. The pellet was washed once more, resuspended to a protein concentration of 5-10 mg/ml in Buffer A ,nd frozen at -70 0 C until use.

The A 2 assays also contained 50 nM cyclopentyladenosine to block the binding of 3 H]-NECA to A 1 receptors (Bruns et al, 1986) and 1 unit/ml adenosine deaminase to degrade endognous adenosines. Varying concentrations of test compounds were incubated with the appropriate radioligand and membrane source for 1 hr at room temperature.

Assays were terminated by filtration over Whatman GF/B filters that had been pre-soaked with 0. polyethyleneimine using a 24 port Brandell cell hawester. The filters were washed three times with 3 ml of ice cold buffer and transfered to plastic scintillation vials to which 4 ml of Beckman Ready-Protein scintillation cocktail was added.

The tubes were shaken and counted in a Beckman 3801 scintillation counter that converted cpm to dpm.

12j WO 90/09178 PCF7/US90/00210 -18- Data were analyzed by utilizing the LigandO commercial computer program (Munson and Rodbard, 1980).

The results of these tet, expressed as the molar concentration of test compound needed to displace 50 percent of the 3 H]-CHA radioligand from rat cortical A 1 receptors, are summarized in Table 3 below: Table 3 Adenosine Antagonists Rat Cortical Sample Binding Constant No. Name Ki (M) 0861 0913 0966 0967 0982 0983 0840 0984 0985 0986 0987 0988 0989 0990 1001 1002 1003 1004 1005 1006

N

6 (endo-.2-.norbornyl) -9-MA

N

6 (endo-2-norbornyl) -2-chloro-9-MA

N

6 -2 ,2-diphe-nylethyl-9-MA

N

6 -2 (2-chlorophenylethyl) 9-MA

N

6 -2-Aminoethyl-9-MA

N

6 2-N-dimethylethyl) -9-MA

N

6 -cyclopentyl-9-MA

N

6 -R-1-phenyl-l-ethyl-9-MA

N

6 -g-1-pheny1-1-ethyl-9-MA

N

6 -S-1-pheny1-2-propyl-9-MA N6 2-thienyl-9-MA N6 (4-chloro-2-methylphenyl) -9-MA

N

6 (3-ethylindole) -9-MA

N

6 (phenethyl) 9-MA

N

6 (endo-2-riorbornyl) -8-oxo-9-HA

N

6 -2 5-trimethoxyphenyl) ethyl--mA

N

6 (endo-2 -norbornyl) -8-bromo-9-mA

N

6 -1-carboxy-l-butyl-9-MA

N

6 (endo-2-florborflYl) -8-amino-9-mA

N

6 (endo-2-norbornYl) -8-carboxy-9-NxA Sodium Salt 11. 6 x 10-8 10. 5 X 10-8 >10-5 >io0- >1o- 17. x 10-8 87>10-8 r _1; WO) 90/09178 PCF/US90/00210 -19- 1059 N 6 -(endo-2-norbornyl)9-[(2 hydroxyethoxy) methyl]adenine 1060 N 6 (endo-2-norbornyl) -8-thio-9-MA 1061 N 6 (endo-2-norbornyl) -8-chloro-9-MA 1062 N 6 -(endo-2-norbornyl)-8-sulfonate- 9-MA Sodium Salt 1063 N 6 -(Endo-2-norbornyl)-2-oxo-9-MA 1064 N 6 (endo-2-norbornyl) -8--yclopentylamine-9-MA 0964 N 6 (endo-2-norbornyl) -8-cyclopentyl-9-MA 0965 N 6 -cyclopentyl-8-cyclopentyl-9-MA 0978 N 6 (exo-2-norbornyl) -9-MA 49 x 10- 8 37 x 10-8 1.5 x 10 8 >10-4 112 x 10-8 190 x 10 8 24 x 10-8 14.1 x 10 8 43 x 10 8 The compounds in Table 3 for which a solution having a concentration greater than 10-"M was required to displace percent of the radioligand are deemed ineffective as A 1 adenosine receptor antagonists.

In further experiments designed to determine the selectivity of N 6 -endo-2-Norbornyl-9-methyl adenine at A 1 receptors, 3 H]-cyclohexyladenosine 3 H]-CHA) was used as the radioligand, rat cortical membranes were the receptor source, and the assay buffer was 50 mM Tris; 2 mM MgCl 2 pH 7.4.

Male Sprague Dawley rats were killed by decapitation and the brains removed. The cerebral cortices were homogenized in 50 mm Tris; 2mm MgC12 (pH and centrifuged at 40,000 x g for 10 minutes. The pellet was washed once, resuspended in Tris/MgCl 2 and incubated with 8 units/ml adenosine deaminase at 37 0 C for 30 minutes. The homogenate was centrifuged, washed once, resuspended to a protein concentration of 5-10 mg/ml and frozen at -70 0 C until use.

The results in Table 4 below show that the test compound has r WO 90/09178 PCT/US90/00210 170 times more affinity for A 1 receptors than for A 2 receptors.

Table 4 Selectivity o' N 6 -endo-2-Norbornyl-9-MA Bovine Caudate Binding Constants At A 1 Receptors At A 2 Receptors 4.1 x 10" 8 M 6.96 x 10-6M

AI/A

2 5.89 x 10 3 170 fold selective for A 1 receptors References Munsro., Peter J. and Rodbard, David (1980). "Ligand: A Versatile Computerized Approach for Characterizing Ligand- Binding Systems." Anal. Biochem. 107:220-239.

Bruns, Robert Lee, Gina and Pugsley, Thomas A.

(1986) "Characterization of the A 2 Adenosine Receptor Labeled by 3 H-NeCA in Rat Striatal Membranes," Mol.

Pharmacol. 29:331-346.

These N 6 -substituted adenines are antagonists of A 2 adenosine receptor-mediated stimulation of adenylate cyclase in A 2 -adenosine receptors and antagonists of A 1 -adenosine receptor-mediated inhibition of adenylate cyclase. These compounds are useful in reversal of adenosine-mediated lipolysis, reversal of adenosine-mediated deleterious cardiovascular effects (conduction defects, hypotension), reversal of adenosine-mediated vascular actions in kidney, bronchodilation, antiarrhythmic action, reversal of adenomediated relaxation of smooth muscle, anti-narcoleptic

I!

r ,I IWO 90/09178 PCF/US90/00210 -21action, CNS stimulation, and blockade of adenosine mediated inhibition of neurotransmitter release.

While particular embodiments of the invention have been described it will be understood of course that the invention is not limited thereto since many obvious modifications can be made and it is intended to include within this invention any such modifications as will fall within the scope of the appended claims.

I!

I

Claims (21)

1. Novel compounds represented by the general formula: \R3 wherein R 2 is selected from the group consisting of cycloalkyl radicals having from 3 to 8 ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13 carbon atoms, aralkyl radicals having from 7 to 14 carbon atoms and halogen- and heteroatom-substituted derivatives of said cycloalkyl, alkyl, aryl and aralkyl radicals wherein said heteroatom may be selected from the group consisting of halogen, nitrogen, phosphorus, sulfur and oxygen; R 1 may be hydrogen or R 2 and R 3 is selected from the group consisting of halogen, amine, carboxy, thio, sulfonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl- or cycloalkyl-substituted Samine and cycloalkyl radicals having from 3 to 8 ring carbon atoms; R 4 is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms wherein said alkyl group can be substituted with oxygen; and R 5 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted .J .with phenyl; and amine wherein said amine can be substituted with members of the group consisting of phenyl, and alkyl and cycloalkyl, having 1 to 6 carbon atoms.

2. The compound of claim 1 wherein R 4 is methyl. 920129,gjndat087,whitby.let,22 WO090/09178 PCr/US90!00210 -23-

3. The compound of claim 2 wherein R, is hydrogen.

4. The compound of claim 3 wherein R 2 is a cycloalkyl having from 4 to 8 carbon atoms in the ring. The compound of claim 3 wherein R 2 is phenyl or a substituted phenyl.

6. The compound of claim 3 wherein R 2 is 2-norbornyl, cyclopentyl and R 3 is selected from the group consisting of iqd~gwi-, cyclopentyl, oxo, bromo, amino, carboxy, thio, chloro, fluoro, sulfonate, sulfonamido, cyclopentylamino, cyclopentyl, and physiologically acceptable salts thereof.

7. The compound of claim 3 wherein R 2 is selected from the group cons~isting of benzyl, phenyl, c-f luorophenyl, 3,4, 5-trimethoxyphenylethyl, 3-pentyl, 2-phenylethyl, 2- (2- chlorophenylethyl) 1-indanyl, 2-aminoethyl, N,N-dimethyl- aminosthyl, 2-thienylbutyl, and cyclohexyl.

8. The compound of claim 3 wherein R 2 is endo-2- Norbornyl and R 4 is phenyl or (2-hydroxyethoxy)methyl.

9. The compound of claim 1 selected from the group consisting of N 6 (endo-2-Norbornyl) (2-hydroxyethoxy) methyl) adenine, N 6 (endo-2-Norbornyl) -8-thio-9-methyl adenine, N 6 (endo-2-Norbornyl)-8-chloro-9-methyl adenine, N 6 -(end o-2 -Norbornyl) -2 -oxo-9 -methyl adenine, N 6 -(endo-2- Norbornyl) -8-cyclopentylamino-9-methyl adenine, N 6 -cyclo- pentyl-9-methyl adenine, N 6 (endo-2-Norbornyl) -9-methyl adenine, N 6 (endo-2-n'rbornyl) -8-bromo-9-MA, N 6 -cyclopentyl- 8- cyclopentyl-9-methyl adenine, N 6 (exo-2-riorbornyl) -9-MA, N 6 -cyclopentyl-2-chloro-9-methyl adenine, N 6 ((3-chioro) A'j'Li,V endo-2-florbornyl)j-9-MA, N 6 -cyclopentyl-9-phenyl ade7nine, N 6 WO 90/09178 PCF/US90/00210 -24- (endo-2-norbornyl) -8-cyclopentyl-9-MA, N 6 -cyclopentyl-9- benzyl adenine, and N 6 -(endo-2-norbornyl)-8-amino-9-MA. The compound of claim 2 wherein Rg is selected from the group consisting of hydrogen, ethoxy, methoxy, propoxy, n-butoxy, isopropoxy, 1-methylpropoxy, 2-methyipropoxy, 2- phenyl-ethoxy, methylamino, butylamino and anilino.

11. The compound of claim 3 wherein R 5 is chloro.

12. The compound of claim 3 wherein R 2 is selected from the group consisting of 3-pentyl, 1-phenyl-2-propyl, and phenyl.

13. The compound of c1i1rn 2 wherein R 2 is hydrogen and R 5 is selected from the group consisting of ethoxy, methoxy, propoxy, n-butoxy, isopropoxy, butyl-2-oxy, 2-methylpropoxy, pentoxy, 2-phenylethoxy, methylamino, butylamino and anilino.

14. The compound of claim 11 wherein R 2 is 2-(3- thienylethyl). The compound of claim 7 wherein R 2 is cyclohexyl.

16. The compound of claim 11 wherein R 2 is 2-(3- pyridylethyl).

17. The method of antagonizing the A 2 -adenosine receptor-mediated stimulation of adenylate cyclase which comprises administering to a subject an effective amount of one or more o7 the compounds of claim 4, 6, 7, 8 or 9. .14_ 1 i- SWO 90/09178 PC/US90/00210

18. The method of claim 17 wherein said subject is a human.

19. The method of antagonizing the A 1 -adenosine receptor-mediated inhibition of adenylate cyclase which comprises administering to a subject an effective amount of a compound of claim 4, 6, 7, 8 or 9. The method of claim 19 wherein said subject is a human.

21. The method of antagonizing the adenosine receptor which comprises administering to a subject an effective amount of a compound selected from the group of compounds represented by the general formula R N .1 x R4 wherein R 2 is selected from the group consisting of cycloalkyl radicals having from 3 to 8 ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13 carbon atoms, aralkyl radicals having from 7 to 14 carbon atoms, and halogen- and heteroatom-substituted derivatives thereof wherein said heteroator may be selected f.rom the group consisting of halogen, nitrogen, phosphorus, sulfur and oxygen; R 1 may be hydrogen or R 2 and R 3 is selected from the group consisting of 4ydeqn, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide phenyl, alkyl- or cycloalkyl-substituted amine, aEli=.r-ladauw l n 1 L-d- 1 WO 90/09178 PCT/US90/00210 -26- a-aie a omc and cycloalkyl radicals having from 3 to 8 ring carbon atoms. R 4 is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms wherein said alkyl group can be substituted with oxygen; and R 5 is selected from the group consisting of hydrogen, hydroxy, amine, halogen, al.oxy and cycloakoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with members of the group consisting of phenyl, alkyl, cycloalkyl, having 1 to 6 carbon atoms.

22. The method cf claim 21 wherein said subject is a human.

23. Novel compounds represented by the general formula R 1 -R2 wherein R 1 is selected fro the group consisting of cycloalkyl radicals having om 3 to 7 ring carbon atoms, alkyl radicals having om 2 to 10 carbon atoms, aryl radicals having from to 10 cazbon atoms, aralkyl radicals having from 7 to carbon atoms and heteroatom substituted derivatives t reof wherein said heteroatom may be selected from the up consisting Df halogen, nitrogen, phosphorus, sulfur nd oxygen; R 2 may b\ hydrogen or R 1 and R 3 is an Sal group comprising from 1 to 4 carbon atoms. L. I. INTERNATIONAL SEARCH REPORT Ineratonl vaictinNo. PCr/US9O/00210 1. CLASSIFICATION OF SUIjECT MATTER (it severalC casirc~uion symbols appoly. idicate all) Accurding to internatint Pa f sd I t ly md 2'1 Aon~2 and [PCa 7~~ j11-S. CL.: -514/261,514/266, 544/277, 544/276,514/262 it 711110S SEARCHIC Minimum Docurnertioton Searched? Documentation searched ethir thanivMinimum DOcumeni.,iion to the Estent that such Documents are Included in the Fields Searcheds II DOCUJMENTS CONSIDERED TO 01 RELEVANT I Categop,, Citation of Document, 11 with indication, wnere aipororiAt.. af the relevant oassages 'a Relevant to Claim No2 x FEI3S Letters vol 2 5, no.2, issued May 1987, 1-42 (Ukena, et al I'NOsubstituted 9-methyladenines: A New Class of Adenosine Receptor Antagonists.", pages 203-208. All pages. X GB, A, 953,897, Shell International 1--16,23-36 Research Maatschappij 02 April 1964, see pages 1,3. X CA, A, 657,337, (Zwahlen), 05 February 1963. 1-16,23-36 X JA, B2, 45,758, (Fujisawa Pharmaceutical Co. Ltd.), 1-16,23-36 17 November 1972. X GB, A, 2,041,359 (Freeman, et al.) 10 September 1-16,23-36

1980. *Soecial Categoeied of cited documents: W later document published after the international rlilng d-ate document deorning the genesral etate of the art ahich is mo Or priority date and mot in conflict with tns Cpolication out. considered to be at Partcular relevance cited to unlderstanid the principle or theory underlying :-e I.E. invention "E earlier document but Published an or after the international X"document of particular relevance; the claimed onnerilon r iting date x cannot be considered novel or cannot be considered -a document which may throw doubts onmpriority claimr(s) or involve an inventive Sten which a9 cited to establish the Publication date ot another document of Particular relevance; the claimed invent on citation or Other s10ecial1 reason (AS specified) cannot be considered to involve an inventive step rem -e document referring to ani oral disclosure, use, exhibition or document is combined with one or more other si~chr other means ments, such combination being obvious to a person s...eo document Published prior to the international iting dae but in the arn. later than the an;~ Miy date claimed document fmember of the sames patent family IV. CV~TIPICATION Oso Actual Completion of the International Search Date of Matting of this International Search Report 29 MARCH 1990 0 5 JUL190 International 3eariching Authority Signature of Authorized Offlcr A 5 ISA/USfoDIN FORSlgwvC t47) 21 12 I International Application NoPCT/US90/00210 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET X X 7 Journal of the American Chemical Society, vol. 79, no. 2, issued 20 January 1957, (Robins, et "Potential Purine Antagonists. IV. Synthesis of Some 9-Methyl-6-substituted-purines", pages 490-494, see pages 490-494. Journal of the American Chemical Society, vol. 79, no. 19, issued 05 October 1957, (Montgxnery, et "Synthesis of Potential Anticancer Agents. IX. 9-Ethyl-6-substituted-purines", pages

5238-5242. See all pages. 1-16,23-36 1-16,23-36 V.L OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE 1 Tis international search report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1.1 Claim numbers because they relate to subject matter 12 not required to be searched by this Authority, namely: Claim numbers because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out L3, specifically: 3. 0 Claim numbers because they are dependent claims not drafted in accordance with the second and third sentences of PCT Rule 6.4(a). VI.0 OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authority found multiple inventions In this international application as follows: As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the International application. As only some of the required additional search fees were timel, paid by the applicant, this international search report covers only those claims of the international application for which fees were paid, specifically claims: 3.1 No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: As all searchableclalms could be searched without effort justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest The additional search fees were accompanied by applicant's protest. O No protet accompanied the payment of additional search fees. Fn PCTISAn210 (supplarnerl shet (Re. 11-87)