NZ199643A - Treatment with immunomodulators and antiviral agents which are 9-(2-hydroxyalkyl) purine amine salt complexes - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number 1 99643 Priority Data(s): .. JP.-j • • • ■ ■ a . y . ft} CosTjpSot'3 (-pacification Filed: / Class: W.K. • ■ • 3 y • ■ Publication Date: - ° P.O. .Joumci i^o: /P58 199643 Under the provisions of Regulation 23 0) lhe "" Spedftotio, has beer, ante^Metf mT$. fRT??9p3! '• •>< . ::'i' * .r " -si fcStai NEW ZEALAND PATENTS ACT, 1953 No.:Divided out of No. 191 483 Date: 5 September 1979 . PATENT -4FEB1982 >] RECEIVED COMPI ETE SPECIFIC ATI ON METHODS OF TREATMENT EMPLOYING IMMUNOMODULATORS AND ANTIVIRAL AGENTS W i:/We, KEKPORT PHARMACEUTICALS INTERNATIONAL^ INC. , a corporation cf the State of California, U.S.A., of 1590 Monrovia Boulevard, Newport Beach, California, U.S.A., and SLOAN-KETTERIHG INSTITUTE & S. FOR CANCER RESEARCH, a corporation of the State of New York, f"** A/e«v Yeirk, U.S.A., of 1275 York Avenue, New York,^U.S.A. hereby declare the invention for which f / we pray that a patent may be granted to itiH/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (followed by page la) % • . -la- This invention relates to methods of treatment employing immunoirodulators and antiviral compounds. This is a divisional of New Zealand patent specification 191,483.

The present invention is based on the discovery that compounds of the formula (Y) ■R where X is OH, NH2/ SH, OR or SR where R is alkyl of 1 to 4 carbon atoms or benzyl, r! is H or alkyl of 1 to 8 carbon atoms, R^ is H or methyl, Y is the salt of an amine of the formula R? R' / N(Cr;H2n)OH where R^ and R^ are lower alkyl, of 1 to 4 carbon atoms and n is an integer of 2 to 4^with p-acetamidobenzoic acid and where z is a number from 0 to 10 t 99643 are useful as immunomodulators, as antiviral agents and in specific cases have antitumor activity. The compounds and compositions where z is 1 to 10 are novel per se.

When is H the presence of Y enhances the immunoregulatory activity and the antiviral activity. If X is the NH2 there is immunoinhibitory activity but no immunostimulatory (immunopotentiatory) activity.

Immunoregulatory activity appears to in crease with increasing chain length for R^, at least from methyl through hexyl. Preferably R^ is n-alkyl, i.e., methyl, ethyl, n-propyl, n-butyl, n-amyl, n-hexyl, n-heptyl or n-octyl. R^ is preferably ' 15 methyl. R can be methyl,' ethyl, n-propyl, n-butyl, isopropyl, etc. When X is NH2 the compound can be present as the free base or as the salt with a nontoxic acid,, i.e., pharmaceutically acceptable acid, e.g., hydrochloric acid, hydrobromic acid, sulfuric 20 acid, phosphoric acid, citric acid, lactic acid, tartaric acid, salicylic acid, acetyl salicylic acid, acetic acid, propionic acid, p-toluene sulfonic acid, methane sulfonic acid, maleic acid, succinic acid, malonic acid ^nd adipic acid.

A preferred class of amines to form the salt with para acetamidobenzoic acid has the formula R5 N(CnH2n)OH R6 199643 where and are lower (C-j_^,) alkyl, e.g. , methyl, ethyl, propyl, isopropyl or butyl and n is an integer of 2 to 4. Typical examples of such amines include 2-dimethylaminoethanol, 1-dimethylaminoisopropanol, 2-diethylaminoethanol, dlethylamxnoisobutanols, 1-diethyl-aminoisopropanol, 2-(methyl) (ethyl) aminoe.th.anol, aiisobutyl-arnino butanols, dimethylaminopropanols, dimethylamino-butanols, 2-diisobutylaminoethanol, dlbutylarainobutanols , 2-dibutylaminoethanol, 2-dipropylaminoethanol, 2-diisopropyl-aminoethanol. The presently preferred amine is 1-dimethylaminoisopropanol. When Y is present, i.e., z is 1 to 10, preferably z is 3. However, z can also be 1, 2, 4, 5, 6, 7, 8, 9 or 10.

While there are preferably used the compounds R\ where Y is the salt of the amine ^N(CnH2n)0H R4X with p-acetamidobenzoic acid there can also be used salts of the formula Y^- wherein the amine is as just defined the acid is a pharmaceutically acceptable acid other than p-acetamidobenzoic acid, e.g., hydrochloric acid, sulfuric acid,.hydrobrcmic acid, phosphoric acid, acetic acid, propionic acid, malonic acid, lactic acid, citric acid, tartaric acid, p-toluene sulfonic acid, adipic acid, maleic acid, succinic acid, methane sulfonic acid, salicylic acid, acetyl salicylic acid. 1 99643 In describing the compounds below, when Y is present the abbreviation DIP'PAcBA stands for dime thylamino-2-propanol-p-acetamido benzoate. Unless a number in parentheses, e.g., (10), follows this abbre-5 viation, then Y is 3. If a number in parentheses follows the abbreviation DIP'PAcBA there the number indicates the number of moles of Y groups present to 1 mole of the 9-(hydroxyalkyl)purine.

In Table 1 below the compounds are believed 10 to be pure except for compound 15443 which is believed to also contain a salt in addition to the compound of the invention.

An immunomodulator is a compound which regulates the immune response. Thus, it covers both im-15 munostimulation (immunopotentiation) and immunoinhibi-tion. Immunostimulation, of course, is useful in building up immunity. Immunoinhibition also has utility in a number of areas. For example, it is useful in organ transplants, e.g., kidney or heart trans-20 plants, to prevent rejection.

In the tables showing the immunopotentiating properties of the compounds, a plus (+) or a minus (-) indicates immunostimulating or immunoinhibiting properties respectively. The number 0 indicates the com-25 pound had neither immunopotentiating activity of immunoinhibiting activity.

There are included in some of the tables several compounds wherein X is not within that claimed. These non-claimed compounds as a rule have 30 relatively low activities and are included to illustrate the fact that the X group can have a significant effect on the properties of the compounds. < 199643 A mitogen is a substance which induces cell proliferation, as occurs during immunization.

Table 1 (excluding compounds 15427 and 15423) shows compounds useful in the invention.

The synthetic procedures A through L men tioned in Table 1 are described in more detail subsequently.

The compositions of the invention are useful in treating mammals (and cells of mammals) including 10 humans, swine, docjs, cats, cattle, horses, sheep, goats, mice, rabbits, rats, guinea pigs, hamsters, monkeys, etc. • • Unless otherwise indicated, all ,parts and percentages are by weight. f All temperatures are in degrees centigrade unless otherwise indicated.

The compositions can comprise, consist essentially of or consist of the materials set forth and the processes can comprise, consist essentially of or 20 consist of the steps set forth with such materials.

The compositions can be administered to the mammals by conventional techniques, e.g., orally, nasally, rectally, vaginally, enterally or parenteral-ly. They can be employed as injectable solutions, 25 e.g., in water, or as tablets, pills, capsules, etc. 199643 Table 1 SUMMARY OF CHEMICAL PROPERTIES OF 9-(HYDROXYLALKYL PURINES No.

, Compound Synthetic / / r1 ' r2 X Y Method 15425 H H OH D 15428 H H OH DIP * PAcBA L ' 15435 H H SH _ C 15437 H H SH DIP•PAcBA L 15446 H CH-j OH A 15447 H CHn OH DIP-PAcBA L 15431 H -J CH-, NH0 B 15432 H CH, NH0 DIP'PAcBA L 15427 CH-, H I E 15423 CH-, H CI _ F 15433 CH_, H NHn _ G 15434 J CH-, H NH^ DIP-PAcBA L 15443 J CH-, H A OH H 15444 CH_, H OH DIP * PAcBA L 15417 J ™ C6H13 H OH I 15418 D "J. J " *"6^13 H OH DIP-PAcBA L 15392 U ^ J *"6^13 CH0 OH J 15410 C6H13 CH, OH DIP-PAcBA L 15426 U X —£s£]3 CHV NHn HC1 Salt K 199643 No.

UV Spectra Elemental Analysis M.Pt.

°C A Max AMin Con_ ~J PH C H N 15425 274 O 250 250 254 222.5 219 221.5 11.93 11.0 12.53 7 1 10 15428 15435 278 -80 323 323 323 251 252 251 23.0 19.9 19.9 7 1 10 - 15437 15446 244 -5 250 250 254 223.5 220 223.5 / 11.0 10.6 12.1 7 1 10 15447 15431 188 O 261 259 261 228 231 225 ■ .8 15.4 15.7 7 1 10 Cal 49.73 5. FD 49.56 5. 62 36 36 .25 .22 15432 15427 178 o 276 276 276 237 237 237 .9 10.9 10.9 7 1 10 Cal 31.60 2. FD 31.53 2. 98 96 18 18 .43 .18 15423 200' -204 265 265 265 228 228 . 228 9.1 9.1 9.1 7 1 10 Cal 45.20 4. FD 45.11 4. 26 27 26 26 .36 .25 15433 215- -16 261. 259 261 228 231 224.5 13.56 13.26 13.80 7 1 10 15434 15443 198- -199 250 250 255 223 218 225.5 7.52 6.91 7.91 7 1 10 15444 15417 226 °C 250 250 255 224 220 223 11.09 10.37 11.96 7 1 10 Cal 59.07 7. FD 59.01 7. 65 55 21 21 .16 .24 15418 15392 202 °C 250 248 254 224 222 220 12.1 13.3 14.1 7 1 10 Cal 60.41 7. FD 60.47 7. 97 86 20 .13 .08 15410 15426 176- -9 °C .261 259 251 230 233 235 9.77 9 .60 9 .77 7 1 10 Cal 53.58 7. FD 53.56 7. 71 67 22 22 .32 .34 199643 Other compounds within the invention are set forth in Table la below wherein the basic formula is the same as that in Table 1. In Tables 1 and la, the alkyl groups for R^- and X are all n-alkyl.

Table la COMPOUND r1 r2 x y c6h13' ch3 oh DIP-PACBA(10) c6h13 ch3 oh DIP'PAcBA(1) h ch3 oh DIP-PAcBA(lO) h ch3 oh DIP•PAcBA(1) ch3 ch3 oh - ch3 ch3 oh DIP-PAcBA c2h5 h oh DIP-PAcBA i i c2h5. h oh - c3h7 h oh - c3h7 h oh DIP;PAcBA c2h5 ch3 oh - C2H5 ch3 oh DIP-PAcBA c2h7 ch3 oh - c3h7 ch3 oh DIP-PAcBA c4h9 h oh - c4h9 h oh DIP-PAcBA c4h9 ch3 oh - c4h9 ch3 oh DIP-PAcBA 1 99643 Table la (cont.) COMPOUND r1 r2 x y C5H11 h oh - C5H11 h oh DIP .PAcBA C5H11 ch3 oh DIP -PAcBA C5H11 ch3 oh - C7H15 h oh - C7H15 h oh DIP •PAcBA C7H15 ch3 oh - C7H15 ch3 oh DIP •PAcBA C8H17 h oh - C8H17 h oh DIP •PAcBA C8H17 ch3 oh - C8H17 ch3 oh DIP •PAcBA C6H13 ch3 och3 - C6H13 ch3 och3 DIP ■PAcBA C6H13 h och3 DIP -PAcBA C6H13 h och3 - CH3 h och3 - ch3 h och3 DIP -PAcBA h h och3 - h • h och3 DIP -PAcBA h ch3 och3 DIP -PAcBA h ch3 och3 - I V t 99643 Table la (cont.) t COMPOUND r1 r2 x y c6h13 ch3 oc2h5 - c6h13 ch3 oc2h5 DIP-PAcBA c6h13 h oc2h5 DIP.PAcBA c6^13 h oc2h5 - c6h13 ch3 oc3h7 — : c6h13 ch3 °c3h7 DIP-PA^cBA ch3 h DIP-PAcBA ch3 h oc3h? - h h oc3h7 DIP-PACBA h ^3 oc3H7 DIP-PAcBA c6h13 ch3 oc4h9 - c6h13 ch3 °c4h9 DIP-PAcBA c6h13 h oc4h9 - c6h13 h oc4h9 DIP-PAcBA h h oc4h9 - h h oc4h9 DIP-PAcBA h ch3 oc4h9 - h ch3 oc4h9 DIP-PAcBA ch3 ch3 oc4h9 - ch3 ch3 oc4hg DIP-PAcBA ch3 h oc4h9 - CH3 h oc4h9 DIP-PAcBA I 99643 Table la (cont.) COMPOUND R1 R2 X y C6H13 CH3 sch3 - C6H13 CH3 sch3 DIP •PAcBA C6H13 H sch3 - C6H13 H sch3 DIP •PAcBA ch3 CH3 sch3 - .

/ ' ■ CH3 CH3 sch3 DIP •PACBA CH3 H sch3 - CH3 H sch3 DIP f •PAcBA H H sch3 - H H sch3 DIP •PAcBA h CH3 sch3 DIP •PAcBA h ch3 sch3 - C6H13 ch3 SC4H9 - C6H13 ch3 DIP •PAcBA C6H13 h sc4h9 DIP •PAcBA C6H13 h sc4h9 - ch3 h sc4h9 - ch3 h SC4H9 DIP •PAcBA H H : SC4H9 - H H sc4h9 DIP •PAcBA H CH3 sc4h9 DIP •PAcBA H CH3 OH DIP •PAcBA(10) H ch3 OH DIP •PAcBA(1) 199643 Table la (cont.) COMPOUND r1 r2 x y C6H13 h O-benzyl - C6H13 h O-benzyl DIP •PAcBA C6H13 ch3 O-benzyl - C6H13 ch3 O-benzyl DIP •PACBA C6H13 ch3 S-benzyl - C6H13 ch3 S-benzyl DIP •PAcBA C6H13 h S-benzyl - C6H13 h S-benzyl DIP •PAcBA 199643 DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference to adenine and hypoxanthine throughout this specification means reference to their respective 9H— purine isomers.

Method A 9-(2-HYDROXY-1-PROPYL)HYPOXANTHINE (NPT OH -2-, CjO AC OH CH—- CH —CH, 2 I OH CH2——CH 3 OH 9-(2-Hydroxy-l-propvl)adenine (I, 4.0 g, 20.7 mmol) was suspended in 50% acetic acid (20 ml) and sodium nitrite (4 g, 58 mmol), was slowly added. The mixture was stirred at 25° for 3 hr. The resulting solution was evaporated to dryness and isopropanol added; this operation was repeated once. The solid residue was boiled in isopropanol and filtered. The filtrate was evaporated and crystallized by addition of acetone. Recrystallization was made from iso-pro-panol/methanol (98:2); a colorless crystalline product was obtained. Yield 3.3 g (82%) M.P. 244-250° uv (H2O; pH 5.5) \ max 250 nm. 1 99643 Method B 9-(2-HYDROXY-l—PROPYL)-6-CHLOROPURINE uCs"ir^ ut> ^ CI OH " u ^ | OH CH / \ fH0 CH3 There were employed the methods of 5 Schaeffer, H.J. Vogel, D. and Vince, R.f J. Med. Chem. 8^502 (1965); and Schaeffer, H.J. and Vince, R., J. Med. Chem. 10, 689 (1967).

A solution of 5-amino-4,6-dichloropyrimidine (I, 20 g, 0.12 mole) in 11% ethanolic solution of iso-10 propanolamine (200 ml) was refluxed for 8 hr. The reaction mixture was evaporated to a syrup, ethanol added and evaporated again; this operation was repeated once. The resulting syrup was poured into water (300 ml) giving a crystalline mass. It was collected 15 by filtration, washed with water and dried to give 19 g of crude 9-(2-hydroxy-l-propylamino) 5-amino-6 chlo-ropyrimidine (II).

The crude compound II was suspended in tri-ethylorthoformate (120 ml) to which ethanesulfonic 20 acid (5 drops) was added. After 15 min. all the solid 199643 dissolved and the solution was kept a 25 overnight. Evaporation in vacuo gave a thick syrup which was submitted to high vacuo evaporation to remove the excess of isopropanolamine. Upon crystallization with 5 xylene, 5 g of crude material was obtained.

Method B 9-(2-HYDROXY-l-PROPYL)ADENINE (NPT 15431) NH.

NH /MeOH b O' ' 130 CH 5— CH—CH _ I 3 OH CHj— CH—CH3 1 9-(2-Hydroxy-l-propyl)-6—chloropyrine (1,9 g, 42.4 (mol) was dissolved in saturated methanolic ammonia and ammonium chloride (50 mg). The mixture was heated at 130° in a bomb for 6 hr. The resulting solution was evaporated to dryness and recrystallized from ethanol/ 15 acetone. Yield = 6.68 g of a colorless crystalline product (81%) mp 193-194° uv (H20; pH 5.5) Amax 260 nm TLC in CHCl3:MeOH (5:1) Rf 0.44 1 99643 Anal. Calc. for C8H11N5O: C, 49.73; H, 5.74? N, 36.25; Found: C, 49.56, H, 5.62; N, 36.22.

Method C 9— (1-HYDR0XYETH-2-YL) -6-MERCAPTOPURINE XNPT 15435) ci nh„ sh There was employed the method of Schaeffer and Bhargava, Biochemistry A_, 71 (1965). 9-( l-Hydroxyeth-2-yl)-6-chloropurine (1,2 g, .01 mol) and thiourea (0.76 g; .01 mol) were dissolved in ethanol (15 ml) and refluxed for 30 min. The resulting precipitate was collected by filtration and suspended in water to form a slurry. Neutralization with sodium acetate gave colorless crystals. Yield 1.5 g (76%).

M.P.•278-280°;- uv (H2O, pH 5.5) A max 320, 230 nm.

CE~2—CH2OH CH-—CH2Otl 199643 Method D 9-(1-HYDROXYETH-2-YL) HYPOXANTHINE (HPT 15425) N NaOH 4 CHJj— CH2OH CH2~CH2OH l There was used the method of Schaeffer, H.J. and Bhargava, P.S., Biochemistry 4_, 71 (1965). 6-Chloro-9'- (l-hydroxyeth-2-yl) purine, III (4 g) was added slowly to warm N NaOH (30 ml) and refluxed for 2 hr. The reaction is cooled in ice and neutralized with glacial acetic acid. After filtration, portions of unreacted III are removed. The product is recrystallized from methanol and washed with acetone. Colorless crystals. Yield, 1 g. (28%); mp 274°; 'uv (H2O, pH 5.5) , X max 250 nm. \99643 Method E 9-(1-HYDROXYL—2-PROPYL)-6—IODOPURINE (NPT hi * -10 / \ h3c ch2oh 9-(l-Hydroxy-2-propyl)-6-chloropurine (I, 1.5 g, 7 mmol) was added to hydroiodic acid (15 ml) at -10° with stirring for 45 min. The precipitate was filtered, neutralized with anhydrous sodium acetate at 5°, and washed with a little cold water (3 times). 10 Recrystallization from ethanol/H20, gave colorless crystals. Yield = 0.9 g (42%) mp = 193-194° uv Xmax 276nm(H20, pH5.5), Anal. Calc. for CgHgN^OI MW = 304.1: C, 31.60; H, 2.98; N, 18.43; I, 41.73. Found: C, 15 31.53; H, 2.96; N, 18.18; I, 41.70. .1 99643 Method F 9-(1-HYDROXY-2-PROPANE)-6-CHLOROPURINE (NPT 15423) CI nh, CI NH, +ch3-ch-ch2ch —> 00 I CH H0C//' ^CHoOH CI nh.

CH nh I /S\ 3 CH20H There was used the method of Schaeffer, H.J. and Schwender, C.F., J. Med. Chem. 1_7, 6 (1974).

A solution of 5-amino-4,6-dichloropyrimidine (I, 6.56 g 40 mmol) and 2-amino-l-propanol (II, 3.3 g, 44 mmol) was refluxed in n-pentanol (288 ml) and tert-10 butylamine (96 ml) for 45 hr. under N2 atmosphere. The solution was evaporated to a syrup and ethanol added 4 times and evaporated. The resulting syrup was suspended in triethylorthoformate (150 ml) and ethane-sulfonic acid (10 drops). The suspension was vigor-15 ously stirred overnight, then evaporated to dryness, ethanol added and this operation repeated three times. Crystallization of colorless product occurs during 199643 evaporation. The crystals were filtered, and the filtrate was evaporated, ethanol added and this operation repeated three times to give a crude material (3.6 g).

Recrystallized from 98% aqueous ethanol. uv (H2O, pH 5.5) \ max 265 nm; mp 201-203°; yield 2.79 (32%).

Anal. C8H9N40C1. Calc. C, 45.20; H, 4.26; N, 26.36; CI, 16.68. Found: C, 45.11; H, 4.27; N, 26.25; CI, 16.71.

Method G 9-(1-HYDR0XY-2-PR0PYL)ADENINE (NPT 15433) NH3/MeOH 130° ch2oh There was used the procedure of Schaeffer, 15 H. and Schwender, C., J. Pharm. Sci., 60^, 1204 (1971). Also Schaeffer et al., J. Med. Chem. 15, 456 (1972). 199643 9-(l-Hydroky-2-propyl)-6-chloropurine (I 2.0 g, 9;4 mmol) was suspended in methanol/ammonia (30 ml) and ammonium chloride (50 mg) added as a catalyst and the mixture heated at 130° for 4.5 hr.; the solu-5 tion was evaporated to dryness. Recrystallization from ethanol of the obtained crude product gave colorless needles. Yield = 1.15 g (63%) mp = 215-216° uv (H20, pH 5.5) Amax 260 nm.

Method H 9— (i—HYDR0XY-;2—PROPYL )HYPOXANTHINE (NPT 15443) NH ■o OH c c CH2OH CH2OH I II 199643 9-(l-Hydroxy-2-propyl)adenine (I, 4 g, 21 mmol) was dissolved in 50% acetic acid (20 ml), sodium nitrite (4 g, 58 mmol) added and the mixture stirred at 25° for 3-1/2 hr. The solution was evaporated to dryness twice with isopropanol. The residue was taken up in isopropanol and filtered, the precipitate discarded, and the filtrate evaporated to form a gel which, upon the addition of acetone, solidified.

Yield = 3.65 (90%) of colorless crystals. Recrystallized from isopropanol/methanol (98:2). mp = 202-207° TLC in CHCl3:MeOH (5:1) 1 spot Rf - 0.30 uy (H2O, pH 5.5) = Xmax 250 nm.

Method I t COMPOUND NPT 15417 There was used the procedure of Schaeffer et al, Journal of Pharmaceutical Sciences 16:1204-1210, Method F.

The product is compound XL in Table III of Schaeffer et al. : Method J ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (NPT 15392) An outline of the synthetic sequence for the preparation of erythro-9-(2-hydroxy-3-nonyl)hypo-xanthine (Nonylhypoxanthine, VIII) is shown in Flow Charts 1 and 2. The improvements over the procedure 199643 of H.J. Schaeffer and C.F. Schwender, J. Med. Chem., 17, 6 (1974 ) in the reaction sequence leading to the erythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) are indicated. The last step, the hydrolysis of the 6- xanthine (VIII) is an adaptation of the method reported by A. Giner-Sorolla, C. Gryte, A. Bendich and G.B. Brown, J. Org . Chem. 34^, 2157 (1969) for the hydrolysis of halogenopurines. to yield Nonylhypoxanthine (VIII) (shown on Flow Chart 2) consists of the preyious conversion by ammonolysis of the chloro derivative (VII) into the aminopurine' 15 (IX, EHNA) followed by its nitrosation to yield Nonylhypoxanthine (VIII). chloropurine derivative (VII), to yield nonylhypo- The alternate route, i.e., the nitrosation of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (IX), Flow Chart 1 OUTLINE OF THE SYNTHESIS OF ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII) Step 1 3-ACETAMIDONONAN—2-ONE (II) Acylation of 2-amino octanoic acid (ch_c0)o0 I II -2k- 199643 Step 2 3-ACETAMIDONONAN-2-ONE HYDROCHLORIDE (III) Formation of the 3—a.cetamidononan-2-one hydrochloride HC1 CH,— [CHj— CH—COCH — —4 CH J—[CH J CH—COCH, 3 2 5 « 3 61% 3 2 5 ' NK:OCH3 NScoca3»Hci ii in Step 3 ERYTHRO-3-ACETAMIDO-2-NONANOL (IV) Reduction of the 3-acetamidorronan-2-one hydrochloride OH KBH, [' CH — [CH ]«— CH—COCH } CH — [CH ] -—CH—CH— CH_ i ^ | 5 15% | MHCOCH:3.HCl 1THC0CH 3 III IV (Figures below the arrow refer to % yield.) Step 4 ERYTHRO—5—AMINO—4-CHLORO—6-(2—HYDROXY-3— NONYLAMINO)PYRIMIDINE (VI) Condensation of erythro-3-acietamido-2-nonanol with 5-amino-4,6-dichloropyrimidine Step 5 ERYTHRO-9—(2-HYDROXY-3-NONYL)-6-CHLOROPURINE (VII) Ring closure of erythro-5-amino-4-chloro-6-(2-hydroxy-3-nonylamino)pyrimid ine (V) VI VII 9 28JUU982 199643 Flow Chart 1 (cont.) Step 6 ERYTHRO-9- ( 2-H YDROXY-3-NONYL )HYPOXANTHINE. (VIII) (By hydrolysis of the 6-chloropurine derivative) CH / N. 100% / \ CH 3-[CH2]5 CHCH <=3 / CH3 ,[CH235 / \ CHOH CH^ VII VIII cr 1 99643 Flow Chart 2 ALTERNATIVE ROUTE FOR THE PREPARATION OF ERYTHRO-9-(2-HYDROXY-3-NONYL HYPOXANTHINE (VIII) Step la ERYTHRO-9-(2-HYDROXY-3-NONYL)ADENINE (IX) Ammonolysis of erythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) CHQH GH3— [GH2]5 CH, VII IX Step 2b ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII) Nitrosation of erythro-9-(2-hydroxy-3- nonyl)adenine (IX) 199643 no. 75% CH,— [CH] _ CHQH J 2 i CH3 ch ch-—[ch] ^chch 3 2 5 | CI^ IX viii 3-ACETAMIDONONAN-2-ONE (II) ch3— [ ch2 ] — ch—cooh kh, (■■ch c0)2q. 70JS ^ ch—- [ch23r—ch— coch, NHQOCH. ii A mixture of 2-amino-l-octanoic acid (I, 200 g, 1.26 mole) in acetic anhydride (960 ml), and pyridine (640 ml) was heated on a boiling water bath for 4 hr. The reaction mixture was evaporated in vacuo, and the residue was partitioned 6-8 times between 5% aqueous solution of NaHC03 (400 ml) and ether (400 ml). .199643 The combined ethereal extracts were dried with anhydrous MgS04 an^ evaporated to dryness to give crude 3-acetamidononan-2-one, 154 g (70%). 3-AMINO-2-NONANONE HYDROCHLORIDE (III) HC1 II NE2-HC1 III The crude product (II) obtained in the preceding operation (154 g). was dissolved in concentrated aqueous HC1 (1,540 ml) and refluxed for 2 hr. and then evaporated to dryness _in vacuo. The resulting solid 10 was recrystallized from a warm solution in EtOH (200 ml) and then cooled to 25°. To this solution ether (6 00 ml) was added. A white crystalline precipitate appears; the suspension is kept at 5° overnight. The precipitate is collected and washed with ether (once 15 with 100 ml) to give 125 g (67%) white crystalline product M.P. 112° dec. or had a lower melting point, it should be recrystallized with charcoal from tetrahydrofuran. In one re-20 peat of this procedure there was used 150: ml of tetrahydro- If the crystalline material were not white furan for 100 g of the crude hydrochloride (III). 199643 ERYTHRO-3-AMINO-2-NONANOL (IV) KBH^ CH— [CHl — CH-COCH ) CH-— [CH_]=— CH~CH — CH. 3 2 5 | 3 752 3 / \ NH2-HC1 NH2 OH III IV 3-Amino-2-nonanone hydrochloride (43.8 g, 0.226 mole) was dissolved in absolute methanol (150' ml) and cooled to -10° in an ice-salt bath. 1/ Potassium borohydride (24.4 g, 0.45 mole) 2/ was added in small portions over a 2-3 hr. period. The mixture is then kept at -10° to -15° for 3 hr. 3,4/ and slowly allowed to reach room temperature (22°), then stirred overnight (20 hr.) at room temperature. The mixture is then evaporated to dryness (syrup) _in vacuo and partitioned between H20 (150 ml) and chloroform (150 ml). The H20 layer was further extracted (3x) with chloroform (100 ml ea.). The chloroform layer was dried with MgS04 and evaporated jln vacuo to give a slightly yellowish, oily product. This liquid was distilled in high vacuo at 95°-100° (0.15 mm Hg) to give pure erythro-3-amino-2-nonanol, 26.4 g, 75% yield, m.p. 81°-86°. ~31- 1 99643 Upon cooling the solution of III, some material precipitates; this has no effect on the outcome of the reaction.

At this point, the present procedure differs from that of Schaeffer et al. Schaeffer adds acetic acid at the same time as KBH4, maintaining the pH at 5-6. It has been found that neutralization entails loss of KBH4 and that a pH above 5 is tolerated.

I More important is the fact that the simul-7-taneous addition of acetic acid and KBH4 (as proposed by Schaeffer) makes the reaction very difficult to control. The ' temperature raises considerably and losses in yield and/or quality of the product occur.

It is recommended to use an efficient ( stirring to insure the proper reaction which 'will be completed when all the small lumps and portions of potassium boro-hydride have disappeared.

Cooling at 0°, as described by Schaeffer et al (Method D, line 4 and ff.) is insufficient. It is an improvement to keep the reaction well below 0°; it is best to keep it below -10° all the time. If the temperature is allowed to go over -10°, substantial loss in yield may result. 1 99643 i ERYTHRO-5-AMINO-4-CHLORO-6— (2-HYDROXY-3-NONYLAMINO)PYRIMIDINE (VI) ••• CI OH +CH, -[CH ]e-CH-CH-CH 3 2 5 NH,- / \ CH— [CH ] * CHOH i 2 5 CH3f V IV VI A mixture of 4, 6-dichloro-5-aminopyrimidine (V, 24.6, 0.15 mole) and eirythro-3-amino-2-nonanol (IV, 26.2 g, 0.164 mole) in 1-pentanol (1.080 ml) and tributylamine (350 ml) was prepared with stirring at 25°. The resulting suspension was heated to reflux under nitrogen atmosphere for 28 hr. (solution took place in about 1/2 hr.). At that time a sample of the reaction product showed a uv X max 267 and 297 nm (H20, pH 5.5).

The resulting solution was concentrated in a hot water bath at 10 mm pressure to a syrup and further- evaporated in an oil bath at 0.1 mm and 100° to 1 99643 yield a viscous liquid to which n-hexane (450 ml) was added. The mixture was refluxed for 1 hr., and the hot, yellowish hexane supernatant was separated from the liquid at the bottom of the round bottom flask.

The resulting light brown oil from which any residual hexane was evaporated _in vacuo and dissolved in chloroform (150 ml). This chloroform solution was extracted 8 times with an aqueous saturated solution of NaHCC>3 (250 ml each time). The chloroform layer 10 was then separated, dried (with sodium or magnesium sulfate) and evaporated under high vacuo (0.1 mm) at 40° (water bath) to give a light brown oil which solidified on cooling. This material can be used j directly in the next step or purified as follows: The 15 resulting oil was dissolved in 75-100 ml chloroform and n-hexane (ca. 300 ml) added to precipitate out a white crystalline solid which was filtered from the cooled solution. (Extraction is carried out 4-8 times, until carbon dioxide is no longer evolved.) 20 This treatment was repeated two more times. Yield: 23.3 g (54%) uv Amax 267, 297 (H20,: pH 5.5) mp 113-116°. 199643 -3U- ERYTHRO-9-(2-HYDROXY-3-NONYL)6-CHLOROPURINE (VII) ^2 (OC2H5)3CH 100% CH—- [CH] / \lH0H D C ? j CH.

VI VII The crude syrup from the preceding operation consisting of erythro-5-amino-4-chloro-6-(2-hydroxy-3-nonylamino)pyrimidine (11.48 g, 40 mmol.) was dissolved in triethylorthoformate (106 ml) and chloroform (34 ml), ethanesulfonic acid (10 drops) was added to effect solution. After standing overnight at 25°, the solution was evaporated to a syrup under vacuo. Yield 11.7 g (quantitative). This syrup consisting of crude erythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) was used in the next step. A Max- 264 nm. 199643 ERYTHRO-9-(2-HYDROXY—3-NONYL)HYPOXANTHINE (VIII) (By hydrolysis of the 6-chloropurine derivative) OH 7 [CH ] _ CHOH , CHj chch VII VIII A suspension of erythro-6-chloro-9-(2-hy-droxy-3-nonyl)purine (VII, 4.0 g, 13.4 mmol) in 0.5 N' NaOH (40 ml) was refluxed for 2 hr. and cooled. Neutralization with glacial acetic acid and cooling gave a crystalline precipitate of erythro-9-(2-hydroxy-3-nonyl)hypoxanthine (VIII) which was filtered and dried. Yield: 3.8 g(guantitative) , m.p. 196° uv Amax (pH 5.5) 251 nm. —36- 199643 The crude product (VIII) thus obtained was homogeneous by paper chromatography (3 solvents) and gave negative test for CI- (copper wire and flame; sodium fusion, acidification and silver nitrate).

Recrystallization of a sample of the crude material 3 times from aqueous ethanol (see Purification) gave colorless crystals, m.p. 202°. Calc. for C14H22N4°2 (VIII): C, 60.41; H, 7.97, N, .13. Found: C, 60.47; H, 7.86; N, 20.08.

% I PURIFICATION OF / ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII) i The crude nonyl hypoxanthine (VIII) is purified by recrystallization. The crude material is dissolved by heating in about 6-10 times its weight in 15 ethyl alcohol, and then an equal volume of H2O is added. The solution is treated with charcoal in an Erlenmeyer and filtered through celite when hot. The I solution is evaporated with continuous stirring on a hot plate.- Water is added in small portions to re-20 place the evaporated volume until an abundant precipitate appears. Keep on evaporating the solvent to remove all the ethyl alcohol while adding repeatedly H2O to reach a volume of 8-12 times the weight of material. The loss in material is about 10% per each 25 recrystallization. Two recrystallizations raised the melting point to 202° and gave a colorless crystalline product while the crude material was somewhat yellow or pink and melted at 192°. 199643 ERYTHRO-9-(2-HYDROXY-3-NONYL)-ADENINE. HC1 (IX) "| • HC1 CH CH^— [CH2] / NcHCH CH^ VII IX The crude oily erythro-9-(2-hydroxy-3-nonyl)-6-chloropurine (VII) (6.15 g) from the preced-5 ing preparations is dissolved in saturated methanolic ammonia (300 ml) and ammonium chloride (1 g) at 80-100° for 1 hr. in a stainless steel bomb (Parr Instruments). After cooling, the solution was evaporated to dryness _in vacuo. Methanol was added and evaporated 10 again (3 times) to eliminate the excess of ammonia.

The syrupy residue was dissolved in absolute methyl alcohol, and dry HCl gas was bubbled, keeping the temperature below 20° (with an ice water bath). . After passing HCl for 1/2 hr., the mixture was cooled 199643 at 5°. The precipitate was collected through a sintered glass funnel, washed with cold methyl alcohol and dried in air.. Yield 6.0 g (92%) m.p. 173-175° dec. uv Amax 260 nm (in H20, pH 5.5).

ALTERNATE ROUTE FOR THE PREPARATION OF ERYTHRO-9-(2-HYDROXY-3-NONYL)HYPOXANTHINE (VIII) / I (By deamination of VII) ch J— [CH0]^ vii viii Sodium nitrite (5.6 g, 71 mmole) was added slowly to a solution of erythro-9-(2-hydroxy-3-nonyl)-adenine (IX, 4.0 g, 14 mmole) in 50% acetic acid (20 ml) and n HCl (3.2 ml) at 25° with stirring. The mixture was stirred for 2 hr. at 25°. After this time, 199643 UV spectrum is monitored. When UV max reached 250 mm, the solution was neutralized with 2 N NaOH. The resulting precipitate was filtered and washed with H20. Yield = 3.03 g (75%) m.p. = 195°.

An analytical sample was recrystallized (3x) from water yielding a product m.p. 202°. Anal. Calc. for C14H22N402: C, 60.40; H, 7.96; N, .13. Found: C, 60.40; H, 7.90; N, 20.12.

Method K ' COMPOUND NPT 154 26 There was used the procedure of H.J. Schaeffer and S.F. Schwender, J. Med. Chem. _17:6 ' (1974).

Method L PREPARATION OF NPT 15410 I ; 0.1 mmoles of 9-(2-hydroxy-3-nonyl)-6-hy-droxy purine, NPT 15392 (27.9 mg) and 0.3 mmoles of 2-hydroxypropyl, dimethylammonium 4-(acetylamino)ben-zoate (DIP•PAcBA) (77.1 mg) were accurately weighed 20 and dissolved in 105 ml of 0.25% sodium carbonate (NaC03) to yield a 0.1% solution of NPT 15410 (the compound formed from NPT 15392 and (DIP-PAcBA) in a 1:3 molar ratio).

EVIDENCE FOR COMPLEX FORMATION 25 Phase solubility studies carried out with NPT 15392 and DIP*PAcBA demonstrate that NPT 15392 has increased solubility at increasing concentrations -4Q- 19 9643 of DIP-PAcBA under conditions of constant pR. This is indicative of an interaction occurring in solution to yield a complex.

In place of th.e mole ratio of 1:3 (NPT 15392 and DIP-PAcBA), other complexes are formed by using mole ratios of 1:1 and 1:10.

Antiviral activity is shown in Tables 2 and 3.

Table 2 INHIBITION OF INFLUENZA VIRUS REPLICATION BY 9-(HYDROXYALKYL) PURINES (Y).

I CH-CH-R^ II Ah Viral Strain: USSR/90 (H-^) I Inf luerxz a A Test Compound % Inhibition of Henad-sorption Foci Cone, (pg/ml) Test Compound Cpd No.

R1 Rl- "X Y <10 -100 ?ioo 15425 H H CH — — I / — 15428 H : H CH DIP-PAcBA - - - 15435 H H SH - 50 15437 H H SH DIP-PAcBA 65 65 15446 H CT3 CH - 2 0 0 15447 H CH3 CH DIP-PAcBA 26 34 15431 H ch3 NH2 - 22 0 15432 H CH3 nh2 DIP-PAcBA 48 62 15427 CH3 H 1 - 15423 H CI - 2 13 6 15433 ca3 H nh2 - 32 0 15434 ay H NH2 DIP-PAcBA 41 62 154 43 ch3 H CH - 0 0 15444 H OH DIP-PAcBA 44 54 15417 C6H13 H CH - 18 58 60 199643 Table 2 (cont.) Test Cpd No. r1 r2 X Y <ao -100 >100 15418 c6h13 h oh DIP•PAcBA 16 46 52 15392 c6h13 ch3 oh - 86 100 100 15410 c6h13 ch3 oh DIP * PAcBA 58 96 96 15426 c6h13 ch3 nh2 - 50 96 100 15110 - - DIP'PAcBA 0 0 Table 3 INHIBITION OF HERPES VIRUS REPLICATION BY 9-(HYDROXYALKYL) PURINES (Y) - R NPT No.

Compound jpiaques (PFU) Percent Inhibition r1 r2 X Y Test Control 15392 c6h13 CH3 OH — 98% 15417 <=6*13 CH3 OH 15418 c6*13 H OH DIP • PAcB2 15410 c6h13 H OH DlP-PAcBi1 98% 199643 BIOLOGICAL ACTIVITY Methods Anti-Influenza Activity - (Hemadsorption Assay) Upon infection of a monolayer of tissue culture cells by influenza virus, the cell surface is altered so that guinea pig erythrocytes can be adsorbed to the cell surface. The number of foci of adsorbed cells (hemadsorption foci forming units HAFFU) is a quantitative measure of infectivity. The method is as follows.

The- monolayers were subcultured in the following manner: The medium was poured off, and the monolayer washed two times with approximately 50 ml per wash of calcium and magnesium free phosphate buffered saline (PBS), (GIBCO #419) at a pH of 7.2. One ml of trypsin-EDTA solution (GIBCO #530L) containing 0.5 g trypsin(1:250) and 2.0 g EDTA/liter of Modified Puck's Saline A was added at 37°C to each flask and dispersed over the monolayer with gentle shaking. The flasks were then placed in an incubator at 37°C. for approximately 3-5 minutes depending on the time required to dislodge the cells. Occasional shaking was required. Ten ml of planting medium was added to each flask and the cells dispersed by aspirating and expelling the suspension from the pipette. The contents of a series of flasks were pooled and the cells in the suspension were diluted with planting medium to 7-8.5 x 10^ cells/ml. The planting medium consisted of the following composition: Minimum Essential Medium Eagles (MEM) with Earle's salts and HEPES buffer (GIBCO #236) supplemented by adding the following substances as specified to 87 ml of MEM: 199643 ml of fetal calf serum (FCS-GIBCO #614HI) 1 ml of L-glutamine (200 Molar-GIBCO #503) 1 ml of Chlortetracycline (5000 g/ml) 5 GIBCO #528) 1 ml of 10,000 units penicillin, 10,000 g streptomycin and 10,000 neomycin mixture (PSN-GIBCO #564) The cells were subcultured into Linbro tis-10 sue culture trays. The trays consisted of 24 flat bottom wells each with a 3 ml capacity per well; the cell culture suspension (1 ml) was added to each well.

The following day the medium was removed and replaced with fresh planting medium. The monolayer^ 15 were used for experimentation when they reached a condition in which they were almost confluent (approximately 3-4 days).

When the Linbro tray HeLa cell cultures were ready for experimentation (see cells), the medium was 20 decanted and 1 ml of maintenance medium (MEM with FCS reduced to 3%) containing the compound being tested at a given concentration was added to 4 replicate cultures within a tray.

A series of different drug concentrations 25 ranging from 2.3 to 150 g/ml were used. Maintenance medium alone was used for control cultures. After the administration of drug and control medium, 0.1 ml of the diluted viral suspension was added to experimental groups and infected control cultures. Saline alone .1 99643 was added to non-infected control cultures. The Linbro trays were then incubated at 37°C. for 18 hours, af^er which media in all groups was aspirated. Each culture was washed once with PBS. The saline was 5 aspirated and 0.5 ml of a 0.4% v/v guinea pig red blood cell suspension in PBS was added to each culture well. The cultures remained at room temperature for 30 minutes after which the medium was decanted and culture washed 2 times with PBS to remove all but the 10 specifically bound red cells. After the third wash, maintenance medium was added to all cultures.

A Howard Micrometer eyepiece (C8385) was inserted within the ocular of a Nikon inverted phase contrast microscope. Each culture was scanned with'a 15 4 x low paper objective and direct counts of hemad- sorbed red cells were counted using the eyepiece grid as a field marker. Partial or complete fields were counted per experimental group depending on the resulting number and density of hemadsorbed cells in the 20 infected control cultures. Magnification of 60 x or 150 x were chosen to obtain the best conditions for enumerating the hemadsorbed cells. Field factors were calculated for counting hemadsorption at 60 x and 150 x.- At 60 x magnification, total field count was 25 calculated using a multiplication factor of 55.5. At 150 x magnification the multiplication factor was 273. The multiplication factors of 55.5 and 273 represent the total number of fields at 60x and 150 x magnifications, respectively. The number of fields counted 30 ranged from 3 to 5 per well with 3 to 4 wells per treatment group employed (see, raw data tables in 1 996 results section for number of fields examined). Means and standard errors were calculated and the data was evaluated using student's t-test analysis.

BIOLOGICAL ACTIVITY 5 Anti-Herpes Activity - (Plaque Assay) The infection of tissue culture cells by Herpes virus causes cell lysis. After a period of time these lysed cells are visualized as a tiny clear area (plaque) on a layer of cells. The incorporation 10 of a test substance into the media will reduce the number of plaques if it is capable of preventing virus replication. The method is as follows: f MATERIALS AND METHODS Virus There was employed herpes hominis type 2 ; purchased from American Type Culture Collection (ATCC), Bethesda, Maryland, ATCC #VR 540, Lot 3D. The lypothilized viral suspension was reconstituted with 1 ml sterile distilled H2O. The virus was passed 20 twice through HeLa-cell monolayers. The tissue-culture supernates were pooled, dispensed in 1-ml ali-quots, and stored at -70°C. The titer of this work-ing-stock suspension was found to be 10"^ TCID5Q/O.I ml (2 days' incubation). 199643 —46— Herpes Virus Plaque Assay Vero cells in log-growth phase were subcul-tured at a concentration of 1x10^ cells/ml in 50-ml Falcon flasks in Eagle's Minimum Essential Medium (MEM), supplemented with 10% fetal calf serum (FCS) and antibiotics. Media were changed the day following planting. The Vero monolayers reached confluence by the second day after planting and with the cells in log phase, the cultures were used for the plaque assay.

Culture media were poured off and the mono- , layers were washed once with phosphate-buffered saline (PBS). Several different dilutions of the working-stock virus suspension were prepared and each cultute flask was infected with 0.5 ml of one of the virus dilutions added to FSC-free medium. This medium contained drug at a concentration of 150 yg/ml. Controls were prepared with medium devoid of drug.

Virus adsprption was allowed to proceed for 2 hours at 37°C, during which time the cultures were rocked gently every 15 minutes. Then Media were poured off and the monolayers were washed once with 10 ml PBS.

Agarose was prepared at a concentration of 6% w/v in 50 ml PBS. A stock medium of MEM supplemented with 2% FCS was prepared. Drug was added to some of the stock medium at 150 yg/ml. The three solutions were maintained at 47°C. In addition, a 1:10 dilution of pooled human anti-herpes sera was readied. Just before the start of treatment, 15 ml of the agarose solution was added to 85 ml of medium. Another 15 ,ml of agarose were added to 85 ml of drug-medium. 1 99643 Each of the washed monolayers in one group of experiments was treated either with 5 ml of aga-rose-medium or with 5 ml of agarose-drug-medium. In another group of experiments, each monolayer was treated either with 0.2 ml of anti-herpes sera in 5 ml of stock medium, or with 0.2 ml of anti-herpes sera in 5 ml of drug-medium. The anti-herpes sera were used in place of agarose to localize plaques by neutralizing any free virus in the medium. The flasks were allowed to remain at room temperature for 5 minutes, after which they were incubated at 37°C for 2 days. Triplicate cultures were used for most treatment groups.

Ten ml of PBS. then were added to each flask. Overlays were shaken gently and then were poured out of the flasks. The monolayers were stained with a solution 0.5% w/v crystal violet in 50% methanol in triple-distilled H2O.

Plaques were counted either directly by transmitted fluorescent light and macroviewing, or by the use of light microscopy for microplaques. Micro-plaques were counted by averaging three fields per experimental group under 150x magnification.

In other tests of antiviral activity the following results were obtained. -48 Compound % Inhibition at yg/ml 0.1-1.0 1.0-10 10-100 >100 15392 30-50 — >70 >70 15417 50-70 >70 15418 >70 >70 15426 15410 -30 30-50 50-70 50-70 50-70 30-50 >70 >70 .1 99643 Virus Influenza A Swine 1976 (Hlsw~Nl) Influenza A Swine 1976 (Hlsw-Nl) Influenza A Swine 1976 <Hlsw-Nl> (Russian) (Swine) t Additional antiviral activity tests of Compound NPT 15410 are shown in Table 3a. 199643 Table 3a INHIBITION OF INFLUENZA.VIRUS REPLICATION BY NPT 15410 Virus Concentration Range (pg/ml) .01-1.0 1.0-10.0 j10.0-100 >100 A/Swine//76 (H2swNl^ +++a ++ i ++++ ++++ A/Texas/77 (H3N2) ++ + ++++ ++++ A/Dunedin/73 (H3N2) NT NT t + ++ A/Jap/305 (H2N2) NT NT ++++ ++++ A/PRg (HqN1) ++ ++ ++++ +++ : 1 A2 Hong Kong (H2N2) NT NT ++ +++ a NT = Not tested + = 10-20% Inhibition + = 20-30% Inhibition ++ = 30-50% Inhibition +++ = 50-70% Inhibition ++++ == >70% Inhibition Immunomodulation activity is shown in Table 4. 199643 50 Table 4 MODULATION OF CELL MEDIATED IMMUNITY BY 9-(HYDROXYALKYL) PURINES Compound . ' npt no.

R1 R2 X ... y . 15425 h h oh — 15428 h h oh DIP•PAcBa 15435 h h sh f 15437 h h sh DIP-PAcBA 15446 h ch., oh - 15447 h ch3 oh Dip-PAcBA 15431 h CH3 nh2 - 15432 h ch3 , nh2 DIP•PAcBA 15427 ch3 h I - 154^3 ch3 h ci . _ 15433 ch3 h nh2 - 15434 ch3 h nh2 DIP -PAcBA . 15443 CH3 h oh - 15444 ch3 h oh DIP•PAcBA 15417 C6H13 h oh - 15418 <=6H13 h oh DIP•PAcBA 15392 c6h13 ch3 oh - 15410 C6H13 ch3 oh DIP * PAcBA 15426 C6H13 CH3 nh2 NPT No 15425 15428 15435 15437 15446 15447 15431 15432 15427 15423 15433 15434 15443 15444 15417 15418 15392 15410 15426 -51' .1 99643 Maximum Percent Change Mitogen Induced (Con A) Mouse Lymph. Prolif. 11 0 100 0 0 55 45 -50 6 12 0 0 0 0 -15 -27 -47 0 0 / 0 +15 -23 -65 53 0 -50 +17 +27 + 27 0 +13 0 0 0 0 0 0 -50 41 73 26 1 172 162 -72 11-15 -50 140 40 40 -50 60 -50 -85 -91 6 -41 Mitogen Induced (PHA) Human Lymp. Prolif. .01-1.0 1.0-10 10-100 199643 Maximum Percent Change (Cont.) NPT No.

Lymphokine Induced (MMF) Guinea Pig Mac. Prolif. .01-1.0 1.0-10 10-100 15425 15428 15435 15437 - 15446 0 0 0 15447 15431 15432 15427 15423 15433 . 15434 f 15443 + 20 0 15444 15417 13 -50 15418 12 80 -50 15392 33 23 15410 12 23 15426 - * 199643 Several compounds were tested for Mitogen Induced Murine Lymphocyte Proliferation with the following results: Compound % Stimulation at y g/ml 01-1.0 1.0-10 -100 >100 15392 >50% -50% -50% not tested 15426 0 0 0 not tested 15410 >50 -50 -50 not tested 15417 o . 0 0 0 15418 -30 >50 -30 not tested BIOLOGICAL ACTIVITY r Immunomodulating Assay The following three assay procedures are used to evaluate the ability of the test substances to modulate the activity of several classes of cells in the immune'system. In these systems it is possible to identify both immunopotentiating activity (evidence by an enhancement of the parameter examined) as wel}. as immunosuppressant activity (evidenced by an inhibition of the parameter examined). ( , 1 99643 1. Mitogen-Induced Mouse Spleen Cell Assay Mouse spleen cells contain a population of both B and T lymphocytes which can be stimulated by a number of foreign substances (e.g., plant mitogens 5 such as Con A) to proliferate. This enhanced proliferation is an indication of enhanced cell mediated immunity. The method below describes the system used to evaluate test substances as immunopotentiators.

MATERIALS Concanavalin A (Calbiochem, La^'Jolla, California), Lot #210073, lyophilized in NaCl, was prepared first as a 1% solution and diluted as a 2X concentration for each dilution (0.5, 1.0, 2.5 yg/ml^) .

Animals Six to eight week old male Balb/c and C3H inbred mice were obtained from the following sources: Flow research Animals, Inc., Dublin, Virginia; Charles River Breeding Laboratories, Wilmington, Massachusetts; Laboratory Supply Company, Indianapolis, Indiana; and 20 Lionel Strong Foundation, San Diego, California.

Cells Three to five mice were sacrificed by cervical dislocation and the spleens aseptically removed. Pooled spleens were minced and teased with sterile 25 forceps; then strained through a double layer of nylon mesh. The cell suspension was washed once with 15 ml of RPMI 1640 supplemented with 5% fetal calf serum and antibiotics. Cells were cultured at a concentration of 106 cells/0.1 ml/well in micro-plates. Cultures 1 99643 were incubated in the presence or absence of mitogen in a humidified atmosphere containing 5% C02 for 48 hours. The test compound was added to cultures at various concentrations concommitant with mitogen.

Proliferation Proliferation was assayed by the degree of incorporation of 1.0 Ci of [3H] thymidine over an 18 hour incubation period. Cultures were harvested by a MASH unit (Otto Hiller Co., Madison, Wisconsin) and 10 thymidine incorporation was assayed by liquid scintillation spectrometry. Cultures were performed in triplicate and data are expressed as means plus or minus 1 the standard error of the experimental means. Drug stimulation indices over control values were also r calculated and portrayed graphically. 2. Mitogen Induced Human Peripheral Blood Lymphocytes - A clinical need exists for therapeutic agents to augment the immune response in patients with deficient or depressed immune states, such as exists 20 in viral diseases or cancer* By studying the ability of agents to augment the proliferation of human peripheral blood lymphocytes in response to a foreign substance one can identify agents with immunopotentiating activity in man. The procedure is that just set forth 25 and that also described by Hadden, J.W., Infect. & Immunity, February, 1976, pages 382-387, especially pages 382-383. 1 99643 3. The macrophage represents a subpopula-tion of white blood cells which is an important component of the immune system in control of both cellular and humoral immunity. The assay system described 5 below evaluates the substances studied as potentiators of macrophage function.

Phytohemagglutinin (PHA) (HA-17) was purchased from Burroughs Wellcome. A preparation containing Macrophage Mitogen Factor (MMF) and Macrophage 10 Activating Factor (MAF) was prepared from antigen-stimulated immune lymph node lymphocytes (guinea pig) as previously described by Hadden et al, Nature 257, 483-485 (1975). Partial purification of this preparation by vacuum dialysis and sephadex G-100 column chroma^o-15 graphy yielded an active.fraction in the range of 35-70,000 daltons exhibiting both mitogenic and activating properties. The active fraction was employed in both the proliferation and activation assays.

' I Methods Ficoll-hypaque purified humah peripheral blood lymphocytes were prepared and PHA-induced lymphocyte proliferation was assayed by the incorporation of tritiated thymidine as described in Hadden et al, Cell. Immunol. 2Q_, 98-103 (1975). Each compound was 25 analyzed in the presence of suboptimal, optimal and supraoptimal concentrations of PHA (.001, .01, 0.1 units/ml respectively). Parafin oil-induced guinea pig peritoneal macrophages were prepared and incubated as monolayer culture (>98% pure macrophages). Lympho-30 kine (MMF)-induced proliferation was assayed by the 1 99643 incorporation of tritiated thymidine at 3 and 5 days of culture as described, Hadden et al, Nature 257, 483-485 (1975). Lymphokine (MAF)-induced macrophage activation to kill Listeria monocytogenes following 5 5 days of culture in the presence or absence of MAF was performed during a 6 hour period as described in Hadden and England, Immunopharmacology, pages 87-100 (Plenum Press, 1977). Phagocytosis was quantitated during a 20-minute exposure to Listeria monocytogenes 10 by counting the number of macrophages containing bacteria and the'number of bacteria per phagocytic cell on gram stained monolayers in Labtek chambers. Intracellular killing of bacteria was evaluated by counting the number of cells containing bacteria and the number 15 of bacteria/cell 6 hours- after the initial 20 minute exposure. Parallel experiments in which macrophages were lysed and intracellular bacteria were cultured confirm the validity of bacterial activity determined by this manner in this system. The drugs were em-20 ployed in each of the three systems over serial log concentration range in triplicate in the presence and absence of mitogen or lymphokine. Each type of experiment was performed at least three times. Previous experiments indicate a parallelism of response to 2 5 pharmacologic modulation in the proliferation and activation assays. 199643 BIOLOGICAL ACTIVITY Anti-Leukemic Activity (Inhibition of L-1210 Growth Leukemic cells isolated from mice bearing 5 the L-1210 tumor are cultured _in vitro and their growth can be measured by counting the number of cells in the culture over a period of time. The incorporation of a test substance into the media will prevent the growth of the leukemic cells, an indication of an 10 effective anti-leukemic agent. 7 I50 (concentration of drug inhibiting growth of L-1210) by % for the tested compounds was as follows: I Concentration r Compound . (micrograms/ml) 15392 28 15410 54 15417 47 15418 70 The assay system used is set forth below.

To Measure Inhibition of Leukemic Cell (L-1210) Growth Check to see that there is adequate cell growth in the stock cultures. Use cells 48-72 hours 25 after transfers are done. 199643 Weigh out the drugs at 50 times the desired final concentration and made serial dilutions.

Make up the final medium using 500 mis McCoy's 5A medium, 15% fetal calf serum, 5 mis penici-5 11 in-streptomycin solution, and 5 mis antibiotic-anti-mycotic solution and let it stand at room temperature.

Using sterile technique, add 0.1 ml of the drug dilutions to each tube.

Add an appropriate quantity of cells to the 10 prepared medium. After mixing, remove a 0.5 ml sample, place it-in a vial containing 9.5 mis of saline, and count it on the Coulter Counter. Multiply the count by 40 to compensate for the 40 fold dilution (0.5 ml into 0.5 ml saline and record the inoculum)^ 15 Add 5 mis of cell suspension to each tube.

Swirl the bottle every 4 tubes to insure a more uniform distribution of cells.

Tighten the caps and place in the CC>2 incubator at 36-38° for 96 hours.

After 96 hours remove the tubes from the incubator and count the contents of each on the Coulter Counter. Multiply all counts by 40 and average the four counts for each drug dilution. If the count is less than the inoculum, record 100% inhibi-25 tion. If the count is greater than the average of the eight control' counts, record 0% inhibition. For all other counts use the following formula: 1 99643 Average cells/ml in treated cultures - inoculum in cells/ml .qq Average cells/ml in control cultures - inoculum in cells/ml 100% - % survival = inhibition of growth due to treatment.

The subject compounds of this invention have been shown to inhibit the replication of a representative sample of both RNA and DNA viruses using stan- , dard tissue culture techniques. In the case of the i RNA viruses, Several strains of influenza virus belonging to both the A and B sub-types were shown to be inhibited, using the hemadsorption technique (Section II, B). The specific compounds found to inhibit influenza virus replication (Type A/USSR 90) are shown in Table 1. Several members of the Series NPT 15392, NPT 15410, NPT 15417, and NPT 15418 were shown to inhibit the replication of at least 4 different strains of influenza virus at concentrations ranging from 1-150 yg/ml.

In addition, several members of the Series, NPT 15410 and 15392, have been shown to inhibit the replication of Herpes Simplex virus, a member of the DNA class of viruses and a virus responsible for severe mucocutaneous lesions in man, along with the fatal Herpes encephalitis. Other members of this class of viruses are responsible for hoof and mouth disease in swine and cattle and infections rhinotra-cheitis in cats and kennel cough in dogs. Even = % Survival I 996 concentrations less than 100 yg/ml of NPT 15392 and 15410 were found to reduce plaque formation caused by Herpes Simplex virus to an extent of >90%. Other members of the RNA and DNA class of viruses are shown in 5 Table 5 and are responsible for the diseases specified. Of all the diseases in the world at least 25% are known to be caused by viruses. In addition, a number of viruses have been isolated that are shown to produce tumors. Thus, antiviral agents may be expec-10 ted to, by themselves, have some antitumor properties.

It is an established fact that many infectious agents, such as viruses (influenza virus, HSV, Friend leukemia virus), bacteria and fungi cause an immune suppressed state in the host, weakening his f 15 defenses to infection by. infectious agents. Most other antiviral antimetabolite substances, like AraC, cause a suppression of host immune defense mechanisms, thereby exhibiting potential to lessen the body's own natural defense mechanisms and enhance secondary in-•2-0 fection. ' An immunopotentiator or immunomodulator is any agent which either restores depressed immune function, or enhances normal immune function, or both. Immune function is defined as the development and ex-25 pression of humoral (antibody-mediated) immunity, cellular (thymocyte-mediated) immunity, or macrophage and granulocyte mediated resistance. It logically includes agents acting directly on the cells involved in the expression of immune response, or on cellular or 30 molecular mechanisms which, in turn, act to modify ( I 9 964 the function of cells involved in immune response. Augmentation of immune function may result from the action of an agent to abrogate suppressive mechanisms derived by negative-feedback influences endogenous or 5 exogenous to the immune system. Thus, immune potentiators have diverse mechanisms of action. Despite the diversity of cell site of action and biochemical mechanism of action of immunopotentiators, their applications are essentially the same; that is, to 10 enhance host resistance.

Applications of Immunopotentiators 1) The principal protective function of the immune system relates to resistance to invasion by pathogens, including viruses, rickettsia, mycoplasma, bacteria, fungi, and parasites of all types. Thus, improvement of immune response, particularly when depressed, would calculatedly improve resistance in infection or infestation by any of the above pathogens. An immunopotentiator alone or in combination with I apti-infective therapy can be applied to any and all infectious diseases. 2) A second protective function of the immune system is thought to be resistance to engraft-' ment of foreign tissue, either natural as in the fetal-maternal relationship; or unnatural as performed by the transplant physician. Immunopotentiators can also be used to facilitate rejection of fetal or placental tissues or to modify or induce tolerance to grafts. ^ 199643 3) A third protective function of the immune system is thought to be resistance to malignant cell development as in cancer. The use of immunopo-tentiators can be used in cancer treatment to enhance tumor rejection and to inhibit tumor recurrences following other forms of therapy. 4) A fourth protective function involves the capacity to recognize foreign-ness and to maintain non-reactivity to self by positive suppressor mech- anisms. In auto-immune and related disorders, immune reactivity directed at self antigens or exaggerated, elevated responses are apparent which are self-destructive. Immunopotentiators can be used to restore I normal suppressor mechanisms, induce tolerance, or t 15 otherwise promote a normal immune response.

Each of the protective functions of the immune system can be modified by non-specific therapy with immunopotentiators alone or in combination with other agents employed to improve resistance or to kill 20 the invading pathogen. In addition, specific resistance can be: augmented by use of immunopotentiators in conjunction with some form of antigen as in a vaccine employing, for example, virus, tumor cell, etc. This use can be to induce either specific immunity or tol-25 erance. The latter might be exemplified by use with antigen in allergy or auto-immune diseases. Use of immunopotentiators may be either therapeutic or prophylactic; the latter particularly in aging, where infection, auto-immunity, and cancer are more common. 30 The timing of administration and routes are variable and may be critical in determining whether a positive 199643 or negative response results. Any agent capable of augmenting immune response may inhibit it depending on timing and dose; thus, under certain circumstances an immunopotentiator could be used as an immunosuppres-5 sive agent for use in allergy, auto-immunity and transplantation.

Table 4 above presents the results of an evaluation of a number of these subject compounds as potentiators of the immune response. Three different 10 test systems were used. The first involves a measure I of the ability of the test compound to enhance the ability of mouse lymphocytes to proliferate in response to a plant mitogen (Con A). The second involves measuring the ability of the test compounds to enhance 15 human lymphocyte proliferation in response to a second plant mitogen (PHA). The third system measures the ability of these test substances to enhance macrophage proliferation in response to a natural lymphokine (MMF, Macrophage Mitogenic Factor). This latter res-20 ponse, the proliferation and activation of macrophages, has been shown to :be involved in the killing of bacteria, viruses and tumor cells by this class of white blood cells.

Significant potentiation of the immune res-25 ponse has been observed by 15392, 15410, and 15418.

Finally, the activity of several of these agents, NPT 15392 and 15410 as inhibitors of the growth of abnormal lymphocytes has been determined. Notably,, both substances are capable of inhibiting the 30 proliferation of mouse leukemic lymphocytes (an L-1210 (996 cell line) in tissue culture. A 50% inhibition of L-1210 cells was effected by NPT 15392 at 28 yg/ml and by NPT 15410 at 54 yg/ml. The ability to inhibit leukemic lymphocytes at concentrations that stimulate 5 normal lymphocytes is a unique property not known to be present in any other class of substances.

The products of the present invention are members of a class of substances, which specifically inhibit the replication of RNA and DNA virus, modulate 10 (potentiate) the immune response and inhibit the I growth of leukemic lymphocytes. Based on _in vitro experiments, which demonstrate activity over a concentration range of 0.01-150 yg/ml, dose ranges effective in mammals are 0.05-500 mg/kg. A lack of toxicity Jias 15 been noted at levels of 1,500 mg/kg in mice for certain numbers of this series.

The immunopotentiators of the invention can be employed, for example, to provide resistance to invasion by the viruses in Table 5. 199643 Virus Arenavirus Influenza 5 Rhinovirus Poliovirus Measles Newcastles Disease Virus 10 Rotavirus Hepatitis Type A Rabies virus ! Arbovirus Vaccinia virus 15 Herpes Simplex Virus Herpes Zoster Varicella Zoster Adenovirus 20 Hepatitis Type B Hoof and Mouth Disease virus Machupo Virus Table 5.

Class Disease RNA Rift Valley Fever RNA Influenza RNA Common Cold RNA Polio RNA Rubella RNA Newcastles disease RNA Gastroenteritis in infants RNA Infectious'Hepatitis RNA Rabies RNA Encephalitis DNA Smallpox DNA Cold sore, Encephalitis, Venereal Disease DNA Shingles DNA Chicken pox DNA Respiratory DNA Chronic Hepatitis, Severe Hepatitis DNA Hoof and Mouth Disease Hemorrhagic Fever 1 9 9643.

POTENTIATION BY DIP PAcBA OF BIOLOGICAL ACTIVITIES Of the substances described in Table lf NPT 15392 and NPT 15446 are new compounds described and .. .. claimed in New Zealand patent specification 191,4 82.

Also new are the DIP.PAcBA salts presented in this table, namely 15428, 15437, 15447, 15432, 15434, 15444, 15418 and 15410. NPT 15392, NPT 15417, NPT 15426 have all been shown to have significant anti-influenza activity by themselves. In one instance (with NPT 15392) the addition of DIP'PAcBA salt to NPT 15392 to form 15410 does not potentiate the anti-influenza activity/ In the case of NPT 15417, addition of DIP•PAcBA salt to form 15418 does potentiate the anti-influenza activity. A summary of the relative ability of DIP-PAcBA salts to potentiate the different biological activities is set forth below.

Table 6 i L DIP'PAcBA Potentiation Immuno-Compound Salt Anti-Influenza Anti-Leukemia pote'ntia- tion 15392 15410 both are equally Yes Yes active 15417 15418 Yes - Yes 15435 15437 Yes - - 15446 15447 Yes - - 15431 15432 Yes - - 15433 15434 Yes - - 15443 15444 Yes 199643 FORMULATIONS The compounds of the present invention can be fed to a mammal at a dosage of 1-1000 mg/kg of body weight and are believed to be active at levels as low 5 as 0.05 mg/kg. The LD^q as determined in mice of NPT 15410 given intraparenterally was 4,300 mg/kg, while subcutaneously was 4,900 mg/kg. NPT 15392 has been given to mice at doses of 1000 mg/kg and no drug related mortality was noted.

They can be administered in tablet or cap sule form to Humans and where solubility permits in the form of syrups or injectable solutions or where insoluble as suspensions. Typical pharmaceutical formulations are described below: r Capsule: NPT 15392 50-500 mg.

Avicel pH 101 (microcrystalline cellulose) to make 800 mg.

Suspension: Aqueous suspensions can be made with a number of suspending agents incorporated with the active drug substances. Included as suspending agents are such substances as sodium carboxymethylcellulose, Na alginate, gum tragacanth, Avicel RC-591 (micro-cellulose), methylcellulose, Veegum, Xanthan gum. In addition to a suspending agent such substances as sweeteners, flavors, colorants, preservatives, protec tive colloids and dispersants may be added. 1 TABLET FORTIULATION NPT 15392 50-500 mg I Avicel pH 101 130 mg Starch, modified 20 mg Magnesiumm stearate U.S.P. 5.5 mg Polivinylpyrrolidone 22 mg I Stearic acid U.S.P. 30 mg 19 SYRUP FORMULATION NPT 15392 -125 mg (or at maximum level of solubility) Corn Sugar Distilled Water 3.25 g .05 g FD and C Red 40 .00175 g Sodium Saccharin .00250 g Alcohol U.S.P. .08 g Methyl paraben U.S.P. .005 g Propyl paraben U.S.P. ^ .001 g Glycerin .31225 g Cherry flavor .00825 g Fruit flavor .00825 g Distilled Water g.s.ad ml 19 9643 IN VIVO TREATMENT OF MICE WITH NPT 15392 AND NPY 15410: EFFECT ON THE IN VITRO STIMULATION OF SPLEEN CELL PROLIFERATION BY CONCANAVALIN A The purpose of this study was to determine 5 the effects of ^n vivo treatment of mice with the compounds NPT 15392 and 15410 on the subsequent activity of spleen cells isolated from these animals and evaluated jin vitro for their proliferative response to the mitogen, Concanavalin A (Con A).

PROCEDURE ' / In Vivo Treatment i Nine male Balb/C mice, 8-9 weeks old, weighing 18-20 gms were divided into three gro.ups. One group was treated twice daily (for 1 day), in the 15 morning and afternoon, with an oral dose of NPT 15392 at 10 mg/kg. The second group was similarly treated with NPT 15410 at 20 mg/kg. A third group, dosed with saline served as a placebo control. ' In Vitro Spleen Cell Assay: Cell Preparation The following day, each group was sacrificed and the spleens removed and pooled. The spleens were minced and the cells washed in RPMI-1640 medium (Grand 1 99643 Island Biologicals) supplemental with 2 mm glutamine and antibiotics. The cell concentration of each preparation was determined by a Coulter counter and adjusted to 5 x 10^ cells/ml with RPMI medium.

Microtiter Plate Assay Microtiter assays were carried out in 0.2 ml incubations, containing 5 x 10^ cells and Con A or Con A and compounds at the indicated concentrations. All assays were performed with 6 replicates and com-10 pared with a blank assay containing only cells. The assay plates were incubated at 37° in 5% CO2 for 4 days. During the final 18-20 hours of incubation, 0.5 ml of 3HTdR (10 yCi/rnl, 6' C^/m mole) were added to each culture. The cultures were harvested with a mul-15 tiple automatic sample harvester (MASH) unit and the incorporated 3HTdR determined with a Beckman LS 8000 liquid scintillation counter, as a measure of cell proliferation. The results are tabulated as the ratio of the activity in the Con A or Con A and compound treated :cultures to the blank cultures.

In vivo treatment with either compound 153 92 or 15410 increases the subsequent response of the spleen cells, Jjri vitro, to Con A stimulation at a sub-optimal mitogen concentration (5 yg/ml). Thus com-25 pound 15410 increased the stimulation ratio to 100:1 compared to 55:1 with the placebo. No significant differences are obtained with either compound 15392 or 15410 treatment when the cells are stimulated with a more optimal concentration of Con A (10 yg/ml). 1 996 There was also tested the effect of subsequent in vitro treatment of Con A stimulated cells with NPT 15392 and 15410 at 1 yg/ml. Both compounds show a marked ability to augment the Con A stimulation, particularly at the suboptimal mitogen concentration (5 y g/ml) and to a lesser extent at 10 g/ml. AT 5 yg/1 of Con A, the stimulation by NPT 15392 is 2.8 fold over Con A alone, while that for NPT 15410 is 3.3 fold.

These results indicate an immunomodulating effect of these compounds on spleen cell proliferation. Pre-treatment of animals with either compounds which sensitize the cells to subsequent mitogenic stimulation while exposure of the cells _in vitro to the compounds following mitogenic stimulation will augment the proliferative response particularly under conditions when the response to mitogen alone is low. 1 99643 EXAMPLE SYNTHESIS OF A ERYTHRO-9-(2-HYDROXY-3-NONYL) 6—ALKOXY PURINE (II) CI \N- I NaQCIi, > OCH.

CH -[CH9], | CH ; • h . i 1 OH - CH.

JO CH3-[CH2]1"j-xCH H L - CH II Compound I (10 mM) and a solution of sodium methoxide (11 mM) in methanol (50 ml) was refluxed for 6 hrs. The reaction flask was cooled, the pH adjusted to 5 with glacial acetic acid and the mixture, evaporated to dryness under reduced pressure. The residue was taken up with a minimum amount of cold water, filtered and dried in vacuo. 1 99643, SYNTHESIS OF ERYTHRO-9-(2-HYDROXY-3-NONYL) 6-METHYLMERCAPTO PURINE (III) nh, S = C \ NH 2 \ 7 sh ICH.

NaOH ch3-[CH2]5' ch - ch.

/CH ch3 - [ch2]^ xch - choh ii SCH ch. - [ch2]5 ch - ch.

Ill Step (1) Compound I — Compound I (10 mM) in ethanol (25 ml) and Compound II thiourea (10 mM) and anhydrous sodium acetate (11 mM) was refluxed for 1 hour. After cooling the resulting product was collected by filtration, suspended in minimum amount of cold water and the pH adjusted to 5 with diluted (20%) acetic acid. The product is washed with minimum amount of cold water, filtered and the precipitate dried under vacuo. 1 99643 Step (2) Compound II > Compound III A solution of Compound II (10 mM) in a 2 N NaClH (25 ml) was cooled at 5°. Methyl iodide (20 mM) was added and the mixture shaken vigorously in a 5 tightly stoppered flask for 15 minutes, at 5°. The mixture was then mechanically stirred at room temperature (25°) for 3 hours, the pH adjusted to 5 with glacial acetic acid. The resulting precipitate was collected by filtration and washed twice with cold 10 water (15 ml) and dried.

I I r - 77 1^4*43

Claims (65)

WHAT WE CLAIM IS:

1. A method of imparting immunomodulating activity, antiviral activity or antitumor activity comprising administering to a mammal excluding a human an effective amount for such purpose of a compound of the formula X HC CH R2 R1 OH where X is OH, NH2, SH, OR or SR (where R is alkyl of 1 to 4 carbon atoms or benzyl) , R"1" is H or alkyl of 1 to 8 carbon 2- • atoms, R is H or methyl, Y is a salt of an amine of the formula 3 4 where R and R are lower alkyl of 1-4 carbon atoms, and n is an integer from 2 to 4 with a pharmaceutically acceptable acid and where z is 0 or a number from 1 to 10. - 77 - _ 78 _ t 9 9643

2. A method according to claim 1- wherein when z is ■ 1 to 10 the acid is p-acetamidobenzoic acid.

3. '' -A method according to clairn^ l wherein z is 1 to 10.

4. A method according to any one of the preceding claims 1 3 where R is H or n-alkyl of 1 to 8 carbon atoms and R and 4 R are alkyl of 1 to 4 carbon atoms.

5. A method according to any one of the preceding claims where R"*" is H or n-alkyl of 1 to 7 carbon atoms.

6. A method according to any one of the preceding claims where R"'" is H or n-alkyl of 1 to 6 carbon atoms.

7. A method according to claim 6 where R"^" is n-hexyl.

8. A method according to claim 3 where X is QH, NH2 f or SH. 2 .

9. A method according to claim 3 where R is H. ' 2

10. . A method according to claim 3 where R is methyl.

11. A method according to any one of claims 8 to.10 where X is OH.

12. A method according to any one of claims 8 to 10 where X is NH2.

13. A method according to any one of claims 8 to 10 where X is SH.

14. A method according to claim 4 where X is OH, NH2 or SH and Y is the salt of a dimethylaminoisopropanol and p-acetamidobenzoic acid.

15. A method according to claim 14 where X is OH. 2

16. A method according to claim 14 where R is hydrogen. 2

17. A method according to claim 14 where R is methyl.

18. A method according to claim 14 or 17 where R"^ is n-hexyl. - 78 - 199643 - 79 -

19. A method according to claim 14 or 17 where R"*" is methyl.

20. a method according to claim 3 where X is OH, NH2 or SH and R"^" is hydrogen.

21. A method according to claim 3 wherein X is OH, 1 9 . R xs n-hexyl and R~ is methyl.

22. A method according to claim 21 wherein z is 3.

23. A method according to claim 20 or 22 where Y is the salt of 1-dimethylaminoisopropanol with p-acetamidobenzoic acid.

24. A method according to claim 3 where X is NH2 and the compound is used in an amount to impart immunoinhibiting activity.

25. A method according to claim 3 wherein thefcompound is used in an amount to impart immunomodulating activity.

26. A method according to claim 2-5 wherein the compound is used in an amount to impart immunostimulating activity.

27. A method according to claim 3 wherein the compound is _used in an amount to impart antiviral activity.

28. A method according to claim 3 wherein the compound \ is used in an amount to impart antitumor activity, X is OH, 1 2 R is alkyl of 1 to 8 carbon atoms and R is methyl.

29. .A method according to claim 28 where R^" is n-hexyl.

30. A method according to claim 29 where Y is the salt Qf a dimethylaminoisopropanol with p-acetamidobenzoic acid.

31. A method according to claim 30 wherein the compound is administered to a mammal having leukemia.

32. A method according to claim 25 wherein the compound is used in an amount to impart immunoinhibiting activitv. '>33. A method according to claim 32 wherein X is OH. * 16 JUL iy§2

X, ~ 79 - - 80 - 199643

34. A method according to claim 33 where R"*" is n-hexyl. 2 .

35. A method according to claim 34 where R is methyl.

36. A method according to claim 1 where z is 0.

37. A method according to claim 36 where R"*" is H or 3 4 n-alkyl of 1 to 8 carbon atoms and R and R are alkyl of 1 to 4 carbon atoms.

38. A method according to claim 36 or 37 where R^" is H or n-alkyl of 1 to 7 carbon atoms.

39. A method according to any one of claims 36 to 38 where R"*" is H or n-alkyl of 1 to 6 carbon atoms.

40. A method according to any one of claims 36 to 39 ]_ . ' where R is n-hexyl.

41. A method according to any one of claims 36 to 40 f where X is OH, NH2 or SH, 2

42. A method according to claim 41 where R is H. 2

43. A method according to claim 41 where R is methyl.

44. A method according to any one of claims 41 to 4 3 where X is OH. '

45. A method according to any one of claims 41 to 43 where X is NH2•

46. A method according to any one of claims 41 to 43 where X is SH.

47. A method according to claim 36 where X is OH, NH2 or SH and R"*" is hydrogen.

48. A method according to claim 47 where X is OH.

49. A method according to claim 36 where X is OH, R1 2 is.n-hexyl and R is methyl.

50. A method according to claim 36 wherein the compound is used in an amount to impart immunomodulating activity. - 80 - - •'81 ~ I 9 9643

51. A method according to claim 50 wherein X is NI^ and the compound is used in an amount to impart immunoin-hibitory activity.

52. A method according to claim 50 wherein the compound is used in an amount to impart immunostimulating activity.

53. A method according to claim 52 where X is OH. 2

54. A method according to claim 53 where R is methyl.

55. A method according to claim 54 where R~*~ is 1 to 6 carbon atoms n-alkyl.

56. A method according to claim 55 where R"*~ is n-hexyl.-

57. A method according to claim 50 wherein the compound is used in an amount to impart immunoinhibiting activity. '

58. A method according to claim 36 where the compound f is used in an amount to impart antiviral activity. ?

59. A method according to claim 57 or 58 where X is OH. 2

60. A method according to claim 59 where R is methyl.

61. A method according to claim 60 where R"*" is n-hexyl.

62. A method according to claim 36 wherein the compound is used in an amount to impart antitumor activity, X is OH, ___ 1 2 R is alkyl of 1 to 8 carbon atoms and R is methyl.

63. A method according to claim 62 where R^" is n-hexyl.

64. A method according to claim 63 wherein the compound is administered to a mammal having leukemia.

65. A method according to any . one of the preceding claims substantially as herein described. r&Ftxtrr PHPfejrAtwtSAJTNOvc, i-s cvjC., o^rvol — By His/their authorised Agents.*. A. J. PARK & SON. VAFE6WS2 a JMjJ - 81 - u