Process for the preparation of tubulysins and intermediates thereof

Abstract

Improved processes for the preparation of tubulysin compounds, tubulysin drug linker compounds, and their intermediates are disclosed.

What is claimed is: 
     
         1 . A compound having the structure of: 
       
         
           
           
               
               
           
         
         optionally in salt form, wherein 
         R 2  is selected from the group consisting of —H, C 1 -C 4  alkyl and R 2A , wherein R 2A  is —C(═O)R 2B , wherein R 2B  is optionally substituted saturated C 1 -C 4  alkyl or optionally substituted C 3 -C 6  unsaturated alkyl; 
         R 3  is —CH 3 , —CH 2 CH 3  or —CH 2 CH 2 CH 3    
         R 7  is —H or C 1 -C 4  saturated alkyl. 
       
     
     
         2 . The compound of  claim 1 , wherein R 2  is —H, —CH 2 CH 3  or —C(═O)R 2B ; R 3  is —CH 3  —CH 2 CH 3  or —CH 2 CH 2 CH 3 ; and R 7  is —H, —CH 3  or —CH 2 CH 3 . 
     
     
         3 . The compound of  claim 1 , wherein R 2  is —C(═O)R 2B , R 3  is —CH 3 , or —CH 2 CH 2 CH 3  and R 7  is —H or —CH 2 CH 3 . 
     
     
         4 . The compound of  claim 1 , wherein R 2  is —CH 2 CH 3 , R 3  is —CH 3 , or —CH 2 CH 2 CH 3  and R 7  is H or —CH 2 CH 3 . 
     
     
         5 . The compound of  claim 1 , wherein R 2  is —H, R 3  is —CH 3 , or —CH 2 CH 2 CH 3  and R 7  is —H or —CH 2 CH 3 . 
     
     
         6 . The compound of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         7 . The compound of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The compound of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The compound of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         10 . The compound of  claim 1 , wherein the compound has the structure of: 
       
         
           
           
               
               
           
         
       
     
     
         11 . A composition comprised of a tubuvaline compound of (R,R)-Formula 1a, optionally in salt form, having the structure of: 
       
         
           
           
               
               
           
         
         and one or more optical isomers thereof as optical impurities, wherein the (R,R)-Formula 1a tubuvaline compound, or salt thereof, is the predominate optical isomer, and wherein its corresponding enantiomer, (S,S)-Formula 1a, optionally in salt form, is the major optical impurity, which has the structure of: 
       
       
         
           
           
               
               
           
         
         wherein: 
         the circled Ar is a 1,3-phenylene or a 5- or 6-membered nitrogen-containing 1,3-heteroarylene, optionally substituted at the remaining positions; 
         R 1  is phenyl, t-butyl, 9-fluorenyl or allyl, optionally substituted, or other moeity so that R 1 —OC(═O)— is a suitable nitrogen protecting group; 
         R 3  is optionally substituted saturated C 1 -C 8  alkyl, optionally substituted unsaturated C 3 -C 8  alkyl or optionally substituted C 3 -C 8  heteroalkyl; 
         R 6  is optionally substituted C 1 -C 8  alkyl; and 
         R 7  is optionally substituted saturated C 1 -C 20  alkyl, optionally substituted unsaturated C 3 -C 20  alkyl, optionally substituted C 3 -C 20  heteroalkyl, optionally substituted C 2 -C 20  alkenyl, optionally substituted C 3 -C 20  heteroalkenyl, optionally substituted C 2 -C 20  alkynyl, optionally substituted C 3 -C 20  heteroalkynyl, optionally substituted C 6 -C 24  aryl, optionally substituted C 5 -C 24  heteroaryl, optionally substituted C 3 -C 20  heterocyclyl, or other moeity so that R 7 —O— provides for a suitable carboxylic acid protecting group, 
         in particular, (R,R)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
         and (S,S)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
         wherein: 
         X 1  is ═N—; and 
         X 2  is S, O, or N(R X2 )—, or 
         X 1  is ═C(R X1 )—; and 
         X 2  is NR X2 , 
         R X1  and R X2  are independently selected from the group consisting of —H, —CH 3  or —CH 2 CH 3 ; and 
         wherein the remaining variable group are as previously defined, 
         more particularly, (R,R)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
         and (S,S)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         12 . The composition of  claim 11 , wherein the tubuvaline composition is comprised of the (R,R)-Formula 1a tubuvaline compound, or salt thereof, as the predominate optical isomer and has no more than about 5% w/w of the diastereomeric tubuvaline compound, (R,S)-Formula 1a, relative to the total amount of optical isomers present in the composition, in particular, no more than about 1.5% w/w, more particularly, no more that about 1.0% w/w, or is essentially free of the diastereomer, as determined by chiral HPLC, wherein the diastereomeric tubuvaline compound, optionally in salt form, has the structure of: 
       
         
           
           
               
               
           
         
         in particular (R,S)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
         wherein the variable groups remain as previously defined for (R,R)-Formula 1a and (S,S)-Formula 1a, 
         more particularly, (R,S)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         13 . The composition of  claim 12 , wherein the composition is essentially free of the diastereomer (R,S)-Formula 1a, and is essentially free of its corresponding enantiomer, (S,R)-Formula 1a, as determined by chiral HPLC, which has the structure of: 
       
         
           
           
               
               
           
         
         in particular, (S,R)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
         wherein the variable groups remain as previously defined for (R,R)-Formula 1a, (S,S)-Formula 1a and (R,S)-Formula 1a, 
         more particularly, (S,R)-Formula 1a has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         14 . The composition of  claim 11 ,  12  or  13 , wherein the composition contains no more that about 3% w/w, in particular no more than about 2% w/w or no more than about 1.5% w/w of the optical impurity of (S,S)-Formula 1a, as determined by chiral HPLC, relative to the total amount of optical isomers present in the composition. 
     
     
         15 . A composition comprised of a tubuvaline compound of (R,R)-Formula 2, optionally in salt form, having the structure of: 
       
         
           
           
               
               
           
         
         and one or more optical isomers thereof as optical impurities, wherein the (R,R)-Formula 2 tubuvaline compound, or salt thereof, is the predominate optical isomer, and wherein its corresponding enantiomer, (S,S)-Formula 2, optionally in salt form, is the major optical impurity, which has the structure of: 
       
       
         
           
           
               
               
           
         
         wherein: 
         the circled Ar is a 1,3-phenylene or a 5- or 6-membered nitrogen-containing 1,3-heteroarylene, optionally substituted at the remaining positions; 
         R 1  is phenyl, t-butyl, 9-fluorenyl or allyl, optionally substituted, or other moeity so that R 1 —OC(═O)— is a suitable nitrogen protecting group; 
         R 3  is optionally substituted saturated C 1 -C 8  alkyl, optionally substituted unsaturated C 3 -C 8  alkyl or optionally substituted C 3 -C 8  heteroalkyl; 
         R 6  is optionally substituted C 1 -C 8  alkyl; and 
         R 7  is optionally substituted saturated C 1 -C 20  alkyl, optionally substituted unsaturated C 3 -C 20  alkyl, optionally substituted C 3 -C 20  heteroalkyl, optionally substituted C 2 -C 20  alkenyl, optionally substituted C 3 -C 20  heteroalkenyl, optionally substituted C 2 -C 20  alkynyl, optionally substituted C 3 -C 20  heteroalkynyl, optionally substituted C F -C 24  aryl, optionally substituted C 5 -C 24  heteroaryl, optionally substituted C 3 -C 20  heterocyclyl, or other moeity so that R 7 —O— provides for a suitable carboxylic acid protecting group, 
         in particular, wherein (R,R)-Formula 2 has the structure of: 
       
       
         
           
           
               
               
           
         
         or a salt thereof, and (S,S)-Formula 2 has the structure of: 
       
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
         X 1  is ═N—; and 
         X 2  is S, O, or N(R X2 )—, or 
         X 1  is ═C(R X1 )—; and 
         X 2  is NR X2 , 
         wherein R X1  and R X2  are independently selected from the group consisting of —H, —CH 3  or —CH 2 CH 3 ; 
         and the remaining variable groups are as previously defined, 
         more particularly, (R,R)-Formula 2 has the structure of: 
       
       
         
           
           
               
               
           
         
         or a salt thereof, and (S,S)-Formula 2, optionally in salt form, has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         16 . The composition of  claim 15 , wherein the tubuvaline composition is comprised of the (R,R)-Formula 2 tubuvaline compound, or salt thereof, as the predominate optical isomer and has no more than about 5% w/w of the diastereomeric tubuvaline compound, (R,S)-Formula 2, relative to the total amount of optical isomers present in the composition, in particular, no more than about 1.5% w/w, more particularly, no more that about 1.0% w/w, or is essentially free of the diastereomer, as determined by chiral HPLC, wherein the diastereomeric tubuvaline compound, optionally in salt form, has the structure of (R,S)-Formula 2: 
       
         
           
           
               
               
           
         
         wherein the variable groups remain as previously defined, 
         in particular, wherein (R,S)-Formula 2 has the structure of: 
       
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
         X 1  is ═N—; and 
         X 2  is S, O, or N(R X2 )—, or 
         X 1  is ═C(R X1 )—; and 
         X 2  is NR X2 , 
         R X1  and R X2  are independently selected from the group consisting of —H, —CH 3  or —CH 2 CH 3 ; and 
         wherein the remaining variable groups are as previously defined, 
         more particularly, wherein (R,S)-Formula 2, optionally in salt form, has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         17 . The composition of  claim 16 , wherein the composition is essentially free of the diastereomer, (R,S)-Formula 2, or salt thereof, and is essentially free, as determined by chiral HPLC, of its corresponding enantiomer, (S,R)-Formula 2, which has the structure of: 
       
         
           
           
               
               
           
         
         or salt thereof, in particular, wherein (S,R)-Formula 2 has the structure of: 
       
       
         
           
           
               
               
           
         
         or salt thereof, wherein the variable groups are as previously defined for (R,R)-Formula 1a, (R,S)-Formula 1a and (S,S)-Formula 1a, 
         more particularly, wherein (S,R)-Formula 2, optionally in salt form, has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         18 . The composition of  claim 15 ,  16  or  17 , wherein the composition contains no more that about 3% w/w, in particular no more than about 2% w/w or no more than about 1.5% w/w of the optical impurity of (S,S)-Formula 2, as determined by chiral HPLC, relative to the total amount of optical isomers present in the composition. 
     
     
         19 . A composition comprised of a tubuvaline compound of (R,R)-Formula 2a, optionally in salt form, having the structure of: 
       
         
           
           
               
               
           
         
         and one or more optical isomers thereof as optical impurities, wherein the (R,R)-Formula 2a tubuvaline compound, or salt thereof, is the predominate optical isomer, and wherein its corresponding enantiomer, (S,S)-Formula 2a, optionally in salt form, is the major optical impurity, which has the structure of: 
       
       
         
           
           
               
               
           
         
         wherein: 
         the circled Ar is a 1,3-phenylene or a 5- or 6-membered nitrogen-containing 1,3-heteroarylene, optionally substituted at the remaining positions; 
         R 1  is phenyl, t-butyl, 9-fluorenyl or allyl, optionally substituted, or other moeity so that R 1 —OC(═O)— is a suitable nitrogen protecting group; 
         R 3  is optionally substituted saturated C 1 -C 8  alkyl, optionally substituted unsaturated C 3 -C 8  alkyl or optionally substituted C 3 -C 8  heteroalkyl; 
         R 6  is optionally substituted C 1 -C 8  alkyl; and 
         R 7  is optionally substituted saturated C 1 -C 20  alkyl, optionally substituted unsaturated C 3 -C 20  alkyl, optionally substituted C 3 -C 23  heteroalkyl, optionally substituted C 2 -C 20  alkenyl, optionally substituted C 3 -C 20  heteroalkenyl, optionally substituted C 2 -C 20  alkynyl, optionally substituted C 3 -C 20  heteroalkynyl, optionally substituted C 6 -C 24  aryl, optionally substituted C 5 -C 24  heteroaryl, optionally substituted C 3 -C 20  heterocyclyl, or other moeity so that R 7 —O— provides for a suitable carboxylic acid protecting group, 
         in particular, wherein (R,R)-Formula 2a has the structure of: 
       
       
         
           
           
               
               
           
         
         or salt thereof, and (S,S)-Formula 2a has the structure of: 
       
       
         
           
           
               
               
           
         
         or salt thereof, wherein: 
         X 1  is ═N—; and 
         X 2  is S, O, or N(R X2 )—, or 
         X 1  is ═C(R X1 )—; and 
         X 2  is NR X2 , 
         wherein R X1  and R X2  are independently selected from the group consisting of —H, —CH 3  or —CH 2 CH 3 ; and 
         wherein the remaining variable groups are as previously defined, 
         more particularly, wherein (R,R)-Formula 2a has the structure of: 
       
       
         
           
           
               
               
           
         
         or salt thereof, and (S,S)-Formula 2a, optionally in salt form, has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         20 . The composition of  claim 19 , wherein the tubuvaline composition is comprised of the (R,R)-Formula 2a tubuvaline compound, or salt thereof, as the predominate optical isomer and has no more than about 5% w/w of the diastereomeric tubuvaline compound, (R,S)-Formula 2a, relative to the total amount of optical isomers present in the composition, in particular, no more than about 1.5% w/w, more particularly, no more that about 1.0% w/w, or is essentially free of the diastereomer, as determined by chiral HPLC, wherein the diastereomeric tubuvaline compound, optionally in salt form, has the structure of (R,S)-Formula 2a: 
       
         
           
           
               
               
           
         
         in particular, wherein (R,S)-Formula 2a has the structure of: 
       
       
         
           
           
               
               
           
         
         or salt thereof, wherein the variable groups remain as previously defined for (R,R)-Formula 2a and (S,S)-Formula 2a, 
         more particularly, (R,S)-Formula 2a, optionally in salt form, has the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         21 . The composition of  claim 20 , wherein the composition is essentially free of the diastereomer, (R,S)-Formula 2a, and is essentially free, as determined by chiral HPLC, of its corresponding enantiomer, (S,R)-Formula 2a, which has the structure of: 
       
         
           
           
               
               
           
         
         in particular, wherein (S,R)-Formula 2a has the structure of: 
       
       
         
           
           
               
               
           
         
         or salt thereof, wherein the variable groups remain as previously defined for (R,R)-Formula 2a, (S,S)-Formula 2a and (R,S)-Formula 2a, 
         more particularly, (S,R)-Formula 2a, optionally in salt form, has the structure of 
       
       
         
           
           
               
               
           
         
       
     
     
         22 . The composition of  claim 19 ,  20  or  21 , wherein the composition contains no more that about 3% w/w, in particular no more than about 2% w/w or no more than about 1.5% w/w of the optical impurity of (S,S)-Formula 2a, as determined by chiral HPLC, relative to the total amount of optical isomers present in the composition. 
     
     
         23 . A method for preparing a composition of any one of  claims 11 - 22 , the method comprising the steps of:
 (a) contacting a compound of Formula A:   
       
         
           
           
               
               
           
         
         wherein R 7  is optionally substituted saturated C 1 -C 20  alkyl, optionally substituted unsaturated C 3 -C 20  alkyl, optionally substituted C 3 -C 20  heteroalkyl, optionally substituted C 2 -C 20  alkenyl, optionally substituted C 3 -C 20  heteroalkenyl, optionally substituted C 2 -C 20  alkynyl, optionally substituted C 3 -C 20  heteroalkynyl, optionally substituted C 6 -C 24  aryl, optionally substituted C 5 -C 24  heteroaryl, optionally substituted C 3 -C 20  heterocyclyl, or other moeity so that R 7 —O— provides for a suitable carboxylic acid protecting group, 
         with a compound of Formula B:
   R 3 NHC(O)OR 1   (B),
 
 
         in a suitable polar, aprotic solvent in the presence of a suitable transition metal (II) or transition metal (III) catalyst, in particular, one comprising a transition metal selected from the group consisting of Cu(II), Zn(II) and Yb(III), so as to form a composition comprised of an enantiomeric mixture of tubuvaline intermediates represented by Formula AB: 
       
       
         
           
           
               
               
           
         
         (b) contacting the enantiomeric mixture with a suitable chiral reducing agent so as to form a composition comprised of essentially an equimolar mixture of diastereomers, wherein the diastereomeric mixture is represented by Formula R-1a, 
       
       
         
           
           
               
               
           
         
         wherein the composition is further comprised of essentially an equimolar mixture of optical impurities that are enantiomers of the diastereomers. 
         (b′) separating the diastereomers from the composition of the Formula R-1a diastereomeric mixture so that the composition, which is comprised of (R,R)-Formula 1a as the predominate optical isomer and having (S,S)-Formula 1a as the major optical impurity, is obtained, wherein the predominate optical isomer and the major optical impurity, each optionally in salt form, have the structures of: 
       
       
         
           
           
               
               
           
         
         respectively, wherein the variable groups of AB, Formula R-1a, R,R-Formula 1a and (S,S)-Formula 1a retain the previous meanings from the Formula A and Formula B compounds. 
       
     
     
         24 . The method of  claim 23 , wherein the transition (II) metal catalyst is comprised of Cu(II), in particular Cu(OTf) 2 , Cu(SbF 6 ) 2 , or CuCl 2 , more particularly Cu(OTf) 2 . 
     
     
         25 . The method of  claim 23  or  24 , wherein the suitable polar, aprotic solvent is acetonitrile, dichloromethane, THF, dioxane, or a mixture of two or three of these solvents, in particular, dichlormethane. 
     
     
         26 . The method of  claim 23 ,  24  or  25 , wherein the chiral reducing agent is a chiral oxazaborolidine prepared from contacting BH 3 -DMS in THF with a suitable chiral ligand, in particular (S)-(−)-CBS. 
     
     
         27 . A method for preparing a composition of any one of  claims 15 - 18 , the method comprising the steps of:
 (c) contacting the composition obtained from steps (a), (b) and step (b′) of any one of  claims 23 - 26  with an suitable hydrolysis agent, wherein the predominate optical isomer of the composition so obtained is (R,R)-Formula 2, optionally in salt form, having the structure of:   
       
         
           
           
               
               
           
         
         and the major optical impurity is (S,S)-Formula 2, optionally in salt form, having the structure of: 
       
       
         
           
           
               
               
           
         
         wherein the variable groups of R,R-Formula 2 and S,S-Formula 2 retain the previous meanings from the Formula A and Formula B compounds. 
       
     
     
         28 . A method for preparing a composition of any one of  claims 19 - 22 , the method comprising the steps of:
 (c) contacting the composition obtained from steps (a), (b) and step (b′) of any one of  claims 23 - 26  with an suitable hydrolysis agent to obtain a composition comprised of (R,R)-Formula 2 as the predominate optical isomer, optionally in salt form, which has the structure of:   
       
         
           
           
               
               
           
         
         and further comprised of its enantiomer, optionally in salt form, as the major optical impurity, which has the structure of: 
       
       
         
           
           
               
               
           
         
         and 
         step (d): contacting the composition so obtained with a suitable acylating agent to obtain a composition comprised (R,R)-Formula 2a as the predominate optical isomer and (S,S)-Formula 2a as the major optical impurity, wherein the predominate optical isomer and the predominate optical isomer, each optionally in salt form, have the structurers of: 
       
       
         
           
           
               
               
           
         
         wherein R 2B  is saturated C 1 -C 6  alkyl, unsaturated C 3 -C 8  alkyl, C 2 -C 8  alkenyl or C 2 -C 4  alkynyl, optionally substituted, in particular —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , —CH 2 CH═CH 2 , —CH 2 CH(CH 3 ) 2 , —CH 2 C(CH 3 ) 3 , —CH 2 C(CH 3 )═CH 2 , —CH═CH 2  or —CHC≡CH, more particularly, —CH 3 , and the remaining variable groups retain the previous meanings of the Formula A and Formula B compounds. 
       
     
     
         29 . The method of any one of  claims 23 - 28 , wherein the optical purity of the composition from step (b′) is substantially or essentially retained by the composition obtained from step (c) and/or step (d). 
     
     
         30 . The method of any one of  claims 23 - 29 , wherein said step (b′) separation is by silica gel flash chromatography. 
     
     
         31 . The method of claim any one  claims 23 - 30 , wherein the circled Ar is a 5-membered nitrogen-containing 1,3-heteroarylene, optionally substituted at the remaining positions. 
     
     
         32 . The method of claim any one  claims 23 - 32 , wherein compound A and compound B of step (a) have the structures of: 
       
         
           
           
               
               
           
         
         respectively, wherein, 
         X 1  is ═N—; and 
         X 2  is S, O, or N(R X2 )—, or 
         X 1  is ═C(R X1 )—; and 
         X 2  is NR X2 , 
         Wherein R X1  and R X2  are independently selected from the group consisting of —H, —CH 3  or —CH 2 CH 3 ; 
         R 1  is phenyl, t-butyl, 9-fluorenyl or allyl, optionally substituted, or other moeity so that R 1 —OC(═O)— is a suitable nitrogen protecting group, in particular t-butyl; and 
         R 3  is optionally substituted saturated C 1 -C 6  alkyl, optionally substituted unsaturated C 3 -C 6  alkyl or optionally substituted C 3 -C 6  heteroalkyl, in particular —CH 3  or —CH 2 CH 2 CH 3 ; 
         R 6  is C 1 -C 6  alkyl, in particular —CH(CH 3 ) 2 ; and 
         R 7  is optionally substituted saturated C 1 -C 20  alkyl, optionally substituted unsaturated C 1 -C 20  alkyl, optionally substituted C 3 -C 20  heteroalkyl, optionally substituted C 2 -C 20  alkenyl, optionally substituted C 3 -C 20  heteroalkenyl, optionally substituted C 2 -C 20  alkynyl, optionally substituted C 3 -C 20  heteroalkynyl, optionally substituted C 6 -C 24  aryl, optionally substituted C 5 -C 24  heteroaryl or optionally substituted C 3 -C 20  heterocyclyl, or other moeity so that R 7 —O— provides a suitable carboxylic acid protecting group, in particular R 7  is —CH 3  or —CH 2 CH 3 . 
         in particular, compound A and compound B have the structures of: 
       
       
         
           
           
               
               
           
         
       
     
     
         33 . A composition comprised of a compound, optionally in salt form, having the structure of: 
       
         
           
           
               
               
           
         
         as the predominate optical isomer, and a corresponding optical impurity, optionally in salt form, having the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         34 . The composition of  claim 33 , wherein the composition is essentially free of the corresponding optical impurity of: 
       
         
           
           
               
               
           
         
         and is essentially free of the corresponding optical impurity having the structure of: 
       
       
         
           
           
               
               
           
         
       
     
     
         35 . The composition of  claim 34 , wherein the composition is prepared from a composition of  claim 21 ,
 wherein the composition of  claim 21  contains no more that about 3% w/w, in particular no more than about 2% w/w or no more than about 1.5% w/w of the optical impurity of (S,S)-Formula 2a, as determined by chiral HPLC, relative to the total amount of optical isomers present in the composition.   
     
     
         36 . A Drug Linker composition, wherein the composition is comprised of a Drug Linker compound having the structure of: 
       
         
           
           
               
               
           
         
         in suitable salt form, and a Drug linker impurity having the structure of: 
       
       
         
           
           
               
               
           
         
         also in salt form, or wherein the Drug Linker compound has the structure of: 
       
       
         
           
           
               
               
           
         
         in suitable salt form, and the Drug linker impurity has the structure of: 
       
       
         
           
           
               
               
           
         
         also in salt form, wherein subscript a is 1 so that A is present, preferably as an α-amino acid or β-amino acid residue; subscript m′ ranges from 1 to 5; R 2  is saturated C 1 -C 6  alkyl, or R 2  is R 2A , wherein R 2A  is —C(═O)R 2B , wherein R 2B  is saturated C 1 -C 6  alkyl or unsaturated C 3 -C 6  alkyl, in particular, R 2  is —CH 3 , —CH 2 CH 3  or —C(═O)CH 3 ; and R 3  is —CH 3  or —CH 2 CH 2 CH 3 . 
       
     
     
         37 . The Drug Linker composition of  claim 36 , wherein R 2  is R 2A , wherein R 2A  is —C(═O)CH 3  and R 3  is —CH 3 , and wherein the composition is prepared from a composition of  claim 33 ,  34  or  35 . 
     
     
         38 . A Ligand Drug Conjugate composition having the structure of Formula 1:
   L-(LU-D + ) p   (1)
   wherein L is a Ligand Unit, LU is a Linker Unit and D +  is a quaternized tubulysin Drug Unit,   and subscript p ranges from 2 to 12,   wherein a plurality of Ligand Drug Conjugate compounds of the composition have identical quaternized tubulysin Drug Units having the structure of (R,R)-Formula D + :   
       
         
           
           
               
               
           
         
         in pharmaceutically acceptable salt form, wherein the wavy line indicate the site of covalent attachment to LU. 
         and wherein at least one compound of the composition has at least one quaternized tubulysin Drug Unit having the structure of (S,S)-Formula D + : 
       
       
         
           
           
               
               
           
         
         with the remainder having identical (R,R)-Formula D +  structures. 
       
     
     
         39 . The Ligand Drug Conjugate composition of  claim 38 , wherein the Ligand Drug Conjugate composition is prepared from a composition of  claim 33 ,  34  or  35 . 
     
     
         40 . The Ligand Drug Conjugate composition of  claim 38  or  39 , wherein the Ligand Unit is an antibody Ligand Unit directed to a cancer cell antigen, in particular CD30, CD33 or CD70.