UA80992C2 - Kahalaide antitumoral compound, use thereof and process for the preparation - Google Patents

Abstract

The present invention is directed to new kahalalide antitumoral compounds, in particular to analogues of kahalalide F, useful as antitumoral, antiviral, antifungal agents and in the treatment of psoriasis.

Description

Description of the invention

The present invention is related to new antitumor compounds of kahalalid, in particular, to analogs 2 of kahalalid E, where aliphatic 5-methylhexanoic acid is replaced by 4-methylhexanoic acid, pharmaceutical compositions containing them, and their use as antitumor, antiviral, antifungal agents and in treatment of psoriasis.

Kahalalide compounds are peptides isolated from the Hawaiian marine herbivorous species of the mollusk EIPusia hyiTesseps and its feed, the green alga Wguorziz. with Kahalalids A-E are described in |Nattap eyi ai., 9). At. Spent 70 os, 1993, 115, 5825-5826).

Kahalalids A-b are described in |Natapp, M. eyi aYi., U. Ogud. Spet, 1996, 61, 6594-6600: "Kapaiaiidev: Bioasiime reriidez ytot a tagipe toiYIISH8K EPiuzia gyTezsepz apa iyez aida! aieei Vguorviv vzr.").

Kahalalids N and u are described in IZspeceg R. U. ey aiYi, 9. Mai Rgod. 1997, 60. 12 Kahalalid O is described in I (Zspetseg R. y). ей айЙ., 9. Mai. Year 2000.

In connection with kahalalid K | see publication Kap, U. ei ai., U. Mai. Year 1999 62(8) 1169-72: "KapNaiaiide

KI: A pemu susiis Oerzireriide itot (Pe pamaiap dgeep aYida Bguorziz zresiev").

For related publications, see also Soei ei aiYi., Teigapedgop, 1999, 55; 7739-7746: "Te arvoishe viegeospetivigh oi KapPaiaryde EE"; AiIregisio0, B. ey a). Teiapedgoop IeTseg5, 2000, 41, 9765-9769: "Kapaiaide V. bZupipeziv oi a paiishga! susioderzireriide"; It's fun, a). 9. He sang. Essays 2001, 27(11), 2287-99: "Spetisa!

Among kahalalide compounds, kahalalide E is the most promising because of its antitumor activity.

It has a complex structure that includes six amino acids in the cyclic part and an exocyclic chain of seven amino acids with a fatty acid end group. Its activity against human cell cultures (3 A-549 cells of human lung carcinoma and HT-29 cells of human intestinal carcinoma was reported in the publication

ER 610078). Kahalalide E has also been shown to have antiviral and antifungal properties.

In preclinical ip mimo studies, it was shown that the maximum tolerated dose (MTD) of a single bolus IV injection of kahalalid E in female mice is 280 μg/kg. Despite the fact that single doses of SC slightly higher than MPD IV were extremely toxic, so that the animals showed signs of neurotoxicity, that Ge"! were fatal, a dose of 280 μg/kg of kahalalid E could be administered repeatedly, according to the scheme of five injections, one injection per day, without any signs of acute toxicity. | See ZyrkKo, R. ei ai., Me.

Rgoseidipoz oii (She 1999 AASK MCI EOKTS Ipiegtaiopa!| Sopiegepse, abzigasi 315: "Rgesiipisaii rpnappasoyudu "3 zitsadiez myp iSpe tagipe paigai! rgodisi Kapaiaiide EE". 3o In | International application MUO 0236145) pharmaceutical compositions containing kahalide P are described new so options for the use of this compound in the treatment of cancer - this publication in its entirety is incorporated herein by reference.

International application UUHO 03330121, from the filing date of which priority is claimed under this application, describes the "clinical use of kahalalide compounds in oncology, and this publication is also incorporated in its entirety into C 70 this description by reference. c In the IPatent of Great Britain ZV 0304367), from the date of filing of which priority is also required according to

With" application, the use of kahalalide compounds in the treatment of psoriasis and related compounds is described, and this publication is also incorporated herein by reference in its entirety.

The synthesis and cytotoxic activities of natural and synthetic compounds of kahalalid are described in International Application 495 to 0158934), which is incorporated in its entirety into this description by reference. UMO document 0158934) describes the synthesis of kahalalide E, as well as compounds with a similar structure, in which the terminal chain is made of fatty acid av | replaced by other fatty acids.

As before, there is still a need to create new antitumor compounds, in particular, additional compounds such as kahalalid with improved properties.

Te) 20 The authors of the invention unexpectedly discovered that one of the compounds-analogues of kahalalid has quite promising activity and shows an increased antitumor effect on IP mimo models. The present invention relates to a compound of formula 1:

F) name) 60 b5

9 M. o

Mn

M o. AMN but oh we

S o n o NN o M o o o

MN chn sa nn o

Ne-o jur a M nn

Formula 1 and its pharmaceutically acceptable salts, prodrugs, tautomers and solvates.

Such a compound corresponds to kahalalide E with a 4-methylhexane chain in the form of a terminal fatty acid, and will be referred to as 4-methylhexane KE in the following description.

In a preferred embodiment, the present invention relates to a compound containing (45)-methylhexanoic acid having the formula 2:

M cm n o h o

And vn zd

Ho) but Mn s u

Lake Mn

Mn | M (o) i nn o o ni mn g) no o o lo, 2 "

Formula 2 o, :z" and its pharmaceutically acceptable salts, prolines, tautomers and solvates. Such a compound will be referred to in this description as (45)-methylhexane KE. 15 The present invention relates to a pharmaceutical composition containing the compound defined above and a pharmaceutically acceptable carrier, excipient or diluent.

The present invention is additionally related to a method of treating any animal, especially a human, affected by (a) a malignant tumor or psoriasis, and this method includes the introduction of a therapeutically effective amount of the compound defined above into the affected individual.

The present invention can be used, in particular, for the treatment of patients with refractory malignant (Ce) tumors, which are characterized by an unsatisfactory response to other therapeutic effects. In particular, the compositions according to the invention can be used after other types of chemotherapy have been used, which did not have an adequate effect.

This invention is directed, in particular, to the treatment of patients affected by a malignant tumor of the prostate gland, a malignant tumor of the breast, hepatocellular carcinoma, melanoma, a malignant tumor of the colon and rectum, a malignant tumor of the kidney, a malignant tumor of the ovary, a malignant tumor

HF) tumor of the MFA. (lung carcinoma), a malignant tumor of the epithelium, a malignant tumor of the pancreas and 7 tumors characterized by increased expression of the oncogene Neg2g/pey.

In another aspect, the present invention is directed to the use of the compound defined above for the production of a medicinal product. In a preferred embodiment, the indicated drug is intended for the treatment of a malignant tumor, psoriasis, viral infection or fungal infection.

The present invention is further related to kits comprising separate containers containing a pharmaceutical composition comprising the compound defined above and a reducing agent. Recovery methods are also provided by the present invention. c The present invention is also directed to a method of obtaining the compound defined above. In the production method, 4-methylhexanoic acid is preferably used as the starting compound. In a preferred embodiment,

4-methylhexanoic acid is (45)-methylhexanoic acid. In the most preferred embodiment, this method is a solid-phase synthesis.

The authors of the invention have identified analogues of kahalalide E, which show significantly more improved activity compared to kahalalide PE. As shown in the comparative examples, 4-methylhexane kahalalide E in an unexplained way shows improved efficacy in IP mimo models of malignant tumors. This is even more surprising in view of the small structural differences between 4-methylhexane cahalalide EC and

Bb-methylhexane kahalalide EC.

The compound according to the invention is kahalalide E with a chain of 4-methylhexane fatty acid instead of 7/0 5-methylhexane. which has a structure according to formula 1: 8 M o

MN

Ma! Mn no M o o t n o NK o M o o o

MN nn sa o n nyu-o yur a Mn s 2 o

Formula 1

In particular, it is preferred that this compound has the stereochemistry defined by formula 2:

M.Sc

We are 9 and Fu

And oh

M o no Mn-Sh (o) s o gYa Ge) ne o z o nn. o bath house

In 4 nn mn |. m i Nn o nn "- s yu "u o r. ch o n i "" i

Formula 2 (ee) o However, the compounds according to the invention have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. The present invention relates to the use of all optical isomers and stereoisomers of the compounds according to the invention and their mixtures, as well as to pharmaceutical compositions containing them, and to methods of treatment in which they are used.

For convenience, the authors of the invention refer to the compounds according to the invention, namely to compounds 1 and 2, as g" compound 4-methylhexylkahalalide EP, or compound 4-mehexKE. Preferably, the 4-mhexKE compounds of the invention are compounds that are largely free, essentially free, or completely free of other cahalalide compounds.

For example, the compound 4-mhexKE according to the invention is preferably free of kahalalide E having a 22 B5-methylhexyl side chain. In particular, the 4-megaxKE according to the invention preferably contains the most 2595, 1095,

GF) Bob, 295, 190 or 0.595, or less than 0.595 of any other kahalalide, especially kahalalide R. In a close aspect, the 4-mhexKE according to the invention is provided in essentially pure form. Such a 4-mhexKE compound, free to some extent from other kahalalides, is particularly suitable for pharmaceutical compositions and methods of treatment according to the invention. 60 The present invention includes compounds according to the invention and their pharmaceutically acceptable salts in which one or more hydrogen, carbon or other atoms are replaced by their isotopes. Such compounds can be used as tools for scientific research and diagnostics in the study of metabolism and pharmacokinetics, as well as in binding assays.

In this description, compounds according to the invention, including compounds of formulas 1 and 2, are defined as compounds that also include pharmaceutically acceptable derivatives or prodrugs. "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, ester salt or other derivative of a compound of the invention which, when administered to a recipient, is capable of providing (directly or indirectly) a compound of the invention or a metabolite or residue thereof. Particularly preferred derivatives and prodrugs are those that increase the bioavailability of the compounds of the invention when such compounds are administered to a patient (for example, by adding a property of increased blood absorption to an orally administered compound), improve delivery of the parent compound to a specific biological compartment, increase solubility, creating the possibility of introduction by injection, change the metabolism or change the rate of excretion.

Salts of compounds of the invention include addition salts derived from the nitrogen in a compound of formula 1 or 7/0 2. Therapeutic activity, as defined herein, is associated with a fragment derived from a compound of the invention, and the identity of the component, what remains is not so important, although for therapeutic and prophylactic purposes it is preferable that it be pharmaceutically acceptable to the patient. Examples of pharmaceutically acceptable acid addition salts include those acid addition salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, organic acids such as tartaric, acetic, trifluoroacetic, citric , malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic and methanesulfonic and arylsulfonic, for example para-toluenesulfonic, acids.

The preferred salt is the trifluoroacetic acid salt.

Compounds according to the invention can be obtained according to the synthetic processes described in

International application UUO 01589341, for example, when adding the corresponding (5) or (K) 4-methylhexanoic acid instead of B5-methylhexanoic acid in example C (international application UUO 0158934). Therefore, the present invention also extends to the method of obtaining the compound of formula 1 or 2. In this method, 4-methylhexanoic acid is used as a starting material. The most preferred starting material is (45)-methylhexanoic acid. The method of synthesis is mainly a method of solid-phase synthesis. Additional details of the synthesis are given in the examples. with

The method according to the invention can be carried out on the basis of starting materials in an enantio-, stereocontrolled and fast mode, using the advantages of the solid-phase synthesis methodology, where i) during all synthesis operations, the molecule in the structure is connected to an insoluble substrate.

Pharmaceutical compositions of the compounds according to the invention can be adapted for any desired route of administration, for example, oral (including buccal or sublingual), rectal, nasal, local

Zo (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes of administration. Such compositions of Me can be obtained by any method known in the field of pharmacy, for example, by association of active He! ingredient with a carrier(s) or filler(s).

Preferred pharmaceutical compositions of the compounds according to the invention include a liquid (solutions, suspensions or emulsions) with a suitable composition for intravenous administration, and they can contain the compounds in their pure form or in combination with any carrier or other pharmaceutically active compounds. International application UMO 0236145), which is included in this description in its entirety as a reference, can serve as a further guide in connection with pharmaceutical compositions.

Thus, the combination of a nonionic surfactant and an organic acid is acceptable for "

Use with a bulking agent to obtain a lyophilized form of a compound according to the invention, with c acceptable for recovery. Recovery is preferably carried out by mixing an emulsifying solubilizer, alkanol and water. ;" Lyophilized compositions preferably include mainly a filler, for example, at least 9095 or at least 9595 bulking agent. Examples of bulking agents are well known and include sucrose and mannitol. Other bulking agents may also be used.

Go! The nonionic surfactant in the lyophilized composition is preferably a sorbitan ester, more preferably a polyethylene sorbitan ester, such as polyoxyethylene sorbitan alkanoate, in particular, polyoxyethylene sorbitan monooleate, for example, polysorbate-80. Nonionic surfactant of course

Ge) is a small percentage of the composition, for example, from O to 595 of the composition, for example, from 2 to Z or 495

Compositions. The organic acid in the composition of the lyophilized composition is usually an aliphatic acid, preferably

Of hydroxycarboxylic acid, and more preferably - hydroxypolycarboxylic acid, in particular, citric acid.

The organic acid is usually a small percentage of the composition, for example, from 0 to 595 of the composition, for example, from 2 to 3 or 495 of the composition.

The amount of the compound according to the invention in the composition of the lyophilized composition is usually less than 195 or more often less than 0.195 of the mixture. An acceptable amount is in the range from 50 to 200 Ωkg, for example, approximately

F) 10 Ohmkg per 100 mg of the composition. The emulsifying solubilizer for the reducing agent preferably contains a polyethylene glycol ester, especially a fatty acid ester, more preferably a PEG-oleate, such as PEG-oleate-Z5. The emulsifying solubilizer is suitably from 0 to 1095 of the reducing agent, usually from about 3 to 795, for example about 595. The alakanol is usually ethanol, which is suitably from 0 to 1095 of the reducing agent, usually from about 3 to 795, for example , approximately 595. Another part of the reducing agent is water, and as a result, a reconstituted solution suitable for intravenous injection is obtained. 65 Further dilution of the reconstituted solution with 0.995 saline can be made for infusion of the kahalalide compound. Suitable infusion equipment preferably includes a glass container, which is preferable to a polyethylene container. Tubes should preferably be made of silicone.

Further, the preferred reducing agent is from 2 to 795, for example, about 595, of an emulsifying solubilizer; from 2 to 795, for example, about 595, of alcohol; and the rest is water.

The compositions may be presented in unit-dose containers or in multiple-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only an additional sterile liquid vehicle, such as water for injection. which is used immediately before consumption.

Thus, the present invention is further related to kits that include individual containers containing a lyophilized composition and a reducing agent. A description of recovery methods is also included in the set.

Administration of compounds or compositions according to the invention is carried out by intravenous infusion. The infusion time can reach 72 hours, more preferably from 1 to 24 hours, most preferably either about 1 hour or about 3 hours. Especially preferred are short infusion times, which allow for a therapeutic effect without the need for the patient to stay in the hospital during the night. However, if necessary, the infusion can be continued for approximately 24 hours or even longer.

Cyclic administration is performed, in the preferred method of administration, patients are given an intravenous infusion of a compound according to the invention in the first week of each cycle, then at other times within the cycle, patients are given time to recover. The preferred duration of each cycle is either 1, 3, or 4 weeks; multiple cycles may be used if necessary. In an alternative dosing protocol, the compounds of the invention are administered, for example, over approximately 1 hour for 5 consecutive days over 3 weeks. Other schemes can be prescribed as options.

If necessary, postpone the dose or reduce the dose and change the administration schedule, depending on the tolerance of the individual patient to treatment, in particular, reducing the dose is recommended for patients whose serum levels of transaminases or alkaline phosphatase are higher than normal. high school

In one of the aspects of the present invention, a method of treating a person affected by a malignant tumor is provided, which includes the administration to the specified patient of the compound according to the invention in a dose below i) 1200 μg/m2/day, preferably below 93 ZOmkg/m2/day, and more mostly lower than 8b0Omkg/m/day. The corresponding dose is at least C20mcg/m/day. The preferred dose is in the range from 400 to 900μg/m/day, preferably from 500 to 800μg/m/day, more preferably from 600 to 750μg/m/day. Particularly preferred are doses of approximately 650 to 700 μg/m2/day. Fo

In a further aspect of the present invention, there is proposed a method of treating a person affected by a malignant tumor, which includes administering to said patient a compound according to the invention daily for 5 days at a dose of less than 900mg/m2/day, followed by a recovery period of 1 to 4 weeks, during which kahalalid compounds are not administered to the patient. The dose is preferably 650-75Omkg/m"/day, more preferably so 5. approximately 700μg/m"/day. The infusion time is preferably between 1 and 24 hours, more preferably between 1 and 24 hours. Particularly preferred is an infusion time of about 1 hour or about 3 hours.

The remaining period is preferably 2-3 weeks, more preferably about 2 weeks.

The present invention also provides a method of treating a person affected by a malignant tumor, which includes "introducing to the specified patient a compound according to the invention every week at a dose below 80 Ωkg/m"/day. The dose is preferably 600-700 μg/m2/day, more preferably - b5Omkg/m2/day. The infusion time is preferably from 1 to 24 hours, more preferably from 1 to 3 hours. The infusion time, which is approximately 1 hour, is particularly preferred.

Despite the above approximate dosage, the exact selection of the dosage of the compound will vary depending on the specific composition, the method of administration and taking into account the specific situation related to the condition of the patient and the tumor being treated. Other factors such as age, body weight, sex, diet, time of administration, speed of excretion, patient's condition, combination of drugs, sensitivity reaction and severity of the disease should also be taken into account. Administration can be carried out continuously or (Ce) periodically, within the limits of the maximum tolerated dose.

This invention is especially directed to the treatment of patients affected by a malignant tumor of the prostate gland, a malignant tumor of the breast, hepatocellular carcinoma, melanoma, malignant

With a rectal tumor, a malignant kidney tumor, a malignant ovarian tumor, a lung carcinoma of the Ministry of Health, a malignant epithelial tumor, a malignant tumor of the pancreas, tumors that overexpress the oncogene Negg/pesh. In the most preferred version, it is directed to the treatment of hepatocellular malignant tumor, melanoma, malignant tumor of the mammary gland and malignant tumor of the prostate gland. o The present invention also relates to a method of treating a skin disease involving hyperproliferation of dermal cells in mammals, wherein said method includes administering to said mammal an effective, non-toxic amount of a compound according to the invention. Especially if the skin disease is psoriasis. This invention is mainly directed to the treatment of a sick person affected by psoriasis, especially severe form of psoriasis.

Compounds and compositions according to the invention can be used in combination with other drugs to provide combined therapy. The specified other medicinal products may be part of the same composition or may be presented in the form of a separate composition for simultaneous administration or 65 I separate administration at different times. The identity of another drug is not particularly limited, although combinations with other chemotherapeutic, hormonal agents and antibodies should be taken into account.

The amounts of the compound according to the invention and the other pharmaceutically active agent or agents and the relative times of administration must be worked out in order to achieve the desired effect of the combination therapy.

Examples

Example 1: obtaining (45)-methylhexane KE

The main procedures and initial stages of the process correspond to those described in the international application UMO 0158934). (45). -Oh-MaI-Oh-You

SiI polymer resin: 70 The Ethos group was removed, and Ethos-MaI-OH (678 mg, 2 mmol, 4 equiv.), Ethos-TykVi)-HIN (992 mg, 2.5 mmol, back equiv.) were sequentially added to the above peptidyl polymer. . ;

ZiOmkl at 2 mmol and 4 equiv.; and Z88μl for 2.5mmol, bekviv.) and NEW (230mg for 1.5mmol and Zekiv.; ZOO7mg for 2mmol and 4equiv.; and Z95mg, 2.5mmol, bekiv.) during UOkhv. In all cases through OOkhv. binding ninhydrin test was negative. Removal of the Ethos group and washing were performed as described in the Sepegai section

Rgosedigev. (45). -O-aPo-Pe-O-MaI-O-TiSiI-polymer resin:

The AHPos group was removed with the help of Pa(PPE z)4 (58 mg, 0.05 mmol, 0.5 eq.) in the presence of RIBiHz (617 μl, mmol, 1 eq.) in an Ar atmosphere, and AlPos-Rpe-2-0Opb-OH ( 66bbmg, 2mmol, 4eq.) Its NOAI (27mg, 2mmol, eq) was dissolved in OME (1.25ml) and added to the peptidyl polymer resin, then SIRSOY (31µg, 2mmol, 4eq) was added, and the mixture was stirred for 5 hours when the ninhydrin test was negative.

After washing with OME and CHCl, an aliquot of the peptidyl polymer resin was treated with a mixture

TFO-H2O (1:99) for a minute, and the resulting product was characterized using the MAIOI-TOR-M5 method, (du calculated for SvvNi46M414O24, 1736.18. Obtained: t/2 1758.67 MAMA", 1774.62 16182 | MKI". (45)-MeNeh-O-MaI-TAIKIVO)-MaI-O-MaI-O-Rgo-Ogp(Vos)-O-aIPo-Pe-O-aPyo- Ty MaI-2-Opr-Rpe- H)-O-aPo-Pe-O-MaI-OH: i9)

After washing with UOME and CH 25Si5, the AIPos group was removed using Pa(PPRI z3)4 (58mg,

O,O5mmol, O,Tequiv.) in the presence of RyBzIiINz (617μl, 5mmol, 1Oequiv.) in an Ag atmosphere. The protected peptide was cleaved from the polymer resin using a mixture of TFO-CHNOCl (1:99) (5x30sec). The filtrate was collected on HO He (4 dml), and the HO was partially removed on a rotary evaporator. Then AFM was added to dissolve the solid substance formed in the process of removing H 20, and the solution was lyophilized, obtaining 63U9mg (387μmol, yield 7790) F of the compound indicated in the title with a degree of purity of 29590, which was confirmed by the NRI C method (Condition A, (p 10 ,5 min). -O-MaI-Ppe-2-OpB-Ma|)-(45)m ethylhexane KE o

The protected peptide (example 6) (b3Omg, 387 μmol) was dissolved in CHCl (390 ml, 1 mM), and NOVI (237 mg, with 1.55 mmol) was dissolved in the minimum volume of OME to dissolve NOVI, OIEA (20 μl, 1, 1bmmol,

eq.) and OIRSOI (24 Ωcl, 1.55 mmol, eq.). The mixture was left on the stirrer for 1 hour, then the result of the cyclization stage was monitored by the NRI C method. The solvent was removed by evaporation under reduced pressure. The protected cyclic peptide was dissolved in a mixture of TFO-H 20 (19:11, 8 Bml), and the resulting mixture was left on a stirrer for 1 hour. The solvent was removed by evaporation under reduced pressure, and dioxane (30 ml) was added, and the solvent was removed by evaporation under reduced pressure (this process was repeated three times), then HO (40 ml) was added, and lyophilization was carried out. The crude product was purified by NRIS (Kgotavii Sv 5μm, 205х5Ωm), isocratically 4495 acetonitrile (0.05906 TFO) in water (0.0595 TFO), 55ml/hour, detected at 22Ωm, obtaining the title product (192mg, 0 /1Zmmol, output 2695, 92.395). MAGOI-TOE-M5, calculating for С75Н424М44Оч46, 14779. со Obtained: t/2 1500.12 |М--Ма!", 1515.97 IMAKI". o H-NMR spectrum (2.5 mM; 500 MHz, HgO-020 (9:1) of this compound is shown in Table I). se) o 50

Co.)

Ф) име) 60 b5 duv ra i 3 Bkeba izhvy 13.19 bd, USNI i p SHYN VYN he I PN

AND? aPe-ei Va; I am 587,000 ov, OS yu same and SHE Tb,ye shSchesve u shi l

Tsianvvei What SE shya zasshe tn t ayu t PIY women and VYMNY PON Vlasii t reak kot and what id, What 8, ba. Y B, 0 of them Sh t, SV y:

PNO ask i MY NIE NN, 1 Vu, Z tb y, BAN I o, ras | zey, V. Yaaschiu Y I ! v ny Z r i i ev I BIVYE fushesyu ( 251 | ! ! B. o i o b-aiio-tiyu y 1.8 idi 13.18 1180 ay; yibbk S i ge V cm z0 : what and t. dyutdek . . y y I 00001; 5 am shcha dev F 35 | Ba ve SHSHKI I! V! I sovek 00000 TEVV, goes | ki re Pot i snu 40 enterit KI noti s nide She Ж tu «Ж «21 fbneth t zo yo s ah ov ee it r uosrev re ta 5,3, 1, im, V-byA", 1585 45. : 1 and shchy ny i so | i ! 1, SN) x 8,83 ne o ve o | Still Isnaui VyBI (IN; we VOYU (r) ref chneneh 01500000 1866 1raU57 (BO); aeibk naOyu o" 1 GO tey bay (bottom of Vona Yae

TN-NMR spectroscopy (IN, MOEBU, TOS5U at (278K)) was performed on the Magap Opyu Riiz (500MHz) facility.

Chemical shifts (4) are expressed in parts per million in decreasing order relative to tetramethylsilane (TM5). Constants

GF) bindings are expressed in Hertz. Example 2: obtaining (4K)-methylhexane KE

Experimental procedures were carried out as in example 1, starting with 1 g of polymer resin, with the only exception that at the appropriate stage (45)-MechNech was replaced by (4K)-MechNech. The obtained product (22Omg, 60 O5mmol, 3095, 92.3956 purity) was characterized using E5-M5, С75Н4124М44Оч1в, 1477.9.

Received: t/2 1499.07 |Ma-Ma!", 1514.94 MKG.

Example C: Cytotoxic activity of ip mygo

The purpose of these studies is to stop the growth of the culture of tumor cells "ip migo" by continuous contact of these cells with the sample being tested.

HUNDREDS OF CRAZY MICE O YANTNNA WAR. lymphoid nebpuyzmi

And Жбы ЮСТЛЯЗ are united about LeNEWYUM erytrolenyuy

AB ObI, 185 lyushynyu 0 orbzh zarnynoma 0 pepbn" zyu VEMENOV NIV Zvnnnoma evil-skinned ground

NTR NTVOE is one of the most important organizations in Russia

Too about some women; t MMoiyu human: Chondroblasts of adenocarcinoma 500 EsSi955 zhedinya - animals: zlenecardinoma with ; back

Td-tzhu NTVZHI yyuidnnia Vfost: Zhaertsinoma. EVOSNVN,

Yew tree EKS.A1740 0 pzodina. Xerostitis plenokyrinomas and prostates; And with kndrogoniivohg

Bk. NIV reduced by receptors:

MAMMARY GLAND; d y Nekm/avai;, (totenral. (o) (o) (av)

З5 и « с з (ог) (ав) (се) со 20

And» 25 o ko 60 bo

MBA-MV-O3I NITVOYU sedino monojuna gland Vdenochviinoma

MAN a divan kih

Your school. ovarian adenocarcinoma of the ovary. nera AR. Obeh "ONE pnashmati ee. (8) matEn; what

Sh rezvlentnyny: F en

D-YAYUV VTV-Y. human kidney carcinoma kidney F

Human pancreatic epithelioid disease Zviloz Zhertsynoma co

A type of colorimetric analysis using the sulforhodamine B reaction (SQV) was adapted for quantitative measurement of cell growth and their viability (according to the technology described by R. May). Sapseg Ipvi.,, 82: 1107-1112). z c In this method of analysis, 96-well culture microplates with a diameter of 1 mm are used (Reaigsioi, 1988; Moztapp, 1983). Most of the cell lines were obtained from the American Type Culture Collection (ATCS), ;" formed from different types of human cancer cells.

The cells were maintained in the culture medium KRMI 1640, 1095 РВ5, enriched with 0.1 g/l penicillin and 45. bAg/l streptomycin sulfate, and then incubated at 372С in an atmosphere of 595 СО» and 9895 humidity. For experiments, cells were collected from subconfluent cultures using trypsin and resuspended in fresh medium before seeding. o Cli I 96- and i mi 5 x103 cli and 195 itins were seeded into well culture microplates, 5 x cells per well, in aliquots of μl (Ce) medium, and left them, allowing them to attach to the surface of the plate under growth conditions in cultural so 50 medium, which does not contain a medicinal product, for 18 hours. Then samples of the tested compound) were added in aliquots of 5 μl in amounts varying from 10 to 10 μg/ml, dissolved in OMZO/EUN/RVZ

Co) (0.5:0.5:99). After a 48-hour exposure, the antitumor effect was measured using the ZKV method: cells were fixed by adding 5 µl of cold 5095 (w/v) trichloroacetic acid (THO) and incubated for 60 minutes at 42. The plates were washed with deionized water and dried. One hundred microliters of ZKV solution (0.495 w/v in 195 acetic acid) was added to each well of the microplate and incubated for 10 minutes at room temperature. Unbound ZKV was removed by washing with 195 acetic acid.

The tablets were air-dried, and the bound dye was solubilized with Tris buffer. Optical densities were read on an automatic spectrophotometric device for reading tablets at one wavelength of 49 Ohm.

On the basis of the data obtained in three parallel wells, the values of the average values of h/- 50 were calculated. 60 Some parameters characterizing the responses of cells were calculated: (zI-growth inhibition, TO|-general growth inhibition (cytostatic effect) and |С- cell death (cytotoxic effect).

The results are given in Table II, there are no significant differences between the given compounds: b5 zE

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Example 4: toxicity of ip mygo

In order to establish the cytotoxicity of the indicated drugs relative to normal cells, 96-well plates were used, seeded at a density of 5,000 cells per well with cells of normal cell b" lines established in accordance with the instructions of the ATCC: AMI-12, normal mouse liver cells, and MKK- 52E, normal rat kidney cells. After seeding the cells in each plate, they were kept overnight before the addition of the test drug. To each well (100 μl medium) was added 1 μl of the drug in the medium in different concentrations (in the final concentration of 1 x 10719-0.01 mg/ml), and then incubated overnight at 372C in the presence of 595 CO. All experiments were repeated at least three times, and two parallel samples were used in each experiment. After 24 hours, an MTZ analysis was performed (aqueous

SeiITieg 96), in accordance with the manufacturer's instructions (RKOMESA) (for all types of cells). Cell viability (mitochondrial activity) was determined by enzymatic conversion of the formazan substrate. "

As can be seen from the results given in Table I, there are significant differences between the compounds of 5-methylhexane

KE and (45)-methylhexane KE is not observed. o) h » Table HER , Liver DIM ISvo (μm)|Kidney MACISvo (μM) (ee) o Example 5: MPD ip mimo in mouse CO-I and in athymic animals

The maximum tolerated dose was determined in CO-I mice and in athymic mice (both sexes) for each (se) drug after a single bolus administration and after 5 daily administrations. The results are shown in table 50 of IM, where no significant differences between the compounds of 5-methylhexane KE and (45)-methylhexane KE are observed.

Co.)

Table IM 00 metypeyuovnovy ke|ovumetiyuvnovy ke

Male mice SO-I Bolus MPD o bo

Example 6: Efficacy of ip mimo in breast xenotransplantation (5003) bB The effectiveness of 5-methylhexane KE and (45)-methylhexane KE, each of which was administered in the amount of three-quarters of the maximum tolerated dose (MTD), was studied in athymic female mice, which after xenotransplantation of the mammary gland intravenously (iv) was administered for five days in a row (i.e. in the mode of

OO x5) bolus injections (days 0-4). The compounds were used in the form of freshly prepared solutions sealed in ampoules in the composition with a carrier (Cremophor-EI "ethanol/water (5:5:90)). Each daily dose (in the ОХОх5 mode) was administered

Intravenously in a volume of 0.2 ml of solution for injections per 20 grams of the animal's weight.

Evaluation of actual tumor size in the appropriately treated groups relative to the vehicle control group (i.e., 95 BP/AU) showed that the lowest (optimal) value was reached on day C after initiation of drug administration in all groups. In addition, pairwise statistical analysis (using the non-parametric Mapp-MUpiipeu method) revealed that there is a significant 70% difference in potency between the two indicated compounds, which is quite surprising considering that these compounds have very similar structures , as well as in the light of the results given in the previous examples.

Based on the efficacy studies described here and the earlier toxicity experiments (Example 4), a therapeutic index of at least 1.33 (1xHMPD/0.75xMPD, the dose at which the drug is toxic/the dose at which the drug is effective) can be approved for compound 75 (45)-methylhexane CR. In addition, another related biological result was that the antitumor effect of (45)-methylhexane KE on mammary gland was longer than on prostate grafts (Example 7) at the same relative dose of MPD. In summary, it can be argued that (45)-methylhexane KE is definitely the more potent isomer against mammary gland transplants, and its duration of biological action suggests that it has a longer-lasting effect on tumors of this type. 00000011 dose (mok) Actual size of the tumor, mmo, pe in ATAS.

Zhentol 11111611 royu sch o

Example 7: effectiveness of ip mimo in prostate xenotransplantation (5003)

The effectiveness of the same dose of each of 5-methylhexane KE and (45)-methylhexane KE was studied in the SM of athymic male mice, which, after xenotransplantation of the prostate gland, were intravenously (iv) administered for five consecutive days (i.e., in the OO x5 mode) boluses of injections (days 0-4). The compounds were used in the form of freshly prepared solutions sealed in ampoules in the composition with the carrier |cremophor-EI /"ethanol/water (5:5:90)|. Ge)

Each daily dose (in the mode of ОХОх5) was administered intravenously in a volume of 0.2 ml of solution for injections per 20 grams of the animal's weight.

Evaluation of actual tumor size in the appropriately treated groups relative to the vehicle control group (ee) (i.e. 95 BP/AU) showed that the lowest (optimal) value was reached on day C after initiation of drug administration in all groups. In addition, with the help of pairwise statistical analysis (using the non-parametric Mapp-MUpiipeu method) it was possible to find out that a significantly greater efficiency was achieved with the help of (45)-methylhexane KE at a dose of 262 μg/kg/day. Based on the efficacy studies described here and earlier toxicity experiments (Example 4), the therapeutic index ts is at least 1.33 (1HMPD/O.75xMPD, the dose at which the drug is toxic/the dose at which the drug is "" effective ) can be approved for the compound (45)-methylhexane KE. The results are shown below" in the MI table. so 00001011 dose (mc) Actual size of the tumor, mm, pelo giv ATS. o kit 00000016 what o 50

Example 8: antitumor activity of analogues of kahalalide ER studied on hollow fibers with

Using a panel of human tumor cell lines

The antitumor activity of the kahalalide E analogues discussed above was tested on the hollow fiber system (HFS) using a panel of human tumor cell lines, namely 5K-Ner-1 (hepatoma), Nerb2 (hepatocellular carcinoma), Raps-1 (pancreatic) cells. glands) and Me!I-28 (melanomas). Human tumor cells were encapsulated in hollow fiber ip migo and then implanted into athymic female ip mimo mice. ime) Doses of 5-methylhexane KE and (45)-methylhexane KE were selected on the basis of previous MPD experiments conducted on athymic mice, as a result of which a dosage of 325 μg/kg/day was selected (see example 5). 60 For five days in a row, the dose was administered intraperitoneally (i.v.) in a volume of 0.2 ml of solution for injections per 20 grams of the animal's weight.

In general, the KE-4(5)-Mei compound exhibits statistically significant antitumor activity against hepatoma cells (subcutaneously, p/sh), hepatocellular carcinoma (both subcutaneously and intraperitoneally), pancreas (intraperitoneally) and shows a trend toward significance of the effect ( i.e. P-0.059) with subcutaneous administration of 65 for pancreatic tumor and melanoma. In contrast to this compound, KE-5-Mei is active against fewer tumor types, namely only pancreatic tumors (both subcutaneous and intraperitoneal) and melanoma (subcutaneous), but not all types of liver cancer that have been tested .

The summary results are shown below in the table, which clearly shows the differences between these compounds. at

Claims (21)

The formula of the invention 1. The compound of the formula The formula of rm. НМ (8) (8) M МН sch shchi 6) n 9 MH c) MH o M (8) 5-5 sya H (8) o o MH shchi a (22) MH den MN o N a g) o x No i s o 2 no s M ch o n i? and its pharmaceutically acceptable salts, esters and ester salts, tautomers and solvates. (uh) 2. The compound according to claim 1, which is about (45)-MeNech-O-Ma!1-I-Tig-I -MaI-O-MaI-O-Rgo-II -Ogp-O- a/o-Pe -cyclo(O-a/o- Tig-O- aio-Pe-O-Ma1I-I-Ppe-2-OpB-I -Mai) and its pharmaceutical acceptable salts, esters and salts of esters, tautomers and solvates. (se) 3. The compound according to claim 1, which is so 50 (45)-MeNeh-O-Ma!-I-Tig-Y -MaI-Yu-MaI-O-R go-I -Ogp-O- a/o- Pe-cyclo(O-alyo-Tig-O-a/o-Pe-O-Ma!-I-Ppe-2-OPpLB-I -Mai) and its pharmaceutically acceptable salts. What) 4. A compound according to any of items 1-3, which contains at most 25 90 of any other kahalalide. 5. A compound according to any of items 1-3, which contains at most 10 95 of any other kahalalide. 6. A compound according to any of items 1-3, which contains at most 5 90 of any other kahalalide. 7. A compound according to any of items 1-3, which contains at most 2 95 of any other kahalalide. o 8. A compound according to any of items 1-3, which contains at most 1 90 of any other kahalalide. 9. A compound according to any one of items 1-3, which contains at most 0.5 95 of any other kahalalide. co 10. A compound according to any one of claims 1-3, which contains less than 0.5 95 of any other kahalalide. 11. The compound of any one of claims 4-10, wherein the other kahalalide is kahalalide ER having a 5-methylhexyl 60 side chain. 12. A compound as defined in any of items 1-3, which is a compound in essentially pure form. 13. A pharmaceutical composition comprising a compound as defined in any of items 1-12 and a pharmaceutically acceptable carrier, excipient or diluent. 14. A method of treating any mammal, preferably a human, affected by a malignant tumor, which comprises administering to the affected individual a therapeutically effective amount of the compound defined in any of items 1-12. 15. The method according to item 14, wherein the mammal is a human. 16. The method according to item 15, where the patient is affected by a resistant form of malignant tumor that does not give a favorable response to other types of therapy. 17. The method according to item 15, where the malignant tumor is selected from a malignant tumor of the prostate gland, a malignant tumor of the breast, a hepatocellular carcinoma, a melanoma, a malignant tumor of the rectum, a malignant tumor of the kidney, a malignant tumor of the ovary, a malignant tumor of the lung, a malignant tumor of the epithelium, a malignant tumor of the pancreas and tumors that express an increased amount of the oncogene Negg/pei. 18. Use of the compound defined in any of items 1-12 in the production of a medicinal product. 70 19. Use of the compound defined in any of items 1-12 in the production of a medicinal product for the treatment of a malignant tumor. 20. A kit comprising individual containers containing a pharmaceutical composition comprising a compound as defined in any of items 1-12 and a reducing agent. 21. The method of obtaining the compound defined in any of items 1-3 by solid-phase synthesis, where 4-methylhexanoic acid or (45)-methylhexanoic acid is used as the starting compound 7/5. Official Bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2007, M 19, 26.11.2007. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. c schi 6) c (o) (o) "c) d) - and? (ee) («c) se) se) Ko) ime) 60 b5