DE2220766B2 - BLEOMYCINE AND MEDICINAL PRODUCTS CONTAINING THESE - Google Patents

Description

2. Medicaments containing one of the bleomycins according to claim 1 as an active ingredient.

The invention relates to the bleomycins defined in claim 1 and medicaments containing them.

The bleomycins according to the invention are produced by reacting

3-aminopropylaminobleomycin (1),

3- (N-4-aminobutyl) -aminopropyl-

aminobleomycin (II) or

3- (N-Methyi-N-3-aminopropyl) -aminopropyl-

aminobleomycin (IIi) with
Ai) carboxylic acid anhydrides of the general formula

R ¹ - C - O - C - R ¹

in which R ^{1 has} the meanings given in claim 1

or with

A2) the anhydrides of succinic acid, glutaric acid, Maleic acid, phthalic acid

or with

A3) carboxylic acids of the general formula R ¹ - COOH, where R ^{1 has} the meanings given in claim 1, in the presence of a carbodiimide,

or with

A4) carboxylic acid halides of the general formula R ¹ —COX, in which R ^{1 has} the meanings given in claim 1, preferably chloromethyl, and X is halogen, preferably chlorine,

or with

A5) carboxylic acid azides of the general formula R ¹ -CON3, in which R ^{1 has} the meanings given in claim 1,

or with

Αβ) carboxylic acid esters of the general formula R ¹ —COOR ⁷ , in which R ^{1 has} the meanings given in claim 1 and R ^{7 is} lower alkyl or substituted or unsubstituted phenyl,

or with

B) an organic halide of the general formula R ² -X, in which R ^{2 has} the meanings given in claim 1 and X is preferably bromine,
or with

C) a sulfonic acid halide of ^{the general formula R 3} _{—SO 2} X, in which R ^{J has} the meanings given in claim 1 and X is preferably chlorine,

or with
D) an isocyanate of the general formula R ⁴ —NCO, in which R ^{4 has} the meanings given in claim 1,

or with

E) an imide ester of the general formula
to

R ⁵ -C OR ⁶

NH

in which R ^{5 has} the meanings given in claim 1 and R ^{6 is} alkyl, preferably methyl.

The bleomycins (I) to (III) used in the production of the bleomycins according to the invention are obtained in high yield by adding an amine to the relevant side chain amine part P is added to a medium in which bleomycin-producing strains for the recovery of Bleomycin can be cultivated.

The bleomycins of the invention are antibiotic substances with high antimicrobial activity and antitumor activity with low toxicity.
Bleomycin (II) is known as "Bleomycin A5".
The process for preparing the compounds of the invention is carried out in one of the solvents

Water; Alcohols such as methanol and ethanol; Ketones such as acetone; or in a mixture of these solvents carried out.

When using alkyl halides such as ethyl iodide, acyl halides, methylsulfonyl halides as a reagent the rate of the reaction is high and the use of such compounds in excess not only causes the desired monosubstitution of the terminal amino group of the side chain of the Bleomycins I to III, but also the substitution of intramolecular amino groups and disubstitution the terminal amino group of the side chain. Therefore, it is usually preferred to include these compounds in to use an amount of 1 to 1.5 moles per mole of the bleomycins I to III. However, less responsive Reagents which do not cause side reactions can be used in an amount of up to about 10 moles each Mol of the bleomycins I to III are used.

The reaction of the invention is called a nucleophilic substitution reaction of the terminal Considered amino group of the bleomycins and they are preferably carried out in an alkaline medium. to pyridine, sodium bicarbonate, triethylamine can be used for this purpose. When implemented with imide esters it is preferred to carry out the reaction by adding triethylamine at a pH of 8.5 to 9.5 perform. However, in view of the stability of the bleomycins, the reaction should last for a long time Periods of time at a pH of 10 or more should be avoided.

The reaction temperature can generally be 10 to 25 ° C. However, if a reactant used is less reactive, the reaction is preferably carried out at about 40 ⁰ C. For example, sulfonylation with various sulfonyl compounds is complete in about 1 to 4 hours at room temperature.

To separate the desired product, the reaction solution is adjusted to a pH of 7 to 5

neutralized and, if necessary, concentrated under reduced pressure. The reaction solution thus treated is then passed through a column which is filled with carboxylmethyl crosslinked dextran. That Once the reaction product has been adsorbed on the adsorbent, it is treated with an aqueous salt solution such as Ammonium chloride solution eluted. The eluate fraction, which contains the desired product, is passed through run a column which is filled with activated carbon or with a weakly acidic cation exchanger. That Product adsorbed once on the adsorbent are eluted with acetone -0.02 η-hydrochloric acid (volume ratio 1: 1) or with methanol - 0.02 N hydrochloric acid (volume ratio 1: 1), concentrated and dried, whereby the desalted, respective bleomycin according to the invention is obtained.

Some bleomycins according to the invention, such as 3- (N-dodecanoyl) aminopropyl aminobleomycin, are not very soluble in water. Therefore, in such a case, the reaction solution can be purified by chromatography be, using a gel filtration and a cross-linked dextran used for this purpose, the against organic solvents, which makes the desalination process unnecessary.

The bleomycins obtained in this way, like the starting bleomycins I to III, are in the form of blue powder. They generally have ultraviolet and infrared adsorption spectra similar to those of the starting substances are similar, although the ultraviolet and infrared adsorption spectra of such derivatives in which Q contain a chromophoric group such as 4-methylphenylsulfonyl and 4-nitrophenyl of those the starting substances are different.

That the products obtained, which differ from the starting bleomycins 1 to III by substitution at the derive terminal amino group, it is confirmed

by the Rr value in thin-layer chromatography furthermore by the fact that it is in the migration region are different from the starting substances in electrophoresis; the fact that they im generally give a negative ninhydrin reaction; and the fact that, according to the van Slyke method, a Reduction in the content of amino nitrogen was found.

According to the process described, the monosubstituted derivatives are obtained as the main product, with depending on the reaction conditions, a small amount of the disubstituted derivatives is also formed.

Bleomycins show no sharp melting or decomposition points, inhibit gram-positive and gram-negative bacteria and have anti-tumor activity; they are soluble in water and aqueous methanol; they form salts with acids and give chelates Copper; they give positive Pauly, Ehrlich, Dragendorff and permanganate reactions, but negative ones Molish, Biuret, Elson-Morgan, Maltol, Fehling, Tollens, Anthron and ferric chloride reactions; she show absorption ranges in the infrared spectrum for the wave numbers: 1040 to 1075 cm- ', 1710 to 1735 cm- ', 1625 to 1680 cm-' and 2910 to 2940 cm- '; they are also characterized by the fact that they become more aqueous when exposed to 6-N for 30 minutes Hydrochloric acid at 105 ° C 1-threonine, 0-amino-j3- (4-amino-6-carboxy-5-methyl-pyrimidin-2-yl) propionic acid, 4-amino-3-hydroxy-2-methyl-n-valeric acid, j3-hydroxy-histidine, L-jS-aminoalanine, L-gulose, 3- (O-carbamoyl) -D-mannose, 2 '- (2-Aminoethyl) -2,4'-bithiazole-4-carboxylic acid and in each case the one corresponding to the side chain part Amino compound yield; they exist as Cu complexes and they correspond to the following formula, in which P and Q have the meanings given in claim 1:

HO

P-Q

OH

OH

The properties of the resulting bleomycins for their hydrochlorides are summarized in the following tables:

Table IA

N- (Seitenkeltc. Cndsländig) -ACYL-Bleomycine I, II, St.

No. Aiis-
Blco-
myctii I. Reaction with
... anhydride I. Il A cct- 2 III A cct- 3 I. Accl- 4th Il Propionic 5 III Propionic 6th I. Propionic 7th Il Butyr X I. But) r- 9 11th Caprylic acid Ml I. Caprylic acid Il Il Lauric acid 12th III Lauric acid 13th I. Lauric acid 14th Il Crotonic acid 15th I. Crotonic acid 16 Il Succinic acid c 17th 111 Succinic acid IX I. Succinic acid 19th Il Malcic acid 20th III Maleic acid 21 I. Maleic acid 22nd Il Phcnoxycssig-
acid- 23 I. Phenoxycssii; -
acidic 24 11th Benzoic acid 25th III Benzocic acid 26th I. Bcnzocsaurc- 27 Il Nicotinic acid 28 III Nicolinic Acid 29 I. Nicotinic acid 30th Il here:
Chloroacctyl
chloriil 31 III 32 I. 33 Il Thi (iphtn-2-C! Ir
bonsiiuie- .1-1 III Ί ΊιίορΐΗΊΐ-2-car
bunsäliia - 35 Thii> phcn-2-ciir
lllMlsillllC-

-CO-CH, -CO-CH, -CO-CH,

-CO-C ₂ H ₅

-CO- (CH ₂ I ₂ -CH, -CO- (CH ₂ ) "- CH, -CO- (CH ₂ )," - CH,

-CO-CH = CH-CH ₁ -CO-CH ₂ -CH ₂ -COOH

-CO-CH = CH-COOH

—CO — CH ₂ - Ο

-CO-CH ₂ CI

_ _ΓΟ

meg Potcnz / mg *
M.smeg- B.sub-
matis tilis 120 ) UV **) DC * (0.64) Elec-
tro-
phore-
se ****; L D-50
ED-50
I. 460 340 243, 292 0.80 (0.21) 0.80 20.4 1400 800 243, 292 0.62 (0.30) 0.77 ~ 43.5 1050 180 242, 292 0.58 (0.66) 0.82 43.5 900 880 243, 292 0.87 (0.21) 0.72 21.8 2000 525 243, 292 0.66 (0.24) 0.78 51.3 I 710 50 242, 292 0.52 (0.62) 0.79 43.3 I 530 780 240, 292 0.86 (0.21) 0.71 25.6 3 9 (K) 1400 243, 292 0.66 (0.68) 0.82 62.8 9600 2,980 243, 292 0.88 (0.23) 0.68 13.3 17 2 (X) 61 243, 292 0.73 (0.55) 0.74 57.9 7 560 I 140 243, 292 0.78 (0.18) 0.44 10 5 700 1 100 243, 292 0.70 (0.24) 0.67 31.5 19 (KX) 108 242, 292 0.58 (0.62) 0.69 51.2 1850 840 243, 292 0.87 (0.21) 0.75 4 3 (K) 96 243, 292 0.70 (0.62) 0.78 47.4 101 225 243, 292 0.85 (0.21) 0.73 32.6 200 68 243, 292 0.62 (0.30) 0.79 113 90 58 243, 292 0.64 (0.62) 0.85 54 88 6 (K) 240, 292 0.87 (0.22) 0.73 10.9 235 1 250 243, 292 0.66 (0.26) 0.75 24 320 220 235 (S), 292 0.61 (0.57) 0.83 60.5 3,500 615 268, 275, 292 0.78 (0.21) 0.76 25.1 8 500 110 268, 275, 292 0.65 (0.57) 0.76 42.1 I 8 (K) 680 238, 295 0.76 (0.21) 0.77 30.8 5 100 I 340 235, 292 0.70 10.30) 0.70 32.1 5 620 760 235, 292 0.61 10.57) 0.86 47.7
(77.7) 2 2 (X) 960 240, 270, 292 0.54 10.23) 1.10 34 2,800 570 240, 292 0.68 (0.24) 0.92 54.3 2 (KX) 9X 292 0.48 (0.62) 0.88 760 960 243, 292 0.90 (0.23) 0.70 18.2 2 (KK) 7 (K) 243,292 0.74 10.24) 0.77 55.1 I 2 (cl 107 242, 292 0.4K (0.62) 0.80 56.4 2 280 I IK) 245, 275 0.87 (0.22) 0.69 15.8 6090 I 170 245, 275 0.71 (0.26) 0.78 37.8 6 X (K) 245, 275 0.58 0.77 39.5

continuation Aiis-
BIcU-
myciii Reaction mil
... anhydrite! O No. I. Furan-2-carbon-
acid- -co- © 36 Il Kuran-2-carbon-
acid- 37 1 4-nile robe
acid- -CO - <^ NO ₂ 38 Il 4-Nilrobenzoc-
acidic 39 11th Isonicotinic acid -CO ^ N 40 I. Phthalic acid ~ ^c ° - <~ y 41 COOH Il Phthalic acid 42 IH Phthalic acid 43 Il 4-nitrophthal-
acid- -CO - ^> NO ₂ 44 COOH III 4-nitrophthal-
acidic 45 III Glutaric acid -CO-ICH ₂ Ij-COOH 46 H Adipic acid —CO— (CH,) ₄ —COOH 47 III Methyl amber
acid --CO-CH ₂ -CH-COO
CH, 48 111 llaconic acid -CO-CH, -C-COOH
Il 49 Il
CH,

mci! Poland // mg * l UV **) M. smey- H. submalis lilis

780 100 247, 292 2.10 1 090 248, 292

16 (KX) 2 300 247, 280

700 I 000 243, 292

710 2 8 (X) 237, 292 620 680 292

390 I20 238.

320 65 237, 287

116 79 243, 292

170 220 24.1. 292

2.10 315 243.292

l) C *** l Elck- LD-50

Im- ED-50 phoresc **** l

0.82 (0.611 0.68 11.6

0.65 (0.211 0.74 47.7

28.8 0.60 10.231 0.76 28.4

0.69 10.231 0.92 84 26.8 0.71 (0.241 0.73 98.4 0.57 (0.24) 0.74 94.7 0.64 (0.25) 0.67 11X1 0.58 10.26) 0.73 0.66 (0.30) 0.83 87.7 76.9 (1.65 (0.29) 0.82 47.7 0.69 (0.301 0.82 44.1

Table I B N- (side chain, terminal) -ALKYL-bleomycins

II, III

No. The end-
iianus-
Blcii-
mycin Reaction mil
... bromite 50 I. Ethyl 51 Il Ethyl- 52 Il AIIyI- 53 III AIIyI-

54 I.

Hcn / yl-

55 I. 4-Nili'oben / yl 56 Il 4-NilrnrH.'ii / .yl 57 III 4-Niliohenzvl

(', 11,

cn, cn cn.

cn,

CH. \ ^ -NO,

niL-μ Polcnz / mp)
M.smeg- H.sub-
malis tilis 2 520 UV ¹ ·) OC "') (0.6.1) Elec-
Iro
phorc-
sc * *) ID-50
ED-50 1 7IK) 7 5 (K) 243. 292 0.70 (0.211 0.96 20.3 .1.1 (St. 8410 248, 292 0.31 (0.211 0.98 27.4 3 450 7WK) 24.1, 292 0.33 (0.21) 0.95 > P 3 4 (K) 2.10 243. 292 0.33 (0.631 0.94 35.8 5 S33 2,760 242.5 . 292.5 0.76 (0.631 0.90 26.2 I I MIKl 3 885 246. 2SO 0.69 10.21) 0.95 28.2 6 950 3,500 246. 28 (1 0.59 (0.231 0.9.1 14.4 705 (1 246, 279 0.58 0.95 31.8

11th Aii.s-
gearing
m \ cin Reaction mil
... sulfonic acid
ehloriil 22 20 766 The end-
gangly
Bleo-
mycin Reaction with
.. .isoeyanat Q Aiis- Rcaklion with the Q 122 896 B.sub-
lilies UV **) 12th (0.62) (0.22) (0.46) Elbow
Uo-
phore-
se ··· *) LD-50
ED-50 > 8 I. Meihan Il Ethyl- -CO-NH-CjH ₅ gangly
Hlco-
myein Mclliylimino-
alhcr-hydro-
ehlorid the
...acid 680 321 74 292 (0.23) (0.63) (0.24) 0.73 5.8 15.4 Table IC N- (Scitenkeite, terminal) - Il Methane- Alkylsulfonyl- ",
Bleomycin
Arylsulfonyl I. Cyclohexyl -C0-NH-Q> I. Amcise
(liienAlhyl-
iminollthcr-) -C- H
Il
NH 230 I 160 866 243, 292 (0.70) (0.22) 0.82 23.0 7.9 No. I. Benzene- Q Il Cyelohexyl Il 830 1052 - 272, 295 (0.22) (0.23) 0.72 5.41 18.3 5 p Il Benzene- -SO, -CH, III Cyclohexyl I 500 225 240, 295 (0.22) (0.63) 0.78 24.6 59 III Benzene- I. Phenyl —CO— NH- \ ~ S 140 I 100 272, 292 (0.60) (0.19) 0.72 26.9 60 I. p-toluene -So ₁ - (^ y meg potency / mg * I
M.smeg- U.sub-
malis ulis Il Phenyl 1,500 230, 290 DG * *) (0.24) 0.67 12.1 LD-50
ED-50 61 Il p-toluene 501 Table I E I 250 232, 293 0.86 (0.30) 0.73 25.6 47.4 62 III p-toluene 1,670 N- (Seilenkellc, cndsländig) -IMIDOACYL-Blcomycine 121 meg potency / mg *) 292 0.63 (0.71) DC * ··) 0.73 20.0 30.8 63 I. 4-chlorobenzene - SO, - '\ ~ y ~ CH., 3 5 (X) No. I 400 M.smcg-
matis 232, 292 0.92 (0.24) 0.63 6.0 > 40 64 11th 4-chlorobenzene 6400 1400 272 230, 295 0.86 (0.26) 0.58 0.75 74.7 65 111 4-chlorobenzene 6 700 77 202 11 229 292 0.59 (0.68) 0.5 0.69 34.9 66 I. Naphthalene-2- - SO, - / ~ V-C1 8,000 78 1,500 228, 286 0.77 (0.24) 0.64 55.3 67 II Naphthalene-2- 19,000 230, 283 0.76 0.71 68 Table 1 D 10,000 0.74 69 ~ ^SO2 XX) 7010 meg potency / mg *)
M.smeg- B.sub-
matis ulis 0.87 DC ***) LD-50 70 23,000 2,388 UV **) 0.84 0.73 Elec-
UO-
phorc—
se ····) ED-50 17 700 8 750 243, 292 0.65 0.78 0.79 22.1 4,350 13 560 243, 292 0.84 0.75 0.81 37.1 26,000 11 500 243, 292 0.75 0.77 0.75 3 236 243, 292 0.79 0.76 N- (side chain, terminal) -N'-subst. CARBAMOYL bleomycin 4 775 244, 283 0.63 0.81 No. 244, 287.5 0.77 71 72 73 UV ··) Elec- 74 Uo-
phorc-
se ··· *) 75 242, 292 0.76 76 243,292 0.81

13th

continuation

No. From reaction with the ^f ) gang- melhylimino-bleoether-hydromycin chloride de. ...acid

1

Il

I.

II 11

1

I) I

1)

I.

II

I.

Il

1

Il

I.

II

1

II

I.

Il

H) I MI

1

Il

III

Vinegar-

Vinegar vinegar

Propionic

Propion isobutler

Valerian

Valerian Pelargon

Pelargon-Bcnzoe-

Benzoin

-C-CH ₁ NH

-CC ₁ H ₁ NH

-CC ₅ H ₇ IiSO)

NH -C-CH,

NH

CC ₈ M _n

NH

NH

4-methoxybenzoene - C.

NH 4-methoxybenzoic

4-methylbenzoene- —C NH

3-Methylbenzoe- —C NH

CH,

3-nitrobenzoic

3-nitrobenzoic phenyl acetic acid

Pherylessgar-Phcnylessig-

4-chlorophenyl acetic acid

4-chlorophenyiacetic acid

4-chlorophenyl aqueous

-CS >

NH

-C-CH;

NH

NO ₂

NH

mcj! Pcitcnz / rng *)
M.smcg- B.sub-
malis lilis 1080 UV **) DC * ") (0.63) Elec-
iro-
phorc-
se **** | LD-50
ED-S) I 867 7 425
6 750 243, 292 0.65 (0.22)
(0.23) 0.96 39.5 3 100
3 640 986 243, 292
242, 292 0.27
0.24 (0.46) 0.91
0.96 34.5
42.1 2 252 19 500 243, 292 0.50 (0.22) 0.94 39.0 3 186 17 640 243, 292 0.36 (0.22) 0.92 37.4 3 725 1 157 243, 292 0.33 (0.67) 0.93 83.6 6 531 15 870 243, 292 0.78 (0.22) 0.92 38.4 10 180 1443 2Λ3, 292 0.43 (0.46) 0.95 69.5 29 370 8 950 242.5, 292 0.59 (0.19) 0.86 17.2 25 500 963 243, 292 0.49 (0.67) 0.90 92.1 4 423 12 900 292 0.80 (0.22) 0.91 24.2 5 199 839 235 (S), 292 0.39 (0.46) 0.94 > 8.2 5 230 9 265 247, 0.60 (0.22) 0.90 27.3 6 500 786 248, 292 0.45 (0.65) 0.97 45.2 7 692 10 462 241, 296 0.76 (0.23) 0.90 26.3 11 890 789 240, 292 0.43 (0.61) 0.88 53.7 5,956 290 0.79 0.90 21.8

10876 13330 235 (S), 291 0.42 (0.18) 0.89 40.0

5 790 1676 287 0.76 (0.68) 0.91 17.9

9 750 17 612 243.288 0.32 (0.22) 0.87 49.5

6,875 1987 243.292.5 0.76 (0.63) 0.95 6.31

11 700 20 900 243, 292.5 0.42 (0.22) 0.98

12 750 7 800 242.292 0.37 (0.26) 0.96

-C-CH, - <f ^ - Cl 14,975 1,629,242.5. 293 0.75 (0.63) 0.93

Il X = /

18920 23 660 242.5. 292.5 0.40 (0.22) 0.97 21

19 (MK) 14 (KK) 292 0.32 (0.22) 0.89 53.3

continuation

No. From reaction with the 0 gangs Methylimino-Blcoäthcr-Hydromycin chloride der
... acid

mcgPolcnz / mg ·) UV ··)

M. smcg- B. submaiis lilis

DC »**) Elek- LD-50 lro- IiD-S) phore se · * · *)

1 (15 I.

IK) I

III Il

Cinammon-

106 Il Ziml- -C-CH ₂ 107 Il 4-nitrophenyl Il
NH vinegar- - C — CH,
Il
NH 108 I. Naphthalene-
I-cssig- 109 Il Naphlhalin
1 vinegar

3- | N, N-dimethyl- -C-CH ₂ -CH ₂ -N amino) -propion- || \

NH

3- (N.N-Dimelhylaminol-propion-

3-chlorophenyl- —C-CH ₂ cssig- II

NH

Cl

11 4-bromophynyl- -C-CH ₂ ^ f V-

pssiu- Il N = /

cssig

9 9 (K) 843 245, 288

12 240 8 450 247, 287

17 6 (X) 24 380 246, 283

IO 170 I 119 291, 300

13 (KX) 8 265 \% \

I 896 5 145 243, 292

I 690 13 760 243, 292

0.60 (0.46) 0.90

0.55 (0.46) 0.85

< ⁰ · ²² )

0.44 (0.22) 0.89 19.5

0.31 (0.22) 0.91> 8.2

0.33 (0.68) 1.18 48.4

0.095 (0.20) 1.10 105.8

NH

Table I F

Bleomycin side chain structure

NH- (CH ₂ Jj-NH ₂

NH- (CH ₂ ) ₃ -NH- (CH ₂ ) ₄ -NH ₂

NH- (CH ₂ Jj-N- (CH ₂ J ₃ -NH ₂

CH ₃

UV
Absorption
maximum Electro
phoresis mcg potency / mg
M. smeg. B. I. subt. DL-50
ED-50 243, 292 I. 1,850 1 800 28.5 243, 293 1 17000 12th 900 20th 243, 292 I. 1900 500 49

These bleomycin derivatives I, II, III also show a positive ninhydrin reaction.

Comments on Tables IA to 1F

*) The effect of the bleomycin derivatives was determined against Myco bacterium smegmatis 607 or Bacillus Subtilis, ATCC No. 6633 using bleomycin Aj hydrochloride (copper-free) as the standard by the cylinder-plate method. The numerical values given represent meg potency in comparison to the selected standard per substance unit (mg).
··) The UV absorption maxima are given in ηΐμ.

· **) TLC is the abbreviation for »thin layer chromatography«. Silica gel served as the adsorbent and was used as the mobile phase a solvent mixture of 10% aqueous methanol, 10% ammonium acetate and ammonia water (10: 9: 1) is used. The Rf values are given. The value in brackets relates to the parent bleomycin.

* ·· ♦) The values given in column 8 show the migration distance compared to bleomycin as the standard in the high-voltage paper trophy at pH 1.9 and 3 (XK) V (comparative blcomycin = 1).

Of the substances mentioned in Tables IA to IE, compounds No. 51, 52, 53, 56, 57, 59, 61, 64, 67, 70, 71, 73, 74 and 76 are positive Ninhydrin reaction.

The IR absorption spectra of 7 representatives of the "> new derivatives which are obtained according to the invention are shown in FIG. 1-7 shown; it shows

Fig. 1 shows the spectrum of 3- (N-benzyl) aminopropylaminobleomycin (No. 54),

2 shows the spectrum of 3- (N-benzenesulfonyl) -amine nopropyl aminobleomycin (No. 60),

F i g. 3 the spectrum of N-acetyl-bleomycin A-, (No. 2).

Fig. 4 is the infrared absorption spectrum of N-succinyl bleomycin A -, (No. 17), n

Fig. 5 is the infrared absorption spectrum of N- (Cyck> hexylcarbamoylj-bleomycin Ai (No. 73),

Fig. 6 is the infrared absorption spectrum of 3- (N-methyl-N-acetimidoylaminopropylI-aminopropyl-aminobleomycin (No. 81),

Fig. 7 is the infrared absorption spectrum of 3- (N-methyl-N-p-chlorophenyl-acetimidoyl-aminopropyljaminopropyl-aminobleomycin (No. 104).

(The numbers in parentheses indicate the compound numbers used in the above-mentioned Tables are shown).

As can be seen from the tables, many of the bleomycins according to the invention show higher antimicrobial properties Efficacy against Mycobacterium smegmatis 607 and Bacillus subitilis as the starting bleomycins I to IM, Jo and they also have remarkable effects in cancer treatment, such as animal studies with Ehrlich's ascitis tumors have shown. Furthermore, the enzymatic attack on the terminal amino group of the side chain can occur the parent bleomycins are prevented is

S-iN-succinylJ-aminopropyi-amir.obleomycin,
3- (N-Acetyl) -aminopropyl-aminobIeomycin, N-Succinylbleomycin A5, N-Acetyibleomycin A5 and

Although 3- (N-methyl-N-3-succinylaminopropyl) aminopropyl-aminobleomysin show stronger Antitii- w morwirkung in the aforementioned animal tests, however, have a better chemotherapeutic index (LDM / ED ™) as compared to 3-aminopropyl aminobleomycin and bleomycin A5 as controls because of their lower toxicity.

The preparation of the bleomycins according to the invention is explained in more detail by the following examples:

example 1
3- (N-crotonyl) aminopropyl aminobleomycin

100 mg of S-aminopropyl-aminobleomycin hydrochloride are dissolved in 5 cm of methanol. ^{0.12 cm 1 of} pyridine and 116 mg of crotonic anhydride are added to the solution with stirring. The mixture is then left to stand at room temperature for 24 hours. The reaction liquid is concentrated under reduced pressure. The residue is dissolved in a small amount of water. The solution is allowed to pass through a column filled with a suspension of 23 cm · t> o carboxymethyl crosslinked dextran in 0.02 M aqueous ammonium chloride in order to adsorb the desired product. The product adsorbed once on the adsorbent is eluted with 0.1 M aqueous ammonium chloride. The eluate fractions which contain the product are collected and then passed through a column which ^{is filled with a suspension of 7 cm 1 of} activated carbon in water.

to adsorb the product. After washing with water, this will be on the adsorbent once adsorbed product with acetone - 0.02-M hydrochloric acid (volume ratio 1: 1) eluted. The eluate is concentrated one to dryness and receives 85.9 mg 3- (N-crotonyl) -aminopropyl-aminobleomycin (1850 meg potency / mg against Mycobacterium smegmaiis 607). Yield 82%.

Example 2
a) 3- (N-Phenoxyacetyl) -aminopropyl-aminobleomycin

I g of 3-aminopropyl-aminobleomycin is dissolved in a mixture of 13 cm 'of water and 120 cm ^{1 of} acetone. 400 mg of sodium bicarbonate and 780 mg of phenoxyacctyl chloride are added to the solution with stirring. The mixture is left to stand at room temperature for 4.5 hours. The pH of the mixture is adjusted to 4.5 by adding hydrochloric acid and the mixture is concentrated under reduced pressure. The concentrate is filtered. The filtrate is passed through a column which ^{is filled with a suspension of 120 cm 1 of} carboxymethyl nuclear dextran in 0.02 M aqueous ammonium chloride, the desired product being adsorbed. The product once absorbed on the adsorbent is eluted with 0.1 M aqueous ammonium chloride. The eluate fraction containing the desired product is collected and passed through a column which ^{is filled with a suspension of 150 cm J} weakly acidic cation exchanger in water, the product being adsorbed. Successively washed with 750 cm ^J 0.5% acetic acid and ¹ 750 cm water. The product, once adsorbed on the adsorbent, is eluted with 0.02 N hydrochloric acid - methanol (volume ratio 1: 1). The eluate is concentrated to dryness under reduced pressure. This gives 390 mg of 3- (N-phenoxyacetyl-aminopropylaminobleomycin (3500 meg potency / mg against Mycobacterium smegmatis 607). Yield: 35.4%.

b) The above-mentioned reaction is repeated, the phenoxyacetyl chloride being replaced by 359 mg Replaced chloroacetyl chloride and 438 mg are obtained

3- (N-chloroacetyl) aminopropy! -; iminobleomycin
(760 meg potency / mg against Mycobacterium smegmatis 607). Yield: 41.5%.

Example 3
3- (N-Propionyl) -aminopropyl-amir, obieomycin

100 mg of 3-aminopropyl-aminobleomycin hydrochloride dissolves one in a mixture of 3 ^cm] of acetone and 2 nm ^s water. 14 mg of 1-ethyl-3-dimethyl-aminopropyl-carbodiimide are added to the solution

Add 10 mg propionic acid. The pH of the mixture is adjusted to 4.8 and the mixture is left for 16 hours stand. The reaction liquid is concentrated under reduced pressure. Add to the residue Add 10 cm 'of water. The mixture is passed through a Column through which with a suspension of 20 cm 'carboxymethyl-crosslinked dextran in a 0.02N aqueous ammonium chloride solution is filled, with the desired product being adsorbed v / ird. That Product adsorbed once on the adsorbent are eluted with a 0.1 M aqueous ammonium chloride solution.

The collected eluate fraction, which contains the product, is passed through a column which filled with 20 cm 'weakly acidic quai ion exchanger

where the product is adsorbed. The once adsorbed on the adsorbent product is washed successively with water 100cm * 100cm '0.5% aqueous acetic acid solution and 100 cm ¹ of water, and then eluted with acetone-0.02 N hydrochloric acid (volume ratio 1: 1). The Elua *. concentrate to dryness under reduced pressure and man

receives 28 mg of 3- (N-propionyl) aminopropyl aminobleomycin (900 meg potency / mg against Mycobacterium smegmalis 607). Yield: 26.5%.

In the same way, starting from 100 mg of bleomycin I hydrochloride in 5 ml of methanol, the Synthesize compounds mentioned in Table II.

Tabel II Deployed reaction time Amount of concentration the end No. Used anhydride and l'yridine amount Carboxymethyl of ammonium prey according to its amount networked chloride- Tab. Dextran Eluent 1Λ-1Ε (Hours.) (ml) 0.14 ml 24 23 0.1-m 50.6 mg I. Acetal 49% 0.11 ml 0.24 ml 4th 23 0.1-m 75.5 mg 7th Butyric acid 72% 0.22 ml 0.15 ml 5 ^2/3 20th 0.1-, 0.2-m 56.4 mg 9 Octanoic acid 52% 0.5 ml - 6th 2O ⁺ .) - 38.4 mg Π Lauric acid (Boil) 34% 314 mg 0.1 ml 4th 10 0.1-m 58 mg 16 Succinic acid 53.9% 100 mg 0.1 ml 3 30th 0.1-m 73.4 mg 19th Maleic acid 68% 52 mg 0.05 ml 3 V ₃ 22nd 0.1-m 35.5 mg 24 Benzoic acid 33% 97 mg 0.15 ml 4th 15th 0.2 m 51.1 mg 27 Nicotinic acid 47% 176 mg 0.1 ml 3 24 0.1-m 66 mg 33 Thiophene-2-carboxylic acid 61% 125 mg 0.48 ml 48 23 0.05, 0.1 m 41.7 mg 36 Furan-2-carboxylic acid 39% 620 mg

⁺ ) The 50% volume of ether is added to the reaction liquid. The resulting precipitate is collected by filtration and dissolved in n-butanol. The solution is passed through a column which ^{is filled with 20 cm 3 of} carboxymethyl-crosslinked dextran, the product being adsorbed. Once the product has been adsorbed on the adsorbent, it is eluted with n-butanol

Example 4
3- (N-benzyl) aminopropyl-aniinobleomycin

200 mg of 3-aminopropyl aminobleomycin hydrochloride are dissolved in a mixture of 4 cm 'of water and 12 cm' of ethanol. ^{0.1 cm 1 of} triethylamine are added to the solution, and 73.5 mg of benzyl bromide are added with stirring. The mixture is left to stand at room temperature for 26 hours and at 3 ° C. for a further 48 hours. The reaction liquid is concentrated and then passed through a column ^{filled with a suspension of 50 cm 3 of} carboxymethyl crosslinked dextran in a 0.02 M aqueous ammonium chloride solution, the desired product being adsorbed. Once the product has been adsorbed on the adsorbent, it is eluted with a 0.1 M aqueous ammonium chloride solution. 270 cm ^{J of} the second half of the eluate is collected and passed through a column which is 35 cm ^! weakly acidic calcium ion exchanger is filled, whereby the product is adsorbed.

The product adsorbed once on the adsorbent is ^{washed successively with 175 cm 3} each of water, 0.5% acetic acid and water and then

-, o eluted with 0.02 η-hydrochloric acid methanol (volume ratio 1: 1). 147 cm ^{3 of} the second half of the eluate (total volume: 217 cm ³ ) are collected and neutralized with a weakly basic exchanger. The treated eluate is under reduced pressure

■ 35 concentrated and dried, giving 80 mg of 3- (N-benzyl) aminopropyl aminobleomycin (5833 meg potency / mg against Mycobacterium 607). Yield: 37.2%.

bo example5

3- (N-p-Toluenesulfonyl) aminopropylaminobleomycin

100 mg of S-aminopropyl aminobleomycin hydrochloride are dissolved in a mixture of 2.5 cm ^J water and 4 cm ³ acetone. 20 mg of sodium bicarbonate are added to the solution. Then 28.4 mg of o-toluenesulfonyl chloride are added while stirring.

The mixture is allowed to react at room temperature for 2 ³ / i hours. The reaction liquid is neutralized and passed through a column which has a dispersion of 5 cm ^! strongly basic anion exchanger (Cl type) is filled in water. The resin layer is then washed with 10 cm ^{1 of} water. The resin eluate and the washing liquids are combined and passed through a column which is filled with a suspension of 20 cm ^! Carboxymethyl crosslinked dextran is filled in a 0.02 M aqueous ammonium chloride solution, the desired product being adsorbed. The product, once adsorbed on the adsorbent, is eiuiert with 139 cm ^{1 of} a 0.1 M aqueous ammonium chloride solution. 76 cm ¹

Table III

The / large half of the tractions of the Eluais samine and passed them through a column which ^{is filled with 10 cm 1 of} weakly acidic cation exchanger, the product being adsorbed. The exchange is washed successively with 50% ¹ water, 0.5% acetic acid and water, and eiuiert with acetone -0.02 η-hydrochloric acid (volume ratio 1: 1 3 mg of 3- (Np-toluenesulfonyl-aniinopropyl-aminobleomycin (80 meg Potcnz / m against Mycobactcrium smegmatis 607) obtained. Yield 47%.

The compounds of Table III are synthesized in the same way:

No. Amount of Deployed lot Some Some Deployed Reaction Amount of concentration the end according to Initial solvent of put it put it Base and Time Carboxy of ammonia prey Tab. bleomycins and its The end- Solution Reagent whose methyl- chlorinated IA-IE I. lot gangly middle and its lot networked Eluent blco- and its lot (Hours.) Dextran 50 100 mg Ethanol 3 ml mycins I lot Ethyl- Triethyl 16 15th 0.1-m 19 mg Water 1.5 ml bromide amine 18.6% 15 mg 0.025 ml 55 550 mg Water 10 ml 4 nitro Triethyl 3 V: 70 0.1 m, 0.2 m 90 mg Ethanol 30 ml benzyl amine 30 (50 C) 16.2% bromide 0.25 ml 235 mg 58 200 mg Water 2 ml Methane- Sodium- 10 20th 0.1-m 29.4 mj Acetone 2 ml sulfone bicarbonate 28% acid 40 mg chloride 20 mg 60 200 mg Water 2.5 ml Benzene- Sodium- l ^3/4 35 0.1-m 89.7 m | Acetone 4 ml sulfone bicarbonate 41% acid- 20 mg chloride 34 mg Table III continued No. Some Some Reaction lot Conc the end according to put it sat Time of carb tration prey Tab. Sulfone Sodium- oxy- to Ammo IA- acid bicar methyl- niumchlo- IE chloride bonat- vcrnet.z- rid-elu- and mcnge tem dextran iermittel whose lot (Hours.) (ml)

100 mg water 2 ml 4-chlorine- 20 mg 3 ¹ A

100 mg

Acetone 4 ml

Water 2 ml
Acetone 4 ml

benzene-13.7 mg
Naphthalene-2-17.6 mg

20 mg 17

20th

0.1-m
0.1-m

49.7 rnt 43.9%

34.9 mi 30.5%

Example b

J- (N-phenyl-carba moyl) -ami nopi'opylaminoblcomycin

200 mg 3-aniinopropyl-aminobleomycin-hydrochloric! dissolve in 10 cm 'of water. M) mg of sodium carbonate are added to the solution. Then 0.04 cm ^{1 of} phenyl isocyanate are added dropwise with stirring ebL'ii. Man liil.il the mixture for 7 hours

Room temperature and then for 40 hours at 3 "(react win adjusted to 3 The pH of the reaction liquid wedge, and then is filtered. The Filtra is passed through a column through which mi of a suspension of 20 cm ^J Carboxymcthyl Road network learning dextran in a 0, 02-m aqueous ammonium chloride solution is filled, whereby the desired prodtik is adsorbable. The product adsorbed once on the adsorption section is eluted with a 0.1-N aqueous ammonium chloride solution. 125 cm ^{1 of} the two

Half of the eluate (total volume: 215 cm ^j ) are collected and passed through a column which ^{is filled with 30 cm J} weakly acidic cation exchanger, the product being adsorbed. The product, which has been adsorbed once on the adsorbent, is ^{washed in succession with 150 cm 1 of} water, 0.5% acetic acid and water, and it is cluted with 0.02 η-hydrochloric acid-acetone (volume ratio 1: 1). The eluate is concentrated to dryness under reduced pressure and 90 mg of 3- (N-phenylcarbamoyl) aminopropyl aminobleomycin (3236 meg potency / mg against Mycobacterium smegmatis 607) are obtained. Yield: 41.6%.

The above-mentioned reaction is repeated with the phenyl isocyanal being replaced by 0.06 cm 'of cyclohexyl isocyanal replaced. 40 mg of 3- (N-cyclohexylcarbamoyl) -aminopropy-aminobleomycin (8751 meg potency / mg against Mycobacterium smegmatis 607). Yield: 18.3%.

Example 7

3- (N-Benzimidoyl) aminopropyl aminobleomycin

100 mg of S-aminopropyl-aminobleomycin hydrochloride are dissolved in 25 cm ^{1 of} water. Add 15

20th

25 mg of methyl benzimidate hydrochloride are added with stirring. The pH of the mixture is adjusted to 9.2 by adding triethylamine. The mixture is kept at room temperature for 5 hours. The reaction liquid is concentrated and the concentrate is adjusted to pH 6.2. The treated concentrate is passed through a column which is filled with a suspension of 20 cm ^! Carboxymethyl crosslinked dextran is filled in a 0.02 M aqueous ammonium chloride solution, the desired product being adsorbed. Once the product has been adsorbed on the adsorbent, it is eluted with a 0.1 M aqueous ammonium chloride solution. The eluate fractions which contain the product are collected and ^{adsorbed on 5 cm 3 of} activated carbon. The adsorbed product is washed with water and eluted with 0.02 η-hydrochloric acid-acetone (volume ratio 1: 1). The eluate is concentrated under reduced pressure and then dried. 45 mg of 3- (N-benzimidoyl) aminopropyl aminobleomycin (4423 meg potency / mg against Mycobacterium smegmatis 607) are obtained. Yield: 40.9%.

In the same way the compounds of Table IV are synthesized:

Table IV

No. Amount of Deployed lot Deployed Deployed Deployed Reaction Amount of concentration the end gcmiil.f Initial water of Solution Methylimino base Time Carboxy of ammonia prey Tab. blcomycins lot The end- medium and ether hydro methyl- chlorinated IA-IK I. gangly whose chloride of networked Eluent blco- lot . . . acid and Dextran mycins I its amount (Hours.) 79 600 mg 15 ml Vinegar- Triethylamine 10 50 0.1, 0.25 m 320 mg 90 mg (pH 8.9) 50% 77 100 mg 5 ml Ant Triethylamine 142 20th 0.02, 0.2 m 73.8 mg here: ethyl (pH 9.25) 70.9% iminoather- 164 mg 82 100 mg 2.5 ml Propionic Triethylamine 5 20th 0.02, 0.2 m 53.5 mg 66 mg (pH 9.4) 50.4% 85 100 mg 2.5 ml Valerian Triethylamine 2 20th 0.1-m 35 mg 22 mg (ph 9.4) 32.3% Table IV continued No. Some Some Reak lot Conc the end according to put it sat functional at tration prey Tab. Methyl- base line Carb to Ammo IA-IE imino- oxy- niumchlo- älher-hydro- methyl- rid-elu- chloride of linked iermittel . . . acid and Dextran its amount (SId.)

87 100 mg

100 mg
100 mg

water
2.5 ml
acetone
2 ml

water
5 ml

water
2.5 ml

acetone
I ml

Pclargon 3 mg

4-methoxybenzoic
54.5 mg
4-methylbenzoc-25 mg

Triethylamine 3 20

(pH 9.1)

Triethylamine 6.5 20

(ph 9.3)

Triethylamine 5 20

(pll 8.9)

0.05-, 0.1-m 22 mg 19.7%

0.02-, 0.1-m 76 mg 68.1%

0.1-m

40 mg 35.9%

continuation

No. lot Deployed Some Some Reak lot Conc the end according to of Solution put it sat ions at tration prey Tab. The end- medium and Methyl- liasc Time Carb to Ammo IA-IE gangly whose imino- oxy- niumchlo- bleo- lot ather hydro melhyl- rid-elu- niycins 1 chloride of vcrnelztcni iermittel . . . acid and Dextran its amount (Hours.) (ml)

95 100 mg

97 100 mg

99 200 mg

102 300 mg

105 100 mg

108 100 mg

110 100 mg

water
2.5 ml
acetone
1 ml
water
2.5 ml
acetone
1 ml

water
5 ml

water
7 ml

water

2.5 ml
acetone

1 ml
water

3 ml
acetone

3 ml

water
2.5 ml

3-methylbenzoene-27 mg

3-nitrobenzoene- 30 mg

Phenylacetic- 60 mg

4-chlorophenyl acetic acid-190 mg, cinnamon-60 mg

Triethylamine (pH 9.1)

Trilithylamine (pH 9.2)

Triethylamine 5.5 (pH 9.0)

Trilithylamine (pH 9.1)

Triethylamine (pH 9.1)

Naphthalene-1-triethylamine vinegar (pH 9.1)

105 mg

3- (N ₁ N-Di- triethylamine methylamino- (pH 9.0) propion-88 mg 20
20th

20th
60
20th

20th
20th

0.1-m

0.1-m

0.1-m

50 mg

44.8%

40 mg 35.3%

70 mg 32.4%

0.1-, 0.15-m 100 mg 29.2%

0.02-, 0.02-m 80.9 mg

72.7%

0.02-, 0.3-m 51.6 mg 45.3%

0.1-, 0.2-m 30 mg 27.2%

Example 8 N-chloroacetyl bleomycin As

1 g of Bleomycin As hydrochloride is dissolved in a mixture of 13 cm ^J water and 20 cm ¹ acetone. 520 mg of sodium bicarbonate and 330 mg of chloroacetyl chloride are added to the solution with stirring. The mixture is left to stand for ^{3 hours.} The pH of the mixture is then adjusted to 4.5 by adding hydrochloric acid. Acetone is distilled off under reduced pressure. The residue is diluted with water and passed through a column which cross-links ^{with a suspension of H0cm J carboxymelhyl!} Dextran is filled in a 0.02 M aqueous ammonium chloride solution, whereby the desired product is adsorbed. The product, once adsorbed on the adsorbent, is eluted successively with 220 cm ^{1 of} a 0.1 M aqueous ammonium chloride solution and 580 cm ^{1 of} a 0.2 M aqueous ammonium chloride solution. The fractions containing the product are collected and desalted in the same manner as described above using weakly acidic cation exchanger. The final is concentrated to dryness. The result is 439.6 mg of N-chloroacetyl-bleomycin A * -, (2000 meg potency / mg against Mycobacleriiim smegmalis b07). Yield: 41.7%.

From p-nitrobenzoyl chloride is obtained in the same way the N- (p-nitrobenzoyl-bleomycin A-, (16,000 mcg potency / mg against Mycobacterium smegmatis 607). Yield: 17.7%.

Example 9 N-acetyl-bleomyein Ai

Ig bleomycin avhydrochloride is dissolved in 50 cm ^{1 of} methanol. ^{0.78 cm 1 of} pyridine and 0.58 cm of acetic anhydride are added to the solution with stirring

W and leaves the mixture at room temperature for 5 hours react. The reaction mixture is then concentrated under reduced pressure and the concentrate dissolved in a small amount of water. The solution is passed through a column, which with

- >> a suspension of 110 cm ^{1 of} carboxymethyl crosslinked) dextran is filled in a 0.02 M aqueous ammonium chloride solution, the desired product being adsorbed. The blue adsorption zone, which contains the product, is mixed with a 0.2 m aqueous

Wi ammonium chloride solution eluted. The fluate fractions which contain the product are collected and passed through a column which ^{is filled with a suspension of 70 cm 1 of} activated carbon in water, on which the product is adsorbed. That once on

The product adsorbed by the adsorbent is washed with water and treated with acetone -0.02 n-salic acid (volume ratio 1: I) eluicrt. The flual is concentrated to dryness. There are 552J mg N-Acelylbleoiny-

-hydrochloric! V 27 Amount of Deployed Amount of Deployed 22 20 766 28 the table V synthesized: Amount of Concentration yield In the same way Amount of (1400 meg Initial Solution Initial Ethanol Carboxy tion an A Tabel Initial will the bleomycins medium and bleomysins lot Deployed Reaction methyl- Ammonium- No. bleomycins II whose Il Pyridine Time networked chloride- according to Il Deployed lot lot Dextran Eluent Tab. Methanol Potency / mg against Mycobacterium smegmatis 607). Yield: 53.6%. (ml) IA-IE lot 300 mg Methanol 100 mg 10 ml links 40 0.2, 0.4 m 107.1 mg 200 mg 10 ml (Hours.) 51.5% Deployed 0.13 ml 2.5 30th 0.1-, 0.2-m 117.3 mg 5 200 mg 200 mg Methanol 300 mg 5 ml . . . anhydride 55.9% 10 ml 10 ml and its 0.24 ml 0.75 20th 0.1, 0.3 m 56.4 mg 8th 100 mg 100 mg water lot 51.0% 10 ml 4 ml 0.1 ml 23 10 acetone 30th 0.1-, 0.2-m 100.9 mg 200 mg 5 ml 4 ml Prop ion- 48.1% 100 mg Methanol 0.15 ml 0.17 ml 1 20th 0.1-m 70 mg 15th 100 mg 5 ml Butyr 65.5% 10 ml 200 mg Methanol 0.34 ml 0.1 ml 2.5 17th 10 ml Caprylic 40 0.1, 0.5 m 133.3 mg 250 mg 5 ml 200 mg Methanol acid- 49.8% 10 ml 295 mg 0.12 ml 2.5 20th Crotonic acid Amount of Conc yield 10 ml Continuation of TabcllcV 0.22 ml Carboxy tration No. Amber- Deployed Reaction methyl-ver Ammonium- Table V continued according to acid- Base and line network chloride elu No. Tab. 20 mg whose Dextran iermittel according to IA-IIi Maleic acid lot (ml) Tab. 87 mg 55 0.1-, 0.2-m 83 mg IA-IE (Hours.) 25.3% 37 Deployed - 52 . . . anhydride (37 ^U C) 30th 0.1, 0.5 m 118.5 mg 23 and its 55.2% 34 lot 0.17 ml 1 20th 0.1, 0.3 m 18.7 mg Pyridine 17.7% 25th Sodium- 1.75 Phenoxyacetic bicarbonate 39 acid- 50 mg 20th 0.1, 0.5 m 39.7 mg 1.13 g 35.1% Benzoic acid Pyridine 5.5 40 0.1, 0.3 m 137.3 mg 323 mg 0.06 ml (65 ⁰ C) 63.9% 12th 4 nitro Pyridine 4th 40 0.1, 0.3 m 120.0 mg benzoc 0.23 ml 55.2% 28 acid- Pyridine 6th 77 mg 0.23 ml 40 Lauric acid Amount of Concentration yield 191 mg Carboxy tion Nicotinic acid Sat down Reaction methyl- Ammonium- 236 mg l'yridine Time networkedcm chloride- Isonicotin lot Dextran Eluent acid- (ml) 236 mg 22nd 0.1, 0.3 m 56.5 mg (Hours.) 52.9% Deployed 0.3 ml 28 . . . anhydride 55 0.1, 0.5 m 233.1 mg and its 72.0% lot 0.24 ml 4.5 Furan-2-car- bonsiiure- 368 mg Thiophene-2- carbonsüurc- 307 mc

continuation

No. Amount of Deployed: Deployed Deployed Reaction Minge on Concentration yield according to Initial Methanol . . . unhvürid Pyridine Time Carboxy lion on Tab. bleomvsins • ■ engc and its lot meihyl- Ammonium- IA-IE II lot vcrnctztcm chloride- Dextran Eluent (Hours.) (ml)

42 200 mg 10 ml phthalic acid- 0.17 ml

146 mg
44 200 mg 10 ml 4-nitro 0.21 ml

phthalic acid

Example 10
N-butyryl-bleoniycin A5

100 g bleomycin As-hydrochloride is dissolved in a mixture of 3 cm ¹ acetone and 2 cm ^J water. 12 mg of i-ethyl-3-dimethylaminopropyl-carbodiimide hydrochloride and 8 mg of butyric acid are added to the solution. The pH of the mixture is adjusted to 4.7 and the mixture is then left to stand for 16 hours. The reaction liquid is concentrated to about 1/3 of its original volume under reduced pressure. 10 cm * of water is added to the concentrate. The mixture is passed through a column through which is filled with a suspension of 20cm ^J carboxymethyl dextran cross-linked in a 0.02 m aqueous solution of ammonium chloride, the product being adsorbed. Once the product has been adsorbed on the adsorbent, it is eluted with a 0.1 M aqueous ammonium chloride solution.

The fractions which contain the product are collected and passed through a column which ^{is filled with 20 cm 3 of} weakly acidic cation exchanger, the desired product being adsorbed. The product adsorbed once on the adsorbent is ^{washed successively with 100 cm 3 of} water, 100 cm ^{3 of} 5% acetic acid, and 100 cm ^{3 of} water and then eluted with acetone -0.02 N hydrochloric acid (volume ratio 1: 1). The eluate is concentrated to dryness. This gives 32 mg of N-butyrylbleomycin A5 (3900 meg potency / mg against Mycobacterium smegmatis 607).

1.5 35 0.05, 0.1 m 133.7 mj

60.5% 3.5 50 0.1-m 117.2 mj

51.5%

Example 11 N- (p-nitrobenzyl) bleomycin A ^

As to 1 g bleomycin hydrochloride was dissolved in a mixture of 20 cm 'of water and 60 cm ^s ethanol. 0.58 cm ^{3 of} triethylamine are added to the solution and 600 mg of p-nitrobenzyl bromide are added with stirring. The mixture is allowed to react at room temperature for 35 hours.

The reaction liquid is concentrated and insoluble substances are filtered off removed. Pass the concentrated reaction liquid

JO is then passed through a column which ^{is filled with a suspension of 150 cm 1 of} carboxylmethyl crosslinked dextran in water, the product being adsorbed. 35 cm ^{3 of} the first run of the elual fractions are discarded and 27 cm ^{3 of} the latter part of the fractions are passed through a column which is filled with 50 cm carboxymethyl crosslinked dextrate, the product being adsorbed. Since the product has been adsorbed once on the adsorbent, it is eluted successively with an aqueous ammonium chloride solution and a 0.25 M aqueous ammonium chloride solution. The eluate fractions, which contain the product, are collected and desalinated with activated charcoal. The resulting solution is concentrated and 120 mg of N- (p-nitrobenzyl) bleomycin As hydrochloride (6950 meg potency / mg of Mycobacterium smegmatis 607) are obtained. Yield: 11%.

The compounds of Table VI are synthesized in the same way:

Tabel VI Deployed
solvent
and its
lot Deployed
. . . bromide
and its
lot Deployed
Triethyl
amine amount Reaction
Time Amount of
Carboxy
methyl-
networked
Dextran concentration
to Ammo
nium chloride
Eluent the end
prey No.
according to
Tab.
IA-IE Amount of
Initial
bleomycins
II (Hours.) (ml) Ethanol
4 ml
water
1.6 ml Ethyl-
60 mg 75 mg 16 25th 0.1-m,
0.25-m 70 mg
34% 52 200 mg Ethanol
4 ml
water
1.6 ml Ethyl-
27 mg 50 mg 8th 25th 0.1-m,
0.25-m 84 mg
41.5% 51 200 mg

Example N- (methylsulfonyl) bleomycin A -,

100 g of bleomycin A ^ hydrochloride are dissolved in a mixture of 4 cm ^{1 of} acetone and 8 cm ^{1 of} distilled water. 160 mg of sodium bicarbonate are added to the solution and 104 mg of methanesulfonyl chloride are added dropwise with stirring. The mixture is allowed to react at room temperature for 19 hours. The reaction liquid is neutralized and then passed through a column which ^{is filled with a suspension of 5 cm 3 of} strongly basic anion exchange ^ (Cl type) in distilled water, whereby the product is adsorbed. That at the adsorption center! adsorbed product is washed with 10% of distilled water. The exchange eluate and the washing liquids are combined and the mixture is passed through a column which ^{is filled with a suspension of 20 cm 3 of} carboxymethyl crosslinked dextran in a 0.02 M aqueous ammonium chloride solution, the desired product being adsorbed. The product, once adsorbed on the adsorbent, is eluted successively with 200 cm ^{3 of} a 0.1 M aqueous ammonium chloride solution and 78 cm ^{3 of} a 0.2 M aqueous ammonium chloride solution. The eluate fractions containing the product are collected and passed through a layer of 5 cm ^{3 of} weakly acidic cation exchanger, the product being adsorbed. The exchange layer is washed successively with 25 cm ^{3 of} water, 0.5% acetic acid, and water, and the product is then eluted with acetone-0.02 N-acid (volume ratio 1: 1). The filuate is concentrated to dryness. This gives 16.2 mg of N- (methylsulfonyl) bleomycin A5 hydrochloride (1670 meg potency / mg against Mycobacterium smegmatis 607). Yield: 15.3%.

In the same way the compounds of Table VII are synthesized:

Table VII

No. amount of

according to initial

Tab. Bleomycins

IA-IE II

Used Reaction solvent used. . . sulfone sodium time and its acid chloride bicarbonate amount and its amount amount

(Hours.)

Amount of concentration yield

Carboxy to Animo-

methyl nium chloride

cross-linked eluant
Dextran

(ml)

200 mg

64 200 mg

200 mg

70 200 mg

water
3 ml
acetone
4 ml Benzoin
34.6 mg 20 mg 19th 13th 30th 0.1-, 0.2-m 27.6 mg
12.6% water
4 ml
acetone
4 ml ToI uol-
81.7 mg 60 mg 22nd 20th 0.1, 0.3 tn 19.5 mg
8.8% water
3.5 ml
acetone
4 ml 4-chlorine
benzoin
45 mg 40 mg 24 40 0.1-, 0.2-m 75.9 mg
33.8% water
3 ml
acetone
4 ml Naphtha-
lin-2-
47 mg 40 mg 4.25 50 0.1-, 0.2-m 47.1 mg
20.7% example

N- (ethylcarbamoyl) bleomycin A5

200 g of bleomycin Aj hydrochloride are dissolved in 10 cm ^{3 of} water. 60 mg of sodium bicarbonate are added to the solution and 0.1 cm ^{3 of} ethyl isocyanate are added dropwise with stirring. The mixture is allowed to react at room temperature for 4½ hours. The reaction liquid is filtered and the pH of the filtrate is adjusted to 4. The filtrate is then passed through a column which is filled with 30 cm carboxymethyl crosslinked dextran, the product being adsorbed. The product, once adsorbed on the adsorbent, is eluted successively with 0.1 M aqueous ammonium chloride and 0.25 M aqueous ammonium chloride. The eluate fractions containing the product are collected and desalted with activated charcoal. The solutions thus obtained are concentrated and 100 mg of N- (ethylcarbamoyl) bleomycin A ₅ hydrochloride (2388 meg potency / mg against Mycobacterium smegmatis 607) are obtained. Yield: 28.6%.

In the same way as described above, the compounds of Table VIII are synthesized:

33

Table VIII

No. Amount of Deployed Deployed Deployed Reaction Amount of concentration the end according to Initial Amount of water . . . ISO Nalrium Time Carboxy to Ammo prey Tab. bleomycins cyanal unü bicarbonate methyl- nium chloride IA-IE Il whose lot networked Eluent lot Dextran (Hours.) (ml)

mg
mg

2 ml 10 ml

Phenyl-0.08 ml

Cyclohexyl 0.05 ml mg 6
mg I '

50
30th

0.01-,
0.15-m
0.01-,
0.25-m

80 mg

15%

60 mg

27.5%

Example N- (acetimidoyl) bleomycin A;

mg of bleomycin A ₅ hydrochloride are dissolved in 15 cm ^{1 of} water. 90 mg of methyl acetate imidate hydrochloride are added to the solution. The pH of the mixture is adjusted to 8.8 by adding triethylamine. The mixture is stirred for 4 hours at room temperature and then concentrated to Vj of its original volume. The pH of the concentrate is adjusted to 7.

The reaction liquid is passed through a column through which cross-linked with 60cm ^J Carboxymethyl Dextran is filled, wherein the desired product is adsorbed. The product, once adsorbed on the adsorbent, is eluted with 417 cm ^{s of} a 0.3 M aqueous ammonium chloride solution, and 320 cm ^{1 of} the second half of the eluate is passed through a column which contains 40 cm ^{1 of} activated carbon. The carbon layer is washed with water and eluted with acetone-0.02 η-hydrochloric acid (volume ratio 1: 1). The eluate is concentrated and 410 mg of N- (methylacetimidoyl) bleomycin As hydrochloride (3100 meg potency / mg against Mycobacterium smegmatis 607) are obtained. Yield: 65%.

In the same way the compounds of Table IX are synthesized:

Table IX

No. Amount of Deployed Deployed Deployed Reaction Amount of Concentration training according to Initial Solution Methylimino base Time Carboxy of ammo prey Tab. bleomycins medium and ether hydro methyl- nium chloride IA-IE II whose chloride- der networked Eluent lot . .. acid and Dextran its amount (Hours.) (ml)

mg
mg
mg
mg

200 mg
100 mg

100 mg

Water 2.5 ml water 2.5 ml water 2.5 ml water 2.5 ml Acetone 2 ml

Water 5 ml water 5 ml

Water 2.5 ml acetone 1 ml

Propion-44 mg

Isobutter

Valerian-21 mg Pelargon-30 mg

Benzoin-50 mg

4-methoxy-benzoin- 54.4 mg 4-methylbenzoene-50 mg

Triethylamine
(pH 9.1) 6.25 15th Triethylamine
(PH 9.4) 6.75 15th Triethylamine
(pH 9.4) 2 20th Triethylamine
(pH 9.0) 2 20th Triethylamine
(pH 9.1) 7th 30th Triethylamine
(pH 9.4) 6.5 15th Triethylamine
(pH 9.1) 4th 20th

0.25-m 70 mg
66% 0.25-m 60 mg
56% 0.25-m 75 mg
75% 0.25-m 52 mg
46.6% 0.1, 0.4 m 130 mg
62.5% 0.25-m 80 mg
71.7% 0.25-m 65 mg
65%

35

continuation

No. Amount of Deployed Deployed Deployed Reaction Amount of concentration the end according to Initial Solution Methylimino base Time Carboxy to Ammo prey Tab. bleomycins medium and üther-llydro- methyl- nium chloride IA-IE Il whose chloride- der networked Fluids lot . . acid and Dextran its amount (Hours.) (ml)

100 mg

100 mg

200 mg

100 mg

100 mg

100 mg

100 mg

100 mg

100 mg

Water 2.5 ml acetone

1 ml

Water 2.5 ml

Water 5 ml of water

2 ml acetone 2 ml water 2.5 ml acetone 2.5 ml water 2.5 ml acetone 1.5 ml

Water 5 ml acetone 5 ml

Water 5 ml

Water 2.5 ml

3-methylbenzoene-27 mg

3-Nitrobenzoe- 32 mg Phenylessig-60 mg 4-Chloiphenylessig-60 mg

4-nitrophenyl acetic acid 137 mg

Cinnamon-60 mg

Triethylamine (pH 9.1)

Triethylamine (pH 9.0)

Triethylamine (pH 9.0)

Triethylamine 3.5 (pH 9.3)

Triethylamine (pH 9.3)

Triethylamine (pH 8.8)

Naphthalene triethylamine 9 ² h 1-vinegar (pH 9.5)

105 mg

3- (N ₅ N -diethylamine

methylamino) - (pH 9.2)

propion

240 mg

Ant triethylamine

(here ethyl (pH 9.2)

imino ether)

82 mg 20

15th

20th
28

20th
20th
14th
40
15th

0.25-m 53 mg
47% 0.1, 0.25 m 60 mg
53.8% 0.25-m 100 mg
45.3% 0.25-m 70 mg
61.9% 0.25-m 60 mg
50.9% 0.25-m 70 mg
62.6% 0.25-m 80 mg
70.2% 0.25, 0.5 m 90 mg
41.4% 0.25-m 30 mg
28.8%

example

S-iN-methyl-N-succinylaminopropyl I-aminopropylaminobleomycin

mg of 3- (N-methyl-N-3-aminopropyl) aminopropyl aminobleomycin hydrochloride are dissolved in 10 cm ^J methanol. ^{0.14 cm 3 of} pyridine is added to the solution and 85 mg of succinic anhydride are added with stirring. The mixture is stirred at room temperature for 2.5 hours, and the reaction liquid is concentrated under reduced pressure. The residue is dissolved in water and the solution is passed through a column which ^{is filled with a suspension of 40 cm J} carboxymethyl crosslinked dextran in a 0.02 m aqueous ammonium chloride solution, the product being adsorbed. The product, once adsorbed on the adsorbent, is eluted successively with 0.1 m and 0.2 m aqueous ammonium chloride solutions. The eluate fractions containing the desired product are collected and passed through a column which ^{is filled with 30 cm 3 of} weakly acidic cation exchanger, whereby the product is adsorbed. The product adsorbed once on the adsorbent is ^{washed successively with 150 cm J} water, 0.5% acetic acid and water, and eluted with 0.02 N hydrochloric acid - acetone (volume ratio 1: 1). The eluate is concentrated to dryness under reduced pressure. This gives 166.9 mg of 3- (N-methyl-N-3-succinylaminopropylj-arninopropyl-arninobloemycin (90 meg potency / mg against Mycobacterium smegmatis 607). Yield: 77.9%.

In the same way the compounds of Table X are synthesized: ^

38

Table X

No. amount of

according to initial

Tab. Bleomycins

IA-IE III

Deployed Deployed Deployed Response

Methanol. . . anhydride pyridine time

amount and its amount

lot

(Hrs.) Amount of

Carboxy

methyl-

networked

Dextran

(ml)

Focus on
Ammonium chloride
Eluent

yield

200 mg
100 mg
100 mg
100 mg
200 mg
100 mg
200 mg

45 200 mg

200 mg
200 mg
100 mg
100 mg

10 ml
5 ml
5 ml
5 ml

10 ml
5 ml

10 ml

10 ml

10 ml

10 ml

5 ml

5 ml

Acet-0.12 ml

Propion-0.12 ml

Lauric acid-270 mg Maleic acid-70 mg

Benzoic acid-320 mg

Nicotinic Acid 175 mg

Thiophene-2-carboxylic acid-340 mg

4 nitro

phthalic acid

275 mg

Phthalic acid

210 mg

Glutaric acid

100 mg

Methyl amber

stinic acid

Itaconic acid

0.23 ml 0.15 ml 0.11 ml 0.28 ml 0.23 ml 0.15 ml 0.35 ml

0.23 ml

0.23 ml 0.14 ml 0.1 ml 0.1 ml

Example 16

3- (N-methyl-N-benzenesulfonylaminopropyl) aminopropyl aminobleomycin

100 mg of 3- (N-methyl-N-3-aminopropyl) aminopropyl-aminobleomycin hydrochloride dissolves one in a mixture of 2 cm to ¹ cm and 7.5 ^J acetone water. 200 mg of sodium bicarbonate and 47 mg of benzene sulfonyl chloride are added to the solution with stirring, and the mixture is allowed to react for 24 hours at room temperature. The reaction liquid is cut off and passed through a column which ^{is filled with a suspension of 5 cm 1 of} strongly basic anion exchanger (Cl type). The resin layer is ^{washed with 10 cm 1 of} water and the oil and the washing liquids are combined with one another. The mixture is passed through a column which is cross-linked with a suspension of 20 cm ^{1 of} carboxymethyl dextran in a

3.5 3 2.5 (reflux)

2.5 2.5 3.5 2.5 39
20th
20th
20th
40
20th
30th

35

35
40
20th
20th

0.1-,
0.2 m

0.1-.
0.2 m

0.1-,
0.2 m
0.1-m

0.1-,
0.2 m

0.1-,
0.2 m

0.1-,
0.2 m

0.1 -m

0.1-m

0.1-,
0.2 m

0.1-,
0.2 m

0.1-,
0.2 m

165.5 mg 80.3%

81.8 mg

78.7%

67.7 mg 60% 75 mg 65.5% 167 mg 77.8%

88.1 mg 82.6% 150 mg 70%

121.9 mg 53.6%

165.2 mg

74.7%

140 mg 64.8% 63 mg

58.3%

52 mg 47.8%

0.02 m aqueous ammonium chloride solution is filled with the product being adsorbed. The product adsorbed once on the adsorbent is eluted successively with 63 cm ^{1 of} a 0.1 M aqueous ammonium chloride solution and 131 cm ^{1 of} a 0.2 M aqueous ammonium chloride solution. 65 cm 'of the second half of the 0.2 m aqueous ammonium chloride compound is obtained and passed through a layer of 10 cm' weakly acidic cation exchange, whereby the product is adsorbed. The product adsorbed on the adsorbent is ^{washed successively with 50 cm 1 of} water, 0.5% acetic acid and water, and then eluted with acetone -0.02 η-hydrochloric acid (volume ratio 1:. The eluate is concentrated to dryness under reduced pressure this gives 60.9 mg of 3- (N-methyl-N-benzenesulfonylaminopropyl / aminopropyl-aminoblocmycin (6700 meg potency / mg against Mycobacteriuiii smegmutis 607). Yield: 55.4%.

The compounds of Table Xl are synthesized in the same way:

40

Table X!

No. Amount of Deployed Deployed Deployed Reaction Amount of Concentration yield according to Initial Solution Reagent and Base and Time Carboxy traticin Tab. blcomycins medium and its amount its ver methyl-ver Ammonium- 1A-1E III whose turned network chloride- lot lot Dextran Eluent (Hours.) (ml)

32 1.5 g acetone chloroacetyl 5.8 g 13 ml sodium chloride water 3 g bicarbonate 145 ml

68 300 mg acetone 4-chlorine- 60 mg 10 ml benzene-sodium-water sulfonic acid bicarbonate 3 ml chloride 45 mg

65 100 mg acetone p-toluene- 120 mg

2 m! sulfonic acid- sodium-water chloride bicarbonate

3 ml 25.2 mg

57 Ig ethanol 4-nitro- 0.58 ml

60 ml of benzyl triethylamine

Water bromide

20 ml 600 mg

53 200 mg ethanol allyl bromide 70 mg

4 ml of 60 mg of triethylamine water

16 ml

Example 17

Synthesis of S-N-methyl-N-S-phenylacetimidoylaminopropyl) -aminopropyl-aminobleomycin

200 mg of 3- (N-methyl-N-3-aminopropyl) aminopropylaminobleomycin hydrochloride are dissolved in 5 cm ^{3 of} water. 60 mg of methyl phenyl octimidate hydrochloride are added to the solution with stirring. The pH of the mixture is adjusted to 9.1 by adding triethylamine and the mixture is left to react for 26 hours at room temperature. The reaction liquid is diluted with water and passed through a column which cross-links ^{with a suspension of 25 cm 3 of carboxymethyl!} Dextran is filled in a 0.02 M aqueous ammonium chloride solution, whereby the desired product is adsorbed. Once the product has been adsorbed on the adsorbent, it is eluted one after the other

Table XII

250

43

20th

150

25th

0.1-,
0.2-,
0.3 m

0.1-,
0.2 m

0.1-,
0.2 m

0.1-,
0.2 m

0.1-,
0.2 m

477 mg 30.2%

220.3 mg 65%

49.2 mg

43.6%

132 mg

12%

77 mg

37.4%

cm ^J 0.1 M aqueous ammonium chloride, 100 cm ³ 0.2 M aqueous ammonium chloride and 260 cm ³ 0.3 M aqueous ammonium chloride. 90 cmj of the last 0.3 M aqueous ammonium chloride eluate fraction is passed through a column which ^{is filled with 30 cm 3 of} weakly acidic cation exchanger, whereby the product is adsorbed. That once on the adsorbent

4 «adsorbed product is washed successively with cm ^{3 of} water, 0.5% acetic acid and water, and then eluted with 0.02 N hydrochloric acid-acetone (volume ratio 1: 1). The eluate is concentrated to dryness under reduced pressure. This gives 170.8 mg of 3- (N-methyl-N-phenylacetimidoyl) -aminopropyl-aminopropyl-aminobleomycin (12,750 mcg potency / mg against Mycobacterium smegmatis 607). Yield: 78.9%.

The compounds of Table XII are synthesized in the same way:

Amount of used Reaction used Amount of concentration

yield

according to the initial solution tab. Neomycins medium and
ΙΛ-ΙΕ III of that

lot

Methyliminoilhcr hydrochloride the . . . acidity and amount of water

base

times carboxy- tration

methyl ammonium

cross-linked chloride

Dcxtran Eluicrmittcl

(Hrs.) (Ml)

81 200 mg

104 200 mg

water
5 ml

acetone
4 ml
water
4 ml

Vinegar-30 mg

4-chlorine

phcnyl-

cssig-

Triethylamine 4 25

(pH 8.8)

Triethylamine 4.5 30

(pH 9.1) 0.1-,
0.2-,
0.3 m

0.1-,
0.2-,
0.3 m

144 mg

70%

£ 193.4m

87%

Example 18

S-iN-methyl-N ^ -cyclohexylcarbamoylaminopropylj-aminopropyl-aminobleomycin

200 mg of 3- (N-methyl-N-3-aminopropyl) aminopropyl aminoblcomycin hydrochloride are dissolved in 10 cm ^{1 of} water. 60 mg of sodium carbonate and 0.05 cm of cyclohexyl isoeyanal are added to the solution with stirring. The mixture is allowed to react for 2 hours. The reaction fluid is nitrated. The pH of the filirate is adjusted to 5.0. The filtrate is diluted with water and passed through a column which is filled with a suspension of 30 cm ^! Cross-link carboxymethyl! Dextran is filled in 0.02 M aqueous ammonium chloride, the product being adsorbed. The adsorbed product

they are eluted successively with 0.1 M aqueous ammonium chloride and 0.25M aqueous ammonium chloride. The eluate fractions containing the product are collected and passed through a column which is filled with 30 cm 'weakly acidic cation exchanger is filled, whereby the desired product is adsorbed. The adsorbed on the adsorbent Product is successively with 150 cm 'of water. 0.5% igcr acetic acid and water, and then washed with 0.02 N-hydrochloric acid acetone (volume ratio 1: 1) eluted. The eluate is concentrated under reduced Pressure to dryness. 88 mg of 3- (N-methyl-NO-cyclohexylcarbamoylaminopropylJ-uminopropyl-aminobleomycin result (Il 500 meg potency / mg against Mycobacterium smegniaiis 607). Yield: 40.4%.

In addition 7 sheets of drawings

Claims (1)

Patent claims: 1. Bleomycins of the general formula 9 NH HO —- / ~~ "—- O -—" W
/ HO / P-Q OH OH O NH ₂ in which P and Q have the following meanings: P = -NH- (CH ₂ J ₃ -NH- —NH- (CH ₂ J ₃ -NH- (CH ₂ J ₄ -NH-— NH— (CH ₂ ) ₃ —N— (CH ₂ J ₃ -NH- CH ₃
Q = -CR ¹ with the following meanings of R ¹ : Methyl, ethyl, n-propyl, n-heptyl, n-undecyl, chloromethyl, propenyl, phenyl, 4-nitrophenyl, phenoxy methyl, 3-pyridyl, 4-pyridyl, 2-furyl, 2-thienyl, or
Q = - C- (CH ₂ ) "- COOH with η = 2 and η = 3
or Q = -C-CH = CH-COOH O 40 45 50 60 or COOH or Q = —R ² with the following meanings of R ² : ethyl, allyl, benzyl, 4-nitrobenzyl, or Q = - SO ₂ R ³ with the following meanings of R ³ : Methyl, phenyl, 4-methylphenyl, 4-chlorophenyl, 2-naphthyl, or Q = —C — NH-R ⁴ Il ο with the following meanings of R ⁴ : Ethyl, cyclohexyl, phenyl,
or Q = -CR ⁵
NH with the following meanings of R ⁵ : Hydrogen, methyl, ethyl, isopropyl, η-butyl, n-octyl, 2- (N, N-dimethylamino) ethyl, Phenyl, 4-methylphenyl, 4-methoxyphenyl, 4-nitrophenyl, 3-methylphenyl, 3-nitrophenyl, benzyl, 4-chlorobenzyl, 4-nitrobenzyl, styryl, 1-naphthylmethyl.