DE2412981A1 - NUCLEOSIDE DERIVATIVES - Google Patents

Description

Folder 23476 - Dr. K / by
Case PH 25974

IMPERIAL CHEMICAL INDUSTRIES LTD. London, UK

Nucleoside derivatives

Priority: March 21, 1973 - Great Britain

The invention relates to nucleoside derivatives and more particularly to newly aylated derivatives of cyclic adenosine 3 ', 5 "monophosphate (cyclic AMP), which has the property of inhibiting the aggregation of blood platelets.

According to the invention a cyclic 9-H-adenine-.beta.-D-ribofuranoside ^3-1, 5'-monophosphate derivative (hereinafter referred to as a cyclic AMP derivative) of formula

409839 / 105Ü

O OR '

OH

proposed, wherein R is an acyl group of the formula -CO.OR · ⁵ or -CO.NHR, where R ^ is an alkyl radical with 1-8 carbon atoms, a haloalkyl radical with 1-4 carbon atoms or an alkenyl radical with up to 4 carbon atoms and R is an alkyl radical with 1-8 carbon atoms, a haloalkyl radical with 1-4 carbon atoms, an alkenyl radical with up to 4 carbon atoms, a phenylalkyl radical with 7-9 carbon atoms, a phenyl radical, a naphthyl radical or a phenyl radical which is a halogen atom or an alkyl or Alkoxy radical with

Carries 1-3 carbon atoms; and R for a hydrogen atom or an acyl group of the formula -CO.OR ^, -CO.UHR

5 3 4

or -CO.R ', where R and R are as defined above and R is an alkyl radical having 1-4 carbon atoms. According to the invention, the base addition salts are also used suggested of these compounds.

In this description the combination of the formula I,

1 2
where R and R both represent hydrogen atoms, cycli-

called AI-IP. It is said of the substituent R that it is in the N position, whereas the sub-

2 2 ¹

substituent R is said to be in the 0 position. The compounds of the invention are thus as N -monoacyl or N, 0 -diacyl derivatives of cyclic AMP called. -

409839/1050

"5 4 Examples of suitable values for R and R when they are is an alkyl radical with 1-8 carbon atoms, are methyl, ethyl, n-propyl, n-butyl, isobutyl, n-hexyl, n-heptyl or n-octyl radicals. Preferred Wer-

■ λ L
te for Rr or R are alkyl radicals with 3-6 carbon atoms, such as n-propyl, η-butyl, isobutyl or n-hexyl radicals.

Examples of suitable values for R or R, if any um.ein haloalkyl or alkenyl radical with up to 4 carbon atoms is, are 2,2,2-trichloroethyl, 2-chloroethyl or allyl radicals, and examples of suitable substituents that may be present on the phenyl radical, when it represents a value for R are o-, m- or p-chloro substituents or p-methoxy radicals.

An example of a suitable value for R when it is a phenylalkyl radical is a benzyl radical.

A special value for R is an n-propyl radical.

Base addition salts according to the invention can be pharmaceutically acceptable base addition salts - such as alkali metal or Alkaline earth metal salts, for example sodium, potassium or calcium salts. Alternatively, it can be a base addition salt which is primarily used in the preparation of compounds according to the invention, namely because of the desirable physical properties of this. Salt, which does not necessarily have to be pharmaceutically acceptable. Examples of such salts are barium salts or salts with inorganic amines, preferably a tertiary amine such as trimethylamine, triethylamine, Diisopropylethylamine and pyridine, or an N-alkylmorpholine, for example N-methylmorpholine.

Particularly useful compounds of the invention are those cyclic AMP derivatives of the formula I, in which

409839 / IOSü

12 2

both R and R are acyl groups, i.e. that R is something represents other than hydrogen. You can, for example Be compounds in which R and R both acyl groups of the formula -CO.OR or both acyl groups of the formula -CO.NEiR are. But they can also be connections,

1 3

in which R stands for an acyl group of the formula -CO.OR or -CO.M-IR and R ″ stands for another acyl group of the formula -CO.OR ³ , -CO.NHR ⁴ or -CO.R ⁵ .

Specific cyclic AMP derivatives of the invention are in explained in the attached examples. Of these will

N, 0 -diphenylcarbamoyl-c.AMP is particularly preferred. Even

N, ^{O 2} '-diisobutoxycarbonyl-c.AMP and N, O ² ' -di-n-butoxycarbonyl-c.AMP are also very powerful.

The cyclic AMP derivatives of the invention can be prepared by methods known per se for the preparation of analogous acylated derivatives of cyclic AMP. Thus, the following methods are proposed as further aspects of the invention, wherein R, R,
genes own:

1 2 " ³ J 4 5
where R, R, R, R and R have the meanings given above

1) For a compound of the formula I in which R is a Acyl group of the formula -CO.OR ^ or -CO.NHR and R

1 2 is hydrogen or R and R are identical acyl groups of the formula -CO.OR ^ or -CO.NHR, reaction of C3>"clic AI ⁴ IP or a base addition salt thereof with at least 1.5 molecular equivalents of an acylating agent which is structurally derived from an acid of the formula R ⁵ CCO.OH or lAjH.CO.OH.

no base addition salt is used as the starting material, then the reaction is preferably carried out in the presence run a base. Appropriate base addition salts are indicated above.

_ 4 409839/105 * 0

A particularly suitable acylating agent is an acid halide, such as an acid chloride, or an anhydride of the acids mentioned above. The base can be volatile organic base, such as pyridine or diisopropylethylamine. When two or more molecular equivalents of an acylating agent, then that is Main product a compound of the formula I, in which both

1 2

Symbols R and R are acyl groups, but smaller amounts of compounds of the formula I are also obtained,

1 2

wherein either R or R is an acyl group. These can also be isolated.

2) For compounds of the formula I in which R is an acyl

L 2

3 group of the formula -CO.NHR 'and R stands for a water

1 2

atom or R and R are both identical acyl groups of the formula -CO.NHR are, implementation of cyclic! AI-IP or a base addition salt thereof with at least 1.5 molar equivalents of an isocyanate of the formula R H = C = O, where R has the meaning given above.

Vienn two or more molecular equivalents of an isocyanate are used, then the main product is a

1 2

bond of the formula I, in which both symbols R and R are acyl groups ^. Smaller amounts of the monoacyl derivatives are also used obtain.

The reaction is conveniently carried out in a diluent or solvent such as N, N-dimethylformamide or pyridine, executed. Appropriate base addition salts are indicated above.

3) For a compound of the formula I in which R is hydrogen, hydrolysis of the corresponding cyclic AMP derivative of the formula I, in which R is an acyl group, with dilute alkali, such as 0.1η sodium hydroxide or Potassium hydroxide, for a period of 15 minutes to 48 hours, at room temperature or hydrolysis of a triacy-

- 5 409839 / 105U

lated derivative of cyclic AMP, commonly known as By-product of process 1) or 2) is obtained under the same conditions.

4) Pure compounds of the formula I, in which R is something else than is hydrogen,

a) Implementation of the corresponding cyclic AMP derivative

2
of the formula I, vrorin R is hydrogen, or a base addition salt thereof with an acylating agent which is structurally different from an acid of the formula R ^ O.CO.OH,

OH or R ⁵ .CO.OH, or with an isocyanate of the formula R .N = C = O; or -

b) Implementation of the corresponding cyclic AMP derivative

1 2

of formula I, wherein R is hydrogen and R is

an acyl group of the formula -CO.OR ⁵ , -CO.NHR or -CO.R ⁵ , or a base addition salt thereof with an acylating agent which is structurally different from an acid

of the formula R- ⁵ CCO-OH or Rffl.CO.OH, or with

4th
an isocyanate of the formula R .1T = C = O.

Process 4) is carried out under the same conditions as processes 1) and 2) and is particularly useful for the production of compounds according to the invention

1 2
genes in which R and R represent different acyl groups.

5) dehydration of an adenosine 5'-phosphate derivative

of formula II: _ "_ _

NHR ¹

HO P 0 ■ CH

II

HO 0R ^£

409839/1050

2412381

A suitable dehydrating agent is, for example

1
a carbodiimide such as N, N -dicyclohexylcarbodiimide

or N, N -di-p-tolylcarbodiimide. The dehydration can be carried out in a diluent or solvent, such as
Formamide, dimethylformamide or pyridine. The dehydration is expediently carried out at a temperature of 80-120 ° C., and the nucleoside 5 ¹ -phosphate
is conveniently used in the form of a salt which is soluble in the reaction medium, such as in the form of a
4 ^! -Morpholine-N, N -dicyclohexylcarboxamidium salts or triethylammonium salts.

6) Implementation of a compound of the formula:

III

HO OR '

wherein R is a radical that is different from an acid
RiEt derives, which is a stronger acid than the undissociated nucleoside, with a base.

A particularly suitable value for R is a halogen atom,
a phenoxy radical, which optionally carries one or more nitro, halogen or methylsulphonyl radicals, an alkoxy radical, in particular a methoxy radical, which carries a cyano radical or a halomethyl radical, or a diaryl phosphate radical. Examples of suitable values for R are a fluorine atom or a 2,4-dinitrophenoxy, p-nitrophenoxy,

409839/1050

2A12981

2,4,5-Trichloroph.enoxy-, p-methylsulphonylphenoxy-, cyanomethoxy-, 2,2,2-trichloroethoxy or diphenyl phosphate radical.

The reaction is conveniently carried out in a diluent or solvents such as a polar organic solvent such as dimethyl sulphoxide, t-butanol or dimethylformamide. Preferably the diluent or solvent should be anhydrous. this

is particularly important when R stands for an acyl group, in order to replace such a radical with hydrogen to avoid due to hydrolysis.

The reaction is suitably carried out at a temperature of -20 ⁰ C to 40 ⁰ C. A suitable base is, for example, an alkali metal alkoxide such as sodium or potassium methoxide or t-butoxide, or an alkali metal hydride such as sodium or potassium hydride.

Methods 5) and 6) are extremely useful when R is hydrogen.

As stated above, the cyclic AMP derivatives of the invention have the property that they are aggregation prevent from platelets. This property is demonstrated in vitro by having the test compound to a stirred sample of a heparinized human platelet rich plasma and the effect measures the test compound in the delay or decrease in the increase in light transmission of the sample, which is caused by the addition of collagen. In this test prevent the cyclic of the invention AMP derivatives promote the aggregation of platelets at concentrations as low as 10 molar. The strongest connections show this effect at concentrations of only

C.

10 ^J to 10 molar. The in vitro inhibition of platelet aggregation is useful in the preservation of

- 8 409839 / 1OSt]

Blood for transfusion purposes without deteriorating its function.

The ability of the cyclic AMP derivatives according to the invention, Preventing platelet aggregation can also be demonstrated in vivo using a standard test used in rabbits in which the test compound is administered to the animal by intravenous injection or infusion and a blood sample is drawn and its capacity, a collagen-induced platelet aggregation to resist is measured by measuring the reduced resistance to flow through a microporous filter and with a comparative sample to which collagen has also been added. In this test, both

6 2 '

for example N, 0 -diphenylcarbamoyl-c.AMP if it is during Was administered by infusion for 20 min at a rate of 10 mg / kg, a 90-100 ^ ige decrease in the Resistance of a blood sample to flow through a microporous filter compared to a control sample. Under under the same conditions this compound had no effect on the concentration of glucose in the blood. This The result is in remarkable contrast to the general

6 2 '
my well-known N, 0 -di-n-butyryl-c.AMP, \ tfelches under the same conditions gives a considerable increase in the concentration of glucose in the blood without having any effect on the aggregation of the platelets. The in vivo inhibition of platelet aggregation is useful in preventing thrombus formation.

The compounds according to the invention do not result in any open ones toxic effects at such doses that an activity

(~ l pt

demonstrate. N, 0 -Diphenylcarbamoyl-c.AKP was tested on rabbits d \ arch intravenous injection administered in doses up to 100 mg / kg, without any toxic phenomena being observed.

- 9 409839 / 105Ü

2A12981

Yfenn a suitable connection to prevent the Aggregation of platelets is used in vitro, then it is appropriate to add them to the blood in an amount

-2-5 that the concentration in the blood is 10 to 10 molar. When used to inhibit platelet aggregation in warm blooded animals, it becomes useful intravenously by infusion or injection at doses from 2 mg / kg to 500 mg / kg or (for the most potent compounds) administered at doses from 2 mg / kg to 50 mg / kg. In humans, this corresponds to a dose of 1 mg / kg bis 20 mg / kg of the preferred compounds, which is expediently repeated every 12 hours.

When used in vivo, a compound of the invention will be useful in the form of a pharmaceutical composition administered containing an inventive cyclic AMP derivative in a sterile, injectable medium.

A preferred composition is a sterile, injectable one Solution of the cyclic AMP derivative. Such a preferred composition can be 7 mg / ml to 140 mg / ml cyclic AMP derivative included.

The invention is further illustrated by the following examples, in which the NPIR spectra using solutions in fully deuterated in dimethyl sulfoxide with tetramethylsilane were measured as an internal standard and the eluate from the chromatography columns by measuring the UV absorption was examined at 260 nm.

Example 1

1 g of c.AI ^ IP was dissolved in 5 ml of water, and then were 5 ml of diisopropylethylamine were added. The mixture was evaporated to a gum and placed in vacuo (12 mm Hg; 40 C) Dried for 24 hours. The gum was in 25 ml dry

- 10 409839 / 105U

Dissolved N, iT-dimethylformamide, which 0.12 ml of diisopropylethylamine and the clear solution was treated with 1.5 ml of phenyl isocyanate. The reaction mixture was Maintained at room temperature for 72 hours. The solvent was removed under reduced pressure and the residue was dissolved in a mixture of 22 parts by volume of acetonitrile and 3 parts by volume of water. The solution was to a Silica column (135 x 2.5 cm) applied, which with the same solvent was eluted .. The eluate fractions (500 - 750 ml) and (1-2 1) were set aside, and the fraction (750 ml - 1000 ml) was evaporated. The residue thus obtained was heated by heating dissolved in acetonitrile and the solution was boiled at 4 ° C. Side put. After 16 hours the precipitate formed was

6 2 ¹

collected by filtration and dried to give N.0 diphenylcarbamoyl-c.AMP-diisopropylethylammonium salt as an almost white powder (500 mg; 31 %) - Found: G 51.0; H 6.2; N 15.4 %. C ₂ ^ H ₂₂ N ₇ OgP- .CgH ₁₉ N.3H ₂ O • requires: C, 51.1; H 6.3; N 15.0% UV X _ (ethanol)

Max

278 mn (<26,900 pounds); NMR 6 6.28 (sH-1 ¹ ) and 5.74 (H-2 ^T ) ppm.

Example 2

The eluate fractions (500-750 ml) and (1-2 l), which had been set aside in Example 1, were combined and evaporated to dryness, and the residue, which consisted of di- and triphenylcarbamoyl-c.AMP, became dissolved in 0.1m sodium hydroxide solution. After 1 hour at room temperature, the resulting solution was ^{neutralized by adding sulfonated polystyrene resin (H +} form), which was stirred with the solution, removed by filtration and washed with water. The combined filtrates and washing liquids were • evaporated to a small volume (4 ml) and placed on a column of diethylaminoethylcellulose (HCO ~ form) (32 χ 4 cm), which was given a linear gradient of triethylammonium bicarbonate (0-> 1 molar

- 11 409839/1050

in 5 1) was eluted. The main product emerged in the eluate between 2.84 and 3 ₅ 7 1. This fraction was evaporated to dryness and the remaining solid was desalted by repeated dissolution and evaporation of ethanol. The final residue was precipitated from ethanol, the N -monophenylcarbamoyl-c.ABEP-triethylamraonium salt being obtained as an almost white powder (400 mg; 24 %).

Found: C, 49.1; H 6.2; N 17.0 %. C ₁₇ H ₁₇ NgO ₇ P-CgH ₁₅ NH ₂ O requires: C, 48.7; H 6.0; N 17.3 % ', UV \ _mn " (ethanol)

Ui OLA.

■ 278 nm (£ 23,500); NMR ό 6.40 (sH-1 ¹⁾ ppm.

Example 3

1 g c.AM? was dissolved in a mixture of 5 ml of water and 5 ml of diisopropylethylamine. The solution was evaporated to a Gumni, then overnight in a vacuum (12 mm Hg; 40 ⁰ C) was dried. The gum was dissolved in 50 ml of dry pyridine and then 0.84 ml of isobutyl chloroformate was added. After 48 hours at room temperature an additional amount of the acid chloride (0.84 ml) was added and the mixture was set aside for an additional 48 hours at room temperature. A crystalline precipitate was filtered off and discarded and the filtrate was evaporated to dryness. The residue was dissolved in 20 ml of water and traces of pyridine were removed by extracting the aqueous layer with 2 × 100 ml of ether. The water layer was then extracted with 5 x 50 ml chloroform. The first three chloroform extracts were combined, evaporated to dryness, and the residue, which was dissolved in chloroform, was applied to a silica column (20 χ 2 cm) using a mixture of chloroform and ethanol in a volume ratio of 4: 1 was used as the eluent. A main product emerged as an eluate between 200-500 ml. This main product was obtained as a white powder after the precipitate had been filtered off, which was obtained by adding petroleum ether

- 12 409839 / 105U

(Bp. 60-80 ⁰ C) had been obtained for the eluate. The final two chloroform extracts contained the same product when it was isolated from the column, so they were combined directly with the precipitated material,

6? ' in order to produce, after repeated precipitation from petroleum ether, N, 0 "-diisobutoxycarbonyl-c.AMP as its diisopropylethylammonium salt (470 mg, 26 %).

Found: C, 51.5; H 7.5; N 12.8 %. C ₂₀ H ₂₈ N ₅ O ₁₀ P-CgH ₁₉ N requires: C, 51.0; H 7.1; N 12.8 %; UV λ _mav (ethanol) 267 nm (£ 17,400); NMR £ 6.34 (sH-1 ¹ ) and 5.68 (H-2 ¹ ) ppm.

Example 4

1 g of c.AMP was dissolved in a mixture of 5 ml of water and 5 ml of diisopropylethylamine. The solution was evaporated to a gum which was dried over power in vacuo (12 mm Hg; 40 C). The gum was dissolved in 50 ml of dry pyridine, followed by the addition of 1 ml of n-propyl chloroformate. Two more 1 ml quantities of the chloroformate were added at 24 hour intervals. After a total of 72 hours at room temperature, the mixture was evaporated into a gum which was dissolved in 20 ml of water. The solution was extracted twice with 50 ml of ether each time, the ethereal extracts were discarded, and then the solution was extracted with 6 × 30 ml of chloroform. The first extract was set aside and the remaining five chloroform extracts were combined and evaporated to dryness. The residue was rubbed with petroleum ether (boiling point 6-8 ° C.). The solid obtained in this way was collected by filtration and air-dried, yielding 600 mg of II, 0 -Di-n-propoxycarbonyl-c.AMP as diisopro-. pyläthylammoniumsalζ were obtained. A portion (450 mg) of the product was' dissolved in water and passed through a column of sulfonated polystyrene ^{resin (H +} form), and the eluate was freeze-dried, where-

6? '
at! Ί, Ο -Di-n-propoxycarbonyl-c.AMP was obtained (370 mg). . 409839/1050

- 13 -

1t

Found: C 42.4} H 5.0; H 14.1; P 6.5 %. ^σ ₁ 8 ^Η 24 ^Ν 5 ° 10 ^Ρ * requires: C 43.1; H 4.85; N 14.0; P 6.0 %; UV λ

HIcDC

(Ethanol) 267 nm (£ 17.450); NMR δ 6.3 (s. H-1 ·) and 5.72 (H. -2 x) ppm. ·

BeisTDJel 5

The first extract that was put aside in Example 4 was evaporated into a gum that contained the

6 2 ¹
Main product of N, 0 -Di-n-propoxycarbonyl-c.AMP with

f \ f \ P '

a second product, known as N, N, 0 -Tri-npropoxycarbonjrl-c.AMP was identified. The gum was dissolved in 9 dl of ethanol, and a solution of 1 ml of 1m Sodium hydroxide was added. After 15 minutes the re- ' action solution neutralized by adding sulfonated polystyrene resin (H -form), which is stirred with the solution, removed by filtration and washed with water. The combined filtrates and washes were freeze-dried, with N -mono-n-propoxycarbonylc.AMP (230 mg) was obtained.

Found: C, 36.3; H 4.3; N 15.1 % for the sodium salt, C ₁₄ H ₁₇ N ₅ O ₈ P Na. 1.5 H ₂ O requires C, 36.2; H 4.3j N 15.1 % ',

UV λ _nnv (ethanol) 267 nm (£ 17,750); NMR c? 6, O6 (see H-1 ') max

Example 6

1 ml of n-3-butyl isocyanate was added to a solution of 1.2 g of c.Ai'IP-di-isopropylethylammonium salt in 40 ml of N, N-dimethylformamide, and the mixture was heated to 100 ° C. for 12 hours. An additional amount of the isocyanate (2 ml) was then added and the solution was heated to 100 ° C. for a further 3 days. The solvent was then evaporated under reduced pressure and the remaining oil was dissolved in 30 ml of water. The solution was extracted with 6 50 ml of chloroform, and the extracts were evaporated to dryness, whereby N, 0 ² '-Di-n-butylcarbamoyl-c.AMP (450 rag)

- 14 409839/1050

4S

as its diisopropylethyl ammonium salt was obtained. Part of this salt (380 mg) was passed through a column of a sulfonated polystyrene resin - (H -form), and the eluate and the washing liquids were

6 2 '
freeze dried to give N., 0 -Di-n-butylcarbamoyl-c.AMP as the free acid (300 mg). Found: C, 44.2; H 5.8 %. C ₂₀ H ₇₀ N ₇ O ₈ PH ₂ O requires:

C 44.0; H 5.9 ⁰ M UV λ _mw (ethanol) 269 nm (£ 20,500); NMR d6.12 (s. H-1 ·) and 5.55 (H-2 ¹ ) ppm.

Example 7

2.5 ml of ethyl chloroformate were added to a solution of 1 g of c.AMP-Triäthylammoniumsalζ in 50 ml of dry pyridine, and the mixture was allowed to stand for 72 hours at room temperature. The solvent was then evaporated under reduced pressure and the residue was dissolved in 30 ml of water and extracted with 6 × 30 ml of chloroform. The first two chloroform extracts were set aside and the remaining four chloroform extracts were combined and evaporated to dryness. The residue was dissolved in 50 ml of water and passed through a column of a sulfonated polystyrene resin (H ⁺ form),

6 2 ¹ and the eluate was freeze-dried, with N, 0 -Dieth-

oxycarbonyl-c.AMP (250 mg) was obtained as the free acid. Found: C, 37.5; H 4.4; N 13.9% C 1 H ₂₀ N ₅ P.2H ₂ O requires C 37.7; H 4.6; N 13.8 %; UV λ _nnv (ethanol) 267 nm

" Max

(£ 16,9OO); NMR ό 6.32 (see H-1 ^! ) And 5.66 (H-2 ¹ ) ppm.

Example 8

1.5 ml of 2,2,2-trichloroethyl chloroformate became one Solution of 1 g of c.AMP triethylammonium salt, which is dissolved in 50 ml dissolved dry pyridine was added. The mixture was left to stand at room temperature for 48 hours and then under evaporated to dryness under reduced pressure. The residue was dissolved in chloroform and on a silica

- 15 409839 / 1OSU

acid column (12 χ 2 cm) fractionated, being a mixture from chloroform and ethanol in a volume ratio of 19: 1 was used as the eluent. Became a major product obtained after 300 ml of eluate had leaked. This was isolated as a white powder after further evaporation (1 g). A portion of it (400 mg) was dissolved in aqueous ethanol and passed through a column of sulfonated polystyrene resin (H form) passed through. The eluate was frozen

6 2 '
dried, N, 0 -Di - ^^^ - trichloroethoxycarbonyl-'c.AIiP (365 mg) was obtained as the free acid. Found: C, 28.6; H 2.2; N 10.3 % - C ₁₆ H ₁₄ N ₅ O _{10 PCl 6} requires C 28.2; H 2.1; N 10.3 % ', UV X _max (ethanol) 267 nm (£ 17,700); NMR 0 ^ 6.48 (s, H-1 ^f ) and 5.81 (H-2 ^T ) ppm.

Example 9

A second portion of the main product (400 mg) obtained as in Example 8 was dissolved in 9 ml of ethanol, and then 1 ml of 1M sodium hydroxide was added. The solution was left to stand at room temperature for 30 minutes. The mixture was neutralized by passing it through a column of sulfonated polystyrene resin (H ⁺ form). The eluate and washings were partially evaporated and then freeze-dried to give N -mono-2,2,2-triethoxycarbonyl-c.AMP (265 mg) as the free acid.

Found: C, 30.4; H 3.1; N 13.2 %. C ₁₃ H ₁₃ N ₅ OgPCl ₃ -H ₂ O requires: C 29.9; H 2.9; N 13.4 %; UV 'λ _max (ethanol) 267 nm (£ 17,100); NMR S 6.03 (s, H-1 ■) ppm.

Example 10

5 ml of n-butyl chloroformate was added to a solution of 1 g c.AMP-Triäthylainmoniumsalz added in 50 ml dry pyridine, whereupon the mixture was left to stand at room temperature for 48 hours. The solvent was reduced

- 16 409839/105 U

The pressure was removed and the residue was dissolved in 30 ml of water and extracted with 6 × 30 ml of chloroform. The combined chloroform extracts were evaporated into a dry powder in the presence of 10 g of silica, which was then applied to a silica column (15 × 1.5 cm). This was followed by fractionation with a mixture of acetonitrile and water in a volume ratio of 88:12. The first main eluate fraction (200-500 ml) was set aside and the second main fraction (500-800 ml) was evaporated to dryness. The residue was dissolved in water and the solution was passed through a column of sulfonated polystyrene resin (Na ⁺ form).

6 2 '

The eluate was freeze-dried, whereby N 0 -Di-n-butoxyrcarbonyl-c.AMP (280 mg) as the sodium salt.

Found: C, 41.8; H 4.9; N 12.6 ^C , L ^c ₂ o ^H 27 ^N 5 ° 10 ^P ' ^Na - ^H 2 ° requires: C 42.2; H 5.1; N 12.3 %. UV λ _mov (ethanol) 267 ran (£ 17,900); NIiR ö 6.28 (s, H-1 ·) and 5.62 (H-2 »)

Example 11

The first main eluate fraction (200-500 ml), which had been set aside in Example 10, contained both di- and tri-n-butoxycarbonyl-c.AMP. It was evaporated and the residue was treated with 10 M aqueous 0.1 M ethanolic sodium hydroxide solution for 4 hours at room temperature. The mixture was neutralized with a sulfonated polystyrene resin (H ⁺ form). The aqueous eluate was freeze-dried to give N -mono-n-butoxycarbonyl-c.AI-IP (220 mg).

Found: C, 39.9; H 4.4; N 15.6 %. C ₁₅ H ₂₀ N ₅ O ₈ PH ₂ O requires: C 40.3; H 4.9; N 15.6% UV λ _max (ethanol) 267 nm (£ 17.45O); IiI-IR S 6.00 (s, H-1 ^! ) Ppm.

Example "12

5 ml of n-heptyl chloroformate was added to a stirred solution

- 17 -409839 / 105U

of 1 g of c.AMP triethylammonium salt in 50 ml of dry pyridine was added. After 43 hours, the solution was treated with 5 ml of water, whereupon the mixture was evaporated to dryness. The residue was dissolved in acetonitrile and poured onto a silica column (30 χ 2 cm) was fractionated with a mixture of acetonitrile and water in a volume ratio of 88:12 as the eluent Silicic acid was fractionated into two fractions A and B by preparative thin-layer chromatography (GFpp-v-plates, 20 χ 20 cdi, acetonitrile water, 88:12). Fraction A (Rp 0.66) was fractionated by EIu ₁ -tion The solid thus obtained was dissolved in water after evaporation under reduced pressure, and the solution was passed through a column of sulfonated polystyrene resin

6 2 ¹ eluate was freeze-dried, N, 0, -Di-n-heptyloxycarbonyl-c.AMP (120 mg) being obtained as the free acid. Found: C, 49.9; H 6.0; N 11.6% C ₂₆ H ₄₀ N ₅ O ₁₀ P requires C 50.8; H 6.5; N 11.4 %. UV λ (ethanol) 267 nm

IHgLa. ·

(£ 17,400); NMR <£ 6.32 (s, H-10 and 5.62 (E-2 ^f ) ppm.

Example 13 **

The fraction obtained after 300 ml of eluate emerged, which was set aside in Example 12, was evaporated. The residue of di- and tri-n-heptyloxycarbonyl-c.AHP was hydrolyzed with 10 ml of aqueous 0.1 M sodium hydroxide at room temperature for 30 minutes. The hydrolyzate was neutralized with sulfonated polystyrene resin (H ⁺ form) and the eluate was freeze-dried to obtain N -monoheptyloxycarbonyl-c.AMP (180 mg). Found: C, 42.7; H 5.3; N 13.5% C ₁₈ H ₂₆ N ₅ O ₈ P-ZK ₂ O requires: C 42.6; H 5.9; N 13.7 %; UV λ _max> (ethanol) 267 nm (£ "17,950); IR CTO III, 03 (s, ¹ H-1) ppm.

- 18 409839 / 105U

Be isT? Iel 14

2 ml of ethyl isocyanate were added to a solution of 1 g of c.AMP triethylammonium salt in 50 ml of dry pyridine. The mixture was refluxed for 16 hours and allowed to cool. The mixture was evaporated under reduced pressure and the gummy residue was fractionated on a silica column (15 x 1.5 cm) with a mixture of acetonitrile and water in a volume ratio of 88:12 as the eluent. A main fraction was isolated after evaporation of the eluate. The residue was dissolved in water and the solution was passed through a column of sulfonated! Polystyrene resin (Na ⁺ form) passed through,

6 2 '

and the eluate was freeze-dried to give N, O -diethylacerbamoyl-c.AMP (610 mg). Found: C, 35.9; H 4.4; N 18.1 %. ₂₁ N ₇ O requires C, ^ ₈ PNa.2H ₂ O: C, 36.3; H 4.7; N 18.5 %. UV λ x 268 nm (£ 21,000); NMR 0 6.13 (s, H-1 ¹ ) and 5.54 (H-2 ¹ ) ppm.

Example 15

A portion (200 mg) of the product obtained in Example 14 was dissolved in 0.1 M potassium hydroxide and kept for 48 hours at room temperature ditched. The solution was neutralized by passing it through a column of sulfonated Polystyrene resin (H -form) was passed through. The eluate and washings were freeze-dried, whereby N -Monoäthylcarbamoyl-c.AMP (120 mg) as free acid was obtained.

Found: C, 33.9; H 4.8; N 19.0% C ₁₃ H ₁₇ N ₆ O ₇ P 1 H ₂ O requires C 34.2; H 5.1; N 18.5 %. UV λ " _nv (ethanol) 268.5

Max.

mn (£ 19,500). NMR ό ~ 6.0 (s, H-1 ^r ) ppm.

' 3example 16

2 g p-chlorophenyl isocyanate was added to a solution of 1 g c.AI IP triethylammonium salt in 50 ml of dry pyridine is added and the mixture was heated for 30 min at 8O ⁰ C.

- 19 - 409839/1050

Then 5 ml of water was added and the solvent was evaporated. The Poickstand was suspended in chloroform, and a precipitation of 1,2-bis-p-chlorophenylurea was removed by filtration. The chloroform filtrate and washes became dry evaporated, and the residue was fractionated on a silica column (10 x 1.5 cm) with acetonitrile as the eluent. After 1.5 l of eluate had emerged, all of the urea had been removed, whereupon the eluate reduced to a mixture from acetonitrile and water in a volume ratio of 22: 3 was changed. The eluate fraction (300-1000 ml) was

6 2 ¹
evaporated to give N, 0 -Di-p-chlorophenylcarbamoyl-c.AMP as a crystalline solid (250 mg) and as an amorphous solid (1.65 g).

Found: C, 42.3; H 3.3; N 13.8 %. C ₂₄ H ₂₀ N ₇ OgPCl ₂ OH ₂ O requires: C, 41.7; H 3.7; N 14.2 %. UV Λ _max (ethanol) 279 nm (£ 29,250); NMR & 6.34 (s, H-1 ^f ) and 5 ^ 74 (H-2 ') ppm.

Example 17

6 2 ¹

800 mg of K, O-di-p-chlorophenylcarbamoyl-c.AMP were dissolved in 9 ml of ethanol, and then 1 ml of 1M sodium hydroxide was added, whereupon the solution was left to stand for 24 hours at room temperature. The solution was made neutral by passing it through a column of sulfonated! Polystyrene resin (H ⁺ form) was passed through. The eluate was freeze-dried to give N -mono-p-chlorophenylcarbamoyl-c.AI-IP as the free acid (240 mg). Found: C, 39.1; H 3.7; N 15.9% C ₁₇ ¹ H ₁₆ N ₆ O ₇ PCl 1 H ₂ O requires: C 39.4; H 3.8; N 16.2 %. UV λ _{m <av} (ethanol) 278 nm (£ 23,500); NMR d 6.09 (s, H-1 ^f ) ppm.

Example 18

2 ml of 1-caphthyl isocyanate were added to a solution of 1 g c.AIiP-Triäthylammoniumsalz, which in 50 ml of dry pyridine was stirred, added. The mixture was 48 hours at room

- 20 - 409839 / 105U

"Let stand temperature, and the precipitate of 1,2-bisnaphthy! urea was filtered off. The pyridine was removed under reduced pressure, and the residue was on a silica column (30 χ 2 cm) with a mixture of acetonitrile and water in a volume ratio of The eluate was evaporated to give 900 g of an amorphous powder, a portion of this material (600 mg) dissolved in aqueous ethanol and passed through a column of sulfonated polystyrene resin (Na ⁺ form) . The eluate was partly

6 2 '

v / ice evaporated and then freeze-dried, whereby N, 0 dinaphth-1-ylcarbamoyl-c.AMP (500 mg) obtained as the sodium salt.

Found: C, 50.8; H 3.6; N 13.1% C ₃₂ H ₂₅ N ₇ OgPNa-JH ₂ O requires: C 51.7; H 4.2; N 13.2 %. UV λ _{m v} (ethanol)

Max.

275 run (wide) (18,500 pounds); NMR δ 6.37 (s, H-1 ') and 5.80 (H-2 ¹ ) ppm.

Example 19 '■'

4 ml of phenyl isocyanate were added to a solution of 1.2 g of c. AI'IP triethylammonium salt in 60 ml of dry pyridine was added, and the mixture was left to stand for 48 hours at room temperature. Then 5 ml of water were added and the mixture was evaporated to dryness. The solid was extracted continuously with ether in a Soxhlet apparatus for 48 hours, whereupon all of the 1,2-bis-pheny! Urea had been removed. The residue in the flask was dissolved in aqueous acetonitrile and the undissolved solid was collected by centrifugation. The viscous supernatant liquid was further diluted with aqueous acetonitrile and the solution was passed through a column of sulfonated polystyrene (H ⁺ form). The eluate and water washes were partially evaporated under reduced pressure and then freeze-dried to give N, 0 -diphenyls arbaiaoyl-c. AMP (1.8 g) was obtained.

- 21 -

409839/1050

Example 20

250 mg of c.AMP were dissolved in a mixture of Λ ml of water and 1 ml of triethylamine. The mixture was evaporated into a gum which was dried in vacuo over concentrated sulfuric acid at room temperature to give the triethylammonium salt. This salt was dissolved in 10 ml of dry pyridine containing 1 ml of octyl isocyanate and the mixture was refluxed for 6 hours. The solution was diluted with 140 ml of water and the mixture was allowed to stand at room temperature for 17 hours. The solid material was filtered off and the filtrate was evaporated under reduced pressure. The residue was dissolved in chloroform and fractionated on a silica column (30 g) with a mixture of chloroform and methanol in a volume ratio of 4: 1 as the eluent. The fractions which contained only the main product were evaporated to dryness. The residue was dissolved in chloroform and the solution was passed through a column of sulfonated polystyrene resin (Na ⁺ form). The eluate was evaporated, the residue was redissolved in water and the solution was

6 · 2 '
de freeze-dried, whereby N, 0 -Di-n-octylcarbamoyl-

c.AI-IP was obtained as the sodium salt (164 mg). Found: C, 50.2; H 7.2; N $ 14.4. C ₂₈ H ^ ₅ H ₇ O ₈ RIa. ^ ₂ O requires: C, 50.1; H 6.9; H 14.6 %. NMR 6.16 (s, H- ¹¹ ) ppm.

3eisniel 21 . ·

100 mg of N, 0 -Di-n-octylcarbamoyl-c.AI-IP were dissolved in 1 ml of dioxane and 1 ml of 1N sodium hydroxide, and the solution was allowed to stand at room temperature for 48 hours. The solution was rendered neutral by passing it through a column of sulfonated polystyrene resin (H ⁺ form). The eluate was freeze-dried to give N -mono-noctylcarbamoyl-c.AMP as the free acid (65 mg).

- 22 409839 / 105Ü

Found: C, 45.4; H 6.3; N 15.7% C ₁₉ H ₂₉ N ₆ O ₇ PH ₂ O requires: C 45.5; H 6.2; N 16.7% UV λ _r _ (water) 269 nm

iUcL / C *

(£ 20,900) inflection 276 nm; NMR at 6.06 (s, H-1 ”) ppm.

Example 22

c.AI-IP triethylammonium salt was prepared from 500 mg of the free acid as described in Example 20. It was dissolved in 20 ml of dry pyridine and 2 ml of o-chlorophenyl isocyanate. The solution was left to stand for 48 hours at room temperature and then diluted with water and left to stand ^{at 4 ° C. for a further 24 hours.} The resulting precipitate was filtered off and the filtrate was evaporated into a gel which was redissolved in acetonitrile. "The solution was passed through a column of sulfonated polystyrene resin (Na ⁺ form). The eluate was in

C. pt

concentrated a gel and freeze-dried, 'where N, 0. Di-o-chlorophenylcarbamoyl-c.AMP as the sodium salt (1.12 g).

Found: C, 43.8; H 3.3; N 14.2 %. C ₂₄ H ₁₉ N ₇ OgPCl ₂ Na requires: C, 43.7; H 2.9; N 14.9 %. UV λ _max (water) 277 nm (£ 23,600); NMR C $ 6.32 (s, H- ¹¹ ) ppm.

Example 23

The procedure described in Example 22 was repeated except that 2 ml of m-chlorophenyl isocyanate was used in place of o-chlorophenyl isocyanate were used. After a total of 48 hours at room temperature, the mixture was washed with water. diluted and stored at 4 ° C for 24 hours. The resulting precipitate was filtered off, the filtrate was evaporated, whereby 1.06 g of the solid was obtained. A portion of this solid (500 mg) was dissolved in acetonitrile and the Solution was applied to a silica column (100 g). The column was filled with a mixture of acetonitrile and water in a volume ratio of 80:20, and the fractions containing the main product were eluted by a

- 23 409839/1050

Jt

Column of sulfonated! Polystyrene resin (Na ⁺ form) passed through. The eluate was partially evaporated and

6 2 ^! then freeze-dried to give H, 0 -Di-m-chlorophenylcarbamoyl-c.AMP as the sodium salt (350 mg). Found: C, 41.0; H 3.9; N 13.1% C ₂₄ H ₁₉ K ₇ O ₈ PCl ₂ Ka.2 ^ 0 requires: C 41.0; H 3.4; N 13.3 %. UV X _ (V / water)

Max.

277 ran (£ 30,100); NMR d 6.31 (s, H-1 ') ppm.

Example 24

The procedure described in Example 22 was repeated except that 2 ml of p-methoxyphenyl isocyanate was used in place of o-chlorophenyl isocyanate and that the starting material was converted into the sodium salt, a mixture of acetonitrile and methanol being used in place of acetonitrile. The eluate was evaporated into a solid (1.15 g). A portion of this solid (0.51 g) was dissolved in methanol and the resulting solution was evaporated to a dry powder in the presence of 5 g of silica. This powder was applied to a silica column (25 g) and fractionated with a mixture of acetonitrile and water / water in a volume ratio of 9: 1 as the eluent. The fractions containing the major product were filtered to remove 100 mg of white crystals and evaporated to a white solid. The solid was dissolved in aqueous methanol and the solution was passed through a column of sulfonated polystyrene resin (Na ⁺ form). The eluate was partly

6 evaporated 2 'wise and freeze-dried, whereby N, 0 -Dirüethoxyphenylcarbamoyl-c.AMP as the sodium salt (285 mg).

Found: C, 46.0; H 4.3; N 14.1 %. C ₂₆ H ₂₅ N ₇ O ₁₀ PNa-SH ₂ O requires: C 45.5; H 4.2; N 14.3 %. UV λ "" (water)

ill CjUv

277mn (£ 28,700); NMR όβ, 29 (s, ¹ H-1) ppm.

- 24 - 409839 / 105ü

JtS

Example 25

3 ml of freshly distilled allyl isocyanate became one Solution of 1.3 g of c.AI - ^ - triethylamnionium salt in 50 nil trokkeris-m Pyridine was added and the mixture was 3 1/2 st heated to reflux. The pyridine was distilled under reduced Omcl-c and the residue was turned on a Silica column (20 χ 3 cm) with a mixture of acetonitrile and water in the ratio of 88:12 by volume as the eluent fractionated. That in the eluate between 170 ml and 380 ml of the contained material was reduced by evaporation Pressure gained, redissolved in 20 ml of water and passed through a column of sulfonated! Polystyrene resin (Ha form, bed volume 15 ml) passed through. Freezer

6 2 '

drying of the eluate and washing gave N, 0 "-diallylcarbamoyl-c.ÄMP as the sodium salt (0.88 g).

Found: G 38.5; H 4.3; N 17.4 %. C ₁₈ H ₂₁ N ₇ O ₈ PNa ^ H ₂ O requires: C 39.0; H 4.6; N 17.7% UV λ _wov (water) 269 nm (£ 22,100); NMR δ 6.14 (s, H-1 x) ppm.

Example 26

3.5 ml of freshly distilled benzyl isocyanate was added a solution of 1.3 g of c.AI-IP triethylammonium salt in 50 ml dry pyridine was added and the mixture was refluxed for 2½ hours. Removal of the pyridine under reduced Pressure left an oil which upon rubbing with 25 ml of ethyl acetate gave a solid. The solid was collected, washed with ethyl acetate and placed on a silica column (20 × 3 cm) with a mixture of acetonitrile and water in a volume ratio of 88:12 as an eluent cleaned. The material contained in the eluate between 150 ml and 550 ml was reduced by evaporation Pressure gained and dissolved as much as possible in 40 ml of water. The solution was clarified by filtration and through a column of sulfonated polystyrene resin (Na -

- 25 .409839 / 1050

Form - bed volume 25 ml) passed through. The eluate and washings were freeze-dried, whereby ίϊ °, Ο * ~ -dibenzylcarbamoyl-c.AMP as sodium salt (0.70 g) was obtained.

Found: C, 47.3; H 4.7; N 14.8 %. C ₂₆ H ₂₅ N ₇ O ₃ PNa ₅ ^ H ₂ O requires: C 47.8; H 4.5; N 15.0 %. UV λ _r _ (water) 269 nni (£ 23,500); NMR 06.16 (s, H-1 x) ppm.

Example 27

3.5 ml of distilled 2-chloroethyl isocyanate were added to a solution of 1.3 g of c.AMP triethylammonium salt in 50 ml of dry pyridine. After 46 st at room temperature wurae the solution below 30 ⁰ C under reduced pressure once 'evaporated. The residue was purified on a silica column (20 × 3 cm) with a mixture of acetonitrile and water in a volume ratio of 88:12 as the eluent. The substance contained in the eluate between 200 ml and 310 ml was obtained by evaporation under reduced pressure, ^{redissolved in 16 ml of water and passed through a column of sulfonated polystyrene (H +} form - bed volume 20 ml). All of the eluate was first concentrated under reduced pressure below 20 ° C. and then

6 2 ¹ freeze-dried, whereby 1.07 g of N, 0 -Di-2-chloroethylcarbarioyl-c. AI-IP was obtained as the free acid.

Found: C, 34.6; H 3.8; N 17.6; Cl 13.4% C ₁₆ H ₂₀ N ₇ OgCl ₂ -P.0.5H ₂ O requires: C, 35.0; H 3.8; N 17.9; Cl 12.9 S ^.

UV λ _mov (water) 268 nm (£ 23,100); NMR 0 ^ 6.34 (s, H-1 ·) -

ppm.
Example 28

5 ml of distilled allyl chloroformate was added dropwise to a solution of 1.3 g of c.AIIP triethylammonium salt in 50 ml of dry pyridine were added, which was stirred in ice / water. The mixture was stirred at room temperature for 42 hours. meanwhile the red solution and the rubbery one

- 26 409839/1050

■ ίΐ

Solid turned into a yellow solution in which crystalline pyridine hydrochloride was suspended. The whole was evaporated under reduced pressure and the residue was redissolved in 25 ml of water. The solution was washed twice with 20 ml of ether each time. Repeated evaporation of the aqueous layer under reduced pressure then gave a gum which was fractionated on a silica column (20 χ 3 cm) with a mixture of acetonitrile and water in a volume ratio of 88:12 as the eluent. The fraction from 130 ml to 160 ml of the eluate V was evaporated under reduced pressure and the residue was redissolved in 10 ml of water. The solution was bottled with about 10 ml of ether and then extracted 12 times with 7 ml of chloroform. The first extract showed two spots on thin layer chromatography, but the other showed only one spot, so they were combined, dried over sodium sulfate and evaporated to give a gum which solidified when rubbed with ether. The solid was redissolved in 5 ml of water and the solution was passed through a column of sulfonated polystyrene ^{resin (Na +} form - bed volume 5 ml). The entire eluate was freeze-dried,

6 2 ¹

where N, 0 -diallyloxycarbonyl-c.AMP as sodium salt (0.13 g) was obtained.

Found: G 38.2; H 3.7; N 12.2 %. C ₁₈ H ₁₉ N ₅ O ₁₀ PNa.2H ₂ O requires: C 38.8; H 4.2; N 12.6 %. UV λ _ηην (water)

ρ max.

267 nin (£ 18,100); IM ο 6.33 (s, H-1 ') ppm.

Example 29

670 mg of N -I-ionophenylcarbamoyl-c.AEP were in a mixture dissolved from 2 ml of water and 2 ml of triethylamine. The solution 'was evaporated into a gum, which was concentrated in vacuo over Sulfuric acid was dried at room temperature, so that the triethylammonium salt was obtained. This Salt was in a mixture of 50 ml of dry pyridine and

- 27 409839 / 105Ü

O j 26 nil η- butyric acid ariliydr id dissolved. Further information n-2utyric anhydride - were added after 24 h (0.5 ml) and after 4S st (1.25 ml) added. After a total of 72 hours, the mixture was treated with 5 ml of water, and after a further 2 hours the mixture was evaporated to dryness. The residue was dissolved in 100 ml of water and 3 times with 100 ml of chloroform extracted. The combined chloroform extracts were concentrated and placed on a silica column (35 g) fractionated with acetonitrile as the eluent. The eluent was converted to a mixture of acetonitrile and water in a volume ratio of 4: 1, and a main fraction (100-150 ml) was collected. A white precipitate in this fraction was collected by filtration, whereby

2 '6

0 -n-Butyryl-N -phenylcarbamoyl-c.AMP was obtained as the free acid (110 mg). The filtrate was evaporated to dryness, the residue was dissolved in a mixture of ethanol and water, and the solution was passed through a column of sulfonated polystyrene resin (Na form). The eluate was partially evaporated and then freeze-dried to give O-n-butyryl-N -phenylcarbamoyl-c.AMP as the sodium salt (357 mg). Found: C, 45.1; H 4.5; N 14.6 %. C ₂₁ H ₂₂ N ₅ O ₈ PNa-H ₂ O requires: C 45.1; H 4.3; N 15.1 %. UV X _mnv (water)

IXIcUa. ·

277 nn (£ 26 ^ 500); NMR S 6.28 (s, H-1 ^f ) ppm.

3 example 30

450 mg N -Honophenylcarbamoyl-c.AMP were according to the instructions of Example 29 converted into the anhydrous triethylammonium salt. The salt was in 10 ml dry Dissolved pyridine and added 2 ml of n-butyl chloroformate Reacted room temperature for 24 hours. The reaction mixture was diluted with water and evaporated into a gum, the v / ieder was dissolved in 50 ml of water. The solution was extracted with 3 x 50 ml chloroform and the extracts were evaporated to dryness. The residue

- 28 4 0 9 8 3 9/1 0 5 0

was redissolved in acetonitrile and fractionated on a silica column (40 g) using 200 ml of acetonitrile as the first eluant and then using a mixture of acetonitrile and water in a volume ratio of 88:12. The main product appeared in the acetonitrile / water fractions. These were passed through a column of sulfonated polystyrene resin (Na ⁺ form). The eluate was partially evaporated and

O ι f.

then freeze-dried to give O ^ -n-butyl oxy carbonyl-N phenylcarbamoyl-c.AMP as the sodium salt (226 mg).
Found: C, 44.5; H 4.0; N 14.1 %. C ₉₉ H ₉₇ NiO ₀ PNa-H ₀ O requires: C 44.9; H 4.4; N 14.3 #. UV λ _ηην (water)

_, Max ·

277th nra (£ 26,800); ' NMR ο 6.35 (s, H-1 ') ppm.

Example 31

230 mg of N -monophenylcarbamoyl-c.AMP were used according to the instructions of Example 29 converted into the triethylammonium salt. The salt was dissolved in 10 ml of dry pyridine and reacted with 2.5 ml of n-hexyl chloroformate. The product was isolated according to the procedure of Example 30,

Ο! f.

v / whether ei 0 -n-hexyloxycarbonyl-N -phenylcarbamoyl-c.AMP was obtained as the sodium salt J [214 mg). Found: C, 44.0; H 5.1; N 12.0 %. C _{2Z |} H ₂₈ Ng0 _g PNa.3H ₂ O requires: C 44.2; H 5.2; N 12.9 %. UV ^ _max (water) 277 nm (£ 24,800); In TMR <^ 6.33 (s, H-1 x) ppm.

Example 32

450 mg L \ i ^D -monophenylcarbarnoyl-c .AMP were converted into the triethylammonium salt according to the procedure of Example 29. The salt was dissolved in 10 ml of dry pyridine and reacted with 3 ml of n-octyl isocyanate at room temperature for 17 days. Then 100 ml of water were added and the mixture was left to stand at 4 ° C. for 24 hours. The solids were filtered off and the filtrate became

- 29 409839 / 105U

SO

Evaporated dry. The residue was dissolved in acetonitrile and placed on a silica column (40 g) Use of 200 ml of acetonitrile and then a mixture of acetonitrile and water in a volume ratio 88:12 fractionated as eluant. The main product appeared in the acetonitrile / water fractions (125 ml - 350 ml). This fraction was passed through a column of sulfonated polystyrene resin (Na form). The IZIuat was partially evaporated and freeze-dried, using ΰ "-n-octylcarbamoyl-N -phenylcarbamoyl-c.AMP as the sodium salt (410 mg) was obtained.

Found: C, 47.3; H 5.8; N 15.1 %. C ₂₆ H ₃₂ N ₇ O ₈ PNa.2H ₂ O requires: G 47.2; H 5.6; N 14.8 %. UV 'λ _max ' (water) 277 nm (£ 28,200); NMR 16, 16 (s, H-1 ') ppm.

Example 33

0.88 g of N ^ mono-p-chlorophenylcarbamoyl-c.Ai'IP-triethylammonium salt were dissolved in a mixture of 20 ml of dry pyridine and 2 ml of phenyl isocyanate. The mixture was allowed to stand at room temperature for 72 hours, after which it was diluted with water and kept at 4 ° C. for 24 hours. The resulting precipitate was filtered off and the filtrate was evaporated into a solid which was dissolved in acetonitrile and fractionated on a silica column (200 g) using acetonitrile and then using a mixture of acetonitrile and water in a volume ratio of 9: 1 as the eluent . The main product appeared in the acetonitrile / water eluate and was isolated as a solid after evaporation. The solid was dissolved in acetonitrile and the solution was passed through a column of sulfonated! Polystyrene resin (iIa ⁺ form) through. The eluate was "partially evaporated and freeze-dried to give N ° -p-chlorophenylcarbamoyl-0 -phenylcarbamoyl-c.AI-I? As the sodium salt (600 mg). Found: C 43.4; H 3.5; N 14 , $ 5. C ₂₄ H ₂₀ N ₇ O ₈ PCl.Na.2H ₂ O

- 30 A09839 / 105Ü

requires: C, 43.7; H '3.6; N 14.9 ti. UV λ _mv (water) 277 nm (£ 30,800)} Kim d 6.32- (β, Η-1 ') ppm.

Example 34

6 2 '

350 mg of K, 0 -diisobutoxycarbonyl-c.AMP were dissolved in 20 ml of a 0.1 M sodium hydroxide solution, whereupon the solution was stored at room temperature for 24 hours. The solution was then sulfonated! Polystyrene resin (H form) neutralized and filtered, and the filtrate was evaporated to dryness in the presence of 10 g of silica. The residue was applied to a silica column (20 × 4 cm) packed in acetonitrile, and the column was eluted with a mixture of acetonitrile and water in a volume ratio of 22: 3. The eluate fraction emerging between 400 and 550 ml was evaporated to determine whether an N -monoisobutoxycarbonyl-c.AMP (250 mg) was obtained, which was converted into the sodium salt ^{by reaction with sulfonated polystyrene resin (Na + form).}

Found: G 37.9; H 4.8; N 14.8 %. C ₁₅ H ₁₉ N ₅ O ₃ PNa-H ₂ O requires: C 38.3; H 4.5; N 15.0% UV λ (ethanol)

ρ ITi cQs. ·

267 nm (£ 16,800); Mim o6.33 (s, H-1 x) ppm.

- 31 409839 / 105U

Claims (1)

Patent claims : it \.) Cyclic AMP derivatives of the formula: NHR ^J 1 3 v / orin R for an acyl group of the formula -CO.DR or 4 ^ -CO.NER, where R ^{J is} an alkyl radical with 1-8 carbon atoms, a haloalkyl radical with 1-4 carbon atoms or an alkenyl radical with up to 4 carbon atoms and R is an alkyl radical with 1-8 carbon atoms, a haloalkyl radical with 1-4 carbon atoms , an alkenyl radical with up to 4 carbon atoms, a phenylalkyl radical with 7-9 carbon atoms, a phenyl radical, a naphthy radical or a phenyl radical bearing a halogen atom or an alkyl or alkoxy radical with 1-3 carbon atoms, 2
is; and R represents a hydrogen atom or an acyl group of the formula -CO.OR ^, -CO.NHR or -CO.R · ', where R ^ and R have the above definitions and R is an alkyl radical having 1-4 carbon atoms, as well the base addition salts thereof. Compounds according to claim 1, characterized in that R ^ or R 'stand for an alkyl radical with 3-6 carbon atoms. 409833/1050 3- compounds according to one of claims 1 or 2, there- ■ 7. Λ characterized in that Rr or R is a methyl, Ethyl, n-propyl, n-butyl, isobutyl, n-hexyl, n-ileptyl, n-octyl, 2,2,2-trichloroethyl, 2-chloroethyl or allyl radical or 'that II is a Ben zyl, phenyl, naphthyl, o-chlorophenyl, m-chlorophenyl, p-chlorophenyl or O-methoxyphenyl radical 5
and R stands for an n-propyl radical. 4. Compounds according to any one of claims 1, 2 or 3, there- 1 2 characterized in that R and R both represent acyl groups of the formula -CO.OR or both stand for acyl groups of the formula -CO.MIR. 5- compounds according to any one of claims 1, 2 or 3> characterized in that R represents an acyl group of Formula -CO. OR ⁵ or -CO.KHR and R ^{2 stands} for another 3 4 their acyl group of the formula -CO.OR, -CO.NHR or -CO.R ⁵ stands. 6. N, 0 -Diphenylcarbamoyl-c.AI-IP and the base addition salts of that. 7. N °, 0 ² '-diisobutoxycarbonyl-c.AMP or N, 0 -di-n-butoxycarbonyl-c.AMP or the base addition salts thereof. 8. "Compounds according to any one of claims 1 to 7, characterized characterized in that they are pharmaceutically acceptable base addition salts. 9. Compounds according to any one of claims 1 to 7, characterized characterized in that it is barium salts or salts with an organic amine. 10. Process for the preparation of the cyclic AMP derivatives according to claim 1, characterized in that one •1 1) for a compound of the formula I in which R is a . - 33 -409839/1050 Acyl group of the formula -CO.OR- or -CO.NHR and 9 A. Ο K ~ stands for a hydrogen atom or R and R ^{stand for identical acyl groups of the formula -CO.OR- 5} or -CO.TIER, reacts a cyclic AIiP or a base addition salt thereof with at least 1.5 molecular equivalents of an acylating agent which structurally derived from an acid of the formula RO.CO.OH or RTiH.CO.OH, where R ^D and R claim I have given meanings; si 2) for a compound of formula I in which R is a 4 2 Acyl group of the formula -CO.IiIIR and R stands for water 1 2 is hydrogen or R and R are identical acyl groups of the formula -00.MiR ^1- , a cyclic AMP or a base addition salt thereof with at least 1.5 molecular equivalents of an isocyanate of the formula 4 4 R .N = C = O, in which R has the meaning given in claim 1; 3) for compounds of the formula I in which R is hydrogen, the corresponding cyclic AMP derivative p
of the formula I, in which R ″ stands for an acyl group, hydrolyzed with dilute alkali or a triacylated derivation of cyclic AMP hydrolyzed; 4) for a compound of the formula I in which R is something other than hydrogen: a) the corresponding cyclic AMP derivative of the formula I, wherein R is hydrogen, or a base addition salt thereof with an acylating agent which structurally differs from an acid of the formula RO.CO.OH, II LIH.CO.OH or R .CO.OH or with an iso- 4 ^ 4 5 cyanate of the formula R ¹ JI = C = O, in which R, R and R ^ have the meanings given in claim 1, or - 34 A09839 / 105Ü b) the corresponding cyclic AMP derivative of the formula 1 2 I, where R is hydrogen and R is an acyl group of the formula -CO.OR, -CO.IiHR or -CO.R- *, or a base addition salt thereof with an acylating agent which is structurally different from an acid of the formula R ³ O.CO.OH or lAsH.CO.OH, or 4 3 with an isocyanate of the formula R .N = C = O, in which R and R have the meanings given in claim 1, implements; 5) an adenosine 5'-phosphate derivative of the formula: NHR ¹ HO- -ΡΟΗ -CH, HO OR * wherein R and R have the meanings given in claim "1 own, dehydrated; or 6) a compound of the formula: -P -.0 OH OR NHR ^J III 409839/1050 1 2 wherein R and R have the meanings given in claim 1 and R represents a radical which
is derived from an acid R ⁰ EI, which is a stronger acid than the undissociated nucleoside, reacts with a base. 11. Pharmaceutical compositions, characterized in that they are a cyclic AMP derivative according to claim 1 contained in sterile injectable medium. 12. Compositions according to claim 11, characterized in that it is a sterile injectable
Solution acts. MIWIANWAMk MMN * .H.nNCKE. DlPt.-INÖ. M.IOH * - 36 -409839/1050