DE102009051799B4 - Bifunctional Prodrugs and Drugs - Google Patents

Abstract

Compounds of the general structure A - L - B where A and B are independently selected from the structures I or II with R1 selected from a halogen and OSO2Ru, where Ru is selected from optionally substituted C1-C6alkyl, C1-6perhaloalkyl, benzyl and phenyl; R2 is selected from hydrogen and optionally substituted C1-8-alkyl; R3, R3 ', R4 and R4' are independently selected from hydrogen and optionally substituted C1-8-alkyl, where two or more of R2, R3, R3 ', R4 and R4' are optionally connected to one another to form one or more optionally substituted carbon cycles or heterocycles X2 is selected from O, C (R14) (R14 ') and NR14', where R14 is selected from hydrogen and optionally substituted C1-8alkyl or C1-8acyl and where R14 'is absent or is selected from hydrogen and optionally substituted C1 -8alkyl or C1-8acyl; R5, R5 ', R6, R6', R7 and R7 'are independently selected from H, OH, SH, CF3, CN, C (O) NH2, C (O) H, C (O ) OH, Halogen, Rk, SRk, S (O) Rk, S (O) 2Rk, S (O) ORk, S (O) 2ORk, OS (O) Rk, OS (O) 2Rk, OS (O) ORk , OS (O) 2Rk, ORk, P (O) (ORk) (ORL), OP (O) (ORk) (ORL), SiRkRLRm, C (O) Rk, C (O) ORk, C (O) N (RL) Rk, OC (O) Rk, OC (O) Rk, OC (O) ORk, OC (O) N (Rk) RL, where Rk, RL and Rm are independently selected from H and optionally substituted tertiary C1-4alkyl, C1-4heteroalkyl, C3-7cycloalkyl, C3-7heterocycloalkyl, C4-12aryl or C4-12heteroaryl groups, two or more of Rk, RL and Rm optionally together form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles; and / or R5 'and R6' and / or R6 'and R7' and / or R7 'and R14' are not present, ie they form a double formation present in the ring between the atoms substituted with the corresponding, namely R5 'and R6 ', and / or R6' and R7 ', and / or R7' and R14 '; two or more of R5, R5', R6 ', R7', R14, and R14 'can optionally be linked to one another to form one or more to form optionally substituted aliphatic or aromatic carbon cycles or heterocycles; and / or R5 + R5 ', and / or R6 + R6' and / or R7 + R7 'are independently = O, = S or = NR12, where R12 is selected from H and optionally substituted C1 -6alkyl; X6 is selected from C or N; RDB is selected from hydrogen and optionally substituted C1-8alkyl or optionally substituted C1-8acyl; X1 is selected from O, S, and NR13, where R13 is selected from H and optionally substituted C1-8alkyl; R is selected from hydrogen or an enzymatically cleavable substrate; a is selected from 0 and 1; b is 1; c is selected from 0, 1 and 2; d is selected from 0 or 1; and L is a linking group to covalently link A and B; and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

Description

The present invention is directed to new compounds, more particularly to new bifunctional prodrugs and drugs. In a further aspect, the present invention is directed to pharmaceutical compositions containing these prodrugs and drugs and their use as cytostatic agents in tumor therapy. The compounds according to the invention are based on CC-1065 analogs.

State of the art

The diverse manifestations of cancer require individual therapy concepts. As a reaction to the complexity of a tumor disease, most of the treatment methods used clinically today represent combinations of different therapeutic approaches. On the one hand, surgical removal of the degenerated tissue is used as the method of choice for easily accessible and clearly delimiting tumors. However, if the tumor is more difficult to access or if it affects vital structures, radiation treatment is the method of choice. At a more advanced stage, when metastases have already formed or at least there is a risk of metastasis, chemotherapy is usually given immediately. Furthermore, the methods of hormone therapy, immunotherapy and therapy with angiogenesis inhibitors and kinase inhibitors are used in tumor therapy.

In the case of an already proven or feared metastasis, as well as in the case of systemic tumors, chemotherapy is currently the most important treatment method, although it is often associated with severe side effects such as blood count disorders, immunodeficiency, mucositis, fever, nausea, vomiting, etc. Typically, chemotherapy drugs are distributed throughout the body through the bloodstream so that they can reach all cells. Chemotherapeutic agents have a cytostatic effect on human cells, i.e. they prevent cells from multiplying, or they have a cytotoxic effect, i.e. they cause the cells to die.

Since most cytostatics show their mechanism of action in proliferating cells and tumor cells belong to the rapidly proliferating cell types, they are used as chemotherapeutic agents. However, the non-tumor cells of the person being treated are also affected, especially those of the bone marrow, hair roots or mucous membrane cells.

The currently known cytostatics used in chemotherapy are divided into the classes of alkylants, antimetabolites, mitosis inhibitors, topoisomerase inhibitors and cytostatic antibiotics according to their mechanisms of action.

The alkylating agents represent a numerically significant, structurally very diverse class of extremely reactive substances. After the medicament has been activated to form a carbocation, the active substance reacts as electrophilic, in particular with nucleic acids, with the formation of covalent bonds. As a result, cross-links of the DNA, abnormal base pairings or strand breaks occur, which prevent replication and ultimately lead to cell death. Typical examples of alkylating agents are cyclophosphamide but also cisplatin. A group of particularly effective alkylating agents also includes the natural antibiotic CC-1065, the duocarmycins, the yatakemycin and derivatives and analogues of this class of natural substances.

Due to the need for chemotherapeutic treatments, the severe side effects of a large number of clinically used active ingredients, and the emergence of resistance to many known chemotherapeutic agents, constant further development in the field of chemotherapeutic agents is necessary.

Chemotherapy for malignant diseases is nowadays associated with serious side effects, since a differentiation between healthy and malignant tissue only takes place via the increased proliferation rate of cancer cells. New concepts have therefore been developed that exploit the genotypic and phenotypic properties of tumor cells and enable the targeted activation of reversible detoxified prodrugs directly at the site of action. Such a targeted activation can take place, for example, by changing the pH value, for example by lowering the pH value. Another option is the so-called ADEPT concept (Antibody-Directed Enzyme Prodrug Therapy). Antibody-enzyme conjugates are used here, which convert the non-toxic prodrug into the drug directly on the tumor and achieve a high level of selectivity. This binary therapy approach consists of two steps. First, a certain amount of an antibody-enzyme conjugate is applied, which is then distributed throughout the organism by the bloodstream. The conjugate binds to specific antigens on tumor cell surfaces or is broken down or broken down by the body. eliminated. If the unbound antibody-enzyme conjugate can no longer be detected, the prodrug is applied in the second step. The prodrug, which is as nontoxic as possible, is also distributed throughout the organism and, due to the enzyme ideally only present on the tumor surface in the form of the antibody-enzyme conjugate, is targeted to toxicity in the tumor tissue. The released drug then unfolds its toxic effect after penetration through the cell membrane, while the enzyme on the outside of the tumor cell remains active and can activate further prodrug molecules. A cleavage of the prodrugs by the body's own enzyme systems should, if possible, not take place in the context of this approach, since otherwise the activity of the therapy would be reduced or eliminated. Disadvantages of previously known prodrugs, however, are too little difference in cytotoxicity between the prodrug and the drug generated from it, and too little cytotoxicity of the drug itself.

As a guideline for the development of compounds for the ADEPT concept, the QIC ₅₀ value (QIC ₅₀ = IC ₅₀ (prodrug) / IC ₅₀ (prodrug in the presence of the enzyme)) should be greater than 1000 and the cytotoxicity of the underlying lying drugs should have an IC ₅₀ value (toxin concentration at which cell growth is inhibited by 50%) less than 10 nM.

Another approach in the context of a targeted treatment of malignant tumors is prodrug monotherapy. This is based on the presence of enzymes that are overexpressed in tumors and that are able to cleave a corresponding prodrug with the release of the corresponding drug. One possible enzyme is, for example, β-D-glucuronidase, which could be detected in increased concentrations in necrotic areas of tumor tissues. Alternatively, conjugates of active ingredients and tumor-specific ligands can also be used for selective targeting in cancer therapy. Here, after selective binding of the ligand to a receptor on the tumor surface, the conjugate is internalized and subsequently released intracellularly.

Other methods besides the ADEPT method described above are the PDEPT (Polymer-Directed Enzyme Prodrug Therapy) or MDEPT (Macromolecular-Directed Enzyme Prodrug Therapy) and VDEPT (Virus-Directed Enzyme Prodrug Therapy) or GDEPT (Gene -Directed Enzyme Prodrug Therapy). The last two mentioned in particular represent possibilities of increasing the above-mentioned prodrug monotherapy.

Clinical studies have already been carried out for the ADEPT concept. It turned out that the ADEPT concept per se is suitable for selective tumor therapy, but that there is still room for improvement with regard to various points in order to enable selective and efficient therapy. One of these essential improvement points comprises the provision of new and effective prodrugs which have a high cytotoxicity difference between the prodrug and the corresponding drug, a high cytotoxicity of the drug and a short plasma half-life of the drug.

There is therefore still a need to provide corresponding prodrugs which have a high cytotoxicity difference from the drug and, as prodrugs themselves, show a low cytotoxicity. The active substance formed (drug) itself should have the highest possible cytotoxicity.

Various attempts have been made for analogues of the antibiotic CC-1065 and the duocarmycins in order to achieve the above-mentioned aim. The CC-1065 itself has an IC ₅₀ value of 20 pmol, but resulted in delayed lethal hepatoxicity in animal experiments and is therefore not suitable for clinical applications. Therefore, an attempt was made to prepare corresponding analogues of this compound. The DNA binding structural unit was changed, but the pharmacophore itself was also modified in various ways. In addition, seco and prodrug compounds were synthesized which generally have similar toxicity and selectivities as the spirocyclopropyl compounds, eg CC-1065 or duocarmycine. For example, Tietze CC-1065 analogs have been described which are reversible by glycosidation of the detoxified anti-methyl-seco-CBI-DMAI-β-D-galactoside (+) - (1S, 10R) compound, an excellent result in terms of cytotoxicity and achieve the quotient of the cytotoxicity of the prodrug and the prodrug in the presence of the activated enzyme. QIC ₅₀ values of over 4500 were achieved.

Bifunctional alkylating agents are those with two reactive centers. They have the ability to cause intra- or inter-strand cross-links in the DNA. With regard to the interstrand cross-links, particularly good damage to the cells and thus cell death are achieved. These compounds are highly cytotoxic. Bifunctional derivatives of, for example, pyrrolobenzodiazidpine are described in the literature. Such bifunctional derivatives are currently in phase 1 clinical studies. Bifunctional compounds were also synthesized for analogues of CC-1050, Mitchell et al., J. Am. Chem. Soc. 1989, 111, 6428-6429; Jia, G., and Lown, W., Bioorg. Med. Chem., 2000, 8, 1607-1617. Fokuda J., et al., Biorganic and Medicinal Chemistry Letters 8, 1998, 2003-2004 , describes new CPI bisalkylators which have 3,3 '- (1,4-phenylene) diacryloyl groups as linkers. However, there is still a need for drugs and prodrugs suitable for chemotherapeutic agents.

Description of the invention

According to the invention, new compounds are provided which are bifunctional alkylants, in particular for use in selective tumor therapy. The novel compounds described herein are distinguished by the fact that these new dimers are more cytotoxic than the monomeric prodrugs or drugs. The IC ₅₀ value of the compounds according to the invention is usually in the pmol range. In addition, a much larger QIC ₅₀ value is achieved. In other words, the quotient between the cytotoxicity of the drug and the cytotoxicity of the prodrug is much greater. In this way, better therapeutic effectiveness combined with lower cytotoxicity of the prodrug and thus fewer side effects when administered to the patient can be achieved.

mit R¹ ausgewählt aus einem Halogen und OSO₂R^u, wobei R^u ausgewählt ist aus gegebenenfalls substituierten C₁-C₆ alkyl, C_1-6 Perhaloalkyl, Benzyl und Phenyl;
R² ist ausgewählt aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl; R³, R^3', R⁴ und R^4' sind unabhängig voneinander ausgewählt aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl, wobei zwei oder mehrere von R², R³, R^3', R⁴ und R^4' gegebenenfalls miteinander verbunden sind, um ein oder mehrere gegebenenfalls substituierte Kohlenstoffzyklen oder Heterozyklen auszubilden;
X² ist ausgewählt aus O, C(R¹⁴)(R^14') und NR^14', wobei R¹⁴ ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl und wobei R^14' nicht vorhanden ist oder ausgewählt ist aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder C_1-8 Acyl;
R⁵, R^5', R⁶, R^6', R⁷ und R^7' sind unabhängig voneinander ausgewählt aus H, OH, SH, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, Halogen, R^k, SR^k, S(O)R^k, S(O)₂R^k, S(O)OR^k, S(O)₂OR^k, OS(O)R^k, OS(O)₂R^k, OS(O)OR^k, OS(O)₂R^k, OR^k, P(O)(OR^k)(OR^L), OP(O)(OR^k)(OR^L), SiR^kR^LR^m, C(O)R^k, C(O)OR^k, C(O)N(R^L)R^k, OC(O)R^k, OC(O)R^k, OC(O)OR^k, OC(O)N(R^k)R^L wobei R^k, R^L und R^m unabhängig voneinander ausgewählt sind aus H und gegebenenfalls substituierte C_1-4 Alkyl, C_1-4 Heteroalkyl, C_3-7 Cycloalkyl, C_3-7 Heterocycloalkyl, C_4-12 Aryl oder C_4-12 Heteroaryl Gruppen, zwei oder mehr von R^k, R^L und R^m bilden gegebenenfalls miteinander ein oder mehrere gegebenenfalls substituierte aliphatische oder aromatische Kohlenstoffzyklen oder Heterozyklen aus;
und/oder R^5' und R^6' und/oder R^6' und R^7' und/oder R^7' und R^14' sind nicht vorhanden, d.h., sie bilden eine im Ring vorhandene Doppelbildung zwischen den mit den entsprechenden substituierten Atomen aus, nämlich R^5' und R^6', und/oder R^6' und R^7', und/oder R^7' und R^14';
zwei oder mehrere von R⁵, R^5', R^6', R^7', R¹⁴, und R^14' können gegebenenfalls miteinander verbunden sein, um ein oder mehrere gegebenenfalls substituierte aliphatische oder aromatische Kohlenstoffzyklen oder Heterozyklen auszubilden;
und/oder R⁵ + R^5', und/oder R⁶ + R^6' und/oder R⁷ + R^7' sind unabhängig voneinander =O, =S oder =NR¹², wobei R¹² ausgewählt ist aus H und gegebenenfalls substituierten C_1-6 Alkyl;
X₆ ist ausgewählt aus C oder N;
R_DB ist ausgewählt aus Wasserstoff und gegebenenfalls substituierten C_1-8 Alkyl oder gegebenenfalls substituierten C_1-8 Acyl;
X₁ ist ausgewählt aus O, S, und NR¹³, wobei R¹³ ausgewählt ist aus H und gegebenenfalls substituierten C_1-8 Alkyl;
R ist ausgewählt aus Wasserstoff oder ein enzymatisch abspaltbares Substrat; a ist ausgewählt aus 0 und 1;
b ist 1;
c ist ausgewählt aus 0, 1 und 2;
d ist ausgewählt aus 0 oder 1;
L ist eine Verbindungsgruppe zur kovalenten Verbindung von A und B; und pharmazeutisch annehmbare Salze oder pharmazeutisch annehmbare Solvate hiervon.In a first aspect, the present invention provides compounds of general structure
ALB
where A and B are independently selected from structures I or II

with R ¹ selected from a halogen and OSO ₂ R ^u , where R ^{u is} selected from optionally substituted C ₁ -C ₆ alkyl, C _1-6 perhaloalkyl, benzyl and phenyl;
R ² is selected from hydrogen and optionally substituted C _1-8 alkyl; R ³ , R ^{3 '} , R ⁴ and R ^4' are independently selected from hydrogen and optionally substituted C _1-8 alkyl, where two or more of R ² , R ³ , R ^{3 '} , R ⁴ and R ^{4' are} optionally are linked together to form one or more optionally substituted carbon cycles or heterocycles;
X ² is selected from O, C (R ¹⁴ ) (R ^{14 '} ) and NR ^14' , where R ^{14 is} selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl and where R ^{14 '} is absent or is selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl;
R ⁵ , R ^{5 '} , R ⁶ , R ^6' , R ⁷ and R ^{7 '} are independently selected from H, OH, SH, CF ₃ , CN, C (O) NH ₂ , C (O) H, C (O) OH, Halogen, R ^k , SR ^k , S (O) R ^k , S (O) ₂ R ^k , S (O) OR ^k , S (O) ₂ OR ^k , OS (O) R ^k , OS (O) ₂ R ^k , OS (O) OR ^k , OS (O) ₂ R ^k , OR ^k , P (O) (OR ^k ) (OR ^L ), OP (O) (OR ^k ) (OR ^L ), SiR ^k R ^L R ^m , C (O) R ^k , C (O) OR ^k , C (O) N (R ^L ) R ^k , OC (O) R ^k , OC (O) R ^k , OC (O) OR ^k , OC (O) N (R ^k ) R ^L where R ^k , R ^L and R ^{m are} independently selected from H and optionally substituted C _1-4 alkyl, C _1-4 heteroalkyl, C _{3- 7} cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl groups, two or more of R ^k , R ^L and R ^m optionally form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles with one another;
and / or R ^{5 '} and R ^6' and / or R ^{6 '} and R ^7' and / or R ^{7 '} and R ^14' are not present, ie they form a double formation present in the ring between the atoms substituted with the corresponding from, namely R ^{5 '} and R ^6' , and / or R ^{6 '} and R ^7' , and / or R ^{7 '} and R ^14' ;
two or more of R ⁵ , R ^{5 '} , R ^6' , R ^{7 '} , R ¹⁴ , and R ^14' can optionally be linked to one another to form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles;
and / or R ⁵ + R ^{5 '} , and / or R ⁶ + R ^6' and / or R ⁷ + R ^{7 '} are independently of one another = O, = S or = NR ¹² , where R ^{12 is} selected from H and optionally substituted C _1-6 alkyl;
X ₆ is selected from C or N;
R _DB is selected from hydrogen and optionally substituted C _1-8 alkyl or optionally substituted C _1-8 acyl;
X ₁ is selected from O, S, and NR ¹³ , where R ^{13 is} selected from H and optionally substituted C _1-8 alkyl;
R is selected from hydrogen or an enzymatically cleavable substrate; a is selected from 0 and 1;
b is 1;
c is selected from 0, 1 and 2;
d is selected from 0 or 1;
L is a linking group to covalently link A and B; and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

In in vitro experiments, the compounds according to the invention showed excellent cytotoxicity _{values with IC 50} values in the pmol range, in some cases below the pmol range. Furthermore, the compounds showed an excellent quotient of the IC ₅₀ . The QIC ₅₀ value of the tested compounds was above 1000, particularly suitable compounds showed values above 100,000.

That is to say, the compounds presented here, which are new dimeric prodrugs and drugs of CC-1065 analogs, have a very high cytotoxicity as a drug, while the prodrugs show only a lower cytotoxicity. As a result, these compounds should be much safer in therapeutic use. The compounds according to the invention from dimers are furthermore significantly more cytotoxic than the monomeric prodrugs or drugs. The compounds thus show themselves to be highly effective with fewer side effects in use.

Earlier bifunctional CC-1065 and duocarmycin analogs showed only a low selectivity and the cytotoxicity values were similar to the monomeric analogs, in some cases even lower. Jia, G. and Lown, J.W., supra, examined bifunctional compounds that exhibited cytotoxicity values in the nanomolar range.

The compounds according to the invention are distinguished by the fact that they show cytotoxicity _{values in the pmol range and have a very large quotient of the IC 50} values of the drug to the prodrug.

In a preferred embodiment, the compounds have a linker L selected from i) Z - (C (R _CH ) ₂ ) _n - Z ', where n is an integer from 1 to 20, Z and Z' are selected independently of one another from C =O, OC =O, SO ₂ , NR _z , NR _z C =O, C =ONR _z where each R _CH and R _{z are} independently selected from hydrogen or optionally substituted alkyl or optionally substituted acyl, preferably from optionally substituted C ₁ -C ₈ alkyl or optionally substituted C ₁ -C ₈ acyl, or ii) L is an oligopeptide with 1 to 10 amino acids; or pharmaceutically acceptable salts or substrates thereof.

In an embodiment preferred therefrom, the linker is one in which Z, Z 'represents a group C = O and R _CH each independently of one another hydrogen. In one embodiment, n is a value from 1 to 6, particularly preferably 3, 4 or 5. In a further embodiment, R _CH is, independently of one another, hydrogen or CH ₃ , n is an integer from 1 to 20 and Z and Z 'are C = O, where X ^{6 is} nitrogen.

It is also preferred that I a is zero in the general structure.

Another preferred embodiment is such that in structures I and II the radicals R ⁶ , R ^{6 '} , R ⁷ , R ^7' and R ² each independently represent a hydrogen or a methyl group - CH ₃ -.

In a further preferred embodiment, structures I and II are CC-1065 analogs such as those described in, for example WO 2007/089149 and WO 01/83448 which are hereby incorporated by reference in their entirety.

The term “substituted”, as used herein in particular in relation to alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, acyl, refers to the fact that these groups have one or more substituents selected from the group comprising OH, = O, = S, = NR ^h , = N-OR ^h , S ^h , NH ₂ , NO ₂ , NO, N ₃ , CF ₃ , CN, OCN, SCN, NCO, NCS, C (O) NH ₂ , C (O ) H, C (O) OH, halogen, R ^h , SR ^h , S (O) R ^h , S (O) OR ^h , S (O) ₂ R ^h , S (O) ₂ OR ^h , OS (O ) R ^h , OS (O) OR ^h , OS (O) ₂ R ^h , OS (O) ₂ OR ^h , OP (O) (OR ^h ) (OR ⁱ ), P (O) (OR ^h ) (OR ⁱ ), OR ^h , NHR ⁱ , N (R ^h ) R ⁱ , ⁺ N (R ^h ) (R ⁱ ) R ^j , Si (R ^h ) (R ⁱ ) R ^j , Si (R ^h ) (R ⁱ ) (R ^j ), C (O) R ^h , C (O) OR ^h , C (O) N (R ⁱ ) R ^h , OC (O) R ^h , OC (O) OR ^h , OC (O) N (R ^h ) R ⁱ , N (R ⁱ ) C (O) R ^h , N (R ⁱ ) C (O) OR ^h , N (R ⁱ ) C (O) N (R ^j ) R ^h , and the thio derivatives of these substituents, or a protonated or deprotonated form of these substituents, where R ^h , R ⁱ , and R ^{j are} independently selected from H and optionally substituted C _1-15 alkyl l, C _1-15 heteroalkyl, C _3-15 cycloalkyl, C _3-15 heterocycloalkyl, C _4-15 aryl, or C _4-15 heteroaryl, or a combination thereof, two or more of R ^h , R ⁱ , and R ^j are optionally linked together to form one or more carbon cycles or heterocycles.

The term "alkyl" as used herein refers to straight or branched chain, saturated or unsaturated hydrocarbon substituents, examples of the alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, isopropyl, sec -Butyl, isobutyl, tert-butyl, isopentyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, pentenyl and the like.
The term “cycloalkyl” or “carbon cycles” as used herein refers to saturated or unsaturated, non-aromatic hydrocarbon cycles that can consist of one, two or more rings. Examples thereof include: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexonyl, etc.

The term “heteroalkyl” as used in the present context relates to straight-chain or branched, saturated or unsaturated hydrocarbon substituents in which at least one carbon has been replaced by a heteroatom. The heteroatoms are preferably selected from S, N, O, and P.

The term “aryl” as used herein refers to aromatic substituents which can consist of one or more rings fused together. Examples of aryl include phenyl, naphthyl, and antracenyl.

The term “heteroaryl” as used in the present context refers to aromatic substituents which can consist of one or more rings fused to one another. At least one carbon atom of the aromatic R group has been replaced by a heteroatom. Examples of heteroaryl groups include pyridinyl, furanyl, pyrrolyl, triazolyl, pyrazolyl, imidazolyl, thiophenyl, indolyl, benzofuranyl, benzimidazolyl, indazolyl, benzotriazolyl, benzisoxazolyl, and quinolinyl.

The term “heterocycloalkyl” or “heterocycles” as used herein refers to saturated or unsaturated, non-aromatic cyclic hydrocarbon substituents which can consist of one or more rings fused together, with at least one carbon in one of the rings replaced by a heteroatom. Examples of heterocycloalkyls include: tetrahydrofuranyl, pyrrolidinyl, piperidinyl, 1,4-dioxanyl, morpholinyl, piperazinyl, oxyzolidinyl, decahydroquinolinyl.

When expressions such as “optionally substituted” are used, these expressions refer to all subsequent radicals unless otherwise stated. That is, the expression “optionally substituted alkyl, heteroalkyl, aryl, acyl” is to be read as “optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted acyl”.
Depending on the substituents, in particular depending on the substituent R, the compounds can be introduced into cells in a simple and directed manner. In one embodiment, R is preferably hydrogen.

In a further preferred embodiment, R represents a substrate which, for example, can be cleaved enzymatically in order to convert prodrugs which have a cleavable product at the substituent R into drugs.
In other words, in a preferred embodiment, the substrate R is a cleavable product.

In a preferred embodiment, the substrate R is a cleavable substrate. This cleavable substrate is preferably one that by proteolytic enzymes, plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase, mannosidase, glycosidase, neuramidase, Sucrose, maltase, fructosidase, glycosylases, prostate-specific antigen (PSA), urokinase-type plasminogen activator (u-PA), metalloproteinase, or an enzyme that is targeted with the help of targeted enzyme prodrug therapy, such as ADEPT, VDEPT, MDEPT , GDEPT, or PDEPT can be cleaved; or a substituent which can be split off or transformed under hypoxic conditions or by reduction by nitroreductase.

The radical R is preferably one selected from the group comprising monosaccharide, disaccharide or oligosaccharide, in particular hexoses, pentoses or heptoses, optionally as a deoxy derivative or amino derivative and optionally substituted by halogen, C _1-8 alkyl, C _{1 -8} acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino or amide groups, or with amino, amido or carboxyl units, the may optionally be substituted by halogen, C _1-8 alkyl, C _1-8 acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino - or amide residues; Dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or an antibody or an antibody fragment, or combinations thereof.

With the aid of the substrate R, the compounds according to the invention can be targeted to target structures. That is, a targeted coupling of the compounds according to the invention to target structures and a corresponding introduction of these compounds according to the invention into e.g. selected cells and cell types is possible. The compounds according to the invention in which R is a substrate other than H have a cleavable or transformable group. The cleavage or transformation of the compound according to the invention with R other than H can take place by chemical, photochemical, physical, biological or enzymatic processes under the appropriate conditions. These conditions include, for example, the provision of appropriate enzymes, a change in the surrounding environment, the action of, for example, radiation, such as UV light, etc. Appropriate methods are known to the person skilled in the art. The substrate is accordingly accessible for known processes such as ADEPT (Antibody Directed Enzyme Prodrug Therapy), PDEPT (Polymer-Directed Enzyme Prodrug Therapy) or MDEPT (Macromolecular-Directed Enzyme Prodrug Therapy), VDEPT (Virus-Directed Enzyme Prodrug Therapy) or GDEPT ( Gene-Directed Enzyme Prodrug Therapy).

wobei R unabhängig voneinander Wasserstoff oder CH₃ ist und n ist eine ganze Zahl aus 3, 4 oder 5.Particularly preferred compounds are the compounds shown below:

where R is independently hydrogen or CH ₃ and n is an integer from 3, 4 or 5.

In a further preferred embodiment of the present invention, components A and B are identical radicals. According to the invention, hybrid compounds are provided from structures I and II, but the compounds are preferably those in which structures A and B represent identical radicals, as in the particularly preferred compounds III and IV, as shown above.

Furthermore, the present application is directed to pharmaceutical compositions which contain the compounds according to the invention, optionally with pharmaceutically acceptable carriers or diluents. The compounds according to the invention can be in the form of pharmaceutically acceptable salts or solvates.

Pharmaceutically acceptable salts are, in particular, acid addition salts which are correspondingly formed on amine groups. Base addition salts or corresponding hybrid addition salts are also possible.

The term “pharmaceutically acceptable solvates” refers to the association of one or more solvent molecules and a compound of the invention. Examples of such solvent molecules that form pharmaceutically acceptable solvates include: water, isopropyl alcohol, ethanol, methanol, DSMO, ethyl acetate, and acetic acid.

The compounds according to the invention are particularly suitable for the production of pharmaceutical compositions suitable in tumor therapy. Monomers of the bifunctional compounds according to the invention are known as cytotoxic compounds suitable for tumor therapy. The present invention includes pharmaceutical compositions which contain the compounds according to the invention in addition to the customary carriers or diluents. The pharmaceutical preparations listed above are produced in the customary manner by known processes, for example by mixing the active ingredient (s) or excipients.
In general, the compounds according to the invention can be administered in total amounts of 0.5 to about 500, preferably 1 to 150 mg / kg of body weight per 24 hours, optionally in the form of several individual doses, in order to achieve the desired results. The possibilities for determining the dose amount are well known to the person skilled in the art. This can take place as a function of the age, the body weight, the type and severity of the patient's disease, the type of preparation and the application of the medicament and the period or interval of administration.

Figure list

• In the 1 describes the synthesis of the β-D-galactosides of the bifunctional (1S) -seco-CBI and the (1S, 10R) -anti-methyl-seco-CBI derivatives, as well as the corresponding seco-drugs: Synthesis of the β-D- Galactosides of the bifunctional (1S) -seco-CBI- and (1S, 10R) -anti-methyl-seco-CBI derivatives and the corresponding seco-drugs: a) 87, BF ₃ · OEt ₂ , CH ₂ Cl ₂ , MS (4 Å), -10 ° C, 3.5 h, then BF ₃ · OEt ₂ , RT, 5.5 h; b) 66a-c (n = 3-5), NEt ₃ , DMF, RT, 20 h; c) Preparative HPLC: Kromasil ^® 100 C18 (250 x 20 mm, 7 microns), A = H ₂ O, B = MeOH, gradient: A / B = 30:70 → 0: 100 in 6.5 min; d) NaOMe / MeOH, RT, 2 h; e) Preparative HPLC: Kromasil ^® 100 C18 (250 x 20 mm, 7 microns), A = H ₂ O, B = MeOH, gradient: A / B = 70:30 → 0: 100 in 15 min; f) 4 N HCI / EtOAc, RT, 3 h; g) 66a-c (n = 3-5), pyridine, DMF, RT, 20 h; h) Preparative HPLC: Kromasil ^® 100 C18 (250 x 20 mm, 7 microns), A = H ₂ O, B = MeOH, gradient: A / B = 30:70 → 0: 100 in 6.5 min.
• The 2 shows the racemic synthesis route for the preparation of the N-Boc-seco-CBI (rac-69) with subsequent separation of the enantiomers via HPLC on a chiral stationary phase.
• The 3 shows the asymmetric synthesis route for the preparation of the (1S) -N-Boc-seco-CBI ((-) - (1S) -69).
• The 4th shows the in vitro cytotoxicity determined in the A549 cell line of the bifunctional (1S, 10R) -anti-methyl-seco-CBI derivatives with n = 3 (oE = without enzyme, mE = with enzyme).
• The 5 shows the in vitro cytotoxicity determined in the A549 cell line of the bifunctional (1S, 10R) -anti-methyl-seco-CBI derivatives with n = 4 (oE = without enzyme, mE = with enzyme).
• The 6th shows the in vitro cytotoxicity determined in the A549 cell line of the bifunctional anti-methyl-seco-CBI derivatives with n = 5 (oE = without enzyme, mE = with enzyme).

The invention is explained in more detail below with the aid of the examples, without the invention being restricted thereto.

Examples

• Die verwendeten Zelllinien sind über ATCC beziehbar und wurden in den empfohlenen Medien kultiviert.
• Kulturmedium für A549: DMEM (Dulbecco's Modified Eagles Medium) mit 4.5 g/l Glucose (Biochrom, T043-10). Das Medium wurde mit 4 mM L-Glutamin und 3.7 g/l Natriumhydrogencarbonat supplementiert.
• Medium-Zusätze: 10% FKS (Fötales Kälberserum) der Firma Biochrom, 30 min inaktiviert bei 56 °C.
• Enzym: β-D-Galactosidase (E.C. 3.2.1.23) von Eschericha coli G 5635 (Sigma), Aktivität: 250-600 Einheiten (Units (U)) pro mg Protein bei pH 7.3 und 37 °C, 1 U = 1 µmol Substratumsatz pro Minute.
• PBS-Puffer: Lösung der PBS-Trockensubstanz (Phosphate Buffered Saline) der Firma Biochrom KG in bidestilliertem Wasser. Salzkonzentrationen in der Pufferlösung mit pH 7.4: 137.0 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.1 mM KH₂PO₄.
• Drehwerte: Polarimeter Modell 241 der Firma Perkin-Elmer. Infrarotspektren: Modell Vector 22 der Firma Bruker. Feststoffe wurden als KBr-Presslinge, Flüssigkeiten als Film zwischen KBr-Platten gemessen. Zur Kalibrierung diente die Polystyrolbande bei 1601 cm^-1.
• UV/VIS-Spektren: Modell Lambda 2 der Firma Perkin-Elmer.
• ¹H-NMR-Spektren: Modelle Mercury-200 (200 MHz), Mercury-300 und Unity-300 (jeweils 300 MHz) sowie Unity Inova-600 (600 MHz) der Firma Varian. Die chemischen Verschiebungen sind in Einheiten der δ-Skala angegeben. Tetramethylsilan (δ_TMS = 0.00 ppm) oder das angegebene Lösungsmittel dienten als interner Standard. Zur Kennzeichnung der Multiplizitäten der Signale werden folgende Abkürzungen verwendet: s (Singulett), d (Dublett), t (Triplett), q (Quartett), m (Multiplett), m_c (zentriertes Multiplett), br (breites Signal). Zur Bezeichnung aromatischer Protonen oder Kohlenstoffatome werden folgende Abkürzungen verwendet: i (ipso), o (ortho), m (meta), p (para). Die Spektren wurden in der Regel erster Ordnung entsprechend interpretiert. Die Kopplungskonstanten J sind in Hertz (Hz) angegeben.
• ¹³C-NMR-Spektren: Modelle Mercury-200 (50 MHz), Mercury-300 und Unity-300 (jeweils 75 MHz) sowie Unity Inova-600 (150 MHz). Als interner Standard diente Tetramethylsilan oder das angegebene Lösungsmittel. Die chemischen Verschiebungen sind den ¹H-breitbandentkoppelten Spektren entnommen, die Multiplizitäten der Signale wurden in multiplett-selection-Experimenten (APT-Pulsfolge) bestimmt.
• Massenspektren: Zur Aufnahme der EI-Spektren diente ein doppelfokussierendes Sektorfeld-Massenspektrometer MAT 95 der Firma Finnigan. ESI-Spektren wurden mit einem Ion-Trap-Massenspektrometer LCQ der Firma Finnigan aufgenommen. Angegeben werden die Quotienten aus Masse zu Ladung sowie in Klammern die relativen Intensitäten bezogen auf den Basispeak (I = 100). Die Messung der ESI-HRMS-Spektren erfolgte an einem 7 Tesla-Fourier Transform Ion Cyclotron Resonance (FTICR)-Massenspektrometer APEX IV der Firma Bruker, das mit einer Apollo-ESI-Quelle der Firma Bruker und einer Spritzenpumpe 74900 series der Firma Cole-Parmer ausgestattet ist. Der Fluss der Spritzenpumpe betrug 2 µL/min. Zur Aufnahme der Spektren und zur Auswertung diente das Programm XMASS der Firma Bruker.
• Dünnschichtchromatographie (DC): Es wurden DC-Fertigfolien SIL G/UV254 der Firma Macherey-Nagel & Co. (Schichtdicke 0.25 mm) verwendet. Angegeben sind R_f-Werte (Laufhöhe relativ zur Laufmittelfront). Als Abkürzungen für die verwendeten Lösungsmittel werden benutzt: DMSO (Dimethylsufoxid), EtOAc (Essigsäureethylester), CH₂Cl₂ (Dichlormethan), MeOH (Methanol). Neben der UV-Detektion dienten eine Vanillin-Schwefelsäure-Lösung (0.5 g Vanillin, 3 mL konz. Schwefelsäure, 85 mL Methanol und 10 mL Essigsäure) und eine Molybdatophosphorsäure-Lösung (5% in Methanol) als Anfärbereagenzien.
• Säulenchromatographie: Alle säulenchromatographischen Trennungen wurden mit Kieselgel 60 (Korngröße: 0.032-0.063 mm) der Firma Merck durchgeführt.
• Hochdruckflüssigkeitschromatographie (HPLC):
  • Analytische HPLC: Analytische Trennungen mit Ausnahme der Oligonukleotid-Experimente wurden auf einer HPLC-Anlage der Firma Jasco, ausgestattet mit einer Lösungsmittelpumpe PU-2080, einer Mischkammer LG-1590-04, einem Multiwellenlängendetektor MD-2010 Plus und der Steuerung LC-Net II/ADC, vorgenommen. Zusätzlich war ein automatischer Probenwechsler (Autosampler AS-2055) derselben Firma angeschlossen. Zur Bedienung, Datenerfassung und Datenauswertung wurden die Computerprogramme Borwin PDA, HSS 2000 und Borwin Chromatography der Firma Jasco eingesetzt. Für die analytischen Messungen wurden die Fertigsäulen Chiralcel^® OD (250x4.6 mm, Partikelgröße: 10 µm) der Firma Daicel Chemical Industries Ltd. sowie Kromasil 100 C18 (5 µm, 250×4 mm) der Firma Jasco verwendet. Als Lösungsmittel dienten n-Hexan, Dichlormethan und Isopropanol in HPLC-Qualität (Chiralcel^® OD) sowie bidestilliertes Wasser (Zusatz von 0.1 Vol.-% Trifluoressigsäure zur Peptidsynthese oder 0.06 Vol.-% konzentrierte Salzsäure) und Acetonitril bzw. Methanol in HPLC-Qualität (Kromasil 100 C18). Alle Proben wurden membranfiltriert und die Lösungsmittel entgast.
• Präparative HPLC: Präparative Trennungen wurden auf einem HPLC-System der Firma Jasco, ausgestattet mit zwei Lösungsmittelpumpen Modell PU-2087 PLUS und einem UV-Detektor Modell UV-2075 PLUS, vorgenommen. Eingesetzt wurden eine Fertigsäule Chiralpak^® IA (250×20 mm, Partikelgröße: 5 µm) sowie eine Fertigsäule des Typs Kromasil 100 C18 (7 µm, 250×20 mm) der Firma Jasco in Verbindung mit einer Vorsäule des Typs Kromasil 100 C18 (5 µm, 50×20 mm) der Firma Jasco. Als Lösungsmittel dienten n-Heptan, n-Hexan und Dichlormethan in HPLC-Qualität (Chiralpak^® IA) sowie bidestilliertes Wasser (Zusatz von 0.1 Vol.-% Trifluoressigsäure zur Peptidsynthese) und Acetonitril bzw. Methanol in HPLC-Qualität (Kromasil 100 C18). Alle Proben wurden membranfiltriert und die Lösungsmittel entgast.

Materials:

• The cell lines used are available from ATCC and were cultured in the recommended media.
• Culture medium for A549: DMEM (Dulbecco's Modified Eagles Medium) with 4.5 g / l glucose (Biochrom, T043-10). The medium was supplemented with 4 mM L-glutamine and 3.7 g / l sodium hydrogen carbonate.
• Medium additives: 10% FCS (fetal calf serum) from Biochrom, inactivated at 56 ° C. for 30 min.
• Enzyme: β-D-galactosidase (EC 3.2.1.23) from Eschericha coli G 5635 (Sigma), activity: 250-600 units (units (U)) per mg protein at pH 7.3 and 37 ° C, 1 U = 1 µmol Substrate turnover per minute.
• PBS buffer: solution of the PBS dry substance (Phosphate Buffered Saline) from Biochrom KG in double-distilled water. Salt concentrations in the buffer solution with pH 7.4: 137.0 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ , 1.1 mM KH ₂ PO ₄ .
• Rotation values: Polarimeter model 241 from Perkin-Elmer. Infrared spectra: Model Vector 22 from Bruker. Solids were measured as KBr pellets, liquids as a film between KBr plates. The polystyrene band at 1601 cm ^{-1 was used for} calibration.
• UV / VIS spectra: Lambda 2 model from Perkin-Elmer.
• ¹ H-NMR spectra: Models Mercury-200 (200 MHz), Mercury-300 and Unity-300 (300 MHz each) and Unity Inova-600 (600 MHz) from Varian. The chemical shifts are given in units on the δ scale. Tetramethylsilane (δ _TMS = 0.00 ppm) or the specified solvent served as an internal standard. The following abbreviations are used to identify the multiplicities of the signals: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), m _c (centered multiplet), br (broad signal). The following abbreviations are used to denote aromatic protons or carbon atoms: i (ipso), o (ortho), m (meta), p (para). The spectra were usually interpreted accordingly in the first order. The coupling constants J are given in Hertz (Hz).
• ¹³ C-NMR spectra: Models Mercury-200 (50 MHz), Mercury-300 and Unity-300 (75 MHz each) and Unity Inova-600 (150 MHz). Tetramethylsilane or the specified solvent was used as an internal standard. The chemical shifts are taken from the ¹ H broadband decoupled spectra, the multiplicities of the signals were determined in multiple selection experiments (APT pulse sequence).
• Mass spectra: A double focusing sector field mass spectrometer MAT 95 from Finnigan was used to record the EI spectra. ESI spectra were recorded with an LCQ ion trap mass spectrometer from Finnigan. The quotients from mass to charge are given as well as the relative intensities in brackets based on the base peak (I = 100). The ESI-HRMS spectra were measured on a 7 Tesla Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer APEX IV from Bruker, which was operated with an Apollo ESI source from Bruker and a 74900 series syringe pump from Cole. Parmer is equipped. The syringe pump flow was 2 µL / min. The XMASS program from Bruker was used to record the spectra and to evaluate them.
• Thin-layer chromatography (TLC): TLC pre-fabricated films SIL G / UV254 from Macherey-Nagel & Co. (layer thickness 0.25 mm) were used. The values given are R _f values (running height relative to the solvent front). The following abbreviations are used for the solvents used: DMSO (dimethyl sulfoxide), EtOAc (ethyl acetate), CH ₂ Cl ₂ (dichloromethane), MeOH (methanol). In addition to UV detection, a vanillin-sulfuric acid solution (0.5 g vanillin, 3 mL conc. Sulfuric acid, 85 mL methanol and 10 mL acetic acid) and a molybdophosphoric acid solution (5% in methanol) were used as staining reagents.
• Column chromatography: All column chromatographic separations were carried out with silica gel 60 (particle size: 0.032-0.063 mm) from Merck.
• High pressure liquid chromatography (HPLC):
  • Analytical HPLC: Analytical separations, with the exception of the oligonucleotide experiments, were carried out on an HPLC system from Jasco, equipped with a PU-2080 solvent pump, a LG-1590-04 mixing chamber, a MD-2010 Plus multi-wavelength detector and the LC-Net II controller / ADC. In addition, an automatic sample changer (autosampler AS-2055) from the same company was connected. The computer programs Borwin PDA, HSS 2000 and Borwin Chromatography from Jasco were used for operation, data acquisition and data evaluation. The ready-to-use columns Chiralcel ^® OD (250x4.6 mm, particle size: 10 µm) from Daicel Chemical Industries Ltd. were used for the analytical measurements. and Kromasil 100 C18 (5 μm, 250 × 4 mm) from Jasco were used. As The solvents used were n-hexane, dichloromethane and isopropanol in HPLC quality (Chiralcel ^® OD) as well as double-distilled water (addition of 0.1% by volume trifluoroacetic acid for peptide synthesis or 0.06% by volume concentrated hydrochloric acid) and acetonitrile or methanol in HPLC quality (Kromasil 100 C18). All samples were membrane filtered and the solvents degassed.
• Preparative HPLC: Preparative separations were carried out on an HPLC system from Jasco, equipped with two solvent pumps, model PU-2087 PLUS and a UV detector, model UV-2075 PLUS. ^{A Chiralpak ®} IA ready-to-use column (250 × 20 mm, particle size: 5 µm) and a Kromasil 100 C18 type (7 µm, 250 × 20 mm) column from Jasco in conjunction with a Kromasil 100 C18 precolumn (5 µm, 50 × 20 mm) from Jasco. The solvents used were n-heptane, n-hexane and dichloromethane in HPLC quality (Chiralpak ^® IA) as well as double-distilled water (addition of 0.1% by volume trifluoroacetic acid for peptide synthesis) and acetonitrile or methanol in HPLC quality (Kromasil 100 C18) . All samples were membrane filtered and the solvents degassed.

example 1

Racemic synthesis of the seco-CBI basic structure, as in 2 shown.

(E / Z) -2-Amino-4-benzyloxy-N- (tert-butyloxycarbonyl) -N- (3-chloro-2-propenyl) -1-iodo-naphthalene (75)

A solution of the amide 73 (2.00 g, 4.21 mmol, 1.0 eq.) In given to absolute DMF (20 mL). The mixture was stirred at room temperature for 2 h, (E / Z) -1,3-dichloropropene (74) (870 μL, 8.42 mmol, 2.0 eq.) Was added and the mixture was stirred at room temperature for a further 6 h. The reaction was ended by careful addition of a saturated NH ₄ Cl solution (50 mL) and ethyl acetate (100 mL) and water (50 mL) were added to the reaction mixture. It was then extracted with ethyl acetate (3 × 150 ml), the combined organic phases were washed with water (4 × 200 ml) and saturated NaCl solution (200 ml _), dried over MgSO 4 and the solvent was removed in vacuo. Column chromatography on silica gel (n-pentane / EtOAc = 10: 1) provided the target compound 75 as a yellow oil (2.29 g, 4.16 mmol, 99%).
R _f = 0.51, 0.44 (n-pentane / EtOAc = 10: 1).

¹ H-NMR (200 MHz, CDCl ₃ ): δ = 1.33 / 1.58 (each s, add. 9 H, C (CH ₃ ) ₃ ), 3.72-4.34 (m, 1 H, 1'-H _a ), 4.47-4.63 (m, 1H, 1'-H _b ), 5.26 (s _br , 2H, OCH ₂ Ph), 5.93-6.18 (m, 2H, 2'-H, 3'-H), 6.65 -6.79 (m, 1 H, 3-H), 7.31-7.63 (m, 7 H, 6-H, 7-H, 5 × Ph-H), 8.10, 8.22 (each d, J = 8.0 Hz, each 1 H, 5-H, 8-H).

¹³ C-NMR (50 MHz, CDCl ₃ ): δ = 28.3 / 28.5 (C (CH ₃ ) ₃ ), 46.0 / 49.1 (C-1 '), 70.3 / 70.4 (OCH ₂ Ph), 80.7 (C (CH ₃ ) ₃ ), 95.0 / 95.1 (C-1), 107.1 / 107.7 (C-3), 120.6 / 120.8 (C-3 '), 122.5 (C-5), 125.5 (C-4a), 126.3, 126.4 , 127.3 (C-6, C-2 ', Ph-C _o ), 128.1 / 128.2 (Ph-C _p ), 128.5 / 128.6 (C-8), 128.7 (Ph-C _m ), 132.7 (C-7 ), 135.3 (C-8a), 136.3 / 136.4 (Ph-C _i ), 142.4 / 142.8 (C-2), 153.7 / 153.9 (C = O), 155.3 (C-4).

rac-5-Benzyloxy-3- (tert-butyloxycarbonyl) -1-chloromethyl-1,2-dihydro-3H-benz [e] indole (rac-58)

The aromatic iodine 75 (2.29 g, 4.16 mmol, 1.0 eq.) Was dissolved in toluene (50 ml) and the solution was thoroughly degassed by introducing a stream of argon gas. Tris (trimethylsilyl) silane (1.46 mL, 4.58 mmol, 1.1 eq.) And AIBN (171 mg, 1.04 mmol, 0.25 eq.) Were added and the mixture was stirred at 80 ° C. for 4 h. After cooling, a 10% aqueous KF solution (50 mL) was added to the reaction mixture and the mixture was stirred at room temperature for 1 h. The organic phase was _{dried over MgSO 4} , the solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (gradient: n-pentane / EtOAc = 50: 1 → 20: 1). The product rac-58 was obtained as a white solid (1.38 g, 3.26 mmol, 78%).
R _f = 0.50 (n-pentane / EtOAc = 10: 1).

¹ H-NMR (300 MHz, CDCl _3): δ = 1.62 (s, 9 H, C (CH _{3) 3),} 3:44 (t, J = 10.9 Hz, 1 H, 10-H _a), 3.92-4.01 (m, 2 H, 1-H, 10-H _b ), 4.13 (t, J = 10.7 Hz, 1 H, 2-H _a ), 4.28 (d, J = 11.1 Hz, 1 H, 2-H _b ), 5.27 (s, 2 H, OCH ₂ Ph), 7.31-7.56 (m, 7 H, 7-H, 8-H, 5xPh-H), 7.65 (d, J = 8.4 Hz, 1 H, 9- H), 7.87 (s _br , 1H, 4-H), 8.30 (d, J = 8.4 Hz, 1H, 6-H).

¹³ C-NMR (50 MHz, CDCl ₃ ): δ = 28.5 (C (CH ₃ ) ₃ ), 41.6 (C-1), 46.5 (C-10), 53.0 (C-2), 70.3 (OCH ₂ Ph ), 81.2 (C (CH ₃ ) ₃ ), 96.4 (C-4), 114.0 (C-9b), 122.4 (C-5a), 121.7, 123.1, 123.6 (C-6, C-7, C-9 ), 127.5 (Ph-C _o ), 127.6 (C-8), 128.0 (Ph-C _p ), 128.5 (Ph-C _m ), 130.2 (C-9a), 136.7 (Ph-C _i ), 143.4 ( C-3a), 152.6 (C = O), 156.0 (C-5).

rac-3- (tert-butyloxycarbonyl) -1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indole (rac 69)

To a solution of the benzyl ether rac-58 (200 mg, 472 μmol, 1.0 eq.) In freshly distilled THF, palladium on activated carbon (10%, 100 mg, 94 μmol, 0.2 eq. With respect to Pd) was first added at 35 ° C. and then ammonium formate (25% aqueous solution, 1.0 mL) was added. After stirring for 25 min at 30 ° C., the solid was immediately filtered off through a layer of Celite, and it was rinsed thoroughly with acetone (400 ml). The solvent was removed in vacuo and, after column chromatography on silica gel (n-pentane / EtOAc = 5: 1), 147 mg of an inseparable mixture (83:17) were obtained from the target compound rac-58 (124 mg, 372 μmol, 79% ) and the dechlorinated racemic by-product (23 mg, 75.5 µmol, 16%).
R _f = 0.54 (n-pentane / EtOAc = 5: 1). C ₁₈ H ₂₀ ClNO ₃ (333.82). ber .: 334.1205 Found: 334.1205, [M + H] ⁺ (ESI-HRMS).

Separation of the enantiomers of rac-3- (tert-butyloxycarbonyl) -1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indole (rac-69)

rac-69 was dissolved in a mixture of n-hexane / isopropanol (1: 1) (c = 25 mg / mL) and 1 mL each of this solution in the preparative HPLC system (column: Chiralpak ^® IA, 250 × 20 mm, particle size: 5 µm, mobile phase: n-hexane / iso-propanol = 97: 3, flow: 18 mL / min, pressure: 5.7 MPa). Fractional collection of the eluate (UV detector: λ = 255 nm) yielded the enantiomers (+) - (1R) -69 and (-) - (1S) -69.

Analytical data for (+) - (1R) -69:

HPLC (preparative): tR: 11.9 min Fraction: 11.1-13.0 min HPLC (analytical): Pillar: Chiralcel ^® OD, 250x4.6 mm, 10 micron Eluent: 2% iso-propanol in n-hexane Flow: 0.8 mL / min t _R : 16.4 min, 99.9% ee

$${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+48.6°\left(\text{c}=\mathrm{0.28,}{\text{CHCl}}_{\text{3}}\right).$$

Analytical data for (-) - (1S) -69:

UV (CH₃CN): λ_max (Ig ε) = 208.5 nm (4.236), 220.0 (4.216), 255.0 (4.853), 303.5 (3.933), 314.5 (3.992), 342.0 (3.489). HPLC (preparative): tR: 14.0 min Fraction: 13.6-15.8 min HPLC (analytical): Pillar: Chiralcel ^® OD, 250x4.6 mm, 10 micron Eluent 2% iso-propanol in n-hexane Flow: 0.8 mL / min t _R : 19.3 min, 99.9% ee $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-45.8°\left(\text{c}=\mathrm{0.24,}{\text{CHCl}}_{\text{3}}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 208.5 nm (4.236), 220.0 (4.216), 255.0 (4.853), 303.5 (3.933), 314.5 (3.992), 342.0 (3.489).

IR (KBr): ṽ (cm ^-1 ) = 3368, 2977, 1682, 1629, 1585, 1522, 1480, 1413, 1339, 1236, 1143, 1058, 911, 852, 758, 674.

¹ H-NMR (300 MHz, CDCl ₃ ): δ = 1.62 (s, 9 H, C (CH ₃ ) ₃ ), 3.41 (t, J = 11.4 Hz, 10-H _a ), 3.90-3.98 (m, 2 H, 1-H, 10-H _b ), 4.12 (t, J = 10.5 Hz, 1 H, 2-H _a ), 4.25 (d, J = 11.5 Hz, 1 H, 2-H _b ), 7.33 (dt, J = 7.6, 1.0 Hz, 1 H, 7-H), 7.49 (dt, J = 7.6, 1.0 Hz, 1 H, 8-H), 7.62 (d, J = 8.4 Hz, 1 H, 9-H), 7.82 (s _br , 1 H , 4-H), 8.21 (d, J = 8.4 Hz, 1H, 6-H).

¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 28.5 (C (CH ₃ ) ₃ ), 41.8 (C-1), 46.5 (C-10), 53.1 (C-2), 81.8 (C (CH ₃ ) ₃ ), 99.1 (C-4), 114.3 (C-9b *), 121.6 (C-5 *), 121.7 (C-9), 122.9 (C-7), 123.6 (C-6), 127.6 (C-8), 130.4 (C-9a), 141.1 (C-3a), 153.3 (C = O), 154.1 (C-5).

MS (ESI): m / z (%) = 334.1 (15) [M + H] ⁺ , 278.1 (100) [MC ₄ H ₈ + H] ⁺ . C ₁₈ H ₂₀ ClNO ₃ (333.82). ber .: 334.1205 Found: 334.1205, [M + H] ⁺ (ESI-HRMS).

Example 2

Asymmetric synthesis of the (1S) -seco-CBI backbone, as in 3 shown.

(+) - (2S) -Glycidyl-3-nitrobenzenesulfonate ((+) - (S) -77)

A solution of (+) - (R) -glycidol (76) (4.85 g, 65.5 mmol, 1.0 eq.) And triethylamine (10.9 ml, 7.95 g, 78.6 mmol, 1.2 eq.) In absolute toluene (130 ml) was cooled to 0 ° C. and m-nitrobenzenesulfonyl chloride (14.5 g, 65.5 mmol, 1.0 eq.) was added in portions. After warming to room temperature, the mixture was stirred at this temperature for 1 h, the mixture was then filtered through Celite and a little silica gel and washed with toluene (250 ml). After removing the solvent in vacuo, the crude product was dissolved in ethanol (max. 20 mL / g) at 40 ° C. and crystallized by slowly cooling to -30 ° C. The target compound (+) - (S) -77 (7.70 g, 29.7 mmol, 45%; (R) -enantiomer undetectable) was obtained in the form of colorless needles.

Analytical data for (-) - (R) -77:

HPLC (analytical): Pillar: Chiralpak ^® IA, 250x4.6 mm, 5 micron Eluent 14% THF in n-hexane Flow: 0.8 mL / min t _R : 52.3 min

Analytical data for (+) - (S) -77:

¹H-NMR (300 MHz, CDCl₃): δ = 2.63 (dd, J = 4.7, 2.5 Hz, 1 H, 3'-H_a), 2.85 (mc, 1 H, 3'-H_b), 3.23 (m_c, 1 H, 2'-H), 4.05 (dd, J = 11.7, 6.6 Hz, 1 H, 1'-H_a), 4.50 (dd, J = 11.6, 2.9 Hz, 1 H, 1'-H_b), 7.83 (t, J = 8.1 Hz, 1 H, 5-H), 8.27 (ddd, J = 7.8, 1.9, 1.2 Hz, 1 H, 6-H), 8.54 (ddd, J = 8.1, 2.2, 1.1 Hz, 1 H, 4-H), 8.78 (m_c, 1 H, 2-H). ¹³C-NMR (75 MHz, CDCl₃): δ = 44.5 (C-3'), 48.7 (C-2'), 71.7 (C-1'), 123.2 (C-2), 128.4 (C-4), 130.8 (C-5), 133.3 (C-6), 138.0 (C-1), 148.3 (C-3). HPLC (analytical): Pillar: Chiralpak ^® IA, 250x4.6 mm, 5 micron Eluent 14% THF in n-hexane Flow: 0.8 mL / min t _R : 48.4 min
R _f = 0.67 (Et ₂ O). $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+22.7°\left(c=\mathrm{2.14,}{\text{CHCl}}_{\text{3}}\right).$$

¹ H-NMR (300 MHz, CDCl ₃ ): δ = 2.63 (dd, J = 4.7, 2.5 Hz _{, 1H, 3'-H a} ), 2.85 (mc, 1 H, 3'-H _b ), 3.23 (m _c , 1H, 2'-H), 4.05 (dd, J = 11.7, 6.6 Hz, 1 H, 1'-H _a ), 4.50 (dd, J = 11.6, 2.9 Hz, 1 H, 1 ' -H _b ), 7.83 (t, J = 8.1 Hz, 1 H, 5-H), 8.27 (ddd, J = 7.8, 1.9, 1.2 Hz, 1 H, 6-H), 8.54 (ddd, J = 8.1 , 2.2, 1.1 Hz, 1H, 4-H), 8.78 (m _c , 1H, 2-H). ¹³ C-NMR (75 MHz, CDCl ₃ ): δ = 44.5 (C-3 '), 48.7 (C-2'), 71.7 (C-1 '), 123.2 (C-2), 128.4 (C-4 ), 130.8 (C-5), 133.3 (C-6), 138.0 (C-1), 148.3 (C-3).

(+) - {(2'R) -2-amino-4-benzyloxy-N- (tert-butyloxycarbonyl) -N- (2,3-epoxypropyl) -1-iodo-naphthalene} ((+) (2 ' R) -78)

UV (CH₃CN): λ_max (Ig ε) = 216.0 nm (4.682), 243.5 (4.437), 304.0 (3.948).The amide 73 (4.76 g, 10.0 mmol, 1.0 eq.) Was dissolved in DMF (125 mL) and warmed to 40 ° C. After adding NaH (721 mg, 30.1 mmol, 3.0 eq .; 60% suspension in mineral oil) and the nosylate (+) - (S) -77 (3.90 g, 15.0 mmol, 1.5 eq.), The mixture was used for 3 Stirred at 40 ° C. for h. After cooling down At room temperature the suspension was combined with saturated NH ₄ Cl solution (250 mL), _{extracted with Et 2} O (3 × 200 mL), the combined organic phases with water (200 mL) and saturated NaCl solution (2 × 200 mL) washed, _{dried over Na 2} SO ₄ and the solvent removed in vacuo. Purification by column chromatography on silica gel (n-pentane / EtOAc = 5: 1) gave the product (+) - (2'R) -78 (5.08 g, 9.56 mmol, 96%) as a mixture of atropisomers in the form of a white solid.
R _f = 0.53 (n-pentane / EtOAc = 7: 3). $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+8.0°\left(\text{c}=\mathrm{0.6,}{\text{CHCl}}_{\text{3}}\right)$$

UV (CH ₃ CN): λ _max (Ig ε) = 216.0 nm (4.682), 243.5 (4.437), 304.0 (3.948).

IR (KBr): ṽ (cm ^-1 ) = 3063, 2978, 2929, 1701, 1617, 1591, 1562, 1503, 1454, 1404, 1370, 1326, 1261, 1231, 1156, 1102, 1029, 973, 912, 879, 841, 760, 736, 697, 642.

¹ H-NMR (300 MHz, tetrachloroethane-d ₂ , 100 ° C; the respective signals of the two atropisomers are labeled (a) and (b)): δ = 1.46 (s, 18 H, 2 × C (CH ₃ ) ₃ ), 2.46 (dt, J = 4.7, 2.6 Hz, 2 H, 3'-H _a (a), 3'-H _a (b)), 2.73 (dt, J = 4.8, 9.0 Hz, 2 H , 3'-H _b (a), 3'-H _b (b)), 3.12-3.19 (m, 1 H, 2'-H (a)), 3.28 (dd, J = 14.4, 6.6 Hz, 1 H, 1'-H _a (a)), 3.35-3.42 (m, 1H, 2'-H (b)); 3.49 (dd, J = 14.7, 4.9 Hz, _{1H, 1'-H a} (b)), 4.09-4.17 (m, 2H, 1'-H _b (a), 1'-H _b (b) ), 5.32, 5.33 (2 × s, 4 H, OCH ₂ Ph (a), OCH ₂ Ph (b)), 6.89, 6.99 (2 × s, 2 H, 3-H (a), 3-H ( b)), 7.34-7.67 (m, 14 H, 6-H (a), 6-H (b), 7-H (a), 7-H (b), 5xPh-H (a) 5xPh-H (b)), 8.25 (m _c , 2 H, 8-H (a), 8-H (b)), 8.36 (m _c , 2 H, 5-H (a) and 5-H (b)

¹³ C-NMR (75 MHz, tetrachloroethane-d ₂ , 100 ° C; the respective signals of the two atropisomers are labeled (a) and (b)): δ = 29.8 (2 signals)
(2xC (CH ₃ ) ₃ ), 47.3, 47.5 (C-3 '(a), C-3' (b)), 51.0 (C-2 '(a)), 51.5 (C-2' (b) ), 52.9 (C-1 '(b)), 54.5 (C-1' (a)), 72.2, 72.3 (2 × OCH ₂ Ph), 82.1 (2 signals) (2 × C (CH ₃ ) ₃ ) , 96.2, 96.3 (C-1 (a), C-1 (b)), 109.6, 109.7 (C-3 (a), C-3 (b)), 123.9 (C-5 (a), C- 5 (b)), 127.2 (C-4a (a), C-4a (b)), 127.7 (2 signals), 128.6, 128.8, 129.4, 129.8, 129.9, 130.0 (2 signals) (C-6 (a ), C-6 (b), C-7 (a), C-7 (b), C-8 (a), C-8 (b), 2 × Ph-C _o (a), 2 × Ph -C _o (b), 2 × Ph-C _m (a), 2 × Ph-C _m (b), Ph-C _p (a), Ph-C _p (a)), 136.9 (2 signals) ( C-8a (a), C-8a (b)), 137.9, 138.0 (Ph-C _i (a), Ph-C _i (b)), 144.8, 145.3 (C-2 (a), C-2 (b)), 155.0, 155.2 (C = O (a), C = O (b)), 157.0, 157.1 (C-4 (a), C-4 (b)).

MS (ESI): m / z (%) = 1085.2 (100) [2M + Na] ⁺ , 554.1 (97) [M + Na] ⁺ . C ₂₅ H ₂₆ INO ₄ (531.38). ber .: 554.0799 Found: 554.0802, [M + Na] ⁺ (ESI-HRMS).

(+) - {(1S) -5-Benzyloxy-3- (tert-butyloxycarbonyl) -1-hydroxymethyl-1,2-dihydro-3H-benz [e] indole} ((+) - (1S) -79)

A solution of freshly sublimed ZnCl ₂ (1.36 g, 9.96 mmol, 2.11 eq.) In absolute THF (80 mL) was cooled to 0 ° C, slowly with methyl lithium (24.9 mL of a 1.6 M solution in Et ₂ O, 39.8 mmol, 8.42 eq.) And stirred for 30 min at this temperature. The solution was then cooled to −78 ° C., TMS-NCS (1.42 mL, 1.31 g, 9.96 mmol, 2.11 eq.) Was added dropwise, the mixture was warmed to 0 ° C. and stirred at this temperature for 30 min. After cooling again to -78 ° C., a solution of the epoxide ((+) - (2'R) -78 (2.51 g, 4.73 mmol, 1.0 eq.) In absolute THF (80 ml) was added dropwise to the reaction mixture and allowed to cool for 30 stirred at this temperature. After warming to 0 ° C, the reaction solution was stirred at this temperature and then warmed to room temperature and stirred for further 12 h, initially for 1.5. the mixture was carefully quenched with saturated NH ₄ Cl solution (250 mL ), _{extracted with CH 2} Cl ₂ (4 × 250 mL), the combined organic phases _{dried over Na 2} SO ₄ and the solvent removed in vacuo. Column chromatographic purification on silica gel (n-pentane / EtOAc = 4: 1) provided the Target compound (+) - (1S) -79 (1.12 g, 2.76 mmol, 58%, 96.1% ee) as a white solid.

Analytical data for (-) - (1R) -79:

HPLC (analytical): Pillar: Chiralcel ^® OD, 250x4.6 mm, 10 micron Eluent: 5% iso-propanol in n-hexane Flow: 0.8 mL / min t _R : 13.6 min

Analytical data for (+) - (1S) -79:

UV (CH₃CN): λ_max (Ig ε) = 207.5 nm (4.447), 217.5 (4.396), 255.0 (4.834), 304.0 (3.994), 315.0 (4.072), 341.0 (3.550). HPLC (analytical): Pillar: Chiralcel ^® OD, 250 x 4.6 mm, 10 micron Eluent: 5% iso-propanol in n-hexane Flow: 0.8 mL / min t _R : 14.3 min
R _f = 0.35 (n-pentane / EtOAc = 3: 1). $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+4.3°\left(\text{c}=\mathrm{1.79,}{\text{CH}}_2{\text{Cl}}_2\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 207.5 nm (4.447), 217.5 (4.396), 255.0 (4.834), 304.0 (3.994), 315.0 (4.072), 341.0 (3.550).

IR (KBr): ṽ (cm ^-1 ) = 3446, 2975, 1699, 1626, 1582, 1517, 1460, 1407, 1332, 1268, 1142, 1032, 906, 845, 760, 696,

¹ H-NMR (300 MHz, CDCl ₃ ): δ = 1.59 (s, 9 H, C (CH ₃ ) ₃ ), 3.72-3.87 (m, 2 H, 1-H, 10-H _a ), 3.91- 3.97 (m, 1 H, 10-H _b ), 4.08-4.15 (m, 1 H, 2-H _a ), 4.19-4.23 (m, 1 H, 2-H _b ), 5.26 (s _br , 2 H , OCH ₂ Ph), 7.28-7.55 (m, 7 H, 7-H, 8-H, 5xPh-H), 7.70 (d, J = 8.4 Hz, 1 H, 9-H), 7.89 (s _br , 1H, 4-H), 8.28 (d, J = 8.2 Hz, 1H, 6-H).

¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 28.5 (C (CH ₃ ) ₃ ), 41.3 (C-1), 52.4 (C-2), 64.7 (C-10), 70.3 (OCH ₂ Ph ), 80.7 (C (CH ₃ ) ₃ ), 96.5 (C-4), 114.4 (C-9b), 122.3 (C-5a), 122.2 (C-9), 122.9 (C-7), 123.4 (C -6), 127.3 (C-8), 127.5, 127.9, 128.5 (5 × Ph-CH), 130.6 (C-9a), 136.9 (Ph-C _i ), 141.9 (C-3a), 152.7 (C = O), 155.6 (C-5).

MS (ESI): m / z (%) = 350.1 (100) [MC ₄ H ₈ + H] ⁺ , 406.2 (44) [M + H] ⁺ , 428.2 (22) [M + Na] ⁺ , 828.4 ( 24) [M + NH ₄ ] ⁺ . C ₂₅ H ₂₇ NO ₄ (405.49). ber .: 406.2013 found: 406.2013, [M + H] ⁺ (ESI-HRMS).

(-) - {(1S) -3- (tert-Butyloxycarbonyl) -1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indole} ((-) (1S) 58)

A solution of (+) - (1S) -58 (2.15 g, 5.30 mmol, 1.0 eq.) In absolute CH ₂ Cl ₂ (20 mL) was at room temperature with triphenylphosphine (4.17 g, 15.9 mmol, 3.0 eq.) And CCl ₄ (4.60 mL, 7.34 g, 47.7 mmol, 9.0 eq.) Was added and the mixture was stirred at this temperature for 3 h. After removing the solvent in vacuo and subsequent purification by column chromatography on silica gel (n-pentane / EtOAc = 20: 1), the target compound (-) (1S) -58 (2.13 g, 5.03 mmol, 95%, 96.1% ee) became white Solid obtained.

Analytical data for (+) - (1R) -58:

HPLC (analytical): Pillar: Chiralcel ^® OD, 250 x 4.6 mm, 10 micron Eluent: 2% iso-propanol in n-hexane Flow: 0.8 mL / min t _R : 9.6 min

Analytical data for (-) - (1S) -58:

HPLC (analytical): Pillar: Chiralcel ^® OD, 250 x 4.6 mm, 10 micron Eluent: 2% iso-propanol in n-hexane Flow: 0.8 mL / min t _R : 13.4 min The further steps were carried out as in Example 1, step C., to obtain the compound enantiomeric -3- (tert-butyloxycarbonyl) -1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indole (rac 69) Get Connections.

Example 3

Synthesis of the bifunctional seco-CBI derivatives and the corresponding β-D-galactosides

- {1,5-bis [(1S) -1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] pentane-1,5-dione]} ((- ) - (1'S) -55a, n = 3)

The phenol (-) - (1S) -69 (61.0 mg, 183 μmol, 2.0 eq.) Was dissolved in 4 M HCl / ethyl acetate (4.5 ml) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was dried in vacuo for 1 h, dissolved in DMF (5 mL), the solution was cooled to 0 ° C and treated with glutaric acid chloride (66a) (12.0 µL, 15.4 mg, 91.4 µmol, 1.0 eq.) And pyridine (29.5 µL, 28.9 mg, 365 µmol, 4.0 eq.). After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S) -55a (38.9 mg, 69.0 µmol, 76%) was obtained as a white solid.

HPLC (preparative):

UV (CH₃CN): λ_max (Ig ε) = 205.5 nm (4.550), 222.5 (4.406), 256.5 (4.909), 264.5 (4.921), 319.0 (4.332). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: - Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.0-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm t _R : 8.4 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-32.7°\left(c=\mathrm{0.257,}\text{DMSO}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 205.5 nm (4.550), 222.5 (4.406), 256.5 (4.909), 264.5 (4.921), 319.0 (4.332).

IR (KBr): ṽ (cm ^-1 ) = 3122, 2926, 1633, 1584, 1522, 1459, 1426, 1394, 1253, 1131, 1075, 1024, 857, 756, 717.

¹ H-NMR (600 MHz, DMSO-d _6): δ = 1.98 (mc, 2 H, 3-H _2), 2.57-2.62 (m, 2 H, 2-Ha, 4-H _a), 2.68- 2.74 (m, _{2H, 2-H b} , 4-H _b ), 3.79 (dd, J = 10.9, 8.3 Hz, 2 H, 2 × 10'-H _a ), 3.99 (dd, J = 10.9, 3.2 Hz, 2 H, 2 × 10'-H _b ), 4.14-4.20 (m, 4 H, 2 × 1'-H, 2 × 2'-H _a ), 4.34 (mc, 2 H, 2 × 2 ' -H _b ), 7.32 (mc, 2 H, 2 × 7'-H), 7.49 (m _c , 2 H, 2 × 8'-H), 7.78 (d, J = 8.3 Hz, 2 H, 2 × 9'-H), 8.02 (s, 2H, 2x4'-H), 8.09 (d, J = 8.4 Hz, 2H, 2x6'-H), 10.31 (s, 2H, 2x OH).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 19.3 (C-3), 34.4 (C-2, C-4), 40.7 (2 × C-1 '), 47.6 (2 × C- 10 '), 52.6 (2 × C-2'), 99.7 (2 × C-4 '), 113.6 (2 × C-5a'), 121.5 (2 × C-9b '), 122.3 (2 × C- 9 '), 122.4 (2 × C-7'), 122.9 (2 × C-6 '), 127.0 (2 × C-8'), 129.8 (2 × C-9a '), 141.8 (2 × C- 3a '), 154.0 (2 × C-5'), 170.4 (2 × C = O).

MS (ESI): m / z (%) = 1125.4 (8) [2M-H] ^- , 561.2 (96) [MH] ^- , 525.2 (100) [M-2H-Cl] ^- . C ₃₁ H ₂₈ Cl ₂ N ₂ O ₄ (563.47). ber .: 585.1318 Found: 585.1317, [M + Na] ⁺ (ESI-HRMS).

Example 4

(-) - {1,5-Bis [(1S) -1-chloromethyl-5- (β-D-galactopyranosyl) -1,2-dihydro-3H-benz [e] indol-3-yl] pentane-1 , 5-dione]} ((-) - (1'S) -53a, n = 3)

Das acetylgeschützte Galactosid (1'S)-71a (148 mg, 121 µmol, 1.0 Äq.) wurde in absolutem Methanol (35 mL) suspendiert und bei 0 °C mit NaOMe in Methanol (90 µL einer 5.4 M Lösung, 484 µmol, 4.0 Äq.) versetzt. Nach Erwärmen auf Raumtemperatur und 30 min Rühren bei dieser Temperatur hatte sich das Substrat vollständig gelöst. Es wurde noch weitere 1.5 h bei Raumtemperatur gerührt, die Reaktionslösung anschließend mit Wasser (3.0 mL) verdünnt und so lange saurer Ionentauscher (Amberlite-IR^® 120) zugegeben, bis die Lösung neutral war (pH = 7). Das Gemisch wurde via Transferkanüle vom Ionenaustauscher abgetrennt, selbiger mit Methanol (2×6 mL) nachgewaschen, die organischen Phasen vereinigt und das Lösungsmittel im Vakuum entfernt. Der erhaltene Rückstand (132 mg) wurde in DMF (5 mL) gelöst und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S)-53a (95.3 mg, 107 µmol, 88%) wurde als hellgelber Feststoff erhalten.

The acetyl-protected galactoside (1'S) -71a (148 mg, 121 μmol, 1.0 eq.) Was suspended in absolute methanol (35 ml) and at 0 ° C. with NaOMe in methanol (90 μL of a 5.4 M solution, 484 μmol, 4.0 eq .) offset. After warming to room temperature and stirring at this temperature for 30 minutes, the substrate had completely dissolved. Stirring was continued for a further 1.5 h at room temperature, then diluting the reaction solution with water (3.0 mL) and a sufficient acidic ion exchanger (Amberlite IR-120 ^®) were added until the solution was neutral (pH = 7). The mixture was separated from the ion exchanger via a transfer cannula, the latter was washed with methanol (2 × 6 mL), the organic phases were combined and the solvent was removed in vacuo. The residue obtained (132 mg) was dissolved in DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S) -53a (95.3 mg, 107 µmol, 88%) was obtained as a light yellow solid.

HPLC (preparative):

UV (CH₃CN): λ_max (Ig ε) = 209.0 nm (4.447), 254.0 (4.702), 262.5 (4.642), 304.0 (4.120), 316.0 (4.200). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-15 70/30 → 0/100 15-22 0/100 22-23 0/100 → 70/30 23-30 30/70 Flow: 16 mL min ^-1 λ: 254 nm t _R : 13.9 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-43.6°\left(c=\mathrm{0.257,}\text{DMSO}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 209.0 nm (4.447), 254.0 (4.702), 262.5 (4.642), 304.0 (4.120), 316.0 (4200).

IR (KBr): ṽ (cm ^-1 ) = 3406, 2927, 1655, 1580, 1469, 1403, 1313, 1263, 1131, 1076, 856, 760, 664. ◯ ◦ ◯

¹ H-NMR (600 MHz, DMSO-d _6): δ = 1.99 (mc, 2 H, 3-H _2), 2.60-2.65 (m, 2 H, 2-H _a, 4-H _a), 2.69 -2.74 (m, 2 H, 2-H _b , 4-H _b ), 3.46-3.49 (m, 2 H, 2 × 3 "-H), 3.52-3.64 (m, 6 H, 2 × 5" - H, _{2x6 "-H 2} ), 3.75-3.79 (m, 4H, 2x2" -H, 2x4 "-H), 3.85-3.88 (m, 2H, 2x10'-H _a ), 4.02 (dd, J = 10.8, 2.4 Hz, 2 H, 2x10'-H _b ), 4.22-4.24 (m, 4 H, 2 × 1'-H, 2 × 2'-H _a ), 4.37 -4.40 (m, 2H, 2 × 2'-H _b ), 4.51 (d, J = 4.6 Hz, 2 H, 2 × OH), 4.54 (m _c , 2 H, 2xOH), 4.82 (d, J = 5.5 Hz, 2 H, 2 × OH), 4.93 (d, J = 7.1 Hz, 2 H, 2 × 1 "-H), 5.27 (d, J = 4.5 Hz, 2 H, 2 × OH), 7.39 (t, J = 7.7 Hz, 2 H, 2 × 7'-H), 7.54 (t, J = 7.5 Hz, 2 H, 2 × 8'-H), 7.85 (d, J = 8.6 Hz, 2 H. , 2 × 9'-H), 8.30 (d, J = 8.6 Hz, 2 H, 2 × 6'-H), 8.23 (s, 2 H, 2 × 4'-H).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 19.2 (C-3), 34.5 (C-2, C-4), 40.7 (2 × C-1 '), 47.6 (2 × C- 10 '), 52.6 (2 × C-2'), 59.7 (2 × C-6 "), 67.6 (2 × C-4"), 70.4 (2 × C-2 "), 73.1 (2 × C- 3 "), 75.2 (2 × C-5"), 101.2 (2 × C-4 '), 101.9 (2 × C-1 "), 116.6 (2 × C-5a'), 122.4 (2 × C- 9 ', 2 × C-9b'), 123.2 (2 × C-6 ', 2 × C-7'), 127.2 (2 × C-8 '), 129.4 (2 × C-9a'), 141.7 ( 2 × C-3a '), 153.7 (2 × C-5'), 170.6 (2xC = O).

MS (ESI): m / z (%) = 909.2 (47) [M + Na] ⁺ , 885.3 (100) [MH] ^- . C ₄₃ H ₄₈ Cl ₂ N ₂ O ₁₄ (887.75). ber .: 909.2375 found: 909.2377, [M + Na] ⁺ (ESI-HRMS).

Example 5

1,6-bis [(1S) -1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] hexane-1,6-dione]} ((1'S) - 55b, n = 4)

Das Phenol (-)-(1S)-69 (58.0 mg, 174 µmol, 2.0 Äq.) wurde in 4 M HCl / Essigester (4.5 mL) gelöst und nach 1 h Rühren bei Raumtemperatur das Lösungsmittel im Vakuum entfernt. Der Rückstand wurde 1 h im Vakuum getrocknet, in DMF (5 mL) gelöst, die Lösung auf 0 °C gekühlt und mit Adipinsäurechlorid (66b) (12.9 µL, 15.9 mg, 86.9 µmol, 1.0 Äq.) sowie Pyridin (28.0 µL, 27.5 mg, 348 µmol, 4.0 Äq.) versetzt. Nach 20 h Rühren bei Raumtemperatur wurde das Reaktionsgemisch mit DMF (5 mL) verdünnt und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Es wurden 3 mg eines Gemisches des Produkts (1'S)-55b (<5.19 µmol, <6%) und nicht eindeutig identifizierter Nebenprodukte als weißer Feststoff erhalten.

The phenol (-) - (1S) -69 (58.0 mg, 174 μmol, 2.0 eq.) Was dissolved in 4 M HCl / ethyl acetate (4.5 ml) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was dried in vacuo for 1 h, dissolved in DMF (5 mL), the solution was cooled to 0 ° C and treated with adipic acid chloride (66b) (12.9 µL, 15.9 mg, 86.9 µmol, 1.0 eq.) And pyridine (28.0 µL, 27.5 mg, 348 µmol, 4.0 eq.). After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. 3 mg of a mixture of the product (1'S) -55b (<5.19 μmol, <6%) and by-products that were not clearly identified were obtained as a white solid.

HPLC (preparative):

Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: - Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.5-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm t _R : 9.4 min ¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.74 (m _c , 4 H, 3-H ₂ , 4-H ₂ ), 2.57 (m _c , 4 H, 2-H ₂ , 5- H ₂ ), 3.79 (dd, J = 10.9, 7.9 Hz, 2 H, 2 × 10'-H _a ), 3.98 (dd, J = 11.1, 3.1 Hz, 2 H, 2x10'-H _b ), 4.13- 4.19 (m, 4 H, 2 × 1'-H, 2 × 2'-H _a ), 4.30-4.36 (m, 2 H, 2x2'-H _b ), 7.31 (t, J = 7.6 Hz, 2 H, 2 × 7'-H), 7.48 (t, J = 7.6 Hz, 2 H, 2 × 8'-H), 7.76 (d, J = 8.3 Hz, 2 H, 2 × 9'-H) , 7.99 (s, 2H, 2x4'-H), 8.07 (d, J = 8.4Hz, 2H, 2x6'-H), 10.32 (s, 2H, 2xOH).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.8 (C-3, C-4), 35.0 (C-2, C-5), 40.7 (2 × C-1 '), 47.7 ( 2 × C-10 '), 52.7 (2 × C-2'), 99.8 (2 × C-4 '), 113.6 (2 × C-5a'), 121.5 (2 × C-9b '), 122.4 ( 2 × C-9 '), 122.5 (2 × C-7'), 123.0 (2 × C-6 '), 127.0 (2 × C-8'), 129.8 (2 × C-9a '), 141.9 ( 2 × C-3a '), 154.1 (2 × C-5'), 170.6 (2xC = O).

MS (ESI): m / z (%) = 575.2 (9) [MH] -, 539.2 (26) [M-2H-Cl] ^- . C ₃₂ H ₃₀ Cl ₂ N ₂ O ₄ (577.50). ber .: 575.1510 found: 575.1520, [MH] ^- (ESI-HRMS).

Example 6

(-) - {1,6-Bis [(1S) -1-chloromethyl-5- (β-d-galactopyranosyl) -1,2-dihydro-3H-benz [e] indol-3-yl] hexane-1 , 6-dione]} ((-) - (1'S) -53b, n = 4)

Das acetylgeschützte Galactosid (1'S)-71b (125 mg, 121 µmol, 1.0 Äq.) wurde in absolutem Methanol (35 mL) suspendiert und bei 0 °C mit NaOMe in Methanol (75 µL einer 5.4 M Lösung, 404 µmol, 4.0 Äq.) versetzt. Nach Erwärmen auf Raumtemperatur und 2 h Rühren bei dieser Temperatur wurde die Suspension mit Wasser (3.0 mL) verdünnt und so lange saurer Ionentauscher (Amberlite-IR^® 120) zugegeben, bis das Gemisch neutral war (pH = 7). Das Gemisch wurde via Transferkanüle vom Ionenaustauscher abgetrennt, selbiger mit DMF (2x6 mL) nachgewaschen, die organischen Phasen vereinigt und das Lösungsmittel im Vakuum entfernt. Der erhaltene Rückstand (123 mg) wurde in DMF (5 mL) gelöst und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S)-53b (76.2 mg, 84.5 µmol, 84%) wurde als hellgelber Feststoff erhalten.

The acetyl-protected galactoside (1'S) -71b (125 mg, 121 µmol, 1.0 eq.) Was suspended in absolute methanol (35 mL) and at 0 ° C with NaOMe in methanol (75 µL of a 5.4 M solution, 404 µmol, 4.0 eq .) offset. After warming to room temperature and stirring for 2 h at this temperature the suspension was diluted with water (3.0 mL) and a sufficient acidic ion exchanger (Amberlite IR-120 ^®) were added, the mixture was until neutral (pH = 7). The mixture was separated from the ion exchanger via a transfer cannula, the latter was washed with DMF (2 × 6 mL), the organic phases were combined and the solvent was removed in vacuo. The residue obtained (123 mg) was dissolved in DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S) -53b (76.2 mg, 84.5 μmol, 84%) was obtained as a light yellow solid.

HPLC (preparative):

UV (CH₃CN): λ_max (Ig ε) = 208.0 nm (4.363), 254.5 (4.623), 262.5 (4.630), 304.0 (3.998), 315.0 (4.055). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-15 70/30 → 0/100 15-22 0/100 22-23 0/100 → 70/30 23-30 30/70 Flow: 16 mL min ^-1 λ: 254 nm t _R : 14.4 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-45.0°\left(c=\mathrm{0.249,}\text{DMSO}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 208.0 nm (4.363), 254.5 (4.623), 262.5 (4.630), 304.0 (3.998), 315.0 (4.055).

IR (KBr): ṽ (cm ^-1 ) = 3406, 2927, 1656, 1580, 1469, 1402, 1316, 1075, 760, 665.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.75 (m _c , 4 H, 3-H ₂ , 4-H ₂ ), 2.60 (m _c , 4 H, 2-H ₂ , 5- H ₂ ), 3.48-3.54 (m, 4 H, 2 × 3 "-H, 2 × 6" -H _a ), 3.56-3.64 (m, 4 H, 2 × 5 "-H, 2 × 6 "-H _b ), 3.75-3.80 (m, 4H, 2 x 2" -H, 2 x 4 "-H), 3.86 (m _c , 2H, 2 x 10'-H _a ), 4.02 (dd, J = 11.0, 2.7 Hz, 2H, 2x10'-H _b ), 4.22-4.23 (m, 4H, 2 × 1'-H, 2 × 2'-H _a ), 4.38-4.41 (m , 2 H, 2x2'-H _b ), 4.50 (d, J = 4.6 Hz, 2 H, 2 × OH), 4.53 (mc, 2 H, 2 × OH), 4.82 (d, J = 5.6 Hz, 2 H, 2 × OH), 4.93 (d, J = 7.1 Hz, 2 H, 2 × 1``-H), 5.26 (d, J = 5.1 Hz, 2 H, 2 × OH), 7.38 (t, J = 7.7 Hz, 2 H, 2 × 7'-H), 7.54 (t, J = 7.6 Hz, 2 H, 2 × 8'-H), 7.85 (d, J = 8.4 Hz, 2 H, 2 × 9 '-H), 8.29-8.30 (m, 4H, 2x4'-H, 2x6'-H).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.6 (C-3, C-4), 35.0 (C-2, C-5), 40.7 (2 × C-1 '), 47.6 ( 2 × C-10 '), 52.6 (2 × C-2'), 59.6 (2 × C-6 "), 67.6 (2 × C-4"), 70.4 (2 × C-2 ") , 73.1 (2 × C-3 ''), 75.2 (2 × C-5 ''), 101.2 (2 × C-4 '), 101.9 (2 × C-1''), 116.5 (2 × C- 5a '), 122.4 (2 × C-9', 2 × C-9b '), 123.1 (2 × C-6', 2 × C-7 '), 127.2 (2 × C-8'), 129.4 ( 2 × C-9a '), 141.7 (2 × C-3a'), 153.6 (2 × C-5 '), 170.7 (2 × C = O).

MS (ESI): m / z (%) = 923.3 (67) [M + Na] ⁺ , 899.3 (100) [MH] ̅̅̅ ^- . C ₄₄ H ₅₀ Cl ₂ N ₂ O ₁₄ (901.78). ber .: 923.2531 found: 923.2530, [M + Na] ⁺ (ESI-HRMS).

Example 7

(-) - {1,7-Bis [(1 S) -1 -chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] heptane-1,7-dione] } ((-) - (1'S) -55c, n = 5)

Das Phenol (-)-(1 S)-69 (62.0 mg, 186 µmοl, 2.0 Äq.) wurde in 4 M HCl / Essigester (4.5 mL) gelöst und nach 1 h Rühren bei Raumtemperatur das Lösungsmittel im Vakuum entfernt. Der Rückstand wurde 1 h im Vakuum getrocknet, in DMF (5 mL) gelöst, die Lösung auf 0 °C gekühlt und mit Pimelinsäurechlorid (66c) (15.5 µL, 18.3 mg, 92.9 µmοl, 1.0 Äq.) sowie Pyridin (30.0 µL, 29.4 mg, 371 µmοl, 4.0 Äq.) versetzt. Nach 20 h Rühren bei Raumtemperatur wurde das Reaktionsgemisch mit DMF (5 mL) verdünnt und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S)-55c (35.9 mg, 60.7 µmοl, 65%) wurde als weißer Feststoff erhalten.

The phenol (-) - (1 S) -69 (62.0 mg, 186 μmol, 2.0 eq.) Was dissolved in 4 M HCl / ethyl acetate (4.5 ml) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was 1 h in vacuo dried, dissolved in DMF (5 mL), the solution cooled to 0 ° C and treated with pimelic acid chloride (66c) (15.5 µL, 18.3 mg, 92.9 µmole, 1.0 eq.) and pyridine (30.0 µL, 29.4 mg, 371 µmole, 4.0 Eq.) Offset. After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S) -55c (35.9 mg, 60.7 µmole, 65%) was obtained as a white solid.

HPLC (preparative):

UV (CH₃CN): λ_max (Ig ε) = 256.0 nm (4.531), 263.5 (4.514), 318.0 (4.006).¹ Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: - Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.0-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 8.9 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-33.6°\left(c=\mathrm{0.268,}\text{DMSO}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 256.0 nm (4.531), 263.5 (4.514), 318.0 (4.006). ¹

IR (KBr): ṽ (cm ^-1 ) = 3193, 2925, 1636, 1582, 1520, 1474, 1424, 1391, 1247, 1129, 1062, 851, 774, 718.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.46-1.51 (m, 2 H, 4-H ₂ ), 1.70 (m _c , 4 H, 3-H2, 5-H2), 2.53 ( m _c , 4 H, 2-H2, 6-H2), 3.78 (dd, J = 10.9, 8.1 Hz, 2 H, 2 × 10'-Ha), 3.98 (dd, J = 11.1, 3.0 Hz, 2 H , 2 × 10'-H _b ), 4.12-4.18 (m, 4 H, 2 × 1'-H, 2 × 2'-Ha), 4.34 (m _c , 2 H, 2 × 2'-Hb), 7.31 (m _c , 2 H, 2 x 7'-H), 7.48 (m _c , 2 H, 2 x 8'-H), 7.77 (d, J = 8.4 Hz, 2 H, 2 x 9'-H ), 8.00 (s, 2H, 2x4'-H), 8.08 (d, J = 8.4 Hz, 2H, 2x6'-H), 10.28 (s, 2H, 2xOH).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 24.0 (C-3, C-5), 28.3 (C-4), 35.0 (C-2, C-6), 40.7 (2 x C -1 '), 47.6 (2 × C-10'), 52.6 (2 × C-2 '), 99.7 (2 × C-4'), 113.5 (2 × C-5a '), 121.4 (2 × C -9b '), 122.3 (2 × C-9'), 122.4 (2 × C-7 '), 122.9 (2 × C-6'), 126.9 (2 × C-8 '), 129.8 (2 × C -9a '), 141.9 (2 × C-3a'), 154.0 (2 × C-5 '), 170.6 (2 × C = O).

MS (ESI): m / z (%) = 1181.5 (6) [2M-H] ^- , 589.2 (89) [MH] ^- , 553.2 (86) [M-2H-Cl] ^- . C ₃₃ H ₃₂ Cl ₂ N ₂ O ₄ (591.52). ber .: 613.1631 found: 613.1634, [M + Na] ⁺ (ESI-HRMS).

Example 8

(-) - {1,7-Bis [(1S) -1-chloromethyl-5- (β-d-galactopyranosyl) -1,2-dihydro-3H-benz [e] indol-3-yl] pentane-1 , 7-dione]} ((-) - (1'S) -53c, n = 5)

Das acetylgeschützte Galactosid (1'S)-71c (152 mg, 121 µmοl, 1.0 Äq.) wurde in absolutem Methanol (35 mL) suspendiert und bei 0 °C mit NaOMe in Methanol (90 µL einer 5.4 M Lösung, 486 µmοl, 4.0 Äq.) versetzt. Nach Erwärmen auf Raumtemperatur und 45 min Rühren bei dieser Temperatur hatte sich das Substrat vollständig gelöst. Es wurde noch weitere 1.5 h bei Raumtemperatur gerührt, die Reaktionslösung anschließend mit Wasser (3.0 mL) verdünnt und so lange saurer lonentauscher (Amberlite-IR^® 120) zugegeben, bis die Lösung neutral war (pH = 7). Das Gemisch wurde via Transferkanüle vom Ionenaustauscher abgetrennt, selbiger mit Methanol (2x6 mL) nachgewaschen, die organischen Phasen vereinigt und das Lösungsmittel im Vakuum entfernt. Der erhaltene Rückstand (142 mg) wurde in DMF (5 mL) gelöst und in aufeinanderfolgenden Injektionen (jeweils 0.3 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S)-53c (98.6 mg, 108 µmοl, 89%) wurde als hellgelber Feststoff erhalten.

The acetyl-protected galactoside (1'S) -71c (152 mg, 121 µmole, 1.0 eq.) Was suspended in absolute methanol (35 mL) and at 0 ° C with NaOMe in methanol (90 µL of a 5.4 M solution, 486 µmole, 4.0 eq .) offset. After warming to room temperature and stirring at this temperature for 45 minutes, the substrate had completely dissolved. Stirring was continued for a further 1.5 h at room temperature, then diluting the reaction solution with water (3.0 mL) and added as long acidic ion exchanger (Amberlite ^® IR-120), was until the solution neutral (pH = 7). The mixture was separated from the ion exchanger via a transfer cannula, the same was washed with methanol (2 × 6 mL), the organic phases were combined and the solvent was removed in vacuo. The residue obtained (142 mg) was dissolved in DMF (5 mL) and purified in successive injections (0.3 mL each) by means of preparative HPLC. The product (-) - (1'S) -53c (98.6 mg, 108 μmol, 89%) was obtained as a light yellow solid.

HPLC (preparative):

UV (CH3CN): λ_max (Ig ε) = 208.0 nm (4.521), 254.5 (4.798), 262.0 (4.801), 303.5 (4.159), 315.0 (4.219). Pillar: Kromasil 100 C18, 250x20 mm, 7 µm Guard column: Kromasil 100 C18, 50x20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-15 70/30 → 0/100 15-22 0/100 22-23 0/100 → 70/30 23-30 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 15.0 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-42.9°\left(c=\mathrm{0.252,}\text{DMSO}\right).$$

UV (CH3CN): λ _max (Ig ε) = 208.0 nm (4.521), 254.5 (4.798), 262.0 (4.801), 303.5 (4.159), 315.0 (4.219).

IR (KBr): ν̃ (cm ^-1 ) = 3406, 2929, 1656, 1580, 1469, 1402, 1313, 1131, 1075, 760, 664.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.49 (m _c , 2 H, 4-H2), 1.71 (m _c , 4 H, 3-H2, 5-H2), 2.55 (m _c , 4 H, 2-H2, 6-H2), 3.48-3.53 (m, 4 H, 2 × 3 "- H, 2 × 6" - Ha), 3.58-3.64 (m, 4 H, 2 × 5 '' - H, 2 × 6 '' - H _b ), 3.75-3.81 (m, 4 H, 2 × 2 '' - H, 2 × 4 '' - H), 3.86 (m _c , 2 H, 2 × 10'-Ha), 4.02 (dd, J = 11.0, 2.7 Hz, 2 H, 2 × 10'-H _b ), 4.21-4.22 (m, 4 H, 2 × 1'-H, 2 × 2 '-Ha), 4.36-4.40 (m, 2 H, 2 × 2'-H _b ), 4.51 (d, J = 4.6 Hz, 2 H, 2 × OH), 4.54 (m _c , 2 H, 2 × OH), 4.83 (d, J = 5.5 Hz, 2 H, 2 × OH), 4.94 (d, J = 7.3 Hz, 2 H, 2 × 1 '' - H), 5.27 (d, J = 5.0 Hz, 2 H, 2 × OH), 7.38 ( t, J = 7.7 Hz, 2 H, 2 × 7'-H), 7.54 (t, J = 7.5 Hz, 2 H, 2 × 8'-H), 7.85 (d, J = 8.4 Hz, 2 H, 2 x 9'-H), 8.29-8.30 (m, 4 H, 2 x 4'-H, 2 x 6'-H).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.9 (C-3, C-5), 28.3 (C-4), 35.1 (C-2, C-6), 40.7 (2 x C -1 '), 47.6 (2 × C-10'), 52.6 (2 × C-2 '), 59.6 (2 × C-6''), 67.6 (2 × C-4''), 70.4 (2 × C-2 "), 73.1 (2 × C-3"), 75.2 (2 × C-5 "), 101.2 (2 × C-4 '), 101.9 (2 × C-1") , 116.6 (2 × C-5a '), 122.4 (2 × C-9', 2 × C-9b '), 123.2 (2 × C-6', 2 × C-7 '), 127.2 (2 × C -8 '), 129.4 (2 × C-9a'), 141.7 (2 × C-3a '), 153.6 (2 × C-5'), 170.8 (2 × C = O).

MS (ESI): m / z (%) = 937.3 (69) [M + Na] ⁺ , 913.3 (100) [MH] ̅̅̅ ^- . C ₄₅ H ₅₂ Cl ₂ N ₂ O ₁₄ (915.81). ber .: 937.2688 found: 937.2691, [M + Na] ⁺ (ESI-HRMS).

Example 9

(+) - {1,5-bis [(1 S, 10R) -1- (10-chloroethyl) -5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] pentane-1,5-dione]} ((+) - (1'S, 10'R) -56a, n = 3)

The phenol (+) - (1S, 10R) -109 (62.1 mg, 179 µmοl, 2.0 eq.)

wurde in 4 M HCl / Essigester (4.5 mL) gelöst und nach 1 h Rühren bei Raumtemperatur das Lösungsmittel im Vakuum entfernt. Der Rückstand wurde 1 h im Vakuum getrocknet, in DMF (5 mL) gelöst, die Lösung auf 0 °C gekühlt und mit Glutarsäurechlorid (66a) (11.7 µL, 15.1 mg, 89.3 µmοl, 1.0 Äq.) sowie Pyridin (28.8 µL, 28.2 mg, 357 µmοl, 4.0 Äq.) versetzt. Nach 20 h Rühren bei Raumtemperatur wurde das Reaktionsgemisch mit DMF (5 mL) verdünnt und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (+)-(1'S,10'R)-56a (42.1 mg, 71.2 µmοl, 80%) wurde als weißer Feststoff erhalten.

was dissolved in 4 M HCl / ethyl acetate (4.5 mL) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was dried in vacuo for 1 h, dissolved in DMF (5 mL), the solution cooled to 0 ° C and treated with glutaric acid chloride (66a) (11.7 µL, 15.1 mg, 89.3 µmοl, 1.0 eq.) And pyridine (28.8 µL, 28.2 mg, 357 µmole, 4.0 eq.) Added. After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (+) - (1'S, 10'R) -56a (42.1 mg, 71.2 µmole, 80%) was obtained as a white solid.

HPLC (preparative):

UV (CH3CN): λ_max (Ig ε) = 206.5 nm (4.576), 222.5 (4.410), 257.0 (4.948), 265.0 (4.977), 320.5 (4.355). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.0-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 8.7 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+23.2°\left(c=\mathrm{0.25,}\text{DMSO}\right).$$

UV (CH3CN): λ _max (Ig ε) = 206.5 nm (4.576), 222.5 (4.410), 257.0 (4.948), 265.0 (4.977), 320.5 (4.355).

IR (KBr): ν̃ (cm ^-1 ) = 3199, 1632, 1684, 1522, 1453, 1415, 1394, 1263, 1158, 1134, 1074, 1024, 859, 757.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.64 (d, J = 6.6 Hz, 6 H, 2 × 10'-CH ₃ ), 1.99 (m _c , 2 H, 3-H2), 2.58-2.63 (m, 2 H, 2-Ha, 4-Ha), 2.70-2.75 (m, 2 H, 2-Hb, 4-Hb), 4.11-4.12 (m, 2 H, 2 × 1'- H), 4.24-4.29 (m, 4H, 2 × 2'-H ₂ ), 4.78 (m _c , 2 H, 2 × 10'-H), 7.31 (t, J = 7.6 Hz, 2 H, 2 × 7'-H), 7.47 (t, J = 7.5 Hz, 2 H, 2 × 8'-H), 7.82 (d, J = 8.4 Hz, 2 H, 2 × 9'-H), 8.01 (s , 2H, 2x4'-H), 8.10 (d, J = 8.4 Hz, 2H, 2x6'-H), 10.25 (s, 2H, 2xOH).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 19.6 (C-3), 23.2 (2 × C-11 '), 34.5 (C-2, C-4), 45.2 (2 × C- 1 '), 49.6 (2 × C-2'), 61.5 (2 × C-10 '), 99.7 (2 × C-4'), 114.7 (2 × C-5a '), 121.5 (2 × C- 9b '), 122.3 (2 × C-7'), 122.5 (2 × C-9 '), 122.9 (2 × C-6'), 126.7 (2 × C-8 '), 129.7 (2 × C- 9a '), 141.8 (2 × C-3a'), 153.7 (2 × C-5 '), 170.1 (2 × C = O).

MS (ESI): m / z (%) = 1205.3 (100) [2M + Na] +, 907.2 (23) [3M + 2Na] ²⁺ , 613.1 (43) [M + Na] ⁺ , 589.2 (100) [MH] ^- . C ₃₃ H ₃₂ Cl ₂ N ₂ O ₄ (591.52). ber .: 589.1666 found: 589.1694, [MH] ^- (ESI-HRMS).

Example 10

(-) - {1,5-Bis [(1S, 10R) -1- (10-chloroethyl) -5- (β-D-galactopyranosyl) -1,2-dihydro-3H-benz [e] indole-3 -yl] pentane-1,5-dione]} ((-) - (1'S, 10'R) -54a, n = 3)

Ein Gemisch (96.3 mg) aus dem acetylgeschützten Galactosid (1'S,10'R)-72a und einem nicht eindeutig identifizierten Nebenprodukt wurde in absolutem Methanol (30 mL) suspendiert und bei 0 °C mit NaOMe in Methanol (57 µL einer 5.4 M Lösung, 308 µmol) versetzt. Nach Erwärmen auf Raumtemperatur und 30 min Rühren bei dieser Temperatur hatte sich das Substanzgemisch vollständig gelöst. Es wurde noch weitere 1.5 h bei Raumtemperatur gerührt, die Reaktionslösung anschließend mit Wasser (3.0 mL) verdünnt und so lange saurer lonentauscher (Amberlite-IR^® 120) zugegeben, bis die Lösung neutral war (pH = 7). Das Gemisch wurde via Transferkanüle vom Ionenaustauscher abgetrennt, selbiger mit Methanol (2x6 mL) nachgewaschen, die organischen Phasen vereinigt und das Lösungsmittel im Vakuum entfernt. Der erhaltene Rückstand (68.2 mg) wurde in DMF (5 mL) gelöst und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S,10'R)-54a (44.7 mg, 48.8 µmοl, 30% über 3 Stufen und zweimaliger Reinigung mittels präparativer HPLC) wurde als hellgelber Feststoff erhalten.

A mixture (96.3 mg) of the acetyl-protected galactoside (1'S, 10'R) -72a and an ambiguous by-product was suspended in absolute methanol (30 mL) and at 0 ° C with NaOMe in methanol (57 µL of a 5.4 M solution , 308 µmol). After warming to room temperature and stirring at this temperature for 30 minutes, the substance mixture had completely dissolved. Stirring was continued for a further 1.5 h at room temperature, then diluting the reaction solution with water (3.0 mL) and added as long acidic ion exchanger (Amberlite ^® IR-120), was until the solution neutral (pH = 7). The mixture was separated from the ion exchanger via a transfer cannula, the same was washed with methanol (2 × 6 mL), the organic phases were combined and the solvent was removed in vacuo. The residue obtained (68.2 mg) was dissolved in DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S, 10'R) -54a (44.7 mg, 48.8 μmol, 30% over 3 steps and two purifications by means of preparative HPLC) was obtained as a pale yellow solid.

HPLC (preparative):

UV (CH3CN): λ_max (Ig ε) = 209.0 nm (4.221), 254.5 (4.531), 263.0 (4.489), 306.5 (3.945), 316.5 (4.021). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-15 70/30 → 0/100 15-22 0/100 22-23 0/100 → 70/30 23-30 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 12.4 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-7.2°\left(c=\mathrm{0.25,}\text{DMSO}\right).$$

UV (CH3CN): λ _max (Ig ε) = 209.0 nm (4.221), 254.5 (4.531), 263.0 (4.489), 306.5 (3.945), 316.5 (4.021).

IR (KBr): ν̃ (cm ^-1 ) = 3406, 2892, 1627, 1581, 1517, 1470, 1402, 1209, 1135, 1075, 854, 760, 618.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.66 (d, J = 6.3 Hz, 6 H, 2 × 10'-CH ₃ ), 2.00 (m _c , 2 H, 3-H2), 2.60-2.68 (m, 2 H, 2-Ha, 4-Ha), 2.69-2.76 (m, 2 H, 2-Hb, 4-Hb), 3.45-3.50 (m, 2 H, 2 × 3 '' -H), 3.52-3.66 (m, 6 H, 2 × 5 "-H, 2 × 6" _{-H 2} ), 3.75-3.82 (m, 4 H, 2 × 2 "-H, 2 × 4 "-H), 4.18-4.22 (m, 2H, 2 × 1'-H), 4.26-4.36 (m, 4H, 2 × 2'-H ₂ ), 4.52 (d, J = 4.6 Hz , 2 H, 2 x OH), 4.55 (m _c , 2 H, 2 x OH), 4.80-4.87 (m, 4 H, 2 x 10'-H, 2 x OH), 4.92 (d, J = 7.5 Hz, 2 H, 2 × 1 '' - H), 5.27 (d, J = 4.9 Hz, 2 H, 2 × OH), 7.38 (t, J = 7.5 Hz, 2 H, 2 × 7'-H) , 7.53 (t, J = 7.5 Hz, 2 H, 2 × 8'-H), 7.90 (d, J = 8.4 Hz, 2 H, 2 × 9'-H), 8.31-8.32 (m, 4 H, 2x4'-H, 2x6'-H).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 19.4 (C-3), 23.1 (2 × C-11 '), 34.5 (C-2, C-4), 45.3 (2 × C- 1 '), 49.5 (2 × C-2'), 59.7 (2 × C-6 ''), 61.3 (2 × C-10 '), 67.6 (2 × C-4''), 70.5 (2 × C-2 "), 73.2 (2 × C-3"), 75.3 (2 × C-5 "), 101.3 (2 × C-4 '), 102.1 (2 × C-1"), 117.8 (2 × C-5a '), 122.6 (2 signals) (2 × C-9', 2 × C-9b '), 123.2 (2 × C-7'), 123.3 (2 × C-6 ') , 127.1 (2 × C-8 '), 129.5 (2 × C-9a'), 141.7 (2 × C-3a '), 153.6 (2 × C-5'), 170.5 (2 × C = O).

MS (ESI): m / z (%) = 1394.4 (8) [3M + 2Na] ²⁺ , 937.3 (100) [M + Na] ⁺ . C ₄₅ H ₅₂ Cl ₂ N ₂ O ₁₄ (915.81). ber .: 937.2685 found: 937.2688, [M + Na] ⁺ (ESI-HRMS).

Example 11

(+) - {1,6-Bis [(1S, 10R) -1- (10-chloroethyl) -5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] hexane-1 , 6-dione]} ((+) - (1'S, 10'R) -56b, n = 4)

Das Phenol (+)-(1 S,10R)-109 (59.7 mg, 172 µmοl, 2.0 Äq.) wurde in 4 M HCl / Essigester (4.5 mL) gelöst und nach 1 h Rühren bei Raumtemperatur das Lösungsmittel im Vakuum entfernt. Der Rückstand wurde 1 h im Vakuum getrocknet, in DMF (5 mL) gelöst, die Lösung auf 0 °C gekühlt und mit Adipinsäurechlorid (66b) (12.5 µL, 15.7 mg, 85.8 µmοl, 1.0 Äq.) sowie Pyridin (27.7 µL, 27.2 mg, 343 µmοl, 4.0 Äq.) versetzt. Nach 20 h Rühren bei Raumtemperatur wurde das Reaktionsgemisch mit DMF (5 mL) verdünnt und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (+)-(1'S,10'R)-56b (39.4 mg, 65.1 µmοl, 76%) wurde als weißer Feststoff erhalten.

The phenol (+) - (1 S, 10R) -109 (59.7 mg, 172 μmol, 2.0 eq.) Was dissolved in 4 M HCl / ethyl acetate (4.5 ml) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was dried in vacuo for 1 h, dissolved in DMF (5 mL), the solution was cooled to 0 ° C and treated with adipic acid chloride (66b) (12.5 µL, 15.7 mg, 85.8 µmοl, 1.0 eq.) And pyridine (27.7 µL, 27.2 mg, 343 µmole, 4.0 eq.). After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (+) - (1'S, 10'R) -56b (39.4 mg, 65.1 µmole, 76%) was obtained as a white solid.

HPLC (preparative):

UV (CH3CN): λ_max (Ig ε) = 196.5 nm (4.295), 256.5 (4.171), 264.5 (4.177), 320.0 (3.541). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: - Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.0-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 8.8 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+8.2°\left(c=\mathrm{0.269,}\text{DMSO}\right).$$

UV (CH3CN): λ _max (Ig ε) = 196.5 nm (4.295), 256.5 (4.171), 264.5 (4.177), 320.0 (3.541).

IR (KBr): ν̃ (cm ^-1 ) = 3210, 2924, 1665, 1633, 1582, 1522, 1394, 1253, 1135, 861, 750.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.63 (d, J = 6.6 Hz, 6 H, 2 x 10'-CH ₃ ), 1.76 (m _c , 4 H, 3-H2, 4 -H2), 2.53-2.59 (m, 2H, 2-Ha, 5-Ha), 2.60-2.66 (m, 2H, 2-Hb, 5-Hb), 4.10 (m _c , 2H, 2x 1'-H), 4.26-4.27 (m, 4 H, 2 × 2'-H2), 4.77 (m _c , 2 H, 2 × 10'-H), 7.30 (t, J = 7.6 Hz, 2 H , 2 × 7'-H), 7.46 (t, J = 7.5 Hz, 2 H, 2 × 8'-H), 7.81 (d, J = 8.6 Hz, 2 H, 2 × 9'-H), 7.99 (s, 2H, 2x4'-H), 8.09 (d, J = 8.4 Hz, 2H, 2x6'-H), 10.22 (s, 2H, 2xOH).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.2 (2 × C-11 '), 23.8 (C-3, C-4), 35.0 (C-2, C-5), 45.2 ( 2 × C-1 '), 49.5 (2 × C-2'), 61.5 (2 × C-10 '), 99.7 (2 × C-4'), 114.6 (2 × C-5a '), 121.5 ( 2 × C-9b '), 122.2 (2 × C-7'), 122.5 (2 × C-9 '), 122.9 (2 × C-6'), 126.6 (2 × C-8 '), 129.7 ( 2 × C-9a '), 141.8 (2 × C-3a'), 153.7 (2 × C-5 '), 170.2 (2 × C = O).

MS (ESI): m / z (%) = 627.2 (26) [M + Na] ⁺ , 603.2 (80) [MH] ^- , 567.3 (100) [M-2H-Cl] ^- . C ₃₄ H ₃₄ Cl ₂ N ₂ O ₄ (605.55). ber .: 603.1823 found: 603.1837, [MH] ^- (ESI-HRMS).

Example 12

(-) - {1,6-bis [(1S, 10R) -1- (10-chloroethyl) -5- (β-d-galactopyranosyl) -1,2-dihydro-3H-benz [e] indole-3 -yl] hexane-1,6-dione]} ((-) - (1'S, 10'R) -54b, n = 4)

Ein Gemisch (97.3 mg) aus dem acetylgeschützten Galactosid (1'S,10'R)-72b und einem nicht eindeutig identifizierten Nebenprodukt wurde in absolutem Methanol (30 mL) suspendiert und bei 0 °C mit NaOMe in Methanol (57 µL einer 5.4 M Lösung, 307 µmοl) versetzt. Nach Erwärmen auf Raumtemperatur und 30 min Rühren bei dieser Temperatur hatten sich das Substanzgemisch vollständig gelöst. Es wurde noch weitere 1.5 h bei Raumtemperatur gerührt, die Reaktionslösung anschließend mit Wasser (3.0 mL) verdünnt und so lange saurer lonentauscher (Amberlite-IR^® 120) zugegeben, bis die Lösung neutral war (pH = 7). Das Gemisch wurde via Transferkanüle vom Ionenaustauscher abgetrennt, selbiger mit Methanol (2x6 mL) nachgewaschen, die organischen Phasen vereinigt und das Lösungsmittel im Vakuum entfernt. Der erhaltene Rückstand (75.9 mg) wurde in DMF (5 mL) gelöst und in aufeinanderfolgenden Injektionen (0.3 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S,10'R)-54b (43.9 mg, 47.2 µmοl, 27% über 3 Stufen) wurde als hellgelber Feststoff erhalten.

A mixture (97.3 mg) of the acetyl-protected galactoside (1'S, 10'R) -72b and a not clearly identified by-product was suspended in absolute methanol (30 mL) and at 0 ° C with NaOMe in methanol (57 µL of a 5.4 M solution , 307 µmole) offset. After warming to room temperature and stirring at this temperature for 30 minutes, the substance mixture had completely dissolved. Stirring was continued for a further 1.5 h at room temperature, then diluting the reaction solution with water (3.0 mL) and added as long acidic ion exchanger (Amberlite ^® IR-120), was until the solution neutral (pH = 7). The mixture was separated from the ion exchanger via a transfer cannula, the same was washed with methanol (2 × 6 mL), the organic phases were combined and the solvent was removed in vacuo. The residue obtained (75.9 mg) was dissolved in DMF (5 mL) and purified in successive injections (0.3 mL) by means of preparative HPLC. The product (-) - (1'S, 10'R) -54b (43.9 mg, 47.2 μmol, 27% over 3 steps) was obtained as a light yellow solid.

HPLC (preparative):

UV (CH3CN): λ_max (Ig ε) = 199.0 nm (3.990), 255.0 (3.268), 262.5 (3.255), 304.0 (2.634), 313.5 (2.649). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-15 70/30 → 0/100 15-22 0/100 22-23 0/100 → 70/30 23-30 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 13.4 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-13.0°\left(c=\mathrm{0.269,}\text{DMSO}\right).$$

UV (CH3CN): λ _max (Ig ε) = 199.0 nm (3.990), 255.0 (3.268), 262.5 (3.255), 304.0 (2.634), 313.5 (2.649).

IR (KBr): ν̃ (cm ^-1 ) = 3406, 2925, 1651, 1579, 1469, 1414, 1210, 1139, 1076, 857, 761.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.65 (d, J = 6.7 Hz, 6 H, 2 × 10'-CH ₃ ), 1.77 (m _c , 4 H, 3-H2, 4 -H2), 2.63 (m _c , 4 H, 2-H2, 5-H2), 3.47-3.55 (m, 4 H, 2 × 3 "- H, 2 × 6" - Ha), 3.57-3.64 (m, 4 H, 2 x 5 '' - H, 2 x 6 '' - H _b), 3.74-3.81 (m, 4 H, 2 x 2 "'- H, 2 x 4''- H), 4:16 to 4:21 (m, 2 H, 2 x 1'-H), 4:28 to 4:34 (m, 4 H, 2 × 2'-H _2), 4:51 (d, J = 4.0 Hz, 2 H, 2 x OH ), 4.54 (mc, 2 H, 2 × OH), 4.80-4.85 (m, 4 H, 2 × 10'-H, 2 × OH), 4.92 (d, J = 7.5 Hz, 2 H, 2 × 1 '' -H), 5.26 (S _br , 2 H, 2 × OH), 7.37 (t, J = 7.6 Hz, 2 H, 2 × 7'-H), 7.52 (t, J = 7.5 Hz, 2 H. , 2 × 8'-H), 7.88 (d, J = 8.4 Hz, 2 H, 2 × 9'-H), 8.29-8.30 (m, 4 H, 2 × 4'-H, 2 × 6'- H).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.2 (2 × C-11 '), 23.7 (C-3, C-4), 35.0 (C-2, C-5), 45.3 ( 2 × C-1 '), 49.5 (2 × C-2'), 59.6 (2 × C-6 ''), 61.3 (2 × C-10 '), 67.6 (2 × C-4''), 70.4 (2 × C-2 "), 73.1 (2 × C-3"), 75.2 (2 × C-5 "), 101.1 (2 × C-4 '), 102.0 (2 × C-1 ''), 117.6 (2 × C-5a '), 122.4 (2 × C-9b'), 122.5 (2 × C-9 '), 123.0 (2 × C-7'), 123.2 (2 × C- 6 '), 126.9 (2 × C-8'), 129.3 (2 × C-9a '), 141.6 (2 × C-3a'), 153.4 (2 × C-5 '), 170.4 (2 × C = O).

MS (ESI): m / z (%) = 1417.4 (4) [3M + 2Na] ²⁺ , 951.3 (100) [M + Na] ⁺ . C ₄₆ H ₅₄ Cl ₂ N ₂ O ₁₄ (929.83). ber .: 951.2844 found: 951.2839, [M + Na] ⁺ (ESI-HRMS).

Example 13

(-) - {1,7-Bis [(1 R, 10S) -1- (10-chloroethyl) -5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] heptane 1,7-dione]} ((-) - (1'R, 10'S) -56c, n = 5)

Das Phenol (-)-(1R,10S)-109 (41.3 mg, 119 µmοl, 2.0 Äq.) wurde in 4 M HCl / Essigester (3.0 mL) gelöst und nach 1 h Rühren bei Raumtemperatur das Lösungsmittel im Vakuum entfernt. Der Rückstand wurde 1 h im Vakuum getrocknet, in DMF (3.5 mL) gelöst, die Lösung auf 0 °C gekühlt und mit Pimelinsäurechlorid (66c) (9.9 µL, 11.7 mg, 59.4 µmοl, 1.0 Äq.) sowie Pyridin (19.2 µL, 18.8 mg, 237 µmοl, 4.0 Äq.) versetzt. Nach 20 h Rühren bei Raumtemperatur wurde das Reaktionsgemisch mit DMF (3.5 mL) verdünnt und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (-)-(1'S,10'R)-56c (28.5 mg, 46.0 µmοl, 77%) wurde als weißer Feststoff erhalten.

The phenol (-) - (1R, 10S) -109 (41.3 mg, 119 μmole, 2.0 eq.) Was dissolved in 4 M HCl / ethyl acetate (3.0 ml) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was dried in vacuo for 1 h, dissolved in DMF (3.5 mL), the solution was cooled to 0 ° C and treated with pimelic acid chloride (66c) (9.9 µL, 11.7 mg, 59.4 µmοl, 1.0 eq.) And pyridine (19.2 µL, 18.8 mg, 237 µmole, 4.0 eq.) Added. After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (3.5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S, 10'R) -56c (28.5 mg, 46.0 µmole, 77%) was obtained as a white solid.

HPLC (preparative):

UV (CH₃CN): λ_max (Ig ε) = 206.5 nm (4.587), 222.5 (4.409), 256.5 (4.950), 264.0 (4.950), 320.0 (4.338). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.0-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 9.1 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-10.7°\left(c=\mathrm{0.252,}\text{DMSO}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 206.5 nm (4.587), 222.5 (4.409), 256.5 (4.950), 264.0 (4.950), 320.0 (4.338).

IR (KBr): ν̃ (cm ^-1 ) = 3191, 2930, 1631, 1584, 1522, 1454, 1425, 1394, 1322, 1263, 1157, 1134, 1071, 1024, 860, 757.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.50 (m _c , 2 H, 4-H ₂ ), 1.62 (d, J = 6.7 Hz, 6 H, 2 × 10'-CH3), 1.71 (m _c , 4 H, 3-H2, 5-H2), 2.54 (m _c , 4 H, 2-H2, 6-H2), 4.09 (m _c , 2 H, 2 × 1'-H), 4.24-4.25 (m, 4 H, 2 × 2'-H ₂ ), 4.76 (m _c , 2 H, 2 × 10'-H), 7.30 (t, J = 7.6 Hz, 2 H, 2 × 7 ' -H), 7.46 (t, J = 7.6 Hz, 2H, 2 × 8'-H), 7.81 (d, J = 8.5 Hz, 2H, 2 × 9'-H), 7.99 (s, 2H , 2x4'-H), 8.09 (d, J = 8.5 Hz, 2H, 2x6'-H), 10.22 (s, 2H, 2xOH).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.2 (2 × C-11 '), 24.1 (C-3, C-5), 28.4 (C-4), 35.1 (C-2, C-6), 45.2 (2 × C-1 '), 49.6 (2 × C-2'), 61.5 (2 × C-10 '), 99.7 (2 × C-4'), 114.6 (2 × C -5a '), 121.5 (2 × C-9b'), 122.2 (2 × C-7 '), 122.5 (2 × C-9'), 122.9 (2 × C-6 '), 126.6 (2 × C -8 '), 129.7 (2 × C-9a'), 141.8 (2 × C-3a '), 153.7 (2 × C-5'), 170.2 (2 × C = O).

MS (ESI): m / z (%) = 1261.4 (43) [2M + Na] +, 641.2 (43) [M + Na] ⁺ , 617.2 (74) [MH] -, 581.3 (80) [M- 2H-Cl] ^- . C ₃₅ H ₃₆ Cl ₂ N ₂ O ₄ (619.58). ber .: 641.1944 found: 641.1956, [M + Na] ⁺ (ESI-HRMS).

Example 14

(+) - {1,7-Bis [(1 S, 10R) -1- (10-chloroethyl) -5-hydroxy-1,2-dihydro-3H-benz [e] indol-3-yl] heptane 1,7-dione]} ((+) - (1'S, 10'R) -56c, n = 5)

Das Phenol (+)-(1S,10R)-109 (59.4 mg, 171 µmοl, 2.0 Äq.) wurde in 4 M HCl / Essigester (4.5 mL) gelöst und nach 1 h Rühren bei Raumtemperatur das Lösungsmittel im Vakuum entfernt. Der Rückstand wurde 1 h im Vakuum getrocknet, in DMF (5 mL) gelöst, die Lösung auf 0 °C gekühlt und mit Pimelinsäurechlorid (66c) (14.2 µL, 16.8 mg, 85.4 µmοl, 1.0 Äq.) sowie Pyridin (27.6 µL, 27.0 mg, 342 µmοl, 4.0 Äq.) versetzt. Nach 20 h Rühren bei Raumtemperatur wurde das Reaktionsgemisch mit DMF (5 mL) verdünnt und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (+)-(1'S,10'R)-56c (41.6 mg, 67.1 µmοl, 79%) wurde als weißer Feststoff erhalten.

The phenol (+) - (1S, 10R) -109 (59.4 mg, 171 μmol, 2.0 eq.) Was dissolved in 4 M HCl / ethyl acetate (4.5 ml) and, after stirring for 1 h at room temperature, the solvent was removed in vacuo. The residue was dried in vacuo for 1 h, dissolved in DMF (5 mL), the solution was cooled to 0 ° C and treated with pimelic acid chloride (66c) (14.2 µL, 16.8 mg, 85.4 µmοl, 1.0 eq.) And pyridine (27.6 µL, 27.0 mg, 342 µmole, 4.0 eq.). After stirring at room temperature for 20 h, the reaction mixture was diluted with DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (+) - (1'S, 10'R) -56c (41.6 mg, 67.1 µmole, 79%) was obtained as a white solid.

HPLC (analytical):

Pillar: Kromasil 100 C18, 250 × 4.0 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6 30/70 → 0/100 6-9.5 0/100 9.5-10.0 0/100 → 30/70 10.0-15 30/70 Flow: 0.8 mL min ^-1 tR: 9.9 min

HPLC (preparative):

UV (CH3CN): λ_max (Ig ε) = 206.5 nm (4.566), 222.5 (4.401), 256.5 (4.953), 264.0 (4.952), 320.0 (4.337). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-6.0 30/70 → 0/100 6.0-8.5 0/100 8.5-9.5 0/100 → 30/70 9.5-15 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 9.1 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=+11.6°\left(c=\mathrm{0.251,}\text{DMSO}\right).$$

UV (CH3CN): λ _max (Ig ε) = 206.5 nm (4.566), 222.5 (4.401), 256.5 (4.953), 264.0 (4.952), 320.0 (4.337).

IR (KBr): ν̃ (cm ^-1 ) = 3184, 2931, 1631, 1584, 1522, 1454, 1424, 1393, 1262, 1157, 1134, 1066, 1024, 860, 756, 632, 413.

¹ H-NMR (600 MHz, CDCl ₃ ): δ = 1.31 (d, J = 6.6 Hz, 6 H, 2 × 10'-CH ₃ ), 1.68-1.77 (m, 4 H, 3-Ha, 4- H2, 5-Ha), 2.07 (m _c , 2H, 3-Hb, 5-Hb), 2.44-2.50 (m, 4H, 2x1'-H, 2-Ha, 6-Ha), 2.56 -2.61 (m, 2H, 2-Hb, 6-Hb), 3.20 (t, J = 9.6 Hz, 2H, 2 × 2'-Ha), 3.77 (dd, J = 10.4, 1.5 Hz, 2H , 2 × 2'-Hb), 4.18 (mc, 2 H, 2 × 10'-H), 7.27 (d, J = 8.2 Hz, 2 H, 2 × 9'-H), 7.34 (t, J = 7.4 Hz, 2 H, 2 × 7'-H), 7.44 (t, J = 7.4 Hz, 2 H, 2 × 8'-H), 8.05 (s, 2 H, 2 × 4'-H), 8.29 (d, J = 8.3 Hz, 2 H, 2 x 6'-H), 10.65 (s, 2 H, 2 x OH).

¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 23.6 (C-3, C-5), 23.8 (2 × C-11 '), 28.6 (C-4), 36.1 (C-2, C- 6), 45.7 (2 × C-1 '), 51.1 (2 × C-2'), 60.0 (2 × C-10 '), 100.7 (2 × C-4'), 114.7 (2 × C-5a '), 122.3 (2 × C-7'), 122.4 (2 signals) (2 × C-9 ', 2 × C-9b'), 123.8 (2 × C-6 '), 126.0 (2 × C- 8 '), 129.7 (2 × C-9a'), 141.3 (2 × C-3a '), 154.4 (2 × C-5'), 171.7 (2 × C = O).

MS (ESI): m / z (%) = 1261.4 (41) [2M + Na] +, 641.2 (57) [M + Na] ⁺ , 617.2 (52) [MH] -, 581.3 (100) [M- 2H-Cl] ^- . C ₃₅ H ₃₆ Cl ₂ N ₂ O ₄ (619.38). ber .: 619.2125 found: 619.2125, [M + H] ⁺ (ESI-HRMS).

Example 15

(-) - {1,7-Bis [(1S, 10R) -1- (10-chloroethyl) -5- (β-D-galactopyranosyl) -1,2-dihydro-3H-benz [e] indole-3 -yl] heptane-1,7-dione]} ((-) - (1'S, 10'R) -54c, n = 5)

Ein Gemisch (126 mg) aus dem acetylgeschützten Galactosid (1'S,10'R)-72c und einem nicht eindeutig identifizierten Nebenprodukt wurde in absolutem Methanol (35 mL) suspendiert und bei 0 °C mit NaOMe in Methanol (73 µL einer 5.4 M Lösung, 394 µmοl versetzt. Nach Erwärmen auf Raumtemperatur und 30 min Rühren bei dieser Temperatur hatte sich das Substanzgemisch vollständig gelöst. Es wurde noch weitere 1.5 h bei Raumtemperatur gerührt, die Reaktionslösung anschließend mit Wasser (3.0 mL) verdünnt und so lange saurer lonentauscher (Amberlite-IR^® 120) zugegeben, bis die Lösung neutral war (pH = 7). Das Gemisch wurde via Transferkanüle vom Ionenaustauscher abgetrennt, selbiger mit Methanol (2x6 mL) nachgewaschen, die organischen Phasen vereinigt und das Lösungsmittel im Vakuum entfernt. Der erhaltene Rückstand (114 mg) wurde in DMF (5 mL) gelöst und in aufeinanderfolgenden Injektionen (jeweils 0.4 mL) mittels präparativer HPLC gereinigt. Das Produkt (- )-(1'S,10'R)-54c (65.8 mg, 69.7 µmοl, 35% über 3 Stufen) wurde als hellgelber Feststoff erhalten.

A mixture (126 mg) of the acetyl-protected galactoside (1'S, 10'R) -72c and a non-clearly identified by-product was suspended in absolute methanol (35 mL) and at 0 ° C with NaOMe in methanol (73 µL of a 5.4 M solution After warming to room temperature and stirring for 30 min at this temperature, the substance mixture had completely dissolved. Stirring was continued for a further 1.5 h at room temperature, the reaction solution was then diluted with water (3.0 ml) and acidic ion exchanger (Amberlite -IR ^® 120) was added until the solution was neutral (pH = 7). The mixture was separated from the ion exchanger via transfer cannula, washed with methanol (2 × 6 mL), the organic phases combined and the solvent removed in vacuo. The residue obtained (114 mg) was dissolved in DMF (5 mL) and purified in successive injections (0.4 mL each) by means of preparative HPLC. The product (-) - (1'S, 10'R) -54c (65.8 mg, 69.7 µmοl, 35% over 3 steps) was obtained as a light yellow solid.

HPLC (analytical):

Pillar: Kromasil 100 C18, 250 × 4.0 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-15 70/30 → 0/100 15-22 0/100 22-23 0/100 → 70/30 23-30 30/70 Flow: 0.8 mL min ^-1 tR: 15.3 min

HPLC (preparative):

UV (CH₃CN): λ_max (Ig ε) = 206.5 nm (4.088), 255.0 (4.275), 262.5 (4.287), 304.5 (3.640), 315.0 (3.700). Pillar: Kromasil 100 C18, 250 x 20 mm, 7 µm Guard column: Kromasil 100 C18, 50 x 20 mm, 5 µm Gradient: Time [min] H ₂ O / MeOH 0 30/70 0-22.5 70/30 → 17.5 / 82.5 22.5-23.5 17.5 / 82.5 → 70/30 23.5-30 30/70 Flow: 16 mL min ^-1 λ: 254 nm tR: 22.8 min $${\left[\mathrm{\alpha }\right]}_{\text{D.}}^{\mathrm{20th}}=-16.5°\left(c=\mathrm{0.254,}\text{DMSO}\right).$$

UV (CH ₃ CN): λ _max (Ig ε) = 206.5 nm (4.088), 255.0 (4.275), 262.5 (4.287), 304.5 (3.640), 315.0 (3.700).

IR (KBr): ν̃ (cm ^-1 ) = 3406, 2927, 1656, 1580, 1470, 1412, 1074, 758.

¹ H-NMR (600 MHz, DMSO-d ₆ ): δ = 1.50 (m _c , 2 H, 4-H ₂ ), 1.65 (d, J = 6.5 Hz, 6 H, 2 × 10'-CH ₃ ) , 1.68-1.74 (m, 4 H, _{3-H2, 5-H2),} 2:57 (m _c, 4 H, _{2-H2, 6-H2),} 3.48-3.65 (m, 8 H, 2 3 × 3 "-H, 2 × 5" _{-H, 2 × 6 "-H 2} ), 3.75-3.81 (m, 4 H, 2 × 2" -H, 2 × 4 "-H) , 4.18 (m _c , 2 H, 2 x 1'-H), 4.27-4.33 (m, 4 H, 2 x 2'-H ₂ ), 4.51 (d, J = 4.5 Hz, 2 H, 2 x OH ), 4.55 (m _c , 2 H, 2 × OH), 4.77-4.86 (m, 4 H, 2 × 10'-H, 2 × OH), 4.93 (d, J = 7.6 Hz, 2 H, 2 × 1 '' - H), 5.26 (d, J = 4.9 Hz, 2 H, 2 × OH), 7.38 (t, J = 7.6 Hz, 2 H, 2 × 7'-H), 7.52 (t, J = 7.5 Hz, 2 H, 2 × 8'-H), 7.89 (d, J = 8.5 Hz, 2 H, 2 × 9'-H), 8.30-8.31 (m, 4 H, 2 × 4'-H, 2 x 6'-H).

¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ = 23.2 (2 × C-11 '), 24.0 (C-3, C-5), 28.3 (C-4), 35.1 (C-2, C-6), 45.3 (2 × C-1 '), 49.5 (2 × C-2'), 59.7 (2 × C-6 ''), 61.3 (2 × C-10 '), 67.6 (2 × C-4 "), 70.4 (2 × C-2"), 73.1 (2 × C-3 "), 75.2 (2 × C-5"), 101.2 (2 × C-4 '), 102.0 (2 × C-1 ''), 117.6 (2 × C-5a '), 122.4 (2 × C-9b'), 122.5 (2 × C-9 '), 123.0 (2 × C-7') , 123.2 (2 × C-6 '), 126.9 (2 × C-8'), 129.4 (2 × C-9a '), 141.6 (2 × C-3a'), 153.4 (2 × C-5 ') , 170.4 (2 x C = O).

MS (ESI): m / z (%) = 965.3 (100) [M + Na] ⁺ , 941.4 (100) [MH] ̅̅̅ ^- . C ₄₇ H ₅₆ Cl ₂ N ₂ O ₁₄ (943.86). ber .: 965.3001 found: 965.3036, [M + Na] ⁺ (ESI-HRMS).

Example 16

Cytotoxicity test

In order to examine the cytotoxic effect of the newly synthesized substances on adherently growing human tumor cells, a clonogenicity test was carried out, which is based on the HTCFA test and reflects the ability of individual cells to proliferate. The adherently growing human bronchial carcinoma of line A549 served as the cell line. The tumor cells were seeded from a stock culture in which they were permanently kept in a culture medium (DMEM with addition of 10% fetal calf serum, 44 mM NaHCO3 and 4 mM glutamine) at 37 ° C and 7.5% CO2 gassing in air, in concentrations from 10 ² to 10 ⁴ cells in 6 well multi-dishes. After 24 hours, the culture medium was removed, the cells were washed once with the serum-free incubation medium UltraCulture and the cells were then incubated in this medium for 24 hours with ten to twelve different concentrations (three orders of magnitude) of the substance to be tested. This was freshly prepared beforehand in a DMSO stock solution and diluted with the incubation medium, so that a DMSO concentration of 1% was ultimately present in the wells. After removing the test substance and a further wash with the culture medium, the remaining cells were cultivated in normal culture medium for 12 days. This was then removed, the clones formed were dried, stained with Löffler's methylene blue and macroscopically counted from a clone size of approx. 500 μm. The relative cloning rates were determined using the formula given in Figure B.14, the number of clones in the control experiment being obtained from untreated cells and being set equal to 100%. The cytotoxic compounds were released from the prodrugs by adding 4.0 U / mL ß-D galactosidase. Each test series was usually carried out at least three times as a duplicate in order to obtain reproducible test results. The results are shown in Table 1. Table 1 Connection number / C ₅₀ Selectivity Q / C ₅₀ 53a 0.15 pmol / L (mE) 146,000 pmol / L (oE) 970,000 53b 5.8 pmol / L (mE) 129,000 pmol / L (oE) 22,000 53c 1.3 pmol / L (mE) 180,000 pmol / L (oE) 140,000 54a 150 pmol / L (mE) 326 000 pmol / l (oE) 2,200 54b 2860 pmol / L (mE) 5,300,000 pmol / L (oE) 1,900 54c 260 pmol / L (mE) 38,300,000 pmol / L (oE) 150,000 55a 0.11 pmol / L 55c 1.0 pmol / L 56a 160 pmol / L 56b 3140 pmol / L 56c 170 pmol / L
(mE) = with enzyme, (oE) = without enzyme

Example 17

In vivo evaluation of the compounds according to the invention in an orthotopic breast tumor SCID mouse model using the ADEPT concept

The female SCID mice are anesthetized by peritoneal injection of 75 mg / g ketamine hydrochloride with 15 mg / g xylazine before the tumor cells are planted. 1 × 10 ⁶ MDR-MB-231 cells (estrogen-independent human breast cancer cell line) suspended in 5 ml of sterile PBS are then implanted. After implantation, the mice are examined daily for loss of body weight, general condition and tumor formation. On day 21, the animals are randomly divided into groups of 5 to 12 mice (control, antibody-enzyme only, prodrug only and ADEPT therapy). On days 22 and 30, the mice are conjugated intravenously with PBS (control, prodrug only) or 50g of a monoclonal anti-human urokinase plasminogen activator receptor (uPAR) antibody with β-galactosidase, uPAR-β-Gal, in PBS ( antibody enzyme only, ADEPT therapy). On days 24, 26, 28, 32, 34 and 36, the mice are injected intravenously with 1% DMSO / NaCL solution (control, only antibody enzyme) of the compound according to the invention 1% DMSO / NaCL solution (only prodrug, ADEPT therapy). The mice are sacrificed on day 38 and the tumors are removed, weighed and fixed in PBS buffer in 4% formalin for 16 hours at room temperature and embedded in paraffin. Tissue sections are examined for cell proliferation, for example with an antibody directed against Ki-67. Furthermore, the tumor size is determined with the help of computed tomography during the treatment.

Both volume reduction and reduction of the formation of metastases can be observed, while no significant influence on weight, leukocytes, hemoglobin and platelets can be seen. The tumor growth rate with ADEPT therapy is lower.

Claims (10)

Compounds of the general structure A - L - B where A and B are selected independently of one another from structures I or II

with R ¹ selected from a halogen and OSO ₂ R, wherein R ^{u u} is selected from optionally substituted C ₁ -C ₆ alkyl, C _1-6 perhaloalkyl, benzyl, and phenyl; R ² is selected from hydrogen and optionally substituted C _1-8 alkyl; R ^3, R ^{3 ',} R ⁴ and R ^4' are independently selected from hydrogen and optionally substituted C _1-8 alkyl, wherein two or more of R ^2, R ^3, R ³ ', R ⁴ and R ^4', if appropriate are linked together to form one or more optionally substituted carbon cycles or heterocycles; X ² is selected from O, C (R ¹⁴ ) (R ^{14 '} ) and NR ^14' , where R ^{14 is} selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl and where R ¹⁴ 'is absent or is selected from hydrogen and optionally substituted C _1-8 alkyl or C _1-8 acyl; R ⁵ , R ^{5 '} , R ⁶ , R ^6' , R ⁷ and R ^{7 '} are independently selected from H, OH, SH, CF ₃ , CN, C (O) NH ₂ , C (O) H, C (O) OH, Halogen, R ^k , SR ^k , S (O) R ^k , S (O) ₂ R ^k , S (O) OR ^k , S (O) ₂ OR ^k , OS (O) R ^k , OS (O) ₂ R ^k , OS (O) OR ^k , OS (O) ₂ R ^k , OR ^k , P (O) (OR ^k ) (OR ^L ), OP (O) (OR ^k ) (OR ^L ), SiR ^k R ^L R ^m , C (O) R ^k , C (O) OR ^k , C (O) N (R ^L ) R ^k , OC (O) R ^k , OC (O) R ^k , OC (O) OR ^k , OC (O) N (R ^k ) R ^L where R ^k , R ^L and R ^{m are} independently selected from H and optionally substituted C _1-4 alkyl, C _1-4 heteroalkyl, C _{3- 7} cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl groups, two or more of R ^k , R ^L and R ^m optionally form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles with one another; and / or R ^{5 '} and R ^6' and / or R ^{6 '} and R ^7' and / or R ^{7 '} and R ^14' are not present, ie they form a double formation present in the ring between the atoms substituted with the corresponding from, namely R ^{5 '} and R ^6' , and / or R ^{6 '} and R ^7' , and / or R ^{7 '} and R ^14' ; two or more of R ⁵ , R ^{5 '} , R ^6' , R ^{7 '} , R ¹⁴ , and R ^14' can optionally be linked to one another to form one or more optionally substituted aliphatic or aromatic carbon cycles or heterocycles; and / or R ⁵ + R ^{5 '} , and / or R ⁶ + R ^6' and / or R ⁷ + R ^{7 '} are independently of one another = O, = S or = NR ¹² , where R ^{12 is} selected from H and optionally substituted C _1-6 alkyl; X ₆ is selected from C or N; R _DB is selected from hydrogen and optionally substituted C _1-8 alkyl or optionally substituted C _1-8 acyl; X ₁ is selected from O, S, and NR ¹³ , where R ^{13 is} selected from H and optionally substituted C _1-8 alkyl; R is selected from hydrogen or an enzymatically cleavable substrate; a is selected from 0 and 1; b is 1; c is selected from 0, 1 and 2; d is selected from 0 or 1; and L is a linking group to covalently link A and B; and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof. Connection after Claim 1 , where L is selected from i) Z - (C (R _CH ) ₂ ) _n - Z ', where n is an integer from 1 to 20, Z and Z' are independently selected from C = O, OC = O , SO ₂ , NR _z , NR _z C =O, C =ONR _z where each R _CH and R _{z are} independently selected from hydrogen or optionally substituted alkyl or optionally substituted acyl, preferably from optionally substituted C ₁ -C ₈ alkyl or optionally substituted C ₁ -C ₈ acyl, or ii) L is an oligopeptide with 1 to 10 amino acids; or pharmaceutically acceptable salts or substrates thereof. Connection according to Claim 1 or 2 , where R is selected from a substrate which is produced by proteolytic enzymes, plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase, mannosidase, glucosidase, neuramidase, sucrose, maltase, fructosidase, glycosylases, prostate-specific antigen (PSA) -Type plasminogen activator (u-PA), metalloproteinase, or an enzyme that can be specifically cleaved with the help of targeted enzyme prodrug therapy, such as ADEPT, VDEPT, MDEPT, GDEPT, or PDEPT; or a substituent that can be split off or transformed under hypoxic conditions or by reduction by nitroreductase, in particular R is selected from a monosaccharide, disaccharide or oligosaccharide, in particular hexoses, pentoses or heptoses, optionally as a deoxy derivative or amino derivative and optionally substituted with Halogen, C _1-8 alkyl, C _1-8 acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _4-12 aryl or C _4-12 heteroaryl, amino or amide groups, or with amino, amido or carboxyl units, which can optionally be substituted with halogen, C _1-8 alkyl, C _1-8 acyl, C _1-8 heteroalkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _{4- 12} aryl or C _4-12 heteroaryl, amino or amide radicals; Dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or an antibody, or combinations thereof. A compound according to any preceding claim, wherein _X ^{6 is} nitrogen and / or L is Z - (C (R _CH ) ₂ ) _n - Z 'with n being an integer from 1 to 20, Z and Z' are C = O and each R _CH is independently hydrogen or CH ₃ . A compound according to any preceding claim, wherein a is zero and / or n is an integer from 3, 4 or 5. A compound according to any one of the preceding claims, wherein R ⁶ , R ^{6 '} , R ⁷ , R ^7' and R ^{2 are} independently hydrogen or CH ₃ . A compound according to any preceding claim, wherein these are:

where R is independently hydrogen or CH ₃ and n is an integer from 3, 4 or 5. A compound according to any preceding claim, wherein A and B are identical. Pharmaceutical composition containing a compound according to one of the Claims 1 to 8th . Using a connection according to one of the Claims 1 to 8th for the treatment of tumor diseases, especially in mammals.