Patent Grant
7320981
This application is a 371 of PCT/GB01/03111 file Jul. 11, 2001.
The present invention relates to antitumoural compounds, and in particular to new antitumoural analogs of variolin B and deoxyvariolin B. The present invention also relates to synthetic processes, and in particular to synthetic processes for producing both the new compounds of the invention and the known compounds variolin B and deoxyvariolin B, including novel intermediates which form a part of such synthetic processes. In addition, the present invention relates to novel, previously undisclosed indications of known variolin compounds.
The variolins are a new class of marine alkaloids isolated from the rare, difficult to access Antarctic sponge Kirkpatricka varialosa, with Variolin B (1) being a typical example.
The variolins all contain a fused pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine core (2), with either a heterocyclic aromatic ring or an ester group attached at C5, as in Variolin B (1) and Variolin D (3).
The variolins are disclosed to have antitumour activity and other useful properties. The complete structure, and antitumour activity, of these and related compounds is described by N. B. Perry et al. Tetrahedron, 1994, 50, 3987-92, and G. Trimurtulu et al, Tetrahedron, 1994, 50, 3993-4000. However, the variolins described in these documents have hitherto only been demonstrated to exhibit a limited range of antitumour activity.
The limited availability of natural material has resulted in the search for alternative synthetic methods being sought for the natural compounds and related analogs.
A synthetic process for producing the related deoxyvariolin B (4) has been described by M. Alvarez et al, Tetrahedron Lett., 2001, 42, 315-317 (which was published before the filing date of the present application but after the priority dates).
The route to deoxyvariolin B described in this reference involves a total of at least fourteen steps, in which the fused tricyclic pyridopyrrolopyrimidine core is constructed from a 7-azaindole and a heteroaryl coupling reaction is then used to introduce the fourth aromatic ring to give intermediate (5). Substitution of the derived sulphone group (6) for an amino group gave deoxyvariolin B (4):
However, as noted above, this synthesis is long and complex. Further, no synthetic process has been reported for variolin B (or any of the natural variolins).
It is therefore desirable to provide a process capable of synthesising deoxyvariolin B and derivatives thereof in a smaller number of steps than the process described above.
It is also desirable to provide a process capable of synthesising variolin B itself as well as the deoxy derivative.
It is further desirable to provide new compounds having antitumour activity comparable or superior to natural variolin B.
The synthetic methods of the present invention provide the first method for preparation of variolin B (1) and provide short, rapid entry to deoxyvariolin B (4) and intermediates such as (5) and (6). These intermediates have been used in the preparation of new antitumour compounds containing the fused tricyclic pyridopyrrolopyrimidine core of the variolins.
According to this invention, new synthetic methods for producing variolin B, deoxyvariolin B and similar compounds have been developed taking advantage of a hidden symmetry element of these compounds. This novel approach allows construction of the core variolin skeleton, consisting of the fused pyridopyrrolopyrimidine core bearing a heterocyclic aromatic ring at C5, in fewer steps than the prior art synthesis.
Thus, it is possible to transform simple monoheteroaromatic molecules into a number of new and known variolin derivatives with potential antitumour therapeutic activity.
Thus, in a first aspect, the invention provides a compound of formula (I):
wherein:
The invention also provides a synthetic process for producing both the new variolin derivatives of formula (I) described above and known variolin derivatives such as those described in the prior art.
Thus, in a second aspect, the invention provides a process for producing a compound of formula (I):
wherein:
wherein:
As described below, the new compounds of formula (I) demonstrate biological activity against mammalian cancer cell lines. Antitumoural activities of these compounds include leukaemias, lung cancer, colon cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, sarcomas and melanomas. Further, the known compounds of formula (I) exhibit previously undisclosed activity against a wide range of cancers.
Thus, in a third aspect, the invention provides a method for the treatment or prophylaxis of cancer in a mammal, which comprises administering to a mammal in need of such treatment an effective amount of a new compound of the invention.
Further, in a fourth aspect, the invention provides a method for the treatment or prophylaxis of cancers selected from ovarian cancer, kidney cancer, prostate cancer, breast cancer and melanoma in a mammal, which comprises administering to a mammal in need of such treatment an effective amount of either a new compound of the invention or a variolin compound of the prior art.
In further aspects, the invention provides synthetic steps to certain preferred compounds, described in more detail later, and to intermediate compounds, especially those of formula (II) above.
In the definitions used in the present application, alkyl groups may be straight or branched chain groups and preferably have from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3 or 4 carbon atoms. Methyl, ethyl and propyl including isopropyl are particularly preferred alkyl groups in the compounds of the present invention. As used herein, the term alkyl, unless otherwise modified, refers to both cyclic and noncyclic groups, although cyclic groups will comprise at least three carbon ring members.
Haloalkyl groups are alkyl groups (including cycloalkyl groups) as defined above which are substituted with one or more halogen atoms (preferably fluorine, chlorine, bromine or iodine) and preferably have from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3 or 4 carbon atoms. Methyl, ethyl and propyl including isopropyl groups which are substituted with 1, 2 or 3 halogen atoms which may be the same or different, especially fluoromethyl, fluorochloromethyl, trifluoromethyl and trichloromethyl, are particularly preferred haloalkyl groups in the compounds of the present invention.
Preferred alkenyl and alkynyl groups in the compounds of the present invention have one or more unsaturated linkages and from 2 to about 12 carbon atoms, more preferably 2 to about 8 carbon atoms, still more preferably 2 to about 6 carbon atoms, even more prefereably 2, 3 or 4 carbon atoms. The terms alkenyl and alkynyl as used herein refer to both cyclic and noncyclic groups, although straight or branched noncyclic groups are generally more preferred.
Preferred alkoxy groups in the compounds of the present invention include groups having one or more (but preferably only one) oxygen linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3 or 4 carbon atoms.
Preferred alkylthio groups in the compounds of the present invention have one or more (but preferably only one) thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Alkylthio groups having 1, 2, 3 or 4 carbon atoms are particularly preferred.
Preferred alkylsulfinyl groups in the compounds of the present invention include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Alkylsulfinyl groups having 1, 2, 3 or 4 carbon atoms are particularly preferred.
Preferred alkylsulfonyl groups in the compounds of the present invention include those groups having one or more sulfonyl (SO2) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Alkylsulfonyl groups having 1, 2, 3 or 4 carbon atoms are particularly preferred.
Preferred alkanoyl groups in the compounds of the present invention include those groups having one or more carbonyl (CO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms (including the carbonyl carbon). Alkanoyl groups having 1, 2, 3 or 4 carbon atoms, especially the formyl, acetyl, propionyl, butyryl and isobutyryl groups, are particularly preferred.
Preferred alkylamino groups in the compounds of the present invention have one or more (but preferably only one) NH linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Alkylamino groups having 1, 2, 3 or 4 carbon atoms, especially the methylamino, ethylamino, propylamino and butylamino groups, are particularly preferred.
Preferred dialkylamino groups in the compounds of the present invention have one or more (but preferably only one) nitrogen atom bonded to two alkyl groups, each of which may from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. The alkyl groups may be the same or different Dialkylamino groups wherein each alkyl group has 1, 2, 3 or 4 carbon atoms, especially the dimethylamino, diethylamino, N-methylethylamino, N-ethylpropylamino, dipropylamino, dibutylamino and N-methylbutylamino groups, are particularly preferred.
Preferred alkanoylamino groups in the compounds of the present invention have one NH—CO— linkage bonded to an alkyl group having from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Alkanoylamino groups having 1, 2, 3 or 4 carbon atoms, especially the formylamino, acetylamino, propionylamino and butyrylamino groups, are particularly preferred. The acetylamino group is especially preferred.
Preferred dialkanoylamino groups in the compounds of the present invention have one nitrogen atom bonded to two alkanoyl groups as defined above, each of which may be the same or different and has from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Dialkanoylamino groups wherein each alkanoyl group has 1, 2, 3 or 4 carbon atoms, especially the diformylamino, formylacetylamino, diacetylamino, dipropionylamino and dibutyrylamino groups, are particularly preferred. The diacetylamino group is especially preferred.
Preferred alkylsulfonylamino groups in the compounds of the present invention have one NH—SO2— linkage bonded to an alkyl group having from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 to about 6 carbon atoms. Alkylsulfonylamino groups having 1, 2, 3 or 4 carbon atoms, especially the methanesulfonylamino, ethanesulfonylamino, propanesulfoylamino and butanesulfonylamino groups, are particularly preferred.
In the compounds of formula (I), R1 is preferably selected from the group consisting of OH, OR′, SH, SR′, SOR′, SO2R′, NH2, NHR′, N(R′)2, NHCOR′, N(COR′)2, NHSO2R′, C(═O)R′, CO2H, CO2R′, C1-C12 alkyl and C1-C12 haloalkyl,
More preferably, R1 is selected from the group consisting of OR′, SR′, SOR′, NH2, NHR′, N(R)2, NHCOR′, N(COR′)2 and NHSO2R′, the or each group R′ being independently selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, aryl (which may optionally be substituted with a group selected from C1-C6 alkyl, C1-C6 alkoxy and halogen), aralkyl (the aryl moiety of which may optionally be substituted with a group selected from C1-C6 alkyl, C1-C6 alkoxy and halogen), aralkenyl (the aryl moiety of which may optionally be substituted with a group selected from C1-C6 alkyl, C1-C6 alkoxy and halogen), and wherein the group R1 is a group of formula N(R′)2 or N(COR′)2, the two R′ groups, together with the nitrogen atom to which they are attached, may form a 5-10 membered heterocyclic ring.
Even more preferably, R1 is selected from the group consisting of C1-C4 alkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, amino, C1-C4 alkylamino, di(C1-C4 alkyl)amino, C1-C4 alkanoylamino, di(C1-C4 alkanoyl)amino, C1-C4 haloalkanoylamino, arylamino (wherein the aryl moiety may optionally be substituted with a C1-C4 alkoxy group), benzylamino (wherein the phenyl part of the benzyl moiety may optionally be substituted with a C1-C4 alkoxy group), cinnamoylamino or dicinnamoylamino (wherein the phenyl part of the or each cinammoyl moiety may optionally be substituted with a C1-C4 alkoxy group), or a 5- to 7-membered nitrogen-containing heterocyclic ring attached to the remainder of the molecule via its nitrogen atom.
Still more preferably, R1 is selected from methoxy, thiomethyl, methylsulfinyl, amino, methylamino, ethylamino, benzylamino, acetylamino, trifluoroacetylamino, diacetylamino, cinnamoylamino, dicinnamoylamino, p-methoxybenzylamino and piperidino.
Most preferably R1 is selected from amino, benzylamino, acetylamino, trifluoroacetylamino, diacetylamino, cinnamoylamino, dicinnamoylamino and p-methoxybenzylamino.
R2 is preferably selected from the group consisting of OH, OR′, SH, SR′, SOR′, SO2R′, NH2, NHR′, N(R′)2, NHCOR′, N(COR′)2, NHSO2R′, C(═O)R′, CO2H, CO2R′, C1-C12 alkyl and C1-C12 haloalkyl,
More preferably, R2 is selected from the group consisting of OR′, SR′, SOR′, NH2, NHR, N(R′)2, NHCOR′, N(COR′)2 and NHSO2R′, the or each group R′ being independently selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, aryl (which may optionally be substituted with a group selected from C1-C6 alkyl, C1-C6 alkoxy and halogen), aralkyl (the aryl moiety of which may optionally be substituted with a group selected from C1-C6 alkyl, C1-C6 alkoxy and halogen), aralkenyl (the aryl moiety of which may optionally be substituted with a group selected from C1-C6 alkyl, C1-C6 alkoxy and halogen), and wherein the group R2 is a group of formula N(R′)2 or N(COR′)2, the two R′ groups, together with the nitrogen atom to which they are attached, may form a 5-10 membered heterocyclic ring.
Even more preferably, R2 is selected from the group consisting of C1-C4 alkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, amino, C1-C4 alkylamino, di(C1-C4 alkyl)amino, C1-C4 alkanoylamino, di(C1-C4 alkanoyl)amino, C1-C4 haloalkanoylamino, arylamino (wherein the aryl moiety may optionally be substituted with a C1-C4 alkoxy group), benzylamino (wherein the phenyl part of the benzyl moiety may optionally be substituted with a C1-C4 alkoxy group), cinnamoylamino or dicinnamoylamino (wherein the phenyl part of the or each cinammoyl moiety may optionally be substituted with a C1-C4 alkoxy group), or a 5- to 7-membered nitrogen-containing heterocyclic ring attached to the remainder of the molecule via its nitrogen atom.
Yet more preferably, R2 is selected from thiomethyl, methylsulfinyl, amino, methylamino, ethylamino, acetylamino, diacetylamino, cinnamoylamino, and p-methoxybenzylamino.
Most preferably, R2 is selected from amino, acetylamino, diacetylamino and p-methoxybenzylamino.
Preferably, R3 is H.
As the person skilled in the art will readily appreciate, the preferred definitions of R1, R2 and R3 above may be combined in various ways, and the compounds covered by such combinations of the above preferred definitions are to be considered as being part of this invention. A combination of two of these definitions is preferred, and a combination of all three preferred definitions is especially preferred.
The following compounds are most preferred:
The compounds of formula (I) contain a basic group, and may therefore form a salt. The nature of such salts is not critical to the present invention, provided that, when the compound is used for therapeutic purposes, the salts are pharmaceutically acceptable, ie more biologically active, about as biologically active or not unduly less biologically active than the free base compound, and less toxic, about as toxic or not unduly more toxic than the free base compound. This can easily be ascertained by simple tests readily apparent to those skilled in the art. However, when the compound is used for other purposes (for example, as an intermediate in the preparation of another compound) even this restriction does not apply. Examples of suitable salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate and phosphate; organic acid salts such as acetate, benzoate, oxalate, maleate, fumarate, tartrate, citrate and succinate; and sulfonic acid salts such as methanesulfonate, benzenesulfonate and p-toluenesulfonate. Preferred salts include hydrochloride, hydrobromide, tartrate and succinate.
Some of the compounds of formula (I) contain a carboxy group, and may therefore form an ester. The nature of such esters is not critical to the present invention, provided that, when the compound is used for therapeutic purposes, the esters are pharmaceutically acceptable, ie more biologically active, about as biologically active or not unduly less biologically active than the free acid compound, and less toxic, about as toxic or not unduly more toxic than the free acid compound. It is preferred that the ester group is physiologically removable, ie the ester can be readily converted in vivo to the free acid. This can easily be ascertained by simple tests readily apparent to those skilled in the art.
The compounds of the present invention contain at least four tertiary amine groups, of which one or more (but preferably only one) may be quaternised to form a quaternary ammonium salt. In this case, a counter ion is also present; examples of suitable counter ions are defined above with reference to salts. The procedure for quaternising the nitrogen atom(s) is readily apparent to those skilled in the art. It is preferred that the group attached to the tertiary amino group to form a quaternary ammonium species is a C1-C6 alkyl group, most preferably a methyl group.
The variolin derivatives of formula (I), including the known variolin compounds, are produced by a novel process which forms part of the present invention.
An element of the process of the invention involves the formation of the intermediate of formula (II) below:
In the intermediate of formula (II), R1a, R2a and R3a represent any of the groups represented by R1, R2 and R3 respectively, and all such groups where reactive functional groups are protected; and Y1 and Y2 are groups capable of being eliminated to produce a fused tricyclic pyridopyrrolopyrimidine ring structure. The intermediates of formula (II) are novel compounds and also form part of the present invention.
Any protecting group known in the art may be used to form the groups R1a, R2a and R3a with reactive functionalities protected. In this regard, reference is made to T. W. Greene et al, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1991.
Any groups capable of being eliminated to produce a fused tricyclic pyridopyrrolo-pyrimidine ring structure may be used as the groups Y1 and Y2. Preferably the group Y, is a hydroxy group or a labile ester group such as acetate, methanesulfonate, p-toluenesulfonate or trifluoromethanesulfonate, more preferably a hydroxy group. Preferably the group Y2 is a halogen atom, more preferably a chlorine atom.
It is preferred that the intermediate of formula (II) is symmetrical, ie R1a and R2a are the same: as described below, this allows the intermediate to be made by addition of two equivalents of reagent to a precursor; this in turn shortens the synthesis. More preferably, R1a and R2a are both methylthio groups.
The intermediate of formula (II) can be made by a number of methods. One preferred method is by reacting an intermediate compound of formula (IV):
wherein R3a and Y2 are as defined above and M is a metal, with a compound of formula (V):
wherein R1a and R2a are as defined above.
In the compound of formula (IV) above, the nature of the metal atom M is not particularly critical, provided that the compound is sufficiently reactive to undergo addition to the compound of formula (V). Examples of suitable metallated species include those where M is Li, Na, K, Mg or Zn; in the case of metallated species with divalent metal ions, a further counterion such as halogen may also be present, or the compound may be in the form of a diorganometallic species. We prefer that M is Li.
The compound of formula (IV) is typically produced in situ by metallating the corresponding halo compound. Suitable reagents are well known in the art, and examples include the metal itself or another more active metallating compound such as an alkylmetal derivative. Alkyllithium derivatives are preferred and butyllithium is especially preferred.
The compound of formula (V) is preferably produced by reacting an intermediate compound of formula (VI):
wherein R1a is as defined above and M is a metal,
with a compound of formula L1-CO-L2, where L1 and L2 are the same or different and each represents a leaving group.
In the compound of formula (VI), the nature of the metal atom M is not particularly critical, provided that the compound is sufficiently reactive to undergo addition to the compound of formula L1-CO-L2. Examples of suitable metallated species include those defined and exemplified above in relation to the compound of formula (IV). We prefer that M is Li.
The compound of formula (VI) is typically produced in situ by metallating the corresponding halo compound, ie a compound of formula (VIII):
wherein R1a is as defined above and X is a halogen atom, preferably bromine or iodine.
The compound of formula (VIII) where R1a is methylthio and X is chloro is commercially available. Corresponding compounds where X is another halogen atom can be prepared from the corresponding chloro compound as described in the literature, see Majeed, A. J.; Antonsen. O.; Benneche, T.; Undheim, K. Tetrahedron, 1989, 45, 993 and Reference Example 1 below.
Suitable reagents and procedures for metallating the compound of formula (VIII) to produce the compound of formula (VI) are known in the art. Examples include the metal itself or another more active metallating compound such as an alkylmetal derivative. Alkyllithium derivatives are preferred and butyllithium is especially preferred.
In the compound of formula L1-CO-L2, L1 and L2 may be the same or different and each represents a leaving group, the precise nature of which is not especially critical. Non-limiting examples of suitable leaving groups include halogen, C1-C6 alkoxy, di(C1-C6 alkyl)amino, nitrogen-containing heterocyclic (especially imidazole) or a labile ester group such as those defined above in relation to Y1. Diethyl carbonate is a particularly preferred example of a compound of formula L1-CO-L2.
In an alternative preferred embodiment, the compound of formula (II) is produced by reacting an intermediate compound of formula (VI), described above, with an intermediate compound of formula (VII):
wherein R3a and Y2 are as defined above, and Z is a leaving group.
In the compound of formula (VI), the group Z is a leaving group, examples of which are defined above with reference to Y1, L1 and L2. It is particularly preferred that Z is a halogen atom, especially chlorine, as two equivalents of the metallated compound of formula (VI) can add cleanly to the compound of formula (VII).
On elimination of the groups Y1 and Y2, the intermediate of formula (II) preferably cyclises to produce an intermediate of formula (III):
wherein R1a, R2a and R3a are as defined above.
The elimination of the groups Y1 and Y2 is preferably carried out by reacting the intermediate of formula (II) with a trialkylsilane of formula RaRbRcSiH wherein Ra, Rb and Rc may be the same or different and each represents a C1-C12 alkyl group. Preferably triethylsilane is used as the reagent.
The reaction is preferably carried out in the presence of acid, the precise nature of which is not particularly critical. A strong organic acid such as p-toluenesulfonic acid or trifluoroacetic acid is preferred and trifluoroacetic acid is especially preferred.
The intermediate compound of formula (III) may then be converted to a compound of formula (I) by functional group interconversions, the general nature of which is known to those skilled in the art By way of example, the amine groups of the known compound deoxyvariolin B (4), prepared by the process of the present invention, may readily be converted into a variety of functionalised derivatives as shown in Scheme I and exemplified below.
In an alternative approach, a further group of analogs may be generated by functionalisation of the C5 heteroaromatic ring of the variolins. This can be readily achieved from intermediate (5) by oxidation to the sulphone (6) or sulphoxide (22) followed by nucleophilic substitution reactions, as shown in Scheme II.
Two particularly important interconversions have not previously been demonstrated for the variolins.
Therefore, in a further aspect, the invention provides a process for producing a compound of formula (I) wherein R1 and R2 are amino groups and R3 is as defined above, said process comprising:
The nature of the amino-protecting group is not especially critical. Examples of suitable protecting groups, their attachment and their removal are given in T. W. Greene et al, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1991, to which reference is made.
It is preferred that the protecting group is a substituted or unsubstituted benzyl group or a phthalimide group, especially a p-methoxybenzyl (PMB) group. The group may be removed by any conventional route, such as under acid conditions (especially a strong organic acid, for example trifluoromethanesulfonic acid or trifluoroacetic acid), under oxidising conditions, for example dichlorodicyanobenzoquinone (DDQ) or reductive conditions, for example with hydrogen and a palladium catalyst.
A preferred embodiment of such a process is illustrated in Scheme III below, in which the conversion of disulphoxide (20) into deoxyvariolin B (4) is achieved in two steps via the bis-amine (29).
In a yet further aspect, the invention provides a process for producing a compound of formula (I) wherein R1 is a methylthio or amino group, R2 is an amino group and R3 is as defined in claim 1, from a compound of formula (III), wherein R1a and R2a are methylthio and R3a is as defined in claim 19, said process comprising:
Any oxidising agent capable of oxidising thioethers to sulfoxides may be used to achieve the optional oxidation step a) of the above process. Non-limiting examples of suitable oxidising agents include hydrogen peroxide, sodium periodate, t-BuOCl, sodium perborate, and peracids such as peracetic acid, m-chloroperbenzoic acid (mCPBA) or magnesium monoperoxyphthalate (MMPP), of which peracids are preferred and mCPBA is especially preferred.
An embodiment of such a process is illustrated in Scheme IV below, in which intermediate (19) is converted to deoxyvariolin B (4) in a single step via the sulfoxide intermediate (20):
The novel methodology employed to construct the core variolin skeleton allows the synthesis of deoxyvariolin B (4) to be completed in a total of only five steps from the simple monoheteroaromatic staring material (7). This synthesis is significantly shorter than the sequence to deoxyvariolin B described in the prior art.
In an alternative embodiment of such a process, illustrated in Scheme V below, dithioether intermediate (19) is converted into thiodeoxyvariolin (5) in a single step by treatment with ammonia solution:
A particularly preferred embodiment of the process of the present invention is illustrated in Scheme VI below. The precise conditions are described in more detail in the Examples, Process Examples and Reference Examples.
The novel synthetic approach of the present invention allows construction of the core variolin skeleton, consisting of the fused pyridopyrrolopyrimidine core bearing a heterocyclic aromatic ring at C5, as in (13), in only four steps from the simple monoheteroaromatic starting material (7).
Straightforward functional group manipulation then allows the conversion of intermediate (13) to the known compound variolin B (1) in four further steps, as illustrated in Scheme VII below:
This short eight step sequence from commercially available starting material (7) represents the first reported synthetic process for the preparation of variolin B.
Similar methodology provides rapid access to the key intermediate (19) useful for the synthesis of deoxy variolin B and related analogs, as illustrated in Scheme VIII below and described in more detail in the Examples, Process Examples and Reference Examples.
The routes described above to variolin B or deoxyvariolin B can be conveniently modified to form other derivatives. In particular, this invention provides new compounds that can be made from intermediates prepared by a new process that is part of this invention.
Thus, according to the present invention, we now provide synthetic routes for the production of variolin B (1), deoxyvariolin B (4) and related intermediates such as (5) and thus for the production of variolin analogs. The synthetic routes of the invention each comprise a number of transformation steps to arrive at the desired product. Each step in itself is a process in accordance with this invention. The invention is not limited to the routes that are exemplified, and alternative routes may be provided by, for example, changing the order of the transformation steps, as appropriate.
In more detail, the synthesis of variolin B according to an especially preferred embodiment of the current invention involves the following eight steps.
This synthesis is illustrated in Scheme IX below. Further details of the processes used are given in the Examples, Process Examples and Reference Examples.
In another preferred modification, starting material (7) is transformed into deoxyvariolin B involving the following five further steps.
This synthesis is illustrated in Scheme X below. Further details of the processes used are given in the Examples, Process Examples and Reference Examples.
As the skilled artisan will readily appreciate, the reaction schemes described herein may be modified and/or combined in various ways, and the compounds generated therefore are to be considered as being part of this invention. In particular the starting material and/or reagents and reactions can be varied to suit other combinations of the substituent groups in the formulae (I) to (VIII).
Pharmaceutical Compositions
Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable composition or oral, topical or parenteral administration, and they may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when administered parenterally.
The correct dosage of the compounds will vary according to the particular formulation, the mode of application, and the particular situs, host and tumour being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.
Administration of the compounds or compositions of the present invention may be by any suitable method, such as intravenous infusion, oral preparations, intraperitoneal and intravenous administration.
Cytotoxic Activity
The compounds of the present invention were tested according to the protocol described below.
A colorimetric type of assay, using sulforhodamine B (SRB) reaction has been adapted for a quantitative measurement of cell growth and viability: see Skehan, P. A. et al. J. Natl. Cancer Inst., 1990, 82, 1107-1112. This form of the assay employs 96 well cell culture microplates of 9 mm diameter (Faircloth, G. T.; Stewart, D. and Clement, J. J., Journal of Tissue and Culture Methods, 1983, 11, 201-205; Mosmann, T. Journal of Immunological Methods, 1983, 65, 55-63.).
Most of the cell lines are obtained from American Type Culture Collection (ATCC) derived from different human cancer types. Cells are maintained in RPMI 1640 10% FBS, supplemented with 0.1 g/l penicillin and 0.1 g/l streptomycin sulfate and then incubated at 37° C., 5% CO2 and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsin and resuspended in fresh medium before plating.
Cells are seeded in 96 well microtiter plates, at 5×103 cells per well in aliquots of 195 μl medium, and they are allowed to attach to the plate surface by growing in drug free medium for 18 hours. Afterward, samples are in aliquots of 5 pi in a ranging from 10 to 10-8 μg/ml dissolved in DMSO/EtOH (0.2% in PS buffer). After 48 hours exposure, the antitumour effect are measured by the SRB methodology: cells are fixed by adding 50 μl of cold 50% (w/v) trichloroacetic acid (TCA) and incubating for 60 minutes at 4° C. Plates are washed with deionized water and dried. 100 μl of SRB solution (0.4% w/v in 1% acetic acid) is added to each microtiter well and incubated for 10 minutes at room temperature. Unbound SRB is removed by washing with 1% acetic acid. Plates are air-dried and bound stain is solubilized with Tris buffer. Optical densities are read on an automated spectrophotometric plate reader at a single wavelength of 490 nm.
The values for mean +/− SD of data from triplicate wells are calculated Some parameters for cellular responses can be calculated: TGI=growth inhibition, TGI=total growth inhibition (cytostatic effect) and LC=cell killing (cytotoxic effect).
The results are shown in Tables 1 and 2 below. Although compounds (1), (4), (5) and (6) are not themselves part of the present invention, the results disclosed in the Tables demonstrate antitumour activity not previously disclosed for these compounds.
This application claims priority from GB application nos. 0017055.5, filed 11 Jul. 2000, and 0030689.4, filed 15 Dec. 2000. The contents of both documents are hereby incorporated by reference to the extent that there is disclosure therein which is not explicitly reproduced in the present specification.
Processes for producing the compounds and intermediates of the present invention are described in the Examples below. In the Process Examples are described processes according to the present invention for producing known compounds. The production of intermediate compounds not part of the present invention is described in the Reference Examples.
General Experimental Details
Unless otherwise stated, all reactions were performed under an inert atmosphere in pre-dried glassware. All organic extracts were washed with water and brine, and dried over MgSO4 prior to concentration in vacuo. Melting points were determined on a Kofler hot-stage apparatus and are uncorrected.
A mixture of triaryl alcohol 12 (100 mg, 0.237 mmol) (prepared as described in Example 16 below) and trifluoroacetic acid (37 μL, 0.48 mmol) were dissolved in 1,2-dichloroethane (0.5 mL). The resulting orange solution was transferred to a Young's tube fitted with a rubber septum, containing triethylsilane (0.30 mL, 1.9 mmol). Under a strong flow of argon, the septum was replaced with a Teflon® screw-cap, and the sealed reaction vessel was heated at 100° C. for 43 h. After cooling, the vessel was opened and the contents diluted with CH2Cl2 (12 mL). The solution was neutralised with 5% NaHCO3 solution (8 mL) and the phases separated. The aqueous layer was repeatedly extracted with CH2Cl2 and the organic extracts were worked up according to *the standard procedure. Purification of the crude material was achieved by flash chromatography on silica gel using gradient elution (48 to 75% EtOAc/hexanes) to afford in order of elution:
(1) the variolin core structure 13 as a yellow solid (41 mg, 47%):
Mp: 192-194° C.; 1H NMR (500 MHz, CDCl3): δ 2.65 (s, 3H), 2.70 (s, 3H), 6.92 (d, J=5.4 Hz, 1H), 7.40 (d, J=5.4 Hz, 1H), 7.71 (d, J=6.8 Hz, 1H), 7.98 (d, J=6.8 Hz, 1H), 8.48 (m, 2H); 13C NMR (75 MHz, CDCl3): δ 14.1, 14.9, 55.6, 101.9, 102.5, 108.5, 110.8, 117.7, 135.9, 138.6, 143.5, 144.3, 154.2, 155.6, 159.6, 161.1, 171.2; HRMS: Calcd for C17H15N5O32S2 (M+) 369.0718, found 369.0720.
and (2) the uncyclised ether 13a as a viscous gum (28 mg, 28%)
1H NMR (500 MHz, CDCl3): δ 2.36 (s, 3H), 2.44 (s, 3H), 3.84 (s, 3H) 6.57 (d, J=5.9 Hz, 1H), 6.80 (d, J=5.9 Hz, 1H), 7.17 (d, J=4.9 Hz, 1H), 7.79 (s, 1H), 8.27 (d, J=5.9 Hz, 1H), 8.30 (d, J=5.9 Hz, 1H), 8.49 (d, J=4.9 Hz, 1H); 13C NMR (75 MHz, CDCl3): δ 14.0 (×2), 56.2, 71.8, 103.5, 106.4, 112.9, 120.8, 150.7, 152.5, 157.1, 158.0, 166.0, 167.1, 167.3, 172.1, 172.4; HRMS: Calcd for C17H1635ClN5O232S2 (M+) 421.0434, found 421.0444.
Bis-sulfide 13 (37 mg, 0.10 mmol) was dissolved in CHCl3 (5 mL) under atmospheric conditions and cooled in a 40° C. bath. A pre-cooled (−40° C.) solution of m-chloroperbenzoic acid in CHCl3 (10 mg/mL) was added dropwise to the solution until TLC analysis indicated the complete consumption of starting material (approx. 2 equiv of m-CPBA was used). The solution was warmed to room temperature and neutralised with saturated NaHCO3 solution. This was repeatedly extracted with CH2Cl2 and after the standard work-up, a yellow solid was obtained, which was predominantly a mixture of diastereomeric bis-sulfoxides. The crude mixture was used without purification, however the bis-sulfoxides 14 had the following spectroscopic characteristics:
1H NMR (500 MHz, CDCl3): (most signals for the diastereoisomers coincide, however, as they represent more than one compound they are all quoted as multiplets) δ 3.02 (m, 3H), 3.19 (m, 3H), 4.12 (m, 3H), 7.00-7.01 (m, 1H), 7.98-7.99 (m, 1H), 8.12-8.14 (m, 1H), 8.48-8.49 (m, 1H), 8.64-8.67 (m, 1H), 8.79-8.81 (m, 1H).
The crude oxidised material was heated with an excess of p-methoxybenzylamine (0.15 mL, 1.1 mmol) at 85° C. for 15 h. The crude red paste was purified by flash chromatography on silica gel using gradient elution (2.5-4% MeOH/CH2Cl2). The yellow fractions were re-chromatographed using gradient elution (50% EtOAc/CH2Cl2 to 100% EtOAc) to give bis-amine 15 as a yellow solid (43 mg, 78% over two steps).
Mp: 74-77° C.; 1H NMR (500 MHz, CDCl3): δ 3.81 (s, 6H), 3.99 (s, 3H), 4 5.6 Hz, 2H), 4.85 (d, J=5.5 Hz, 2H), 5.51 (m, 1H), 6.82 (d, J=5.6 Hz, 1H), 6.89-6.91 (m, 4H), 7.00 (d, J=5.2 Hz, 1H), 7.29 (m, 1H), 7.34 (d, J=8.5 Hz, 2H), 7.39 (d, J=8.5 Hz, 2H), 7.43 (m, 1H), 8.16 (d, J=5.6 Hz, 1H), 8.26 (d, J=5.2 Hz, 1H), 10.39 (m, 1H); 13C NMR (75 MHz, CDCl3): δ 44.3, 44.9, 55.3 (2×CH3), 55.5, 101.3, 101.6, 101.8, 111.4, 112.2, 114.0 (×2), 128.5, 128.8, 130.5, 131.4, 137.5, 141.5, 141.9, 144.7, 148.7, 154.6 (br), 158.7, 158.9, 159.4, 160.9, 162.6; HRMS: Calcd for C31H29N7O3 (M+) 547.2332, found 547.2334.
NaH (60%, 60 mg, 1.5 mmol) was washed three times with petroleum ether, and suspended in dry DMF (1.5 mL). The stirred suspension was cooled in ice, and ethanethiol (0.14 mL, 1.9 mmol) was added dropwise. After the gas evolution had subsided, the clear solution was stirred at room temperature for 10 min. A portion of the NaSEt solution (1.1 mL) was added to a solution of bis-amine 15 (40 mg, 0.073 mmol) in dry DMF (1.5 mL) and the mixture was stirred at 50° C. for 7 h. After cooling, aqueous NH4Cl solution was added and the mixture was extracted with EtOAc (×3). The organic extracts were washed three times with water to remove DMF and then worked up as usual. The yellow solid produced was purified by flash chromatography on silica gel using 3% MeOH/CH2Cl2 as the eluant to afford alcohol 16 as a yellow solid (34 mg, 87%).
1H NMR (500 MHz, CDCl3): δ 3.80 (s, 3H), 3.81 (s, 3H), 4.61 (d, J=5.5 Hz 2H), 485 (d, J=5.4 Hz, 2H), 5.35 (m, 1H), 6.76 (d, J=5.5 Hz, 1H), 6.88-6.92 (m, 4H), 7.03 (d, J=6.8 Hz, 1H), 7.06 (d, J=5.7 Hz, 1H), 7.32 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.4 Hz, 2H), 7.68 (d, J=6.8 Hz, 1H), 8.05 (d, J=5.5 Hz, 1H, 8.29 (d, J=5.7 Hz, 1H), 10.94 (m, 1H), 15.75 (br s, 1H); 13C NMR (75 MHz, CDCl3): a 44.3, 452, 55.3 (2×CH3), 100.3, 100.5, 106.9, 107.5, 111.4, 114.1 (2×C), 128.8, 129.2, 130.2 (2×C), 137.5, 142.8, 143.8, 145.4, 149.7, 158.6, 158.9, 159.0, 159.5, 159.8, 160.0; HRMS: Calcd for C30H27N7O3 (M+) 533.2175, found 533.2185.
A mixture of alcohol 18 (prepared as described in Example 17 below) (1.04 g, 2.65 mmol), triethylsilane (3.4 ml, 21.5 mmol) and trifluoroacetic acid (0.81 ml, 10.6 mmol) was refluxed for 3 h. After cooling, the red residue was dissolved in CH2Cl2 (40 ml) and a saturated solution of NaHCO3 was added. The brown mixture was stirred for 1 h at room temperature and the layers were separated. The aqueous layer was extracted with CH2Cl2 (3×50 ml) and the combined organic layers were dried, filtered and concentrated under reduced pressure. The red residue was purified by flash chromatography using ethyl acetate:hexane 1:4 to ethyl acetate:hexane 1:3 as eluent to afford the pyridopyrrolopyrimidine 19 (0.3 g, 33%) as a pale yellow solid. 1H NMR (300 MHz, CDCl3): 8.64 (dd, J=8.1 and 1.7 Hz, 1H), 8.60 (dd, J=4.6 and 1.7 Hz, 1H), 8.51 (d, J=5.4 Hz, 1H), 8.06 (d, J=6.4 Hz, 1H), 7.82 (d, J=6.6 Hz, 1H), 7.51 (dd, J=8.5 and 4.6 Hz, 1H), 7.34 (d, J=5.4 Hz, 1H), 2.73 (s, 3H), 2.68 (s, 3H).
Oxidation of bis-sulfide 19 to bis-sulfoxide 20 was carried out by the same procedure described above in Example 2.
A mixture of p-methoxybenzylamine (2 ml) and bis-sulfinyldeoxyvariolin 20 (30 mg, 8.1×105 mol) was stirred at 95° C. for 2 h and evaporated at reduced pressure. The red residue was purified by flash chromatography using DCM/MeOH (0.2%) to DCM/MeOH (2%) as eluent to afford N′,N-bis(p-methoxybenzyl)deoxyvariolin 29 (21 mg, 49%) as a yellow oil. 1H NMR (300 MHz, CDCl3): 10.4 (brs, 1H), 8.56 (d, J=7.8 Hz, 1H), 8.31 (d, J=5.4, 1H), 8.27 (dd, J=5.2 and 1.1 Hz, 1H), 7.63 (d, J=6.8 Hz, 1H), 7.42-7.33 (m, 12H), 6.98 (d, J=5.4 Hz, 1H), 6.93-6.89 (m, 4H), 5.52 (brs, 1H), 4.89 (d, J=5.4 Hz, 2H), 4.69 (d, J=5.9 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H).
Piperidine (0.04 ml, 0.4 mmol) was added to a solution of sulfinyldeoxyvariolin 22 (7 mg, 2.1×10−5 mol) (prepared ae described in Process Example 5 below) in THF (2 ml).
The yellow solution was stirred at 70° C. overnight and evaporated at reduced pressure.
The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (3%) as eluent to afford piperidinyldeoxyvariolin 28a (5.6 mg, 78%) as a yellow oil. 1H NMR (300 MHz, CDCl3): 8.63 (dd, J=8.4 and 1.5 Hz, 1H), 8.38 (dd, J=4.8 and 1.7 Hz, 1H), 8.35 (d, J=5.4, 1H), 7.60 (d, J=6.6 Hz, 1H), 7.50 (d, J=6.6 Hz, 1H), 7.46 (dd, J=8.0 and 4.6 Hz, 1H), 6.84 (d, J=5.4 Hz, 1H), 3.92 (brs, 4H), 1.73 (brs, 6H). MS (electrospray ionisation, ESI) 346 (M+1).
Ethylamine (0.34 ml, 2M in MeOH) was added to a solution of sulfinyldeoxyvariolin 22 (prepared as described in Process Example 5 below) (11 mg, 3.4×10−5 mol) in THF (2 ml). The yellow solution was stirred at 70° C. overnight and evaporated at reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (4%) as eluent to afford N′-ethyldeoxyvariolin 28b (5.5 mg, 53%) as a yellow oil. 1H NMR (300 MHz, CDCl3): 8.67 (dd, J=7.9 and 1.4 Hz, 1H), 8.36 (d, J=4.4, 1H), 8.22 (d, J=4.8, 1H), 7.52 (brs, 2H), 7.45 (dd, J=7.8 and 4.1 Hz, 1H), 6.93 (d, J=5.1 Hz, 1H), 3.54 (d, J=6.8 Hz, 2H), 1.29 (t, J=7.0, 3H). (ESI) 306 (M+1).
Butylmethylamine (0.029 ml, 0.24 mmol) was added to a solution of sulfinyldeoxyvarolin 22 (prepared as described in Process Example 5 below) (8 mg, 2.4×10−5 mol) in THF (2 ml). The yellow solution was stirred at 70° C. overnight and evaporated at reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (4%) as eluent to afford N′-butylmethyldeoxyvariolin 28c (2 mg, 62% based on recovered starting material) as a yellow oil. 1H NMR (300 MHz, CDCl3): 8.75 (d, 1H), 8.41 (dd, 1H), 8.39 (d, J=5.5, 1H), 7.78 (d, J=6.5 Hz, 1H), 7.68 (d, J=6.5 Hz, 1H), 7.42 (dd, J=8.0 and 4.5 Hz, 1H), 6.89 (d, J=5.6 Hz, 1H), 3.60 (brs, 2H), 3.41 (s, 3H), 1.62 (brs, 4H), 1.05 (brs, 3H). (EST) 348 (M+1).
Benzylamine (0.050 ml, 0.45 mmol) was added to a solution of sulfinyldeoxyvariolin 22 (4 mg, 1.2×105 mol) in THF (1.5 ml). The yellow solution was stirred at 70° C. overnight and evaporated at reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (4%) as eluent to afford N′-benzyldeoxyvariolin 28d (2.1 mg, 47%) as a yellow oil. 1H NMR (300 MHz, CDCl3): 8.81 (brs, 1H), 8.75 (d, J=7.1, 1H), 8.64 (d, J=6.0 Hz, 1H), 7.49-7.31 (m, 8H), 6.99 (d, J=6.2 Hz, 1H), 4.78 (d, J=5.8 Hz, 2H). (ESI) 368 (M+1).
A solution of sulfonyldeoxyvariolin 6 (prepared as described in Process Example 5 below) (5.8 mg, 1.7×10−5 mol) in MeOH (2 ml) was added to a solution of sodium methoxide in MeOH (2 ml) at 0° C. The yellow solution was stirred at 24° C. for 4 h, quenched with a saturated solution of NH4Cl and extracted with ethyl acetate (3×10 ml). The combined organic layers were dried, filtered and evaporated under reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (1%) to DCM/MeOH (3%) as eluent to afford methoxydeoxyvariolin 27 (2.6 mg, 53%) as a yellow solid 1H NMR (300 MHz, CDCl3): 8.78 (dd, J=8.1 and 1.5 Hz, 1H), 8.51 (d, J=5.43 Hz, 1H), 8.41 (dd, J=4.6 and 1.5 Hz, 1H), 7.69 (d, J=6.6 Hz, 1H), 7.63 (d, J=6.6 Hz, 1H), 7.50 (dd, J=8.1 and 4.6 Hz, 1H), 7.34 (d, J=5.4 Hz, 1H), 4.14 (s, 3H). (ESI) 293 (M+1).
Trifluoroacetic anhydride (6 nil, 4.3×105 mol) was added to a solution of deoxyvariolin 4 (4 mg, 1.4×1015 mol) in THF (1.5 ml). The yellow solution was stirred at 24° C. overnight and evaporated at reduced pressure. The yellow residue was dissolved in DCM (5 ml) and washed with a saturated solution of NaHCO3 (4 ml). The organic layer was dried, filtered and evaporated under reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (4%) as eluant to afford N′-trifluoroacetyldeoxyvariolin 26 (0.9 mg, 43% based on recovered starting material) as a yellow oil. 1H NMR (300 MHz, CDCl3): 8.99 (dd, J=8.4 and 1.1 Hz, 1H), 8.57 (d, J=5.6 Hz, 1H), 8.42 (dd, J=4.6 and 1.3 Hz, 1H), 7.91 (d, J=6.7 Hz, 1H), 7.79 (d, J=6.6 Hz, 1H), 7.58 (dd, J=8.3 and 4.3 Hz, 1H), 7.52 (d, J=5.6 Hz, 1H). (ESI) 374 (M+1).
Methanesulfonyl chloride (5.5 μl, 5×10−5 mol) was added to a solution of deoxyvariolin 4 (prepared as described in Process Example 2 or 4 below) (5 mg, 1.8×10−5 mol) and Et3N (5 μl, 3.6×10−5 mol) in THF (1.5 ml). The yellow solution was stirred at 240° C. overnight and evaporated at reduced pressure. The yellow residue was dissolved in DCM (5 ml) and washed with a saturated solution of NaHCO3 (4 ml). The organic layer was dried, filtered and evaporated under reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (4%) as eluant to afford N′-methanesulfonyldeoxyvariolin 25 (1.5 mg, 46% based on recovered starting material) as a yellow oil. 1H NMR (300 MHz, CDCl3): 8.89 (dd, J=7.9 and 1.1 Hz, 1H), 8.76 (d, J=5.7 Hz, 1H), 8.42 (dd, J=42 and 1.2 Hz, 1H), 7.78 (d, J=6.4 Hz, 1H), 7.72 (d, J=6.5 Hz, 1H), 7.64 (d, J=5.6 Hz, 1H), 7.57 (dd, J 8.3 and 4.3 Hz, 1H), 3.15 (s, 3H).
Cinnamoyl chloride (9 μl, 5.4×10−5 mol) was added to a solution of deoxyvariolin 4 (5 mg, 1.8×105 mol) (prepared as described in Process Example 2 or 4 below) and Et3N (12 μl, 5.4×10−5 mol) in THF (2 ml). Immediately, DMAP (1 mg, 0.9×10−5 mol) was added in one portion, the yellow solution was stirred at 24° C. overnight and evaporated at reduced pressure. The yellow residue was dissolved in DCM (5 ml) and washed with a saturated solution of NaHCO3 (4 ml). The organic layer was dried, filtered and evaporated under reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (1%) to DCM/MeOH (4%) as eluent to afford N′-biscinnamoyldeoxyvariolin 24a (1.1 mg, 21% based on recovered starting material) and N′-biscinnamoyl-N-cinnamoyldeoxyvariolin 24b (0.6 mg, 9% based on recovered starting material) as yellow oils.
1H NMR (300 MHz, CDCl3): 8.78 (d, J=6.4, 1H), 8.69 (dd, J=7.4 and 1.1 Hz, 1H), 8.38 (dd, J=4.6 and 1.2 Hz, 1H), 7.91 (d, J=15.3 Hz, 2H), 7.64-7.32 (m, 14H), 6.90 (d, J=15.6 Hz, 2H). (ESI) 560 (M+Na), 538 (M+1).
1H NMR (300 MHz, CDCl3): 1H NMR (300 MHz, CDCl3): 8.82 (d, J=6.4, 1H), 8.79 (dd, J=7.4 and 1.1 Hz, 1H), 8.58 (dd, J=4.6 and 1.3 Hz, 1H), 8.01-7.82 (m, 4H), 7.71 (d, J=15.3 Hz, 2H), 7.64 (dd, J=8.3 and 4.2 Hz, 1H), 7.58-7.31 (m, 16H), 6.90 (d, J=15.6 Hz, 3H). (ESI) 668 (4+1), 690 (M+Na).
Acetyl chloride (3.5 μl, 4.8×10−5 mol) was added to a solution of deoxyvariolin 4 (9 mg, 3.2×10−5 mol) (prepared as described in Process Example 2 or 4 below) and Et3N (9 μl, 6.5×10−5 mol) in THF (2 ml). The orange slurry was stirred at 24° C. overnight and evaporated at reduced pressure. The yellow residue was dissolved in DCM (5 ml) and washed with a saturated solution of NaHCO3(4 ml). The organic layer was dried, filtered and evaporated under reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (5%) as eluent to afford N′-bisacetyldeoxyvariolin 23a (1 mg, 26% based on recovered starting material), N′-bisacetyl-N-acetyldeoxyvariolin 23b (1 mg, 23% based on recovered starting material) and N′-bisacetyl-N-bisacetyldeoxyvariolin 23c (0.5 mg, 10% based on recovered starting material) as yellow oils.
1H NMR (300 MHz, CDCl3): 8.76 (d, J=5.6, 1H), 8.68 (dd, J=6.9 and 1.1 Hz, 1H), 8.42 (dd, J=4.1 and 1.2 Hz, 1H), 7.73 (d, J=6.6 Hz, 1H), 7.66 (d, J=6.6 Hz, 1H), 7.54-7.42 (m, 2H), 2.41 (s, 6H). (ESI) 384 (M+Na).
1H NMR (300 MHz, CDCl3): 8.82 (d, J=5.6 Hz, 1H), 8.72 (dd, J 7.8 and 1.2 Hz, 1H), 8.53 (dd, J=4.4 and 1.2 Hz, 1H), 7.90 (d, J=6.4 Hz, 1H), 7.87 (d, J=6.5 Hz, 1H), 7.70 (d, J=5.4 Hz, 1H), 7.60 (dd, J=8.3 and 4.9 Hz, 1H), 2.68 (s, 3H), 2.40 (s, 6H). (ESI) 426 (M+Na), 404 (M+1).
1H NMR (300 MHz, CDCl3): 8.88 (d, J=5.4 Hz, 1H), 8.63 (dd, J=8.3 and 1.7 Hz, 1H), 8.56 (dd, J=4.6 and 1.3 Hz, 1H), 8.36 (d, J=6.7 Hz, 1H), 7.99 (d, J=6.6 Hz, 1H), 7.75 (d, J=5.5 Hz, 1H), 7.58 (dd, J=8.3 and 4.6 Hz, 1H), 2.43 (s, 12H). (ESI) 468 (M+Na).
Acetyl chloride (1.5 μl, 1.8×10−5 mol) was added to a solution of deoxyvariolin 4 (prepared as described in Process Example 2 or 4 below) (5 mg, 1.8×10−5 mol) and Et3N (4 μl, 2.7×10−5 mol) in THF (1.5 ml) at −78° C. The orange slurry was stirred overnight increasing the temperature very slowly until room temperature and afterwards evaporated at reduced pressure. The yellow residue was dissolved in DCM (5 ml) and washed with a saturated solution of NaHCO3 (4 ml). The organic layer was dried, filtered and evaporated under reduced pressure. The yellow residue was purified by flash chromatography using DCM/MeOH (1%) to DCM/MeOH (4%) as eluent to afford N′-acetyl-N-acetyldeoxyvariolin 23d (1 mg, 26%) and N′-acetyldeoxyvariolin 23e (0.5 mg, 10%) as yellow oils.
1H NMR (300 MHz, CDCl3): 8.83 (dd, J=7.4 and 1.4 Hz, 1H), 8.52 (d, J=6.2, 1H), 8.41 (dd, J=4.2 and 1.4 Hz, 1H), 7.75-7.71 (m, 2H), 7.56-7.48 (m, 1H), 7.39 (d, J=6.3 Hz, 1H), 2.43 (s, 3H). (ESI) 342 (M+Na).
1H NMR (300 MHz, CDCl3): 8.91 (dd, J=7.4 and 1.4 Hz, 1H), 8.58 (d, J=6.2, 1H), 8.52 (dd, J=4.2 and 1.4 Hz, 1H), 8.19 (d, J=6.4 Hz, 1H), 8.03 (brs, 1H), 7.85 (d, J=6.5 Hz, 1H), 7.61 (dd, J=8.3 and 4.9 Hz, 1H), 7.39 (d, J=6.1 Hz, 1H), 2.65 (s, 3H), 2.43 (s, 3H). (EST) 384 (M+Na).
2-Chloro-4-methoxypyridine (11) (Reference Example 3) (0.633 g, 4.41 mmol) was dissolved in freshly distilled THF (18 mL) and the reaction cooled to below −90° C. n-BuLi in hexanes (1.6 M, 2.9 mL, 4.5 mmol) was added over a period of 17 min to the stirred solution, keeping the temperature below −97° C. The orange solution was then stirred at −78° C. for 1 h, by which time it had become a wine-red colour. The reaction mixture was re-cooled to below −90° C. and a solution of ketone (9) (1.14 g, 4.09 mmol) in THF (10 mL) was added over 11 min, keeping the temperature below −90° C. The dark mixture was stirred at −78° C. for 3.5 h, then quenched with methanol and allowed to warm to room temperature. The reaction mixture was shaken with aqueous NH4Cl solution, extracted with ethyl acetate (×3) and subjected to standard workup. The crude mixture was purified by flash chromatography on silica gel using gradient elution (70 to 75% EtOAc/hexanes) to give the triaryl alcohol 12 as a cream solid (1.32 g, 76%).
1H NMR (500 MHz, CDCl3): δ 2.49 (s, 6H), 3.43 (s, 3H), 6.55 (s, 1H), 6.76 Hz, 1H), 7.39 (d, J=5.4 Hz, 2H), 8.25 (d, J=5.4 Hz, 1H), 8.46 (d, J=5.4 Hz, 2H); 13C NMR (75 ME CDCl3): δ 14.1, 55.8, 78.0, 107.1, 113.7, 124.8, 149.8, 152.3, 157.2, 165.9, 171.0, 171.1; IR (CDCl3 solution): 3344 cm−1; HRMS: Calcd for C17H1635ClN5O232S2 (M+) 421.0434, found 421.0448.
BuLi (6.9 ml, 2.5 M in hexane) was added dropwise to a solution of iodopyrimidine 8 (Reference Example 1) (4.3 g, 17 mmol) in THF (50 ml) at −100° C. The black solution was stirred for 30 min at the same temperature. A solution of 2-chloronicotinoyl chloride 17 (1 g, 5.7 mmol) in THF (7 ml), previously cooled at −78° C., was added via cannula. The intense red mixture was stirred for 3 h at −95° C. and a saturated solution of NH4Cl (50 ml) was added. The layers were separated and the aqueous layer was extracted with diethyl ether (3×100 ml). The combined organic layers were dried, filtered and concentrated under reduced pressure. The red residue was purified by flash chromatography using ethyl acetate:hexane 1:3.5 to ethyl acetate:hexane 1:1.5 as eluent to afford the alcohol 18 (1.3 g, 58%) as a pale orange solid. 1H NMR (300 MHz, CDCl3): 8.56 (d, J=5.1H. 2H), 8.37 (dd, J 4.7 and 1.5, 1H), 7.39 (d, J=5.1 Hz, 2H), 7.22 (dd, J=7.8 and 1.9, 1H), 7.17 (dd, J=7.8 and 4.4 Hz, 1H), 2.48 (s, 6H).
(This compound is not part of the present invention)
Alcohol 16 (prepared as described in Example 3 above) (33 mg, 0.062 mmol) was dissolved in neat triflic acid (0.4 mL) under atmospheric conditions. The flask was sealed and the deep red solution was left at room temperature for 5 h. The flask was cooled in ice and MeOH (2 mL) was added dropwise. Addition of concentrated aqueous ammonia (2 mL) produced a bright yellow precipitate. The suspension was applied to the top of a chromatography column containing reverse-phase silica, which had been equilibrated with 50% MeOH/water. The yellow suspension was applied to the column with 20% MeOH/water (50 mL). The polarity of the eluting solvent system was decreased to 80% MeOH/water (50 mL), and then to 85% MeOH/water containing 0.1% TFA, whereupon the yellow product began to elute. The bright yellow fractions were combined and concentrated in vacuo to give variolin B as its trifluoroacetate salt MeOH (10 mL) was added, followed by concentrated aqueous ammonia (1-2 mL) to give the free base. Removal of the solvents under reduced pressure, followed by drying (35° C., 0.03 mm Hg) overnight gave variolin B (1) (10 mg, 55%), which was identical in all aspects with the natural material.
(This compound is not part of the present invention)
A solution of mCPBA (Aldrich 70%) (98 mg, 0.39 mmol) in DCM (4 ml), previously dried over Na2SO4, was added dropwise to a cooled (−30° C.) solution of dithioether 19 (Example 4) (61 mg, 0.18 mmol) in DCM (5 ml). The yellow solution was stirred for 15 min at 0° C. A saturated aqueous Na2S2O3 solution (5 ml) was added and the organic layer was washed with a saturated solution of NaHCO3 (5 ml). The combined aqueous layers were extracted with DCM (3×10 ml). The combined organic extracts were dried, filtered and concentrated. The yellow residue was poured in a sealed tube with dioxane (4 ml) and ammonia solution 32% (8 ml) was added. The brown mixture was stirred for 14 h at 85° C. The resulting yellow mixture was evaporated in vacuo and DCM/MeOH (10:1) (11 ml) were added, the solution dried and the solvent evaporated at reduced pressure. The yellow solid was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (5%) as eluent to afford deoxyvariolin 4 (14 mg, 29%, 2 steps) as a yellow solid. 1H NMR (300 MHz, DMSO): 8.92 (dd, J=8.1 and 1.5 Hz, 1H), 8.45 (dd, J=4.6 and 1.4 Hz, 1H), 8.22 (d, J=5.5, 1H), 7.68 (d, J=6.6 Hz, 1H), 7.63 (d, J=6.6 Hz, 1H), 7.58 (dd, J=8.1 and 4.6 Hz, 1H), 7.06 (d, J=5.4 Hz, 1H). (ESI) 278 (M+1).
(This compound is not part of the present invention)
Ammonia solution 32% (3 ml) was added to a solution of dithioether 19 (Example 4) (12 mg, 0.035 mmol) in dioxane (2 ml). The brown mixture was stirred for 14 h at 85° C. in a sealed tube. The resulting yellow mixture was evaporated in vacuo, DCM (5 ml) was added, the solution dried and the solvent evaporated at reduced pressure. The yellow solid was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (3%) as eluent to afford thiodeoxyvariolin 5 (8 mg, 73%) as a yellow solid. 1H NMR (300 MHz CDCl3): 8.72 (dd, J=8.1 and 1.5 Hz, 1H), 8.48 (d, J=5.4 Hz, 1H), 8.39 (dd, J=4.8 and 1.6 Hz, 1H), 7.66 (d, J=6.8 Hz, 1H), 7.56 (d, J=6.7 Hz, 1H), 7.48 (dd, J=8.1 and 4.6 Hz, 1H), 7.32 (d, J=5.3 Hz, 1H), 2.67 (s, 3H). (ESI) 309 (M+1).
(This compound is not part of the present invention)
N′,N-bis (p-methoxybenzyl)deoxyvariolin 29 (Example 5) (15 mg, 2.9×10−5 mol) was treated with neat triflic acid (1.5 ml) and stirred for 17 h at 24° C. The black solution was evaporated at reduced pressure and the black slurry was dissolved in DCM (4 ml) and washed with a saturated solution of NaHCO3 (5 ml). The aqueous layer was extracted with DCM (3×5 ml) and the combined organic layers were dried, filtered and evaporated. The brown residue was purified by flash chromatography using DCM/MeOH (1%) to DCM/MeOH (5%) as eluent to afford deoxyvariolin 4 (1.5 mg, 19%) as a yellow solid. 1H NMR (300 MHz, DMSO): 8.92 (dd, J 8.1 and 1.5 Hz, 1H), 8.45 (dd, J=4.6 and 1.4 Hz, 1H), 8.22 (d, J=5.5, 1H), 7.68 (d, J=6.6 Hz, 1H), 7.63 (d, J=6.6 Hz, 1H), 7.58 (dd, J=8.1 and 4.6 Hz, 1H), 7.06 (d, J=5.4 Hz, 1H). (ESI) 278 (M+1).
(These compounds are not part of the present invention)
A solution of mCPBA (Aldrich 70%) (70 mg, 0.30 mmol) in DCM (3 ml), previously dried over Na2SO4, was added dropwise to a solution of thiodeoxyvariolin 5 (39 mg, 0.13 mmol) in DCM (7 ml). The yellow solution was stirred for 2 h at 24° C. A saturated aqueous Na2S2O3 solution (5 ml) was added and the organic layer was washed with a saturated solution of NaHCO3 (5 ml). The combined aqueous layers were extracted with DCM (3×20 ml). The combined organic extracts were dried, filtered and concentrated. The yellow residue was purified by flash chromatography using DCM/MeOH (2%) to DCM/MeOH (5%) as eluent to afford sulfinyldeoxyvariolin 22 (15 mg, 35%) as a yellow oil and sulfonyldeoxyvariolin 6 (25 mg, 58%) as a yellow solid.
1H NMR (300 MHz, CDCl3): 8.86 (dd, J=8.1 and 1.5 Hz, 1H), 8.74 (d, J=5.6 Hz, 1H), 8.43 (dd, J=4.6 and 1.3 Hz, 1H), 7.77 (d, J=6.8 Hz, 1H), 7.72 (d, J=6.6 Hz, 1H), 7.67 (d, J=5.8 Hz, 1H), 7.54 (dd, J=8.0 and 4.6 Hz, 1H), 3.02 (s, 3H). (ESI) 325 (M+1).
1H NMR (300 MHz, CDCl3): 8.80 (dd, J=8.1 and 1.4 Hz, 1H), 8.71 (d, J=5.6 Hz, 1H), 8.41 (dd, J=4.8 and 1.4 Hz, 1H), 7.76 (d, J=5.6 Hz, 1H), 7.74 (d, J=6.4 Hz, 1H), 7.64 (d, J=6.4 Hz, 1H), 7.52 (dd, J=8.2 and 4.8 Hz, 1H), 3.40 (s, 3H).
Iodopyrimidine 8 was prepared following the experimental procedure described in the literature: Majeed, A. J.; Antonsen. O.; Benneche, T.; Undheim, K. Tetrahedron 1989, 45, 993.
A pre-cooled (−97° C.) solution of n-BuLi in hexanes (1.55 M, 10.0 mL, 15.5 mmol) was added slowly over a period of 21 min to a solution of 4-iodo-2-methylthiopyrimidine (8) (3.90 g, 15.5 mmol) in freshly distilled THF (47 mL) at −97° C. (methanol/liquid N2 bath). Care was taken to prevent the temperature from rising above −97° C. After addition was complete, the dark mixture was stirred for 30 min at −97° C. and then, a pre-cooled (−97° C.) solution of diethyl carbonate (0.94 mL, 7.8 mmol) in THF (4 mL) was added over a period of approx. 3 min. After 15 min at −97° C. the bath was allowed to warm to −35° C. over 2 h, and then to room temperature. The reaction mixture was shaken with aqueous NH4Cl and extracted with EtOAc (×3). After the usual workup, the crude material was partially purified by vacuum distillation in a Kügelrohr apparatus (160° C., 0.03 mm Hg). Further purification was achieved by flash chromatography on silica gel using gradient elution (25, 30 and then 50% EtOAc/hexanes) to afford pure ketone 9 as a yellow solid (1.14 g, 53%).
Mp: 106-107° C.; 1H NMR (500 MHz, CDCl3): δ 2.51 (s, 6H), 7.54 (d, J=4.9 Hz, 2H), 8.79 (d, J=4.9 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ 14.2, 114.9, 158.8, 159.2, 173.2, 190.7; IR (KBr disc): 1695 cm−1; HRMS: Calcd for C11H10N4O32S2 (M+) 278.0296, found 278.0289.
4-Methoxy-2-pyridone (10) (0.805 g, 6.43 mmol) and freshly distilled POCl3 (8 mL) were heated at reflux for 15 h. Excess POCl3 was removed in vacuo and the resultant viscous oil was cooled in ice and carefully neutralised with saturated NaHCO3 solution. The mixture was extracted with EtOAc (×3) and the extracts were worked up in the standard manner to give a brown oil. This material was partially purified by vacuum distillation in a Kugelrohr apparatus (100° C., 0.07 mm Hg). The distillate was triturated with petroleum ether and a white precipitate was filtered off. The filtrate was concentrated and final purification by flash chromatography on silica gel using 30% EtOAc/hexanes as the eluant gave 2-chloro-4-methoxypyridine (11) as a colourless oil (0.586 g, 63%).
| Number | Date | Country | Kind |
|---|---|---|---|
| 0017055.5 | Jul 2000 | GB | national |
| 0030689.4 | Dec 2000 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB01/03111 | 7/11/2001 | WO | 00 | 6/25/2003 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO02/04447 | 1/17/2002 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040058939 | Alvarez et al. | Mar 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 0212240 | Feb 2002 | WO |
| WO 03006457 | Jan 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20050014778 A1 | Jan 2005 | US |
