CA1265160A

CA1265160A - Gem-dihalo and tetrahalo-1,12-diamino-4,9-diaza- dodecanes

Info

Publication number: CA1265160A
Application number: CA000516066A
Authority: CA
Inventors: Fritz Gerhart; Pierre Mamont
Original assignee: Merrell Dow Pharmaceuticals Inc
Current assignee: Aventis Pharmaceuticals Inc
Priority date: 1985-08-21
Filing date: 1986-08-15
Publication date: 1990-01-30
Anticipated expiration: 2007-01-30
Also published as: CA1265160C

Abstract

PATENTS
ABSTRACT
Gem-diahlo and gem-tetrahalo-1,12-diamino-4,9-diaza-dodecane derivatives useful as antiproliferative or antitumor agents.
C-34,435 US

Description

PATENTS

i31,Eiff.``

GEM-DIHALO AND TETRAHALO-1!12-DIAMINO-4,9-DIAZA-DODECANES

It is a well known observation that the biosynthesis of natural polyamines, such as putrescine, spermidine and 5 spermine, is elevated in rapidl~ proli~erating cells relative to norma~ quiescent cel1s. Conversely, it is also known that the depletion of putrescine and spermidine leads to a reduction in cell proliferation.

Ornithine is the metabolic precursor o~ putrescine, 10 which in turn, is the metabolic precursor of spermidine, which in turn, is the metabolic precursor of spermin~.
Metabolically, these biochemical conversions are catalysed by the enzymes ornithine decarboxylase, spermidine synthase and spermine synthase, respectively. Additionally, spermi-15 dine and spermine synthase enzymes utilize decarboxylated-S-adenosyl-L-methionine as a co-substrate, the reaction product of the S-adenosyl-~-methionine decarboxylase enzyme. Inhibitors of these enzymes, including inhibitors of S-adenosyl-L-methionine decarboxylase therefore, should 20 serve to prevent the biosynthesis of putrescine and the higher polyamines derived therefrom, viz, spermidine and spermine, and should, theoretically, be effective as antiproliferative agents and~or an~itumor agents.

However, in the past, the use of irreversible ; 25 ornithine decarboxylase inhibitors or inhibitors of S-adenosyl-L-methionine decarboxylase, spermidine synthase and spermine synthase have not proven to b~ totally effective. Thus, for example, putrescine and spermidine are ; C-34,435 US

PATENTS

not essential for the maintenance of cell viability as long as the preexisting spermine pool i~ maintained above a certain critical level. M~reover, a total in vivo inhibi~ion of the decarboxylase enzymes is difficult due to 5 their rapid turnover.

Applicants have discovered a class of compounds which antagonize spermine functions in the cell. These compounds are highly effective inhibitors o~ cell growth in rapidly proliferatiny cells. Accordingly, the compounds of this 10 invention are use~ul as antiproliferative and antitumor agents.

SUMMARY OF THE INVENTION

The present invention relates to certain selective gem-dihalo and tetrahalo derivatives of spermine. More 15 particularly this invention relates to gem-dihalo or tetrahalo-1,12-diamino-4,9-diaza-dodecane derivatives having the ~ormula X Y X Y
~ / CH NH-~H -C-( CH -N \ ~

2 2 2 2~ 2)2H2 C CH2 NH2 X Y
(~) wherein X and Y represent hydrogen or halogen, with the proviso that in the case of the dihalo derivatives both halogens are present on one and only one carbon atom, and 25 in the case of the ~etrahalo derivatives the compounds are 2,2,11,11-halo-substituted; and the pharmaceutically acceptable salts thereof.

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~S~4 PATENTS

Additionally, certain aspects o~ this invention are directed to a process for the preparation o~ the compounds herein described, pharmaceutical compositions containing the same, and the use o~ these compounds as antitumor 5 agents.

DETAILED DESCRIPTION OF THE INYENTION

As indicated in general formula ~1) above the compounds o~ ~his invention ~orm a speci~ic class of gem-dihalo or gem-tetrahalo-spermines together with their 10 pharmaceutically acceptable salts. As used throughout, however, a more definitive nomenclature will be employed, and the compounds will be designated as derivatives of gem-dihalo or gem-tetrahalo-1,12-diamino-4t9-diaza-dodecanes. When used herein, the term halo refers solely to 15 chloro and fluoro derivatives.

All of the compounds encompassed within this invention are specific gem-dihalo derivatives or specific gem-tetrahalo derivatives. That is to say, the compounds of this invention are limited to either the 2,2-dihalo, the 20 6,6-dihalo or the 2,2,11,11 tetrahalo derivatives of 1,12-diamino-4,9-diaza-dodecane, as indicated by the proviso limitations. In view o~ the symmetrical nature of the molecule, the 2,2-dihalo deriva~ives are identical with the 11,11-dihalo derivatives, and such derivatives shall be 25 designated throughout as 2,2-dihalo derivatives.

It will be noted that in the case the 2,2,11,11-tetrahalo derivatives due to their method of preparation, the substitutions at the 2 and 11 carbon atoms will be symme~rical. That is to say, the same substitution occuring 30 at the 2-position must occur also at the ~1-position.

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65~ PATENTS

The compounds of this invention encompassed within the scope of Claim 1 include:

2,2-difluoro-1,12-diamino-4,9-diaza-dodecane, 2,2-dichloro-1,12-diamino-4,9-diaza-dodecane, 2-chloro-2-~luoro-1,12-diamino-4,9-diaza-dodecane, 6,6-difluoro-1,12-diamino-4,9-diaza-dodecane, 6,6-dichloro-1,~2-diamino-4,9-diaza-dodecane, 6-chloro-6-fluoro-1,12-di~mino-4,9-diaza-dodecane, 2,2,11,11-tetra~luoro-1,12-diamino-4,9-diaza-dodecane, 2,2,11,11-tetrachloro-1,12 diamino-4,9-diaza-dodecane, and 2,11-dichloro-2,11-difluoro-1,12-diamino-4,9-diaza-dodecane.

The pharmaceutically acceptable salts include those non-toxic organic or inorganic acid addition salts of the base compounds o~ Formula (1) above. Illustrative inorganic acids include hydrochloric, hydrobromic, sulfuric and phosphoric acids as well as metal salts such as sodium 20 monohydrogen orthophosphate and potassium hydrogen sulfate.
Illustrative organic acids which form suitable salts include the mono, di and tricarboxylic acids, such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaricg malic, tartaric, citric, ascorbic, 25 maleic, hydroxymaleic, ben~oic, p-hydroxybenzoic, phenyl- -acetic, cinnamic, salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sul~onic acid and 2 hydroxyethane sulfonic acid. Such salts can exist in either a hydrated or substantially anhydrous form.

All of the compounds of this invention are prepared in a logical sequence starting with the correspondiny 2,2-dihalo-1,4-butanediols having the ~ormula C-34,435 US

PATE~TS

HO-C~2-C-(CH ) -OH

(2) wherein X1 and Y1 represent chlorine or fluorine. These 5 compounds are readily prepared by reacting the appropriate 2,2-dihalosuccinic acid with trifluoroacetic acid to form the corresponding 2,2-dihalosuccinic anhydrides. Cleavage of the anhydrides with methanol results in the formation of the corresponding methyl 2,2-dihalosuccinates. Reduction of 10 the corresponding free acid function with borane-methyl sulfide complex results in the formation of the corresponding alcohols, i.e., the methyl 2,2-dihalo-4-hydroxybutanoates. ~eduction of the ester function, for example using sodium borohydride, results in the formation 15 of the desired starting materials, i.e., the 2,2-dihalo-1,4-butanediols (2~.

The 6,6-dihalo-1,12-diamino-4,9-diaza-dodecane derivatives of this invention can be prepared in accordance with the following synthetic sequence wherein:
20 X1 and Y1 = chlorine or fluorine O
Pht =

25 Mes = CH3-S-O
Tos = CH3 ~ S

~ ~B C~34,435 US

PATENTS

X,~ Y~ X~ Y~ X~ Y1 ~0-CH2-C-~ C~2 )2-8 - ~ MesO-CN2 C ~ CN2 )2 PhtN-C112-C-~ CN2 )2-NPht 2 X~ Y1 x~
IlN-cH2-c-~c~2)2N~8 t' N2N Cli2-~--~C82)2 N~2 Tos Tos \Br( Cit2 )3NP-nt

3 PhtN-~ C82 )3--~--C~2--C-~ CN2 ~2-N ( C}12 )3 Tos Tos

4 H2N-~ CN2 )3--NU--CH2--C--~ CH2 )--NH--( CH2 )3-NH2 .

C-34,43~ US -6 ; PATE~ITS

~ J
The compounds 2,2-dihalo-1,4'bu~anediol (2) are treated wi-th two equivalents of methanesulfonyl chloride in the presence of pyridine to form the corresponding 1,4-bis-methanesul~onyloxy-2,2-dihalobutanes (3). Reaction of

5 (3) with two equivalen-ts of potassium phthalimide in a solvent such as dimethylformamide results in the formation of the corresponding 2,2-dihalo-1,4-diphthalimido-butanes (_). Heating of (~) with hydrazine in a solvent such as ethanol removes the phthaloyl protecting groups to form the 10 key in-termediates, 2,2-dihalo-1,4-diaminobutanes (5~, which can be readily isolated as their dihydrohalide salts. The compound 2,2-difluoro-1,4-diaminobutane is a key interme-diate useful in the preparation of 6,6-difluoro-1,12-diamino-4,9-diaza-dodecanes. Inasmuch as these 2,2-dihalo-15 1,4-diaminobutanes, represent such useful and novel compounds, they are specifically claimed herein as a part of the instant invention.

To avoid obtaining higher alkylated products, the primary amines of (5) are protected in a standard manner 20 with p-toluenesulfonyl chloride to yield the corresponding 2,2-dihalo-1,4-di-p-toluenesulfonylamino-butanes (6).
Alkylation of the protected amines (6) with two equivalents of 3-bromopropylphthalimide under anhydrous conditions in a suitable aprotic solvent, such as dimethylformamide in the 25 presence of sodium iodide and potassium tert-butoxide, yields the corresponding 6,6-dihalo-1,12-diphthalimido-4,9-di-p-toluenesulfonyl-4,9-diaza-dodecanes (7).

These 4,9-diaza-dodecane derivatives (7) are deprotected in two steps, the first step involves heating 30 with hydrazine in a solvent such as ethanol to remove the phthaloyl groups. The products so obtained are then heated with aqueous HBr to remove the protecting tosyl moieties.
The final products obtained in this manner are the corresponding 6,6-dihalo-1,12-diamino-4,9-diaza-dodecanes g ~ C-34,435 US

~L~:~i53L~, PATENS3 t1), ~hich can b~ readlly ~sol~tet a~ thelr corresponding tetrahydroh~lid~ ~lts, pre~erably a8 thelr tetrahydro-brom:~d~ salt~.

The 2, 2-dlhalo-1 ,12-diamino~,9-diaza-dodecane deriYa-tives o~ the present invention are prepared in accordanc~
wl~h the ~ollowin~ reactlon sequence, start~ng again with the appropriate 2, -dihalo-1 "4-butanedio~ (2~. In this react~o~ sequenc~ ~he symbcls X~, Y1, Pht D Mes and Tos have the sa~e meanings previollsly indicated~

X~ ,Y1 ~ cH2-c -( c~2 )~ sO-C~2--C-~ C~2 )2 OUe5 ~ i~esO-C~2~=c~2 2 3 e I

~ /~ x~, y~ ~ y BN-C~2-C~2 ~ - H211-C82-C-CN'c82 ~ Pb'tN-CH2-C-C~=CU2 ~0.
11 1~ 9 ~( Cfl2 )4NP~t ~tN-(C12)~ -~2-'C-C'd~A2 ~ ~2~ 12)~-r-~2-C-CN~2 rO,i, To~

C-34,435 ~

0, PAT~1TS

X~
2 )3 ;`J ( CH2 )4 ~-CH2-C-cH=cH2 ~r( CH2 )3~Pht H~-( CH ) -;~-CH2-C-CH=CH2 1`5 14 X1 Y1 x~ Y~
Pht~ ~ CH2 )3 ~ ( C~2 )4 ~ C 2 Pht~l--( CH2 )3--;\1--( CH2 )4--~--CH2--C--CH20H

Pht~-( CH2 )3-~-( CH2 )4-:1-CH2-C CH2 Tos Tos X;l Y1 Pht~-( CH2 )3-~1-( CH2 )4-~1-CH2 C CH2 ;1 Tos Tos ~ , ~/1 35 H2;`1-( CH2 )3-;lH-( CH2 )4-~H-CH2 C CH2 ;~H2 _ g _ C-34, 435 US

. PATE~TS

6~
The 2,2-dihalo-1,4-butanediols t2) are treated with two equivalents of methanesulfonyl chloride in the presence of pyridine to form the corresponding 1,4-bis-methane-sul~onyloxy-2,2-dihalobutanes (33. When (3) is heated with 5 diazabicycloundecen in a solvent such as dimethylformamide, there is obtained the corresponding 1-methanesulfonyloxy-2,2--dihalo-3-butenes (8).

Reaction of (8) with potassium phthalimide in a solvent such as dimethyl~ormamide results in the formation 10 of the corresponding 1-phthalimido-2,2-dihalo-3-butenes (9). Cleavage of the phthaloyl derivative (93 with hydra~ine in an alcoholic solvent provides the correspond-ing 1-amino-2,2-dihalo-3-butenes ~10).

The primary amines t10) are protected by reaction with 15 p-toluenesulfonyl chloride in a standard manner to obtain the corresponding N-(2,2-dihalo-3-butenyl)-p-toluene-sulfonamides ~11). Alkylation of the protected amine (11) with 4-bromobutylphthalimide in the presence of potassium tert.-butoxide under anhydrous conditions in a suitable 20 solvent, such as dimethylformamide results in the formation o~ the corresponding N-(4-phthalimidobutyl)-N-(2,2-dihalo-3-butenyl)-p~toluenesulfonamides (12).

The action of hydra~ine in a solvent such as ethanol upon (12) ~orms the corresponding 9-amino-3,3-dihalo-5-p-25 toluenesulfonyl-5-aza-1-nonenes (13), which upon treatment with p~toluenesulfonyl chloride yield the corresponding 9-p-toluenesulfonylamino-3,3-dihalo-5-p-toluenesulfonyl-5-aza-l-nonenes (14).

Alkylation of the protected amines ~14~ takes place 30 with 3-bromopropylphthalimide in the presence of potassium tert-butoxide under anhydrous conditions in a solvent such as dimethylformam.ide to yield the corresponding 3,3-C-34,435 U~

~26S~ PATENTS

" ~
dihalo-13-phthalimido-5/10-di-~-toluenesul~onyl-5,10-diaza-1-tridecenes (15~. Oxidation o~ the double bond t15) with KMnO4 in an aqueous acetic acid solution results in the formation of the corresponding 2,2-dihalo-12-5 phthalimido-4,9-di-p-toluenesulfonyl-4,9-diaza-dodecanoic acids (16). Reduction o~ (16) with borane-methylsulfide complex ~orms the corresponding 2,2-dihalo-12-phthalimido-4,9-di-p~toluenesulfonyl-4,9~diaza-dodeeanols (17).

Reaction of (17) with methanesulfonyl chloride in the 10 presence of pyridine utilizing an anhydrous solvent such as dichloromethane results in the ~ormation o~ the correspond-ing 2,2-dihalo-1-methanesulfonyloxy-12-phthalimido-4,9-di-p-toluenesulfonyl-4,9-diaza-dodecanes (183.

Further reaction of (18) with potassium phthalimide in 15 anhydrous dimethylformamide results in the ~ormation of the corresponding 2,2-dihalo-1,12-diphthalimido-4,9-di-p-toluenesulfonyl-4,9-diaza-dodecanes (193, which can be be deprotected in two stages, first by heating with hydrazine in a solvent such as ethanol to remo~e the phthaloyl 20 groups, and then followed by heating the products so obtained with aqueous HBr in order to remove the protecting tosyl moieties. The desired products resulting therefrom are the corresponding 2,2-dihalo-1,12-diamino-4,9-diaza-dodecanes (1), which can be isolated as their corresponding 25 tetrahydrohalide, and more particularly, as their tetra-hydrobromide salts.

The 2~2,11,11-tetrahalo-1,12-diamino-4,9-diaza-dodecane derivatives of the present invention can be readily prepared in accordance with the following reaction 30 sequence. In this sequence, the symbols Xl1 Y~, Pht, Mes and Tos have the same meanings as previously indicated with the proviso that both values for X1 and bo~h ~alues for Y
must be the same.

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r( C-H2 )1~r ) CH:2=CN-C-C82-~-( C~12 ).~ -CH2-C-C~I=CH2 11 ~19 X~ ~1 X~, Y~ X~ Y1 I X~ Y~
2H0CH2-C-C~I2-N-( CH2 )4--N--CH2 C CH208 ~ HOOC--C--CH2-N-( CN2 )4-N CH2 C

X,~ Y1 X;~ /Y1 Meso-cN2-c-c~32-N-( CH2 )4-N-CH2 C C82 e Tos Tos ll PhtN-CN2-C--C~2-~3-( CH2 )4--7-C82-C-CH2-WPht Tos Tos Xj~
H2~1--CH2--C~C~2~N8-~ CH2 )4-NH--CN2 C CH2 2 C-34, 435 US-12~.

PATENTS

, Two equivalents of the compounds, N-( 2, 2-dihalo-3-butenyl)-~-toluenesulfonylamide ~11), prepared as indicated in accordance ~ith the previous reaction sequence, can be symmetrically dialkylated with one e~uivalent of 1,4-5 dibromobutane under anhydrous conditions in the presence ofpotassium tert.-butoxide, utilizing a solvent such as dimethylformamide, to ~orm the corresponding 3,3,12,12-tetrahalo-5,10-di-p-toluenesulfonyl-5,10-diaza-1,13-tetradecadienes ( _ ).

Oxidation of the double bonds of ~19~, utilizin~ KMnO4 in an aqueous acetic acid solution results in the formation of the corresponding 2,2,11,11-tetrahalo-4,9-di-~-toluene sulfonyl-4,9-diaza-1,12-dodecanedioic acids (20). Reduction of (20) with borane-methylsulfide complex forms the 15 corresponding 2,2,11,11-tetrahalo-4,9-di p-toluene-sulfonyl-4,9-diaza-1,12-dodecanediols (21).

Reaction of (21) with methanesulfonyl chloride in the presence of pyridine, utilizing an anhydrous solvent such as dichloromethane, results in the preparation of the 20 corresponding 1,12-methanesul~onyloxy-2,2,11,11-tetrahalo-4,9-di-p-toluenesul~onyloxy-4,9-diaza-dodecanes t22).
Fur~her reaction of (22) with potassium phthalimide in anhydrous dimethylformamide yields the corresponding 1,12-diphthali~ido-2,2,11,11-tetrahalo-4,9-di-p-toluene-25 sulfonyl-4,9-diaza-dodecanes (23).

The compounds (23) can be deprotected in two stages, ~irst by hea~ing with hydrazine in a solvent such as ethanol to remoYe the phthaloyl groups, and secondly by heating the products so obtained with aqueous HBr so as to 30 remo~e the protecting tosyl moieties. The products so obtained are the corresponding desired 2,2,11,11-tetrahalo-1,12-diamino-4,9-diaza-dodecanes (1), which can be readily isolated as their tetrahydrobromide salts ~1).

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~ he compounds o~ the present invention are useful as antiproliferative and antitumor agents. The mechanism by which these compounds function is not known. What is known, however, is that when the compounds o~ ~his invention are 5 added to a culture medium of growing rat hepa~oma tissue culture (HTC) cells, a marked reduction o~ cell growth occurs. In combination with known ornithine decarboxylase inhibitors, such as 2-difluoromethyl~2,5-diaminopentanoic acid (DFMO) or ~2R,5~ -6-heptyne-2,5-diamine (R,R-MAP), a 10 ~urther inhibition of cell growth occurs.

rhe compounds of this invention have also been found to be capable of slowing neoplastic cell proliferation when tested in standard animal tumor models. A preferred manner of utilizing these compounds is in combination with DFMO or 15 ~R,R~-MAP, or in combination with other t~erapeutic methods or agents ~nown to affect polyamine metabolism in the treatment o~ neoplasms in animals. As used herein, the term animals is taken to me~n warm blooded mammals, such as mice, rats/ dogs, cats, guinea pigs, co~s, horses and 20 humans.

The compounds of this invention can be utilized both prophylactically and therapeutically. The amount of active ingredient ~or therapeutic administration can vary over a wide range and is dependent upon such factors as the 25 species of animal to be treated, its age, health, sex, weight, nature and the severity of the condition being treated. Generally, a therapeutically ef~ective amount of the active ingredient to be administered will range from about O.2 to 5 grams, and pre~erably from O.5 to 3 grams 30 per day. For prophylactic administration, corresponding lower doses of from 0.05 to 2 grams per day can be utili 7.e~ ~ .

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PATEN1'S

When administered in combination with other ornithine decarboxylase inhibitors, such as DFM0 or CR,~-MAP, an amount of from 0.1 to 4 grams o~ the particular gem-dihalo or tetrahalo-1,12-diamino-4,9~diaza-dodecane and from 0,5 5 to 10 gr~ms o~ the ornithine decarboxylase inhibitor are administer~d per day.

The compounds o~ this invention can be ora~ly admin-istered. Illustrative dosage levels for oral a~ministration range from 2 to 50 mg per kg of body weight. Preferably, 10 from 10 to 20 mg per kg o~ the gem-dihalo or tetrahalo-1~12-diamino-4~9-diaza-dodecane are orally administered per day in divided doses. In those instances where the drug is administered via the parenteral route, corresponding lower doses are employed~ When administered in combination with 15 ornithine decarboxylase inhibitors, the compounds can be administered in standard dosage unit forms, such as tablets, capsules, dragees, lozenges, elixirs, emulsions, suspensions and various intravenous, intramuscular or intradermal suspensions.

When administered orally, the preferred dosage form is that of a tablet or capsule. The amount of active ingredient contained in each dosage unit ~ill, of course, vary depending upon the particular species of the gem-dihalo or tetrahalo-1,12-diamino-4,9-diaza-dodecane 25 employed, and the particular dosage form utilized.
Generally, a given dosage unit will contain from 10 to 500 mg of the active ingredient in addition to the various pharmaceutical excipients contained therein. Tablets containing from 100 to 400 mg of the active ingredient, are 30 the preferred dosage unit and can be administered b.i.d., or t.i.d. or q.i.d.

C-34,~35 US

~Z~5~6~ PATENTS

In preparing solid dose forms such as tab~ets, the active in~redient is ~enerally blended with conventional pharmaceutical carriers or excipients such as gelatin, various starches, lactose, calcium phosphate or powdered 5 sugar. ~he term pharmaceutical carrier as used herein also includes lubricants employed to improve the ~low of tablet granulations and which prevent adhesion of tablet material to the surfaces of tablet dies and punches. Suitable lubri-cants include, ~or example, talc, stearic acid, calcium 1~ stearate, magnesium stearate and zinc stearate. Also included within the de~inition of a pharmaceutical carrier as used herein, are disintegrating agents added to assist the breakup and dissolution of tablets following adminis-tration, as well as coloring and/or flavoring agents to 15 enhance the aesthetic qualities of the tablets and make them more acceptable to the patient.

Suitable liquid excipients for the preparation of liquid dosage unit forms include water and alcohols such as ethanol, benzyl alcohol and the polyethylene glycols, 20 either with or without the addition of a surfactant. In general, the preferred liquid excipients, particularly for injectable preparations, include water9 saline solution, dextrose and glycol solutions such as an aqueous propylene glycol or an aqueous solution of polyethylene glycol.

Liquid preparations to be used as sterile injectable solutions will ordinarily contain ~rom about 0.5 to about 25% by weightl and preferably from about 1 to about 10% by weight, of the active ingredient in solution. In certain topical and parenteral preparations, various oils can be 30 utilized as carriers or excipients. Illustrative o~ such oils are mineral oils, glyceride oils such as lard oil, cod liver oil, peanut oil, sesame oil, corn oil and soybean oil. For insoluble compounds, suspending agents may be added as well as agents to control the viscosity, as for C-34,435 US

i.~S~60 PATENTS

example, magnesium aluminum silicat~ or carboxymethyl-cellulose. In addition ~o these excipien~s, buf~ers, preservatives and emulsi~ying agents may ~lso be added.

The proportion of the active ingredient employed in 5 parenteral dosage unit forms ranges ~rom 0.05 to about 20%
by ~eight, preferably from abou~ 0.1 to about 10% by weight of the total liquid composition, the remaining component or components comprising any of the various pharmaceutical excipients previously mentioned. In order to minimize or 10 elimina~e irritation at the site of injection, such compositions may contain a non-ionic surfactant ha~ing a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations - ranges ~rom about 5 to about 15% by weight. The surfactant 15 can be a single component having the above-identified HLB, or a mixture o~ two or more components having the desired HLB. Illustrative of surfactants useful in parenteral .
formulations are the class of polyoxyethylene sorbitan fatty acid esters as, for example, sorbitan monooleate and 20 the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The invention described herein is more particularly illustrated in conjun~tion with the followin~ specific 25 preparation, but is not necessarily limited thereto.

C-34,435 ~S
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~ PATENTS

PREPARATXO~ OF 6,6-DIFLUORO-1,12-DIAMINO-4~9-D~AZA-DODECANE
2,2-Difluoro-1,4-butanediol . . .
2,2-Difluorosuccinic acid t120 g/ 0.78 moles) and trifluoroacetic anhydride (540 mL) are reflux~d (bath 5 temperature 80C) for 2 hours. Most of the tri~luoroacetic acid is distilled utilizing a short Vigreux column, the ~inal traces are removed under vacuum t12 mm Hg, 50C) and finally by stripping twice with carbontetrachloride. The - oily residue solidifies on scratching with petroleum ether.
10 Filtration and washing with petroleum ether yields 2,2-difluorosuccinic anhydride as slightly violet crystals:
98 g (92~).

The 2,2-difluorosuccinic anhydride (98 g, 0.72 moles) is dissolved in dichloromethane and slowly added with 15 stirring to methanol, cooled in an ice bath. The mixture is kept at room temperature overnight, evaporated and stripped twice with carbon tetrachloride to yield methyl 2, 2-difluorosuccinate as a slightly brownish oil: 121 g t100%).

In a 4 L flask equipped with a reflux condenser and 20 1 L dropping funnel, a solution of BH3.Me2S complex (10 Ml 88 mL) in dry dichloromethane (1 L) is slowly added o~er a 2 hour period to a stirred solution o~ the methyl 2, 2-di~luorosuccinate (120 g, 0.714 moles) in dry tetrahydro-furan at 20C. After re~luxing (bath temperature 80C) for 25 about 15 hours, the mixture is allowed to cool to room temperature and methanol t1 L) is slo~ly added. Evaporation yields methyl 2,2-difluoro-4-hydroxybutyrate as an oil which is stripped with methanol (1 L) and finally with CCl4 to yield a yellow oil: 100 g t92%).

C-34,4~5 US

~ O PATENTS

To a cold (0C) solution of sodium borohydride (10.3 g, 0.272 moles) in ethanol, a solution of the methyl ~ difluoro-4-hydroxybutyrate (55 g, 0.36 moles) in ethanol is slowly added, while maintaining the internal 5 temperature of the reaction mixture between - 5C and 0C.
The mixture is stirred ~or 1 hour at 0C, then, an approx-imately 4 N solution of HCl gas in methanol (200 mL) is carefully added. Sodium chloride is filtered, the methanol is removed under vacuum, and the residue is dissolved in 10 ethanol. Additional NaCl is again removed by filtration (membrane filter) and evaporation of the ~iltrate yields the compound 2,2-difluoro-1,4-butanediol as a colorless oil when distilled in a Kugelrohr at 150C/0.05 mm H~; 41 g ~ 90% ) .

15 2,2-Difluoro-1,4-diaminobutane To a stirred mixture o~ 2,2-di~luoro-1,4-butanediol (12.97 g, 103 mM), dry pyridine (65 mL) and dichloromethane (200 mL), is slowly added a solution of methanesul~onyl chloride (23.6 g) in dichloromethane (50 mL). Stirring is 20 continued overnight, and the mixture is washed with 2 N HCl (twice), 10% aqueous NaHC03 until neutral, and finally with water. The organic solution is dried (MgS04) and evaporated to yield 1,4-bis-methanesulfonyloxy-2,2-difluorobutane as a slightly yellow oil ~25.4 g, 87.5%) which crystallizes on 25 scratching with dry ether: white crystals, 22.3 g (77%).

The compound 1,4 bis-methanesulfonyloxy-2,2-difluoro-butane (66 g, 234 m~), potassium phthalimide (97 g, 10%
excess) and dry DMF (700 mL) are stirred and heated (bath temperature ~10C) under nitro~en for 110 hours. Most of 30 the DMF is removed under vacuum (oil pump) and water is added to the residue. The precipitated material is filtered, washed three times with water, dissolved in chloroform, and washed with 1 N NaOH (twice) and brine C-3~,435 US

~ PATENTS

(twice). After dryin~ the organic solution (Na2S04) and concentrating, the cornpound 2,2-di~luoro 1,4-diphthalimido-butane begins t.p crystalliz~. Hexane is added, the solid material which results i5 collected and 5 washed with ether to yield white crystals: 69.55 g t77%).

Under nitro3en and with e~ficient stirring a mixture of 2,2-difluoro-1,4-diphthalimido-butane: (69.55 g (180,6 mM), tetrahydrofuran (361 mL) and 361 mL of a 1 N
solution of hydrazine hydrate in ethanol are heated 10 overnight at 90C ~bath temperature~. After addition of 6 N
HCl (700 mL), stirring and heating are continued for 1~ hours. The reaction mixture is cooled tice bath), and the precipitate is filtered (ethanol wash). The filtrate is evaporated, the residue is taken up in water, ~iltered, and 15 evaporated again. This procedure is repeated once again using a membrane filter (Millipore). The residue is stripped with isopropanol (twice) and ethanol (twice) to remove the final traces of water. Digestion with ethanol and subsequent filtration of the product so obtained, 20 results in the formation o~ white crystals that are successively washed with ethanol, acetone and ether: 30.9 g (87%~. This material is recrystallized by dissolving in the minimum amount of hot water (approximately 50 mL), filtering and adding ethanol to form pure 2,2-difluoro-25 1,4-diaminobutane as the dihydrochloride salt: 22 g (61.8%).

Calc~d for C4H12C12F2N2 C,24.38; H 6 14; N 14 22 Found: C,24.33 H,5.19; N,14.24 C 34,435 US
..

~2~5~6~ PATENTS

6,6-Dl~luoro-1,12-dlphthalimido-4,9-di-E~-toluenesulfonyl-4,9-diaza-dodecane To a mixture o~ 2,2-difluoro-1,4-diaminobutane dihydrochloride ~1.57 g, 8 mM), triethylamine (3.23 g, 5 4 equivalents) and dry dichloromethane is slowly added with stirring a solution o~ toluenesulfonyl chloride ~3.04 g, 2 equivalentsl in dichloromethane. After stirring overnight at room temperature, additional dichloromethane is added.
The reaction mixture is washed with 1 N HCl ( twice), brine 10 (twice), dried (Na2S04) and evaporated to yield white crystals: 3 g. The resulting material is digested with ether and thoroughly washed with ether to yield pure 2,2-difluoro-1,4-di-p-toluenesulfonyl~mino-butane: 2.45 g (71%).

Under nitrogen, potassium tert-butoxide (1.3 g, 2 equiYalents) is added to a stirred solution of 2,2-difluoro-1,4-di-p-toluenesulfonylamino-butane (2.45 g, 5.67 mM) in dry dimethyl~ormamide (5 mL). After 30 minutes at room temperature, additional dimethylformamide (5 mL), 20 N-3-bromopropylphthalimide (3.04 g, 2 equiYalents~, and sodium iodide (170 mg) are added. The mixture is stirred overnight, the dimethylformamide is removed under vacuum (oil pump). Water ( 250 mL ) is added to the residue, and the precipitate is filtered, washed with water and dried over 25 P205: 4.57 g. The crude material is recrystallized ~rom chloroform/ether to yield pure 6,6-difluoro-1,12-diphthalimido-4,9-di-p-toluenesulfonyl-4,~-diaza-dodecane as whi~e crys~als: 3.45 g (75%).

6 ? 6-Difluoro-1,12-diamino-4,9-diaza-dodecane A mixture of 6,6-difluoro-1,12-diphthalimido-4,9-di-p-toluenesul~onyl-4,9-diaza-dodecane (3.4 g, 4.22 m~), ethanol (8.5 mL) and 8.5 mL of a 1 N solution of hydrazine C-34,435 US

~2~ PATENTS

hydrate in ethanol is s~irred and heated ~ bath temperature:
90C) overnight. Evaporation and stripping twice with rnethanol yields a residue which is dissolved in a mixture of water ~20 mL), methanol (20 mL), and concentrated HCl (40 mL) t and heated ( bath temperature: 90C) under reflux for 3 hours. The reaction mixture is cooled, filtered and evaporated, and the residue i's dissolYed in hot 1 N HCl.
The solution is filtered, the filtrate is evaporated, and the residue is stripped with isopropanol ~twice) and 10 ethanol (twice). The residue is dissolved i~ ethanol 9 ether is added, and upon evaporation of the solvent, there is obtained 1,12-diamino-6,6-difluoro-4,9-di-p-toluene-sulfonyl-4,9-diaza-dodecane as a white crystalline ~oam:
2.7 g (quantitative yield).

The compound 1,12-diamino-6,6-difluoro-4,9-di-p-toluenesulfonyl-4,9-diaza-dodecané (2.7 g, 4.36 mM) and 47%
aqueous HBr are refluxed (bath temperature: 110C) for 17 hours. After cooling to room temperature, the solution is carefully extracted with ether (three times) and finally 20 with chloroform. Evaporation of the aqueous phase, followed by stripping with isopropanol (twice~ and ethanol (twice) produces a solid residue which is digested with acetone, collected, and washed with acetone (three times), ethanol (three times) and finally with ether: white crystals, 1.8 g 25 (74%). This materi~l is recrystallized by dissolving in hot (80C) aqueous ethanol (100 mL, H20: ethanol 1:9), filtering through paper, and adding additional ethanol (10-20 mL) to yield the desired 6,6-difluoro-1,12-diamino-4,9~diaza-dodecane as the tetrahydrobromide salt: white 30 crystals 1.2 g, (49%).

10~28Br4F2N4 C~21.37; H,5.02; N,9,g7 ~ound: C,20.97; H,4.75; N,10,01 C-34,435 US

~ PATENTS

PREPARATION OF 2,2,11,11~TETRA~LUORO-4,12-DIAMINO-4~9-DIAZA-OCTANE
, . _ 1-Phthalimido-2,2-difluoro-3-butene To a stirred mixture o~ 2,2-di~luoro-1,4-butanediol 5 (12097 g, 103 mM), dry pyridine t55 mL) and dichloromethane (200 mL) is slowly added a solution of methanesulfonyl-chloride (23.6 g) in dichloromethane (50 mL). Stirring is continued overnight. The reaction mixture is wasbed with 2 N HCl (twice)l ~ollowed by 10~ aqueous NaHC03 until 10 neutral, and ~hen with water. The organic layer is dried (MgS04) and upon evaporation o~ the solvents, a slightly yellow oil is obtained. Crystallization is induced by scratching the oil ~ith dry ether, to yield the compound 1,4-bis-methanesul~onyloxy-2,2-difluorobutane as white 15 crystals: 22.3 g (77%).

The compound 1,4-bis-methanesul~onyloxy-2,2-difluoro-butane (20 g, 71 mM) and diazabicycloundecene (21.6 g, 142 mM) are stirred with dry tetrahydrofuran ~150 mL) and heated overnight at 80C under nitrogen. The solvents are 20 removed under vacuum3 and the residual oil is dissolved in dichloromethane, washed ~ith 1 N HCl (twice), brine (twice) and dried (Na2S04). E~aporation of the solvents yields the compound 1-methanesulfonyl-2,2-difluoro-3-butene as an oil:
11.2 g (85%).

The compound 1~methanesulfonyl-2,2-difluoro-3-butene (11.2 g, 60.2 mM), potassium phthalide (12.3 g, 66.4 mM) and dry dimethylformamide (30 mL) are stirred and heated (bath temperature 110C) under nitrogen ~or 120 hours.
After cooling to room temperature, the crude reaction 30 product is precipitated by the addi~ion of water (approximately 300 mL), ~iltered and dissolved in C-34,435 US

, "

A~ ~ 5 ~ Q PATENTS

dichloromethane. The dichloromethane solution is washed with l N potassium hydroxide ~twice3, water (twice), dried (Na2S04) and evapora~ed to yield 1-phthalimido-2,2-difluoro-3-butene as beige-colored crystals: 11.9 g (83%).

5 1-p-Toluenesul~onamino-2,2-dlfluoro-3-butene The compound 1-phthalimido-2,2-difluoro-3-butene (~1.4 g, 48.1 mM) is heated for 20 hours. After cooling in an ice bath, the phthalic acid is removed via filtration and the filtrate e~aporated. The residue so obtained is 10 dissolved in water, extracted with ether (twice), evaporated to dryness and stripped with isopropanol.
Trituration with ether yields hygroscopic crystals of 1-amino-2,2-di~luoro-3-butene as the hydrochloride salt:
6.12 g, (88%).

To a stirred mixture of 1-amino-2,2-difluoro-3-butene hydrochloride (6.1 g, 42.5 ~M), 50 mL of dry dichloro-methane and triethylamine t8.74 g, 2 equivalents), all of which have been cooled in an ice bath, is slowly added a solution of tosyl chloride (8.1 g, 1 eq~ivalent) in 50 mL
20 of dichloromethane. Stirring is continued overnight at room temperature, additional dichloromethane is added to the ~ reaction mixture, and the resulting organic layer is washed ; with 1 N HCl (twice), water (twice) and dried (Na2S04).
Evaporation of the solvent yields a brown semi-solid 25 material (~.66 g~, which is purified via ~lash chromatography on silica (300 g, eluent: ethyl acetate/petroleum ether 20-80; fraction size 100 mL).
Fractions 17-25 are combined and evaporated to yield 1-p-toluenesulfonamino-2,2-di~luoro-3-butene as white crystals;
30 4.8 g (43%). The desired compound can be recrystallized from ether/petroleum ether to yield very ~ine, cotton-like, needles.

C-34,435 US

~ ~ S ~ ~ ~ PATENTS

. .
Anal- Calc'd ~or C11H13F2N2S C,50.56; H95-02; N~5-36-Found: C,50.93; H,5.02; N,5.45.

Following essentially the same procedure ~or the preparation of the 2,2-difluoro-4912-diamino-4,9-diaza-5 dodecane, two equivalents of the 1-~-toluenesulfonamino-2,2-difluoro-3-butene, prepared above, is alkylated with one equ~valent of 1,4-dibromobutane; the double bonds are oxidized to the corresponding dicarboxylic acid with ~MnO4 in aqueous acetic acid; the resulting dicarboxylic acids 10 are reduced to the corresponding primary diols with borane-methylsulfide complex; the diols are mesylated with methanesulfonyl chloride; the mesyl derivative is reacted with potassium phthalimide to obtain the corresponding 1,12-diphthalimido deri~ative; and the phthaloyl and tosyl 15 protecting groups are removed to obtain the desired 2,2,11,11-tetrafluoro-1,12-diamino-4 9 9-diaza-dodecane.

DEMONSTRATION OF THE ANTIPROLIFERATIVE EFFECT OF ~,6-DIFLUORO-l,12-DIAMINO-4,9-DIAZA-DODECANE

.
Morris rat hepatoma rl288C (HTC) cells are routinely 20 grown as a suspension culture in Swim's 77 medium supple-mented with 10% (V/V) dialysed horse serum, 11.0 mM
glucose, 2 mM glutamine, 0.057 mM cystine, 5.g mM NaHC03 and 50 mM o~ N-tris(hydroxymethyl)methylglycine. The HTC
cell cultures are incubated in the presence or absence of 25 1 m or 10 m of the compound 6,6-difluoro~1,12-diamino-4,9-diaza-dodecane and observed ~or a period of 11 days.

The cell culture medium is changed at day 2, to maintain cells in a logarithmic phase of gro~th. The actual cell numbers are determined by cell-counting and the C-34,435 US

.

", ~q ~ PATENTS
. i ~ !
relative cell growth is calculated taking into account the various dilution factors employed. The per cent inhibition of cell ~rowth is calculated according to the equation~
rNtn-NtO
100 - 100 . _ N n- N O

wherein NcO is the relatiYe growth of control cultures at time = 0, Ncn is the relative growth of control cultures at time = n, NtO is the relative growth of test cultures at time = 0, and Ntn is the relative growth of test cultures at time = n.
:
:; 15 Table I illustrates that administration of 1 ~ m of 6,6-difluoro-1,12-diamino-4,9-diaza-dodecane to the culture medium inhibits cell growth by 54% at the end of 4 days, whereas the administration of 10 ~m of the drug inhibits cell growth by 77% at the end of the same period of time.

C-34,435 US

.
... .

o ___ ___ _______ o ~oo ~

C~J h P O ~9 o ~ a~
,_ C!s E~ o c~> u~ C'J cr~

Q ~ ~ N
l O O ~
~ ~ O co ~
O O
~ __ ___ _______ 4~ ' ~1 _ O~

a _ ____~

Another aspect o~ this invention i~ the u8e of the fluorinated compounds disclosed herein a6 Nuclear Magnetic Resonance (NMR) ima~ing agents useful for the detection and diagnosis of tumor tissue by means of Fl9 NMR in ViYo spectroscopy and tumor imaging by Fl9 NMR tomography.
Although all of the fluorinated compounds of this invention are useful as such NMR agents ~or the detection an~ for the accurate determination of the precise location of any tumor existing in the mammal sought to be diagnosed and/or treated, those most active compou~ds useful as anti-proliferative and anti-tumor agents, are most preferred as NMR tumor imaging agents. Another preferred class of NMR imaging agents useful for the ~etection and location of any mammalian tumors are the difluoro intermediates used to prepare the fluorinated compounds of formula I, especially 2,2-difluoro-1,4-butane diamine.
For this end-use application the compounds may be administered in the range of 0~2 to 5 g.

C-3~,435 -28-. .

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1 . A gem-dihalo or tetrahalo-1,12-diamino-4,9-diaza-dodecane compound having the formula wherein x and Y represent hydrogen or halogen, with the proviso that in the case of a dihalo compound both halogens are present on one and only one carbon atom, and in the case of a tetrahalo compound the compound is 2,2,11,11-halo-substituted, or a pharmaceutically acceptable salt thereof, said halogen being selected from chloro and fluoro.

2. A compound according to claim 1 wherein the halogens are at the 6,6-position, or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1 wherein the halogens are at the 2,2-position, or a pharmaceutically acceptable salt thereof.

4. A compound according to claim 1 wherein the halogens are at the 2,2,11,11-positions, or a pharmaceutically acceptable salt thereof.

5. A compound according to claim 1 which is 6,6-difluoro-1,12-diamino-4,9-diaza-dodecane or a pharmaceutically acceptable salt thereof.

6. A process for the preparation of a gem 2,2-dihalo-, a gem 6,6-dihalo or a gem 2,2,11,11-tetrahalo-1,12-diamino-4,9-diaza-dodecane derivative having the formula wherein X and Y represent hydrogen, chloro or fluoro, or their pharmaceutically acceptable salts, which comprises heating a compound of the formulae wherein X1 and Y1 are chloro or fluoro, with hydrazine in an inert solvent to remove the phthaloyl protecting groups and by heating with aqueous HBr to remove the p-toluene-sulfonyl pro-tecting groups, and optionally converting the so-deprotected compounds to their pharmaceutically acceptable salts.

7. The process according to claim 6 wherein the gem-dihalo-compound prepared is a 6,6-dihalo-1,12-diamino-4,9-diaza-dodecane or a pharmaceutical acceptable salt thereof.

8. The process according to claim 6 wherein the gem-dihalo derivative prepared is a 2,2-dihalo-1,12-diamino-4,9-diaza-dodecane or a pharmaceutical acceptable salt thereof.

9. The process according to claim 6 wherein the gem-tetrahalo derivative is a 2,2,11,11-tetrahalo-4,12-diamino-4,9-diaza-dodecane or a pharmaceutical acceptable salt thereof.

10. An antiproliferative or antitumor composition comprising a therapeutically effective amount of a gem-dihalo or tetrahalo-1,12-diamino-4,9-diaza-dodecane compound having the formula wherein X and Y represent hydrogen or halogen, with the proviso that in the case of a dihalo compound both halogens are present on one and only one carbon atom, and in the case of a tetrahalo compound the compound is 2,2,11,11-halo-substituted; or a pharmaceutically acceptable salt thereof, said halogen being selected from chloro and fluoro, in admixture with a therapeutically acceptable carrier therefor.

11. A composition according to claim 10 wherein the halogens are at the 6,6-position, or a pharmaceutically acceptable salt thereof.

12. A composition according to claim 10 wherein the halogens are at the 2,2-position, or a pharmaceutically acceptable salt thereof.

13. A composition according to claim 10 wherein the halogens are at the 2,2,11,11-positions, or a pharmaceutically acceptable salt thereof.

14. A composition according to claim 10 which is 6,6-difluoro-1,12-diamino-4,9-diaza-dodecane or a pharmaceutically acceptable salt thereof.

15. An antiproliferative or antitumor composition according to claim 10 which contains, in addition, a therapeutically effective amount of 2-difluoromethyl-2,5-diaminopentanoic acid or [2R,SR]-6-heptyne-2,5-diamine.