HK1171741B - Conjugates of pyrrolo[1,4]benzodiazepine dimers as anticancer agents - Google Patents
Conjugates of pyrrolo[1,4]benzodiazepine dimers as anticancer agents Download PDFInfo
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Description
The present invention relates to pyrrolo dimer conjugates[1,4]benzodiazepine (PBD), the compounds containing them and their therapeutic application, including as anticancer agents. The invention also relates to the process of preparing the conjugates for their anticancer applications and to the dimers themselves.
Pyrrolo dimers[1,4]benzodiazepines are anticancer agents that act by covalently binding to the DNA of cells. These derivatives have been described in applications WO 00/12508 and WO 2005/085260 as well as in the following publications: Eur.J.Med.Chem. 2005, 40, 641-654; Tetrahedron Letters 1988, 29(40), 5105110-58.
The chemistry of conjugates has been known for many years and has been applied to several families of cytotoxic agents such as maytansinoids (WO 04103272), taxanes (WO 06061258), leptomycins (WO 07144709), CC-1065 and its analogues (WO 2007102069); see also about conjugates, Monneret C., et al., Cancer Bulletin 2000, 87(11), 829-38; Ricart A.D., et al., Clinical Practice Oncology 2007, 4, 245-255; Singh R. and Rickson H.K., Therapeutic Antibodies Methods and Protocols, 2009, 525, 445-467.
Pyrrolo dimer conjugates[1,4]benzodiazepine have already been described in applications WO 07085930 or WO 2009/016516.
- What?
wherein T can represent an aryl or heteroaryl group replaced by -G-D-(Z) p-SZa or -G-D-(Z) p-C(=O) ZbRb. G represents a single or double bond or either -O-, -S- or -NR-. Diar represents a single bond or either of the following: -E-, -E-NR-, -E-NR-F-, -E-O-, -E-O-F-, -E-NR-CO-, -E-NR-CO-F-, -E-CO-, -CO-E-CO-F-, -E-Sero-, -E-S-C-F-, -E-C-C-C-C-O-, -E-C-C-C-Rb. Eiar represents a single or double bond or -O-, -S- or -NR-cyclic, wherein Diar represents a single bond or one of the following groups: -E-, -E-NR- or -NR-C-Rb. Eihar and Fjhycylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkylkyl
The group L2= -CH2C(=O)NR3-(CH2CH2O) i-ALK- which characterizes some of the compounds of the present invention includes the amide motif (-CONR3-) and can only correspond in WO 07085930 or WO 2009/016516 to the motif -E-CONR-F- with E= alkyl and F= -(CH2CH2O) i-alkyl. However, the group L1 which is linked to the phenyl or pyridinyl cycle and which is linked to L2 is not described or suggested by these two applications. Indeed, only G-pattern could correspond. However, G can be only a single bond (single, double, triple) or -O-, -S-NR- or -S-NR-link. The following compounds of the invention are described in the combination of WO 0CH2CH-ALK-016-016-016-09-060-ALK-016-060-ALK-016-060-ALK-016-060-ALK-016-060-ALK-060-016-05-060-ALK-060-016-060-ALK-060-016-016-016-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-06-0
- What?
but none of these dimers include a linker similar to those described in the present invention (in particular, no -ALK-S- pattern).
Thus, both applications WO 07085930 and WO 2009/016516 do not describe or suggest the compounds of the present invention.
The technical problem to be solved by the present invention is to propose new pyrrolo dimer conjugates[1,4]benzodiazepine.
We mean:
conjugate: a cell targeting agent to which at least one molecule of a cytotoxic compound is covalently bound; cell targeting agent (or cell binding agent in English): a molecule with an affinity for a biological target: it may be for example a ligand, a protein, an antibody, more specifically a monoclonal one, a protein or antibody fragment, a peptide, an oligonucleotide, an oligosaccharide. The targeting agent has the function of directing the biologically active compound as a cytotoxic to a biologically identifiable biological target: an antigen (or group of antigens); these antigens may be located preferably on the surface of cancer cells or tumor cells; for example, these antigens may be associated with,growth factor receptor, a product of an oncogene or gene mutated tumor suppressor a molecule related to angiogenesis, an adhesion molecule; alkyl group: a saturated aliphatic hydrocarbon group obtained by removing a hydrogen atom from an alkane. The alkyl group may be linear or branched. Examples include the methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tertiary-butyl, penteroyl, 2,2-dimethylpropyl, hexyl group; cyclic group: a cyclic alkyl group containing between 3 and 8 carbons involved in the structure. Examples include cyclic groups: cyclopropyl, cycloproyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl.- or bicyclic. These are in particular the phenyl and naphthyl groups; heteroarylic group: an aromatic group comprising at least one heteroatom (O, S, N) involved in the cycle and connected to the carbon atoms forming the cycle, mono- or bicyclic. These are in particular the pyridinyl, pyrrolyl, thienyl, furanyl, pyrimidinyl, triazoyl groups; heterocycloalkyl group: a cycloalkyl group comprising at least one heteroatom (O, S, N) involved in the cycle and connected to the carbon atoms forming the cycle; monoxy group: an -O-alkyl group, the alkyl group being as defined above; alkylo group: an -O-alkyl group,where the alkyl group is as defined above; alkyl group: a saturated divalent group of the raw formula -CmH2m-, obtained by removing two hydrogen atoms from an alkane. The alkane can be linear or branched.
- What?
A linear alkyl group may be more specifically of the formula - ((CH2) m-, m representing an integer; in the range of values, the limits are included (e.g. a range of type i from 1 to 6 includes the limits 1 and 6.
AcOEt: ethyl acetate; ALK: alkylene group (C1-C12) and more specifically (C1-C6) alkylene; CCM: thin-layer chromatography (TLC); DAR: drug antibody ratio; DBU 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC: N,N'-dicyclohexylcarbodiimide DCM: dichloromethane; DEAD: N-diethyl acetylate; N-dihydrocarboxylate DIC: N,N'-dihydroxylcarbodiimide EDEA; DIP: N,N-dihydroxylcarbodiimide DMA; PRMA: dimethylcarbonyl acetate; DMMP: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate: dimethylcarbonyl acetate of dimethylcarbonyl acetate of dimethylcarbonyl acetate of dimethylcarbonyl acetate of dimethylcarbonyl acetate of dimethylcarbonyl acetate of dimethylcarbonyl acetylcarbonyl acetate of dimethylcarbonyl acetate of dimethylcarbonyl carbonyl acetate of dimethylcarbonyl carbonyl carbonyl carbonyl car
Fig.1 : high resolution mass spectrum of ex.1 conjugate after deglycosylation;Fig.2 : high resolution mass spectrum of ex.2 conjugate after deglycosylation;Fig.3 : high resolution mass spectrum of ex.3 conjugate after deglycosylation;Fig.4 : high resolution mass spectrum of ex.4 conjugate after deglycosylation;Fig.5 : high resolution mass spectrum of ex.6 non-deglycosylated conjugate.
These figures show for each conjugate after deconvolution the distribution of species containing 0 to 8 tomycyn dimers (D0: no dimer; Dx: x dimers).
The invention relates to compounds with formula:
- What?
in which:
---- represents a single bond or a double bond with the condition that if---- represents a single bond then:❖ ---- represents a single bond;❖ U and/or U' identical or different, represents: ■nt) independently of each other: H;❖ W and/or W' identical or different, represents: ■nt) independently of each other: OH, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR, -OR-NRR', a cyclic amine such as N10 and C11 are included in a cycle, -NROR', -NRCOR', -N3, -CN, Hal, a trialkylphosphonium or triarylphosphonium group;R1, R2, R1', R2' identical or different, represent, independently of each other: H, Hal or an (C1-C6) alkyl group possibly substituted by one or more substituents (s) selected from among: Hal, CN, NRR', CF3, OR, an aryl or heteroaryl group, S(O) qR with q =0,1 or 2;
or R1 and R2 and/or R1' and R2' together form a double bond =CH2 or =CH-CH3 respectively; Y and Y', identical or different, independently represent each other H or OR; M represents CH or N; ALK and ALK' identical or different,independently represent a (C1-C6) alkyl group; R and R' independently represent each other, H or a (C1-C6) alkyl or aryl group possibly substituted by one or more substituents chosen from: Hal, CN, NRR', CF3, OR, an aryl or heteroaryl group; L represents:
❖ the -L1-L2- group in which L1 is linked to the aromatic cycle comprising M by the ALK or OALK group and represents one of the following groups:
- What ?
- What?
- What is it?
- What?
and L2 is the group -CH2C(=O) -NR3-(CH2CH2O) i-ALK- connected to L1 by -CH2C(=O) -
or❖ the -O-ALK-NR3-ALK-S-(CH2CH2O) i-ALK- group attached to the aromatic cycle containing M by the OALKR3 group represents H or a (C1-C6) alkyl group;i represents an integer from 1 to 40,rather than 1 to 20, preferably 1 to 10;Zb represents a single bond, -O- or -NH- and Rb represents H or a group (C1-C6) alkyl, (C3-C7) cycloalkyl, aryl, heteroaryl or (C3-C7) heterocycloalkyl or Zb represents a single bond and Rb represents Hal.
Compounds of formula (I), including those exemplified, may exist as bases or salts of addition to pharmaceutically acceptable acids or as hydrates or solvates of these bases or salts.
In particular, the two ALK and ALK' groups attached to the phenyl or pyridinyl nucleus both designate a methylene group:
- What?
In particular, the following formula (I) compounds are distinguished from formula (IA) or formula (IB):
Y and Y' more specifically represent a (C1-C4) alcoxy group, in particular the methoxy group. R and R' may more specifically represent independently of each other, H or a (C1-C6) alkyl group. Depending on a particular mode, U=U' and/or W=W' and/or R1=R1' and/or R2=R2' and/or Y=Y' and/or the two ALK and ALK' groups attached to the phenyl or pyridinyl nucleus are identical.
In particular, L may be chosen from among the following:
The following shall be reported in the table of the Annex to Implementing Regulation (EU) No 540/2011 for the purposes of the calculation of the CO2 savings:
Among these, ALK is most notably a (C1-C4) alkylene group. In particular, ALK can be any of the following: -CH2CH2-, -CH2CMe2-, -CH2CH2CMe2-.
i represents an integer from 1 to 40, rather than 1 to 20, preferably 1 to 10. i can take any value from 1 to 40, in particular i can be 3, 4, 5, 6, 7, 8, 9 or 10.
Table I describes representative examples of compounds according to the formula (IA). Each compound in this table can exist in the form with M=CH (benzene) or M=N (pyridine). Compounds with M=N are more soluble in water.
- What?
The compounds of the invention include the chemical group -C(=O)ZbRb (GCR1) which is reactive to a reactive chemical group (GCR2) present on the target agent. The reaction between GCR1 and GCR2 ensures the attachment of the compound to the target agent by forming a covalent bond. Thus, the compound is suitable for conjugation to a target agent. More specifically, Zb represents O; in this case, GCR1 represents an acid (Rb=H) or ester. More specifically, -C=O)ZbRb represents -COOH, -COO(((C1-C6) an alkyl, notably -COOCH3, -COOCH2 or -CH2. The following are examples of amino groups that are particularly active on the target compound:
- What?
or the group
- What?
where GI represents at least one inductive group such as -NO2 or -Hal, including -F. These may be, for example, the following groups:
- What?
Another type of group-C(=O)ZbRb is as follows:
- What?
Examples of GCR2 include the lysine ε-amino groups carried by the side chains of lysine residues that are present on the surface of an antibody, the saccharide groups of the hinge region or the cystine thiols by reduction of intra-chain disulfide bonds (Garnett M.C., et al., Advanced Drug Delivery Reviews 2001, 53, 171-216). More recently, other approaches have been considered such as the introduction of cystine by mutation (Junutula J.R., et al., Nature Biotechnology 2008, 26, 925-932; WO09026274) or the introduction of non-natural amino acids by reduction of intra-chain disulfide bonds (Graafde J., Chemical and al., February 3, 2009, J.C., 2009; Reviews 2009; and 2009; and DOC 2009: J.C., 2009; and 2009; these are applicable to all types of non-natural amino acids using their combined structure and function (Chemical and Biotechnology Reviews 2009; J.C., 2009; and 2009; and 2009; and 2009; J.C., 2009; and 2009; and 2009; and 2009; and 2009).
The compounds of the invention can therefore be used for the preparation of a targeting agent to which the dimer is covalently bound in the M position of the formula:
- What?
In particular, the targeting agent is an antibody.
- What?
The compounds of formula (I) may be prepared as shown in Figure 1:
LG and LG' denote a leaving group. L3 can represent the group -L-C(=O)ZbRb, in which case P3 is therefore a compound of formula (I). In the case where P3 does not represent the group -L-C(=O)ZbRb, it is necessary to transform L3 into a group -L-C=O)ZbRb by one or more reactions.
In particular, in the case of
- What?
the L3 group terminated by the -C(=O) ZbRb= -C(=O) C1-C4-alkyl or -C(=O) O-alkyl group can be introduced, which is then transformed into the -C(=O) O-OH group which finally reacts with the N-N'-disuccinimide carbonate or NHS. The transformation of -COOalkyle/alkyl into -COOH can be achieved by a lithine treatment. It is particularly advantageous to use a methyl ester in particular. The reaction with the N,N'-disuccinimide carbonate is conducted in the presence of a base, e.g. DEA; with the NHS in the presence of a coupling agent, e.g. DCC.
The same applies in the case of
- What?
a -C(=O)ZbRb=-COOH group may be introduced which then reacts with N,N'-carbonyldiimidazole (JACS 1958, 80, 4423; JACS 1960, 82, 4596).
Err1:Expecting ',' delimiter: line 1 column 558 (char 557)
These are as follows:
- What?
in which:
■ L, M, ALK, ALK', Zb and Rb are as defined above; ■ E and E' independently represent a -OH group or a starting group.
Err1:Expecting property name enclosed in double quotes: line 1 column 138 (char 137)
A distinction is also made between intermediates of formula:
- What?
where L* is chosen from: -ALK-SH; -O-ALK-NR3-ALK-SH;
- What?
In the present invention, leaving group is an atom or group of atoms which, in the heterolytic reaction between P2 and P1 or P'1, leaves with the electron doublings of the covalent bond linking ALK and LG or LG. The leaving group is chosen more specifically from a halogen atom, notably chlorine or bromine, a mesylate, tosylate, nosylate or -OPPh3+ group.
For the other Schemes below, the following abbreviations are used for simplification:
Preparation of P2 P2 is obtained from the corresponding halogenated diol of formula
- What?
According to the teaching of ex.3 (Sheet 2):
i. protection of both alcohol functions by means of a protective group such as tert-butyl-dimethyl-silyloxy (TBS) ii. preparation of the corresponding organolithium or organomagnesium by means of n-BuLi or magnesium respectively iii. nucleophilic addition to a ketone to form an alcohol function iv. preparation of thiol by forming the corresponding thioacetate (see ex.3.7 and 3.8) v. deprotection vi. introduction of LG and LG. In the case of a mesylate group, methasulfonyl chloride is used in the presence of a base such as a tertiary amine (see ex. TEA or DIP); ex.1.4.
An example of halogenated diol and corresponding protected diol is that described in Figure 1 on page 48 of WO 2009/016516 (compounds 2 and 3 of Figure 1). Two examples of protected diols are those of CAS Nos 181225-40-1 and 181225-41-2.
The halogenated diol can be obtained by reduction of the corresponding diacid or diester compound, e.g. that of CAS No 193010-40-1. See also in the case of a pyridine (M=N): Liebigs Annalen der Chemie 1991, 10, 987-988 or Tetrahedron 2005, 61(7), 1755-1763 (compound 3 of Scheme 1).
Step (i): formation of the amide and activation of the acid; the two steps are performed successively in an aprotic polar solvent such as DCM: reaction between the amine function and the N-hydroxysuccinimide halogen acetate and then addition in situ of a coupling agent such as DIC.
Step (ii): protection of the carboxylic acid as a methyl ester and the amine as a trifluoroacetamide; the reaction is carried out in two successive steps in an aprotic polar solvent such as DCM: protection of the acid by treatment with trimethylsilyldiazomethane in the presence of methanol and then protection of the amine by addition of trifluoroacetic anhydride and a base such as TEA;
Step (iii): alkylation of the amine and saponification of the ester; the reaction is carried out in two successive steps in an anhydrous aprotic polar solvent such as THF: alkylation of the amine by treatment with a base such as NaH in the presence of a reagent carrying a nucleophile group such as an R3Hal alkyl halides and then addition of lithine and water;
Step (i): following step (iii), the reactions of step (i) are repeated for the case R3=H.
Step (iv): elongation of the PEG chain; the reaction is carried out in an anhydrous aprotic polar solvent such as THF or DMF by treatment of a halogen ester with the alcoholate of a benzophenone-imine-PEG alcohol generated by the action of NaH or potassium naphthalene as described in WO 2007/127440;
Step (v): selective cleavage of imine by hydrogenation in the presence of palladium on coal according to Wessjohann, L. et al., Synthesis 1989, 5, 359-63;
Step (vi): protection of the amine by addition of trifluoroacetic anhydride and a base such as TEA.
Amino-PEG alcohols are commercially available for e.g. i=3,4,7,8 or can be prepared from PEG diols, commercially available for i=3 to 12, according to the procedure described in US 7230101.
- What?
i. nucleophilic reaction between one of the -OH functions (the other two being protected by GP and GP' which designate protective groups) and a bromoamine protected by a bucc of formula Br-ALK-NHboc in the presence of a base such as K2CO3 in a polar solvent such as DMF, THF or MeCN (see e.g. conditions on page 63 of WO 07085930).
One variant is the nucleophilic substitution of bromoamine by hydroxy diester with the formula:
- What?
then reduce the ester function to the CH2OH function, e.g. with sodium borohydride; the conditions for nucleophilic substitution and reduction given on pages 62-63 of WO 2007/085930 can be applied;reducing amination with the aldehyde of formula HC ((=O) -ALK-SSMe in the presence of titanium isopropoxide; the reaction is carried out at room temperature in an aprotic polar solvent such as anhydrous THF;iv. an intermediate complex is formed which is reduced in situ with a reducing agent such as sodium cyanoborohydride;v. introduction of LG and LG. In the case of a mesylate group, methanesulfonyl chloride is used in the presence of a base such as a tertiary amine (e.g. TEA); see ex.1.4.
Err1:Expecting ',' delimiter: line 1 column 84 (char 83)
According to this variant, alkylation is carried out using a Hal-ALK-SCOMe intermediate (Hal=I for example) and then the thiol is released by treatment in a basic medium:
vi. alkylation by an alkyl halides carrier of a thioacetyl group in the presence of caesium carbonate in a polar aprotic solvent such as DMF;vii. selective cleavage of the acetyl group in a weak base medium;viii. formation of the -SSMe group by reaction of the intermediate thiol with MeSSO2Meix. cleavage of the protective groups GP and GP'x.; transformation of the hydroxyl groups into nucleophile groups LG/LG', preferably mesylate groups.
- What?
Err1:Expecting ',' delimiter: line 1 column 189 (char 188)
The companies Thermo Fisher Scientific, Rockford, IL. 61105, USA, Jenkem Technology USA Inc., 2033, W. McDermott Dr., Allen Tx 75013-4675, USA and Quanta BioDesign, Ltd.195, West Olentangy Street, Suite O, Powell, Ohio 43065-8720, USA are the companies which market compounds with the general formula
- What?
This may be in particular the compound with CAS No 756525-99-2.
The addition of thiol on the maleimide pattern is described on page 721 of Bioconjugate Techniques , Greg T. Hermanson, 2nd Ed, Elsevier Inc. (isbn-13: 978-0-12-370501-3; isbn-10:0-12-370501-0).
- What?
The preparation is similar to that described in Figure 3 by replacing P5 by P4.
A variant described in Schedule 4 ' corresponds to a preparation similar to that described in Schedules 2 and 2 ':
i. Deprotection by disulfide reduction
- What?
We use the compound P2 of formula:
- What?
which is obtained according to Scheme 5' below:
i. alkylation of the hydroxyl of the aromatic cycle by a piperazine mono protected at position 1 and carrying in 4 an alkyl chain functionalized at the terminal position by a nucleofuge LG group. Preferably the nucleofuge group is a mesylate group and the Williamson reaction is carried out in the presence of a hydride in an anhydrous aprotic polar solvent such as THF or DMFii. unprotection of the boc, GP and GP' groups; preferably in acidic medium, e.g. in the presence of hydrochloric acid or TFA when the GP and GP' groups are TBDMS.- What?
A variant of steps i and ii can be used to perform nucleophilic substitution of bromoamine by hydroxy diester with the formula:
- What?
then subsequently reduce the ester function to -CH2OH, e.g. with sodium borohydride; the conditions for nucleophilic substitution and reduction given on pages 62-63 of WO 2007/085930 can be applied;The Commission has
We use the compound P2 of formula:
- What?
which is obtained by reducing amining according to Scheme 6' below.v. introduction of the -ALK-SSMe group by reducing amining e.g. in the presence of cyanoborohydride and titanium isopropoxide;
Err1:Expecting ',' delimiter: line 1 column 102 (char 101)
This Scheme is based on the previous Scheme 3. The compound P6 may be, for example, the compound of CAS No 564476-32-0 which is prepared according to WO 03068144 (see compound 10a in Figure 7) or the compound of CAS No 309916-91-4.
- What?
may be prepared according to the following schemes:
Step (i): activation of the alcohol as mesylate; the reaction is carried out in an anhydrous aprotic polar solvent such as DCM by treatment with mesylic chloride in the presence of a base such as TEA.
Step (ii): mesylate/halogen exchange; the reaction is carried out by reflux of an aprotic polar solvent such as acetone with a sodium halides such as sodium iodide.
Step (iii): deprotection with a solution of hydrochloric acid (e.g. solution in dioxane) or trifluoroacetic acid.
Step (iv): acid activation; the reaction is carried out at room temperature in an aprotic polar solvent such as DCM by treatment with NHS in the presence of a coupling agent such as DCC.
Step (v): elongation of the PEG chain; the reaction is carried out in an anhydrous aprotic polar solvent such as THF or DMF by treatment of a halogen ester with the alcoholate of a monoprotected PEG diol in the form of tetrahydropyrane ether (THP).
The following examples will be useful to the professional in order to draw his inspiration from the operating conditions.
The conjugate is obtained by the process of:
(i) to contact and react an aqueous solution of the targeting agent, if buffered, and a solution of a compound of formula (I);
One variant is to separate the conjugate that formed in step (i) from the unreacted targeting agent and any aggregates present in the solution at step (ii) and to separate the unreacted compound of formula (I) from any aggregates that formed in step (i) and to leave the unreacted targeting agent in the solution at step (ii).
The compound of formula (I) preferably has an activated function, -C(=O)ZbRb, reactive to GCR2 groups, especially to amino groups present on antibodies. A chemical group that is not very reactive or not sufficiently reactive can be easily transformed into a more reactive group by one or more known chemical reactions; e.g. -COOH + N-hydroxysuccinimide →
- What?
or -COO(C1-C6)alkyl → -COOH → -COOH + N-hydroxysuccinimide →
- What?
An example of a process applicable to an antibody and a compound of formula (I) is given in Example 1.
The aqueous solution of the targeting agent can be buffered using e.g. buffers such as potassium phosphate or N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES buffer). The buffer depends on the nature of the targeting agent. The compound of formula (I) is dissolved in a polar organic solvent, e.g. DMSO or DMA.
The reaction between the targeting agent and the formula (I) compound can be monitored by ESA with a refractometric and/or ultraviolet detector to determine its progress. If the grafting rate is insufficient, the reaction can be allowed to continue and/or formula (I) compound can be added. The general method given in the examples section can be used for more details on the special conditions for conjugation.
The skilled person has different chromatographic techniques for the separation of step (ii): the conjugate can be purified for example by steric exclusion chromatography (SEC), adsorption chromatography (such as the ion exchanger, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, chromatography on mixed media such as ceramic hydroxyapatite or HPLC.
Aggregates are likely to form under the influence of a large number of parameters such as a high concentration of target agent in solution, pH of solution, high shear forces, number of grafted dimers and their hydrophobic nature, temperature (see references cited in the introduction to J.Membrane Sci. 2008, 318, 311-316), the influence of some of which is not precisely elucidated. In the case of proteins or antibodies, the number of grafted dimers and their hydrophobicity can sometimes be determined by reference to AAPS, Protein and Biochemistry Journal, EE82-E9803 (see EE947-492); in the case of proteins or antibodies, the techniques known for this purpose may be used, such as the Aggregate Analysis (AAPS, 1993).
After step (i) or (ii), the solution of the conjugate may undergo step (iii) of ultrafiltration and/or diafiltration, resulting in the conjugate in aqueous solution.
The antibody (see Janeway et al. Immunobiology , 5th edition, 2001, Garland Publishing, New York) may be selected from those described in applications WO 04043344, WO 08010101, WO 08047242, WO 05009369 (anti-CA6). The antibody may be monoclonal, polyclonal or multispecific, may be a fragment of an antibody, or may be a mouse, human, humanised or chimeric antibody.
Err1:Expecting ',' delimiter: line 1 column 204 (char 203)
In the case where the targeting agent is an antibody, UV spectroscopy may be a method used to determine DAR. This method is based on that presented in Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols , vol. 525, 445, Springer Science. It consists of measuring the absorbance of a solution of conjugate after the separation step (ii) at two wavelengths notated LO1 and LO2.
Err1:Expecting ',' delimiter: line 1 column 204 (char 203) - What?
equations for which:
cD and cA are the concentrations in solution of the pyrrolo dimer[1,4]benzodiazepine and antibody parts of the conjugate; eDLO1 and eDLO2 are the molar absorption coefficients of pyrrolo dimer[1,4]benzodiazepine prior to conjugation at both wavelengths LO1 and LO2; LOeALO1 and eALO2 are the molar absorption coefficients of the antibody at both wavelengths LO1 and LO2.
Naked antibodies are antibodies to which no pyrrolo dimer is attached to benzodiazepine, i.e. antibodies before the conjugation stage.
The solution of these two equations leads to:
The mean DAR is then cD/cA. In the case of pyrrolo dimers[1,4]benzodiazepine, the two wavelengths are considered: LO1= 280 nm and LO2= 320 nm. The mean DAR measured on SEC spectrum is preferably between 1 and 10, preferably between 1.5 and 7.
Conjugate can be used as an anticancer agent. Due to the presence of the targeting agent, the conjugate is made very selective towards tumor cells rather than healthy cells. This allows the targeted compound of formula (I) that has anticancer activity to be directed in an environment close to or directly within tumor cells (for this, see the following publications describing the use of monoclonal antibody conjugates in the treatment of cancers: Antibody drug conjugates for cancer therapy Carter P.J., et al., J., 2008, 14, 154-169; Targeted cancer therapy: conferring cytotoxicity specificity drugs R. Chem., Chari., 2008, 41, 98-107). It is possible to treat solid or liquid residues of cancer.
The conjugate is formulated as a buffered aqueous solution at a concentration generally between 1 and 10 mg/ ml. This solution can be injected as an infusion or reconstituted to form a solution for infusion.
Chemical displacements (in ppm) are expressed in ppm.
The spectra were obtained on a Waters UPLC-SQD apparatus in positive and/or negative electrospray mode (ES+/-). Chromatographic conditions: column: ACQUITY BEH C18 1.7 μm-2,1x50 mm; solvents: A: H2O (0.1% formic acid) B: CH3CN (0.1% formic acid); column temperature: 50°C; flow rate: 1 ml/min gradient (2 min): 5 to 50% of B in 0.8 min; 1.2 min: 100 % of B; 1.85 min: 100 % of B; 1.95 : 5 % of B.
The spectra were obtained on a Waters ZQ apparatus in positive and/or negative electrospray mode (ES+/-). Chromatographic conditions: column: XBridge C18 2.5 μm 3x50 mm; solvents: A: H2O (0.1% formic acid) B: CH3CN (0.1% formic acid); column temperature: 70°C; flow rate: 0.9 ml/min; gradient (7 min): from 5 to 100% B in 5.3 min; 5.5 min ; 100 % B 6.3 min: 5 % B.
The spectra were obtained by chemical ionization (reacting gas: ammonia) on the WATERS GCT of (direct introduction without LC).
The spectra were obtained on a Waters UPLC-SQD apparatus in positive and/or negative electrospray mode (ES+/-). Chromatographic conditions: column: ACQUITY BEH C18 1.7 μm-2,1x50 mm; solvents: A: H2O (0.1% formic acid) B: CH3CN (0.1% formic acid); column temperature: 70°C; flow rate: 1 ml/min; gradient (2 min) : 5 to 50% of B in 1 min; min: 100% of B 1.3 min: 1.45 min: 100% of B 1.75: 5% of B.
The spectra were obtained on a Waters UPLC-SQD apparatus in ionization mode: electrospray in positive and/or negative mode (ES+/-). Chromatographic conditions: column: ACQUITY BEH C18 1.7 μm - 2.1x50 mm; solvents: A: H2O (0.1 % formic acid) B: CH3CN (0.1% formic acid); column temperature: 70 °C: flow rate: 1 ml/min; gradient (4 min) ; 5 to 100% of B in 3.15 min; 3.75: 5% of B.
The spectra were obtained on a Waters UPLC-SQD apparatus in ionization mode electrospray in positive and/or negative mode (ES+/-). Chromatographic conditions: column: ACQUITY BEH C18, 1.7 μm - 2.1x30 mm; solvents: A: H2O (0.1% formic acid) B: CH3CN (0.1% formic acid); column temperature: 45°C; flow rate: 0.6 ml/min; gradient (2 min) : 5 to 50% of B in 1 min; 1.3 min: 100% of B; 1.45 min: 100% of B; 1.75 min: 5% of B.
Deglycosylation is a technique of enzymatic digestion using glycosidase. It is made from 500 μl of conjugate + 100 μl of Tris HCl 50 mM buffer + 10 μl of glycanase-F (100 units of lyophilized enzyme/100 μl of water). The mixture is passed to the vortex and kept overnight at 37°C. The deglycosylated sample is then ready for analysis in HRMS. Depending on the case, HRMS analysis of the sample can also be performed without prior deglycosylation. In both cases, the mass spectra were formed on a Waters Q-Tof-2 solvent in electroscopic mode (ES). Positive chromatography: 30%: 4 μm; Color: 4 μm; Color: 4 μm; Color: 4 μm; Color: 0.6 μm; Color: 250 μm; Chemical isomer: 250 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.3 μm; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: 0.0; Chemical isomer: Chemical isomer: Chemical isotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherotherother
All intermediate compounds described in this application are claimed for use in the preparation of a compound of formula (I). More specifically for each example, all intermediate compounds described are claimed for use in the preparation of the respective compound of formula (I).
A conjugate is prepared by reacting hu2H11 (also referred to as hu53 2H11 on page 15 of WO 2008010101; this is an antibody comprising a Vh with the amino acid sequence SED ID No. 24)) and 3-((2-{[2-[2-({3-([3-(2-{[2-(2,6-bis-[(S)-2-eth-E) -ylidene-7-dimethoxy-1,2,3,11-a-tetrahydro-pyrrolo[2,1c][1,4]benzphenyl-5-one-sulphylloxymethyl]-pyrrodiaxyl-4-yloxy) -ethyl]methyl-aminoxo-1,1-, dimethyl-ethyl-2-propanoxy-2-pyrrodiol-pyrroxy-1-in-hydroxy-propanoxy-propanoxy-hoxy-propanoxy-hoxy-propanoxy-propanoxy)
8.19 mg of hu2H11 in 2.16 mL of an aqueous buffer of 0.05 M N- ((2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES), 0.05 M NaCl and 2 mM ethylene-diamine-tetraacetic acid (EDTA) pH=8, are added to 516 μg of 3-(2- ((2-[2-2-{3-(((2-(((((2-(2,6-bis-[S) 2-eth-E)) -yl-7-dimethoxy-1, pre-hydroxy-tetrahydroxyprol (GEPS), pre-hydroxyprol (GEPS), pre-hydroxyprol (GEPS), pre-hydroxyprol (GEPS), pre-hydroxyprol (GEPS), pre-hydroxyprol (GEPS), pre-hydroxyprol (GEPS), pre-hydroxyprol (PHP), pre-hydroxyprol (PHP), pre-hydroxyprol (PHP), pre-hydroxyprol (PHP) -HIP) -HIP) in a solution of N-hydroxyprol (PHP) and N-hydroxyprol (PHP) -HIP) are added to a solution of N-hydroxyprol (PHP) -HDP (PHP) and pre-HDP-HDP-HDP (PHP) in a concentration of 10 to 10 mg/ml (PHP) and a concentration of 10 to 10 to 10 to 10 mg (M/ml (PHP) and a concentration of N-HDP-HDP-HDP-HDP-HDP-HDP-HDP-R (PHDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-HDP-H
The resulting conjugate (2.5 mL) is dosed by spectrophotometry using the extinction coefficients of 4-{2-[methyl-(2-methyl-2-mercapto-propyl) -amino]-ethoxy}-2,6-bis-[(S)-2-eth-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]-pyridine (ε319 nm=8848 M-1 cm-1 and ε280 nm=8634 M-1 cm-1) and hu2H11 (ε280 nm=208380 M-1 cm-1); an average of 3.8 tommycins per L-arginone antibody molecule at a concentration of 1.5 mg/m2 was determined.
A 3,3 mg resin-based diisopropylethylamine (3,72 mmol/g) benzodiazepine-5-ylloxymidyl]pyridine solution containing 4,2-[methyl- ((2-methyl-2-mercaptopropyl) -amino]-ethoxy}-2,6-bis-[(S)-2-eth-E) -ylidene-7-dimethoxy-1,2,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,3-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,4-,
A solution of 4-{2-[methyl-(2-methyl-2-methyldisulphanyl-propyl) -amino]-ethyxy}-2,6-bis-[(S)-2-ethy-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]pyridine in solution in 2,1 mL methanol and 930 μL DMF is added to 40 mg of 4-{2-[methyl-(2-methyl-methyl-disulphanyl-propyl) -amino]-ethyxy}-2,6-bis-bis-[(S)-2-ethy-(E) -ylidene-7-dimethoxy-1,2,2,3,11a-tetrahydro-pyrrolo[1,2,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,2,2,2,2,2,2,2,2,2,3,2,3,2,3,2,3,2,3,3,3,4,4,4,4,5,4,5,5,5,5,5,5,5,5,5,5,5,5,5,5,5,6,5,6,8,8,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,9,
After 30 min of agitation, the mixture is hydrolysed and the organic phase washed with water and then dried on MgSO4 and concentrated under PR. The resulting residue (22 mg) is added to a solution of 20 mg tomycin in 0.7 mL DMF, as well as 30 mg K2CO3 and 12 mg KI. The mixture is agitated at 30°C by 4 mL hydrolysed water and then precipitated into the water.The fractions containing the test product are assembled and concentrated under PR to obtain 8 mg of 4-{2-[methyl-(2-methyl-2-methyldisulphanyl-propyl) -amino]-ethyloxy}-2,6-bis-[S) -2-ethylo-E) -ylidene-7-dimethroxy-1,2,3,11a-tetrahydro-pyrolo[2,1c][1,4]benzodiazide-5-benzodiazide-8-yloxy-pyrol]pyrol. 1 MHz; ; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; H; HThe following is a list of the most commonly used methods of measuring the sound level of a sound level:
The mixture is heated for 1 hour and a quarter at 100°C. After return to ambient air, the mixture is hydrolysed and then a 5N aqueous solution of soda added until a pH=12 is reached. The aqueous phase is extracted 3 times with AcO2, and the organic phases are removed at MgSO4 and concentrated under PR. This results in 310 mg of 4-methylmethylmethylmethacrylate (H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3, H2O3) (H2O) (H2O) (H) (
To a 390 mg suspension of 4-[2-amino-ethoxy]-2,6-bis- ((hydroxymethyl) pyridine (prepared after unprotection of the 4-(2-tert-butoxycarbonylamino-ethoxy)-2,6-bis- ((hydroxymethyl) pyridine boc group described on page 101 of WO 07085930) in 2 mL of THF are added 270 μL of 2- ((methyl) isobutyraldehyde and then 730 μL of titanium isopropoxide. After 20 min, an additional 270 μL of 2-methyl) isobutyraldehyde and 730 μL of titanium isopropoxide are added and the mixture is agitated for 2 min. Then an additional 124 μL of sodium cyanide is added to the mixture, after 1 min, an additional 6 mg of TA is agitated and the mixture is agitated.The resulting precipitate is filtered, dissolved in an aqueous solution HCl 1M. The aqueous phase is brought to basic pH with an aqueous solution of 5M sodium, extracted three times with DCM and the combined organic phases are concentrated under PR. This gives 322 mg of 4-[2-(methyl-2-methyl-methyldisulfanyl-propylamino) -ethoxy]-2,6-bis-(hydroxymethyl) -pyridine. RMN 1H (400 MHz, DMSO-d6) : 1,26 (s, 6 H) ; 1,81 (m, wide H) wide 1 2,39 (s, 3 672 H) ; 2 , 942 H; 2 , 2,115, J = 2,115, J = 2,115, H = 2,115, H = 2,115, H = 2,94The time taken for the measurement of the measurement is the time taken for the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the meas
A conjugate is prepared by reacting hu2H11 with 3-{2-[2-(2-{2-(2-{2-(2-{[[2-(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))
12 mg of 3-{2-[2-(2-{2-[2-(2-(2-{[2-{[2-(([2-(([2-((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))
18 mg of 3-{2-[2-({2-[2-[2-(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))
Prepared in accordance with ex.1 from 3-{2-[2-(2-{2-[2-(2-{[2-(2-[2,6-bis-hydroxymethyl-pyridine-4-yloxy) -ethyl]-methyl-amino}-1,1-dimethyl-ethyl sulphanyl) -acetylamino]-ethylenediamine:
- What?
The test is performed on a sample of the test chemical (s) and the test chemical (s) is performed on a sample of the test chemical (s).
45 mg of 4-{2-[(2-mercapto-methyl-propyl) -amino-ethy}-2,6-bis- ((hydroxymethyl) -pyridine in solution in 1 mL DMF, are added to 73.7 mg of 3-[(2-{2-[2-(2-iodo-acetylamino) -ethy]-ethy]-ethy) -ethy) -methylprate in solution in 1 mL DMF and 39 μL DIPMEA. After 24 h at TA, the mixture is concentrated by PR and purified by flash chromatography on silica (Analogue Super Flash SFDO225-8g), using a 10% gradient of methanol base in DCM. The gradient is: 0,05 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0 m; 3,0; 3,0 m; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 3,0; 2,0; 2,0; 2,
After 2 h at TA, 330 μL of MeOH is added and the mixture is cooled to 0°C. 360 μL of a 2M solution of 3-(2-{2-[2-(2-amino-ethylsilyldiazomethane) is added to the hexane. After 1 h, the mixture is neutralized by adding 50 μL of acetic acid and added of saturated N-hydroxysuccinimidyl acetate in solution in 3 mL of DCM. After 2 h at TA, 330 μL of MeOH is added and the mixture is cooled to 0°C. 360 μL of a 2M solution of 3-(2-{2-[2-(2-amino-ethylsilyldiazomethane) in hexane is added. After 1 h, the mixture is neutralized by adding 50 μL of acetic acid and then added of saturated NaHCO3 iodide until the pH is reached. The organic phase is concentrated at a frequency of 0.48 Hz: < 0,04 Hz; < 0,04 Hz; < 0,04 Hz; < 0,04 Hz; < 0,04 Hz; < 0,04 Hz; < 0,04 Hz; < 0,04 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 Hz; < 0,08 JH2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H
80 mg of 4-{2-[methyl-(2-methyl-2-methyldisulfanyl-propyl) -amino]-ethoxy)-2,6-bis-(hydroxymethyl) -pyridine in solution in 1.95 mL of methanol, is added to a 198 mg solution of tris2-carboxyl) phosphine hydrochloride in 730 μL of water. After 2 h at TA, the mixture is concentrated under PR and taken up again in 10 mL of water. The aqueous phase is brought to pH=8 by addition of an aqueous solution of soda extract and then 2x of AcO6. The organic phases are then mixed with an aqueous solution saturated in NaOH and concentrated under PR. The result is 68 mg of: NaCl;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;
A conjugate is prepared as in ex. 1 by reacting hu2H11 and 3-(2-{[2-(2-{2-[1-(3,5-bis-[(S)-2-eth-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]-1-methyl-ethylsulfanyl]-acetylamino{-ethimidhoxy) -ethoxy]-ethoxy}-ethoxy) -propanoate of N-hydroxysuccinyl.
The resulting conjugate is dosed by spectrophotometry using the extinction coefficients of 1-(1-methyl-1-methyldisulfanyl-ethyl)-3,5-bis-[(S)-2-eth-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl] -benzene (ε319 nm= 8460 M-1 cm-1 and ε280 nm= 10531 M-1 cm-1) and hu2H11 (ε280 nm= 208380 M-1 cm-1) - with an average of 4.2 nm. - derivatives of tomycin by an antibody molecule were determined.
Prepared as in ex.2, from 3-(2-{2-[2-(2-{2-[1-(3,5-bis-(((S)-2-eth-E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]-1-methyl-ethylsulphanyl]-acetylamino acid-ethoxy-ethoxy]-ethoxy-ethoxy-ethoxy-propanoic acid:
- What?
The test shall be carried out in accordance with the instructions given in Appendix 2 to this annex.
Prepared as in ex 2, from 3-(2-{2-[2-(2-{2-[1-(3,5-bis-[(S)-2-eth-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]-1-methyl-ethylsulfanyl]-acetylamino}-ethoxy) -ethoxy]-ethoxy}-ethoxy) -methyl propanoate:
The test shall be carried out in accordance with the instructions given in Appendix 2 to this annex.
Prepared as in ex.2, from 3-(2-{2-[2-(2-{2-[1-(3,5-bis-hydroxymethyl-phenyl)-1-methyl-ethyl-sulphanyl]-acetyllamino-ethoxy) -ethoxy]-ethoxy-ethoxy) -methyl propanoate:
- What?
The test shall be carried out in accordance with the instructions given in Appendix 2 to this annex.
Prepared as described in ex.2, from 3,5-bis-hydroxymethyl-1- ((1-mercapto-1-methyl-ethyl) benzenes:
- What?
The frequency range of the measurement is defined as the frequency range of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measur
A 43 mg solution of 5-hydroxymethyl-1-(1-mercapto-1-methyl-ethyl) -3-tert-butyl-dimethyl-silanyloxy-methyl) -benzene in 1 mL of a mixture of acetic acid/THF/water (3/1/1) is agitated at TA for 4.5 h, concentrated under PR and then resumed in 3 mL of water. The pH of the aqueous phase is brought to 7 by addition of a 10% aqueous solution of Na2CO3 and then the aqueous phase is extracted with AcOEt. The organic phase is dried on Mg4 and concentrated under PRSO. This gives 18 mg of 3,5-hydroxymethyl-1-methyl-benzene.
After 3 h at TA, the mixture is concentrated under PR and purified by flash chromatography on silica (Biotage 25+M), using a gradient of 0 to 55% AcOEt in heptane. 230 mg of 5-hydroxymethyl-1-mercapto-1-methyl-ethyl) acetonitrile in solution in 3.7 mL acetonitrile is added to 114 μL hydrazine. After 3 h at TA, the mixture is concentrated under PR and purified by flash chromatography on silica (Biotage 25+M), using a gradient of 0 to 55% AcOEt in heptane. 230 mg of 5-hydroxymethyl-1-methyl-1-hydroxymethyl) acetonitrile is added to 114 μL hydrazine.
1 g of 1-(1-Hydroxy-1-methyl-ethyl)-3,5-bis-(tert-butyl-dimethyl-silanyloxymethyl) benzene in solution in 4,7 mL of 1,2-dichloroethane is added to 404 μL of thioacetic acid and 376 mg of zinc iodide. The mixture is heated to 50 °C for 40 min. After return to TA, the salts are removed by filtration and the organic phase is concentrated and purified by flash chromatography on silica (Bio 40+M), using a gradient of 0 to 30% AcO in heptane. This results in 248 μl of 1-thyoacetic acid[d]: 1-((((dimethyl-ethyl-ethyl-ethyl-ethyl-ethyl; ; 4,11 mHg; ; 4,11 mHg; 4,11 mHg; 4,11 mHg; 4,11 mHg; 4,11 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 2,16 mHg; 1,17 mHg; 1,17 mHg; 2,16 mHg; 1,17 mHg; 1,17 mHg; 2,16 mHg; 1,17 mHg; 1,17 mHg; 2,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 2,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 2,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 2,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,17 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 1,16 mHg; 2,16 mHg; 1,16 mH
After 1 h 30, 4,27 mL of acetone is added by drip. The mixture is allowed to rise to TA and then waterrolysed by a saturated aqueous solution of NH4Cl. The extraction phase is triturated with 100 mL of AcOther and the organic phases are dried on SO4, concentrated and purified by PRM: 10,5 mL of n-BuLi in 1.6 M solution in hexane. After 1 h 30, 4,27 mL of acetone is added by drip. The mixture is allowed to rise to TA and then waterrolysed by a saturated aqueous solution of NH4Cl. The extraction phase is triturated with 100 mL of AcOther and the organic phases are dried on SO4, concentrated and purified by chromatography: 0,11 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,9 mL; 0,11 mL; 0,11 mL; 0,11 mL; 0,9 mL; 0,11 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 mL; 0,9 m
A conjugate is prepared by reacting hu2H11 with 3-{2-[2-(2-{2-[2-(4-{4-{4-[2-(2,6-bis-[(S)-2-eth-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1 c][1,4]benzodiazepine-5-one-8-yloxymethyl]-pyridin-4-yloxy) -ethyl]-piperazine-1-yl}-1,1-dimethyl-4-oxo-butylmethyl) -acetyllamino]-ethylamino]-methyl-hoxy-nm-hoxy-nm-hoxy-nm-hoxy-nm-hoxy-nm-eth-n-nm-hydroxy-nm-hydroxy-pyrropoin. The conjugate is dosed by spectrometry to determine the coefficients of the two molecules and the average size of the sample is 78-180 cm2 and the average size is 43-180 cm2 M-180-113-113-113 mm.
Prepared by the following method, from 3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis-[(S)-2-eth-E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1c][1,4]benzo diazepine-5-one-8-yloxymethyl]-pyridin-4-yloxy) -ethyl]-piperazineyl}-1,1-dimethyl-4-oxo-butyl sulphanyl) -acetyllamino]-ethetoxy-ethetoxy-ethetoxy-ethetoxy-ethetoxy-ethetoxy-ethetoxy-propanoic
- What?
The test is performed on a single test vessel.
Prepared by the following method, starting from 3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis-[(S)-2-eth-(E) -ylidene-7-dimethoxy-1,2,3,11a-tetra hydro-pyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]-pyridine-4-yloxy) -ethyl]-piperazine-1-yl}-1,1-dimethyl-4-oxo-butyl sulfanyl) -acetyllamino]-ethyoxy-hoxy-hoxy-ethy]-hoxy-methylpropanoate
- What?
The test is performed on a single test vessel.
Prepared as follows from 3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis-hydroxymethyl-pyridine-4-yloxy) -ethyl]-piperazine-1-yl}-1,1-dimethyl-4-oxo-butyl sulphanyl) -acetylamino]-ethoxy}-ethoxy) -ethoxy]-ethoxy}-methyl propanoate
- What?
The following are the values of the values of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the
Prepared as shown in Example 2 from 4- ((2-[4-(4-mercapto-4-methyl) -pentanoyl) -piperazine-1-yl]-ethoxy)-2,6-bis- ((hydroxymethyl) -pyridine
RMN 1H (500 MHz, DMSO-d6): all signals are wide with 1.22 (s, 6 H) ; 1.71 (m, 2 H) ; 2.20 to 2.58 (m partially masked, 8 H) ; 2.75 (m, 2 H) ; 3.12 (m, 2 H) ; 3.19 (m, 2 H) ; 3.47 to 3.53 (m, 18 H) ; 3.60 (m, 5 H) ; 4.18 (m, 2 H) ; 4.46 (m, 4 H) ; 5.31 (m, 2 H) ; 6.86 (s, 2 H) ; 8.00 (m, 1 H).
Prepared as shown in Example 2 from 4- ((2-[4-(4-methyl-4-methyldisulfanyl) -pentanoyl) -piperazine-1-yl]-ethoxy) --2,6-bis- ((hydroxymethyl) -pyridine
- What?
The frequency range of the measurement is defined as the frequency range of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measur
600 mg of 4- ((182-piperazine-1-yl-ethoxy)-2,6-bis- ((hydroxymethyl) pyridine in solution in 12 mL DMF are added to 344 μL of TEA and then, after 10 min of agitation, 748 mg of 4-methyl-4-methyl disulfanyl-pentanoic acid, 417 μL of diisopropylcarbodiimide and 69 mg of 1-hydroxybenzotriazole hydrate. After 15 h at TA, the mixture is concentrated under PREt, added to 15 mL of water and extracted 2x with AcOMS. The organic phases are partially concentrated under PRSO4, concentrated and separated by superphysic photography (Analogue: Flash: 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4
After 12 h at TA, the resulting precipitate is recovered by filtration on a sintered glass, then taken up in MeOH concentrated under PR and diluted in 40 mL of a mixture of MeOH/water 1/1. The resulting solution is deposited on Mega-SCX, 25 150GMML (Varian). After washing the phase with MeOH, the base of interest is removed with a solution of 2N ammonia in the trymethyl methanol. The phase is: : PROH: 3 m; ; ; ; ; ; ; 4,0 m; ; ; 4,0 m; ; ; 4,0 m; ; 4,0 m; ; 4,0 m; ; 4,0 m; ; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0 m; 4,0; 4,0 m; 4,0; 4,0 m; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 4,0; 2,0; 2,0; 2,0; 2,0; 2,0; 2,0; 2,0; 2,0; 2,0; 2,0; 2,0
3,1 g of 4-[2-(4-tert-butoxycarbonyl-piperazine-1-yl) ethoxy]-pyridine-2,6-dicarboxylate of diethyl in solution in 105 mL of EtOH, 779 mg of sodium borohydride and 2,29 g of CaCl2 are added. After 3 h at TA, the mixture is hydrolysed and concentrated under PR. The resulting residue is added with water and the resulting aqueous phase is extracted 4x AcOEt. The organic phases collected with the diethyl are then concentrated with an aqueous solution saturated with NaCl under PR. 2,4 g of 4-(2-(4-tert-butoxycarbonyl-pipyridine-1-yl are obtained: : 2,6-d-hydroxy-2-d; 4,6-d-hydroxy-2-d; 4,6-d-hydroxy-d; 4,6-d-hydroxy-d; 4,6-d-hydroxy-d; 4,6-d-hydroxy-d; 4,6-d-hydroxy-d; 4,6-d-hydroxy-d; 4,6-d-hydroxy; 4,6-d-hydroxy; 4,6-d-hydroxy; 4,6-d; 4,6-d-hydroxy; 4,6-d-hydroxy; 4,6-d; 4,6-d-hydroxy; 4,6-d (H, 4,5-H, 4,6-H, 2,6-H, 2,6-H, 2,6-H, 2,6-H, 2,6-H, 2,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 4,6-H, 2,6-H, 2,6-H, 2,6-H, 2,6-H, (H, 4,5-H, 4,6-H, 4,6-H, 2,6-H, 2,6-H, 2,6-H, 2,6-H, 2-H, 3-H, 3-H, 3-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H, 4-H
After 1 hour, the mixture is reduced to TA. After 1 hour of further agitation, the mixture is hydrolysed and the organic phase washed 2 times with water and then dried on MgSO4 and concentrated under PR. The resulting residue (6.7 g) is added with 140 mL of DMF and brought to 60°C. 190 mg of ethyl ethyl dister of chelidic acid (Scriminella, P. P., Tecto, U.; U.; Tecto, V.; Org.; J. Chemella, 1989, T.After concentration under PR, the mixture is hydrolysed, extracted 3x with AcOEt and the combined organic phases are washed with a saturated NaCl solution, concentrated under PR and purified by flash chromatography on silica (Analogix Super Flash SiO2 SF25-150g) using a gradient of 60 to 85% OEt in heptane, yielding 3.1 g of 4-[4-Actert-butoxycarbonyl-piperazin-1-yl) -oxy]pyridine-2,6-dicarboxylate of RMH 1 (400 MHz, DMSO-d = 1.34; Jt, 1.34; Jt, 1.37; Jt, 2.39; H, 2.44; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 1.34; Jt, 2.39; Jt, 2.39; Jt, 2.4; Jt, 2.4; Jt, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.t, 2.The time taken for the measurement of the measurement is the time taken for the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the meas
Prepared as shown in Example 1 from 4- ((2-piperazine-1-yl-ethoxy)-2,6-bis- ((hydroxymethyl) pyridine.
- What?
The frequency range of the measurement is defined as the frequency range of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measur
A conjugate is prepared by reacting hu2H11 and 3-{2-[2-(2-{2-[2-(2-{2-{[2-{[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-])]) 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-]) 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-]) 2-[2-[2-[2-[2-[2-[2-[2-]) 2-[2-[2-[2-[2-[2-[2-[2-[2-]) 2-[2-[2-[2-[2-[2-[2-]) 2-[2-[2-[2-[2-[2-[2-[2-]) 2-[2-[2-[2-[2-[2-[2-]]]]-[2-[2-[2-[2-]-]-[2-[2-[2-]-[3-]-[3-[4-]-[4-]-[4-]-[4-]-[4-]-[4-]-[4-]-[4-]-[5-]-[5-]-[5-]-[5-]-[5-]-[6-]-[6-]-[6-]-[6-]-[6-]-[6-]-[7-]-[7-]-[7-]-[7-]-[7-]-[7-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[8-]-[
Prepared by the reaction of 3-{2-[2-(2-{2-[2-(2-(2-{[[2-(2,6-bis-[(S)-2-eth-E) -ylidene-7-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1c][1,4]benzodiazepine-5-one-8-yloxymethyl]-pyridine-4-yloxy) -ethyl]-methyl-amino acid with:
- What?
The test is performed on a sample of the test chemical (s) and the test chemical (s) is performed on a sample of the test chemical (s).
Prepared as follows from 3-{2-[2-((2-{2-[2-(2-({[[2-(2-(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((())))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))
- What?
The test shall be carried out in accordance with the instructions given in Appendix 2 to this annex.
Prepared in the form of a solution in which the total content by weight of the active substance is less than 0,5% by weight of the active substance:
- What?
The frequency range of the measurement is as follows: RMN 1H (500 MHz, DMSO-d6): absorptions are wide with 1.20 (s, 6 H) ; 1.58 to 1.73 (m, 6 H) ; 2.38 (s, 3 H) ; 2.52 to 3.07 (m, 13 H) ; 3.36 to 3.63 (m, 23 H) ; 3.67 to 3.91 (m, 8 H) ; 4.06 to 4.26 (m, 6 H) ; 5.02 to 5.26 (m, 4 H) ; 5.33 to 5.60 (m, 2 H) ; 6.39 to 7.42 (m, 6 H) ; 7.76 (m, 2 H).
Prepared as specified in note 2 to this chapter from 3-{2-[2-(2-{2-[2-(2-iodo-acetyl) -methyl amino]-ethoxy}-ethoxy) -ethoxy]-ethoxy}-methyl propanoate.
- What?
The following are the parameters to be taken into account for the calculation of the maximum frequency of the rotary dial: RMN 1H (400 MHz, DMSO-d6): 50-50 rotamers with: 1.22 (s, 3H) ; 1.24 (s, 3H) ; 2.41 (s, 3H) ; 2.53 (t, J=6.2 Hz, 2H) ; 2.56 (m, 2H) ; 2.80 (s, 1H) ; 2.88 (t, J=5.9 Hz, 2H) ; 3.05 (s, 2H) ; 3.35 to 3.58 (m, 18 H) ; 3.59 (s, 3H) ; 3.62 (t, J=6.2 Hz, 2H) ; 4.13 (t, J=5.9 Hz, 2H) ; 4.45 (d, J=5.9 Hz, 4H) ; 5.29 (t, J=5.9 Hz, 2H) ; 6.85 (s, 2H) ; 2.C: L/B ; 3.35 to 3.58 (m, 18 H) ; 3.34 (s, 3H) ; 3.13 (t, J=6.2 Hz, 2H) ; 4.45 (d, J=5.9 Hz, 2H) ; 4.45 (t, J=5.9 Hz, 2H) ; 6.85 (m, J=5.9 Hz, 2H) ; 2.85 (s, 2H) ; 2.C: L/B ; 3.18 (m, 3H) ; 3.18 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
After 2 hours at TA, the mixture is concentrated under PR and purified by flash chromatography on silica (Analogix Super Flash SiO2 SF25-24g), using a gradient of 0 to 6% of Me in the DCM. 210 mg of 3-{2-[2-(((2-[2-iodo-acyl) -thyl-amino]-hoxy-methoxy-methoxy-methoxy-methoxy-hoxy-methoxy-methoxy-methoxy-methoxyprate is obtained: 1 μm; 0.83 μm; 3.83 μm; 3.63 μm; 3.63 μm; 3.63 μm; 3.63 μm; 3.56 μm; 3.63 μm; 3.63 μm; 3.56 μm; 3.63 μm; 3.56 μm; 3.63 μm; 3.56 μm; 3.63 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μm; 3.65 μs; 3.65 m; 3.65 m; 3.65 m; 3.65 m; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.65; 3.6; 3.6; 3.
After 12 h at TA, the mixture is concentrated under PR dissolved in a minimum of methanol and deposited on Mega BE-SCX, 10GM 60ML (Varian). After washing the phase with MeOH, the product of interest is elevated with a 2N ammonia solution in the methanol. The methanol/NH3 phase is concentrated under PR. This gives 270 mg of 3-methyl-4-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-methyl-3-
To a 500 mg solution of 3-[2-(2-{2-(tert-butoxycarbonyl-methyl-amino) -ethoxy]-ethoxy}-ethoxy) -ethoxy]-propanoic acid in 4.8 mL MeOH cooled to 0°C, add 2 mL of a 2M solution of trimethylsilyldiazomethane in the hexane. After 2 h, the mixture is neutralized by addition of 120 μL of silicon acetic acid and then concentrated PR and purified by flash chromatography on the flash (Analogix Super Flash SiO2 SF25-40g), using a gradient of 05% methanol in the DCM base. This results in 400 mg of 3-methylsilyldiazomethane at a frequency of 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.2-; 0.3-; 0.3-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4; 0.4-0.4
After 5 min of stirring, 150 μL of iodomethane are added. The mixture is then stirred 2 h to TA and then added 150 μL of additional iodomethane. After 12 h to TA, the mixture is hydrolysed and then brought to acid pH by addition of acetic acid at 0 °C. The aqueous phase is extracted with 3 trominoxyl acetone, the organic solvents: 3 mg of NaH; 3 mg of phenol + 38 mg of NaOH; and the resulting mixture is obtained by the same reaction with 3-OH-methyl acetone (OH-methyl-methyl-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl) = 1,2-OH-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl-methyl-methyl) = 1,2-OH-methyl-methyl-methyl-methyl) (OH-methyl-methyl-methyl-methyl-methyl-methyl) = 1,2-methyl-methyl-methyl-methyl-methyl-methyl-methyl) = 1,2-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-
The MDA-MB-231, MDA-A1 or HCT116 cells in their exponential growth phase are trypsinated and resuspended in their respective culture media (DMEM/F12 Gibco #21331, 10% SVF Gibco #10500-056, 2 nM Glutamine Gibco #25030 for MDA cells; DMEM Gibco #11960, 10% SVF Gibco #10500-056, 2 mM Glutamine Gibco #25030 for HCT116 cells). The cell suspension is seeded in 96 well culture plates Cytostar (GE Healthcare Europe, #RPN0163) in the culture medium containing complete serum at a concentration of 5000 cell/well (MDA-MBQ-231, MDA-ACT111, HQ116). After 4 successive incubations, the tomylicate is added to the triplets in a dilution of 4 drops.The cells are cultured for 3 days at 37°C in a 5% CO2 atmosphere in the presence of cytotoxic agents. On the 4th day, 10 μl of a thymidine 14C solution (0.1 μCi/well, Perkin Elmer #NEC56825000) is added to each well. Thymidine 14C incorporation is measured 96 h after the start of the experiment with a microbeta (Perkin Elmer) radioactive meter. The data are expressed as a % survival in the ratio between the count obtained with cytotoxically treated cells and that obtained with cells from wells (controlled by the culture medium alone).
| IC50 [nM] | |||
| Structure du composé de formule (IA) | HCT116 | MDA-A1 | MDA-MB231 |
| 0,030 | 1,991 | 0,009 | |
| 0,120 | 2,260 | 0,070 | |
| 0,127 | >10 | 0,084 | |
| 1,250 | >10 | 0,450 | |
The compounds tested for which ZbRb= -OMe are found to have potent anticancer activity suggesting that similar compounds with another ZbRb group may exhibit similar activity.
The cells in their exponential growth phase are trypsinated and resuspended in their culture medium (DMEM/F12 Gibco #21331, 10% SVF Gibco #10500-056, 2 nM Glutamine Gibco #25030). The cell suspension is seeded in 96-well Cytostar (GE Healthcare Europe, #RPNQ0163) culture plates in the complete culture medium containing serum at a density of 5000 cells/well. After 4 h incubation, successive dilutions of the cytotoxic antibody-immune conjugates are added to the wells at decreasing concentrations of 10-7 to 10-12 M (triple enlicate for each concentration). The cells are cultured at 37°C in an atmosphere with 5% of the cytotoxic antibody-immune conjugates for 3 days.On day 4, 10 μl of a thymidine 14C solution (0.1 μCi/well, Perkin Elmer #NEC56825000) is added to each well. Thymidine 14C incorporation is measured 96 h after the start of the experiment with a microbeta (Perkin Elmer) radioactive meter. The data is expressed as a % survival by the ratio of the count from cells treated by the immunoconjugate to cells from control wells (treated by the culture medium alone). In some experiments*, the hu2H11 nubular antibody was added to the wells at a concentration of 1 μM at the start of the experiment and the proliferation of measurements was as described above.
| IC50 [pM] | |||
| Structure | IC50 moyen | IC50 moyen (+hu2H11 nu *) | rapport IC50 |
| 31 | 540 | 17 | |
| 910 | 5095 | 6 | |
| 54 | 3981 | 74 | |
| 1255 | 9125 | 7 | |
| 68,5 | 1119 | 16 | |
The non-cleavable hu2H11 conjugates prepared with tomamycin dimers described in examples 3 and 5 of international application WO 09016516 tend to have their monomer purity decrease over time after several months of storage at 3-5°C. These conjugates, formulated in an aqueous buffer at pH 6.5 of 10 mM histidine concentration containing 10% sucrose and 5% NMP, with an average of 3 to 3,5 tomamycin dimers per antibody molecule, with an initial monomer purity of 97.5%, can lose 6 to 15% monomer purity in 6-8 months. This phenomenon was not observed with the conjugate in example 2 which has an average of 3.5 monomer tomamycin dimers in a general storage area of 4 months, thus suggesting that the objective is to maintain a better stability of 99% of the monomer purity, while maintaining the same general storage conditions.
Two conjugates of the same antibody hu2H11, rated C1 (prepared with tomycin dimer as described in example 5 of international application WO 09016516) and C2 (conjugated in example 2), were evaluated at 4 dose levels on measurable breast tumours MDA-MB-231 implanted in female SCID mice. The control groups were not treated. The doses of both conjugates were given in μg tomycin dimer/kg. They were administered at 80, 40, 20 and 10 μg/kg by intravenous bolus (IV) injection on day 13 for C1 and day 24 for C2.
For the assessment of conjugate anti-tumor activity, the animals were weighed daily and the tumors were measured twice weekly using a backstage foot. The evaluation of anti-tumor activity was performed at the Maximum Tolerated Dose (MTD). A dose that produced a 20% loss in nadir weight or 10% (or more) of conjugate-related mortality is considered toxic. The body weight of the animals includes the weight of the tumors. The tumor weight is calculated using the formula: mass (mg) = [length (mm) x width (mm) ]/2. The efficacy parameters are ΔT/C, percentage of regression, mean and partial regressions (ΔRP and partial regressions) and the number of tumors at the end of the study (ΔSST).
The evolution of tumour volume for each treated mouse (T) and control (C) is calculated for each tumour by subtracting tumour volume on study start day from tumour volume on the specified observation day. The mean ΔT is calculated for the treated group and the mean ΔC is calculated for the control group. The ratio ΔT/ΔC is then calculated and expressed as a percentage: ΔT/ΔC = (delta T/delta C) x 100
A dose is considered to be therapeutically active for ΔT/ΔC less than 40% and very active for ATIAC less than 10%. When ΔT/ΔC is less than 0, the dose is considered to be highly active and the percentage of regression is then calculated (according to Plowman J, Dykes DJ, Hollingshead M, Simpson-Herren L and Alley MC.
The percentage of tumour regression is defined as the percentage of tumour volume decrease in the treated group on the specified observation day compared to its volume on the first day of treatment. At a specific time t and for each animal, the percentage of tumour regression is calculated. The average percentage of regression is then calculated for the group.
Partial regression (RP): Regressions are defined as partial when the decrease in tumor volume reaches 50% of the tumor volume at the start of treatment.
Complete regression (CR): Complete regression is obtained when the tumor volume = 0 mm3 (CR is considered to be present when the tumor volume cannot be measured)
SST: Tumor-free mice are defined as mice with no detectable tumour at the end of the study (beyond 100 days of treatment).
| Conjugué | Voie d'administration/ Dose en mL/kg par injection | Dose en µg/kg de dimère de tomaymycine (mg prot/kg) | Jour d'administration | Mortalité | Perte de poids moyenne en % au nadir (jour) | ΔT/ΔC moyen en %(jour) | Pourcentage de régression moyen (jour) | Régressions | |||
| Partielle | Complète | ||||||||||
| C2 | IV | 80 (2,98) | 24 | 4/6 | - | - | - | - | - | - | Toxique |
| DAR 3,5 | 10 mL/kg | 40 (1,49) | 0/6 | -5,0(31) | <0 (46) | 96,2(46) | 6/6 | 1/6 | 1/6 | DMT.hautemement actif | |
| 20 (0,75) | 0/6 | -4,4(31) | <0 (46) | 66,4 (46) | 4/6 | 0/6 | 0/6 | Hautemement actif | |||
| 10 (0,37) | 0/6 | -4,0 (31) | 14 (46) | - | 0/6 | 0/6 | 0/6 | Actif | |||
| Abbréviations: DMT = Dose Maximale Tolérée, SST = Souris sans tumeur à la fin de l'étude |
Using a single dose regimen, the highest dose tested for these two conjugates in this study (80 μg/ kg) was toxic, inducing weight loss and mortality.
At DMT (40 μg/kg), C1 is highly active and induces a weight loss of 9.6% at nadir, a ΔT/ΔC of 7% and 3 RP for 5 SCID mice.
At DMT (40 μg/kg), C2 is highly active and induces a nadir 5% weight loss, 96.2% tumor regression with 6 RP for 6 mice including 1 SST. It is also highly active at the lower dose of 20 μg/kg with 66.4% tumor regression and 4 RP for 6 mice. Antirumor activity was also observed at the lowest dose of 10 μg/kg without inducing regression or RP.
In conclusion, C2 is found to have strong anticancer activity and to have shown better activity than C1 with tumor regression, RP and SST to DMT, which were not observed with C1 at the same doses.
Claims (34)
- Compound of formula (I): in which:• represents a single bond or a double bond with the condition that, if represents a single bond, then:❖---- represents a single bond;❖ U and/or U', which are identical or different, represent(s), independently of one another, H;❖ W and/or W', which are identical or different, represent(s), independently of one another: OH, -OR, -OCOR, -COOR, -OCOOR, -OCONRR', a cyclic carbamate such that N10 and C11 are included in a ring, -NRCONRR', -OCSNHR, a cyclic thiocarbamate such that N10 and C11 are included in a ring, - SH, -SR, -SOR, -SOOR, -SO3 -, -NRSOOR', -NRR', a cyclic amine such that N10 and C11 are included in a ring, -NROR', -NRCOR', -N3, -CN, Hal or a trialkylphosphonium or triarylphosphonium group;• R1, R2, R1' and R2' , which are identical or different, represent, independently of one another: H, Hal or a (C1-C6)alkyl group optionally substituted by one or more substituent(s) chosen from: Hal, CN, NRR', CF3, OR, an aryl or heteroaryl group, or S(O)qR with q = 0, 1 or 2; or else• R1 and R2 and/or R1' and R2' together form respectively a double bond =CH2 or =CH-CH3;• Y and Y', which are identical or different, represent, independently of one another, H or OR;• M represents CH or N;• ALK and ALK', which are identical or different, represent, independently of one another, a (C1-C6)alkylene group;• R and R' represent, independently of one another, H or a (C1-C6)alkyl or aryl group optionally substituted by one or more substituent(s) chosen from: Hal, CN, NRR', CF3, OR or an aryl or heteroaryl group;• L represents:❖ the -L1-L2- group in which L1 is attached to the aromatic ring comprising M via the ALK or OALK group and represents one of the following groups: -ALK-S- -O-ALK-NR3-ALK-S- and L2 represents the -CH2C (=O) -NR3- (CH2CH2O)i-ALK-group attached to L1 via -CH2C(=O)-; or else❖ the -O-ALK-NR3-ALK-S- (CH2CH2O)i-ALK- group attached to the aromatic ring comprising M via the OALK group;• R3 represents H or a (C1-C6)alkyl group;• i represents an integer ranging from 1 to 40, rather from 1 to 20, preferably from 1 to 10;• Zb represents a single bond, -0- or -NH- and Rb represents H or a (C1-C6)alkyl, (C3-C7)cycloalkyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl group or else Zb represents a single bond and Rb represents Hal.
- Compound according to Claim 1 of formula:
- Compound according to Claim 1 of formula (IA) or (IB) :
- Compound according to Claim 1 to Claim 3, in which Y and Y' represent a (C1-C4)alkoxy group, more particularly a methoxy group.
- Compound according to Claim 1 to Claim 4, in which L is chosen from: -ALK-S-CH2C (=O) -NH- (CH2CH2O)i-CH2CH2- -O-ALK-NR3-ALK-S-CH2C (=0) -NH- (CH2CH2O) i-CH2CH2 -O-ALK-NR3-ALK-S-(CH2CH2O)i-CH2CH2-
- Compound according to Claim 1 or Claim 5, in which L is chosen from:
- Compound according to Claim 1 or Claim 5, in which L is chosen from:
- Compound according to Claim 1 to Claim 7, in which -COZbRb represents -COOH, -COO(C1-C6)alkyl, -COOCH3, -COOCH2CH=CH2, the group, in which IG represents at least one inductive group, more particularly or
- Compound according to Claim 1, chosen from one of the following:
- Process for the preparation of a conjugate, consisting in:(i) bringing into contact and allowing to react an optionally buffered aqueous solution of a binding agent and a solution of a compound as defined in one of Claims 1 to 9;(ii) and then optionally separating the conjugate formed in stage (i) from the compound of formula (I) and/or from the binding agent not having reacted and/or from the aggregates which might be formed.
- Process according to Claim 10, in which the - C(=O)ZbRb group is reactive with regard to the chemical groups present on the binding agent, in particular with regard to the amino groups present on an antibody, so as to ensure the attachment of the compound of formula (I) to the binding agent by formation of a covalent bond.
- Process according to Claim 10 or Claim 11, in which stage (ii) consists in:- separating the conjugate which has formed in stage (i) from the unreacted binding agent and from the aggregates possibly present in the solution; or else- separating the conjugate of stage (i) only from the unreacted compound of formula (I) and from the aggregates which might be formed and leaving in the solution the binding agent which might not have reacted.
- Process according to one of Claims 10 to 12, in which the binding agent is a ligand, a protein, an antibody, more particularly a monoclonal antibody, a protein or antibody fragment, a peptide, an oligonucleotide or an oligosaccharide.
- Process according to Claim 10 to Claim 13, in which the reaction takes place at a temperature of between 20 and 40°C and/or the duration of the reaction varies between 1 and 24 h.
- Process according to one of Claims 10 to 14, in which, after stage (i) or (ii), the solution of the conjugate is subjected to a stage (iii) of ultrafiltration and/or of diafiltration.
- Conjugate capable of being obtained by the process according to one of Claims 10 to 15.
- Conjugate according to Claim 16, in which the binding agent is an antibody, preferably a monoclonal antibody, characterized by a mean DAR of between 1 and 10, preferably between 1.5 and 7, the DAR being calculated by the equation DAR = cD/cA with:
- Use of a compound according to one of Claims 1 to 9 in the preparation of a binding agent to which is covalently attached, in the position para to M, the dimer of formula:
- Use according to Claim 18, in which the binding agent is an antibody, preferably a monoclonal antibody.
- Use according to Claim 18 or Claim 19, in which the dimer has the formula:
- Use according to Claim 18 to Claim 20, in which Y and Y' represent a (C1-C4)alkoxy group, more particularly a methoxy group.
- Binding agent to which was covalently attached, in the position para to M, the dimer of formula: after reaction of a compound as defined in one of Claims 1 to 9 with the binding agent.
- Binding agent according to Claim 22, in which the dimer is as defined in either of Claims 20 and 21.
- Binding agent according to Claim 23, having affinity for an antigen or group of antigens located on cancer cells or stromal cells associated with a tumour.
- Binding agent according to Claim 22 to Claim 24, which is an antibody, preferably a monoclonal antibody.
- Solution of conjugate capable of being obtained by the process according to one of Claims 10 to 15.
- Compound according to one of Claims 1 to 9 for use as anticancer agent.
- Conjugate according to Claim 16 or Claim 17 for use as anticancer agent.
- Pharmaceutical composition comprising a compound according to one of Claims 1 to 9 or else a conjugate according to Claim 16 or Claim 17 and at least one excipient.
- Use of a compound of formula P2 or else a compound of formula: in which L* is chosen from: -ALK-SH ; -O-ALK-NR3-ALK-SH ; in which formulae:■ L, M, ALK, ALK' , R3, Zb and Rb are as defined in one of Claims 1 to 9;■ E and E' represent, independently of one another, an -OH group or a leaving group;as intermediate in the preparation of a compound of formula (I) as defined in one of Claims 1 to 9.
- Use according to Claim 30, in which the leaving group is chosen from a halogen atom or a mesylate, tosylate, nosylate or -OPPh3 + group.
- Use of a compound chosen from one of the following: it being understood that the -COOH end functional group can be replaced by a -COO(C1-C6)alkyl end functional group, in particular a -COOMe end functional group, and that the -SH end functional group can be replaced by a disulphide functional group, in particular an -SSMe functional group, as intermediate in the preparation of a compound of formula (I) as defined in one of Claims 1 to 9.
- Use of a compound according to one of Claims 1 to 9 in the manufacture of an anticancer agent.
- Use of a conjugate according to Claim 16 or Claim 17 in the manufacture of an anticancer agent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0904043 | 2009-08-25 | ||
| FR0904368 | 2009-09-11 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1171741A HK1171741A (en) | 2013-04-05 |
| HK1171741B true HK1171741B (en) | 2015-01-23 |
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