BUTENOLIDE DERIVATIVES AS ANTI-CANCER AGENTS BACKGROUND OF THE INVENTION
Technical Field
The present invention relates generally to chemical compounds having anti-tumour effects and, more particularly, to new butenolides: 3,4 dichloro-5 ( 1'- methyl-1 ' formylamino) -2 (5H) furanone, 3,4 dichloro-5 (1 ' - methyl-1 ' formylamino) -2 (3H) furanone and derivatives thereof, which may be synthesized by the reaction of an alkylformamide (e.g., N-methylformamide) and a halogenated acylchloride (e.g., dichloroacetylchloride) or a di-halogenated carboxylic acid anhydride.
Description of the Related Art
Chlorinated compounds are potential immunomodulators in the management of both neoplastic and viral diseases. For example, it is known that certain halogenated carboxylic acids, such as trichloro- and dichloro-acetic acid, are capable of producing halogen that reacts with macromolecules within a cell such as protein. N-methylformamide is a known anti- cancer agent, but its use clinically as an anti- neoplastic agent has been limited because of associated hepatotoxicity .
It would be desirable to provide other novel compositions and treatment methods using chlorinated compounds .
BRIEF SUMMARY OF THE INVENTION A principal objective of the present invention is to provide a novel chemical compound having anti-tumour effects . Another object of the invention is to provide a butenolide (furandione) compound having anti-tumour effects that can be easily produced in large quantities
and that is water soluble.
A further object of the present invention is to provide an anti-cancer agent that has a broad spectrum of tumour-reduction activity against syngeneic mouse tumours .
Yet a further object of the invention is to provide a novel compound that has anti-tumour activity against chemoresistant xenografts.
Still another object of this invention is to provide methods for treating tumours in mammals using such novel compounds .
These and other objects of the invention are accomplished by a butenolide (furandione) anti-cancer agent or compound of the formula as set out in the accompanying claims .
Thus, according to a first aspect, the present invention provides a compound of formula:
Rl -C CH -R2
R3 -C C=0
O
or ts isomer:
Rl C -R2
R3 -CM C=0
- 3 - wherein
Rl and R2 are independently hydrogen, chlorine, bromine, fluorine, hydroxyl, amino, alkyl or aryl group or any functional group; and
R3 is independently alkyl, aryl, hydroxyl, or any functional group.
R4
/
Preferably, Rl and/or R3 is N and
\
R5
R4 and R5 are independently alkyl, aryl, hydroxyl, alkoxy or any functional group. In particular, a compound of the formula:
Rl -C CH -R2
R3 N C=0
is provided, wherein Rl and R2 are independently hydrogen, chlorine, bromine, fluorine, hydroxyl, amino, alkyl or aryl group or any functional group and R3 and R4 are independently alkyl, aryl, hydroxyl, alkoxy or any other group (preferably functional) . When each of Rl and R2 are chlorine, R3 is methyl, and R4 is formyl (-CH0) , the compound is 3,4 dichloro -5(1 '-methyl - 1 ' formylamino) -2 (5H) furanone or is 3,4 dichloro -5(1'- methyl - 1 ' formylamino) -2 (3H) furanone.
In the present invention, the term "functional group" is understood to include: a hydrogen atom, a halogen atom, alkyl, hydroxy, aryl, heteroaryl, alkoxy,
acyloxy, aryloxy, amino, alkylamino, arylamino, acyl, amide, imido, isocyanate, epoxide and vinyl group, and more complex heterocyclic groups such as hydantoin or rhodanine. Where Rl is amino it may be substituted by any of the above functional groups, or it may itself form part of one of the more complex R3 heterocyclic groups, thus forming a bicyclic system. Alkyl groups in any of the above, which of course include within their scope cyclo-alkyl groups, may be: replaced by (cyclo) alkenyl or (cyclo) akynyl ; substituted by an oxo group or hydrazone thereof;
Other preferred compounds of the invention are: a compound as claimed in the first aspect of the invention wherein one of Rl or R2 is chlorine, and the other is hydrogen; a compound as claimed in the first aspect of the invention wherein one of Rl or R2 is chlorine, and the other is OH; a compound as claimed in the first aspect of the invention wherein one of Rl or R2 is hydrogen, and the other is OH; a compound as claimed in the first aspect of the invention wherein both Rl and R2 are hydrogen; and a compound as claimed in the first aspect of the invention wherein both Rl and R2 are OH.
According to a second aspect, the present invention provides a composition comprising a compound, as set out in the first aspect of the invention, and a pharmaceutically acceptable carrier or diluent. Preferably the composition is a pharmaceutical composition.
One or more compounds as set out in the first aspect of the invention may be present in association with one or more non-toxic pharmaceutically and/or veterinarily acceptable carriers and/or diluents and/or adjuvants and if desired other active ingredients. Suitable carriers or diluents include water, buffers,
sodium bicarbonate, oil, polyethylene glycol (PEG) , glycerol and liposomes, or any which are known in the art (e.g. Handbook of Pharmaceutical Excipients (1994) 2nd Edition, Eds. A. Wade/PJ Weller, The Pharmaceutical Press, American Pharmaceutical Association) . The precise ingredients of a composition will vary depending on the use and nature of the composition; for example, if the composition is to be administered parenterally, adjuvants such as a local anaesthetic, preservative and buffering agents may be advantageously included.
The composition may be in the form suitable for oral, rectal, topical or parenteral administration. Suitable forms may be, for example, aqueous or oily suspensions, powders, granules, emulsions, solutions, cream, ointment, gels and liposomes.
All preferred features of the first aspect of the invention also apply to the second.
According to a third aspect, the present invention provides a method of making a compound as set out in the first aspect of the present invention, by reacting an alkylformamide and either a di-halogenated carboxylic anhydride or a halogenated acylchloride .
A general method of this aspect of the present invention involves reacting an alkylformamide with a halogenated acylchloride, such as dichloroacetylchloride, or a di-halogenated carboxylic acid anhydride, such as dichloroacetic anhydride, in a suitable solvent such as dichloromethane or chloroform. The reaction is cooled in an ice bath. Cold water is added with excess sodium bicarbonate powder and stirred for several hours. The bicarbonate powder is removed and the solvent evaporated and purified by column chromatography. The product is further evaporated and dried under vacuum. The third aspect of the invention also covers a method of making a composition as set out according to the second aspect of the invention. This method comprises mixing together the compound (according to the
first aspect of the invention) and a carrier or diluent. The mixing can be carried out using techniques known in the art .
Preferred features of aspects one and two also apply to the third aspect of the invention.
According to a fourth aspect, the present invention provides a method of treating a tumor in a mammal which comprises administering to said mammal an amount of a compound, as set out in the first aspect of the present invention, effective to reduce the size of said tumor. This aspect of the invention is particularly relevant to the treatment of humans, but is also applicable to general veterinery industry, in particular domestic pets such as dogs and cats and farm animals such as horses, pigs, cattle, sheep, etc.
Tumors treated with the compounds or compositions of the present invention may be solid tumors such as colon, ovary, or lymphomas etc, and/or other types of tumors such as leukaemias . The particular dosage regime will ultimately be determined by the attending physician and will take into consideration such factors as the medication being used, animal type, age, weight, severity of symptoms and/or severity of treatment being or to be applied, method of administration of the medication, adverse reactions and/or other contraindications. Specific defined dosage ranges can be determined by standard designed clinical trials with patient progress and recovery being fully monitored. Such trials may comprise an escalating dose design using a low percentage of the maximum tolerated dose in animals as the starting dose in man. Preliminary guidance for dosage ranges can be taken from the results given in the experimental section of this text and by the following ranges which have been extrapolated to humans.
The medication according to this aspect of the invention may be given to a patient in combination with
other suitable treatment, such as radiotherapy and/or surgical procedures .
Preferred features of aspects one to three also apply to the fourth aspect of the invention. According to a fifth aspect, the present invention provides a compound or a composition as set out in the first or the second aspect of the invention for use in medicine .
All relevant features of the first to fourth aspects of the invention also apply to the fifth aspect. According to a sixth aspect, the present invention provides a compound or a composition as set out in the first or the second aspect of the invention for use in the treatment of a tumor, preferably to reduce the size of the tumor.
All relevant features of the first to fifth aspects of the invention also apply to the sixth aspect. According to a seventh aspect, the present invention provides the use of a compound as set out in the first aspect of the invention in the manufacture of a medicament for the treatment of a tumor.
All relevant features of the first to sixth aspects of the invention also apply to the seventh aspect .
In one particular preferred embodiment, this novel butenolide is synthesized by the reaction of N- methylformamide with dichloroacetylchloride. It has been found that this compound interacts with nucleophilic sites within a cell and has effective antitumour activity against, e.g., murine colon adenocarcinomas (MAC13 and MAC15A) , M5076-recticulum cell sarcoma, and human colon xenografts, SW620, HCT116 and D D-1 (which are normally chemoresistant) .
Generalizing, the present invention is directed toward a novel composition preferably formed by reacting an alkylformamide and an halogenated acylchloride or anhydride to generate the novel butenolide (furandione) compound, and use of such compounds as anti-cancer
- 8 - agents. Compounds of the present invention may be water- or lipid-soluble .
In the formulae of the present invention, alkyl groups include a chain length of 1 to 20 carbon atoms, preferably 1 to 6 carbon atoms .
BRIEF DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the present invention and the advantages thereof, reference should be made to the following Detailed Description taken in connection with the accompanying drawings in which:
FIGURE 1 is a diagram illustrating the formation of a preferred butenolide composition in accordance with the invention; FIGURE 2 is diagram illustrating the mechanism by which the preferred butenolide composition is believed to react with nucleophilic sites in cells to effect the purposes of the present invention;
FIGURE 3 is a table showing anti-tumor activity of the butenolide compound against M5 tumours;
FIGURE 4 is a table showing anti-tumor activity of the butenolide against MAC 13 tumours;
FIGURE 5 is a table showing anti-tumor activity of the butenolide composition and 5-Fluorouracil ("5FU") against MAC 15 tumours;
FIGURE 6 is a table showing anti-tumor activity of the butenolide composition against MAC 15 tumours;
FIGURE 7 is a table showing anti-tumor activity of the inventive composition and 5FU against MAC 15 tumours ;
FIGURE 8 is a table showing anti-tumor activity of the inventive composition against MAC 15A tumours with the drug being dissolved in 0.1% sodium bicarbonate;
FIGURE 9 is a table showing anti-tumor activity of the inventive composition against MAC 15 tumours with the drug being dissolved in 0.2% sodium bicarbonate;
FIGURE 10 is a table showing anti-tumor activity of
- 9 - the inventive composition against MAC 15 tumours with the drug being dissolved in water;
FIGURE 11 is a table showing anti-tumor activity of the preferred butenolide composition and several variations thereof against MAC 13 tumours;
FIGURE 12 is a table showing anti-tumor activity of several test compositions against MS tumours;
FIGURE 13 is a chart showing anti-tumor activity of the inventive composition against SW620 xenografts; FIGURE 14 is a chart showing anti-tumor activity of inventive composition against HCT116 xenografts;
FIGURE 15 is a chart showing anti-tumor activity of the inventive composition against DLD-1 xenografts;
FIGURE 16 shows a route by which butenolides may be made, the form in which it may exist in equilibrium, as well as some example compounds;
FIGURE 17 shows examples of compounds which can be made by altering the side chain in order to manipulate lipophilicity; FIGURE 18 shows how the biological activity of compounds maybe manipulated and studied by varying the halogen in the 3 , 4 -position;
FIGURE 19 shows a sketch of how butenolide building blocks maybe prepared, starting with furfural; FIGURE 20 shows further reactions which provide some other example compounds; and
FIGURE 21 shows how structure-activity relationships of the compounds of the present invention may be investigated. FIGURE 22 shows a scheme for the synthesis of some example compounds of the present invention.
FIGURE 23 is a chart showing the results of treating MAC 15A tumor sc transplants with WOA081/96B, 5-fluorouracil and N-methylformamide. FIGURE 24 shows the reaction of WOA081/96B with nitrophenylhydrazine . This reaction may be used for the structural investigation of the compounds of the present
- 10 - invention, and is an example of a reaction of a compound of the present invention with a nucleophilic substance.
FIGURE 25 is a flow-chart showing a general process of producing a compound of the present invention. FIGURES 26 to 33 show further reaction schemes for preparing compound according to the invention, and the compounds produced thereby.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention relates to a preferred butenolide (furandione) composition (sometimes referred to herein for convenience as "WOA 081/96B") and its anti -tumour activity against tumour lines with different histology, growth characters and spectra of chemosensitivity to standard agents. Several variations of this butenolide composition are also described as set forth below and are provided with different reference identifiers for convenience of discussion. By way of brief background, the mouse adenocarcinoma of the colon ("MAC") series of transplantable tumours has been used in a variety of chemotherapy studies. The MAC tumours are similar to human colon cancer. It has also been demonstrated that the spectrum of chemotherapeutic sensitivity shows good correlation with the response rates of standard chemotherapeutic agents against colorectal cancers . The anti-tumour activity is usually close to maximum tolerated dose indicating the general insensitivity of the tumour system. Similarly, human colon tumour xenografts SW620, HCT116 and DLD-1 are chemoresistant, particularly the slow growing DLD-1 tumour. The M5076 is a reticulum cell sarcoma and is generally chemosensitive .
- 11 -
Preparation of WOA 081 /96B
FIGURE 1 illustrates a method of preparing WOA 081/96B in accordance with the present invention. WOA 081/96B is prepared preferably by the reaction between equimolar amounts of dichloroacetylchloride or dichloroacetic anhydride with dry N-methylformamide in 500 mis of dry dichloromethane. The reaction preferably is carried out in the following steps. The dry N- methylformamide is dissolved in dry dichloromethane (or in any other suitable solvent, e.g., chloroform) . The dichloroacetylchloride or dichloroacetic anhydride in dichloromethane is added dropwise with stirring while the solution is cooled in an ice-bath maintaining reaction temperature between 60-80°C. Use reflux condenser and stir for 6-24h. Cold water is added, and the solution is stirred for another 1 hour. Add in small portions excess sodium bicarbonate powder and stir overnight (or for a similar time period) .
The excess bicarbonate powder is removed by a filtering process using a sintered glass funnel with a vacuum pump. The dichloromethane is evaporated, leaving a yellow or yellow-orange residue. This is purified by column chromatography using petroleum ether:dichloromethane : ethanol (2:2:1) or petroleum ether :diethyl ether (1:1) . The purity of the product is checked on a silica TLC plate using petroleum ether :diethyl ether (1:1) as solvent. The solvent is evaporated and the product dried under vacuum overnight (or for such a time period) with magnesium sulphate or calcium chloride. The resulting product is stored below 4°C and protected from both light and moisture.
Spectroscopic and elemental analysis of the 2- nitrophenylhydrazone derivative suggest the formation of WOA 081/96B. The above-described method is useful to prepare
- 12 - other derivatives of halogenated acylchloride or halogenated carboxylic acid anhydride with N- methylformamide or any other amides. The present invention includes such resulting derivatives. Thus, for example, compositions referred to herein as WOA 080/96 and WOA 082/96 are products formed from the reaction between trichloro- and chloroacetyl chloride with N-methylformamide, respectively. These two products as will be seen also demonstrate significant anti-tumour activity, thereby further validating the efficacy of the inventive technique.
The compounds of the present invention may assume different equilibrium forms depending on the pH of the medium. Without being bound, the following equation is proposed to be a possible equilibrium which occurs for a compound of the present invention:
H Rl— C — C — R2 OH- Rl C — C — R2
R3— C C = O H+ R3 CH C = O
\ / O OH OH
The scope of the invention is understood to include all such alternative forms of the compounds of the present invention.
Example
Test Protocol
Pure strain NMRI and BDFj (age 6-8 weeks) from an inbred colony were used for transplanting MAC and M5076 tumours, respectively. They were fed on an RM3E diet (Lillco-England) and water ad libitum.
MAC 13 and MAC 16 tumours were transplanted into mice by sc implantation of tumour fragments (about 1 x 2
- 13 - mm) in the flank. MAC 15A ascites tumours were transplanted in male mice by sc inoculation of 1 x 106 tumour cells in 0.2 ml of physiological saline.
An evaluation of WOA 081/96B against SW620, HCT116 and DLD-1 human colon tumour xenografts was carried out by independent testing. The results are shown in FIGURES 13-15. In the independent testing, the following protocol was devised and used.
Tumour fragments excised from donor animals were first transplanted subcutaneously in the flank of the male NCR-nude mice.
WOA 081/96B was diluted either in PBS or 1% sodium bicarbonate solution and treatment was by a once daily intraperitoneal (ip) , subcutaneous (sc) , peroral (po) or intravenous (iv) administration.
Each group contained a minimum of 5 tumour-bearing mice . With the more rapidly growing MAC13 , MAC15A and M5076 sc tumours, chemotherapy commenced 2 days after implantation. MAC13 and MAC15A sc tumours are palpable at this stage and anti-tumour responses were assessed about 14 days later by recording tumour weights. With the slower growing MAC16, SW620, HCT1 16 and DLD-1 tumours, chemotherapy did not conunence until tumours could be reliably measured. Tumour volume was calculated from the formula a2 x b/2, where a is the smaller diameter and b is the larger.
Statistical significance was determined by either Mann-Whitney U test or a computerised method (statview 512+) .
Experimental Results
The WOA 081/96B compound interacts with nucleophilic sites within a cell as shown in FIGURE 2 and was thus tested for anti-tumour activity. As shown in FIGURES 3-15, WOA 081/96B has significant activity against MAC1-3, MAC15A, M5076 and human colon tumour xenografts SW620, HCT116 and DLD-1.
- 14 -
It was active when given daily or on alternate days independent of the route of administration.
FIGURE 3 is a table showing anti-tumor activity of WOA 081/96B against M5 tumours transplanted sc . The treatment was given ip daily, with the drug being dissolved in water. 8 animals per group, sacrificed on day 17 after transplant.
FIGURE 4 is a table showing anti-tumor activity of WOA 08 1/96B against MAC 13 tumours 14 days after sc transplants. 5 animals per group. The drug was dissolved in water and given ip.
FIGURE 5 is a table showing anti-tumor activity of WOA 081/96B and 5FU against MAC 15 tumours. The tumours were transplanted sc and treatment given ip daily. The drugs were dissolved in water.
FIGURE 6 is a table showing anti-tumor activity of WOA 081/96B against MAC15 tumours transplanted sc . Treatment was provided using various routes. The drug was dissolved in water. 5 animals per group. FIGURE 7 is a table showing anti-tumor activity of
WOA 081/96B and 5FU against MAC 15 tumours transplanted sc . The treatment was given ip and the drugs were dissolved in water. 6 animals per group.
FIGURE 8 is a table showing anti-tumor activity of WOA 081/96B against MAC15A tumours transplanted sc.
Treatment was given either ip or orally. The drug was dissolved in 0.1% sodium bicarbonate and administered using various schedules. 5 animals per group.
FIGURE 9 is a table showing anti-tumor activity of WOA 081/96B against MAC 15 tumours transplanted sc .
Treatment was given ip . The drug was dissolved in 0.2% sodium bicarbonate and Vitamin E dissolved in olive oil . 5 animals per group.
FIGURE 10 is a table showing anti-tumor activity of WOA 081/96B against MAC15 tumours transplanted sc .
Treatment was given ip, either daily or on alternate days. The drug was dissolved in water. 5 animals per
- 15 - group .
FIGURE 11 is a table showing anti-tumor activity of WOA 080/96, 081/96B and 082/96 against MAC13 tumours 14 days after sc transplant . The drugs were dissolved in water and given ip on days 1,2,3,4,5 and 8. 5 animals per group .
FIGURE 12 is a table showing anti-tumor activity of WOA 080/96, 081/96B, 082/96, 083/96, and 084/96 against MS tumours 20 days after sc transplant. The drugs were dissolved in water and administered ip . 5 animals per group .
FIGURE 13 is a chart showing anti-tumor activity of WOA 081/96B against SW620 xenografts.
FIGURE 14 is a chart showing anti-tumor activity of WOA 081/96B against HCT116 xenografts.
FIGURE 15 is a chart showing anti-tumor activity of WOA 081/96B against DLD-1 xenografts.
FIGURE 20 shows further reactions which provide some other example compounds. The reaction of mucochloric acid and hydrazine will provide diaza lactones and the reaction with isocyanates will furnish carbamates which are closer to the original lead structure. A wide variety of isocyanates or isothiocyanates are commercially available to synthesize a series of analog compounds. Umpolung in the 5- position: numerous N nucleophiles which are either prepared by using thionyl chloride or DEAD will be connected to the 5-position.
FIGURE 23 is a chart showing the results of treating MAC 15A tumor sc transplants with WOA081/96B, 5-fluorouracil and N-methylformamide.
As the results show, this compound demonstrates significant anti-tumour activity over a wide range of chemoresistant tumours. Dose-limiting body weight loss is preventable by formulating it in 1% sodium bicarbonate solution.
FIGURES 26 to 33 show further reaction schemes for
- 16 - preparing compound according to the invention, and the compounds produced thereby.
Having thus described our invention, what we claim as new and desire to secure by Letters Patent is set forth in the following claims.
Other variations and modifications of this invention will be apparent to those skilled in the art alter careful study of this application. This invention is not to be limited except as set forth in the following claims. All references disclosed herein are incorporated by reference.