WO1997013748A1

WO1997013748A1 - Sulfoxide derivatives of nitrogen-mustard and anticancer agent containing the same

Info

Publication number: WO1997013748A1
Application number: PCT/KR1996/000173
Authority: WO
Inventors: Jung Woo Kim; Chul-Hoon Kwon; Koo Hun Chung; Joon Kyum Kim; Jae Soo Shin; Kwan Kee Min
Original assignee: Chong Kun Dang Corp
Current assignee: Chong Kun Dang Corp
Priority date: 1995-10-09
Filing date: 1996-10-09
Publication date: 1997-04-17
Anticipated expiration: 1998-04-09
Also published as: KR970020099A; AU7340696A

Abstract

According to the present invention, are provided sulfoxide derivatives of nitrogen-mustard represented by general formula (I), wherein R1 represents a hydrogen, or haloethyl; R2 represents a lower alkyl or phenyl group substituted or non-substituted; and X represents a halogen atom. Provided that R1 is a chloroethyl and X is a chlorine, R2 does not represent methyl group; and an anticancer agent comprising the same as a prodrug. The sulfoxide derivatives according to the present invention convert to a sulfide in a hypoxic condition to show cytotoxicity, so that it is very useful as an anticancer agent selectively functions on a solid cancer which is in a hypoxic condition.

Description

TITLE OF INVENTION

SULFOXIDE DERIVATIVES OF NITROGEN-MUSTARD AND ANTICANCER

AGENT CONTAINING THE SAME

TECHNICAL FIELD

The present invention relates to sulfoxide derivatives of nitrogen-mustard which are represented by a general formula:

wherein, R₁ represents a hydrogen, or haloethyl; R₂ .represents a lower alkyl or phenyl group substituted or non-substituted; and X represents a halogen atom. Provided that R₁ is a chloroethyl and X is a chlorine, R₂ does not represent methyl group; and pharmaceutically acceptable salts thereof, and their use as an anticancer agent.

BACKGROUND ART

Despite improvements in chemotherapy for cancer in the past few decades, due care have been required in the clinical application of conventional anticancer agents in many cases due to their non-specific selectivity, including undesirable side effects. With their physiological properties (e.g., cellular heter ogeneity and inadequate blood flow in the local tumor site) , solid tumors have shown little responsiveness on chemotherapy and are also resistant to radiotherapy (Warren, 1978; Poste, 1986) . Now that said solid tumor cells have been reported to be in localized hypoxia, nutrient deprivation, and low pH (Kennedy et al., 1980), some selective-specific drugs on target sites have been suggested in consideration of said tumor's specific physiological and environmental factors (Denny and Wilson, 1986; Stratford et al., 1986; Sartorelli, 1988; Wilson et al., 1989a and 1989b; Denny et al., 1990) . In view of these situations, sulfoxide derivatives have been the remarkable prominent. Said compound is converted into sulfide, a physiologically active metabolite, by redox enzyme present in the mammal tissues, especially under hypoxic conditions.

In the meanwhile, nitrogen mustard forms aziridinium ion based upon the following reaction formula (A) , and combines with a DNA base by alkylation reaction to form the structure as illustrated in Example (B) . Since nitrogen mustard can optionally attack any position of oxygen or nitrogen having lone pair electrons of DNA base molecule such as cytocine, guanine, thymine and adenine, it demonstrates a strong toxicity to cells. Further, with its non-selectivity property in distinguishing tumor cells from normal cells, nitrogen mustard shows a strong toxicity to normal cells.

( N i t rogen mustard) ( Format i on of az i r i d i n i um sa l t !

( A l ky l at i on)

(A) [In the formula, X represents DNA base.]

In order to alleviate the toxicity of nitrogen mustard, attempts have been made to delocalize the lone pair electrons of nitrogen. Among these, melphalan is an antineoplastic agent having phenylalanine substituent on R group of nitrogen mustard. The active form of this compound is delivered to the cells in a transport system of amino acid, which is indicated for the treatment of osteoma, ovarian cancer or mastocarcinoma by the therapy of a single agent or with concurrent administration. However, some side effects associated with the overdose of melphalan are myelosuppression, gastrointestinal disturbances, and hypersensitivity; it has relatively low selectivity with negligible effects. Chlorambusil with a similar structure to melphalan has been used for a long term treatment of leukemia but its high toxicity has led to some side-effects such as myelosuppression, nausea, hepatic disorder and hepatica tocixity.

me i pnaIan chlorambusi

To be free from the aforementioned disadvantages the conventional arts face, the present inventors have endeavored to conduct the research and development of the compounds having strong activity and high selectivity to tumor cells thus, designing and synthesizing sulfoxide derived from nitrogen mustard, which is converted to the corresponding sulfide, a physiologically active metabolite as mentioned above, to complete this invention.

In general, sulfoxide derivatives of nitrogen mustard do not form a bond with a DNA base by aziridine ring formation as mentioned above, so that it has no antineoplastic activity. Also, sulfone derivatives, metabolites of the sulfoxide derivatives, have no antineoplastic activity in a normal state because of the same reason. However, sulfoxide derivatives are metabolized to a sulfide derivatives under anaerobic (hypoxic) conditions, and the sulfide group readily go through aziridine ring formation, whereby showing very selective cytotoxicity. As the fact that the inner tumor cells, especially a solid cancer tissue has extremely hypoxic condition is well known, the sulfoxide derivatives having the above-described property are very useful for the treatment of solid cancers.

R

l f ox i de( no act i v i ty) sulfone (no act ivi ty,

[o: [ H ]

====> aziridinium ring Tormaiion sul f iύe (high act IVI ty) In 1992, p-(methylsulfinyl)phenyl nitrogen mustard as a sulfoxide prodrug of an anti-cancer agent, which is converted to a sulfide, under hypoxic conditions, designed to specifically attack the tumor cells, has been developed by the inventor of the present invention. However, the compound has insufficient cytotoxicity (specific action to the tumor cells) , so that an anticancer agent having more selectivity and more excellent effect.

DISCLOSURE OF THE INVENTION

As a result of intensive study to develop the prodrug of an anti-cancer medicine having excellent selectivity to tumor cells, the present inventor unexpectedly found that the sulfoxide derivatives of nitrogen mustard represented by general formula (I) can fulfill these requirements.

(I)

In the formula, R, , R- and X are defined as above.

Among the compounds, p- (n-propylsulf inyl) -N,N-bis(2- chloroethyl) aniline, p- (phenylsulf inyl ) -N,N-bis(2- chloroethyl) aniline, 4,4'-N,N,N' ,N'-tetrakis(2- chloroethyl) dianiline sulfoxide, 4 , 4 ' -N, N' -bis (2- chloroethy 1 ) dianil ine sulfoxide and 4-N,N- bis (chloroethyl) amino-4 ' - (9-acridinyl) aminodi phenyl sulfoxide are mentioned as desirable compounds. The compounds of the present invention can be synthesized by the use of known methods. For example, the compounds, wherein R₂ is lower alkyl or phenyl group substituted or non-substituted and R₁ is haloethyl, can be prepared by reducing the nitro group at p-position to introduce an amino group; reacting with 2-chloroethanol or ethylene oxide/ acetone to introduce a hydroxyethyl group at N- position; reacting the product with halogenating agent; and then oxidizing the resultant sulfide group, as illustrated in Scheme I.

Scheme

(In the formula, R₂ is lower alkyl or phenyl group substituted or non-substituted.) The compounds, wherein R₂ is lower alkyl or phenyl group substituted or non-substituted and R₁ is H, can be systhesized by concentration control of C1CH-CH-.OH used at the second step of the above scheme I.

The compounds wherein R₂ is an aniline group having one or more lower alkyl group with halogen substituent at N- position can be obtained by firstly preparing 4,4'- dia inodiphenyl sulfide; introducing one or more hydroxyethyl group to the amino group of the compound; halogenating the introduced hydroxy group; and oxidizing the sulfide group, as illustrated in Scheme II.

(In the formula, n and m are 0, 1, 2, but on the other hand n and m can not be 0 simultaneously)

Depending upon the features of the substituent

20 included in R₂ group, various derivatives may be synthesized as shown, for example, in the following reaction schemes :

⁵ Scheme

5

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1 - 4 are graphs of plotting the survival rate of V-79 cells under normal condition or hypoxic condition with regard to the concentration of medicines, when the compound of Example 5, 10, 14 or 17 is administered (Experimental Example 1) .

Figs. 5 and 6 show the result of the test for tumor inhibition in vivo (Experimental Example 2) .

BEST MODE FOR CARRYING OUT THE INVENTION

Here-in-after, the present invention will be described in detail by referring to the following Examples and Experimental Examples. However, it should be noted that the present invention is not restricted to these Examples.

Example 1 : p-(n-Propylthio)nitrobenzene

1-Bromopropane (7.5 ml, 8.24 g, 83 mmol) was added with stirring to a solution of p-nitrothiophenol (10.70 g, 69 mmol) in 50 ml of ethyl alcohol. To the clear orange- color solution, 40% NaOH solution (6.9 ml, 69 mmol) was added and the solution was heated under reflux for 40 min. The clear orange solution was cooled, and ice water was added thereto. The mixture was extracted with methylene chloride (4 x 100 ml) . The combined organic layer was dried over anhydrous Na₂S0₄ and evaporated to give 14.92 g of brown oil which contained 4 substances detected by UV after TLC operation. This brown oil was applied to a silica gel column (40 μm; 5 x 17 cm) packed dry. Then it was wet and eluted with hexane/EtOAc (4:1) under a positive pressure (15 psi) . The corresponding fractions were collected, and the solvent was removed by evaporation to give 11.20 g (yield: 86.3%) of pure compound as orange crystalline solid. m.p. = 102-103 °C

TLC : R_f = 0.71 in hexane/EtOAc (4:1)

¹H NMR (CDC1-) <_ (ppm)

7.32-7.60 & 8.15-8.44 (dd, 4H, -C₆H₄~) , 3.01-3.33 (t, 2H, -CH₂S-) , 1.66-2.19 (m, 2H, CH₂) ,

1.08-1.50 (t, 3H, CH₃)

Example 2 : p-(n-Propylthio)aniline

To a boiling suspension of p-(n-propylthio)nitro benzene (11.20 g, 67 mmol) suspended in a solution of 37% HCl (35 ml) and water (30 ml), tin turnings (11.0 g) were added upon which the suspension became a clear light brown solution. The solution was heated under reflux with vigorous stirring for 15 min. Activated charcoal (0.1 g) was added and the mixture was allowed to continue refluxing for an additional 15 min. The mixture was filtered hot and the filtrate was cooled and diluted with cold water (100 ml) . Aqueous NaOH (40%) was added till the mixture became strongly alkaline. The resulting gray product was filtered. The filtered product was suspended in boiling ethyl alcohol (60 ml) , and charcoal was added thereto. The suspension was heated under reflux for 30 min. The mixture was filtered hot, and the filtrate was quickly added to ice cold water (500 ml) causing the formation of a white precipitate. The mixture was then filtered and dried to give 10.1 g of crude white powder which contained 3 substances detected by UV after TLC operation. This product was dissolved in hexane/EtOAc (4:1) (25 ml) and applied to a silica gel column (40 μm; 5 x 17 cm) packed dry. Then it was wet and eluted with the same solvent. The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to give 8.13 g (yield: 85.6%) of pure compound as yellow oil.

TLC : R_f = 0.37 [hexane/ ethyl acetate (4:1)] H NMR (CDCl_j) δ (ppm)

6.55-6.82 & 7.19-7.50 (dd, 4H, -C₄H₆-) , 3.58 (s, 2H, NH₂) , 2.68-3.08 (t, 2H, CH-.S-) , 1.32-1.93 (m, 2H, CH₂) , 0.85-1.30 (t, 3H, CH₃)

Example 3 : p-(n-Propylthio) -N.N-bis(2-hvdroxyethyl)aniline p-(n-Propylthio) aniline (8.12 g, 49 mmol) was added to a suspension of CaCO- (10.00 g, 100 mmol) in water (250 ml) .

The cloudy mixture was heated under reflux with vigorous stirring, protected from light, for 24 h. An additional four equivalents of 2-chloroethanol (14.3 ml, 196 mmol) and CaCO- (10.00 g, 100 mmol) were added in two(2) equivalent portions over the following 48 h. The reaction mixture was then cooled, pH (5.1) adjusted to 7.0 with 10% aqueous NaOH, and the mixture was extracted with EtOAc (4 x 200 ml) . The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give 8.18 g of a brown oil. The crude material was applied to a silica gel column (40 μm, 5 x 17 cm) packed dry. Then it was wet and eluted with EtOAc/hexane (2:1) under a positive pressure (15 psi) . The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to give 3.80 g (yield: 30.6%) of the product as a pale brown oil. TLC : R_f = 0.46 [hexane/ ethyl acetate (1:3)] ¹H NMR (CDCl_j) (ppm) 6.38-6.73 & 7.05-7.40 (dd, 4H, -C₆H₄~) , 3.15-3.94

[m, 10H, -N(CH₂CH₂OH)₂] , 3.14-2.95 (t, 2H, CH-S- ), 1.32-1.68 (m, 2H, -CH₂-) , 0.73-1.12 (t, 3H, CH₃).

Example 4 : p-(n-Propylthio. -N.N-bis(2-chloroethyl) aniline p-(n-Propylthio) -N,N-bis(2-hydroxyethyl) aniline (3.80 g, 15 mmol) was dissolved in P0C1- (9.0 ml) with stirring. The solution was heated under reflux at 100°C in a water bath for 40 min, then the hot reaction mixture was cooled to room temperature. The mixture was extracted with ethyl acetate (4 x 100 ml) and washed with 10% sodium bicarbonate solution (3 x 200 ml) . The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give 3.23 g of the product as a brown oil. TLC : R_f = 0.84 [hexane/ ethyl acetate (2:1)] ¹H NMR (CDCl_j) <S (ppm)

6.48-6.73 & 7.07-7.46 (dd, 4H, -C₆H₄-) , 3.32-3.75 [m, 8H, -N(CH₂CH₂C1)₂] , 2.58-2.89 (t, 2H, CH-S-) , 1.32-1.82 (m, 2H, -CH₂~) , 0.76-1.15 (t, 3H, CH₃-)

Elemental analysis C₁₃H₁₉C1₂NS

Calcd . : C , 53 . 42 ; H , 6 . 55 ; N , 4 . 79 ; S , 10 . 97 ; Cl ,

24.26 Found : C, 53.44; H, 6.53; N, 4.81; S, 10.87; Cl , 24.18 Example 5 : p-(n-Propylsulfinyl) -N.N-bis ( 2-chloroethyl) aniline

To a solution of p-(n-propylthio)-N,N-bis(2- chloroethyl)aniline (1.00 g, 3.4 mmol) in trifluoroacetic acid (5 ml), H₂0₂/trifluoroacetic acid (1.0 ml, 1 equivalent), prepared by adding 30% H₂0₂ (8.6 ml) to trifluoroacetic acid (16.4 ml) , was added while stirring at 0°C. The reaction was allowed to proceed for 2h. The ice bath was removed and the reaction was allowed to continue for an additional 30 min. Trifluoroacetic acid was evaporated to give 0.78 g (yield : 74.3 %) of pure product as a light yellow solid.

TLC : R_f = 0.41 [hexane/ ethyl acetate (1:3)] H NMR (CDC1-) <_ (ppm) 6.30-6.61 & 6.98-7.39 (dd, 4H, -C_fiH₄-) , 3.38-3.79

[m, 8H, -N(CH₂CH₂C1)₂] , 2.71-3.00 (t, 2H, CH-SO- ), 1.40-1.84 (m, 2H, -CH₂~) , 0.78-1.15 (t, 3H, CH--) Elemental analysis C₁₃H₁₉C1₂NS0 Calcd. : C, 50.65; H, 6.21; N, 4.54; S, 10.40;

Cl, 23.00 Found : C, 50.39; H, 6.09; N, 4.63; S, 10.47; Cl, 22.85

Example 6 : p-(4-nitrophenyl) henyl sulfide

Thiophenol (3.0 ml, 4.73 g, 43 mmol) was added to a boiling solution of l-bromo-4-nitrobenzene (8.70 g, 43 mmol) in ethyl alcohol (50 ml). Aqueous NaOH (40%) (3.0 ml, 43 mmol) was then added drop-wise to the solution and the solution heated under reflux for 1 h. The solution was cooled, diluted with water (200 ml) , and then extracted with CH-C1-. (4 x 150 ml) . The combined organic layer was washed with 10% NaOH, dried over anhydrous Na₂S0₄ and evaporated to give 8.63 g (85% yield) of the product as chro atographically pure yellow solid. m . p . : 89 - 91 °C

TLC : R_f = 0.69 [hexane/ ethyl acetate (20:1)]

¹H NMR (CDC1₃) δ (ppm)

7.41 (s, 5H, C₆H₅S) , 7.02-7.73 (dd, 4H, C₆H₄N0₂~) IR (Nujol, cm^"1)

3030, 1600-1500 (C_&H₄) , 1530, 1350 (N0₂)

Example 7 : p-Phenylthioaniline

With the use of p-(4-nitrophenyl)phenyl sulfide (59.7 mmol, 13.80 g) prepared in Example 6, the procedure of

Example 2 was repeated to give 11.65 g of white solid powder which contained 4 substances detected by UV after

TLC operation. The solid was dissolved in 30 ml of hexane/EtOAc (3:1), applied to a silica gel column (40 μm; 5 x 17 cm) packed dry. Then it was wet and eluted with hexane/EtOAc (3:1) at a positive pressure (15 psi) . The collected corresponding fractions were evaporated to obtain

10.23 g (yield: 85.3%) of the product as while solid crystalline product. m.p. : 91-93 °C

TLC : R_f = 0 . 48 [ hexane/ ethyl acetate ( 3 : 1 ) ] ¹H NMR ( CDC1₃) (ppm)

7 . 13 ( s , 5H , C₆H₅S ) , 6 . 68 - 7 . 50 (dd , 4H , -C₆H₄NH₂)

Example 8 : p-(Phenylthio) -N.N-bis(2-hydroxyethyl)aniline

With the use of p-phenylthioaniline (50.0 mmol, 10.05 g) prepared in Example 7, the procedure of Example 3 was repeated to give 9.60 g of the crude product as a brown oil. The oil was applied to a silica gel column (40 μm, 5 x 17 cm) packed dry. Then it was wet and eluted with ethyl acetate / hexane (1:1) under a positive pressure (15 psi).

The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to obtain 2.26 g (yield: 16.2%) of the product as brown oil. TLC : R_f = 0 . 25 [hexane/ ethyl acetate ( 1 : 2 ) ] H NMR ( CDC1₃) δ (ppm)

7 . 19 (s , 5H , C₆H₅S-) , 6 . 52-6 . 77 (dd , 4H , C₆H₄~ ) , 3 . 26-4 . 00 [m, 10H , N (CH₂CH-.OH) ₂ ]

Example 9 : p-(Phenylthio)-N.N-bis(2-chloroethyl_ aniline

With the use of p-(phenylthio) -N,N-bis(2- hydroxyethyl)aniline (7.82 mmol, 2.26 g) prepared in

Example 8, the procedure of Example 4 was repeated to give 2.11 g of the crude product as a light brown viscous oil.

The crude product was applied to a silica gel column (40 μm, 2.5 x 12.5 cm) packed dry. Then it was wet and eluted with ethyl acetate / hexane (1:4) under a positive pressure

(15 psi) . The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to obtain 2.03 g (yield: 80%) of the product as light brown oil.

TLC : R_f = 0.69 [hexane/ ethyl acetate (4:1)], UV quenching and NBP positive ¹H NMR (CDC1₃) <S (ppm) 7.11 (s, 5H, C₆H₅S-) , 6.40-7.48 (dd, 4H, C₆H₄N-) ,

3.32-3.78 [m, 8H, N(CH-CH₂C1)₂] Elemental analysis C₁₆H₁₇C1-NS

Calcd. : C, 58.90; H, 5.25; N, 4.29; S, 9.83; Cl, 21.73 Found : C, 58.77; H, 5.16; N, 4.23; S, 9.72;

Cl, 21.70

Example 10 : p-(Phenylsulfinyl) -N.N-bis(2-chloroethyl) aniline With the use of p-(phenylthio)-N,N-bis(2- chloroethyl)aniline (3.1 mmol, 1.00 g) prepared in Example 9, the procedure of Example 5 was repeated to give 0.84 g of the crude product as dark brown viscous oil. The crude product was applied to a silica gel column (40 μm, 2.5 x 12.5 cm) packed dry. Then it was wet and eluted with ethyl acetate / hexane (3:1) under a positive pressure (15 psi). The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to obtain 0.71 g (yield: 68%) of the product as an light yellow crystalline product. m.p. : 122-124 °C

TLC : R_f = 0.68 [hexane/ ethyl acetate (1:3)]

¹H NMR (CDC1₃) δ (ppm)

7.20 (s, 5H, C₆H₅S-) , 6.51-7.59 (dd, 4H, -C₆H_N- ) , 3.36-3.77 [m, 8H, N(CH₂CH-C1) ₂]

Elemental analysis C₁₆H₁₇C1₂NS0

Calcd. : C, 56.15; H, 5.01; N, 4.09; S, 9.37;

Cl, 20.72 Found : C, 56.20; H, 5.03; N, 4.04; S, 9.45; Cl, 20.64

Example 11 : 4 ,4 '-Diaminodiphenyl sulfide

4-Amino-4'-nitrodiphenyl sulfide (10.00 g, 40 mmol) was suspended in a solution of cone. HCl (37%) (26.5 ml) and water (25 ml) , and the light yellow suspension was heated to 100°C. To the boiling suspension, thus obtained tin turnings (9.00 g) were added to form a clear dark yellow solution. Additional HCl (2.0 ml) and tin turnings (1.00 g) were added and the solution was allowed to boil under reflux for 15 min. Activated charcoal (0.1 g) was added to the mixture and the reaction was allowed to boil for an additional 10 min. The mixture was filtered hot and the filtrate was allowed to cool. To the cooled filtrate, water (150 ml) and 40% aqueous NaOH was added till the solution became strongly alkaline (pH 12-13) and the resulting precipitate was filtered. The filtered grey solid product was dissolved in boiling alcohol (60 ml) and heated with activated charcoal (0.25 g) under reflux for 30 min. The mixture was filtered hot and the hot filtrate was added to ice cold water (500 ml) which resulted in the precipitation of a white solid. The solid was filtered and dried to give 6.51 g (yield: 71.2 %) of the product as a white crystalline solid, m.p. : 104 - 107 °C TLC : R_f = 0.50 [hexane / ethyl acetate (1:1)]

Example 12 : 4.4 ' -N . N . N ' . N ' -Tetrakis ( 2 - hydroxyethyl)dianiline sulfide

4,4'-Diaminodiphenyl sulfide (6.50 g, 30 mmol) and 2- chloroethanol (16.1 ml, 19.32 g, 240 mmol) were added to a suspension of CaC0₃ (12.00 g, 120 mmol) in water (200 ml). The cloudy mixture was heated under reflux with vigorous stirring, while being protected from light for 24 h. An additional 4 equivalents of 2-chloroethanol (8.0 ml, 9.66 g, 120 mmol) and CaC0₃ (6.00 g, 60 mmol) were added and the reaction was allowed to continue for 24 h. The reaction mixture was then cooled, and the pH (2.6) was adjusted to 7.0 with 10% NaOH. The mixture was then extracted with ethyl acetate (4 x 100 ml) . The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give 6.60 g of a dark brown viscous oil which contained 5 substances detected by UV after TLC operation. The crude oil was then applied to a silica gel column (40 μm, 5 x 15 cm) packed dry. Then it was wet and eluted with ethyl acetate under a positive pressure (15 psi) . The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to obtain 3.15 g (yield: 26.8%) of the product as clear viscous oil.

TLC : R_f = 0.30 (ethyl acetate) ¹H NMR (CDC1₃) δ (ppm)

6.32-6.72 & 6.90-7.30 [dd, 8H, S(C₆H₄)2], 3.16-3.68 [m, 20H, N(CH-.CH-.0H)-]

Example 13 : 4.4 ' -N . . N ' . N ' -Tetraki s ( 2 - chloroethyl)dianiline sulfide With the use of 4,4 '-N,N,N' ,N'-tetrakis(2- hydroxyethyl)dianiline sulfide (7.4 mmol, 2.90 g) prepared in Example 12, the procedure of Example 4 was repeated to give 1.24 g of the crude product as a light brown oil. The crude product was applied to a silica gel column (40 μm, 5 x 12.5 cm) packed dry. Then it was wet and eluted with hexane / ethyl acetate (5:1) under a positive pressure (15 psi) . The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to obtain 0.82 g (yield: 23.7%) of the product as yellow oil.

TLC : R_f = 0.51 [hexane/ ethyl acetate (5:1)], UV quenching and NBP positive ¹H NMR (CDC1₃) δ (ppm)

6.48-6.80 & 7.10-7.51 [dd, 8H, S(C₆H₄)₂], 3.46-3.89 [m, 16H, N(CH-.CH₂C1)₂]

Elemental analysis C₂₀H₂₄C1₄N₂S

Calcd. : C, 51.52; H, 5.19; N, 6.01; S, 6.88;

Cl, 30.41 Found : C, 51.43; H, 5.16; N, 5.99; S, 6.92; Cl, 30.37

Example 14 : 4.5 ' -N . N . N ' . N ' -Te t r ak i s ( 2 - chloroethyl) dianiline sulfoxide

With the use of 4, 4 '-N,N,N' ,N'-tetrakis (2- chloroethyl)dianiline sulfide (0.73 mmol, 0.35 g) prepared in Example 13, the procedure of Example 5 was repeated to give 0.31 g of the crude product as grey viscous oil. The crude oil was diluted in ethyl acetate (2 ml) and applied to three preparative TLC plates, and was then eluted with ethyl acetate / hexane (3:2). The regions with R_f = 0.35 were scraped, dissolved in ethyl acetate / ethanol (1:1), filtered and evaporated to give 220 mg (yield : 60.8%) of the product as a light grey solid.

TLC : R_f = 0.34 [hexane/ ethyl acetate (2:3)] ¹H NMR (CDC1₃) δ (ppm) 6.41-6.90 & 7.29-7.67 [dd, 8H, S(C₆H₄)₂], 3.55-4.02 [m, 16H, N(CH₂CH₂Cl)₂] Elemental analysis C₂₀H₂₄C1₄N₂SO

Calcd. : C, 49.81; H, 5.02; N, 5.81; S, 6.65; Cl, 29.40

Found : C, 49.83; H, 4.98; N, 5.77; S, 6.72; Cl, 29.34

Example 15 : 4.4'-N.N'-bis(2-hvdroxyethyl. dianiline sulfide 4,4'-Diaminodiphenyl sulfide (5.00 g, 23.1 mmol) prepared in Example 11 and 2-chloroethanol (6.2 ml, 7.44 g, 92.4 mmol) were added to a suspension of CaC0₃ (4.10 g, 41.0 mmol) in water (150 ml) . The cloudy mixture was heated under reflux, while being protected from light for 24 h. An additional 2 equivalents of 2-chloroethanol (3.1 ml, 3.72 g, 46.2 mmol) and CaC0₃ (4.62 g, 46.2 mmol) were added and the reaction mixture was heated under reflux for 24 h. The reaction mixture was then cooled, and the pH (4.2) was adjusted to 7.0 with 10% NaOH. The mixture was then extracted with ethyl acetate (4 x 100 ml) . The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give 6.23 g of brown oil which contained 7 substances detected by UV after TLC operation. The crude oil was then applied to a silica gel column (40 μm, 5 x 15 cm) packed dry. Then it was wet and eluted with ethyl acetate / hexane (5:1) under a positive pressure (15 psi). The collected corresponding fractions were dried over anhydrous sodium sulfate and evaporated to obtain 0.77 g (yield: 11.0%) of the product as pale yellow oil. TLC : R = 0.23 (ethyl acetate / hexane = 4:1) ¹H NMR (CDC1₃) <S (ppm)

6.37-6.70 & 6.92-7.31 [dd, 8H, S(C₆H₄)₂], 3.76 [s, 2H, (NH-)₂], 2.92-3.45 [m, 10H, N(CH₂CH₂OH)₂]

Example 16 : 4.4'-N_rN'-Bis(2-chloroethyl)dianiline sulfide With the use of 4,4'-N,N'-bis(2-hydroxyethyl)dianiline sulfide (21.4 mmol, 0.75 g) prepared in Example 15, the procedure of Example 12 was repeated to give 0.63 g of the crude product as golden yellow oil. The crude oil was diluted in ethyl acetate (3 ml) and applied to three preparative TLC plates, and was then eluted with ethyl acetate / hexane (1:3). The regions with R_f = 0.43 were scraped, dissolved in ethyl acetate / ethanol (1:1), filtered and evaporated to give 0.46 g (yield : 52.3%) of the product as a charcoal colored oil.

TLC : R_f = 0.43 [hexane/ ethyl acetate (3:1)], UV quenching and NBP positive ¹H NMR (CDC1₃) δ (ppm)

6.51-6.89 & 7.04-7.41 [dd, 8H, S(C₆H₄)₂], 3.96 [S, 2H, (NH-)₂], 3.18-3.68 [m, 8H, N(CH₂CH₂C1)₂]

Elemental analysis C₁₆H₁₈C1-N₂S

Calcd. : C, 56.31; H, 5.32; N, 8.21; S, 9.39;

Cl, 20.78 Found : C, 56.22; H, 5.28; N, 8.14; S, 9.40; Cl, 20.81

Example 17 : 4.4 -N.N -Bis(2-chloroethyl.dianiline sulfoxide

With the use of 4,4'-N,N'-bis(2-chloroethyl)dianiline sulfide (0.91 mmol, 0.31 g) prepared in Example 16, 0.29 g of the crude product was obtained as light grey viscous oil. The crude oil was diluted in ethyl acetate (2 ml) and applied to three preparative TLC plates, and was then eluted with ethyl acetate / hexane (1:1). The regions with R_f = 0.35 were scraped, dissolved in ethyl acetate / ethanol (1:1), filtered and evaporated to give 0.23 g (yield : 70.1%) of the product as a white crystalline solid.

TLC : R_f = 0.35 [hexane/ ethyl acetate (1:1)] ¹H NMR (CDC1₃) δ (ppm) 6.58-6.98 & 7.16-7.51 [dd, 8H, S(C₆H₄)₂], 4.01 [s, 2H, (NH-)₂], 3.22-3.75 [m, 8H, N(CH-CH₂C1)₂] Elemental analysis C₁₆H₁₈C1₂N₂S0

Calcd. : C, 53.79; H, 5.08; N, 7.84; S, 8.97; Cl, 19.85

Found : C, 53.71; H, 4.97; N, 7.77; S, 8.95; Cl, 19.80

Example 18 : 4-[N.N-Bis(2-hydroxyethyl) 1amino-4 - nitrodiphenyl sulfide

To a solution of 4-amino-4'-nitrodiphenyl sulfide (25.00 g, 101.5 mmol) in 200 ml of THF and 250 ml of AcOH, 45 ml of 10% (w/v) of ethylene oxide in THF (102.2 mmol) was added at 5°C. The reaction mixture was stirred at room temperature for several days during which 45 ml of the ethylene oxide solution was added every day until mole ratio of ethylene oxide to the starting compound was about 7. The mixture was then neutralized with aqueous Na₂C0₃, and extracted with ethyl acetate three times. The ethyl acetate extract was washed with water, dried over anhydrous sodium sulfate, and evaporated in vacuo to remove the solvent. The crude product was treated with charcoal in ethyl acetate solution to give 19.5 g of yellow product. The residue after evaporation of mother liquid was purified on a flash column using methylene chloride / acetone (8:2) as the eluant to obtain 7.8 g of the product. [Total yield : 80.43%] m.p. : 99.5 - 100.5 °C

TLC : R_f = 0.41 [methylene chloride / acetone (8:2)] ¹H NMR (CDC1₃) δ (ppm)

6.63-8.03 (m, d, 8H, 0₂NC₆H₄SC₆H₄N) , 3.63-3.90 [d_, 8H, N(CH₂CH₂OH)₂] Elemental analysis C₁₆H₁₈N₂0₄S

Calcd. : C, 57.47; H, 5.42; N, 8.38; S, 9.59 Found : C, 57.62; H, 5.51; N, 8.30; S, 9.48 Example 19 : 4-[N,N-Bis(2-chloroethyl) 1amino-4'- nitrodiphenyl sulfide

A mixture of 10.00 g (29.91 mmol) of 4-[N,N-bis(2- hydroxyethyl) ]amino-4'-nitrodiphenyl sulfide prepared in Example 18 and 65 ml of P0C1₃ was heated under reflux for 1 h, then the hot reaction mixture was poured to chopped ice with stirring. The resultant mixture was extracted with methylene chloride. The extract was treated with activated charcoal, washed with saturated aqueous sodium bicarbonate to pH 6 and washed with water, and dried over anhydrous

Na₂S0₄, then evaporated in vacuo to give the crude product.

This crude product was purified with activated charcoal and crystallized with ethyl acetate / hexane to give 8.90 g of yellow product (yield: 80.15%).

m.p. : 112.5 - 113.5 °C

TLC : R_f = 0.51 [hexane / ethyl acetate (5:1)]

¹H NMR (CDC1₃) δ (ppm)

6.63-8.06 (m, d, 8H, 0-NC₆H₄SC₆H₄N) , 3.73 [s, 8H, N(CH₂CH₂C1)₂]

Elemental analysis C₁₆H₁₆N₂0₂C1₂S

Calcd. : C, 51.76; H, 4.34; N, 7.55; Cl , 19.10;

S, 8.64 Found : C, 51.87; H, 4.31; N, 7.59; Cl , 18.98; S, 8.69

Example 20 : 4 [N. N-Bis (2 -chloroethyl) laroino-4 '- nitrodiphenyl sulfoxide

To a solution of 4-[N,N-bis(2-chloroethyl) ]amino-4 '- nitrodiphenyl sulfide (13.50 g, 36.36 mmol) in trifluoroacetic acid (TFA, 64 ml) , 15.9 ml of H₂0₂ / TFA (36.36 mmol, prepared by mixing 4.3 ml of 30% H₂0₂ with 14.1 ml of TFA) was added while stirring at 5°C. The reaction was allowed to proceed for 6.5 h. After adding ethyl acetate and water, the reaction mixture was neutralized to pH 6-7 with aqueous sodium carbonate. The separated aqueous layer was extracted with ethyl acetate three times. Ethyl acetate extracts were combined and washed with water, dried over anhydrous sodium sulfate, and evaporated in vacuo. The crude product was crystallized with ethyl acetate / hexane to give 13.67 g (yield : 97.08%) of a yellow product. m.p. : 107 - 108 °C

TLC : R_f = 0.45 [hexane / ethyl acetate (1:1)] ¹H NMR (CDC1-) δ (ppm)

6.63-8.40 (d, m, and d, 8H, 0₂NC₆H₄S0C₆H₄N) , 3.60-3.77 [t, 8H, N(CH₂CH₂C1)₂] Elemental analysis C₁₆H₁₆N₂0₃C1₂S

Calcd. : C, 49.62; H, 4.16; N, 7.23; Cl, 18.31; S, 8.28

Found : C, 49.77; H, 4.19; N, 7.17; Cl, 18.30; S, 8.21

Example 21 : 4-fN.N-Bis(2-chloroethyl) 1amino-4 '- aminodiphenyl sulfoxide

Iron (19.14 g, 100 mesh) was activated by refluxing it with 1.5 ml of distilled water and 1 drop of concentrated hydrochloric acid for 0.5 h. Ethanol (68.4 ml) and 4 - [N,N - bis (2 - chloroethyl) amino - 4' - nitrodiphenyl sulfoxide (13.67 g, 35.30 mmol) prepared in Example 20 were added into the mixture at 40 - 50 °C. The reaction mixture was heated under reflux for 15 min. Several drops of 10% NaOH was added to the reaction mixture to pH 8. The hot reaction mixture was then filtered, and the residue was washed with ethanol and water. The filtrate was evaporated in vacuo to remove ethanol, and extracted with ethyl acetate three times. The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated in vacuo. The crude product was purified on a flash column using ethyl acetate / methylene chloride (6:4) as an eluant, and then recrystallized from ethyl acetate / hexane to give 9.09 g of off-white crystalline title compound (yield : 72.07%) and 0.8 g (6.64%) of off-white solid identified as 4-[N,N- bis (2-chloroethyl) ]amino-4 '-aminodiphenyl sulfide.

4- [N,N-bis( 2-chloroethyl) ]amino-4 '-aminodiphenyl sulfoxide m.p. : 136.5 - 138 °C TLC : R_f = 0.49 (ethyl acetate) H NMR (CDC1₃) δ (ppm) 6.60-7.50 (d, m, 8H, NC₆H₄S0C₆H₄N) , 3.60-3.73 [t,

8H, N(CH₂CH₂C1)₂] Elemental analysis C₁₆H₁₈N₂0C1₂S

Calcd. : C, 53.79; H, 5.08; N, 7.84; Cl, 19.85; S, 8.97 Found : C, 53.88; H, 5.10; N, 7.89; Cl, 19.90;

S, 9.03

4- [N, N-Bis (2-chloroethyl) lamino-4 '-aminodiphenyl sulfide m.p. : 77 - 79 °C TLC : R_f = 0.64 [methylene chloride / acetone (196:4)]

¹H NMR (CDC1₃) <_ (ppm)

6.50-7.30 (d, d, 8H, NC₆H₄SC₆H₄N) , 3.68 [s, 8H, N(CH₂CH₂C1)₂] Elemental analysis C₁₆H₁₈N₂C1₂S Calcd. : C, 56.13; H, 5.32; N, 8.21; Cl , 20.78;

S, 9.39 Found : C, 56.30; H, 5.33; N, 8.17; Cl , 20.68; S, 9.42

Example 22 : 4-N.N-Bis(chloroethyl)amino-4'-(9-acridinyl. aminodiphenyl sulfide hydrochloride

1) 9-Chloroacridine

A mixture of 5.00 g (5.12 mmol) of 9(10H)-acridone,

25.0 ml of S0C1-. and 0.1 ml of dimethylformamide was heated under reflux until a clear solution resulted and for additional 0.5 h. After removal of S0C1₂ in vacuo, the residue was dissolved in 70 ml of CHC1₃ and then stirred into chopped ice. Saturated aqueous NaHC0₃ was added to adjust the pH value to 7. The aqueous layer was extracted with CHC1₃. Then the combined CHC1₃ extract was washed with water, dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was crystallized from CC1₄ to give 3.1 g (yield: 73.09%) of the product as greenish crystals. m.p. : 118.5 - 120.5 °C

TLC : R_f = 0.67 [methylene chloride / ethyl acetate

(92:8)] IR showed disappearance of the peak of C=0 and appearance of the peak of C-Cl.

2) 4- N,N-Bis (chloroethyl) amino-4 '- (9-acridinyl) aminodiphenyl sulfide hydrochloride

A solution of 4-[N,N-bis(2-chloroethyl) ]amino-4'- aminodiphenyl sulfide (0.52 g, 1.53 mmol) prepared in Example 21 and 9-chloroacridine (0.30 g, 1.40 mmol) prepared as above in N-methyl-2-pyrrolidinone (13.5 ml) was treated with a few drops of concentrated HCl and then stirred at room temperature for 1.5 h. The mixture was diluted with ethyl acetate, and the resultant precipitate was collected and washed with the same solvent to give a crude product. The crude product was mixed with 10 ml of

N-methyl-2-pyrrolidinone and the mixture stirred for 1 h.

Ethyl acetate (250 ml) was gradually added to complete the precipitation, and the resultant precipitate was collected and washed with ethyl acetate to give the pure title compound (0.70 g, yield: 89.4%) as reddish solid. m.p. : 261-262 °C

TLC : R_f=0 . 73 [ hexane/Et0Ac ( l : 1 ) ] ¹H NMR ( CDC1₃ / DMS0-d₆ 1 : 4 ) <S (ppm) 6 . 70-8 . 40 (m , m , m , 16H , ArH) , 3 . 75 [ s , 8H , N(CH₂CH₂C1)₂] Elemental analysis C₂₉H₂₆N₃C1₃S

Calcd. : C, 62.76; H, 4.72; N, 7.57; Cl , 19.17; S, 5.78 Found : C, 62.83; H, 4.74; N, 7.48; Cl , 19.06;

S, 5.86

Example 23 : 4-ΓN.N-Bis(chloroethyl) ]amino-4'-(9-acridinyl) aminodiphenyl sulfoxide hydrochloride By the use of 4-[N,N-bis(2-chloroethyl) ]amino-4'- aminodiphenyl sulfoxide (1.00 g, 2.81 mmol) prepared in Example 21 and 9-chloroacridine (0.55 g, 2.57 mmol) prepared in Example 22, the procedure of Example 22 2) was repeated to give 1.3 g (yield: 88.45%) of the title compound as yellow solid, m.p. : 250-251 °C TLC : R_f = 0.81 (ethyl acetate) ¹H NMR (CDC1₃ / DMS0-d₆ 1:4) 6 (ppm)

6.75-8.40 (m, m, m, 16H, ArH) , 3.75 [s, 8H, N(CH₂CH₂C1)₂]

Elemental analysis C₂₉H₂₆N₃Cl₃OS

Calcd. : C, 61.01; H, 4.59; N, 7.36; Cl, 18.63;

S, 5.62 Found : C, 60.84; H, 4.86; N, 7.34; Cl , 18.47; S, 5.53

Experimental Example 1 : In Vitro Cytotoxicity Assessment A confluent flask of Chinese Hamster Lung V-79 transformed cells was scraped to collect the monolayer and the cells were counted by trypan blue dye exclusion. Glass milk dilution bottles were then inoculated with 500,000 cells in 10 ml of medium (minimum essential medium eagle supplemented with 10% fetal bovine serum, Penicilline G sodium, streptomycin sulfate, amphotericin and essential and non-essential amino acids and vitamins) and incubated for 20 hours in a 95% air/ 5% humidified atmosphere. One set of six bottles was sealed with sterilized rubber septa and fitted with 21-gauge needles for gas inflow and outflow. Each bottle was gassed individually, all outlet tubings and additive tubings immersed in water were connected to a flow meter (average flow velocity = 0.15 liter/min) to allow visible monitoring of gas flow as well as to prevent back flow of air into the cultures. To produce hypoxia, cultures were gassed continuously for 2h at 37°C with a humidified mixture of 95% N₂ / 5% C0₂. Normally aerated cultures were maintained in air until drug treatment. At this time, appropriate dilutions of the drug in ethanol/water were added directly to the cultures, without breaking the hypoxia, by injecting 0.25 ml of drug through the rubber septa. In the aerobic set, cells were exposed to drug for 3h under normal aerated conditions. In hypoxic set, cells were gassed 10 min after injection of the drug, needles were pulled out and the sealed bottles were incubated for 3h in a normally aerated incubator. After drug exposure, the cells were washed twice with 5 ml sterile HBSS, and treated with 2.5% trypsin (Gibed® labs) for 2-3 min. Cells were collected by centrifugation, resuspended in 5 ml fresh solvent/or buffer and counted by trypan blue dye exclusion. Appropriate dilutions were made, and 500 cells were cultured in triplicate in a total of 10 ml medium. Dishes were incubated for 6-8 days to allow colony formation. Colonies were then rinsed twice with 0.9% saline, fixed with ethanol, stained with crystal violet and counted. Results were reported as the number of colonies surviving chemical treatment per number of colonies in the solvent treated control. The IC-₀ values were determined by semi-logarithm plotting the drug concentration versus cell viability as determined by the number of colonies surviving the treatments. The results of the plotting with regard to the compounds prepared in the Examples are shown in Figs. 1 - 4. The IC₅₀ and IC-₀ values under normal or hypoxic condition are shown in Table 1 below.

Table 1

As can be shown from the above results, the sulfoxide derivatives according to the present invention have much lower cytotoxicity than the corresponding sulfide. It is found that the sulfoxides have rather higher cytotoxicity under hypoxic condition than under normal condition so that they have selective effects on a solid cancer having hypoxic condition as compared to the normal cells.

Experimental Example 2 : Tumor Inhibition in vivo

The B16 transformed cells cultured in vitro were treated with 0.25% trypsin, collected, washed with sterilized phosphate buffered saline (PBS) . And then the cell density was adjusted to 10⁷ cells/ml. After 0.2 ml of cells (2 x 10⁶ cells/ 0.2 ml/ mouse) were implanted into the subcutaneous tissue of the shoulder's upper part of BDF1 male mouse whose fur was previously eliminated, they were passaged in vivo per 14 days. B16 tumors were carefully isolated from the BDF1 male mouse tissue that had been passaged, necrosis cite was removed, and the weight of fresh tumor was measured. After adding sterilized physiological saline solution of 10-folds of the tumor weight, the mixture was ground by using a glass homogenizer to prepare a supernatant. The supernatant was transplanted to a subcutaneous tissue of the shoulder's upper part of BDF1 male mouse (0.2 ml/ mouse) (0th day). After 8 days, 45 animals having tumor tissue of 100 - 300 mm³ volume were selected and divided into the experimental groups at random (6 animals per each group) . The experimental compound(s) was administered into the abdominal cavity at the day 8 , 12, 16, and 20. As the experimental compound, 200 mg/kg of cyclophosphamide and 60 mg/kg of 2-hydroxypyrimidine were firstly administered into the abdominal cavity, and after 1 hr the compound according to the present invention(300, 150 mg/Kg) was administered. The tumor inhibition effect was evaluated by the body weight and the tumor growth inhibition rate (TGI) determined by the volumes of tumor in administered group and cotrol group at the day 8, 12, 16 and 20.

* Volume of tumor (mm³) = a x b x b x l/2 a : the longest diameter of the tumor b : the shortest diameter of the tumor

* TGI (Tumor Growth Inhibition rate, %) = (1 - Vt / Vc) x 100

Vt : tumor volume of the administered group (mm³) Vc : tumor volume of the vehicle control group (mm³)

The results of the plotting with regard to the compounds prepared in Examples are shown in Figs. 5 and 6. The change of body weight of the animals and tumor growth inhibition rate are shown in Tables 2 and 3 below.

Table 2 : Change of body weight of the BDF. mice transplanted with B16 tumor tissue

(* : PO.05, ** : PO.01 statistically significant CPM : cyclophosphamide) Table 3 : Results of tumor growth inhibition test in BDFi mice transplanted with B16 tumor tissue

(- ^' P<0.05, ** • P<0.01 statistically significant CPM : cyclophosphamide)

As can be shown from the results, the growth of tumor can be inhibited by about 80% in case of administrating the sulfoxides of the present invention. The tumor inhibition effect rises by 10% when using the compound of the present invention with the conventional compounds as compared to the case using only cyclo phosphamide (70%) , which whows the inhibition of growth of the solid cancer transplanted to mice.

Claims

What is claimed is :

1. Sulfoxide derivatives of nitrogen-mustard represented by a general formula (I) and pharmaceutically acceptable salts thereof :

wherein, R₁ represents a hydrogen, or haloethyl; R₂ represents a lower alkyl or phenyl group substituted or non-substituted; and X represents a halogen atom, provided that R₁ is a chloroethyl and X is a chlorine, R₂ does not represent methyl group.

2 . Sulfoxide derivatives and salts thereof according to claim 1, wherein the compound is p-(n-propylεulfinyl)-N,N-bis(2-chloroethyl)aniline.

3. Sulfoxide derivatives and salts thereof according to claim 1, wherein the compound is p-(phenylsulfinyl)-N,N-bis (2-chloroethyl)aniline.

4. Sulfoxide derivatives and salts thereof according to claim 1, wherein the compound is 4,4'-N,N,N',N'-tetrakis(2-chloroethyl)dianiline sulfoxide.

5. Sulfoxide derivatives and salts thereof according to claim 1, wherein the compound is 4,4'-N,N'-bis(2-chloroethyl)dianiline sulfoxide.

6. Sulfoxide derivatives and salts thereof according to claim 1, wherein the compound is 4-[N,N-bis(chloroethyl)] amino-4'-(9-acridinyl) aminodiphenyl sulfoxide.

7. An anticancer agent comprising sulfoxide derivatives of nitrogen-mustard represented by a general formula (I) and pharmaceutically acceptable salts thereof as an active ingredient, with pharmaceutically acceptable carrier(s) :