Patent Application
20160107999
The present invention relates to substituted azadibenzocyclooctyne compounds and to a method for their preparation. The substituted azadibenzocyclooctyne compounds according to the invention may be used in metal-free click reactions. The invention therefore also relates to a method for the modification of a target molecule by reaction of an azadibenzocyclooctyne conjugate with a target molecule comprising a 1,3-dipole or a 1,3-(hetero)diene.
A revolutionary development in the rapidly expanding field of “chemical biology” is related to chemistry in living systems. Chemistry in living systems concerns chemical reactions that are mild in nature, yet so rapid and high-yielding that they work at about physiological pH, in water, and in the vicinity of biomolecular functionalities. Such reactions may be grouped under the term “bioorthogonal chemistry”. In the field of bioorthogonal chemistry there are two main challenges: first, the development of suitable chemistry, and second, the application thereof in living organisms (in vivo).
In the field of chemistry, an enormous toolbox of chemical reactions is available that may be applied to the construction of complex organic molecules. However, the vast majority of such reactions can only be performed under strictly anhydrous conditions, in other words, in the complete absence of water. Although still a good minority of chemical reactions may be performed in, or in the presence of, water, most of these reactions can still only be applied in vitro because the interference of other compounds present in the living organism with the chemicals involved can not be excluded. At present, only a handful of chemical reactions is fully compatible with other functional groups present in the living organism.
An example of such a reaction is the cycloaddition of cyclic alkynes and azides, one of the reactions known as “click reactions”. This reaction has become a versatile tool for bioorthogonal labeling and imaging of biomolecules (e.g. proteins, lipids, glycans and the like), proteomics and materials science. In essence, two separate molecular entities, one charged with an azide and one charged with a strained cycloalkyne, will spontaneously combine into a single molecule by a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). The power of SPAAC for bioorthogonal labeling lies in the fact that an isolated cyclic alkyne or azide is fully inert to biological functionalities, such as for example amines, thiols, acids or carbonyls, but in combination undergo rapid and irreversible cycloaddition leading to a stable triazole conjugate. For example, azido-modified proteins, obtained by expression in auxotrophic bacteria, genetic engineering or chemical conversion, can be cleanly labeled with biotin, fluorophores, PEG-chains or other functionalities upon simply stirring the azido-protein with a cyclooctyne conjugate. Moreover, the small size of azide has proven highly useful for application of SPAAC in the imaging of specific biomolecules by means of the chemical reporter strategy.
Apart from azides, cyclooctynes also show high reactivity with other dipoles, such as nitrones and nitrile oxides. For example, the strain-promoted alkyne-nitrone cycloaddition (SPANC) was applied for the N-terminal modification of proteins.
SPAAC and SPANC cycloaddition reactions (Scheme 1) proceed spontaneously, hence in the absence of a (metal) catalyst, and these and a select number of additional cycloadditions are also referred to as “metal-free click reactions”.
Several cyclic alkynes and their application in bioorthogonal labeling are described in the prior art. US 2009/0068738 (Bertozzi et al.), incorporated by reference, relates to modified cycloalkyne compounds and their use in modifying biomolecules via a cycloaddition reaction that may be carried out under physiological conditions. The cycloaddition involves reacting a modified cycloalkyne, such as for example difluorinated cyclooctyne compounds DIFO, DIFO2 and DIFO3, with an azide moiety on a target biomolecule, generating a covalently modified biomolecule. It was observed that fluoride substitution has a strongly accelerating effect on the cycloaddition with azide. For example DIFO3 displays a significantly improved (30× faster) reaction rate constant of up to k=0.076 M−1 s−1, versus a maximum of 0.0024 M−1 s−1 for non-fluorinated systems.
In WO 2011/136645 (van Delft and Rutjes et al.), incorporated by reference, a bicyclic compound wherein a cyclopropyl moiety is fused to a cyclooctyne moiety is disclosed. This fused cyclooctyne compound is used in metal-free click reactions. For example the cycloaddition with an organic azide in aqueous conditions proceeds with a rate constant in the range of k=0.09-0.28 M−1 s−1 (depending on the solvent), whereas the rate constant for the cycloaddition with nitrones increases up to k=1.25 M−1 s−1.
In a specific class of cyclic alkynes used in metal-free click reactions, the cyclooctyne moiety is fused to aryl groups (benzoannulated systems). An example of a benzoannulated system is disclosed in WO 2009/067663 (Boons et al.), incorporated by reference. The cycloaddition with azides of these dibenzocyclooctyne compounds DIBO (1) proceeds with a rate constant of k=0.12 M−1 s−1.
Another benzoannulated system, biarylazacyclooctynone BARAC (2), was reported by Bertozzi et al. (J. Am. Chem. Soc. 2010, 132, 3688-3690), incorporated by reference. By placing an amide functionality in the ring, the reaction kinetics of the cycloaddition of BARAC with azides is improved significantly (k=0.96 M1 s−1). A range of substituted derivatives of BARAC (Me, F, MeO) was also reported (J. Am. Chem. Soc. 2012, 134, 9199-9208) and the influence on reaction rate constant determined. However, only negligible difference in reactivity with azide was noticed (range for k=0.9-1.2 M1 s−1) by attachment of a single substituent on the aryl moiety, even for the strongly electron-withdrawing fluoride. An important disadvantage of BARAC and derivatives thereof is the relatively low stability, leading to short shelf-life (Org. Biomol. Chem., 2013, 11, 3436-3441). In addition, BARAC is known to be susceptible to Michael addition by thiols.
Azadibenzocyclooctyne DIBAC was developed earlier by Rutjes and van Delft et al. (Chem. Commun. 2010, 46, 97-99, incorporated by reference,) and shows somewhat lower reaction kinetics in the cycloaddition with azides (k=0.31 M−1 s−1) with respect to BARAC, but in contrast DIBAC displays high stability and excellent shelf-life, and no Michael addition side-products. In addition, DIBAC can be readily synthesized in good yield by a variety of synthetic strategies. Based on this beneficial combination of properties, DIBAC (also often called DBCO or ADIBO) has become the standard cyclooctyne in research applications for copper-free click reactions with 1,3-dipoles.
One paper by Starke et al. (Arkivoc 2010, 11, 350-359) describes the preparation and evaluation of the tetramethoxy-substituted DIBAC analogue (MeO)4-DIBAC, but aryl substitution in this case led to significant decrease in reactivity (factor 40 with respect to plain DIBAC).
Another substituted DIBAC analogue is described in US 2012/0029186 (Popik et al.). In this analogue one or more C1-C12 organic groups, i.e. C1-C12 hydrocarbon moieties, may be present on the aryl groups. However, no specific examples of these substituted DIBAC analogues are disclosed.
There exists a continuing need for novel, readily accessible and reactive bioorthogonal probes for use in metal-free click reactions, such as 1,3-dipolar cycloaddition with azides, nitrones and other 1,3-dipoles.
The present invention relates to a compound of the Formula (5):
wherein:
R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen, C1-C12 haloalkyl, —CN, —N3, —NO2, —NCX, —XCN, —N(R9)2, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)R9, —OS(O)2R9, —OS(O)OR9, —OS(O)2OR9, —P(O)(R9)(OR9), —P(O)(OR9)2, —OP(O)(OR9)2, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, —N(R9)C(X)R9, —N(R9)C(X)XR9 and —N(R9)C(X)N(R9)2, wherein X is oxygen or sulfur and wherein R9 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups;
with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen, C1-C12 haloalkyl, —CN, —N3, —NO2, —NCX, —XCN, —N(R9)2, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)R9, —OS(O)2R9, —OS(O)OR9, —OS(O)2OR9, —P(O)(R9)(OR9), —P(O)(OR9)2, —OP(O)(OR9)2, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, —N(R9)C(X)R9, —N(R9)C(X)XR9 and —N(R9)C(X)N(R9)2, wherein X and R9 are as defined above;
p is 0 or 1;
L is a linking group selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C2-C200 (hetero)arylene groups, C3-C200 alkyl(hetero)arylene groups, C3-C200 (hetero)arylalkylene groups, C4-C200 (hetero)arylalkenylene groups, C5-C200 (hetero)arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, (hetero)arylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, (hetero)arylalkenylene groups, (hetero)arylalkynylene groups optionally being substituted and/or optionally interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably being selected from the group consisting of O, S, N and NR12, wherein R12 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups; and
Q is a functional group selected from the group consisting of hydrogen, halogen, R11, —CH═C(R11)2, —C≡CR11, —[C(R11)2C(R11)2O]q—R11 wherein q is in the range of 1 to 200, —CN, —N3, —NCX, —XCN, —XR11, —N(R11)2, —+N(R11)3, —C(X)N(R11)2, —C(R11)2XR11, —C(X)R11, —C(X)XR11, —S(O)R11, —S(O)2R11, —S(O)OR11, —S(O)2OR11, —S(O)N(R11)2, —S(O)2N(R11)2, —OS(O)R11, —OS(O)2R11, —OS(O)OR11, —OS(O)2OR11, —P(O)(R11)(OR11), —P(O)(OR11)2, —OP(O)(OR11)2, —Si(R11)3, —XC(X)R11, —XC(X)XR11, —XC(X)N(R11)2, —N(R11)C(X)R11, —N(R11)C(X)XR11 and —N(R11)C(X)N(R11)2, wherein X is oxygen or sulphur and wherein R11 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups.
The invention also relates to a conjugate wherein a compound according to Formula (5) is conjugated to a label via a functional group Q, and to the use of said conjugate for bioorthogonal labeling, imaging or modification of a target molecule. The invention further relates to a method for the modification of a target molecule, wherein a conjugate according to the invention is reacted with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene.
The verb “to comprise” as is used in this description and in the claims and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
The compounds disclosed in this description and in the claims may be described as benzoannulated azacyclooctyne compounds, i.e. cyclooctyne compounds wherein two aromatic moieties are fused to the cyclooctyne moiety and the cyclooctyne contains a nitrogen.
The compounds disclosed in this description and in the claims may further exist as positional isomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include positional isomers of DIBAC, with substituents on either or both of the two aromatic rings, as well as mixtures thereof.
An unsubstituted alkyl group has the general formula CnH2n+1 and may be linear or branched. Unsubstituted alkyl groups may also contain a cyclic moiety, and thus have the concomitant general formula CnH2n−1. Optionally, the alkyl groups are substituted by one or more substituents further specified in this document. Examples of suitable alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl and 1-dodecyl.
A haloalkyl group has the general formula CnYqH(2n+1)−q, wherein Y is selected from the group consisting of F, Cl, Br and I and wherein q=in the range of 1-25. A haloalkyl group may be linear or branched. Examples of suitable haloalkyl groups include trifluoromethyl (—CF3), pentafluoroethyl (—CF2CF3), tribromomethyl (—CBr3) and pentabromoethyl (—CF2CF3), dibromomethyl (—CHBr2), dichloromomethyl (—CHCl2), difluoromethyl (—CHF2), bromomethyl (—CH2Br), chloromomethyl (—CH2Cl) and fluoromethyl (—CH2F).
Unsubstituted alkenyl groups have the general formula CnH2n−1, and may be linear or branched. Examples of suitable alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, pentenyl, decenyl, octadecenyl and eicosenyl. Unsubstituted alkenyl groups may also contain a cyclic moiety, and thus have the concomitant general formula CnH2n−3. Optionally, the alkenyl groups may be substituted by one or more substituents further specified in this document.
Unsubstituted alkenes have the general formula CnH2n whereas unsubstituted alkynes have the general formula CnH2n−2. Optionally, the alkenes and alkynes may be substituted by one or more substituents further specified in this document.
Aryl groups comprise at least six carbon atoms (i.e. at least C6) and may include monocyclic, bicyclic and polycyclic structures. Optionally, the aryl groups may be substituted by one or more substituents further specified in this document. Examples of aryl groups include groups such as for example phenyl, naphthyl and anthracyl.
Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms (i.e. at least C7) and may include monocyclic and bicyclic structures. Optionally, the aryl groups may be substituted by one or more substituents further specified in this document. An arylalkyl group is for example benzyl and the like. An alkylaryl group is for example 4-t-butylphenyl and the like.
Heteroaryl groups comprise at least two carbon atoms (i.e. at least C2) and one or more heteroatoms N, O, P or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document. Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.
Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C3) and may include monocyclic and bicyclic structures. Optionally, the heteroaryl groups may be substituted by one or more substituents further specified in this document.
Where an aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group. Similarly, an alkyl(hetero)aryl group is meant to include an alkylaryl group and a alkylheteroaryl group, and (hetero)arylalkyl is meant to include an arylalkyl group and a heteroarylalkyl group. A C2-C24 (hetero)aryl group is thus to be interpreted as including a C2-C24 heteroaryl group and a C6-C24 aryl group. Similarly, a C3-C24 alkyl(hetero)aryl group is meant to include a C7-C24 alkylaryl group and a C3-C24 alkylheteroaryl group, and a C3-C24 (hetero)arylalkyl is meant to include a C7-C24 arylalkyl group and a C3-C24 heteroarylalkyl group.
Unless stated otherwise, alkyl groups, alkenyl groups, alkenes, alkynes, (hetero)aryl groups, (hetero)arylalkyl groups and alkyl(hetero)aryl groups may be substituted with one or more substituents selected from the group consisting of, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C3-C12 cycloalkyl groups, C5-C12 cycloalkenyl groups, C8-C12 cycloalkynyl groups, C1-C12 alkoxy groups, C2-C12 alkenyloxy groups, C2-C12 alkynyloxy groups, C3-C12 cycloalkyloxy groups, halogens, amino groups, oxo and silyl groups, wherein the silyl groups can be represented by the formula (R10)3Si—, wherein R10 is independently selected from the group consisting of C1-C12 alkyl groups, C2-C12 alkenyl groups, C2-C12 alkynyl groups, C3-C12 cycloalkyl groups, C1-C12 alkoxy groups, C2-C12 alkenyloxy groups, C2-C12 alkynyloxy groups and C3-C12 cycloalkyloxy groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, alkoxy groups, alkenyloxy groups, alkynyloxy groups and cycloalkyloxy groups are optionally substituted, the alkyl groups, the alkoxy groups, the cycloalkyl groups and the cycloalkoxy groups being optionally interrupted by one of more hetero-atoms selected from the group consisting of O, N and S.
In a first aspect, the present invention relates to substituted azadibenzocyclooctyne compounds. These substituted azadibenzocyclooctyne compounds, which may also be referred to as substituted DIBAC analogues, comprise one or more substituents on the aryl rings. The invention therefore relates to a compound of Formula (5):
wherein:
R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen, C1-C12 haloalkyl, —CN, —N3, —NO2, —NCX, —XCN, —N(R9)2, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)R9, —OS(O)2R9, —OS(O)OR9, —OS(O)2OR9, —P(O)(R9)(OR9), —P(O)(OR9)2, —OP(O)(OR9)2, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, —N(R9)C(X)R9, —N(R9)C(X)XR9 and —N(R9)C(X)N(R9)2, wherein X is oxygen or sulphur and wherein R9 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups;
with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen, C1-C12 haloalkyl, —CN, —N3, —NO2, —NCX, —XCN, —N(R9)2, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)R9, —OS(O)2R9, —OS(O)OR9, —OS(O)2OR9, —P(O)(R9)(OR9), —P(O)(OR9)2, —OP(O)(OR9)2, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, —N(R9)C(X)R9, —N(R9)C(X)XR9 and —N(R9)C(X)N(R9)2, wherein X and R9 are as defined above;
p is 0 or 1;
L is a linking group selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C2-C200 (hetero)arylene groups, C3-C200 alkyl(hetero)arylene groups, C3-C200 (hetero)arylalkylene groups, C4-C200 (hetero)arylalkenylene groups, C5-C200 (hetero)arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, (hetero)arylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, (hetero)arylalkenylene groups, (hetero)arylalkynylene groups optionally being substituted and/or optionally interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably being selected from the group consisting of O, S, N and NR12, wherein R12 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups; and Q is a functional group selected from the group consisting of hydrogen, halogen, R11, —CH═C(R11)2, —C≡CR11, —[C(R11)2C(R11)2O]q—R11 wherein q is in the range of 1 to 200, —CN, —N3, —NCX, —XCN, —XR11, —N(R11)2, —+N(R11)3, —C(X)N(R11)2, —C(R11)2XR11, —C(X)R11, —C(X)XR11, —S(O)R11, —S(O)2R11, —S(O)OR11, —S(O)2OR11, —S(O)N(R11)2, —S(O)2N(R11)2, —OS(O)R11, —OS(O)2R11, —OS(O)OR11, —OS(O)2OR11, —P(O)(R11)(OR11), —P(O)(OR11)2, —OP(O)(OR11)2, —Si(R11)3, —XC(X)R11, —XC(X)XR11, —XC(X)N(R11)2, —N(R11)C(X)R11, —N(R11)C(X)XR11 and —N(R11)C(X)N(R11)2, wherein X is oxygen or sulphur and wherein R11 is independently selected from the group consisting of hydrogen, halogen, C1-C24 alkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups.
In a preferred embodiment, X is O.
In another preferred embodiment, R9 is independently selected from the group consisting of hydrogen, halogen and C1-C12 alkyl groups, more preferably from the group consisting of hydrogen, halogen and C1-C6 alkyl groups, even more preferably from the group consisting of hydrogen, halogen and C1-C4 alkyl groups. Most preferably, R9 is independently selected from the group consisting of hydrogen, F, Cl, Br, methyl, ethyl, propyl, i-propyl, butyl and t-butyl.
When R1, R2, R3, R4, R5, R6, R7 and/or R8 is a C1-C12 haloalkyl group, the haloalkyl group is preferably selected from the group consisting of —CF3, —CBr3, —CCl3, —CHBr2, —CHCl2, —CHF2, —CH2Br, —CH2Cl and —CH2F.
As specified above, at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen, C1-C12 haloalkyl, —CN, —N3, —NO2, —NCX, —XCN, —N(R9)2, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)R9, —OS(O)2R9, —OS(O)OR9, —OS(O)2OR9, —P(O)(R9)(OR9), —P(O)(OR9)2, —OP(O)(OR9)2, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, —N(R9)C(X)R9, —N(R9)C(X)XR9 and —N(R9)C(X)N(R9)2, wherein X and R9 are as defined above. The substituted DIBAC analogues according to the Formula (5) thus comprise one or more substituents. The term “substituent” in this context relates to a group that is present on an aryl moiety of the substituted DIBAC analogue, or an atom that is present on said aryl moiety wherein said atom is not a hydrogen atom. In other words, at least one of R1, R2, R3, R4, R5, R6, R7 and R8 in the compounds according to Formula (5) does not equal hydrogen. Preferably, the substituted DIBAC analogues according to the invention comprise 1, 2, 3 or 4, more preferably 1 or 2, substituents.
In a preferred embodiment, the one or more substituents present on the aryl groups of the substituted DIBAC analogues (5) are electron-withdrawing substituents having a positive value for the para-Hammett substituent constant σp and/or the meta-Hammett substituent σm. Groups with a positive value for σp and/or σm include for example F, Cl, Br, I, NO2, CN and many others. para-Hammett substituent constants σp and meta-Hammett substituents σm are known for a large number of substituents (see for example C. Hansch et al., Chem. Rev. 1991, 91, 165-195, incorporated by reference). In a preferred embodiment, R1, R2, R3, R4, R5, R6, R7 and R8 are therefore independently selected from the group consisting of hydrogen and electron-withdrawing substituents having a positive value for the para-Hammett substituent constant σp and/or the meta-Hammett substituent σm, with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of electron-withdrawing substituents having a positive value for the para-Hammett substituent constant σp and/or the meta-Hammett substituent σm. In other words, at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is not hydrogen.
In another preferred embodiment, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen (preferably —F, —Cl, —Br), C1-C12 haloalkyl, —CN, —NO2, —NCO, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)2R9, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, wherein X and R9 are as defined above; with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen (preferably —F, —Cl, —Br), C1-C12 haloalkyl, —CN, —NO2, —NCO, —N+(R9)3, —C(X)N(R9)2, —C(X)R9, —C(X)XR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2, —OS(O)2R9, —XC(X)R9, —XC(X)XR9, —XC(X)N(R9)2, wherein X and R9 are as defined above.
In a more preferred embodiment, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen (preferably —F, —Cl, —Br), —NO2, —CN, —N+(R9)3, —C(O)N(R9)2, —C(O)R9, —C(O)OR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2 and —OS(O)2R9, wherein R9 is as defined above; with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen (preferably —F, —Cl, —Br), —NO2, —CN, —N+(R9)3, —C(O)N(R9)2, —C(O)R9, —C(O)OR9, —S(O)R9, —S(O)2R9, —S(O)OR9, —S(O)2OR9, —S(O)N(R9)2, —S(O)2N(R9)2 and —OS(O)2R9, wherein R9 is as defined above.
In a further preferred embodiment, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen (preferably —F, —Cl, —Br), —NO2, —CN, —N+(R9)3, —C(O)R9, —C(O)OR9, —S(O)R9 and —S(O)2R9, wherein R9 is as defined above; with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of —F, —Cl, —Br, —NO2, —CN, —N+(R9)3, —C(O)R9, —C(O)OR9, —S(O)R9 and —S(O)2R9, wherein R9 is as defined above.
Even more preferably, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen (preferably —F, —Cl, —Br), —NO2, —N+(R9)3 and —CN; with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen (preferably —F, —Cl, —Br), —NO2, —N+(R9)3 and —CN. Most preferably, R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from the group consisting of hydrogen, halogen (preferably —F, —Cl, —Br); with the proviso that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is selected from the group consisting of halogen (preferably —F, —Cl, —Br). As was already described above, also in these preferred embodiments at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is not hydrogen.
In one specific embodiment, R7 and R8 are hydrogen. In another specific embodiment, R5 and R6 are hydrogen. In another specific embodiment, R5, R6, R7 and R8 are hydrogen.
In one embodiment, R1 is equal to R3, and/or R2 is equal to R4, and/or R7 is equal to R8, and/or R5 is equal to R6. In a preferred embodiment, R1 is equal to R3, and R2 is equal to R4, and R7 is equal to R8, and R5 is equal to R6.
In a preferred embodiment, R1 and R3 are selected from the group consisting of F, Cl and Br, and it is further preferred that R1 is equal to R3. More preferably, R2 is equal to R4, and R7 is equal to R8, and R5 is equal to R6. Even more preferably, R2, R4, R5, R6, R7 and R8 are all hydrogen. Yet even more preferably, R1 is equal to R3; and R1 and R3 are selected from the group consisting of F, Cl and Br; and R2, R4, R5, R6, R7 and R8 are all hydrogen.
In another preferred embodiment, R2 and R4 are selected from the group consisting of F, Cl and Br, and it is further preferred that R2 is equal to R4. More preferably, R1 is equal to R3, and R7 is equal to R8, and R5 is equal to R6. Even more preferably, R1, R3, R5, R6, R7 and R8 are all hydrogen. Yet even more preferably, R2 is equal to R4; and R2 and R4 are selected from the group consisting of F, Cl and Br; and R1, R3, R5, R6, R7 and R8 are all hydrogen.
In yet another preferred embodiment, R3 is selected from the group consisting of F, Cl and Br. In this embodiment it is preferred that R5 and R6 are hydrogen. It is also preferred that that R7 and R8 are hydrogen. More preferably, R1, R2 and R4 are hydrogen. The invention thus also relates to a compound according to Formula (5a), wherein R3 is selected from the group consisting of F, Cl and Br; R1, R2 and R4, R5, R6, R7 and R8 are hydrogen; and wherein L, p and Q are as defined above. In this embodiment, it is further preferred that R3 is Cl.
In yet another preferred embodiment, R4 is selected from the group consisting of F, Cl and Br. In this embodiment it is preferred that R5 and R6 are hydrogen. It is also preferred that R7 and R8 are hydrogen. More preferably, R1, R2 and R3 are hydrogen. The invention thus also relates to a compound according to Formula (5c), wherein R4 is selected from the group consisting of F, Cl and Br; R1, R2, R4, R5, R6, R7 and R8 are hydrogen; and wherein L, p and Q are as defined above. In this embodiment, it is further preferred that R4 is Br.
Substituted azadibenzocyclooctyne compounds (5a) and (5c) are shown below.
In one embodiment of the substituted azadibenzocyclooctyne compounds according the invention, p is 0, i.e. Q is bonded directly to the amide carbonyl group. In another embodiment, p is 1, in other words, Q is connected to the amide carbonyl group via linking unit L.
Linkers (L), also referred to as linking units or linking groups, are well known in the art. Examples of suitable linking units include (poly)ethylene glycol diamines (e.g. 1,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethylene glycol chains), polyethylene glycol or polyethylene oxide chains, polypropylene glycol or polypropylene oxide chains and 1,x-diaminoalkanes wherein x is the number of carbon atoms in the alkane.
Another class of suitable linkers comprises cleavable linkers. Cleavable linkers are well known in the art. For example Shabat et al., Soft Matter 2012, 6, 1073, incorporated by reference herein, discloses cleavable linkers comprising self-immolative moieties that are released upon a biological trigger, e.g. an enzymatic cleavage or an oxidation event. Some examples of suitable cleavable linkers are peptide-linkers that are cleaved upon specific recognition by a protease, e.g. cathepsin, plasmin or metalloproteases, or glycoside-based linkers that are cleaved upon specific recognition by a glycosidase, e.g. glucoronidase, or nitroaromatics that are reduced in oxygen-poor, hypoxic areas
In a preferred embodiment of the substituted azadibenzocyclooctyne compounds according to the invention, the linking group L is selected from the group consisting of linear or branched C1-C24 alkylene groups (preferably linear C1-C24 alkylene groups), (poly)ethylene glycol diamines, polyethylene glycol chains, polyethylene oxide chains, polypropylene glycol chains, polypropylene oxide chains and 1,x-diaminoalkanes wherein x is the number of carbon atoms in the alkane and wherein x is in the range of 1-20. Said C1-C24 alkylene group is preferably a C1-C12 alkylene group, even more preferably a C1-C8 alkylene group, yet even more preferably a C1-C6 alkylene group, and most preferably a C1-C4 alkylene group. Said C1-C24 alkylene group may for example be a methylene, ethylene, propylene or butylene group.
Preferably, Q is selected from the group consisting of —CN, —N3, —NCX, —XCN, —XR11, —N(R11)2, —+N(R11)3, —C(X)N(R11)2, —C(R11)2XR11, —C(X)R11, —C(X)XR11, —XC(X)R11, —XC(X)XR11, —XC(X)N(R11)2, —N(R11)C(X)R11, —N(R11)C(X)XR11 and —N(R11)C(X)N(R11)2, wherein X and R11 are as defined above. More preferably, X is oxygen. Most preferably, Q is selected from the group consisting of —OR11, —SR11, —N(R11)2, —+N(R11)3, —C(O)N(R11)2, —C(O)OR11, —OC(O)R11, —OC(O)OR11, —OC(O)N(R11)2, —N(R11)C(O)R11, —N(R11)C(O)OR11 and —N(R11)C(O)N(R11)2. Furthermore, the functional group Q may optionally be masked or protected. The R11 groups may be selected independently from each other, which means that the two R1 groups present in for example a —N(R11)2 substituent may be different from each other.
Specific examples of the substituted DIBAC analogues according to the invention include compounds of the Formula (16a), (16c), (17a), (17b), (18a) and (18b), as shown above.
The substituted azadibenzocyclooctyne compounds according to the present invention are readily synthesized. A general route for the synthesis of the compounds of Formula (5) according to the invention is shown in
The invention thus also relates to a method for the manufacturing of a compound of the Formula (5), said method comprising the steps of:
Steps (a)-(h) of said method for the manufacturing of a compound of the Formula (5) are also shown in Scheme 2. Steps (a)-(h) are discussed in more detail below.
Step (a): Sonogashira Coupling of a Substituted 2-G-Benzyl Alcohol (I) with a Substituted Ortho-Ethynylaniline (II) in Order to Obtain a Compound of Formula (III)
Step (a) is a Sonogashira coupling of a substituted 2-G-benzyl alcohol (I), wherein G is selected from the group consisting of F, Cl, Br, I and OTf (wherein OTf is triflate, i.e. trifluoromethanesulfonate), with a substituted ortho-ethynylaniline (II). In a preferred embodiment, G is Cl, Br, I or OTf, more preferably, Cl, Br or I and most preferably I.
Typical reaction conditions for a Sonogashira coupling are known to a person skilled in the art. Said coupling takes preferably place in the presence of a palladium catalyst (e.g. Pd(PPh3)2Cl2), a copper catalyst (preferably a copper(I) halide, e.g. CuI) and a base (e.g. triethylamine). Said coupling is preferably executed with tetrahydrofuran (THF) as a solvent and under a mixed hydrogen-nitrogen atmosphere.
Step (b): Protection of the Amine Hydrogen Atom in (III) with a Protecting Group (PG) in Order to Obtain an Alkyne of Formula (IV)
In step (b), the amine hydrogen atom in (III) is protected with a protecting group (PG). Protecting groups are known to a person skilled in the art, as are protecting groups that are suitable for the protection of an amine. Reaction conditions for the protection reaction strongly depend on the type of protecting group that is introduced, and are also known to the person skilled in the art.
In a preferred embodiment, the protecting group is selected from the group consisting of t-butyloxycarbonyl (BOC), fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), trichloroethyloxycarbonyl (Troc), nitrobenzenesulfonyl (Ns), and trifluoroacetyl (TFA).
The partial hydrogenation of the alkyne bond in (IV) in order to obtain alkene (V) is executed in the presence of a suitable hydrogenation catalyst, preferably a palladium catalyst, more preferably a palladium catalyst in the presence of a poisonous additive, preferably quinoline. Palladium hydrogenation catalysts are known in the art. Preferred palladium catalysts include Pd/BaSO4 (e.g. 10%), Pd/CaCO3 or Pd/C. Preferred reaction conditions strongly depend on the type of Pd-catalyst used in the hydrogenation, and are known to a person skilled in the art.
The oxidation of the primary alcohol in (V) in order to obtain aldehyde (VI) is executed by the use of suitable oxidant, preferably Dess-Martin periodinane in dichloromethane. Oxidants for the selective transformation of alcohols into aldehydes are known in the art. Preferred oxidants and/or oxidative conditions include Dess-Martin reagent, chromium-based reagents (PCC, PDC), Ley oxidation (Pr4NRuO4, NMO), Swern oxidant. Preferred reaction conditions strongly depend on the starting material.
In step (e), the protective group on the amine hydrogen atom in (VI) is removed. Reaction conditions for deprotection strongly depend on the type of protecting group that is introduced, and are also known to the person skilled in the art.
Upon deprotection of the amine, a spontaneous ring-closure takes place by attack of the liberated amine onto the aldehyde and expulsion of water, thereby generating a cyclic imine.
The resulting imine may be in situ reduced to an amine (VII) by addition of a suitable reductive agent or after a work-up procedure, for example to neutralize the reagents applied to remove the protective group. Reductive methods for the selective reduction of imines into aldehydes are known in the art. Preferred reductive conditions include H2/Pd—C, NaBH4, LiBH4, NaCNBH3 in the presence of HOAc, Na(tBuO)3BH, LiEt3BH, Li(AcO)3BH. Preferred reaction conditions strongly depend on the starting material.
Step (f): Introduction of (L)p-Q into Azadibenzocyclooctene (VII) in Order to Obtain an Azadibenzocyclooctene of Formula (VIII)
The generated amine (VII) may be acylated by a suitable electrophilic reagent in order to convert the amine into an amide functionality (as in compound VIII) and thereby at the same time introducing one (or more) functionalities at the other side of the chain. Methods for the acylation of amines are known in the art. Preferred reagents include acid anhydrides (symmetrical or mixed) acid halogenides (F, Cl, Br, I), acid in the presence of an activating reagents (DCC, EDC, BOP, PyBOP, T3P, HATU or the like). Suitable chains involve alkyl chains, aryl chains, oligomers of ethyleneglycol or ethylenediamine or the like, which may or may not be substituted. The functionality at the other side of the chain may involve a suitable reactive group (Q) for follow-up conjugation reactions, for example a carboxylic acid, an amine, an alcohol, a thiol. Such reactive groups are known in the art and are typically introduced in protected form in order to avoid undesired side-reactions during acylation of the amine or during subsequent transformations. Suitable protective groups for the functionalities mentioned are known to a person skilled in the art. The functionality at the other side of the chain may alternatively already contain the desired property, i.e. a “label”, for example a reporter molecule for detection (radionuclide, fluorophore, NMR contrast agent, chelating moiety such as DTPA, DOTA, NOTA, a stable radical) or a solid phase. The term “label” is described in more detail below. Suitable reporter molecules are known in the art. Multiple such functional moieties may be present in a single linker.
The bromination of the alkene bond in (VIII) in order to obtain dibromide (IX) is executed in the presence of elemental bromine, preferably in dichloromethane.
Elimination of the cyclooctene bromine atoms in dibromide (IX) in order to obtain the cyclic alkyne (5) is executed by treatment with excess of a suitable strong base, preferably KOtBu in THF. Bases suitable for elimination of bromide are known to a person skilled in the art. Preferred bases include KOtBu, NaOtBu, LiHMDS, KHMDS, NaHMDS, LDA, KH, NaH. Preferred solvents for such elimination involve THF, Et2O, dioxane, DMF or DMSO. Preferred reaction conditions strongly depend on the type of base used and the ease of elimination and may be performed with varying stoichiometries of base (from 2 up to >10 equivalents) and temperature (from −78 C to reflux).
As is shown in Scheme 3, substituted 2-G-benzyl alcohols (I), wherein G is selected from the group consisting of F, Cl, Br, I and OTf, more preferably Cl, Br, I and OTf, may be prepared starting from the corresponding anthranilic acid derivative (XI) via diazonium salt (XII) formation, followed by substitution with G (wherein G is selected from the group consisting of F, Cl, Br, I and OTf) in order to obtain (XIII). Subsequent reduction of the acids (XIII) provides the substituted 2-G-benzyl alcohols (I).
The substituted azadibenzocyclooctyne compounds according to the present invention are very suitable for use in metal-free click reactions, and consequently these compounds are versatile tools in applications such as for example bioorthogonal labeling, imaging and/or modification, including surface modification, of a large range of target molecules. The present invention therefore also relates to a conjugate wherein a substituted azadibenzocyclooctyne compound according to the invention is conjugated to a label via a functional group Q.
The term “label” refers to any tag (including identifying tags or reporter molecules) that may be conjugated to a compound of the Formula (5). A wide variety of labels are known in the art, for a wide variety of different applications. Depending on the specific application, a suitable label for that specific application may be selected. Suitable labels for specific applications are known to the person skilled in the art, and include e.g. all kinds of fluorophores, biotin, polyethylene glycol (PEG) chains, polypropylene glycol (PPG) chains, mixed polyethylene/polypropylene glycol chains, radioactive isotopes, steroids, pharmaceutical compounds, lipids, amino acids, peptides, polypeptides (proteins), glycans (including oligo- and polysaccharides), nucleotides (including oligo- and polynucleotides), peptide tags and solid phases. Examples of suitable fluorophores are for example all kinds of Alexa Fluor (e.g. Alexa Fluor 555), cyanine dyes (e.g. Cy3 or Cy5), coumarin derivatives, fluorescein, rhodamine, allophycocyanin, chromomycin, and so on. Examples of suitable peptide tags include FLAG or HIS tags. Examples of suitable radioactive isotopes include 18F, 126I, 64Cu, 111In, 99Tc and 68Ga. An example of a suitable glycan is concanavalin.
In a preferred embodiment, the label is selected from the group consisting of fluorophores, biotin, polyethylene glycol chains, polypropylene glycol chains, mixed polyethylene/polypropylene glycol chains, metal chelator complexes (with or without enclosed (radioactive) metal), radioactive isotopes, steroids, pharmaceutical compounds, lipids, amino acids, peptides, polypeptides, glycans, nucleotides, peptide tags and solid phases. A peptide is herein defined as a chain of amino acid monomers linked by peptide bonds, said chain comprising 2 to 50 amino acid monomers. A polypeptide is herein defined as a chain of amino acid monomers linked by peptide bonds, said chain comprising 51 or more amino acid monomers.
Functional group Q may be connected to the label directly, or indirectly via a linker or linking unit. Linking units are well know in the art. Linkers may e.g. have the general structure Q-L-Q, wherein Q and L are as defined above. Examples of suitable linking groups L are described above.
The present invention further relates to the use of a conjugate according to the invention for bioorthogonal labeling, imaging or modification of a target molecule.
The conjugates according to the present invention are successfully applied in bioorthogonal labeling, imaging or modification, including surface modification, of target molecules such as e.g. amino acids, peptides, polypeptides (i.e. proteins), lipids and glycans.
The present invention therefore also relates to a method for the modification of a target molecule, wherein a conjugate according to the present invention is reacted with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene. As an example, the strain-promoted cycloaddition of a cycloalkyne with an azide (SPAAC) or with a nitrone (SPANC) was depicted in Scheme 1. The reaction of a cyclooctyne with a 1,3-(hetero)diene is known as a (hetero) Diels-Alder reaction. These reactions are also referred to as metal-free click reactions.
1,3-Dipolar compounds are well known in the art (cf. for example F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 3rd Ed., 1990, p. 635-637), and include nitrile oxides, azides, diazomethane, nitrones, nitrilamines, etc. Preferably, the compound comprising a 1,3-dipole is an azide-comprising compound, a nitrone-comprising compound or a nitrile oxide-comprising compound.
(Hetero) Diels-Alder reactions and 1,3-(hetero)dienes are also well known in the state of the art. Examples of 1,3-dienes include, amongst others, 1,3-butadiene, 1,3-cyclopentadiene, 1,3-cyclohexadiene, furan, pyrrole, and their substituted varieties. Examples of 1,3-heterodienes include amongst others 1-oxa-1,3-butadiene, 1-aza-1,3-butadiene, 2-aza-1,3-butadiene, 3-aza-1,3-butadiene, and their substituted varieties.
A large variety of target molecules, i.e. compounds comprising a 1,3-dipole or a 1,3-(hetero)diene, may be modified by the method according to the invention. Suitable target molecules are well known in the art and include, but are not limited to, biomolecules such as for example amino acids, proteins, peptides, glycans, lipids, nucleic acids, enzymes and hormones. In principle, any compound comprising a 1,3-dipole or a 1,3-(hetero)diene may be suitable as a target molecule.
Applications of the method for the modification of target molecules according to the present invention include diagnostic and therapeutic applications, cell labeling of living cells, for example MV3 cells, fibroblasts, Jurkat, CHO or HEK cells, modification of biopolymers (proteins, lipids, nucleic acids, glycans), enrichment of proteins and glycans for mass spectrometric analysis, tuning of polymer properties, surface modifications etc.
In one embodiment, the reaction of the conjugate is performed in vitro. In a preferred embodiment, the reaction is performed in vivo, i.e. under physiological conditions.
The conjugates according to the present invention that are applied in the modification of a target molecule are described above in great detail. One of the large advantages of these conjugates is that they may be applied both in vivo and in vitro. In addition, the here described conjugates suffer less from undesired aspecific lipophilic interactions, and show good reaction kinetics in metal-free click reactions. Another advantage is that the here described conjugates are easily synthesized and amenable to simple and straightforward modification of various parts of the conjugate. This makes it possible to “fine tune” a conjugate for a specific application, and optimise reaction kinetics for this application.
In a preferred embodiment, a compound of the Formula (5), or a conjugate thereof as described above, is reacted with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene. Said reaction proceeds cleanly and rapidly, with excellent rate constants. For example the cycloaddition of (16a) or (16c) with benzyl azide in deuterated methabol proceeds rapidly and cleanly to the corresponding triazole adducts with excellent reaction kinetics (k=0.9 M−1 s−1 for Cl-DIBAC (16a) and 0.8 for Br-DIBAC (16c)).
Cl-DIBAC (16a) and Br-DIBAC (16c) show an excellent rate constant comparable to BARAC (2), which is the fastest non-substituted benzannulated cyclooctyne currently known. The increase in rate as observed for the DIBAC analogues with electron-withdrawing groups Cl-DIBAC (16a) and Br-DIBAC (16c) is relatively high, with an over two-fold rate enhancement. The reactivity of the azadibenzocyclooctyne compounds according to the invention may be modulated by the presence of the substituents.
Interestingly, substituents on DIBAC appear to have a larger effect on the rate than observed for BARAC. For example, for BARAC a fluoride at the meta-position relative to the alkyne results in a 1.1 to 1.3-fold rate enhancement, for DIBAC a chloride, which is less electron-withdrawing than fluoride, results in a more than two-fold rate increase. The same pattern is observed for substituents at the para-position.
The invention further relates to a composition comprising a conjugate according to the invention, further comprising a pharmaceutically acceptable carrier. Conjugates according to the invention are described in detail above. A wide variety of suitable pharmaceutically acceptable carriers are known in the art (cf. for example R. C. Rowe, P. J. Sheskey and P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, 4th Ed. 2003).
Unless stated otherwise all chemicals were obtained from commercial sources and used without further purification. 1 M KOtBu in THF solution was purchased from Sigma-Aldrich. If no further details are given the reaction was performed under ambient atmosphere and temperature. Analytical thin layer chromatography (TLC) was performed on silica gel-coated plates (Merck 60 F254) with the indicated solvent mixture, visualization was done using ultraviolet (UV) irradiation (λ=254 nm) and/or staining with KMnO4. Purification by column chromatography was carried out using Silicycle silica gel (0.040-0.063 mm, and ca. 6 nm pore diameter). THF and CH2Cl2 were dried over an activated alumina column using an MBraun SPS800 solvent purification system. NEt3 was distilled under N2-atmosphere from CaH2.
Infrared (IR) Spectroscopy:
IR spectra were recorded on an ATI Matson Genesis Series FTIR spectrometer fitted with an ATR cell. The vibrations (ν) are given in cm−1.
Nuclear Magnetic Resonance (NMR) Spectroscopy:
1H-NMR spectra were recorded on a Varian Inova 400 (400 MHz) for room temperature measurements and a Varian Inova 500 (500 MHz) for low temperature measurements. 13C-NMR spectra were recorded on a Bruker DMX300 (75 MHz) spectrometer. Unless stated otherwise all spectra were taken at ambient temperature. 1H-NMR chemical shifts (δ) are reported in parts per million (ppm) relative to a residual proton peak of the solvent, 6=3.31 for CD3OD and 6=7.26 for CDCl3. Broad peaks are indicated by the addition of br. Coupling constants are reported as a J-value in Hertz (Hz). In case of rotamers the spectrum was taken at lower temperature to freeze the compound in its two rotamer states, causing separate peaks for each rotamer. In these cases shifts, coupling constants and integrals are given of each separate peak. 13C-NMR chemical shifts (δ) are reported in ppm relative to CD3OD (δ=49.0) or CDCl3 (δ=77.0). If rotamers are observed in the spectrum, the minor rotamer peaks are labeled with *.
Mass Spectrometry (MS):
High Resolution Mass Analyses were performed using Electrospray Ionization on a JEOL AccuToF.
2-Amino-4-chlorobenzoic acid (6a, 10.0 g, 58.2 mmol) was dissolved in DMSO (100 mL), and 30% H2SO4 was added (100 mL). The solution was cooled to 0° C., whereupon NaNO2 (8.8 g, 129 mmol) was added. The reaction was stirred for two hours at room temperature, after which a solution of KI (19.3 g, 106 mmol) in H2O (50 mL) was added. After one hour, an additional portion of KI (9.7 g, 58.2 mmol) in H2O (25 mL) was added. In addition, DMSO (50 mL) was added to keep the reaction mixture solubilized. After one additional hour, EtOAc (300 mL) was added, and the organic layer was washed with H2O (3×200 mL) and brine (200 mL), and subsequently dried over MgSO4. The solvents were removed in vacuo to obtain crude 7a as white solid. 7a was not further purified and used as a crude in the following reaction. 1H-NMR (400 MHz, CD3OD) δ: 8.01 (d, J=2.1 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.45 (dd, J=8.4, 2.1 Hz, 1H). 13C-NMR (75 MHz, CD3OD) δ: 168.9, 141.7, 138.6, 136.0, 132.7, 129.3, 95.0. HRMS (EI+) m/z calcd for C7H4O2ClI [M]•+ 281.8945. found 281.8936.
2-Amino-4-chlorobenzoic acid (1.1 g, 6.4 mmol) was dissolved in acetic acid (8 mL) and Br2 (0.33 mL, 6.4 mmol) was added. The mixture was stirred at room temperature for 4 hours and subsequently poured into saturated aqueous NaHSO3 (50 mL). The H2O-layer was extracted with EtOAc (2×50 mL), and the combined organic layers were washed with water (2×50 mL), brine (50 mL), and subsequently dried over MgSO4. The solvents were evaporated under reduced pressure to obtain 6b as a mixture of two products. 6b was not further purified and used as a crude in the following reaction. 1H-NMR (400 MHz, CD3OD) δ: 8.00 (s, 1H), 6.92 (s, 1H).
Crude 2-amino-5-bromo-4-chlorobenzoic acid 6b (6.0 g, 24 mmol) was dissolved in DMSO (100 mL) and 30% H2SO4 (100 mL) and the resulting mixture was cooled to 0° C. NaNO2 (3.6 g, 53 mmol) was added and the mixture was stirred for 2 hours at room temperature. After this time, a solution of KI (8.0 g, 48 mmol) in H2O (40 mL) was added. After one hour an additional portion of KI (4.0 g, 24 mmol) in H2O (20 mL) was added. After one more hour, EtOAc (200 mL) was added, and the organic layer was washed with H2O (2×200 mL) and brine (200 mL), and dried over MgSO4. The solvents were removed in vacuo to obtain 7b as a mixture of two products. 7b was used as a crude in the following reaction. 1H-NMR (400 MHz, CD3OD) δ: 8.16 (s, 1H), 8.07 (s, 1H).
2-Amino-5-bromobenzoic acid (6c, 2.0 g, 9.2 mmol) was dissolved in DMSO (50 mL) and 30% H2SO4 (50 mL) and NaNO2 (0.89 g, 13 mmol) were added. The reaction mixture was stirred for 1 hour at room temperature, whereupon a solution of KI (3.1 g, 19 mmol) in H2O (20 mL) was added and the reaction mixture was stirred for another hour. Next, another portion of KI (3.1 g, 19 mmol) in H2O (10 mL) was added and the reaction mixture was stirred for an additional hour. The reaction mixture was quenched with a saturated aqueous Na2SO3-solution (75 mL), EtOAc (100 mL) was added and the layers were separated. Hereupon, the H2O-layer was extracted with EtOAc (100 mL). The combined organic layers were washed with H2O (2×100 mL) and brine (100 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to afford compound 7c as a yellow solid. 7c was not further purified further and used as a crude in the following reaction. RF=0.05 (EtOAc/n-heptane, 1:4). 1H-NMR (400 MHz, CDCl3) δ: 8.13 (d, J=2.4 Hz, 1H), 7.90 (d, J=8.4 Hz, 1H), 7.33 (dd, J=8.4, 2.4 Hz, 1H).
2-Amino-5-nitrobenzoic acid (6d, 1.82 g, 10 mmol) was dissolved in DMSO (50 mL) and 30% H2SO4 (50 mL) was added. The resulting mixture was heated for two hours at 50° C. The reaction was cooled to 0° C. and a solution of NaNO2 (970 mg, 14 mmol) in water (25 mL) was added. The mixture was stirred at 0° C. for one hour, whereupon a solution of KI (5.0 g, 30 mmol) in H2O (10 mL) was added and the mixture was stirred for 1 hour at room temperature. Next, another portion of KI (5 g, 30 mmol) in H2O (10 mL) was added and the mixture was stirred for an additional hour. EtOAc (100 mL) was added and the reaction was quenched with saturated aqueous NaHSO3 (100 mL). The organic layer was washed with water (2×100 mL) and brine (100 mL) and subsequently dried over MgSO4. The solvents were evaporated under reduced pressure and the crude product was obtained as yellow solid (12.0 g, 120%). 7d was not further purified and used as a crude in the following reaction. 1H-NMR (400 MHz, CD3OD) δ: 8.54 (d, J=2.7 Hz, 1H), 8.29 (d, J=8.6 Hz, 1H), 8.01 (dd, J=8.7, 2.7 Hz, 1H). 13C-NMR (75 MHz, CD3OD) δ: 168.0, 149.2, 144.1, 139.2, 127.1, 125.8, 103.0, 49.6, 49.3, 49.1, 48.8, 48.5. FT-IR νmax (cm−1): 2932, 1722, 1588, 151, 1342, 1295, 1022, 1234, 728. HRMS (EI+) m/z calcd for C7H4NO4I [M]•+ 292.9185. found 292.9184.
4-chloro-2-iodobenzoic acid 7a (15 g, 53 mmol) was dissolved in dry THF (250 mL) and the solution was cooled to 0° C. Hereupon, NEt3 (11 mL, 80 mmol) and ethyl chloroformate (7.6 mL, 80 mmol) were added. The reaction was stirred for 1.5 hour and subsequently NaBH4 (8.0 g, 210 mmol) was added in four portions. After 1.5 hour, additional NaBH4 (4.0 g, 105 mmol) was added and the reaction was stirred for another hour. Hereupon, the reaction was quenched with H2O (100 mL) and EtOAc (200 mL) was added. The organic layer was washed with H2O (3×150 mL), brine (100 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was obtained by gradient column chromatography (EtOAc/n-heptane, 1:9 to 1:6). Compound 8a was obtained as white solid (8.4 g, 75% over 2 steps). 1H-NMR (400 MHz, CDCl3) δ: 7.82 (s, 1H), 7.45-7.33 (m, 2H), 4.65 (d, J=6.2 Hz, 2H), 1.94 (t, J=6.2 Hz, 1H). 13C-NMR (75 MHz, CDCl3) δ: 141.1, 138.3, 133.8, 128.8, 128.6, 96.9, 68.6. HRMS (EI+) m/z calcd for C7H6OClI [M]•+ 267.9152. found 267.9160.
Crude 5-bromo-4-chloro-2-iodobenzoic acid (7b, 11 g, 30 mmol) was dissolved in dry THF (100 mL) and the solution was cooled to 0° C. Next, NEt3 (6.2 mL, 44 mmol) and ethyl chloroformate (4.3 mL, 44 mmol) were added. The reaction mixture was stirred for 1 hour and subsequently a solution of NaBH4 (2.24 g, 59 mmol) in H2O (10 mL) was added. The mixture was stirred another hour, prior to quenching with H2O (100 mL). The H2O-layer was extracted with EtOAc (2×100 mL), and the combined organic layers were washed with H2O (2×150 mL) and brine (150 mL). The organic layer was dried over MgSO4 and the solvents were removed under reduced pressure. The crude product was purified using column chromatography (EtOAc/n-heptane, 1:6) to obtain 7b as a white solid (3.84 g, 38% over 3 steps). 1H-NMR (400 MHz, CDCl3) δ: 7.88 (s, 1H), 7.72 (s, 1H), 4.62 (dd, J=6.0, 0.7 Hz, 2H), 1.96 (t, J=6.1 Hz, 1H). 13C-NMR (75 MHz, CDCl3) δ: 142.8, 139.5, 139.2, 135.7, 132.4, 122.9, 94.3, 68.0, 52.4.
5-Bromo-2-iodobenzoic acid (7c) (750 mg, 2.3 mmol) was dissolved in dry THF (25 mL) and the reaction mixture was cooled to 0° C. NEt3 (0.48 mL, 3.4 mmol) and ethyl chloroformate (0.33 mL, 3.4 mmol) were added and the reaction mixture was stirred for 1 hour. Next a solution of NaBH4 (130 mg, 3.4 mmol) in H2O (2 mL) was added and the mixture was stirred for 1.5 hour. The reaction was quenched with H2O (15 mL), whereupon CH2Cl2 (20 mL) was added and the layers were separated. Hereupon, the H2O-layer was extracted with CH2Cl2 (20 mL). Subsequently, the combined organic layers were washed with H2O (25 mL) and brine (25 mL), dried over MgSO4 and concentrated in vacuo. The crude product was purified by gradient column chromatography (n-heptane/EtOAc, 19:1 to 9:1) to obtain compound 7c as a white solid (410 mg, 54% over 2 steps). RF=0.40 (EtOAc/n-heptane, 1:4). 1H-NMR (400 MHz, CDCl3) δ: 7.65 (d, J=8.3 Hz, 1H), 7.63 (d, J=2.4 Hz, 1H), 7.14 (dd, J=8.3, 2.5 Hz, 1H), 4.64 (d, J=6.1 Hz, 2H), 1.96 (t, J=6.2 Hz, 1H).
2-Iodo-5-nitrobenzoic acid (7d) (3.0 g, 10.2 mmol) was dissolved in dry THF (100 mL) and the reaction was cooled to 0° C. NEt3 (2.1 mL, 15.4 mmol) and ethyl chloroformate (1.5 mL, 15.4 mmol) were added and the reaction was stirred for 1 hour. Next, a solution of NaBH4 (0.78 g, 20.5 mmol) in H2O (5 mL) was added and the reaction was stirred for 1.5 hour. After this time, an additional portion of NaBH4 (0.78 g, 20.5 mmol) in H2O (5 mL) was added and the reaction was stirred for an additional 30 minutes. The reaction was then quenched by the addition of H2O (20 mL). The reaction was diluted with EtOAc (150 mL) and the organic layer was washed with H2O (2×100 mL) and brine (100 mL) and subsequently dried over MgSO4. The solvents were removed in vacuo and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:9 to 1:3). Compound 8d was obtained as an orange solid (1.32 g, 55% over 2 steps). 1H-NMR (400 MHz, CDCl3) δ: 8.36 (d, J=2.8 Hz, 1H), 8.01 (d, J=8.5 Hz, 1H), 7.85 (dd, J=8.6, 2.8 Hz, 1H), 4.75 (d, J=3.4 Hz, 2H), 2.10 (t, J=5.0 Hz, 1H). HRMS (EI+) m/z calcd for C7H6NO3I [M]•+ 278.9393. found 278.9396.
Compound 8a (8.5 g, 29.9 mmol), Pd(PPh3)2Cl2 (430 mg, 0.60 mmol), and CuI (57 mg, 0.30 mmol) were added to a flame-dried flask. The flask was evacuated and refilled with an N2/H2-mixture (3:2) three times. THF (150 mL) and NEt3 (12.4 mL, 89 mmol) were bubbled through with an N2/H2-mixture (3/2) for 10 minutes and subsequently added to the reaction mixture. After this time, 2-ethynylaniline (3.75 mL, 33 mmol) was added, and the mixture was stirred overnight under N2/H2-atmosphere. The reaction mixture was diluted with CH2Cl2 (250 mL) and the organic layer was washed with H2O (3×250 mL). The H2O-layers were combined and back-extracted with CH2Cl2 (150 mL). The organic layers were combined and washed with brine (150 mL). The solvents were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:4 to 1:1) to obtain 9a as a yellow solid (7.3 g, 95%). 1H-NMR (400 MHz, CDCl3) δ: 7.52 (d, J=1.9 Hz, 1H), 7.35 (t, J=8.7 Hz, 2H), 7.29 (dd, J=8.2, 2.1 Hz, 1H), 7.19-7.14 (m, 1H), 6.74-6.70 (m, 2H), 4.83 (d, J=4.8 Hz, 2H), 4.41 (br s, 2H), 2.06 (t, J=5.6 Hz, 1H). 13C-NMR (75 MHz, CDCl3) δ: 148.34, 140.24, 133.36, 132.08, 131.58, 130.35, 128.92, 128.47, 123.70, 117.92, 114.53, 107.03, 92.26, 90.84, 63.69. HRMS (ESI+) m/z calcd for C15H13ClNO [M+H]+ 258.0686. found 258.0677.
Compound 8b (3 g, 8.6 mmol), Pd(PPh3)2Cl2 (0.121 g, 0.17 mmol) and CuI (0.016 g, 0.086 mmol) were added to a flame-dried Schlenk flask. The flask was subsequently evacuated and refilled with an N2/H2-mixture (3:2) three times. At the same time, dry THF (150 mL) and dry NEt3 (3.6 mL, 25.9 mmol) were bubbled with a N2/H2-mixture for 10 minutes. The bubbled solutions were subsequently added to the Schlenk flask. Next, 2-ethynylaniline (1.08 mL, 9.5 mmol) was added and the mixture was stirred for 4 hours under N2/H2-atmosphere. Hereupon, CH2Cl2 (150 mL) was added and the organic layer was washed with H2O (3×100 mL). The H2O-layers were combined and back-extracted with CH2Cl2 (150 mL). The organic layers were combined and dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:4 to 1:2). Compound 9b was obtained as a white solid (2.74 g, 94%). 1H-NMR (400 MHz, CDCl3) δ 7.78-7.69 (m, 1H), 7.60 (s, 1H), 7.33 (dd, J=8.0, 1.6 Hz, 1H), 7.18 (ddd, J=8.2, 7.4, 1.6 Hz, 1H), 6.80-6.57 (m, 2H), 4.83 (d, J=5.7 Hz, 2H), 4.37 (br s, 2H), 2.01 (t, J=6.2 Hz, 1H). 13C-NMR (75 MHz, CD3OD) δ: 150.7, 144.3, 133.7 (2C), 133.2, 133.0, 131.4, 123.7, 122.9, 118.2, 115.7, 107.8, 94.5, 90.5, 62.7. HRMS (ESI+) m/z calcd for C15H12BrClNO [M+H]+ 335.9791. found 335.9781.
(5-Bromo-2-iodophenyl)methanol (8c) (106 mg, 0.34 mmol), Pd(PPh3)2Cl2 (7.0 mg, 0.01 mmol) and CuI (1.2 mg, 6.3 μmol) were added to a flame-dried Schlenk flask. The flask was evacuated and refilled with an N2/H2-mixture (3:2) three times. Dry THF (3 mL) and dry NEt3 (71 μL, 0.51 mmol) were bubbled through with an N2/H2 mixture (3:2) for 10 minutes and subsequently added to the mixture. After this time, 2-ethynylaniline (0.060 mL, 0.58 mmol) was added and the mixture was stirred for 16 hours under an N2/H2-atmosphere. The reaction mixture was diluted with CH2Cl2 (5 mL) and the organic layer was washed with H2O (5 mL). The water layer was then back-extracted with CH2Cl2 (2×5 mL). The combined organic layers were washed with H2O (15 mL) and brine (20 mL). Next, the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:6 to 1:2) to obtain 9c as a yellow solid (102 mg, 100%). RF=0.40 (EtOAc/n-heptane, 1:2). 1H-NMR (300 MHz, CD3OD) δ: 7.68-7.67 (m, 1H), 7.43-7.42 (m, 2H), 7.28 (ddd, J=7.7, 1.5, 0.4 Hz, 1H), 7.12 (ddd, J=7.3, 6.6, 1.6 Hz, 1H) 6.79-6.76 (m, 1H) 6.64 (td, J=7.7, 1.1 Hz, 1H), 4.80 (d, J=0.6 Hz, 2H). 13C-NMR (75 MHz, CD3OD) δ: 148.6, 144.2, 132.4, 131.0, 129.4, 129.2, 129.1, 121.4, 120.0, 116.5, 113.8, 106.5, 91.6, 89.8, 61.3. FT-IR νmax film (cm−1): 3360, 2923, 2850, 2362, 2202, 1610, 1489, 1450, 815, 750. HRMS (ESI+) m/z calcd for C15H113brNO [M+H]+ 302.0181. found 302.0169.
(2-Iodo-5-nitrophenyl)methanol (9d) (1.32 g, 4.73 mmol), Pd(PPh3)2Cl2 (66 mg, 0.095 mmol) and CuI (5 mg, 0.047 mmol) were added to a flame-dried flask. The flask was evacuated and refilled with an N2/H2-mixture (3/2). Dry THF (70 mL) and dry NEt3 (2.0 mL, 14 mmol) were bubbled through with an N2/H2 mixture (3:2) for 10 minutes and subsequently added to the mixture. Hereupon, 2-ethynylaniline (0.81 mL, 7.1 mmol) was added, and the mixture was stirred for 3 hours. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with H2O (3×100 mL). The H2O-layers were combined and back-extracted with CH2Cl2 (100 mL). The organic layers were combined and dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:4 to 2:1). Compound 9d was obtained as a red solid (1.13 g, 89% yield). 1H-NMR (400 MHz, CDCl3) δ: 8.38 (d, J=2.0 Hz, 1H), 8.16 (dd, J=8.5, 2.0 Hz, 1H), 7.67 (d, J=8.5 Hz, 1H), 7.50-7.32 (m, 1H), 7.24-7.16 (m, 1H), 6.87-6.53 (m, 2H), 4.98 (d, J=4.0 Hz, 2H), 4.44 (br s, 2H), 2.10 (t, J=6.1 Hz, 1H). 13C-NMR (75 MHz, CDCl3) δ: 177.2, 165.2, 143.4, 132.5, 132.3, 131.1, 128.5, 127.9, 122.6, 122.3, 118.1, 114.7, 96.7, 96.2, 63.4. HRMS (ESI+) m/z calcd for C15H13N2O3 [M+H]+ 269.0926. found 269.0916.
Compound 9a (7.3 g, 28.4 mmol) was dissolved in THF (34 mL) and Boc2O (7.4 g, 33.9 mmol) was added. The mixture was heated to 70° C. and stirred for three days. The mixture was diluted with EtOAc (300 mL) and the organic layer was washed with H2O (3×200 mL), and brine (200 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the thus obtained crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:7 to 1:4) yielding 10a as a white solid (8.24 g, 81%). 1H-NMR (400 MHz, CDCl3) δ: 8.13 (br d, J=7.0 Hz, 1H), 7.83 (s, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.45 (dd, J=7.7, 1.4 Hz, 1H), 7.39-7.31 (m, 3H), 7.01 (t, J=7.3 Hz, 1H), 4.88 (d, J=4.5 Hz, 2H), 2.38 (br s, 1H), 1.57 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 152.6, 140.2, 139.8, 133.6, 131.8, 131.5, 130.2, 129.5, 128.8, 123.6, 122.3, 118.1, 111.0, 92.6, 90.5, 81.3, 63.7, 28.3 (3C). HRMS (ESI+) m/z calcd [M+Na]+ for C20H20ClNNaO3 380.1029. found 380.1019.
Compound 9b (1.8 g, 5.35 mmol) was dissolved in THF (5.4 mL) and Boc2O (1.17 g, 5.35 mmol) was added. The mixture was heated to 70° C. and stirred overnight. The reaction mixture was diluted with EtOAc (100 mL) and the organic layer was washed with H2O (3×100 mL), and brine (100 mL), and was subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:8 to 1:2). Compound 10b was obtained as a white solid (1.34 g, 57%), also starting material 9b was re-obtained (540 mg, 30%). 1H-NMR (400 MHz, CDCl3) δ: 8.12 (d, J=5.8 Hz, 1H), 7.74 (br s, 1H), 7.72 (s, 1H), 7.63 (s, 1H), 7.44 (ddd, J=7.7, 1.6, 0.5 Hz, 1H), 7.40-7.31 (m, 1H), 7.02 (dt, J=7.6, 1.1 Hz, 1H), 4.85 (d, J=4.8 Hz, 2H), 2.43 (br s, 1H), 1.57 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 151.7, 141.2, 140.2, 133.8, 133.2, 133.1, 131.5, 130.4, 122.9, 122.3, 118.2, 118.0, 110.7, 91.8, 91.3, 81.4, 63.1, 28.3 (3C). HRMS (ESI+) m/z calcd [M+H]+ for C20H20BrClNO3 436.0301. found 436.0315.
Compound 9c (381 mg, 1.26 mmol) was dissolved in THF (1.2 mL) and Boc2O (275 mg, 1.26 mmol) was added. The reaction was stirred for two days at 70° C. in a sealed tube. The reaction mixture was diluted with CH2Cl2 (10 mL) and the organic layer was washed with H2O (15 mL). The H2O-layer was extracted with CH2Cl2 (10 mL). The combined organic layers were washed with H2O (2×10 mL) and brine (10 mL). Next, the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-heptane, 1:4) to obtain compound 10c as yellow oil (444 mg, 87%). RF=0.55 (EtOAc/n-heptane, 1:2). 1H-NMR (300 MHz, CD3OD) δ: 7.87 (d, J=8.3 Hz, 1H), 7.71 (m, 1H), 7.50-7.48 (m, 1H), 7.46-7.41 (m, 2H), 7.38-7.32 (m, 1H), 7.08 (td, J=8.7, 1.2 Hz, 1H), 4.85 (s, 2H), 1.54 (s, 9H). 13C-NMR (75 MHz, CD3OD) δ: 154.9, 146.8, 140.8, 134.4, 132.9, 131.3, 131.0, 130.7, 124.4, 124.1, 121.7, 121.0, 115.0, 92.9, 91.9, 81.8, 63.2, 28.7 (3C). FT-IR νmax film (cm−1): 3395, 2976, 2928, 2366, 1735, 1519, 1498, 1455, 1394, 1243, 1161, 1044, 746. HRMS (ESI+) m/z calcd for C20H20BrNNaO3 [M+Na]+ 424.0524. found 424.0513.
Compound 9d (100 mg, 0.37 mmol) was dissolved in THF (370 μL) and Boc2O (81 mg, 0.37 mmol) was added. The reaction was stirred in a sealed tube at 70° C. overnight. The reaction mixture was diluted with CH2Cl2 (10 mL) and the organic layer was washed with H2O (3×10 mL) and brine (10 mL) and subsequently dried over MgSO4. The solvents were removed in vacuo and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:6 to 1:4). Compound 10d was obtained as red solid (70 mg, 51%). In addition 9d was reobtained (40 mg, 40%). 1H-NMR (400 MHz, CDCl3) δ: 8.36 (d, J=1.6 Hz, 1H), 8.20 (dd, J=8.5, 2.3 Hz, 1H), 8.15 (br s, 1H), 7.80 (s, 1H), 7.71 (d, J=8.5 Hz, 1H), 7.49 (d, J=7.7 Hz, 1H), 7.40 (t, J=7.9 Hz, 1H), 7.04 (t, J=7.6 Hz, 1H), 5.00 (d, J=4.6 Hz, 2H), 2.59 (br s, 1H), 1.58 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 152.5, 147.1, 142.9, 140.5, 132.7, 131.8, 130.9, 123.1, 122.9, 122.4, 118.3, 114.6, 110.4, 94.6, 92.3, 81.6, 63.5, 28.3 (3C). HRMS (ESI+) m/z calcd for C20H20N2NaO5 [M+Na]+ 391.1270. found 391.1265.
Compound 10a (8.24 g, 23.1 mmol) was dissolved in methanol (100 mL). After addition of quinoline (273 μl, 2.31 mmol) and 10% Pd/BaSO4 (492 mg, 0.231 mmol), the reaction was stirred under H2-atmosphere for 2 hours. The reaction mixture was then filtered over celite and diluted with CH2Cl2 (150 mL). The organic layer was washed with 2 M aqueous HCl (2×100 mL), H2O (100 mL), and brine (100 mL). The organic layer was dried over MgSO4 and the volatiles were removed under reduced pressure to obtain compound 11a (7.91 g, 95%). 1H-NMR (400 MHz, CDCl3) δ: 7.26-7.18 (m, 2H), 7.14 (dd, J=8.2, 1.9 Hz, 1H), 7.11 (s, 1H), 6.99 (t, J=7.4 Hz, 1H), 6.92 (s, 1H), 6.90 (d, J=12.0 Hz, 1H), 6.69 (d, J=12.0 Hz, 1H), 6.62 (br s, 1H), 4.67 (d, J=6.2 Hz, 2H), 1.43 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 150.3, 137.2, 137.0, 134.9, 133.3, 130.0, 129.8, 129.5, 129.3, 128.8, 128.4, 127.8 (2C), 125.8, 123.1, 120.4, 81.0, 63.2, 28.2. HRMS (ESI+) m/z calcd for C20H23ClNO3 [M+H]+ 360.1367. found 360.1387.
Compound 10b (470 mg, 1.1 mmol) was dissolved in methanol (20 mL) and 10% Pd/BaSO4 (15 mg, 14 μmol) and quinoline (13 al, 0.11 mmol) were added. The reaction was stirred under H2-atmosphere for two hours. Additional 10% Pd/BaSO4 (15 mg, 14 μmol) was added, and after 1 hour again 10% Pd/BaSO4 (15 mg, 14 μmol) was added. After 1 additional hour the reaction was completed and filtered over celite. The celite was washed with CH2Cl2 (50 mL), and the thus obtained organic layer was washed with 2M aqueous HCl (2×50 mL), H2O (50 mL), and brine (50 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure to obtain 11b as a single product (470 mg, 100%). 1H-NMR (400 MHz, CDCl3) δ: 8.12 (d, J=8.5 Hz, 1H), 7.74 (br s, 1H), 7.72 (m, 1H), 7.63 (s, 1H), 7.44 (ddd, J=7.7, 1.6, 0.5 Hz, 1H), 7.36 (dddd, J=7.5, 1.6, 0.5 Hz, 8.5 Hz, 1H), 7.01 (dt, J=7.5, 1.1 Hz, 1H), 4.85 (d, J=4.8 Hz, 2H), 2.45 (br s, 1H), 1.57 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 152.8, 138.7, 135.9, 134.8, 133.3, 130.3, 129.2, 129.0, 128.6, 128.3 (2C), 125.7, 123.4, 121.4, 120.8, 81.2, 62.5, 28.2 (3C). HRMS (ESI+) m/z calcd for C20H22BrClNO3 [M+H]+ 438.0472. found 438.0495.
Compound 10c (720 mg, 1.8 mmol) was dissolved in methanol (12 mL) and 10% Pd/BaSO4 (35 mg, 33 μmol) and quinoline (21 μL, 0.18 mmol) were added. The reaction mixture was stirred under H2-atmosphere for 1.5 hour. Next, the mixture was filtered over celite and the solvents were removed under reduced pressure. The crude product was purified by column chromatography (EtOAc/n-heptane, 1:9) to obtain compound 11c as orange oil (650 mg, 90%). RF=0.40 (EtOAc/n-heptane, 1:2). 1H-NMR (400 MHz, CDCl3) δ: 7.48 (s, 1H), 7.21 (t, J=7.5 Hz, 1H), 7.14-7.12 (m, 2H), 6.99 (t, J=7.3 Hz, 1H), 6.88 (d, J=12.1 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H), 6.64 (d, J=11.9 Hz, 1H), 6.62 (s, 1H), 4.70 (d, J=6.4 Hz, 2H), 1.43 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 152.6, 140.5, 134.8, 134.3, 131.3, 130.7, 130.5, 130.2, 129.4, 128.3, 128.0, 127.3, 123.0, 121.7, 120.2, 81.2, 63.3, 28.2 (3C). FT-IR νmax film (cm−1): 3421, 2976, 2933, 2362, 2327, 1705, 1576, 1519, 1472, 1446, 1398, 1364, 1308, 1230, 1156, 1053, 1022, 767, 763. HRMS (ESI+) m/z calcd for C20H23BrNO3 [M+H]+ 404.0861. found 404.0865.
Compound 10d (70 mg, 0.19 mmol) was dissolved in methanol (10 mL) and quinoline (11 μL, 95 μmol) and 10% Pd/BaSO4 (2.66 mg, 2 μmol) were added. The reaction was stirred under H2-atmosphere for 3 hours after which the mixture was filtered over celite. The celite was washed with CH2Cl2 (20 mL) and the organic layer was washed with H2O (2×20 mL) and brine (20 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:4) to obtain 11d as a red solid (55 mg, 78%). 1H-NMR (300 MHz, CDCl3) δ: 8.24 (d, J=2.4 Hz, 1H), 7.84 (dd, J=8.5, 2.4 Hz, 1H), 7.73 (br s, 1H), 7.26-7.18 (m, 1H), 7.10 (d, J=8.5 Hz, 1H)), 7.06 (s, 1H), 6.98 (d, J=7.4 Hz, 1H), 6.93 (d, J=12.0 Hz, 1H), 6.81 (d, J=12.0 Hz, 1H), 6.56 (s, 1H), 4.79 (s, 2H), 1.40 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 152.6, 147.0, 142.2, 140.3, 135.0, 129.9, 129.7, 129.3 (2C), 128.8, 125.7, 123.4, 123.1, 122.4, 120.8, 81.3, 62.9, 28.13 (3C). HRMS (ESI+) m/z calcd for C20H2N2NaO5 [M+Na]+ 393.14264. found 393.14315.
Compound 11a (7.91 g, 22 mmol) was dissolved in dry CH2Cl2 (150 mL) under Ar-atmosphere in a flame-dried flask. Dess-Martin periodinane (11.2 g, 26.4 mmol) and NaHCO3 (5.54 g, 66 mmol) were added and the mixture was stirred for 45 minutes. The reaction was quenched by the addition of saturated aqueous NaHSO3 (100 mL). The layers were separated and the H2O-layer was extracted with CH2Cl2 (100 mL). The organic layers were combined and washed with saturated aqueous NaHSO3 (200 mL), H2O (2×200 mL) and brine (200 mL) and then dried over MgSO4. The organic solvents were evaporated and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:9). Compound 12a was obtained as a yellow solid (7.36 g, 95%). 1H-NMR (300 MHz, CDCl3) δ: 10.13 (s, 1H), 7.87 (d, J=8.2 Hz, 1H), 7.74 (d, J=8.3 Hz, 1H), 7.33 (dd, J=8.1, 1.9 Hz, 1H), 7.25-7.18 (m, 1H), 7.16 (d, J=12.0 Hz, 1H), 7.09 (d, J=2.0 Hz, 1H), 7.00-6.87 (m, 2H), 6.84 (d, J=11.9 Hz, 1H), 6.44 (br s, 1H), 1.46 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 190.6, 152.4, 140.4, 140.0, 135.6, 132.3, 131.7, 130.3, 129.7, 129.5, 129.0, 128.9, 128.3, 125.1, 123.1, 120.3, 80.5, 28.3 (3C). HRMS (ESI+) m/z calcd for C20H20ClNNaO3 [M+Na]+ 380.1029. found 380.1032.
Compound 11b (1.17 g, 2.67 mmol) was dissolved in dry CH2Cl2 (40 mL) under Ar-atmosphere in a flame-dried flask. NaHCO3 (670 mg, 8.0 mmol) and Dess-Martin periodinane (1.47 g, 3.47 mmol) were added and the reaction was stirred for 1 hour. Hereupon, the reaction was quenched with saturated aqueous NaHSO3 and diluted with CH2Cl2 (20 mL). The organic layer was washed with H2O (3×50 mL) and brine (50 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:9). Compound 12b was obtained as a yellow solid (1.02 g, 89%). 1H-NMR (400 MHz, CDCl3) δ: 10.07 (s, 1H), 8.02 (s, 1H), 7.85 (d, J=8.2 Hz, 1H), 7.26-7.22 (m, 1H), 7.21 (s, 1H), 7.07 (d, J=11.9 Hz, 1H), 6.98-6.90 (m, 2H), 6.87 (d, J=11.9 Hz, 1H), 6.39 (s, 1H), 1.47 (d, J=1.0 Hz, 9H). 13C-NMR (75 MHz, CDCl3) δ: 189.3, 152.4, 140.1, 138.8, 135.6 (2C), 132.7, 131.9, 130.5, 129.5, 129.1, 127.6, 125.1, 123.4, 122.2, 120.7, 80.7, 28.3 (3C). HRMS (ESI+) m/z calcd for C20H19BrClNNaO3 [M+Na]+ 458.0135. found 458.0123.
Compound 11c (651 mg, 1.62 mmol) was dissolved in dry CH2Cl2 (15 mL) and placed under an Ar-atmosphere in a flame-dried flask. Subsequently, Dess-Martin periodinane (888 mg, 2.09 mmol) and NaHCO3 (406 mg, 4.83 mmol) were added and the mixture was stirred for 40 minutes. The reaction was quenched with saturated aqueous Na2SO3 (15 mL). The mixture was diluted with CH2Cl2 (25 mL), washed with saturated aqueous NaHSO3 (15 mL), H2O (15 mL) and brine (15 mL). Next, the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-heptane, 1:9) to obtain compound 12c as a yellow oil which solidified upon storage at −20° C. (560 mg, 86%). RF=0.40 (EtOAc/n-heptane, 1:4). 1H-NMR (400 MHz, CDCl3) δ: 10.16 (s, 1H), 7.93 (d, J=2.2 Hz, 1H), 7.87 (d, J=7.7 Hz, 1H), 7.43 (dd, J=8.3, 2.2 Hz, 1H), 7.23-7.19 (m, 1H), 7.17 (d, J=12.0 Hz, 1H), 6.99-6.96 (m, 2H), 6.92 (d, J=7.3 Hz, 1H), 6.82 (d, J=11.9 Hz, 1H), 6.42, (br s, 1H), 1.45 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 190.4, 152.4, 137.5, 136.4, 135.5, 134.7, 133.8, 131.9, 129.6, 129.3, 129.2, 128.7, 125.5, 123.1, 122.0, 120.3, 80.6, 28.2 (3C). FT-IR νmax film (cm−1): 2982, 1729, 1695, 1584, 1515, 1445, 1238, 1369, 1148, 1051, 1016, 780, 746. HRMS (ESI+) m/z calcd for C20H20BrNNaO3 [M+Na]+ 424.0524. found 424.0516.
Compound 11d (170 mg, 0.46 mmol) was dissolved in dry CH2Cl2 (5 mL) under Ar-atmosphere in a flame-dried flask. Dess-Martin periodinane (234 mg, 0.55 mmol) and NaHCO3 (116 mg, 1.38 mmol) were added. The reaction was stirred for 30 minutes whereupon saturated aqueous NaHSO3 (10 mL) was added. The mixture was diluted with CH2Cl2 (15 mL) and the organic layer was washed with saturated aqueous NaHSO3 (30 mL), water (2×30 mL) and brine (30 mL) before drying over MgSO4. The solvents were removed in vacuo and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:19 to 1:6) to obtain 12d as a red solid (145 mg, 86%). 1H-NMR (400 MHz, CDCl3) δ: 10.27 (s, 1H), 8.66 (d, J=2.4 Hz, 1H), 8.14 (dd, J=8.5, 2.5 Hz, 1H), 7.82 (d, J=8.3 Hz, 1H), 7.32 (d, J=8.6 Hz, 1H), 7.25 (d, J=11.8 Hz, 1H), 7.26-7.21 (m, 1H), 6.97 (d, J=11.9 Hz, 1H), 6.93-6.88 (m, 2H), 6.44-6.35 (br s, 1H), 1.45 (s, 9H). 13C-NMR (75 MHz, CDCl3) δ: 189.7, 152.4, 144.8, 135.7, 134.1, 133.0, 131.9, 131.6, 129.6, 129.3, 128.4, 127.4, 126.0, 125.4, 123.5, 121.0, 80.8, 28.3 (3C). HRMS (ESI+) m/z calcd for C20H20N2NaO5 [M+Na]+ 391.1270. found 391.1259.
Compound 12a (7.34 g, 20.6 mmol) was dissolved in 2M HCl in EtOAc (100 ml, 200.00 mmol) and the reaction was stirred for 1 hour. Then, NaBH4 (3.11 g, 82.4 mmol) in H2O (10 mL) was added and the reaction was stirred overnight. As reduction of the imine was not completed yet, another portion of NaBH4 (2.25 g, 60 mmol) was added and the reaction was stirred for 1.5 hour. Hereupon, the reaction was quenched by the addition of H2O (50 mL) and the product was extracted with EtOAc (100 mL). The organic layer was washed with H2O (3×100 mL) and brine (100 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure to obtain 13a as a yellow solid (4.7 g, 95%). 1H-NMR (300 MHz, CDCl3) δ: 7.18-7.09 (m, 3H), 7.06-6.84 (m, 2H), 6.69-6.60 (m, 1H), 6.58 (d, J=13.2 Hz, 1H), 6.46 (dd, J=8.1, 0.6 Hz, 1H), 6.27 (d, J=13.1 Hz, 1H), 4.55 (s, 2H), 4.22 (br s, 1H). 13C-NMR (75 MHz, CDCl3) δ: 146.8, 141.1, 136.6, 134.7, 134.0, 133.3, 130.3, 129.7, 128.3, 127.3, 126.1, 121.3, 118.0, 117.9, 48.8. HRMS (ESI+) m/z calcd for C15H13ClN [M+H]+ 242.0737. found 242.0726.
Compound 12b (920 mg, 2.1 mmol) was dissolved in 2M HCl in EtOAc (40 mL, 80 mmol). After 30 minutes, NaBH4 (240 mg, 6.3 mmol) in H2O (2 mL) was added. After two hours another portion of NaBH4 (240 mg, 6.3 mmol) in H2O (2 mL) was added, followed by another portion of NaBH4 (80 mg, 2.1 mmol) in H2O (1 mL) after 1 hour. After an extra 30 minutes, the reaction was quenched by the addition of H2O (40 mL). The layers were separated, and the H2O-layer was extracted with EtOAc (50 mL). The organic layers were combined and washed with H2O (2×100 mL), and brine (100 mL), and subsequently dried over MgSO4. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:6). Compound 13b was obtained as yellow solid (340 mg, 50%). 1H-NMR (300 MHz, CDCl3) δ: 7.44 (s, 1H), 7.24 (s, 1H), 6.99-6.87 (m, 2H), 6.63 (dt, J=7.7, 1.3 Hz, 1H), 6.59 (d, J=13.0 Hz, 1H), 6.47 (dd, J=8.0, 1.2 Hz, 1H), 6.20 (d, J=13.1 Hz, 1H), 4.52 (s), 4.24 (s). 13C-NMR (75 MHz, CDCl3) δ: 146.6, 140.1, 138.4, 134.7, 134.4, 134.0, 133.4, 131.5, 128.5, 125.1, 121.3, 120.1, 118.1, 118.0, 48.6. HRMS (ESI+) m/z calcd for C15H12BrClN [M+H]+ 319.9842. found 319.9825.
Compound 12c (560 mg, 1.4 mmol) was dissolved in 2M HCl in EtOAc (20 mL, 40 mmol) and stirred for 1.5 hour. Next, NaBH4 (196 mg, 5.2 mmol) and a few drops of water were added. The reaction was stirred overnight whereupon an additional portion of NaBH4 (196 mg, 5.2 mmol) was added and, after an additional 90 minutes, the reaction was quenched with H2O (15 mL). The H2O-layer was extracted with EtOAc (2×15 mL). The organic layers were combined and washed with 2M aqueous NaOH (2×20 mL), H2O (2×20 mL) and brine (20 mL). Next, the organic layer was dried over MgSO4 and concentrated in vacuo to obtain compound 13c as a yellow solid (360 mg, 91%). RF=0.55 (EtOAc/n-heptane, 1:2). 1H-NMR (400 MHz, CDCl3) δ: 7.36 (dd, J=8.7, 2.1 Hz, 1H), 7.34 (d, J=2.1 Hz, 1H), 7.02 (d, J=8.1 Hz, 1H), 6.96 (dd, J=7.7, 1.7 Hz, 1H), 6.90 (ddd, J=7.2, 6.5, 1.6 Hz, 1H), 6.62 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 6.55 (d, J=13.1 Hz, 1H), 6.47 (dd, J=8.1, 1.2 Hz, 1H), 6.26 (d, J=13.1 Hz, 1H), 4.54 (s, 2H). 13C-NMR (75 MHz, CDCl3) δ: 146.3, 139.8, 137.7, 134.3, 132.9, 131.3 (2C), 130.2, 127.7, 126.9, 125.8, 121.1, 119.8, 117.5, 48.7. FT-IR νmax film (cm−1): 3391, 3002, 2924, 2850, 2362, 2098, 1593, 1485, 1325, 1269, 901, 828, 776, 750. HRMS (ESI+) m/z calcd for C15H113brN [M+H]+ 286.02314 found 286.0219.
Compound 12d (200 mg, 0.54 mmol) was dissolved in HCl in EtOAc (10 mL, 20 mmol). After 30 minutes NaBH4 (470 mg, 12.4 mmol) in water (1 mL) was added and after stirring overnight the reaction was quenched with H2O (10 mL). The H2O-layer was extracted with EtOAc (20 mL), and the combined organic layers were washed with H2O (3×20 mL), brine (20 mL) and subsequently dried over MgSO4. The solvents were removed in vacuo to obtain 13d as a red solid (140 mg, 100%) 1H-NMR (400 MHz, CDCl3) δ: 8.11 (dd, J=8.5, 2.4 Hz, 1H), 8.05 (d, J=2.3 Hz, 1H), 7.29 (d, J=8.6 Hz, 1H), 7.01 (d, J=7.7 Hz, 1H), 6.97-6.91 (m, 1H), 6.71-6.62 (m, 2H), 6.51 (d, J=8.1 Hz, 1H), 6.36 (d, J=13.4 Hz, 1H), 4.69 (s, 2H). 13C-NMR (75 MHz, CDCl3) δ: 147.3, 146.7, 145.9, 139.5, 135.9, 135.4, 131.3, 128.8, 125.0, 124.1, 122.8, 121.2, 118.4, 118.0, 49.6. HRMS (ESI+) m/z calcd for C15H13N2O2 [M+H]+ 253.0977 found 253.0965.
Compound 13a (3 g, 12.4 mmol) was dissolved in dry CH2Cl2 (100 mL) and NEt3 (3.46 mL, 24.8 mmol) was added. After cooling the mixture to 0° C., methyl 5-chloro-5-oxopentanoate (2.13 mL, 15 mmol) was added and the reaction was stirred overnight. Hereupon, the reaction was quenched with H2O (100 mL) and the layers were separated. The organic layer was washed with 2M aqueous NaOH (2×70 mL), water (2×70 mL) and brine (70 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:2) to obtain 14a as a yellow solid (2.11 g, 46%). 1H-NMR (400 MHz, CDCl3) δ: 7.31-7.27 (m, 3H), 7.23 (d, J=8.8 Hz, 1H), 7.19-7.10 (m, 3H), 6.69 (d, J=13.1 Hz, 1H), 6.62 (d, J=13.1 Hz, 1H), 5.43 (d, J=14.9 Hz, 1H), 4.17 (d, J=14.9 Hz, 1H), 3.59 (s, 3H), 2.25-2.03 (m, 3H), 1.94-1.76 (m, 3H). 13C-NMR (75 MHz, CDCl3) δ: 173.5, 171.8, 140.8, 137.6, 135.8, 133.3, 132.7, 131.7, 131.5, 131.4, 131.1, 128.7, 128.5, 128.1, 128.1, 127.3, 53.9, 51.4, 33.5, 33.0, 20.4. HRMS (ESI+) m/z calcd for C21H21ClNO3 [M+H]+ 370.1210. found 370.1203.
Compound 13b (200 mg, 0.62 mmol) was dissolved in dry CH2Cl2 (15 mL) and the solution was cooled to 0° C. Subsequently, NEt3 (174 μL, 1.25 mmol) and methyl 5-chloro-5-oxopentanoate (133 al, 0.94 mmol) were added. The reaction was stirred overnight and then quenched with H2O (10 mL). The layers were separated, and the H2O-layer was extracted with CH2Cl2 (20 mL). The organic layers were combined and washed with H2O (2×30 mL), and brine (30 mL) and dried over MgSO4. The solvents were removed in vacuo and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:2). Compound 14b was obtained as yellow solid (230 mg, 82%). 1H-NMR (400 MHz, CDCl3) δ: 7.53 (s, 1H), 7.34-7.29 (m, 3H), 7.22 (s, 1H), 7.18 (dd, J=6.4, 2.6 Hz, 1H), 6.64 (d, J=13.3 Hz, 1H), 6.60 (d, J=13.3 Hz, 1H), 5.45 (d, J=15.2 Hz, 1H), 4.12 (d, J=15.2 Hz, 1H), 3.59 (s, 3H), 2.62-2.29 (m, 1H), 2.26-1.96 (m, 2H), 1.93-1.76 (m, 3H). 13C-NMR (75 MHz, CDCl3) δ: 173.4, 171.8, 140.5, 136.2, 135.5, 135.0, 134.9, 133.2, 132.7, 131.4, 130.0, 129.0, 128.7, 128.2, 128.1, 120.8, 53.4, 51.3, 33.3, 32.9, 20.3. HRMS (ESI+) m/z calcd for C21H20BrClNO3 [M+H]+ 448.0321. found 448.0315.
Compound 13c (360 mg, 1.26 mmol) was dissolved in dry CH2Cl2 (8 mL) and NEt3 (351 μL, 2.52 mmol) was added. The mixture was cooled to 0° C., whereupon methyl 5-chloro-5-oxopentanoate (221 μL, 1.89 mmol) was added. The reaction was stirred for 90 minutes, after which it was quenched with H2O (5 mL). The mixture was diluted with CH2Cl2 (10 mL) and washed with 2M aqueous NaOH (2×10 mL), 2M aqueous HCl (2×10 mL), H2O (2×10 mL) and brine (10 mL). Next, the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-heptane, 1:2) to obtain compound 14c as a yellow solid (466 mg, 90%). RF=0.30 (EtOAc/n-heptane, 1:2). 1H-NMR (400 MHz, CDCl3) δ: 7.42 (d, J=2.1 Hz, 1H), 7.31-7.27 (m, 4H), 7.19-7.15 (m, 1H), 7.00 (d, J=8.3 Hz, 1H), 6.69 (d, J=13.1 Hz, 1H), 6.59 (d, J=13.1 Hz, 1H), 5.49 (d, J=15.2 Hz, 1H), 4.16 (d, J=15.2 Hz, 1H), 3.59 (s, 3H), 2.22-2.17 (m, 2H), 2.13-2.04 (m, 2H), 1.85-1.79 (m, 2H). 13C-NMR (75 MHz, CDCl3) δ: 173.5, 171.8, 140.7, 136.9, 136.1, 134.5, 133.7, 132.9, 131.4, 131.1, 130.1, 128.8, 128.1 (2C), 127.6, 121.2, 54.0, 51.4, 33.3, 33.1, 20.4. FT-IR νmax film (cm−1): 3473, 2947, 2868, 2150, 1874, 1731, 1653, 1498, 1437, 1403, 1195, 1169, 1018, 832, 776. HRMS (ESI+) m/z calcd for C21H21BrNO3 [M+H]+ 414.0705. found 414.0699.
Compound 13d (45 mg, 0.18 mmol) was dissolved in dry CH2Cl2 (5 ml) and the solution was cooled to 0° C. Subsequently, NEt3 (50 μL, 0.36 mmol) and methyl 5-chloro-5-oxopentanoate (37 al, 0.27 mmol) were added. The reaction was stirred overnight and quenched by the addition of 0.1 M aqueous NaOH (5 mL). The reaction was diluted with CH2Cl2 (10 mL) and the organic layer was washed with 2M aqueous NaOH (2×20 mL), H2O (20 mL) and brine (20 mL), and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:6 to 2:3). Compound 14d was obtained as a red solid (25 mg, 37%). 1H-NMR (300 MHz, CDCl3) δ: 8.20 (d, J=2.4 Hz, 1H), 8.02 (dd, J=8.5, 2.4 Hz, 1H), 7.32 (dt, J=11.5, 4.1 Hz, 5H), 7.24-7.17 (m, 1H), 6.81 (d, J=13.4 Hz, 1H), 6.73 (d, J=13.3 Hz, 1H), 5.55 (d, J=15.1 Hz, 1H), 4.23 (d, J=15.0 Hz, 1H), 3.58 (s, 3H), 2.25-2.08 (m, 2H), 2.06-1.91 (m, 2H), 1.86-1.66 (m, 2H). HRMS (ESI+) m/z calcd for C21H21N2O5 [M+H]+ 381.1451. found 381.1446.
Compound 14a (1 g, 2.7 mmol) was dissolved in CH2Cl2 (50 mL) and the solution was cooled to 0° C. A solution of Br2 (154 μl, 3 mmol) in CH2Cl2 (5 mL) was added dropwise and after 2 hours at 0° C. additional Br2 (20 μL, 0.39 mmol) in CH2Cl2 (1 mL) was added. After 1 hour the reaction was quenched by addition of saturated aqueous NaHSO3 (50 mL), and layers were separated. The organic layer was washed with saturated aqueous NaHSO3-solution (50 mL), water (2×50 mL) and brine (50 mL) and subsequently dried over MgSO4. The volatiles were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:4 to 1:2) to obtain compound 15a (1.02 g, 71%) as mixture of two diastereoisomers (X:Y, 1:0.8). The isomers could be separated however due to slow isomerization of X to Y, no full analysis of 15aY was performed. Analytical data for 15aX: RF=0.30 (EtOAc/n-heptane, 1:2). 1H-NMR (300 MHz, CDCl3) δ: 7.72 (d, J=2.1 Hz, 1H), 7.28-7.15 (m, 2H), 7.12-6.94 (m, 3H), 6.83 (d, J=8.3 Hz, 1H), 5.85 (d, J=9.9 Hz, 1H), 5.77 (d, J=14.8 Hz, 1H), 5.12 (d, J=10.0 Hz, 1H), 4.14 (d, J=14.8 Hz, 1H), 3.61 (s, 3H), 2.97-2.27 (m, 3H), 2.27-2.13 (m, 1H), 2.08-1.92 (m, 2H). 13C-NMR (75 MHz, CDCl3) δ: 173.6, 172.7, 138.9, 137.8, 137.0, 134.7, 131.5, 130.9 (2C), 130.7, 130.4, 129.6, 129.0, 128.9, 59.5, 54.5, 51.8, 51.5, 34.8, 33.3, 20.3. HRMS (ESI+) m/z calcd for C21H21Br2ClNO3 [M+H]527.9577. found 527.9564. 15aY: RF=0.35 (EtOAc/n-heptane, 1:2). 1H-NMR (400 MHz, CDCl) δ 7.65 (dd, J=7.8, 1.3 Hz, 1H), 7.25-6.98 (m, 4H), 6.88-6.78 (m, 2H), 5.71 (d, J=9.6 Hz, 1H), 5.14 (d, J=9.6 Hz, 1H), 5.13 (d, J=14.5 Hz, 1H), 4.95 (d, J=14.0 Hz, 1H), 3.63 (s, 3H), 2.48-2.31 (m, 3H), 2.26-2.05 (m, 1H), 2.04-1.80 (m, 2H).
Compound 14b (100 mg, 0.22 mmol) was dissolved in CH2Cl2 (10 mL) and the solution was cooled to 0° C. A solution of Br2 (11 μl, 0.22 mmol) in CH2Cl2 (1 mL) was added dropwise and the reaction was stirred at 0° C. After 2 hours, the reaction was quenched with saturated aqueous NaHSO3 (10 mL). The layers were separated and the H2O-layer was extracted with CH2Cl2 (10 mL). The combined organic layers were washed with saturated aqueous NaHSO3 (20 mL), water (2×20 mL) and brine (20 mL) and dried over MgSO4. The solvents were removed under reduced pressure to obtain 15b as white solid (130 mg, 96%). 15b was obtained as a mixture of two diastereoisomers (15bX:15bY, 0.32:1). For the 1H-NMR signals from 15bX are designated with *, signals from 15bY with o. In the 13C-NMR data, only peaks are given from major isomer 15bX. 1H-NMR (400 MHz, CDCl3) δ 7.82* (s, 0.3H), 7.65o (d, J=7.7 Hz, 1H), 7.35o (s, 1H), 7.32-7.08*,o (m, 3.4H), 7.02o (s, 1H), 7.00* (s, 0.3H), 6.90o (d, J=7.8 Hz, 1H), 5.80* (d, J=9.8 Hz, 0.3H), 5.79* (d, J=15.1 Hz, 0.3H), 5.69o (d, J=9.6 Hz, 1H), 5.12*,o (d, J=9.5 Hz, 1.3H), 5.08o (d, J=14.6 Hz, 1H), 4.92° (d, J=14.3 Hz, 1H), 4.09* (d, J=15.3 Hz, 0.3H), 3.63o (d, J=0.7 Hz, 3H), 3.62* (s, 1H), 2.60-2.29*,o (m, 4H), 2.21-2.02*,o (m, 1.3H), 2.01-1.83*,o (m, 2.6H). 13C-NMR (75 MHz, CDCl3) δ: 173.6, 172.8, 137.9, 137.7, 136.8, 134.9, 134.2, 133.2, 131.0 (2C), 130.8, 130.5, 130.0, 122.7, 59.1, 53.9, 51.5, 51.3, 34.7, 33.2, 20.3. HRMS (ESI+) m/z calcd for C21H20Br3ClNO3 [M+H]+605.8682. found 605.8686.
Compound 14c (100 mg, 0.24 mmol) was dissolved in dry CH2Cl2 (5 mL). The solution was cooled to 0° C. and Br2 (13 μL, 0.24 mmol) was added. After stirring for 1.5 hour, additional Br2 (13 μL, 0.24 mmol) was added and the reaction was stirred for an additional hour. Hereupon, the reaction was quenched with saturated aqueous Na2SO3 (5 mL). The mixture was diluted with CH2Cl2 (10 mL) and washed with saturated aqueous Na2SO3 (2×10 mL), H2O (2×10 mL) and brine (10 mL). Next, the organic layer was dried over MgSO4 and concentrated in vacuo. The crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:4 to 1:2) to obtain compound 15c as a white solid (111 mg, 80%). Compound 15c was obtained as mixture of diastereoisomers (15cX:15cY, 1:0.47). The major isomer (15cX) could be obtained pure, due to slow isomerization of 15cY to 15cX, no spectrum of pure 15cY was obtained. Analytical data for 15cX: RF=0.25 (EtOAc/n-heptane, 1:2). 1H-NMR (400 MHz, CDCl3) 6: 1H-NMR (400 MHz, CDCl3) δ: 7.61 (d, J=8.4 Hz, 1H), 7.36-7.26 (m, 2H), 7.19 (td, J=7.6, 1.4 Hz, 1H), 7.11-7.03 (m, 2H), 7.00 (dd, J=7.8, 1.4 Hz, 1H), 5.84 (d, J=10.0 Hz, 1H), 5.82 (d, J=15.1 Hz, 1H), 5.12 (d, J=10.0 Hz, 1H), 4.09 (d, J=15.2 Hz, 1H), 3.62 (s, 3H), 2.41-2.27 (m, 3H), 2.25-2.14 (m, 1H), 2.09-1.94 (m, 2H). 13C-NMR (75 MHz, CDCl3) δ: 173.6, 172.8, 137.8, 136.8, 136.1, 135.0, 132.2, 131.8, 130.8 (2C), 130.5, 130.4, 129.7, 122.6, 59.4, 54.8, 51.9, 51.5, 34.7, 33.2, 20.3. FT-IR νmax film (cm−1): 3453, 2953, 2367, 1734, 1660, 1593, 1484, 1441, 1398, 1254, 1203, 1179, 1152, 1015, 875, 844, 801, 769. HRMS (ESI+) m/z calcd for C21H21Br3NO3 [M+H]+ 571.9090. found 571.9080. Analytical data for 15cY: 1H-NMR (400 MHz, CDCl3) δ: 7.68-7.61 (m, 1H), 7.38-7.12 (m, 4H), 6.88 (dd, J=7.7, 1.3 Hz, 1H), 6.79 (d, J=8.2 Hz, 1H), 5.72 (d, J=9.4 Hz, 1H), 5.19 (d, J=9.6 Hz, 1H), 5.09 (d, J=14.7 Hz, 2H), 4.96 (d, J=14.1 Hz, 1H), 3.63 (s, 3H), 2.45-2.30 (m, 3H), 2.24-2.11 (m, 1H), 2.12-1.83 (m, 2H).
Compound 14d (25 mg, 66 μmol) was dissolved in CH2Cl2 (2 mL) and the reaction was cooled to 0° C. A solution of Br2 (4.1 μl, 79 μmol) in CH2Cl2 (2 mL) was added dropwise. The reaction was stirred at 0° C. for 1 hour, and subsequently quenched with saturated aqueous NaHSO3 (10 mL) and diluted with CH2Cl2 (10 mL). The organic layer was washed with saturated aqueous NaHSO3 (10 mL), water (2×10 mL) and brine (10 mL) and dried over MgSO4. The solvents were evaporated under reduced pressure to obtain 15d as a red solid (30 mg, 84%). 15d was obtained as a mixture of two diastereoisomers (15dX:15dY, 0.42:1). For the 1H-NMR signals from 15dX are designated with *, signals from 15dY with o. 1H-NMR (400 MHz, CDCl3) δ: 8.04* (dd, J=8.6, 2.0 Hz, 0.43H), 7.99-7.90*,o (m, 2.5H), 7.79* (d, J=2.0 Hz, 0.4H), 7.66o (dd, J=7.8, 1.4 Hz, 1H), 7.34-7.14*,o (m, 3.1H), 7.12*,o (d, J=8.4 Hz, 1.2H), 7.09-6.990 (m, 1H), 6.910 (dd, J=7.8, 1.3 Hz, 1H), 5.93* (d, J=11.6 Hz, 0.43H), 5.92* (d, J=13.7 Hz, 0.43H), 5.75o (d, J=9.5 Hz, 1H), 5.28o (d, J=9.6 Hz, 1H), 5.15* (d, J=9.9 Hz, 0.43H), 5.04o (d, J=14.3 Hz, 1H), 4.28* (d, J=15.3 Hz, 0.43H), 3.63o (s, 3H), 3.62* (s, 1H), 2.48-2.39*,o (m, 1.45H), 2.39-2.32*o (m, 3H), 2.17-2.05*,o (m, 1.3H), 2.04-1.89*,o (m, 3.4H). HRMS (ESI+) m/z calcd for C21H21Br2N2O5 [M+H]+ 538.9817. found 538.9822.
Compound 15aX (90 mg, 0.17 mmol) was dissolved in dry THF (3 mL) under Ar-atmosphere in a flame-dried flask. The solution was cooled to −40° C. and a KOtBu-solution in THF (1M, 340 μL, 0.34 mmol) was added dropwise. After stirring at −40° C. for 1.5 hour, additional KOtBu-solution in THF (1M, 50 μL, 0.05 mmol) was added. After 30 minutes, the reaction was quenched with H2O (5 mL) and diluted with CH2Cl2 (10 mL). The layers were separated and the organic layer was washed with H2O (3×15 mL) and brine (15 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by column chromatography (EtOAc/n-heptane, 1:3). Compound 16a was obtained as a white solid (23 mg, 37%), with a 5% contamination of compound 15a. 1H-NMR (300 MHz, CDCl3) δ: 7.60 (d, J=8.2 Hz, 1H), 7.48-7.35 (m, 3H), 7.34-7.27 (m, 2H), 7.21 (d, J=2.2 Hz, 1H), 5.11 (d, J=13.8 Hz. 1H), 3.59 (d, J=13.9 Hz, 1H), 3.55 (s, 3H), 2.43-2.21 (m, 1H), 2.20-1.98 (m, 2H), 1.98-1.82 (m, 1H), 1.82-1.64 (m, 2H). 13C-NMR (75 MHz, CDCl3) δ: 173.4, 172.5, 151.7, 146.2, 133.5, 133.3, 129.0, 128.7, 128.2, 128.1, 127.2, 125.4, 124.6, 122.0, 113.3, 109.2, 54.7, 51.4, 33.6, 32.8, 20.5. HRMS (ESI+) m/z calcd for C21H19ClNO3 [M+H]+ 368.1054. found 368.1043.
Compound 15cX (75 mg, 0.13 mmol) was dissolved in dry THF in a flame-dried flask under Ar-atmosphere, and the solution was cooled to −40° C. Next, a solution of KOtBu in THF (1M, 260 μL, 0.26 mmol) was added dropwise. After 2 hours, only one bromide was eliminated, whereupon additional KOtBu (130 μL, 0.13 mmol) was added. After each subsequent hour an additional amount of KOtBu (30 μL, 0.03 mmol) was added, while maintaining the reaction at −40° C. After 5.5 hours the reaction was completed and quenched by the addition of H2O (5 mL). The H2O-layer was extracted with EtOAc (3×10 mL). The combined organic layers were washed with H2O (20 mL), and brine (20 mL) and subsequently dried over MgSO4. The solvents were removed under reduced pressure and the crude product was purified by gradient column chromatography (EtOAc/n-heptane, 1:4 to 1:2) to obtain compound 16c as a brown oil (20 mg, 37%). 1H-NMR (400 MHz, CDCl3) δ: 7.84 (d, J=2.0 Hz, 1H), 7.44 (ddd, J=8.1, 2.0, 0.4 Hz, 1H), 7.42-7.36 (m, 3H), 7.35-7.30 (m, 1H), 7.10 (d, J=7.6 Hz, 1H), 5.08 (d, J=13.9 Hz, 1H), 3.61 (d, J=13.9 Hz, 1H), 3.56 (s, 3H), 2.36-2.24 (m, 1H), 2.21-2.05 (m, 2H), 1.97-1.85 (m, 1H), 1.83-1.69 (m, 2H). 13C-NMR (75 MHz, CDCl3) δ: 173.4, 172.5, 151.5, 149.6, 135.3, 131.0, 129.0, 128.6, 128.2, 127.1, 126.5, 122.5, 122.3, 122.0, 113.9, 108.8, 54.8, 51.4, 33.6, 32.8, 20.5. HRMS (ESI+) m/z calcd for C21H18BrNNaO3 [M+Na]+434.0368. found 434.0366.
Kinetic experiments for 3, 16a and 16c were performed as follows. First, 2.25 μmol alkyne was mixed with 2.25 μmol benzylazide in 0.5 mL CD3OD. The exact ratio between benzyl azide and alkyne was determined by comparison of the integrals of the aromatic signals and the benzylic protons of the alkyne. For 3, 16a and 16c, the rate constant was determined by comparing the signal from the methyl ester of the starting material (δ: 3.52 ppm), to the signal from the methyl-ester of the product (δ: 3.60 ppm). Product formation was confirmed by mass spectrometry. For 16a: HRMS (ESI+) m/z calcd for C28H26ClN4O3[M+H]+ 501.1693. found 501.1693. For 16c: HRMS (ESI+) m/z calcd for C28H26BrN4O3[M+H]+ 545.1188. found 545.1189.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13169208.9 | May 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/NL2014/050319 | 5/21/2014 | WO | 00 |
