Abstract:
A process is disclosed for nitrating photocleavable compounds comprising a photocleavable group linked to an effector moiety which are capable of releasing the effector moiety on irradiation with light or a group is capable of linkage to an effector moiety. The process employs copper (II) nitrate and acetic anhydride to nitrate substituted indolines at the 7-position. This reaction which proceeds in good yield and selectivity and can be carried out in the presence of the functional groups at the 4-position and/or the effector moiety which is typically linked via an acyl linkage to the heterocyclic nitrogen at the 1-position. These nitration conditions have the advantages of a favourable isomeric ratio of products and leaving the protecting groups intact, thereby giving additional scope for isomer separation. In a preferred embodiment, the present invention employs clay supported copper (II) nitrate (“claycop”) and acetic anhydride which provides improved yield and isomeric ratio as compared to the use of homogeneous, soluble copper (II) nitrate.

Description:
FIELD OF INVENTION  
         [0001]    The present invention relates to a process for producing photocleavable compounds capable of releasing effector moieties on irradiation with light, and in particular to a process of nitrating indolines.  
         BACKGROUND OF INVENTION  
         [0002]    Photocleavable (caged) reagents that release biologically active compounds rapidly upon flash irradiation with near-UV light are potentially valuable tools for study of biological processes. However, reagents that can photorelease effectors such as neuroactive amino acids rapidly and efficiently in aqueous solution have proved elusive. Thus, while there are reports in the prior art of approaches to the problem based on a wide range of different photolabile protecting groups, overall these have met with limited success.  
           [0003]    In WO 00/55133, a family of photolabile 1-acyl-7-nitroindolines are disclosed which are capable of rapidly and stoichiometrically releasing effectors, such as neuroactive amino acids, peptides and metal ion chelators, where the acyl linkage between the effector and the nitroindoline is broken upon photolysis as exmplified in Scheme 1.  
                         
 
           [0004]    Among the best of the compounds disclosed in this application are compounds having a methoxy group on the 4-position of the heterocyclic ring. Compounds of structure 2 were shown to be ˜3-fold more photosensitive than other useful compounds disclosed in this application such as compounds 1. 2  These quantitative comparisons of photolysis efficiency were made with the 1-acetyl compounds 1a and 2a and the advantage was maintained for the corresponding L-glutamate derivative 2b. 3   
                         
 
           [0005]    Despite the significant improvement in photosensitivity of 2b and related compounds, the process for making these compounds suffers from the disadvantage of a poor yield for introduction of the nitro group. In this step, the precursor 3b was treated with sodium nitrate in TFA solution 2  and the reaction product was a complex mixture of products, from which the 7-nitro isomer 2b was isolated by reversed phase HPLC in ˜4% yield. The corresponding 5-nitro isomer was obtained in similar yield and the same 1:1 ratio of 5- and 7-nitro isomers was obtained for the 1-acetyl compound 3a, where isomers 2a and 4 could be easily separated by silica gel chromatography. Although the detailed photolysis mechanism is not known, oxygen transfer takes place from the 7-nitro group to the adjacent 1-acyl group, 1  so the 5-isomer is not useful. The previous nitration conditions result in concomitant loss of the protecting groups on the amino acid moiety in 3b, so separation options were limited to methods appropriate to the zwitterion products.  
         SUMMARY OF THE INVENTION  
         [0006]    Broadly, the present invention relates to an improved process for nitrating photocleavable compounds comprising a photocleavable group linked to an effector moiety which are capable of releasing the effector moiety on irradiation with light or a group is capable of linkage to an effector moiety. In particular, the present invention relates to a process of nitrating precursor compounds having a sensitive substituent at the 4-position, such as an alkoxy group. The process can also be used where the effector is initially linked to the precursor compound, e.g. linked via an acyl linkage to the nitrogen atom at the one position on the heterocyclic ring. This is particularly useful as the prior art nitration methods had the disadvantage of removing protecting groups of present on effector moieties such as amino acids.  
           [0007]    The present invention employs copper (II) nitrate and acetic anhydride to nitrate substituted indolines at the 7-position. This reaction which proceeds in good yield and selectivity and can be carried out in the presence of the functional groups at the 4-position and/or the effector moiety which is typically linked via an acyl linkage to the heterocyclic nitrogen at the 1-position. These nitration conditions have the 2-fold advantage of a much more favourable isomeric ratio of products and leaving the protecting groups intact, thereby giving additional scope for isomer separation. In a preferred embodiment, the present invention employs clay supported copper (II) nitrate (“claycop”) and acetic-anhydride which provides improved yield and isomeric ratio as compared to the use of homogeneous, soluble copper (II) nitrate.  
           [0008]    Accordingly, in a first aspect, the present invention provides a process for producing a 7-nitroindoline, the process comprising reacting copper (II) nitrate and acetic anhydride and a compound represented by the formula:  
                         
 
           [0009]    wherein:  
           [0010]    R 1  is an alkoxy or substituted alkoxy group;  
           [0011]    R 2  and R 3  are independently selected from:  
           [0012]    hydrogen;  
           [0013]    alkyl or substituted alkyl; or  
           [0014]    R 2  and R 3  together are cycloalkyl;  
           [0015]    R 4  is hydrogen;  
           [0016]    alkyl or substituted alkyl;  
           [0017]    phenyl or substituted phenyl;  
           [0018]    (CH 2 ) n Y; or  
           [0019]    (CH 2 ) m O(CH 2 ) n Y;  
           [0020]    m and n are independently between 1 and 10;  
           [0021]    Y is selected from hydrogen, CO 2 H or salts thereof, OPO 3   2 − or salts thereof, or CO 2 R 5 , wherein  
           [0022]    R 5  is an alkyl or substituted alkyl group; and  
           [0023]    X is an effector moiety linked to the nitrogen atom at the 1-position of the indoline ring via an acyl linkage, or is a group which capable of linkage to an effector moiety;  
           [0024]    to produce the 7-nitroindoline.  
           [0025]    In the above definition, preferably the alkyl or substituted alkyl groups are C 1 -10 and the alkoxy group is represented by O(CH 2 ) n —Y, where n and Y are defined above. Particularly preferred classes of compounds are those in which R 4  is hydrogen and R 1  is methoxy.  
           [0026]    Preferably, the process is selective for the 7-nitro isomer, that is other isomers are produced in minor amounts. For example, preferably the reaction yields greater than a 5:1 ratio, and more preferably greater than a 12:1 ratio, favouring the production of the 7-nitro isomer.  
           [0027]    The nitration reaction can be carried out before or after the reaction to link the effector moiety to the indoline, depending on the sensitivity of the effector moiety or group for linking to the effector moiety to conditions employed in the synthesis and particularly the nitration reaction.  
           [0028]    Conveniently, the reaction is carried out at about room temperature. Preferred solvents for the reaction are halogenated alkanes, such as CCl 4  or CH 2 Cl 2 , or dichloroethane. The use of halogenated alkanes is particularly preferred as they favour the production of the desired 7-nitro isomer. Ethers such as diethyl ether are also suitable solvents for the reaction, with the results with diethyl ether reported herein producing a similar isomeric ratio of products, but having a slower rate of reaction.  
           [0029]    The process may comprise the additional step of separating the 7-nitroindoline and optionally, formulating it for further use, e.g. for delivery.  
           [0030]    Optionally, the process may comprise the initial step of linking a carboxylic acid group present on an effector moiety to the nitrogen atom at the 1-position of the indoline so that it is linked via an acyl group.  
           [0031]    Embodiments of the present invention will now be described in more detail by way of example and not limitation. 
       
    
    
     DETAILED DESCRIPTION  
       [0032]    Effector Moieties  
         [0033]    The X group can be an effector moiety or a group capable of being coupled to or converted into an effector moiety. The effector moiety is preferably linked to the indoline via an acyl linkage, which may be derived either from a carboxylate moiety inherently present in the effector or as part of a carbamate or urea linkage between the effector and the indoline, for example:  
                         
 
         [0034]    Depending on particular cases, the linkage to a hydroxyl or amino group on the effector may be performed either before or after the nitration reaction according to the present invention. Where the linkage of effector to indoline is carried out post nitration, it can be accomplished as follows:  
                         
 
         [0035]    In cases where the effector species is resistant to the nitration conditions, the non-nitrated indoline can be taken through the same steps to create the acyl linkage to the effector moiety.  
         [0036]    Examples of effector moieties include labels, drugs, toxins, or carrier or transport molecules. Effectors preferably have a carboxylic acid group which can be coupled to the nitrogen atom at the 1-position on the indoline to form an acyl linked effector.  
         [0037]    Techniques for coupling the photocleavable group to both peptidyl and non-peptidyl coupling partners are well known in the art. In preferred embodiments, the effector moiety is a biologically active compound such as an amino acid (either L or D-amino acids), and more particularly neuroactive amino acids such as L-glutamate, GABA and glycine. The procedures described herein can also be readily adapted to linking larger effector groups such as oligopeptides or polypeptides to the photocleavable group. Examples of especially suitable peptides are as follows: thyrotrophin releasing hormone TRH; enkephalins (locally acting endogenous opiates); bradykinin; and angiotensin II. Generally, the methods described herein are applicable to any oligopeptides with non-amidated C-termini.  
         [0038]    In further embodiments, the photocleavable group is attached to an effector moiety which is a metal ion chelator thereby providing compounds which on photolysis release the chelator to bind metal ions, e.g. to reduce the metal ion concentration in a system. Thus, by way of example, a photocleavable group can be attached to a carboxylate group containing chelator such as BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), EDTA (ethylenediaminetetraacetate) or EGTA (ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid). These compounds can then be photolysed to chelate Ca 2 + or other metal ions. An example of a prior art use of compounds capable of releasing metal ion chelators is provided in Adams et al (12), although the effectiveness of the 5,7-dinitroindoline compounds described in this paper is poor.  
         [0039]    The synthesis of photoreleasable compounds including oligo or polypeptide effector moieties can be achieved by linking a terminal amino acid (i.e. the C-terminal amino acid) to the photocleavable group and then using polypeptide chain extension techniques to build up the peptide chain stepwise, or by coupling an oligopeptide or polypeptide to the terminal amino acid linked to the photocleavable group. Standard peptide synthesis techniques could also be adapted by linking to the synthesis resin a suitably substituted alkoxy group at position 4. This would give a resin containing the protecting group and C-terminal residue which could be elaborated and eventually cleaved from the resin by standard techniques.  
         [0040]    If the photoreleasable compound is synthesized using the scheme described in the examples below, it is preferred that the effector moiety is stable to the nitration reaction carried out after it is attached to the nitrogen of the indole ring. In the case of amino acids, this means that the use of amino acids other than tryptophan, tyrosine, cysteine or methionine is preferred.  
         [0041]    In other aspects, the present invention provides precursor compounds in which the effector moiety has not been linked to the photocleavable group (e.g. X is H, COY as defined above, or COCL) and/or in which the nitration reaction has not been carried out (e.g. the substituent at the 7-position is hydrogen).  
         [0042]    Compositions  
         [0043]    The photoreleasable compounds described above can be formulated as compositions. These compositions may comprise, in addition to one or more of the compounds, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.  
         [0044]    In some embodiments, the compounds can be formulated as pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the photoreleasable compound can be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer&#39;s injection, lactated Ringer&#39;s injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.  
         [0045]    After administration, the photoreleasable compound can be activated to release the effector moiety, conveniently by exposure to a flash of UV light. Preferably, the photoreleasable compound according to the present invention is given to an individual in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful effect in the patient. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, the data on the efficiency and kinetics of release of the effector moiety and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington&#39;s Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.  
         [0046]    Discussion  
         [0047]    Exploratory nitrations were carried out on 1-acetyl-4-methoxyindoline 3a. We showed previously 2  that 3a underwent exclusive Friedel-Crafts acetylation at C-7 and hence predicted that nitrosation would also occur at the desired 7-position. Greater regiospecificity than for nitration was expected, as the nitrosonium ion is many orders of magnitude less reactive than the nitronium ion: 4  a detailed study of nitrosation reactions of electron-rich aromatic compounds with nitrosonium tetrafluoroborate has been reported. 5  In the event, we were able to reproduce the reported 5  high-yielding nitrosation of anisole with excess NO + BF 4   −  in dry acetonitrile, but similar treatment of 3a gave a mixture that still contained 50% starting material, together with the nitrated isomers 2a and 4. The 5-nitro isomer 4 was the more abundant of the two (ratio ˜2:1). Nitration of other substrates under these conditions has been observed when oxygen was present 6  but the successful control experiment on anisole suggests this was not the case here. While the result is interesting, we chose instead to examine alternative methods for selective 7-nitration.  
         [0048]    Among the wide range of nitration reagents,  7  the clay-supported copper(II) nitrate (claycop) developed by Laszlo and Cornélis 8  that effects nitration in the presence of acetic anhydride appeared particularly suitable, as the reaction proceeds under conditions unlikely to perturb the tert-butyl protecting groups of 3b. Furthermore, nitration of acetanilide proceeds with extremely high ortho-selectivity. 8 b We hoped that similar selectivity might obtain for our N-acylated substrates 3.  
         [0049]    In either CCl 4  or CH 2 Cl 2  as solvent, 3a was consumed in 4 h and cleanly yielded a mixture of 2a and 4 in a 5:1 ratio, i.e. strongly favouring the 7-nitro isomer. In diethyl ether as solvent 9  the reaction was much more sluggish: only ˜20% of 3a reacted in 4 h and there was no benefit to the product ratio. Gratifyingly, the same reaction on 3b in CCl 4  overnight cleanly gave the nitrated compounds 5a and 6, with the desired isomer 6 as the dominant product (˜1:12 ratio) and the protecting groups intact. Although 2a and 4 were readily separated by silica gel chromatography, isomers 5a and 6 were less easily resolved. However, pure 6 was obtained by fractional crystallisation of the mixed isomers, followed by TFA treatment and simple desalting by preparative HPLC to obtain 2b. The 5a+6 mixture could alternatively be treated with TFA to remove the protecting groups and 2b isolated from the resulting mixture by the 2-stage reversed phase HPLC procedure previously reported. 2  However, the former procedure was more convenient and the overall yield of 2b from 3b was ˜40%.  
                         
 
         [0050]    The claycop nitration provides a satisfactory solution to the problem but we also evaluated homogeneous nitration with copper(II) nitrate-acetic anhydride to assess the effect of the clay matrix. Under typical conditions 10  3a was completely transformed and the nitrated isomers 2a and 4 were obtained in 3:2 ratio, i.e. 40% 5-nitro compared to 17% with claycop. Thus the claycop procedure is more favourable and convenient. These results greatly facilitate access to 2b and related compounds: further applications will be reported elsewhere.  
       EXPERIMENTAL  
       [0051]    General details were as previously described. 2  Claycop was prepared with a 2:3 (w/w) ratio of Cu(NO 3 ) 2  and montmorillonite K10 as described. 8 a Compounds 1-4 were available from previous work. 1,2  Only the experimental data for the successful claycop nitration of 3b and subsequent isolation of 2b are reported here.  
         [0052]    Claycop nitration of 1-{S-[4-t-butoxycarbonyl)-4-(t-butoxycarbonylamino)]-butanoyl-4-methoxyindoline.  
         [0053]    Compound 3b (2.17 g, 5 mmol) was added at room temp to a suspension of claycop (3.2 g) in CCl 4  (20 mL) and acetic anhydride (10 mL) and the mixture was stirred overnight. The solid was filtered and washed with EtOAc and the filtrate was washed with saturated aq. NaHCO 3  and brine, dried and evaporated. Flash chromatography [EtOAc-petroleum ether 45:55] gave two fractions. The first gave a pale yellow gum (322 mg) which was discarded. The second fraction gave a pale solid (1.61 g) that contained a mixture of 5- and 7-nitro isomers 5a and 6. Fractional crystallisation (CHCl 3 -petroleum ether) gave 1-{S-[4-t-butoxycarbonyl)-4-(t-butoxycarbonylamino)]butanoyl}-4-methoxy-7-nitroindoline 6 (1.03 g, 43%) as yellow crystals, mp 145-147° C.; (Found: C, 57.70; H, 6.98; N, 8.76. C 23 H 33 N 3 08 requires C, 57.61; H, 6.94; N, 8.76%);  1 H NMR: (CDCl 3 ) δ 7.74 (1H, d, J 8.8 Hz, 6-H), 6.64 (1H, d, 5-H), 5.15 (1H, d, J 7.2 Hz, NH), 4.17-4.25 (3H, m, CH and H-2), 3.91 (3H, s, OMe), 3.04-3.12 (2H, m, 3-H), 2.50-2.56 and 2.58-2.64 (2H, 2×m, COCH 2 ), 2.25-2.30 and 1.96-2.03 (2H, 2×m, CH 2 ), 1.46 (9H, s, CMe 3 ), 1.43 (9H, s, CMe 3 ). The residue in the mother liquor was recrystallised (EtOAc-petroleum ether) to give 1-{S-[4-t-butoxycarbonyl)-4-(t-butoxycarbonylamino)]butanoyl}-4-methoxy-5-nitroindoline 5a (57 mg, 2%) as yellow crystals, mp 102-103° C.; (Found: C, 57.41; H, 7.12; N, 8.12. C 23 H 33 N 3 O 8 +½ EtOAc requires C, 57.35; H, 7.12; N, 8.03%);  1 H NMR: (CDCl 3 ) δ 8.02 (1H, d, J 9.0 Hz, 7-H), 7.86 (1H, d, 5-H), 5.17 (1H, br s, NH), 4.17-4.26 (1H, m, CH), 4.16 (2H, t, J 8.2, H-2), 3.92 (3H, s, OMe), 3.26 (2H, t, 3-H), 2.46-2.66 (2H, m, COCH 2 ), 2.26-2.34 and 1.96-2.04 (2H, m, CH 2 ), 1.48 (9H, s, CMe 3 ) and 1.42 (9H, s, CMe 3 ). Signals from the solvent of crystallisation were present at δ4.12, 2.04 and 1.26.  
         [0054]    1-[S-(4-Amino-4-carboxybutanoyl)]-4-methoxy-7-nitroindoline 2b. A solution of 6 (479 mg, 1 mmol) in TFA (15 mL) was stirred at room temp for 1 h and concentrated in vacuo. The residue was dissolved in water (60 mL) and adjusted to pH 7.0 with 1 M aq. NaOH. The solution was washed with ether and analysed by reversed phase HPLC (mobile phase 25 mM Na phosphate, pH 6.0+25% MeCN (v/v); 1.5 mL/min), t R  4.2 min. The solution was lyophilised, redissolved in 25 mM Na phosphate, pH 6.0 (60 mL) and pumped onto a preparative reversed phase HPLC column (details as described 2 ). The column was first washed with water for 2 h, then the product was eluted with water+20% MeCN (v/v; all flow rates 2.5 mL/min). Fractions containing the product were analysed, combined and quantified (UV spectroscopy) to give a solution of 2b (867 μmol, 87%). The solution was passed through a 0.2 μm filter, lyophilised and stored at −20° C. as a pale yellow powder. Fractions from the very end of the eluted peak contained a trace amount of 5b, t R  5.6 min, and were discarded without significant loss of 2b.  
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