WO1996021665A1 - Turcasarins, novel expanded porphyrins, and uses thereof - Google Patents

Abstract

Compounds of Formula (I), wherein R?1, R2, R3¿ may be alkyl or a variety of functional groups and their use as radiation sensitizers.

Description

DESCRIPTION

TURCASARINS, NOVEL EXPANDED PORPHYRINS,

AND USES THEREOF

FIELD OF THE INVENTION The present invention relates generally to the field of expanded porphyrins. More particularly, it concerns a decapyrrolic expanded porphyrin, named turcasarin, its characterization and its use as an anion chelating agent and as a photodynamic agent.

BACKGROUND OF THE INVENTION

In recent years, the chemistry of large pyrrole-containing macrocycles has been receiving increasing attention (References 1-8). Much of this interest reflects the fact this class of compounds, known

generically as "expanded porphyrins", bears important analogies to the well -studied porphyrins, yet display chemical properties such as anion binding, that have no parallel in the chemistry of porphyrins or other

naturally occurring tetrapyrrolic macrocycles. Further interest in expanded porphyrins is derived from the study of extended π-conjugation pathways. Indeed, much

attention has been devoted to making ever larger

π-conjugated systems that display far-red visible light absorption. For instance, the octavinylogous porphyrin 1 prepared by Franck and coworkers (FIG. 2) has 34

π-electrons in a conjugated path (Knϋbel and Franck, 1988). Also in the all-pyrrole category, expanded porphyrins with as many as six pyrrole subunits have been prepared (Sessler, J.L. et al., 1991 and Gossauer, A., 1983). One such example is rubyrin 2 which has 26 π-electrons in its conjugation pathway (Sessler, J.L. et al . , 1991). Other even larger expanded porphyrins which rely on additional non-pyrrole subunits to define the macrocycle core have also been prepared. For

instance, the eight-nitrogen torrand 3 (Bell, T.W.

et al . ) and the Schiff base derived macrocycle 4

(Sessler, J.L., et al . ) each have extended π-conjugation pathways with visible absorptions considerably red shifted relative to the porphyrins.

To date, there remains relatively little documented information concerning the chemistry of the

above-mentioned expanded porphyrin systems. Indeed, at present, structural information is available only for derivatives of sapphyrin (e.g., structure 5), rubyrin (e.g., structure 2) and pentaphyrin (e.g., structure 6) in the all-pyrrole series. Therefore, numerous

fundamental questions concerning these molecules still remain to be answered, such as those pertaining to ring size, aromaticity, and effective macrocycle stability. The synthesis and structural characterization of larger macrocycles would be a particular advance in this area, allowing the answers to such inter-related questions to be elucidated.

It has long been appreciated that a considerable number of ionic (e.g. phosphorylated) nucleotide analogues exhibit antiviral activity in cell-free extracts, yet are inactive in vivo due to their inability to cross lipophilic cell membranes. For example, the

anti-herpetic agent, acyclovir (FIG. 7A, structure a; 9- [(2-hydroxyethoxy)methyl]-9H-guanine), is typical in that it is able to enter the cell only in its uncharged nucleoside-like form. Upon gaining entry to the

cytoplasm it is phosphorylated, first by a viral-encoded enzyme, thymidine kinase (FIG. 7A, structure b), and then by relatively nonspecific cellular enzymes to produce the active, ionic triphosphate nucleotide-like species (FIG. 7A, structure c). There, it functions both as an

inhibitor of the viral DNA polymerase and as a chain terminator for newly synthesized herpes simplex DNA.

Many other potential antiviral agents, including, for instance, the anti-HIV agent, Xylo-G (FIG. 7B, structure d; 9-(β-D-xylofuranosyl)guanine), on the other hand, are not phosphorylated by a viral enzyme and are, therefore, largely or completely inactive. If, however, the active monophosphorylated forms of these putative drugs (such as in FIG. 7B, structure e) could be transported into cells, it would be possible to fight viral infections with a large battery of otherwise inactive materials. If such specific into-cell transport were to be achieved, it would therefore greatly augment the treatment of such debilitating diseases as, for example, AIDS, herpes, hepatitis and measles. Given the fact that AIDS is currently a major national health problem of frightening proportions, and that something so nominally benign as measles still claims over 100,000 lives per year world-wide, treatment of these diseases would be particularly timely and worthwhile.

There continues to exist a need for nucleotide transport agents. In early work, Tabushi was able to effect adenosine nucleotide transport using a lipophilic, diazabicyclooctane-derived, quaternary amine system.

However, this same system failed to mediate the transport of guanosine 5'-monophosphate (GMP) or other

guanosine-derived nucleotides. Since then, considerable effort has been devoted to the generalized problem of nucleic acid base ("nucleobase") recognition, and various binding systems have been reported. Currently known nucleotide binding systems include acyclic, macrocyclic, and macrobicyclic polyaza systems; nucleotide-binding bis-intercalands; guanidinium-based receptors; and various rationally designed H-bonding receptors. These latter H-bonding receptors have been shown to be effective for the chelation of neutral nucleobase and/or nucleoside derived substrates but, without exception, have also all proved unsatisfactory for the important task of charged nucleotide recognition. Large macrocyclic compounds, particularly macrocyclic compounds larger than sapphyrins, which could be

relatively easily protonated could prove to be useful in anion binding and transport. Despite intensive efforts in this field, there continues to exist a need for new synthetic systems capable of effecting the recognition and through-membrane transport of phosphate-bearing species such as anti -viral compounds. Furthermore, there continues to exist a need for rationally designed receptors which are "tunable" for the selective complexation of a given nucleobase-derived system.

There is clearly, therefore, a need for novel drug delivery systems to be developed. Compounds which would allow negatively-charged (anionic) structures,

particularly specifically-recognized nucleotides, to be transported across naturally lipophilic cellular

membranes would represent an important scientific and medical advance. The development of such anionic

carriers may also prove to be important in the clinical treatment of cystic fibrosis, in that such compounds would likely facilitate the out-of-cell diffusion of chloride anions. There also exists a continuing need for new chelating molecules and, particularly, chelating molecules that bind metals for use in various procedures such as photodynamic therapy. One of the main motivations, then, justifying the preparation of new metal-chelating molecules is to obtain macrocycles designed such that metal coordination may be facilitated through rational structural design. Such molecules may be used to form strong, non-labile

complexes of many of the metals or semi-metals on the Periodic Table, including large metal ions, such as lanthanides and actinides. Such a goal derives in part from the general lack of understanding that continues to exist for the metal coordination of the lanthanides and actinides. Also, molecules of sufficiently large size and/or suitable shape may act as bimetallic or even oligometallic receptors. Such metal coordinating molecules may also find use in applications involving physiological (i.e., aqueous) environments. One such application is as a

photosensitizer in the photodynamic therapy (PDT) of tumors. For example, by virtue of tumor localizing capability, singlet oxygen production, and long

wavelength (i.e., 700-800 nanometer) absorption of visible light (where bodily tissues are readily

transparent), metal complexes of these novel molecules would likely be well suited for such an application.

SUMMARY OF THE INVENTION

The present invention provides a new class of expanded porphyrins containing 10 pyrrole units. The present invention also provides uses of and a method of manufacture for this new class of macrocycles. In a first respect, the present invention is a compound of formula I:

wherein, individually in each occurrence, each of R¹, R² or R³ is a monoradical moiety of hydrogen, alkyl, alkyl halide, alkene, alkyne, aryl, amino, hydroxyl, alkoxy, carboxy, carboxamide, ester, amide, sulfonate, hydroxy substituted alkyl, glycol, polyglycol, alkoxy substituted alkyl, carboxy substituted alkyl, amino substituted alkyl, sulfonate substituted alkyl, ester substituted alkyl, amide substituted alkyl, substituted aryl, substituted alkyl, substituted ester, substituted ether, substituted amide, thiol, alkyl thiol, alkoxycarbonyl, aryloxycarbonyl, aldehyde, ketone, carboxylic acid, phosphate, phosphate substituted alkyl, phosphonate, phosphonate substituted alkyl, sulfate, sulfate

substituted alkyl, or is of the formula (CH₂)_m-A-(CH-)_n-B wherein A is CH₂ , O, S, NH or NR⁴ or wherein A is a diradical moiety selected from any of the R^1-3 groups oxy, sulfide, carbonyl, alkene, alkyne, alkyl halide, hydroxyalkyl, glycol, polyglycol, alkyl thiol,

substituted alkyl, phosphate, phosphonate, sulfate, phosphate substituted alkyl, phosphonate substituted alkyl, sulfate substituted alkyl, carboxy, carboxyamide, thiol-substituted carboxyamide, derivatized carboxyamide of formula CONR⁴ where R⁴ may be alkyl, alkyl halide, hydroxyalkyl, glycol, polyglycol, or alkyl thiol; wherein B independently in each occurrence is any of the R^1-3 groups, nucleobase, saccharide, nucleotide, an expanded porphyrin, steroid, amino acid, peptide or polypeptide, turcasarin or polymeric or solid support matrix, wherein n is 0-10, wherein m is 0-10, and wherein the total number of carbon atoms in each of R^1-4 is less than or equal to 20, or salt thereof.

The present invention provides a new class of

expanded porphyrins exemplified by the 10-pyrrole

containing macrocycle 11 (FIG. 1) ( [40] decaphyrin- [1.0.1.0.0.1.0.1.0.0]). This compound 11 is prepared by the acid catalyzed condensation of 4,4'-diethyl-5,5'-diformyl-3,3'-dimethyl-2,2'-bipyrrole 10 (Sessler,

et al . , 1990) with 2,5-bis(4-propyl-2-pyrrolyl)pyrrole 9. Compound 11, depending on its state of protonation, displays an intense purple to blue to green color in organic solution. Thus, in view of its color, compound 11 is termed "turcasarin" (from Middle English turcas) and, as used herein, this class of compounds is referred to as "turcasarins." Thus, for instance, compound 11, having a systematic name of 15, 20, 37, 42-tetraethyl-

16,19,38,41-tetramethyl-2,11,24,33-tetrapropyl- 45,46,47,48,49,50,51,52,53,54-d,caazaundecacyclo- [40.2.1.1^5,8.1^9,12.1^14,17.1^18,21 .1^23,26 .1^27,30.1^31,34.1^36,39.1⁴

⁰,⁴³]-tetrapentaconta-1,3,5,7,9(47),10,12,

14,16,18(49),19,21,23,25,27,29,31(52),32,34,36,38,40(54),41,43-tetracosaene, can be renamed as 15,20,37,42-tetraethyl-16,19,38,41-tetramethyl-2,11,24,33- tetrapropylturcasarin. Turcasarin 11 itself is a macrocycle which can be generally characterized by the presence of ten py rolic subunits contained within a 40 π-electron macrocyclic framework and by UV/VIS absorption bands that are

considerably red-shifted as compared to that of other porphyrins. Generally, the turcasarins of the present invention may be prepared by acid catalyzed condensation of a 5,5'-diformyl-bipyrrole with a terpyrrole. A generalized scheme of this condensation is shown in FIG. 3 wherein a substituted or unsubstituted 5, 5' -diformyl-bipyrrole 12 and a substituted or unsubstituted terpyrrole 13 are condensed in an acid catalyzed reaction to prepare a turcasarin 14 corresponding to a compound of formula I. The condensation is typically run in an organic solvent such as an alcohol solution.

Thus, in a second respect, the present invention is a process for the production of turcasarins, comprising condensing a bipyrrole and a terpyrrole in the presence of an acid under conditions such that a turcasarin is formed. In this aspect of the invention, the bipyrrole may be of formula II:

wherein R^2-3 are as defined above; and wherein Z is H or -CHO, and the terpyrrole can be of formula III:

wherein R¹ is as defined above. The present invention provides a solution to the needs described above by providing a novel composition for use in specific anion binding and transport.

Turcasarin and turcasarin analogues are able to bind negatively charged substances, anions, at near-neutral pH, and would be expected to have the ability to

transport anionic compounds across cell membranes. The turcasarin macrocyclic compounds of this invention are particularly contemplated for use in transporting

antiviral nucleotide analogues into cells, and in facilitating chloride anion exit from cells. They may thus find use in the treatment of a variety of viral diseases and also cystic fibrosis. It is contemplated that the compounds of this invention have a wide variety of uses. In particular, the turcasarins may be useful as a chelating agent and as a photodynamic agent. For example , the turcasarins are useful as chelating agents of metal cations such as uranium oxide or zinc cations. In Example 4, the use of turcasarin 11 to form a uranyl complex is demonstrated. Advantageously, the turcasarin is capable of binding two uranyl cations. This provides another advantage of this invention since the chelating capacity is double that of most molecules. Thus, the turcasarins may be used to treat solutions contaminated with uranium cations to thereby remove the uranium cations from solution.

Additionally, uranium is diamagnetic and is itself useful in photodynamic therapy; therefore, one skilled in this art would expect that the turcasarin/uranyl complex would be useful in photodynamic therapy also. In addition, both the red-shifted wavelength of the lower energy absorption and the high extinction coefficient for turcasarin 11 indicates that turcasarin is a likely candidate for use as a photodynamic agent and,

accordingly, the turcasarins may have utility as

photodynamic agents per se In addition, the macrocycles may also be useful as carriers for the through-membrane transport of nucleotide monophosphates such as guanosine-5' monophosphate. Turcasarin and analogues thereof may be further characterized by the ability to undergo facile

protonation at the pyrrolic nitrogens and, once

protonated, by an ability to form complexes with anions. It will also be understood that any of the above

turcasarin macrocycles may be either singly, doubly, triply or four- fold protonated. An advantageous

functional characteristic of turcasarin and analogues thereof is the ability to bind anions at near-neutral pH and possibly to transport anions across lipophilic structures such as biological membranes. Preferred compounds of the present invention therefore have the capacity to bind anions and yet the ability to retain overall supramolecular charge neutrality. In particular, the fact that turcasarins are larger than sapphyrins, the only other class of compounds known to display such behavior, makes them considerably easier to protonate and thus much more effective in the recognition and transport of anions than this other class of molecules. This then represents a significant advance embodied in the present invention.

A range of compounds with a wide variety of

substituents in the R^1-3 groups fall within the scope of the present invention. In one embodiment of this

invention, at least one of R¹, R² or R³ is other than alkyl of 1 to 3 carbons. The precursor molecules may be derivatized or the turcasarin macrocycle may be modified after condensation to yield turcasarins having a variety of substituents. For example, when an R^1-3 group is an ester, the ester may be converted through well known synthetic procedures to produce a carboxylic acid which itself may be converted to a cyano group, an ether, another ester by transesterification, an aldehyde, an alcohol, a halide or an amide which itself can be

converted to an amine. The of-positions of the starting bipyrroles may be carboxyl-, ester-, formyl-, and/or unsubstituted. Likewise, the alpha-position on the terpyrroles may be carboxyl-, ester-, formyl-, and/or unsubstituted.

It is contemplated that one of skill in the art will be able to prepare various turcasarin conjugates, including those listed above, without undue

experimentation given the readily-available starting materials and in light of the synthetic methodology disclosed in the present application.

In certain embodiments, turcasarin compounds are contemplated that contain the turcasarin macrocycle core for phosphate binding and also nucleobase "appendages" for specific nucleic acid recognition. These are

referred to as turcasarin-nucleobase conjugates, which term is intended to include any conjugate formed by the covalent conjugation of any turcasarin macrocycle to any nucleic acid base ("nucleobase"). The synthesis of such conjugates is described in Example 8 and specific

turcasarin-nucleobase conjugates are exemplified in structures 51, 55, 59 and 63 of Schemes F through I.

The term "nucleobase", as used herein, refers generally to any moiety that includes within its

structure a purine or pyrimidine, a nucleic acid, nucleoside, nucleotide, or any derivative of any of these, such as a "protected" nucleobase. Thus, the term nucleobase includes adenine, cytosine, guanine,

thymidine, uridine, inosine, and the like; bases, nucleotides or nucleosides, as well as any base,

nucleotide or nucleoside derivative based upon these or related structures. Nucleotides can be readily coupled onto a turcasarin through manipulation of amide, ether and thioether linkages. For example, a turcasarin of formula I wherein R¹, R² and/or R³ contains a functional group of formula - Y-CO-CH₂Br, wherein Y is NH or O, may be reacted with an hydroxyl group of an oligonucleotide in the presence of a Lewis acid such as FeBr₃ to form an ether linkage between the turcasarin linker and the oligonucleotide.

Alternatively, oligonucleotide analogues containing one or more thiophosphate or thiol groups are selectively alkylated at the sulfur atom with an alkyl halide

derivative of the turcasarin complex.

Oligonucleotides are used to bind selectively compounds which include the complementary nucleotide or oligo- or poly- nucleotides containing substantially complementary sequences. As used herein, a substantially complementary sequence is one in which the nucleotides generally base pair with the complementary nucleotide and in which there are very few base pair mismatches. The oligonucleotide may be large enough to bind probably at least 9 nucleotides of complementary nucleic acid.

A particular example of a useful nucleobase are the so-called antimetabolites that are based upon purine or pyrimidine structure. These structures typically exert their biological activity as antimetabolites through competing for enzyme sites and, thereby, inhibiting vital metabolic pathways. However, in the context of the present invention, the term "antimetabolite nucleobase" is used quite broadly to refer to any purine or

pyrimidine-based molecule that will effect an

anticellular, antiviral, antitumor, antiproliferative or antienzymatic effect, regardless of the underlying mechanism. Exemplary structures are shown in Table 1, and include the antimetabolites FU, AraC, AZT, ddl , xylo-GMP, Ara-AMP, PFA and LOMDP . It is contemplated that turcasarin-nucleobase conjugates will have a wide variety of applications, including their use as carriers for the delivery of antiviral drugs to a particular body or even subcellular locale. In the case of antimetabolite nucleobases, it is known that many nucleobase antimetabolites can not be readily employed in therapy due to the fact that their charged nature inhibits their uptake by target cells, or otherwise inhibits or suppresses their unencumbered movement across biological membranes. Typically, this shortcoming is due to the presence of charged structures such as phosphates, phosphonates, sulfates or sulfonates on the nucleobase that, due to their charged nature, prevents or inhibits their crossing of a biological membrane. It is proposed that the turcasarins of the present invention may be employed as transport agents for carrying such nucleobases across membranes, (whether the nucleobase is directly conjugated to the macrocycle or simply complexed with it). This point is elaborated in further detail in Sessler et al. (1992), (reference 8m) incorporated herein by reference.

Generally speaking, in the context of turcasarin- nucleobase constructs designed for drug delivery, it will usually be the case that one will employ only one or two nucleobase-containing substituents for each turcasarin macrocycle . Turcasarin derivatives with a single

nucleobase are termed "ditopic receptors", whereas those with two nucleobases are termed "tritopic receptors". However, the invention is not limited to compounds containing one or two nucleobase units, indeed, mono- or di-substituted turcasarin-nucleobase conjugates may have any number of nucleobases or nucleobase oligomers or polymers attached. The ultimate number of such residues that are attached will, of course, depend upon the application. One may employ a turcasarin derivative with 10 or so bases attached to bind and transport complementary oligo- or poly-nucleotides. Of course, there is no limitation to the particular position (s) within the turcasarin macrocycle to which the

nucleobase(s) may be attached to construct a conjugate.

Further examples of turcasarin derivatives or conjugates encompassed by the present invention are turcasarin saccharide derivatives, wherein the macrocycle is appended to a saccharide-based unit, such as a sugar, sugar derivative or polysaccharide. The synthesis of turcasarin-saccharide compounds is described in Example IX and specific turcasarin saccharide conjugates are represented by structures 66, 68, 76 and 84 in reaction schemes J, K, M and O, respectively. A non-exhaustive, exemplary list of sugars which may be conjugated to turcasarin in this manner is set forth in Table 2. Of course, any sugar or modified sugar may be employed including sugars having additional phosphate, methyl or amino groups, and the like. Moreover, the use of both D- and L-forms, as well as the α and β forms, are also contemplated. The use of sugars such as glucose, galactose, galactosamine, glucosamine and mannose is particularly contemplated. Turcaεarin-saccharide

conjugates are envisioned to be of use where one would like, to control, or otherwise modify, the water

solubility of the resultant turcasarin-based compound, for example, in exploiting its novel properties in connection with human or animal applications. In still further embodiments, the invention concerns compositions which are composed of a turcasarin

derivative in accordance with any one of the embodiments discussed above complexed to a second substance, wherein the second substance includes within its structure a negatively charged moiety. More particularly, the second substance will include a negatively charged component such as a chloride, phosphate, phosphonate, sulfate, or sulfonate moiety, of which, turcasarin-chloride ion complexes are a particular example.

In other preferred embodiments, the second substance will include a purine or pyrimidine, or an analog of either, within its structure. As mentioned above, these nucleobase structures include, for example, adenine, cytosine, guanine, thymidine, uridine and inosine;

various antimetabolic or antienzymatic nucleobase

compounds; and also oligo or polynucleotides such as DNA or RNA. Antimetabolic and antienzymatic compounds include those with antitumor, anticellular,

antiproliferative and antiviral activity, examples of which are represented in Table 1.

In further related embodiments, the invention concerns a method for forming a complex between a

turcasarin macrocycle and a negatively charged second substance, or selected agent. The method involves preparing a turcasarin or turcasarin analogue or

conjugate, such as any one of the turcasarin derivatives as described above, and contacting this turcasarin or turcasarin derivative with a negatively charged substance or selected agent under conditions effective to allow the formation of a complex between the turcasarin macrocycle and the negatively charged substance.

This method is appropriate for binding, or

complexing, a range of negatively charged substances or selected agents, such as for example, chloride ions and other halides, pseudohalides such as azide or cyanide anions, and anionic clusters such as ferricyanide. The complexing of phosphate-containing compounds, including, simple alkyl or aryl phosphates, nucleotides, oligo- and polynucleotides, such as DNA, RNA and anti-sense

constructs, and nucleotide analogues is particularly contemplated. Even more preferable, is the complexing of antiviral compounds such as phosphonate derivatives and simple species such as the pyrophosphate derivatives PFA and COMDP; the antiviral agents of FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F and Table 1, and

particularly, acyclovir monophosphate, Xylo-GMP, and Ara-AMP. Here, as throughout this invention, a key

embodiment is the high basicity of the turcasarin, which allows this class of compounds and its derivatives to act as vastly improved anion chelators or carriers in

comparison to expanded porphyrins such as sapphyrins or any other extant system.

Still further methodological embodiments concern methods for the cellular transport of a given substance, generally a negatively charged substance. This may be employed as a means of, for example, successfully

introducing a negatively charged substance into a cell, or alternatively, as a means of facilitating the removal of a negatively charged substance from a cell. To achieve this, one would prepare a turcasarin macrocycle in accordance with the present invention, contact the macrocycle with the negatively charged substance under conditions effective to allow complex formation, and then simply contact the cell, either in vi tro or in vivo, with the macrocycle-bound substance.

Any one of a variety of negatively charged

substances may be delivered to a cell, including a cell within an organism, in this manner. The delivery of oligonucleotides, including anti-sense constructs, and nucleotide analogues, such as antiviral compounds, is particularly envisioned. One example concerns the introduction of a turcasarin-complex composition which includes an antiviral antimetabolic or antienzymatic compound into a cell suspected of being a virally

infected target cell. Another example is the

introduction of an antitumor antimetabolic or antienzymatic compound into a cell suspected of being a tumor or proliferating cell.

Of course, it is contemplated that such target cells may be located within an animal or human patient, in which case an effective amount of the complex, in

pharmacologically acceptable form, would be administered to the patient. Generally speaking, it is contemplated that useful pharmaceutical compositions of the present invention will include the selected turcasarin derivative in a convenient amount that is diluted in a

pharmacologically or physiologically acceptable buffer, such as, for example, phosphate buffered saline. The route of administration and ultimate amount of material that is administered to the patient or animal under such circumstances will depend upon the intended application and will be apparent to those of skill in the art.

The capacity of turcasarin and analogues thereof to effect specific into-cell transport of anti-viral

compounds is contemplated to be of use against a wide variety of debilitating diseases such as AIDS, herpes, hepatitis and measles. As a mediator of DNA import, turcasarins may conceivably be employed in the treatment of any disease in which the delivery of an

oligonucleotide or DNA fragment would be advantageous, such as in supplying a functioning gene, or in inhibiting an aberrant gene, for example, by employing an antisense DNA construct. As discussed above, the larger size, high basicity, and relative ease with which turcasarins may be protonated, renders them particularly effective molecules for use in anion transport.

Additionally, certain turcasarins with advantageous chloride ion transporting properties may be employed as synthetic carriers capable of facilitating out-of-cell diffusion of chloride anions, and are therefore contemplated for use as therapeutic agents for the treatment of cystic fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed

description of specific embodiments presented herein.

FIG. 1 shows a synthetic scheme for the synthesis of turcasarin 11.

FIG. 2 shows structures of expanded porphyrins described in the Background of the Invention. FIG. 3 shows a generalized synthetic scheme for the synthesis of turcasarin macrocycles of the present invention.

FIG. 4 shows a schematic representation of the

interconversion between the two limiting enantiomeric conformations of turcasarin 11. These conformations may be considered as left- or right-handed twists along the long axis of a presumed open, pseudo-circular

intermediate.

FIG. 5 shows a two-dimensional H,H-correlated spectrum (H,H COSY) of the alkyl region of turcasarin 11 that allowed definitive peak assignments to be made. Paired sets of crosspeaks indicate near C₂ symmetry in the molecule and provide evidence for diastereotopic protons in the propyl side chains.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and 7F show the structures for potential antiviral agents a through i.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The turcasarins of the present invention are of formula I shown hereinabove. The structure has substituent groups R^1-3, each of R^1-3 being defined as above.

Representative examples of alkanes used as such monoradicals include methane, ethane, straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonaneand decane, with methane, ethane and propane being preferred. Representative examples of alkenes useful as such monoradicals or diradicals include ethene, straight-chain, branched or cyclic isomers of propene, butene, pentene, hexene, heptene, octene, nonene and decene, with ethene and propene being preferred. Representative examples of alkynes useful as such monoradicals or diradicals include ethyne, straight-chain, branched or cyclic isomers of propyne, butyne, pentyne, hexyne, heptyne, octyne, nonyne and decyne, with ethyne and propyne being preferred.

Among the halides, chlorine and bromine are preferred in the practice of this invention. Representative examples of alkyl halides used in this invention include halides of methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane and decane, with halides,

preferably chlorides or bromides, of methane, ethane and propane being preferred. Representative examples of hydroxyalkyls include alcohols of methane, ethane, straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with alcohols of methane, ethane or propane being preferred. Representative examples of useful glycols of this invention include diols of ethane, straight -chain, branched or cyclic isomers propane, butane, pentane, hexane, heptane, octane, nonane and decane, with diols of ethane or propane being preferred. Representative examples of polyglycols include polyethylene glycol, polypropylene glycol and polybutylene glycol as well as polyalkylene glycols containing combinations of ethylene, propylene and butylene. Representative examples of alkyl thiols include thiols of ethane, thiols of

straight-chain, branched or cyclic isomers of propane, butane, pentane, hexane, heptane, octane, nonane and decane, with thiols of ethane (ethanethiol, C₂H₅SH) or propane (propanethiol, C₃H₇SH) being preferred.

Representative examples of substituted alkyls include alkyls substituted by two or more functional groups described herein. Representative examples of phosphates include phosphate or polyphosphate groups .

Representative examples of phosphate substituted alkyls include alkyls as described above substituted by one or more phosphate or polyphosphate groups. Representative examples of phosphonate substituted alkyls include alkyls as described above substituted by one or more phosphonate groups. Sulfate substituted alkyls include alkyls as described above substituted by one or more sulfate groups, a representative example of which is diethyl sulfate ((C₂H₅)₂SO₄). Representative examples of carboxy groups include carboxylic acids of the alkyls described above as well as aryl carboxylic acids such as benzoic acid. Representative examples of carboxyamides include primary carboxyamides (CONH₂), secondary (CONHR⁴) and tertiary (CONR⁴R⁴) R⁴ carboxyamides where R⁴ is a

functional group as described herein. Representative examples of useful amines include a primary, secondary or tertiary amine of an alkyl as described hereinabove.

Representative examples of useful oligonucleotides include nucleotides, oligonucleotides and polynucleotides composed of adenine, cytosine, guanine, thymine or uracil bases. It is understood that the term nucleotide as used herein refers to both naturally-occurring and synthetic nucleotides, poly- and oligonucleotides and to analogs and derivatives thereof such as methylphosphonates, phosphotriesters, phosphorothioates and phosphoramidates. Representative examples of useful steroids include any of the steroid hormones of the following five categories: progestins (e.g. progesterone), glucocorticoids (e.g., cortisol), mineralocorticoids (e.g., aldosterone), androgens (e.g., testosterone) and estrogens (e.g., estradiol). Representative examples of useful amino acids include amino acids with simple aliphatic side chains (e.g., glycine, alanine, valine, leucine, and isoleucine), amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine, and histidine), amino acids with oxygen and sulfur-containing side chains (e.g., serine, threonine, methionine, and cysteine), amino acids with side chains containing carboxylic acid or amide groups (e.g., aspartic acid, glutamic acid, asparagine, and glutamine), and amino acids with side chains containing strongly basic groups (e.g., lysine and arginine), and proline. Representative examples of useful peptides include any of both naturally occurring and synthetic di-, tri-, tetra-, and pentapeptides derived from any of the above described amino acids .

Representative examples of useful polypeptides include both naturally occurring and synthetic polypeptides

(e.g., insulin, ribonuclease, and endorphins) derived from the above described amino acids and peptides.

Representative examples of polymeric or solid support matrixes include resin-type polystyrene derived solid support matrixes, aminopropyl-functionalized silica gel or natural polymers such as polysaccharides.

Representative examples of nucleobases useful in this invention include those shown in Table 1 hereinbelow. Representative examples of sugars useful in the practice of this invention include those shown in Table 2

hereinbelow. Representative examples of useful chelates include EDTA, EGTA, DTPA, DOTA, ethylene diamine, bipyridine, 1,10-phenanthralene, crown ether, aza crown and catechols. The bipyrroles that are used in the practice of this invention to prepare turcasarins contain two formyl groups at the alpha positions. The R^2-3 substituents may be introduced on the pyrrole ring before or after the condensation reaction to synthesize a turcasarin

depending on the nature of the substituent. If the substituent impairs the formation of the turcasarin macrocycle, for example, due to steric hindrance, then the substituent may be incorporated after the

condensation. The bipyrroles are well known compounds which can be readily prepared by conventional techniques well known to those of skill in the art.

The terpyrroles used in the practice of this

invention may be unsubstituted at the alpha positions. The terpyrroles used in this invention are well known materials which can be readily prepared by conventional techniques well known to those skill in the art . One method of terpyrrole preparation involves the synthesis as shown in Example 2. Other methods include those described by Chierici et al., Gaz. Chim. Ital.. volume 86, pages 1278-1283 (1956).

In the condensation to form the turcasarins of this invention, the most preferred acid is hydrogen chloride. Suitable solvents for the condensation include ethanol or methanol with cosolvents such as chloroform or methylene chloride. The condensation is generally run at room temperature.

The turcasarin and turcasarin analogues of the present invention are characterized by the capacity to bind anions and yet retain overall supramolecular charge neutrality. A particular advantage to turcasarin

molecules is their large size. This property renders turcasarins easier to protonate than other macrocycles and makes them considerably more effective at anion recognition and transport than any other classes of expanded porphyrins, including the sapphyrins. The increased basicity of turcasarin relative to sapphyrin is thus of considerable importance and represents a

significant advance relative to the existing art.

Additionally, several other turcasarin analogues are contemplated by the present invention. Different

combinations of bipyrroles and terpyrroles may be

employed in the condensation step to yield a variety of different macrocyclic products. The generation of further turcasarin analogues is particularly described in Example VII, and represented in reaction schemes A through E. A range of compounds with a wide variety of alkyl and/or aryl substituents in the meso and/or

β-pyrrolic positions, as exemplified by structures 27, 35, 40 and 47, thus fall within the scope of the present invention.

It is contemplated that turcasarins of the present invention will be of use as drug delivery agents. It is contemplated that they will find utility in mediating the cross-membrane transport of negatively charged compounds or molecules, including halides, pseudohalides, such as azide or cyanide anions, or anionic clusters such as ferricyanide. The anion carrying properties of

turcasarins make them ideal candidates for the

development of synthetic carriers capable of facilitating out-of-cell diffusion of chloride anions, and hence for use as therapeutic agents for the treatment of cystic fibrosis. Importantly, turcasarin and analogues thereof are contemplated for transporting phosphate-containing compounds into cells. Phosphate-containing compounds which may be transported in this manner include, for example, simple alkyl or aryl phosphate, nucleotides such as AMP or GMP, oligonucleotides and DNA or RNA, including anti-sense DNA or RNA constructs, and more particularly, antiviral compounds such as those depicted in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F structures b,e,f,g,h,i, Table 1, and equivalents thereof.

Any one of a variety of antiviral agents may be delivered to a cell using turcasarin or a turcasarin analogue in accordance herewith. These agents include, for example, the anti-HSV and anti-HIV agents acyclovir monophosphate, Xylo-GMP, Ara-AMP, and/or phosphonate derivatives that also have documented anti-HSV and anti-HIV activity in vi tro (e.g., FIG. 7F and FIG. 7G), and simple species such as the pyrophosphate derivatites PFA (FIG. 7E) and COMDP (FIG. 7F) that have demonstrated anti-HIV reverse transcriptase activity in cell-free media.

As mentioned above, the large size, increased basicity and ease of protonation of turcasarins makes this class of macrocycles considerably more effective at the recognition and transport of anions than other classes of molecules. The capacity of turcasarin, and analogues thereof, to effect specific into-cell transport of anti-viral compounds is contemplated to be of

advantageous use against a wide variety of debilitating diseases, including, for example, herpes, hepatitis, measles, and AIDS. Such diseases are of major medical and economic importance with AIDS being an international health problem and even measles claiming over 100,000 lives per year world-wide. Furthermore, it is contemplated that the turcasarin compounds of the present invention may be rendered useful as nucleotide transporters if one or more nucleobase recognition units were to be "appended" directly onto the phosphate-chelating macrocyclic core. This would impart a further degree of nucleotide specificity to binding and transport reactions. Accordingly, turcasarin-nucleobase conjugates which have been derivatized by the addition of one or more nucleobase compounds form an important aspect of the present invention.

Turcasarin mononucleobase derivatives may include any of the naturally-occurring purine or pyrimidine nucleobases, namely, cytosine, guanine, thymidine, adenine, uridine or inosine. Equally, they may include modified versions of any of these, such as the

heterocyclic components of those nucleoside/nucleotide analogues listed in Table 1. Also included within the invention are the turcasarin mononucleobase derivatives including chemically modified nucleobases such as

"protected" bases. Protecting groups are used to protect reactive groups, such as amino and carboxyl groups, from inappropriate chemical reactions. Turcasarin-nucleobase conjugates with protected bases include, for example, conjugates wherein one or more base has a protecting group, such as 9-fluorenylmethylcarbonyl,

benzyloxycarbonyl, 4-methoxyphenacyloxycarbonyl, t-butyloxycarbonyl, 1-adamantyloxycarbonyl, benzoyl, N- triphenylmethyl or N-di- (4-methoxyphenyl)phenylmethyl on the amino group of the nucleobase.

It is contemplated that various chemical means may he used to covalently bond nucleobases to turcasarin macrocycles. Various spacers may be used for the

binding, such as, for example, oligomethylene bridges with terminal amino, or hydroxy function, which allow formation of an amide or ester bond for the connection of the turcasarin and nucleobase units. This bridge may also be modified, for example, by the reduction of the amide bond to give the amine function. Specific examples of the synthesis of turcasarin-nucleobase conjugates are described in Example 7 and the resultant compounds are represented by structures 51, 55, 59 and 63 of reaction schemes F through I.

Turcasarin nucleobase conjugates would be useful as antiviral agents, capable of binding and solubilizing nucleotides and of effecting their selective through- membrane transport at or near physiologic pH. Turcasarin nucleobase conjugates with appended oligonucleotides are also contemplated by the present invention, and would be of use in binding and transporting oligo- or

polynucleotides, including antisense constructs, into cells. As a mediator of DNA import, turcasarins may conceivably be employed in the treatment of any disease in which the delivery of an oligonucleotide or DNA fragment would be advantageous, such as in supplying a functioning gene, or in inhibiting an aberrant gene, for example, by employing an antisense DNA construct.

Another class of turcasarin derivatives or

conjugates contemplated are the turcasarin saccharide derivatives which comprise a turcasarin macrocycle conjugated to a sugar, sugar derivative or

polysaccharide. The synthesis of turcasarin-saccharide compounds, as represented by structures 66, 68, 76 and 84 in reaction schemes J, K, M and O, respectively, is described in Example IX. It will be understood that any one of a variety of individual sugar units, such as those set forth in Table 2, or polymers thereof, may be

conjugated to turcasarin in accordance herewith. Table 2 is intended to include modified versions of the sugar units, such as sugars having additional phosphate, methyl or amino groups and the like, and also includes D- and L-isomers and α and β forms.

In addition to the turcasarin analogues described above and the nucleobase and saccharide conjugates, it will be appreciated that a variety of other substituents, of desirable chemical function, may be appended to a functionalized turcasarin moiety to construct a turcasarin-based conjugate. It is contemplated that the synthesis of turcasarin conjugates includes, for example: metal chelator moieties such as EDTA, EGTA, 1,10-phenanthralene, DTPA, DOTA, crown ether, azacrown, catecholate and ethylene diamine; alkylating agents such as ethylene diamine, epoxide and bromoacetamide; steroids and steroid derivatives; amino acids, peptides and polypeptides; other turcasarins, turcasarin derivatives, polymeric turcasarin, or other macrocyclic compounds such as sapphyrins, texaphyrins or derivatives thereof; and polymeric matrices or solid supports such as polymers, glasses, agarose, polyacrylamide , controlled pore glass, silica gel, polystyrene and sepharose. Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. For example, it is anticipated that the open beta positions on the residual terpyrrole on the turcasarin ring, once the ring is formed, can be functionalized using standard Friedal-Crafts alkylation or acylation, with the resulting compounds potentially being further manipulated to prepare functionalized turcasarins. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the

techniques disclosed in the examples which follow

represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

SYNTHESIS OF TERPYRROLE TETRAESTER 8 The synthesis of terpyrrole tetraester 8 (FIG. l)was carried out as follows. Dipyrroylbutanedione 7 (23.54 g., 40m mol), prepared according to the procedure of Johnson et al . (1992), ammonium acetate (vacuum dried at 50°C, 91. lg, 1 mol), acetic anhydride (32 mL, 0.33 mol) and glacial acetic acid (150 mL) were held at reflux under argon for 18 hours, poured into stirred ice water (400 mL), and stirred for 1 hour. The solid was

collected by filtration, washed with water, and dissolved in CH₂Cl₂. The CH₂Cl₂ solution was washed with water, diluted with methanol, and concentrated on the rotary evaporator until the volume was approximately 50 mL. The flask contents were cooled in ice for 1 hour and the solid collected by filtration, washed (MeOH, Et₂O), and dried to give the pure product as light yellow needles or powder (17.85 g, 31.3 mm mol, 78%).

Characterization Data for 8:

Mp 150-152 °C. ¹H NMR (300 MHz, CDCl₃) d = 0.99 (t, J_HH = 7.4 Hz, 6H, CH₂ CH₂CH₃) ; 1.36 (t, J_HH = 7.1 Hz , 6H,

OCH₂CH₃) ; 1.40 (t, J_HH = 7.1 Hz, 6H, OCH₂CH₃) ; 1.62

(sextet, J_HH = 7 Hz, 4H, CH₂CH₂CH₃) ; 3.06 (m, 4H,

CH₂CH₂CH₃) ; 4.37 (q, J_HH = 7.1 Hz, 8H, OCH₂CH3) 6.61 (d, J_HH = 2.5 Hz, 2H, pyrrole CH) ; 9.05 (bs, 2H, NH) ; 9.10 (bs, 1H, NH) . ¹³C NMR (75.5 MHz, CDCl³)d = 14.2, 14.3,

14.4, 24.5, 28.0, 60.5, 60.5, 109.4, 111.9, 119.0, 124.3, 132.2, 135.7, 161.3, 166.0 MS (CHCl₃) : 569 (100%) , 523 (83%), 477 (64%). HRMS : Calculated for C₃₀H₃₉N₃O₈: m/z = 569.2720. Found: 569.2720.

EXAMPLE 2

SYNTHESIS OF TERPYRROLE 9

The synthesis of terpyrrole 9 (FIG. 1) was carried out as follows. Terpyrrole tetraester 8 (5.7 g, 10 mmol), NaOH (10 g, 400 mmol), and ethylene glycol (100 mL) were placed in a three-necked 250 mL round bottomed flask equipped with an argon inlet, thermometer,

condenser, and stir bar, and brought to reflux under a gentle flow of argon through the flask. When the

solution reached 180°C, the flask contents were heated for another 30 minutes and cooled. When the solution reached 100-120°C, the flask contents were diluted with water (70 mL, degassed by the freeze-pump-thaw method) and allowed to cool to room temperature. The flask contents were diluted to 400 mL with ice water. The solid so obtained was separated by filtration under an argon blanket (an aluminum foil cover and a pipette blower works fine), washed with water, and dried in vaeuo to give the pure product as a tan to light green powder (2.62 g, 9.3 mmole, 93%).

Characterization Data for 9

M.P.: (sealed tube) 141-144 °C. ¹H NMR (300 MHz, DMSO- d₆) d = 0.91 (t, J_HH = 7.4 Hz, 6H, CH-CH-CH-) ; 1.53

(sextet, J_HH = 7.4 Hz, 4H, CH₂CH₂CH₃) ; 2.35 (t, J_HH = 7.4

Hz, 4H, CH₂CH₂CH₃); 6.13 (m, 2H, CHC (n-Pr) CHNH) ; 6.44 (s, 2H, CH); 10.47 (s, 2H, NH); 10.56 (s, 1H, NH) . ¹³C NMR (75.5 MHz, DMSO-d₆)d = 14.0, 23.9, 28.9, 103.1, 103.3, 114.1, 123.3, 125.5, 125.7. MS (CHCl₃): 281 (100%), 252 (25%), 238 (8%). HRMS: Calculated for C₁₈H₂₃N₃: m/z = 281.1892. Found: 281.1878. EXAMPLE 3

SYNTHESIS OF TURCASARIN 11

The synthesis of turcasarin 11 (FIG. 1) was carried out as follows. Bipyrrole 10 (72.5 mg, 2.67 × 10^-4 mol) was dissolved in 20 mL absolute ethanol with the aid of a heat gun. CHCl₃ was added (500 mL), followed by

terpyrrole 9 (74.9 mg, 2.67 × 10^-4 mol). HCl gas was bubbled into the stirred reaction mixture for five

minutes. The reaction was then stirred under an argon blanket for four hours. The CHCl₃ was removed using a rotary evaporator. The ethanolic residue was treated with concentrated aqueous NaHCO₃ until effervescence ceased, and then extracted into chloroform in a

separatory funnel. The organic layer was separated off, dried over Na₂SO₄, and taken to dryness in vaeuo. The residue was taken up in a minimum amount of CH₂Cl₂, and NH₃ gas bubbled into the solution for one minute. The resulting solution was purified chromatographically on a pre-basified 2 cm × 20 cm silica column (achieved by bubbling NH₃ through a CH₂Cl₂-silica slurry before

packing). The first major purple band was collected using CH₂Cl₂ as the eluent placed in a separatory funnel, and washed first with water (2 × 25 mL) and then 1N HCl (3x 25 mL). The organic layer was then separated, dried over Na₂SO₄, and taken to dryness in vaeuo .

Recrystallization from CH₂Cl₂ layered with hexanes gives pure 11 as shiny green metallic crystals (62.9 mg, 5.34 × 10^-5 mol, 20%).

Characterization data for 11:

M.P.: dec. above 215 °C; ¹H NMR (500 MHz, CDCl₃): d = 0.99 (t, J _HH = 7 Hz, 6H, CH-CH-), 1.05 (t, J _HH = 7 Hz, 6H, CH₂CH₃), 1.08 (t, J_HH= 7 Hz, 6H, CH₂CH₂CH₃), 1.27 (t, J _HH = 7 Hz, 6H, CH₂CH₂CH₃), 1.77 (m, 2H, CH₂CH₂CH₃), 1.85 (s, 6H, CH₃), 1.89 (m, 2H, CH₂CH₂CH₃), 2.06 (s, 6H, CH₃), 2.10 (m, 2H, CH₂CH₂CH₃), 2.19 (m, 2H, CH₂CH₂CH₃), 2.25 (q, J_HH = 7 Hz, 4H, CH₂CH₃), 2.36 (m, 4H, CH₂CH₃), 2.64 (m, 2H, CH₂CH₂CH₃) , 2.87 (m, 2H, CH₂CH₂CH₃), 2.89 (m, 2H,

CH₂CH₂CH₃), 3.13 (m, 2H, CH₂CH₂CH₃), 6.73 (s, 2H, meso-H), 6.84 (s, 2H, meso-H), 7.00 (s, 2H, pyrrole CH), 7.65 (bs, 2H, pyrrole CH), 8.68 (s, 2H, pyrrole CH), 9.73 (bs, 2H, pyrrole CH), 14.32 (s, 2H, NH), 14.56 (s, 2H, NH), 14.75 (s, 2H, NH), 15.37 (s, 2H, NH), 15.74 (s, 2H, NH); ¹³C NMR (75.5 MHz, CDCl₃): d = 11.4, 11.5, 14.1, 14.4, 14.9, 15.0, 18.0, 18.2, 23.0, 23.2, 28.2, 28.9, 119.1, 119.7, 121.4, 122.8, 123.7, 124.7, 125.1, 127.0, 128.6, 132.7, 133.0, 133.9, 136.4, 137.2, 137.6, 139.6, 141.6, 143.1, 147.8, 148.17, 149.0, 151.3; UV/vis (CH₂Cl₂): l_-max [nm] (e) = 369 (46 500), 399 (49 000), 445 (51 500), 642 (312 500); HRMS (FAB): Calculated for C₆₈H₇₉N₁₀: m/z =

1035.6489. Found: 1035.6505 ((M + 1)⁺ - 4 HCl);

Elemental Analysis: Calculated for C₆₈H₈₂Cl₄N₁₀●H₂O: C 68.10, H 7.06, N 11.68, Cl 11.82. Found: C 68.55, H 7.08, N 11.55, Cl 11.79.

Crystallographic summary for 11 (C₆₈H₇₄N₁₀)⁴⁺ (Cl-)₄ - 3CH₂Cl₂ - 3/2 (C₆H₁₄), Mr = 1557.29; crystallized as dark green blocks from methylene chloride layered with

n-hexane in the triclinic, space group P\¦o(1,-) (no. 2), with a = 14.221(6), b = 15.374(3), c = 20.531(5)A, a =

100.03(2), b - 101.71(3), g = 106.23(3)°, V = 4091(2) ų, r_calc = 1.26 g-cm^-3 for Z = 2, F(000) = 1642, m (Mo Ka) = 3.888 cm^-1, 1 = 0.71073 A. FIG. 6 shows top and side views of turcasarin 11, the views derived from the crystallographic data. Data were collected on a Nicolet R3 diffractometer at -90°C, equipped with a Nicolet LT-2 low-temperature device, using the omega scan technique at 5 - 12°/min out to a 2q limit of 45°. A total of 12144 reflections were collected, of which 10699 were unique. The R for averaging symmetry equivalent reflections was equal to 0.034. Data were corrected for L_p effects and decay but not for absorption. The structure was solved by the direct methods and refined by full-matrix least- squares using SHELXTL-Plus (Sheldrick, 1991). The hydrogen atoms were calculated in ideal positions, with Uiso set to 1.2×U_eq of the relevant atom. One molecule of n-hexane and one of methylene chloride are disordered. The tetrahydrochloric acid salt of 11, (C₆₈H₇₄N₁₀)⁺⁴ (Cl-)₄ - 3Ch₂Cl₂ - 3/2 (C₆C₁₄), Mr = 1557.29, had a triclinic, space group P\¦o(1,-) (no. 2), with a = 14.221(6), b = 15.374(3), c = 20.531(5)A, a = 100.03(2), b = 101.71(3), g = 106.23(3)° V = 4091 (2)A³ r_calc = 1.26 g-cm^-3 for Z = 2, F(000) = 1642, m (Mo Ka) = 3.888 cm^-3, 1 = 0.71073 A. One molecule of n-hexane and one of methylene chloride are disordered within the lattice. A total of 853 parameters were refined in blocks to a final R - 0.0824, wR = 0.100 and a goodness-of-fit = 2.607 using 4869 reflections having F > 4s(F).

In accord with what was found in the solution phase NMR experiments, the tetraprotonated form of turcasarin 11 is found to adopt a nearly C₂ symmetric twisted

"figure-eight" conformation in the solid state, and not an open circular conformation. This twist conformation serves to define two enantiomers (both of which are seen in the X-ray structure) such that the molecular geometry of 11 in the solid state is closer to that of the

"twisted ribbons" in FIG. 4, rather than that of the more open circle-like "loop."

As can be seen from an inspection of FIG. 4, the twist observed in the solid state also serves to define two pentaphyrin-like "hemi-macrocycles" as well as found smaller "pockets." Occupying each of these latter pockets are two chloride anions held within hydrogen bonding distance to the nitrogens. These hydrogen bonding interactions, as implied above, are currently considered to account for the slow conformational

interconversion observed at room temperature in solution on the NMR time scale. However, it is of interest to note that at the point of "ribbon crossing" the two pyrrole-containing π systems are separated from one another by a van der Waals distance of 3.268 A.

Turcasarin 11 may be considered as being a 40 TΓ- electron annulene. Thus, turcasarin should not be aromatic as predicted by the (4n + 2) rule for aromatic systems. However, it displays a Soret-like absorption in the visible portion of the electronic spectrum that is considerably red-shifted compared to that of the

porphyrins. On the other hand, no evidence of ring current effects are seen for the pyrrolic proton

resonances in the proton NMR spectrum. Thus, 11 is considered as being π delocalized, but not aromatic.

NMR studies were also used to obtain solution state structural information. The ¹³C NMR spectrum shows, for instance, apparent C₂ symmetry in the macrocycle frame as judged by the number of observable peaks. The ¹H NMR spectrum is also consistent with C symmetry as judged by the number of signals in the interior NH and exterior b- pyrrolic and meso-proton region. The signals in thealkyl region, on the other hand, proved more complex, with no clear assignment of symmetry or structure being possible from immediate inspection. In fact, complex splitting patterns for the methylene protons of the alkyl sidechains were observed. The complex splitting patterns mentioned above lead to the suggestion that the macrocycle may exist in two limiting conformations. These two conformations result from a twist along a long axis of the molecule as shown in FIG. 4. Turcasarin 11 could therefore exist as a pair of enantiomers. It would thus be dissymmetric even though it possesses no formal stereogenic centers. Such a postulate of conformational chirality, even though unexpected for a fully conjugated macrocycle, would, of course, account for the observed complex splitting patterns which would arise as the result of an imposed diastereotopicity on the alkyl side chains.

Definitive ¹H NMR peak assignments were made by COSY (FIG. 5) and NOESY. The NOESY spectrum also showed significant exchange crosspeaks in all regions indicating that the two enantiomers can readily interconvert, at least on the time scale of such experiments. However, variable temperature ¹H NMR (CDCl₃, 50°C to -50°C), by virtue of very little change in the chemical shifts, indicated that this interconversion is slow on the NMR time scale at room temperature. This slow

interconversion could reflect conformational

stabilization imposed by the presumably strong hydrogen bonding interactions between the macrocycle and the chloride anions observed in the solid state. Additionally, the data indicates that the rate of interconversion between the two limiting enantiomeric figure-eight conformations is slow on the NMR time scale at room temperature. This slow interconversion is considered to reflect the presumably strong hydrogen bonding interactions found in the solid state, where the four chloride counter ions are observed to be in intimate hydrogen bonding contact with certain pyrrole NH groups of the protonated with four hydrogens macrocycle. The present data demonstrate that conjugated, polypyrrolic macrocycles need not be flat (or near-flat) in their overall three dimensional structure but rather can adopt conformations that make them potentially chiral. EXAMPLE 4

SYNTHESIS OF THE BIS-URANYL CHELATE OF TURCASARIN 11

In a 25 mL round-bottomed flask equipped with an argon inlet and a magnetic stirrer, turcasarin 11

hydrochloride (6.2 mg, 5.25 × 10^-6 mol) was dissolved in 10 mL 2-propanol. To the resulting blue solution, pyridine (5 mL) was added followed by UO₂(OAc)₂-2H₂O (20 mg, excess). The blue mixture was stirred at reflux under an argon blanket for three hours. The solvent was removed in vaeuo . The residue was taken up in CH₂Cl₂ and purified chromatographically (silica gel, 3 × 15 cm, CH₂CL₂ as eluent). The only band (green) to elute was collected, and the solvent was removed in vaeuo to afford a green solid. This solid can be recrystallized from diffusion of isopropyl ether into a CH₂Cl₂ solution of 2 (or from hexanes layered on a CHCl₃ solution of 2) to afford fine green needles. Yield: 5.7 mg (69%). M.p.: dec. > 250°C; H NMR (300 MHz, CD₂Cl₂) δ 0.38 (t, J = 6.4 Hz, 6H, CH₂CH₂CH₃) , 1.33 (t, J = 7.3 Hz, 6H, CH₂CH-CH₃), 1.51 (s, H₂O) , 1.66 (t, J = 7.4 Hz, 6H, CH-CH-CH-), 1.86 (t, J = 7.4 Hz, 6H, CH₂CH₂CH₃) , 2.09 (m, 4H, CH₂CH₂CH₃), 2.26 (m, 8H, CH₂CH₂CH₃ & CH₂CH₂CH₃) , 2.80 (m, 4H,

CH₂CH₂CH₃), 3.35 (s, 6H, CH₃), 3.47 (m, 4H, CH₂CH₃), 3.59 (s, 6H, CH₃), 3.84 (m, 4H, CH₂CH₃), 4.36 (s, 2H, pyrrole-CH), 4.70 (d, J = 4 Hz., 2H, pyrrole-CH), 7.34 (d, J = 4.2 Hz., 2H, pyrrole-CH), 8.15 (s, 2H, pyrrole-CH), 8.21 (s, 2H, pyrrole-CH), 9.29 (s, 2H, NH); UV/vis (CH₂Cl₂) λ_max 445.5, 681.5 nm; MS (FAB) m/e 1570; HRMS (FAB) m/e calc'd. for C₆₈H₇₂N₁₀O₄ ²³⁸U₂; 1568.6754, found: 1568.6754. The H NMR spectrum of complex appears similar to that of the parent compound 11, including diastereotopic

splitting patterns in the alkyl region. This may suggest a coordination mode in which the "figure-eight"

macrocyclic frame observed in the metal-free ligand is retained more or less unchanged in the case of this particular metallo-adduct. Characterization data: UV/vis (CH₂Cl₂): λ_max [nm] = 445, 682; LRMS (FAB): Calculated for C₆₈H₇₄N₁₀U₂O₄; m/z = 1570 Found: 1570; ¹H NMR spectrum: Similar to that of the parent compound 11, including diasteroetopic splitting patterns in the alkyl region. This example demonstrates the use of turcasarins as metal ion chelating agents and particularly as a chelating agent of uranyl cations. It is contemplated that further chelates may be used as photodynamic agents, for example, chelates with diamagnetic lanthanide cations, such as La(III), Lu(III) or In(III).

EXAMPLE 5

SYNTHESIS OF TURCASARIN HAVING

DIFFERENT SUBSTITUENTS THAN TURCASARIN 11

15,20,37,42-Tetraethyl-2,11,16,19,24,33,38,41-octamethylturcasarin has also been prepared. 4,4'-Diethyl-5,5'-diformyl-3,3'dimethyl-2,2'bipyrrole (71.6 mg., 0.00026 mol) was dissolved in 20 mL absolute ethanol with the aide of a heat gun. CHCL₃ was added ( 500 mL) , followed by 2 , 5 -bis (4 -methyl-2 -pyrrolyl ) pyrrole ( 59 . 2 mg . , 0 . 000263 mol ) . HCl gas was bubbled into the stirred reaction mixture for five minutes . The reaction was then stirred under an argon blanket for four hours . The CHCl₃ was removed using a rotary evaporator . The ethanolic residue was treated with concentrated aqueous NaHCO₃ until effervescence ceased and the extracted into

chloroform in a separatory funnel. The organic layer was separated off, dried over Na₂SO₄ and taken to dryness in vaeuo. The residue was taken up in a minimum amount of methylene chloride and ammonia gas was bubbled into the solution for one minute. The resulting solution was purified chromatographically on a pre-basified 2 × 20 centimeter silica gel column (achieved by bubbling ammonia through a methylene chloride-silica slurry before packing). The first major purple band using methylene chloride as the eluent was collected, placed in a separatory funnel, and washed first with water (2 x 25 mL) and then 1N HCl (3 × 25 mL). The organic layer was separated off, dried over Na₂SO₄, and taken to dryness in vaeuo. Yield: 16.1 mg (11.5%). The ¹H NMR spectrum of this newer system, while retaining spectral features characteristic of the NH, b-pyrrolic and meso proton resonances, remained essentially unchanged relative to 11, and proved far less complex in the alkyl region.

Characterization data: ¹H NMR (300 MHz, CDC1₃) : d = 1.03 (t, J_HH = 7 Hz, 6H, CH₂CH₂), 1.09 (t, J_HH = 7 Hz, 6H,

CH₂CH₃), 1.67 (s, H₂O), 1.90 (s, 6H, CH₃), 2.09 (s, 6H, CH₃), 2.33 (q, J_HH = 7 Hz, 4H, CH₂CH₃), 2.39 (q, J_HH = 7 Hz, 4H, CH₂CH₃) , 2.45 (s, 6H, CH₃), 2.80 (s, 6H, CH₃), 6.76 (s, 2H, meso-H), 6.95 (s, 2H, meso-H), 7.04 (s, 2H, pyrrole CH), 7.62 (d, 2H, pyrrole CH), 8.60 (s, 2H, pyrrole CH), 9.68 (d, 2H, pyrrole CH), 14.23 (s, 2H, NH), 14.44 (S, 2H, NH), 14.67 (s, 2H, NH), 15.21 (s, 2H, NH), 15.58 (s, 2H, NH); UV/vis (CH₂Cl₂): l_max = 644 nm; HRMS (FAB): Calculated for C₆₀H₆₃N₁₀ :m/z = 923.5237. Found: 923.5252 ((M + 1)⁺ - 4 HCl).

Using the procedure of this example, it is

contemplated that compounds of formula I may be prepared where R¹, R² or R³ is hydroxy substituted methyl, ethyl, or propyl; carboxy or ester substituted methyl, ethyl or propyl; polyglycol substituted methyl, ethyl, or propyl, by employing the appropriate substituted bipyrroles and terpyrroles. EXAMPLE 6

TURCASARINS AS BINDING AGENTS

The turcasarins may be used as receptors for anions, cations or neutral molecules provided the turcasarin is of appropriate charge. For instance, anionic turcasarin may be prepared for use as a metal cation chelant by removing protons from the secondary amine groups in the macrocycle in organic solution using strong bases such as butyl lithium.

Due to the presence of four chloride anion binding pockets in the tetraprotonated form of 11, it is expected that turcasarin, in analogy to several other expanded porphyrins, could serve as an anion binding and transport agent under solution phase conditions. EXAMPLE 7

SYNTHESIS OF SUBSTITUTED TURCASARINS AND FURTHER ANALOGUES

It will also be apparent to one of skill in the art that many turcasarins may be obtained within the context of this synthetic methodology. For example, it is also envisioned that different combinations of bipyrroles and terpyrroles may be employed in the condensation step to produce various analogues of 11.

A preliminary example concerns the use of

5,5'-diformyl-4,4'-dipropyl-2,2'-bipyrrole as a starting material for use in reaction sequences in accordance with the present invention. This synthetic process involves reaction with compound 9 in a condensation which will produce an analogue of compound 11, corresponding to the turcasarin of Formula I wherein R¹ and R² are propyl, and R³ and R⁴ is hydrogen. Accordingly, it is contemplated that a variety of approaches may be employed, in accordance with the present invention, to prepare systems bearing a wide variety of alkyl and/or aryl substituents in the meso and/or β-pyrrolic positions.

For example, meso substituted compounds may be prepared to test the extent to which the presence of different groups, such as, for example, 4'-phenyl and/or 2'-phenyl donating groups may augment effective nitrogen lone pair basicity and/or enhance higher pH phosphate transport capability. Extensions to systems bearing two (or more) meso substituents are also contemplated within the scope of the invention. In any event, it is

important to appreciate that adding further meso

substituents will, in all likelihood, induce substantial distortions of the macrocycle. Thus, these syntheses should provide compounds that will allow steric effects to be assessed. It is contemplated that transport and pK_a' tests will be conducted to determine the effects of the various substitutions. It is contemplated that phenyl-bearing turcasarins may be prepared by a "direct insertion" procedure. This procedure (a specific example of which is shown in Scheme A) involves the condensation of a bis-α-free bipyrrole (e.g., 25) with an α, ω-free bis(pyrrolyl)-pyrrole (e.g., 26) in the presence of benzaldehyde or substituted benzaldehyde under turcasarin forming conditions, as described herein, which will afford bisarylturcasarin 27. In Scheme A, a range of other macrocyclic and non- macrocyclic products may also be obtained.

Other syntheses can be envisioned such as, for instance, the synthesis shown in Scheme B in which an α-free, ω-protected bipyrrole (e.g., structure 28 in Scheme B), is co-condensed with an α- free terpyrrole (e.g., 29) in the presence of an aromatic aldehyde. The benzyl ester of the resulting terpyrrolyl-bipyrrole 30 can then be selectively cleaved under standard

debenzylation conditions and decarboxylated with

trifluoroacetic acid to give the α-free terpyrrolyl -bipyrrole, structure 31. Acid catalyzed condensation of terpyrrolyl-bipyrrole 31 with another molecule of α-free terpyrrole 29 in the presence of an aromatic aldehyde yields an ethyl ester-protected diaryl, bis(terpyrrolyl)bipyrrole 32, which can be deprotected to form the α, ω- free bis(terpyrrolyl)-bipyrrole 33. Acid catalyzed co-condensation of 33 with an a, ω-free bipyrrole (e.g., 34) in the presence of an aromatic aldehyde, under

turcasarin-forming conditions such as those described in Example 3, affords a turcasarin with each of its four "meso" positions substituted with aryl groups (e.g., 27).

³

It will, of course, be understood that in syntheses such as those exemplified in Schemes A and B,

substituents in the starting compounds, such as those groups represented by R^1-3 may be varied as desired.

Thus, by using the appropriately substituted starting units, a turcasarin product may be produced in which groups R^1-3 (as represented by structure 27) may be substituted with any of the R^1-3 groups listed above. Extensions of known methods may also be used that would allow one of skill in the art, in light of the present disclosure, to prepare functionalized turcasarins bearing one or more non-alkyl substituents in the β positions. One procedure for entry into such systems involves the preparation of carboxyalkyl substituted systems, for example, compounds bearing substituents such as -(CH₂)_n-CO₂H, and then further elaborating these to form the corresponding hydroxyalkyl, aminoalkyl, thiol, sulfonate, ether, amide, ester, or formylalkyl

derivatives.

In addition to the direct conversions described above, it is important to appreciate that carboxyalkyl substituted turcasarins may also be used as the basis for obtaining other, more complex, functionalized systems. For instance, the carboxylic acid group of the

carboxyalkyl-bearing turcasarins may be activated using standard reagents (such as, for example, thionyl chloride or DCC) and used to prepare either ester- or amide-linked conjugates.

Said conjugates could include compounds that contain one or more nucleic acid base ("nucleobase") or sugar ("saccharide") subunits, as is described in detail in the following Examples. For example, in such syntheses, one may condense a protected aminoalkyl nucleobase, for instance, the known material, 1-(2-aminoethyl)-4- triphenylmethyl)amino]-pyrimidine-2-one, with the

activated turcasarin carboxylic acid and subsequently effect nucleobase deprotection. Similarly, one may employ an amino-bearing protected sugar, such as per-O-acetylated glucosamine, and subsequent deprotection. In addition, one may use such a conjugation approach to prepare complex ethers, esters, or amides where the ether, ester, or amide linkages are used to append a wide variety of polyfunctional substituted alcohol and/or amine fragments on to the turcasarin periphery.

The carboxyalkyl substituted turcasarins may be prepared by several routes. For example, as shown in Scheme C, condensation of bipyrrole 36 with terpyrrole 37 may be used to obtain, following debenzylation,

bis (dicarboxyterpyrrolyl) bipyrrole 38b. Following condensation of compound 38b with bipyrrole 39, under turcasarin-forming conditions such as those described in Example 3, there will be provided turcasarin 40 bearing four carboxyalkyl substituents.

Secondly, in retrosynthetic analogy to the

preparation of 3,8,17,22-tetraethyl-12-(carboxyethyl)-2,7,13,18,23-pentamethylsapphyrin, benzyl 3,5-dimethyl-4- (methoxycarbonylethyl)-pyrrole-2-carboxylate 41 may be converted, as shown in reaction Scheme D, to its

corresponding bipyrrole 44a (3,3'-bis(methoxycarbonylethyl)-5,5'-bis(benzyloxycarbonyl)-4,4'-dimethyl-2,2'-bipyrrole) via sulfuryl chloride oxidation to acid 42, followed by standard iodination (43), and copper bronze mediated Ullman coupling.

Following standard debenzylation to produce 44b and Clezy formulation with trifluoroacetic acid (TFA) and

triethylorthoformate, condensation of the resulting bipyrrole 45 (3,3'-bis(methoxycarbonylethyl)-5,5'- diformyl-4,4'-dimethyl-2,2'-bipyrrole) with the

terpyrrole 46 of reaction Scheme E under turcasarin- forming conditions will provide a turcasarin containing at least four carboxyalkyl substituents protected as their corresponding methyl esters, structure 47.

EXAMPLE 8

SYNTHESIS OF TURCASARIN-NUCLEOBASE CONJUGATES

It is contemplated that turcasarin-based dibasic phosphate chelators may be modified so as to obtain ditopic binding systems that display high inherent specificity for a given purine or pyrimidine derived nucleotide. This may be achieved by adding a synthetic appendage of a nucleobase moiety. For example, the known aminobutyl cytosine derivative may be employed with an acid-catalyzed detritylation procedure.

Alternatively, the mode of base attachment may be varied, for example, at the level of coupling, protecting group, precursors, and the length, nature, and

orientation of any linking groups. The effects of such variations on yield, selective binding, and other

properties of the resultant turcasarin-based molecule, such as, for example, effective through-membrane

transport capability may then be determined and any adjustments made accordingly. For example, the nature of R groups may be changed, or secondary amides converted to tertiary amides. It is contemplated nucleobase "chelation" may be achieved via complementary Watson-Crick type base-pairing interactions. These compounds should display base-selective transport capability. This may be specifically examined by various structural, static binding, and dynamic transport analyses. In particular, it will be determined whether the cytosine-for-guanine selectivity holds in the case of suitably designed synthetic

conjugates. In addition, it will be tested whether this same base-pairing approach suffices to engender

nucleobase selectivity in the case of adenine-thymine pairing. As an extension of the above analyses, doubly functionalized systems may be synthesized for use in the selective binding of dinucleotides, some of which have interesting antiviral properties, as well as for the recognition and transport of mononucleotides. In the latter case, the possible combination of both

Watson-Crick and Hoogsteen type base-pairing interactions could confer a degree of specificity not available using simpler systems. To the extent this proves true, it is possible that the doubly functionalized turcasarin system could serve as a viable antiviral adjuvant, capable not only of binding and solubilizing the phosphate portion of a nucleotide monophosphate, but also of effecting its selective through-membrane transport at or near physiologic pH.

In view of the successful studies carried out in the sapphyrin series, (for example, it was found that the attachment of a nucleobase to a sapphyrin core greatly enhanced the nucleotide recognition selectivity for transport), it is expected that the functionalization of turcasarins which because of their larger core size and increased basicity are inherently much better for anion recognition and phosphate anion chelation than

sapphyrins, will lead to superior systems relative to any produced to date.

Reaction Schemes F through I represent examples of the synthesis of turcasarin nucleobase conjugates. The synthetic methodology represented in these reaction schemes may be straightforwardly adapted for the

synthesis of any given turcasarin nucleobase conjugate by employing the desired starting materials. Groups R^1-3 are as defined hereinabove in Schemes F through I.

An example of the preparation of a turcasarin nucleobase conjugate is shown in reaction Scheme F. Here, a tetracarboxyalkyl bearing turcasarin, tetra (ethoxycarbonylethyl)-turcasarin 40 (as prepared hereinabove in Scheme C) is saponified to its diacid form 49 by treatment with a 1:1 mixture of HCl and

trifluoroacetic acid. DCC coupling of turcasarin diacid 49 with trityl protected aminoethyl cytosine 50 in methylene chloride at 0°C followed by deprotection with TFA affords the amide linked bis (aminoethyl) cytosine turcasarin conjugate 51.

Similarly, as shown in Scheme G, the amide linked bis (aminoethyl) guanosine turcasarin conjugate 55 is prepared by DDC coupling of turcasarin diacid 49 with benzoyl-protected aminoethyl guanosine 54 in DMF at 0°C followed by deprotection with TFA.

In another example of a dicarboxyalkyl bearing turcasarin, as shown in Scheme H, the

tetra (methoxycarbonylethyl) -turcasarin 47 is saponified to diacid 57 by treatment with a 1:1 mixture of HCl and trifluoroacetic acid. DCC coupling of turcasarin diacid 57 with trityl-protected aminoethyl cytosine 58 in methylene chloride at 0°C followed by deprotection with TFA affords the amide linked bis (aminoethyl) cytosine turcasarin conjugate 59.

The amide linked bis (aminoethyl) guanosine turcasarin conjugate 63 of Scheme I is prepared by DDC coupling of turcasarin diacid 61 with benzoyl-protected aminoethyl guanosine 62 in DMF at 0°C followed by deprotection with TFA.

EXAMPLE 9

SYNTHESIS OF TURCASARIN-SACCHARIDE CONJUGATES

Turcasarin derivatives or conjugates including one or more saccharide units may be prepared according to the synthetic methodology described hereinbelow. The sugar units (represented originally by structures 65 and 75) in Schemes J through O are intended to represent any

individual sugar or sugar derivative, such as those set forth in Table 2, or polymers thereof, and include modified sugars, such as methyl, amino, and phosphate sugars, and D-, L-, α and β forms of said sugars.

The synthetic methodology represented in the

following reaction schemes may be straightforwardly adapted for the synthesis of any turcasarin saccharide compound as desired. Groups R^1-3 in Schemes J through O are as defined hereinabove. Referring now to Scheme J, amide-linked turcasarin saccharide conjugates such as those represented by structure 66b, are prepared by coupling diacid chloride substituted turcasarin 64 (prepared by treating its respective diacid turcasarin with thionyl chloride) with the acetoxy protected HBr salt of amino saccharide 65 in methylene chloride and pyridine, and deprotecting with KOH in methanol.

In the same fashion, the bis (acid

chloride) turcasarin 67 as shown in Scheme K is prepared by treating its respective diacid turcasarin, formed as shown in Scheme H, with thionyl chloride. The amide- linked bis(saccharide) turcasarin 68b may be prepared by coupling turcasarin diacid chloride 67 with the acetoxy protected HBr salt of amino saccharide 65 in methylene chloride and pyridine, followed by deprotection with KOH in methanol. To prepare ether-linked bis(saccharide)turcasarin conjugates, the dihydroxy functionalized turcasarins, such as those represented by structure 73b in Scheme L, may be prepared. For example, reduction of the methyl esters of macrocycle 69 with borane-THF to the

corresponding alcohols followed by protection with acetic anhydride will provide the acetoxy-protected diol 71a. Standard debenzylation to afford 71b followed by

condensation with diformyl bipyrrole 72, under

turcasarin-forming conditions such as those described in Example 1, will provide the acetoxy-protected dihydroxy turcasarin 73a. Deprotection of the hydroxyl groups is achieved by treatment with HCl in methanol to afford dihydroxyturcasarin 73b.

Dihydroxyturcasarins, formed as described above (Scheme L), can be coupled with acetoxy- and/or benzoyl-protected bromo-substituted saccharide units, such as structure 75 as shown in Scheme M, in methylene chloride with a catalyst such as silver triflate and barium carbonate. This results in the production of acetoxy- and/or benzoyl-protected bis (saccharide) turcasarin conjugates such as 76a in Scheme M. Treatment of the acetoxy and/or benzoyl protected

bis (saccharide) turcasarin 76a with KOH in methanol yields the corresponding deprotected bis(saccharide)turcasarin conjuaate 76b (Scheme M).

To prepare an ether linked bis(saccharide)turcasarin conjugate with the ether linkages in the beta positions of the residual bipyrrole units on the turcasarin

periphery, the appropriate acetoxy-protected diformyl bipyrrole is prepared as shown in Scheme N. Reduction of the methyl esters of bipyrrole 77 with borane-THF to the corresponding alcohols followed by protection with acetic anhydride provides the acetoxy-protected diol 79a.

Standard debenzylation affords diacid 79b, followed by Clezy formulation with TFA and triethylorthoformate to yield the acetoxy-protected bipyrrole 80. Condensation of bipyrrole 80 with diacid 81 (prepared in accordance with the procedure shown in Scheme C) under turcasarin-forming conditions will provide the acetoxy-protected dihydroxy turcasarin 82a. Deprotection of the hydroxyl groups is achieved by treatment with HCl in methanol to afford dihydroxyturcasarin 82b.

Referring now to Scheme O, the dihydroxyturcasarin thus formed can be coupled with acetoxy- and/or benzoyl-protected bromo-substituted saccharide units, such as structure 75, in methylene chloride with silver triflate and barium carbonate to afford the acetoxy- and/or benzoyl-protected bis(saccharide)turcasarin conjugate 84a. Treatment of the acetoxy and/or benzoyl protected bis(saccharide)turcasarin 84a with KOH in methanol will yield the corresponding deprotected

bis(saccharide)turcasarin conjugate 84b.

I

EXAMPLE 10

TURCASARIN AND TURCASARIN ANALOGUES AS CHELATING

RECEPTORS It is contemplated that the diprotonated turcasarin systems will act as effective receptors for a variety of anions. For instance, it is contemplated that

diprotonated turcasarin may bind both fluoride and phosphate anions in a strong and non-labile manner.

Furthermore, it is believed that the monoprotonated form of turcasarin will act as an effective carrier for the through-membrane transport of guanosine-5'- monophosphate in Pressman type model system. Thus, protonated turcasarins are proposed to be of use in molecular recognition.

Once a range of turcasarin analogues have been generated, as described herein in the foregoing detailed examples, the thermodynamics and kinetics of anion binding under a range of conditions and with an array of different anions may be determined. The structure and function of the most promising turcasarin compounds may then be optimized such that they bind either

phosphate-bearing nucleotides, or chloride ions, with high affinity and selectivity at neutral pH.

As a complement to structural studies, quantitative analyses may also be conducted. For example, the

relevant .pK ' values for various turcasarins may be determined by employing the methods previously used to determine the pK_a' values for sapphyrin and anthraphyrin. One should beware of possible "artifacts" arising from anion chelation, which can be avoided by using

non-chelating buffers and solvents. Such initial studies should be followed by studies in which the various pK_a' values are recorded in different solvents and in the presence of different anions such as hydrosulfate, bicarbonate, azide, cyanide, fluoride, bromide, and iodide, that are of biological relevance. Other

qualitative tests (e.g. UV/vis, FABMS) may also be used to reconfirm that the turcasarins do not form complexes under physiological conditions with Na⁺, K⁺, Ca²⁺, or other bio-cations. The reason for these latter studies is that such cation complex formation could preclude efficient anion binding and transport.

Once pK_a' values are recorded, a second set of quantitative analyses may be performed to determine the actual affinity constants for each and every relevant receptor-to-anionic interaction. Thus, for instance, K_S for [H₆Tur²⁺●GMP^2-] formation in a variety of solvent systems may be measured in a similar manner to that for the hydrohalide salts of sapphyrin and anthraphyrin.

Standard methods as quantitative UV/vis titrations and concentration dependent NMR chemical shift analyses may be used, along with more sophisticated techniques such as those involving static and time-resolved fluorescence. The latter methods offer considerable advantages and are particularly useful for measuring high affinity constants (i.e. those in the K_s ≥ 10⁶ M^-1 range). These

fluorescence-based methods require highly colored

materials with good singlet state emission

characteristics, but these criteria are clearly met by the turcasarins. Quantitative kinetic studies may also be carried out and used to determine whether the rate limiting step in GMP (or chloride) transport involves initial

receptor-anion complex formation, through-membrane carrier-complex diffusion, product release, and/or rate of carrier back-diffusion. On- and off-rates for complex formation may be measured, for example, by dynamic NMR, UV/vis, or time-resolved fluorescence, in, e.g., simple water-saturated dichloromethane solutions. More precise analyses of receptor-mediated transport may also be made, again with a mind to determining the dynamics of

complexation and decomplexation. The U-tube model system may be employed, and when appropriate, more sophisticated membrane analogues such as mixed phosphatidylcholine- cholesterol liposomes may be used. For the latter studies, it may prove most convenient to prepare nucleotide or halide encapsulating liposomes and then determine the kinetics of anion extrusion as a function of carrier concentration and/or external

solution pH. Here again, either UV/vis or time-resolved fluorescence analyses may be used. Here, it might prove necessary to add a specific fluorophore, such as

6-methoxy-N-(3-sulphopropyl)quinolinium (a halide

selective reagent) so as to be able to detect small quantities of any escaped anions.

EXAMPLE 11

TURCASARIN AND ANALOGUES AS CELLULAR ANION TRANSPORTERS

Using a H₂O-CH₂Cl₂-H₂O Pressman-type U-tube model system, it is believed turcasarin will be found to be capable of effecting through-membrane transport of GMP and other nucleotides at near-neutral pH (i.e. in the pH 6.0 to 6.5 regime). The effective transport by turcasarin, a larger, more basic system, may derive from lower in-core

NH⁺-to-NH⁺ repulsions. In any event, these properties make turcasarin an ideal candidate for use in the

delivery of phosphorylated compounds such as antivirals. The ability of turcasarin to effect nucleotide transport in a manner that is free of any chloride (or other halide) anion inhibition (even though this same material binds chloride anion quite effectively at very low pH and in the solid state) is particularly important.

Furthermore, it is contemplated that transport effected by this latter turcasarin carrier could be made somewhat nucleobase selective by adding the appropriate complementary TIPS derivative to the organic membrane phase. For example, in the presence of C-Tips

(triisopropylsilyl-protected cytidine), the rate of turcasarin-mediated GMP transport was observed to be substantially enhanced. It is also contemplated that it should be possible to effect base-specific phosphate (or phosphonate) entity transport under physiological

conditions and also through synthetic selection and thereby design turcasarin anion receptors that are selective for either phosphate-derived antivirals, or chloride anion, or both.

In further studies, those kinetic and thermodynamic factors that militate both for and against rapid,

selective transport of phosphorylated nucleotides and nucleotide analogues can be precisely determined. Both the use of simple U-tube and more elaborate liposomal test systems is contemplated.

EXAMPLE 12

USE OF TURCASARIN AND ANALOGUES AS THERAPEUTIC AGENTS Turcasarin compounds of the present invention are contemplated to be of use as anion transporters in various embodiments relating to human treatment. The turcasarins are particularly contemplated for use as delivery agents for antiviral compounds and may thus be employed to combat a variety of diseases including AIDS, herpes, hepatitis and measles. Turcasarin compounds optimized for chloride transport are also contemplated for use in the treatment of cystic fibrosis.

In developing the turcasarin compounds of the present invention for therapeutic use as anti-viral transporters, in vitro tests will first be conducted. These will follow protocols similar to those used earlier to screen the photodynamic antiviral activity of

sapphyrin and several other expanded porphyrins. In brief, a monolayer of Vero cells will be infected with HSV-1, coated with an overlay culture medium, and then exposed to various relative and absolute concentrations of both putative carrier and known active antiviral.

Then, following incubation at 37°C, adjuvant efficacy will be determined by counting the number of plaque forming units (PFU) obtained in the presence and absence of a given carrier.

Following such in vitro tests, the activity of promising turcasarin receptors will be followed-up, for example, in anti-HIV screens, and then in in vivo animal studies. These studies will be conducted according to the standard practice for such animal trials, the

execution of which will be known to those of skill in the art.

During the animal trial stage, the turcasarin compounds, whether used in antiviral delivery, or for chloride transport in cystic fibrosis treatment, may be modified further if required. They might, for instance, be modified to overcome poor water solubility or

susceptibility to in vivo degradation. Alternatively, if such problems occur, the turcasarins could be enveloped within a bio-compatible liposome (made, e.g. from

Cremophor) and then administered intravenously. Such an approach has previously resulted in good in vivo murine adenocarcinoma photodynamic tumor killing with a water- insoluble texaphyrin-type expanded porphyrin. The "Trojan Horse" method could also be employed to deliver the turcasarin antiviral carrier in vivo to the desired locus of biological activity. Here, the idea would be to use non-infectious viral membrane material to produce liposomes and then use these in turn as transport

vehicles to get the putative carrier to the site of cellular infectivity. Toxicity studies will also be carried out at this stage. The methods for determining both acute and chronic toxicity will be known to those of skill in the art. Available evidence indicates that turcasarins, like sapphyrins, will be relatively nontoxic. Toxicity can be investigated in relation to solubility, net charge at physiologic pH, and changes in appended β-pyrrolic and/or meso substituents.

Furthermore, the phosphate-binding turcasarin compounds of the present invention may act as receptors and transporters for other biologically important

molecules with negative charges, particularly,

polynucleic acids such as DNA, RNA and oligonucleotides. Another dimension to the invention, therefore, concerns the possibility of using turcasarins in the transport of DNA molecules, such as antisense DNA constructs, into cells for use in so-called gene therapy programs.

Normal cellular uptake of negatively-charged DNA is known to be limited. Current in vitro methods rely on severe cellular modifications which often cause excessive cell damage, and as a result, are not viable in vivo.

The "coating" of nucleic acid phosphate groups with turcasarins, thus rendering them suitable for diffusional uptake in vivo, is therefore very attractive and even has implications for chromosomal gene therapy. EXAMPLE 13

ANION EXCHANGE PROCEDURES Tetrahydrobromide salt of Turcasarin

In a 125 ml separatory funnel, turcasarin 11

hydrochloride (20.1 mg, 1.70 × 10^-5 mol) was dissolved in

50 ml dichloromethane. The deep green solution was washed with 10% aqueous NaOH(3 × 50 ml). Washing the resulting deep purple organic phase with 1N HBr afforded a deep green solution that was separated off and dried over anhydrous sodium sulfate. The solvent was removed with the aid of a rotary evaporator to afford the

tetrahydrobromide salt of turcasarin (23.1 mg, 1.70 × 10^-5 mol, 100%) as a gold colored solid with a metallic luster. UV/visλ_maχ (CH₂Cl₂):646.6 nm.

Tetraperchlorate salt of Turcasarin

In a 125 ml separatory funnel, turcasarin 11

hydrochloride (18.2 mg, 1.54 × 10^-5 mol) was dissolved in 50 ml dichloromethane. The deep green solution was washed with 10% aqueous NaOH(3 × 50 ml). Washing the resulting deep purple organic phase with 1N HClO₄

afforded a deep green solution that was separated off and dried over anhydrous sodium sulfate. The solvent was removed with the aid of a rotary evaporator to afford the tetraperchlorate salt of turcasarin (22.1 mg, 1.54 x 10^-5 mol, 100%) as a gold colored solid with a metallic luster. UV/visλ_maχ (CH₂Cl₂):636.8 nm.

Tetratrifluoroacetate salt of Turcasarin

In a 125 ml separatory funnel, turcasarin 11

hydrochloride (19.6 mg, 1.65 × 10^-5 mold) was dissolved in

50 ml dichloromethane. The deep green solution was washed with 10% aqueous NaOH(3 × 50 ml). The resulting deep purple organic phase was separated off and dried over anhydrous sodium sulfate. One drop of

trifluoroacetic acid (TFA) was added and the solution changed to a deep green color. The solvent was removed with the aid of a rotary evaporator to afford the

tetratrifluoroacetate salt of turcasarin (24.6 mg, 1.65 × 10^-3 mol, 100%) as a gold colored solid with a metallic luster. UV/visλ_maχ (CH₂Cl₂):564.5 nm and 628.5 nm.

The UV/visλ_maχ peak shifts of these examples

demonstrate that the anion(s) bound by turcasarin may be replaced by a different anion(s), thereby providing a method for the production of a variety of turcasarin anion complexes and demonstrating a property of

"exchanging" of anions.

EXAMPLE 14

ZINC CHELATION PROCEDURE

In a 50 ml round bottomed flask equipped with a magnetic stirrer and an argon inlet, turcasarin 11

hydrochloride (5.0 mg, 4.23 × 10^-6 mol) was dissolved in 10 ml anhydrous isopropyl alcohol. Pyridine (5 ml) was then added to the blue solution. (UV/visλ_maχ of the resulting purple (free base) solution in CH₂Cl₂:603.5 nm.) ZnCl₂ (10 mg, excess) was dissolved in 5 ml isopropyl alcohol and added to the stirred reaction mixture. The reaction was allowed to stir at room temperature

overnight. The resulting precipitate was collected via vacuum filtration and washed with isopropyl alcohol. The product was recrystallized from CH₂Cl₂ layered with n-hexane to afford gold needles. Yield: 3.3 mg.

UV/visλ_maχ(CH₂Cl₂):585.5 nm.

This example demonstrates that the turcasarins are useful as chelants for a variety of metals such as zinc. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES The following references, to the extent that they provide exemplary procedural or other details

supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Sessler, J.L. and Burrell, A.K., Top . Curr. Chem. , 161:177-273, 1991.

2. Knϋbel, G. and Franck, B. Angew. Chem. , 100:1203- 1211, 1988; Angew. Chem . In t . Ed . Engl . , 27:1170- 1172, 1988.

3. Beckmann, S. et al., Angew. Chem . Int . Ed . Engl . , 102:1439-1441, 1990 and 29:1395-1397, 1990. 4. Bell, T.W. et al., Pure & Appl . Chem. , 65:361-366, 1993. 5. Sessler, J.L. et al., Angew. Chem., 103:1018-1021, 1991; Angew. Chem. Int. Ed. Eng., 30:977-980, 1991.

6. Bell, T.W. et al., Pure Appl . Chem., 65:361-366,

1993.

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et al., Angew. Chem. Int. Ed. Engl., 102:1431-1434, 1990 and 29:1387-1390, 1990; j) Dolphin, D. et al., J. Am. Chem. Soc, 115:9301-9302, 1993; k) Carre, F.H. et al., Organomet, 12:2748-2486, 1993; 1)

Markl, G. and Striebl, U. , Angrew. Chem., 105:1387- 1389, 1993; Angew. Chem. Int. Ed. Engl., 32:1333- 1335, 1993; m) Sessler, J.L. et al., J. Am. Chem. Soc, 114:8306-8307, 1992; n) Hu, Z. et al., Tet. Lett., 34:1879-1882, 1993.

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13. U.S. Patent 5,159,065

14. U.S. Patent 5,120,411

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18. WO 94/09003

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Claims

CLAIMS :

1. A compound of formula I

wherein, individually in each occurrence, each of R^1-3 is a monoradical moiety of hydrogen, alkyl, alkyl halide, alkene, alkyne, aryl, amino, hydroxyl, alkoxy, carboxy, carboxamide, ester, amide, sulfonato, hydroxy substituted alkyl, glycol, polyglycol, alkoxy substituted alkyl, carboxy substituted alkyl, amino substituted alkyl, sulfonato substituted alkyl, ester substituted alkyl, amide substituted alkyl, substituted aryl, substituted alkyl, substituted ester, substituted ether, substituted amide, thiol, alkyl thiol, alkoxycarbonyl,

aryloxycarbonyl, aldehyde, ketone, carboxylic acid, phosphate, phosphate substituted alkyl, phosphonate, phosphonate substituted alkyl, sulfate, sulfate

substituted alkyl, combination thereof, or is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂, O, S, NH or NR⁴ or wherein A is a diradical moiety selected from any of the R^1-3 groups, oxy, sulfide, carbonyl, alkene, alkyne, alkyl halide, hydroxyalkyl, glycol, polyglycol, alkyl thiol, substituted alkyl, phosphate, phosphonate, sulfate, phosphate substituted alkyl, phosphonate

substituted alkyl, sulfate substituted alkyl, carboxy, carboxyamide, thiol-substituted carboxyamide, derivatized carboxyamide of formula CONR⁴ where R⁴ may be alkyl, alkyl halide, hydroxyalkyl, glycol, polyglycol, or alkyl thiol; wherein B independently in each occurrence is a nucleobase, saccharide, nucleotide, expanded porphyrin, steroid, amino acid, peptide, polypeptide, turcasarin or polymeric or solid support matrix, wherein n is 0-10, wherein m is 0-10, and wherein the total number of carbon atoms in each of R^1-4 is less than or equal to 20, or salt thereof.

2. The compound of claim 1, wherein each R¹ is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂, and B is a nucleobase.

3. The compound of claim 2, wherein the nucleobase is AraC, erythrohydroxynonyladenine, AraAMP, Floxuridine, Azaribine, Fluorouracil (5-FU), Azathioprine,

Idoxuridine, Azauridine, LOMPD, AZT, Mercaptopurine, Bromodeoxyuridine, PFA, Chlorodeoxyuridine, Thioguanine, Cytarabine, Trifluoromethylde-oxyuridine, Deoxyuridine, Xylo-GMP, Dideoxylnosine DDL

4. The compound of claim 2, wherein the nucleobase is any of the nucleobases shown in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and 7F.

5. The compound of claim 1, wherein each R¹ is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂, and B is an nucleotide.

6. The compound of claim 5, wherein the nucleotide is an oligonucleotide or a polynucleotide.

7. The compound of claim 1, wherein each R¹ is hydroxy substituted alkyl.

8. The compound of claim 1, wherein each R¹ is carboxy substituted alkyl.

9. The compound of claim 1, wherein each R¹ is a

saccharide.

10. The compound of claim 1, further defined as being the salt of hydrogen chloride.

11. The compound of claim 1, further defined as having structure 11 of FIG. 1.

12. The compound of claim 1, wherein each R² is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂, and B is a nucleobase.

13. The compound of claim 12, wherein the nucleobase is AraC, erythrohydroxynonyladenine, AraAMP, Floxuridine, Azaribine, Fluorouracil (5-FU), Azathioprine,

Idoxuridine, Azauridine, LOMPD, AZT, Mercaptopurine, Bromodeoxyuridine, PFA, Chlorodeoxyuridine, Thioguanine, Cytarabine, Trifluoromethylde-oxyuridine, Deoxyuridine, Xylo GMP, Dideoxylnosine DDI.

14. The compound of claim 12, wherein the nucleobase is any of the nucleobases shown in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F.

15. The compound of claim 1, wherein each R² is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂, and B is an nucleotide.

16. The compound of claim 15, wherein the nucleotide is an oligonucleotide or a polynucleotide.

17. The compound of claim 1, wherein each R² is hydroxy substituted alkyl.

18. The compound of claim 1, wherein each R² is carboxy substituted alkyl.

19. The compound of claim 1, wherein each R² is a saccharide.

20. The compound of claim 1, wherein each R³ is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂, and B is a nucleobase.

21. The compound of claim 20, wherein the nucleobase is AraC, erythrohydroxynonyladenine, AraAMP, Floxuridine, Azaribine, Fluorouracil (5-FU), Azathioprine,

Idoxuridine, Azauridine, LOMPD, AZT, Mercaptopurine, Bromodeoxyuridine, PFA, Chlorodeoxyuridine, Thioguanine, Cytarabine, Trifluoromethylde-oxyuridine, Deoxyuridine, Xylo-GMP, Dideoxylnosine DDL

22. The compound of claim 20, wherein the nucleobase is any of the nucleobases shown in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F.

23. The compound of claim 1, wherein each R³ is of the formula (CH₂)_m-A-(CH₂)_n-B wherein A is CH₂ , and B is an nucleotide.

24. The compound of claim 23, wherein the nucleotide is an oligonucleotide or a polynucleotide.

25. The compound of claim 1, wherein each R³ is hydroxy substituted alkyl.

26. The compound of claim 1, wherein each R³ is carboxy substituted alkyl.

27. The compound of claim 1, wherein each R³ is a saccharide.

28. The compound of claim 1, wherein R^1-3 are each alkyl

29. The compound of claim 1, wherein the total number of carbon atoms in each of R^1-4 is less than or equal to 10.

30. A metal chelate of the compound of claim 1.

31. The metal chelate of claim 30, further defined as being a uranyl chelate.

32. The use of the metal chelate of claim 30 in the preparation of a pharmaceutical composition for use as a radiation sensitizer.

33. The use of the metal chelate of claim 31 in the preparation of a pharmaceutical composition for use as a radiation sensitizer.

34. The use of the compound of claim 1 in the

preparation of a pharmaceutical composition for use as a radiation sensitizer.

35. The use of the compound of claim 1 as a metal chelating agent.

36. A process for the production of turcasarins, comprising: condensing a bipyrrole and a terpyrrole in the presence of an acid under conditions such that a turcasarin is formed.

37. The process of claim 36, wherein the bipyrrole is of formula II:

wherein Z is H or -CHO and the terpyrrole is of formula III:

wherein for both bipyrrole and terpyrrole, individually in each occurrence, each of R^1-3 is a monoradical moiety of hydrogen, alkyl, alkyl halide, alkene, alkyne, aryl, amino, hydroxyl, alkoxy, carboxy, carboxamide, ester, amide, sulfonato, hydroxy substituted alkyl, glycol, polyglycol, alkoxy substituted alkyl, carboxy substituted alkyl, amino substituted alkyl, sulfonato substituted alkyl, ester substituted alkyl, amide substituted alkyl, substituted aryl, substituted alkyl, substituted ester, substituted ether, substituted amide, thiol, alkyl thiol, alkoxycarbonyl, aryloxycarbonyl, aldehyde, ketone, carboxylic acid, phosphate, phosphate substituted alkyl, phosphonate, phosphonate substituted alkyl, sulfate, sulfate substituted alkyl, combination thereof or is of the formula (CH₂)_m-A- (CH₂)_n-B wherein A is CH₂, O, S, NH or NR⁴ or wherein A is a diradical moiety selected from any of the R^1-3 groups, oxy, sulfide, carbonyl, alkene, alkyne, alkyl halide, hydroxyalkyl, glycol, polyglycol, alkyl thiol, substituted alkyl, phosphate, phosphonate, sulfate, phosphate substituted alkyl, phosphonate

substituted alkyl, sulfate substituted alkyl, carboxy, carboxyamide, thiol-substituted carboxyamide, derivatized carboxyamide of formula CONR⁴ where R⁴ may be alkyl, alkyl halide, hydroxyalkyl, glycol, polyglycol, or alkyl thiol; wherein B independently in each occurrence is a nucleobase, saccharide, nucleotide, expanded porphyrin, steroid, amino acid, peptide, polypeptide, turcasarin or polymeric or solid support matrix, wherein n is 0-10, wherein m is 0-10, and wherein the total number of carbon atoms in each of R^1-4 is less than or equal to 20, or salt thereof.

38. The process of claim 36, wherein the acid is

hydrogen chloride.