Claims:CLAIMS
What is claimed is:

1. A pharmaceutically active compound comprises:

a plurality of small molecules as canonical Wnt signaling pathway inhibitors, wherein the plurality of small molecules target and inhibit key proteins of nuclear transcription complex to inactivate the Wnt signaling pathway in growth of a tumor, and

a macrocyclic compound having tetrahydrofuran moiety sub-structure as a Wnt/b-catenin inhibitors to suppress the growth of the tumor,

wherein the macrocyclic compound involves in synthesis of trans-2, 5-disubstituted tetrahydrofuran through an iodocyclization reaction of resultant product from the reaction of R, R-tartaric acid,

wherein the small molecules utilize trans-2,5-disubstituted groups at C17 and C19 to form the macrocyclic with the 17-membered ring.

2. The compound of claim 1, further an amino acid moiety is incorporated in the macrocyclic ring that introduces a chiral diversity site for obtaining further structural analogs.

3. The compound of claim 1, wherein the macrocyclic compound is a 17-membered ring macrocycle with a single olefin geometry.

4. The compound of claim 1, wherein the iodocyclization produces trans- and cis-2,5-disubstituted tetrahydrofuran derivatives that are used to develop 17-membered macrocyclic compounds.

5. The compound of claim 1, further five different alloc amino acids are coupled to the hydrolyzed trans- and Cis-2,5-disubstituted tetrahydrofuran that produces five different macrocyclic precursors numbered as SM-61(L-valine), SM-62(L-phenyl alanine), SM-63(L-Isoleucine), SM-64(L-proline) and SM-65(L-pipecolic acid) to develop the 17-membered macrocyclic compounds with tetrahydrofuran substructure.

6. The compound of claim 5, wherein the five macrocyclic precursors are reacted with a catalyst for the crucial ring-closing metathesis to produce five 17-membered ring macrocycle compounds numbered as SM-66, SM-67, SM-68, SM-69, and SM-70. , Description:BACKGROUND OF THE INVENTION
A. Technical field
[0001] The present invention generally relates to the biologically active pharmaceutical composition targeting Wnt receptors, and more particularly, to the synthesis of pharmaceutically relevant, small molecules as Wnt/b-catenin pathway inhibitors. More specifically, the present invention relates to a method of synthesis of furan-based, novel macrocyclic compounds, and the methods of preparation thereof.

B. Description of related art
[0002] Wnt is an acronym in the field of genetics that stands for “Wingless/Integrated”; Wnt/b-catenin pathway starts with the cell surface protein interactions, passing the signals to the cytoplasmic and then to nuclear machinery. Its clinical importance is demonstrated by mutations that lead to various diseases, including breast and prostate cancer and glioblastoma. The dysregulation of the Wnt-b-catenin signaling pathway is commonly found across many tumor types. It is involved in several developmental processes and plays a central role in maintaining the adult tissue homeostasis by regulating cell proliferation, differentiation, migration, genetic stability, and apoptosis. In addition, it is also known to be highly crucial to maintain adult stem cells in a pluripotent state.

[0003] The Wnt-b-catenin signaling pathway activation is commonly found in colorectal, breast, lung, cancers, hematopoietic malignancies, and also, known to contribute to tumor recurrence. There are significant challenges in targeting the Wnt pathway, including finding efficacious agents without damaging the system of normal somatic stem cell function in cellular repair and tissue homeostasis.

[0004] Wnt/b-catenin pathway is critical to cell repair and maintenance of stem cell functions. One of the key mechanisms of this pathway’s activation is the loss of function of APC (anaphase-promoting complex) which functions as a negative regulator. Wnt/b-catenin signaling is activated by truncated APC protein that negates the destruction of complex-mediated b-catenin ubiquitination. Several mutations are known to cause the loss of this function; they prevent the removal of the Wnt receptor in the intestinal crypt, thereby causing Wnt signaling activation. In general, patients with breast cancers have “activation of Wnt” and that is associated with lower overall survival. In a transgenic mouse model, b-catenin-dependent signaling inhibition in ErbB2-derived cells is known to impair tumor initiation and metastasis. Further, ERBB2-overexpressing tumor cells on treatment with small molecule b-catenin/CBP (CREB Binding Protein) inhibitor are shown to significantly decrease the proliferation and ErbB2 expression. There is an increase in active Wnt signaling in breast cancer stem cells which is confirmed by increased expression of the activated b-catenin protein, both downstream targets AXIN2 and LEF1, and decreased expression of DKK1 protein.

[0005] Currently, few existing patent references attempted to address the aforementioned problems are cited in the background as prior arts over the presently disclosed subject matter and are explained as follows.

[0006] A prior art WO2011014973 to Daniel Obrecht, et al., entitled “Conformationally constrained, fully synthetic macrocyclic compounds” discloses a Conformationally restricted, spatially defined 12-30 membered macrocyclic ring systems of formulae Ia and Ib are constituted by three distinct molecular parts such as template A, conformation modulator B and bridge C. These macrocycles Ia and Ib are readily manufactured by parallel synthesis or combinatorial chemistry in solution or on a solid phase. They are designed to interact with a variety of specific biological target classes, examples being the agonistic or antagonistic activity on G-protein coupled receptors (GPCRs), ion channels, and signal transduction pathways. In particular, these macrocycles act as antagonists of the motilin receptor, the FP receptor and the purinergic receptors P2Y1, as modulators of the serotonin receptor of subtype 5-HT2B, as blockers of the voltage-gated potassium channel Kv1.3 and as inhibitors of the ß-catenin-dependent "canonical" Wnt pathway.

[0007] Another prior art US20180296680A1 to Matthew J. Webber, et al., entitled “Supramolecular modification of proteins” discloses the modification of biomolecules, small molecules, and other agents of via conjugation of excipients, tags, or labels is of great importance. Supramolecular chemistry utilizes specific, directional, reversible, non-covalent molecular recognition motifs in order to achieve the organization of molecules, and could be used to complex tags to agents of interest (e.g., insulin, glucagon, antibodies). It provides useful supramolecular complexes wherein an agent of interest is specifically bound to a host via non-covalent interactions, and wherein the host is conjugated to a tag. It also provides methods and compounds useful in preparing supramolecular complexes, and methods of treating diseases using the supramolecular complexes.

[0008] Though the above mentioned prior arts disclose a method of synthesis of pharmaceutically active 14 or 17 members macrocyclic ring with tetrahydrofuran substructure that acts as a signal pathway inhibitor to suppress the growth of tumors, none of the prior art discloses the synthesis of the complex furan-based macrocyclic compound. Therefore, there is a need for screening this set of macrocyclic compounds in search of Wnt/b-catenin inhibitors related to modulation of nuclear complex dealing with multiple nuclear protein-protein interactions / DNA-protein interactions. Also, there is a need for finding small molecules that could inhibit the Wnt/beta (b)-catenin pathway, especially at the level of the nuclear complex between b-catenin and TCF (T-Cell Factor) or LEF (Lymphoid Enhancer Factor). In addition, these novel small molecules which are attractive for building an IP portfolio, could also easily lead to obtaining several structurally-related analogs for further enhancing their biological potential. Moreover, these compounds could also be synthesized on a large scale in a time-efficient manner, which is also an attractive feature of these types of small molecules. Further, there is a need for an approach that is useful for interfering with tumor growth.

SUMMARY OF THE INVENTION
[0009] The present invention generally discloses the field of the synthesis of pharmaceutically relevant, small molecules as Wnt/b-catenin pathway inhibitors. Further, the present invention discloses a method of synthesis of furan-based, novel macrocyclic compounds, and the methods of preparation thereof.

[0010] According to the present invention, the pharmaceutically active compound is developed to inhibit Wnt/beta (b)-catenin pathway, especially at the level of the nuclear complex between b-catenin and TCF (T-Cell Factor) or LEF (Lymphoid Enhancer Factor), and an approach which is useful to interfering with the tumor growth. In one embodiment, the pharmaceutically active compound comprises a plurality of small molecules and a macrocyclic compound. In one embodiment, the small molecules act as canonical Wnt signaling pathway inhibitors. In one embodiment, the small molecules could inhibit the Wnt/beta (b)-catenin pathway to suppress the growth of the tumor.

[0011] In one embodiment, the small molecules target and inhibit the key proteins of (b)-catenin, TCF (T-Cell Factor) or LEF (Lymphoid Enhancer Factor), and CBP (chronic bacterial prostatitis). The small molecules target and inhibit the key proteins of the nuclear transcription complex to inactivate the Wnt signaling pathway in the growth of tumors such as in breast cancer patients. In one embodiment, the small molecules utilize trans-2,5-disubstituted groups at C17 and C19, which could lead to a macrocycle with the 17-membered ring. The utilization of trans-2,5-disubstituted groups at C17 and C19 leads to a macrocycle with the 17-membered ring. In one embodiment, an amino acid moiety is incorporated in the macrocyclic ring that allows the introduction of a chiral diversity site for obtaining further structural analogs.

[0012] Due to the presence of trans-2,5-disubstituted tetrahydrofuran moiety in eribulin and several other bioactive natural products and related analogs, this is considered as a privileged scaffold, and it could serve as a good starting point in building a chemical toolbox with a diverse set of macrocyclic compounds. With this goal, the invention has two objectives. In one embodiment, the first objective of the present invention is to develop a practical and scalable synthesis of trans-2,5-disubstituted tetrahydrofuran as the key scaffolds. In one embodiment, the second objective of the present invention is to develop the synthesis for obtaining different macrocyclic compounds with the 17-membered ring.

[0013] In one embodiment, the macrocyclic compound is a 17-membered ring macrocycle with a single olefin geometry. In one embodiment, the 17-membered macrocyclic compound has a tetrahydrofuran moiety sub-structure. The 17-membered macrocyclic compound with tetrahydrofuran acts as a Wnt/b-catenin inhibitor to suppress the growth of the tumor. In one embodiment, the 17-membered macrocyclic compound involves in synthesis of trans-2,5-disubstituted tetrahydrofuran through the iodocyclization reaction of resultant product from the reaction of R, R-tartaric acid. In one embodiment, the use of R,R-tartaric acid is attractive and also provides an opportunity for obtaining the other enantiomeric product by starting with the S,S-isomer.

[0014] In one embodiment, the R, R-tartaric acid is converted into diol by using one pot esterification and acetonide protection followed by the reduction with LiAlH4. The selective mono-protection of the diol affords the mono O-benzyl ether, and it is then converted to its corresponding iodo compound. In one embodiment, the R, R-tartaric acid is treated with vinyl MgBr/CuI, and acetonide is removed followed by an iodocyclization reaction that produces trans- and cis-2,5-disubstituted tetrahydrofuran derivatives. This trans- and cis-2,5-disubstituted tetrahydrofuran derivatives are then used to develop 17-membered macrocyclic compounds using the Grubbs catalyst.

[0015] In one embodiment, the 17-membered macrocyclic compounds are synthesized with tetrahydrofuran substructure. In one embodiment, the synthesis of 17-membered macrocyclic compounds having tetrahydrofuran substructure uses five different alloc amino acids. The alloc amino acids are coupled to the hydrolyzed trans- and Cis-2,5-disubstituted tetrahydrofuran that produces five different macrocyclic precursors numbered as SM-61(L-valine), SM-62(L-phenyl alanine), SM-63(L-Isoleucine), SM-64(L-proline) and SM-65(L-pipecolic acid). In one embodiment, the five macrocyclic precursors are then reacted with the Grubbs catalyst for the crucial ring-closing metathesis to produce five 17-membered ring macrocycle compounds. The five 17-membered ring macrocycle compounds are numbered as SM-66, SM-67, SM-68, SM-69, and SM-70.

[0016] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF DRAWINGS
[0017] The foregoing summary, as well as the following detailed description of the invention, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, exemplary constructions of the invention are shown in the drawings. However, the invention is not limited to the specific methods and structures disclosed herein. The description of a method step or a structure referenced by a numeral in a drawing is applicable to the description of that method step or structure shown by that same numeral in any subsequent drawing herein.

[0018] FIG. 1 shows a schematic diagram of the Wnt/b-catenin pathway in the absence and presence of the Wnt ligand in an embodiment of the present invention.

[0019] FIG. 2 shows four representative examples of bioactive natural products having one or more than one furan rings in the architectures in one embodiment of the present invention.

[0020] FIG. 3A shows the generation luciferase-based primary screening assay to identify Wnt-b-catenin inhibitors in one embodiment of the present invention.

[0021] FIG. 3B shows the luciferase-based screening data with SM-61, SM-62, SM-63, SM-64, SM-65 (all precursor to macrocyclic compounds), and SM-66, SM-67, SM-68, SM-69, SM-70 in different embodiments of the present invention.

[0022] FIG. 3C shows the difference in the luciferase-based screening data obtained with SM-61, SM-66, and SM-67 in different embodiments of the present invention.

[0023] FIG. 4A shows the RT-PCR data related to genes, TCF, and LEF with small molecules SM-61 (open chain precursor) and SM-66 (macrocyclic compound) in one embodiment of the present invention.

[0024] FIG. 4B shows the RT-PCR data related to genes, Axin, b-catenin (also known as CTNBB-1), and TCF with small molecules SM-61 (open chain precursor) and SM-66 (macrocyclic compound) in one embodiment of the present invention.

[0025] FIG. 4C shows the RT-PCR data with specific genes, SNAIL, CKS-2, and HEF-1, related to cell migration and cell invasion with small molecules SM-61 and SM-66 in one embodiment of the present invention.

[0026] FIG. 4D shows the RT-PCR data with specific genes, VIMENTIN, and CCND-1, related to cell metastasis and proliferation with small molecules SM-61 and SM-66 in one embodiment of the present invention.

[0027] FIG. 5A shows the western blots related to phosphor-b-catenin, b-catenin, TCF-1/TCF-7, after treatment with IWR-1, SM-61, and SM-66 in different embodiments of the present invention.

[0028] FIG. 5B shows the western blots related to AXIN-2, LEF, and CBP, after treatment with IWR-1, SM-61, and SM-66 in different embodiments of the present invention

[0029] FIG. 5C shows the western blots related to TCF-1 and cyclin D1 after treatment with IWR-1, SM-61, and SM-66 in different embodiments of the present invention.

[0030] FIG. 6A shows the RNA sequencing data with “cell migration gene set” upon treatment with small molecules, IWR1 (positive control from literature), SM-61 (open structure, a precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in one embodiment of the present invention.

[0031] FIG. 6B shows the RNA sequencing data with “metastasis, breast cancer gene set” upon treatment with small molecules, IWR1 (positive control from literature), SM-61 (open structure, the precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in one embodiment of the present invention.

[0032] FIG. 6C shows the RNA sequencing data with “epithelial to mesenchymal gene set” upon treatment with small molecules, IWR1 (positive control from literature), SM-61 (open structure, the precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in one embodiment of the present invention.

[0033] FIG. 6D shows the RNA sequencing data with “stemness gene set” upon treatment with small molecules, IWR1 (positive control from literature), SM-61 (open structure, the precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in one embodiment of the present invention.

[0034] FIG. 6E shows the RNA sequencing data with “transcription gene set” upon treatment with small molecules, IWR1 (positive control from literature), SM-61 (open structure, the precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in one embodiment of the present invention.

[0035] FIG. 6F shows the RNA sequencing data with “transcription gene set” upon treatment with small molecules, IWR1 (positive control from literature), SM-61 (open structure, the precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in another embodiment of the present invention.

[0036] FIG. 7 shows an effect of small molecules, IWR-1, SM-61, and SM-66 on the formation of tumorspheres (G2; 2nd generation) from an established cancer cell line, HCT116 in one embodiment of the present invention.

[0037] FIG. 8A shows the effect of small molecules, IWR-1, SM-66 on the formation of tumorspheres (G2; 2nd generation/patient 1) from Indian patient 1 buccal mucosa in one embodiment of the present invention.

[0038] FIG. 8B shows the effect of small molecules, IWR-1, SM-66 on the formation of tumorspheres (G2; 2nd generation/patient 2) from Indian patient 2 buccal mucosa in one embodiment of the present invention.

[0039] FIG. 9A shows the RT-PCR for the metastasis gene, vimentin, on the formation of tumorspheres (G2; 2nd generation/patient 2) from Indian patient 2 buccal mucosa in the presence of SM-61 and SM-66 in one embodiment of the present invention.

[0040] FIG. 9B shows the RT-PCR for the cell migration and invasion genes (for ex SNAIL and HEF1), on the formation of tumorspheres (G2; 2nd generation/patient 2) from Indian patient 2 buccal mucosa in the presence of SM-61 and SM-66 in one embodiment of the present invention.

[0041] FIG. 9C shows the RT-PCR for the stemness genes (for ex CD44 and CD133), on the formation of tumorspheres (G2; 2nd generation/patient 2) from Indian patient 2 buccal mucosa in the presence of SM-61 and SM-6 in one embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS
[0042] A description of embodiments of the present invention will now be given with reference to the Figures. It is expected that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive.

[0043] According to the present invention, an improved and effective pharmaceutically small molecules as Wnt/b-catenin pathway inhibitors and a method of synthesis of furan-based, novel macrocyclic compounds thereof, are disclosed. Referring to FIG. 1, a schematic diagram of the Wnt/b-catenin pathway in the absence and presence of the Wnt ligand, according to one embodiment of the present invention. In one embodiment, the pathway is activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, which then transmits the biological signal inside the cell. The Wnt/b-catenin signaling pathway is generally highly evolutionarily conserved in animals. Wnt/b-catenin signaling pathway is first identified for its role in carcinogenesis, then for its function in embryonic development, including cell fate specification, cell proliferation and, cell migration.

[0044] In one embodiment, the pharmaceutically active compound is developed to inhibit Wnt/beta (b)-catenin pathway, especially at the level of the nuclear complex between b-catenin and TCF (T-Cell Factor) or LEF (Lymphoid Enhancer Factor), and an approach which is useful to interfering with the tumor growth. In one embodiment, the pharmaceutically active compound comprises a plurality of small molecules and a macrocyclic compound. In one embodiment, the small molecules act as canonical Wnt signaling pathway inhibitors. In one embodiment, the small molecules could inhibit the Wnt/beta (b)-catenin pathway to suppress the growth of the tumor. In one embodiment, a 17-membered macrocyclic compound having a tetrahydrofuran moiety as the privileged sub-structure is synthesized to identify the small molecules as Wnt/b-catenin inhibitors. In one embodiment, the tetrahydrofuran moiety is commonly found in several bioactive plants, marine natural products, and natural product-derived compounds.

[0045] Referring to FIG. 2, four representative examples of bioactive natural products having one or more than one furan rings in the architectures, according to one embodiment of the present invention. In one embodiment, the bioactive natural products include eribulin or compound 1, amphidinolide E or compound 2, amphidinolide F or compound 3, and haterumalides or compound 4. In one embodiment, eribulin is an approved drug for the treatment of metastatic breast cancer. It results from extensive synthesis efforts that are made over the years toward halichondrain.

[0046] In one embodiment, the tetrahydrofuran-based sub-structures along with the macrocyclic rings are also commonly found in several other bioactive natural products. For example, amphidinolide E and amphidinolide F, both belong to the family of amphidinolides. The amphidinolide E and amphidinolide F are known as highly potent cytotoxic agents. In the case of amphidinolide E, cis-2,5-disubstituted tetrahydrofuran is embedded in the functionalized 18-membered macrocyclic ring, whereas, two trans-2,5-disubstituted tetrahydrofuran moieties are a part of a densely functionalized 23-membered ring in amphidinolide F. In another embodiment, the haterumalides are also natural products from another family that contain the tetrahydrofuran moiety along with a macrocyclic ring. The haterumalides are also known for their potent cytotoxicity.

A. Synthesis Planning of Furan-derived Macrocyclic Compounds and Execution:

[0047] The fascinating architecture of eribulin could serve as an excellent starting point in developing a diversity synthesis program utilizing one of the key fragments building a chemical toolbox to hunt for small molecule modulators of protein-protein interaction, and in other, selected signaling pathways. With the continuous interest in building a toolbox having compounds with different types of natural product-inspired macrocyclic rings, the present invention is started with the “stereo-defined furan moiety” or compound 5 as highlighted in Scheme 1. The generic structure as per the present invention to explore the additional macrocyclic chemical space is also shown as compound 6. In one embodiment, compound 6 is a specific diversity-based, 17-macrocyclic synthesis target. In addition, the macrocyclic compound 6 has several attractive features in this design.

[0048] Due to the presence of trans-2,5-disubstituted tetrahydrofuran moiety in eribulin and several other bioactive natural products and related analogs, this is considered as a privileged scaffold, and it could serve as a good starting point in building a chemical toolbox with a diverse set of macrocyclic compounds. With this goal, the invention has two objectives. In one embodiment, the first objective is to develop a practical and scalable synthesis of trans-2,5-disubstituted tetrahydrofuran as the key scaffolds 11 and 12 as shown in Scheme 2. In one embodiment, the second objective is to develop the synthesis for obtaining different macrocyclic compounds with the 17-membered ring. In an approach, the utilization of trans-2,5-disubstituted groups at C17 and C19 leads to a macrocycle with the 17-membered ring. In one embodiment, the incorporation of an amino acid moiety in the macrocyclic ring allows the introduction of a chiral diversity site for obtaining further structural analogs. In an exemplary embodiment, the present invention aims to obtain different types of macrocyclic compounds with the privileged trans-2,5-disubstituted tetrahydrofuran moiety and to explore their properties in finding the inhibitors of the Wnt/b-catenin pathway. Upon the identification of functional chemical probes, the present invention aims to explore the value of these different macrocyclic compounds functioning as the inhibitors of tumor cell migration/metastasis and cell invasion.

[0049] In one embodiment, the present approach that utilizes the iodocyclization in providing the key scaffolds 11 and 12, which could then lead to producing 13, as shown in Schemes 2 and 3. An advantage of this approach is the use of a cheap chiral starting material R,R-tartaric acid or compound 7, which could results compound 10 in a few simple steps. The use of R,R-tartaric acid is attractive and also provides an opportunity for obtaining the other enantiomeric product by starting with the S,S-isomer.

[0050] As shown in Scheme 2, R,R- tartaric acid or compound 7, is converted into compound 8 by using one pot esterification and acetonide protection. In one embodiment, this is further followed by reduction with LiAlH4; selective mono-protection of the diol or compound 8 afforded the mono O-benzyl ether. This is then converted to its corresponding iodo compound 9 upon treatment with vinyl MgBr/CuI. Then the compound 9 is converted into compound 10 followed by the removal of an acetonide. An iodocyclization reaction then produces trans- and cis-2,5-disubstituted tetrahydrofuran derivatives or key scaffolds 11 and 12 as the major and minor products. In one embodiment, the key scaffolds 11 and 12 are derived in the ratio of about 4:1. The key scaffolds 11 and 12 are easily separable and the pure products are then thoroughly subjected to the detailed structural analysis by 1D- and 2D-NMR studies.

[0051] Having a method for obtaining a sufficient amount of the key intermediate 11, it is then utilized for developing the synthesis of 17-membered macrocyclic compounds. In one embodiment, Scheme 3 shows an approach to synthesis a macrocyclic compound 16. In one embodiment, compound 13 is hydrolyzed to obtain the free acid which is then allylated and produces compound 14 upon the removal of the hydroxyl protection. In one embodiment, the compound 14 is then coupled with five different Alloc amino acids (Amino acid are commercially available inexpensive enantioenriched building blocks. Amino acid/derivatives are widely present in several macrocyclic natural products as well as bioactive synthetic macrocycles). In one embodiment, the amino acid-derived building blocks are incorporated into 17-membered macrocyclic skeletons or compounds 15 as a key diversity site. In one embodiment, the compounds 15 are numbered as SM-61(L-valine), SM-62(L-phenyl alanine), SM-63(L-Isoleucine), SM-64(L-proline) and SM-65(L-pipecolic acid) for the crucial ring-closing metathesis.

[0052] In one embodiment, the use of the 2nd generation Grubbs catalyst successfully produces the 17-membered ring macrocycle with a single olefin geometry or compound 16. The olefin geometry is not assigned due to an overlapping of signals in NMR, in the region of interest. In one embodiment, this approach produces five macrocyclic compounds. The five macrocyclic compounds are numbered as 16a or SM-66, 16b or SM-67, 16c or SM-68, 16d or SM-69, and 16e or SM-70 as shown in Scheme 4. Also, the approach validates the feasibility of the ring formation strategy that is independent of the utilized amino acid. As a test case, in three examples, the hydrogenation conditions led to producing the de-protected compounds and the removal of the double bond produces compound 17 as shown in Schemes 3 and 4. All the products are thoroughly purified and well-characterized by MS and 1D- and 2D-NMR studies.

B. Biological Applications Using Substituted Furan-based Macrocyclic Compounds and their Acyclic Precursors:

1. Luciferase Assay (FIGs. 3A-3C):

Wnt Reporter Activity Assay:

[0053] Wnt Reporter Activity Assay is used in a 96 well format to identify active small molecules that have the potential in modulating modulate the Wnt signaling pathway. The LEADING LIGHT Wnt Reporter Assay Starter Kit is obtained from Enzo Life Sciences (cat. no. ENZ-61001-0001). The system contains an engineered 3T3 mouse fibroblast cell line, which expresses the firefly luciferase reporter gene under the control of Wnt-responsive promoters (TCF/LEF). Wnt target genes that contain consensus TCF/LEF binding element, which could be transcriptionally induced by the Canonical Wnt signaling pathway.

Protocol:

[0054] In one embodiment, the engineered 3T3 mouse fibroblast cells are seeded into 96 well plates or 3T3 at a density of 10,000 cells/well. After 24 hours, cells are treated with the small molecules at a final concentration of 10µM re-suspended in the assay medium. After the 24 hours post-treatment, the firefly luciferase reagent is added to the cells and it is then subjected to immediately for reading the luminescence using a plate reader (Promega E1910). The top and bottom reads of the plate are taken and analyzed as the ratio of firefly luciferase and renilla luciferase. The Wnt reporter activity is plotted as a sample versus luciferase units that also include positive and negative controls.

2. RT-PCR Studies (FIGs. 4A-4C):

[0055] The effect on several genes is measured for a better understanding of the role of the active small molecules on the Wnt/b-catenin pathway. In one embodiment, the present invention majorly focuses on the genes, such as, AXIN and APC that are involved in the protein destruction complex. In addition, the present invention also focuses on the genes that are central to nuclear multi-protein transcription complex (i.e. b-catenin, LEF, TCF, and CBP) and control the b-catenin-mediated transcription machinery. The RT-PCR study analysis indicates that AXIN and APC genes are not affected by small molecules SM-61, SM-66, and the positive control, IWR-1 (as shown in FIG. 4B). It appears that as with the positive control IWR-1, the small molecules, SM-61, and SM-66 stabilize the protein destruction complex for inhibiting the b-catenin migration to the nucleus.

[0056] It is well known that Wnt/b-catenin signaling is actively involved in various biological processes including cell proliferation, metastases, migration, and invasion. Further, the effect of small molecules (SM-61 and SM-66) on cell proliferation, metastases, migration, and invasion are also determined. In one embodiment, several candidate genes related to these pathways are examined and the expression of genes upon exposure is measured. In one embodiment, the analysis of RT-PCR data clearly shows that SM-66 significantly reduced the expression CCND1, Vimentin and HEF1, CKS2 and SNAIL after the 24h of treatment (FIG. 4C and 4D). The results suggest that small molecule SM66 inhibits the Wnt/b-catenin signaling and its further effects in HCT116 cells.

[0057] The quantitative PCR (QuantStudio 5 Real-Time PCR System, USA) is performed using the STB GreenTM Premix Ex Taq TM II according to the manufacturer’s instruction (One-Step TB Green™ PrimeScript™ RT-PCR Kit II, USA). The cDNA is then used as the template for real-time PCR with gene-specific primers as shown below:
CCND1:
5'-ATGCCAACCTCCTCAACGAC-3'

VIMENTIN:
5'-TCTACGAGGAGGAGATGCGG-3'
5'-GGTCAAGACGTGCCAGAGAC-3

SNAIL2:
5'-ACCACTATGCCGCGCTCTT-3'

CKS2:
5’-CGCTCTCGTTTCATTTTCTGC-3’,
5’-TGGAAAGTTCTCTGGGTAACATAACA-3’

HEF1:
5'-CATAACCCGCCAGATGCTAAA-3'
5'-CCGGGTGCTGCCTGTACT-3'

CTNNB1:
5'-TCTGAGGACAAGCCACAAGATTACA-3'
5'-TGGGCACCAATATCAAGTCCAA-3'

APC:
5'-CATGATGCTGAGCGGCAGA-3'
5'-GCTGTTTCATGGTCCATTCGTG-3'

AXIN2:
5'-GAGTGGACTTGTGCCGACTTCA-3'
5'-GGTGGCTGGTGCAAAGACATAG-3'

TCF4:
5'-CTGCCTTAGGGACGGACAAAG-3'
5'-TGCCAAAGAAGTTGGTCCATTTT-3'

LEF1:
5'-AATGAGAGCGAATGTCGTTGC-3'
5'-GCTGTCTTTCTTTCCGTGCTA-3'

RNA isolation:

[0058] 0.5x 106 HCC116 Cells are cultured in a 6 well plate and cells are exposed to DMSO, IWR1, and small molecules (SM-61 and SM-66) for 24 h. RNA extraction is done using the Nucleo spin RNA isolation kit according to manufacturer protocol (MACHEREY-NAGEL GmbH & Co. KG). The quality and concentration of RNA are measured by nanodrop.

cDNA synthesis:

[0059] RNA is converted to cDNA using the PrimeScript 1st strand cDNA Synthesis kit (Takara, USA), 3 µg of mRNA, 1 µL of Oligo dT Primers, 1 µL dNTPs and 7 µL of RNAase free water are mixed in a polymerase chain reaction (PCR) tube and incubated at 65°C for 10 min, followed by cooling at 4 °C.

3. Western Blots (FIG. 5A-5C):

Cell seeding and compound treatments:

[0060] HCT116 cells (ATCC Cat. No. CCL-247) representative of colorectal carcinoma are seeded into 6 well plates (cell seeding density: 0.3 million cells/well). After 24 hrs, the cells are treated with the compounds to achieve a final concentration of 10µM along with DMSO and the positive control. After the 24 hrs post-treatment, the cells are harvested for protein expression analysis by western blotting.

Whole-cell lysate preparation:

[0061] The treated cells are lifted and washed with PBS buffer. Then the cells are lysed used RIPA buffer (Sigma, cat. no. R0278) supplemented with 1X protease inhibitor cocktail (Sigma, cat. no. S8820), 100µM Sodium ortho-vanadate (Sigma, cat. no. S6508), 50mM ß-Glycerophosphate (Sigma, cat. no. G9422), 1mM 1,4-Dithiothreitol (Sigma, Cat. no. 10197777001). The lysis step is performed on ice for 30min with a gentle vortex at regular intervals. The lysed cell suspension is centrifuged at 14000 rpm for 10 min and the supernatant is collected and quantified by the Bicinchoninic acid method (Pierce BCA Assay Kit, Thermo Scientific, Cat. No. 23227).

Western blotting:

[0062] 50µg of total protein is used for separation on 10% SDS-PAGE; transferred to PVDF membrane at 100V, 2hrs. The blot is blocked with a 7% non-fat milk solution for 1hour. The primary antibody is added to the blots and incubated overnight at 4 0C under the rocking condition. The horseradish peroxidase (HRP) tagged secondary antibody is added and incubated at room temperature under rocking conditions. All the antibodies are procured from Cell Signalling Technologies. HRP-tagged GAPDH antibody (Santacruz, cat. no. sc-47724) is used as the loading control probing agent. The blots are developed with Femto LUCENT PLUS-HRP (G-Biosciences, cat. no. 786003). The results are captured using the iBright Western blot imaging system (Thermoscientific, iBright CL1000 Imaging system). The imaging details are shown in FIGs. 5A-5C.

4. RNA Sequencing Studies (FIGs. 6A-6F):

Methods:

RNA Sequencing:

[0063] The total RNA (300 ng) is utilized for preparing RNA libraries using the NEB ultra RNA library kit (NEB #E7770S/#E7775S). The mRNA is fragmented by the enzyme and then converted to cDNA according to the literature protocol. The cDNA is end-repaired and further purified using Ampure XP beads. The cleaned DNA is adapter-ligated and also purified using Ampure XP beads. These adapter-ligated fragments are then subjected to 12 cycles of PCR using primers provided in the kit. The PCR products are purified using Ampure XP beads. The quantification and size distribution of the prepared library is determined using Qubit fluorometer and Agilent Tape station D1000 Kit (Agilent Technologies) according to the manufacturer’s instructions.

Data Analysis:

[0064] The transcriptome libraries (mRNA) are constructed using the NEB adapters and are sequenced on Illumina HiSeq at 150 nt read length using the paired-end chemistry. The raw data obtained is then processed for the low quality bases and adapters contamination. The raw reads subject to contamination (structural RNA/low complexity sequences) removal by mapping with bowtie 2-2.2.1. The decontaminated data set is mapped to the human genome (hg19) using hisat 2-2.1.0. Reads mapping to genes (hg19 gene list GTF) are counted using the feature count module of the sub-reads package. The read counts are normalized in DESeq2-3.5 and subjected to differential expression analysis.

Differential Gene Expression Analysis:

[0065] Significantly affected differentially expressed genes are selected based on 1.5x fold change, and then adjusted P-value of 0.05. Higherchial clustering heatmaps are created for the differentially expressed genes, using heatmap.2 function in R. Euclidian distance is used to calculate the distance between genes [rows] and samples [columns]. As shown in Figure, different gene clusters could be distinguished for SM-66, SM-61, and IWR-1 based on their gene expression pattern in response to small molecules.

Results:

Differential Gene Expression:

[0066] HCT116 cells are exposed to 10 µM SM-61, SM-66, and IWR-1 for 24 h and then subjected to RNA sequencing analysis. To obtain an impression of the number of genes affected per treatment, the genes are selected using the criteria =2-fold up- or down regulation with adjusted p-value <0.05.

Pathway Analysis:

[0067] In order to seek more insight into the mechanism of action of SM61, SM66, and IWR1, the differentially expressed genes are analyzed for over-representation of the genes representative of WNT dependent biological processes. In one embodiment, the gene sets are selected from Molecular signature databases. As shown in Figure, most of the genes down-regulated by SM-66 and SM-61 are involved in WNT dependent biological processes including EMT, stemness, metastasis, cell migration, and invasion. The effect is more pronounced with SM-66 than SM-61 and it is not observed with IWR-1.

5. Effect on Tumorspheres Obtained from HCT 116 (FIG. 7) and Indian Patient Buccal Mucosa (FIGs. 8A-8B; the RT-PCR Studies, FIGs. 9A-9C):

Cell culture and tumorsphere formation:

[0068] The colorectal HCT116 cell lines are cultured in DMEM medium with 10% fetal bovine serum (FBS) and 5% antibiotics. For the tumorsphere formation, 1300 single cells per well are cultured in ultra-low attached 24-well plates with tumorsphere medium (Promo cell USA) with 2% penicillin–streptomycin. Cells are incubated for 7-10 days at 37 °C and 5% of CO2. The spheres are observed using an inverted microscope and Floid imaging system (Life sciences technologies, USA).

Patient derived tumorspheres:

[0069] Tissue biopsy samples are cleaned with PBS buffer, treated with antibiotic solution and necrotic or dead tissue is removed. Then, clean tissue samples are subjected to enzymatic digestion using collagenase and trypsin. After digestion, cells are transferred in DMEM medium with F12 and B27 and cultured for primary cancer cells (PCCs). After characterization, 1300 PCCs per well are seeded in 24 well ultra-low attachment plates with tumorsphere medium and incubated for 7-10 days at 37 °C and 5% of CO2 for deriving patient-derived tumorspheres. The spheres are captured using an inverted microscope and a Floid imaging system (Life sciences technologies).

Tumorsphere formation (from HCT116) in the presence of small molecules:

[0070] To check the effect small molecules on tumorspheres formation, the first and second generation HCT116 tumorsphere are treated with the positive control IWR1 (and in some cases, XAV 939) and small molecules SM-61 and SM-66 for 10 days. For primary generation spheres, the HCT116 cells (1300 cells/well) are seeded in 24 well ultra-low attachment plates with tumorsphere medium and then exposed to small molecules on the same day of initiation. After 7 days, the first generation (G1) spheres are collected and further re-seeded 1300 primary spheres for second-generation (G2) sphere formation. The effect of small molecules on second-generation spheres is measured by an inverted microscope. Further, the Floid image system records appropriate images at different time intervals.

Patient-derived Tumorsphere formation in the presence of small molecules:

[0071] To check the effect of small molecules on patient-derived tumorspheres formation, the first and second generation patient-derived tumorspheres of two Indian patients Buccal Mucosa are formed. The patient-derived tumorspheres are then exposed to positive control IWR-1 and SM-66 for 10 days. For the first-generation spheres, the PCCs are seeded in 24 ultra-low attachment plates with the tumorsphere medium. After 7 days, the first-generation spheres (G1) are collected and further re-seeded to single spheres for the second-generation (G2) sphere formation and then exposed to small molecules. The effect of small molecules on second-generation spheres is measured by an inverted microscope and a Floid image system record images at different time intervals.

[0072] As performed with HCT cell lines, several genes are analyzed on the formation of 2nd generation tumorsphere in the presence of small molecules SM-61, SM-66, and IWR-1. The genes selected for this study are related to metastasis (Vimentin gene), migration and cell invasion (SNAIL and HEF1 gene), and stemness (CD44 and CD133). These results are shown in FIGs. 9A-9C.

[0073] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. It should be understood that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the invention.

[0074] The foregoing description comprise illustrative embodiments of the present invention. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only, and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Merely listing or numbering the steps of a method in a certain order does not constitute any limitation on the order of the steps of that method. Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings in the foregoing descriptions. Although specific terms may be employed herein, they are used only in generic and descriptive sense and not for purposes of limitation. Accordingly, the present invention is not limited to the specific embodiments illustrated herein.