CZ200132A3 - Feeding system - Google Patents

Abstract

The carrier system, comprising the homeobox peptide or a fragment or derivative thereof, and the active agent is a therapeutically active non-peptide and non-oligonucleotide active agent. The active agent is bound to the carrier directly or can be linked via a linker. In addition, the feed system may also contain a system targeting agent.

Description

Feeding system

Technical field

The invention relates to a novel drug delivery system for target cells. The system can be used to improve the distribution, metabolism and excretion of the active ingredients. The system is effective both intact and dissociated.

BACKGROUND OF THE INVENTION

For many years, the pharmaceutical industry has been concerned with the effective administration of substances with therapeutic effect in such a way that they are delivered to the site of action and their effect is as high as possible. Some of the difficulties lie in the short biological half-life with which these substances are excreted from the body, the disadvantageous deposition of the target tissue or some disadvantageous properties of the active substance itself, such as insufficient solubility, hydrophobicity and the like. For this reason, efforts have been made to improve in this regard, for example, the active substances have been protected from the environment on the way to the site of action, for example, enteric coated tablets, controlled release pharmaceutical dosage forms and the like.

In the development of peptides and pharmaceutical compositions containing them, a further problem arose because of the possible degradation of these substances by enzymes not only in the digestive system but also in the bloodstream. An example of how these problems have been addressed is the inclusion of peptides in liposomes or polymeric microspheres in which the peptides can be delivered to the lymphatic system.

Another problem, especially in the case of substances whose function develops within cells, is the barrier formed by the cell membrane. Thus, it may be possible to increase the half-life of the active agent so that it passes through the organism without degradation, but this procedure is ineffective if it is a substance acting only within the cells and the active substance is not delivered to the cells.

EP 485578 discloses that a homeodomain derived from Drosophila antennapedia, in particular helix 3 of this peptide, may be used as a cell delivery vector. It is said to be a specific chain of 57 amino acid residues from said homeopeptide, referred to as the pAntp peptide. This peptide is said to have the ability to penetrate fibroblasts and embryonic cells in vivo. Emphasis is placed on the last 27 amino acid residues in the sequence that correspond to helix 3 and 4. However, no binding of the pAntp peptides to another peptide or to a drug of another position is described.

In many of the following publications, for example, Derossi D. et al., J. Biol. Chem., 1994, 269, 10444-10450, Joliot AH et al., 1991, The New Biol. 3, 1121-1134 and PNAS, 1991, 88, 1864-1868 as well as Perez F. et al., J. Cell. Sci., 1992, 102, 712-722 describes how a synthetic peptide of 16 amino acid residues derived from the third helix of the above peptide may be used to introduce biologically active products or antisense oligonucleotides into cells. The amino acid sequence used was RQIKIWFQNRRMKWKK (SEQ ID No. 1), also referred to as Penetratin ^R.

• · · «···· · · ·

To prevent enzymatic cleavage of this peptide, Brugidou J. et al., Biochem. Biophys. Res. Comm. 1995, 214 (2), 685-693, a so-called retro-inverso form consisting of D amino acids in reverse order was prepared, with two isoleucine residues at positions 3 and 5 of penetratin replaced by valine and a glycine residue added at the C terminus to facilitate binding to resin. Another retro-inversion form was prepared by replacing said glycine residue with a cholesterol group linked via a sulfhydryl group as a linker. The addition of the cholesterol group improved penetration due to the increased hydrophobicity of the molecule.

This development of the retro-inverso form of penetratin has given rise to the proposal of International Application WO 97/12912 which discloses peptides of 16 amino acid residues containing 6 to 10 hydrophobic amino acids, the sixth amino acid from either end being tryptophan. This defines the minimum properties of the sequences useful as internalization vectors.

Penetratin, its analogs and its retro-inverso forms are described as carriers to facilitate cellular internalization of conjugated peptides or oligonucleotides.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a delivery system for therapeutic agents to facilitate the internalization of an active agent into cells and hence to facilitate the therapeutic effect of the agent. The system can also improve the half-life of the active substance in the body. 4 4 4 4 4 4 4 4 4 4 4

4444 44 44 44 44 444 human or other animal, improve the solubility of substances in biological fluids, reduce known toxic and other undesirable side effects of the active substances, improve the desired therapeutic effect and provide alternative routes of administration while improving metabolism and reduce the risk of resistance substances.

The delivery system of the present invention consists of an active agent bound to a carrier which is a peptide homeobox or a fragment or derivative thereof, wherein the active agent is a non-peptide and non-nucleotide substance.

The invention will now be described with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the stabilization of microtubule formation using the system of the invention.

Figure 2 shows a comparison of internalization by the system of the invention compared to the internalization of the carrier alone.

Fig. 3 also shows the internalization of the system according to the invention.

Giant. 4 illustrates the intracellular stability of the delivery system of the present invention.

The invention will be further described in connection with some preferred embodiments.

• ·

According to a first embodiment, the system according to the invention consists of an active substance which is bound to a carrier. The active ingredient may be bound directly or indirectly to the carrier group. In a preferred embodiment, the bond is indirect through a linking group, for example a sulfhydryl group, carboxyl group, or any larger group, as described below, such a group is usually referred to as a linker.

According to the invention, suitable active substances for this use are any active substances which are not peptide or oligonucleotide in nature. These may include cytotoxic agents, antineoplastic agents, antihypertensive agents, cardioprotective agents, antiarrhythmic agents, ACE inhibitors, anti-inflammatory agents, diuretics, muscle relaxants, local anesthetic agents, hormones, cholesterol lowering agents, anticoagulants, antidepressants, tranquillizers, neuroleptics, analgesics including narcotics and anti-fever agents, anti-viral agents, antibacterial agents, antifungal agents, bacteriostatic agents, CNS agents, anti-convulsants, anti-anxiety agents, antacids, narcotics, antibiotics, respiratory agents, antihistamines, immunosuppressants, immunoactivating agents, nutritional supplements, cough suppressants, diagnostic agents, emetics and antiemetics.

Preferably, the active agent used is a cytotoxic or antineoplastic agent, particularly for use in the treatment of cancer, or in a light-activatable form. These substances generally include compounds, DNA destroyers, antimetabolites, antitumor antibiotics, natural products and analogs thereof, dihydrofolate reductase inhibitors, pyrimidine analogs, purine analogs, cyclin-dependent kinase inhibitors, thymidylate synthetase inhibitors, DNA intercalators, DNA splitting agents, topoisomerase inhibitors, anthracyclines, Vinca family, mitomycins, bleomycins, cytotoxic nucleosides, pteridine compounds, diynenes, podophyllotoxins, platinum-containing substances, differentiators and taxanes. Particularly suitable substances from the above groups are, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, trisubstituted purines such as olomoucine, roscovitine, bohemin and purvalanol, flavopiridol, staurosporine, cytosinarabine, melosinarabine, melphine, mine, , mitomycin A, carninomycin, aminopterin, tallysomycin, podophyllotoxin and its derivatives, etoposide, cisplatin, carboplatin, vinblastine, vincristine, vindesine, paclitaxel, docetaxel, taxoterretinic acid, butyric acid, acetylspermidine, tamoxecin, and tamoxecin, camoxin, camoxin, camoxin, camoxin. Preferably, the active agent is selected from the group of methotrexate, podophyllotoxin and derivatives thereof, etoposide, camptothecin, paclitaxel, doxorubicin, roscovitin and bohemin.

The carrier in the system of the invention may be any moiety capable of facilitating the internalization of the active agent into cells. Particularly suitable carriers are peptide homeoboxes or derivatives thereof, for example, helix 3 of such homeobox. The homeobox-type peptide is preferably derived from the Drosophila Antennapedia homeoprotein, homologues or derivatives of this sequence can also be used. Preferably, the carrier is penetratin or a derivative thereof. Penetratin derivatives which have been described in the literature, for example in EP 485578B, describe sequences homologous to pAntp. Other penetratin derivatives which may be used for the purposes of the invention include the truncated forms and / or modified forms of penetratin as described in WO 97/12912, UK Publication No. 9825000.4, filed November 13, 1998 and 9902522.3, filed February 4, 1998. 1999. A preferred truncated form of penetratin is the sequence RRMKWKK (SEQ ID No. 2). Other truncated forms include sequences of up to 15 amino acid residues, including NRRMKWKK, QNRRMKWKK, and FQNRRMKWKK, preferred sequences are peptides containing 7 amino acid residues of the group KRMKWKK, RKMKWKK, RRKKKK, RRKKWKK, RRKKWKKK, RRKKWKKK, RRKKWKKK, RRKKWKKK standard features are used for amino acids, especially for ornithine (O), diamino butyric acid (B) and norleucine (N).

With respect to penetratin or derivatives thereof, another modification which is preferred within the scope of the invention is the conversion of the free carboxylic acid at the carboxyterminal end of the amino acid into a carboxamide group. By way of example, when the carrier is penetratin, the carboxyl group of the terminal lysine can be converted to the carboxamide group. Such modification will increase the stability of the entire group and hence the delivery system as such.

The carrier can be in the optically active form L or D. Unless otherwise indicated, the carrier is in the L form. D-penetratin has been described by Brugidou J. et al., Biochem. Biophys. Res. Comm. 1995, 214 (2), 685693. The carrier can also be converted to a retroform, that is, the amino acid residues are in reverse order.

9 9 9 9 9 9

999999 9 9 9 9 9 9 9 relative to the original sequence. Also, these retroforms may exist as forms L and D. In one preferred embodiment, the carrier may be a D-penetratin or a D-form of a truncated and / or modified form of penetratin.

The active ingredient may be attached to either end of the carrier. For example, if the carrier is penetratin, corresponding to SEQ ID NO. 1 or a derivative thereof, the active ingredient may be bound directly or indirectly to the terminal lysine or arginine. Preferably, the active ingredient is bound to the amino terminal terminus of the carrier.

As mentioned above, the active ingredient and the carrier can be coupled directly or indirectly via a linker. The direct bond may be any conventional functional bond through conventional groups, for example, hydroxy, carboxy, or amino. The indirect bond, which is more preferably, takes place via a linking group. Such a group may be a bi- and multifunctional alkyl, aryl, aralkyl or peptide group, alkyl, aryl or aralkylaldehyde, acid esters and their anhydrides, sulfhydryl or carboxyl groups such as maleimidobenzoic, maleimidopropionic, succinimidoderivatives or derivatives derived from bromide or acid chloride cyanuric, carbonyldiimidazole, succinimidyl esters, sulfonic acid halides and the like. The functional groups used to form covalent bonds between the linker and the active agent, on the one hand, and between the linker and the carrier, on the other hand, may be amino, hydrazine, hydroxyl, thiol, maleimido, carbonyl or carboxyl groups and the like. two or more groups. The linker may be a short sequence of 1 to 4 amino acid residues, or alternatively, the linker may be a linker to the carrier.

Preferably, the linker comprises a cysteine residue that readily binds to the carrier to form an active (linker-Cys) -carrier type linkage. In the course of the specification, this cysteine residue is considered to be part of the linker. Thus, a complete linker can only be formed due to the binding of the active ingredient to the carrier, since this cysteine residue can also be prepared as part of the carrier. In a preferred embodiment, the linker is selected from (methylamino) benzoyl-Cys, succinimidobenzoyl-Cys, succinimidopropionoyl-Cys, beta-alanyl-succinyl, actyl-Cys, and (4''-aminoanilino) -succinimidopropionoyl-Cys. In these preferred embodiments, the cysteine residue is preferably initially a terminal residue of the carrier, whereas the non-cysteine component of the linker binds to the active agent prior to reaction with the carrier. Thus, the complete linker is only formed after the reaction of the active ingredient and the carrier.

In the same way as the cysteine residue is incorporated into the linker, other amino acids can also be included in the linker which, like cysteine, form a link with the carrier. For example, 3 or 4 amino acid residues may be included, and one of these residues is again preferably a cysteine residue. However, any amino acid residues may be used, preferably cysteine, beta-alanine and glycine. The inclusion of these residues is particularly advantageous when the carrier is a truncated form of penetratin, for example RRMKWKK.

In use, the delivery system can dissociate by chemical or enzymatic cleavage between

4 4 4 4 4

44

4445 44 4 4

444 444 4444

4 4 4 ·· ·· ίο ···· · * ·· ·· · * · active ingredient and carrier. Where the linker contains amino acid residues, cleavage can occur directly in the linker.

According to the invention, each carrier molecule is bound to at least one active agent molecule. In another embodiment, the carrier is formulated to facilitate binding to more than one moiety derived from the active agent, which moieties may be the same or different. For example, the carrier may contain components which may themselves facilitate binding of more than one active agent, for example, natural amino acid derivatives or polyvalent synthetic amino acids, or the carrier may be specifically modified, for example, using a network of branched lysine residues which may be by a carrier linked as a linking group, wherein each lysine residue may be linked to the active ingredient. In this way, a single carrier molecule can carry up to 32 groups of active ingredient, preferably 2 to 10 groups, and in particular 4 to 5 groups. In another embodiment, each drug moiety may be directly or indirectly bound to the carrier by the same or different linker. In the case of the binding of a plurality of types of active substances, the proportions and doses of the individual active substances can be adjusted to facilitate the administration of a specific combination of substances.

Preferred examples of this embodiment are. a penetratin carrier having a network of lysine residues attached to at least one end to facilitate binding of up to 32 active agent moieties, or wherein the carrier is penetratin or a derivative thereof, for example a truncated derivative, corresponding to SEQ ID NO. The active ingredient may be a podophyllotoxin at both ends of the carrier or an epipodophyllotoxin having a linker of succinimidopropionyl group.

9 9 9 9 9 · ··· «···« · · ·

9 9 9 9 9

9999 99 99 99 99 999 camptothecin or paclitaxel.

In a particularly preferred embodiment of the invention, the nodic agent is penetratin or a derivative thereof, which is indirectly bound to the active agent of the group of doxorubicin, methotrexate, podophyllotoxin and derivatives thereof, etoposide, camptothecin, paclitaxel, doxorubicin, roscovitin and bohemin.

According to another embodiment of the invention, the system may further comprise a group for targeting the system. This group brings the system to a specific cell type in which the function of the active ingredient is most advantageous. Thus, this group affects the natural distribution of the active ingredients towards a particular cell type. This system targeting moiety may be bound to the active agent, but is preferably bound to a carrier.

Suitable groups of this type are, for example, the peptide sequences described in E. Ruoslahti et al., U.S. Pat. No. 5,622,699, Pasqualini, R. Ruoslahti,

E. Nature (London), 1996, 380, 364-366, Ruoslahti, E.

Ann. Roar. Cell. Dev. Biol., 1996, 12, 697-715 and Arap, W., Pasqualini, R., Ruoslahti, E. Science 1998, 279, 377-380.

These publications describe certain peptides that can deliver the system to a particular cell type. Upon delivery to the appropriate cells, the internalization of the active agent by the carrier is facilitated.

The systems described are new chemicals, their specific composition is shown in the following table.

«· ···· • 9 ·· 99

9 9 9

9 9

999 9 ·

• 99

•

9 9

# Active substance Linker Carrier (methotrexate) < ((methylamino) benzoyl-EGpA) ₄ (L ^ pARQKIWFQNRRMKWKK- OH doxonibicin succinimidobenzoyl-C RQIKIWFQNRRMKWKK-OH doxorubicin succinimidobenzoyl-C (DK) (DK) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) DR- _NH2) paciitaxei 2 &apos; -succinimidopropionoyi-C RQIKIWFQNRRMKWKK-OH N-tenn C-tetm paclitaxel carboxyfluorescein 2’-sU <cinimidopropionoyl- GCG BYE RQIKIWFQNRRMKWKK paciitaxei 2 'succininiidopropionoyl-C rqikiwfqnrrmkwkk-nh ₂ paciitaxei 2’-succininndoproptonoyl- C0A rrmkwkk-nh ₂ paciitaxei 7-succininopropionoyl-C RQIKIWFQNRRMKWKK-OH podo; philotoxin 4-succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH N-tenn C-tenn by philotoxin · biotinamidocaproyi 4-succinimidopropionoyl GCG BYE RQIKIWFQNRRMKWKK by philotoxin 4-succinimidopropionoyl-C rqikiwfqnrrmkwkk-nh ₂ podo phyilotoxin 4-succinimidoproptonoyl-C (D) (D) (D-QXD-I) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) ) (DK - NH ₂ ) by philotoxin 4-Succinimidopropionoyl-CpA rrmkwkk-nh ₂ pod fsyllotoxin 4-succinimidopropionoyl-CpA (DR) (DR) (DM) (D-W-KXD) (DK) _(DK-NH2) epipodo philotoxin 4 'succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH epipodo philotoxin 4 &apos; -succinimidopropionoyl-C RQIKIWFQNRRMKWKK-NH ₂ . epipodo philotoxin 4’-sufccininiidopropíonoylC0A. RRMKWKK-NHj 4'-demethyi epipodo philotoxin 4-Succininudopropionoyl-C RQIKIWFQNRRMKWKK-OH etoposide (G2, G3 and £) succindmidopropionoyl-C RQIKIWFQNRRMKWKK-OH roscovotin sudfinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH bahemm PA-succinyl-βΑ RQIKIWFQNRRMKWKK-OH bohemin Succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH by philotoxin 4-acetyl-C RQIKIWFQNRRMKWKK-OH by philotoxin 4-Acetyl-CpA RRMKWKK-NHi 4'-demethyl epipodo phyilotoxin 4-acetyl-COA RRMKWKK-NHi 4'-demethyi epipodo, phiotoxin 4-acetyl-C rqikiwfqnrrmkwkk-nh ₂

··· · ··

pod, phyllotoxin 4-succinimidopropionoyl GCpA rrmkwkk-nh ₂ camptothecin IO-O-suRcinimidopropionoyl C RQIKIWFQNRRMKWKK-NH2 C-tenn N-tenn podo ¹ phyllotoxin podo phyllotoxin 4-succinimidopropionoyl-C 4-Succinopropionoyl-C RRMKWKK N-temt C-term epipodo phyllotoxin camptothecin 4’-suJ (CiniiniopropionoyI-C 10-O-su cinimidopropionoylC RRMKWKK N-tenn C-tenn epipodotoxicin paclitaxel 4’-succinimidopropionoyI-C 2 ’- (succinunido) propionoyl-C RRMKWKK 4 &apos; -methoxyepipodcylllotoxin 4- (4 ”-aminoanilino) sutetninudopropionoyi-C RQEKIWFQNRRMKWKK-NHj 4´-methoxyepipodo phyllotoxin 4- {4 ”-ajnnioanilino) succinimidopropionoyl-CPA YYMKWKK-NHz 4 &apos; -demethylepipodo phyllotoxin 4- (4 ”-aminoaniIino) succinimidopropionoyl-CPA YYMKWKK-NHz

The therapeutic effect may be induced by the intact system or its dissociated components after administration of the delivery system of the invention, which may include the active agent as such or bound to a linker, a linker or linker moiety and a carrier moiety. That is, the term "delivery system" means all or any part of the system that has the desired effect.

Said systems may be prepared by any known method. For example, the pAntp peptide can be assembled by conventional solution or solid phase peptide synthesis to provide a protected precursor in which only the deprotected terminal amino group is reactive. This group can then be directly reacted with the active ingredient or a suitable reactive derivative thereof. This amino group can also be converted into a different functional group suitable for reaction with the active ingredient or linker. For example, an amino group is possible.

It is allowed to react with succinic anhydride to give a carboxyl end group suitable for some types of linkages, while the cysteine derivative yields a thiol group capable of various selective linkages. Once the desired functional group has been obtained, the active ingredient or derivative thereof can be coupled, for example by forming an amide, ester or disulfide bond. It is also possible to link a linker, for example a mmaleimidobenzoyl group, by reacting a linker precursor with a suitable function, followed by forming a covalent bond between the linker and the active agent. In this way, more binding structures can be prepared, for example by progressively extending the precursor using trivalent chemical groups. For example, elongation of the peptide chain using N ^{alpha, epsilon} -Fmoc-protected lysine derivatives leads to the formation of di-, tetra- and octavalent precursor constructs after 1, 2 or 3 binding cycles, each associated with deprotection.

Using these procedures, one of ordinary skill in the art can prepare a wide variety of conjugates of the active ingredients and carriers using a variety of linkers. As will be described hereinafter, the appropriate active ingredient may be selected for association with a particular carrier and optionally a linker, wherein the linker may be coupled to the active ingredient or carrier prior to binding of the two components.

The systems of the invention may be formulated, together with a physiologically acceptable diluent or carrier, into pharmaceutical compositions for use in mammals, particularly in human or veterinary medicine. These compositions may be administered, for example, together with a liquid diluent or carrier, which may be a solution, suspension, or.

Emulsions in water or oil, often as a solution for injection, suitable for parenteral administration, which must be sterile and pyrogen-free. Oral administration also involves a liquid diluent or carrier, but more usually is a solid carrier such as starch, lactose, dextrin or magnesium stearate. These solid compositions may be in powder form, but are usually formulated into more convenient dosage forms, such as tablets or capsules. Liposomes and nanoparticles can also be used.

In addition to injection and oral administration, the active compounds may also be administered in the form of suppositories or pessaries. Other useful pharmaceutical forms are substances for administration through the oral mucosa or nasal mucosa or through the entire airways, including the alveoli. Topical formulations include emulsions, ointments, creams, gels and sprays.

The compositions of the invention may be formulated for single administration, i.e., formulated in divided or divided doses.

As will be further described in the Examples section, the delivery system of the invention provides a number of advantages over the present invention

Also known systems for administration of non-peptide and neoligonucleotide agents. These advantages are higher efficacy compared to conventional formulations, increased cell uptake, increased water solubility, reduced side effects, good bioavailability and reduced incidence of drug resistance.

The invention is illustrated by the following examples, which are not intended to limit the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

The amino acid and peptide nomenclature complies with the IUPAC-IUB rules of Eur. J. Biochem. 1984, 138, 937. Other abbreviations used are: AcOH = acetic acid, Boc = tert-butyloxycarbonyl, Bu ^t = tert-butyl, DE MALDI-TOF MS = desorption time in mass spectrometry after laser extraction of the matrix, DIC = 1, 3-diisopropylcarbodiimide, DIEA = diisopropylethylamine, DMAP = 4-dimethylaminopyridine, DMEM = modified Dulbecco's Eagle medium, DMF = dimethylformamide, Et ₃ M = triethylamine, EtOAc = ethyl acetate, Et ₂ O = diethyl ether, FCS = fetal calf serum, HOBt 1-hydroxybenzotriazole, MeCN = acetonitrile, MeOH = methanol, NMR = nuclear magnetic resonance, PE = petroleum ether fraction boiling at 40 to 60 ° C, PBS = physiological saline solution with phosphate buffer, Pmc = 2,2,5, 7,8-pentamethylchroman-6-sulfonyl, PyBOP = benzotriazol-1-yloxytrispyrrolidinephosphonium hexafluorophosphate, RP-HPLC = reverse-phase high performance liquid chromatography,

TFA = trifluoroacetic acid, Trt = triphenylmethyl.

General procedures

RP-HPLC was performed using Vydac 218TP54 (4.5 x 250 mm) and 218TP1022 (22 x 250 mm) columns for analytical and preparative purposes. The flow rate was 1 ml / min for analytical purposes and 9 ml / min for preparative purposes. Elution at 25 ° C was performed using an increasing MeCN gradient in water containing a constant concentration of 0.1% TFA, elution time being 20 minutes for analytical procedures or 40 minutes for preparative procedures. Flash chromatography was performed according to the method of W. C. Still, M. Kahn, A. Mitra, J. Org. Chem., 1978, 43, 2923-2925 using Merck 60 silica gel with a particle size of 230 to 240 mesh. Peptide synthesis was performed on an ABI 433A (Perkin-Elmer Applied Biosystems). Amino acid derivatives were obtained from Novabiochem AG, Laufelfingen, Switzerland, with the exception of Fmoc-D-Ile-OH, which was obtained from Bachem AG, Bubendorf, Switzerland. Standard synthesis protocols of 0.1 mmol or 0.25 mmol were used (FastMoc MonPrevPk programs) using the Fmoc protecting group of G. B. Fiels, R.L. Noble, Intl. J. Peptide Protein Res., 1990, 35, 161. Peptidyl resins were cleaved and deprotected using phenol, water, thioanisole, 1,2-ethanedithiol and TFA in a ratio of 0.75: 0.5: 0. 5: 0.25: 10 (w / v / v / v / v / v) according to DS King, CG Fields, GB Fields, Intl. J. Peptide Protein Res., 1990, 36, 255. DE MALDI-TOF MS was performed using a Dynamo spectrometer (Thermo BioAnalysis, Hemel Hempstead, UK). The matrix used was alpha-cyano-4-hydroxy cinnamic acid. The spectrometer was calibrated using an appropriate amount of authentic peptide. NMR spectra were recorded on a Brucker DPX300. Paclitaxel, podophyllotoxin and 10-hydroxycamptothecin were obtained from Hande Tech Development Co. USA, Houston, TX, USA.

4'-demethylepipodophyllotoxin was prepared according to M. Kuhn, C. Keller-Juslen, A. von Warburg,

Helv. Chim. Acta, 1969, 52,944). Roscovitin was 4 4 4 4 4 4 4 4 4 4 4 4 4 4

IQ · 4 4 4 4 4 4

Připraven * 44 * 44 44 4 * »· 4 prepared according to the publication L. Havlíček, J. Hanuš, J.

Vesely, S. Leclerc, L. Meijer, G. Shaw, M. Strnad, J.

Copper. Chem. 1997, 40, 408. Bohemin, chemically 6- (benzylamino) -2 - [(3- (hydroxypropyl) amino] -9-isopropylpurine) was synthesized in a similar manner.

Anhydrous DMF, CICH 2 CH 2 Cl and CH 2 Cl 2 were stored over a 4A molecular sieve.

Example 1

H-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) ) -Trp-Lys (Boc) -Lys (Boc) -resin

The peptide sequence was initiated using 0.5 mmol / g Fmoc-Lys (Boc) - [(4- (hydroxymethyl) phenoxyacetyl) resin], ABI 401425, 0.5 mmol / g of the following amino acid was added each time. The final peptidyl resin was obtained at a yield of 100% in an amount of 1.37 g. The material was washed with Et 2 O and dried under vacuum. To demonstrate the chemical integrity of this intermediate, a small proportion of the peptidyl resin was cleaved and deprotected, and the crude product H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met was analyzed. -Lys-Trp-Lys-Lys-OH, product purity was greater than 90% when analyzed by RP-HPLC, chemical identity was demonstrated by DE MALDI-TOF MS and quantitative amino acid analysis.

[H-Glu (OBu ¹¹ ) -Gly-bAla] ₄ -Lys2-Lys-bAla-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) -Resins • · · · · ·

44 4

137 mg, 25 micromoles of the above peptidyl resin was acylated using 47 mg, 0.15 mmol Fmoc-betaAla-OH, 78 mg, 0.15 mmol PyBOP, 20 mg, 0.15 mmol HOBt and 39 microliters, 0.225 mmol DIEA in 2 ml DMF over 2 hours. The Fmoc protecting group was then removed by treatment with 20% piperidine in DMF for 20 minutes and the material was washed thoroughly with DMF. The product was then elongated sequentially by double acylation and deprotection using Fmoc-Lys (Fmoc) -OH at 0.15 mmol in the first cycle and 0.3 mmol in the second cycle using a similar bond followed by deprotection. The chain was then extended again using 0.6 mmol of Fmoc-Gly-OH and 0.6 mmol of Fmoc-Glu (OBuA -OH again using acylation followed by removal of the Fmoc protecting group. The resulting Fmoc group and product were removed. was thoroughly washed sequentially with DMF, CH 2 Cl 2 and Et ₂ O, then dried under vacuum To demonstrate the chemical integrity of this intermediate, a small proportion of the peptidyl resin was cleaved and the side chain was deprotected, and the crude product analyzed The product was found to be &gt; 89% pure by RP-HPLC, dradient 20-25% MeCN at 17.7 min, lambda = 200-300 nm, resulting in [H-GluGly-betaAla] ₄ -Lys ₂ - Lys-betaAla-Arg-Gln-Ile-Lys-Ile-TrpPhe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH, the identity of this product was demonstrated by DE MALDI-TPF MS:

[M + H] ⁺ = 3732, Formula C ₁₆ H ₂₇ 69 N ₅₃ O ₄₄ S = 3731.30.

{[4 [N- (2,4 'diamino-6-pteridinylmethyl) -N-methylamino] benzoyl] -Glu (OBu ^t) -Gly-betaAla} ₄ -Lys ₂ -Lys ····. · <· «·· ······

9Π 9,999 9 9 · 9 · 9 9 9

ZU · 9999 ·· ^ 99 · 99 99 99 99

-bAla-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys ( Boc) -Trp-Lys (Boc) -Lys (Boc) - Resin mg, 25 µmol of the above peptidyl resin was reacted overnight with 76 mg, 0.2 mmol of acid hemihydrochloride dihydrate ([4 [N- (2 4-diamino-6-pteridinylmethyl) -N-methylamino] benzoic acid and 104 mg, 0.2 mmol PyBOP, 27 mg,

0.2 mmol HOBt and 70 microliters, 0.4 mmol DIEA in 2 mL DMF. The product was washed sequentially with DMF, CH ₂ Cl ₂ and Et ₂ O, then dried under vacuum to give 85 mg of the resultant peptidyl resin as an orange solid.

{[4 [N- (2,4-diamino-6-pteridinylmethyl) -N-methylamino] benzoyl] -Glu-Gly-betaAla] 4-Lys- _2- Lys-bAla-Arg-Gln-Ile-Lys-Ile- Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH

I i * c η M J 1 * Ϊ s * Ao «

* ·

W \

I · »/

···································

The product obtained above is cleaved and deprotected using 12 ml of protecting group cleavage reagent, reaction time 1.5 hours. The residual resin was filtered off and washed on the sinter with small portions of pure TFA. The filtrate was combined with the wash liquid, 100 mL of Et ₂ O was added and the mixture was cooled. The precipitated product is collected by centrifugation and the ether supernatant is decanted. The product was washed 3 more times with Et ₂ O in a similar manner. The resulting crude product was dried under vacuum to give 61 mg of an orange powder. This material was redissolved in 4 mL of a 0.1% aqueous TFA solution and then filtered. The resulting solution was applied in two separate portions to a preparative RP-HPLC column using a gradient of 17.5 to 27.5% MeCN. The fractions containing the title compound were collected, monitored by analytical RP-HPLC and the appropriate fractions were combined. After centrifugation in vacuo, 13.5 mg of the title compound are obtained. Identity was confirmed by analytical RP-HPLC eluting for 17.8 minutes with a gradient of 17.5 to 27.5% MeCN, product purity greater than 99%, lambda = 200-300 nm. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 4962. Formula C ₂₂₅ H ₃₂ N ₈ O _{4 8} S = 4960.54.

Example 2

H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-MetLys-Trp-Lys-Lys-OH

411 mg, 75 micromole H-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) - The Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) resin according to Example 1 was acylated using 264 mg, 0.45 mmol Fmoc-Cys (Trt) -OH, 234 mg, 0.45 mmol PyBOP, 61 mg, 0.45

9,999 9

99

9

9 9 9

99999

9 9

9999 9 9 99

9 9

9 99

9

9

9 9 9 9 mmol HOBt and 0.12 mL, 0.675 mmol DIEA in 3 mL DMF over 3 hours. The resulting peptidyl resin was washed 3 times with 25 ml DMF for 5 minutes each and then 20% piperidine in DMF was added over 20 minutes. After filtration of the reagent, the product H-Cys (Trt) -Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg ( The Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) resin was washed sequentially with DMF, CH 2 Cl 2 and Et ₂ O and dried in vacuo. The product is then cleaved off within 2 hours and deprotected. The residual resin was filtered off and washed on the sinter with a small amount of TFA. To the filtrate combined with the wash was added 100 mL of Et ₂ O and the mixture was cooled. The precipitated product is collected by centrifugation and the heterogeneous supernatant is decanted. The product was washed 3 more times with Et ₂ O in a similar manner. 238 mg of crude product is obtained, which is dried under vacuum. A portion of 119 mg of this material was redissolved in 2 mL of a 0.1% aqueous TFA solution and filtered. The resulting solution was purified by preparative RPHPLC using a gradient of 17.5 to 27.5 MeCN. The appropriate fractions were separated and monitored by analytical RP-HPLC, whereupon the appropriate fractions were combined. After centrifugation in vacuo, 60.9 mg of pure title compound is obtained. Analytical RP-HPLC: t _R = 15.8 minutes (17.5 to 27.5% MeCN gradient, purity greater than 99%, lambda = 214 nm), DE MALDI-TOF MS:

[M + H] ⁺ = 2351, Formula C ₁₇ H 17 N ₃ O ₂ S ₂ = 2349.87.

N- [3- (meleimido) benzoyl] doxorubicin

OUeO ·· ♦ · 9 9 9 9 9 9 9 9 9

9 9 9 9 9

99999 9 9 9 9 9 9 • 9 9999 99 9

9999 99 99 99 99 99

Dissolve 5.9 mg, 10 µmol of doxorubicin hydrochloride in 1 mL of water and 0.5 mL of DMF. 0.5 ml of 0.1 M aqueous phosphate buffer, pH 7.2 are added with stirring. To the resulting suspension was added 3-maleimidobenzoic acid N-hydroxysuccinimide ester (12.9 mg, 40 µmol) dropwise in 1 mL DMF. The red-colored reaction mixture gradually becomes clear and a precipitate is observed after about 10 minutes. The progress of the reaction was monitored by analytical RP-HPLC, after 2 hours the reaction of doxorubicin was complete. The mixture is diluted with 1.5 ml of water, cooled to 4 ° C and centrifuged. Decant the supernatant. The resulting pellet was redissolved in 1 mL of dimethylformamide and diluted with 2 mL of a 0.1% aqueous TFA solution. This solution was applied to a pre-filled solid phase column (500 mg LiChrolut RP-18 Merck, the column was pre-equilibrated in MeOH with 0.1% aqueous TFA). The column is washed with 4 ml of a 0.1% aqueous TFA solution and then eluted with a 6: 4 MeCN / water mixture, water containing 0.1% TFA, using 2 washings, 2 x 4 ml. The first fraction contained the title compound and was used directly in the next step.

N- {3- [3- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH) Succinimido] Benzoyl } doxorubicin

♦ ♦ © © © © © © © © © © © © © © © © © © © © ©

The above solution of N- [3 (maleimido) benzoyl] doxorubicin was diluted with 1 ml DMF and 50 microliters of Et ₃ N was added. The color of the solution turned dark brown. H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in solution in 1 mL of DMF was then added. The mixture was stirred while the brown color turned to light red as the mixture was stirred. The reaction was monitored by analytical RP-HPLC. After 1.5 hours the reaction is complete

3- (maleimidobenzoyl) doxorubicin terminated. The solution is acidified with 0.5 ml of acetic acid, diluted with 3 ml of water and applied to a pre-filled solid phase extraction column, taking 500 mg of LiChrolut RP-18 (Merck). The column is washed with 6 ml of a 0.1% aqueous TFA solution and then eluted with 6 ml of a 6: 4 mixture of MeCN and water containing 0.1% TFA. The eluate is dried by centrifugation in vacuo. The residue was redissolved in 2 mL of a 0.1% aqueous TFA solution, filtered and purified by preparative RP-HPLC using a gradient of 20 to 40% MeCN. The fractions containing the title compound were collected, analyzed by analytical RP-HPLC and pooled as needed. The material is centrifuged again in vacuo to thereby precipitate. 1.2 mg of pure title compound are obtained. Analytical RP-HPLC: t _R = 15.6 and 15.8 minutes (partially separated thioether diastereomers), gradient 0 to 60% MeCN, purity greater than 95%, lambda = 200 to 300 nm, DE MALDI-TOF MS:

[M + H] ⁺ - 3094, [M + 2H] ²⁺ = 1548, Formula C145B207N37O35S2 = 3092, 56.

Example 3

H-Cy with D-Lys-D-Lys-D-Trp-D-Lys-D-Met-D-Ar gD-Arg-D-Asn-DGln-D-Phe-D-Trp-D-Ile-D- _{Lys-D-Ile-D-Gln-D-Arg-NH2}

Starting from 0.64 mmol / g Rink Amide AM resin (Novabiochem) to give H-Cys (Trt) -D-Lys (Boc) -DLys (Boc) -D-Trp-D-Lys (Boc) - D-Met-D-Arg (Pmc) -D-Arg (Pnc) -Dsn (Trt) -D-Gln (Trt) -D-Phe-D-Trp-D-Ile-D-Lys (Boc) -D -Ile-DGln (Trt) -D-Arg (Pmc) -resins in quantitative yield. The peptide resin was cleaved and deprotected using 10 ml cleavage reagent / g over 2 hours and the crude peptide obtained was isolated by precipitation from Et ₂ O, centrifuged by decanting the supernatant and drying. A portion of 100 ml of this material was redissolved in 2 ml of a 0.1% aqueous TFA solution and filtered. The resulting solution was purified by preparative RP-HPLC using a gradient of 17.5 to 27.5% MeCN to give 36.4 mg of pure final product after centrifugation in vacuo. Analytical RP-HPLC: t _R = 16.3 minutes (gradient 17.5 to 27.5% MeCN, purity greater than 99%, lambda = 214 nm). DE MALDI-TOF MS: [M + H] ⁺ = 2350.1, Formula C107H174N36O20S2 - 2348.89.

N- {3- [3- (H-Cys-D-Lys-D-Lys-D-Trp-D-Lys-D-Met-D-Arg-D-Arg-D-Asn-D-Gln-D)] -Phe-D-Trp-D-Ile-D-Lys-D-Ile-D-Gln-D-Arg-NH ₂ ) succinimido] benzoyl} doxorubicin

99 • 9 9 • 9

9 9 9

9.9 9

9 • 9 9

9

998 9 99

12.6 mg, 17 micromoles of N- [3- (maleimido) benzoyl] doxorubicin and 20 mg, 8.5 micromoles of H-Cys-D-Lys-D-Lys-D-Trp-D-Lys-D-Met- D-Arg-D-Arg-D-Asn-D-Gln-D-Phe-D-Trp-D-Ile-D-Lys-D-Ile-D-Gln-D-Arg-NH2 is dissolved in 1 mL DMF and 100 microliters of Et ₃ N. The mixture was stirred for 2 hours and then quenched by the addition of 0.5 mL of AcOH and 0.5 mL of water, and the resulting mixture was filtered. The filtrate was purified by preparative RP-HPLC using a 20-40% MeCN gradient to afford 6.3 mg of the product as a red solid. Analytical RP-HPLC: t _R = 16.3 min, gradient 0 to 60% MeCN, purity greater than 95%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 3092.7, C145H2O8N38O34S2 = 3091.57.

Example 4

2 '- (maleimidopropionoyl) paclitaxel

A mixture of 29.2 micromoles, 25 mg of paclitaxel, 0.120 mmol, 20.3 mg of 3-maleimidopropionic acid, 66 micromoles, 10.3 microliters of DIC and 1 ml of pyridine was stirred for 1 hour. Then the solvent was evaporated, the residue was dissolved in water and the solution was extracted with methylene chloride. The organic phase was washed with water and saturated sodium chloride solution and then

0 0 · 0 ·

0 0

000 0 0 0 0 0

Dried over magnesium sulphate, the solvent was evaporated to dryness to give 22.2 mg of a colorless solid (76%) which was recrystallized from ethyl acetate / hexane to give pure product.

¹ H-NMR (300 MHz, CDCl ₃ ), δ: 1.13, 1.22, 1.68, 1.91 (s, each 3H, CH 3), 2.23, 2.47 (s, each 3H) , Ac-CH ₃₎

2.35 (m, 2H, H 6), 2.78 (t, 4H, J = 5.40 Hz, CH ₂ ), 2.84 (m, 2H, H 14), 3.81 (m, 2H, CH ₂ ), 3.87 (m, 1H, H 3), 4.26 (m, 2H, H 2 O), 4.44 (dd, 1H, J = 10.87, 4.25 Hz, H 7), 4, 98 (d, 1H, J = 7.69 Hz, H 5), 5.47 (d, 1H, J = 3.45 Hz, H 2 '), 5.68 (d, 1H, J = 7.09 Hz, H 3 ', 6.05 (dd, 1H, J = 9.28, 5.86 Hz, H 2), 6.28 (s, 1H, H 10), 6.18 (t, 1H, J = 8.77) Hz, H13), 6.49 (s, 2H, CH.dbd.CH), 8.16-7.34 (m, 15H) Ph). ¹³ C-NMR (75 MHz; CDCl ₃ ) δ: 10.01, 15.20, 21.22, 22, 54, 23.09, 27.18, 32.90, 33.71, 35.90, 43 , 54, 45.96, 52.86,

58.89, 72.18, 72.53, 74.86, 75.51, 76.02, 79.52, 81.42,

84.89, 126.94, .127.91, 128.74, 128.94, 129.14, 129.45, 129.59, 130.65, 132.39, 133.11, 133.85, 134, 09, 134.46, 137.17, 143.25, 167.45, 168.01, 168.10, 169.77, 170.29, 171.10, 171.69, 204.24.

2 '- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH) paclitaxel'

about me

O

H

'O • ·· 9 ··· 9 9 9 9 9 • ···· · 99 · ···· · ♦ ·· 9 9 9 9 ···

A solution of 10 micromoles, 10.05 mg of 2 '- (maleimidopropionoyl) paclitaxel and 10 micromoles,

23.5 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in 1 mL of DMF are mixed with 1, 39 microliters, 10 micromol Et3N. The reaction mixture was stirred for 1 hour then diluted with 0.5 mL. A 0.1% aqueous solution of TFA and the resulting solution was purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN. 20.5 mg of product are obtained in the form of a colorless solid in 62% yield. Analytical RP-HPLC: t _R = 17.4 min, using a 0 to 60% MeCN gradient, product purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 3355.9, C161H229N37O38S2 = 3354.90.

Example 5

4 (5) -carboxyfluorescein-beta-Ala-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH2

The peptide sequence was generated using Rink Amide AM resin, 385 mg, 0.65 mmol / g (Novabiochem) was obtained, yielding 1.50 g of H-betaAla-Arg (Pmc) -Gin (Trt) - in quantitative yield. Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) ) -Gly-Cys (Trt) -Gly-Resins. A portion of 50 mg, 75 micromoles of this peptidyl resin was stirred for 18 hours in the dark with a solution of 113 mg, 0.3 mmol of 4 (S) -carboxyfluorescein, 156 mg, 0.3 mmol PyBOP, 41 mg, 0.3 mmol HOBt and 78 microliters, 0.45 mmol DIEA in 4 mL DMF. The resin was isolated by sintering and washed sequentially with DMF, CH 2 Cl 2 and Et ₂ O. After drying, the resin was treated for 1.5 hours with 5 ml of the light-free cleavage reagent. Product was isolated ·· ·· * ·

precipitation of Et 2 O and centrifugation gave 237 mg of a yellow powder. A portion of 100 mg of this powder was purified by preparative RP-HPLC using a gradient of 22.5 to 32.5% MeCN to give 36.9 mg of pure title compound as a yellow film after isolation by centrifugation in vacuo. Analytical RP-HPLC: t _R = 18.6 and 19.2 minutes (separated 4- and 5-carboxyfluorescein geometric isomers), gradient 22.5 to 32.5% MeCN, purity greater than 99%, lambda = 214 nm. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2892.2, [M + Na] &lt; ⁺ &^gt;

2913.7, Formula C135H195N39O29S2 = 2892.4.

2 '- [Succinimidopropionoyl- (4 (S) -carboxyfluorescein-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly- Cys-Gly-NH 2) paclitaxel

To a solution of 12.3 micromoles, 12.4 mg of 2 '- (maleimidopropionoyl) paclitaxel and 4.3 micromoles, 12.5 mg of betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg- Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH 2 in 1 mL DMF was added 1.8 µL of Et 3 N. The reaction mixture was stirred for 1 hour then diluted with 0.5 mL of a 0.1% aqueous TFA solution, filtered and purified by RP-HPLC using a gradient of 10 to 70% MeCN to give 3.2 mg of pure title compound as a white solid. ·>

· ♦ ·· «·

· Colorless solid. Analytical RP-HPLC: t _R = 21.6 min, gradient 0 to 60% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 3397.35, Formula C189H251N41O46S2 ⁼ 3397.40.

Example 6

H-Cys-Arg-Gln-Ile-Lys-Ip-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂

Starting from 0.69 mmol / g of Rink Amide AM resin, H-Cys (Trt) -Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) was sequentially assembled. -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) -Resin. After deprotection within 1.5 hours, the reaction was quenched. the crude peptide is obtained by precipitation from Et ₂ O followed by centrifugation and collecting the supernatant, then the product is dried. A total of 472 mg was purified by preparative RP-HPLC using a gradient of 16.5 to 26.5% MeCN to give 109.9 mg of pure product. Analytical RP-HPLC: t _R = 16.0 min, gradient 17.5 to 27.5% MeCN, purity greater than 99%, lambda = 214 nm. DE MALDI-TOF MS: (M + H ⁺ )

2349, 3, C107H174N36O20S2 = 2348.89.

2 '- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2)] paclitaxel

To a solution of 9 micromoles, 9 mg of 2 '- (maleimidopropionoyl) paclitaxel and 9 micromoles, 20.9 mg of H-Cys-Arg-Gln-Ile-Lys. 9

-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH 2 in 1 mL of DMF was added 1.8 µL of ΕϋβΝ. The mixture was stirred for 1 hour, then diluted with 1.5 mL of a 0.1% aqueous TFA solution, filtered and purified by preparative RP-HPLC using a gradient of 10 to 80% MeCN. 15.9 mg of pure title compound are obtained as a colorless solid in a yield of 53%. Analytical RP-HPLC: t _R = 18.5 min, gradient 0 to 60% MeCN, purity greater than 97%.

DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 3353, 6, C3.61H230N38O37S2 - 3353.91.

Example 7

H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2

Starting from 0.69 mmol / g Ring'Amide AM resin (Novabiochem), H-Cys (Trt) -BetaAla-Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys ( Boc) -Trp-Lys (Boc) -Lys (Boc) -resins. After deprotection within 1.5 hours, the crude peptide is precipitated from Et2 / D after centrifugation, decanting the supernatant and drying.

A total of 246 mg was purified by preparative RP-HPLC using a gradient of 6.5 to 16.5% MeCN to give 106.4 mg of pure product. Analytical RP-HPLC: t _R = 15.8 minutes, gradient 6.5 to 16.5 MeCN, purity greater than 95%, lambda = 214 nm. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 1205.4. C52H92N2O9S2 = 1205.55.

2 '- [Succinimidopropionoyl- (H-Cys-beta-Ala-Arg-Arg-Met-Lys)]

-Trp-Lys-Lys-NH ₂ ) paclitaxel 9 9

9

9 9

9 9 9

99 ·· ·

9 99 · ·

9 9

9 9

999

To a solution of 17 micromoles, 17.4 mg of 2 '- (maleimidoproinoyl) paclitaxel and 15 micromoles, 18.1 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1 ml DMF 2.0 µl of Et ₃ N is added. The mixture is stirred for 1 hour, then filtered and purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN. 9.4 mg of product are obtained as a colorless solid. Analytical RP-HPLC: t _R = 17.2 min, gradient 0 to 60% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 2211.7, Formula C106H14SN22O26S2 = 2210.57.

Example 8

2'-methoxyacetyl-7- (maleimidopropionoyl) paclitaxel

A solution of 29 micromol, 25 mg of paclitaxel, 0.176 mmol, 32.8 mg of methoxyacetic acid N-hydroxysuccinimidyl ester and 0.176 mmol, 30.6 microliters of DIEA in 1 ml of methylene chloride is refluxed for 4 hours. cooler. Then 1.6 microliters of methanol are added. The mixture was stirred for 10 minutes and then washed with 0.1 M aqueous HCl, water, saturated sodium chloride solution, and then dried over magnesium sulfate. The solvent was evaporated in vacuo to give 24.8 mg (91%)

2 '- (methoxyacetyl) paclitaxel as a white solid. 30 [mu] mol of this material together with 3-maleimidopripionic acid, 4.1 [mu] l, 90 mmol of DIC and 20 [mu] mol, 2.6 mg of DMAP are dissolved in 2.5 ml of methylene chloride and stirred for 40 minutes. The mixture was washed with water and dried over magnesium sulfate. The solvent was evaporated in vacuo to give a pale yellow solid which was dissolved in a mixture of DMF and methanol, filtered and purified by preparative RP-HPLC using a gradient of 20 to 70% MeCN to give pure product as a 76% yield. colorless solid 24.4 mg. Analytical RP-HPLC: t _R = 21.8 min, gradient 10 to 70% MeCN, purity greater than 98%.

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 1, 15, 1.20, 1.79, 1.96 (s, each 3H, CH ₃ x 4), 2.20, 2.45 (s, each 2H, Ac-CH ₃ x 2)

2.34 (m, 2H, H 6), 2.63 (m, 4H, H 14, CH ₂ ), 3.40 (s, 3H, OCH ₃ ), 3.73-3.94 (m, 3H, CH ₂ , H 3), 4.16-4.21 (m, 2H,

H20) 1, 4, 97 (d, 1 H, J = 8.06 • Hz, H5) 5.54-5.69 (m, 3H, H7, H2, H 3 ', 5.98 (m, 1H, H2 '), 6 22 (s, 1H, H 10), 6.24 (m, 1H, H13), 6.68 (with, 2H, CH = CH) 7.12-8.13 (m, 15H,

Ph).

2'-Methoxyacetyl-7- [succinimidiopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] paclitaxel

To a solution of 11 micromol, 12.3 mg of 2'-methoxyacetyl-7- (maleimidopropionoyl) paclitaxel and 11 micromol, 26.8 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln- Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in 1 ml DMF was added 1.58 microliters, 11 micromol Et3N. The mixture was stirred for 2 hours, then diluted with 0.5 mL of a 0.1% aqueous TFA solution and purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN. 15.5 mg of pure product are thus obtained as a colorless solid in a yield of 40%. Analytical RP-HPLC: t _R = 15.1 minutes (gradient 10 to 70% MeCN, purity greater than 97%). DE MALDI-TOF MS: (M + H] ⁺ = 3425, 99, the molecular formula Ci64H 33N370 _{2 4} A ₂ = 3424.96.

7- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Paclitaxel · · · · ·

K 35 micromol, 11.9 mg of 2'-methoxyacetyl-7- [succinimidiopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp) -Lys-Lys-OH) paclitaxel in 1 mL of methanol was added 0.21 microliter of methanolamine. The mixture was stirred for 1 hour, diluted with 0.5 mL of a 0.1% aqueous TFA solution, filtered and purified by preparative RPHPLC using a gradient of 10 to 70% MeCN. 5.6 mg (48%) of pure product are thus obtained as a colorless solid. Analytical RP-HPLC: t _R = 14.3 min, gradient 10 to 70% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 3355.7, C161H229N37O38S2 = 3354.90.

Example 9

2 '- (p-methoxytrityl) paclitaxel

O

A solution of paclitaxel (0.632 mmol, 540 mg) and p-methoxytrityl chloride (10 molar equivalents) in methylene chloride (10 ml) was treated with pyridine (1.3 ml) under nitrogen. The mixture was stirred for 22 hours and then the solvent was evaporated

• · · · · · ·

O r · · · · ³⁶ .. · vacuum. The residue was redissolved in ethyl acetate, washed with water and saturated sodium chloride solution and then dried over magnesium sulfate. The solvent was evaporated to give a pale yellow solid which was purified by flash chromatography using EtOAc / PE 8: 9 to give pure product in quantitative yield. Recrystallization from ethyl acetate / methylene chloride gave a pale yellow crystalline solid.

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 1.08, 1, 15, 1.51, 1.65 (s, each 3H, CH ₃ x 4), 1.90 (m, 1H, H6), 2.25, 2.29 (s, each 3H, Ac-CH ₃ x ₂ ), 2.55 (m, 1H, H 6), 2.54 (m, 2H, H 14), 3.75 (s, 3H, OCH ₃ ), 3.66 (m, 1H, H 3), 4.20 (m, 2H, H 2 O), 4.40 (m, 1H, H 7), 4.62 (m, 1H, H 2 '), 4 94 (d, 1H, J = 8.06 Hz, H 5), 5.61 (m, 1H, H 2), 5.70 (m, 2H, H 13, H 3 '), 6.19 (s,

1H, H10), 6.72-8.08 (m, 29H, Ph).

2 '- (p-methoxytrityl) -7- (maleimidopropionoyl) paclitaxel

micromol, 38.4 mg of 2 '- (p-methoxytrityl) paclitaxel and 125 microliters of pyridine are dissolved in 2 ml of methylene chloride. A solution of 1.48 mmol, 250.5 mg of 3-maleimidopropionic acid, 0.80 mmol, 101.5 mg of DIC and 10 mg of DMAP in 2 ml of methylene chloride was then added and the mixture was stirred for 1 hour. The solvent was evaporated and the slurry was partitioned between water and methylene chloride. The organic layer was washed with water and saturated sodium chloride solution and dried over sulfate.

magnesium. Evaporate the solvent and purify the residue by thin layer chromatography by centrifugation (Chromatrotron ^R ) using EtOAc / PE 5: 4. Recrystallization from ethyl acetate / methylene chloride yielded 22 mg of the product as a colorless solid (49% yield).

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 1, 18, 1, 12, 1.76, 1.96 (s, each 3H, CH ₃ x 4), 2.17, 2.26 (s, each 3H, Ac-CH ₃ x 2)

2.10, 2.34 (m, 2H, H6), 2.62 (m, 4H, H14, _CH2 -Mim), 3.75 (s, 3H, _OCH3), 3, 73-3,79 (m, 3H, _CH2 -Mim. H3), 4.06 (m,

2H, H 2 O), 4.61 (d, 1H, J = 3.47 Hz, H 2 '), 4.76 (d, 1H, J = 9.52 Hz, H 5), 5.53 (m, 1H, H7), 5.60 (d, 1H, J = 6.98 Hz, H 3 '), 5.71 (m, 1H, H 2), 6.14 (s, 1H, H 10), 6.60 (m 3H, H 13, CH = CH), 6.75-7.79 (m, 29H, Ph).

7- (maleimidopropionoyl) paclitaxel

A solution of 17 micromol, 22 mg of 2 '- (p-methoxytrityl) -7- (maleimidopropionoyl) paclitaxel, 1.72 mmol, 186.4 mg of anisole and 0.172 mmol, 16.3 mg of chloroacetic acid in 10 mL of methylene chloride was stirred for 5 hours . The reaction mixture was then washed with a 1% aqueous sodium carbonate solution

Water, a saturated sodium chloride solution, and then dried over magnesium sulfate. The solvent was evaporated to dryness and the residue was purified by thin layer chromatography by centrifugation (Chromatotron ^R ) using EtOAc / PE 1: 1 to give 24 mg of pure product as a white solid which was recrystallized from ethyl acetate / methylene chloride. .

¹ H-NMR (300 MHz, CDC1 ₃₎ delta: 1, 15, 1.18, 1.20, 1.76,

2.04 (s, each 3H, CH 3 X 4), 2.18, 2.37 (s, each 3H, AcCH ₃ x ₂ ), 2.34 (m, 2H, H 6), 2.64 (m, 4H, H 14, CH ₂ ), 3.783.91 (m, 3H, CH ₂ , H 3), 4.12 (m, 2H, H 2 O), 4.71 (d, 1H, J.

= 3.25 Hz, H 2 '), 4.94 (d, 1H, J = 8.17 Hz, H 5), 5.54 (dd,

1H, J = 10.46, 7.21 Hz, H7), 5.66 (d, 1H, J = 6.88 Hz,

H3 '), 5.80 (dd, 1H, J = 8.92, 2.42 Hz, H2), 6.15 (m, 1H, H13), 6.18 (s, 1H, H10), 6, 68 (s, 2H, CH-CH), 7.10-8.12 (m, 15H, Ph).

7- [succinimidiopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] paclitaxel

To a solution of 4.8 micromol, 4.8 mg of 7- (maleimidopropionoyl) paclitaxel and 4.8 micromol, 11.2 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg -Arg-Met-Lys-Trp-Lys-Lys-OH in 1 mL DMF was added 0.67 microliters of Et ₃ N. The mixture was stirred for 30 minutes, then filtered and purified by preparative RPHPLC using a gradient of 10 to 70% MeCN. which in the

yield 54% yields 8.6 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 14.3 min, gradient 10 to 70% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 3355.0, C161H229N37O38S2 ⁼ 3354.90.

Example 10

4- (maleimidopropionoyl) podophyllotoxin

A solution of 60 µmol, 25.6 mg podophyllotoxin, 0.31 mmol, 52.4 mg 3-maleimidopropionic acid, 0.17 mmol, 21.5 mg DIC and 80 µmol, 10 mg DMAP in 2 mL methylene chloride was stirred for 1 hour. The solvent was evaporated in vacuo and the residue redissolved in 1 ml DMF / methanol and purified by preparative RP-HPLC using a gradient of 20 to 70% MeCN to give 7.3 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 20.1 minutes, gradient 0 to 60% MeCN, purity greater than 95%.

¹ H-NMR (300 MHz, CDC1 ₃₎ delta: 2.66-2.71 (t, J = 6.3 Hz, 2H, CH _2), 2.82 to 2.84 (m, 2H, H2 and H3), 3.69 (s, 6H, _OCH3 x2), 3.75 (s, 3H, _OCH3), 3.83 (t, J = 6.3 Hz, 2H, _CH2), 4.12 (t, J = 9.92 Hz, 1H, H11), 4.31 (m, 1H, H11),

4.53 (d, J = 11.4 Hz, 1H, H 1), 5.80 (d, J = 8.7 Hz, 1H, H 4), 5.92 (dd, J = 5.49, 1, 17 Hz, 2H, OCH ₂ O), 6.32 (s, 2H, H 2 '6'), 6.47 (s, 1H, H 8), 6.66 (s, 2H, CH = CH), 6, 74 (s, 1H, H 5 ').

4- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Podophyllotoxin

To a solution of 8 micromoles, 5 mg of 4- (maleimidopropionoyl) podophyllotoxin and 7.7 micromoles, 18 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys -Trp-Lys-Lys-OH in 1 ml DMF was added 1.06 microliters, 11.4 micromoles Et ₃ N.

The mixture was stirred for 30 minutes, then diluted with 0.5 mL of a 0.1% aqueous TFA solution, filtered and purified by preparative RP-HPLC using a gradient of 10 to 60% MeCN to give pure product as 7.8 in 35% yield. mg of a colorless solid. Analytical RP-HPLC: t _R = 12.8 min, gradient 0 to 60% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 2915.34, the molecular formula C ₃₆ H ₃₂ S ₂ ooN360 ₂ = 2915.40.

Example 11

Biotinamidocaproyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH ₂

450 mg, 75 micromole H-betaAla-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) ) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) -Gly-Cys (Trt) -Gly-resin was mixed with a solution of 136 mg, 0.3 mmol of biotinamido-caproic acid N-hydroxysuccinimidyl ester, 41 mg, 0.3 mmol HOBt and 105 microliters, 0.6 mmol DIEA in 3 mL DMF for 18 hours. The peptide resin was separated using a sinter and washed successively with DMF, CH ₂ Cl ₂ and Et ₂ O. After drying in vacuo, the resin was treated with 5 mL of the cleavage reagent for 1.5 hours. The biotinylated peptide was isolated by Et ₂ O precipitation and centrifugation to give 244 mg of product. A 120 mg portion of this product was purified by preparative RP-HPLC using a 20-30% MeCN gradient to afford 63.8 mg of the product as a colorless solid. Analytical RP-HPLC: t _R = 16.7 minutes, 20-30% MeCN gradient, purity greater than 99%, lambda = 214 nm. DE MALDI-TOF MS: [M + H] ⁺ = 2874.3, [2M + H] ⁺ = 5738.7 [M + 2H] ²⁺ = 1437.8, the molecular formula Ci3oH _2X oN4 _{2 2} 0 6 S ₂ = 2873.52.

4- [Succinimidopropionoyl- (biotinamido-caproyl-betaAla-Arg-Gln-11 le-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Giy-NH ₂ )] podophyllotoxin

99 · 9 · 9 ···· · · ·

9 9

9

9 9

To a solution of 7 micromol, 4 mg of 4- (maleimidopropionoyl) podophyllotoxin and 7 micromol, 27.7 mg of biotinamidocaproyl-beta-Ala-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys -Trp-Lys-Lys-Gly-Cys-Gly-NH ₂ in 0.5 mL DMF was added 10 µL ΕίβΝ. The mixture was stirred for 1 hour, then diluted with 0.5 mL of a 0.1% aqueous TFA solution, filtered and purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN. 2.2 mg of pure product are thus obtained as a colorless solid. Analytical RP-HPLC: t _R = 17.2 min, gradient 0 to 60% MeCN, purity greater than 97%.

DE MALDI-TOF MS: [M + H] ⁺ = 3438.9, Formula C159H237N43O37S2-3439, 05.

Example 12

4- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] Podophyllotoxin

NHj

NH, ^{0 M} vs ° ° ° in y ° in °> ^b ° HN ^ NH, n * «4 4444

4 4 4 ·

To a solution of 20 µmol, 12.2 mg of 4- (maleimidopropionoyl) podophyllotoxin and 15 µmol, 34.7 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met -Lys-Trp-Lys-Lys-NH ₂ in 1.5 mL DMF was added 5 µL of Et 3 N.

The mixture was stirred for 40 minutes and then purified by preparative RPHPLC using a 0 to 60% MeCN gradient to give 30.1 mg of pure product as a colorless solid in 69% yield. Analytical RP-HPLC: t _R = 15.8 min, gradient 0 to 50% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 2914.4, total formula

C36H20lN'37C3S2 = 2914.41.

Example 13

H-Cys-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe-D-Gln-D-Asn-D-Arg-D-Arg-D- Nle-D-Lys-D-Trp-D-Lys-D-Lys-NH ₂

Starting from 0.69 mmol / g Rink Amide AM resin (Novabiochem), H-Cys (Trt) -D-Arg (Pmc) -DGln (Trt) -D-Ile-D-Lys (Boc) is assembled - D-Ile-D-Trp-D-Phe-D-Gln (Trt) -Dsn (Trt) -D-Arg (Pmc) -D-Arg (Pmc) -D-Nle-D-Lys (Boc) -D -Trp-DLys (Boc) -D-Lys (Boc) -resin. After deprotection within 1.5 hours, the crude peptide is obtained by precipitation from Et ₂ O by centrifugation, decanting the supernatant and drying. Aliquots with a total weight of 246 were purified by preparative RP-HPLC using a gradient of 17.5 to 27.5% MeCN to give 45.9 mg of pure product. Analytical RP-HPLC: t _R = 16.9 minutes, gradient 17.5 to 27.5% MeCN, purity greater than 99%, lambda = 214 nm. DE MALDI-TOF MS: [M + H] ⁺ = 2330.3, the molecular formula C _{2 0} 8Hi76N360 OS = 2330.85.

9 99 »

4- [Succinimidoproionoyl- (H-Cys-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe-D-Gln-D-Asn-D-Arg- D-Arg-D-Nle-D-Lys-D-Trp-D-Lys-D-Lys-NH 2)] podophyllotoxin

To a solution of 11 micromoles, 6.2 mg of 4- (maleimidopropionoyl) podophyllotoxin and 7 micromoles, 17 mg of H-Cys-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp- D-Phe-D-Gln-D-Asn-D-Arg-D-Arg-D-Nle-D-Lys-D-Trp-D-Lys-D-Lys-NH 2 in 1 mL DMF was added 1.4 microliters of Et3N. The mixture was stirred for 30 minutes, then filtered and purified by preparative RP-HPLC using a 0 to 60% MeCN gradient to give 10.5 mg of pure product as a colorless solid in 52% yield. Analytical RP-HPLC: t _R = 15.8 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2895, 66, C137H2O3N37O31S2 = 2896.37.

Example 14

4- [Succinimidopropionoyl- (H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2)] Podophyllotoxin

To a solution of 17.7 micromoles, 10 mg of 4- (maleimidopropionoyl) podophyllotoxin and 25 micromoles, 30.4 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1.5 mL DMF was added 3.5 microliters of Et ₃ N. The mixture was stirred for 40 minutes, then filtered and purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN. In this way, 17.8 mg of pure product is obtained as a colorless solid in a yield of 57%. Analytical RP-HPLC: t _R = 14.8 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 1772.3, C81H119N21O20S2 = 1771.07.

Example 15

H-Cys-beta-Ala-D-Arg-D-Arg-D-Met-Lys-D-Trp-D-Lys-D-Lys-NH ₂

Starting from 0.69 mmol / g Rink Amide AM resin (Novabiochem), H-Cys- (Trt) -betaAla-D-Arg (Pmc) -D-Arg (Pmc) -D-Met-D-Lys is assembled. (Boc) - D - Trp - D - Lys (Boc) - D - Lys (Boc) - Resins. After deprotection within 1.5 hours, the crude peptide is obtained by precipitation from Et ₂ O, centrifugation, decanting the supernatant and drying.

Aliquots with a total weight of 237 mg were purified by preparative RP-HPLC using a gradient of 8 to 18% MeCN to give 66 mg of pure product. Analytical RP-HPLC: t _R = 12.9 min, gradient 9 to 19% MeCN, purity greater than ·· ·· ♦ ·

99 ♦ 9 · 9 • 9 9 9

9 99 • 9 9

9 9 • 9 9 • 9 9 ♦ ♦ 99%, lambda = 214 nm. DE MALDI-TOF MS: [M + H] ⁺ = 1207.2, the molecular formula C 5 H 9 _{2 2 9} N2o0 S2 = 1205.55.

4- [Succinimidopropionoyl- (H-Cys-beta-Ala-D-Arg-D-Arg-D-Met-Lys-D-Trp-D-Lys-D-Lys-NH2)] Podophyllotoxin

To a solution of 18.9 micromol, 10.7 mg of 4- (maleimidopropionoyl) podophyllotoxin and 28 micromol, 33.8 mg of H-Cys-beta-Ala-D-Arg-D-Arg-D-Met-Lys-D-Trp- D-Lys-D-Lys-NH ₂ in 1.5 mL DMF was added 1.5 microliters of Et ₂ N. The mixture was stirred for 40 minutes, then filtered and purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN. 6.9 mg of pure product are thus obtained in a yield of 21% as a colorless solid. Analytical RP-HPLC: t _R = 14.8 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 1771.5, the molecular formula C8iHn N2i02oS2 ₉ ~ 1771.07.

Example 16

4 '- (maleimidopropionoyl) epipodophyllotoxin

OH

* · ·· • · 00000 · 0 0 • 000 0 ·

A solution of 12 mmol, 5 mg of 4'-demethylepipodophyllotoxin, 50 µmol, 12.2 mg of 3-maleimidopropionic acid and 28 µmol, 3.47 mg of DIC in 1 ml of pyridine was stirred for 30 minutes. Then 0.5 ml of methanol was added and the mixture was purified by preparative RP-HPLC using a gradient of 0 to 60%

MeCN to give 4.2 mg of pure product as a colorless solid in 62% yield. Analytical RP-HPLC: t R = 17.6 minutes, gradient 0 to 60% MeCN, purity greater than 95%.

1 H-NMR (300 MHz, CDCl ₃ ), δ: 2.84 (m, 1H, H 2), 2.99 (t, J = 7.44 Hz, 2H, CH ₂ -Mim), 3.32 (dd) , J = 14.04, 5,0.7 Hz, 1H, H2), 3.69 (s, 6H, _OCH3 x2), 3.95 (t, J = 7.44 Hz, 2H, CH ₂ -Mim) 4.39 (dd, J = 8.13, 4.28 Hz, 2H, H11), 4.66 (d, J = 5.00 Hz), 1H H1), 4.89 (d, J = 3) 32 Hz, 1H, H 4), 6.01 (d, J = 6.42 Hz, 2H, OCH ₂ O), 6.32 (s, 2H, H 2 '6'), 6.57 (s, 1H H, 6.74, (s, 2H, CH.dbd.CH), 6.90 (s, 1H, H5). ¹³ C-NMR (75 MHz, CDCl ₃ ) δ: 28.64, 31.02, 32.55, 37.33, 39, 53, 42, 99, 55.15, 65, 78, 66, 56, 100.65, 106.54,

107, 97, 109, 65, 130, 68, 130.92, 133.21, 136, 96, 146, 62, 147.61, 150.39, 167.36, 169.30, 173.89.

4 '- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Epipodophyllotoxin ·· 9 9

9 99 99

OH

Oa ^

C ^ f '

Ύ Ύ / x> Υ * Γ / ° \ O ⁰ 30 ° E ⁰ \

J. MjN

HfcI NHj

To a solution of 2.3 micromol, 1.3 mg of 4 '- (maleimidopropionoyl) epipodophyllotoxin and 2.3 micromol, 5.4 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn- Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in 0.5 ml DMF was added 0.21 microliters, 2.3 micromol Et ₃ N. The mixture was stirred for 40 minutes and then diluted with 1 ml 0.1 % aqueous TFA solution, the solution was filtered and purified by preparative RP-HPLC using a gradient of 0 to 60%

MeCN to give pure product as 3.2 mg of a colorless solid in 48% yield. Analytical RP-HPLC: t _R =

14.6 minutes, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2,902.2. molecular formula C ₃ N 6 O ₃ 5Hi9gN 2S2 ₃ = 2901.37.

Example 17

4 '- [sukcinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH _2)] epipodophyllotoxin

4 ·

4444

To a solution of Ί micromol, 4 mg of 4 '- (maleimidopropionoyl) epipodophyllotoxin and 6 micromol, 15 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys- Trp-Lys-Lys-NH ₂ in 0.5 mL DMF was added 1 µL of Et 3 N. The mixture was stirred for 40 minutes and then purified by preparative RP-HPLC to give the pure product as a 14.1 mg colorless solid in 81% yield. Analytical RP-HPLC: t _R = 19.7 minutes, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 2900.4

C ₁₃ H 19 N ₃ O ₃ Si ₂ = 2900.39.

Example 18

4 '- [Succinimidopropionoyl- (H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] epipodophyllotoxin

HjN

O

ÍLA λ

NHj φφ φφφφ

ΦΦ φ φ φ «« • • • • φ φ φ φ

φ φ

φ φ

Κ solution of 14 micromoles, 7.9 mg of 4 '- (maleimidopropionoyl) epipodophyllotoxin and 26 micromoles, 31.5 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1 ml DMF was added 1.2 microliters of Et ₃ N. the mixture was stirred for 40 min and then purified by preparative RP-HPLC using a gradient of 0-60% MeCN to give a yield of 63% gave 15.8 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 13.3 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ 1757.2, C ₈ H ₁₁ N ₂ O ₂ O ₂ S ₂ = 1757.05.

Example 19

4 '- (chloroacetyl) epipodophyllotoxin

To a stirred solution of 0.50 mmol, 200 mg of 4'-demethylepipodophyllotoxin and 40 microliters of pyridine in 2 ml of methylene chloride was added dropwise at 0 ° C 0.50 mmol, 56.5 mg of chloroacetyl chloride. According to analytical RP-HPLC, approximately 60% of the starting 4'-demethylepipodophyllotoxin was consumed within 1 hour of stirring at 0 ° C. The reaction mixture was poured into ice-water and the resulting mixture was extracted with methylene chloride. Wash the organic layer with water and a saturated aqueous solution

9 9 9 9 9 9 9 9 9 9 9

99999 9

9 9 * 9 sodium chloride, then dried over magnesium sulphate. The solvent was evaporated in vacuo and the residue was purified by flash chromatography using EtOAc / PE 5: 4 to 3: 2. 81.5 mg of pure product are obtained after recrystallization from EtOAc / PE as a colorless solid in a yield of 34%.

¹ H-NMR (300 MHz, CDCl ₃ δ: 2.78 (m, 1H, H 3), 3.25 (dd,

J = 14.12, 5.07 Hz, 1H, H2) , 3.68 (s, 6H) OCH ₃ x 2) 4.30 (m, 2H, H11), 4.35 (s, 2H, CH2Cl2), 4.57 (d, J = 5.12 Hz) 1H, H1), 4.83 (d, J = 3.37) Hz, H4) , 5.96 (d, J = 4.10Hz, 2H, OCH ₂ O), 6.32 (s, 2H, H 2 ') 6 ') 50 (s, 1H, H8), 6.87 (with, 1H, H5).

4'-chloroacetyl-4- (maleimidopropionoyl) epipodophyllotoxin

A solution of 0.17 mmol, 81.5 mg of 4 '- (chloroacetyl) epipodophyllotoxin, 0.68 mmol, 115.6 mg of 3-maleimidopropionic acid, 0.376 mmol, 47.5 mg of DIC, 73 micromol, 9 mg of DMAP and 20 microliters pyridine in 2 mL methylene chloride was stirred for 1 hour. Then the solvent was evaporated to dryness and the resulting pale yellow solid was redissolved in 1 mL DMF and the solution was purified by preparative RP-HPLC using a 30-70% MeCN gradient to give 54.3 mg of a colorless solid in 51% yield. . Analytical RP-HPLC: t _R = ·· · * «·

22.0 minutes, gradient 0 to 60% MeCN, purity greater than 97%. ¹ H-NMR (300 MHz, CDC1 ₃₎ delta: 2.71 (t, 2H, J = 6.80 Hz, _CH2 -Mim), 2.98 (m, 1H, H3), 3.25 (dd , J = 14.20, 5.13 Hz, 1H, H2), 3.69 (s, 6H, 0CH ₃ x2), 3.87 (t, J = 6.83 Hz, 2H, CH ₂ -Mim) , 3.88 (m, 1H, Hll), 4.33 (s, 2H, CH ₂ C1), 4.35 (m, 1H, Hll), 4.70 (d, J = 5.10 Hz, 1H , H), 6.01 (d, J = 4.23 Hz, 2H, _OCH2 O), 6.13 (d, J = 3.50 Hz, 1H, H4), 6.31 (s, 2H, H 2 '6'), 6.56 (s, 1H, H 8), 6.71 (s, 2H, CH = CH), 6.92 (s, 1H, H 5).

4'-Chloroacetyl-4- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] epipodophyllotoxin

To a solution of 6.8 micromol, 43 mg of 4'-chloroacetyl-4s - (maleimidopropionoyl) epipodophyllotoxin and 10 micromol,

25.4 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in 1.5 mL of DMF was added 2 5 .mu.l of Et ₃ N. the mixture was stirred for 30 minutes and then purified by preparative RP-HPLC using a gradient of 10-70% MeCN to give a yield of 67% gave 22 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 16.7 min, gradient 0 to 60% MeCN, higher purity

9 · * 9

than 99%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2978.3, C137H199CIN36O33S2 = 2977.85.

4'-Demethyl-4- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] epipodophyllotoxin

Solution 5.5 micromol, 16.4 mg 4'-chloroacetyl-4- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys) -Trp-Lys-Lys-OH)] epipodophyllotoxin in 1 ml DMF and 0.5 ml water is treated at 0 ° C with 20 microliters of concentrated aqueous NH 3 solution. After 2 minutes, the reaction mixture was acidified by the addition of 0.1 mL of 5% aqueous acetic acid. The solution was then purified by preparative RP-HPLC using a 0 to 60% MeCN gradient to give 11.4 mg of pure product as a colorless solid in 73% yield. Analytical RP-HPLC: t _R = 14.9 min, gradient 0 to 60% MeCN, purity greater than 99%. DE MALDI-TOF MS: [M + H] ⁺ - 2902.2, the molecular formula C _{8 135} Hi9 N36O32S ₂ = 2901.37.

Example 20

G2- (maleimidopropionoyl) etoposide, G3- (maleimidopropionoyl) etoposide and 4 (maleimidopropionoyl) etoposide

Solution 37.4 micromol, 22 mg etoposide, 78 micromol, 13.2 mg 3-maleimidopropionic acid and 39.6 micromol •

mg of DIC in methylene chloride / pyridine (2: 0.15) was stirred for 30 minutes. Then the solvent was evaporated in vacuo. The resulting light yellow solid was dissolved in 1.5 mL of methanol and the solution was purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to give 3.4 mg of G2- (maleimidopropionoyl) etoposide, 2.4 mg of G3 - (maleimidopropionoyl) etoposide and 7.7 mg of 4 '- (maleimidopropionoyl) etoposide as colorless solids, overall yield 48%.

G2- (maleimidopropionoyl) etoposide

Analytical RP-HPLC: t _R = 16.7 min, gradient 0 to 60% MeCN, purity greater than 99%.

¹ H-NMR (300 MHz, CDC1 ₃₎ delta: 1.39 (d, J = 4.99 Hz, 3H, _CH3), 2.39 (t, J = 7.30 Hz, 2H, CH ₂ - Mim), 2.87 (m, 1H, H 3), 3.14 (dd, J = 14.20, 5.09 Hz, 1H, H 2), 3.39 (m, 2H, G 4.5), 3 63 (m, 2H, G2,6), 3.72 (m, 2H, CH ₂ -Mim), 3.76 (s, 6H, _OCH3 x2), 3.84 (t, J = 8.9 Hz, 1H, G3), 4.19 (m, 2H, H11, G6), 4.38 (m, 1H, H11), 4.60 (d, J = 4.10 Hz,

1H, H4), 4.74-4.84 (m, 2H, H1, G6). 6.00 (d, J = 5.10 Hz, 2H, OCH ₂ O), 6.24 (s, 2H, H 2 '6'), 6.54 (s, 1H, H 8), 6.70 (S 2 H, CH = CH), 6.75 (s, 1H, H 5). ¹³ C-NMR (75 MHz, CDCl ₃ ) δ: 20.66, 33.48, 33.95, 37.86, 41.46, 44.00, 56.87, 66.75, 68 , 07, 68.34, 72.15, 74.61, 75.26, 80.24, 100.29, 100.434, 102.07, 108.33, 109.00, 111.33, 128.75, - 130.90, 133.40, 134.50, 134.66, 146.80, 147.27, 149.09, 169.94, 170.78, 175.08.

G3- (maleimidopropionoyl) etoposide

Analytical RP-HPLC: t _R = 18.4 min, gradient 0 to 60% MeCN, purity greater than 99%.

¹ H-NMR (300 MHz, CDC1 ₃₎ delta: 1.33 (d, J = 5.0 Hz, 3H, _CH3), 2.74 (t, J = 7.35 Hz, 2H, CH ₂ - Mim), 2.91 (m, 1 H,

H3), 3.28 (dd, J = 14.01, 5.26 Hz, 1H, H2), 3.38 (m, 2H,

G4.5), 3.54 (m, 2H, G2.6), 3.87 (m, 2H, CH ₂ -Mim), 3.7 6 d * (s, 6H, OCH ₃ x ₂ ), 4.16-4.26 (m, 2H, H11, G3), 4.42 (t, J = 8.98 Hz, 1H), 4.61 (d, J = 5.09 Hz, 1H, H1), 4.68 (m, 1H, and GI), 4.91 (d, J = 3.34 Hz, H4), 5.13 (m, 1H, G 3). 6.00

(d, J = 11.25 Hz, 2H, OCH ₂ O), 6.26 (s, 2H, H 2 '6'), 6.54 (s, 1H, H 8), 6.71 (s, 2H, CH = CH), 6.83 (s, 1H, H 5). ¹³ C NMR (75 MHz, CDC1 ₃₎ delta: 0 _(67, 33.59, 34.13, 37.93, 41.62, 44.11, 56.84, 66.87, 68.26, 68, 38, 73.39, 74.38, 74.49, 100.17, 102.01, 102.54, 108.25, 109.51, 111.10, 128.38, 130.89, 133.28, 134.50, 134.66, 146.84, 147.59, 149.29, 170.64, 170.80, 175.38.

4 '- (maleimidopropionoyl) etoposide

M

AND

Analytical RP-HPLC: t _R = 17.7 min, gradient 0 to 60% MeCN, purity greater than 99%.

1 H-NMR (300 MHz, CDCl ₃ ) δ: 1.39 (d, J = 4.82 Hz, 3H, CH ₃ ), 2.88 (m, 1H, H 3), 2.96 (t, J = 7.24 Hz, 2H, CH ₂ Mim, 2.91 (m, 1H, H3), 3.34 (dd, J = 14.01, 5.26 Hz, 1H, H2), 3.36 (m, 2H, G4,5), 3.45-3.58 (m, 2H, G2,6), 3.92 (t, J = 3.20 Hz, 2H, CH ₂ -Mim), 3.65 (s 6H, OCH ₃ x ₂ ), 3.7 6 (m, 1H, G 3), 4.15-4.27 (m, 2H, H 11, G 6), 4.43 (m, 1H, H 11), 4, 62-4, 67 (m, 2H, H1, G1), 4.75 (m, 1H, G7), 4.91 (d, J = 3.27 Hz, H 4), 6.00 (d, J = 6.68 Hz, 2H, _OCH2 O), 6.25 (s, 2Ή, H2'6 '), 6.54. (s, 1H, H8), 6.71 (s, 2H,

CH = CH), 6.82 (s, 1H, H5). ¹³ C-NMR (75 MHz, CDCl 3) δ: 20.62, 32.41, 33.94, 37.87, 41.61, 44.31, 56.52, 66.84, 68.44, 73, 47, 74.13, 74.88, 80.06, 100.24, 102.10, m 102.30 107.89, 109.36, 111.22, 128.65, 132.68, 134.61, 138.28, 147.74, 149.29, 151.76, 168.90, 170.76, 175.56.

G2- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Etoposide

To a solution of 4.4 micromol, 3.3 mg G2- (maleimidopropionoyl) etoposide and 5.7 micromol, 13.4 mg H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg -Arg-Met-Lys-Trp-Lys-Lys-OH in 0.5 mL DMF was added 0.7 µL, 4.9 µM Et 3 N. The mixture was stirred for 30 minutes, then diluted with 1 mL of a 0.1% aqueous TFA solution and purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to give pure product in 80% yield as 10.8 mg of a colorless solid substances. Analytical RP-HPLC: t _R = 14.6 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 3091 1, the molecular formula C 3 H _{2 4 6} ion3 037S2 = 3089, 55th

G3- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Etoposide

To a solution of 3.1 micromol, 2.3 mg (G (Hmaleimidopropionoyl) etoposide) and 4.3 micromol, 10.2 mg H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg -Met-Lys-Trp-Lys-Lys-OH in 0.5 ml DMF was added 0.6 microliters, 4.4 micromol Et ₃ N. The mixture was stirred for 30 minutes, then diluted with 1 ml of a 0.1% aqueous solution. TFA and purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to give 7.4 mg of pure product as a colorless solid in 79% yield. Analytical RP-HPLC: t _R = 14.7 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 3090.3, C143H210N36O37S2 = 3089, 55.

4 '- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Etoposide

1 Y 'S' in r ', OH

I ^X NH

Κ of a solution of 4.8 micromol, 3.6 mg of 4 ^Λ - (maleimidopropionoyl) etoposide and 5.9 micromol, 13.9 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn- Arg-Arg-Met-Lys-Trp-Lys-Lys -OH in 0.5 ml DMF was added 0.7 microliters, 5.1 micromol Et ₃ N. The mixture was stirred for 30 minutes, then diluted with 1 ml 0.1 % aqueous TFA solution and purified by preparative RP-HPLC using a 0 to 60% MeCN gradient to give 11.2 mg of pure product as a colorless solid in 77% yield. Analytical RP-HPLC: t _R = 14.6 min, gradient 0 to 60% MeCN, purity greater than 99%. DE MALDI-TOF MS: [M + H] ⁺ = 3090.9, C143H210N36O37S2 = 3089, 55.

Example 21

O- (maleimidopropionoyl) roscovitine

A solution of 29 µmol, 10.3 mg roscovitine, 64 µmol, 10.8 mg 3-maleimidopropionic acid, 35 µmol, 4.4 mg DIC and 2 µmol, 0.35 mg DMAP in 1 ml pyridine was stirred for a total of 40 minutes. Then the solvent was evaporated in vacuo and the resulting pale yellow solid was redissolved in methylene chloride, washed with water and saturated sodium chloride solution and dried over magnesium sulfate. The solvent was evaporated to give 14.1 mg of the product as a pale yellow solid in 96% yield. This material was used for the next reaction without further purification.

• »•

4 4

O- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] roscovitine

To a solution of 28 µmol, 14.1 mg O- (maleimidopropionoyl) roscovitine and 14.9 µmol, 35 mg H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met -Lys-Trp-LysLys-OH in 1.5 ml DMF was added 2 microliters, 14.5 micromol EtsN. The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient of 10 to 60% MeCN to yield 7.2 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 15.5 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 2856.1

C33H204N42Cl2S2 ⁼ 2855, 44.

Example 22

O-betaAla bohemin ··· · • ·

A solution of 58.8 micromol, 20 mg bohemine, 0.128 mmol, 24.2 mg Boc-betaAla-OH, 79 micromol, 8.8 mg DIC and 9.8 micromol, 1.2 mg DMAP in 2 mL methylene chloride is stirred for 2 hours. 5 hours. The solvent was evaporated in vacuo and the resulting white solid was purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to give 30 mg of O- (Boc-betaAla) bohemin as a colorless solid. Analytical RP-HPLC: t _R = 19.8 min, gradient 0 to 60% MeCN, purity greater than 99%. A solution of 7.6 mg of O- (Boc-betaAla) bohemine in 1 mL of 9: 1 TFA / water was stirred for 1 hour. Then the solvent was evaporated to dryness and the residue was used for the next reaction without further purification, the purity of the title compound by analytical RP-HPLC was greater than 98%.

O- (betaAla-succinyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH) bohemine

14.9 micromol, 81.7 mg succinyl-betaAlaArg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) Asn (Trt) -Arg (Pmc) - Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) Lys (Boc) -Resin, 14.9 micromol, 7.6 mg O-betaAla • ·

Bohemine, 14.9 micromol, 7.8 mg PyBOP, 14.9 micromol, 2.4 mg HOBt and 0.2295 mmol, 29.7 mg DIEA in 2 mL DMF is stirred for 2 hours. The peptide resin was filtered off, washed with DMF, CH ₂ Cl ₂ and Et ₂ O and then dried under vacuum to give a total of 82 mg of resin which was treated with 5 mL of cleavage agent for 2 hours. 42 mg of crude product is obtained by precipitation of Et ₂ O and centrifugation and collecting the supernatant. The product was purified by preparative RPHPLC using a gradient of 0 to 60% MeCN to give 14.3 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 14.8 min, gradient 0 to 60%.

MeCN, purity greater than 93%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2812.7, C132H2O4N4O25S = 2811.37.

Example 23 (maleimidopropionoyl) bohemine

HN

12.8 mg, 76 micromoles of 3-maleimidopropionic acid are dissolved in 1 ml of methylene chloride. The mixture was stirred and 5.3 mg, 42 micromol of DIC in 0.5 mL of methylene chloride was added. The reaction was allowed to proceed with stirring for 40 minutes. Then the solvent was evaporated under reduced pressure. The residue of 3-maleimidopropionic anhydride is redissolved in 0.5 ml of anhydrous pyridine. A solution of 10.3 mg, 30 micromol of bohemin and 0.35 mg, 2 micromol of DMAP in 0.5 ml of pyridine was added and the mixture was stirred under nitrogen for one hour. The mixture was then evaporated to dryness under reduced pressure. The residue was redissolved in 1 mL DMF and the solution was purified by preparative RP-HPLC on a column using a gradient of 10 to 60% MeCN to give the pure product as a 14.7 mg colorless solid in 88% yield. Analytical RP-HPLC: t _R = 17.7 minutes, gradient 0 to 60% MeCN, purity greater than 95%. ¹ H-NMR (CDC1 ₃₎ and DE MALDI-TOF MS were consistent with the structure of the title compound. Formula C25H29N7O4 = 491.54.

O- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Bohemin

Dissolve 0.74 mg, 1.5 micromol (maleimidopropionoyl) bohemine in 0.3 ml DMF and add 50 microliter Et ₃ N. Then add a solution of 3.5 mg, 1.5 micromol H-Cys-Arg- Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in 0.25 mL DMF. The mixture was stirred under nitrogen and monitored by analytical RP-HPLC. After one hour, the reaction is complete. The mixture was filtered and the resulting product was purified by preparative RP-HPLC using a gradient of 10 to 10 using a gradient of 10 to 10 ° C. % MeCn to give the pure product in a yield of 40% as a colorless solid (1.7 mg). Analytical RP-HPLC: t _R = 15.0 min, gradient 0 to 60% MeCN, purity greater than 95%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2842, C132H2O2N42O25S2 ⁼ 2841.42.

Example 24

4- (iodoacetyl) podophyllotoxin

> 3

A mixture of 0.49 mmol, 204 mg podophyllotoxin, 1.03 mmol, 192 mg iodoacetic acid, 0.552 mmol, 69.7 mg, 0.164 mmol DIC and 20 mg of DMAP in 5 ml of anhydrous methylene chloride _at d, cooled to 0 ° C was added with 0.2 ml of pyridine and stirred at 0 ° C for 1 hour. The mixture was evaporated to dryness. The resulting pale yellow residue was redissolved in MeCN and purified by preparative RP-HPLC using a gradient of 20 to 70% MeCN to give 89.5 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 22.3 min, gradient 0 to 60% MeCN, purity greater than 95%.

¹ H-NMR (300 MHz, CDCl 3) δ 2.85 (m, 2H, H 2, 3), 3.70 (s, 6H, OCH ₃ x ₂ ), 3.72 (s, 2H, CH ₂ I) , 3.74 (s, 3H, _OCH3), 4.13 (m, 1H, Hll), 4.34 (m, 1H, Hll), 4.53 (d, 1H, J = 3.60 Hz H1), 5.83 (d, 1H, J = 8.43 Hz, H 4), 5.93 (dd,

2H, J = 4.35, 1.17 Hz, OCH ₂ O), 6.31 (s, 2H, H 2 '6'), 6.48

4

4 •

44 (s, 1H, H8), 6.77 (s, 1H, H5).

4- [acetyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] podophyllotoxin

Q ”Q

Y

MH + 4

MjN

H? <

To a solution of 17 micromoles, 10 mg, 4- (iodoacetyl) podophyllotoxin and 6 micromoles, 14 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys -Trp-Lys-Lys-OH in 1 ml DMF was added 0.9 µl, 6 µmol Et 3 N. After stirring for 1 hour, 0.5 mL of MeCN was added and the solution was purified by preparative RP-HPLC using a 10-60% MeCN gradient to give 9.9 mg of pure product as a colorless solid in 59% yield. Analytical RP-HPLC: t _R = 15.4 minutes, gradient 0 to 60% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2806, 8, C131H195N35O30S2 = 2804.30.

Example 25

4- [Acetyl- (H-Cys-betaAla-Arg-Met-Lys-Trp-Lys-Lys-NH _2)] podophyllotoxin

9. 9

OF

A solution of 17 micromol, 10 mg of 4- (iodoacetyl) podophyllotoxin and 23 micromol, 28.6 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1 mL of DMF was added with 2. 4 microliters, 17 micromol Et3N. After stirring for 1 hour, 0.15 mL of MeCN was added and the mixture was purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to give 29.4 mg of pure product as a colorless solid in 100% yield. Analytical RP-HPLC: t _R = 14.1 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 1661.0, Formula C76H114N2O18S2 = 1659.97.

Example 26

4'-Demethyl-4- (iodoacetyl) epipodophyllotoxin

OH * · · «

To a solution of 0.26 mmol, 104 mg of 4'-demethylepipodophyllotoxin, 0.53 mmol, 98.8 mg of iodoacetic acid and 0.32 mmol, 40.1 mg of DIC in two ml of methylene chloride was added 50 µl of pyridine at 0 ° C. and 0.1 mmol,

12.8 mg DMAP. The mixture was stirred for 1 hour and then the solvent was evaporated. The residue was dissolved in 1 mL DMF and the solution was purified by preparative RP-HPLC using a gradient of 20 to 60% MeCN to give 35.7 mg of pure product as a colorless solid in 24% yield. Analytical RP-HPLC: t _R = 20.3 min, gradient 0 to 60% MeCN, purity greater than 96%.

1 H-NMR (300 MHz, CDCl ₃ ) δ: 3.02 (m, 1H, H 3), 3.20 (m, 1H, H 2), 3.71 (s, 6H, OCH ₃ x 2), 3.63 (s, 2H, _CH2 I), 3.74 (s, 3H, _OCH3), 4.05 (m, IH, Hll), 4.27 (m, IH, Hll), 4.60 (d, 1 H, J = 4.94 Hz, H 1), 6.06 (d, 1 H, J = 3.41 Hz, H 4), 5.92 (m, 2H, OCH ₂ O), 6.21 (s, 2H , H2'6 '), 6.49 (s, 1H,

H8), 6.80 (s, 1H, H5).

4'-Demethyl-4- [acetyl- (H-Cys-betaAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] epipodophyllotoxin

9 · ····

Κ solution of 17.6 micromol, 10 mg of 4'-demethyl-4- (iodoacetyl) epipodophyllotoxin and 14.9 micromol, 18 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1 mL of DMF, 2.1 mL, 15 micromol of Et ₃ N was added. The mixture was stirred for 1 hour, then the reaction mixture was purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to yield 11, 2 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 12.8 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt;

1647.2, the molecular formula C75H N ₂ oOigS _{u2 2} = 1645.95.

Example 27

4'-Demethyl-4- [acetyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ ) epipodophyllotoxin

H ¹

To a solution of 22 micromoles, 12.6 mg of 4'-demethyl-4 (iodoacetyl) epipodophyllotoxin and 8 micromoles, 20 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg -Met-Lys-TrpLys-Lys-NH ₂ in 1 ml DMF was added 1.2 microliters, 9 micromol Et ₃ N. The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient of 0 to 60%

9

69.

9 · 9 · 9

9 9

999 ·

9

9999 99

MeCN to give 13.3 mg of pure product as a colorless solid in 56% yield. Analytical RP-HPLC: t _R = 14.5 min, gradient 0 to 60% MeCN, purity greater than 96%. DE MALDI-TOF MS: [M + H] ⁺ = 2789.5, C30H194N36O29S2 = 2789.29.

Example 28

4- (Boc-Gly) podophyllotoxin

A mixture of 400 mg, 0.97 mmol podophyllotoxin, 510 mg, 2.91 mmol Boc-Gly-OH, 1.73 mmol, 273 microliters DIC, 0.41 mmol, 50 mg DMAP and 173 microliters pyridine in 5 ml methylene chloride stir for one hour. The solvent was evaporated, the residue redissolved in 1.5 ml DMF and purified by RP-HPLC using a gradient of 20 to 70% MeCN, ₄ to give 502.6 mg of pure product as a colorless solid in 91% yield. Analytical RP-HPLC: t _R = * 22.1 minutes, gradient 0 to 60% MeCN, purity greater than 20%;

%.

4- (H-Gly) podophyllotoxin

'0' · ♦ ··

To a solution of 0.24 mmol, 137 mg of 4- (Boc-Gly) podophyllotoxin in 8 mL of methylene chloride was added 0.5 mL of TFA. The mixture was stirred for 1 hour and then the solvent was evaporated. The resulting light yellow solid residue was purified by preparative RP-HPLC using a 10 to 70% MeCN gradient to give 41.7 mg of pure product as a colorless solid in 37% yield. Analytical RP-HPLC: t _R = 15.2 min, gradient 0 to 60% MeCN, purity greater than 97%.

4- (maleimidopropionoyl-Gly) podophyllotoxin

To a solution of 70 micromol, 11.8 mg of 3-maleimidopropionic acid and 38 micromol, 4.83 mg of DIC in 1 ml of DMF was added 17 micromole, 8 mg, 4- (H-Gly) podophyllotoxin, micromole, 1.2 mg of DMAP and 20 mg. microliters of pyridine. The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to give 1.1 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 18.2 min, gradient 0 to 60% MeCN, purity greater than 97%.

· »« «· · ·

4 - [(Succinimidopropionoyl-Gly) - (H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] podophyllotoxin o ^ o

To a solution of 1.8 micromol, 1.1 mg of 4- (maleimidopropionoyl-Gly) podophyllotoxin and 4 micromol, 5 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1 ml DMF was added 0.5 microliters, 4 micromol of 13N. The mixture was stirred for 1 hour, then diluted with 0.5 mL MeCN and purified by preparative RPHPLC using a 0 to 60% MeCN gradient to give 1.1 mg of the title compound as a colorless solid in 33% yield. Analytical RP-HPLC: t _R = 14.7 min, gradient 0 to 60% MeCN, purity greater than 97%. DE MALDI-TOF MS: [M + H] ⁺ = 1829.8, Formula C ₈₃ Hi 22 N ₂₂ O 2 Si 2 = 1828.12.

Example 29

10-O- (maleimidopropionoyl) camptothecin

0 '· * · © · © © · © © © © © * * * * © © © © © © © © © © © · © © © · ·· · «

Κ of a solution of 40 micromol, 14.7 mg of 10-hydroxycamptothecin, 0.228 mmol, 38.5 mg of 3-maleimidopropionic acid and 0.125 mmol, 15.8 mg of DIC in 2 ml of methylene chloride are added 0.2 ml of pyridine. The mixture was stirred for 1 hour and then evaporated to dryness. The resulting pale yellow solid was redissolved in 1 mL DMF and purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to give 9.2 mg of pure product as a pale yellow solid in 45% yield. Analytical RPHPCL: t _R = 15.7 min, gradient 0 to 60% MeCN, purity greater than 97%.

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 1.05 (t, 3H, J = 7.5 Hz, CH ₃ ), 1.91 (m, 2H, J = 7.8 Hz, CH ₂ ) 2.98 (t, 2H, J = 7.8)

Hz, _CH2), 4.04 (t, 2H, J = 7.8 Hz, _CH2), 5.32 (m, 3H, H5,

H17), 6.77 (s, 2H, CH.dbd.CH), 7.60 (m, 1H, H11), 7.72 (m,

2H, H 14, H 9), 8.24 (d, 1H, J = 9.2 Hz, H 12), 8.36 (s, 1H,

H7).

10-0- [sukcinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH _2)] camptothecin

·· 9 · ^ ·

9 9

9

9 9

9

9 «· 9

9 · 9 999 9 9 9 99

9

9

9 9

To a solution of 9 micromol, 4.6 mg of 10-O- (maleimidopropionoyl) camptothecin and 4 micromol, 10 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met -Lys-Trp-LysLys-NH ₂ in 1 ml DMF was added 0.55 microliters, 4 micromol Et ₃ N. The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to give in a yield of 57%, 6.5 mg of pure product was obtained as a colorless solid. Analytical RP-HPLC: t _R = 14.0 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 2864.7, the molecular formula C ₃ 4Hi95N ₃ gO28S2 - 2864.36.

Example 30

H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH 2

Starting from 0.69 mmol / g of Rink Amide AM resin, H-Cys (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) and Trp-Lys (Boc) -Lys (Boc) was prepared. -Cys (Trt) -resin. After deprotection within 1.5 hours, the crude peptide is precipitated from Et ₂ O, centrifuged, discarded the supernatant, and dried. Portions with a total weight of 258 mg were purified by preparative RP-HPLC using a gradient of 9 to 19% MeCN to give 132.4 mg of pure product. Analytical RP-HPLC: t _R = 20.3 min, gradient 8 to 18% MeCN, purity greater than 99%, lambda = 214 nm. DE MALDI-TOF MS: [M + H] ⁺ = 1238.6, the molecular formula C 5 H 9 _{2 2} N _{2 3} o09S = 1237.63.

Bis- [4- (succinimidopropionoyl) podophyllotoxin] - (H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH ₂ )

To a solution of 19 micromoles, 11 mg of 4- (maleimidopropionoyl) podophyllotoxin and 12 micromoles, 15 mg of H-Cys-ArgArg-Met-Lys-Trp-Lys-Lys-Cys-NH ₂ in 1 ml DMF was added 2.8 microliters of Et 3 N . The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to give the pure product as a mg of a colorless solid in 32% yield. Analytical RP-HPLC: t _R =

17.4 minutes, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 2369, 7, the molecular formula CnoH ₁₄₆ N ₂₂ 03iS3 - 2368.66.

Example 31

4 '- (succinimidopropionoyl) epipodophyllotoxin- (H-Cys-ArgArg-Met-Lys-Trp-Lys-Lys-Cys-NH ₂ ) -10-0- (succinimidopropionoyl) camptothecin • · · ·

To a solution of 0.005 mmol, 2.6 mg of 10-O- (maleimidopropionoyl) camptothecin, 5.6 micromol, 3.1 mg of 4 '- (maleimidopropionoyl) epipodophyllotoxin and 11 micromol, 13 mg of H-Cys-Arg-Arg-Met- Lys-Trp-Lys-Lys-Cys-NH ₂ in 1.5 ml DMF was added 1.5 microliters of Et ₃ N. The mixture was stirred for 1.5 hours and then purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to give 1.9 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 14.8 min, gradient 0 to 60% MeCN, purity greater than 96%.

DE MALDI-TOF MS: [Μ + H] ⁺ = 2304.6, the molecular formula Ol07Hl ₃ 8N24O28S ₃ = 2304.58.

Example 32

4 '- (succinimidopropionoyl) epipodophyllotoxin- (H-Cys-Arg-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH ₂ ) -2' - (succinimidopropionyl) paclitaxel

OH

To a solution of 2 micromoles, 3.5 mg of 4 '- [succinimidopropionoyl- (H-Cys-Arg-Arg-Met-Lys-Trp-Lys-Lys-Cys-NH ₂ )] epipodophyllotoxin, 2 micromoles, 2 mg of 2'- (maleimidopropionyl) paclitaxel in 1 ml DMF was added 0.3 microliters of βίβΝ. The mixture was stirred for 1.5 hours and then purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to give 1.5 mg of pure product as a colorless solid. Analytical RP-HPLC: t _R = 17.8 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2794.5, Formula C134H173N23O37S3 = 2794.14.

Example 33

4'-methoxy- (4''-aminoanilino) epipodophyllotoxin a

4'-Demethyl- (4''-aminoanilino) epipodophyllotoxin

A solution of 3.6 mmol, 1.5 g of podophyllotoxin in 15 ml of ethylene dichloride was kept at 0 ° C while hydrogen bromide gas was passed through the solution. After 45 minutes, the reaction mixture was purged with nitrogen to remove excess hydrogen bromide. Then 4.32 mmol, 0.85 g of anhydrous barium carbonate and 4.32 mmol, 0.6 g of 4-nitroaniline are added to the solution. The mixture was stirred for 18 hours at room temperature under nitrogen. The mixture was diluted with ethyl acetate and filtered. The filtrate was washed with water, dried over magnesium sulfate and purified by flash chromatography using methylene chloride / ethyl acetate / acetone (10: 5: 5) to give > crude 4'-methoxy- (4''-aminoanilino) epipodophyllotoxin and

4'-demethyl- (4''-aminoanilino) epipodophyllotoxin. Further purification by preparative RP-HPLC using a gradient of 10 to 70% MeCN gave pure products as yellow solids.

4'-methoxy-4- (4''-nitroanilino) epipodophyllotoxin

Ananylitic RP-HPLC: t _R = 22.3 min, gradient 0 to 60% MeCN, purity greater than 95%.

HNMR (300 MHz, CDC1 ₃₎ delta: 3.09 (m, 2H, H2,3), 3.77 (s, 6H, _OCH3), 3.83 (s, 3H, OCH ₃₎ 3 86 (m, 1H, H 11),

4.42 (m, 1H, Hll), 4.63 (m, 2H, H-4), 4.84 (m, 1H, NH), 6.00 (m, 2H, OCH ₂ O) 6 31 (s, 2H, H 2 ', 6'), 6.57 (m, 3H, H 8, Ar), 6.76 (s, 1H, H 5), 8.16 (d, 2H, J = 9.08) Hz, Ar).

4'-Demethyl-4- (4''-nitroanilino) epipodophyllotoxin

Analytical RP-HPLC: t _R = 20.5 minutes, gradient 0 to% MeCN, purity greater than 95%. ¹ H-NMR (300 MHz, CDCl ₃ ) δ: 3.07 (m,

(WITH, 6H, och ₃ : ), 3.81 (m (1H, H11), 4.40 (m, 2H, Hl), 4.73. (m, 1H, H4), 4.83 (m, (br, 1H, OH) 5.98 (m, 2H, OCH ₂ O), 6.31 6.57 (m, 3H, H8, Ar), 6.76 (s, 1H, H 5); 9.04 Hz, Ar)

2H, H2.3), 3.79 (m, 1H, H11), 4.60

1H, NH), 5.45 (s, 2H, H 2 ', 6'), 8.14 (d, 2H, J =

To a solution of 4'-methoxy- (4''-aminoanilino) epipodophyllotoxin and 4'-demethyl- (4''-aminoanilino) epipodophyllotoxin 10: 1 in ethyl acetate / methanol was added 10% palladium on activated carbon. The mixture was stirred under a hydrogen atmosphere for 3 hours. The catalyst is then filtered off and washed several times with methanol. The filtrates and washings were combined and evaporated to dryness to give a pale yellow solid which was redissolved in MeCN and purified by preparative RPHPLC using a 10-70% MeCN gradient to give the title compounds as yellow solids in quantitative yield. .

9 9 9 9 9 9 9 9 9

9 9 9 9 9 • 99999 9 9 * * 9 9 9 9

9999 99 9

4'-methoxy-4- (4''-aminoanilino) epipodophyllotoxin

Analytical RP-HPLC: t _R = 16.1 min, gradient 0 to 60% MeCN, purity greater than 95%.

1 H-NMR (300 MHz, CDCl 3) δ: 287 (m, 1H, H 3), 3.11 9m, 1H, H 2), 3.68 (s, 6H, OCH ₃ ), 3.73 (s, 3H, OCH _3), 3.61 (m, 1H, Hll), 4.15 (m, 1H, Hll), 4,52-4, .62 (m, 2H, H-4), 5.86 (m, 2H, OCH ₂ O), 6.28 (s, 2H, H 2 ', 6'), 6.37 (m, 2H, Ar), 6.45 (s, 1H, H 8), 6.69 (s, 1 H, H 5), 7.03 (m, 2H,

Ar).

4'-Demethyl-4- (4''-aminoanilino) epipodophyllotoxin

Ό *

Analytical RP-HPLC: t _R = 14.2 min, gradient 0 to 60% MeCN, purity greater than 97%.

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 3.05 (m, 1H, H 3), 3.18 (m, 1H, H 2), 3.78 (s, 6H, OCH ₃ ), 3.93 (m, 1H, H11), 4.38 (m, 1H, H11), 4.60 (d, 1H, J = 5.91 Hz, H1), 4.70 (d, 1H, J = 3.86) Hz, H 4), 5.96 (m, 2H, OCH ₂ O), 6.33 (s, 2H, H 2 '6'), 9 9 9 9 9 9 '* ·:

9999 99

6.53 (s, 1H, H 8), 6.62 (d, 2H, J = 8.66 Hz, Ar), 6.75 (s, 1H, H 5), 7.19 (d, 2H, J) = 8.60 Hz, Ar).

4'-Methoxy-4- [4''-aminoanilino- (maleimidopropionoyl)] epipodophyllotoxin

To a solution of 41 micromoles, 20.8 mg of 4'-methoxy-4- (4''-aminoanilino) epipodophyllotoxin, 0.226 mmol, 38.2 mg of 3-maleimidopropionic acid, 0.124 mmol, 15.7 mg of DIC and 40 micromoles, 4 9 mg of DMAP in 2 ml of methylene chloride was added 0.2 ml of pyridine. The mixture was stirred for 1 hour and then evaporated to dryness. The resulting pale yellow solid was redissolved in 1 mL DMF and then purified by preparative RPHPLC using a 20-70% MeCN gradient to give 10.1 mg of pure product as a colorless solid in 38% yield. Analytical RP-HPLC: t _R = 19.5 min, gradient 0 to 60% MeCN, purity greater than 96%.

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 2.71 (t, 2H, J = 7.0 Hz, CH ₂ ), 2.91 (m, 1H, H 3), 3.14 (m, 1H) , H2), 3.76 (s, 6H, _OCH3), 3.82 (s, 3H, _OCH3), 3.93 (t, 2H, J = 7.0 Hz, _CH2), 3.97 (m, 1H, H5, Hll), 3.49 (m, 1H, Hll), 4.63 (m, 2H, H-4), 5.97 (m, 2H, _OCH2 O), 6.32 (s, 2H, H 2 '6'), 6.50 (m, 2H, Ar), 6.53 (s, 1H, H 8), 6.73 (s, 2H, CH = CH), 6.74 ( s, 1H, H5), 7.32 (m, 2H, Ar).

4'-Methoxy-4- [4 '' -aminoanilino- (succinimidopropionoyl) - (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp -Lys-Lys-NH 2)] epipodophyllotoxin

0 ^ 0

To a solution of 6 micromoles, 4.1 mg of 4'-methoxy-4- [4 '' aminoanilino- (maleimidopropionoyl)] epipodophyllotoxin and 6 micromoles, 14 mg of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp- Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ in 1 mL DMF was added 2 mL Et ₃ N. The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient 0 to 60% MeCN to give 5.8 mg of pure product as a colorless solid in 32% yield. Analytical RP-HPLC: t _R = 16.0 min, gradient 0 to 60% MeCN, purity greater than 99%. DE MALDI-TOF MS: [M + H] ⁺ = 3003.9, the molecular formula H ₂ CI4 o7N _{3 2 3} 90 A ₂ = 3004.54.

Example 34

4 ' -methoxy-4- [4 -aminoanilino- (sukcinimidopropionoyl) - (H-Cys-betaAla-Arg-Met-Lys-Trp-Lys-Lys-NH ₂₎ 3epipodofyllotoxin

9 ·

To a solution of 7 micromoles, 4.6 mg of 4'-methoxy- [4 '' - aminoanilino- (maleimidopropionoyl) epipodophyllotoxin and 14 micromoles, 16.3 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-TrpLys-Lys -NH 2 in 1 microliters of DMF was added 1 mL Et 3 N. The mixture was stirred for 1 hour and then purified by preparative RP-HPLC using a gradient of 0 to 60% MeCN to give 6.4 mg of pure product as a colorless solid in 49% yield. Analytical RP-HPLC: t _R = 15.2 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: (M + H] ⁺ = 1861.6, C87H125N23O19S2 = 1861.20.

Example 35

4'-Demethyl-4- [4 '' -aminoanilino- (maleimidopropionoyl) epipodophyllotoxin

OH • 9 • 9 · 9

9 · ·· · · ·

To a solution of 24 micromoles, 12 mg of 4'-demethyl-4- (4''-aminoanilino) epipodophyllotoxin, 49 micromoles, 8.3 mg of 3-maleimidopropionic acid and 27 micromoles, 3.4 mg of DIC in 2 ml of a mixture of DMF and methylene chloride 1: 1 is added 10 microliters of pyridine. The mixture was stirred for 1 hour and then evaporated. The resulting pale yellow solid was purified by preparative RP-HPLC using a gradient of 10 to 70% MeCN to yield 5.3 mg of pure product as a colorless solid in 34% yield. Analytical RPHPLC: t _R = 19.5 min, gradient 0 to 60% MeCN, purity greater than 96%.

¹ H-NMR (300 MHz, CDCl 3) δ 2.65 (t, 2H, J = 7.3 Hz, CH ₂ ), 2.98 (m, 1H, H 3), 3.17 (m, 1H, H2), 3.79 (s, 6H, _OCH3), 3.93 (t, 2H, J = 7.0 Hz, _CH2), 3.99 (m, 1H, H5, Hll), 4.38 (m, 1H, H 11), 4.58 (d, 1H, J = 4.95 Hz, H 1), 4.64 (d, 1H, J = 3.95 Hz, H 4) 5.96 (m, 2H) OCH ₂ O), 6.33 (s, 2H, H 2 '6'), 6.49-6.53 (m, 3H, H 8, Ar), 6.74 (s, 2H, CH = CH), 6.75 (s, 1H, H 5), 7.33 (m, 2Ή, Ar).

4'-Demethyl-4- [4-aminoanilino- (succinimidopropionoyl) (H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] epipodophyllotoxin

0 ^ 0

0

To a solution of 8.3 micromol, 5.3 mg of 4'-demethyl- [4-aminoanilino- (maleimidopropionoyl)] epipodophyllotoxin and 13 micromol, 15.6 mg of H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp- Lys-Lys-NFL in 1.5 ml DMF was added with 2 microliters of Et ₃ N. The mixture was stirred for 1 hour and then purified by preparative RPHPLC using a gradient of 0 to 60% MeCN to give 14.9 mg in 97% yield. pure product as a colorless solid. Analytical RP-HPLC: t _R = 13.7 min, gradient 0 to 60% MeCN, purity greater than 98%. DE MALDI-TOF MS: [M + H] ⁺ = 1847.1

C86H123N23O19S2 = 1847.17.

Example 36

Cytotoxic activity of {[4 [N- (2,4-diamino-6-pteridinylmethyl) -N-methylamino] benzoyl] -Glu-Gly-betaAla} _4- Lys ₂ -Lys-betaAla-Arg-Gln-Ile-Lys- In-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in vitro

The aforementioned substance is abbreviated as MTX-Pen in the following tables and evaluated for its ability to induce cell proliferation inhibition in normal (immortalized) human cells, in particular HaCaT cells in Tables 1 and 2 as well as human colorectal carcinoma cells. HT29 cells in Table 3. Methotrexate, designated MTX in Tables 1-3, and the free vector H-Ala-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-AsnArg-Arg- Met-Lys-Trp-Lys-Lys-OH, this vector is designated Pen in Table 1.

• · 9

Assay: cells were seeded in 96 well plates at 2500 cells / well in DMEM with 10% FCS and antibiotics. After overnight incubation, the test substances were prepared in the same medium at various dilutions and the solutions were added to the cells. Samples were then taken 1, 2, 3 and 4 days after the addition of the solution. Nucleotide Releasing Reagent (LumiTéch ViaLight) was then added to disrupt cells and release ATP.

After incubation for 5 minutes at room temperature, the mixture was transferred to 96-well opaque plates and stored at -20 ° C until analysis. Plates were then transferred to a luminometer (Lucy 1, Labtech International) and ATP Monitoring Reagent (LumiTéch ViaLight) was added to each well at 20 microliter / well and light intensity was immediately measured. Six readings were made for each sample. In addition, appropriate checks were also carried out. It has been shown that ATP bioluminescence was proportional to the number of viable cells over a range of cells in all wells. The following tables show the results, with statistically significant results being indicated by stronger printing.

Table 1. HaCaT cells

Dose (mM)% cell death

Day 1 Day 2 Day 3 Day ⁴ MTX MTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen 40.0 4 29 ! 6 15 Dec 82 -22 79 97 5 92 98 12 13.3 22nd -42 1S 35 63 0 82 97 -17 92 98 -6 4.4 4 -8 8 24 45 -4 77 95 -1 93 98 10 1.3 13 -24 16 31 82 -31 77 82 2 94 88 -14 0 ~ 5 -4 -19 6 31 2 -6 75 29 -29 93 49 -26 0-2 7 14 26 11 21 0 79 20 May -3 93 51 2!

»«

4 •

494

944

Table 2. HaCaT cells

Dose (gM) ·% cell death

Day 1 Day 2 Day 3 Day 4 Λί7%. MTX-Pen MTX MTX-Pen MTX. MTX-Pen MTX MTX-Pen 40.0 42 88 95 94 13.3 '27 87 95 94 4.4 21 15 Dec 70 52 97 95 92 83 1.5 14 19 Dec 67 12 96 -16 91 17 0.5 0 13 59 24 96 -27 91 2 0.2 3 41 94 86 0.1 19 Dec 7 45 65

Table 3. HT 29 cells

Dose (µM)% cell death

Day ¹ Day - Day. 3 Day 4 MTX MTX-Pen MTX MTX-Pen MTX MTX-Pen MTX MTX-Pen 40.0 31 79 96 98 13.3 3 45 88 96 4.4 -14 10 -4 6 ⁵⁸ 46 86 77 1.5 17 16 -5 9 48 15 Dec 84 45 0.5 15 Dec 14 -12 8 52 17 88 16 OJ 10 -5 54 85 0.1 6 -17 52 84

Example 37

Stabilization of microtubule formation by paclitaxel and 2 '- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH) paclitaxel

Performance of the test - Tetramethylrhodamine labeled bovine tubulin and tubulin solution (total

9 * «9 · 98 ·· * 9 9 9» · ·

9 9 9

99999 9 9

9 9 9 φ «· ♦ ·· 99 9« 99

9 (0.5 mg / ml concentration) in G-PEM buffer containing 80 mM PIPES pH 6.8, 1 mM EDTA and 1 mM GTP is incubated in the presence of 10 microM paclitaxel, designated A in Fig. 1, 10 microM ^of 2 - [sukcinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-LysLys-OH)] paclitaxel (B) in the presence or absence of the test substance (C) for a total of 30 minutes at 37 ° C. Microtubule formation was followed by a Nikon Eclipse E800 fluorescence microscope. Photos were taken with a Kodak DCS 420 camera (digital camera) and analyzed using Adobe 5.0 software.

Example 38

Internalization of 4- [succinimidopropionoyl (biotinamido-caproyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH2) ] of podophyllotoxin into cells

A549 cells were seeded in 96-well plates at 50,000 cells / well in DMEM with 10% FCS and antibiotics. After overnight incubation, solutions of 4- [succinimidopropionoyl (biotinamido-caproyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-) were prepared. Cys-Gly-NH ₂ )] podophyllotoxin ("conjugate" in Fig. 2) and biotinamidocaproyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp -Lys-Lys-Gly-Cys-Gly-NH2 (&quot; vector &quot; in FIG. 2) as a serial dilution of 6 decreasing concentrations in cell culture medium, solutions were added to the wells of the cells. At the end of the 60 minute incubation period, the cells were rinsed 3 times with PBS and then fixed for 20 minutes at a temperature of ·············

-20 ° C with 95: 5 ethanol: acetic acid. After fixation, cells were permeabilized for 10 minutes with PBS containing 3% Tween-20. Endogenous alkaline phosphatase was neutralized by incubating the plates at 65 ° C for 60 minutes. Cells were then incubated for 30 minutes at room temperature with alkaline phosphatase-streptavidin conjugate (Pierce Chemical Co) in 0.1% BSA in PBS, then the cells were washed extensively with PBS. A freshly prepared solution of 1 mg / ml * nitrophenyl phosphate in 10 mM diethanolamine, pH 9.5, with 0.5 mM magnesium chloride was added to each well, and the material was incubated until sufficient color development, about 30 minutes. The enzymatic reaction was stopped by adding 50 microliters of a 2 M aqueous sodium hydroxide solution. Alkaline phosphatase activity was measured spectrophotometrically at 405 nm.

Example 39

Visualization of internalization of 4- [succinimidopropionoyl (biotinamidocaproyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Cys-Gly-NH ₂ )] podophyllotoxin into cells

Cells were seeded onto 8-well slides at 50,000 cells per well in DMEM with 10% fetal calf serum and antibiotics. After overnight incubation, 4- [succinimidopropionoyl (biotinamido caproyl-betaAla-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-) was dissolved in the cell medium. Gly-Cys-Gly-NH ₂ )] podophyllotoxin at 10 µM and the solution was added to the cells. At the end of the incubation period of 60 minutes, the cells were washed 3 times with PBS and fixed for 20 minutes at a temperature of 20 ° C.

-20 ° C with 95: 5 ethanol: acetic acid. After fixation, cells were permeabilized for 10 minutes with PBS containing 3% Tween-20. The slides were then incubated for 30 minutes with streptavidin-FITC conjugate (Pierce Chemical Co.) after dilution in PBS at room temperature, the cell material should be washed thoroughly with PBS and then placed in Hydromount (BDH). Fluorescence distribution was analyzed using a Nikon Eclipse E800 fluorescence microscope. Photos were taken with a Kodak DCS 420 digital camera and analyzed with Adobe 5.0 software. A representative representation is shown in Figure 3.

Example 40

Stability of 4- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] Podophyllotoxin x 10 ⁶ HL60 cells were incubated for 1 hour with 15 microM 4- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys) -OH)] podophyllotoxin (Compound A in Figure 4) in the presence or absence of the test compound (B) in DMEM. After incubation, the cell material is washed thoroughly with PBS until no more test substance can be detected in the wash liquid. The cells are then incubated for an additional hour with pure culture medium. After centrifugation of the cell material, the pellet is resuspended in 50 mM Tris pH 7.5 containing a protease inhibitor mixture, and the cell material is solubilized by sonication for 1 minute. The insoluble fraction was centrifuged for 15 minutes using an Eppendorf centrifuge, and the supernatant was analyzed by analytical RP-HPLC at '333 s with a n-g concentration.

444

4

4444 44

4 44 using a gradient of 0 to 60% MeCN, lambda = 254 nm. The intact test substance was identified by comparison with chromatograms obtained using the authentic test substance and analysis of DE MALDI-TOF MS material, indicated by the arrow in Figure 4 and taken from the main fractions. The pellets were further extracted with DMSO and the extracts were analyzed in a similar manner, showing no test substance.

Example 41

Serum peptide vector stability

The test substances were dissolved in cell culture medium, 10% FCS in DMEM, at concentrations ranging from 1 to 40 microM. The solutions were incubated at 37 ° C and samples were taken at various intervals. After filtration, aliquots were analyzed by RPHPLC using photodiode UV detectors. Intact vectors were identified by comparison with chromatograms prepared using authentic peptides and analysis of DE MALDI-TOF of the respective main fractions. Half-lives for the 4 different vectors are summarized in Table 4. Similar results were obtained when human or mouse serum was used instead of bovine serum, FCS. Mouse serum is particularly preferred because cell culture conditions are mimicked. In all cases, the major metabolic product of the 16-amino acid peptide H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH with 15 amino acid residues amino acid residues resulting from cleavage of the Lys residue from the C-terminus. This peptide survives in the serum for several hours until further degradation at the C-terminus. Peptide-type vectors containing L-amino acids were

degraded much slower and it was not possible to identify individual metabolites. All peptide vectors containing D-amino acids were very stable and would usually be detectable after 72 hours of incubation.

Table 4

VeAtor Serum H-Arg-Gln-He-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp- Lys-Lys-OH 10 min H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ > 12 h H-D-Arg-D-Gln-D-Ile-D-Lys-D-Ile-D-Trp-D-Phe-D-Gln-D-Asn-D- Arg-D-Arg-D-Me-D-Lys-D-Trp-D-Lys-D-Lys-NH 3 > 24h H-Ara-Arg-Met-Lys-Trp-Lys-Lys-NHn 3h

Example 42

Stability of binding of active substances and esters in serum

Test substances identified as A: 4 in Table 5 [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys -NH ₂ )] podophyllotoxin, B: 4- [Acetyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys -H)] podophyllotoxin, C: 2 '- [Succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys -NH 2 O] paclitaxel is dissolved in cell culture medium, 10% FCS in DMEM at a concentration ranging from 1 to 40 microM. The solutions are incubated at 37 ° C and samples are taken at specified intervals. After filtering, 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9

9

9

Aliquots of these samples are analyzed using RP-HPLC and UV detector with photodiode. Hydrolysis of the ester linkages between the hydroxy group of the drug and the carboxyl group of the linker was followed by detection of free podophyllotoxin or paclitaxel. Half-lives for the three different combinations of drug and linker are summarized in Table 5. Similar results were obtained when human or mouse serum was used instead of bovine serum. The serum of the mouse is very advantageous since the procedure can be carried out under similar conditions to that in which cell cultures are grown to demonstrate cytotoxicity.

Table 5

The half-life of the ester bond between the compound and the linker in the serum> 24h

min

o> 12 h

Í.ťffjPÍMM ^ irtrgiSŽÍgBgtMag ···· 9 9 4

99 ·· 99 • 9 9 9

999 9

9 g β 999999

Example 43

Comparison of cytotoxic effect of paclitaxel and its conjugates with vectors

To demonstrate cytotoxic biological effects on A54 9 lung carcinoma cells and MCF7 mammary carcinoma cell lines, paclitaxel-vector conjugates were a vector containing 16 amino acid residues, 2 '- [succinimidopropionoyl- (H-Cys-Arg) -Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] paclitaxel and also with a vector containing 7 amino acid residues, 2 '- [ succinimidopropionoyl- (H-Cys-betaAla-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ ) paclitaxel used to monitor that the cell material was exposed to the complexes for 1 hour, i.e., at wherein the conjugates are metabolically stable under the conditions under which the assay is performed as set forth in Examples 41 and 42. IC50 values at 1 hour and 3 days are summarized in Table 6 and compared to those obtained using free paclitaxel. . It should be noted that due to the negligible solubility of unconjugated paclitaxel in water, the elution of non-internalized cell compounds was far less effective than conjugates whose solubility in physiological environments is greater than 10 mg / ml. It can be concluded that after 1 hour of treatment the cytotoxic efficacy is due to the presence of intact paclitaxel conjugates, as also evident from Example 37.

Test run - Cell material was seeded onto 96 well plates at 2500 cells / well in DMED medium containing 10% FCS and antibiotics. Po «

• 9 • · ©

· © * * * * * * * * * * * * * * © * * © * * © © © © © © © © © Test substances were prepared at various dilutions in a cell culture medium overnight, in the case of free paclitaxel dimethyl sulfoxide was added to at least partially dissolve, solutions were added to the cell material. After 1 hour of treatment, the plate was incubated for an additional hour, then the supernatants were removed and the wells were washed with cell culture medium for 5 times 2 minutes. After a total incubation of 72 hours, total viable cells were quantitated using a standard MTT procedure.

Table 6

Test substance 72h, IC50 (microM) Cell line A 549 MCF7 Exposure time 1 h 2 d 1 h 2 d Paclitaxel 0,028 <0.015 0.04 <0.015 Paclitaxel-vector (16-mer) 0,618 <0.015 0.202 0.017 Paclitaxel-vector (7-mer) 0,043 <0.015 0.325 <0.015

Example 44

Evaluation of paclitaxel-vector and podophyllotoxin-vector conjugates on cancer cell lines

The cell lines were treated with serial dilutions of the test substances, in Table 7, the following substances: 2'-paclitaxel-vector conjugate, 2 [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln) -Asn-Arg ^; ί 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ··

-Arg-Met-Lys-Trp-Lys-Lys-OH)] paclitaxel, 7-paclitaxel-vector conjugate, 7- [succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn) -Arg-Arg-Met-Lys-Trp-Lys-Lys-OH)] paclitaxel and a 4-podophyllotoxin-4-succinimidopropionoyl- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-) conjugate Phe-Gln-Asn-Ar-Arg-Met-Lys-Trp-Lys-Lys-OH)] podophyllotoxin. After incubation for 96 hours, cytotoxicity was determined using a standard SRB assay.

Table 7. Evaluation of cytotoxicity on cell lines at exposure time of 96 hours, IC ₅₀ (microM)

Cellular line Vector Conjugate 2'-paclita- xel-vector Conjugate 7-paclita- xel-vector Paclitaxel Conjugate 4-podophyll- lotoxin- -vector Etoposide BE > 25 0.0305 1.6 < 0.0025 0.55 1.1 COLO205 > 25 0.074 1.9 0.0026 0.495 0.8 DLD-1 > 25 0.6 25 0.054 0.65 0.57 HCT116 > 25 0.096 2.4 < 0.0025 0.53 1.9 HT29 > 25 0.092 2.25 < 0.0025 0.53 2.6 KM12 > 25 0.105 2.95 0.0028 5 0.0058 0.58 0.58 LIM1215 > 25 0.12 3.65 1.1 0.33 LS174T > 25 0.195 7.4 0.0085 1.25 0.46 A2780 > 25 0.105 2.8 < 0.0025 0.54 0.21 A2780Cis ^K > 25 0.125 4.3 0.0051 0.54 0.68 CH1 > 25 0.115 6.6 0.0041 0.51 0.165 CHIDox " > 25 4.6 > 25 5 0.54 0.51 6.6 CH1Taxol ^K > 25 0.13 8.7 0.0058 0.52 0.145 SKOV-3 > 25 0.235 22nd 0.01 0.74 13

Example 45

Evaluation of etoposide and podophyllotoxin derivatives for inhibition of topoisomerase II

Topoisomerase II Assay - 0.3 microgram of plasmid DNA is incubated at 37 ° C with 4 units of purified recombinant human topoisomerase II in digestion buffer containing 30 mM Tris.HCl at pH 7.6, 60 mM NaCl, 3 mM ΆΤΡ, 15 mM mercaptoethanol and 8 mM magnesium chloride, with or without addition of the test substance at a final concentration of 1 mM, 100 microM or 10 microM. The reaction is stopped by adding SDS to a final concentration of 1% by weight. The samples were treated with proteinase K for 30 minutes at 37 ° C and then extracted 2 times with an equal volume of a 42: 1 mixture of chloroform and isoamyl alcohol. After addition of the dye, the samples were run on 4 x TAE, 1% agarose gel containing 0.5 mg / ml ethidium bromide and electrophoresed for 16-24 hours. Inhibition of topoisomerase II is determined by the production of linear plasmid DNA and the captured cleavage intermediate, and the ratio of substrate (helical DNA) to product (released DNA) is determined. The relaxation assay is performed in the same manner, except that the reaction buffer is optimized for detecting catalysis rather than detecting cleavage so that only 2 units of enzyme per sample are used. The reaction buffer used is 50 mM tris.HCl, pH 8, 120 mM KCl, 0.5 mM ATP,

0.5 mM childhiothreitol and 10 mM magnesium chloride. Inhibition of topoisomerase II can be inferred from the ratio of substrate (helical DNA) to product (released DNA).

• ·

Table 8

Test substance efficacy ³ Etoposide IC Podophyllotoxin - 4'-demethylepipodophyllotoxin IC 4'-Demethyl-4- (4''aminoanilino) - epipodophyllotoxin AND BetaAla H-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ - 4- [sukcinimidopropionoyl- (H-Cys-Arg-betaAla -Arg-Met-Lys-Trp-Lys-Lys-NH _2)] podophyllotoxin 4 '- [Succinimidopropionoyl- (H-Cys-betaAla-Arg- -Arg-Met-Lys-Trp-Lys-Lys-H2)] epipodophyllotoxin IC 4'-Demethyl-4- [acetyl- (H-Cys-beta-Ala-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH ₂ )] epipodophyllotoxin IC 4'-Demethyl-4- [4''-Aminoanilino- (succinimidopropionoyl) - (H-Cys-betaAla-Arg-Arg-Met-Lys-Trp-Lys-Lys--NH ₂ )] epipodophyllotoxin AND

a) I means inhibition of helix plasmid relaxation by topoisomerase II. C means the buildup of the reaction intermediate topoisomerase II.

Abbreviations:

Boc t-butyloxycarbonyl

Bu ¹³ tert-butyl

CF3COOH trifluoroacetic acid

CH 2 Cl 2 dichloromethane

DE MALDI-TOF MS laser desorption ionization mass spectrometry time

DMF

Et ₂ O

N, N-dimethylformamide diethyl ether ·· · ·

Fmoc 9-fluorenylmethyloxycarbonyl HMBA p-hydroxymethylbenzoyl HOBt 1-hydroxybenzotriazole MeCN acetonitrile Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl Pr ^ NEt N, N-diisopropylethylamine PyBOP benzotriazol-1-yloxytrispyrrolidinophosphonium- hexafluorophosphate RP-HPLC high pressure liquid chromatography reverse phase Trt trityl (triphenylmethyl)

Example 1a

H-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) ) -Trp-Lys (Boc) -Lys (Boc) -resin

The peptide was obtained using the ABI 433A peptide synthesizer (Perkin-Elmer Applied Biosystems). A standard procedure ("FastMoc 0.25 mmol MonPrevPk") was used.

The starting resin was 0.5 mmol / g resin FmocLys (Boc) - [(4- (hydroxymethyl) phenoxyacetyl) resin] (ABI 401425). The resulting peptidyl resin, obtained in 100% yield and 1.37 g, was washed with Et ₂ O and dried under vacuum.

To demonstrate the chemical integrity of this intermediate, a small proportion of the peptidyl resin was cleaved and deprotected and the crude product analyzed, H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg -Met-Lys-Trp-Lys-Lys-OH with purity greater than 90% by analytical RP-HPLC, the identity of the compound was confirmed by DE MALDI-TOF MS and quantitative amino acid analysis.

[H-Glu (OBu *) -Gly-bAla] ₄ -Lys ₂ -Lys-bAla -Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) - Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc)-Resin * 137 mg, 0.025 mmol of the above peptidyl resin was acylated using 47 mg, 0.15 mmol Fmoc *

-betaAla-OH, 78 mg, 0.15 mmol PyBOP, 25 mg, 0.15 mmol HOBt and 39 mg, 0.225 mmol Pr 2 NEt in 2 mL DMF over 2 h. Then the Fmoc group is cleaved for 20 minutes with 20% piperidine in DMF and the material is washed extensively with DMF. The product was extended 2 more times by double acylation and deprotection using Fmoc-Lys (Fmoc) -OH (0.15 mmol in the first cycle and 0.3 mmol in the second cycle) using similar coupling cycles and subsequent deprotection. The chain was then extended again using 0.6 mmol of Fmoc-Gly-OH and 0.6 mmol of Fmoc-Glu (OBu ¹¹ ) -OH again using similar degrees of acylation and cleavage of the Fmoc protecting group. The product was then deprotected and thoroughly washed successively with DMF, CH ₂ Cl ₂ and Et ₂ O, then dried in vacuo.

t

To demonstrate the chemical integrity of this t intermediate, a small proportion of the peptidyl resin was cleaved and the side chain was deprotected, and the crude product was analyzed, [H-Glu-Gly-betaAla] ₄ -Lys ₂ -Lys-betaAla-Arg- Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH, a purity of greater than 89% was shown by RP-HPLC on a Vydac 218TP54 column, flow 1 ml / min, 25 ° C, gradient of 15 to 25% MeCN in 0.1% aqueous CF 3 COOH over 20 minutes, t _R = 17.7 minutes,

100 lambda = 200-300 nm, identity was confirmed by DE MALDI-TOF MS: [M + H] ⁺ = 3732, Formula C165H269N53O44S = 3731.30.

{[4 [N- (2,4-diamino-6-pteridinylmethyl) -N-methylamino] benzoyl] -Glu (OBu ¹¹ ) -Gly-betaAla} ₄ -Lys ₂ -Lys-bAla-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) Resin mg, 0.025 mmol of the above peptidyl resin was reacted overnight with 76 mg of 0.2 mmol of hemihydrochloride dihydrate [4 [N- (2,4-diamino-6-pteridinylmethyl) -N] at room temperature. methylamino] benzoic acid (Aldrich 86, 155-3) and 104 mg, 0.2 mmole of PyBOP, 27 mg, 0.2 mmol of HOBt and 70 .mu.l, 0.4 mmol Pr ¹ 2 NEt in 2 mL of DMF. The product was washed sequentially with DMF, CH ₂ Cl ₂ and Et ₂ O, then dried under vacuum to give 85 mg of the resultant peptidyl resin as an orange solid.

{[4 [N- (2,4-diamino-6-pteridinylmethyl) -N-methylamino] benzoyl] -Glu-Gly-betaAla} 4-Lys- _2- Lys-bAla-Arg-Gln-Ile-Lys-Ile- Trp-Phe-Gn-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH

O

101

The above product is processed. 1.5 hours at room temperature with phenol, water, thioanisole, 1,2-ethanedithiol and TFA in a ratio of 0.75: 0.5: 0.5: 0.25: 10. 12 ml of this reagent are used. The resin residue is then filtered off and washed on the sinter with small portions of pure trifluoroacetic acid. The filtrates and the washings were combined, 100 mL of diethyl ether was added and the mixture was cooled. The precipitated product is centrifuged and the ether supernatant is decanted. The product was washed 3 more times with diethyl ether. The resulting crude product was dried under vacuum to give 61 mg of an orange powder. This material was redissolved in 4 mL of a 0.1% aqueous trifluoroacetic acid solution and filtered. The resulting solution was applied in two portions to an RP-HPLC column (Vydac 218TP1022, 22 x 250 mm). The column was eluted at 9 ml / min using a gradient of 17.5 to 27.5% MeCN in 0.1% aqueous trifluoroacetic acid over 25 minutes at 25 ° C. The main fractions were collected, monitored by analytical RP-HPLC and pooled as needed. After centrifugation in vacuo, 13.5 mg of pure product is obtained. Analytical RP-HPLC: t _R = 17.8 min (Vydac 218TP54, gradient 17.5 to 27.5% MeCN in 0.1% aqueous trifluoroacetic acid solution, 20 min, flow rate 1 mL / min, 25 ° C, purity higher than 99%, lambda = 200-300 nm). DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 4962, C225H321N81O48S = 4960.54.

Example 2a · · · · · · · · · ««

102

H-Cys-Arg-Gln-Ile-Lys-Ip-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH

411 mg, 0.075 mmol H-Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) -Arg (Pmc) -Arg (Pmc) - Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) resin was acylated by treatment with 264 mg, 0.45 mmol of Fmoc-Cys (Trt) -OH, 234 mg,

0.45 mmol PyBOP, 61 mg, 0.45 mmol HOBt and 0.12 mL, 0.675 mmol N, N-diisopropylethylamine in 3 mL DMF for a total of 3 hours. The resulting peptidyl resin was washed for 3 x 5 min with 25 mL DMF, then allowed to drip and 20% piperidine in DMF was added for 20 min. After filtering off the reagent, the resulting product, H-Cys (Trt) -Arg (Pmc) -Gin (Trt) -Ile-Lys (Boc) -Ile-Trp-Phe-Gln (Trt) -Asn (Trt) - The Arg (Pmc) -Arg (Pmc) -Met-Lys (Boc) -Trp-Lys (Boc) -Lys (Boc) resin was sequentially washed with DMF, CH 2 Cl 2 and Et ₂ O, then dried in vacuo.

The above product is treated for 2 hours at room temperature with 12 ml of a mixture of phenol, water, thioanisole, 1,2-dithioethane and trifluoroacetic acid in a ratio of 0.75: 0.5: 0.5: 0.25: 10. The residual resin is then filtered off and washed on the sinter with small portions of pure trifluoroacetic acid. The filtrate and washings were combined, 100 mL of diethyl ether was added and the mixture was cooled. The precipitated product is isolated by centrifugation and the ether supernatant is decanted. The product was washed 3 more times with diethyl ether in a similar manner. 238 mg of crude product are obtained, which is dried under vacuum. A portion of 119 mg of this material was redissolved in 2 mL of 0.1% aqueous trifluoroacetic acid and filtered. The filtered solution was applied to a RPHPLC column (Vydac 218TP1022, 22 x 250 mm). The column is eluted ·· ♦ 9 9

9 9

103 at 9 mL / min using a gradient of 17.5 to 27.5% MeCN in 0.1% aqueous trifluoroacetic acid over 25 min at 25 ° C. The main fractions were collected, monitored by analytical RP-HPLC and pooled as needed. After centrifugation in vacuo, 60.9 mg of pure product is obtained. Analytical RP-HPLC: t _R = 15.8 min (Vydac 218TP54, gradient 17.5 to 27.5% MeCN in 0.1% trifluoroacetic acid aqueous solution, 20 min, mL / min, 25 ° C, purity higher than 99%, lambda = 214 nm), DE MALDI-TOF MS: [M + H] ⁺ = 2351, Formula C107H173N35O21S2 = 2349, 87.

N- [3- (maleimido) benzoyl] -doxorubicin

5.9 mg, 10 micromole of doxorubicin hydrochloride (Aldrich, 86.036-0) was dissolved in 1 mL of water and 0.5 mL of DMF. 0.5 ml of 0.1 M aqueous phosphate buffer, pH 7.2 are added with stirring. To the resulting suspension was added 12.9 mg, 40 µmol of N-hydroxysuccinimide acid ester

3-maleimidobenzoic acid dropwise in 1 mL DMF. The red-colored reaction mixture gradually becomes clear and a precipitate is observed after about 10 minutes. The progress of the reaction was monitored by analytical RP-HPLC, after 2 hours the reaction of doxorubicin was complete. The mixture is diluted with 1.5 ml of water, cooled to 4 ° C and centrifuged. Decant the supernatant. The resulting pellet was redissolved in 1 mL of dimethylformamide and diluted with 2 mL of a 0.1% aqueous TFA solution. This solution was applied to a pre-filled solid phase column (500 mg LiChrolut RP-18 Merck, the column was pre-equilibrated in MeOH with 0.1% aqueous TFA). The column is washed with 4 ml of a 0.1% aqueous TFA solution and then eluted with 6: 4 MeCN / water, water containing 0.1% TFA, 2 fractions are used.

· ♦ • © ©

104 washings, 2 x 4 ml. The first fraction contained the title compound and was used directly in the next step.

N- {3- [3- (H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-0H) Succinimido] Benzoyl] -doxorubicin

The above solution of N- [3 (maleimido) benzoyl] doxorubicin was diluted with 1 ml DMF and 50 microliters of Et ₃ N was added. The color of the solution turned dark brown. H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in solution in 1 mL of DMF was then added. The mixture was stirred while the brown color turned to light red as the mixture was stirred. The reaction was monitored by analytical RP-HPLC. After 1.5 hours the reaction is complete

3- (maleimidobenzoyl) doxorubicin terminated. The solution is acidified with 0.5 ml of acetic acid, diluted with 3 ml of water and applied to a pre-filled solid phase extraction column, taking 500 mg of LiChrolut RP-18 (Merck). The column is washed with 6 ml of a 0.1% aqueous TFA solution and then eluted with 6 ml of a 6: 4 mixture of MeCN and water containing 0.1% TFA. The eluate is dried by centrifugation in vacuo. The rest is again • · »· ····

105 was dissolved in 2 ml of 0.1% aqueous TFA solution, filtered and purified by preparative RP-HPLC (Vydac 218TP1022, 22 x 250 mm) using a gradient of 20 to 40% MeCN. The fractions containing the title compound were collected, analyzed by analytical RPHPLC and pooled as needed. The material was centrifuged again in vacuo to give 1.2 mg of pure title compound. Analytical RP-HPLC: t _R = 15.6 and 15.8 minutes (partially separated thioether diastereomers), gradient 0 to 60% MeCN, purity greater than 95%, lambda = 200 to 300 nm, DE MALDI-TOF MS:

[M + H] ⁺ - 3094, [M + 2H] ²⁺ = 1548, Formula C 14 H 20 7 N 37 O 35 S 2 = 3092.56.

Example 3a

2 '- [(3-maleimidopropionoyl)] paclitaxel

3-Maleimidopropionic acid (5.7 mg, 0.034 mmol) was dissolved in dichloromethane (0.2 mL). The mixture was stirred while 2.4 mg (0.019 mmol) of diisopropylcarboldiimide in 0.5 ml of anhydrous dichloromethane was added. The reaction was allowed to proceed for 30 minutes with stirring, then the solvent was evaporated under reduced pressure. The residue of 3-maleimidopropionic anhydride is redissolved in 0.5 ml of anhydrous pyridine. A solution of 1 mg, 0.0012 mmol of paclitaxel (Aldrych 41.701-7) in 0.5 mL of anhydrous pyridine was then added and the mixture was stirred under nitrogen for 3 hours. The mixture was then evaporated to dryness under reduced pressure. The residue is treated with 1.5 ml of water. After 10 minutes, the mixture was extracted with 3 x 5 mL dichloromethane. The extracts were combined, washed with 3 x 1 mL water, dried over magnesium sulfate and evaporated to dryness to give the title product as a white residue.

· * Φφφφ

106

2 '- {3- [3- (Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH) Succinimido] Propionoyl} paclitaxeí

The product from the previous reaction was redissolved in 0.25 mL of DMF and 2.5 mg, 0.0011 mmol of H-Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg was added. -Met-Lys-Trp-Lys-Lys-OH in solution in 0.25 mL of DMF along with approximately 0.05 mL of Et ₃ N.

The mixture was stirred under nitrogen and monitored by analytical RP-HPLC. After 45 minutes the reaction is complete. The mixture was diluted to 2 mL with 0.1% aqueous trifluoroacetic acid, filtered and applied to an RP-HPLC column (Vydac 218TP1022, 22 x 250 mm). The column was eluted at 9 mm / min using a gradient of 0 to 60% MeCN in a 0.1% aqueous trifluoroacetic acid solution at 25 ° C for 40 minutes. The main fractions are analyzed by RP-HPLC and combined as necessary. After centrifugation in vacuo, 1.2 mg of pure product is obtained. Analytical RPHPLC: t _R = 17.4 and 17.5 min (partially separated thioether diastereomers), (Vydac 218TP54, gradient 0 to 60% MeCN in 0.1% trifluoroacetic acid aqueous solution, 20 min, 1 mL / min, 25 min ° C, purity greater than 95%, lambda = 200 to 300 nm DE MALDI-TOF MS: [M + f]

107 ·· 0 ··· 00 * 0 0-0 0 0 ♦ ··· • ·

0000 00

H] ⁺ = 1679, Formula C161H229N37O38S2 = 3354.90.

Example 4a

Cytotoxic activity of {[4 [N- (2,4-diamino-6-pteridinylmethyl) -N-methylamino] benzoyl] -Glu-Gly-betaAla} _4- Lys2-Lys-betaAla-Arg-Gln-Ile-Lys-Ile -Trp-Phe-Gn-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH

This substance, which will be abbreviated as “MTX-Pen” in the following tables, was evaluated for its ability to inhibit the proliferation of normal (immortalized) human cells, HaCaT cells in Tables 1 and 2, in addition to human colorectal carcinoma cell line assays. HT29 in Table 3. For comparison, the free methotrexate was used in Tables 1 to 3 and the free vector H-Ala-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met- Lys-Trp-Lys-Lys-OH, this vector is designated Pen in Table 1.

Assay - Cells were seeded in 96 well plates at 2500 cells / well in DMEM with 10% fetal calf serum and antibiotics. After overnight incubation, the test substances were prepared in the same medium at various dilutions and the solutions were added to the cells. Samples were then taken 1, 2, 3 and 4 days after the addition of the solution. Nucleotide Releasing Reagent (LumiTech ViaLight) was then added to disrupt cells and release ATP. After incubation for 5 minutes at room temperature, the mixture was transferred to 96-well opaque plates and stored at -20 ° C until analysis. Then, the plates were transferred to a luminometer (Lucy 1, Labtech International) and φ * φφφφφφφφφφφφφφφφφφ

108

ATP Monitoring Reagent (LumiTech ViaLight) at 20 microliters / well and instantly measured light intensity. Six readings were made for each sample. In addition, appropriate checks were also carried out. It has been shown that ATP bioluminescence was proportional to the number of viable cells over a range of cells in all wells.

The following tables show the results, with statistically significant results being indicated by stronger printing.

Table 1. HaCaT cells

( _U M) * ^{/ o} cell death Day 1 Den2 Day 3 Day ⁴ MTX Λ / 7Ύ-Ατ / τ Pen MTX MTX-Pen Pen MTX MTX-Pen Pen MTX MTX-Pen Pen 40.0 4 29 16 15 Dec 82 -22 . 79 97 5 92 98 12 12.3 22nd -42 18 35 63 0 82 97 -17 92 98 -6 4.4 4 -8 8 24 45 -4 77 95 -1 93 98 10 1.5 13 -24 16 31 82 -31 77 82 2 94 88 -14 0.5 -4 -19 6 31 2 -6 75 29 -29 93 49 -26 0.2 7 14 26 It 21 0 79 20 May -3 93 51 21

Table 2. HaCaT cells

Dose (µM)% cell death

Day AND Day 2 Day 3 Day 4 MTX. MTX-Pen MTX MTX-Pen MTX MTX-Pen MTX MTX-Pen 40.0 42 88 95 94 13.3 27 Mar: 87 95 94 4.4 21 15 Dec 70 52 97 95 92 88 1.5 14 19 Dec 67 12 96 -16 91 17 0.5 0 13 59 24 96 -27 91 2 0.2 3 41 94 86 0.1 ..... 19 7 45 65

109

· «·· • · · · 9 99 9 999 9 • 9 99 9 9 9 9 • 999 9 9 9 • 9 9 9 9 9 9 9 9 9 9 9 9 9 9999 99 9 9 9 9 99 99

Table. 3. H3J cells 29

Dose (μΜ)% cell death

Day ¹ Day 2 Day. 3 Day 4 MTX MTX-Pen MTX. MTX-Pen MTX. MTX-Pen MTX. MTX-Pen 40.0 31 79 96 98 13.3 3 45 88 96 4.4 -14 10 -4 6 58 46 86 77 1.5 17 16 -5 9 48 15 Dec 84 45 0.5 15 Dec 14 -12 WITH 52 17 88 16 0.3 10 -5 54 85 0.1 6 -17 52 84

Example 5a [(3-maleimidopropionoyl)] bohemine

12.8 mg, 76 micromoles of 3-maleimidopropionic acid are dissolved in 1 ml of methylene chloride. The mixture was stirred and 5.3 mg, 42 micromol of DIC in 0.5 mL of methylene chloride was added. The reaction was allowed to proceed with stirring for 40 minutes. Then the solvent was evaporated under reduced pressure. The residue of 3-maleimidopropionic anhydride is redissolved in 0.5 ml of anhydrous pyridine. A solution of 10.3 mg, 30 micromol of bohemin and 0.35 mg, 2 micromol of DMAP in 0.5 ml of pyridine was added and the mixture was stirred under nitrogen for one hour. The mixture was then evaporated to dryness under reduced pressure. The residue was redissolved in 1 ml DMF and the solution was purified by preparative RP-HPLC on a column (Vydac).

110

9 9

999 · 9 ► 999 99

218TP1022, 22 χ 250 mm) using a gradient of 10 to 60% MeCN to give the pure product as a 88% yield

14.7 mg of a colorless solid. Analytical RP-HPLC: t _R =

17.7 minutes (Vydac 218TP54 column), gradient 0 to 60% MeCN, purity greater than 95%. ¹ H-NMR (CDCl ₃ ) and DE MALDITOF MS were consistent with the proposed structure for the title compound. Formula C25H29N7O4 = 491.54.

O {3- [3- (Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH) Succinimido] Propionoyl} Bohemine

O. «V

with.

π - τ ^T

R / **:><4 ΝΗζγΝΗζ S> γ Y ⁴ Sy y ⁰ \

NH *

NH

^υ ^ 'ΐιΐϋ.

The product from the previous reaction (0.74 mg, 1.5 mmol) was dissolved in DMF (0.3 mL) and Et ₃ N (50 mL ₎ was added. H-Cys-Arg-Gln (3.5 mg, 1.5 mmol) was added. -Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH in solution in 0.25 mL of DMF. The mixture was stirred under nitrogen and monitored by analytical RP-HPLC. After one hour, the reaction is complete. The mixture was filtered and applied to a RPHPLC column (Vydac 218TP1022, 22 x 250 mm). The column was eluted at 9 ml / min using a gradient of 10 to 60% MeCN in 0.1% aqueous trifluoroacetic acid over a period of 40 minutes at 25 ° C. The main fractions were monitored by analytical RP-HPLC and pooled as needed. After centrifugation in vacuo, 1.7 mg of pure product is obtained in a yield of 40%. Analytical · 9 · 9 99 • 999 · 9 · 9 ·· · 9 · 9 9999 9

-1-1-1 · · * · 999 99 ·

1X1 ·························· 99 ···

RP-HPLC: t _R = 15.0 min (Vydac 218TP54, gradient 0 to 60% MeCN in 0.1% aqueous trifluoroacetic acid, 20 min, 1 mL / min, temperature 25 ° C, purity greater than 95%, lambda = 200-300 nm). DE MALDI-TOF MS: [M + H] &lt; ⁺ &^gt; = 2842, C132H2O2N42O25S2 = 2841, 42.

Represented by:

• · · ·

Sequence Listing <110> Cyclacel Limited Fischer, M Peter Mang, Shudong <120> Pure Feed <130> P004618WO NJN <140> PCT / GB99 / 01957

5141> 1999-06-22 <150> GB 9814527.9 <151> 1998-07-03 <160> 27 <170> Patentin Ver. 2.1 <210> 1 <211 »16 • <212> PRT <213> Drosophila melanogaster

Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Lys Trp Lys Lys 2 5 10 15 <210 »2 <211» 7 <212> PRT <213 »Artificial Creator • · · <220>

<223> Artificial sequence description: synthetic peptide <400> 2

Arg Arg Met Lys Trp Lys Lys 1.5 <210> 3 <211> 8 <212> PRT <213> Artificially created sequence <220>

<223> Description, man-made sequences: synthetic peptide <400> 3

Asn Arg Arg Met Lys Trp Lys Lys 1 5 <210> 4 <212> 9 <212> PRT <2135 Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 4

Gin Asn Arg Arg Met Lys Trp Lys Lys 9999 9999 9999 9

9 ·· ·· ·· "· _e" <210> 5 <211> 10 <212> PRT <213> The artificial sequence <22Q>

<223> Artificial sequence description: synthetic peptide <400> S

Phe Gin Asn Arg Arg Met Lys Trp Lys Lys 10 <210> 6 <211> 7 <212> PRT <213> man-made sequence <220>

<223> Description of sealed sequence: synthetic peptide <400> 6

Lys Arg Met Lys Trp Lys Lys 1 5 <210> 7 <211> 7 <212> PRT <213> Artificially created sequence <220>

<223> Description of man-made sequence: synthetic peptide <400> 7

Arg Lys Met Lys Trp Lys Lys <210> 8 <211> 7 <212> PRT <213> Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 8

Arg Arg Glu Lys Trp Lys Lys 1 5 <210> 9 <211> · 7 <212> PRT <213> Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 9

Arg Gin Lys Trp Lys Lys

···· <210> 10 <211> 7 <212> PRT <213> Artificially created sequence <220>

<221> MOD_RES <222> (3) <223> Orn <220>

<223> Artificial sequence description: synthetic peptide <400> 10

Arg Arg Xaa Lys Trp Lys Lys 1 5 <210> 11 <211> 7 <212> · PRT <213> Specifically created sequence <220>

<223> description of engineered sequence: synthetic peptide <400> 11

Arg Arg Met Lys Gin Lys Lys 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 9 99 9 9 9 9 <210> 12 <211> 7 <212> PRT <213> Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 12

Arg Arg Met Lys Trp Phe Lys 1 S <210> 13 <211> 7 <212> PRT <213> Artificially created sequence <220>

<221> MOD_RES <222> (2) <223> Orn <220>

<223> Artificial sequence description: synthetic peptide <400> 13

Arg Xaa Arg Lys Trp Lys Lys 1 5 <210> 14 <2ii> 7 <212> PRT <213> Etaele generated, sequence

9 · «

«<220>

<223> Artificial sequence description: synthetic peptide <400> 14

Arg Arg Met Trp Lys Lys Lys 1 5 <210> 15 <211> 7 <212> PRT <213> Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 15

Arg Arg Met Lys Lys Trp Lys 1 5 <210> 16 <211> 20 <212> PRT <213> Artificially created sequence <220>

<221> MOD_RES <222> (4) <223> bAla <220>

<223> Description of man-made sequence: synthetic peptide • 4 4 44 44 4 4 444 444 <400> 16

Leu Leu Leu Gin Ile Lys Ile Trp Phe Gin As Arg Arg Met, c 10 15

Lys Trp Lys Lys 20 <210> 17 <211> 17 <212> PRT <213> Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 17 cys Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 15 1 ° ¹⁵

Lys <210> 18 <211> 20 <212> PRT <213> Artificially created sequence <220>

<221> MOD__RES <222> (1) <223> bAla ·· ······· · · · 9 9 9 ♦ 9 • 99 9 9

999 99 ·

9 9 9

9900 99 99 <220>

<223> p ₀ pi _S artificial sequences: synthetic peptide <220>

<221> MOD_RES <222> (20) <223> Amidation <400> 18

Ala Arg Gin Ile Lp Ie Trp Phe Gin Asn Arg Arg Lys Trp Lys 1 5 I® ¹⁵

Lys Gly Cys Gly 20 <210> 19 <211> 9 <212> PRT <213> Artificially created sequence <220>

<221> MOD_RES <222> (2) <223> bAla <220>

<223> Artificial sequence description: synthetic peptide <220>

<221> MODJERS <222> (9) <223> Amidation

0 0 φ

0

0000 00

0 00 0

100> 19 rs Ala Arg Arg Lys Trp Lys Lys 1 5

210> 20

211> 17

212> PRT

213> Artificially created sequence: 220>

: 221> MOD_RES: 222> (1): 223> bAla120>

<223> Artificial sequence description: synthetic peptide <400> 20

Ala Arg Gin II. Lys IU « ^{G1 Aan Met LyS T} ” ^Ly '! 5 1 ° ¹⁴

Lys <210> 21 <211> 9 <212> PRT <213> Artificially created sequence <220>

<223> Artificial sequence description: synthetic peptide

»9 9 9 • 9

999 • * 99 · 9 9 9 <220>

<221> MOD_R2S <222> (9) <223> Amidation <400> 21

Cys Arg Arg Met Lys Trp Lys Lys Cys 1 5 <210> 22 <211> 17 <212> PRT <213> Artificial sequence <220>

<223> Artificial sequence description: synthetic peptide <400> 22

Ala Arg Gin Ile Lp Ie Trp Phe Gin Asn Arg Arg Lys Trp Lys 1 5 10 ¹⁵

Lys <210> 23 <211> 19 <212> PRT <213> Artificially created sequence <220>

<223> description of a wholly generated sequence: synthetic peptide · ΦΦ · • φ φ

φ · · • · • • • • • •

φ <220>

<221> MOD_RES <222> (19) ^<223> Amidation <400> 23

Arg Gin Ile Lp Ie Trp Phe Gin Asn Arg Arg Lys Ip Lys Lys 1 5 10 ¹⁵

Gly Cys Gly <210> 24 <211> 8 <212> PRT <2i3> Artificially created sequence <220>

<221> MOD_RES <222> (1) <223> bAla <22G>

<223> description of engineered sequence: synthetic peptide <220>

<221> MOD_RES <222> (8) <223> Amidation <400> 24

Ala Arg Arg Lys Trp Lys Lys

·· »· 9« <210> 25 <211> 16 <212> PRT ^<213> Artificial sequence <220>

<223> Artificial sequence description: synthetic peptide <220>

<221> MOD_RSS <222> (16) <223> Amidation <400> 25

Arg Gin Ile Trp Phe Gin Asn Arg Arg Lys Trp Lys Lys <210> 26 <211> 7

<221> MOD__RES <222> (7) <223> Amidation <220>

<223> Artificial sequence description: synthetic peptide <400> 26

Arg Arg Met Lys Trp Lys Lys ··································· <210> 27 <211> 17 <212> PRT <213> Artificially created sequence <220>

<221> MOD_RES <222> (17) <223> Amidation <220>

<223> Artificial sequence description: synthetic peptide <400> 27

Cys Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Met Lys Trp Lys

5. 10 15

Claims (31)

PATENT CLAIMS CLAIMS 1. A delivery system comprising an active ingredient bound to a carrier, wherein the carrier is a homeobox of a peptide or a fragment or derivative thereof and the active ingredient is a therapeutically active non-peptide and a non-oligonucleotide active ingredient. A delivery system according to claim 1, characterized in that it is effective in an intact condition. The delivery system of claim 1 or 2, wherein the homeobox peptide is derived from helix 3 of the homeobox sequence of the peptide or a fragment or derivative thereof. The delivery system of claim 3, wherein the homeobox of the peptide is derived from the Prosophila antennapedia peptide or a fragment or derivative thereof. A delivery system according to claim 3 or 4, wherein the carrier is penetratin or a fragment or derivative thereof. 6. The delivery system of claim 5 wherein the carrier comprises the sequence of SEQ ID NO. 1. A delivery system according to claim 5, characterized in that it comprises a truncated sequence of SEQ ID NO. 1. 9 «« 99 113 9 9 99 99 • · »» 9 9 · · 9,999 9 9 9 9 9 9999 98 • 9 • 9 9 ♦ »·· a 9 9 A delivery system according to claim 5 or 7, characterized in that it comprises the sequence SEQ ID NO. 2. A delivery system according to claims 5 to 8, wherein the free carboxyl group at the carboxyterminal end of the carrier amino acid is converted to a carboxamide group. A delivery system according to any one of the preceding claims, characterized in that it comprises D-amino acids. 11. A delivery system according to any one of the preceding claims, comprising a cytotoxic agent. from claims 3 to 9 that. The homeobox peptide of claims 1 to 10, wherein the active agent is A delivery system according to claim 11, characterized in that the active substance is selected from the group of DNA damaging or antimetabolites, antitumor antibiotics, natural products and analogs thereof, dihydrofolate reductase inhibitors, pyrimidine analogs, purine analogs, inhibitors cyclin-dependent kinases, thymidylate synthetase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, anthracyclines, Vinca family, mitomycins, bleomycins, cytotoxic nucleosides, pteridine compounds, diynenes, podophyllotoxins, platinum-inducing substances differentiation and taxanes. ·· 9999 9 9 9 9 9 9 9 9 9 99 9 114 99 99 • · · · 9 9 9 • 999 9 9 9999 98 • * 9 9 9 9 9 9 9 · · 99 99 A delivery system according to claim 12, characterized in that the active ingredient is selected from the group consisting of methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, trisubstituted purines such as olomoucine, roscovitine, bohemine and purvalanol, flavopirabine, stosurosparidine, stosurosparidine , melphalan, leurosine, actinomycin, daunorubicin, doxorubicin, mitomycin D, mitomycin A, carninomycin, aminopterin, tallysomycin, podophylllotoxin and its derivatives, etoposide, cisplatin, carboplatin, vinblastine, vincristin, pithelin, vindesel, vindesel, vindesel, acetylspermidine, tamoxifen, irinotecan and camptothecin. The delivery system according to claim 13, characterized in that the active substance is selected from the group of methotrexate, podophyllotoxin and its derivatives, etoposide, camptothecin, paclitaxel, doxorubicin, roscovitin and bohemin. 15. A delivery system according to any one of the preceding claims, directly bound to the carrier. of claims 1 to 14, wherein the active ingredient is A delivery system according to any one of claims 1 to 14, characterized in that the active ingredient is bound indirectly to the carrier via a linker. The delivery system of claim 16, wherein the linker is selected from (methylamino) benzoyl-Cys, succinimidobenzoyl-Cys, succinimidopropionoyl-Cys, beta-alanyl-succinyl, acetyl. 115 • ·· · · -Cys and (4 '' -aminoaniline) -succinimidopropionoyl-Cys. A delivery system according to any one of claims 1 to 17, characterized in that 1 carrier group binds more than one active substance group. A delivery system according to claim 18, characterized in that the active substance groups are different. A delivery system according to claim 18 or 19, characterized in that each active substance group is linked to the carrier via a linker. The delivery system of claim 20, wherein each active agent group is bound to the carrier by the same linker group. 22. The delivery system of claim 20, wherein each active agent group is bound to a carrier by a different linker group. 23. The delivery system of claim 21, wherein more than one group of active agent is bound to the carrier by a network of lysine residues. A delivery system according to any one of claims 20 to 22, characterized in that more than 1 drug group is bound to the carrier via a linker group selected from (methylamino) benzoylCys, succinimidobenzoyl-Cys, succinimidopropionoyl-Cys. · • · - x- - W »W W 116 .......... beta-alanyl-succinyl, acetyl-Cys and (4'-aminoaniline) succinimidopropionoyl-Cys. The delivery system of claim 24 wherein the linker group is succinimidopropionoyl-Cys. The delivery system of claim 24, wherein the carrier group is a truncated form of penetratin and the linker further comprises 1 to 4 amino acid residues. A delivery system according to claim 26, wherein the amino acid residues are selected from the group of cysteine, glycine, glutamic acid and beta-alanine. A delivery system according to any one of claims 1 to 27, v y z n and no at emerging t and m, that further contains group for system targeting. 29. Feeders The system of claim 26, v y z n and no at emerging t and m, that group for the targeting is associated with the carrier. The delivery system of claim 26, characterized by and s e that skuping for targeting is linked with effective substance. 31. Macromolecule in y z n a c i se by A delivery system according to any one of claims 1 to 30. gstesa ·· ♦ · · « 117 32. A macromolecule selected from the following delivery systems: # Active substance Linker Carrier (methotrexate) < ((methylamino) benzoyl- EGpA) " (LhpARQIKIWFQNRRMKWKK- OH doxorubicin succinimidobenzoyl-C RQIKIWFQNRRMKWKK-OH doxorubicin succinimidobenzoyl-C (D-K) (D-K) (D-W) (D) (D-R) (D-N) (D-Q) (D-F) W) (D-D (D-KXD-1)) NHfr paclitaxel 2 'succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH N-tenn C-tenn paciitaxel carboxyfluorescein 2’-stk cinimidopropionoylGCG BYE RQIKIWFQNRRMKWKK paclitaxel 2'-succinimidopropionoyl-C RQIKIWFQNRRMKWKK-NH ₂ paclitaxel 2’-succininidopropionoyl- CpA RRMKWKK-NHj paclitaxel 7-succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH podo-phyllotoxin 4-succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH N-tenn C-tenn by phytotoxin biotinamidocaproyl 4-succinimidopropionoyl- GCG BYE RQIKIWFQNRRMKWKK according to phllotoxin 4-succinimidopropionoyl-C RQIKIWFQNRRMKWKK-NHj according to phllotoxin 4-succinimidopropionoyl-C (D) (D-QXD-I) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (D) (DK- NH ₂₎ according to phllotoxin 4-Succinimidopropionoyl-CpA rrmkwkk-nh ₂ according to yllotoxin 4-succinixidopropionoyl-CpA (DR) (DR) (DM) (D-W-KXD) (DK) _(DK-NH2) epipodo phylotoxin 4 &apos; -succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH epipodo phylotoxin 4 &apos; -succinimidopropionoyl-C RQIKIWFQNRRMKWKK-NH ₂ . epipodo phylotoxin 4’-succinimidopropionoyl- CPA YYMKWKK-NH ₂ 4 * -Demethyl epipodotoxotoxin 4-succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH etoposide (G2, G3 and 4 ’) succinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH roscovotin sukiinimidopropionoyi-C RQIKIWFQNRRMKWKK-OH bohemin PA-sulfinyl-pA RQIKIWFQNRRMKWKK-OH 118 • · · ··· · · bohemin suitsinimidopropionoyl-C RQIKIWFQNRRMKWKK-OH according to phllotoxin 4-acetyl-C RQIKIWFQNRRMKWKK-OH according to phllotoxin 4-Acetyl-CpA RRMKWKK-NHj 4'-demethyl epipodo f (yllotoxin 4-Acetyl-CpA YYMKWKK-NH ₂ 4'-demethyl epipodo, yllotoxin 4-acetyl-C rqíkiwfqnrrmkwkk-nh ₂ podophyllotoxin 4-suteinimidopropionoyi- ΌϋβΑ rrmkwkk-nh ₂ camptothecin 10-O-suftcinimidapropionoyl- C rqikiwfqnrrmkwkk-nh ₂ C-term N-tenn pod phylllotoxin pod phylllotoxin 4-succinimidopropionoyl-C 4-succinimidopropionoyl-C rrmkwkk N-term C-tenn epipodo f.yllotoxin camptothecin 4 &apos; -succinimidopropionoyl-C 10-O-su cinimidopropionoylC RRMKWKK N-tenn C-tenn epipodo phylotoxin paclitaxel 4'-succinimidopropionoyl-C 2 '- (succinimido) propionoyl-C RRMKWKK 4'-methoxyepipodophyllotoxin 4- (4 * ’- aniinoanilino) sufecinimidooropionoyl-C RQKIWFQNRRMKWKK-NH _Z 4´-methoxyepipodo phylotoxin 4- (4 ”-aminoaniline) succinimidopropionoyl-COA YYMKWKK-NH ₂ 4'-demethylepipodophyllotoxin 4- (4 ”-aminoaniIino) succinimidopropionoyl-CpA rrmkwkk-nh ₂ 33. A delivery system as described in the description and in the example section.