JPH0555508B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to mitomycin analogs with disulfide groups. These compounds are organically substituted 7-
It is a mitomycin C derivative with an amino group. This compound is an experimental animal tumor suppressor. Nomenclature - Systematic chemical abstract name of mitomycin C [1aR-(1aα, 8β, 8aα, 8bα)]-6-amino-8-[((aminocarbonyl)oxy)methyl]
-1,1a,2,8,8a,8b-hexahydro-8a
-Methoxy-5-methyl-alidino [2', 3',
3,4]pyrrolo[1,2- a ]indole-4,
7-dione and therefore the azilinopyrroloindole ring system is numbered as follows: Mitomycin A commonly used nomenclature in the literature identifies the above ring system containing various characteristic substituents of mitomycin as mitosan. Applicants have used this system herein and have chosen the azilino nitrogen atom to be designated N ^1a and the ring amino nitrogen atom to be designated N ⁷ . With respect to the stereochemical form of the products of the present invention, when identifying the product by its original name mitosan or by its structural formula, it is intended that its stereochemical form be identified as that of mitomycin C. Mitomycin C is an antibiotic produced by fermentation that is currently approved by the Food and Drug Administration to be used in combination with other recognized chemotherapeutic agents to treat infectious cancers of the stomach and pancreas. It is commercially available as a palliative treatment. (Myutamicin R○, Physicians' Desk, manufactured by Laboratories, Cealaki Youth, Bristol, New York 13201)
^{Peference35th} Edition, 1981, pages 717-718)
Mitomycin C and its fermentation production method were introduced on May 2, 1972.
U.S. Patent No. 3,660,578 (April 6, 1957)
This is the subject of a priority claim (including a Japanese application). The structures of mitomycins A, B, C and porphyromycin C were given by J. Amer, J.S. Webb et al., Radar Laboratory Division, American Cyanamid.
Chem.Soc., 84 , 3185-3187 (1962). One of the chemical transformations used in this structural study of mitomycins A and C was the former, the latter by reaction of 7-9a-dimethoxymitosane with ammonia,
The conversion was to 7-amino-9a-methoxymitosane. Substitution of the 7-methoxy group of mitomycin A has been found to be a very interesting reaction for producing derivatives of mitomycin C with anticancer activity. The following reports and patents each have the anticancer activity of mitomycin A.
-Conversion to substituted aminomitomycin C derivatives. The purpose of this study was to produce a derivative that is active and, in particular, less toxic than mitomycin C. “The Journal of Antibiotics” by Mitsui et al.
XXI, 189–198 (1968). Kinoshita et al. J.Med.Chem. 14 , 103-109
(1971). J.Med.Chem. 24 , 975-981.
(1981). Jenger, Sami, Remmers and Bradner, Amer.Chem.Soc., March 1982, 183rd Annual Journal.
No.MEDI72. Jenger et al. J.Med.Chem. 1983 , 26, 16~
20. Jenger et al., Amer.Chem.Soc., 1983, 3
Month, 185th Annual Journal, No.MEDI82. The following patents relate to the preparation of 7-substituted aminomitosan derivatives by reaction of mitomycin A, mitomycin B or their N ^1a -substituted derivatives with primary or secondary amines: Koslitsch et al., U.S. Pat.
No. 3332944, Mitsui et al., issued on January 1, 1969, U.S. Patent No. 3420846, Mitsui et al., issued on June 17, 1969, U.S. Pat.
U.S. Patent No. 3514452 published on the 26th, Nakano et al. 1980
U.S. Patent No. 4,231,936 published on November 4, 1981; US Pat.
issue. Mitomycin C with substituted amino group at 7-position
Derivatives have also been produced by direct biosynthesis, ie by adding a series of primary amines to the fermented broth and carrying out a conventional mitomycin fermentation. (Amer.Soc.of
Microbiology. 1982 Annual Journal 028, CA Klarituji et al.) Mitomycin C is the major mitomycin produced by fermentation and is the commercially available form. The recent technology for converting mitomycin C to mitomycin A used in the production of semi-synthetic substituted amino analogues of mitomycin C in the above patents and reports involves hydrolysis of mitomycin C to the corresponding highly unstable 7-hydroxymitosane and then handling. This method involves methylation using a difficult substance, diazomethane. One way to avoid the use of diazomethane for methylation is the use of 7-alkyloxymitosans for the synthesis of mitomycin C analogues. (Kyowa Hakko Co., Ltd., Japanese Patent No. J5 6073−085,
Farmdoc No. 56227 D/31) The present invention relates to a group of mitomycin C homologs having a dithioorganic substituent on the amino nitrogen atom in the 7-position. These compounds have the following formula: It is indicated by. In the formula, R ⁹ is an organic group, ie, a structural component of an organic thiol having the formula R ⁹ SH, and Alk ₂ and R have the meanings given below. This compound also has the formula: and the formula: It is expressed as In the above formula, Alk ₁ has 1 to 6 carbon atoms when R ⁷ is connected through its carbon atom, and 2 carbon atoms when R ⁷ is connected through its S, O or N. Alk 2 represents a straight-chain or branched alkylene group having from 2 to 6 carbon atoms (in which case R ⁷ and -SS- are connected to different carbon atoms); Alk ₂ has from 2 to 6 carbon atoms with an optional R ⁷ substituent; Represents a straight chain or branched alkylene group,
and the sulfur or nitrogen atom connected to it and the R ⁷ substituent connected to it through the oxygen, sulfur or nitrogen, if any, are bonded to different carbon atoms in Alk ₂ ; Alk ₁ and Alk ₂ are May have a double bond; R is hydrogen, lower alkyl, lower alkanoyl,
benzoyl; or benzoyl substituted with lower alkyl, lower alkoxy, halo, amino or nitro; R ⁷ is hydroxy, halo, amino, alkylamino or dialkylamino having 1 to 12 atoms;
Alkanoylamino, benzoylamino or A-
Substituted benzoylamino, naphthoylamino or A
-substituted naphthoylamino, cycloalkyl each having 3 to 8 ring members or A-substituted cycloalkyl, 5
Cycloalkenyl or A-substituted cycloalkenyl, phenyl or A-substituted phenyl, naphthyl or A-substituted naphthyl having from 1 to 2 ring members;
rings, heterocyclic groups selected from the group consisting of heteroaromatic groups and heteroalicyclic groups having 3 to 8 ring members and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur in each ring; carbon atoms; alkoxy or alkylthio, carboxy, each having 1 to 6;
alkoxycarbonyl having 1 to 7 carbon atoms,
phenoxycarbonyl or A-substituted phenoxycarbonyl, phenoxy or A-substituted phenoxy,
naphthoxy or A-substituted naphthoxy, 2 carbon atoms
Alkoxycarbonylamino, guanidino, ureido (-NHCONH ₂ ) with 6 to 6, 2 carbon atoms
N-alkylurein (-
NHCONH alkyl), having 3 to 7 carbon atoms
N ³ -haloalkylureylene, N ³ -haloalkyl-N ³ -nitrosourylene having 3 to 7 carbon atoms, and dialkylaminocarbonyl having 3 to 13 carbon atoms, provided that the above A substituent is 1 or 2 lower alkyl , lower alkanoyl, lower alkoxy, halo, amino, hydroxy, or nitro group; R ⁶ is alkyl having 1 to 12 carbon atoms; , alkenyl or alkynyl having 3 to 12 carbon atoms,
Cycloalkyl and A-substituted cycloalkyl having 3 to 8 ring members, cycloalkenyl having 5 to 8 ring members, phenyl, A-substituted phenyl, naphthyl,
A-substituted naphthyl, 1 to 2 rings, 3 to 8 in each ring
A heterocyclic group selected from the group consisting of a heteroaromatic group and a heteroalicyclic group having a ring member and 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulfur (provided that the above A-substituent has 1 or 2 lower alkyl, lower alkanoyl, lower alkoxy, halo, amino, hydroxy or nitro), and R ⁸ can be esterified with the adjacent sulfur atom. , constitutes an S-cysteinyl group that can be salt-formed or attached within a peptide bond. The substance of the invention is a tumor suppressor based on animal experiments. These are amino disulfides with the formula or: R ⁷ Alk ₁ -SS-Alk ₂ NH ₂ or R ⁸ -SS-Alk ₂ NH ₂ with the formula: It is produced by reacting with a mitosan derivative having
Y in the above formula is a group that is easily substituted by reaction with a primary amine group to form an N ⁷ -substituted mitomycin C derivative. The mitosan derivatives include mitomycin A and its homologs in the formula in which Y is lower alkoxy having 1 to 6 carbon atoms. The value is the formula where Y is the formula: It is a derivative with a formula such that it is an amidino group with provided that R ⁵ in the formula represents hydrogen (preferred), lower alkyl, phenyl, lower alkyl phenyl, lower alkoxyphenyl, halofenyl, aminophenyl, or ^nitrophenyl ;
R ⁴ is each independently lower alkyl or together with the nitrogen atom to which they are attached pyrrolidine, 2
- or 3-lower alkyl-pyrrolidine, piperidine, 2-, 3- or 4-lower alkyl-piperidine, 2,6-di-lower alkylpiperidine, piperazine, 4-substituted piperazine (however, the 4-substituent is alkyl or carbalkoxy, phenyl, methylphenyl, methoxyphenyl, halofenyl, nitrophenyl, or benzyl), azepine, 2-, 3-, 4- or 5-lower alkylazepine, morpholin, thiomorpholin, thiomorpholin-1 -oxide, or thiomorpholine-1,1-dioxide. The lower alkyl, lower alkanoyl and lower alkoxy groups mentioned above have 1 to 6 carbon atoms. The latter is an amidino derivative of mitomycin C and is described in Application Serial No. 385149 filed June 4, 1982. The reaction between a mitosan derivative having the formula and an amino disulfide having the formula or is carried out in an anhydrous organic liquid medium at approximately -
Occurs in a temperature range of 15 to +15°C. Temperatures between 0 and 20°C are preferred. Any anhydrous organic liquid medium can be used as long as it is stable under the reaction conditions and is not detrimental to the reaction. A 1 to 4 molar formula or amino disulfide is used for the mitosan reactant having the formula. The use of equimolar amounts of the two reactants is preferred. Reaction times of several hours, preferably about 8 to 50 hours, are used. The product is recovered by evaporating the liquid organic reaction medium and chromatographically treating the residue. Amino disulfides having the formula and formula are known compounds and can be prepared in a variety of ways. For example, these include the appropriate thiol R ⁷ Alk ₁ SH or R ⁸ SH as a butene salt with the formula: NH ₂ Alk ₂ SSO ₃ Na or the formula: It can be produced by reacting with sulfenylthiocarbonate having Clayman et al. (J.of Org.Chem., 29 , 3737~
3738 (1964)) produced the following compounds by the butene salt method: 2-aminoethyl n-butyl disulfide 2-aminoethyl n-hexyl disulfide 2-aminoethyl n-octyl disulfide 2 -aminoethyl n-decyl disulfide 2-aminoethyl phenyl disulfide 2-aminoethyl benzyl disulfide Methanol has been found to be a preferred reaction solvent for the reaction of Bunte salt and thiol. It has been found that reaction temperatures of 0 to -10°C are preferred when using this solvent.
Other solvents required high temperatures. The main drawback of this process is the formation of symmetrical disulfides as a by-product, possibly as a result of the disproportionation of the desired mixed disulfides. The starting mixed disulfide having the formula and may be prepared by reaction of the appropriate thymol with a sulfenylthiocarbonate of the formula. This is S.
J. Breuys et al. (J.of the Amer.Chem.Soc., 92 ,
7269-7270 (1970)). Generally, this process involves adding a thiol to a methanol solution of an amino-alkylsulfenylthiocarbonate having the formula and allowing the reaction to proceed at a temperature ranging from 0 to 25°C. Reaction times vary from virtually instantaneous to several hours depending on the particular thiol used. The progress of the reaction can be followed by measuring the presence of unreacted thiol in the reaction vessel. If the reaction is slow, a contact amount of triethylamine can be added as a reaction accelerator. The formula R ⁷ Alk ₁ SH or R ⁸ SH which is converted to the intermediate formula by reaction with a butene salt or sulfenylthiocarbonate and then converted to the product of the invention as described above. It is a large number of representative thiols. In the case of the amino-containing thiols in the following list, generally they are first converted into a protected form, preferably β-(trimethylsilyl), before conversion to an intermediate having the formula or and reaction with a mitosan intermediate having the formula It is desirable to change to ethoxycarbonylamino derivatives. Then β-
The (trimethylsilyl)ethoxycarbonylamino group is subsequently cleaved by treatment with tetraethylammonium fluoride in acetonitrile (LA
Carpino et al., JCSChem.Comm., 358 (1978))
A free amino compound with the desired formula or formula can be generated.

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[Table] Activity against P-388 murine leukemia The table shows _CDF1 female mice implanted intraperitoneally with a tumor inoculum of ¹⁰⁶ ascitic cells of P-388 murine leukemia using either the formula or the test compound with Figure 2 shows the results of experiments on treatment with various doses of or mitomycin C. Compounds were administered by intraperitoneal injection. Groups of 6 mice were used for each dose and were treated with a single dose of compound on the day of inoculation. A group of 10 saline control mice were included in each experimental series. The mitomycin C treatment group was included as a positive control. A 30-day test chart was used that allowed for the mean survival days measured for each group of mice and the recorded number of survivors at the end of the 30-day period. Mice were weighed before treatment and again on day 6. Changes in body weight were taken as a measure of drug toxicity. Using mice weighing 20 g each, a weight loss of up to about 2 g was not considered to be significant. The result is T/C% (the ratio of the average survival time of the treatment group to the average survival time of the saline control group multiplied by 100)
It was measured with Saline treated control animals usually died within 9 days. The “maximum effect” in the table below is T/C%
It is expressed as , and the dose that achieves the effect is shown. Values in brackets are values obtained with mitomycin C as a positive control in the same experiment. A measure of the comparative activity of the compounds of the invention against mitomycin C can therefore be deduced. The minimum effective dose at T/C°C is
It appeared to be 125. The minimum effective dose shown in the table below is the dose that gives a T/C% of about 125. The two numbers in each case in the "Average Body Weight Change" column are the average body weight changes of the mice at the maximum and minimum effective doses, respectively.

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[Table] The table shows the results of an antitumor test using B16 melanoma grown in mice. BDF ₁ was inoculated subcutaneously in mice by intratumoral implantation. A 60-day report card was used. 10 for each test drug dose
The average survival days were determined using groups of animals. Control animals were inoculated and treated with injection vehicle in the same manner as the test animals, and no drug was treated with the injection vehicle.
The average survival time was 24.5 days. Survival time of test mice relative to control (T/C%) was used as a measure of efficacy to determine the maximum and minimum effective doses of each test compound. The minimum effective dose was determined to be the dose that gave a T/C% value of 125. Test animals received each dose of test compound by intravenous injection on days 1.5 and 9.

[Table] BDF ₁ was able to examine the effects of compound No. 3 (BL-6796) on B16 melanoma grown in mice. The animals in this experiment were inoculated with the tumor intraperitoneally and the test compound was also injected intraperitoneally. As before, they were treated on days 1, 5 and 9 and the mean survival days were determined. Again, the minimum effective dose was considered to be the dose that produced a T/C% of 125.
At a dose of 1 mg/Kg of compounds No. 3, 6 and 10, animals survived the entire 60-day study period (T/C% 235) and 5
The animals showed an average weight gain of +0.1 g on day 1. 0.5mg/Kg
At this dose, the average survival time was 42.0 days (T/C% 165), indicating that the minimum effective dose was 0.5 mg/Kg or less.
In the same study, mitomycin C showed a T/C% of 165 at a dose of 3 mg/Kg. This was the maximum effective dose of mitomycin C in this experiment. It is therefore clear that Compound No. 3 is several times more effective than mitomycin against B16 melanoma in mice. Rat total and specific white blood cells (WBC)
Compounds 1 (BL-6787) and 3 administered intravenously to
(BL-6796) was tested. Both compounds were found to be spinal depressant, but significantly less so than mitomycin C. The results are shown in the table. Compound 1
The results for Mitomycin C and Mitomycin C were obtained in the same experiment and can therefore be directly compared. Compound 1 is somewhat less spinally depressing. Compound 3 seems to have less spinal depressive properties based on other experiments, but a direct comparison with the other two compounds could not be obtained.

[Table] Considering the antitumor activity observed in experimental animal tumors and the extremely insufficient spinal depressant activity compared to mitomycin C, the present invention has applications for suppressing mammalian tumors. For this purpose, substantially non-toxic antitumor effective doses of the substances of the invention are systematically administered to tumor-bearing mammals. The compounds of this invention are primarily used for injections in much the same way as mitomycin C and in some cases for the same purpose as mitomycin C. The dosage may be increased or decreased depending on the sensitivity of the particular tumor. The compounds are readily dispensed as dry pharmaceutical compositions containing diluents, buffers, stabilizers, solubilizers, and other necessary ingredients for formulation. The composition consists of a liquid vehicle that is ready for injection immediately prior to use. Suitable injectable fluids include water, isotonic salt solutions, and the like. In the following experimental procedures, temperatures are given in degrees Celsius (°C). Floton magnetic resonance spectra ( ¹ HNMR) were performed on a Varian XL100 or Jeol FX-90Q (90MHz) in pyridine _b5 unless otherwise noted.
recorded on the spectrum. Infrared (IR) spectra were obtained with a Bechman 4240 spectrophotometer.
The IR values are expressed as ν _nax in cm ^-1 . Thin layer chromatography (TLC) was performed on 0.25 mm E. Merck precoated silica gel plates using ultraviolet light and/or iodine vapor as embodying agents. The flash chromatography method (J.Org.Chem., 14, 2923 (1978) was carried out using silica wool (32-63um). The solvent was evaporated under reduced pressure below 50°C. Method 1 Mitomycin From A 7-[2-(benzyldithio)ethylamino]-9a-methoxymitosane (compound 1) 2 ml methanol containing 300 mg triethylamine
200mg of S-benzyldithioethylamine in
A solution of 99 mg (28.3 mM) of Mitomycin A in 10 ml of methanol was added to the solution of Mitomycin A (0.28 mM) at 0 to 4°C.
The mixture was stirred at 20°C for 2.5 hours and the reaction progressed for 10:
Using a 90v/v methanol-chloroform solvent system
Monitored with TLC. The reaction mixture was concentrated under reduced pressure, and the dried solid residue was collected using a flash chromatograph (35 g
Silica gel) using 7:93 v/v methanol-chloroform as eluent gave 87 mg (59%) of the title compound as an amorphous pure solid. ¹ HNMR (90Hz, pyridine d ₅ , δ): 2.08 (s,
3H), 2.74 (m, 3H), 3.16 (d, 1H, J=6
Hz), 3.24 (s, 3H), 3.40−4.20 (m, 6H),
4.04 (s, 2H), 4.56 (d, 1H, J=14Hz),
5.08 (t, 1H, J = 12Hz), 5.40 (dd, 1H, J =
6, 12Hz), 7.44 (bs, 5H), IR (BKr), ν _nax , cm ^-1 : 3440, 3350, 3290,
Analytical values for 3060, 3020, 1720, 1635, 1560, 1325, 1060, C ₂₄ H ₂₈ N ₄ O ₅ S ₂ : Calculated values: C, 55.80; H, 5.46; N, 10.84; S, 12.41; Measured values: C, 55.08; H, 5.31; N, 10.52; S, 12.10. Method 2 (reference example) N-[2-(2-aminoethyldithio)ethyl]
Atomamide (compound 2) a Bunte salt method 695 2-aminoethanethiethyl sulfate in 7 ml methanol containing 318 mg (7.95 mM) sodium hydroxide
cm (4.59mM) solution was stirred at nitrogen temperature for 10 minutes and then
It was subcooled to ℃. 357 mg (3.43 mM) of N -acetylcysteamine was added to this solution, and the mixture was further stirred for 10 minutes. After diluting the sticky liquid with about 60 ml of methanol, 624 mg (7.95 mM) of acetyl chloride was carefully added to make it acidic. The waxy solid obtained by evaporation of the clear liquid was used directly for conversion to compound 3. b Sulfenylthiocarbonate method SJ Preuss et al. (used the method described above. Methyl-2-aminoethylsulfenylthiocarbonate hydrochloride in methanol solution was treated with acetylcysteamine at 0°C. without triethylamine promoter. The reaction was practically instantaneous. Method 3 (Reference Example) 7-[2-(2-acetylaminoethyldithio)
Ethylamino]-9a-methoxymitosan (Compound 3) Mitomycin A 100 mg (0.29 mM) in 4 ml methanol containing 200 mg (198 mM) triethylamine
A solution of about 400 mg of compound 2 in 2 ml of methanol was added to the solution at about 0°C. The resulting precipitate was separated and the solution was left at room temperature for 4 hours. The solution was evaporated to dryness and flash chromatographed on 10 g of silica gel using gradient elution (4-8% methanol in methylene chloride). The desired product is the faster-moving blue zone component, which as an amorphous solid
45 mg (31.5%) isolated. Solid in hexane 5
% methylene chloride and dried to obtain an analytical sample. ¹ HNMR (100MHz, pyridine d ₅ , δ): 2.06 (s,
3H), 2.10 (s, 3H), 2.76 (bs, 1H), 2.80−
3.20 (m, 5H), 3.24 (s, 3H), 3.60 (dd, 1H,
J=12 and 2Hz), 3.70−4.10 (m, 5H), 4.54
(d, 1H, J=12Hz), 5.04(t, 1H, J=10
Hz), 5.38 (dd, 1H, J=4 and 10Hz), IR (KBr), ν _nax , cm ^-1 : 3420, 3350, 3290,
Analytical values for 1720, 1635, 1656, 1330, 1060, C ₂₁ H ₂₉ N ₅ O ₆ S ₂ : Calculated: C, 49.26; H, 5.67; N, 13.68; S, 12.51; Measured: C, 49.33; H, 6.04; N, 13.20; S, 11.48. Method 4 (Reference example) 7-[2-(octyldithio)ethylamino] from 7-substituted amidino-9a-methoxymitosane
-9a-methoxymitosan (compound 4) 1 ml methanol containing 0.2 ml triethylamine
7-(dimethylaminomethylene)amino-9a-
2-
(Octyldithio)-ethylamine hydrochloride 139mg
(0.54mM) was added. 10:90v/v methanol-
Thin layer chromatography using methylene chloride showed consumption of the green starting material and a main blue zone (RF=0.6) was visible. The solid residue obtained by evaporation of the liquid under reduced pressure was subjected to flash chromatography on 12 g of silica gel and eluted with 20:1 v/v methylene chloride-methanol to give 27 mg (34%) of the title compound as an amorphous solid. Part ¹ HNMR spectrum (100MHz) is shown in the table. Methods 5-22 Applications of various dithioamines The amines shown in the table are
can be reacted to produce products having the formula and as shown in the table as described in any of the following. Analysis and spectral data for some of the above substances and some listed in the table are shown in the table.

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toric acid methyl ester

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[Table] Various thiols and 7-[2-(3-nitro-2-pyridyldithio)
ethylamino]-9a-methoxymitosane to obtain the following compound. 7-[2-(3-nitro-2-pyridyldithio)ethylamino]-9a-methoxymitosane 7-dimethylaminomethyleneamino-9a-methoxy-mitosane (1.0 g, 2.61 mM) in deoxygenated methanol ( Add triethylamine (1.1 ml,
7.83mM) was added. The reaction mixture was sonicated and stirred at approximately 22° C. for 24 hours. Thin layer chromatography (silica gel, 10% CH ₃ OH (CH ₂ Cl ₂ liquid))
The results showed that more than 90% of the starting compound (green) had been converted to the desired blue compound. The reaction mixture was concentrated under reduced pressure and the resulting residue was washed 1″×
5% of CH ₂ Cl ₂ in a 16″ silica gel packed column
It was subjected to MeOH chromatography. _{CH2Cl2 _}
Gradient elution with 1-5% v/v MeOH in methanol gave the title product as a pure blue amorphous solid (430 mg). The spectrum of this product was consistent with the spectrum described above. Method A 7-[2-(3-nitro-2-pyridyldithio)
To a deoxygenated solution of ethylamino]-9a-methoxymitosane (approximately 1.1 equivalents) in acetone (3-5 ml) was added triethylamine (approximately 1.1 equivalents) under stirring and an argon or nitrogen atmosphere, followed by thiol reagent ( 1 eq.) in acetone (1-2 ml) was added dropwise. Thin layer chromatography on silica gel (10% MeOH in _CH2Cl2 ) to monitor the progress of the reaction _.
was monitored until the starting nitropyridyl dithiomitosane reactant and the product no longer had the same Rf value. For example, μBendpack C ₁₈ was used for HPLC monitoring. When the reaction is complete,
The raw reagent disappears and the product appears. here,
The reaction mixture was concentrated under reduced pressure (approximately 30 °C) and the residue was slurried on a neutral Woelm alumina column (1/4″ x 10″) using 2-5% MeOH in _{CH2Cl2 .} It was subjected to packed column chromatography.
This separated the desired mitosane product from the pyridylthione by-product remaining in the column. The product thus obtained, eluted with 2% MeOH in CH ₂ Cl ₂ , was further subjected to flash chromatography on silica gel and eluted with 5-7% MeOH in CH ₂ Cl ₂ .
Eluted with MeOH. The main band corresponding to the product was separated and the properties of the amorphous product obtained were determined. Method B 7-[2-(3-nitro-2-pyridylthio)ethylamino]-9a-methoxymitosane (approx.
2-5% v/v acetone (or 2
- saturated NaHCO ₃ in methanol (10 ml) (containing 5% v/v methylene chloride)
Add aqueous solution (6 drops) to make methanol solution 1
A chemical equivalent of thiol reagent (about 1 ml of water is used as a solution of thiol reagent if the water solubility of the thiol reagent is sufficient) was added. The progress of the reaction was monitored by thin layer chromatography (silica gel, CH ₂ Cl ₂
(10% MeOH). After the reaction is completed,
The reaction mixture was diluted with water (15ml) and concentrated to approximately 10ml in a rotary evaporator at 30°C. The resulting solution was applied to a reverse phase _C18 column and gradually eluted with a gradient (100% _H2O -80% MeOH aqueous solution). The product was eluted after the by-product thione was eluted by increasing the methanol concentration. The product is obtained primarily as a blue fraction and is concentrated to give an amorphous solid.
If further purification was required, the above chromatography steps were repeated. 7-[2-(4-chlorophenyldithio)ethylamino]-9a-methoxymitosane (39). -4-
¹ H NMR data (pyridine d ₅ ) prepared by method A using chlorothiophenol:

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[Table] 7-[2-(4-bromophenyldithio)ethylamino]-9a-methoxymitosane (47). -4-
¹ H NMR data (pyridine d ₅ ) prepared by method A using bromothiophenol:

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[Table] 7-[2-(4-fluorophenyldithio)ethylamino]-9a-methoxymitosane (46). -4
- prepared by method A using fluorothiophenol ^1H NMR data (pyridine d ₅ ):

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[Table] 7-[2-(2-chlorophenyldithio)ethylamino]-9a-methoxymitosane (55). -2-
¹ H NMR data (pyridine d ₅ ) prepared by method A using chlorothiophenol:

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[Table] 7-[2-(2-bromophenyldithio)ethylamino]-9a-methoxymitosane (65). -2-
¹ H NMR data (pyridine d ₅ ) prepared by method A using bromothiophenyl:

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[Table] 7-[2-(2,6-dichlorophenyldithio)
ethylamino]-9a-methoxymitosane (52).
¹ H NMR data (pyridine d ₅ ) prepared by method A using -2,6-dichlorothiophenol:

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[Table] 7-[2-(2,4-dichlorophenyldithio)
ethylamino]-9a-methoxymitosane (50).
¹ H NMR data (pyridine d ₅ ) prepared by method A using -2,4-dichlorothiophenol:

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[Table] 7-[2-(3-chlorophenyldithio)ethylamino]-9a-methoxymitosane (58). -3-
¹ H NMR data (pyridine d ₅ ) prepared by method A using chlorothiophenol:

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[Table] 7-[2-(2,5-dichlorophenyldithio)
ethylamino]-9a-methoxymitosane (57).
¹ H NMR data (pyridine d ₅ ) prepared by method A using -2,5-dichlorothiophenol:

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[Table] 7-[2-(3,4-dichlorophenyldithio)
ethylamino]-9a-methoxymitosane (61).
¹ H NMR data (pyridine d ₅ ) prepared by method A using -3,4-dichlorothiophenol:

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[Table] 7-[2-(3-trifluoromethylphenyldithio)ethylamino]-9a-methoxymitosane (51). ¹ H NMR data (pyridine d ₅ ) prepared by method A using -3-trifluoromethylthiophenol:

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[Table] 7-[2-(3-methoxyphenyldithio)ethylamino]-9a-methoxymitosane (54). -3
-Prepared by method A using methoxythiophenol ^1H NMR data (pyridine d ₅ ):

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[Table] 7-[2-(2-methoxyphenyldithio)ethylamino]-9a-methoxymitosane (56). -3
-Prepared by method A using methoxythiophenol ^1H NMR data (pyridine d ₅ ):

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[Table] 7-[2-(2-aminophenyldithio)ethylamino]-9a-methoxymitosane (62). -2-
¹ H NMR data (pyridine d ₅ ) prepared by method A using aminothiophenol:

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[Table] 7-[2-(4-aminophenyldithio)ethylamino]-9a-methoxymitosane (43). -4-
¹ H NMR data (pyridine d ₅ ) prepared by method A using aminothiophenol:

【table】

[Table] 7-[2-(3-aminophenyldithio)ethylamino]-9a-methoxymitosane (53). -3-
¹ H NMR data (pyridine d ₅ ) prepared by method A using aminothiophenol:

【table】

[Table] 7-[2-(4-hydroxyphenyldithio)ethylamino]-9a-methoxymitosane (42). −
¹ H NMR data (pyridine d ₅ ) prepared by method A using 4-hydroxythiophenol:

【table】

[Table] 7-[2-(1-phenylethyldithio)ethylamino]-9a-methoxymitosane (48). -1-
Prepared by method A using phenylethanethiol ^1H NMR data (partial, pyridine d ₅ ):

【table】

[Table] 7-[2-(4-pyridylmethylthio)ethylamino]-9a-methoxymitosane (64). ¹ H NMR data (pyridine d ₅ ) prepared by method A using -4-pyridylmethanethiol:

【table】

[Table] 7-[2-(4-methoxy-2-pyridylmethyldithio)ethylamino]-9a-methoxymitosane (63). ¹ H NMR data (pyridine d ₅ ) prepared by method A using -4-methyl-2-pyridylmethanethiol:

【table】

[Table] 7-[2-(4-pyridyldithio)ethylamino]-9a-methoxymitosane (60). ¹ H NMR data (pyridine d ₅ ) prepared by method A using -4-pyridinethiol:

【table】

[Table] 7-[2-(3-Methyl-2-imidazolylmethyldithio)ethylamino]-9a-methoxymitosane (59). -Produced by method A using 3-methyl-2-imidazolylmethanethiol ^1H NMR data (pyridine d ₅ ):

【table】

[Table] 7-[2-(2-amino-2-(ethoxycarbonyl)ethyldithio)ethylamino]-9a-methoxymitosane (40). - Manufactured by method A using ethylcystenate ¹ H NMR data (pyridine d ₅ , δ): 1.16 (t,
3H, J=8Hz), 2.00 (m, 1H), 2.08 (s,
3H), 2.72 (m, 1H), 3.00 (m, 4H), 3.20 (s,
3H), 3.56 (bd, 1H, J=16Hz), 3.72−4.12
(m, 3H), 4.20 (q, 2H, J=8Hz), 4.52
(d, 1H, J=16Hz), 5.04 (t, 1H, J=12
Hz), 5.36 (dd, 1H, J=4, 12Hz). IR (KBr, ν _nax , cm ^-1 ): 3420, 3290, 2920,
1720, 1630, 1555, 1510, 1445, 1320, 1210,
1055. UV (MeOH, λ _nax , nm): 220, 368. 7-[2-(2-(methoxycarbonyl)ethyldithio)ethylamino]-9a-methoxymitosane (32). -Prepared by method A using methyl 2-mercaptopropionate ^1H NMR data (pyridine d ₅ ):

【table】

[Table] 7-[2-(2-dimethylaminoethyldithio)
ethylamino]-9a-methoxymitosane (33).
Produced by method A using -2-dimethylaminoethanethiol ^1H NMR data (pyridine d ₅ , δ): 1.98 (s,
9H), 2.00 (bs, 1H), 2.42 (m, 2H), 2.58 (bs,
1H), 2.75 (m, 4H), 2.99 (bs, 1H), 3.06 (s,
3H), 3.45 (bs, 1H), 3.85 (m, 3H), 4.39
(dd, 1H, J=4, 10Hz), 4.70 (m, 1H),
5.23 (dd, 1H, J=4, 10Hz), 7.16 (t, 1H). 7-[2-(2-carboxyphenyldithio)ethylamino]-9a-methoxymitosane (34). −
¹ H NMR data (pyridine d ₅ ) prepared by method B using 2-mercaptobenzoic acid:

【table】

[Table] 7-[2-(4-nitro-3-carboxyphenyldithio)ethylamino]-9a-methoxymitosane (35). ¹ H NMR data (pyridine d ₅ ) prepared by method B using -5-mercapto-2-nitrobenzoic acid:

[Table] 7-[2-(2-amino-2-carboxyethyldithio)ethylamino]-9a-methoxymitosane (36). - produced by method B using cysteine ^1H NMR data (D ₂ O):

[Table] 7-[(2-(γ-glutamylamino)-2-(carboxymethylaminocarbonyl)ethyldithio)ethylamino 9a-methoxymitosane (37). - produced by method B using glutathione ^1H NMR data (D ₂ O):

[Table] 7-[2-(2-amino-2-((1-carboxy-3-methyl-1-butyl)aminocarbonyl)ethyldithio)ethylamino]-9a-methoxymitosane (38). -L-cysteinyl-(L)-
^1H NMR data ( _D2O ) prepared by method B using leucine:

【table】

[Table] 7-[2-(2-carboxyethyldithio)ethylamino]-9a-methoxymitosane (49). -2
-Prepared by method B using mercaptopropionic acid ^1H NMR data (pyridine d ₅ ):

【table】

[Table] 7-[2-(4-nitrophenyldithio)ethylamino]-9a-methoxy-1a-methylmitosane (41) 1a-Methylmitomycin A (98mg, 0.28mM)
Add p-nitrophenyldithioethylamine hydrochloride (81 mg) to a deoxygenated methanol (5 ml) solution of L. Cheng et al., J. Med. Chem. 20, 767 (1977) at 0°C under an argon atmosphere. , then triethylamine (70 μ) was added. The reaction mixture was warmed to room temperature and the reaction was almost complete by thin layer chromatography (silica gel, 5% v/v methanol in CH ₂ Cl ₂ ) after 3.5 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was chromatographed twice on silica gel.
Elution with 5% v/v MeOH in CH ₂ Cl ₂ gave pure desired product as a blue amorphous solid (68 mg, 43%). ^1H NMR data ( _pyridine d5 ):

【table】

【table】

[Table] 7-[2-(3-nitro-2-pyridyldithio)
[Propylamino]-9a-methoxymitosane (44) 7-dimethylaminomethyleneamino-9a-methoxymitosane (744 mg, 1.91 mM) was added to a decarbonized methanol (12 ml) solution at about 0°C for 3 hours with stirring.
-(4-Nitrophenyldithio)propylamine hydrochloride (1.55g) (prepared from 3-mercaptopropylamine using the de Brois method and using methoxycarbonylphenyl chloride) and triethylamine (774μ) were added. The reaction mixture was sonicated, brought to room temperature and reacted for 16 hours. Thin layer chromatography (silica gel, 10% MeOH in CH ₂ Cl ₂ ) gave a predominantly blue fast-migrating component with traces of the starting compound (green) and mitomycin C. The reaction mixture was concentrated and the residue was chromatographed twice on silica gel, eluting with 5-10% v/v _MeOH in _CH2Cl2 . The blue target compound was obtained as an amorphous solid. ^1H NMR data ₍ pyridine d5 )

【table】

【table】

[Table] 7-[2-(4-nitrophenyldithio)propylamino]-9a-methoxymitosane (45). - Prepared by method A using compound number 33 and 4-nitrothiophenol ^1H NMR data (pyridine d ₅ ):

【table】

【table】

[Table] A drug efficacy test similar to that described above was conducted and the following results were obtained.

【table】

【table】

【table】

【table】

【table】

【table】

【table】

Claims (1)

[Claims] 1 Formula: (In the formula, Alk ₂ represents a linear or branched alkylene group having 2 to 6 carbon atoms; R ⁹ is -Alk ₁ -R ⁷ , R ⁸ or a residue of an amino acid or a peptide, and Alk ₁ is a group having 1 carbon atom. R represents a straight-chain or branched alkylene group having from 2 to 6 carbon atoms; R represents hydrogen or lower alkyl; R ⁷ represents dialkylamino, phenyl or A-substituted phenyl having from 2 to 12 carbon atoms; R 8 is a group consisting of a heterocyclic group selected from the group consisting of a monocyclic heteroaromatic group or a heteroalicyclic group having 1 to 4 nitrogen heteroatoms, and R ⁸ is phenyl, A-substituted phenyl, and selected from the group consisting of nitro-substituted pyridyl, and substituent A is lower alkyl, lower alkoxy, halogen,
1 to 3 of NH ₂ , hydroxy or nitro groups). 2 formula: Or formula: (In the above formula, Alk ₁ represents a straight chain or branched alkylene having 1 to 6 carbon atoms, and in that case R ⁷ and
SS is linked to a different carbon atom; Alk ₂ is optionally linked to R ⁷
Represents a straight-chain or branched alkylene group having 2 to 6 carbon atoms with substituents, the sulfur and nitrogen atoms attached to it and any R ⁷ substituents attached to different carbon atoms of Alk ₂ ; Alk ₁ and Alk ₂ may have a double bond; R represents hydrogen or lower alkyl; R ⁷ is dialkylamino, phenyl or A-substituted phenyl, naphthyl or A-substituted naphthyl, having 2 to 12 carbon atoms; Represents a group selected from the group consisting of a heterocyclic group selected from the group consisting of a heteroaromatic group or a heteroalicyclic group that is a 5- to 7-membered monocyclic ring and has 1 to 4 nitrogen heteroatoms, provided that The A substituents above shall be selected from the group consisting of 1 or 3 lower alkyl, lower alkoxy, halo, NH ₂ , hydroxy or nitro groups; and R ⁸ is phenyl, A-substituted phenyl, and nitro substituted represents a group selected from the group consisting of pyridyl, provided that said A substituent is selected from the group consisting of one or three lower alkyl, lower alkoxy, halo, NH ₂ , hydroxy or nitro groups;
Additionally, R ⁸ and the adjacent sulfur atom together constitute an S-cysteinyl group that can be esterified, formed into a salt, or bonded within a peptide bond. ) The compound according to claim 1, characterized in that it is represented by: 3 Alk ₂ in the formula is ethylene and R
Claim 2 having a formula such that is hydrogen
Compounds described in Section. 4 Claim 3 in which Alk ₁ is ethylene
Compounds described in Section. 5 In the formula, Alk ₂ is ethylene and R
Claim 2 having a formula such that is hydrogen
Compounds described in Section. 6. A compound according to claim 2 having the chemical name 7-[2-(benzyldithio)ethylamino]-9a-methoxymitosane. 7. A compound according to claim 2 having the chemical name 7-[2-(phenyldithio)ethylamino]-9a-methoxymitosane. 8. A compound according to claim 2 having the chemical name 7-[2-(4-methoxyphenyldithio)ethylamino]-9a-methoxymitosane. 9. A compound according to claim 2 having the chemical name 7-[2-(4-nitrophenyldithio)ethylamino]-9a-methoxymitosane. 10. A compound according to claim 2 having the chemical name 7-[2-(2-nitrophenyldithio)ethylamino]-9a-methoxymitosane. 11. The compound according to claim 2 having the chemical name 7-[2-(2,4-dinitrophenyldithio)ethylamino]-9a-methoxymitosane. 12. A compound according to claim 2 having the chemical name 7-[2-(3-nitro-2-pyridyldithio)ethylamino]-9a-methoxymitosane. 13. A compound according to claim 2 having the chemical name 7-[2-(5-nitro-2-pyridyldithio)ethylamino]-9a-methoxymitosane. 14 1 to 4 mole parts of the amine represented by the formula: R ⁷ -Alk ₁ -SS-Alk ₁ NH ₁ or the formula: R ⁸ -SS-Alk ₁ NH ₂ by the formula: [Formula, and in which Alk ₁ has 1 to 6 carbon atoms, has 1 to 2 phenyl or A-substituted phenyl rings, and has 3 in each ring.
1 to 4 nitrogen heteroatoms having 8-membered rings
Alk ₂ represents a straight-chain or branched alkylene group having a heterocyclic group selected from the group consisting of a heteroaromatic group or a heteroalicyclic group having 2 to 6 carbon atoms; Representation: Alk ₁ and Alk ₂ may have a double bond; R represents hydrogen or lower alkyl; R ⁷ represents a group selected from the group consisting of dialkylamino having 2 to 12 carbon atoms, However, above A
The substituents shall be selected from the group consisting of one or three lower alkyl, lower alkoxy, halo, NH ₂ , hydroxy or nitro groups; and R ⁸ is selected from phenyl, A-substituted phenyl, and nitro-substituted pyridyl. with the proviso that the A substituent is selected from the group consisting of one or three lower alkyl, lower alkoxy, halo, NH ₂ , hydroxy or nitro groups;
In addition, R ⁸ and the adjacent sulfur atom together constitute an S-cysteinyl group that can be combined in esterification, salt formation, or peptide bonding, and Y is lower alkoxy having 1 to 6 carbon atoms or the formula: (In the above formula, R ⁵ is hydrogen, lower alkyl, phenyl,
represents lower alkyl phenyl, lower alkoxyphenyl, halofenyl, aminophenyl, or nitrophenyl; R ³ and R ⁴ independently represent lower alkyl, or together with the nitrogen atom to which they are bonded represent pyrrolidine, 2- or 3-lower Alkyl-pyrrolidine, piperidine, 2-, 3- or 4-lower alkyl-piperidine, 2,6-di-lower alkylpiperidine,
piperazine; alkyl or carbalkoxy having 1 to 8 carbon atoms, phenyl, methylphenyl;
Piperazine 4-substituted with methoxyphenyl, halophenyl, nitrophenyl or benzyl; azepine, 2-, 3-, 4- or 5-lower alkyl azepine, morpholin, thiomorpholin, thiomorpholin-1-oxide, or thiomorpholin-1,1 -represents a carbon dioxide; and each lower alkyl, lower alkanoyl and lower alkoxy mentioned above has 1 to 6 carbon atoms. ) represents a group represented by ] in an organic solution inert to the reaction with 1 mol part of mitosan represented by the formula: Or formula: (wherein Alk ₁ , Alk ₂ , R, R ⁷ and R ⁸ are as defined above) under reaction conditions at a temperature of about -15 to +50°C for a sufficient period of time to produce a suitable amount of a product having the formula: A method for producing a compound represented by the above formula or formula, characterized by contacting the compound with the above formula or formula.