WO2010005737A2

WO2010005737A2 - Artificial carbohydrate receptors and methods of use thereof

Info

Publication number: WO2010005737A2
Application number: PCT/US2009/047544
Authority: WO
Inventors: Predrag Cudic
Original assignee: Florida Atlantic University
Priority date: 2008-06-16
Filing date: 2009-06-16
Publication date: 2010-01-14
Also published as: WO2010005737A3

Abstract

Artificial (e.g., synthetic) carbohydrate receptors that specifically bind sialic acid and cancer cell surface carbohydrate antigens containing terminal sialic moieties (e.g., Lewis oligosacharide antigens) and methods of use were developed. The artificial carbohydrate receptors and methods can be used to detect and identify biologically important carbohydrates, selectively label cancer cells, to modulate cancer cell growth, and to modulate inflammation. Because the artificial carbohydrate receptors demonstrate a strong affinity for sialylated Lewis oligosacharide antigens (e.g., sialyl Lewis x (sLe^x) and sialyl Lewis a (sLe^a)) which are overexpressed on tumor cells during the onset and progression of cancer, and are overexpressed on the endothelium of chronically inflamed tissues, the artificial carbohydrate receptors described herein can find use as cancer diagnostic and therapeutic agents.

Description

ARTIFICIAL CARBOHYDRATE RECEPTORS AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the priority of U.S. provisional patent application no. 61/061,752 filed June 16, 2008 which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to the fields of chemistry, cell biology and oncology. More particularly, the invention relates to artificial carbohydrate receptors.

BACKGROUND

[0003] The structure of carbohydrates, which decorate the cell surface of higher organisms, change with the onset of cancer and inflammation, representing therefore highly promising targets for early diagnosis and selective anticancer therapy. Clinically most useful cell surface carbohydrates are sialylated Lewis antigens, sialyl Lewis x (sLe^x) and sialyl Lewis a (sLe^a). sLe^x antigen is overexpressed on lung, breast, and ovary cancer cells, while sLe^a is preferentially present on the cells of colon, pancreas and stomach cancers. However, these antigens are not merely markers of cancer cells. sLe^x and sLe^a expression on cancer cells mediates their adhesion to vascular endothelial cells and promotes hematogenous metastasis of cancer. Therefore, agents that inhibit this process have the potentials to be of considerable therapeutic value. Traditionally, most detection and therapeutic methods that target carbohydrate antigens have used monoclonal antibodies (mAbs) and lectins. Methods involving monoclonal antibodies, however, are associated with a number of disadvantages including high costs, laborious productions, and potential immunogenic side effects.

SUMMARY

[0004] The invention relates to artificial (e.g., synthetic) carbohydrate receptors that specifically bind sialic acid and cancer cell surface carbohydrate antigens containing terminal sialic moieties (e.g., sialylated Lewis oligosacharide antigens) and methods of use. Described herein are artificial polyvalent receptors, including 1,8-naphthyridine-based receptors, with different tether lengths and cancer cell lines that overexpress Lewis oligosaccharides. The artificial carbohydrate receptors and methods described herein can be used to detect and identify biologically important carbohydrates, to modulate cancer cell adhesion to epithelial cells, to modulate cancer cell growth, and to modulate inflammation. Because the artificial carbohydrate receptors described herein demonstrate a strong affinity for sialylated Lewis oligosacharide antigens (e.g., sialyl Lewis x (sLe^x) and sialyl Lewis a (sLe^a)) which are overexpressed on certain tumor cells during the onset and progression of cancer, the artificial carbohydrate receptors described herein can find use as cancer diagnostic and therapeutic agents. Further, because sLe^x and 6-sulfo-sLe^x epitopes are absent from non- inflamed endothelial tissues but are overexpressed on the endothelium of chronically inflamed tissues, they represent promising targets for new and selective anti-inflammatory drugs and for the development of non-invasive diagnostic agents that identify sites of chronic inflammation or cancer prior to the presentation of disease symptoms.

[0005] The synthetic (i.e., artificial) receptors described herein provide a number of advantages over known detection and treatment methods. Advantages include the relatively small size of the receptors, their desirable three-dimensional structure, and their hydrogen- bonding site and lipophilic binding pocket to promote hydrophobic and hydrogen-bonding interactions. A major obstacle in the design of receptors for binding of carbohydrates in water lies in the structural complexity of carbohydrates and the fact that hydrogen-bonding interactions in water are weak. Tumor cell extracellular pH is acidic, and the novel carbohydrate receptors described herein bind carbohydrate substrates at the acidic pH found on cancer cells. Additional advantages offered by the compositions and methods described herein include stability, increased half-life, lack of immunogenicity, ease of synthesis, low cost, and compatibility with automation. [0006] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0007] As used herein, "bind," "binds," or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or in an organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample or organism. Generally, a first molecule that "specifically binds" to a second molecule has a binding affinity at least one order of magnitude greater for the second molecule than for other molecules to which it is exposed.

[0008] As used herein, an "isolated" or "substantially pure" substance is one that has been separated from components which naturally accompany it. Typically, a small molecule or peptide is substantially pure when it is at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, and 99%) by weight free from the other organic molecules with which it is associated. [0009] As used herein, "artificial carbohydrate receptors" and "synthetic carbohydrate receptors" mean organic structures held together by covalent bonds capable of selective binding to molecular substrates by means of various intermolecular interaction, leading to an assembly of two or more species.

[00010] As used herein, a "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

[00011] By "therapeutically effective amount" is meant an amount of a compound of the present invention effective to yield the desired therapeutic response, for example, an amount effective to delay the growth of a cancer, or to shrink the cancer or prevent metastasis.

[00012] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals.

[00013] The terms "patient" or "individual" are used interchangeably herein, and refer to a mammalian subject to be treated or diagnosed, with human patients being preferred. In some cases, the compositions and methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

[00014] "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. "Treatment" may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.

[00015] Although compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[00016] The invention is pointed out with particularity in the appended claims. The above and further advantages of this invention may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which: [0010] FIG. 1 is a schematic illustration of the chemical formulas for 1,8-naphthyridine- based carbohydrate receptors 1-3.

[0011] FIG. 2 is a schematic diagram of the synthesis of receptors 1-3. [0012] FIG. 3 is a schematic illustration including two graphs of the qualitative discrimination of closely related monosaccharides by receptor 2.

[0013] FIG. 4 is a series of fluorescent microscopy images of Lewis antigen-expressing cells in the presence of receptor 2. (A): Le^y expressing Hep 3B cells, (B): normal fibroblast cells (C): sLe^x expressing Hep G2 cells, (D): Hep G2 cells in the absence of receptor 2. [0014] FIG. 5 is a series of fluorescent microscopy images of leukocytes. (A): Leukocytes isolated from a breast cancer mouse model incubated with receptor 2, (B): Control leukocytes isolated from a breast cancer mouse model, (C): Leukocytes isolated from a healthy mouse incubated with receptor 2, (D): Control leukocytes isolated from a healthy mouse. [0015] FIG. 6 is a graph of results from a competitive ELISA assay. [0016] FIG. 7A is an HPLC chromatogram of receptor 2. [0017] FIG. 7B is a MALDI TOF spectrum of receptor 2. [0018] FIG. 8 A is an HPLC chromatogram of receptor 3. [0019] FIG. 8B is a MALDI TOF spectrum of receptor 3. [0020] FIG. 9 is an NMR titration of receptor 2 with sialic acid.

[0021] FIG. 10 is a graph of fluorescence emission spectra of receptor 2 recorded in H₂O and D₂O.

[0022] FIG. 11 is a schematic illustration of solid-phase synthesis of polyvalent receptors 20- 28.

[0023] FIG. 12A is a schematic illustration of synthesis of bis(aminoethyl)aminoacetic acid or any other poly-amino acid bridge 14.

[0024] FIG. 12B is a schematic illustration of synthesis of scaffolds 17 and 18. [0025] FIG. 13 is a schematic illustration of solid-phase synthesis of peptidic spacers 19 and 20. [0026] FIG. 14 is a schematic illustration of solid-phase synthesis of control compounds 29-

36.

[0027] FIG. 15 is a schematic illustration of synthesis of divalent (A) and triivalent receptors

(B) in solution.

[0028] FIG. 16 is a graph showing inhibition of HepG2 adhesion to E-selectin.

DETAILED DESCRIPTION

[0029] Described herein are artificial carbohydrate receptors, compositions, and methods for detecting and identifying biologically important carbohydrates, and for detecting and treating cancer and inflammation. The compositions and methods described herein address the challenges of identifying novel cancer cell-specific labeling agents that distinguish diseased cells from healthy cells, that are capable of facilitating early diagnosis of cancer, and are capable of identifying cancer cell-specific agents that can be used to treat cancer without toxic side effects. The below described preferred embodiments illustrate adaptations of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Chemical and Biological Methods

[0030] Methods involving conventional chemistry and biochemistry techniques are described herein. Protocols for synthesizing, purifying, and characterizing compounds, peptides and proteins as well as combinatorial libraries are described in treatises such as HPLC of Biological Macromolecules Revised & Expanded (Chromatographic Science) by Karen M. Gooding and Fred E. Regnier, 2002 CRC Press, Boca Raton, FL; HPLC Method Development for Pharmaceuticals, by Satinder Ahuja and Henrik Rasmussen, vol., 8, 1^st ed., 2007, Academic Press, Burlington, MA; Chemistry of Peptide Synthesis by N. Leo Benoiton, 1^st ed., 2005, CRC Press, Boca Raton, FL; Cell-Free Protein Synthesis (Methods and Protocols) by Alexander S. Spirin and James R. Swartz, 1^st ed., 2007 Wiley- VCH Press, Weinheim, Germany; and Fmoc Solid Phase Peptide Synthesis, A Practical Approach, W. C. Chan, P.D. White, Eds., Oxford University Press, New York 2004. Methods involving conventional cellular biology and molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Optical Imaging Techniques in Cell Biology by Guy C. Cox, 1^st ed., 2006, CRC Press, Boca Raton, FL; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 2003 (with periodic updates).

Artificial Carbohydrate Receptors and Cancer

[0031] Described herein are artificial (e.g., synthetic) carbohydrate receptors that specifically bind sialic acid and cancer cell surface carbohydrate antigens containing terminal sialic moieties (e.g., sialylated Lewis oligosacharide antigens). The structure of carbohydrates, which decorate the cell surfaces of higher organisms, change with the onset of cancer and inflammation, representing therefore highly promising targets for early cancer diagnosis and selective anticancer therapy. Carbohydrate structures that are most clinically useful are sialylated oligosaccharides such as sialyl Lewis x (sLe^x) and sialyl Lewis a (sLe^a). sLe^x antigen is overexpressed on lung, breast, and ovary cancer cells, while sLe^a is preferentially overexpressed on colon, pancreas and stomach cancer cells. The fact that these antigens are present on several different tumor cell proteins, that they are overexpressed in most malignant cells, that they occur in large quantities in serum from many cancer patients, and that they are resistant for proteolytic degradation make them good candidates for detection in pre-malignant tissue or in blood serum. [0032] These antigens are not merely markers of cancer cells, but are functionally related to the malignant behavior of cancer cells as indicated by the studies of their interactions with selectins. For instance, sLe^x and sLe^a expression on cancer cells mediates their adhesion to vascular endothelial cells and promotes hematogenous metastasis of cancer. Therefore, the development of new therapeutic and diagnostic agents that target sialylated Lewis antigens may have broad applicability to a variety of cancers.

[0033] Synthetic mimetics of carbohydrate binding proteins as described herein have a variety of biomedical applications, among which the development of new cancer diagnostic and therapeutic agents is particularly important. Compositions and methods for the preparation of monovalent 1,8-naphthyridine based receptor molecules (e.g., receptor 2, FIG. 1) as well as multivalent 1,8-naphthyridine based receptor molecules (e.g., receptors 21-28, FIG. 11) are described below. With regard to polyvalent receptor molecules, incorporation of multiple copies of binding and recognition elements within a receptor's three-dimensional structure allows simultaneous multiple interactions of the receptor molecule with multivalent oligosaccharide ligands such as sLe^x and sLe^a, thus enhancing the receptor's selectivity and affinity. The relatively long peptidic spacers between monovalent binding units may also promote the receptors' selective binding to the cell surface where multivalent oligosaccharide ligands are present in multiple copies. This approach also allows further modification of the receptor structure for optimal targeted therapy. In addition, fluorinated amino acids can be incorporated into receptors' peptide sequence for noninvasive in vivo cancer cell 19F magnetic resonance imaging (MRI). The expected advantages of such mimetics are non-immunogenicity due to low molecular weight, enhanced stability, relatively simple synthesis and low cost, and potential rapid blood clearance. Another advantage is that since these artificial receptors are designed to target terminal sialic acid present in sialylated Lewis carbohydrate antigens, they will be less likely affected by antigen modifications. Small molecule mimics of carbohydrate binding proteins will elucidate the role of carbohydrate recognition in the onset and progression of cancer, and lead to the development of new cancer diagnostic and therapeutic agents. [0034] The receptors, compositions and methods described herein can also be used in nanotechnology platforms for cancer diagnosis and treatment, e.g. as nanomedecines. The receptors, compositions and methods may be particularly suitable for the design of highly integrated nanomedicine platforms that incorporate multiple functions such as cancer cell specific targeting, delivery of drugs or imaging agents, and therapy monitoring. The integration of diagnostic imaging and therapeutic capabilities is critical to addressing the challenges of cancer heterogeneity (both inter- and intra-tumor) and adaptation. Diagnostic imaging is used first to characterize cancer cell phenotype(s) in order to deliver target specific therapy. After cancer cell detection, the ability to target the identified cancer cell markers to eradicate all of the diverse phenotypes of cancer is important. Receptors and compositions (e.g., synthetic mimetics of carbohydrate binding proteins) as described herein can be used as nanomedicines for providing synergistic targeted therapy by molecular recognition of the cancer cell antigens and reduction of chemotherapeutic toxicity by minimizing anticancer drugs circulation in the vascular system and targeted drug delivery, thus improving the therapeutic index. Overexpression of oligosacharide antigens (e.g., sLex and sLea antigens) during the onset and progression of cancer could be used for diagnostic and therapeutic purposes. Synthetic mimetics of carbohydrate binding proteins as described herein have a variety of biomedical applications, among which is the development of new agents for early cancer diagnosis, therapy, and follow- up (theranostics). The Artificial Carbohydrate Receptors and their Synthesis [0035] The artificial carbohydrate receptor compounds are represented by the formula

wherein

Xi independently of one another are hydrogen, hydroxyl (OH), amino (NH₂) or carboxyl

(CO₂H);

X₂ independently of one another are amino (NH₂) or carboxyl (CO₂H); or both X₂ together form a group of formula

or one of X₂ is carboxyl (CO₂H) and the other X₂ is a di- or polyvalent bridging moiety comprising at least two α,ω-amino acid segments and at least two linker segments of formula

-NH-Z-C(=O)-O-

wherein Z is a divalent chain having from 3 to 20 chain members selected from the group consisting of: 3 to 15 CH₂ groups, 1 to 3 C(=O) groups, 1 to 3 NH groups and 1 to 6 non- adjacent -O- groups; which bridging moiety may be terminated by NH₂ in the case of polyvalent bridging moieties, and/or binds to at least one terminal group of the formula

X₃ together with the carbon atom to which it is bonded and independently of one another are methylene (CH₂) or carbonyl (C(=O)); and

n is 1, 2 or 3;

or a salt of the receptor compound.

[0036] In a first embodiment of the receptor compounds, the two variables X₂ together form the group of the formula

[0037] Preference is given to those receptor compounds of the first embodiment in which n is 1. Further, receptor compounds of the first embodiment are preferred in which Xj independently of one another denote hydrogen, hydroxyl or amino. Particularly preferred receptor compounds of the first embodiment are those in which n is 1 and one or both of Xi are hydrogen. Also preferred receptor compounds of the first embodiment are those in which X₃ together with the carbon to which they are bonded denote CH₂. Particularly preferred receptor compounds of the first embodiment are those in which n is 1 and one or both of Xi are hydrogen. [0038] In a second embodiment of the receptor compounds both variables X₂ denote amino. Preference is given to those receptor compounds of the second embodiment in which n is 1. Further, receptor compounds of the second embodiment are preferred in which X] independently of one another denote hydrogen, hydroxyl or amino. Also preferred receptor compounds of the second embodiment are those in which X₃ together with the carbon to which they are bonded denote CH₂. Particularly preferred receptor compounds of the second embodiment are those in which n is 1 and one or both of Xi are hydrogen.

[0039] In a third embodiment of the receptor compounds one of X₂ denotes carboxyl and the other X₂ is a di- or polyvalent bridging moiety comprising at least two α,ω-amino acid segments and at least two linker segments of formula

-NH-Z-C(=O)-O-

wherein Z is a divalent chain having from 3 to 20 chain members selected from the group consisting of: 3 to 15 CH₂ groups, 1 to 3 C(=O) groups, 1 to 3 NH groups and 1 to 6 non- adjacent -O- groups.

[0040] The bridging moiety of this embodiment preferably has two, three, four or five valances, and is in particular di- or trivalent.

[0041] The α,ω-amino acid segments of this embodiment are in particular derived from lysine (Lys). However, α,ω-amino acid segments having shorter or longer alkylene chains than lysine between the α- and the ω-position, e.g., chains having from 2 to 8 methylene groups, preferably from 3 to 6 methylene groups, are generally suited. Alternatively, α,ω-amino acid segments of the bridging moiety may be derived from amino acids such as arginine (Arg), asparagine (Asn) and/or glutamine (GIn), for example. Preference is given to bridging moieties comprising two or three segments derived from lysine.

[0042] The linker segments of the bridging moiety may be derived from any compound of the general formula

H₂N-Z-C(=O)-OH

in which the chain represented by Z may be straight or branched. The arrangement of the CH₂- groups and where present the C(=O), NH and/or -O- groups of Z is generally not critical so long as -O- groups are non-adjacent, i.e., no -O-O- segments are present. Otherwise, the C(=O), NH and -O- groups may be separated from one another by alkylene groups, such as, for example, methylene, ethylene and propylene, and the C(=O), NH and -O- groups may form substructures such as, for example, -NH-C(=O)-, -CC=O)-NH-, -NH-O-, -0-NH, -NH-NH-, -0-CC=O), -C(=O)-O-, -NH-C(=O)-O-, -O-C(=O)-NH-, -NH-C(O)-NH-, -NH-C(O)-NH-, -CC=O)-NH-O-, -C(=0)-0-NH, -NH-O-C(O)-, -0-NH-C(O)-, -NH-NH-C(O)- and -CC=O)-NH-NH- within the chain or in combination with the terminal -NH- and/or -C(=0)-0- group of the linker segment.

[0043] Preference is given to linker segments in which Z represents a straight-chain or branched, in particular a straight-chain, -C_xH_2x- group in which x is from 3 to 15, in particular from 5 to 12.

[0044] Further preference is given to linker segments in which Z comprises oxyalkylene groupings such as -(CH₂CH₂O)_2-4-, -(CH₂CH₂CH₂O)_2-4-, and -(CH₂CH(CH₃)O)_2-4-.

[0045] Especially preferred are those receptor compounds of the third embodiment in which the bridging moiety comprises two or three lysine segments and two or three linker segments selected from the group consisting of :

-NH-(CH₂)_S-CO-O-, -NH-(CH₂)π-C0-0- and

-NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(=O)-CH₂OCH₂-C(=O)-O-.

[0046] Preference is given to those receptor compounds of the third embodiment in which n is 1. Further, receptor compounds of the third embodiment are preferred in which Xi independently of one another denote hydrogen, hydroxyl or amino. Also preferred receptor compounds of the third embodiment are those in which X₃ together with the carbon to which they are bonded denote CH₂. Particularly preferred receptor compounds of the third embodiment are those in which n is 1 and one or both of Xi are hydrogen.

[0047] In a fourth embodiment, the receptor compounds are represented by the formula

[0048] Preference is given to those receptor compounds of the fourth embodiment in which n is 1. Further, receptor compounds of the fourth embodiment are preferred in which Xi independently of one another denote hydrogen, hydroxyl or amino. Particularly preferred receptor compounds of the fourth embodiment are those in which n is 1 and one or more, especially all, of Xi are hydrogen. Also preferred receptor compounds of the fourth embodiment are those in which one or more, especially all, of X₃ together with the carbon to which they are bonded denote CH₂. Particularly preferred receptor compounds of the fourth embodiment are those in which n is i and one or more, especially all, of Xi are hydrogen. Particularly preferred receptor compounds of the fourth embodiment are further those in which n is 1 and one or more, especially all, of Xi are hydrogen, and one or more, especially all, of X₃ together with the carbon to which they are bonded denote CH₂.

[0049] Further preference is given to those receptor compounds of the fourth embodiment in which X] denotes hydrogen, amino (NH₂) or carboxyl (CO₂H) and the linker segment is selected from the group consisting of

-NH-(CH₂)₅-CO-O-

-NH-(CH₂)_H-CO-O-

-NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(==O)-CH₂OCH₂-C(=O)-O-.

[0050] Particularly preferred among the receptor compounds of the fourth embodiment are those in which Xi independently of one another denote hydrogen, hydroxyl or amino, especially hydrogen; X₃ together with the carbon to which they are bonded denote CH₂; n is 1 and the linker segment is selected from the group consisting of

-NH-(CH₂)₅-CO-O-

-NH-(CH₂)_H-CO-O-

-NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(=O)-CH₂OCH₂-C(=O)-O-.

[0051] In a fifth embodiment, the receptor compounds are represented by the formula

[0052] Preference is given to those receptor compounds of the fifth embodiment in which n is 1. Further, receptor compounds of the fifth embodiment are preferred in which Xi independently of one another denote hydrogen, hydroxyl or amino. Particularly preferred receptor compounds of the fifth embodiment are those in which n is 1 and one or more, especially all, of Xi are hydrogen. Also preferred receptor compounds of the fifth embodiment are those in which one or more, especially all, of X₃ together with the carbon to which they are bonded denote CH₂. Particularly preferred receptor compounds of the fifth embodiment are those in which n is 1 and one or more, especially all, of Xi are hydrogen. Particularly preferred receptor compounds of the fifth embodiment are further those in which n is 1 and one or more, especially all, of Xj are hydrogen, and one or more, especially all, of X₃ together with the carbon to which they are bonded denote CH₂. Further preference is given to those receptor compounds of the fifth embodiment in which Xi denotes hydrogen, amino (NH₂) or carboxyl (CO₂H) and the linker segment is selected from the group consisting of

-NH-(CH₂)_S-CO-O-

-NH-(CH₂)H-CO-O-

-NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(=O)-CH₂OCH₂-C(=O)-O-.

[0053] Particularly preferred among the receptor compounds of the fifth embodiment are those in which Xi independently of one another denote hydrogen, hydroxyl or amino, especially hydrogen; X₃ together with the carbon to which they are bonded denote CH₂; n is 1 and the linker segment is selected from the group consisting of

-NH-(CH₂)₅-CO-O-

-NH-(CH₂)H-CO-O-

-NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(=O)-CH₂OCH₂-C(=O)-O-.

[0054] The receptor compounds can be prepared by methods involving conventional chemistry and biochemistry techniques as, for example, outlined in the references cited above.

[0055] The 1,8-naphthyridine-comprising macrocyclus is a key structure of the receptor compounds. The macrocyclus is formed by reacting 1,8-naphthyridine dicarbaldehyde with a such as tris(2-aminoethyl)amine (TREN) (see Figure 2) or a bis(2-aminoethyl)amino derivative of a linker (see Figure 12B, compound 15).

[0056] Condensation of 1,8-naphthyridine dicarbaldehyde with an equimolar amount of tris(2-aminoethyl)amine (TREN) followed by treatment with NaBH₄ yields a mixture of receptor compounds 2 and 3 (FIG. 1). However, if a 2:3 ratio of 1,8-naphthyridine dicarbaldehyde and TREN is used, the major product formed is receptor compound 3 while receptor compound 2 cannot be detected. The receptor compounds can be purified by RP-HPLC purification of crude reaction mixtures.

[0057] 1,8-Naphthyridine dicarbaldehyde is known in the art and can be prepared in five steps as outlined in Figure 2 (see He and Lippard, Tetrahedron 56:8245-8252, 2000).

[0058] Receptor compounds comprising aminoacid segments are conveniently prepared by way of solid-phase synthesis. A general solid-phase synthetic route to di- and trivalent receptors

2, 21, 26-28, and 37-39 is outlined in Figure 11. A strategy for the synthesis of these di- and trivalent receptor molecules is summarized as follows: synthesis of bis(aminoethyl)aminoacetic acid or any other poly-amino acid 14 that is necessary for completion of the 1,8-naphthyridine macrocyclic scaffold containing carboxylic groups, amino groups or amino acid functionalities; solution synthesis of 1,8-naphthyridine macrocyclic scaffolds 17 and 18 with suitable arms for solid-phase attachment to peptidic spacers according to the protocols already developed (Figure 12); solid-phase synthesis of different length lysine or other amino acid based spacers that include 6-aminohexanoic, 12-aminododecanoic acid or other fatty acids; coupling of 1,8- naphthyridine macrocyclic scaffolds 17 and 18 using standard SPPS protocols; and cleavage of the final product from the resin, product purification and characterization, (Figure 13). [0059] By incorporating bis(aminoethyl)aminovaleric acid or any other poly-amino acids 14 (Figure 12A) into a 1,8-naphthyridine macrocyclic framework, the required monovalent receptor's flexibility and overall positive charge is maintained. In addition, the presence of the carboxylic groups make this naphthyridine macrocyclic scaffold suitable for attaching to a desired peptide tether using standard solid-phase methodology.

[0060] Bis(aminoethyl)aminoacetic acid or any other poly-amino acid bridge can be prepared from commercially available diethyltriamine 11 (Figure 12A) as described by J. Zubeta et al. (Inorg. Chem. Commun. 7:481-484, 2004). According to this protocol, the selective protection of the primary amine groups of diethyltriamine with phthalic anhydride in acetic acid, followed by reaction with appropriate bromo acidic acids or side chain activated amino acid (e.g. Ser, Thr, Cys) in the presence of KI, yields fully protected poly-amino acid bridges 14. Removal of the protecting groups with concentrated HCl yield the desired crude poly-amino acid bridge 12 as the hydrochloride salt. The crude product 12 is then purified by extraction with ether to remove all organic impurities, and fully characterized by mass spectrometry and NMR spectroscopy and MS spectrometry.

[0061] Synthesis of 1,8-naphthyridine monovalent binding units 17 and 18 suitable for solid- phase modification: According to standard SPPS protocols, carboxylic group of the amino acid needs to be activated in solution prior to its coupling to free primary amine already attached to the resins. Since receptor 2 has two "arms" with free primary amino groups, this compound is modified for solid-phase synthesis. Therefore, 1,8-naphthyridine units are bridged with bis(aminoethyl)aminoacetic acid or any other poly-amino acid 14, that generate the desired compound with free carboxylic groups instead (Figure 12B). In addition, a significant increase in the macrocyclization yields is expected since a macrobicyclic compound, such as receptor 3, cannot be formed. Macrocyclic compound containing 1,8-naphthyridine units bridged with appropriate poly-amino acid 14 can be prepared according to the same protocol as described for compound 2.

[0062] Condensation of equimolar amounts of bis(aminoethyl)aminoacetic acid or any other poly-amino acid 14 and 1,8-naphthyridine dicarbaldehyde 10 under high dilution conditions yield imine precursor. Reduction of this precursor with NaBH₄, followed by deprotection of the carboxylic group, and purification by RP-HPLC results in compound 17 suitable for incorporation into a desired peptidic chain(s) on the solid-support. Scaffold 18 is prepared in the same way by reacting 10 with GIy, and is used for the construction of control compounds 29-36.

Administration of Compositions

[0063] The artificial carbohydrate receptors and compositions described herein can be delivered to the cell surface or administered to a subject (e.g., a human) in any suitable formulation. For example, artificial carbohydrate receptors may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice. A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP/NF. Other substances may be added to the compositions to stabilize and/or preserve the compositions.

[0064] The compositions described herein may be administered to mammals by any conventional technique. Typically, such administration will be parenteral (e.g., intravenous, subcutaneous). The compositions may also be administered directly to a target site by, for example, surgical delivery to an internal or external target site, or by catheter to a site accessible by a blood vessel. The compositions may be administered in a single bolus, multiple injections, subcutaneously or by continuous infusion (e.g., intravenously or pump infusion). For parenteral administration, the compositions are preferably formulated in a sterilized pyrogen-free form.

Effective Doses

[0065] The compositions containing artificial carbohydrate receptors are preferably administered to a mammal (e.g., rodent, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., detecting or treating cancer and/or inflammation). Such a therapeutically effective amount can be determined as described below.

[0066] Toxicity and therapeutic efficacy of the compositions utilized in the methods described herein can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of preferred compositions lies preferably within a range that includes an ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0067] As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.

Kits

[0068] Described herein are kits for diagnosing cancer in a mammalian (e.g., human) subject. Kits can be used to detect the presence of cancerous cells in a biological sample obtained from a subject. Any suitable biological sample can be assayed for the presence of cancerous cells, including blood, plasma, serum, cerebral spinal fluid, saliva, urine, etc. In one embodiment, a kit as described herein includes an enzyme-linked immunosorbent assay (ELISA) in which antibodies are replaced with receptors as described herein. In such an embodiment, the kit includes a solid substrate, at least one receptor as described herein, and instructions for using the kit to detect cancer in a subject. ELISA methods are well known in the art and are described, for example, in The ELISA Guidebook (Methods in Molecular Biology) by John R. Crowther, 1^st ed., Humana Press, 2000.

EXAMPLES

[0069] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way. Example 1 - Naphthiridine-based artificial receptors for identification and detection of biologically important carbohydrates at a physiologically relevant pH.

[0070] Macrocyclic structures are particularly interesting for the design of artificial carbohydrate receptor molecules mainly because they are large enough to contain spherical binding site (lipophylic cavity) of appropriate size and shape. Incorporation of condensed polypyridine units into such macrocyclic structures could result in construction of artificial receptors capable of selective carbohydrate binding in water through a combination of electrostatic, hydrophobic and hydrogen bonding interactions. In addition, condensed polypridine compounds exhibit strong absorption and fluorescence emission bands, and complexation-induced changes in their absorption or fluorescence spectra will allow carbohydrate substrates discrimination and determination of the stability constants. [0071] To demonstrate suitability of macrocyclic polypyridine structures for design of artificial receptors capable of binding carbohydrate substrates in water, three types of 1, 8-naphthyridine based receptor molecules (i.e., compounds 1-3 of Figure 1) were examined. Synthesized bi-bracchial macrocyclic 2 and cage-type macrobicyclic receptor 3 possess a 1, 8-naphthyridine core moiety bridged with two flexible tris (2-aminoethyl) amines (TREN). hi these receptors two adjacent pyridinic nitrogen atoms acts as the proton acceptors (AA), while lateral MEN secondary amines function as the proton donors (DD). The relay of hydroxyl groups in monosaccharides offers an ADDA motif complementary to the DAAD array of naphthyridine receptors to form multiple H-bonds. hi addition, synthesized receptor molecules differ in their flexibility and therefore conformational adaptability toward carbohydrate substrates.

[0072] Synthesis and structural characterization of carbohydrate sensors 1-3: 1,8- Naphthyridine dicarbaldehyde, a key sensor intermediate, was synthesized over four steps as reported previously (He and Lippard, Tetrahedron 56:8245-8252, 2000) (see Figure 2). Condensation of 1,8 naphthyridine dicarbaldehyde with an equimolar amount of TREN followed by treatment with NaBH₄ yields a mixture of sensors 2 (30%) and 3 (15%). However, if the 3:2 ratio of 1, 8-naphthyridine dicarbaldehyde and TREN is used, the major product formed is receptor 3 (45%) while receptor 2 can not be detected. In both cases pure receptor 2 and receptor 3 were obtained after RP-HPLC purification of crude reaction mixtures (Fig. 7 and Fig. 8). The compound 1 was prepared for control studies by condensation of 1,8 naphthyridine dicarbaldehyde with two equivalents of propylamine using the same synthetic strategy. After solvent evaporation, the oily residue was redissolved in water containing 0.1 % TFA and the final product 1 was purified by RP-HPLC. The product 1 was obtained in 60% yield. Since recent studies showed that tumor cells have a lower extracellular pH (~ 6.8) than normal cells, all spectroscopic and binding studies were performed at pH 6.5. The change of the UV absorption of receptor 2 as the function of pH was used as an indicator of the suitability of this pH for the binding studies. Lowering the pH of the receptor 2 solution from 11-8 caused decrease of the absorption intensity at 315 nm, while further lowering the pH of the solution fiorn 8-1 resulted with the opposite effect In this case 1he absorption intensity increased with a bathochromic shift of the maximum wavelength (X_1113x=S 19 nm, pH 1), associated with the formation of an isosbestic point at 274 ram. Similar dependence of a receptor's UV/vis spectra on pH was observed for receptor 3 as well. These experimental findings are in accordance with the data previously reported in the literature for similar compounds indicating that protonation occurs firstly at secondary nitrogen sites in TREN bridges, while protonation of remaining aromatic nitrogens require much lower pH (pK_Α for unsubstituted 1,8-naphthyridine is 3.39). Based on these experiments, it can be assumed that at pH 6.5 in the receptors 1-3, only lateral TREN primary and secondary amines will be protonated, allowing therefore a desired DAAD H-bonding arrangement for optimal interactions with monosaccharide substrates. The repulsive interactions between positively charged TREN bridges may be expected to prevent complete self-stacking of 1,8-naphthyridine units in both receptors 2, 3 and create a hydrophobic binding pocket. Although the TREN bridges in both macrocyclic receptors are flexible enough to allow the binding pocket to adjust to carbohydrate substrate for optimal interactions, the greater degree of structural flexibility and therefore conformational adaptability may be expected for the receptors.

[0073] It may thus be expected that these receptor molecules can complex carbohydrates in water by a combination of hydrophobic, electrostatic and hydrogen bonding interactions. The 1,8- naphthyridine receptors 1-3 showed pronounced differences in their UV/vis and fluorescence spectra. As expected, comparison of the UV/vis spectra of reference compound 1 and receptors 2 and 3 taken in an aqueous buffer (sodium cacodylate, pH 6.5; C_bUffe_r ⁼ 0.05 M, c\= 2xlO⁴ M, λ_maxl= 312 nm, ε_x= 4725.7 M^-1Cm^'1, C₂= IxIO^"4 M, λ_max2= 315 nm, β_∑= 7372.5 M^-1Cm^"1, C₃= 6.7xl O^"5 ,

an absorption band broadening and bathochromic shift for 2 and 3. Observed effects are indicative of weak intramolecular π-π stacking interactions presence between the 1 ,8-naphthyridine units; strong stacking interactions are reported to produce more pronounced hypochromicity and bathochromic shift. [0074] In the fluorescence emission spectra of receptors 1-3 taken also in aqueous buffer and at comparable fluorophore concentrations (sodium cacodylate, pH 6.5; c_\= 6x10^~6 M, λ_exCrtl⁼320 nm, λ_emissl=375 nm, /_maxl=767, c₂=3xl0^"6 M, λ_exolt2=320 nm, λ_emiss2= 375 nm, /_max2= 392, c₃=2xl0^" M, λ_eXci_t3=320 nm, λ_emiss3=375 nm, /_max3=189), the emission intensity (/_max) observed for 2 and 3 is two and four times lower than the emission intensity of reference compound 1, supporting our previous finding of weak intramolecular stacking interactions. Intermolecular interactions can be eliminated as a possible cause for the fluorescence quenching since fluorescence emission of 1-3 is linearly dependent on their concentrations within the range of IxIO^"6 - IxKr⁵ M.

[0075] In contrast to H₂O solutions, D₂O solutions of 2 and 3 exhibited a 34% and 30% increase in the fluorescence intensities, respectively, suggesting the possibility of other fluorescence quenching mechanism in addition to the intramolecular stacking interactions, (Figure 10). An identical effect of D₂O on fluorescence intensity was reported for ethidium bromide (a tricyclic aromatic amine) and it was interpreted by excited state quenching caused with solute to solvent proton transfer. In this case, the low fluorescence intensity of free ethidium bromide in water was attributed to proton transfer from the excited singlet state to water molecules, while the increase of fluorescence intensity in D₂O was attributed to a reduction in the proton transfer rate.

[0076] Carbohydrate Binding in Solution: All examined carbohydrate substrates are commercially available. The binding affinities of receptors 1-3 toward a variety of monosaccharide substrates were examined by UV/vis, and fluorometric titration experiments in cacodylate buffer (0.05 M) at pH 6.5, and room temperature. In all experiments, the concentration of receptor was kept constant (approximately 10⁴ M for UV/vis, and approximately 10^"6 M or fluorimetric titration), whereas the concentration of carbohydrate substrates was varied from approximately 10^"5-10^"2 M in the case of UV/vis and approximately 10^"5-10^"3 M in the case of fluorimetric titration experiments. The dissociation constants (IQ) were calculated using the SPECFIT global analysis software (Fig. 3). The dissociation constants are shown in Table 1, and a reasonable good agreement between the two methods was obtained.

Table 1. Dissociation constants (iζj/mM) for 1 :1 complexes between various carbohydrate substrates and 1,8-naphthyridine receptors in water (pH 6.5).

[0077] For the binding experiments in which K^ was determinable, the Job's plot indicated formation of 1 :1 stoichiometry complexes. All the substrates tested did not change appreciably the absorption spectra of the reference compound 1, even at high substrate concentrations (500-fold excess with the respect to concentration of 1). As shown in Table 1, receptor 3 binds negatively charged monosaccharide substrates with millimolar affinities in aqueous solution. Affinities toward neutral substrates are much lower, and thus not determinable by florescence or UV/vis titration experiments under the same experimental conditions. These results indicate that receptor 3 binds negatively charged monosaccharides mainly through charge-charge interactions, and that hydrogen bonding and/or hydrophobic interactions play a negligible role or play no role in the complex stabilization. In support of this, no significant differences in binding of negatively charged substrates was observed. Obtained results can be explained by receptor 3 lacking flexible 2- aminoethyl "arms" and being a somewhat more sterically hindered binding pocket, so that complete substrate encapsulation cannot occur. Qn the other hand, conformationally more flexible receptor 2 showed millimolar affinities toward neutral hexoses such as glucose, galactose, and fructose, as well as negatively-charged glucose-1-phosphate, glucose-6-phosphate and sialic acid. However, receptor 2 has the highest affinity for negatively charged sialic acid (K_d = - 0.3 mM, Table 1), roughly an order of magnitude higher than for all other substrates tested. This result indicates that hydrogen bonding and hydrophobic interactions, in addition to charge-charge interactions, significantly contribute to the overall complex stability. [0078] In order to examine the role of hydrogen bonds in stabilization of the complexes, various neutral pyranose and deoxy-pyranose substrates were investigated. Among these substrates, receptor 2 showed binding preferences for glucose over deoxy-glucose substrates (Table 1), clearly indicating that the number of hydroxyl groups which the receptor can interact with is crucial for the complex stability. The stability constants for the complexes with deoxy-glucoses were not determinable by fluorimetric or UV/vis titrations under the same experimental conditions. [0079] An additional confirmation for hydrogen bonding was found in the titration of receptor 2 with somewhat smaller pentoses such as D-xylose and D-ribose. As in previous cases, the stability constants for these complexes also were not determinable by fluorimetric and UV/vis titrations experiments. This could be due to the smaller ring size of the pentose substrates, but also (as in the case of the deoxy-glucoses) due to the number of hydroxyl groups available for interaction.

[0080] Besides binding to monosaccharides in water, receptors 2 and 3 displayed the ability to qualitatively (opposite variation of the florescence emission intensity) discriminate monosaccharide substrates as illustrated in Fig. 3. In the case of receptor 2, florescence quenching was observed for galactose, glucose-6-phosphate, fructose and sailic acid, while florescence enhancement was observed for glucose, and glucose- 1 -phosphate substrates. A similar trend in florescence intensity was observed for receptor 3 as well. Observed variations in florescence intensities of receptor 2 and receptor 3 upon complexation of closely related monosaccharides such as glucose/galactose or glucose- l-phosphate/glucose-6-phosphate make these two receptor molecules particularly attractive as lead structures for further development of more efficient and selective carbohydrate sensors.

[0081] The observed 1:1 stoichiometry for complexes of receptor 2 and receptor 3 with monosaccharide substrates, and the fact that the stability constants for 1 are very low and thus not determinable by UV/vis or fluorescence titrations, indicate that complexation occurs by insertion of the substrate molecule into the receptor's lipophylic pocket. Additional evidences for the formation of inclusion type of complexes were found in ¹H NMR titration of receptor 2 with sialic acid, the receptor/substrate system that forms one of the most stable complexes in water, Table 1. The ¹HNMR titration experiment was carried out in deuterated cacodylate buffer (0.1 M, pD=6.0, Figure 9). The concentration of receptor 2 was kept constant at 0.007 M while sialic acid concentration was varied form 0.006-0.075 M. Marked up-field chemical shifts was observed in the ¹H NMR spectra of receptor 2's aromatic protons of up to 0.3 ppm upon addition of sialic acid substrate, accompanied with the sharpening of receptor 2's aromatic signals (possibly caused by changing receptor 2's conformation and increasing the distance between aromatic moieties to accommodate sialic acid substrate). Complexation of sialic acid caused not only changes in the ¹H NMR spectra of receptor 2, but also the appearance of sialic acid amide proton and its down-field chemical shift, Figure 9. Sialic acid's slow amide hydrogen/deuterium exchange rate and down-field chemical shift of this particular hydrogen in D₂O was possible only if the inclusion complex was formed. However, due to the complex precipitation at higher shale acid concentrations, obtained H-NMR chemical shifts could not be used for the IQ determination. Due to overlap of the signals, the aliphatic region of spectra could not be used for analysis.

[0082] Because receptor 2 shows the highest affinity toward sialic acid (K_d =0.3 mM, Table 1), receptor 2 was tested for cell labeling studies. As a model system, sLe^x-expresssing hepatocellular carcinoma HepG2 cells (ATCC HB-8065), control Le^y-expresssing hepatocellular carcinoma Hep3B cells (ATCC HB-8064), and normal fibrolast cells (ATCC CCL-201) were chosen. In all three cases, cells were incubated with receptor 2 at concentrations ranging between 0.1 mM and 10 mM with immobilized cells (60000 cells) under the same conditions required for the standard immunohistochemistry assay. The cells were then observed with a fluorescent microscope. Figure 4 shows florescence microscope images of the cells incubated with 2 at 1 mM concentration. Receptor 2 labeled only HepG2 cells, but not fibroblast or Le^y expressing Hep3B cells even at a higher concentration (10 mM) and prolonged incubation time. In addition to this, an MTT cell proliferation assay showed that compound 2 is not appreciably toxic toward HepG2 cells after 24 h of incubation and at ImM concentration. Considering the fact that malnourished or dying cells often start to leak proteins into surroundings, which can potentially interfere with binding of a carbohydrate receptors to their cell specific substrates, low cell toxicity is among the highly desirable properties of new sensory or drug delivery systems. Carbohydrate binding proteins, E-, P- and L-selectins, are known to mediate leukocyte adhesion to the endothelial surface by binding to oligosaccharide sialylated and fucosylated epitopes such as sialyl Lewis X (sLe^x) and 6-sulfo-sLe^x, which decorate the surface of most leukocytes, endothelial cells in the lymph node and the endothelium of inflamed tissues. However, sLe^x and 6-sulfo-sLe^x epitopes are absent from non-inflamed endothelial tissues but are overexpressed on the endothelium of chronically inflamed tissues representing therefore highly promising targets for new and more-selective anti-inflammatory drugs and for the development of a non-invasive diagnostic that might identify sites of chronic inflammation or cancer prior to the presentation of disease symptoms. To more broadly probe cell labeling properties of receptor 2, this compound was incubated with leukocytes isolated from the blood of a healthy mouse and from a mouse model of breast cancer. As shown in Figure 5, receptor 2 labeled only leukocytes isolated from a mouse model of breast cancer but not from a healthy mouse. In addition, in a competitive ELISA assay, receptor 2 at 10 mM concentration inhibited anti-sLe^x mAb KM93 binding to sLe^x-BSA conjugate, indicating that synthetic receptor 2 and this sLe^x-specific mAb share the same target (Figure 6). E-selectin promoted adhesion of HepG2 cells was almost completely inhibited by receptor 2 at 1OmM concentration. The extent of inhibition was comparable to anti-sLe^x mAb at 10 μg/mL concentration. See FIG. 16. [0083] The results described above demonstrate the feasibility of constructing artificial receptor molecules capable of binding to specific cancer cells. Receptors described herein can be used for the design of new and more efficient artificial carbohydrate receptors that may be used as lead structures for the development of a novel type of diagnostic agents, drug delivery systems or new therapeutic agents.

Example 2 - Design, synthesis and binding properties of K8-naphthiridine-based macrocycles: new selective sialic acid receptors.

[0084] Synthesis and structural characterization of receptors 1-3: 1,8-Naphthyridine dicarbaldehyde (compound 10 in Fig. 2), a key receptor intermediate, was synthesized over five steps as previously reported (Le and Hippard, Tetrahedron 56:8245-8252, 2000). Condensation of 1,8-naphthyridine dicarbaldehyde (compound 10 in Fig. 2) with an equimolar amount of tris(2-aminoethyl)amine (TREN) followed by treatment with NaBH₄ yields mixture of receptors 2 (30%) and 3 (15%), (Figure 2). However, if the 2:3 ratio of 1,8-naphthyridine dicarbaldehyde (compound 10 in Fig. 2) and TREN is used, the major product formed is receptor 3 (45%) while 2 cannot be detected. In both cases pure receptor 2 and receptor 3 were obtained after RP-HPLC purification of crude reaction mixtures. The compound 1 was prepared for control studies by condensation of compound 10 with two equivalents of propylamine using the same synthetic strategy. After solvent evaporation, the oily residue was redissolved in water containing 0.1 % TFA and the final product 1 was purified by RP-HPLC. The product 1 was obtained in 60% yield. Because tumor cells have a lower extracellular pH (~6.8) than normal cells, it was decided to perform all spectroscopic measurements in cacodylate buffer at pH 6.5. The pH titration and changes of the UV/vis absorption of receptor 2 as the function of pH were used as an indicator of the suitability of this pH for the binding studies. The pK_Λ values obtained for aromatic, tertiary, secondary and primary nitrogens of the receptor 2 were 2.8, 5.7, 8.6, and 10.7, respectively. In the case of UV/vis experiments, lowering the pH of the receptor 2 solution from 11 to 8 caused a decrease of the absorption intensity at 315 nm while further lowering the pH of the solution from 8 to 1 resulted in the opposite effect. In this case, the absorption intensity increased with a bathochromic shift of the maximum wavelength (λ_max=319 nm, pH 1), associated with the formation of an isosbestic point at 274 nm. Similar dependence of UV/vis spectra on pH was observed for 3. The experimental findings described herein indicate that protonation occurs firstly at primary and secondary nitrogen sites in TREN bridges, while protonation of remaining aromatic nitrogens requires a much lower pH (jpK_a for unsubstituted 1,8-naphthyridine is 3.39). Based on these experiments it can be assumed that at pH 6.5, in receptors 1-3, only lateral TREN primary and secondary amines will be protonated, allowing therefore the desired DAAD H- bonding arrangement for optimal interactions with monosaccharide substrates. The repulsive interactions between positively charged TREN bridges may be expected to prevent complete self-stacking of 1,8-naphthyridine units in both receptors 2 and 3 thus creating a hydrophobic binding pocket. Although the TREN bridges in both macrocyclic receptors are flexible enough to allow the binding pocket to adjust to a carbohydrate substrate for optimal interactions, the greater degree of structural flexibility and conformational adaptability may be expected for receptor 2. Therefore, it is expected that synthesized receptor molecules 2 and 3 can complex carbohydrates in water by a combination of hydrophobic, electrostatic and hydrogen bonding interactions. [0085] The 1,8-naphthyridine receptors 1-3 showed pronounced differences in their UV/vis and fluorescence spectra. As expected, comparison of the UV/vis spectra of reference compound 1 and receptors 2 and 3 taken in an aqueous buffer (sodium cacodylate, pH 6.5; co_ffer" 0.05 M, Ci= 2xlO^"4 M, λ_maxl= 312 nm, ε_x= 4725.7 M^-1Cm^'1, C₂= IxIO^"4 M, λ_max2= 315 nm, S₁= 7372.5 M^" 'cm^"1, C₃= 6.7xlO^"5 , λ_max3= 316 nm, £₃= 13713.0 JVf¹Cm^'1), shows a hypochromicity (^₃Os₁, £^> ₂ ^<2£^>i), and an absorption band broadening and bathochromic shift for receptor 2 and receptor 3. Observed effects are indicative of weak intramolecular π-π stacking interactions present between the 1,8-naphthyridine units; strong stacking interactions are reported to produce more pronounced hypochromicity and bathochromic shift. In the fluorescence emission spectra of compounds 1-3 taken also in aqueous buffer and at comparable fluorophore concentrations (sodium cacodylate, pH 6.5; C₁= 6x10^" M, λ_exci_tl=320 nm, λ_emj_sSl⁼375 nm, /_maχl⁼767, c₂=3xl0^" M, λ_excit2=320 nm, λ_emi_ss2= 375 nm, /_max2= 392, c₃=2xl0^"6 M, λ_excit3=320 nm, λ_emi_ss3=375 nm,

the emission intensity (/_max) observed for receptors 2 and 3 is two and four times lower than the emission intensity of reference compound 1 , supporting a previous finding of weak intramolecular stacking interactions. Intermolecular interactions can be eliminated as a possible cause for the fluorescence quenching since fluorescence emission of receptors 1 -3 is linearly dependent on their concentrations within the range of 1x10^" - 1x10^" M. [0086] In contrast to H₂O solutions, D₂O solutions of receptors 2 and 3 exhibited 34% and 30% increases, respectively, in the fluorescence intensities, respectively, suggesting the possibility of other fluorescence quenching mechanism in addition to the intramolecular stacking interactions (Figure 10).

[0087] Binding studies: All examined carbohydrate substrates are commercially available. The binding affinities of receptors 1-3 toward a variety of monosaccharide substrates were examined by UV/vis, and fiuorometric titration experiments in cacodylate buffer (0.05 M) at pH 6.5, and room temperature. In all experiments the concentration of receptor was kept constant xIO^"4 M, c₃=6 ^χlθ^"5 M for UV/vis, and

xIO^"6 M, c₃=2^χ l0^'6 for fiuorimetrie titrations), whereas the concentration of carbohydrate substrates was varied from approximately 1 x 10^" - 4^χlO^"2 M in the case of UV/vis and approximately l ^χlθ^"5-l ^χlθ^"3 M in the case of fiuorimetrie titration experiments. The dissociation constants (K_d) were calculated using the SPECFIT global analysis software.

[0088] The dissociation constants are shown in Table 2, and a reasonably good agreement between the two methods was obtained.

Table 2. Dissociation constants for 1 :1 complexes between various carbohydrate substrates and receptors 2 and 3 determined in cacodylate buffer at pH 6.5.

[0089] For the binding experiments in which K_d was determinable, the Job plot indicated formation of 1 :1 stoichiometry complexes. All the substrates tested did not change appreciably the fluorescence or absorption spectra of reference compound 1 even at high substrate concentrations (500-fold excess with the respect to concentration of 1). As shown in Table 2, receptor 3 binds negatively charged monosaccharide substrates with millimolar affinities in aqueous solution. Affinities toward neutral substrates are much lower, and thus not determinable by fluorescence or UV/vis titration experiments under the same experimental conditions. These results indicate that receptor 3 binds negatively-charged monosaccharides mainly through charge-charge interactions and that hydrogen bonding and/or hydrophobic interactions play a negligible role or no role in the complex stabilization. In support of this, no significant differences in affinities of receptor 3 toward negatively-charged substrates was observed. Obtained results can be explained by the lack of flexible 2-aminoethyl "arms" in the receptor 3 and somewhat more sterically hindered binding pocket so that complete substrate encapsulation cannot occur. On the other hand, conformationally more flexible receptor 2 showed millimolar affinities toward neutral hexoses such as glucose, galactose, and fructose, as well as negatively charged glucose- 1 -phosphate, glucose-6-phosphate and sialic acid. However, receptor 2 had the highest affinity for negatively-charged sialic acid (K_d = ~ 0.3 mM, Table 2), roughly an order of magnitude higher than for all other substrates tested. These results suggest that the hydrogen bonding and hydrophobic interactions, in addition to the charge-charge interactions, significantly contribute to the overall complex stability.

[0090] In order to examine the role of hydrogen bonds in stabilization of the complexes, various neutral pyranose and deoxy-pyranose substrates were investigated. Among these substrates, receptor 2 shows binding preferences for glucose over deoxy-glucose substrates (Table 2) clearly indicating that the number of hydroxyl groups which receptor can interact with is crucial for the complex stability. The stability constants for the complexes with deoxy- glucoses were not determinable by fluorimetric or UV/vis titrations under the same experimental conditions. Additional evidence for hydrogen bonding was found in titration of 2 with somewhat smaller pentoses such as D-xylose and D-ribose. As in the previous cases, the stability constants for these complexes also were not determinable by fluorimetric and UV/vis titrations experiments. This could be due to the smaller ring size of the pentose substrates, but also (as in the case of the deoxy-glucoses) due to the number of hydroxyl groups available for interaction. [0091] In the case of larger disaccharide substrates D-trehalose and D-gentiobiose (both composed of two glucose units), the K_& values determined for receptor 2 were comparable to those of glucose, suggesting that only one glucose unit of these disaccharides participate in the binding. It is hypothesized that receptor 2's relatively flexible and less sterically hindered structure allows formation of such complexes.

[0092] Besides binding to monosaccharides in water with different affinities, receptors 2 and 3 also display the ability to qualitatively discriminate between closely related monosaccharide substrates by opposite variation of the fluorescence emission intensity. Fluorescence quenching was observed for titration of 2 with galactose and glucose-6-phosphate, while fluorescence enhancement was observed for glucose and glucose- 1 -phosphate substrates. A similar trend in fluorescence intensity was observed for the titration of 3 with negatively-charged glucose- 1- phosphate and glucose-6-phosphate. Complexation induced conformational changes in receptors 2 and 3 and/or a reduction in the rate of proton transfer from the secondary nitrogens of each receptor molecule to solvent caused by possible participation of these nitrogens in complexation can possibly explain observed variations in the fluorescence emission intensities. [0093] The observed 1 : 1 stoichiometry for complexes of receptors 2 and 3 with monosaccharide substrates and the fact that the stability constants for receptor 1 are very low and thus not determinable by UV/vis or fluorescence titrations indicate that complexation occurs by insertion of the substrate molecule into each receptor's lipophilic pocket. Additional evidence for the formation of inclusion types of complexes were found in ¹H NMR titration of receptor 2 with sialic acid, the receptor/substrate system that forms one of the most stable complexes in water, Table 2. ¹H NMR titration experiment was carried out in deuterated cacodylate buffer (0.1 M, pD=6.0) at room temperature. The concentration of 2 was kept constant at 6.5 mM while sialic acid concentration was varied form 0.006-0.075 M. Marked downfield chemical shifts were observed in the ¹H NMR spectra of receptor 2's aromatic protons of up to 0.3 ppm upon addition of sialic acid substrate accompanied with the sharpening of receptor 2's aromatic signals. ¹H NMR spectra of sialic acid were also changed due to complex formation. The most dramatic change was observed for sialic acid amide proton. Observed downfield chemical shift of this particular proton is possible only if the inclusion complex with receptor 2 is formed. On the other hand, increase in the intensity of amide signal in D₂O cannot be only attributed to the complex formation because it parallels concentration increase of the free sialic acid in solution. Overlap of the signals in the aliphatic region of the spectra prevents its use for the complex analysis. Also, due to the complex precipitation at higher sialic acid concentrations, obtained ¹H-NMR chemical shifts could not be used for the K^ determination.

[0094] The present results demonstrate that macrocyclic bi-bracchial 1,8-naphthyridine receptor 2 is capable of binding and recognition of monosaccharide substrates in water through multiple noncovalent interactions. The K_& of these complexes, determined by fluorimetrie and UV/vis titrations, cover a wide range from ~ 0.3 to >10 mM, thus displaying selectivity with respect to substrate charge, size and number hydrogen bonding pattern. Significantly, among monosaccharide substrates tested receptor 2 showed the strongest binding affinity for sialic acid (K_d = ~0.3 mM), a monosaccharide found at the termini of cell surface glycoconjugates that plays many important roles in a wide variety of physiological and pathological processes. Macrocyclic receptor 2 represents the first example of artificial receptor molecules that can not only bind specific carbohydrates in solution with millimolar affinities, but also recognize this structure on the cell surface, mimicking the monosaccharide binding site in lectins. Receptor molecule 2 represents a promising basis for the development of new and more efficient carbohydrate receptors that may have broad applications in bio-analytical and medicinal fields.

Experimental Methods

[0095] General Information: All commercially available chemicals were reagent grade and used without further purification. ¹H and ¹³C NMR spectra were recorded on a Varian Mercury 400 MHz spectrometer and referenced according to a residual solvent peak as an internal standard (DMSO 2.49 ppm, and CD₃OD, 4.78 ppm). RP-HPLC analyses of reaction mixtures and products purification were performed on Finnigan SpectraSYSTEM liquid chromatography system. For RP-HPLC separation, a Phenomenex Jupiter C-18 preparative column, (250 x 21.2 mm, 10 μm particle size, 300 A pore size, flow rate 7 mL/min with linear gradient from 0% - 100% B over 70 min.) or Vydac C-18 analytical column (250 x 4.6 mM, 5 μm particle size, 120 A pore size, flow rate 1 mL/min with linear gradient from 0% -100% B over 70 min.) was employed, and eluting products were detected by UV at 315 nm. A solvent system consisting of 0.1% TFA in H₂O (v/v) as A and 0.1% TFA in CH₃CN (v/v) as B was used for HPLC elution. UV/vis spectra were recorded on a Varian Cary 3 spectrophotometer, and fluorescence spectra on a Perkin Elmer LS50B Luminescence Spectrometer.

[0096] Preparation of control compound 1 : n-Propylamine (0.191 mL, 1.32 mmol) was added to a solution of l,8-naphthyridine-2,7-carboxaldehyde (200mg, 1.16 mmol) in dry CH₂Cl₂MeOH (150 mL, 1 : 1 v/v) under argon. The reaction mixture was stirred at room temperature overnight. The solvent mixture was evaporated without heating under reduced pressure. The oily residue was redissolved in dry CH₂Cl₂ZMeOH (150 mL), cooled to 0 ⁰C, and NaBH₄ (51 mg, 1.3 mmol) was slowly added. After stirring for 4 hours at 0 ⁰C and 30 min at room temperature, the solvent was evaporated and the residue purified by RP-HPLC to obtain 1 (R_t=28.5 min., 100 mg, 32% yield). ESI HR-MS m/z: calculated [M+H⁺] 273.2074, observed [M+H⁺] 273.2079; ¹H NMR (CD₃OD, 400 MHz) δ: 1.08 (m, CH₃, 6H) 1.86 (m, CH₂, 4H), 3.19 (q, CH₂, 4H), 4.68 (s, CH₂, 4H), 7.70 (s, Ar, 2H), 8.54 (s, Ar, 2H). ¹³C NMR (CD₃OD, 100.75 MHz) δ: 10.10, 19.56, 49.40, 50.77, 121.44, 122.16, 139.31, 154.19, 156.67. [0097] Preparation of macrocyclic bi-bracchial receptor 2: A solution of tris(2- aminoethyl)amine (0.17 mL, 1.7 mmol) in dry CH₃CN/MeOH (20 ml, 1 :1 v/v) was added dropwise over 30 minutes at room temperature and under argon atmosphere to a well-stirred solution of l,8-naphthyridine-2,7-dicarboxyaldehyde (300 mg, 1.7 mmol) in dry CH₂C1₂/CH₃OH (150 mL, 1 :1 v/v). The reaction mixture was stirred at the same temperature for 18 hours. Evaporation of the solvent under reduced pressure without heating left a solid residue which was collected, washed with diethyl ether and dried under high vacuum to yield a yellow powder (270 mg) corresponding to the tetraimino intermediate. The yellow powder was redissolved in CH₂CVCH₃OH (150 mL, 2:1 v/v), cooled to 0 ⁰C, and NaBH₄ (87mg, 2.3mmol) was slowly added. The reaction mixture was stirred for an additional 4 hours at the same temperature. The solvent was evaporated, the residue dissolved in water (30 mL) and extracted with CH₂Cl₂/Me0H (3 x 50 mL, 9:1 v/v). The organic layer was dried with MgSO₄ and evaporated. The residue was purified by preparative RP-HPLC (R_t=41.7 min., 234 mg, 23% yield). ESI HR- MS m/z: calculated [M+H⁺] 601.4198, observed [M+H⁺] 601.4241. ¹H NMR (D₂O, 400 MHz) δ: 2.84 (t, 8H) 2.88 (t, 4H) 3.08 (t, 4H) , 3.30 (t, 8H), 4.59 (s, CH₂, 12H), 7.57 (d, Ar, 6H), 8.33 (d, Ar, 6H). ¹³C NMR (D₂O, 100.75 MHz) δ: 35.35, 44.67, 48.92, 51.53, 114.88, 117.78, 122.62, 123.43, 140.55, 153.78, 155.67. [0098] Preparation of macrobicyclic receptor 3: Receptor 3 was prepared according to the procedure outlined above for receptor 2. Only in this case 1.5 equivalent of 1,8-naphthyridine- 2,7-dicarboxyaldehyde was used. R_t=44.5 min., 397 mg, 31% yield. ESI HR-MS m/z: calculated [M+H⁺] 755.4734, observed [M+H⁺] 755.4742. ¹H NMR (D₂O, 400 MHz) δ: 3.15 (t, CH₂, 12H) 3.37 (t, CH₂, 12H), 4.52 (s, CH₂, 12H), 7.47 (d, Ar, 6H), 8.30 (d, Ar, 6H). ¹³C NMR (D₂O, 100.75 MHz) δ: 44.31, 48.75, 51.13, 114.87, 117.77, 121.96, 122.42, 140.19, 153.56, 155.62. [0099] UV/vis and fluorescence spectra of 1-3: For the UV/vis experiments solutions of compounds 1 - 3 were prepared in cacodylate buffer (c= 0.05 mol dm^"3, pH 6.5) each. The concentration of 1 was 2 x 10^"4 M, 2 was 10^"4 M, and 3 was 6.7x 10^"5 M respectively. The spectra were taken at room temperature. The florescence emission spectra for both compounds were taken under the same conditions. Only in this case, the concentration of 1 was 6 x 10^"7 M, and receptor 2 was 3 x 10^"7 M, respectively. All the concentrations tested were within the linear response of absorbance or fluorescence intensity versus concentration.

[00100] Determination of pK_a: For the pH titration experiment 6.5 mg of receptor 2 was dissolved in 0.21 mL of water (0.0515 M), and titrated with 0.08843 M solution of NaOH. The NaOH solution was standardized with potassium hydrogen phthalate using phenolphthalein as the indicator. The change of the UV/vis absorption spectra of receptor 2 as the function of pH was determined in the following buffers: for pH 1-3 phosphate buffer, pH 4-5 acetate buffer, pH 6-8 phosphate buffer, pH 9 Tris HCl buffer, pH 10-11 piperidine, pH 12 and 13 phosphate buffer. In all cases buffer concentration was 0.05 M. The concentration of 2 was 3.6 x 10^"5 M. [00101] Binding Studies: The stability constant of the complexes between the 1,8- naphthyridine receptor 2 and various monosaccharides were determined by UV/vis titration experiments in cacodylate buffer (0.05 M, pH 6.5) at room temperature. In these experiments the concentration of receptor 2 or 3 was kept constant (8 x 10^"5 M) while the concentration of monosaccharide substrates was varied from 3 x 10^"5 to 1.8 x 10^"3 M. The monosaccharide substrates were prepared in aqueous solution of the receptor (8 x 10^"5 M) in order to keep the receptor concentration constant. Under these conditions the UV/vis absorbance of the receptor 2 was proportional to its concentrations. The stability constants (log K₁) for the complex of 1 :1 stoichiometry were calculated using SPECFIT software. In all calculations, the substrate concentration range corresponding to cca. 20-80% complexation was used. All binding experiments were performed in triplicate. [00102] ¹H-NMR titration experiment: The ¹H-NMR spectra were recorded in cacodylate buffer (100 mM, pD 6.0) at room temperature. In these experiments the concentration of receptor 2 was kept constant (6.5 mM), while the concentration of sialic acid was varied from 6.3-75.3 mM. The signal of the cacodylate protons in the ¹H NMR spectrum was used as an internal standard (0.00 ppm).

[00103] Cell labeling assay: The cells were cultured in modified Eagle's Minimum Essential Media (EMEM, ATCC) supplemented with 10% fetal bovine serum (ATCC) in a humidified atmosphere of 5% CO₂ in air. For microscopy experiments the cells were harvested from sub- confluent (-80%) cultures using a trypsin-EDTA solution and re-suspended in fresh medium. The cells (5 x 10⁵) with a >90% viability, as determined by trypan blue exclusion, were plated in 35 mm plates in complete medium and incubated overnight at 37 ⁰C in a humidified atmosphere of 5% CO₂ in air. After removal of the medium, the cells were fixed with MeOH, and the plate blocked with 10% BSA in PBS at 4 ⁰C. In the next step, the blocking solution was removed, and the cells gently washed with serum free media (3 x 2 mL) before adding an additional aliquot of serum free media (2 mL) containing receptor 2 (1 mM). The cells were incubated with 2 for 2 hours at 37 ⁰C. Non-bound 2 was washed with serum free media (3 x 2 mL). The bound 2 accumulation was captured with the Olympus 1X70 Inverted Fluorescence Microscope camera (370 nm excitation, 420 nm emission).

Example 3 - Polyvalent Artificial carbohydrate receptors

[00104] A major obstacle in the design of receptors for carbohydrate binding in water lies in the three-dimensional complexity of carbohydrate structure and the fact that hydrogen-bonding interactions in water are weak. One of the possible solutions to overcome this obstacle could be achieved by construction of a water-soluble system containing a spherical intermolecular cavity into which the carbohydrate substrate may be included. In addition to this, by introducing chirality via amino acid side chains into the binding site, the resulting synthetic receptor could carry out the enantioselective recognition of chiral carbohydrate substrates. On the other hand, synthetic polyvalent receptors possess multiple copies of monosaccharide recognition elements, which are found quite regularly in biological systems. The advantages of polyvalent receptors over their monovalent counterparts include an ability to create conformal contact between large biological surfaces, to produce graded responses with a single type of interaction and to increase the specificity and affinity toward carbohydrate substrates. In addition, compared to monovalent binding, multivalent interactions exhibit greater reversibility in the presence of competing receptors. Therefore, low affinity multivalent interactions are less likely to entrap cells in unproductive binding events. Also, binding events mediated by multiple weak interactions are expected to be more resistant to shear stress, such as that encountered when cells interact in the blood stream. Polyvalent synthetic mimetics of carbohydrate binding proteins, e.g., compound 21-28 (Figure 11), could have a variety of biomedical applications, among which the development of new cancer diagnostic and therapeutic agents are particularly important. These molecules are soluble in water, they posses the necessary three-dimensional structure, suitable flexibility and multiple copies of lipophilic (naphthyridine) binding pockets, where binding of monosaccharides can occur through a combination of hydrophobic interaction and hydrogen bonds. In addition, naphthyridine aromatic units, besides participation in binding, exhibit strong absorption and fluorescence emission bands, and complexation-induced changes in the intensity of their fluorescence emission can be exploited to assess their affinities toward Lewis oligosaccharides in solution and on the cancer cell-surface.

[00105] The spectrophotometric titration experiments performed with the variety of monosaccharide substrates revealed that the monovalent receptor 2 (Figure 1) has the highest affinity toward sialic acid. In addition, the results described herein showed that monovalent receptor 2 can specifically label the sLex-expressing HepG2 cells, it can inhibit anti-sLex mAb binding to sLex antigen in a competitive ELISA assay, and it can inhibit adhesion of the sLex- expressing HepG2 cells to E-selectin suggesting that the terminal sialic acid moiety from sLex antigen is recognized by receptor 2.

[00106] Since multiple interactions with multivalent targets are required for physiologically relevant binding, it is anticipated that incorporation of multiple copies of monovalent binding and recognition elements (e.g. 2) into the receptors' three-dimensional structure will enhance the receptors selectivity and affinity. In addition, the relatively long spacers between monovalent binding units may also promote the receptors selective binding to the cell-surface where multivalent oligosaccharide ligands are present in multiple copies. Advantages of this approach include relatively small size of the receptor molecule, stability (through non-natural composition), increased half-life, lack of immunogenicity, ease of synthesis, and low cost. [00107] Synthesized bi-bracchial macrocyclic receptor 2 (Figure 1) and cage-type macrobicyclic receptor 3 (Figure 1) possess a naphthyridine core moiety bridged with two flexible TRENs. In these receptors, two adjacent pyridinic nitrogen atoms acts as the proton acceptors (AA), while lateral TREN secondary amines function as the proton donors (DD). The relay of hydroxyl groups in monosaccharides offers an ADDA motif complementary to the DAAD array of naphthyridine receptors to form multiple H-bonds. In addition to this, synthesized receptor molecules differ in their flexibility and conformational adaptability toward carbohydrate substrates. Since naphthyridine moieties act as the sensing chromophores as well, fluorescence spectroscopic methods were used to detect saccharides complexation. 1,8- Naphthyridine dicarbaldehyde, a key receptor intermediate, was synthesized as previously reported (He and Lippard, Tetrahedron 56:8245-8252, 2000). The macrocyclic compound 2 (receptor 2, Figure 1) was obtained by condensation of equimolar amount of TREN and 1,8- naphthyridine dicarbaldehyde, while this ratio was 2:3 for the synthesis of macrobicyclic receptor 3 (Figure 2). Corresponding final products were purified by RP-HPLC, and fully characterized by HR-MS, and NMR spectroscopy. Receptor 1 (Figure 1) was prepared for comparison purposes using the same synthetic strategy. All spectroscopic measurements were performed in cacodylate buffer at pH 6.5, similar to the characteristic tumor cell extracellular pH (~6.8). [00108] In order to determine the suitability of pH 6.5 for the binding studies, the pK_Α values for receptors 2 and 3 were estimated based on pH-induced changes in its absorption spectra. In both cases, lowering the pH of the receptor's solution from 11 to 8 caused a decrease of the absorption intensity at 315 nm, while further lowering the pH of the solution from 8 to 1 resulted in the opposite effect. The findings described herein indicate that protonation occurs firstly at primary and secondary nitrogen sites in TREN bridges, while protonation of remaining nitrogens require much lower pH (pK_Α for unsubstituted 1,8 -naphthyridine is 3.39). Therefore, it can be assumed that at pH 6.5, only lateral TREN primary and secondary amines are protonated. The repulsive interactions between positively-charged secondary nitrogen atoms from TREN bridges may be expected to prevent self-stacking of 1,8- naphthyridine units and create a hydrophobic binding pocket. This was confirmed by observed hypochromic effect in the UV/Vis and fluorescence emission spectra of receptors 2 and 3 compared to reference compound 1. The dissociation constants (Ki) for the complexes between 1,8-naphthyridine receptors 1-3 with a variety of neutral and negatively-charged monosaccharides were determined by fluorimetric and UV-Vis titration, and the observed values are in the range from ~ 0.3 to ≥IO mM (Table 3), similar to the A^ determined for lectin/monosaccharide complexes. The highest affinities were obtained for the complexes between negatively-charged monosaccharides and receptor 2. Upfield chemical shifts in the ¹H NMR spectrum of receptor 2 aromatic protons in the presence of sialic acid confirmed that complexation occurred. All carbohydrate substrates tested did not change appreciably the fluorescence of UV -vis spectra of the reference compound 1, even at high substrate concentrations (500-fold excess with the respect to concentration of compound 1). [00109] The results described herein demonstrate the feasibility of constructing an artificial receptor molecule capable of binding to specific cancer cells. Compound 2 can be used for the design of new and more efficient artificial carbohydrate receptors that may be used as lead structures for the development of novel types of diagnostic agents, drug delivery systems or new therapeutic agents.

Table 3. Dissociation constants (AVmM) for 1 :1 complexes between various carbohydrate substrates and 1,8-naphthyridine receptors in water (pH 6.5)

Example 4 - Bivalent and trivalent artificial carbohydrate receptors.

[00110] The results described above demonstrated that monovalent receptor molecules designed from 1,8-naphthyridine units are capable of binding various monosaccharides in aqueous media, displaying structure selectivity with respect to the ring size and charge. Particularly interesting is macrocyclic receptor 2, which not only displayed the highest affinity toward sialic acid in water, but also showed an interesting activity on a cellular level. The flexible structure and presence of the side chains carrying carboxylic or amino functional groups in receptor 2 make this molecular framework suitable for further chemical modification. [00111] The cage-like molecules, such as monovalent receptor 2, represent particularly interesting topology for "enveloping" molecular species. The presence of hydrogen donor/acceptor groups within a three-dimensional structure would permit monosaccharide substrates not only to be complexed but to be encapsulated, allowing therefore monosaccharide binding in water. Furthermore, in this type of monovalent receptors, the three-dimensional lipophilic cavity is already pre-organized for the three-dimensional substrate binding, meaning that there is less entropically, and often enthalpically, unfavorable conformational rearrangements that must take place in order to adopt the optimum complex geometry. [00112] High specificity of multivalent receptors relies not only on the complementarity of the individual binding sites with a particular carbohydrate substrate but also on the relative position of the binding sites to each other. Considering this, the design of the multivalent artificial receptors is based on the findings that in typical E-selectin, the distances that separate carbohydrate binding sites are about 40 A (Stahn et al., Glycobiol. 8:311-319, 1998). Therefore, long spacers are preferred for linking monovalent binding sites for interactions with multiple ligands at the cell surface. In the multivalent receptors, the length and conformation of the spacer may influence a receptor's binding to carbohydrate substrates. For example, the structural features of the multivalent receptors such as rigidity of the scaffold displaying the binding determinants and their relative position affect the entropy of binding.

[00113] Because most spacers do have conformational degrees of freedom and it is unlikely that the spacing between a substrate and receptor binding sites can be precisely matched, the functional affinity of multivalent receptors will therefore reflect both the advantages of conformational entropy gains and the disadvantages of conformational entropy penalties. To assess the effect of the spacer length on a receptor's affinity and selectivity toward Lewis oligosaccharides and sLe^x expressing HepG2 cells, di-, tri- or other multivalent 1,8- naphthiridine-based artificial receptors exhibiting distances >40 A and <40 A between their naphthiridine mononeric binding sites can be prepared. Taking into consideration that a typical carbon-carbon covalent bond has a bond length of 1.54 A, and carbon-nitrogen 1.47 A, proposed (Fig. 13) peptidic spacers composed of Lys core matrix and 6-aminohexanoic, 8-aminooctanoic or 12-aminododecanoic acids will result in -40 A, -50 A and -60 A distances between the two receptor binding sites. Shorter distances can be obtained by attaching naphthiridine binding units directly to the Lys core matrix (-25 A). Conformational changes (defolding) associated with much longer spacer arms then those proposed herein may reduce a receptor's binding potency. Incorporation of proposed spacers into a multivalent receptor's framework allows more insight into their structure-activity relationship. The receptor with the highest binding affinity and increased inhibition potency serves as a lead scaffold for further optimization using a combinatorial chemistry approach. In addition, introduction of the Lys residue into a multivalent receptor's framework also assures its chirality and may improve its selectivity. This concept is extended to peptides with different sequences, different number of binding units, and spacer optimization using a combinatorial chemistry approach.

[00114] A general solid-phase synthetic route to di- and trivalent receptors 21-28 (Figure 11) is outlined. To synthesize these multivalent receptors, the facile assembly, high purity, and potential for combinatorial diversity afforded by solid-phase peptide synthesis (SPPS) is exploited. A strategy for the synthesis of these di- and trivalent receptor molecules is summarized as follows: synthesis of bis(aminoethyl)aminoacetic acid or any other poly-amino acid 14 that is necessary for completion of the 1,8-naphthyridine macrocyclic scaffold containing carboxylic groups, amino groups or amino acid functionalities; solution synthesis of 1,8-naphthyridine macrocyclic scaffolds 17 and 18 with suitable arms for solid-phase attachment to peptidic spacers according to the protocols already developed (Figure 12); solid-phase synthesis of different length lysine or other amino acid based spacers that include 6- aminohexanoic, 12-aminododecanoic acid or other fatty acids; coupling of 1,8-naphthyridine macrocyclic scaffolds 17 and 18 using standard SPPS protocols; and cleavage of the final product from the resin, product purification and characterization, (Figure 13). [00115] Advantage is taken of polyvalent receptors 21-28 relatively simple total solid-phase synthesis and a general route for the synthesis of their soluble combinatorial libraries is developed. Given the 20 amino acids building blocks, even short peptidic spacers between naphthiridine binding and recognition elements offer enormous diversity and potential for design and development of new and efficient carbohydrate receptors. In addition, this approach also permits further modification of the receptor molecules in order to develop novel carrier systems for anticancer drugs that will specifically distinguish diseased from healthy cell/tissue. [00116] Synthesis of poly-amino acid bridges: By incorporation of bis(aminoethyl)amino valeric acid or any other poly-amino acids 14 (Figure 12A) into a 1,8- naphthyridine macrocyclic framework, the required monovalent receptor's flexibility and overall positive charge is maintained. In addition, the presence of the carboxylic groups make this naphthyridine macrocyclic scaffold suitable for attaching to a desired peptide tether using standard solid-phase methodology. Bis(aminoethyl)aminoacetic acid or any other poly-amino acid bridge can be prepared from commercially available diethyltriamine 11 (Figure 12A) as described by J. Zubeta et al.(Inorg. Chem. Commun. 7:481-484, 2004) According to this protocol, the selective protection of the primary amine groups of diethyltriamine with phthalic anhydride in acetic acid, followed by reaction with appropriate bromo acidic acids or side chain activated amino acid (e.g. Ser, Thr, Cys) in the presence of KI, yields fully protected poly-amino acid bridges 14. Removal of the protecting groups with concentrated HCl yield the desired crude poly-amino acid bridge 12 as the hydrochloride salt. The crude product 12 is then purified by extraction with ether to remove all organic impurities, and fully characterized by mass spectrometry and NMR spectroscopy and MS spectrometry.

[00117] Synthesis of 1,8 -naphthyridine monovalent binding units 17 and 18 suitable for solid- phase modification: According to standard SPPS protocols, carboxylic group of the amino acid needs to be activated in solution prior to its coupling to free primary amine already attached to the resins. Since receptor 2 has two "arms" with free primary amino groups, this compound is modified for solid-phase synthesis. Therefore, 1,8-naphthyridine units are bridged with bis(aminoethyl)aminoacetic acid or any other poly-amino acid 14, that generate the desired compound with free carboxylic groups instead (Figure 12B). In addition, a significant increase in the macrocyclization yields is expected since a macrobicyclic compound, such as receptor 3, cannot be formed. Macrocyclic compound containing 1,8-naphthyridine units bridged with appropriate poly-amino acid 14 are prepared according to the same protocol as described for compound 2.

[00118] Condensation of equimolar amounts of bis(aminoethyl)aminoacetic acid or any other poly-amino acid 14 and 1,8-naphthyridine dicarbaldehyde 10 under high dilution conditions yield imine precursor. Reduction of this precursor with NaBFLt₁ followed by carboxylic group deprotection, and purification by RP-HPLC results in compound 17 suitable for incorporation into a desired peptidic chain(s) on the solid-support. Scaffold 18 is prepared in the same way by reacting 10 with GIy, and is used for the construction of control compounds 29-36. Control compounds are based on compound 1 which, according to the results described herein, does not bind sialic acid in water.

[00119] Solid-phase synthesis of 1,8-naphthyridine based di- and trivalent receptors 21-28 and control compounds 29-36: A central component defining the branched architecture is the core matrix which multimerizes monovalent binding units to give them the desired arrangement. Amino acid lysine is commonly used in the core matrix because it has two amino groups available for the branching reactions. The first step in total SPPS of these receptor molecules is anchoring of orthogonally protected Fmoc-Lys(Alloc)-OH (Chem-Impex International, Inc.) to the resins. The Alloc group can be selectively removed in the presence of other protective groups by treatment with PdCl₂(PPh₃)₂, making this derivative an extremely useful tool for the preparation of modified peptides by Fmoc SPPS (Grieco et al., Pept. Res. 57:250-256, 2001). Taking into consideration the compatibility of the deprotection and cleavage conditions with the resin linkage, Rink amide PEGA resins (substitution level 0.2-0.5 mmol/g, Novabiochem) are used. Lysine attachment to amide resins, and assembly of Lys core matrix are performed according to the standard HBTU/HOBt/NMM coupling protocol. To assure proper distance within the receptor's binding units for optimal interaction with polyvalent carbohydrate substrates, lysine's side chain is elongated with commercially available Fmoc-6-aminohexanoic acid (Anaspec), Fmoc-8-aminooctanoic acid (Anaspec) or Fmoc-12-aminododecanoic acid (Focus Synthesis LLC) (Figure 13).

[00120] During this elongation, only side chain £--NH₂ groups of the Lys residues need to be deprotected, having the Qf-NH₂ group of the last Lys residue protected throughout the solid-phase assembly of the naphthiridine polyvalent receptors 21-28 and controls 29-36. After selective Fmoc removal with 20% piperidine in DMF, compound 17 is coupled to 19 or 20 using the same HBTU/HOBt/NMM coupling protocol (Figure 15). To assure coupling of the bulky compound 17 to the Lys-based spacer, low substitution level resins are used. In addition, to minimize the possibility of coupling of one molecule of 17 to two tether's side chains, 17 is added to the reaction mixture in a large excess with the half equivalent of the coupling reagents. Cleavage of the assembled receptors from the resins using a 95% TFA, 2.5% TIS, 2.5% H₂O mixture yields crude final products 21-28 (Figure 11). Sterical hindrance can pose a problem and affect attachments of bulky 1,8-naphthyridine carbohydrate binding units 17 to the poly-Lys spacer. [00121] In order to establish the optimal conditions for coupling of 17, different methods for activation of the carboxylic group are tested such as activation with (9-(7-azabenzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), (2-(benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU), 1-benzotriazolyloxytris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP), 1,3-diisopropylearbodiimide (DIC), etc. This strategy affords unimpeded peptide tether assembly by standard solid-phase Fmoc chemistry, yet also leaves the NH₂ group of the amino fatty acids for the 1,8-naphthyridine binding units, 17, attachment.

[00122] The efficiency of the coupling reactions are monitored by Kaiser test and MALDI- TOF MS analysis. The control compounds 29-36 are synthesized in a similar manner (Figure 14). In the advent of the solid-phase synthesis failure, solution synthesis is investigated (Figure 15). Lys-based spacers are assembled on the Rink amide PEGA resins as previously described (J.P. Tarn, J. Immunol. Methods 196:17-32, 1996). However, in this case, Z-Lys(Alloc)-OH is coupled as the last amino acid in order to keep the protected iV-terminus of the spacer(s) throughout the entire receptor synthesis. After selective Alloc removal, peptidic spacers are cleaved from the resins and purified by RP-HPLC. Naphthyridine compound 17 is then coupled in DMF according to the standard SPPS protocol. Final deprotection is achieved by catalytic hydrogenation with 10% Pd/C as a catalyst. In both cases, crude products 21-36 are purified by RP-HPLC, and fully characterized by analytical RP-HPLC and MALDI-TOF mass spectrometry. [00123] Determine the affinity of divalent 21-24 (FIG. 15) and trivalent 25-28 (FIG. 15) receptors toward Lewis oligosaccharide antigens: Since polyvalent binding is important for high affinity and selective binding of carbohydrate substrates, the effect of the number of monovalent binding units and the role of tether's length in receptor binding to Lewis oligosaccharides is investigated. In addition, defining the structural requirements (e.g., the number of binding units or the length of the tether which multimerizes binding units) responsible for receptor specific carbohydrate binding facilitates solid-phase synthesis of artificial polyvalent receptors for Lewis oligosaccharides by reducing structural complexity and also promotes judicious chemical modifications for structure-function analyses and modulation of their biological or physiochemical properties (Kragol et al, Eur. J. Biochem. 269:4226-4237, 2002). The information gained from these studies assists the design of new and more selective polyvalent receptors leading the way to a new generation of sensors for tumor-associated carbohydrate antigens, drug delivery systems or new chemotherapeutics that target these antigens. [00124] Determine the affinity of polyvalent receptors 21-28 (FIG. 15) and control compounds 29-36 (FIG. 14) for Lewis oligosaccharides in solution by fluorescence titration and/or NMR spectroscopy: Fluorescence titration experiments are used to determine the affinities of 21-36 toward sLe^x, sLe^a, Le^x and Le^y oligosaccharides in solution. All four Lewis oligosaccharides are commercially available from Sigma. The titration experiments are performed as described above. Concentration of 21-36 are kept constant while concentration of Lewis oligosaccharide substrates vary. All titrations are carried out in phosphate buffer at pH 6.8 (tumor cell extracellular pH) and at room temperature. As an alternative method, NMR spectroscopy is also used to determine affinity of these receptors toward Lewis oligosaccharides. In this case, the affinity constants of the complexes between polyvalent receptors 21-36 and Lewis oligosaccharides are determined by ¹FI NMR titration experiments in deuterated aqueous buffer (e.g. cacodylate buffer pD = 6.8). In general, the concentration of the Lewis oligosaccharide substrate is kept constant, while the concentration of the polyvalent receptor is varied. The data is fitted by nonlinear least-squares analysis.

[00125] Determine the binding efficacy of polyvalent receptors 21-28 and control compounds 29-36 toward BSA bound sLe^x (modeling cell surface-expressed sLe^x) by competitive ELISA assay: The role of sLe^x oligosaccharide in binding of receptors 21-36 is examined by competitive ELISA assay. Solution of sLe^x-BSA (EMD Biosciences) (0.1 μg/mL) and control BSA (1 μg/mL) in 0.1 M NaHCO₃ (pH 8.6) is added to a 96-well microplate (Costar). Each well is brought to 100 μL and incubated overnight at 4 ⁰C. The supernatant is then removed, and 200 μL of blocking buffer (1 mg/mL BSA in 0.1 M NaHCO₃, pH 8.6) is added to each well. After incubation for 1 h at 4 ⁰C, each well is washed with 200 μL of PBS. To measure specific binding of 21-36 to sLe^x-BSA, mixtures of anti-sLe^x monoclonal antibody (5 μg/mL, KM93; EMD Biosciences) and 21-36 (various concentrations) in PBS with 1% BSA are added to each well and incubated for Ih at room temperature. Wells are washed five times with PBS. In the next step, the goat anti-mouse IgM-horseradish peroxidase conjugate (1 :10,000 dilution in PBS) is added to each well (100 μL) and resulting wells are incubated for Ih at room temperature. Wells are then washed five times with PBS and 3,3',5,5'-tetramethylbenzidine (TMB) substrate (ready- to-use peroxydase substrate in a mild acidic buffer, Sigma) is added (100 μL). After 10-15 min of incubation at room temperature, the reaction is quenched by adding 100 μL of stop reagent (Sigma). Absorbance of a blue reaction product is read at 630 nm using an Awareness Technology Stat Fax-2100 96-well microtiter plate reader (the stop reagent does not produce a spectral change from blue to yellow). In this way, potential overlap in absorbances between receptors 21-36 and blue peroxidase product is avoided. Concentration dependent changes in absorbance at 630 nm indicate that receptors 21-36 and anti-sLe^x monoclonal antibody share the same ligand.

[00126] Assess divalent 21-24 and trivalent 25-28 receptor ability to bind sLe^x expressing cells and inhibit cell adhesion and invasion: The results described above showed that monovalent receptor 2 (Figure 1) binds preferentially to the sLe^x-expressing HepG2 cells. Cell binding studies are performed with receptors 21-36 in order to assess their affinity and specificity toward sLex-expressing HepG2 cells qualitatively by fluorescence microscopy and quantitatively by immunocytochemistry assay. However, sLe^x antigen is not merely a marker of cancer cells, but also mediates cancer cell adhesion to vascular endothelial cells and promotes hematogenous metastasis of cancer. The interaction of cancer cells and endothelium through sLe^x epitopes provides a target for inhibition by receptors 2 and 21-36, and could therefore be useful in the prevention of cancer cell metastasis.

[00127] Determine the ability of polyvalent receptors 21-28 and control compounds 29-36 to bind model sLe^x expressing (HepG2) cells qualitatively by fluorescence microscopy: The ability of synthesized polyvalent receptors 21-36 to label sLe^x-expressing HepG2 cells (ATCC HB- 8065), as a model system, is tested using fluorescence microscopy. The microscopy experiments are performed in the same way as the experiments described above. Briefly, HepG2 cells (ATCC) are cultured in modified Eagle's Minimum Essential Media (EMEM) (ATCC) supplemented with 10% fetal bovine serum (ATCC) in a humidified atmosphere of 5% CO₂ in air. For microscopy experiments, cells are harvested from sub-confluent (-80%) cultures using a trypsin-EDTA solution and then re-suspended in fresh medium. Cells (5 x 10⁵) with a >90% viability, as determined by trypan blue exclusion, are plated in 35 mm plates in complete medium and incubated overnight at 37 ⁰C in a humidified atmosphere of 5% CO₂ in air. The cells are then washed with serum-free media (3 x 2 mL) before adding an additional aliquot of serum free media (2 mL) containing polyvalent receptors (final concentration is determined). The cells are incubated with 21-36 for 2 h at 37 ⁰C. Non-bound polyvalent receptors 21-36 are washed with serum-free media (3 x 2 mL). The bound fluorophore accumulation is captured with the Olympus 1X70 Inverted Fluorescence Microscope camera (370 nm excitation, 420 nm emission). For controls, the same experiment is performed with Le^y-expressing Hep3B cells and control fibroblast cells.

[00128] Determine the ability of polyvalent receptors 21-28 and control compounds 29-36 to bind model sLe^x expressing (HepG2) cells quantitatively by immunocytochemistry assay: To examine whether sLe^x antigen is essential for binding of 21-36 to sLe^x-expressing HepG2 cells, a cell based ELISA competition assay is performed. The assay is carried out in a 96-well plate format. The cells (number of cells/well to be determined) are seeded in complete medium and incubated overnight at 37 ⁰C in a humidified atmosphere of 5% CO₂ in air. After removal of the medium, the cells are fixed with 4% paraformaldehyde, and the plate blocked with 10% BSA in PBS at 4 °C. In the next step the blocking solution is removed, and the cells are gently washed with washing buffer (PBS with 1 mM CaCl₂ and 0.5 niM MgCl₂). To measure specific binding of polyvalent receptors to HepG2 cells, mixtures of anti-sLe^x monoclonal antibody (KM93; 1.0 nM) and 21-36 (various concentrations) in PBS with 1% BSA are added to each well and incubated for 1 hour at room temperature. Each well is then washed with washing buffer and the goat anti-mouse IgM-horseradish peroxidase conjugated secondary antibody (1 :10,000 dilution in PBS) is added. The plate is incubated for an additional 1 hour at room temperature. Wells are then washed with washing buffer and TMB substrate (100 μL, ready-to-use peroxydase substrate in a mild acidic buffer, Sigma) is added. After 10-15 min of incubation at room temperature, the reaction is quenched by adding 100 μL of stop reagent (Sigma) and absorbance is measured using an Awareness Technology Stat Fax-2100 96-well microtiter plate reader at 630 nm. A concentration-dependent decrease in absorbance at 630 nm indicates that receptors 21-36 and anti-sLe^x monoclonal antibody have a common target on the cell surface. As a control, the same experiment is performed with Le^y-expressing Hep3B cells and anti-Le^y monoclonal antibody (F3, EMD Biosciences). In this way, the hypothesis that artificial polyvalent receptors 21-36 preferentially bind sLe^x antigens on the cell surface is confirmed.

[00129] Toxicity assay: Malnourished or dying cells can leak proteins into their surroundings. This process can interfere with the binding of 21-36 to the cell surface sLe^x antigen and produce false negative results. In order to assess this possibility, toxicity of synthesized polyvalent receptors toward HepG2 cells is determined using an MTT cell proliferation assay. Cells (5000 cells/well) are seeded in 96- well plates in complete medium and incubated overnight at 37 ⁰C in a humidified atmosphere of 5% CO₂ in air. The medium is removed and each well treated with lOOμl medium containing different concentration of 21-36. The cells are treated for 1-2 days at 37⁰C in a humidified atmosphere of 5% CO₂ in air. At the end of the incubation time period, the medium is removed from the cells and 100 μl of MTT (Sigma, dissolved in serum free-medium at 1 mg/ml) is added per well. The cells are incubated for 3 h at 37°C. The MTT medium is removed and 100 μl of DMSO is added to each well. The plate is shaken at 60 rpm for 5 min before the reading at 540 nm in a microplate reader (SpectraMax). A decrease in absorbance compared to the control cells indicates a reduction in the rate of cell proliferation. [0100] Cell adhesion assay: Since sLe^x and sLe^a expression on cancer cells mediates their adhesion to vascular endothelial cells and promotes hematogenous metastasis of cancer, agents that inhibit this process have the potentials to be of considerable therapeutic value. With this assay, receptors 21-36 are investigated for their ability to inhibit tumor cell adhesion to endothelium, and further confirmation of their affinity and selectivity toward sialylated Lewis antigens is obtained.

[0101] Cell adhesion assays are performed on the plates precoated with the matrix components and plates coated with human umbilical endothelial cells (HUVEC, ATCC CRL- 1730). Pro-Bind 96 well plates (BD Biosciences) are pre-coated overnight at 4⁰C with E- selectin-BSA conjugate (2 μg/ml). Control wells are coated with 1% BSA. After coating, the wells are incubated with 1% BSA in PBS for 1 hour at 37°C, then washed with PBS before use. Cells (sLe^x expressing HepG2 and breast cancer cell lines) are labeled for 1 hour with 1 mM Calcein AM at 37°C, washed with PBS, and then detached with 5 mM EDTA in PBS. Detached cells are washed once more in PBS, resuspended in serum free media, and added to the wells (5 x 10⁴ cells per well). Various concentrations of 21-36 in PBS with 1% BSA are added to each well. The plates are then incubated at 37°C for 1 hour. Each well is washed three times in PBS, and then overlaid with serum free media prior to quantification. The percentage of adherent cells is determined using a microplate spectrofluorometer (Molecular Devices SpectraMax Gemini EM), with excitation wavelength 485 nm and emission wavelength 530 nm. Results are expressed as the ratio of fluorescence from wells containing E-selectin to uncoated control wells containing 5 x 10⁴ cells in the presence and absence of receptors 21-36. In the case of adhesion assays with the HUVEC cells (activated and non-activated), plates are seeded on pre-coated plates with 1% gelatin in complete medium and incubated overnight at 37 ⁰C in a humidified atmosphere of 5% CO₂ in air. The medium is removed and each well treated with lOOμl medium containing different concentration of 21-36, followed by cells (sLe^x expressing HepG2 and breast cancer cell lines) pre-labeled with calcein. The plates are incubated at 37⁰C for 1 hour and are treated as described above.

[0102] Cell invasion assay: Tumor proliferation, invasion and metastasis are characteristic features of the malignant phenotype, while benign solid tumors show no signs of invasion and metastasis. Therefore, a cell invasion assay is used to assess receptors 21-36 potentials as lead structures in the development of new cancer diagnostic and/or anti-metastatic agents. [0103] Invasion assays are performed using the cell invasion assay kit (Chemicon Int.) and protocol recommended by the manufacturer. In short, a 24-well tissue culture plate containing inserts with 8 μm pore size filters precoated on the upper side with ECMatrix™ is used. 300 μL of the serum free medium is placed into inserts to allow rehydration of the ECM layer for 1 h at room temperature. After removal of rehydration medium, 500 μL of medium containing 10% fetal bovine serum (chemoattractant) is added to the lower chamber. 300 μL of HepG2 cell suspension without and with compounds 21-36 at various concentrations (previously prepared in serum free medium) are added to each insert. After incubation for 24 hours, the medium is removed and non invading cells as well as the ECM gel layer are removed by a cotton-tipped swab. Invasive cells on the lower surface of the membrane are stained by dipping inserts into the staining solution for 20 min. After rinsing the inserts with water, and air drying, stained cells are dissolved in 10% acetic acid (200 μL/insert), and transferred to a 96-well plate for colorimetric reading at 560 nm.

Example 5 - Additional Embodiments of Artificial Carbohydrate Receptors.

[0104] Described herein are polyvalent artificial carbohydrate receptors having monomeric naphthididine binding units (e.g. receptor 2 of FIG. 1 or its derivatives) linked by peptidic spacers. These molecules are soluble in water, they possess the necessary three-dimensional structure, suitable conformational flexibility and multiple multiple binding and recognition sites where binding can occur through a combination of hydrophobic interaction and hydrogen bonds between receptor molecules and carbohydrate substrates. Advantages of this approach include relatively small size of the receptor molecules, stability (through non-natural composition), ease of synthesis and low cost.

[0105] Results described here showed that monomeric macrocyclic receptor 2 in Scheme 1 below is capable of binding and recognition of monosaccharide substrates in water through multiple noncovalent interactions. Since receptor 2 not only has the highest affinity toward sialic acid in water, but also showed an interesting activity on a cellular level, its structure-binding relationship was further elucidated. For this purpose, three analogs 37-39 of this receptor were synthesized. While the main structural characteristic such as structural flexibility, and presence of 1,8-naphthyridine aromatic units remain the same, these receptors differ in the connectivity of naphthyridine aromatic units, overall molecular charge at pH 6.5, and capabilities to create H- bonds, Scheme 1.

[0106] Scheme 1. Receptor 2 synthetic analogs.

[0107] As previously mentioned, bridging units for receptors 37 and 38 (Scheme 1) were prepared from commercially available diethyltriamine as described by J. Zubeta et al. (Inorg. Chem. Commun. Vol. 7:481-484, 2004), Scheme 2. According to this protocol, the selective protection of the primary amine groups of diethyltriamine with phthalic anhydride in acetic acid, followed by reaction with the 5-bromopropionate in the presence of KI, resulted in fully protected compound 40 (Scheme T). Removal of the phthalimide and ethyl protecting groups with concentrated HCl gave the desired crude bis(aminoethyl)aminopropionic acid 41 (Scheme 2) as the hydrochloride salt. The crude product 41 (Scheme 2) was fully characterized by mass spectrometry and NMR spectroscopy. Receptor 37 (Scheme 1) was prepared according to the same protocol as previously described for 2 (Scheme 1). However, preparation of receptors 38 and 39 (Scheme 1) required slight protocol modification. In this case in order to incorporate an amide functionality into receptors structure, macrocyclization was performed using 1,8- naphthyridine-2,7-dicarbonyl chloride and corresponding polyamine bridge. In all cases resulting amide receptors 38 and 39 (Scheme 1) were obtained in good yields.

[0109] Scheme 2. Molar absorption coefficients of receptors 37-39 (ε37 =6800 M^"1 ε 38

=8000 M^"1 ε 39 =7500 M^"1) are similar to the parent compound 2 (ε 2= 7372.5 M^"1 cm^"1) indicating no occurrence of significant conformational changes (e.g. absence of intramolecular stacking interactions). However, the observed additional absorption band at 325 nm in the UV/vis spectra of receptors 38 and 39 (Scheme 1) can be explained by electron transitions (n-π) in the carbonyl groups, FIG. 1. The absence of significant intramolecular stacking interactions was confirmed by comparison of receptors 37-39 (Scheme 1) fluorescence emission spectra. In all cases no fluorescence quenching was observed. The shape of receptor 39 's fluorescence emission spectra and observed bathochromic shift for 38 and 39 indicates a different fluorescence emission mechanism involving resonance stabilization and derealization of amide π-electrons over the naphthyridine aromatic ring. The resonance stabilization energy may cause these analogs to emit at a much higher wavelength and display additional emission maximum. Fluorescence emission spectra of receptors 2, 37-39 (Scheme 1) were determined. [0110] Since receptor 2 showed selectivity toward sialic acid substrate, the role of different functional groups in receptors 37-39 (Scheme 1) in binding of this substrate was investigated. Despite the fact that the carboxylate and amide functional groups play prominent roles in carbohydrate substrate binding in natural systems, receptors 37-39 (Scheme 1) displayed much weaker affinities toward sialic acid compared to 2 (Scheme 1). No significant changes in UV/vis or fluorescence emission spectra of receptors 37-39 (Scheme 1) were observed upon addition of sialic acid substrate (up to 100 equivalents) that would indicate complexation. This observation clearly indicates the importance of H-bond donors (e.g. secondary amines in receptor's bridges) and positive charge (e.g. primary amines in receptor's arms) in the receptors structure for selective negatively charged carbohydrate binding.

[0111] Synthesis of Receptor 37 (Scheme 1): A solution of compound 41 (Scheme 2) (300mg , 1.7 mmol) in dry CH₃CN/MeOH (20 ml, 1 :1 v/v) was added dropwise over 30 minutes at room temperature and under argon atmosphere to a well-stirred solution of l,8-naphthyridine-2,7- dicarboxyaldehyde (300 mg, 1.7 mmol) in dry CH₃CN/MeOH (150 mL, 1 :1 v/v). The reaction mixture was stirred at the same temperature for 18 hours. Evaporation of the solvent under reduced pressure without heating left a solid residue which was collected, washed with diethyl ether and dried under high vacuum to yield a yellow powder (270 mg) corresponding to the tetraimino intermediate. The yellow powder was redissolved in CH₂Cl₂ZMeOH (150 mL, 2:1 v/v), cooled to 0 ⁰C, and NaBH4 (87mg, 2.3mmol) was slowly added. The reaction mixture was stirred for additional 4 hours at the same temperature. The solvent was evaporated, the residue dissolved in water (30 mL) and extracted with CH₂Cl₂ZMeOH (3 x 50 mL, 9:1 v/v). The organic layer was dried with MgSO₄ and evaporated. The residue was purified by preparative RP-HPLC (Rt=37.7min), 33% yield (100 mg). Brown solid, mp= 207-208 ⁰C). ESI-MS: expected 657.3; observed [M+H] 658; ¹HNMR (D₂O, 400 MHz) δ: 7.42 (d, Ar, 4H), 6.57 (d, 4H), 4.56 (s, 4H), 3.65 (t, 8H), 3.52 (t, 4H), 2.62 (t, 8H), 1.88 (t, 4H).

[0112] Synthesis of Receptor 38 (Scheme 1): l,8-napthyridine-2,7-dicarbonyl chloride (200 mg, 0.78 mmol) was dissolved in 20OmL anhydrous acetone and compound 27 (130 mg, 0.78 mmol) was added dropwise for 2 hours and the mixture allowed to stir for 16 more hours. The resulting mixture was evaporated and the desired compound (receptor 38 Scheme 1) was purified by HPLC and characterized by IHNMR and Mass spectrometry. Yield 50% (130 mg) mp. 200- 202⁰C. IHNMR (400 MHz, D2O) δ: 8.42 (d, Ar, 4H), 7.97 (d, 4H), 3.35 (t, 8H), 3.22 (m, 4H), 2.92 (t, 8H), 1.88 (t, 4H).ESI-MS expected 714.2, observed [M+Na] 736.2. [0113] Synthesis of Receptor 39 (Scheme l):l,8-Napthyridine-2,7-dicarbonyl chloride (200 mg, was dissolved in 20OmL anhydrous acetone and TREN (120 mg, 0.80 mg) was added dropwise over 2 hours. The mixture was allowed to stir for 16 hours. The resulting mixture was evaporated and the desired compound 28 (Scheme 1) was obtained in 80% yield. Brown solid (160 mg), mp. 222-223°C. ¹H NMR (D₂O, 400 MHz) δ: 8.10 (d, Ar, 8H), 7.71 (d, Ar, 4H), 2.86 (t, CH₂, 4H), 2.80 (t, CH₂, 4H), 2.59 (t, CH₂, 8H), 2.41 (t, CH₂, 4H). ESI-MS:; expected 656.74, Observed [M+Na] 678.74. l,8-Napthyridine-2,7-dicarbonyl chloride was prepared according to the protocols described in the following references: He C. & Lippard SJ. Design and Synthesis of Multidentate Dinucleating Ligands Based on 1,8-Naphthyridine. Tetrahedron, (2000), 56, 8245- 8252; and Chandler CJ et al. The synthesis of macrocyclic polyether-diesters incorporating 1, 10-phenanthrolino and 1,8-naphthyridino subunits. J. Heterocyclic Chem. (1982), 19, 1017-1017.

Example 6 - Lectins, a Model System for the Development of Artificial Synthetic Receptors [0114] Lectins were shown to be valuable tools for the structural and functional investigation of complex carbohydrates, especially glycoproteins, and for the examination of the changes that occur on cell surfaces during physiological and pathological processes, from cell differentiation to cancer. Some lectins are studied as active anticancer compounds (e.g. mistletoe-lectins), and their potential for specific sugar interaction has attracted a great deal of attention for the development of novel carrier systems to target drugs specifically to different cells and tissues. In spite of the lectins' interesting biomedical potentials, their disadvantage is their relatively large size (immunogenicity), susceptibility to proteolytic degradation and potential toxicity. Smaller molecules which mimic the lectin function represent attractive alternatives. The expected advantages of such lectinomimetics are non-immunogenicity due to low molecular weight, enhanced stability, relatively simple synthesis and low cost, and potential rapid blood clearance. [0115] A major obstacle in the design of receptors for carbohydrate binding in water lies in the three-dimensional complexity of carbohydrate structure and the fact that hydrogen-bonding interactions in water are weak. One of the possible solutions to overcome this obstacle is achieved by construction of a water-soluble system containing a spherical intermolecular cavity into which the carbohydrate substrate may be included. In addition to this, by introducing chirality via amino acid side chains into the binding site, the resulting synthetic receptor could carry out the enantioselective recognition of chiral carbohydrate substrates. On the other hand, synthetic polyvalent receptors possess multiple copies of monosaccharide recognition elements, which are found quite regularly in biological systems. The advantages of polyvalent receptors over their monovalent counterparts include an ability to create conformal contact between large biological surfaces, to produce graded responses with a single type of interaction and to increase the specificity and affinity toward carbohydrate substrates. In addition, compared to monovalent binding, multivalent interactions exhibit greater reversibility in the presence of competing receptors. Therefore, low affinity multivalent interactions are less likely to entrap cells in unproductive binding events. Also, binding events mediated by multiple weak interactions are expected to be more resistant to shear stress, such as that encountered when cells interact in the blood stream. These molecules are soluble in water, they possess the necessary three-dimensional structure, suitable conformational flexibility and lipophilic (naphthyridine) binding pockets where binding can occur through a combination of hydrophobic interaction and hydrogen bonds between receptor molecules and carbohydrate substrates. In addition, naphthyridine aromatic units, besides participating in binding, exhibit strong absorption and fluorescence emission bands, and complexation-induced changes in the intensity of their fluorescence emission can be exploited to assess their affinities toward Lewis oligosaccharides in solution and on the cancer cell-surface. Advantages of this approach include relatively the small size of the receptor molecules, stability (through non-natural composition), ease of synthesis, and low costs. [0116] In the experiments described above, monovalent receptor molecules designed from 1,8- naphthyridine units were shown to be capable of binding various monosaccharides in aqueous media, displaying structure selectivity with respect to the ring size and charge. Particularly interesting is maerocyclic receptor 2 (FIG. 1), which not only displayed highest affinity toward sialic acid in water, but also showed an interesting activity on a cellular level. Monomeric receptor 2 (FIG. 1) labeled the sLe^x-expressing HepG2 cells, inhibited anti-sLe^x mAb binding to sLex antigen in a competitive ELISA assay, and inhibited adhesion of the sLe^x-expressing HepG2 cells. Since multiple interactions with multivalent ligands are required for physiologically relevant binding, oligomerization of receptor 2 (FIG. 1) within the receptor's core structure may be performed for improving the receptor's selectivity and affinity. Flexible structure and presence of the side chains carrying carboxylic or amino functional groups make this molecular framework suitable for further chemical modification. Analogs of receptor 2 (FIG. 1) containing multiple binding and recognition sites are synthesized. The cage-like molecules, such as monovalent receptor 2 (FIG. 1), represent particularly interesting topology for "enveloping" molecular species. The presence of hydrogen donor/acceptor groups within a three- dimensional structure would permit monosaccharide substrates not only to be complexed but to be encapsulated, allowing therefore monosaccharide binding in water. Furthermore, in this type of monovalent receptor, the three-dimensional lipophilic cavity is already preorganized for the three-dimensional substrate binding, meaning that there is less entropically, and often enthalpically, unfavorable conformational rearrangements that must take place in order to adopt the optimum complex geometry. On the other hand, high specificity of multivalent receptors relies not only on the complementarity of the individual binding sites with a particular carbohydrate substrate but also on the relative position of the binding sites to each other. Design of the multivalent artificial receptors described herein is based on the findings that in typical E- selectin, the distances that separate carbohydrate binding sites were estimated to be about 40 A. Therefore, long spacers are required to link monovalent binding sites for interactions with multiple ligands at the cell surface. To assess the effect of the spacer length on receptors' affinity and selectivity toward Lewis oligosaccharides and sLe^x expressing HepG2 cells, di- and trivalent 1,8-naphthyridine based artificial receptors exhibiting distances >40 A and <40 A between their naphthyridine mononeric binding sites are synthesized. Taking into consideration that a typical carbon-carbon covalent bond has a bond length of 1.54 A, and carbon-nitrogen 1.47 A, proposed peptidic spacers composed of Lys core matrix and 6-aminohexanoic, 8-aminooctanoic or 12- aminododecanoic acids will result in -40 A, ~50 A and -60 A distances between the two receptor binding sites. Shorter distances will be obtained by attaching naphthiridine binding units directly to the Lys core matrix (~25 A). Incorporation of proposed spacers into multivalent receptors' framework allows additional insights into their structure-activity relationship. The receptor with the highest binding affinity and increased inhibition potency serves as a lead scaffold for further optimization using a combinatorial chemistry approach. In addition, introduction of the Lys residue into multivalent receptors' framework assures their chirality and may improve their selectivity. This concept can be extended to peptides with different sequences, and different numbers of binding units and spacer optimization using a combinatorial chemistry approach.

[0117] A general solid-phase synthetic route to di- and trivalent receptors 49-56 is designed. To synthesize these receptor molecules, advantage is taken of the facile assembly, high purity, and potential for combinatorial diversity afforded by solid-phase peptide synthesis (SPPS). The strategy for the synthesis of these di- and trivalent receptor molecules is summarized as three steps. [0118] Step 1 : synthesis of bis(aminoethyl)serine bridge 44 as outlined in Scheme 4 below.

43 44

[0119] Scheme 3. Synthesis of bis(aminoethyl)serine bridge 44.

[0120] Step 2: synthesis of monovalent binding units 46 and 48 suitable for solid-phase coupling according to the protocol outlined in Scheme 4.

[0121] Scheme 4: Synthesis of receptors binding units 46 and 48.

[0122] Step 3: Solid-phase synthesis of 1,8-naphthyridine based di- and trivalent receptors

49-56 and control compounds 57-64 in Scheme 6 below.

d-<

Lysine (K)

20% piperidine/DMF; b) Fmoc-Lys(Alloc)-OH or Boc-Lys(Alloc)-OH, HBTU, HOBt, NMM; c) PdC12(PPh3)2; d) Fmoc-NH-(CH2)nCOOH (n=5, 7, 11), HBTU, HOBt, NMM; e) 20% piperidine/DMF; f) 10, or 12, HBTU, HOBt, NMM; g) 95% TFA, 2.5% TIS, 2.5% H2O; h) H2, 5% Pd/C. [0123] Scheme 5. Solid-phase synthesis of polyvalent receptors 49-56, and control compounds 57-64.

[0124] In the event of the solid-phase synthesis failure, solution synthesis is investigated. Lys- based spacers are assembled on the Rink amide PEGA resins as previously described. However, in this case, Z-Lys(Alloc)-OH is coupled as the last amino acid in order to keep the protected N- terminus of the spacer(s) throughout the entire receptor synthesis. In both cases, crude products 16-31 (Scheme 5) are purified by RP-HPLC, and fully characterized by analytical RP-HPLC and MALDI-TOF mass spectrometry.

[0125] Advantage is taken of polyvalent receptors 49-56 (Scheme 5) relatively simple total solid-phase synthesis and develop a general route for the synthesis of their soluble combinatorial libraries. Given the 20 amino acids building blocks, even short peptidic spacers between naphthyridine binding and recognition elements offer enormous diversity and potential for design and development of new and efficient carbohydrate receptors. This approach will also permit further modification of the receptor molecules in order to develop novel carrier systems for anticancer drugs that will specifically distinguish diseased from healthy cell/tissue. In addition, fluorinated amino acids can be also incorporated into receptors peptide sequence for noninvasive in vivo cancer cell 19F magnetic resonance imaging (MRI).

Example 7 - Characterization of Polyvalent Receptors

[0126] Determine the affinity of polyvalent receptors 49-56 and control compounds 57-64 (Scheme 5) toward Lewis oligosaccharide antigens: Since polyvalent binding is important for high affinity and selective binding of carbohydrate substrates, the effect of the number of monovalent binding units and the role of tether's length in receptor binding to Lewis oligosaccharides is investigated. In addition, defining the structural requirements (e.g. the number of binding units or the length of the tether which multimerizes binding units) responsible for receptor specific carbohydrate binding facilitates solid-phase synthesis of artificial polyvalent receptors for Lewis oligosaccharides by reducing structural complexity and also promotes judicious chemical modifications for structure-function analyses and modulation of their biological or physiochemical properties. The information gained from these studies helps guide the design of new and more selective polyvalent receptors leading the way to a new generation of sensors for tumor-associated carbohydrate antigens, drug delivery systems or new chemotherapeutics that target these antigens.

[0127] Determine the affinity of polyvalent receptors 49-56 (Scheme 5) and control compounds 57-64 (Scheme 5) for Lewis oligosaccharides in solution by fluorescence titration and/or NMR spectroscopy: Fluorescence titration experiments are used to determine the affinities of 49-64 (Scheme 5) toward sLex, sLea, Lex and Ley oligosaccharides in solution. All four Lewis oligosaccharides are commercially available (Sigma). The titration experiments are performed as described above. As an alternative method, NMR spectroscopy is also used to determine affinity of these receptors toward Lewis oligosaccharides.

[0128] Determine the binding efficacy of polyvalent receptors 49-56 (Scheme 5) and control compounds 49-64 (Scheme 5) toward BSA bound sLex (modeling cell surface-expressed sLex) by competitive ELISA assay: The role of sLex oligosaccharide in binding of receptors 18-33 is examined by competitive ELISA assay according to the standard protocol. For this assay, sLex- BSA conjugate (EMD Biosciences), control BSA, anti-sLex monoclonal antibody (KM93; EMD Biosciences), and the goat anti-mouse IgM-horseradish peroxidase conjugate are used. According to the protocol, absorbance of a blue reaction product is read at 630 nm using an Awareness Technology Stat Fax-2100 96-well microtiter plate reader (the stop reagent does not produce a spectral change from blue to yellow). In this way, potential overlap in absorbances between receptors 49-64 (Scheme 5) and blue peroxidase product are avoided. Concentration- dependent decreases in absorbance at 630 nm indicate that receptors 49-64 (Scheme 5) and anti- sLex monoclonal antibody share the same ligand.

[0129] Assess polyvalent receptors 49-56 and control compounds 57-64 ability to bind sLex expressing cells and inhibit cell adhesion and invasion: Described results showed that monovalent receptor 2 (Scheme 3) binds preferentially to the sLex-expressing HepG2 cells. Binding studies are performed with receptors 49-64 (Scheme 5) in order to assess their affinity and specificity toward sLex expressing HepG2 cells qualitatively by fluorescence microscopy and quantitatively by immunocytochemistry assay. However, sLex antigen is not merely a marker of cancer cells, but also mediates cancer cell adhesion to vascular endothelial cells and promotes hematogenous metastasis of cancer. The interaction of cancer cells and endothelium through sLex epitopes could provide a target for inhibition by receptors 2 (FIG. 1), and 49-64 (Scheme 5), and it could therefore have implications in the prevention of cancer cell metastasis. For the cell adhesion assays, sLex expressing HepG2 cells 49-56 (Scheme 5) and control compounds 57-64 (Scheme 5) to bind model sLex expressing (HepG2) cells qualitatively by fluorescence microscopy: The ability of synthesized polyvalent receptors 57-64 (Scheme 5) to label sLex-expressing HepG2 cells (ATCC HB-8065), as a model system, is tested using fluorescence microscopy. The microscopy experiments are performed as described above. The bound fluorophore accumulation is captured with the Olympus 1X70 Inverted Fluorescence Microscope camera (370 nm excitation, 420 nm emission). For controls, the same experiment is performed with Ley-expressing Hep3B cells (ATCC HB-8064) and control fibroblast cells (ATCC CCL-201).

[0130] Determine the ability of polyvalent receptors 49-56 (Scheme 5) and control compounds 57-64 (Scheme 5) to bind model sLex expressing (HepG2) cells quantitatively by immunocytochemistry assay: To examine whether sLex antigen is essential for binding of 49-64 (Scheme 5) to sLex-expressing HepG2 cells, a cell based ELISA competition assay is performed. The assay is carried out in a 96-well plate format according to standard protocols. A concentration-dependent decrease in absorbance at 630 nm indicates that receptors 49-64 (Scheme 5) and anti-sLex monoclonal antibody have a common target on the cell surface. As a control, the same experiment is performed with Ley-expressing Hep3B cells (ATCC HB-8064) and anti-Ley monoclonal antibody (F3, EMD Biosciences). In this way, artificial polyvalent receptors 49-64 (Scheme 5) preferentially binding sLex antigens on the cell surface is confirmed. [0131] Toxicity assay: Malnourished or dying cells can leak proteins into their surroundings. This process can interfere with the binding of 49-64 (Scheme 5) to the cell surface sLex antigen and produce false negative results. In order to assess this possibility, toxicity of synthesized polyvalent receptors toward HepG2 cells is determined using MTT cell proliferation assay. [0132] Cell adhesion assay: Since sLex and sLea expression on cancer cells mediates their adhesion to vascular endothelial cells and promotes hematogenous metastasis of cancer, agents that inhibit this process have the potentials to be of considerable therapeutic value. With this assay, the ability of receptors 49-64 (Scheme 5) to inhibit tumor cell adhesion to endothelium is examined, further proving the receptors' affinity and selectivity toward sialylated Lewis antigens. Cell adhesion assays are performed on the plates precoated with the matrix components and plates coated with human umbilical endothelial cells (HUVEC, ATCC CRL- 1730) according to the standard protocols. Pro-Bind 96 well plates (BD Biosciences) are precoated with E- selectin-BSA conjugate, while control wells are coated with BSA. Previously Calcein AM- labeled sLex expressing HepG2 cells are added to the wells, following by addition of various concentrations of 49-64 (Scheme 5) in PBS to each well. After plate incubation for 1 h at 37⁰C, the percentage of adherent cells is determined using a microplate spectrofluorometer (Molecular Devices SpectraMax Gemini EM), with excitation wavelength 485 nm and emission wavelength 530 nm. Results will be expressed as the ratio of fluorescence from wells containing E-selectin to uncoated control wells containing 5 x 10⁴ cells in the presence and absence of receptors 49-64 (Scheme 5).

[0133] Cell invasion assay: Tumor proliferation, invasion and metastasis are characteristic features of the malignant phenotype, while benign solid tumors show no signs of invasion and metastasis. Therefore, cell invasion assays represent an important additional step in assessing receptors 16-31 (Scheme 5) potentials as lead structures in the development of new cancer diagnostic and/or anti-metastatic agents. Invasion assays are performed using the cell invasion assay kit (Chemicon Int.) and protocol recommended by the manufacturer.

Other Embodiments

[0134] Any improvement may be made in part or all of the compositions and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the present disclosure and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the present disclosure or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention.

Claims

1. A receptor compound of the formula

wherein

Xi independently of one another are hydrogen, hydroxyl (OH), amino (NH₂) or carboxyl (CO₂H);

X₂ independently of one another are amino (NH₂) or carboxyl (CO₂H); or together form a group of formula

or one of X₂ is carboxyl (CO₂H) and the other one is a di- or polyvalent bridging moiety comprising at least two α,ω-amino acid segments and at least two linker segments of formula

-NH-Z-C(=0)-0-

wherein Z is a divalent chain having from 3 to 20 chain members selected from the group consisting of: 3 to 15 CH₂ groups, 1 to 3 C(=O) groups, 1 to 3 NH groups and 1 to 6 non- adjacent -O- groups; which bridging moiety binds to at least one terminal group of the formula

X₃ together with the carbon atom to which it is bonded and independently of one another methylene (CH₂) or carbonyl (C(=O)); and

n is 1, 2 or 3; or a salt of the receptor compound.

2. The receptor compound of Claim 1 or the salt thereof wherein the two variables X₂ together form the group of the formula

3. The receptor compound of Claim 1 or the salt thereof wherein the two variables X₂ denote amino.

4. The receptor compound of Claim 1 or the salt thereof wherein one of X₂ denotes carboxyl and the other is a di- or tri-valent bridging moiety formed from two or three lysine segments and two or three linker segments selected from the group consisting of : -NH-(CH₂)₅-CO-O-

-NH-(CH₂)_H-CO-O- -NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(=O)-CH₂OCH₂-C(=O)-O-.

5. The receptor compound of Claim 1 having the formula

in which X₂ independently of one another are amino (NH₂) or carboxyl (CO₂H), "Linker" represents the linker segment, and X₁, X₃ and n are as defined in Claim 1, or a salt thereof.

6. The receptor compound of Claim 5 or the salt thereof, wherein the designation "Linker" represents a linker segment selected from the group consisting of: -NH-(CH₂)₅-CO-O-

7. The receptor compound of Claim 1 having the formula

8. The receptor compound of Claim 7 or the salt thereof, wherein the designation "Linker" represents a linker segment selected from the group consisting of:

-NH-(CH₂)_S-CO-O-

-NH-(CH₂)H-CO-O-

-NH-(CH₂)₂-(CH₂OCH₂)₃-(CH₂)₂-NH-C(=O)-CH₂OCH₂-C(=O)-O-.

9. A composition comprising a receptor having the following structure:

10. The composition of claim 9, wherein the receptor binds sialic acid.

11. The composition of claim 9, wherein the receptor binds sialyl Lewis x and sialyl Lewis a monosaccharides.

12. The composition of claim 9, wherein the receptor specifically binds cancer cells.

13. A method of detecting a cancer cell in a sample comprising the steps of: providing a sample comprising a plurality of cells; contacting the sample with at least one receptor having the following formula:

detecting binding of the at least one receptor to a sialyl lewis x monosaccharide or a sialyl lewis a monosaccharide; and correlating binding of the receptor to the sialyl lewis x monosaccharide or sialyl lewis a monosaccharide with the presence of at least one cancer cell in the sample.

14. The method of claim 13, wherein the at least one cancer cell is a human cancer cell.

15. An artificial polyvalent 1,8-naphthiridine-based receptor that specifically binds sialic acid.

16. The artificial receptor of claim 15, wherein the artificial receptor is a carbohydrate receptor.

17. The artificial receptor of claim 15, comprising a plurality of units for sialic acid binding and recognition.

18. The artificial receptor of claim 17, wherein at least one of the units for sialic acid binding and recognition comprises compound 2 or a derivative thereof, and wherein compound 2 is:

19. The artificial receptor of claim 17, wherein at least one unit for sialic acid binding and recognition is linked by peptidic spacers.

20. The artificial receptor of claim 15, wherein the artificial receptor is soluble in water and possess the necessary three-dimensional structure for sialic acid binding and recognition.

21. A kit for detecting cancerous cells in a biological sample, the kit comprising: a) a receptor as in claims 1-8; b) a solid substrate; and c) instructions for use.