This is a continuation-in-part application of U.S. Serial No. 374,343, filed June 30, 1989, which in turn is a continuation-in-part of application U.S. Serial No. 270,963, filed November 14, 1988, the contents of which are hereby incorporated by reference into the present application.

Background of the Invention;

Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms which normally govern proliferation and differentiation. For many years there have been two principal strategies for chemotherapeutic treatment of cancer: a) blocking hormone-dependent tumor cell proliferation by interference with the production or peripheral action of sex hormones; and b) killing cancer cells directly by exposing them to cytotoxic substances, which injure both neoplastic and normal cell populations.

Relatively recently, cancer therapy is also being attempted

by the induction of terminal differentiation of the neoplastic ^' cells (1). In cell culture models differentiation has been reported by exposure of cells to a variety of stimuli, including: cyclic AMP and retinoic b _. acid (2,3), aclarubicin and other anthracyclines (4).

There is abundant evidence that neoplastic transformation does not necessarily destroy the potential of cancer cells to differentiate (1,5,6). There are many examples of tumor 0 cells which do not respond to the normal regulators of proliferation and appear to be blocked in the expression of their differentiation program, and yet can be induced to differentiate and cease replicating. A variety of agents, including some relatively simple polar compounds (5,7-9), 5 derivatives of vitamin D and retinoic acid (10-12) , steroid hormones (13), growth factors (6,14), proteases (15,16), tumor promoters (17,18), and inhibitors of DNA or RNA synthesis (4,19-24), can induce various transformed cell lines and primary human tumor explants to express more 0 differentiated characteristics.

Early studies by the present inventors identified a series of polar compounds that were effective inducers of differentiation in a number of transformed cell lines (8,9) . 5 of these, the most effective inducer, until recently, was the hybrid polar/apolar compound N,N'-hexamethylene bisacetamide (HMBA) (9) . The use of polar/apolar compounds to induce murine erythroleukemia cells (MELC) to undergo erythroid differentiation with suppression of oncogenicity 0 has proved a useful model to study inducer-mediated differentiation of transformed cells (5,7-9). HMBA-induced

MELC terminal erythroid differentiation is a multistep process. Upon addition of HMBA to MELC (745A-DS19) in 5

culture, there is a latent period of 10 to 12 hours before commitment to terminal differentiation is detected. Commitment is defined as the capacity of cells to express terminal differentiation despite removal of inducer (25) . Upon continued exposure to HMBA there is progressive recruitment of cells to differentiate. Recently, the present inventors reported that MELC cell lines made resistant to relatively low levels of vincristine become markedly more sensitive to the inducing action of HMBA an can be induced to differentiate with little or no laten period (26) .

HMBA is capable of inducing phenotypic changes consisten with differentiation in a broad variety of cells lines (5) . The characteristics of the drug induced effect have bee most extensively studied in the murine erythroleukemia cel system (MELC) (5,25,27,28). MELC induction o differentiation is both time and concentration dependent. The minimum concentration required to demonstrate an effec iji vitro in most strains is 2 to 3 mM; the minimum duratio of continuous exposure generally required to induc differentiation in a substantial portion (>20%) of th population without continuing drug exposure is about 3 hours.

The primary target of action of HMBA is not known. Ther is evidence that protein kinase C is involved in the pathwa of inducer-mediated differentiation (29) . The in vitr studies provided a basis for evaluating the potential o HMBA as a cytodifferentiation agent in the treatment of human cancers (30) . Several phase I clinical trials wit

HMBA have been completed (31-36) . Recently, the firs

evidence was reported that this compound can induce a therapeutic response in patients with cancer (35,36) . These phase I clinical trials demonstrate that the potential efficacy of HMBA is limited, in part, by dose-related toxicity which prevents achieving optimal blood levels and by the need for intravenous administration of large quantities of the agent, over prolonged periods.

The present invention provides new chemical inducers which are 2-20 times more active than HMBA. It has unexpectedly been found that compounds having two or more nonpolar components connected by a polar group and having groups on the termini of the compound are effective inducers of terminal differentiation. For instance, bis-hexamethylene triacetamide, which comprises three acetamide groups connected by two six-carbon chains, has been found to be a potent inducer of terminal differentiation in MELC.

This new class of compounds of the present invention may be useful for selectively inducing terminal differentiation of neoplastic cells and therefore aid in treatment of tumors in patients.

FW^TY ^ft f he Invention:

The invention provides a class of compounds having the structure:

[R-A]-B-A ₁ -B ₁ -[A ₂ -B ₂ -] _a [A ₃ -B ₃ -] _b [A ₄ -R ₁ 3

wherein each of A, A _χ , A ₂ , A ₃ , and A ₄ represent a polar group which comprises a nitrogen, sulfur or oxygen ato wherein each of A, A _j , A ₂ , A ₃ , and A ₄ may independently b the same as, or different from, the others of A, A _χ , A ₂ , A ₃ , and A ₄ ;

wherein each of R and R _λ is a hydrogen atom; a lower alkyl, alkenyl, or alkynyl group; or a group having the structure:

-N

I R,

each of R ₂ and R ₃ being a hydrogen atom or a lower alkyl alkenyl, or alkynyl group; and wherein each of R, R _χ , R ₂ an R ₃ may independently be the same as, or different from th other of R, R _j , R ₂ and R ₃ ;

wherein each of [R-A] and [A ₄ -R ₁ ] have a dipole momen greater than about 2.7 Debye units;

wherein each of B, B _j^ , B ₂ , and B ₃ represents a nonpola group which comprises at least 4 atoms in a chain, th termini of which chains are attached to A and A _χ , A _χ and A ₂ A ₂ and A ₃ , and A ₃ and A ₄ , respectively; wherein each suc atom is oxygen, nitrogen, carbon, or sulfur and wherein eac of B, B _x , B ₂ , and B ₃ ;

and wherein each of a and b is independently 0 or 1.

The invention also concerns a method of selectively inducing terminal differentiation of neoplastic cells and thereby inhibiting proliferation of such cells which comprises contacting the cells under suitable condition with an amount of the compound effective to selectively induce terminal differentiation.

Moreover, the invention provides a method of treating a patient having a tumor characterized by proliferation of neoplastic cells which comprises administering to the patient an amount of the compound effective to selectively induce terminal differentiation of such neoplastic cells, thereby inhibiting their proliferation, and suppressing oncogenicity.

Lastly, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound in an amount less than an amount which cause toxicity in the patient.

Brief Description of the Figures;

Figure 1: Comparison of the hybrid polar/apolar compounds, (A) hexa ethylene bisacetamide

_ (HMBA) ; (B) suberic acid bis-N-methyl b diacetamide (SBDA) ; and (C) bis- hexamethylene triacetamide (BHTA) as inducers of differentiation of vincristine-sensitive (745A-DS19) (•) and ₀ vincristine-resistant (VCR.C(2) 15) (A)

MELC for HMBA and SBDA and (V3.17) for BHTA. Each compound was added to the cells in culture at the final concentration indicated. Benzidine reactive cells (left panel of each section of this figure) were determined after 4 d. of culture and commitment (right panel of each section of this figure) after 2 d. of culture. 0

Figure 2: Concentration dependent curves of inducer activity of compound 12. with vincristine- sensitive (745A-DS19) (•) andvincristine- resistant (VCR.C(2)15) (A) MELC. The 5 compound was added to the cultures at the final concentrations indicated. Benzidine reactive cells were determined after 5 d. in culture and commitment after 2 d. of culture. 0

Figure 3: optimal concentration of IC-135 for inducement of terminal differentiation.

Figure 4: Comparison of HMBA and IC-135 on DS19 5

cells.

Figure 5A and B: Comparison of % cell committed for HMBA and IC- 135 on V3.17 and DS19 cell lines.

Figure 6A and B: Comparison of B+% for HMBA and IC-135 on V3.17 and DS19 cell lines.

Detailed Description of the Invention:

The invention provides a class of compounds having the structure:

[R-A]-B-A ₁ -B ₁ -[A ₂ -B ₂ -] _a [A ₃ -B ₃ -] _b [A ₄ -R ₁ ]

wherein each A, A _x , A ₂ , A ₃ , and A ₄ represent a polar group which comprises a nitrogen, sulfur or oxygen atom and wherein each of A, A _χ , A ₂ , A ₃ , and A ₄ may independently be the same as, or different from, the others of A, A _χ , A ₂ , A ₃ , and A ₄ ;

wherein each of R and R _j^ is a hydrogen atom; a lower alkyl, alkenyl, or alkynyl group; or a group having the structure:

-N r

I

each of R ₂ and R ₃ being a hydrogen atom or a lower alkyl, alkenyl, or alkynyl group; and wherein each of R, R _j ^ R ₂ an

R ₃ may independently be the same as, or different from, th others of R, R _{l f} R ₂ and ₃ \,>

wherein each of [R-A] and [A ₄ -R _j ] have a dipolar momen greater than about 2.7 Debye units;

wherein each of B, B _χ , B ₂ , and B ₃ represents a nonpolar group which comprises at least 4 atoms in a chain, th termini of which chains are attached to A and A _χ , A _χ and A ₂ , A ₂ and A ₃ , and A ₃ and A ₄ , respectively; wherein each suc

atom is oxygen, nitrogen, carbon, or sulfur and wherein each of B, B _χ , B ₂ , and B ₃ ;

and wherein each of a and b is independently 0 or 1.

The compounds of the present invention are made up of two components. One component comprises a polar group, i.e. functional groups with significant dipole moments, such as amides, sulfoxides, a ine oxides and related functional groups.

The terminal portions of the compound, represented by R-A and A ₄ -R _χ , each have dipole moments greater than about 2.7 debye units. The polar groups within the compound, represented by - _1# -A ₂ - and -A ₃ -, have significant dipolar moments but not necessarily in excess of 2.7 debye units. In the preferred embodiments, the polar groups are carbonyl radicals or bivalent radicals of an amide, a sulfoxide or a amine oxide. Each polar group need not necessarily be the same as the other polar groups. In the most preferred embodiments, the poplar groups within the compound are the same as each other and the terminal polar groups are the same. Preferably, all the polar groups are amide groups attached to the compound at the nitrogen atom or at the carbon atom of the carbonyl radical. The amide group may comprise one or more hydrocarbon substituents, such as a lower alkyl or alkenyl groups, including branched or unbranched groups. The term "lower alkyl or alkenyl group" is intended to include saturated and unsaturated hydrocarbon groups with 1 to about 5 carbon atoms.

The embodiments where a and b are 0 and A is a carbonyl radical or a group having the structure:

I

^R 4 wherein R ₄ is a hydrogen atom or a lower alkyl or alkenyl group, have proven to be most useful embodiments to date.

Particularly preferred are compounds where a and b are 0, A is a carbonyl radical and R has the structure:

wherein R ₂ and R ₃ each is hydrogen atom, a methyl group or a ethyl group.

The compound also requires at least two nonpolar sections, designated B and B _{l t} which are attached to and connect polar groups. Additional nonpolar sections may also be present, e.g. B ₂ when a is 1 and B ₃ when b is 1. The nonpolar sections may comprise linear saturated hydrocarbon chains, linear unsaturated hydrocarbon chains containing one or more double or triple bonds, or saturated or unsaturated hydrocarbon chains containing one or more lower alkyl or alkenyl groups or small carbocyclic rings as substituents. In one of the preferred embodiments, the nonpolar groups are hydrocarbon chains comprising 4 to 7 methylene groups, especially preferred are hydrocarbon chains containing 6 carbon atoms.

The preferred compounds for the practice of the present invention are those having the structures:

(CH ₃ ) ₂ -N- -N-(CH ₃ ) ₂

O H O

II I 'I

(CH ₃ ) ₂ -N-C-(CH ₂ ) ₅ -C-(CH ₂ ) ₅ -C-N-(CH ₃ ) ₂

C=0

I ^N -( ^CH 3>2

R R

\ /

N

I

O C=0 o M | ||

R-N-C- ( CH, ) -CH- ( CH, ) -C-N-R

I ml X -S X I

R R

0 0 0

II II H or R-N-C- ( CH ₂ ) _χ -C-NR- ( CH ₂ ) _χ -C-N-R

R R

wherein R is hydrogen or a methyl group and x is 5 or 6.

Other compounds of this invention include a compound having the structure:

a compound having the structure:

CH-

wherein Rl and R2 may be the same or different and each is a lower alkyl group; a compound having the structure:

^{2 5} U ^{3 2}

wherein R is H or a lower alkyl group; a compound having the structure:

wherein n is an integer which is greater than 3 and ₁ and R ₂ may be the same or different and each is H or a lower alkyl group;

a compound having the structure:

wherein XI and X2 may be the same or different and each is N(CH3)2 or HNCH ₃ ; a compound having the structure:

wherein Rl and R2 may be the same or different and is H or CH3;

a compound having the structure:

CH3- N — C 5-C-N-CH3 I II II I

CH3 O 0 CH3

wherein Rl and R2 may be the same or different and each is H or CH3; a compound having the structure:

or

HO-NH-C—(CH ) —C-NH-OH

II * * I III o o a compound having the structure:

wherein Rl and R2 may be the same or different and is H or a lower alkyl group;

a compound having the structure:

0 Rl R2 0 l I U

2((CH3)2—N- -C)—C—(CH2)4 C—(C—N—(CH3)2))2

wherein Rl and R2 may be the smae or different and is H or a lower alkyl group.

The invention also concerns a method of selectively inducing terminal differentiation of neoplastic cells and thereb inhibiting proliferation of such cells which comprises contacting the cells under suitable condition with an amoun of the compounds shown above effective to selectively induc terminal differentiation in the cells.

The contacting must be performed continuously for prolonged period of time, i.e. for at least 48 hours, preferably for abut 4-5 days or longer.

The method may be practiced in vivo or n vitro. If th method is practiced in vitro, contacting may be effectiv by incubating the cells with the compound. Th concentration of the compound in contact with the cell should be from about 0.5 to 25 mM, preferably from about mM to about 5 mM. However, for most preferred compoun having the structure: 8 spaces, an amount of about 0.01 m to about 10 mM has been shown to be effective. Th concentration depends upon the individual compound. Fo example, compound 12 of Table 1 should have a concentratio from about 0.1 to about 2 mM, preferably from about 0.5 t

However, for the most preferred compound having the structure:

HO-NH-C—(CH-) _β —C-NH-OH II ^a • II

an amount of about 0.01 mM to about 10 mM has been shown to be effective.

The concentration depends upon the individual compound. For example, compound 12 of Table 1 should have a concentration from about 0.1 to about 2 mM, preferably from about 0.5 to about 0.7 mM. Another factor determining the preferable range is the state of the tumor cells. Thus, in cells which have low levels of vincristine resistance, the range of effective concentration of compound 12 from Table 1 is from about 0.01 to about 0.3 mM with a preferable range of about 0.05 to about 0.1 mM.

The invention also concerns a method of treating a patient having a tumor characterized by proliferation of neoplastic cells which comprises administering to the patient an amount of the compounds shown above effective to selectively induce terminal differentiation of such neoplastic cells thereby inhibiting their proliferation, and suppressing oncogenicity.

The method of the present invention is intended for the treatment of human patients with tumors. However, it is also likely that the method would be effective in the

treatment of tumors in other animals. The term tumor is intended to include any cancer caused by the proliferation of neoplastic cells, such as lung cancer, acute lymphoid myeloma, bladder melanoma, renal carcinoma, breast carcinoma, or colorectal carcinoma. The administration of the compound to the patient may be effected orally or parenterally. To date, administration intravenously has proven to be effective. The administration of the compound must be performed continuously for a prolonged period of time, such as for at least 3 days preferably more than 5 days. In the most preferred embodiments, the administration is effected continuously for at least 10 days and is repeated at intervals wherein at each interval the administration is continuously effected for at least 10 days. For example, the administration may be effected at intervals as short as 5-10 days, up to about 25-35 days and continuously for at least 10 days during each such interval. The optimal interval period will vary depending on the type of patient and tumor. For example, in the incidence of acute leukemia, the so called myelodysplastic syndrome, continuous infusion would seem to be indicated so long as the patient tolerated the drug without toxicity and there was a positive response.

The amount of the compound administered to the patient i less than an amount which would cause toxicity in th patient. In the certain embodiments, wherein the compoun has the structure:

II

(CH ₃ ) ₂ -N-C-(CH ₂ ) ₅ -C-(CH ₂ ) ₅ -C-N-(CH ₃ ) ₂

< ^" θCH ₂ CH ₃

O H O iι n

(CH ₃ ) ₂ -N-C-(CH ₂ ) ₅ -C-N-(CH ₃ ) ₂

the amount of the compound which is administered to the patient is less than the amount which causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound. Preferably, the concentration of the compound in the patient's plasma is maintained at about 1.0 mM. It has been found with HMBA that administration of the compound in an amount from about 5 gm/m ² /day to about 30 gm/m ² /day, particularly about 20 gm/m ² /day, is effective without producing toxicity in the patient. For the compound shown above, in vitro studies have shown that the optimal amount of the compounds is substantially lower than 30 gm/m ² /day, and may even be lower than 1 gm/m ² /day. The optimal amount of the compound which should be administered to the patient in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

This invention, in addition to the above listed compounds, is intended to encompass the use of homologs and analogs of such compounds. In this context, homologs are molecules having substantial structural similarities to the above-

described compounds and analogs are molecules havin substantial biological similarities regardless of structura similarities.

Tabla 1

CPD I Structure

0 0

II H H If

1. CH _j -C N- (CH ₂ ), ^■ M-C-CH, 200 >95 >95

O H H CH _j -M-C-CH _j 75 SO •70 NO

0 CH. O

It H « II

CH ₃ -C-H- (CH _j ) ₂ -C- (CH2) ₂ -M-C-CH ₃ 200 >»5 >95

O CH. O li H H M

CH ₃ -C-M- (CH ₂ ) _j -C-CH _j -M-C-CH _j 200 >»5 >90

4. II

0 H,C O

* II H H H N s. CH, -C-U-CH, -C- -CC--CH, -K-C-CH, 200 >ts NO

CH„

0 0 II H I II

«. CH _j CHj-C-N- (CHj) ₅ -M-C-CH _a CH ₃ 2 It >90 >90

O O

■ II II ■ __

7. CH ₃ -»-C- (CH ₂ ) ,-C-H-CH _j 200 >95 >90

(CH ₃ )-*-e- (CH,) ₄ -C-»-(CH,)

^» 2'» 3'2 22t >»0 >90 o o fl II

9. (CH ₃ ) ₂ -M-C- (CH ₂ ) ₇ -C-»- (CH _j ) _a 242 >90 >90

10.

HM- (CH, ) - -K- (CH.) --HH 341 >90 >90 i ^a • i ^{3 ■}

C-CH, C-CH, C-CH, II '

O 0 o O

11. II N

(CH ₃ ) _a -H-C- -("CVH ₃ )s ₅ --C-(CH ₂ ) ₅ -C-H-(CH ₃ ) ₂

C-0

I

H-(CH ₃ ) 3(» 2.5 >»0 >90

n I 1

(CH ₃ ) ₂ -N-C-(CH ₂ ) ₅ -C-(CH ₂ ) ₅ -C-H-(CH ₃ ) ₂

12. o* ^S OOI ₂ CH ₃ 442 O.C >90 >90

The invention also concerns a pharmaceutical composition comprising a pharmaceutically acceptable carrier, such as sterile pyrogen-free water, and the compound in an amoun less than an amount which if administered intravenously o orally to a patient would cause the patient's blood plasm concentration of the compound to exceed toxic levels.

The invention is illustrated in the Experimental Detai section which follows. This section is set forth to aid i an understanding of the invention but is not intended to, and should not be construed to, limit in any way th invention as set forth in the claims which follo thereafter.

Experimental Details: Methods Cells and Materials

MELC 745A-DS19 cells and the variants of MELC derived fro this cell line, namely, the vincristine-resistant MELC V3.1 and VCR.C(2)15 cell lines (26), and the dimethylsulfoxide resistant cell line, DR10 (37) , were maintained in alph minimal essential medium containing 10% fetal calf seru (16) . Cell cultures for all experiments were initiated wit cells in logarithmic growth phase (day 2 cultured cells) a a density of 10 cells/ml. Inducer compounds were added i the final concentrations indicated below, dissolved i culture medium without fetal calf serum unless otherwis indicated. Cell density and benzidine reactively wer determined as described (16) .

Commitment to terminal differentiation, characterized b limited cell division (colony size <32 cells) an accumulation of hemoglobin (benzidine reactive colonies) wa

assayed by a colony cloning assay using 2% methylcellulose as described (25) .

Chemistry

HMBA, compound 1. of Table 1, (9) was obtained from Aldrich

Chemical Co.

The preparation and characterization of compounds 1., 2, 6_, 2' -ϋ ^and ■£ °f Table 1 (9,27) was previously described. All new amides were purified by chromatography on alumina with 5% methanol in methylene chloride, and were judged pure by thin layer chromatography (single spot) and "^H NMR spectroscopy. Final products were characterized by ¹ H NMR, infrared, and Cl mass spectroscopy, while intermediates were characterized by ¹ H NMR spectra. The data were consistent with the assigned structures (expected infrared and NMR signals, M + 1 mass spectra) .

For the synthesis of compound 3_ (Table 1) , the known 3- methyl-1 ,5-dibromopentane (38) was converted to the bis- phthali ide derivative, and this was cleaved with hydrazine to afford 3-methyl-l,5-diaminopentane, isolated as the dihydrochloride (m.p. 123-126°). This was converted to compound 2 with acetic anhydride and triethylamine in dioxane.

Compound 4. (Table 1) (m.p. 67-68°) was obtained in quantitative yield by similar acetylation of commerically available 2-methyl-l,5-diaminopentane.

Compound 5, (Table 1) was synthesized from meso-2,3- dimethylsuccinic acid in six steps. Reduction of the dimethyl ester with LiAlH ₄ afforded the meso-2,3-

dimethylbutanediol (92% yield) . This was converted to the bis-tosylate, and then to the bis-phtali ide. Deprotection and acetylation as before gave compound 5_ as an oil (61% yield from the diol) .

Compound 10, (Table 1) was prepared by making a solution of

19.8gm commercial bis-hexamethylenetriamine in 500 ml of

1,4-dioxane at room temperature under argon. Then 44.8 ml. of triethylamine was added, and 20.3 ml. of acetyl chloride was slowly added with stirring. After two hours of stirring at room temperature the triethylamine hydrochloride was removed by filtration and the filtrate was concentrated in vacuo. The product triacetyl compound was isolated as a clear viscous oil at about 90% yield by chromatography o basic alumina using isopropano1 / ethy1 acetate/dichloromethane in the ratio 2/3/5. On thin laye plates of basic alumina with this solvent mixture th product had an R _f of ca. 0.6.

The mass spectrum (chemical ionization, NH ₃ carrier) showe peaks at 342 (100%, M + 1) , 227 (10%) and 115 (22%). Th infrared spectrum (thin film on NaCl) had bands at 3288, 2931, 2858, 1627, 1560, 1437, 1373, and 1292 cm ^"1 . In th proton NMR (CDC1 ₃ ) the acetyl groups appeared at 6.10 as broad signal, while the methylene protons appeared as

multiples with the expected intensities in the regions of 3.12 to 3.30 and 1.21 to 1.54.

bis-Hexamethylenetriacetamide (IC-135)

For preparation of the triamide compound ϋ (Table 1) , dimethyl malonate was dialkylated with ethyl 6- bromohexanoate under standard conditions. The resulting tetraester was then hydrolyzed and thermally monodecarboxylated to 1,7,13-tridecanetricarboxylic acid. Treatment with thionyl chloride followed by diethylamine afforded compound .11 (Table 1) .

Compound i2. (Table 1) was prepared by simple dialkylation of dimethyl malonate with the know N,N'-dimethyl-6- bromohexanecarboxamide (39) .

Each compound in Table 1 was assayed 3 or more times to determine effectiveness as an inducer of MELC (745A-DS19) cell line. The cell density of MELC in culture for 5 d. without inducer was 2.0 to 2.6 x 10 ₆ cells/ml. The cell density of MELC in culture for 5 d. with inducer was 1.2 to 2.0 x 10 _g cells/ml. Compounds 11 and JL2. were dissolved in absolute ethyl alcohol. The final concentration of ethyl alcohol in the cultured medium ranged between 0.1 and 3%. This concentration of ethyl alcohol had no effect on cell growth of MELC in culture without inducer. All other compounds were dissolved in culture medium without fetal calf serum. The indicated optimal concentration represents the final concentration in the culture medium.

Results

New Hybrid Polar/Apolar compounds Active as Cytodifferentiation Agents

In earlier studies (8) , the present inventors reported that fairly high concentrations of certain polar organic solvents induce MELC to undergo erythroid differentiation. The question arose as to how the effectiveness of polar compounds might be increased so that similar concentrations of these solvent-like molecules could induce differentiation. Although the mechanisms were not clear, and are still incompletely understood, the followin hypothesis was considered: perhaps, at the target site of action, more than one solvent molecule must bind o interact. if this were so, the well-known chelate effec could provide more effective compounds. Instead of bindin two or more independent solvent molecules, the cellula target might interact more efficiently with a singl molecule carrying two or more solvent-like groups. Provide these groups were held in the right relationship one to th other, binding of one would carry the other into the targe region providing a high, effective concentration. Such chelate effect is well precedented; it accounts for th strong binding of metal ions by chelating ligands, and i the basis for much of the catalytic activity of enzyme.

This concept led the present inventors to new effectiv cytodifferentiating agents. The best studied to data is hexamethylene bisacetamide (HMBA, Table 1, compound i) , consisting of two acetamide molecules linked at nitrogen b a six carbon polyethylene chain (5,9). N-methyl acetamid

(Table 1, compound 2.) , another of the polar organi

solvents, was shown to be effective, but only at high concentration (50 mM) , whereas HMBA induces erythroid differentiation in MELC at an optimal concentration of 5 mM (8) . The present inventors previously showed that the optimum number of methylene groups in the apolar chain is six (27) . In the present invention, it was found that a four or five carbon chain is comparably effective if extra carbons are provided as branching methyl groups (Table l, compounds 3., 4. and 5.) . This suggests that the important 0 factor is not simply the length of the chain but also the number of hydrophobic hydrocarbon units in it.

Acetamide can also be dimerized by linking the methyl groups of the molecule. Suberic acid bis-N-methylamide (SBDA;

15 Table 1, compound 2) , can be thought of as N-methylacetamide linked at the acetyl group by four methylene units. SBDA is comparable in activity as an inducer to HMBA (Figure 1A and B) . Since the structures of SBDA and HMBA are different, it is likely that the metabolic fates of these 0 two compounds are completely different, and their similarity in effectiveness means that the compounds themselves are the principal active agents, rather than their catabolic products.

25 HMBA and SBDA have their polar groups separated by identical methylene bridges, and they have a similar ratio of polar to apolar hydrophobic moieties. Many structurally related compounds have been examined, but only a few showed comparable or greater activity (Table 1, compounds 6. through

30. 12) • It is clear that the structures of active compounds may differ sufficiently from HMBA to make it likely that they will also display different pharmacokinetics. One of the more active of these hybrid compounds is a dimmer of HMBA. Bishexamethylene triacetamide (BHTA; compound 10) is

35

about 2 fold more active as inducer, on a molar basis, than HMBA (Figure 1C) . The most active of the hybrid compounds assayed in this study is one in which two pentamethylene carboxyamides are linked by the dimethyl ester of malonic acid (compound 12) . This compound, with 4 exposed polar groups balanced by two apolar pentamethylene domains, is roughly 10 fold more active than HMBA. For example, 0.6 mM compound 12. is about as effective as 5.0 mM HMBA, inducing over 90% of cells to differentiate after 5 d. in culture (Fig. 2).

Polymethylenediamine derivatives carrying propionyl groups instead of the acetyl groups of HMBA are also active, but methoxycarbonyl groups are less effective and bulky pivaloyl groups lead to loss of activity. The present inventors previously showed that HMBA can have a double bond (cis or trans) or a triple bond in the center and retain its activity (27) . Replacement of the polymethylene chain with a cyclohexane ring leads to inactivity (27) , although compound 9., with a longer chain interrupted by an amide group, is active.

Diamides of dicarboxylic acids, such as suberic acid, are active with either one or two methyl groups on each nitrogen (compounds _» 2. - and ) , but not with a methyl and a methoxyl substituent, and they are also active with one (but not two) ethyl groups on each nitrogen. Suberic diamides of pyrollidine, of morpholine, or of piperazine ar inactive. This shows that there is a limit to the amoun of carbon tolerated on the ends of the polar groups.

These findings indicate that for optimal activity, two, o even better, three or four uncharged polar groups of limite bulk must be connected by chains of about 5 to 6 carbons.

Increased Sensitivity of Vincristine-Resistant MELC to Polar/Apolar Compounds

As previously reported, MELC resistant to relatively low concentrations of vincristine show a marked increase in sensitivity to HMBA (Fig. 1A) (26) . The dose-response of MELC to several of the new polar compounds identified as being as or more active than HMBA was examined. The compounds assayed for inducing activity with vincristine- resistant MELC include compounds 1, 3. _/ 4., 8., Ifi, ϋ and 12 (Table 1) . In each instance, vincristine-resistant MELC were induced at lower concentrations than were required for induction of vincristine-resistant MELC were induced more rapidly than the vincristine-sensitive cells. For the most active of the compounds, 0.1 mM compound 12 was the optimal concentration for inducing vincristine-resistant MELC (VCR.C(2)15) . For example, 0.1 mM compound 12 induced only 6% commitment of over 95% of V.C.R. (2) 15 cells after 2 d. and the accumulation of over 80% benzidine-sensitive (DS- 19) MELC, 0.1 mM compound 12 induced only 6% commitment by d. 2 and 4% benzidine reactive cells by d. 5. At concentrations of compound 12 as low as 0.05, 35% of VCR.C(2)15 became benzidine reactive by d. 2, compared to only 2% of DS-19.

Effect of Polar/Apolar Compounds on MELC Resistant to inducer Mediated Terminal Cell Division

Several of the new polar/apolar compounds were evaluated as inducers of MELC cell line DR10, which is resistant to induction by dimethylsulfoxide and can be induced by HMBA to accumulate hemoglobin but not commit to terminal cell division (37) . Compounds 1_, 8. and 10, assayed as inducers of DR10, caused accumulation of hemoglobin, but not commitment to terminal cell division. Thus, these new hybrid polar/apolar compounds are similar to HMBA in their effect on DR10 cells.

Cell cultures were grown in the presence of different concentrations of Hexa ethylenebisacetamide (HMBA)

and compound 10, bis-Hexamethylenetriacetamide (IC-135) .

At 1, 2 and 5 days, the cell densities and the percentag of cells that were benzidine reactive (B+) were measured. Table 2 shows the cell densities, B+%, and percent of cell committed for cell lines DS19 and V3.17 grown in 1 mM to mM of HMBA and IC-135. Figures 3, 4, 5A and B, and 6A an B are graphical representations of the data presented i Table 2.

Table 3 shows cell counts for days 1, 2 and 5 and percentag of cells committed and benzidine reactive (B+) at day 5 fo cell liens DS-19, V3.17 and DR-10 grown in 5 mM of HMBA an 0.5 to 3 mM of IC-135.

As can be seen in Table 2 and 3 and Figures 3-6, IC-135 is more reactive in the tested cell lines at lower concentrations than HMBA.

Table 2

24h 48h 120h commitmen

IC-135 50 mM Stock. 40 λ - ImM

200 λ • • 5mM

HMBA 200 mM Stock 10 A - ImM 35 50 λ • 5mM.

Table 3

TESTING OF IC-135 ON DIFFERENT

MELC LINES

Discussion

The development of agents which can induce transformed cells to differentiate and suppress their oncogenicity has important implications for the treatment of cancer. While a number of agents have been identified that can induce tumor cells in vitro to express features of their differentiated phenotype and to decrease their proliferative capacity (4,10-24), these agents have generally proved to be relatively ineffective or two toxic when evaluated in clinical trials (40) .

Among cytodifferentiation agents, the hybrid polar/apolar compound, HMBA, has been one of the best characterized with respect to its in vitro inducing activity in MELC and in a number of other transformed cell lines, as well as for certain human tumor explants (30) . It can trigger a differentiation program in transformed cells which is similar to that of their normal lineage (5) .

The recent finding that vincristine-resistant MELC are more sensitive to HMBA and to other active hybrid polar/apolar compounds provides an approach toward identifying the mechanisms of HMBA action (26) . The lack of a latent period for inducer-mediated differentiation of vincristine- resistant cells suggests that an early effect of inducers may involve alternations (e.g. new proteins, or modification of existing proteins such as phosphorylation) which are

constitutively expressed in the vincristine-resistant cell lines, and may, therefore, be identified and characterized.

The observed positive therapeutic responses to HMBA, albeit

largely transient, occurred despite relatively low serum concentrations of HMBA (<1 mM) , compared to the optimum demonstrated in vitro (4 to 5 mM) (35,36). The present invention provides a new group of hybrid polar/apolar compounds which are as active or even more active, on a molar basis, than HMBA.

References:

1. Sporn, M.B., Roberts, A.B., Driscoll, J.S. (1985) in Cancer: Principles and Practice of Oncology. eds. Hellman, S., Rosenberg, S.A. & DeVita, V.T. ,

Jr., Ed. 2, (J.B. Lippincott, Philadelphia), P. 49.

2. Breitman, T.R., Selonick, S.E., and Collins, S.J. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Natl. Acad. Sci. USA., 77:2936-2940, 1980.

3. Olsson, I.L., and Breitman, T.R. Induction of differentiation of the human histiocytic lymphoma cell line U.937 by retinoic acid and cyclic adenosine 3' :5'-monophosphate-inducing agents. Cancer Res., 42:3924-3927, 1982.

4. Schwartx, E.L. & Sartorelli, A.C. (1982) Cance Res.. 42. 2651-2655.

5. Marks, P.A., Sheffery, M. & Rifkind, R.A. (1987) Cancer Res. 47. 659.

6. Sachs, L. (1978) Nature (Lond.) 221, 535.

7. Friend, C. , Scher, W. , Holland, J.W. & Sato, T. (1971) Proc. Natl. Acad. Sci. (USA), 68 _/ 378-382.

8. Tanaka, M. , Levy, J. , Terada, M. , Breslow, R. Rifkind, R.A. & Marks, P.A. (1975) Proc. Natl. Acad Sci. (USA), 22, 1003-1006.

9. Reuben, R.C., Wife, R.L. , Breslow, R. , Rifkind, R.A. & Marks, P.A. (1976) Proc. Natl. Acad. Sci. (USA) , 73. 862-866.

10. Abe, E., Miyaura, C. , Sakagami, H. , Takeda, M. , Konno, K. , Yamazaki, T. , Yoshika, S. & Suda, T. (1981) Proc. Natl. Acad. Sci. (USA), 21, 4990-4994.

11. Schwartz, E.L., Snoddy, J.R. , Kreutter, D., Rasmussen, H. & Sartorelli, A.C. (1983) Proc. Am. Assoc. Cancer Res.. 24. 18.

12. Tanenaga, K., Hozumi, M. & Sakagami, Y. (1980) Cancer Res.. 40. 914-919.

13. Lotem, J. & Sachs, L. (1975) Int. J. Cancer. 15. 731-740.

14. Metcalf, D. (1985) Scienct. 229. 16-22.

15. Scher, W. , Scher, B.M. & Waxman, S. (1983) Exp. Hematol.. H, 490-498.

16. Scher, W. , Scher, B.M. & Waxman, S. (1982) Biochem. & Biophvs. Res. Corom. _f 109. 348-354.

17. Huberman, E. & Callaham, M.F. (1979) Proc. Natl. Acad. Sci. (USA), 26, 1293-1297.

18. Lottem, J. & Sachs, L. (1979) Proc. Natl. Acad. Sci. (USA), 26, 5158-5162.

19. Terada, M. , Epner, E. , Nudel, U. , Salmon, J. ,

Fibach, E., Rifkind, R.A. & Marks, p.A. (1978) Proc. Natl. Acad. Sci. (USA), 2 , 2795-2799.

20. Morin, M.J. & Sartorelli, A.C. (1984) Cancer Res.. 44., 2807-2812.

21. Schwartz, E.L., Brown, B.J., Nierenberg, M. , Marsh J.C. & Sartorelli, A.C. (1983) Cancer Res.. 43 2725-2730.

22. Sugano, H. , Furusawa, M. , Kawaguchi, T. & Ikawa, Y (1973) Bibl. Hematol.. 39., 943-954.

23. Ebert, P.S., Wars, I. & Buell, D.N. (1976) Cance Res.. 36, 1809-1813.

24. Hayashi, M. , Okabe, J. & Hozumi, M. (1979) Gann, 70 235-238.

25. Fibach, E. , Reuben, R.C., Rifkind, R.A. & Marks P.A. (1977) Cancer Res.. 32, 440-444.

26. Melloni, E. , Pontre oli, S., Damiani, G., Viotti P., Weich, N., Rifkind, R.A. & Marks, P.A. (1988 Proc. Natl. Acad. Sci. (USA), £5, 3835-3839.

27. Reuben, R. , Khanna, P.L., Gazitt, Y., Breslow, R. Rifkind, R.A. & Marks, P.A. (1978) J. Biol. Chem. 253. 4214-4218.

28. Marks, P.A. and Rifkind, R.A. Hexamethylen

Biscetamide Induced Differentiation of Transforme

Cells: Molecular and Cellular Effects an

Therapeutic Application. International Journal o

Cell Cloning, .6:230-240, 1988.

29. Melloni, E. , Pontremoli, S., Michetti, M. , Sacco, 0., Cakiroglu, A.G. , Jackson, J.F., Rifkind, R.A. & Marks, P.A. Proc. Natl. Acad. Sciences (USA) 84, 5282-5286, 1987.

30. Marks, P.A. & Rifkind, R.A. (1984) Cancer. 54, 2766- 2769.

31. Egorin, M.J., Sigman, L.M. VanEcho, D.A. , Forrest,

A., Whitacre, M.Y. & Aisner, J. (1987) Cancer Res.. 47. 617-623.

32. Rowinsky, E.W. , Ettinger, D.S., Grochow, L.B., Brundrett, R.B. , Cates, A.E. & Donehower, R.C. (1986) J. Clin. Oncol.. 4., 1835-1844.

33. Rowinsky, E.L. Ettinger, D.S., McGuire, W.P., Noe, D.A. , Grochow, L.B. & Donehower, R.C. (1987) Cancer

Res.. 42, 5788-5795.

34. Callery, P.S., Egorin, M.J., Geelhaar, L.A. & Nayer, M.S.B. (1986) Cancer Res.. 46. 4900-4903.

35. Young, C.W. Fanucchi, M.P. , Walsh, T.B. , Blatzer, L. , Yaldaie, S., Stevens, Y.W. , Gordon, C. , Tong, W. , Rifkind, R.A. & Marks, P.A. (1988) Cancer Res. , 48, 7304-7309.

36. Andreeff, M. , Young, C., Clarkson, B. , Fetten, J. , Rifkind, R.A. & Marks, P.A. (1988) Blood. 72, 186a.

37. Ohta, Y., Tanaka, M. , Terada, M. , Miller, O.J.,

Bank, A., Marks, P.A. & Rifkind, R.A. (1976) Proc. Natl. Acad. Sci. (USA) 21, 1232-1236.

38. Cartledge, F.K. & Nguyen, T.B. (1986) J. Org. Chem.. H, 2206-2210.

39. Hoffmann-LaRoche and Co., A.G. British Patent l, 138-529 (1969).

40. Dmitrovsky, E., Markman, M. & Marks, P.A. (1989) i Cancer Chemotherapy and Biological Therapy. Annua 11, eds., D.L. Longo, H.M. Pinedo, B.A. Chabner, eds. , Excepta Medica, Publisher, in press.