FI73131B - ANALYSIS OF ALKALOIDDERIVAT OCH SALTER DAERAV VID FRAMSTAELLNING AV DIAGNOSTISKA PREPARAT. - Google Patents

Description

1 73131

Use of alkaloid derivatives and their salts in the manufacture of diagnostic preparations

The invention relates to the use of alkaloid derivatives as defined in claim 1 and their salts for the preparation of diagnostic preparations, in particular preparations for the diagnosis of tumors.

A process for the preparation of thiophosphoric acid salts of alkaloid derivatives which have a recognized pharmacological cytostatic effect and which have been proposed for use as water-soluble compounds is known from the prior art. Suitable water-soluble alkaloid salts are berberine, sanguinarine and salts of yellow alkaloids and salts of bisbenzylisoquinoline alkaloids, for example: curine, fangoquinoline, tetrandine, pendulin, thalidazine, aporphin-benzyl-isoquinoline, alkali-alkali-alkali-alkali-alkali. , e.g. 20-hydroxy-voacamidine, indole-indoline alkaloids, e.g. leoricidin, lurosine, vincaleucoblastine, leurocristine, tropo-20 Ion alkaloids, e.g. colchicine, isoquinoline alkaloids, e.g. liriodenine, o-methylteroline oxopurpurine, chelidonine, protpine, stylopine, allocryptopine, copticin, chelerythrine, chorysamine, chelidimerin, homokelidonine, methoxycelidonine, kelilulitin, kelirubin, narcicazine-25, talicarpine, Pakistanin, pasistanamine , sanguinarine, caffeine, nitydine, pheasantan, steroid alkaloids, indole-iso-quinoline alkaloids, e.g. 9-methoxy thicine, ellipticine, indole alkaloids, e.g. reserpine, quinoline-indo-lycidine alkaloids, e.g. compotesin, pyroline alkaloids, e.g. tatrophan, pyrolizidine alkaloids, e.g. heliotrine, acrinodial alkaloids, e.g. melicopine, acromycin, normelioepidine, phenantroindolizidine alkaloids, e.g. tyloferin, tylocrebine, imidazole alkaloids, e.g. pilocarpine, quinolizidine alkaloids, e.g. matrix, oxymarin, cryptoleurin, quinazolone alkaloids, e.g.

2,731 31 febrifugin, benzazepine alkaloids, e.g., cephalotaxine, deoxyharringtonine, homoharringtonine, harringtonine, and others.

Also of interest in this connection are the known thiophosphorus derivatives of the free base alkaloids. Examples of such known derivatives are thiophosphoric acid di (ethyleneimido) -N-berberinol ethylamide, thiophosphoric acid tri (N-sangvinarinol) ethylamide and thiophosphoric acid of all alkaloids comprising the condensed isoquinoline system of the yellowing plant.

As is known, these known compounds with cytostatic activity are sparingly soluble in water, so that it is preferable to use organic solvents for the same purpose, especially in the preparation of injection solutions, instead of using an aqueous solvent.

The desired aqueous solubility is then achieved by losing the active ingredients without losing their cytostatic effect or undesired side effects, by converting the active ingredients into salts with pharmaceutically acceptable acids by reacting the alkaloid in question, which may itself be carcinostatically active. or antibiotics or other nitrogen- or phosphorus-containing organic compounds, and the product obtained is optionally converted into a pharmaceutically acceptable salt, with the exception of the bases of the alkaloids of the condensed isoquinoline system of the yellow mill. -30 ethylamine and N, N ', N "-triethylenethiophosphoramide derivatives. The above-mentioned compounds have proved to be particularly suitable as alkaloid components.

Suitable as carcinostatic agents for use in the reaction are in particular: 3,731 31 CH, -Nil CH.CII.CI ^ Η, -, Ιη CH.CB.CI "* 9- \ _ A" · ° CU.

- / - i I «V-«> “C-.CN. ^ T" '-.

CHj-O CH ^ HjCl CU, - O CH.CK.CI '' / \ H «.C —CH, H, c— CH, c txxxixj (XL) 5 fLIV) (LV) oo" .CHt i CH- CHtCH, OC NH - P - N 2 -. ('') - CH-OCNH - P - | *

II 1 CH \ -_ / ^ Il I

10 is * · '-' a n c / N. / \ H? C— CH, HtC — CH 2 (LVI) (LVII) ° ^ -CH, n Cl ·), 15 (7-nh-p-nJ> -? - <| n CHt · CH, —CH . ^ CH, / \ / \

HjC “CHt H, C —CH, (.evin) (cnc> 20 CH ^ CH. ° ^ Η '^" * CHC- CH-v M .CH, ° V Mp- <! I NPW> -PN | CHt -CH 2 J, CH, H, c J, ^ CH-CH 2 J, "cH, / \ / \ / \ H» C - CH, H 2 C - CH 2 H 2 C - CH 2 (LX) (LV.J ) 25 H / ΓΛ n CH,

I N-P-O— (N) —O-P— N

H.C "1 \ - / I e» * N - N "»> \ / \

HjC-CH, HtC-CH, (LXD) 30 H 2 Cl 2 CH, CH 2 -CH, || „CH, | K-P-Nj N— P - N | · "CH, C ^ -CH," 'CH.

/ \ '\ H, C — CH, \ C— CH, 35 LXIII) (UXJV) 4 731 31 CH, - C' Vu-ΓΗ ^ · Crt- CH - CM> N '/ \ 5 M'C "CH * ILXV1

/ ~ * \ x7 Y Ύ V

10 \ O ^ -NiCHaCH2CH2.H2O | vX / ^ N-iU <T

vo ^ «VV Ö N sws ^ C7 / N <] C.HjCOWM-BN ^ l>« - 5 - * <l HfV ™ ° N <is ^ 15 nitrogen mustard gas, cyclophosphamide, triamcinone, chlorambucil, busulfan, 20 H2C “CH2 H2C ^ ^ H2 Γ-Λ ^ / CH2CH2C1

-P- S GHCH - {V — N

u I I \ = / \

2 N COOH CHOCH_CL

/ \ Z 2 25 H2C -CH2 nitomine, mannitol nitrogen mustard gas, amethopterin, 6-mercaptopurine, 5-fluorouracil, cytosine arabinoside, podophylline, actinomycin C, actinomycin D, mitramycin, mitomycin C, adriamycin, asparaginase, benzomethicin, N (CH-.) -, / T ~ \ XCH3 I 3 "

H.C-NH-NH-CH

(ch3) 2h-J ^ VA.n (ch3) 2 5 73131 ft ΓΗ 0

(C1CH2CH2) 2 ^ Y ^ NH (C1CH2CH2) 2Ny ^ N F NH

3 H

5

Cl / - \ / —W- \

UnAn <] ClCH2CH2-B ^ Ä ^ y ^ J, -CH2CH2Cl2Cl-10 [> N N ^ N <] ch2ch2ci V 'CH7CHCl1 - N Y. HCl n ^ n 2 2 \ Y 'CH2CH2C1

OF

15 Δ

SH? H 0H

N -NH N jj N ^

^ N ^ ^ N ^ H2 ° hcY ^ N ^ HCK ^ N ^ CO2H

20 (XXI) (XXIII) nh2 n <Y \ Yf .conh

I T / NY

where R = H or (XDCV) CH3

R

Salt formation can be performed with virtually any pharmaceutically acceptable acid that is sufficiently water soluble and thus produces sufficiently water soluble salts; for economic reasons, hydrochloric acid is preferably used to give the corresponding hydrochlorides.

6,731 31

The alkaloid thiophosphoric acid amide salts do not differ from their bases in their cytostatic activity and thus in their pharmacological activity; however, due to their substantially better water solubility, they are easier to dispense accurately and, until now, they do not necessarily have the detrimental side effects caused by the necessary organic solvents.

Suitable alkaloid salts are also the salts of berberine, sanguinarine and, in addition, yellow alkaloids and other alkaloids (see also the structural formulas in Figures 1-17), in particular the hydrochlorides.

The reaction of the alkaloid salts with the cytostatic agents is conveniently carried out in a solvent or solvent mixture at elevated temperature; however, the alkaloid base can first be reacted with a thiphosphoric acid amide and then the reaction product converted to a salt. The reaction of the reaction product of the alkaloid compound and the cytostat with the desired acid is conveniently carried out in an organic solvent, whereby after salt formation, the salt in question precipitates or is extracted with water or an aqueous acid into an aqueous solution.

The preparation composition, for example a preparation consisting of Chelidonium Majus L. alkaloids, is based on reaction products between alkaloids and alkylating agents such as thiophosphoric acid triaziridide 25 (ti-TEPA).

This compound has three reactive groups that are either attached to alkaloid molecules or are substituted with and OH or NH 2 groups. In this way, when this compound is combined with a single pure alkaloid, a wide variety of reaction products can be formed. To study simpler reactions in series of experiments, thio-TEPA, cyclophosphoramide, and other organic compounds were added to the individual alkaloids. In the case of chelidonine, at least 12 could be detected by thin layer chromatography

II

7,731 31 reaction products. To obtain an accurate analysis of these reaction products, some of them were isolated chromatographically and crystallized.

The chemical formulas obtained from elemental analyzes of these components are shown as possible molecular structure formulas in Figures 1-17.

When thio-TEPA is added to a mixture containing different alkaloids, it is possible that the molecules of the three different alkaloids are linked together via the thio-TEPA molecule, resulting in a very large number of different components in the synthesis. This highly complex mixture of reaction products contains some that can be considered to cause the unique biological activity obtained with the mixture.

In fact, separation of the reaction products of the alkaloid extract of Chelidonium Majus L. and thio-TEPA by two-dimensional thin layer chromatography gave more than 50 spots fluorescing under UV light.

In addition, it could be shown that the Ukrainian preparation 20 (in which case the reaction was carried out between the alkaloids Chelidonium Majus L. and thiophosphoric acid triaziridide, usually called thio-TEPA) did not contain any free ethylene groups of thio-TEPA.

This was demonstrated as follows: 1. by chemical analysis, and 2. so that in a biological test, the lethal dose of thio-TEPA is 1 mg / kg body weight, but the dose of the preparation is 250 mg / kg body weight and is not toxic.

In addition, the product has no effect on leukemia-30 mia 1210 or hematology. From this it can be concluded that the ethylene groups of thio-TEPA, which also account for its toxicity, are blocked by alkaloids.

The figures show the chemical structure of the 17 different compounds mentioned above, obtained completely by analyzing some of the reaction products obtained from the reaction of thio-TEPA and cyclophosphamide with pure alkaloids.

8,731 31

Figure 1: Kelilutin + thiophosphoric acid triaziridide (1-kelilutin, 2-thiophosphoric acid triaziridide, 3-kelilutin + thiophosphoric acid triaziridide).

Calculated: C 49.44 H 6.36 N 11.53 P 8.49% 5 Found: C 49.41 H 6.34 N 10.65 P 8.67%

Figure 2: Chelerythrine + thiophosphoric acid triaziridide C66H69N6 ° 15PS-

Calculated: C 63.45 H 5.56 N 6.72 P 2.47%

Found: C 62.69 H 5.37 N 5.37 P 2.35% 10 Figure 3: Copticin + thiophosphoric acid triaziride C24H27N4U5PS ·

Calculated: C 56.02 H 5.28 N 10.88 P 6.01 S 6.23%

Found: C 55.94 H 5.12 N 11.10 P 5.89 S 6.10%

Figure 4: Celidonine + thiophosphoric acid triaziridide C66H75N6 ° 18PS ’

Calculated: C 60.82 H 5.79 N 6.44 P 2.37 S 2.45%

Found: C 61.14 H 5.76 N 5.94 P 2.40 S 2.39%

Figure 5: Protopine + thiophosphoric acid triaziride C32H55N11 ° 3P4S3 '20 Calculated: C 44.59 H 6.43 N 17.87 P 14.37 S 11.16%

Found: C 44.58 H 6.14 N 17.76 C 44.72 H 6.30 N 17.77 P 14.04 S 12.71% Figure 6: Reserpine + thiophosphoric acid triaziridide C27H92N7PS018'2H2O 'mp 120-125 °.

25 Calculated: C 59.95 H 6.70 N 6.79 S 2.22%

Found: C 59.89 H 6.62 N 6.82 S 2.26%

Figure 7: Reserpine + thiophosphoric acid triaziridide C35H46N3 ° 11PS ·

Calculated: C 56.21 H 6.20 N 5.61 P 4.14 S 4.28% 30 Found: C 56.30 H 6.22 N 4.11%

Figure 8: Caffeine + thiophosphoric acid triaziride C 1 H 2 N 2 O 2. , sp. 110-112 °; 215-216 °.

304215 6

Calculated: C 46.68 H 5.48 N 27.22 P 4.01 S 4.15%

Found: C 47.47 H 5.44 N 27.25 P 4.02 S 4.15% 35 Figure 9: Narcotinin + thiophosphoric acid triaziride C28H35N4PS07 'SP * 225-226 ° · 9 73131

Calculated: C 55.80 H 5.85 N 9.29 P 5.13 S 5.29%

Found: C 55.34 H 5.69 N 9.52 P 4.80 S 5.29%

Figure 10: Aconitine + thiophosphoric acid triaziridide C74H106N5 ° 22PS'M * 197-200 ° · 5 Calculated: C 60.02 H 7.21 N 4.72 P 2.09 S 2.16%

Found: C 60.30 H 7.22 N 4.38 P 2.09 S 2.16%

Figure 11: Aconitine + thiophosphoric acid triaziridide C39H59N4 ° 11PS 'mp' 210 "211 ° ·

Calculated: C 56.92 H 7.22 N 6.80 P 3.76 S 3.86% 10 Found: C 56.91 H 7.12 N 6.89 P 3.60 S 3.73%

Figure 12: Aconitine + thiophosphoric acid triaziridide C35H55N4PS011, mp * 190-192 °.

Calculated: C 54.83 H 7.19 N 7.26%

Found: C 54.83 H 6.98 N 8.74% Figure 13: Pilocarpine + thiophosphoric acid triaziridide C26H31N3 ° 7 *

Figure 14: Allocryptopine + cyclophosphamide (N, N-bis - (β-chloroethyl) -N ', O-propylene phosphoric acid ester diamide) C49H65N4 ° 12C12P' Sp '159-160 °.

20 Calculated: C 58.22 H 6.27 N 5.58 P 3.08 Cl 7.06%

Found: C 58.25 H 6.25 N 5.40 P 2.53 Cl 7.41% C 54.84 H 6.16 N 5.62 P 2.51 Cl 7.26%

Figure 15: Protopine + cyclophosphamide / N, N-bis - (β-chloroethyl) -N10-propylene phosphoric acid ester diamide / c47H55N4012PC14, m.p. 239-242 °.

Calculated: C 54.24 H 5.32 N 5.38 P 2.97 Cl 13.62%

Found: C 54.04 H 5.25 N 4.85 P 2.72 Cl 10.13% C 54.48 H 5.22 N 4.69 P Cl 9.91%

Figure 15a: Protopine + cyclophosphamide / N, N-bis ([3-30 chloroethyl) -N ', O-propylene phosphoric acid ester diamide] C47H59N4 ° 12PC14' Sp '239-242 °.

Calculated: C 54.03 H 5.69 N 5.36 P 2.96 Cl 13.57%

Found: C 54.04 H 5.25 N 4.85 P 2.27 Cl 10.13% C 54.48 H 5.22 N 4.69 Cl 9.91% 35 Figure 15b: Protopine + cyclophosphamide / N, N-bis- (phloroethyl) -N ', O-propylene phosphoric acid ester idamide_7 C47H59N4 ° 12PC14' mp 239-242 °.

10,731 31

Calculated: C 54.03 H 5.69 N 5.36 P 2.96 Cl 13.57%

Found: C 54.04 H 5.25 N 4.85 P 2.27 Cl 10.13% C 54.48 H 5.22 H 4.69 Cl 9.91%

Figure 16: Chelerythrine + cyclophosphamide / N, N-bis (/ 3-5 chloroethyl) -N ', O-propylene phosphoric acid ester diamide / C27H37N309PC12 / m.p. 188-192 °.

Calculated: C 49.94 H 5.74 N 6.64 P 4.76 Cl 10.91%

Found: C 49.85 H 5.31 N 6.06 P 4.95 Cl 13.23% C 49.84 H 5.24 N 5.96 Cl 14.24% Figure 17: Celidonine + cyclophosphamide / N, N-bis (3-chloroethyl) -N ', O-propylene phosphoric acid ester diamide_7 C27H35N3 ° 7PC13' mp 273-276 °

Calculated: C 49.82 H 5.41 N 6.45 P 4.75 Cl 16.33%

Obtained: C 49.26 H 5.07 N 5.12 P 3.50 Cl 16.50% 15 For diagnostic purposes, it is relevant that the preparation fluorinates under UV light Ukrainian (Chelidonium Majus L. alkaloid-thiophosphoric acid triziridide derivative) a yellow-green. The activation frequencies are in the frequency range 220-490 nm. The fluorescence spectrum ranges from 410 to 665 nm, with a maximum at 550 nm. The wide spectral range is explained by the fact that the preparation consists of a group of different alkaloid derivatives. The limit of visibility of the fluorescence phenomenon at UV 366 nm on a thin layer chromatography plate is 0.000007 mg / ml at dilution, 0.0000003 mg per 1 mm2. This property of finished 25 tea is also retained in the body, so the distribution of the Ukrainian preparation in the body could also be observed in clinical trials.

The following are some of the clinical findings: The patient reports January 30 M. B., 64 years old, male, diagnosis: adenocarcinoma of the rectum and large regional metastaasimuodostusta LWS-la, IL OS. process. et Femur dext. The patient received two sets of Ukrainian preparations with the previously described therapy regimen. The dose is 140 mg and 160 mg. Disease course: general condition 35 improved, analgesic use decreased. Subjectively, the patient felt better, with varying amounts of heat waves felt in the tumor area. Fluorescence factor: Six days 11 73131 after the end of the second series of treatments, clear limited fluorescence was seen in and around the skin area above the tumor. In addition, fluorescence occurred in one limited clinically tumor-free skin area.

5 These points are the same ones that felt warm. Greater local heat generation can be seen with fever strips. After 13 days, the fluorescence is clearly less intense. It appears as irregular spots.

1 0 Case 2

Patient R.N. 68-year-old man, diagnosis: bronchiic carcinoma, upper left lobe. Histology: large cell, infiltrated N. bronchi. Jaundice, maximal stasis. Three times diagnostically in Ukraine, a total of 130 mg. Käm-15 is a high-fluorescence region on the sword attachment. Questionable fluorescence on the left side of the chest. After one week, no fluorescence was observed.

Case 3

Patient M.L., 72-year-old woman, diagnosis: pancreatic-20 carcinoma, numerous maximal metastases. Operation: Probatoria and P.E. liver metastases. Histology: metastasis of multicellular, partially mucus-forming adenocarcinoma. Persistent pain and strong alkaloid use, epidural catheter, carbostesin instillation, visceral blockade. Ultrasound: pay-25 metastases, large pancreatic carcinoma. Treatment with Ukraine discontinued while receiving placebo. By palpation: the left lobe of the liver shrank so that under the chest it could fit five transversely instead of three. The metastases appeared softer. After Ukrainian injections, white blood cells rise to 13,000. Fluorescence: a few days after the start of therapy in the middle of the surgical wound, once 30 mm wide fluorescence, which was not subsequently recovered.

Case 5

Patient R.M., 73-year-old woman, diagnosis: no Lei-35 comprehensive gastric carcinoma. Surgery: Probatoria and partial abdominal resection. In the evening before surgery 2 x 20 mg of ukraine i.v. Histology: widespread 12,731 31

Lymphagiosis carcinomatosa in the large abdomen of gastric adenocarcinoma. Note: Abdominal fluorescence does not occur in healthy patients. Breast nodules (breast vesicular disease) do not fluoresce after Ukrainian administration.

5 Case 9

Patient W.S., a 26-year-old man, diagnosis: scar tumor (above the sword appendix). 0.5 mg of ukrainin in 1 ml of 0.9% physiological NaCl solution subcutaneously 50 cm from the scar tumor (havan region), about one minute after 10 injections the scar tumor fluoresces strongly, the fluorescence was maintained for two days.

Case 10

Patient N.U., a 58-year-old woman, diagnosis: breast cancer. Histology: Carsinosis pleurae, multiple lumen-15 stasis. Status after recurrent pleural effusions, cytostatic instillation. 2.5 mg Ukrainian injection. After a short time, the fluorescence is clear at the pleural fluid discharge site. (4 RFI below the operating horn). Weak fluorescence in the scar removal area of the breast.

20 Description of clinical results 60 patients were treated with Ukrainian, of whom 59 had advanced cancer. In the course of clinical observations, it was shown that the preparation has a different effect on tumor growth, but in the case of mis-25 weather, it did not result in bone marrow damage worth mentioning.

Patients could be distinguished from 3 different groups:

Group I consisted of 8 patients: 2 patients with breast cancer, 2 patients with malignant melanoma, 1 patient with 30 Ia basocellular epithelioma, 1 patient with cylindrical cell carcinoma of the parotid gland, two patients probably had breast cancer, in the armpit and the upper 35-side of the clavicle, but the primary tumor could not be located. In all these eight cases, a clear temporary decrease in tumor tissue was obtained, with 13 731 31 of tumor tissue partially regressing in the opposite order to that at birth, i. those metastases that had recently appeared were the first to disappear. In this case, no necrosis occurred, but first the tumor tissue was clearly confined to the environment and the local edema and possibly also the permanent lymphatic edema due to the obstruction of the fluid flow was reduced. Then the tumor nodule gradually decreased. These patients showed a clear effect as a subjective sensation immediately after the first injection, such as: pain was reduced in one case as early as 1 to 2 hours after injection; the tumor appeared to be warming, which could be demonstrated as local warming in one patient; tension and itching in the tumor area; hot flushes, tachycardia, mild angulation, or headache; elevated virt-15 sanitation, fatigue, negativity, nausea, and into depression. Not all of these symptoms occurred simultaneously, but all patients experienced some of them as individual episodes after each injection.

The occurrence of said subjective phenomena can be clearly seen from the clear correlation of pulse, blood pressure and temperature values measured in one patient.

Group III comprised 27 patients: 10 cases of bronchial carcinoma, 6 cases of breast cancer, 2 cases of ovarian cancer-25 cases, 2 cases of peripheral cancer, 1 case of pancreatic cancer, 2 cases of rectal cancer, 1 case of gastric cancer and 1 case of advanced gastric cancer, unambiguously locate.

In contrast to group I, no effect on the relationship between the tumor and the side effects described was obtained here. Even with longer treatment times and higher doses, no changes were observed that could be attributed to the Ukrainian preparation.

Group II was the so-called. intermediate group, it comprised 25 patients, 35 with very advanced disease: 9 cases of breast cancer, 7 cases of bronchial cancer, case of rectal cancer, 2 cases of osteosarcoma, 1 case of prostate cancer, 14,731 31 1 case of thyroid cancer, 1 case of colon cancer and 1 colon cancer. In this group, the effect of the preparation was very variable. All of these patients experienced the same sequelae after long-term treatment as described in group I, but at the same rate.

The effect on the tumor in these patients was not so clear, which, however, must also be attributed to e.g. that the cancer was already very widespread in part.

10 In two patients with bronchial cancer, there was a temporary short-term reduction in the tumor, and in two patients there was a temporary cessation of growth. In four breast cancer patients, the ulcers on the metastases healed and in eight patients, the pain caused by the tumor was partially reduced. In one patient with infiltrated recurrent thyroid cancer, the tumor changed shape so that it was clearly confined to healthy tissue. In almost all of these patients, the general condition improved in part, contrary to expectations, even when tumor growth could not be stopped. To clarify the matter, one representative medical report from each group is attached.

Group I

Patient T.J., a 40-year-old woman, diagnosis: malignant-grade 25 melanoma. Histologically found. (In 1977, a patient was diagnosed with malignant melanoma in the left tibia, which was removed by surgery.) In 1980, a lymph node metastasis of 5 x 5 cm was detected in the left groin and the patient was then treated with Ukrainian. He received a total of 680 mg over three treatments.

Depression, muscle pain and malaise occasionally occurred during the first course of treatment. After the second treatment period, the lymph node was only the size of a walnut and after the third treatment period it was the size of a bean; it was hard and yet difficult to feel by palpable. The condition was good.

15 73131

Group II

Patient H.G., a 63-year-old woman, diagnosis: breast cancer in the left breast, lymph node metastases and bone metastases on the clavicle. Previous systemic therapy: Nolvadex from June 1977 to January 1978; polychemotherapy with endoxane, methotrexate, fluorouracil, and prednisolone, followed by combination therapy with adriblastine-endoxane from March 1978 to April 1979. Elipten (aminoglutethimide) from April 1979 to January 10, 1980.

Ukrainian therapy was started on February 29, 1980. Finding at the beginning of therapy: 5 x 5 cm ulcerated metastasis on the clavicle. In the area of the eyes, a subjective feeling of pressure and a feeling of tension on the left clavicle 15. Therapy was started with 2.5 mg. Right at the beginning, the patient noticed a feeling of warmth in the tumor on the left side of the body and on the clavicle. Already after six injections, the ulcer was no longer visible, however, the size of the tumor itself was unchanged. Subjective sensation of tension had also disappeared, as did perception of pressure in the eye area for two years. At the last examination on April 8, 1980, the patient was subjectively free from ailments. He complained of a reasonable weight loss. The local finding on the left clavicle was unchanged. At that time, there were no 25 detectable ulcers. According to the laboratory study (blood count, SMA, serum calcium) there were no pathological findings. No serious side effects could be identified.

Group III

Patient K.G., 58-year-old woman, diagnosis: adenoso-30 lidi ovarian carcinoma; removed by surgery.

Previous systemic therapy: Endodoxane 1,200 mg per infusion. Cobalt irradiation 4,500 rd / g. Prophylactic therapy in Ukraine. No side effects or subjective sensations such as dizziness, fever, frequent heartbeat, or depression were observed during treatment. General condition and appetite improved. One month after the start of therapy, a lump was found in the left chest 16 73131, at which point a week later, breast removal was performed according to Patey. Histological examination revealed a partially necrotized solid carcinoma. Ukrainian processing was suspended.

5 The study of the mechanism of action was supplemented by in vitro studies of the immunostimulatory effect of ukrain. lymphocyte-transformation experiments. Lymphocytes are known to be immunocompetent in that they can recognize or react with specific antigens. Such cells are termed immunocytes and their precursors as immunoblasts.

Immunoblast formation can also occur in vitro. Immunoblasts are 20-30 μm in size, while normal lymphocytes are only 5-15 μm in size.

15 The following immunological studies were performed to determine the biological mode of action of Ukrainian. Lymphocytes isolated from guinea pigs after Ukrainian addition were used for blast transformation. A mixture of compound 20 and EDTA available under the tradename "Ficoll 4000" (0.9 EDTA + 0.1 Ficoll 4000) was used for isolation.

Isolated lymphocytes were grown in vitro in Parker's solution at an addition of 1.6 μg, 0.16 μg and 0.016 μg. At the same time, lymphocytes were cultured in Parker's solution with the unspecified stimulator phytohemagglutinin-25 (PHA, 5 μg / ml) and as a control without addition. To avoid bacterial infection, the required amount of antibiotics was added: penicillin, streptomycin, and nysatin. Cultures were incubated at 37 ° C and the number of lymphocytes transformed under the microscope was counted once a day for three days.

For each preparation, 100 lymphocytes were counted and those larger than 15 μm for human lymphocytes and greater than 20 μm for guinea pig lymphocytes were evaluated as transformed. In all cases, the number of transformed cells before the start of culture was at most 10.1%. The experiments were performed using 17,731 31 lymphocytes from 10 healthy humans and 10 healthy guinea pigs. These results clearly indicate that the Ukrainian preparation affects lymphocyte transformation.

The dose of ukrain was selected based on the following criteria: since the ukrainin treatment series uses an average of 5 single doses of 16 mg / ml, the experiments were performed on 1 / 10,000, 1 / 100,000, and 1 / 10,000,000 of this amount. The results are summarized in the following Tables I and II.

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Table I

% Of transformed guinea pig lymphocytes

No. Culture medium and Incubation time 5 additive 24 h_48 h_72 h_ 1. 1.6 yug / ml ukrainin ^ __ 45, & _40, 2_ 0.16 yug / ml "42.3 29.3 28.5 0.016 ^, ug / ml" 35, 6 33.6 25.0 PHA 32.2 28.3 27.9 10 Without stimulator 19.2 15.3 1.0 2. 1.6 ug / ml ukraine 53.9 30.2 18.3 0, 16 μg / ml "43, / 32.2 32.0 (?) 0.016 μg / ml" 27.3 30.2 10.5 PHA 20.8 17.2 1 /, 9 15 Without stimulator 13.3 12 , 2 19.2 (?) 3. 1.6 ug / ml Ukrainian 50.2 40.3 42.8 0.16 ug / ml "49.3 51.1 33.9 0.016 / ug / ml" 41 .0 29.3 30.1 PHa 32.5 29.8 22.3 20 Without stimulator 18.0 11.2 10.3 4. 1.6 ug / ml ukrainian 55.0 42.3 0.16 ^ , ug / ml "50.0 40, / 43.8 0.016 ug / ml" 41.2 30.5 39.5 PHA 33.2 27.0 10.0 25 Without stimulator 18.2 18.0 16, 2 5. 1.6 μg / ml Ukrainian 51.3 43.5 31.5 0.16 μg / ml "48.2 33.2 33.5 19 - 73131

Table I (continued)

No. Culture medium and Incubation time additive 24 h 48 h 72 h 0.016 yug / ml Ukrainian 39.9 31.5 - 30.2 PHA 33.4 20.3 11.3

Without stimulator 12.5 12.5 8.4 6. 1.6 μg / ml Ukrainian 58.5 57.9 41.8 0.16 μg / ml 48.3 50.0 43.2 0.016 μg / ml "40.1 35.2 30.0 PHA 30.2 22.1 17.3

Without stimulator * 9.0 18.2 13.5 7. 1.6 yug / ml Ukraine 43.3 41.2 39.0 0.16 yug / ml "42.3 36.6 36.2 0.016 yug / ml "41.2 37.2 35.5 PHA 33.3 33.4 27.2

Without stimulator 15.1 16.1 13.2 8. 1.6 ug / ml ukraine 48.7 45.1 40.5 0.16 ug / ml "43.1 39.2 31.1 0.016 ug / ml" ml "35.6 29.8 24.2 PHA 31.3 27.0 24.3

Without stimulator 12.2 10.1 10.5 9. 1.6 ug / ml ukraine 53.3 51.5 41.2 0.16 ug / ml 49.6 47.2 42.2 0.016 ug / ml ml "39.2 36.7 34.6 PHA 38.3 32.8 25.2

Table I (continued) 20. 731 31

No. Culture medium and Incubation time additive 24 h 48 h 72 h

Without stimulator 10.1 16.7 11, 3 5 10. 1.6 yug / ml Ukrainian 56.2 53.6 40.7 0.16 ug / ml 49.3 42.0 34.5 0.016 ug / ml "39.0 37.3 20.3 pha 34.2 33.2 2 /, 5

Without stimulator 12.0 10.2 8.4 2i. 73131

Table II

% Of transformed human lymphocytes 1. culture medium and X incubation time additive 21 h 48 h 72 h_ 5 1.6 ug / ml ukrain 47.2 45.4 41.2 0.16 ug / ml "43.8 39.2 38.6 0.016 ug / ml "36'7 3ί'2_ 36.6 24.8 20.8 PHA 5 yug / ml, _____ 111 <4 2 0 0

Without stimulator __'______ 10 2. 1.6 ug / ml ukraine 49.9_41, 2_37.2__ 0.16 ug / ml "43.2 40.3 33.2 0.016 ug / ml" "8> 6_36.2_26 , 8_ PHA 5 μg / ml 31, 2 27.0 _21.2

Without stimulator 18.4 14.1 8.4 15 3. 1.6 yug / ml Ukraine 1__18> 8_18.2_ 0.16 yug / ml "19.5 18.2 15.3 0.016 / Ug / ml - 14> 2_ 1 ^ 13> 2,, 33.1 28.2 14.1 PHA 5 ^ ug / ml -------- -

Without stimulator 1 ^ _8 '^^ ^ _ 20 4. 1, 6 ug / ral ukraine 43.9 _____________ 0.16 ug ug / ml "41.2 40.6 41.2 0.016 ug / nl" ^ · 6 ? 1 · 2__,, 40.2 53.0 23.3 PHA 5 yUg / ml -------: ---

Without stimulator ^ 9.6_ ^ 2, 'j_10.8__ 25 5. 1, 6 yug / ml Ukrainian 9j, 9 - 51, 5_47.8_ 0.16 7ug / ml "48, 6 43.6 41, 0

Table II (continued). 731 31

No. Culture medium and Incubation time additive 24 h 48 h 72 h 0.016 yug / ml Ukrainian 37.2 37.8 36.2 5 PHA 5 yug / ml 36.4 23.3 25.4

Without stimulator 15.3 14.4 12.8 6. 1.6 ug / ml Ukrainian 55.5 53.4 51.5 0.16 ug / ml "51.2 49.1 47.3 0.016 / ug / ml "43.2 36.2 36.0 10 PHA 5 ug / ml 37.1 36.2 34.7

Without stimulator 19.3 18.4 16.5 7. 1.6 yUg / ml Ukrainian 40.6 46.0 42.5 0.16 yug / ml "47.2 45.2 24.5 0.016 y, ug / ml" ml "^ 9.8__32.0_21.2 _ 15 PHA 5 yug / ml 30.1 26, 8 19.0

Without stimulator 14.7 13.1 8.5 8. 1.6 ug / ml Ukrainian 52.3 37.4 21.8 0.16 ug / ml "43.3 20.3 19.3 0.016 ug / ml "41.2 19.4 W, 4 PHA 5 μg / ml 37.3 19.5 20.1

Without stimulator ^ ^ _8 'J_8' 2_ 9. 1.6 μg / ml Ukrainian 4 /, 2 32.8 29.1 0.16 μg / ml "43.4 36.4 34.2 0.016 μg / ml "^ 8.6_28.0_2Q> ° _ 25 PHA 5 μg / ml 36.6 28.6 21.4

Table II (cont'd)

No. Culture medium and Incubation time additive 24 h 48 h 72

Without stimulator 15.3 13.3 10.7 5 10. 1.6 ug / ml ukraine 40.33 37.4 33.3 0.16 ug / ml "43> 4_4-1> 2_3" _ 0.016 ug / ml "37.1 36.2 29.1 PHA 5 μg / ml 30.0_19.4_18.4_

Without stimulator 9.3 9.6 5.3 24 73131

The experiments showed a statistically significant difference in the numbers of transformed lymphocytes from animals and humans and control groups in the presence of Ukrainian. From this it can be concluded that the Ukrainian preparation has an immunological effect and that it stimulates the human control system.

In only one case out of 10 (see Table II, No. 3) did no clear lymphocyte transformation be obtained. It is about human lymphocytes. Thus, in certain conditions, patient No. 3 may experience an "in vitro equivalent" phenomenon with the clinical phenomenon, in which an individual patient does not react at all to Ukrainian.

All other patients had a higher transformation ratio than PHAR.

15 Finally, agar electrophoresis for ukraine, known per se, using 0.05 mg of ukrainin placed in a small groove in an agar gel (Sörensen buffer + NaCl) and run for four hours at 70 V and a current of 2 mA was obtained in surface-printed filter-tin paper, which indicated the starting point and polarity, when viewed under a UV lamp, a fluorescent zone directed at the negative pole. Fluorescent farcts were positively charged.

It is clear from the above that the preparations described at the beginning and the preparations and the compounds, respectively, can be used for the diagnosis of tumors of all kinds (in addition to therapeutic treatment), in which case the fluorescence of the preparations is of great importance. In this case, in order to make a diagnosis, it is possible to make a rapid and reliable early diagnosis by injecting the preparation or preparations after the preparations 30 have accumulated in the diseased tissue after injection, in which the injection fluid rapidly spreads from the injection and accumulates in the affected area.

This observation also extends to all endoscopic methods, such as bronchoscopy, mediastinocopy, thoroscopy, esophagoscopy, otorinoscopy, laryngoscopy, rectoscopy, proctoscopy, where, for example, cold light can be replaced by UV light.

The diagnostic method can be supplemented by incorporating a radioactive isotope into the preparation so that the radio-5 activity can be measured in a manner known per se with corresponding detectors and the accumulation of radioactivity is plotted, for example, with a gamma radiation scanner.

Finally, in addition to the labeling of said preparations with fluorescent substances and radioactive isotopes, it is also conceivable to provide the preparations with radiation-absorbing compounds or radicals so that the accumulation of the preparation on a certain tissue area can be identified due to the increased X-ray absorption.

Claims (17)

26 731 31 Use of alkaloid compounds of formula (I) and their salts R7-7R'R1. I; (I) R 1 R 4 R 5 wherein R 1 = H; 0CH-. ; Y Ά. _ 13 ^ / 1 ^ R0 = OCH.,; OH; ΟΗ -, - ΟΗ ,, - Ν-Ρ-Ν ^ -3 1 1 I I \ 15 R9? R 10 or and R e together form a group; ^ 0 V 20 ^ \ Y. Rn f / 12 R0 - H; 0CHo; CHn-CHO-N-P-N 3. z ^ | j v R9f NR „ R 10 or R 2 and R 2 together form a group: - °) 30 'v / O = R 4 = H; R 4 = «, · or R 4 + = O 35 or R 1 and R 4 together form a group: 27,731 31 cht OH 5 Y, R19 t / 1 2 Rc = CH0; OH; 0Ho-CHo-N-P-N U \ 9i R11 Rio 10 R '= CH0; OH; D 3 or R 2 and R 8 together form a group: OOH 3 raC). JOO O 'cc oa ° -R14 20 3 Rg = H; Rg = H; or together with R 1 = O; or Rg and Rg together form a group; 30 i ^ fCf0). · 1 'X ^ T0' * 14 no-r13 provided that at least one of P-2, R3 and R8 (alone or in combination with R1 or R1) represents a group: 28 7 31 31 Υ / R1 0 t / 12 -CH „-CH - NPN 2 2 II \ r9x Rn R10 5 or contain such a group, R? = H; Ry = H; or Rg and together form a group: 10. H "'"' 'OOO · 000 · 15. H, CO h3co ^ ^ \ ^^ 3 20 r ° "R15 0 / Rg = H; CH2-CH2-C1; alkaloid; q = H; OH; alkaloid; R 1 = H; alkaloid; or R11 + R12 = CH2-CH2? 30 Y = S; O; X = NH; O; R 2 = H; alkaloid; Ϊ / R12 R1 ^ = OH; CH „-CH„ -N-P-N IJ Z Z | | v 35 R9 | R10 29 7 31 31 H N - CH »5/2 CH» -CH »-N-P -> O CH»; 2. I \ / 2 CH »O-CH» I 2 2 cich2h2o c ^ 2 ch3 1 5 γ t / ^ 2 R .. = OH; CH »-Cl; CH »-CH» -N-P-N 14 z £ £ j | \ M R11 R1 o 20 H 25 N-CH / V CH -CH »-N-P— * O CH»; 2. CH \ / | 2 O -CH CH2 ch2 30 O v o -N) '\ / CH2-' 35 3 30 731 31 H N-CH »/ X CH» -CH »-N-P -» O CH »; 2 2 | v / 2 CH 2 /, 2 O-CH »5 1 1 CH»! 2 Cl 10 f N-CH 2 - / R 15 = CH 2 -CH 2 -? - pC 0/2; CI? 2 O-CH 15 1 1 CH »i2 Cl as active ingredient for the manufacture of diagnostic preparations. Use of compounds as defined in claim 1 for the preparation of preparations for the diagnosis of tumors. 31 73131