WO2017196261A1

WO2017196261A1 - Jak and hdac dual-inhibitor compounds

Info

Publication number: WO2017196261A1
Application number: PCT/SG2017/050248
Authority: WO
Inventors: Brian William Dymock; Eugene Guorong YANG; Jong-Young YEN; Eng Chong TAN
Original assignee: National University Of Singapore; Academia Sinica
Priority date: 2016-05-11
Filing date: 2017-05-11
Publication date: 2017-11-16

Abstract

The present disclosure described provides novel dual inhibition compounds that specifically target the JAK-STAT and HDAC pathways, two distinct cellular pathways that are useful in the treatment of various diseases and disorders. In certain aspects, the compounds described herein are useful in the treatment of cellular proliferative and inflammatory diseases or disorders, such as graft vs. host disease. In particular embodiments, the compounds described herein inhibit JAK2 and HDAC6.

Description

JAK AND HDAC DUAL-INHIBITOR COMPOUNDS

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/334,970 (filed May 11, 2016). This application is hereby incorporated by reference in its entirety for all purposes.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

[0002] The Sequence Listing written in file SEQ_097520- 1047938-002710PC_ST25.TXT, created on May 10, 2017; 1,065 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[0003] Targeted treatment of cellular proliferative and inflammatory diseases typically focus on inhibiting a singular cellular pathway or enzyme. Targeting a singular cellular pathway or enzyme, however, is limited in that many diseases or disorders are caused by dysregulation of more than a single pathway or overexpression of more than a single enzyme. To combat these issues, treatments have expanded to formulating multiple drugs into a single dosage or administering a cocktail of drugs.

[0004] The Janus kinase/signal transducer and activator of transcription (JAK-STAT) and histone deacetylase (HDAC) pathways are two well-studied cellular pathways that are intimately associated with gene regulation and cellular function. Dysregulation of one or both pathways has been strongly linked to particular cellular proliferative and inflammatory diseases or disorders.

[0005] In recent studies, scientists have demonstrated that JAK-STAT and HDAC compounds play an important role in immune-related diseases, such as graft vs. host disease (GvHD). llogeneic bone marrow transplantation (alloBMT) is a crucial therapy for hematologic diseases, but it is limited by the development of GvHD in which alloreactive T cells cause cytokines from the transplanted tissue to storm and to reject the transplant recipient's body. Allogeneic response normally occurs after donor T cells interact with recipient's antigen-presenting cells (APC) post-transplantation. Activation of donor T cells, through the engagement of mismatched T cell receptors (TCRs)-MHC molecules on APCs, induces proliferation and causes damage to the recipient's tissue. Mismatched TCR-MHC interaction promotes activation of the JAK-STAT signaling pathway in T cells, which in turn leads to deregulation of histone acetylation and deacetylation in alloreactive T cells. JAK2 inhibitors (e.g., Choi, Jaebok et al. PloS 2014, 9(10), el09799) and HDAC inhibitors (e.g., Reddy, Pavan et al. Proceedings of the Nat'l Acad, of Sciences of the United States of America 2004, 101(11), 3921-26) have been shown to have potential as treatments for GvHD.

[0006] Single agent JAK or HDAC inhibitors have also been successfully launched in the JAK2-driven hematological malignancy myelofibrosis (ruxolitinib) and HDAC inhibitor sensitive T-cell lymphomas (1, FK228 and belinostat). Recently, panobinostat in combination with bortezomib and dexamethasone was approved as the first targeted HDAC agent to treat multiple myeloma. Garnock- Jones, K. P., Drugs 2015, 75, 695-704. Broadening of therapy into leukemias/lymphomas and solid tumors is highly desirable, and with this aim in mind, much effort has been expended in the clinic to evaluate compounds inhibiting either the JAK or HDAC families with varying degrees of isoform selectivity. Notably, ruxolitinib has received wide coverage of its encouraging initial responses in combination with capecitabine (a prodrug of 5-fluorouracil) in patients with recurrent or treatment-refractory metastatic pancreatic cancer, hinting at the potential value of JAK inhibitors in difficult-to-treat solid tumors. "Ruxolitinib Plus Capecitabine Improves Survival in Pancreatic Cancer," available on the worldwide web at onclive.com/web-exclusives/Ruxolitinib-Plus-Capecitabine- Improves-Survival-in-Pancreatic-Cancer. Combination of ruxolitinib with panobinostat is being studied in the clinic for myelofibrosis with the expectation that this combination could be extended to other indications if successful. "Panobinostat and Ruxolitinib in MPN clinical trial," available on the worldwide web at clinicaltrials.gov/ct2/show/NCT01433445.

[0007] Current methods of treating cellular proliferative disorders, for example, often require multiple therapeutic interventions for successful long term outcomes. Traditional approaches to combating cancer with radiation or chemotherapy are poorly selective. See, e.g., Al-Lazikani, B. et al., Nat Biotechnol 2012, 30, 679-92. At the other extreme, targeted treatment of cancer typically aims to target cancer cells via blockage of a single biological target. However, more intensive evaluation of typical 'targeted' drugs often reveals multiple mechanisms need to be targeted to achieve a sufficient clinical response. Accordingly, typical targeted cancer treatments generally include cocktails of separate drugs or a multicomponent drug (i.e., two or more targeted therapies in a single formulation). Both of these approaches, however, have drawbacks that include poor patient compliance, complex pharmacokinetics, and the challenge of drug-drug interactions.

[0008] A recently emerging trend in small-molecule cancer therapy is designed multiple ligand (DML) inhibitors— i.e., a single small-molecule drug that can bind to more than one cellular target, affecting more than one important disease pathway simultaneously, and that exclude multi-kinase drugs, which are typically ATP-competitive inhibitors that are structurally similar. This approach has several advantages. For example, DMLs do not suffer from potential drug-drug interactions (unless combined with another drug). Once the potency ratio is determined, it is fixed within the molecule, hence variable pharmacokinetics does not affect multiple pathway inhibition as it commonly does with combinations of two single agents.

[0009] Although prior work has developed small molecules that can inhibit JAK kinases or HDAC enzymes individually, dual inhibitors of JAK kinases and HDAC enzymes are very rare. See Ning et al., Design and synthesis of multi-acting inhibitors against HDAC, FLT3 and JAK2. European J. Med. Chem. 2015, 95, 104-15. However, the parent compound for the Ning et al. series, SB 1317, is also active against cyclin dependent kinases (CDKs; e.g., CDK1), and Ning et al. does not specify the selectivity of their inhibitor.

[0010] As such, there is a need for compounds that can inhibit both JAK kinases and HDAC enzymes, as such compounds could be useful in the treatment of, e.g., cancers and inflammatory diseases, such as GvHD. The present invention satisfies this need and provides other advantages as well.

BRIEF SUMMARY

[0011] In certain aspects, the present disclosure provides novel dual inhibition compounds that specifically target two distinct cellular pathways that are useful in the treatment of various diseases and disorders. In certain aspects, the present disclosure describes novel dual inhibition compounds of the JAK-STAT and HDAC pathways. In other aspects, the compounds described herein are useful in the treatment of cellular proliferative and inflammatory diseases or disorders. In particular embodiments, the compounds described herein inhibit JAK2 and a specific HDAC (e.g., HDAC6). The potency of synergistic inhibition of the JAK-STAT and HDAC pathways with a single compound provides opportunities for advancing the treatment of various diseases and disorders including cancer and inflammation, such as GvHD.

[0012] In certain embodiments, the present invention provides a compound having Formula I

or a pharmaceutically acceptable salt thereof,

wherein

n is an integer from 1 to 4;

1 2

C¹ and Cr are each independently C₆-ioaryl or C2-₉heteroaryl;

L 1 is a group of formula -X 1 -Y-X2 -, wherein X 1 is attached to C 1 and X2 is attached to C 2 ; and wherein X 1 , X2 , and Y are selected such that the group L 1 comprises from 5 to 15 ring atoms;

X 1¹ and X 2" are each independently a Ci_₈alkyl group or heteroCi_₇alkyl group, Y is a group of formula -CR^a=CR^b- or -CHR^aCHR^b-;

Z is a bond, CH₂, O, S, N(R^a), C(0)N(R^a), or N(R^a)C(0);

R^a and R^b are each independently selected from the group consisting of H, Ci_₃alkyl, fluoroCi_₃alkyl, and heteroCi_₃alkyl; or R^a and R^b together with the carbon atoms to which they are attached form a ring-fused C5_₇cycloalkenyl or C₄_₆heterocycloalkenyl group;

wherein the R^aR^b ring-fused group has from 0 to 2 ring substituents that are each

independently selected from the group consisting of halogen, Ci_₃alkyl, Ci_₃alkyloxy, fluoroCi_₃alkyl, and fluoroCi_₃alkyloxy;

R 1 and R 2" are each independently selected from the group consisting of H, halogen, Ci_₈alkyl, haloCi_₈alkyl, heteroCi_₇alkyl, hydroxyl, amino, Ci_₈acylamino, Ci_₈alkylamino, sulfonylamino, thio, -C(0)OR^c, -C(0)N(H)R^c, and -C(0)N(R^c)OR^c; or R¹ and R² together with the carbon atoms to which they are attached form a ring-fused C5_₇cycloalkenyl,

C₄_₇heterocycloalkenyl, or C₂-₅heteroaryl group; wherein the R 1 R 2 ring-fused group has from 0 to 2 ring substituents that are each independently selected from the group consisting of halogen,

Ci_₃alkyl, Ci_₃alkyloxy, fluoroCi_₃alkyl, and f uoroCi_₃alkyloxy;

R³ is selected from the group consisting of -OR^5a, -N(R^5a)R^5b, and -L²R^5c;

each L is selected from the group consisting of a bond, Ci_₆alkylene, and

-N(R^c)C(0)Ci_₆alkylene;

R⁴ is selected from the group consisting of H, Ci_₃alkyl, and trifluoromethyl;

R^5a is selected from the group consisting of C_6-1oaryl, Ci_₉heteroaryl, C₃_₈cycloalkyl, and

C₃_₇heterocyclyl, wherein the C_6-1oaryl, Ci_₉heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group includes a substituent that is selected from the group consisting of -C(0)OR^c, - C(0)NHR^c,

-C(0)N(R^c)OR^c, and -L²R⁶; or R^5a and R^5b together with the amino atom to which they are attached form a C₄_₈heterocyclyl ring with a substituent that is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c,-C(0)N(R^c)OR^c, and -L²R⁶;

R^5b is selected from the group consisting of R^c, -C(0)OR^d, and -C(0)NHR^d; or R^5b and R^5a together with the amino atom to which they are attached form a C₄_₈heterocyclyl ring with a substituent that is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c, -C(0)N(R^c)OR^c, and -L²R⁶;

R^5c is selected from the group consisting of C_6-1oaryl, Ci_₉heteroaryl, C₃_₈cycloalkyl, C₃_₇heterocyclyl, -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c , wherein the C₆-i₀aryl, Ci_₉heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group includes an -L²R⁶ substituent; each R⁶ is independently selected from the group consisting of -C(0)NHR^c, -C(0)N(R^c)OR^c, -C(0)OR^c, Ci_₈alkyleneC(0)N(R^c)OR^c, and Ci_₈alkyleneC(0)OR^c;

each R^c is independently selected from the group consisting of H and Ci_₆alkyl; and each R^d is independently selected from the group consisting of Ci_₈alkyl, C₇_i₂arylalkyl, Ci_ gheteroaryl, C₂-iiheteroarylalkyl, C₃_₆cycloalkyl, and C₄_iocycloalkylalkyl.

[0013] In certain aspects, the invention provides a method of treating a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of a compound as set forth herein (i.e., any aspect or combination of aspects) to a subject in need thereof.

[0014] In certain aspects, the disease or disorder is selected from the group consisting of cancer and inflammation. In certain aspects, the disease is cancer. In certain aspects, the disease or disorder is inflammation. [0015] In certain aspects, the disease or disorder is inflammation. In certain aspects, the disease or disorder is graft vs. host disease.

[0016] In certain aspects, the disease or disorder is selected from the group consisting of a hematologic cancer, a hyperproliferative condition, a gynecologic cancer, a gastrointestinal tract cancer, a urinary tract cancer, a skin cancer, a brain tumor, a head and neck cancer, a respiratory tract cancer, an ocular cancer, and a musculoskeletal cancer. In certain aspects, the hyperproliferative condition is psoriasis or restenosis.

[0017] In certain preferred aspects, the administration of the compound dually inhibits JAK-STAT and HDAC pathways. In certain aspects, administration of the compound dually inhibits JAK2 and HDAC pathways. In certain aspects, administration of the compound dually inhibits JAK2 and HDAC 6.

[0018] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS [0019] FIGS. 1A and IB summarize enzyme inhibition and solid tumor cell proliferation data for selected carboxylic acids. FIG. 1A provides enzyme inhibition data, while FIG. IB provides solid tumor cell proliferation data.

[0020] FIGS. 2A-2C summarize enzyme inhibition and solid tumor cell proliferation data for selected hydroxamic acids. FIG. 2A provides enzyme inhibition data, while FIGS. 2B and 2C provide solid tumor cell proliferation data. The same molecule structure shown in FIGS. 2B and 2C was also used in the experiments of FIG. 2A.

[0021] FIG. 3A shows a model for HDAC6 and its calculated binding mode with compound 51. FIG. 3B shows more detailed interactions between compound 51 and HDAC6.

[0022] FIG. 4A shows a model for HDACl and its calculated binding mode with compound 51. FIG. 4B shows more detailed interactions between compound 51 and HDACl.

[0023] FIG. 5A shows a model for JAK2 and its calculated binding mode with compound 51. FIG. 5B shows more detailed interactions between compound 51 and JAK2. [0024] FIG. 6A shows a selectivity index (SI) plot for compound 51 for the Class I, II and IV HDAC isoforms plotted with a log scale. The black bars are HDAC/JAK2; the white bars, HDAC/HDAC6.

[0025] FIG. 6B illustrates the selectivity screening data of compound 51 against a panel of 97 kinases.

[0026] FIGS. 7 A and 7B shows that compound 51 effectively blocks colony formation in the erythroleukemia cell line HEL92.1.7 expressing endogenous JAK2V617F. FIG. 7A shows a bar graph indicating dose response based on total HEL92.1.7 cell populations, with data presented as mean + SEM. FIG. 7B shows photographs of selected colonies indicating controls (al, a2), treatment with compound 51 at 0.2 μΜ (bl, b2), 1.0 μΜ (cl, c2) and 2.0 μΜ ((11, d2).

[0027] FIGS. 8A-8D show that compound 51 effectively blocks dual signalling pathways in MM and AML cell lines. FIGS. 8A and 8B show the detection of acetylated tubulin (Ac- Tubulin) and acetylated histone 3 (Ac-H3) in cell lines KMS-12-BM (FIG. 8A) and MOLM- 14 (FIG. 8B) after treatment with compound 51. FIGS. 8C and 8D show the detection of p- STAT3 in cell lines KMS-12-BM (FIG. 8C) and MOLM-14 (FIG. 8D) after treatment with compound 51.

[0028] FIGS. 9A-9D show that compound 51 effectively blocks dual signalling pathways in the erythroleukemia cell line HEL92.1.7 expressing endogenous JAK2^V617F. FIG. 9A shows the detection of acetylated tubulin (Ac-Tubulin) and acetylated histone 3 (Ac-H3) in HEL92.1.7 cells that were treated with compounds 1 and 51. FIG. 9B shows the

quantification of HDAC inhibitory responses by densitometry. FIG. 9C shows the detection of p-JAK2 and p-STAT5 (Ac-H3) in HEL92.1.7 cells that were treated with compound 51. FIG. 9D shows the quantification of JAK inhibitory responses by densitometry. [0029] FIGS. lOA-lOC show that compound 51 triggers apoptosis in the AML cell line, MOLM-14. FIG. 10A shows MOLM-14 cells that were treated with compound 51 and subsequently stained by annexin-V FITC and propidium iodide(PI). FIG. 10B shows annexin V FITC vs. PI plots showing the populations corresponding to viable and non- apoptotic (Annexin V-PI-), early (Annexin V+PI-), and late (Annexin V+PI+) apoptotic cells following 48 h treatment of compound 51. FIG. IOC shows MOLM-14 cells that were treated with compound 51, lysed, and subjected to immunoblotting to detect PARP cleavage. [0030] FIGS. 11A and 1 IB illustrate the rat microsomal stability data for compounds 51 and 52. FIG. 11 A shows a plot of parent remaining against time for male and female mirosomes. FIG. 1 IB shows the parameters of Ti_/2 and Clin_t, app values (mean + SD) for compounds 51 and 52 (3 μΜ) upon incubation at 37°C for 45 min in male (MRLM) and female (FRLM) rat liver microsomes.

[0031] FIGS. 12A and 12B illustrate the reduction of allogeneic T cell proliferation in murine and human mixed lymphocyte reaction (MLR) by various inhibitors. FIG. 12A shows a murine MLR, while FIG. 12B shows a human MLR.

[0032] FIGS. 13A-13C illustrate the results of targeting JAK2 and HDAC6 activities, which reduced T cell viability at early and late stages of activation. FIG. 13A shows the results from early T cell activation; FIG. 13B shows the results from late T cell activation. FIG. 13C shows the results of a CD3 bypass assay.

[0033] FIGS. 14A and 14B show how treatment with JAK2-HDAC6 dual inhibitor reduced pro-inflammatory cytokine production by T cells. FIG. 14A shows the effects on interferon- gamma (ΠΤΝΓγ), while FIG. 14B shows the effects on tumor necrosis factor alpha (TNFa).

[0034] FIGS. 15A-15F show how the suppression of JAK2 and HDAC6 activities attenuated GvHD effects and prolonged survival in a murine model for severe GvHD.

BALB/cJNarl recipient mice were lethally irradiated with a severe dose (FIGS. 15A-15C) or a mild dose (FIGS. 15D-15F). FIG. 15A and 15D show the effects on percentage body weight change, FIG. 15B and 15F show the percentage of survival , and FIGS. 15C and 15F show the GvHD scores for the experiments.

DETAILED DESCRIPTION

I. DEFINITIONS

[0035] The terms "a," "an," or "the" as used herein not only includes aspects with one member, but also includes aspects with more than one member. For example, an

embodiment including "a diluent and a binder" should be understood to present certain aspects with at least a second diluent, at least a second binder, or both. An embodiment including "an active agent" should be understood to present certain aspects with at least a second active agent, which may be of a different class (e.g., a JAK-STAT/HDAC inhibitor with a different class of anti-cancer or anti-inflammatory drug). [0036] The term "about" as used herein to modify a numerical value indicates a defined range around that value. If "X" were the value, "about X" would generally indicate a value from 0.95X to 1.05X. Any reference to "about X" specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" is intended to teach and provide written description support for a claim limitation of, e.g., "0.98X."

[0037] In compositions comprising an "additional" or "second" component, the second component as used herein is chemically different from the other components or first component. A "third" component is different from the other, first, and second components, and further enumerated or "additional" components are similarly different.

[0038] "Agent" as used herein indicates a compound or mixture of compounds that, when added to a pharmaceutical composition, tend to produce a particular effect on the

composition's properties. For example, a composition comprising a thickening agent is likely to be more viscous than an otherwise identical comparative composition that lacks the thickening agent.

[0039] "Alkyl," by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C_1-2, C_1-3, C_1-4, C_1-5, C_1-6, C_1-7, C_1-8, C_1-9, CMO, C_2-3, C₂^, C₂_₅, C₂-₆, C₃_₄, C₃-5, C₃_₆, C₄-5, C₄_₆ and C_5-6. For example, C_1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 12 or up to 20 carbon atoms, such as heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted, but in a preferred embodiment, are unsubstituted unless the substitution is expressly described.

[0040] "Alkylene" refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group. For instance, a straight chain alkylene can be the bivalent radical of -(CH₂)_N-_I where n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene. Alkylene groups can be substituted or unsubstituted, but in a preferred embodiment, are unsubstituted unless the substitution is expressly described. [0041] "Alkenyl" refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C₂, C₂_3, C₂_₄, C₂_5, C₂_6, C₂_7, C₂_8, C₂_9, C₂_io, C₃, C₃_₄, C₃-5, C₃_6, C₄, C₄-5, C₄_6, C5, C5-6, and C₆. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.

Alkenyl groups can be substituted or unsubstituted, but in a preferred embodiment, are unsubstituted unless the substitution is expressly described.

[0042] As used herein, the phrase "comprising X" does not exclude other elements besides X. For example, a composition comprising X and Y may include other components besides X and Y.

[0043] As used herein, the phrase "effective amount" or "effective dose" means an amount sufficient to achieve the desired result and accordingly will depend on the ingredient and its desired result. Nonetheless, once the desired effect is known, determining the effective amount is typically within the skill of a person skilled in the art.

[0044] "Haloalkyl" refers to alkyl, as defined above, where some or all of the hydrogen atoms are replaced with halogen atoms (e.g., -CH₂F). As with the alkyl group, haloalkyl groups can have any suitable number of carbon atoms, such as Ci_₃ or Ci_6. For example, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In some instances, the term "perfluoro" can be used to define a compound or radical where all the hydrogens are replaced with fluorine. For example, perfluoromethyl refers to 1,1,1 -trifluoromethyl.

[0045] "Heteroalkyl" refers to an alkyl group having from 1 to 3 heteroatoms (e.g., N, O and S). Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as -S(O)- and -S(0)₂-. For example, heteroalkyl can include ethers, thioethers and alkylamines. The heteroatom portion of the heteroalkyl can replace a hydrogen of the alkyl group to form a hydroxy, thio or amino group. Alternatively, the heteroartom portion can be the connecting atom, or be inserted between two carbon atoms.

[0046] "Cycloalkyl" refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C₃_6, C₄_₆, Cs_6, C₃_₈, C₄_₈, C5_₈, C_6-8, C₃_9, C₃_io, C₃_ii, and C₃_i₂. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2]

bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene. When cycloalkyl is a saturated monocyclic C₃_₈ cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C₃_₆ cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted, but in a preferred embodiment, are unsubstituted unless the substitution is expressly described.

[0047] "Heterocycloalkyl" refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms of N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as -S(O)- and -S(0)₂-. Heterocycloalkyl groups can include any number of ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in the heterocycloalkyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The

heterocycloalkyl group can include, for example, aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4- isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine,

thiomorpholine, dioxane, or dithiane. The heterocycloalkyl groups can also be fused to aromatic or non-aromatic ring systems to form members including, but not limited to, indoline. Heterocycloalkyl groups can be unsubstituted or substituted. For example, heterocycloalkyl groups can be substituted with Ci_6 alkyl or oxo (=0), among many others.

[0048] "Aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of ring atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be substituted or unsubstituted, but in a preferred embodiment, are unsubstituted unless the substitution is expressly described. [0049] In certain aspects, substitution (e.g., of an aryl or a heteroaryl group) includes one, two, or three substituents, wherein each substituent is independently selected from halogen, cyano, hydroxy, C_1-6 alkyl, C₂-6 alkenyl, C₂_₆ alkynyl, C_1-6 alkoxy, C_1-6 haloalkyl, C_1-6 haloalkoxy, C3-5 cycloalkyl- alkoxy, amino, C_1-6 alkylamino, di-C_1-6 alkylamino, and -

C(0)R 7 , wherein each R 7 is a member independently selected from H, hydroxy, Ci_₆ alkyl, Ci_6 alkoxy, amino, Ci_₆ alkylamino and di- Ci_₆ alkylamino.

[0050] "Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom, such as N, O or S. Additional heteroatoms can also be useful, such as B, Al, Si and P. The heteroatoms can also be oxidized, such as -S(O)- and -S(0)₂-. Heteroaryl groups can include any number of ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8 ring members and from 1 to 4 heteroatoms, or from 5 to 8 ring members and from 1 to 3 heteroatoms, or from 5 to 6 ring members and from 1 to 4 heteroatoms, or from 5 to 6 ring members and from 1 to 3 heteroatoms. The heteroaryl group can include, e.g., pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5- isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroaryl groups can also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles (e.g., indole or isoindole), benzopyridines (e.g., quinoline or isoquinoline), benzopyrazine (e.g., quinoxaline), benzopyridazines (e.g., phthalazine or cinnoline), benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted, but in a preferred embodiment, are unsubstituted unless the substitution is expressly described.

[0051] "Cycloalkylalkyl" refers to a cycloalkyl-alkyl group in which the cycloalkyl and alkyl moieties are as previously described. Exemplary monocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl. The group may be a terminal group or a bridging group.

[0052] "Heterocycloalkylalkyl" refers to a heterocycloalkyl-alkyl group in which the heterocycloalkyl and alkyl moieties are as previously described. Exemplary

heterocycloalkylalkyl groups include (2-tetrahydrofuryl)methyl, (2- tetrahydrothiofuranyl)methyl. The group may be a terminal group or a bridging group.

[0053] "Arylalkyl" means an aryl-alkyl-group in which the aryl and alkyl moieties are as previously described. Preferred arylalkyl groups contain a Ci_5 alkyl moiety. Arylalkyl groups include, but are not limited to, benzyl, phenethyl and naphthelenemethyl.

[0054] The term "or" as used herein should in general be construed non-exclusively (i.e., as an inclusive disjunction). For example, a composition comprising A or B would typically present an aspect with a composition comprising both A and B. "Or" should, however, be construed to exclude those aspects presented that cannot be combined without contradiction (e.g., a composition pH that is between 9 and 10 or between 7 and 8).

[0055] The terms "such as," "for example," and "e.g." do not imply limitation to the examples expressly disclosed, at least not in every aspect of the invention. For example, the statement "a group X, such as A, B, or C" implies an aspect with "a group X selected from the group consisting of A, B, and C," but does not limit the group X to the species A, B, or C in every aspect of the invention.

[0056] "Formulation," "pharmaceutical composition," and "composition" as used interchangeably herein are equivalent terms referring to a composition of matter for pharmaceutical use.

[0057] The term "pharmaceutically acceptable" means compatible with the treatment of animals, and in particular, humans.

[0058] The term "pharmaceutically acceptable salt" is meant to include a salt of an active compound that is prepared with a relatively nontoxic acid or base, depending on the particular substituents found on the compounds described herein. When a compound of the present invention contains a relatively acidic functionality, a base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonium (e.g., tetraalkylammonium), zinc or magnesium salt, or a similar salt. When a compound of the present invention contains a relatively basic functionality, an acid addition salt can be obtained by contacting the neutral form of rhe compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,

monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogen sulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,

methanesulfonic, and the like. Also included are salts of amino acids, such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S.M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0059] Certain neutral forms of the compounds may be regenerated by contacting a derivative salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0060] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are intended to be encompassed within the scope of the present invention.

[0061] Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are intended to be within the scope of the present invention. [0062] Certain compounds of the present invention may possess asymmetric carbon atoms (i.e., chiral centers) or cis/trans stereoisomers (i.e., geometric isomers). In general, the racemates, diastereomers, geometric isomers, and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. In certain aspects, a composition is enriched in one isomer (e.g., entiomerically or

diastereomerically enriched).

[0063] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes at greater than natural abundance, such as deuterium ( 2 H), tritium ( 3 H), iodine- 125 ( 125 I), phosphorous-32 ( 32 P), or carbon- 14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0064] The term "pharmaceutically acceptable" means compatible with the treatment of animals, and in particular, humans. [0065] The term "subject" as used herein includes all members of the animal kingdom, such as mammals, e.g., humans.

[0066] The term "treating" or "treatment" as used herein (and as well understood in the art) means an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.

[0067] Treatment methods comprise administering to a subject a therapeutically effective amount of an active agent. In certain aspects, the administering step may consist of a single administration or may comprise a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.

[0068] The term "w/w" or "wt/wt" means a percentage expressed in terms of the weight the ingredient or agent over the total weight of the composition multiplied by 100.

II. DETAILED DESCRIPTION OF THE EMBODIMENTS

A. Dual Inhibition Compounds [0069] In certain aspects the present invention provides a compound having Formula I

(I)

or a pharmaceutically acceptable salt thereof,

wherein

n is an integer from 1 to 4;

1 2

C¹ and CT are each independently C6-ioaryl or C2-9heteroaryl;

1 1 2 1 1 2

L is a group of formula -X -Y-X -, wherein X is attached to C and X is attached

2 1 2 1

to C ; and wherein X , X , and Y are selected such that the group L comprises from 5 to 15 ring atoms;

1 2

X¹ and X" are each independently a Ci_₈alkyl group or heteroCi_₇alkyl group, Y is a group of formula -CR^a=CR^b- or -CHR^aCHR^b-;

Z is a bond, CH₂, O, S, N(R^a), C(0)N(R^a), or N(R^a)C(0);

independently selected from the group consisting of halogen, Ci_₃alkyl, Ci_₃alkyloxy, fluoroCi_₃alkyl, and fluoroCi_₃alkyloxy; R 1 and R 2" are each independently selected from the group consisting of H, halogen, Ci-galkyl, haloCi_₈alkyl, heteroCi_₇alkyl, hydroxyl, amino, Ci_₈acylamino, Ci_₈alkylamino, sulfonylamino, thio, -C(0)OR^c, -C(0)N(H)R^c, and -C(0)N(R^c)OR^c; or R¹ and R² together with the carbon atoms to which they are attached form a ring-fused C5_₇cycloalkenyl, C₄_₇heterocycloalkenyl, or d-sheteroaryl group; wherein the R 1 R 2 ring-fused group has from 0 to 2 ring substituents that are each independently selected from the group consisting of halogen,

Ci_₃alkyl, Ci_₃alkyloxy, fluoroCi_₃alkyl, and fluoroCi_₃alkyloxy;

each L is selected from the group consisting of a bond, Ci_₆alkylene, and

-N(R^c)C(0)Ci_₆alkylene;

R^5c is selected from the group consisting of C_6-1oaryl, Ci_₉heteroaryl, C₃_₈cycloalkyl,

C₃_₇heterocyclyl, -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c , wherein the C₆_i₀aryl, Ci-₉heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group includes an -L²R⁶ substituent; each R⁶ is independently selected from the group consisting of -C(0)NHR^c, -C(0)N(R^c)OR^c, -C(0)OR^c, Ci_₈alkyleneC(0)N(R^c)OR^c, and Ci_₈alkyleneC(0)OR^c;

each R^c is independently selected from the group consisting of H and Ci_₆alkyl; and each R^d is independently selected from the group consisting of Ci_₈alkyl, C_7- i₂arylalkyl, Ci_₉heteroaryl, C₂-iiheteroarylalkyl, C₃_₆cycloalkyl, and C₄_iocycloalkylalkyl. [0070] In certain aspects, if one or more L 2 groups were present, exactly one L 2 group is not a bond. In certain aspects, if one or more L 2 groups were present, all L 2 groups are a bond.

[0071] In certain aspects, L¹ comprises at least one oxygen as a ring atom. In certain aspects, L¹ comprises one, two, or three oxygen atoms as ring atoms (e.g., two ether oxygens, as in Formulae la, lb, and Ic).

[0072] In certain aspects, L¹ comprises at least one amine or amide nitrogen as a ring atom. In certain aspects, L¹ comprises one, two, or three amine or amide nitrogens as ring atoms.

[0073] In certain aspects, Z is a bond, CH₂, O, S, N(R^a), C(0)N(R^a), or N(R^a)C(0). In certain aspects, Z is O, S, or N(R^a). In certain aspects, Z is O. In certain aspects, Z is S. In certain aspects, Z is -S(O)-, or -S0₂-. In certain aspects, Z is N(R^a). In certain aspects, Z is - NH-. In certain aspects, Z is -C(0)N(R^a)-. In certain aspects, Z is -N(R^a)C(0)-. . In certain aspects, Z is -C(0)NH-. In certain aspects, Z is -N(H)C(0)-. In certain aspect, Z is a bond. In certain aspects, Z is CH₂. [0074] In certain aspects, n is 2. In certain aspects, n is 3. In certain aspects, n is 4. In certain aspects, n is 1, and R³ is -L²R^5c.

[0075] In certain aspects, R³ is -OR^5a; and R^5a is selected from the group consisting of C₆aryl, C₃_₅heteroaryl, C₃_₈cycloalkyl, and C3_₇heterocyclyl, wherein the Cearyl, C₃_

5heteroaryl,

C₃_₈cycloalkyl, or C₃_₇heterocyclyl group substituent is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c. In certain aspects, R^5a is selected from the group consisting of Cearyl (i.e., phenyl) and C₃_₅heteroaryl (e.g., pyridyl, pyrrolyl, or imidazolyl).

[0076] In certain aspects, R³ is selected from the group consisting of -N(R^5a)R^5b; wherein R^5a is selected from the group consisting of Cearyl, C₃_₅heteroaryl, C₃_₈cycloalkyl, and C₃_

₇heterocyclyl, wherein the Cearyl, C₃_₅heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group substituent is -L²R⁶; and wherein R^5b is R^c.

[0077] In certain aspects, R³ is selected from the group consisting of -N(R^5a)R^5b; wherein R^5a and R^5b together with the amino atom to which they are attached form a C₄_₈heterocyclyl ring with a substituent that is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c, -C(0)N(R^c)OR^c, and -L²R. [0078] In certain aspects, R³ is -Ci_₆alkyleneR^5c; and R^5c is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c. In certain aspects, R³ is ₅alkyleneR^5c; and R^5c is -C(0)N(H)OH.

[0079] In certain aspects, the compound has Formula la

(la).

[0080] In certain aspects, C¹ is selected from the group consisting of

In certain aspects, C 1 includes 1 to 3 R 7 substituents.

[0081] In certain aspects, C¹ is selected from the group consisting of

In certain aspects, C 1 includes 1 to 3 R 7 substituents. [0082] In certain aspects, C¹ is

In certain aspects, C 1 includes 1 to 2 R 7 substituents. [0083] In certain aspects, C¹ is

In certain aspects, C 2 includes 1 to 3 R 7 substituents.

[0084] In certain

In certain aspects, C 2 includes 1 to 3 R 7 substituents.

[0085] In certain aspects, C is selected from the group consisting of

In certain aspects, C 2 includes 1 to 2 R 7 substituents.

2

[0086] In certain aspects, C is selected from the group consisting

In certain aspects, C 1 includes 1 to 3 R 7 substituents. [0087] In certain aspects, the compound has Formula lb

(lb).

[0088] In certain aspects, the compound has Formula Ic

(Ic).

[0089] In certain aspects, R 1 and R 2 are each independently selected from the group consisting of H, halogen, Ci_₃alkyl, haloCi_₃alkyl, heteroCi_₃alkyl, hydroxyl, amino, and thio; or R 1 and R 2 together with the carbon atoms to which they are attached form a ring-fused pyrrole or imidazole group.

[0090] In certain aspects, R 1 and R 2 are each H.

[0091] In certain aspects, R 1 is H and R2 is CH₃. In certain aspects, R 1 is H and R2 is CF₃.

In certain aspects, R 1 is F and R2 is CH₃. In certain aspects, R 1 is F and R2 is CF₃. [0092] In certain aspects, R is selected from the group consisting of [0

[0094] In certain aspects, R is selected from the group consisting of

In certain aspects, R is selected from the group consisting of

[0096] In certain aspects, R is selected from the group consisting of

and H [0097] In certain aspects, R is selected from the group consisting of

[

[0099] In certain aspects, R comprises a -C(0)N(H)OH group or a salt thereof.

[0100] In certain aspects, R⁴ is H or C_1-6 alkyl. In certain aspects, R⁴ is H. [0101] In certain aspects, the compound has a molecular weight of 750 or less when not in salt form. In certain aspects, the compound has a molecular weight of 700 or less when not in salt form. In certain aspects, the compound has a molecular weight of 650 or less when not in salt form. In certain aspects, the compound has a molecular weight of 625 or less when not in salt form. In certain aspects, the compound has a molecular weight of 600 or less when not in salt form. In certain aspects, the compound has a molecular weight of 575 or less when not in salt form. In certain aspects, the compound has a molecular weight of 550 or less when not in salt form. In certain aspects, the compound has a molecular weight of 525 or less when not in salt form. In certain aspects, the compound has a molecular weight of 500 or less when not in salt form.

[0102] In certain aspects, the compound has a calculated logP of 6 or less. In certain aspects, the compound has a cLogP of 5.5 or less. In certain aspects, the compound has a calculated logP of 5 or less. In certain aspects, the compound has a cLogP of 4.5 or less. In certain aspects, the compound has a cLogP of 4 or less. [0103] In certain aspects, the compound has 6 or fewer hydrogen bond donors. In certain aspects, the compound has 5 or fewer hydrogen bond donors. In certain aspects, the compound has 4 or fewer hydrogen bond donors. In certain aspects, the compound has 3 hydrogen bond donors.

[0104] In certain aspects, the compound has the structure

[0105] In certain aspects, the compound has the structure

B. Diseases and Conditions

[0106] Administering a dual inhibition compound with a therapeutic agent is useful in treating or preventing many diseases including cancers and inflammation.

[0107] In certain aspects, the invention provides a method of treating a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of a compound as set forth herein (i.e., any aspect or combination or aspects) to a subject in need thereof. [0108] In certain aspects, the disease or disorder is selected from the group consisting of cancer and inflammation. In certain aspects, the disease is cancer. In certain aspects, the disease or disorder is inflammation.

[0109] In certain aspects, the disease or disorder is selected from the group consisting of a hematologic cancer, a hyperproliferative condition, a gynecologic cancer, a gastrointestinal tract cancer, a urinary tract cancer, a skin cancer, a brain tumor, a head and neck cancer, a respiratory tract cancer, an ocular cancer, and a musculoskeletal cancer. In certain aspects, the hyperproliferative condition is psoriasis or restenosis.

[0110] In certain aspects, the administration of the compound dually inhibits JAK-STAT and HDAC pathways. In certain aspects, administration of the compound dually inhibits JAK2 and HDAC pathways. In certain aspects, administration of the compound dually inhibits JAK2 and HDAC 6.

1. Cancer

[0111] In certain aspects, cancer can be treated or prevented by administering a dual inhibition compound described herein to inhibit the JAK-STAT and HDAC pathways. In some embodiments, administration of said compounds dually inhibits JAK2 and HDAC6. Cancer generally includes any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites. Non-limiting examples of different types of cancer suitable for treatment using the compositions of the present invention include leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, or hairy cell leukemia), lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell lymphoma, or Burkitt's lymphoma)_ovarian cancer, breast cancer, lung cancer (e.g., non- small-cell lung carcinoma), bladder cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, colon cancer, skin cancer, and multiple myeloma.

[0112] In particular embodiments, the cancer may be selected from the group consisting of idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasma cell disorder, hairy cell leukemia, Kaposi's sarcoma, lymphoma, breast carcinoma, ovarian cancer, cervical cancer, vaginal or vulva cancer, endometrial hyperplasia, colorectal carcinoma, polyps, liver cancer, gastric cancer, pancreatic cancer, gall bladder cancer, prostate cancer, kidney or renal cancer, urinary bladder cancer, urethral cancer, penile cancer, melanoma, glioblastoma, neuroblastoma, astrocytoma, ependynoma, brain-stem glioma, medulloblastoma, menigioma, astrocytoma, oligodendroglioma, nasopharyngeal carcinoma, laryngeal carcinoma, small-cell lung carcinoma, non-small-cell lung carcinoma, mesothelioma, retinoblastoma, osteosarcoma, musculoskeleletal neoplasm, squamous cell carcinoma, and a fibroid tumour. [0113] In some embodiments, the dual inhibition compounds describe herein are useful in preventing solid tumors. In some embodiments, the dual inhibition compounds described herein are useful in treating resistant myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML).

2. Inflammation [0114] In certain aspects, inflammation can be reduced or prevented by administering a compound of Formula I to inhibit the JAK-STAT and HDAC pathways. In some

embodiments, administration of said compounds dually inhibits JAK2 and HDAC6. Inflammation generally includes any of various conditions characterized by a localized protective response elicited by injury, infection, or destruction of tissues, and which is manifest by heat, swelling, pain, redness, dilation of blood vessels or increased blood flow, invasion of the affected area by white blood cells, loss of function, or any other symptoms known to be associated with the inflammatory condition. Non-limiting examples of different types of inflammatory diseases suitable for treatment using the compositions of the present invention include inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, spastic colon, rheumatoid arthritis, amyotrophic lateral sclerosis, multiple sclerosis, psoraiasis, restenosis, lupus, vascular infection, myocardial infarction, graft vs. host disease (GVHD), and Sjogren's syndrome. In certain embodiments, the inflammatory disease is psoriasis or restenosis.

[0115] In certain preferred embodiments, the inflammatory disease is GVHD. C. Pharmaceutical Compositions

[0116] The dual inhibition compounds described herein are useful in the manufacture of a pharmaceutical composition or a medicament for modulating the immune system of a subject with cancer or an infectious disease. In certain aspects, a pharmaceutical composition or medicament comprising a dual inhibition compound can be administered to a subject for the treatment of a cancer or an inflammatory disease.

[0117] Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques or methods well-known in the art of pharmacy using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in, e.g., "Remington's Pharmaceutical Sciences" by E.W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including, but not limited to, orally, topically, nasally, rectally, pulmonary, parenterally (e.g., intravenously, subcutaneously, intramuscularly, etc.), and combinations thereof. The most suitable route of administration for a dual inhibition compound in any given case will depend, in part, on the disease or disorder being treated. The most suitable route of administration can will also depend on the nature, severity, and optionally, the stage of the cancer or inflammatory disease being treated. [0118] In certain embodiments, the pharmaceutical compositions or medicaments described herein are suitable for systemic administration. Systemic administration includes enteral administration (e.g., absorption of the compound through the gastrointestinal tract) or parenteral administration (e.g., injection, infusion, or implantation). In some embodiments, the pharmaceutical compositions or medicaments may be administered via a syringe or intravenously. In preferred embodiments, the pharmaceutical compositions or medicaments are injected subcutaneously.

[0119] In certain embodiments, the pharmaceutical compositions or medicaments described herein are suitable for oral administration. For oral administration, a pharmaceutical composition or a medicament can take the form of, e.g., a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient. Preferred are tablets and gelatin capsules comprising the active ingredient(s), together with (a) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, or hydroxypropyl methylcellulose; if desired (b) lubricants, e.g., silica, anhydrous colloidal silica, talcum, stearic acid, corn starch, sodium benzoate, sodium acetate, or polyethyleneglycol; for tablets also (c) disintegrants, e.g., starches (e.g., potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (d) diluents or fillers, e.g., lactose, dextrose, sucrose, or calcium hydrogen phosphate, calcium sulfate, (e) absorbents, colorants, flavors, or sweeteners, or (f) wetting agents, e.g., sodium lauryl sulfate. When a dosage form is a capsule, in addition to the above materials it may also contain liquid carriers, such as water, saline, or a fatty oil. Tablets or capsules may be either film-coated or enteric-coated according to methods known in the art.

[0120] Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound.

[0121] Formulation for administration by inhalation (e.g., aerosol), or for oral, rectal, or vaginal administration is also contemplated. [0122] In some embodiments, the compounds are prepared with a polysaccharide, such as chitosan or derivatives thereof (e.g., chitosan succinate, chitosan phthalate, etc.), pectin and derivatives thereof (e.g., amidated pectin, calcium pectinate, etc.), chondroitin and derivatives thereof (e.g., chondroitin sulfate), and alginates.

[0123] In some embodiments, the compositions further include a pharmaceutical surfactant. In other embodiments, the compositions further include a cryoprotectant. Non-limiting examples of cryoprotectants include glucose, sucrose, trehalose, lactose, sodium glutamate, PVP, cyclodextrin, 2-hydroxypropyl-13-cyclodextrin (HPI3CD) glycerol, maltose, mannitol, saccharose, and mixtures thereof.

D. Methods of Administration [0124] Pharmaceutical compositions or medicaments comprising a dual inhibition compound can be administered to a subject at a therapeutically effective dose, as described herein. In some embodiments, the pharmaceutical composition or medicament comprising a dual inhibition compound described herein is administered to a subject in an amount sufficient in combination with an effective amount of a therapeutic agent to elicit an effective therapeutic response in the subject. In some embodiments, the pharmaceutical composition or medicament comprising a dual inhibition compound described herein can be administered to a subject at a therapeutically effective dose to elicit inhibition of the JAK-STAT and HDAC pathways. In some embodiments, the pharmaceutical composition or medicament comprising a dual inhibition compound described herein can be administered at a therapeutically effective dose to elicit inhibition of JAK2 and HDAC6.

[0125] The pharmaceutical composition or medicament comprising a dual inhibition compound described herein may be administered on a routine schedule (e.g., hourly, daily, every 3 days, weekly, monthly, yearly). Alternatively, the pharmaceutical composition or medicament comprising a dual inhibition compound described herein I may be administered according to a repeating schedule (e.g., 3 days of daily administration, 3 days without administration, or 1 week of daily administration, 2 consecutive weeks without administration, etc.). The timing of administration (i.e., routine or repeating schedule) of the dual inhibition compounds described herein can readily be determined by a person of ordinary skill in the art.

[0126] In certain aspects, the administering step may consist of a single administration or may comprise a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the

concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.

E. Kits, Containers, Devices, and Systems

[0127] A wide variety of kits and systems can be prepared according to the present invention, depending upon the intended user of the kit and system and the particular needs of the user. In some aspects, the present invention provides a kit that includes dual inhibition compounds described herein.

[0128] Some of the kits described herein include a label describing a method of administering dual inhibition compounds described herein. Some of the kits described herein include a label describing a method of inhibiting a disease or disorder in a subject. In some embodiments, the disease or disorder is cancer or inflammation.

[0129] The compositions of the present invention, including but not limited to, compositions comprising dual inhibition compounds described herein may, if desired, be presented in a bottle, jar, vial, tube, or other container-closure system approved by the Food and Drug Administration or other regulatory body, which may provide one or more dosages containing the compounds. The package may also include a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice indicating approval by the agency. In some embodiments, the kit includes a composition as described herein, a container closure system including the composition or a dosage unit form including the formulation, and a notice or instructions describing a method of use as described herein. [0130] In some embodiments, the kit includes a container which is compartmentalized for holding the various elements of a formulation (e.g., the dry ingredients and the liquid ingredients) or composition, instructions for making the formulation or composition, and instructions for administering the formulation or composition for enhancing the immune response in a subject with a cancer or infectious disease.

[0131] Kits with unit doses of the compounds described herein, e.g., in oral, rectal, transdermal, or injectable doses, are provided. In such kits, an informational package insert describing the use and attendant benefits of the composition for dual inhibition of JAK-STAT and HDAC pathways in a subject with a cancer or inflammatory disease may be included in addition to the containers containing the unit doses.

III. EXAMPLES

[0132] The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.

[0133] Example 1: General Synthetic Strategy

[0134] Starting from macrocycle chloride 3 (William, A. D. et al. J. Med. Chem. 2011, 54, 4638-58) (Scheme 1), substitution with Boc-protected anilines 4a, 5a and 7a, phenol 6a and piperidine-4-methyl ester 8a gave esters 4-6 and 8 and nitro 7. The substitution reaction was carried out at 100 °C for 24 h or at 120 °C for 18 h in DMF.(Scheme 1). The reaction with 8a was heated for 3 days at 80 °C in acetonitrile. Nitro -substituted aniline 7 provided a route to install a longer linker for the hydroxamic acid, similar to compound 1. Reduction of nitro 7, followed by coupling with monoester acids 10a- 12a bearing chain lengths of 4-6 methyenes gave Boc-protected esters 10-12. [0135] Scheme 1. Preparation of Intermediates 4-8^a

4a 5a 6a 7a 8a ^aReagents and Conditions: (a) 4a-7a, Cs₂C0₃, TBAI, DMF, 120 °C, 18 h, 45 to 82% yield; (b) 8a, TBAI, MeCN, reflux, 72 h, 62% yield.

[0136] Compounds with long-chain hydroxamic acids connected directly to the macrocycle ring were synthesised from commercially available phenol 13 (Scheme 3). The common intermediate phenol 14 was prepared using two strategies. The first was a three-step protocol, without purification or protecting groups, entailing the reduction of aldehyde 13 to the primary benzyl alcohol followed by chlorination using thionyl chloride. Refluxing in neat allyl alcohol gives the desired allyl ether 14 in 35% yield over three steps.

[

aReagents and Conditions: (a) Fe, NH₄C1, EtOH/H₂0 (2:1), reflux, 3 h; (b) 10a - 12a, NMM, ClC0₂Et, DCM, O °C to RT, 18 h, 59 to 79% (over 2 steps). [0138] A four-step, preferred method was to use methoxymethyl (MOM) as a temporary phenol protecting group, with concomitant reduction of the aldehyde, followed by alkylation of the benzyl alcohol with allyl bromide under phase-transfer conditions. Finally, deprotection of the phenol by refluxing in aqueous 4 M hydrochloric acid in THF. This four- step protocol, while longer, gives a better overall yield of 72%. [0139] As per the reported synthesis of compound 3 (William, A. D. et al. J. Med. Chem. 2011, 54, 4638-58), the nitro group was reduced using iron and ammonium chloride. The crude aniline was stirred with the pyrimidine at 95 °C in dioxane for 18 h to give the diallyl compounds 21-25. Ring-closing metathesis with the second-generation Grubbs catalyst (Grubbs II catalyst) (Nolan, S. P.; Clavier, H. Chem. Soc. Rev. 2010, 39, 3305-16) in DCM with aqueous 4 M hydrochloric acid gave macrocycles 26-30 in good yields as approximately 85: 15 mixtures of trans to cis isomers of the alkene.

18a 19a

aReagents and Conditions: (a) NaBH₄, MeOH, RT, 2 h; (b) SOCl₂, DCM, RT, 18 h ; (c) AUyl alcohol, reflux, 3 h, 35% (3 steps); (d) MOMC1, NaBH₄, MeOH, RT, 2 h; (e) c.NaOH, NEt₃BnCl, AUyl bromide, RT, 72 h; (f) 4M HC1, THF, reflux, 3 h, 72% yield (3 steps); (g) NaBH₄, MeOH, RT, 2 h; (h) PPh₃, CC1₃CN, DCM, RT, 16 h; (i) AUyl alcohol, reflux, 4 h, 35% (3 steps); (j) K₂C0₃, MeCN, reflux, 18 h, 84-100%; (k) 20, Fe, NH₄C1, EtOH/H₂0 (2: 1), reflux, 3 h; (1) TsOH.H₂0, Dioxane, 95 °C, 18 h, 28-62% (2 steps); (m) second-generation Grubbs catalyst, DCM, 4M HC1, reflux, 3 h, 62 to 83% yield.

[0141] Esters 26-30 were converted to the acids by treating the ester with potassium trimethylsilanoate. Attempts to couple the acids with O-THP protected hydroxylamine were ultimately successful using (l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- b]pyridinium 3-oxidhexafluorophosphate (HATU). Deprotection of the THP group using anhydrous hydrogen chloride in dioxane released the free hydroxamic acids in low to moderate yield. [0142] Scheme 4. Preparation of Test Compounds 3 l-52^a

aReagents and Conditions: (a) KOTMS, THF, RT, 2 h; (b) c.HCl, RT, 18 h, 44 to 83% yield (2 steps); (c) HATU, H₂OTHP, Et₃, DMSO, RT, 3 d; (d) 4M HC1 in dioxane, RT, 18 h, 12- 62% yield.

[0143] Example 2: General Biological Methods Cell growth inhibition assays

[0144] Human breast cancer MCF-7 cells, human breast cancer MDA-MB231 cells, prostate cancer cell line PC-3 and colon cell line HCT-116 cells were grown in media that was supplemented with 10% fetal bovine serum, 50 μg / mL penicillin and 50 μg/mL streptomycin at 37 °C with 5% C0₂. The cells were sub-cultured to 80-90% confluency and used within 15-20 passages for the assay. Cell viability was assessed with MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide).

[0145] For the multiple myeloma cell lines (i.e., KMS 12BM, OPM2, AML Cell lines KG1 and MOLM14 and NKT cell lymphoma cell lines), NKYS and KHYG were cultured in RPMI 1640 supplemented with 10% Fetal Bovine Serum, 100 units/mL penicillin and 100 μg/mL streptomycin. An additional supplementation of IL-2 was required for NKYS (100 units/mL of IL-2) and (200 units/mL of IL-2) for KHYG. All cells were grown in a humidified atmosphere at 37 °C with 5% C0_2.

[0146] MCF7, MDA-MB231, PC-3 and HCT-116 cells were seeded at 2500 cells per well in a 96-well plate for 24 h. The media was removed and aliquots of test compounds (initially prepared as 10 mM stock solutions in DMSO) were added to each well and the plates were incubated for 72 h. The final amount of DMSO per well was maintained at 0.5% v/v. At the end of the incubation period, the media was removed and replaced with FBS free media (150 μΐ,) and MTT (50 μΙ_, of 2 mg/mL solution in phosphate buffer saline, pH 7.4). After incubation for 3 h at 37 °C with 5% C0₂, the supernatant was removed and a solution of 100 μΐ, DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm on a micro plate reader. Cell viability was determined from readings of treated wells compared to control wells (absence of test compound) with correction of background absorbance. The IC₅₀ (concentration required to reduce cell viability to 50% of

control/untreated cells) was determined in triplicates on separate occasions, using two different stock solutions. Percentage viability readings for each test compound were plotted against log concentration on GraphPad Prism (Version 5.0, GraphPad Software, San Diego, CA), with constraints set at > 0 and < 100%. A sigmoidal curve was generated from which IC₅₀ was obtained.

[0147] KMS 12BM, OPM2, KG1, MOLM14, NKYS and KHYG cells were treated in 96 well plates at a density of 25,000 cells per well in triplicates for 48 h in a 37 °C incubator with 5% C0_2. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Proliferation Assay. The luminescence was measured with a Tecan spectrophotometer. Dose response curves were plotted as above.

Immunoblotting [0148] For immunoblotting analysis, at least 3xl0⁶ cells were seeded for each condition. Cells were treated with the compound (e.g., compound 51) at the stated concentrations and harvested at the indicated timepoints. Equal amounts of protein were separated on an SDS polyacrylamide gel and transferred onto a PVDF membrane. The membrane was then probed with the following antibodies: phosho-STAT3, STAT3, acetylated alpha tubulin, alpha tubulin, acetylated histone 3, and histone. Determination of Toxicity in TAMH Cells

[0149] Transforming growth factor-alpha mouse hepatocyte (TAMH) cells were seeded at a cell density of 12,000 cells/well (60,000 cells/mL) in a 96-well plate (NUNC) a day before drug treatment. Cells were then treated test compounds starting from a concentration of 100 μΜ (0.5% DMSO concentration: 1.0 μΐ of compound 51 and 52 of a 10 mM stock solution in 99 μΐ_^ of media in each well; drug stock concentration is 10 mM). Drug-treated cells were then incubated at 37 °C for 24 hours after which cell viability was determined with the CellTiter-Glo Cell Viability Assay, which is described on the worldwide web at promega.eom/~/media/files/resources/protocols/technical%20bulletins/0/celltiter%20glo%201 uminescent%20cell%20viability%20assay%20protocol.pdf. The cell-reagent mixture was then transferred to a solid white flat-bottom 96-well plate. Luminescence was then recorded with an integration time of 0.25 seconds. Either percentage inhibition at the top

concentration or an IC₅₀ was calculated as for the cell assays above.

Determination of in vitro metabolic stability in male and female rat liver microsomes [0150] Liver microsomal incubations were conducted in triplicate. Incubation mixtures consisted of 7.5 μΐ_^ of 20 mg/mL in female rat liver microsomes (FRLM) and in male rat liver microsomes (MRLM) (final: 0.3 mg microsome protein/mL), 2.5 μΐ_^ of 600 μΜ compound 51/52 in acetonitrile (final: 3 μΜ), 440 μΐ, of 0.1 M phosphate buffer (pH 7.4). The mixture was first shaken for 5 min for pre-incubation in a shaking water bath at 37 °C. Reaction was by adding 50 μί of 10 mM NADPH to obtain a final concentration of ImM NADPH in the mixture. The total volume of the reaction mixture was 500 μΐ_^. For metabolic stability studies, aliquots of 50 μΐ_^ of the incubation sample mixture were collected at 0, 5, 10, 15, 30, and 45 min. After collection of samples, the reaction was terminated with 100 μΐ_^ of chilled acetonitrile containing the internal standard (1.5 μΜ compound 1). The mixture was then centrifuged at 10,000 x g to remove the protein, and the supernatant was

subsequently applied to LC-MS/MS analysis.

[0151] Positive control (PC) samples were prepared as described above, except the test compound was replaced with the known P450 substrate (Midazolam, 3 μΜ). The samples were assayed for the degradation of midazolam to evaluate the adequacy of the experimental conditions for drug metabolism study. Negative control samples were also prepared as described above but without NADPH. Enzyme Assays

[0152] Enzyme inhibition assays were carried out by Reaction Biology Corporation (RBC). Lobera, M.; et al. Nature chemical biology 2013, 9, 319-25. For HDAC assays: 2X of HDAC enzyme was added into reaction plate except for the control wells (no enzyme), where buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KC1, and 1 mM MgCl₂) was added instead. Inhibitors in 100% DMSO were added into the enzyme mixture via acoustic technology, then spun down and pre-incubated. 2X of the Substrate Mixture was then added in all reaction wells to initiate the reaction: For Fluorogenic HDAC General Substrate— 50 μΜ, Arg-His-Lys-Lys(Ac) (SEQ ID NO: l); for HDAC8-only substrate— 50 μΜ, Arg-His- Lys(Ac)-Lys(Ac) (SEQ ID NO:2); and for Class2A Substrate— Acetyl-Lys(trifluoroacetyl)- AMC. The mixture was spun, shaken, and incubated for 2 h at 30 °C with seal. Developer with Trichostatin A was added to stop the reaction and to generate the fluorescent color. A kinetic measurement was carried out for 1.5 h with Envision with 15 min interval. (Ex/Em= 360/460 nm). An endpoint reading was taken for analysis after the development reached a plateau.

[0153] Kinase assays were carried out according to published procedures. Anastassiadis, T. et al. Nature biotechnology 2011, 29, 1039-45. Briefly, kinase profiling was performed using the "HotSpot" assay platform. Specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer: 20 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na₃V0₄, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed about 20 min later by addition of a mixture of ATP and

33 P ATP to a final concentration of 10 μΜ. Reactions were carried out at 25 °C for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of the background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC₅₀ values and curve fits were obtained using Prism (GraphPad Software). Cellular Assays

Cell proliferation inhibition assays

[0154] Human breast cancer MCF-7 cells, human breast cancer MDA-MB231 cells, and prostate cancer cell line PC-3 were grown in DMEM Media. Colon cell line HCT-116 cells were grown in Mccoys Media. They were supplemented with 10% fetal bovine serum, 50 μg / mL penicillin and 50 μg/mL streptomycin at 37 °C with 5% C0₂. The cells were sub- cultured to 80-90% confluency and used within 15-20 passages for the assay. Cell viability was assessed with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) as follows: The cells were seeded at 2500 cells per well in a 96-well plate for 24 h. The media was removed and aliquots of test compounds (initially prepared as 10 mM stock solutions in DMSO) were added to each well and the plates were incubated for 72 h. The final amount of DMSO per well was maintained at 0.5% v/v. At the end of the incubation period, the media was removed and replaced with FBS free media (150 μΐ,) and MTT (50 μΙ_, of 2 mg/mL solution in phosphate buffer saline, pH 7.4). After incubation for 3 h at 37 °C with 5% C0₂, the supernatant was removed, and a solution of 100 μΙ_, DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm on a micro plate reader. Cell viability was determined from readings of treated wells compared to control wells (absence of test compound) with correction of background absorbance. The IC₅₀ (concentration required to reduce cell viability to 50% of control/untreated cells) was determined in triplicates on separate occasions, using two different stock solutions. Percentage viability readings for each test compound were plotted against log concentration on GraphPad Prism (Version 5.0, GraphPad Software, San Diego, CA), with constraints set at > 0 and < 100%. A sigmoidal curve was generated from which the IC₅₀ was obtained.

Multiple myeloma cell lines: KMS 12BM, OPM2, AML Cell lines KG1 and MOLM14 and NKT-cell lymphoma cell lines

[0155] NKYS and KHYG were cultured in RPMI 1640 supplemented with 10% Fetal Bovine Serum, 100 units/mL penicillin, and 100 μ§/ηιΙ. streptomycin. An additional supplementation of IL-2 was required for NKYS (100 units/mL of IL-2) and (200 units/mL of IL-2) for KHYG. All cells were grown in a humidified atmosphere at 37 °C with 5% C0₂. KMS 12BM, OPM2, KG1, MOLM14, NKYS and KHYG cells were treated in 96-well plates at a density of 25,000 cells per well in triplicates for 48 h in a 37 °C incubator with 5% C0_2. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Proliferation Assay as referenced above. The luminescence was measured with a Tecan spectrophotometer. Dose response curves were plotted as above.

[0156] HEL cells were cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum and 55 μΜ β-mercaptoethanol. HEL cells (2.5xl0⁴/ 90 μί) were seeded in 96-well plates and inhibitors of various concentrations were added accordingly. As negative control, cells were treated with vehicle (DMSO) only. After 36 h of incubation, 10 μΐ_^ of PrestoBlue dye were added into each well and further incubated for 2.5 h. Measurement was done at 570 nm against 600 nm according to the standard PrestoBlue dye protocol (available on the worldwide web at thermofisher.com/us/en/home/references/protocols/cell-and-tissue- analysis/protocols/prestoblue-cell- viability-reagent-for-microplates-protocol.html). This experiment was performed in triplicates and repeated twice. Mean values + SD for each concentration were determined. Calculation of cell viability was done according to standard methods.

Immunoblotting [0157] HEL92.1.7 cells: SDS-PAGE and Western blot analyses were performed according to a known procedure. Chen, P.-H. et al. Cell Cycle 2012, 11, 3611-26. HEL cells (5xl0⁵/ mL) were seeded into a six- well plate and treated with inhibitor (0, 0.1, 0.5 and 1 μΜ) for 1 hr. Cells were collected and washed with PBS. The pellet was lysed in RIP A buffer supplemented with phosphatase inhibitor cocktail and protease inhibitor cocktail. The lysate was loaded into 10% (HEL cells) or 8% (HeLa stable clone) polyacrylamide gel. The proteins were then transferred to PVDF membrane and detected through specific antibodies: anti-acTubulin (6-11B-1) (Biolegend), anti-STAT5 (Santa Cruz Biotechnology); anti-H3 (9715S), anti-acH3 (9677S), anti-pJAK2 (Y1007/1008), anti-JAK2 (D2E12), anti-pSTAT5 (Tyr694) and anti-Tubulin (Cell Signaling Technology). [0158] KMS-12-BM and MOLM-14: For immunoblotting analysis, at least 3xl0⁶ cells were seeded for each condition. Cells were treated with compound 51 at the stated concentrations and harvested at the indicated timepoints. Equal amounts of protein were separated on an SDS polyacrylamide gel and transferred onto a PVDF membrane. The membrane was then probed with the following antibodies: phopho-STAT3, STAT3, acetylated alpha tubulin, alpha tubulin, acetylated histone 3, and histone. Colony forming unit assay

[0159] HEL cells (200 cells/mL) were mixed vigorously with methylcellulose semi-solid medium per the procedure for ClonaCell-TCS Medium (available on the worldwide web at stemcell.com/~/media/Technical%20Resources/7/E/3/E/5/28372_clonacell_tcs.pdf?la=en). Inhibitors of various concentrations were added accordingly. The mixture was vortexed and aliquoted into six-well plates for 14-16 days incubation, after which colonies werer counted.

[0160] Example 3: Enzyme Inhibitory Activity

[0161] Select carboxylic and hydroxamic acids of the present disclosure were both tested in enzymatic and cellular assays. Reference compounds include 1 (pan-HDAC), 2 (JAK2 selective) and Tubastatin A (HDAC6 selective). Data for carboxylic acids is shown below in FIG. 1A-B, and for hydroxamates, in FIG. 2A-C. JAK2, HDACl and HDAC6 were the proteins selected for further studies. Significant inhibition of JAK2 enzymatic activity was observed with acids 31-34 (ICso <39 nM). Of these compounds, only aminobenzoic acids 31 and 32 afforded weak HDAC6 inhibition and were completely inactive against HDACl at the highest concentration tested (10 μΜ). Likewise, ether 33 and piperidine 34 were not HDAC active.

[0162] The notes in FIGS 1A and IB indicate the following: (a) The reported IC₅₀ values for compound 1 are: MCF-7 (c.6 μΜ) (Yi, X. et al., Biochemical pharmacology 2008, 75, 1697-1705); MDA-MB-231 (c.4.5 μΜ) (Carlisi, D. et al. J. Cellular Physiology 2015, 230, 1276-89); HCT-116 (2.85 μΜ) and PC-3 (1.21 μΜ) (Wang, H. et al. J. Med. Chem. 2011, 54, 4694-4720). (b) This control was tested in the same assay as compounds 1-52, but reported literature values vary depending on assay format, (c) The reported IC₅₀ values for compound 2 are 1.69, 0.77 and 0.85 μΜ for HCT-116, PC-3 and MCF-7, respectively (Hart, S. et al. Leukemia 2011, 25, 1751-59). Compound 2 has not been reported for MDA-MB231. "NC" indicates no dose response curve, (d) The lowest concentration tested was 39 nM.

[0163] Without intending to be limited by theory, certain embodiments of the hybrid molecules were designed to place the zinc -chelating functional group in the solvent-exposed region of the JAK2 enzyme, while binding features of pacritinib were retained in the JAK2 enzyme binding pocket. Lengthening the distance between the Zn chelating group and macrocycle using an aniline linker yielded progressively more HDAC6 inhibition with a 5- methylene spacer (35, 20% inhibition at 10 μΜ) and with 6 methylenes (36, IC₅₀ = 2.6 μΜ). Compound 36 has HDAC activity; however, it is completely inactive against HDACl at 10 μΜ. Compound 36 was potent against JAK2 (IC₅₀ = 2.3 nM), similar to 2, indicating that a wide range of HDAC-binding side chains were fully compatible with the targeted levels of JAK2 activity.

[0164] Activity seen with compounds 35 and 36 was mirrored in compounds 37-41 with aliphatic linkers of increasing length directly attached to the macrocycle. Again, compound 40 (HDAC6 IC₅₀ = 2.8 μΜ) with 6 methylene groups provided the best results for HDAC6 activity.¹⁶ As with 35/36, the inhibition of HDAC6 activity increased as the chain length increased to 6 methylenes before it decreased with an additional methylene. No HDAC inhibition was detected with the shortest 3 and 4 carbon linkers in compounds 37 and 38, whereas the 5-carbon-linked 39 was weakly active (HDAC6 IC₅₀ = 45 μΜ). Increasing the chain length to 7 methylenes decreased the HDAC6 activity to 10 μΜ. Potency against JAK2 was maintained with some variations. Shortening the linker as in 37 decreases JAK2 potency nearly 20-fold from 2.6 nM (40) to 47 nM (37).

[0165] This HDAC6 selectivity and activity profile is mirrored in the enzymatic activity of the hydroxamic acids, but with significantly greater HDAC6 activity (>1000-fold in the most active cases). Aminobenzhydroxamates 42 and 43 had much improved HDAC6 activity as did piperidine 45; however, phenol ether 44 had sub-micromolar potency against HDAC6 (IC₅₀ = 66 nM) and weak HDACl activity (56% at 10 μΜ), representing at least a 1,500-fold improvement over the acid derivative 33 (HDAC6 ICso >100 μΜ). The phenol ether also had JAK2 IC₅₀ of 1.3 nM.

[0166] Longer, vorinastat-type side chains in compounds 46 and 47 conferred high HDAC6 potency, with IC50S of 2.5 and 2.3 nM, respectively. HDACl potency was still not detectable at the highest concentration tested (10 μΜ). Furthermore, 46 displayed a high level of JAK2 potency with an IC₅₀ of 6.9 nM, while 47 was approximately 4-fold lower at 30 nM.

However, compounds 46 and 47 were not active in cells (with the exception of MDA-MB231 breast cancer cells for 47 (GI₅₀ = 3.74 μΜ) (Table 2; NC = no dose response curve, ND = not determined). Without intending to be bound by theory, although compound 46 had a desirable LLE [LLE = -LogIC₅₀-cLogP (Hopkins, A. L. et al. Nat. Rev. Drug Discov. 2014, 13, 105-21)] of 5.2, it had a molecular weight of 666 and 5 hydrogen bond donors, likely resulting in poor solubility and permeability.

[0167] In certain embodiments, direct linking of the hydroxamate to the macrocycle (e.g., compounds 48-52) improved solubility and permeability while maintaining a favorable enzyme inhibition profile. With the long-chain aliphatic hydroxamic acids 48-52, JAK2 activity was similar, with ICso's from 1 to 5 nM, but HDAC6 IC50S ranged widely from 2.1 to 510 nM. HDAC1 potency roughly tracked that of HDAC6, with compound 51 having the highest (IC₅₀ = 221 nM) and an HDAC6/1 selectivity of 100-fold. The compounds also maintain very strong JAK2 potency with compound 51 the most potent, with an IC₅₀ of 1.4 nM.

[0168] Example 4: Modeling Studies

[0169] Compound 51 was docked into a homology model of the HDAC6 protein. The model used was derived from a crystal structure of the HDAC4 catalytic domain (PDB code 2VQW) (FIGS. 3A and 3B).

[0170] FIG. 3A shows an electrostatic surface of HDAC6 with blue indicating positively charged areas and red indicating negatively charged areas. Without intending to be limited by theory, the model hydroxamate is bound deep in the Zn pocket while the large macrocyclic cap has surface complementarity. A pocket unique to HDAC6 is formed by Pro352 and Lys353 into which a hydrophobic linker of the macrocyle is bound. FIG. 3B shows that compound 51 and HDAC6 residues Tyr386, His215 and His216 are calculated to form hydrogen bonds with the hydroxamate that also coordinates the catalytic Zn.

[0171] A similar model docking was conducted for HD AC 1 (FIGS . 4 A and 4B ) and JAK2 (FIGS. 5A and 5B). [0172] FIG. 4A shows an electrostatic surface of HDAC1 with blue indicating positively charged areas and red indicating negatively charged areas. Without intending to be limited by theory, the model hydroxamate is bound in the Zn pocket, while the large macrocyclic cap is positioned on the highly solvent-exposed surface. FIG. 4B shows that compound 51 and HDAC1 residues Tyr303, Hisl40, and Hisl41 are calculated to form hydrogen bonds with the hydroxamate that also coordinates the catalytic Zn.

[0173] FIG. 5A shows an electrostatic surface of JAK2 with blue indicating positively charged areas and red indicating negatively charged areas. Without intending to be limited by theory, in this aspect, the macrocycle binds in the ATP cavity with very good surface complementarity, very similar to the reported orientation of pacritinib. Poulsen, A.et al. Comput. Aided Mol. Des. 2012, 26, 437-50. FIG. 5B shows the model's detailed interactions with protein residues ^positions 930-934 - SEQ ID NO:3). The aminopyrimidine is calculated to bind to the backbone of the hinge residue Leu932 with a donor-acceptor hydrogen bond arrangement. The oxygen of the macrocyclic linker of compound 51 is calculated to make close contacts, possibly hydrogen bonds, with both sidechain hydroxyl and backbone NH of Ser936. Modeling Methods

[0174] To prepare the models, the HDAC1 X-ray structure (ID: 4BKX), HDAC2 structure (4LXZ) and HDAC4 structure (2VQW) was downloaded from the Protein Data Bank (available on the worldwide web at rcsb.org) and prepared using the protein preparation wizard in Maestro version 10 using standard settings. Schrodinger, LLC, 2014. New York, NY, USA (available on the worldwide web at schrodinger.com). This included the addition of hydrogen atoms, bond assignments, removal of water molecules further than 5 A from the ligand, protonation state assignment, optimization of the hydrogen bond network and restrained minimization using the OPLS2005 force field. Kaminski, G. A. et al. J. Phys. Chem. B 2001, 105, 6474-87. The proteins were superimposed using structural alignment and the HDAC2 structure ligand SAHA was duplicated into HDAC1 and HDAC4. The HDAC6 protein sequence (ID: Q9UBN7) was downloaded from Uniprot (available on the worldwide web at uniprot.org). An HDAC6 homology model was built using the HDAC4- SAHA complex as template and Prime version 3.8 with default settings. Schrodinger, LLC, 2014. New York, NY, USA (available on the worldwide web at schrodinger.com). JAK2 modeling was performed according to the procedure of Poulsen et al., J. Comput. Aided Mol. Des. 2012, 26, 437-50.

[0175] A conformational analysis of each inhibitor was done to obtain a conformational ensemble for docking. Poulsen, A. et al. J. Comput. Aided Mol. Des. 2012, 26, 437-50. Grids for docking were prepared for HDAC1 and HDAC6 using Glide version 6.5 with standard settings. Constraints for the zinc atom and the residues that hydrogen bond with the hydroxamate were included. Docking into JAK2 was carried out as previously described. Schrodinger, LLC, 2014. New York, NY, USA (available on the worldwide web at schrodinger.com). Docking of the hybrid inhibitors into HDAC1 and HDAC6 with constraints to the catalytic Zn atom did not produce low energy poses. Docking of 2 without the solubiliing group and without constraints resulted in reasonable poses of low energy conformations binding to the cap-region of HDAC1 and HDAC6. Poses of 2 were then merged with the linker and hydroxamate part of 1. [0176] The HDAC1 and HDAC6 inhibitor-protein complexes were finally minimized using MacromodellO.6. Schrodinger, LLC, 2014. New York, NY, USA (available on the worldwide web at schrodinger.com). Distance constraints between both the hydroxamate oxygen atoms and Zinc (2.3 A) as well as the 3 residues that hydrogen bond with the hydroxamate (2.8 A) were included. All residues more than 9 A from the ligand were constrained before the complex was subjected to 500 steps of Polak-Ribiere-Conjugate- Gradient (Polak, E.; Ribiere, G. Revue frangaise d'informatique et de recherche

operationnelle, serie rouge 1969, 3, 35-43.) minimization using the OPLS2005 force field and GB/SA continuum solvation model (Hasel et al., 1988). Hasel, W. et al. Tetrahedron Computer Methodology 1988, 1, 103-16.

[0177] Example 5: Isoform Selectivity

[0178] Compound 51 was tested against the 4 JAK isoforms JAK1, JAK2, JAK3 and TYK2 as well as FLT3, an important kinase in AML and targeted by 2 (Table 1). Compound 51 was also tested against 11 HDAC isoforms covering the four classes of zinc-utilising deacetylases: class I, IIA, IIB and IV (Table 2 and FIG. 6A).

[0179] Table 1. Isoform selectivity of compound 51 against the JAK and FLT3 kinases. ^a

ICso (μΜ) or ^"

Isoform JAK2 SI HDAC6 SI^C

% inhibition

JAK1 8.7% ( a 0.1 μΜ >100 >100

JAK2 0.0014 1 1.5

JAK3 55% a ¾ 0.1 μΜ >50 >25

TYK2 56% a ¾ 0.1 μΜ >50 >25

FLT3 67% e ¾ 0.1 μΜ >50 >25 kinases were tested in duplicate at 100 nM concentration. JAK2 SI = fold less potent compared to JAK2 IC₅₀ (1.4 nM). ^c HDAC6 SI = fold less potent compared to HDAC6 IC₅₀ (2.1 nM).

[0180] Table 2. Isoform selectivity of compound 51 against class I, II, and IV HDAC enzymes.³

IC₅₀ (μΜ) or ^"

Isoform Class JAK2 SI HDAC6 Sf

% inhibition

FID AC 1

I 0.222 158 111 IC₅₀ (μΜ) or

Isoform Class JAK2 SI HDAC6 Sf

% inhibition

HDAC2

I 0.049 35 23

HDAC3

I 2.17 1,550 1,033

HDAC4

IIA 8.6% @ 10 μΜ >7,000 >5,000

HDAC5

IIA 16.1% @ 10 μΜ >7,000 >5,000

HDAC6

IIB 0.0021 1.5 1

HDAC7

IIA 1.3% @ 10 μΜ >7,000 >5,000

HDAC8

I 0.74 μΜ 528 352

HDAC9

IIA 14% @ 10 μΜ >7,000 >5,000

HDAC10

IIB 0.080 μΜ 57 38

HDAC11

IV 0.93 μΜ 664 443

a HDAC enzymes were tested in a 10-dose IC₅₀. ^b JAK2 SI = fold less potent compared to JAK2 IC₅₀ (1.4 nM). ^c HDAC6 SI = fold less potent compared to HDAC6 IC₅₀ (2.1 nM).

[0181] Compound 51 is selective for JAK2 against all the tested kinases. JAK1 was especially insensitive to compound 51, with less than 10% inhibition at the test concentration of 100 nM corresponding to a selectivity of much greater than 100-fold against both JAK2 and HDAC 6. Potency for JAK3 and TYK2 is 55% and 56% at 100 nM, greater than for JAK1. Selectivities for JAK3 and TYK2 over JAK2 are still higher than pacritinib at over 50-fold, and 25-fold over HDAC6. A low potency against FLT3 of only 67% inhibition at 100 nM corresponded to over 50-fold selectivity and over 25-fold against HDAC6. Within the five tested kinases, compound 51 was over 50-fold selective in favour of JAK2.

[0182] Of the 11 tested HDAC isoforms, compound 51 was very weakly active against the four class IIA HDACs 4, 5, 7 and 9 with less than 20% inhibition at 10 μΜ corresponding to >7, 000-fold selectivity over JAK2 and over 5,000-fold over HDAC6 (Table 2). Low micromolar potency against the class I isoform HDAC3 (IC₅₀ = 2.17 μΜ) still represented a high selectivity of >l,000-fold for both JAK2 and HDAC6. The next-most active isoforms were HDACs 11 and 8, with IC₅₀ of 0.93 μΜ and 0.74 μΜ, respectively (SI of 352-664 for JAK2 and HDAC6). HDACl was next most potently inhibited followed by HDACs 2, 10 and 1 with IC50S of 49, 80 and 222 nM, respectively. Figure 6A indicates the similar selectivity between HDAC isoforms and JAK2/HDAC6.

[0183] FIG. 6B provides the results of a broader kinase selectivity test for compound 51. This compound was screened against a panel of 97 kinases (DiscoveRx). The TREE spot™ map indicates the very high selectivity of compound 51 (FIG. 6B). From 90 non-mutated kinases, only 9 displayed <35% of control binding remaining, which approximately equates to an IC₅₀ of around 10 μΜ) (Table 3).

[0184] Table 3. Selectivity of compound 51 against various kinase enzymes.

Kinase Percent Control²¹

JAK2 0

TYK2 4.9

ΡΒΚ-λ 5.7

JAK3 7.9

NTRK1 13

KIT 21

MAPK8 26

MAPK10 29

KIT(V559D,T670I) 31

alower numbers equate to stronger hits; values of 5-10 approximately equate to IC₅₀S of 100- ΙΟΟΟηΜ; values above 10 approximately equate to IC₅₀S of 1 ->10 μΜ.

[0185] Table 4 summarizes the selectivity of the panel results. The ' S' scores are all less than 0.1, indicating a high degree of kinase selectivity. The 'S,' or selectivity score, is defined as the number of kinases bound by the compound divided by the total number of kinases tested, excluding mutant kinases. This S score is very favorable in comparison with other well-known kinase inhibitors, such as sunitinib (0.65), sorafenib (0.22) and imatinib (0.12). "Panobinostat and Ruxolitinib in MPN clinical trial," available online at

clinicaltrials .gov/ct2/show/NCT01433445.

[0186] Table 4. Selectivity of compound 51 against various kinase enzymes.

Selectivity Score (S) ,^:a Number of Hits Number of Non- Calculated S(x)

Mutant Kinases

S(35) 8 90 0.089

S(10) 4 90 0.044 S(l) 1 90 0.011 ^a 'S(x)' for a given percentage x is defined as the number of kinases bound by the compound at x% divided by the total number of kinases tested, excluding mutant kinases. ^bThe selectivity score can be understood by comparing with other well-known kinase inhibitors, such as sunitinib (0.65), sorafenib (0.22) and imatinib (0.12). [0187] Example 6: Cellular Assays: Solid Tumor Cell Lines

[0188] Selected JAK-HDAC dual inhibitors were evaluated in four solid tumor cell lines: breast cancer (MDA-MB-231, MCF-7), colorectal cancer (HCT-116) and prostate cancer (PC-3). Each compound was evaluated in a dose response study to establish their ability to inhibit cell proliferation (FIGS. 1 and 2). Compounds 1 and 2 were used as references, with tubastatin as an HDAC6 selective inhibitor. IC₅₀ values obtained for these three reference compounds were generally in the low to sub-micromolar range and were well within 3 -fold of the reported values.

[0189] Even the poorly soluble J AK2- selective acids 31-33 had measurable ICso's in two cell lines. However, the zwitterionic piperidine acid 34 was more active. Acid 36, potent against JAK2 (IC₅₀ = 2.3nM) and inhibiting HDAC6 in the low micromolar range (IC₅₀ =

2.6 μΜ) was more active against HCT-116 cells, but in other cell lines, the dose response was poor, with no sigmoidal curve fit possible. Short aliphatic-chain acids 37-39 were tested in two cell lines and found to be weakly active. A longer 6 carbon linker (40) conferred low micromolar HDAC6 inhibition and slightly improved cell activity, while one carbon more (41) was less active against HDAC6 and slightly less active than compound 40 in all four cell lines.

[0190] Selected hydroxamic acids were then tested in the same four cell lines. Aromatic hydroxamate 42 was active, whereas its meta analogue, 43, was only active against HCT-116 cells. Although phenolic ether 44 was potent against PC-3 cells, its poor solubility hampered studies in other cells. In general, a similar trend was seen as for the carboxylic acid analogues: the basic piperidine 45 was quite potent across all cell lines. However, it had very weak HDAC6 inhibition (HDAC6 IC₅₀ = 899 nM) compared to the other compounds with similar length. Despite being highly potent against both JAK2 and HDAC6, long-chain analogues 46 and 47 were only weakly active in these cells. The only exception was compound 47 with an IC₅₀ of 3.74 μΜ in MB-MDA-231 cells. Precipitation of these compounds in the cell assay was observed. More positive results were obtained with directly attached aliphatic side chains of 3 to 7 carbons for compounds 48 to 52, respectively. The best solid tumor cell potency of these analogues was obtained for six-carbon-linked compound 51, also the most potent inhibitor of JAK2 and HDAC6 with IC₅₀ values of 1.4 and 2.1 nM, respectively. Compound 51 had the lowest IC₅₀ values in all cell lines tested with the exception of HCT- 116, where piperidine 45 was the most potent.

[0191] Example 7: Cellular Assays: Hematological Cell Lines

[0192] Selected compounds were tested in a range of blood/bone-marrow-derived cancer cells. Compounds 46, 47, 50, 51, and 52 were assessed in three hematological cells lines: the AML cell line HL-60, the erythroleukemia (EL) cell line HEL92.1.7 (expressing mutated JAK2^V617F) and the acute T-cell leukemia cell line Jurkat (Table 5). All compounds were more potent in these cell lines than in solid tumor cell lines. Compound 51, having the strongest profile in vitro, was the most potent compound with around 1 μΜ IC₅₀ values in the HEL and Jurkat cell lines and about 4 μΜ in the FLT-3 sensitive HL-60. The JAK1/2 selective inhibitor ruxolitinib was not potent in these cells indicating a lack of sensitivity to JAK pathway inhibition. However they are sensitive to HDAC blockade indicated by the activity of 1. Tubastatin is not potent indicating lack of sensitivty to HDAC6. Compound 51 has similar potency to compounds 1 and 2 in HEL and Jurkat cells. This data suggets that the broad potency range of compound 51 is due to its ability to block multiple pathways but not necessarily eliciting synergy between those pathways in terms of inhibition of proliferation.

[0193] Table 5. Cell proliferation inhibition assay data of selected compounds in three leukemia cell lines

Compound IC₅₀ (μΜ) ^a

HL-60 HEL92.1.7^C Jurka ¹ l^e

1.34 0.58 0.66

Tubastatin »4ⁱ »2ⁱ »4ⁱ

2

1.14 1.24 1.09

Ruxolitinib^h

>4¹ >2¹ »4ⁱ

46 »4ⁱ »2ⁱ >4ⁱ 47 >4ⁱ »2ⁱ >4ⁱ 50 >4ⁱ c.2¹ 2.49 51 _{c 4}iJ 1.07 1.20 52 >4ⁱ >2ⁱ 2.89 ^aAnti-proliferative inhibitory activities are the average of at least 3 determinations. ^b Acute Myeloid Leumekia. ^cErythroleukemia (JAK2^V617F). ^dAcute T-Cell Leukemia. ^epan-HDAC. ^fHDAC6 selective. ^gJAK2/FLT3.

hJAKl/JAK2. 'top concentration tested. ^J47% inhibition at 4 uM.

[0194] Compound 51 was studied in an expanded range of cell lines. Hence compound 51 was tested against the multiple myeloma (MM) cell lines KMS-12-BM and OPM-2, with different translocations in MM (t4: 14 and ti l: 14) respectively, both being translocations which correspond to poor prognosis in MM (Table 6). Katagiri, S. et al. Int. J. Cancer 1985, 36, 241-46; Otsuki, T. et al. Int. J. Hematol. 2000, 72, 216-22 An additional two AML cell lines, KG-1 and MOLM-14 were selected, where MOLM-14 has a FLT3-ITD mutation while KG1 does not. MOLM-14 is a M5 stage (FAB) cancer while KG-1 is a M6 stage (FAB) cancer. Matsuo, Y. et al. Leukemia 1997, 11, 1469-77; Koeffler, H. et al. Blood 1980, 56, 344-50. Finally, two natural killer T-cell lymphoma (NKTCL) cell lines NKYS and KHYG, with positive and negative EBV status, respectively, were tested against compound 51. Iqbal, J. et al. Leukemia 2009, 23, 1139-51; Chiang, A. K. et al. International J. Cancer 1997, 73, 332-38. NK cell neoplasms have been shown to be sensitive to compound 1 due to suppression of the JAK-STAT pathway suggesting that a combination of both JAK and HDAC inhihibtion could be an effective strategy. Karube, K. et al. Cancer letters 2013, 333, 47-55. Anti-proliferative potency of compound 51 in NK cell neoplasms ranged from 1.08 to 2.11 uM with the most potent cell line being the NKTCL. For the MM cell line KMS-12-BM, compound 51 was more potent than both compounds 1 (7.86 μΜ) and 2 (3.68 μΜ), potentially supporting dual pathway blockage as a more efficacious strategy.

[0195] Table 6. Cell proliferation inhibition assay data of compound 51 and reference standards in a range of hematological cell lines

Compound IC₅₀ (μΜ)^Β

KMS-12-BM^b OPM-2^b KG-1^C MOLM-14^c NKYS^d KHYG^d l^e 7.86 3.33 - 5.00 - -

2^f 3.68 1.21 1.48 0.08 1.60 1.24

51 2.11 2.0 1.63 1.14 1.08 1.09 a^aAnti-proliferative inhibitory activities are the average of at least 3 determinations. b^b"M» *ultiple Myeloma. ^cAcute Myeloid Leumekia. ^dNatural Killer T-Cell Lymphoma. ^epan-HDAC. ^hHDAC6 selective. ^fJAK2/FLT3. Blank cells = not tested.

Table 7. Cell proliferation assay data of compounds in a normal cell line

Therapeutic window (TAMH IC₅₀ /

Compound TAMH IC₅₀ (u,M)^a

Cancer cell IC₅₀)^d

l^b 7.86 + 2.2 13.1

2^C 3.68 + 0.88 3.1

51 9.57+ 1.00 8.7

52 5.26+ 0.24 1.8

aAverage of 4 determinations. ^Bpan-HDAC. ^CJAK2/FLT3. °based on the most sensitive cell lines with average IC50S of 0.6 μΜ for 1 , 1 .2 μΜ for 2 (excluding MOLM-14), 1 .1 μΜ for compound 51 and 2.9 μΜ for compound 52

[0196] Another relevant measure of a compound's anti -proliferative potential is its effects in blocking the formation of colonies of tumor cells growing in a 3D matrix. Compound 51 was tested against HEL92.1.7 cells grown over 14-16 days in methylcellulose (ClonaCell™- TCS Medium). It effectively blocked colony formation in a dose-dependent manner and at concentrations similar to its IC₅₀ against cells growing in 2D culture (FIG. 7A). FIG. 7A provides a bar graph indicating dose response based on total cell populations, with data presented as mean + SEM. FIG. 7B provides photographs of selected colonies indicating controls (al, a2), treatment with compound 51 at 0.2 μΜ (bl, b2), 1.0 μΜ (cl, c2) and 2.0 μΜ (dl, d2). The photographs of selected colonies further demonstrate the potent anti- growth effects of compound 51.

[0197] To determine their toxicity in normal cells, compounds 51 and 52 were tested Transforming growth factor-a Mouse Hepatocytes (TAMH) (Table 7), comparing with 1 and 2 as controls. Compound 51 inhibited the proliferation of TAMH cells with an IC₅₀ of 9.57 μΜ, whereas 2 was nearly (x3) more potent (IC₅₀ of 3.68 μΜ). Compounds 1 and 52 were also slightly more potent than compound 51. This data suggests that dual inhibitors are not simply toxic to every cell line. [0198] Example 8: Intracellular Mechanism of Action

[0199] To investigate whether the dual enzyme inhibition profile of compound 51 translates into intracellluar inhibition against both the JAK-STAT and HDAC pathways, the effects of compound 51 in the MM cell line KMS 12BM (FIG. 8A) and the AML cell line MOLM-14. (FIG. 8B) were tested. The cell lines were treated with compound 51 at the respective concentrations and timepoints. Compound 51 was tested at approximately 2 x IC₅₀ where it showed increased histone-3 acetylation (Ac-H3) and potent increase of acetylated tubulin (Ac-tubulin), mediated by HDAC6.¹⁰⁵ The positive control tubastatin (15 μΜ)⁵⁵ indicated a significant increase in Ac-tubulin but no increase in Ac-H3 (FIGS. 8 A and B).

[0200] After lysis, acetylated tubulin (Ac-Tubulin) and acetylated histone 3 (Ac-H3) were detected by immunoblotting. Subsequently, as a loading control the same membranes were re-probed with Tubulin and H3 respectively. Exposure of the cells to compound 51, in a time course from 0 to 48 h, led to an increase of Ac-H3 indicating strong blockade of HDAC signalling. The in vitro selectivity profile of compound 51, showing sub-micromolar activity for HDACs 1, 2, 8, 10 and 11, is apparently sufficient to induce inhibition of histone deacetylation in cells. Increase of compound 51 -induced Ac-H3 peaked at 16 and 4 h in

KMS 12BM and MOLM-14 cells, respectively, and was still detectable at 48 hours (Figures 8A and B). Similar effects were seen in both cell lines for Ac-tubulin where compound 51 showed strong and constant induction of Ac-tubulin up to 48 h in KMS-12-BM cells and peaking at 16 hours in MOLM-14. [0201] The effects of compound 51 on the JAK-STAT pathway were then tested. KMS- 12-BM (FIG. 8C) and MOLM-14 (FIG. 8D) were pre-treated with compound 51 for 3 h and then were treated with lOng/mL of IL-6 for 15 minutes. Pacritinib was used as a positive control for the inhibition of the JAK2 pathway in each cell line at approximately its IC₅₀ concentration (2 μΜ for KMS-12-BM and 0.1 μΜ for MOLM-14). After lysis, p- STAT3(TY705) was detected by immunoblotting. The same membranes were re -probed with STAT3 to detect total protein levels. Under these conditions, STAT3 levels were

significantly reduced (FIGS. 8C and D). Similarly, a dose response study with compound 51 revealed progressively increased suppression of STAT3 levels with increasing concentrations of the dual inhibitor. At concentrations around the proliferation IC₅₀ level, 1.5 μΜ, STAT3 was completely inhibited in both cell lines.

[0202] The effects of compound 51 in V617F-expressing HEL92.1.7 cells were also evaluated (FIGS. 9A-9D). Using 1 as a positive control, a strong increase in Ac-H3 was seen at 0.1, 0.5 and 1.0 μΜ. HEL92.1.7 cells were treated with compounds 1 and 51 at the respective concentrations for 1 hour. After lysis, acetylated tubulin (Ac -Tubulin) and acetylated histone 3 (Ac-H3) were detected by immunoblotting (FIG. 9A). Subsequently, as a loading control the same membranes were re-probed with Tubulin and H3 respectively. Compound 51 gave a dose response with similar responses at the same concentrations (ratio of total histones : Ac-H3), indicating effective HDAC pathway blockade. As expected from its potent HDAC6 inhibition, Ac-tubulin was more sensitive to the effects of compound 51 with significant increases at the lowest concentration tested and a clear dose response. Even at 0.1 μΜ, increase of Ac-tubulin could be seen with compound 51 but not with compound 1. FIG. 9B shows the quantification of HDAC inhibitory responses by densitometry.

[0203] JAK-STAT pathway inhibition is clearly evident upon treatment of HEL92.1.7 cells with compound 51 (FIG. 9C). HEL92.1.7 cells were treated with compound 51 at the respective concentrations for 1 hour. After lysis, p-JAK2 and p-STAT5 were detected by immunoblotting. The same membranes were re-probed with JAK2 and STAT5 to detect total protein levels. Upregulation of phopho-JAK2 (p-JAK2) has been reported using the Y1007/8 p-JAK2 antibody described in studies with pacritnib in HEL92.1.7 cells. 28 Although phosphorylation of the specific Y1007/8 residue(s) increases, phosphorylation of the critical

Y221 decreases as does total phosphorylation levels. 28 Compound 51 gives the same increase in p-JAK2 (Y1007/8) as 2 in this assay. Furthermore, upon treatment with compound 51, STAT5 is decreased in this cell line at around its proliferation IC₅₀, providing direct evidence for JAK pathway inhibition in HEL92.1.7 cells. FIG. 9D shows the quantification of JAK inhibitory responses by densitometry.

[0204] Taken together, these data in three hematological cell lines provides stong evidence for dual JAK-STAT and HDAC pathway inhibition in cells induced by a single compound, 51. [0205] Example 9: Mechanism of Cell Death

[0206] The phenotypic observations of cell death induced by compound 51 were then confirmed to be because of apoptosis. Early apoptosis involves translocation of membrane phosphatidylserine (PS) from the inner side of the plasma membrane to the surface. Annexin V, a Ca²⁺-dependent phospholipid -binding protein, has high affinity for PS, and

fluorochrome-labeled Annexin V can be used for the detection of exposed PS using flow cytometry. In the early stages of apoptosis the cell membranes are still largely intact hence Annexin V can enter and bind exposed PS, whereas propidium iodide (PI) cannot. In the later stages of apoptosis, the cell membrane is not intact and propidium iodide (PI) can also enter giving a specific readout for dead cells. Conversely, viable cells with intact membranes exclude PI, therefore, cells that are considered viable are both Annexin V and PI negative, while cells that are in early apoptosis are Annexin V positive and PI negative, and cells that are in late apoptosis or already dead are both Annexin V and PI positive. An effective strategy is to track the progress of cells over time through early to later phases of apoptosis which further supports the assertion that the mechanism of cell death is indeed via apoptosis. Compound 51 was hence studied in a time course dose response experiment in the AML cell line MOLM-14 using doxorubicin as a positive control (FIG. 10). FIG. 10B shows the 48 h data. Both an increase in dose and time led to an increase in Annexin V positive cells indicating early apoptotic events. At 24 h it became evident that cells were dying shown by the increased levels of PI+ cells. At this timepoint a dose response was achieved for both

Annexin V and PI, whereas at the earlier timepoint of 4 h, no dose response was observed. At the later timepoint of 48 h, more cells were PI+ indicating late apoptosis (Figures 10A, 10B).

[0207] Compound 51 triggers apoptosis in AML cell line, MOLM-14. As shown in FIG. 10A, MOLM-14 cells were treated with compound 51 for 4h, 24h, and 48h at the

concentrations indicated and subsequently stained by Annexin-V FITC and Propidium

Iodide(PI). Fluorescence was measured via flow cytometry(LSRII) and at least 10,000 events were analysed. As provided in FIG. 10B, Annexin V FITC vs Propidium Iodide -plots from the gated cells show the populations corresponding to viable and non-apoptotic (Annexin V- PI-), early (Annexin V+PI-), and late (Annexin V+PI+) apoptotic cells following 48 h treatment of compound 51. MOLM-14 cells were treated with compound 51 at the respective concentrations and timepoints and then lysed. Doxorubicin (ΙμΜ) was utilised as a positive control for induction of PARP cleavage. As shown in FIG. IOC, cleaved PARP was detected by immunoblotting. [0208] Poly(ADP-ribose) polymerase (PARP) has been implicated in cell death pathways via apoptosis. D'Amours, D. et al. J. Cell. Sci. 2001, 114, 3771-78; Danial, N. N.;

Korsmeyer, S. J. Cell 2004, 116, 205-19. During apoptosis, PARP is cleaved by caspases to give a N-terminal DNA binding domain. Increase in cleaved PARP upon drug treatment indicates cell death via apoptosis. Treatment of MOLM-14 cells with compound 51 led to dose related increases in cleaved PARP at both the 8 h and 24 h timepoints (FIG. IOC).

Taking all the data together, compound 51 has been shown to induce cell death via apoptosis.

[0209] Example 10: Assessment of In Vitro Metabolism

[0210] Because compound 51 was a suitable dual inhibitor template, it and its homologue 52 were tested in male and female rat liver microsomes to assess the propensity of the template towards degradation by phase I metabolism. In female rat liver microsomes, compound 51 had a moderate half life of 26.3 minutes (apparent clearance of 10.7 L/h/kg) but was cleared more rapidly in male microsomes. Compound 52 showed no significant difference between male and female microsomes (FIGS. 11A and 11B). [0211] Compound 51 has single digit nanomolar potency against JAK2 and HDAC6 with good potency (<100 nM) against HDACs 2 and 10 and sub-micromolar potency against HDACs 1, 8 and 11. Approximately 50-fold selectivity within the JAK family, and FLT-3, was also seen. Broad antiproliferative potency was observed for compound 51 across a range of solid and hematological cell lines. This activity translated into inhibition of tumor cell colony formation. In detailed studies in AML, MM and EL cell lines, compound 51 was shown to block both the JAK-STAT and HDAC pathways at concentrations around or below its IC50. Apoptosis was shown to be the mechanism of cell death via Annexin V/PI studies and dose and time related increases in cleaved PARP. Compound 51 was also shown to be moderately stable in rat liver microsomes with a half life of just over 25 minutes. This first detailed demonstration of isoform-selective JAK-HDAC bispecific inhibitors provides useful tool compounds for further studies of multiple pathway inhibition achieved with a single DML molecule.

[0212] Example 11 : Synthesis of Specific Compounds

General Synthetic Information [0213] The starting materials and reagents used in preparing these compounds generally are either available from commercial suppliers, such as Aldrich Chemical Co., or are prepared by methods known to those skilled in the art following procedures set forth in references, such as Fieser and Fieser's Reagents for Organic Synthesis; Wiley & Sons: New York, 1991, Volumes 1-15; Rodd's Chemistry of Carbon Compounds, Elsevier Science Publishers, 1989, Volumes 1-5 and Supplemental; and Organic Reactions, Wiley & Sons: New York, 1991, Volumes 1-40. The following synthetic reaction schemes are merely illustrative of some methods by which the compounds of the present invention can be synthesised, and various modifications to these synthetic reaction schemes can be made and will be suggested to one skilled in the art having referred to the disclosure contained in this application.

[0214] The starting materials and the intermediates of the synthetic reaction schemes can be isolated and purified if desired using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data.

[0215] Unless specified to the contrary, the reactions described herein preferably are conducted at atmospheric pressure (e.g., under an inert atmosphere) at a reaction temperature range of from about -25 °C to about 150 °C, more preferably from about 0 °C to about 125 °C, and most preferably and conveniently at about room (or ambient) temperature, e.g., about 20 °C. Unless stated otherwise, all non-aqueous reactions were performed in oven-dried round bottom flasks under an inert nitrogen atmosphere with commercially available anhydrous solvents. All reaction temperatures stated in the procedures are external bath temperatures. Flash chromatography was performed on silica gel 60 (0.040 - 0.063 mm).

[0216] Yields refer to chromatographically and spectroscopically homogeneous materials, unless otherwise stated. Purity of the compounds were assessed by high pressure liquid chromatography by detection at 254 nm using an Agilent 1200 series HPLC system with a Zorbax SB-C18 5 micron 4.6 x 250 mm column using a gradient elution starting from a 5% solution of acetonitrile and 1% trifluoroacetic acid (TFA) and a 95% solution of water and 1% TFA to a 100% solution of acetonitrile and 1% TFA at 0.5 mL per minute over 15 min. HPLC purity is above 99% unless stated.

[0217] ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AMX400 (400 MHz) NMR spectrometer at ambient atmosphere. The deuterated solvent used was CDC1₃ unless otherwise stated. Chemical shifts are reported in parts per million (ppm), and residual undeuterated solvent peaks were used as internal reference: proton (7.26ppm for CDCI₃, 2.50ppm for DMSO-d6), carbon (77.0ppm for CDC1₃, 39.52ppm for DMSO-d6). 1H NMR coupling constants (J) are reported in Hertz (Hz), and multiplicities are presented as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad). Low-resolution mass spectra were obtained on an Agilent 6130B Quadrupole LC/MS in ESI mode with an Agilent 1260 Infinity LC system using a ThermoScientific Hypersil 150 x 2.1mm 5 micron column or a Shimadzu LCMS-2020 in ESI mode.

Synthesis and Spectroscopic Data

[0218] 1 l-(2-Chloroethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (3). Following a literature procedure (William, A. D. et al. J. Med. Chem. 2011, 54, 4638-58), 29.4 mg (0.0631 mmol) of the diallyl precursor compound and 6.7 mg (0.00789 mmol,

0.13 eq.) of the second-generation Grubbs catalyst was dissolved in 14 mL of degassed DCM (5 mM concentration) to obtain an orange solution. 1 mL of 4M hydrochloric acid was added changing the solution to a yellow colour. The reaction mixture was heated at reflux for 3 h. After cooling to rt, the reaction was quenched with saturated sodium bicarbonate solution and extracted with DCM (x3). The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The organic layer was concentrated and purified by column chromatography using 3:2 Hex/EtOAc to obtain 17.1 mg (62%) in a 87: 13 E/Z ratio of macrocycle 3 as a yellow solid. LRMS (ESI) m/z 438.0 (M+H⁺). E isomer only: 1H NMR δ 8.70 (s, 1 H), 8.47 - 8.40 (m, 1 H), 8.30 (s, 1 H), 7.81 (d, J = 7.8 Hz, 1 H), 7.60 (d, J = 7.7 Hz, 1 H), 7.49 (t, J = 7.7 Hz, 1 H), 7.39 (br. s., 1 H), 7.17 (d, J = 5.3 Hz, 1 H), 6.85 (s, 2 H), 5.92 - 5.77 (m, 2 H), 4.66 (d, J = 2.6 Hz, 4 H), 4.26 (t, J = 5.8 Hz, 3 H), 4.17 (d, J = 4.9 Hz, 2 H), 4.07 (d, J = 4.4 Hz, 2 H), 3.87 - 3.80 (m, 2 H). ¹³C NMR δ 164.3, 160.2, 158.7, 151.5, 138.6, 137.1, 134.1, 132.0, 130.9, 129.9, 128.9, 127.9, 127.7, 126.3, 121.4, 119.2, 113.7, 107.9, 70.2, 69.5, 69.3, 67.6, 65.6, 42.2, 29.7. [0219] l l-(2-N-(tert-Butyl 4-(methoxycarbonyl)phenylcarbamate)-ethoxy)-14,19-dioxa- 5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa- l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (4). 0.147 g (0.335 mmol) of macrocycle chloride (3), 0.183 g (0.562 mmol, 1.7 eq.) of caesium carbonate, 0.122 g (0.330 mmol, 1.0 eq.) of tetrabutylammonium iodide, and 0.129 g (0.514 mmol, 1.5 eq.) of compound 4a was mixed, and 3 mL of DMF was added. The mixture was stirred at 120 °C for 16 h. The mixture was cooled to rt and diluted with water. It was extracted with ethyl acetate (x3). The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The organic layer was concentrated and purified by column chromatography using 2: 1 to 1: 1 to 2:3 Hex/EtOAc solution to obtain 117 mg (53%) of compound 4 as a yellow solid. TLC (1: 1 Hex/EtOAc) R_f = 0.31. LRMS (ESI) m/z 653.3 (M+H⁺). 1H MR δ 8.60 (d, J = 2.6 Hz, 1 H), 8.38 (d, J = 5.1 Hz, 1 H), 8.25 (s, 1 H), 8.06 - 8.01 (m, 2 H), 7.82 - 7.76 (m, 2 H), 7.58 (d, J = 7.7 Hz, 1 H), 7.47 (t, J = 7.7 Hz, 1 H), 7.42 - 7.37 (m, J = 8.7 Hz, 2 H), 7.13 (d, J = 5.3 Hz, 1 H), 6.83 (dd, J = 2.6, 8.7 Hz, 1 H), 6.77 (d, J = 8.7 Hz, 1 H), 5.81 - 5.76 (m, 2 H), 4.62 (s, 2 H), 4.33 (s, 2 H), 4.19 - 4.14 (m, 2 H), 4.04 (d, J = 4.0 Hz, 2 H), 4.01 (d, J = 4.5 Hz, 2 H), 3.91 (s, 3 H), 1.45 (s, 9 H). ¹³C NMR 5 166.5, 164.3, 160.0, 158.4, 154.0, 151.6, 147.3, 138.5, 137.0, 133.4, 132.0, 130.9, 130.1, 129.8, 128.8, 127.6, 127.1, 126.9, 126.3, 126.2, 121.4, 119.2, 111.9, 107.6, 81.1, 70.1, 69.2, 67.4, 66.8, 65.3, 52.0, 49.6, 28.2.

[0220] 1 l-(2-(tert-Butyl 3-(methoxycarbonyl)phenylcarbamate)-ethoxy)-14,19-dioxa- 5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa-

1(25),2(26), 3,5, 8, 10,12(27), 16,21,23-decaene (5). This compound was synthesized following the procedure for compound 4 using 190 mg (0.434 mmol) of compound 3, 447 mg

(1.37 mmol, 3.2 eq.) of caesium carbonate, 201 mg (0.544 mmol, 1.3 eq.) of

tetrabutylammonium iodide and 171 mg (0.678 mmol, 1.6 eq.) of 5a. The compound was purified by column chromatography using 2: 1 to 1: 1 Hex/EtOAc solution to obtain 127 mg (45%) of compound 5 as a yellow solid. TLC (1:1 Hex/EtOAc) R_f = 0.24. LRMS (ESI) m/z 653.3 (M+H⁺). 1H NMR δ 8.44 (d, J = 2.64 Hz, 1H), 8.25 (d, J = 5.15 Hz, 1H), 8.13 (s, 1H), 7.83 (s, 1H), 7.75 - 7.80 (m, 1H), 7.68 (d, J = 7.91 Hz, 1H), 7.48 (d, J = 9.29 Hz, 2H), 7.32 - 7.40 (m, 2H), 7.30 (d, J = 7.78 Hz, 1H), 7.13 (s, 1H), 7.03 (d, J = 5.27 Hz, 1H), 6.72 (dd, J = 2.70, 8.72 Hz, 1H), 6.66 (d, J = 8.66 Hz, 1H), 5.66 (q, J = 5.14 Hz, 2H), 4.50 (s, 2H), 4.23 (s, 2H), 4.01 - 4.06 (m, 2H), 3.95 - 4.01 (m, 3H), 3.92 (d, J = 4.52 Hz, 2H), 3.88 (d, J = 4.77 Hz, 2H), 3.79 (s, 3H), 1.28 - 1.36 (m, 9H). ¹³C NMR δ 166.5, 164.7, 159.7, 157.8, 154.3, 151.8, 143.2, 138.6, 136.9, 133.1, 132.1, 131.8, 131.1, 130.9, 129.8, 129.0, 128.7, 127.9, 127.7,

127.1, 127.1, 126.3, 121.4, 119.4, 112.0, 107.6, 81.0, 70.1, 69.2, 67.5, 66.7, 65.3, 52.2, 49.6, 28.3.

[0221] 1 l-(2-(Methyl 4-hydroxybenzoate))-14,19-dioxa-5,7,26-triaza-tetracyclo

[19.3.1.1(2,6).1(8,12)] heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (6). This compound was synthesised following the procedure for compound 4 using 201 mg (0.459 mmol) of compound 3, 307 mg (0.943 mmol, 2.0 eq.) of caesium carbonate, 169 mg

(0.457 mmol, 1.0 eq.) of tetrabutylammonium iodide and 142 mg (0.932 mmol, 2.0 eq.) of compound 6a. The crude product was purified by column chromatography using 2:1 to 2:3 to 1:2 Hex/EtOAc solution to obtain 206 mg (82%) of compound 6 as a yellow solid. TLC (1: 1 Hex/EtOAc) R_f = 0.22. LRMS (ESI) m/z 554.2 (M+H⁺). 1H NMR δ 8.65 (d, J = 2.3 Hz, 1 H), 8.40 (br. s., 1 H), 8.28 (s, 1 H), 8.05 - 7.99 (m, 2 H), 7.81 (d, J = 7.7 Hz, 1 H), 7.60 (d, J = 7.3 Hz, 2 H), 7.52 - 7.45 (m, 1 H), 7.17 (d, J = 5.3 Hz, 1 H), 7.02 - 6.96 (m, 2 H), 6.95 - 6.84 (m, 2 H), 5.87 - 5.78 (m, 2 H), 4.66 - 4.60 (m, 2 H), 4.59 (s, 2 H), 4.43 - 4.33 (m, 4 H), 4.09 (d, J = 4.5 Hz, 2 H), 4.05 (d, J = 4.0 Hz, 2 H), 3.93 - 3.84 (m, 3 H). ¹³C NMR δ 166.8, 164.7,

162.5, 159.7, 157.9, 151.9, 138.7, 136.9, 133.7, 131.9, 131.6, 131.1, 129.9, 129.0, 127.9, 127.7, 126.4, 122.9, 121.4, 119.5, 114.3, 113.6, 107.8, 70.1, 69.3, 68.0, 67.6, 66.7, 65.6.

[0222] 1 l-(2-N-(tert-Butyl 4-nitrophenylcarbamate)-ethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (7). This compound was synthesised following the procedure for compound 4 using 432 mg (0.988 mmol) of compound 3, 387 mg (1.19 mmol, 1.2 eq.) of caesium carbonate, 185 mg (0.502 mmol, 0.51 eq.) of tetrabutylammonium iodide and 275 mg (1.15 mmol, 1.2 eq.) of compound 7a in 4 mL of DMF. The crude product was purified by column chromatography using 1: 1 Hex/EtOAc solution to obtain 395 mg (62%) of compound 7 as a light yellow solid. TLC (1: 1 Hex/EtOAc) R_f = 0.18. LRMS (ESI) m/z 640.3 (M+H⁺). 1H NMR δ 8.64 (d, J = 2.4 Hz, 1 H), 8.40 (d, J = 5.3 Hz, 1 H), 8.26 (s, 1 H), 8.25 - 8.19 (m, 2 H), 7.80 (d, J = 7.8 Hz, 1 H), 7.62 - 7.52 (m, 4 H), 7.51 - 7.45 (m, 1 H), 7.15 (d, J = 5.3 Hz, 1 H), 6.86 - 6.77 (m, 2 H), 5.82 - 5.77 (m, 2 H), 4.63 (s, 2 H), 4.34 (s, 2 H), 4.24 - 4.19 (m, 2 H), 4.17 - 4.11 (m, 2 H), 4.06 (d, J = 3.9 Hz, 2 H), 4.01 (d, J = 4.3 Hz, 2 H), 1.52 - 1.45 (m, 9 H). ¹³C NMR δ 164.4, 160.1, 158.5, 153.5, 151.6, 149.1, 144.7, 138.6, 137.1, 133.7, 131.6, 131.0, 130.1, 128.9,

127.6, 126.9, 126.6, 126.3, 124.1, 121.5, 119.2, 112.2, 107.8, 82.0, 70.1, 69.4, 67.6, 67.0, 65.4, 49.9, 28.2.

[0223] l l-(2-(Methyl isonipecotate)-ethoxy)- 14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (8). 1.00 g (2.29 mmol) of compound 3 was dissolved in 20 mL of acetonitrile. 0.90 mL (6.66 mmol, 2.9 eq.) of compound 8a was added to the solution, and it was heated at reflux for 64 h. The solution was cooled to rt and concentrated. The crude product was purified by column chromatography using 45:55 to 3:7 Hex/EtOAc to EtOAc to 9: 1 EtOAc/MeOH to give 255 mg (25%) of recovered compound 1 and 771 mg (62%) of compound 8 as a yellow solid. TLC (10: 1 EtOAc/MeOH) R_f = 0.16. LRMS (ESI) m/z 544.3 (M+H⁺). 1H NMR δ 8.68 (s, 1 H), 8.40 (d, J = 5.1 Hz, 1 H), 8.29 (s, 1 H), 7.79 (d, J = 7.8 Hz, 1 H), 7.61 - 7.55 (m, 2 H), 7.51 - 7.44 (m, 1 H), 7.14 (d, J = 5.3 Hz, 1 H), 6.86 - 6.82 (m, 2 H), 5.91 - 5.77 (m, 2 H), 4.64 (s, 2 H), 4.60 (s, 2 H), 4.20 - 4.10 (m, 4 H), 4.06 (d, J = 4.5 Hz, 2 H), 3.70 - 3.65 (m, 3 H), 3.09 - 3.00 (m, 2 H), 2.89 (t, J = 5.6 Hz, 2 H), 2.41 - 2.30 (m, 3 H), 2.02 - 1.93 (m, 2 H), 1.92 - 1.80 (m, 2 H). ¹³C NMR δ 175.1, 164.2, 160.2, 158.7, 151.9, 138.6, 137.1, 133.5, 131.9, 130.9, 129.9, 128.9, 127.6, 127.0, 126.2, 121.4, 119.2, 112.7, 107.7, 77.2, 70.0, 69.3, 67.6, 66.7, 65.8, 57.2, 53.1, 51.6, 40.3, 27.8.

[0224] 1 l-(2-N-(tert-Butyl 4-aminophenylcarbamate)-ethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (9). 395 mg (0.617 mmol) of 7, 140 mg (2.51 mmol, 4.0 eq.) of iron and 219 mg (4.10 mmol, 6.6 eq.) of ammonium chloride in 10 mL of ethanol, 5 mL of ethyl acetate and 3 mL of water. Compound 9 was used without further purification. TLC (1: 1 Hex/EtOAc) R_f = 0.09. LRMS (ESI) m/z 610.3 (M+H⁺).

[0225] 1 l-(2-(tert-Butyl 4-(methyl 7-carbamoylpentanoyl)phenylcarbamate))-ethoxy)- 14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa- 1(25),2(26),3,5,8,10,12(27),16,21, 23-decaene (10). 100.3 mg (0.1645 mmol) of aniline 9, 30 (0.2025 mmol, 1.2 eq.) of monomethyl adipate (10a), 85.1 mg (0.224 mmol, 1.4 eq.) of HATU and 43 μΐ_^ (0.307 mmol, 1.9 eq.) of triethylamine were dissolved in 2 mL of DMF and stirred at 60 °C for 18 h. The reaction was cooled to rt, diluted with water and extracted with ethyl acetate (x3). The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The crude product was purified by column chromatography using 2:3 to 1:4 Hex/EtOAc to EtOAc solution to obtain 97.5 mg (79%) of compound 10 as a yellow amorphous solid (contains 1: 1 DMF). TLC (2:3 Hex/EtOAc) R_f = 0.51. LRMS (ESI) m/z 752.4 (M+H⁺). 1H NMR δ 8.48 (d, J = 2.51 Hz, 1H), 8.35 (d, J = 5.65 Hz, 1H), 8.23 (s, 1H), 8.10 (s, 1H), 7.78 - 7.87 (m, 2H), 7.62 (d, J = 7.78 Hz, 1H), 7.47 - 7.57 (m, 3H), 7.16 - 7.23 (m, 3H), 6.87 (dd, J = 2.70, 8.72 Hz, 1H), 6.78 (d, J = 8.78 Hz, 1H), 5.73 - 5.87 (m, 2H), 4.62 (s, 2H), 4.48 (s, 2H), 4.08 - 4.15 (m, 4H), 3.99 - 4.06 (m, 4H), 3.67 (s, 3H), 2.32 - 2.43 (m, 4H), 1.63 - 1.80 (m, 4H). ¹³C NMR δ 174.0, 171.0, 162.7, 158.1, 155.5, 154.8, 152.3, 138.8, 136.2, 131.8, 129.7, 129.1, 127.9, 127.7, 127.3, 126.7, 121.8, 120.0, 112.0, 107.5, 80.6, 70.2, 69.2, 67.6, 66.3, 65.4, 51.6, 49.5, 38.6, 37.0, 36.5, 33.6, 28.3, 24.9, 24.4, 24.2.

[0226] 1 l-(2-(tert-Butyl 4-(methyl 7-carbamoylhexanoyl)phenylcarbamate))-ethoxy)- 14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa-

1(25),2(26), 3,5, 8, 10,12(27), 16,21,23-decaene (11). This compound was synthesised using the method for compound 10 with 83.3 mg (0.137 mmol) of compound 9, 36 μΐ_^ (0.203 mmol, 1.5 eq.) of ethyl hydrogen pimelate (11a), 63.3 mg (0.166 mmol, 1.2 eq.) of HATU and 40

(0.285 mmol, 2.1 eq.) of triethylamine. The crude product was purified by column chromatography using 3:7 to 1:3 Hex/EtOAc solution to obtain 78.0 mg (73%) of compound 11 as a pale yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.36. LRMS (ESI) m/z 780.4 (M+H⁺). 1H NMR δ 8.58 (d, J = 2.51 Hz, 1H), 8.33 - 8.42 (m, 1H), 8.27 (s, 1H), 7.75 - 7.85 (m, 2H), 7.55 - 7.67 (m, 2H), 7.43 - 7.55 (m, 3H), 7.11 - 7.23 (m, 3H), 6.85 (dd, J = 2.76, 8.66 Hz, 1H), 6.78 (d, J = 8.78 Hz, 1H), 5.72 - 5.89 (m, 2H), 4.63 (s, 2H), 4.48 (s, 2H), 3.97 - 4.20 (m, 12H), 3.25 - 3.42 (m, 1H), 2.24 - 2.42 (m, 7H), 1.56 - 1.78 (m, 7H), 1.32 - 1.56 (m, 13H), 1.19 - 1.31 (m, 5H). ¹³C NMR δ 173.7, 171.2, 164.8, 159.7, 157.7, 154.8, 151.9, 138.6, 136.9, 136.2, 132.1, 131.1, 129.7, 128.9, 127.7, 127.7, 127.1, 126.3, 121.4, 119.9, 119.5, 112.1, 107.6, 70.1, 69.2, 67.5, 66.4, 65.5, 60.2, 60.1, 49.6, 37.2, 34.1, 34.0, 32.8, 28.9, 28.5, 28.3, 25.1, 25.0, 24.7, 24.6, 24.4, 14.2.

[0227] 1 l-(2-(tert-Butyl 4-(methyl 7-carbamoylheptanoyl)phenylcarbamate))-ethoxy)- 14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa- 1(25),2(26),3,5,8,10,12(27), 16,21, 23 -decaene (12). 73.5 mg (0.391 mmol, 1.7 eq.) of monomethyl suberate, 140 mg (0.229 mmol), aniline 9, 158 mg (0.414 mmol, 1.8 eq.) and 90

(0.642 mmol, 2.8 eq.) of triethylamine. The crude product was purified by column chromatography using 2:3 (12a) to 1:4 Hex/EtOAc solution to obtain 105 mg (59%) of compound 12 as a pale yellow solid. TLC (3:7 Hex/EtOAc) R_f = 0.33. LRMS (ESI) m/z 780.4 (M+H⁺). 1H NMR δ 8.60 (d, J = 2.4 Hz, 1 H), 8.38 (d, J = 4.5 Hz, 1 H), 8.27 (s, 1 H), 7.80 (d, J = 7.8 Hz, 1 H), 7.63 (s, 1 H), 7.59 (d, J = 7.7 Hz, 1 H), 7.53 - 7.45 (m, 4 H), 7.19 (d, J = 7.7 Hz, 2 H), 7.14 (d, J = 5.3 Hz, 1 H), 6.83 (dd, J = 2.6, 8.7 Hz, 1 H), 6.78 (d, J = 8.7 Hz, 1 H), 5.80 (q, J = 4.8 Hz, 2 H), 4.63 (s, 2 H), 4.48 (s, 2 H), 4.15 - 4.07 (m, 4 H), 4.07 - 3.98 (m, 4 H), 3.65 (s, 3 H), 2.35 - 2.27 (m, 4 H), 1.76 - 1.66 (m, 2 H), 1.62 (td, J = 7.3, 14.3 Hz, 2 H), 1.49 - 1.40 (m, 9 H), 1.40 - 1.32 (m, 6 H). ¹³C NMR δ 174.2, 171.2, 164.5, 160.0, 158.2, 154.8, 151.8, 138.6, 137.0, 133.2, 132.1, 131.0, 129.8, 128.9, 127.7, 127.1, 126.3, 121.4, 119.9, 119.4, 112.1, 107.6, 80.5, 70.2, 69.2, 67.5, 66.4, 65.5, 51.5, 49.6, 37.4, 33.9, 29.6, 28.7, 28.7, 28.3, 25.3, 24.6.

[0228] 2-((Allyloxy)methyl)-4-nitrophenol (14). 2.00 g (12.0 mmol) of 2-hydroxy-5- nitrobenzaldehyde (13) was dissolved in 30 mL of DCM and cooled to 0 °C. 2.5 mL

(17.8 mmol, 1.5 eq.) of trimethylamine. 1.1 mL (14.5 mmol, 1.2 eq.) of chloromethyl methyl ether was added, and the solution was allowed to warm to rt and stirred for 18 h at rt. The solution was concentrated and the crude product used directly in the next step. TLC (4: 1 Hex/EtOAc) R_f = 0.13. The crude mixture was redissolved in 30 mL of methanol and cooled to 0 °C. 0.461 g (12.2 mmol, 1.0 eq.) of sodium borohydride was added portionwise, and the mixture was stirred at rt for 1 h. The reaction was quenched with saturated ammonium chloride solution. This was extracted (x4) with dichloromethane. The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The solution was concentrated and the crude product used directly in the next step. TLC (1: 1 Hex/EtOAc) R_f = 0.54. The crude product was redissolved in 10 mL of DCM. 0.845 g (3.71 mmol, 0.3 eq.) of triethylbenzylammonium chloride, 3.1 mL (35.8 mmol, 3.0 eq.) of allyl bromide and 19 mL (363 mmol, 30 eq.) of concentrated sodium hydroxide solution was added sequentially at rt. The biphasic mixture was stirred at rt for 66 h. This was extracted (x3) with dichloromethane. The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The solution was concentrated and the crude product used directly in the next step. TLC (3: 1 Hex/EtOAc) R_f = 0.59. The crude product was redissolved in 20 mL of THF and 10 mL of 4 M HC1 was added to the solution. The solution was heated at reflux for 3 h after which it was cooled to rt. This was extracted (x3) with dichloromethane. The combined organic layer was washed with saturated sodium bicarbonate solution and dried with anhydrous sodium sulfate. The solution was concentrated, and the product was purified using column chromatography starting from 9: 1 to 85: 15 to 3: 1 to 2: 1 Hex/EtOAc solutions to obtain 1.81g (72% over 4 steps) of the product as a yellow oil. TLC (2: 1 Hex/EtOAc) R_f = 0.30; LRMS (ESI) m/z 152.0 (M+H⁺). 1H NMR δ 8.09 (dd, J = 2.7, 9.0 Hz, 1 H), 7.98 (d, J = 2.6 Hz, 1 H), 6.92 (d, J = 9.0 Hz, 1 H), 6.00 - 5.86 (m, 1 H), 5.38 - 5.25 (m, 2 H), 4.75 (s, 2 H), 4.12 (d, J = 5.8 Hz, 2 H). ¹³C NMR δ 161.9, 140.7, 132.7, 125.4, 124.1, 122.7, 119.0, 116.8, 71.9, 70.3.

[0229] Methyl 4-(2-((allyloxy)methyl)-4-nitrophenoxy)butanoate (15). 0.126 g (0.602 mmol) of phenol 14 1065 was dissolved in 6 mL of acetonitrile. 0.172 g (1.24 mmol, 2.1 eq.) of potassium carbonate and 0.11 mL (0.888 mmol, 1.5 eq.) of methyl 4-bromobutyrate (15a) was added to the solution. The mixture was heated at reflux for 18 h with stirring. The solution was cooled to rt, diluted with water, and extracted (x3) with DCM. The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The organic layer was concentrated and purified by column chromatography using 3:1 to 2: 1 Hex/EtOAc to obtain 0.151 g (100%) of compound 15 as a light yellow oil. TLC (2: 1 Hex/EtOAc) R_f = 0.28; LRMS (ESI) m/z 310.1 (M+H⁺), 332.1 (M+Na⁺). 1H MR δ 8.29 (d, J = 2.9 Hz, 1 H), 8.12 (dd, J = 2.8, 9.0 Hz, 1 H), 6.87 (d, J = 9.0 Hz, 1 H), 6.02 - 5.90 (m, 1 H), 5.33 (qd, J = 1.6, 17.2 Hz, 1 H), 5.22 (qd, J = 1.3, 10.4 Hz, 1 H), 4.52 (s, 2 H), 4.15 - 4.08 (m, 4 H), 3.67 (s, 3 H), 2.52 (t, J = 7.2 Hz, 2 H), 2.20 - 2.10 (m, 2 H). ¹³C NMR δ 173.1, 160.6, 141.3, 134.3, 128.3, 124.7, 123.9, 117.3, 110.3, 71.9, 67.6, 65.9, 51.6, 30.1, 24.2.

[0230] Methyl 5-(2-((allyloxy)methyl)-4-nitrophenoxy)pentanoate (16). Compound 16 was synthesised using the procedure for compound 15 using 145 mg (0.691 mmol) of 14, 0.15 mL (1.05 mmol, 1.5eq.) of methyl 5-bromopentanoate (16a) and 209 mg (1.51 mmol, 2.2eq.) of potassium carbonate in 6 mL of acetonitrile. The compound was purified by flash column chromatography using 4: 1 to 2: 1 Hex/EtOAc solution to obtain 165 mg (90%) of compound 16 as a light yellow oil. TLC (7:3 Hex/EtOAc) R_f = 0.32. LRMS (ESI) m/z 346.1 (M+Na⁺). 1H NMR δ 8.27 (d, J = 2.8 Hz, 1 H), 8.10 (dd, J = 2.8, 9.0 Hz, 1 H), 6.85 (d, J = 9.2 Hz, 1 H), 6.01 - 5.89 (m, 1 H), 5.32 (qd, J = 1.6, 17.2 Hz, 1 H), 5.20 (qd, J = 1.4, 10.4 Hz, 1 H), 4.51 (s, 2 H), 4.12 - 4.03 (m, 4 H), 3.64 (s, 3 H), 2.38 (t, J = 7.0 Hz, 2 H), 1.91 - 1.76 (m, 4 H). ¹³C MR 5 173.5, 160.8, 141.2, 134.3, 128.3, 124.6, 123.8, 117.2, 110.2, 71.8, 68.3, 65.9, 51.4, 33.3, 28.2, 21.3. [0231] Ethyl 6-(2-((allyloxy)methyl)-4-nitrophenoxy)hexanoate (17). This compound was synthesised using the procedure for compound 15 using 429 mg (2.05 mmol) of 14, 0.45 mL (2.53 mmol, 1.2eq.) of ethyl 6-bromohexanoate (17a) and 730 mg (5.28 mmol, 2.6eq.) of potassium carbonate in 10 mL of acetonitrile. The compound was purified by flash column chromatography using 9: 1 to 85: 15 Hex/EtOAc solution to obtain 631 mg (88%) of compound 17 as a yellow solid. TLC (7:3 Hex/EtOAc) R_f = 0.34. LRMS (ESI) m/z 374.1 (M+Na⁺). ¹H MR δ 8.31 - 8.25 (m, 1 H), 8.10 (td, J = 3.2, 9.0 Hz, 1 H), 6.85 (dd, J = 1.6, 9.0 Hz, 1 H), 6.01 - 5.89 (m, 1 H), 5.37 - 5.28 (m, 1 H), 5.24 - 5.17 (m, 1 H), 4.51 (d, J = 2.1 Hz, 2 H), 4.14 - 4.01 (m, 6 H), 2.31 (dt, J = 1.9, 7.4 Hz, 2 H), 1.88 - 1.77 (m, 2 H), 1.74 - 1.63 (m, 2 H), 1.54 - 1.44 (m, 2 H), 1.22 (dt, J = 2.3, 7.2 Hz, 3 H). ¹³C NMR δ 173.3, 160.9, 141.2, 134.3, 128.3, 124.6, 123.8, 117.2, 110.2, 71.8, 68.5, 65.9, 60.1, 34.0, 28.5, 25.4, 24.5, 14.1.

[0232] Methyl 7-(2-((allyloxy)methyl)-4-nitrophenoxy)heptanoate (18). This compound was synthesised using the procedure for compound 15 using 409 mg (1.96 mmol) of compound 14, 0.42 mL (2.37 mmol, 1.2eq.) of methyl 7-bromoheptanoate (18a) and 579 mg (4.19 mmol, 2.1eq.) of potassium carbonate in 15 mL of acetonitrile. The crude product was purified using flash column chromatography using 9:1 to 4: 1 Hex/EtOAc solution to obtain 576 mg (84%) of compound 18 as a light yellow oil. TLC (2:1 Hex/EtOAc) R_f = 0.42.

LRMS (ESI) m/z 374.1 (M+Na⁺). 1H NMR δ 8.33 - 8.28 (m, 1 H), 8.16 - 8.10 (m, 1 H), 6.86 (d, J = 9.0 Hz, 1 H), 6.03 - 5.91 (m, 1 H), 5.34 (qd, J = 1.6, 17.3 Hz, 1 H), 5.26 - 5.20 (m, 1 H), 4.53 (s, 2 H), 4.15 - 4.09 (m, 2 H), 4.06 (t, J = 6.3 Hz, 2 H), 3.68 - 3.63 (m, 3 H), 2.35 - 2.28 (m, 2 H), 1.87 - 1.78 (m, 2 H), 1.70 - 1.59 (m, 2 H), 1.53 - 1.44 (m, 2 H), 1.44 - 1.35 (m, 3 H). ¹³C MR 5 174.0, 161.0, 141.2, 134.4, 128.3, 124.7, 123.9, 117.3, 110.2, 71.9, 68.7, 66.0, 51.4, 33.8, 28.7, 25.6, 24.7.

[0233] Methyl 8-(2-((allyloxy)methyl)-4-nitrophenoxy)octanoate (19). This compound was synthesised using the procedure for compound 15 using 188 mg (0.901 mmol) of 14, 254 mg (1.07 mmol, 1.2eq.) of methyl 8-bromooctanoate (19a) and 284 mg (2.06 mmol, 2.3eq.) of potassium carbonate in 5 mL of acetonitrile. The crude product was purified by flash column chromatography using 5: 1 to 4: 1 Hex/EtOAc solution to obtain 286 mg (87%) of compound

19 as a yellow oil. TLC (2: 1 Hex/EtOAc) R_f = 0.54; LRMS (ESI) m z 366.1 (M+H⁺). 1H NMR δ 8.30 - 8.24 (m, 1 H), 8.13 - 8.06 (m, 1 H), 6.84 (d, J = 9.2 Hz, 1 H), 6.01 - 5.87 (m, 1 H), 5.36 - 5.27 (m, 1 H), 5.23 - 5.15 (m, 1 H), 4.51 (s, 2 H), 4.09 (dd, J = 1.3, 5.6 Hz, 2 H), 4.07 - 4.00 (m, 2 H), 3.63 (s, 3 H), 2.28 (t, J = 7.5 Hz, 2 H), 1.85 - 1.76 (m, 2 H), 1.61 (quin, J = 7.2 Hz, 2 H), 1.50 - 1.41 (m, 2 H), 1.41 - 1.28 (m, 4 H). ¹³C NMR δ 174.0, 160.9, 141.1, 134.3, 128.3, 124.6, 123.7, 117.1, 110.2, 71.8, 68.7, 65.9, 51.3, 33.8, 28.8, 28.8, 28.7, 25.6, 24.7.

[0234] Methyl 4-(4-(4-(3-((allyloxy)methyl)phenyl)pyrimidin-2-ylamino)-2- ((allyloxy)methyl) phenoxy)butanoate (21). 196 mg (0.634 mmol) of 15 was dissolved in 10 mL of methanol and 5 mL of water (on bigger scale, a small volume of ethyl acetate was added to aid the dissolution of the compound). 108 mg (1.94 mmol, 3.1 eq.) of iron and 216 mg (4.04 mmol, 6.4 eq.) of ammonium chloride was added and the reaction mixture was heated at reflux for 4 h. The reaction mixture was cooled to rt and filtered through a pad of diatomaceous earth, washing with DCM. Saturated sodium bicarbonate was added to the filtrate and it was extracted (x3) with DCM. The combined organic layers were washed with brine solution and dried over anhydrous sodium sulfate. The combined organic layer was concentrated and dissolved in 10 mL of dioxane. 459 mg (1.76 mmol, 2.8 eq.) of pyrimidine

20 (see J. Med. Chem. 2011, 54, 4638) and 97.4 mg (0.512 mmol, 0.81 eq.) of PTSA was added to the solution. The reaction mixture was stirred at 95 °C for 16 h. The reaction was cooled to rt and quenched with saturated sodium bicarbonate solution and extracted (x3) with DCM. The combined organic layers were washed with brine solution and dried over anhydrous sodium sulfate. The crude product was purified using flash column

chromatography using 3: 1 to 7:3 to 3:2 Hex/EtOAc solution to obtain 186 mg (58%) of compound 21 as a brown amorphous solid and 107 mg of pyrimidine starting material. TLC (2:1 Hex/EtOAc) R_f = 0.23; LRMS (ESI) m/z 504.2 (M+H⁺). 1H NMR δ 8.41 (d, J = 5.3 Hz, 1 H), 8.05 - 7.98 (m, 2 H), 7.63 (s, 2 H), 7.61 - 7.59 (m, 1 H), 7.50 - 7.43 (m, 3 H), 7.12 (d, J = 5.3 Hz, 1 H), 6.85 (d, J = 8.8 Hz, 1 H), 6.04 - 5.91 (m, 2 H), 5.35 (q, J = 1.6 Hz, 1 H), 5.30 (q, J = 1.6 Hz, 1 H), 5.20 (dddd, J = 1.3, 3.0, 10.6, 14.0 Hz, 2 H), 4.60 (s, 2 H), 4.58 (s, 2 H), 4.50 - 4.46 (m, 1 H), 4.36 (s, 1 H), 4.08 (tdd, J = 1.4, 5.6, 12.4 Hz, 4 H), 4.02 (t, J = 6.0 Hz, 2 H), 3.88 - 3.83 (m, 1 H), 3.69 (s, 3 H), 2.54 (t, J = 7.3 Hz, 2 H), 2.17 - 2.08 (m, 2 H). ¹³C NMR δ 173.6, 164.9, 160.3, 158.1, 152.0, 139.0, 137.2, 134.9, 134.6, 132.6, 130.0, 128.9, 127.5, 126.4, 126.3, 121.2, 120.1, 117.2, 116.8, 111.7, 107.9, 71.8, 71.5, 71.3, 68.5, 67.2, 66.8, 66.2, 62.6, 51.6, 30.5, 24.7.

[0235] Methyl 5-(4-(4-(3-((allyloxy)methyl)phenyl)pyrimidin-2-ylamino)-2- ((allyloxy)methyl)phenoxy) pentanoate (22). This compound was synthesised using the procedure for 21 using 209 mg (0.646 mmol) of compound 16, 110 mg (1.97 mmol, 3.0eq.) of iron and 315 mg (5.89 mmol, 9.1 eq.) of ammonium chloride in 6 mL of methanol and 3 mL of water; for the second step, 264 mg (1.01 mmol, 1.5 eq.) of compound 20 and 110 mg (0.577 mmol, 0.89 eq.) of PTSA in 5 mL of dioxane was used. The crude product was purified by flash column chromatography using 3: 1 to 3:2 Hex/EtOAc solution to obtain 93.7mg (28%) of compound 22 as a yellow oil. TLC (aniline 16) (7:3 Hex/EtOAc) R_f = 0.11; TLC (22) (1: 1 Hex/EtOAc) R_f = 0.43; LRMS (ESI) m/z 518.2 (M+H⁺). ¹H MR δ 8.42 (d, J = 5.3 Hz, 1 H), 8.05 - 7.97 (m, 2 H), 7.66 - 7.60 (m, 2 H), 7.50 - 7.44 (m, 3 H), 7.10 (d, J = 5.1 Hz, 1 H), 6.87 - 6.80 (m, 1 H), 5.97 (tdd, J = 5.4, 10.6, 17.3 Hz, 2 H), 5.37 - 5.28 (m, 2 H), 5.25 - 5.15 (m, 2 H), 4.59 (d, J = 3.6 Hz, 4 H), 4.49 - 4.43 (m, 1 H), 4.36 (s, 1 H), 4.08 (tdd, J = 1.3, 5.6, 14.4 Hz, 4 H), 3.98 (br. s., 2 H), 3.84 (dd, J = 4.2, 5.2 Hz, 1 H), 3.67 (s, 3 H), 2.40 (t, J = 6.8 Hz, 2 H), 1.87 - 1.79 (m, 4 H). ¹³C NMR δ 173.8, 164.6, 160.5, 158.5, 152.0, 138.9, 137.3, 134.9, 134.6, 132.6, 129.8, 128.8, 127.4, 126.3, 126.2, 121.0, 120.0, 117.1, 116.7, 111.6, 107.8, 71.8, 71.4, 71.2, 68.4, 67.8, 66.8, 66.2, 62.5, 51.4, 33.6, 28.7, 21.6.

[0236] Methyl 5-(4-(4-(3-((allyloxy)methyl)phenyl)pyrimidin-2-ylamino)-2- ((allyloxy)methyl)phenoxy)hexanoate (23). This compound was synthesised using the procedure for compound 21 using 193 mg (0.549 mmol) of compound 17, 115 mg

(2.05 mmol, 3.7 eq.) of iron and 162 mg (3.02 mmol, 5.5 eq.) of ammonium chloride in 6 mL of methanol and 3 mL of water; for the second step, 244 mg (0.934 mmol, 1.7 eq.) of 20 and 81.2 mg (0.427 mmol, 0.78 eq.) of PTSA in 5 mL of dioxane was used. The crude product was purified by flash column chromatography using 7:3 to 3:2 Hex/EtOAc solution to obtain 122 mg (41%) of 23 as a yellow oil. TLC (aniline 17) (1: 1 Hex/EtOAc) R_f = 0.33; TLC (23) (1:1 Hex/EtOAc) R_f = 0.33; LRMS (ESI) m/z 545.4 (M+H⁺). 1H MR δ 8.43 (d, J = 5.1 Hz, 1 H), 8.06 - 7.97 (m, 2 H), 7.66 - 7.60 (m, 2 H), 7.51 - 7.42 (m, 3 H), 7.11 (d, J = 5.1 Hz, 1 H), 6.87 - 6.81 (m, 1 H), 6.04 - 5.92 (m, 2 H), 5.35 (d, J = 1.4 Hz, 1 H), 5.31 (d, J = 1.3 Hz, 1 H), 5.25 - 5.15 (m, 2 H), 4.60 (d, J = 4.1 Hz, 4 H), 4.18 - 4.04 (m, 6 H), 3.97 (t, J = 6.3 Hz, 2 H), 2.34 (t, J = 7.5 Hz, 2 H), 1.86 - 1.76 (m, 2 H), 1.76 - 1.66 (m, 2 H), 1.57 - 1.49 (m, 2 H), 1.30 - 1.21 (m, 2 H). ¹³C NMR δ 173.5, 164.7, 160.6, 158.5, 152.2, 138.9, 137.3, 135.0, 134.6, 132.6, 129.8, 128.8, 127.4, 126.3, 126.2, 121.0, 120.0, 117.1, 116.7, 111.7, 107.8, 71.8, 71.4, 71.2, 68.1, 66.8, 60.1, 34.2, 29.0, 25.7, 24.7, 14.2.

[0237] Methyl 5-(4-(4-(3-((allyloxy)methyl)phenyl)pyrimidin-2-ylamino)-2- ((allyloxy)methyl)phenoxy)heptanoate (24). This compound was synthesised using the procedure for compound 21 using 440 mg (1.25 mmol) of compound 18, 212 mg (3.79 mmol, 3.0 eq.) of iron and 407 mg (7.61 mmol, 6.1 eq.) of ammonium chloride in 6 mL of methanol and 6 mL of water; for the second step, 496 mg (1.90 mmol, 1.5 eq.) of 20 and 195 mg

(1.02 mmol, 0.82 eq.) of PTSA in 6 mL of dioxane was used. The crude product was purified by flash column chromatography using 3: 1 to 7:3 to 3:2 to 1: 1 Hex/EtOAc solution to obtain 390 mg (57%) of 24 as a dark yellow oil and 97.9 mg (24%) of the aniline 18 as a yellow oil. TLC (aniline 18) (1: 1 Hex/EtOAc) R_f = 0.27; TLC (23) (1: 1 Hex/EtOAc) R_f = 0.46; LRMS (ESI) m/z 546.3 (M+H⁺). ¹H MR δ 8.41 (d, J = 5.1 Hz, 1 H), 8.02 (s, 1 H), 7.99 (td, J = 2.0, 6.4 Hz, 1 H), 7.76 (br. s., 1 H), 7.66 - 7.57 (m, 2 H), 7.47 - 7.41 (m, 2 H), 7.08 (d, J = 5.3 Hz, 1 H), 6.82 (d, J = 8.5 Hz, 1 H), 6.02 - 5.89 (m, 2 H), 5.31 (tdd, J = 1.7, 3.7, 17.2 Hz, 2 H), 5.22 - 5.13 (m, 2 H), 4.61 - 4.55 (m, 4 H), 4.13 - 4.07 (m, 2 H), 4.07 - 4.02 (m, 2 H), 3.93 (t, J = 6.3 Hz, 2 H), 3.64 (s, 3 H), 2.35 - 2.27 (m, 2 H), 1.81 - 1.72 (m, 2 H), 1.65 (td, J = 7.5, 15.0 Hz, 2 H), 1.52 - 1.42 (m, 2 H), 1.42 - 1.33 (m, 2 H). ¹³C NMR δ 173.9, 170.8, 164.5, 160.5, 158.4, 152.0, 138.8, 137.2, 134.9, 134.5, 132.6, 129.7, 128.7, 127.3, 126.2, 126.1, 120.9, 119.9, 117.0, 116.5, 111.5, 107.6, 71.7, 71.3, 71.2, 71.1, 68.1, 66.7, 60.1, 51.2, 33.8, 29.0, 28.7, 25.6, 24.7, 20.8, 14.0.

[0238] Methyl 5-(4-(4-(3-((allyloxy)methyl)phenyl)pyrimidin-2-ylamino)-2- ((allyloxy)methyl)phenoxy)octanoate (25). This compound was synthesised using the procedure for compound 21 using 328 mg (0.896 mmol) of compound 19, 188 mg

(3.37 mmol, 3.8 eq.) of iron, and 407 mg (5.60 mmol, 6.3 eq.) of ammonium chloride in 5 mL of methanol and 5 mL of water; for the second step, 351 mg (1.35 mmol, 1.5 eq.) of 20 and 136 mg (0.715 mmol, 0.80 eq.) of PTSA in 5 mL of dioxane was used. The crude product was purified by flash column chromatography using 3:1 to 7:3 to 65:35 Hex/EtOAc solution to obtain 311 mg (62%) of 25 as a yellow solid. TLC (aniline 19) (1: 1 Hex/EtOAc) R_f =; TLC (25) (1: 1 Hex/EtOAc) R_f = 0.45; LRMS (ESI) m/z 560.3 (M+H⁺). 1H MR δ 8.43 (d, J = 5.3 Hz, 1 H), 8.04 (s, 1 H), 8.03 - 7.98 (m, 1 H), 7.72 (br. s., 1 H), 7.66 - 7.60 (m, 2 H), 7.49 - 7.41 (m, 2 H), 7.09 (d, J = 5.1 Hz, 1 H), 6.84 (d, J = 8.8 Hz, 1 H), 5.97 (dtdd, J = 2.6, 5.5, 10.6, 17.2 Hz, 2 H), 5.33 (td, J = 1.6, 17.3 Hz, 2 H), 5.20 (ddd, J = 1.6, 10.5, 13.6 Hz, 2 H), 4.59 (d, J = 2.0 Hz, 4 H), 4.11 (td, J = 1.3, 5.5 Hz, 2 H), 4.06 (td, J = 1.3, 5.6 Hz, 2 H), 3.95 (t, J = 6.4 Hz, 2 H), 3.66 (s, 3 H), 2.32 (t, J = 7.5 Hz, 2 H), 1.82 - 1.73 (m, 2 H), 1.69 - 1.61 (m, 2 H), 1.52 - 1.43 (m, 2 H), 1.43 - 1.31 (m, 4 H). ¹³C NMR δ 174.0, 164.5, 160.5, 158.4, 152.1, 138.8, 137.3, 134.9, 134.5, 132.5, 129.7, 128.7, 127.3, 126.3, 126.2, 120.9, 119.9, 117.0, 116.6, 111.6, 107.7, 71.8, 71.3, 71.1, 68.3, 66.7, 51.3, 33.9, 29.2, 28.9, 28.9, 25.8, 24.7.

[0239] 1 l-(4-(Butanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene methyl ester (26). Following the literature procedure (J. Med. Chem. 2011, 54, 4638), 29.4 mg (0.0631 mmol) of compound 21 and 6.7 mg (0.00789 mmol, 0.13 eq.) of the second- generation Grubbs catalyst was dissolved in 14 mL of degassed DCM (5 mM concentration) to obtain an orange solution. 1 mL of 4 M hydrochloric acid was added, changing it to a yellow solution. The reaction mixture was heated at reflux for 3 h. After cooling to rt, the reaction was quenched with saturated sodium bicarbonate solution and extracted with DCM (x3). The combined organic layer was washed with brine solution and dried with anhydrous sodium sulfate. The organic layer was concentrated and purified by column chromatography using 3:2 Hex/EtOAc to obtain 17.1 mg (62%) of 26 in an 87: 13 E/Z ratio as a yellow solid. LRMS (ESI) m/z 476.2 (M+H⁺).

(E isomer only) 1H NMR δ 8.64 (d, J = 2.4 Hz, 1 H), 8.43 - 8.37 (m, 1 H), 8.29 (s, 1 H), 7.80 (d, J = 7.8 Hz, 1 H), 7.71 - 7.67 (m, 1 H), 7.59 (d, J = 7.7 Hz, 1 H), 7.48 (t, J = 7.7 Hz, 1 H), 7.14 (d, J = 5.1 Hz, 1 H), 6.88 - 6.79 (m, 2 H), 5.88 - 5.82 (m, 2 H), 4.64 (s, 2 H), 4.62 (s, 2 H), 4.16 (d, J = 4.8 Hz, 2 H), 4.08 - 4.00 (m, 4 H), 3.70 (s, 3 H), 2.57 (t, J = 7.3 Hz, 2 H), 2.18 - 2.10 (m, 2 H). ¹³C NMR δ 173.7, 164.4, 160.1, 158.3, 152.0, 138.6, 137.1, 133.2,

132.0, 131.0, 129.8, 128.9, 127.7, 127.1, 126.3, 121.4, 119.3, 112.4, 107.6, 70.1, 69.3, 67.6, 67.5, 65.7, 51.6, 30.6, 24.7. [0240] 1 l-(5-(Pentanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene methyl ester (27). This compound was synthesised using the procedure for compound 26 using 156.5 mg (0.302 mmol) of compound 22 and 25.9 mg (0.0305 mmol, 0.10 eq.) of second-generation Grubbs catalyst in 30 mL of DCM and 3 mL of 4 M HC1. The crude product was purified by flash column chromatography using 1: 1 to 2:3 Hex/EtOAc solution to obtain 104 mg (70%) (5:1 E/Z isomers) of compound 27 as a yellowish green solid. TLC (1:1 Hex/EtOAc) R_f = 0.23. LRMS (ESI) m/z 490.2 (M+H⁺). (E isomer only) 1H NMR δ 8.65 (d, J = 2.4 Hz, 1 H), 8.39 (d, J = 5.3 Hz, 1 H), 8.29 (s, 1 H), 7.95 (s, 1 H), 7.78 (d, J = 7.8 Hz, 1 H), 7.58 (d, J = 7.7 Hz, 1 H), 7.49 - 7.45 (m, 1 H), 7.12 (d, J = 5.3 Hz, 1 H), 6.87 - 6.77 (m, 2 H), 5.87 - 5.82 (m, 2 H), 4.63 (s, 4 H), 4.16 (d, J = 4.4 Hz, 2 H), 4.05 (d, J = 4.1 Hz, 2 H), 3.98 (m., 2 H), 3.69 - 3.65 (s, 3 H), 2.42 (t, J = 7.0 Hz, 2 H), 1.88 - 1.81 (m, 4 H). ¹³C NMR δ 173.8, 164.1, 160.2, 158.5, 152.0, 138.5, 137.1, 133.2, 132.0, 130.8, 129.7, 128.8, 127.6, 126.9, 126.1, 121.5, 119.2, 112.2, 107.5, 70.0, 69.2, 68.1, 67.5, 65.6, 51.4, 33.6, 28.6, 21.6.

[0241] 1 l-(6-(Hexanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene methyl ester (28). This compound was synthesised using the procedure for compound 26 using 151 mg (0.277 mmol) of compound 23 and 26.4 mg (0.0311 mmol, 0.11 eq.) of second- generation Grubbs catalyst in 30 mL of DCM and 3 mL of 4 M HC1. The crude product was purified by flash column chromatography using 1: 1 to 2:3 Hex/EtOAc solution to obtain 113 mg (79%) (5: 1 E/Z isomers) of compound 28 as a green amorphous solid. TLC (1: 1 Hex/EtOAc) R_f = 0.20; LRMS (ESI) m/z 518.3 (M+H⁺). (E isomer only) 1H NMR δ 8.64 (d, J = 2.0 Hz, 1 H), 8.40 (d, J = 5.1 Hz, 1 H), 8.29 (s, 1 H), 7.80 (d, J = 7.7 Hz, 1 H), 7.67 (s, 1 H), 7.58 (d, J = 7.7 Hz, 1 H), 7.51 - 7.41 (m, 1 H), 7.13 (d, J = 5.1 Hz, 1 H), 6.88 - 6.78 (m, 2 H), 5.88 - 5.81 (m, 2 H), 4.68 - 4.60 (m, 4 H), 4.19 - 4.09 (m, 4 H), 4.06 (d, J = 4.0 Hz, 2 H), 3.98 (t, J = 6.3 Hz, 2 H), 2.35 (t, J = 7.5 Hz, 3 H), 1.88 - 1.78 (m, 2 H), 1.72 (quin, J = 7.6 Hz, 2 H), 1.59 - 1.47 (m, 2 H), 1.31 - 1.21 (m, 3 H). ¹³C NMR 5 173.6, 164.2, 160.2, 158.6, 152.2, 138.5, 137.1, 133.1, 132.1, 130.8, 129.7, 128.8, 127.7, 127.0, 126.2, 121.4, 119.3, 112.4, 107.6, 70.1, 69.2, 68.5, 67.5, 65.7, 60.2, 34.2, 29.0, 25.7, 24.7, 14.2.

[0242] 1 l-(7-(Heptanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene methyl ester (29). This compound was synthesised using the procedure for compound 26 using 390 mg (0.715 mmol) of compound 24 and 31.3 mg (0.0369 mmol, 0.05 eq.) of second- generation Grubbs catalyst in 40 mL of DCM and 4 mL of 4M HCl. The crude product was purified by flash column chromatography using 65:35 to 3:2 to 1: 1 Hex/EtOAc solution to obtain 266 mg (72%) (10:3 E/Z isomers) of compound 29 as a green amorphous solid. TLC (1:1 Hex/EtOAc) R_f = 0.24; LRMS (ESI) m/z 518.2 (M+H⁺). (E isomer only) 1H NMR δ

8.63 (d, J = 2.3 Hz, 1 H), 8.42 - 8.37 (m, 1 H), 8.29 (s, 1 H), 7.79 (d, J = 7.8 Hz, 1 H), 7.73 (s, 1 H), 7.58 (d, J = 7.7 Hz, 1 H), 7.47 (t, J = 7.7 Hz, 1 H), 7.13 (d, J = 5.1 Hz, 1 H), 6.89 - 6.78 (m, 2 H), 5.84 (q, J = 4.7 Hz, 2 H), 4.66 - 4.60 (m, 4 H), 4.16 (d, J = 4.8 Hz, 2 H), 4.05 (d, J = 4.1 Hz, 2 H), 3.97 (t, J = 6.4 Hz, 2 H), 3.66 (s, 3 H), 2.36 - 2.29 (m, 2 H), 1.85 - 1.76 (m, 2 H), 1.72 - 1.63 (m, 2 H), 1.54 - 1.45 (m, 2 H), 1.45 - 1.36 (m, 2 H). ¹³C NMR δ 174.1, 164.1,

160.3, 158.6, 152.2, 138.5, 137.1, 133.0, 132.1, 130.8, 129.7, 128.8, 127.6, 127.0, 126.2,

121.4, 119.3, 112.4, 107.5, 70.1, 69.2, 68.6, 67.5, 65.6, 60.3, 51.4, 33.9, 29.1, 28.8, 25.7, 24.8, 14.1.

[0243] 1 l-(8-(Octanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene methyl ester (30). This compound was synthesised using the procedure for compound 26 using 311 mg (0.555 mmol) of compound 25 and 48.3 mg (0.0569 mmol, 0.10 eq.) of second- generation Grubbs catalyst in 55 mL of DCM and 5.5 mL of 4M HCl. The crude product was purified using flash column chromatography using 1:1 to 45:55 Hex/EtOAc solution to obtain 246 mg (83%) (4: 1 E/Z isomers) of compound 30 as a brown amorphous solid. TLC (1: 1 Hex/EtOAc) R_f = 0.29; LRMS (ESI) m/z 532.3 (M+H⁺). (E isomer only) 1H NMR δ 8.66 - 8.62 (m, 1 H), 8.40 (d, J = 5.1 Hz, 1 H), 8.29 (s, 1 H), 7.83 - 7.71 (m, 2 H), 7.58 (d, J = 7.5 Hz, 1 H), 7.47 (t, J = 7.7 Hz, 1 H), 7.13 (d, J = 5.1 Hz, 1 H), 6.88 - 6.78 (m, 2 H), 5.91 - 5.81 (m, 2 H), 4.66 - 4.60 (m, 3 H), 4.16 (d, J = 4.5 Hz, 2 H), 4.05 (d, J = 4.0 Hz, 2 H), 3.96 (t, J = 6.4 Hz, 2 H), 3.66 (s, 3 H), 2.32 (t, J = 7.5 Hz, 2 H), 1.85 - 1.75 (m, 2 H), 1.69 - 1.60 (m, 2 H), 1.54 - 1.44 (m, 2 H), 1.44 - 1.31 (m, 4 H). ¹³C NMR 5 174.1, 164.1, 160.2, 158.6, 152.3,

138.5, 137.1, 133.0, 132.1, 130.8, 129.7, 128.8, 127.6, 127.0, 126.2, 121.4, 119.2, 112.3, 107.5, 70.0, 69.2, 68.7, 67.5, 65.6, 51.3, 34.0, 29.2, 29.0, 28.9, 25.8, 24.8.

[0244] 1 l-(4-(4-Aminobenzoic acid)ethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (31). 117 mg (0.179 mmol) of compound 4 was dissolved in 4 mL of THF and 0.811 g (5.69 mmol, 32 eq.) of 90% potassium trimethylsilanoate was added. The resultant orange suspension was stirred at rt for 2 h, after which 1.5 mL of concentrated hydrochloric acid was added to the reaction and a yellow precipitate was formed. The reaction was stirred for a further 24 h at rt. 5 mL of water was added to the mixture and stirred at rt for 1 h. The solid was filtered and washed successively (x3) each with water, ethyl acetate and acetone. The resulting solid was dried under vacuum overnight to obtain 69.9 mg (56%) of compound 31 as a yellow solid. LRMS (ESI) m/z 539.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO- d₆) δ 9.54 (s, 1 H), 8.51 (d, J = 5.0 Hz, 1 H), 8.47 (br. s., 1 H), 8.15 (s, 1 H), 8.01 (d, J = 4.0 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 2 H), 7.55 (br. s., 2 H), 7.39 (d, J = 5.0 Hz, 1 H), 7.10 (d, J = 6.3 Hz, 1 H), 7.00 - 6.95 (m, 1 H), 6.68 (d, J = 8.5 Hz, 2 H), 5.79 (d, J = 14.9 Hz, 1 H), 5.66 (d, J = 15.2 Hz, 1 H), 4.53 (s, 2 H), 4.43 (s, 3 H), 4.11 (br. s., 3 H), 3.99 (br. s., 4 H), 3.52 (br. s., 2 H). ¹³C NMR (DMSO-d₆) δ 167.4, 163.0, 160.0, 159.2, 152.6, 151.1, 138.5, 136.7, 133.9, 131.1, 130.8, 129.8, 129.1, 126.6, 126.5, 120.6, 119.6, 117.1, 113.1, 111.0, 107.4, 69.1, 68.9, 67.9, 67.0, 65.0, 41.8.

[0245] 1 l-(4-(3-Aminobenzoic acid)ethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (32). This compound was synthesised using the procedure for compound 31, using 58.2 mg (0.0892 mmol) of ester 5, 416 mg of potassium trimethylsilanoate (2.92 mmol, 33 eq.). 37.7 mg (72%) of compound 32 was isolated as a light yellow solid. LRMS (ESI) m/z 539.2 (M+H⁺). HPLC purity 95.6%. 1H NMR (DMSO-d₆) δ 9.55 (s, 1 H), 8.51 (d, J = 5.1 Hz, 1 H), 8.46 (d, J = 2.5 Hz, 1 H), 8.15 (s, 1 H), 8.02 (dd, J = 1.8, 5.9 Hz, 1 H), 7.58 - 7.53 (m, 2 H), 7.39 (d, J = 5.3 Hz, 1 H), 7.27 (s, 1 H), 7.21 (t, J = 7.7 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1 H), 7.10 (dd, J = 2.6, 8.8 Hz, 1 H), 6.98 (d, J = 8.8 Hz, 1 H), 6.93 - 6.88 (m, 1 H), 5.83 - 5.75 (m, 1 H), 5.71 - 5.61 (m, 1 H), 4.53 (s, 2 H), 4.44 (s, 2 H), 4.11 (t, J = 5.3 Hz, 2 H), 3.99 (d, J = 5.0 Hz, 4 H), 3.48 (t, J = 5.3 Hz, 2 H). ¹³C NMR (DMSO-d₆) δ 167.9, 163.0, 160.0, 159.1, 151.1, 148.8, 138.6, 136.7, 133.8, 131.4, 130.8, 129.8, 129.1, 129.0, 126.6, 126.5, 126.5, 120.6, 119.6, 116.9, 116.4, 113.1, 112.8, 107.4, 69.1, 68.9, 67.9, 67.0, 64.9, 54.9, 42.3.

[0246] 1 l-(2-(4-Hydroxybenzoate))-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (33). This compound was synthesised using the procedure for compound 31 using 133 mg (0.241 mmol) of compound 6 and 1.06 g of potassium trimethylsilanoate (7.43 mmol, 31 eq.) in 5 mL of THF and 1.5 mL of cone. HC1. 66.8 mg (47%) of compound 33 was isolated as a light yellow solid. LRMS (ESI) m/z 540.2 (M+H⁺). HPLC purity 95.7%. 1H NMR (DMSO-d₆) δ 9.67 (s, 1 H), 8.53 (d, J = 5.3 Hz, 1 H), 8.45 (d, J = 2.6 Hz, 1 H), 8.14 (s, 1 H), 8.03 (d, J = 5.8 Hz, 1 H), 7.91 (d, J = 8.8 Hz, 2 H), 7.59 - 7.54 (m, 2 H), 7.43 (d, J = 5.4 Hz, 1 H), 7.15 - 7.08 (m, 3 H), 7.07 - 7.02 (m, 1 H), 5.83 - 5.74 (m, 1 H), 5.69 - 5.60 (m, 1 H), 4.52 (s, 2 H), 4.44 - 4.39 (m, 4 H), 4.36 - 4.31 (m, 2 H), 3.98 (d, J = 4.9 Hz, 4 H). ¹³C NMR (DMSO-d₆) δ 166.9, 163.4, 162.1, 159.5, 158.5, 151.0, 138.6, 136.5, 133.8, 131.3, 131.0, 130.7, 129.8, 129.1, 126.9, 126.6, 126.6, 123.1, 120.7, 119.8, 114.4, 113.7, 107.4, 69.2, 68.9, 67.8, 67.0, 66.7, 64.8.

[0247] l l-(2-(Isonipecotate)-ethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (34). This compound was synthesised using the procedure for compound 31 using 182 mg (0.335 mmol) of 8 and 1.30 g of potassium trimethylsilanoate (9.12 mmol, 27 eq.). 157 mg (83%) of compound 31 was isolated as a light yellow solid. LRMS (ESI) m/z 531.2 (M+H⁺). 1H NMR (DMSO-d₆) δ 11.21 (br. s., 1 H), 9.89 (s, 1 H), 8.56 - 8.49 (m, 2 H), 8.14 (s, 1 H), 8.01 (d, J = 6.9 Hz, 1 H), 7.60 - 7.49 (m, 2 H), 7.43 (d, J = 5.5 Hz, 1 H), 7.16 (dd, J = 2.3, 8.7 Hz, 1 H), 7.00 (dd, J = 3.9, 8.7 Hz, 1 H), 5.88 - 5.75 (m, 1 H), 5.71 - 5.63 (m, 1 H), 4.52 (s, 2 H), 4.47 (s, 2 H), 4.40 (br. s., 3 H), 4.03 (d, J = 5.1 Hz, 4 H), 3.97 (d, J = 5.0 Hz, 2 H), 3.65 - 3.52 (m, 2 H), 3.47 - 3.38 (m, 2 H), 3.16 - 3.01 (m, 2 H), 2.07 - 1.92 (m, 4 H). ¹³C NMR (DMSO-d₆) δ 174.6, 164.2, 158.8, 157.6, 150.7, 138.7, 136.4, 133.8, 131.2, 130.9, 129.9, 129.2, 126.9, 126.8, 126.3, 120.9, 119.8, 112.9, 107.5, 69.0, 68.9, 67.2, 67.0, 65.6, 63.2, 55.0, 54.9, 51.6, 37.8, 25.2, 25.1. [0248] l l-(2-(7-(4-Aminophenylcarbamoyl)hexanoic acid))-ethoxy)-14,19-dioxa-5,7,26- triaza-tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23- decaene (35). This compound was synthesised using the procedure for 31 using 82.0 mg (0.105 mmol) of compound 11 and 461 mg (3.23 mmol, 31 eq.) of potassium

trimethylsilanoate in 5 mL of THF and 1.5 mL of concentrated HC1. 52.2 mg (76%) of compound 35 was obtained as a light yellow solid. LRMS (ESI) m/z 652.3 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.98 (br. s., 1H), 9.62 (s, 1H), 8.52 (d, J = 5.14 Hz, 1H), 8.46 (d, J = 2.76 Hz, 1H), 8.14 (s, 1H), 7.99 - 8.05 (m, 1H), 7.62 (d, J = 8.66 Hz, 2H), 7.52 - 7.59 (m, 2H), 7.41 (d, J = 5.40 Hz, 1H), 7.25 (d, J = 6.65 Hz, 2H), 7.10 (dd, J = 2.64, 8.78 Hz, 1H), 6.96 (d, J = 8.91 Hz, 1H), 5.81 (td, J = 5.47, 15.65 Hz, 1H), 5.67 (td, J = 6.04, 15.65 Hz, 1H), 4.54 (s, 2H), 4.43 (s, 2H), 4.18 (t, J = 4.89 Hz, 2H), 4.01 (dd, J = 5.58, 15.62 Hz, 4H), 2.28 (t, J = 7.40 Hz, 2H), 2.19 (t, J = 7.34 Hz, 2H), 1.46 - 1.61 (m, 4H), 1.26 - 1.34 (m, 2H). ¹³C NMR (DMSO-d₆) δ 174.4, 163.3, 159.7, 158.8, 150.6, 138.6, 136.6, 134.1, 131.0, 129.8, 129.2, 126.7, 126.6, 126.6, 120.7, 120.1, 119.7, 113.1, 107.5, 69.2, 68.8, 67.0, 64.8, 36.2, 33.5, 28.2, 24.8, 24.3.

[0249] 1 l-(2-(7-(4-Aminophenylcarbamoyl)heptanoic acid))-ethoxy)-14,19-dioxa-5,7,26- triaza-tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23- decaene (36). This compound was synthesised using the procedure for compound 31 using 41.6 mg (0.0533 mmol) of compound 12 and 231 mg (1.62 mmol, 30 eq.) of potassium trimethylsilanoate in 3 mL of THF and 1 mL of concentrated HC1. 26.7 mg (75%) of compound 36 was obtained as a light yellow solid. LRMS (ESI) m/z 666.3 (M+H⁺). HPLC purity 91%. 1H NMR (DMSO-d₆) δ 12.25 (br. s., 2 H), 9.53 (s, 1 H), 9.47 (s, 1 H), 8.54 - 8.47 (m, 2 H), 8.16 (s, 1 H), 8.04 - 7.98 (m, 1 H), 7.59 - 7.52 (m, 2 H), 7.41 - 7.35 (m, 1 H), 7.32 (d, J = 8.8 Hz, 1 H), 7.16 - 7.07 (m, 1 H), 6.96 (d, J = 8.9 Hz, 1 H), 6.60 (d, J = 8.7 Hz, 2 H), 5.91 - 5.78 (m, 1 H), 5.73 - 5.63 (m, 1 H), 4.54 (s, 2 H), 4.50 - 4.43 (m, 2 H), 4.12 - 3.94 (m, 6 H), 3.46 - 3.38 (m, 2 H), 2.76 (d, J = 15.4 Hz, 2 H), 2.66 (d, J = 15.4 Hz, 2 H), 2.20 (q, J = 7.6 Hz, 2 H), 1.62 - 1.43 (m, 4 H), 1.28 (br. s., 4 H). ¹³C NMR (DMSO-d₆) δ 174.5, 174.4, 171.2, 170.2, 162.9, 160.2, 159.4, 151.2, 138.6, 136.8, 133.9, 131.0, 130.8, 129.8, 129.1, 126.6, 126.5, 126.5, 120.8, 120.5, 119.6, 113.2, 112.2, 107.4, 72.4, 69.1, 68.9, 68.0, 67.0, 65.1, 42.7, 42.6, 36.2, 33.6, 28.4, 28.3, 25.1, 24.4.

[0250] 1 l-(4-(Butanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (37). This compound was synthesised using the procedure for compound 31 using 58.5 mg (0.123 mmol) of compound 26 and 527 mg (3.70 mmol, 30 eq.) of potassium trimethylsilanoate in 5 mL of THF and 1.5 mL of concentrated HC1. 50.1 mg (80%) of compound 37 was obtained as an orange solid. LRMS (ESI) m/z 462.3 (M+H⁺). HPLC purity >99% (ratio of 77.9%. acid to 22.1% ester) 1H NMR (DMSO-d₆) δ 10.01 (br. s., 1 H), 8.56 (d, J = 5.3 Hz, 1 H), 8.41 - 8.36 (m, 1 H), 8.18 - 8.10 (m, 1 H), 8.05 (d, J = 6.7 Hz, 1 H), 7.60

- 7.54 (m, 2 H), 7.53 - 7.44 (m, 1 H), 7.11 (dd, J = 2.3, 8.7 Hz, 1 H), 6.95 (d, J = 8.8 Hz, 1 H), 5.87 - 5.75 (m, 1 H), 5.67 (td, J = 5.8, 15.8 Hz, 1 H), 4.52 (s, 2 H), 4.51 - 4.44 (m, 2 H), 4.11

- 4.03 (m, 2 H), 4.02 - 3.93 (m, 4 H), 1.97 (quin, J = 6.4 Hz, 2 H). ¹³C NMR (DMSO-d₆) δ 173.1, 164.9, 158.0, 156.3, 151.6, 138.7, 136.1, 132.5, 131.5, 130.8, 129.8, 129.2, 127.0, 126.8, 126.5, 121.1, 120.1, 112.6, 107.3, 69.1, 68.8, 67.2, 67.1, 64.9, 51.3, 29.9, 24.3.

[0251] 1 l-(5-(Pentanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (38). This compound was synthesised using the procedure for compound 31 using 330 mg (0.674 mmol) of compound 27 and 2.90 g (20.3 mmol, 30 eq.) of potassium trimethylsilanoate in 10 mL of THF and 2 mL of concentrated HCl. 288 mg (83%) of 38 was obtained as a yellow soild. LRMS (ESI) m/z 476.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.60 (s, 1 H), 8.52 (d, J = 5.3 Hz, 1 H), 8.48 (d, J = 2.6 Hz, 1 H), 8.17 (s, 1 H),

8.03 (td, J = 2.2, 6.2 Hz, 1 H), 7.58 - 7.54 (m, 2 H), 7.41 (d, J = 5.4 Hz, 1 H), 7.11 (dd, J = 2.7, 8.7 Hz, 1 H), 6.98 - 6.93 (m, 1 H), 5.88 - 5.80 (m, 1 H), 5.74 - 5.65 (m, 1 H), 4.55 (s, 2 H), 4.51 - 4.47 (m, 2 H), 4.06 (d, J = 6.0 Hz, 3 H), 3.98 (dd, J = 5.7, 13.7 Hz, 7 H), 2.31 (t, J = 7.1 Hz, 2 H), 1.80 - 1.65 (m, 5 H). ¹³C NMR (DMSO-d₆) δ 174.4, 163.3, 159.7, 158.7, 151.5, 138.6, 136.6, 133.4, 131.0, 130.9, 129.8, 129.1, 126.6, 126.3, 120.7, 119.7, 112.7, 107.3, 69.1, 68.9, 68.0, 67.1, 65.1, 33.3, 28.2, 21.3.

[0252] 1 l-(6-(Hexanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (39). This compound was synthesised using the procedure for compound 31 using 113 mg (0.218 mmol) of 28 and 951 mg (6.67 mmol, 31 eq.) of potassium

trimethylsilanoate in 5 mL of THF and 1.5 mL of concentrated HCl. 51.4 mg (44%) of compound 39 was obtained as a yellow solid. LRMS (ESI) m/z 490.2 (M+H⁺). HPLC purity 91.7%. 1H NMR (DMSO-d₆) δ 9.81 (s, 1 H), 8.57 - 8.53 (m, 1 H), 8.43 (d, J = 2.6 Hz, 1 H), 8.16 (s, 1 H), 8.04 (td, J = 1.9, 6.8 Hz, 1 H), 7.61 - 7.54 (m, 2 H), 7.45 (d, J = 5.5 Hz, 1 H), 7.11 (dd, J = 2.8, 8.8 Hz, 1 H), 6.99 - 6.92 (m, 1 H), 5.88 - 5.79 (m, 1 H), 5.74 - 5.64 (m, 1 H), 4.54 (s, 2 H), 4.52 - 4.47 (m, 2 H), 4.06 (d, J = 5.8 Hz, 2 H), 4.02 - 3.93 (m, 4 H), 2.25 (t, J = 7.3 Hz, 2 H), 1.72 (quin, J = 6.9 Hz, 2 H), 1.58 (td, J = 7.3, 15.0 Hz, 2 H), 1.51 - 1.40 (m, 2 H). ¹³C NMR (DMSO-d₆) 5 174.4, 164.1, 158.9, 157.5, 151.7, 138.7, 136.4, 132.9, 131.2, 130.9, 129.8, 129.2, 126.8, 126.7, 126.4, 120.9, 119.9, 112.7, 107.3, 69.1, 68.8, 68.2, 67.1, 65.1, 33.7, 28.5, 25.2, 24.2.

[0253] 1 l-(7-(Heptanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (40). This compound was synthesised using the procedure for compound 31 using 266 mg (0.514 mmol) of 29 and 2.24 g (15.7 mmol, 30 eq.) of potassium

trimethylsilanoate in 10 mL of THF and 2 mL of concentrated HCl. 203 mg (72%) of compound 40 was obtained as a light yellow solid. LRMS (ESI) m/z 504.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.85 (br. s., 1 H), 8.55 (d, J = 5.4 Hz, 1 H), 8.41 (d, J =

2.4 Hz, 1 H), 8.23 - 8.12 (m, 2 H), 8.05 (d, J = 6.8 Hz, 1 H), 7.62 - 7.55 (m, 2 H), 7.55 - 7.50 (m, 1 H), 7.49 - 7.42 (m, 1 H), 7.16 - 7.05 (m, 1 H), 6.96 (d, J = 8.8 Hz, 1 H), 5.89 - 5.78 (m, 1 H), 5.74 - 5.63 (m, 1 H), 4.58 - 4.44 (m, 4 H), 4.12 - 4.02 (m, 2 H), 4.02 - 3.91 (m, 4 H), 2.22 (t, J = 7.3 Hz, 2 H), 1.78 - 1.65 (m, 2 H), 1.53 (quin, J = 7.3 Hz, 2 H), 1.44 (td, J = 7.2, 14.4 Hz, 2 H), 1.38 - 1.28 (m, 2 H). ¹³C NMR (DMSO-d₆) δ 174.4, 164.4, 158.5, 157.1, 151.8, 138.7, 136.3, 132.7, 131.3, 130.8, 130.0, 129.8, 129.2, 128.7, 126.9, 126.8, 126.4, 121.0, 120.1, 112.6, 107.3, 69.2, 68.8, 68.2, 67.1, 65.0, 33.6, 28.6, 28.2, 25.3, 24.4.

[0254] 1 l-(8-(Octanoate)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (41). This compound was synthesised using the procedure for compound 31 using 246 mg (0.463 mmol) of compound 30 and 2.00 g (14.0 mmol, 30 eq.) of potassium trimethylsilanoate in 10 mL of THF and 2 mL of concentrated HC1. 136 mg (51%) of compound 41 was obtained as a light yellow solid. LRMS (ESI) m/z 518.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d6) δ 9.93 (br. s., 1 H), 8.56 (d, J = 5.4 Hz, 1 H), 8.39 (d, J = 2.5 Hz, 1 H), 8.14 (s, 1 H), 8.06 (d, J = 6.9 Hz, 1 H), 7.62 - 7.53 (m, 2 H), 7.53 - 7.44 (m, 1 H), 7.11 (dd, J = 2.6, 8.8 Hz, 1 H), 6.96 (d, J = 8.8 Hz, 1 H), 5.88 - 5.77 (m, 1 H), 5.74 - 5.62 (m, 1 H), 4.53 (s, 2 H), 4.51 - 4.45 (m, 2 H), 4.06 (d, J = 5.9 Hz, 2 H), 4.01 - 3.91 (m, 4 H), 2.21 (t, J = 7.3 Hz, 2 H), 1.78 - 1.64 (m, 2 H), 1.57 - 1.48 (m, 2 H), 1.48 - 1.39 (m, 2 H), 1.39 - 1.26 (m, 4 H). ¹³C NMR (DMSO-d6) δ 174.4, 164.7, 158.2, 156.7, 151.9, 138.7, 136.2, 132.5, 131.5, 130.8, 129.8, 129.2, 127.0, 126.8, 126.5, 121.1, 120.2, 112.6, 107.3, 69.2, 68.8, 68.2, 67.1, 65.0, 33.7, 28.7, 28.5, 28.4, 25.4, 24.4.

[0255] 1 l-(2-(4-Aminophenylhydroxamate)ethoxy)- 14, 19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (42). 39.4 mg (0.0732 mmol) of 31, 12.0 mg (0.102 mmol, 1.4 eq.) of 0-(tetrahydro-2H- pyran-2-yl)hydroxylamine, 33.8 mg (0.0889 mmol, 1.2 eq.) of HATU and 21 (0.150 mmol, 2.0 eq.) of triethylamine were dissolved in 2 mL of DMF. The solution was stirred at rt for 24 h after which it was diluted with water to precipitate out a yellow solid. There were two methods to obtain the crude product. The solid obtained could be filtered under vacuum to obtain the product. However, if the precipitate cannot be filtered easily, extraction of the mixture with 10% methanol in ethyl acetate (x3) was performed. The combined organic layer was washed with brine and dried with anhydrous sodium sulfate. The crude product was purified by flash column chromatography using 2:3 Hex/EtOAC to EtOAc to 40: 1 to 20: 1 DCM/MeOH to obtain 11.8 mg of the THP protected hydroxamate as a pale yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.17. LRMS (ESI) m/z 638.4 (M+H⁺). This product was dissolved in 5 niL of dioxane and 1 niL of 4 M HC1 in dioxane was added. An orange precipitate formed immediately upon addition of the acid solution. The mixture was stirred for 18 h at rt. The reaction was quenched with saturated sodium bicarbonate solution to precipitate out a yellow solid. The mixture was concentrated to remove the volatile organic solvent. The precipitate was then filtered and dried under vacuum to obtain 6.7 mg (23%) of compound 42 as a yellow solid. LRMS (ESI) m/z 554.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.53 (br. s., 1H), 8.42 - 8.59 (m, 2H), 8.16 (s, 1H), 8.01 (d, J = 6.15 Hz, 1H), 7.49 - 7.61 (m, 4H), 7.39 (d, J = 4.52 Hz, 1H), 7.07 - 7.14 (m, 1H), 6.97 (d, J = 8.78 Hz, 1H), 6.60 - 6.73 (m, 2H), 6.29 (br. s., 1H), 5.79 (br. s., 1H), 5.69 (s, 1H), 4.50 - 4.61 (m, 2H), 4.40 - 4.50 (m, 2H), 4.10 (t, J = 5.08 Hz, 2H), 4.00 (d, J = 4.27 Hz, 4H), 3.49 (d, J = 5.27 Hz, 2H). ¹³C NMR (DMSO-d₆) δ 162.8, 160.2, 159.4, 151.2, 151.1, 138.6, 136.8, 134.0, 130.9, 130.9, 130.8, 129.8, 129.1, 128.2, 126.6, 126.5, 126.4, 120.5, 119.6, 119.3, 115.4, 114.0, 113.2, 111.0, 107.4, 69.1, 68.9, 67.9, 67.0, 65.0, 41.9.

[0256] 1 l-(2-(3-Aminophenylhydroxamate)ethoxy)- 14, 19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (43). This compound was synthesised following the procedure for compound 42. 163 mg (0.302 mmol) of compound 32, 36.3 mg (0.310 mmol, 1.0 eq.) of 0-(tetrahydro-2H-pyran-2- yl)hydroxylamine, 128 mg (0.336 mmol, 1.1 eq.) of HATU and 130 μΐ. (0.928 mmol, 3.1 eq.) of triethylamine was dissolved in 3 mL of DMF. The crude product was purified by flash column chromatography using 1 :4 Hex/EtOAC to 25: 1 to 25: 1.5 to 25:2 DCM/MeOH to obtain 17.6 mg of the THP protected hydroxamate as a yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.16. LRMS (ESI) m/z 638.4 (M+H⁺). This product was dissolved in 4 mL of dioxane and 0.5 mL of 4 M HC1 in dioxane was added to give 12.4 mg (7%) of compound 43 as a yellow solid. LRMS (ESI) m/z 554.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.52 (s, 1H), 8.46 - 8.54 (m, 2H), 8.16 (s, 1H), 8.01 (d, J = 3.89 Hz, 1H), 7.52 - 7.60 (m, 2H), 7.39 (d, J = 5.14 Hz, 1H), 7.08 - 7.17 (m, 2H), 7.04 (s, 1H), 6.98 (d, J = 8.78 Hz, 1H), 6.93 (d, J = 7.53 Hz, 1H), 6.77 (dd, J = 1.69, 7.97 Hz, 1H), 5.92 (t, J = 5.40 Hz, 1H), 5.73 - 5.88 (m, 1H), 5.61 - 5.73 (m, 1H), 4.54 (s, 2H), 4.47 (s, 2H), 4.12 (t, J = 5.27 Hz, 2H), 3.96 - 4.04 (m, 4H), 3.43 - 3.50 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.9, 160.2, 159.4, 151.1, 148.7, 138.6, 136.8, 133.9, 130.9, 130.8, 129.8, 129.1, 128.8, 126.6, 126.5, 126.4, 120.5, 119.6, 114.9, 114.2, 113.2, 110.3, 107.4, 69.1, 68.9, 67.8, 67.0, 66.3, 65.0, 42.0.

[0257] 1 l-(2-(4-Hydroxyphenyl hydroxamate))-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (44). This compound was synthesised following the procedure for compound 42. 33.9 mg (0.0628 mmol) of compound 33, 11.0 mg (0.0.0939 mmol, 1.5 eq.) of O- (tetrahydro-2H-pyran-2-yl)hydroxylamine, 47.7 mg (0.125 mmol, 2.0 eq.) of HATU and 26 μΐ_^ (0.186 mmol, 3.0 eq.) of triethylamine was dissolved in 2 mL of DMF. The crude product was purified by flash column chromatography using DCM to 30: 1 to 20: 1 DCM/MeOH to obtain 31.0 mg of the THP protected hydroxamate as a yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.19; LRMS (ESI) m/z 639.4 (M+H⁺). This product was dissolved in 5 mL of dioxane and 0.5 mL of 4M HC1 in dioxane was added to give 6.5 mg (18%) of compound 44 as a yellow solid. LRMS (ESI) m/z 555.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.55 (s, 1H), 8.47 - 8.56 (m, 2H), 8.17 (s, 1H), 8.02 (d, J = 3.39 Hz, 1H), 7.77 (t, J = 9.35 Hz, 2H), 7.53 - 7.61 (m, 2H), 7.40 (d, J = 5.15 Hz, 1H), 7.00 - 7.18 (m, 4H), 5.75 - 5.86 (m, 1H), 5.61 - 5.72 (m, 1H), 4.54 (s, 2H), 4.45 (d, J = 3.14 Hz, 2H), 4.39 (br. s., 2H), 4.34 (br. s., 2H), 3.99 (d, J = 5.14 Hz, 4H). ¹³C NMR (DMSO-d₆) δ 162.9, 161.1, 160.2, 159.4, 150.9, 138.6, 136.7, 134.2, 130.8, 130.8, 129.9, 129.1, 129.0, 128.6, 126.8, 126.5, 126.5, 120.5, 119.7, 114.3, 114.3, 113.8, 107.4, 101.0, 69.1, 68.9, 67.9, 67.0, 66.7, 66.3, 64.9, 61.4.

[0258] l l-(2-(N-Hydroxy-l-piperidine-4-carboxamide)-ethoxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene hydrochloride (45). This compound was synthesised following the procedure for compound 42. 70.9 mg (0.134 mmol) of 34, 24.6 mg (0.210 mmol, 1.6 eq.) of 0-(tetrahydro-2H-pyran- 2-yl)hydroxylamine, 78.1 mg (0.205 mmol, 1.5 eq.) of HATU and 100 (0.714 mmol, 5.3 eq.) of triethylamine was dissolved in 5 mL of DMF. The crude product was purified by flash column chromatography using EtOAC to 25: 1 to 22:3 DCM/MeOH to obtain 68.8 mg of the THP protected hydroxamate as a yellow solid. TLC (9: 1 DCM/MeOH) R_f = 0.18; LRMS (ESI) m/z 630.5 (M+H⁺). This product was dissolved in 5 mL of dioxane and 0.5 mL of 4M HC1 in dioxane was added to give 16.4 mg (23%) of compound 45 as a yellow solid. LRMS (ESI) m/z 546.3 (M+H⁺). HPLC purity 81% (19% carboxylic acid). 1H NMR (DMSO-d₆) δ 10.38 (br. s., 1H), 9.53 (s, 1H), 8.48 - 8.55 (m, 2H), 8.18 (s, 1H), 7.96 - 8.04 (m, 1H), 7.51 - 7.59 (m, 2H), 7.39 (d, J = 5.14 Hz, 1H), 7.08 - 7.15 (m, 1H), 6.97 (d, J = 8.78 Hz, 1H), 5.79 - 5.88 (m, 1H), 5.63 - 5.74 (m, 1H), 4.55 (s, 2H), 4.48 (s, 2H), 4.02 - 4.10 (m, 4H), 4.00 (d, J = 5.14 Hz, 2H), 2.98 (d, J = 11.54 Hz, 1H), 2.68 (t, J = 5.58 Hz, 2H), 1.91 - 2.20 (m, 3H), 1.56 - 1.69 (m, 3H). ¹³C NMR (DMSO-d₆) δ 162.9, 160.2, 159.3, 151.2, 138.6, 136.8, 133.8, 131.0, 130.7, 129.8, 129.1, 126.5, 126.5, 126.3, 120.6, 119.6, 113.1, 107.3, 69.1, 68.9, 67.1, 65.3, 56.9, 53.1, 28.5. [0259] l l-(2-(Nl-(4-Aminophenyl)-N7-hydroxyheptanediamide)ethoxy)-14,19-dioxa- 5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa- l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (46). This compound was synthesised following the procedure for compound 42. 25.9 mg (0.0397 mmol) of 35, 7.0 mg (0.0600 mmol, 1.5 eq.) of 0-(tetrahydro-2H-pyran-2-yl)hydroxylamine, 27.2 mg (0.0715 mmol,

1.8 eq.) of HATU and 20 μΐ_^ (0.143 mmol, 3.6 eq.) of triethylamine was dissolved in 1 mL of DMF. The crude product was purified by flash column chromatography using 1:4

Hex/EtOAC to 25: 1 to 25: 1.5 DCM/MeOH to obtain 10.5 mg of the THP protected hydroxamate as an off white solid. TLC (2:3 Hex/EtOAc) R_f = 0.05; LRMS (ESI) m/z 751.6 (M+H⁺). This product was dissolved in 5 mL of dioxane and 0.3 mL of 4 M HC1 in dioxane was added to give 3.2 mg (12%) of compound 46 as a pale brown solid. LRMS (ESI) m/z 666.4 (M+H⁺). HPLC purity 93.5%. 1H NMR (DMSO-d₆) δ 9.52 (br. s., 1H), 9.47 (br. s., 1H), 8.45 - 8.55 (m, 2H), 8.17 (br. s., 1H), 8.01 (br. s., 1H), 7.56 (br. s., 2H), 7.39 (d, J = 4.39 Hz, 1H), 7.25 - 7.35 (m, J = 8.28 Hz, 2H), 7.10 (d, J = 8.91 Hz, 1H), 6.97 (d, J = 8.66 Hz, 1H), 6.48 - 6.68 (m, J = 8.28 Hz, 2H), 5.83 (d, J = 15.81 Hz, 1H), 5.68 (d, J = 15.94 Hz, 1H), 4.55 (br. s., 2H), 4.47 (s, 2H), 4.10 (br. s., 2H), 3.90 - 4.07 (m, 5H), 2.21 (t, J = 6.90 Hz, 2H), 1.94 (t, J = 7.34 Hz, 2H), 1.40 - 1.69 (m, 4H), 1.24 (d, J = 6.90 Hz, 2H). ¹³C NMR (DMSO- d₆) δ 162.8, 162.4, 160.2, 159.3, 151.2, 138.6, 131.0, 129.8, 129.1, 126.5, 120.8, 120.5, 119.6, 115.7, 113.2, 112.1, 107.3, 68.9, 67.0, 65.1, 42.7, 28.7, 28.3. [0260] 1 l-(2-(Nl-(4-Aminophenyl)-N8-hydroxyoctanediamide)ethoxy)-14,19-dioxa- 5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8, 12)]heptacosa- l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (47). This compound was synthesised following the procedure for compound 42. 26.7 mg (0.0496 mmol) of compound 36, 11.6 mg (0.0990 mmol, 2.0 eq.) of 0-(tetrahydro-2H-pyran-2-yl)hydroxylamine, 31.1 mg (0.818 mmol, 1.6 eq.) of HATU and 30 μΐ_^ (0.214mmol, 4.3 eq.) of triethylamine was dissolved in 2 mL of DMF. The crude product was purified by flash column chromatography using 2:3 Hex/EtOAC to 50: 1 to 25: 1 DCM/MeOH to obtain 29.8 mg of the THP protected

hydroxamate as a yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.18. LRMS (ESI) m/z 765.4 (M+H⁺). This product was dissolved in 4 mL of dioxane, and 0.5 mL of 4 M HC1 in dioxane was added to give 21.0 mg (62%) of compound 47 as a yellow solid. LRMS (ESI) m/z 681.6 (M+H⁺). 1H NMR (DMSO-d₆) δ 9.44 - 9.54 (m, 2H), 8.46 - 8.55 (m, 2H), 8.16 (s, 1H), 8.01 (d, J = 5.27 Hz, 1H), 7.50 - 7.59 (m, 2H), 7.35 - 7.42 (m, 1H), 7.31 (d, J = 8.78 Hz, 2H), 7.10 (dd, J = 2.38, 8.78 Hz, 1H), 6.96 (d, J = 8.78 Hz, 1H), 6.54 - 6.64 (m, 2H), 5.75 - 5.89 (m, 1H), 5.61 - 5.75 (m, 1H), 4.54 (s, 2H), 4.47 (s, 2H), 4.06 - 4.12 (m, 2H), 4.01 (dd, J = 5.14, 14.68 Hz, 4H), 2.13 - 2.25 (m, 2H), 1.79 - 1.98 (m, 2H), 1.37 - 1.61 (m, 4H), 1.16 - 1.33 (m, 4H). ¹³C NMR (DMSO-d₆) δ 162.9, 160.2, 159.4, 151.2, 144.8, 138.6, 136.8, 133.9, 131.0, 130.8, 129.8, 129.1, 128.9, 126.6, 126.5, 126.5, 120.8, 120.5, 119.6, 113.2, 112.1, 107.4, 69.1, 68.9, 68.0, 67.1, 65.1, 48.6, 42.7, 36.2, 32.2, 29.0, 28.4, 25.2, 25.0.

[0261 ] 11 -(4-(N-Hydroxybutanamide)oxy)- 14,19-dioxa-5 ,7 ,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene

(48) . This compound was synthesised following the procedure for compound 42. 170 mg (0.368 mmol) of compound 37, 123 mg (1.05 mmol, 2.9 eq.) of 0-(tetrahydro-2H-pyran-2- yl)hydroxylamine, 228 mg (0.600 mmol, 1.6 eq.) of HATU and 0.24 mL (1.71 mmol, 4.7 eq.) of triethylamine was dissolved in 5 mL of DMF. The crude product was purified by flash column chromatography using 2:3 Hex/EtOAC to 25: 1 EOAc/MeOH to obtain 100 mg of the THP protected hydroxamate as a yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.11; LRMS (ESI) m/z 561.2 (M+H⁺). This product was dissolved in 5 mL of dioxane and 1.2 mL of 4 M HCl in dioxane was added to give 78.2 mg (45%) of compound 48 as a yellow solid. LRMS (ESI) m/z 477.2 (M+H⁺). HPLC purity >99%. Major E isomer only 1H NMR (400 MHz, DMSO- d₆) δ 10.42 (br. s., 1H), 9.52 (s, 1H), 8.45 - 8.58 (m, 2H), 8.13 - 8.23 (m, 1H), 8.01 (d, J = 5.90 Hz, 1H), 7.52 - 7.62 (m, 2H), 7.45 - 7.52 (m, 1H), 7.38 (d, J = 5.27 Hz, 1H), 7.06 - 7.15 (m, 1H), 6.89 - 6.98 (m, 1H), 5.76 - 5.90 (m, 1H), 5.62 - 5.76 (m, 1H), 4.55 (s, 2H), 4.47 - 4.53 (m, 2H), 4.07 (d, J = 5.77 Hz, 2H), 3.92 - 4.03 (m, 5H), 2.17 (t, J = 7.22 Hz, 1H), 1.94 (quin, J = 6.68 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.7, 163.0, 160.1, 159.2, 151.2, 138.6, 136.8, 133.7, 131.0, 130.8, 129.7, 129.1, 126.5, 126.5, 126.3, 120.5, 119.6, 112.8, 107.3, 69.1, 68.8, 67.7, 67.0, 65.1, 28.8, 24.9.

[0262] l l-(4-(N-Hydroxypentanamide)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene

(49) . This compound was synthesised following the procedure for compound 42. 103 mg (0.217 mmol) of acid 38, 38.0 mg (0.324 mmol, 1.5 eq.) of 0-(tetrahydro-2H-pyran-2- yl)hydroxylamine, 128 mg (0.336 mmol, 1.6 eq.) of HATU and 91 (0.649 mmol, 3.0 eq.) of triethylamine was dissolved in 3 mL of DMF. The crude product was purified by flash column chromatography using 2:3 Hex/EtOAC to 25:0.5 to 25: 1 DCM/MeOH to obtain 131 mg of the THP protected hydroxamate as a yellow oil. TLC (2:3 Hex/EtOAc) R_f = 0.18; LRMS (ESI) m/z 575.4 (M+H⁺). This product was dissolved in 8 mL of dioxane and 1 mL of 4 M HCl in dioxane was added to give 81.9 mg (77%) of 49 as a pale yellow solid. LRMS (ESI) m/z 491.3 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.52 (br. s., 1H), 8.44 - 8.60 (m, 2H), 8.12 - 8.21 (m, 1H), 7.99 (d, J = 5.77 Hz, 1H), 7.50 - 7.61 (m, 2H), 7.37 (d, J = 5.02 Hz, 1H), 7.04 - 7.20 (m, 2H), 6.88 - 6.99 (m, 1H), 5.75 - 5.89 (m, 1H), 5.61 - 5.74 (m, 1H), 4.54 (s, 2H), 4.48 (s, 2H), 4.05 (d, J = 5.52 Hz, 2H), 3.90 - 4.01 (m, 4H), 2.02 (t, J = 6.15 Hz, 2H), 1.60 - 1.78 (m, 4H). ¹³C NMR (DMSO-d₆) δ 173.3, 168.7, 162.9, 160.2, 159.3,

151.3, 138.6, 136.8, 133.6, 131.0, 130.7, 130.1, 129.8, 129.1, 128.9, 128.6, 128.2, 126.6,

126.4, 126.2, 126.2, 125.3, 120.6, 119.6, 112.8, 107.3, 69.1, 68.9, 68.0, 67.9, 67.1, 65.8, 65.2, 51.2, 32.9, 32.1, 28.4, 28.2, 21.9, 21.3, 21.0.

[0263] l l-(4-(N-Hydroxyhexanamide)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene

(50) . This compound was synthesised following the procedure for compound 42. 292 mg (0.597 mmol) of compound 39, 106 mg (0.905 mmol, 1.5 eq.) of 0-(tetrahydro-2H-pyran-2- yl)hydroxylamine, 342 mg (0.901 mmol, 1.5 eq.) of HATU and 250 μΐ. (1.78 mmol, 3.0 eq.) of triethylamine was dissolved in 3 mL of DMF. The crude product was purified by flash column chromatography using 1 :4 Hex/EtOAC to 50: 1 to 25: 1 DCM/MeOH to obtain 686 mg of the THP protected hydroxamate as a yellow oil with DMF. TLC (1:3 Hex/EtOAc) R_f = 0.38; LRMS (ESI) m/z 589.4 (M+H⁺). This product was dissolved in 10 mL of dioxane and 2 mL of 4 M HC1 in dioxane was added to give 111 mg (37%) of compound 50 as a yellow solid. LRMS (ESI) m/z 505.2 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.52 (br. s., 1H), 8.44 - 8.55 (m, 2H), 8.12 - 8.20 (m, 1H), 7.88 - 8.05 (m, 1H), 7.43 - 7.61 (m, 2H), 7.28 - 7.42 (m, 1H), 7.11 (dd, J = 2.57, 8.72 Hz, 1H), 6.85 - 6.98 (m, 1H), 5.74 - 5.89 (m, 1H), 5.68 (td, J = 5.93, 15.62 Hz, 1H), 4.53 (s, 2H), 4.48 (s, 2H), 4.01 - 4.09 (m, 2H), 3.79 - 4.01 (m, 4H), 1.84 - 2.02 (m, 2H), 1.62 - 1.78 (m, 2H), 1.48 - 1.62 (m, 2H), 1.32 - 1.48 (m, 2H). ¹³C NMR (DMSO-d₆) δ 168.7, 162.9, 162.3, 160.2, 159.3, 151.4, 138.6, 136.8, 133.7, 131.1, 130.8, 129.8, 129.1, 126.6, 126.5, 126.3, 120.6, 119.6, 112.8, 107.3, 69.1, 68.9, 68.3, 67.1, 65.2, 40.4, 38.2, 35.8, 32.4, 28.6, 25.3, 25.1.

[0264] l l-(4-(N-Hydroxyheptanamide)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene

(51) . This compound was synthesised following the procedure for compound 42. 46.6 mg (0.0925 mmol) of compound 40, 11.2 mg (0.0956 mmol, 1.0 eq.) of 0-(Tetrahydro-2H- pyran-2-yl)hydroxylamine, 49.4 mg (0.130 mmol, 1.4 eq.) of HATU and 20 (0.143 mmol, 1.5 eq.) of triethylamine was dissolved in 1.5 mL of DMF. The crude product was purified by flash column chromatography using 2:3 Hex/EtOAC to 50:1 EOAc/MeOH to obtain 30.5 mg of the THP protected hydroxamate as a yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.18.

LRMS (ESI) m/z 603.3 (M+H⁺). This product was dissolved in 2 mL of dioxane and 0.5 mL of 4 M HC1 in dioxane was added to give 13.4 mg (36%) of compound 51 as a yellow solid. LRMS (ESI) m/z 519.3 (M+H⁺). HPLC purity >99%. 1H NMR (DMSO-d₆) δ 9.51 (s, 1H), 8.46 - 8.54 (m, 2H), 8.13 - 8.23 (m, 1H), 8.01 (d, J = 6.15 Hz, 1H), 7.50 - 7.60 (m, 2H), 7.38 (d, J = 5.14 Hz, 1H), 7.10 (dd, J = 2.57, 8.72 Hz, 1H), 6.94 (d, J = 8.78 Hz, 1H), 5.78 - 5.89 (m, 1H), 5.61 - 5.75 (m, 1H), 4.55 (s, 2H), 4.43 - 4.53 (m, 3H), 4.06 (d, J = 5.77 Hz, 2H), 3.90 - 4.03 (m, 4H), 1.96 (t, J = 7.34 Hz, 2H), 1.64 - 1.79 (m, 2H), 1.52 (td, J = 7.26, 14.71 Hz, 2H), 1.43 (td, J = 7.39, 14.46 Hz, 2H), 1.26 - 1.37 (m, 2H). ¹³C NMR (DMSO-d₆) δ 169.1, 162.9, 160.2, 159.3, 151.4, 138.6, 136.8, 133.6, 131.0, 130.7, 129.8, 129.1, 126.5, 126.5, 126.2, 120.6, 119.6, 112.8, 107.3, 69.1, 68.8, 68.2, 67.0, 66.3, 65.2, 40.1, 39.9, 39.7, 39.3, 39.1, 38.9, 32.2, 28.7, 28.3, 25.3, 25.1. HRMS (ESI) m/z 541.2425, calc. 541.2421 (diff 0.7 ppm).

[0265] l l-(4-(N-Hydroxyoctanamide)oxy)-14,19-dioxa-5,7,26-triaza- tetracyclo[19.3.1.1(2,6). l(8,12)]heptacosa-l(25),2(26),3,5,8,10,12(27),16,21,23-decaene (52). This compound was synthesised following the procedure for compound 42. 58.9 mg (0.114 mmol) of compound 41, 16.6 mg (0.142 mmol, 1.2 eq.) of 0-(tetrahydro-2H-pyran-2- yl)hydroxylamine, 56.3 mg (0.148 mmol, 1.3 eq.) of HATU and 30 (0.214mmol, 1.9 eq.) of triethylamine was dissolved in 1 mL of DMF. The crude product was purified by flash column chromatography using 2:3 Hex/EtOAC to 50: 1 EOAc/MeOH to obtain 41.6 mg of the THP protected hydroxamate as a yellow solid. TLC (2:3 Hex/EtOAc) R_f = 0.18; LRMS (ESI) m/z 617.4 (M+H⁺). This product was dissolved in 2 mL of dioxane and 0.5 mL of 4M HC1 in dioxane was added to give 24.7 mg (41%) of compound 52 as a yellow solid. LRMS (ESI) m/z 533.3 (M+H⁺). HPLC purity >99%. 1H NMR (400 MHz, DMSO-d₆) δ 10.33 (br. s., 1H), 9.51 (s, 1H), 8.66 (br. s., 1H), 8.44 - 8.57 (m, 2H), 8.11 - 8.22 (m, 1H), 8.01 (d, J =

5.77 Hz, 1H), 7.48 - 7.59 (m, 2H), 7.38 (d, J = 5.02 Hz, 1H), 7.06 - 7.14 (m, 1H), 6.94 (d, J =

8.78 Hz, 1H), 5.85 (td, J = 5.43, 15.75 Hz, 1H), 5.69 (td, J = 5.93, 15.62 Hz, 1H), 4.55 (s, 2H), 4.41 - 4.53 (m, 2H), 4.06 (d, J = 5.77 Hz, 2H), 3.88 - 4.02 (m, 4H), 1.95 (t, J = 7.34 Hz, 2H), 1.64 - 1.77 (m, 2H), 1.38 - 1.58 (m, 4H), 1.22 - 1.38 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.0, 162.9, 160.2, 159.3, 151.4, 138.6, 136.8, 133.6, 131.0, 130.7, 129.8,

129.1, 126.5, 126.4, 126.2, 120.6, 119.6, 112.8, 107.3, 69.1, 68.8, 68.3, 67.0, 66.3, 65.2, 32.2, 28.8, 28.5, 28.4, 25.4, 25.0. [0264] Example 12: Inhibition of JAK and HDAC Activity Attenuated GvHD Effect

[0264] Investigations of T cell alloresponse indicated that the inhibition of JAK and HDAC activity reduced both mouse and human T cell alloresponse in vitro and attenuated GvHD effect in a murine model. [0264] For this series of experiments, seven- to ten-week-old female C57BL/6JNarl (H-2^b) and BALB/cJNarl (H-2^d) mice were purchased from National Laboratory Animal Center (NLAC), Taipei, Taiwan and used as donors and recipients, respectively. All mice were maintained in a specific pathogen free animal facility and provided with sterilized food and water by Taiwan Mouse Clinic, Taipei, Taiwan. All experimental procedures in this study were approved by the Institutional Animal Care and Use Ethics Committee (IACUC) of Academia Sinica. Inhibitors were dissolved in PBS containing final concentration of 21% Kolliphor^® EL (Sigma Aldrich, St. Louis, Missouri, United States), 7% ethanol (Merck, Kenilworth, New Jersey, United States) and 2% DMSO (Sigma Aldrich, St. Louis, Missouri, United States), and were injected intraperitoneally at a dose of 25 μg/kg or 100 μg/kg daily from day 0 to day 4. For the in vitro assay, inhibitors were dissolved in DMSO (100%).

[0269] All data were expressed as mean + standard deviation (SD) of three independent experiments. Statistical analyses were performed using Prism 5 (GraphPad Prism software, San Diego, CA). Student's two-tailed t-test was used to determine statistical significance between groups, where P values < 0.05 were considered to be significant. Mixed lymphocytes reaction (MLR)

[0270] A mouse mixed lymphocytes reaction was done by using splenocytes derived from spleen of BALB/cJNarl (H2^d) (Stimulator) and C57BL/6JNarl (H2^b) mice (Responder) (FIG. 12A). Red blood cells were lysed using ACK lysis buffer. Splenocytes were then washed and resuspended in complete RPMI 1640 culture medium. Stimulator splenocytes were treated with mitomycin C at a final concentration of 50 μ ηύ to block their proliferation and washed at least 3 times to completely remove mitomycin C. Responder splenocytes (2xl0⁵ cells) were co-cultured with stimulator splenocytes (4xl0⁵ cells) at a 2: 1 (S:R) ratio in 96- well round-bottom plates containing complete medium at 37°C in a humidified 5% C0₂ atmosphere. The cells were treated with inhibitors at the indicated concentrations on DO. On D3, cells were pulsed with 1 μθ of [³H]-TdR (Moravek Inc., USA) for 18 h before harvesting, and counted using Packard TopCount NXT Gamma Counter (Perkin Elmer, USA). [0271] For a human mixed lymphocytes reaction (FIG. 12B), human PBMC from healthy donors (IRB number: AS-IRBOl-11153) were isolated using Leucosep™ (Greiner Bio-One GmBH, Frickenhausen, Germany). Total PBMC from a donor was mixed with irradiated (20 Gy) PBMC from another donor at a ratio of 1: 1. Mixed culture was incubated in complete medium for 5 days in 96-well round-bottom plates at 37°C in a humidified 5% C0₂ atmosphere. Inhibitors at various concentrations were added on DO. Cells were pulsed with 1 μθ of [ H]-TdR (Moravek Inc., USA) on D5 for 18 h before harvesting, and they were counted using Packard TopCount NXT Gamma Counter (Perkin Elmer, USA).

T cell activation and viability assay [0272] Early and late T cell activation protocols were modified from Thompson CB et al. (1989). Briefly, T cells were isolated from spleen and lymph nodes of C57BL/6JNarl mouse and purified by using CD90.2 microbeads kit. For early activation, 2.5xl0⁵ T cells were activated with plate-bound anti-CD3 (145-2C11) (Biolegend, San Diego, California) and anti- CD28 (37.51) (Biolegend, San Diego, California) monoclonal antibodies (mAbs) at the concentration of 5 μg/ml for 18 h. In the late response assay, 5xl0⁴ T cells were activated with plate-bound anti-CD3/28 mAbs for 48 h to obtain complete activation. Inhibitors at various concentrations were then added for both assays and the cells were incubated in complete medium for 24 h in 96-well flat bottom plates at 37°C in a humidified 5% C0₂ atmosphere. Viability assay was carried out according to PrestoBlue™ cell viability reagent protocol (Molecular Probes, Life Technologies). Results from the early T cell activation are shown in FIG. 13A; results from the late T cell activation are shown in FIG. 13B.

CD3 bypass assay

[0273] A CD3 bypass assay was performed according to the protocol described by

Mansour Haeryfar SM et al. (2005). Briefly, isolated mouse T cells were stimulated with anti-CD3/28 mAbs, anti-CD3/28 mAbs in combination with PMA/ionomycin or

PMA/ionomycin alone (PMA and ionomycin concentrations were 15 ng/mL and 500 ng/mL, respectively) on DO. Inhibitors at various concentrations were added on the same day and the cells were further incubated in complete RPMI for 48 hr in 96-well flat-bottom plates at 37°C in a humidified 5% C0₂ atmosphere. Cell viability was then determined according to the protocol described previously, with the results shown in FIG. 13C. Cytokine quantification by ELISA

[0274] T cells were activated as previously described in the late response assay and the inhibitors were added at the concentrations indicated in FIGS. 14A and 14B. Cells were further incubated in complete medium and T-cell culture supernatants were harvested after 24 hr. The levels of interferon-gamma (IFNy) and tumor necrosis factor alpha (TNFa) in cell- free supernatants were immediately measured by sandwich ELISA using capture and detection antibodies, recombinant cytokines, and assay protocols all supplied by Biolegend, San Diego, California. Results for IFNy are shown in FIG. 14A; results for TNFa are shown in FIG. 14B. Bone marrow transplantation and GVHD induction

[0275] GVHD was induced by intravenous administration of 5xl0⁶ bone marrow cells (>95% purity) and 1X10⁶ or 2.5xl0⁶ T cells (>90% purity) prepared as described below. T cells were obtained from spleen and lymph nodes of C57BL/6JNarl mice. Red blood cells were lysed with ACK (Ammonium-Chloride-Potassium) lysis buffer and lymphocyte suspension was filtered through a 70 μπι nylon filter mesh and washed with PBS containing 2% FBS. T cells were further purified by using CD90.2 microbeads kit (Miltenyi, Bergisch Gladbach, Germany). Bone marrow cells were obtained from the femurs and tibias of C57BL/6JNarl mice and processed in a similar manner as T cells. Bone marrow cells were depleted of T cells through CD90.2 microbeads. Purity of both fractions was determined by using flow cytometry (BD, Franklin Lakes, New Jersey, United States).

[0276] Prior to bone marrow transplantation, BALB/cJNarl recipient mice (H-2^d) were lethally irradiated with 6.8 Gy (Severe dose) or 5.6 Gy (Mild dose) using an RS-2000 X-Ray irradiator (Suwanee, Georgia, United States). Recipients were then injected with 200 μΐ_^ of the above described cell suspension. The severity of GVHD was assessed with a clinical scoring system based on weight loss, posture, activity, fur, skin, and diarrhea. Mice were observed daily for general health throughout the course of the experiment. Experiments were terminated after day 30 and all surviving mice were euthanized to prevent animal suffering.

[0277] Results for the severe dose are shown in FIGS. 15A-15C, and results for the mild dose are shown in FIGS. 15D-15F. The effects on percentage body weight change (FIG. 15A and 15D), percentage of survival (FIG. 15B and 15E), and GvHD score (FIG. 15C and 15F) are shown for both experiments. The data shown are pooled from two independent experiments. [0278] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS

or a pharmaceutically acceptable salt thereof,

wherein

n is an integer from 1 to 4;

1 2

C¹ and Cr are each independently C6-ioaryl or C2-9heteroaryl;

Z is a bond, CH₂, O, S, N(R^a), C(0)N(R^a), or N(R^a)C(0);

R 1 and R 2" are each independently selected from the group consisting of H, halogen, Ci_₈alkyl, haloCi_₈alkyl, heteroCi_₇alkyl, hydroxyl, amino, Ci_₈acylamino, Ci_₈alkylamino, sulfonylamino, thio, -C(0)OR^c, -C(0)N(H)R^c, and -C(0)N(R^c)OR^c; or R¹ and R² together with the carbon atoms to which they are attached form a ring-fused Cs^cycloalkenyl, C 1 2

4_₇heterocycloalkenyl, or C₂-₅heteroaryl group; wherein the R R ring-fused group has from 0 to 2 ring substituents that are each independently selected from the group consisting of halogen,

Ci_₃alkyl, Ci_₃alkyloxy, fluoroCi_₃alkyl, and fluoroCi_₃alkyloxy;

R³ is selected from the group consisting of -OR^5a, -N(R^5a)R^5b, and -L²R^5c; 2

each L is selected from the group consisting of a bond, Ci_₆alkylene, and

-N(R^c)C(0)Ci_₆alkylene;

-C(0)N(R^c)OR^c, and -L²R⁶; or R^5a and R^5b together with the amino atom to which they are attached form a C₄_₈heterocyclyl ring with a substituent that is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c ,-C(0)N(R^c)OR^c, and -L²R⁶;

R^5c is selected from the group consisting of C_6-1oaryl, Ci_₉heteroaryl, C₃_₈cycloalkyl, C₃-₇heterocyclyl, -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c , wherein the C₆-i₀aryl, Ci-₉heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group includes an -L²R⁶ substituent; each R⁶ is independently selected from the group consisting of -C(0)NHR^c, -C(0)N(R^c)OR^c, -C(0)OR^c, Ci_₈alkyleneC(0)N(R^c)OR^c, and Ci_₈alkyleneC(0)OR^c;

each R^c is independently selected from the group consisting of H and Ci_₆alkyl; and each R^d is independently selected from the group consisting of Ci_₈alkyl, C₇_ narylalkyl, Ci_₉heteroaryl, C2-iiheteroarylalkyl, C₃_₆cycloalkyl, and C₄_iocycloalkylalkyl. 2. The compound of claim 1, wherein if one or more L groups were

2

present, exactly one L group is not a bond. 3. The compound of claim 1 or 2, wherein L¹ comprises at least one oxygen as a ring atom. 4. The compound of any one of claims 1 to 3, wherein Z is O. 5. The compound of any one of claims 1 to 4, wherein n is 2. 6. The compound of any one of claims 1 to 5, wherein R³ is -OR^5a; and wherein R ^a is selected from the group consisting of Cearyl, C₃_₅heteroaryl,

C₃_₈cycloalkyl, and C₃_₇heterocyclyl, wherein the Cearyl, C₃_₅heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group substituent is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c. 7. The compound of claim 6, wherein R^5a is selected from the group consisting of Cearyl and C₃-₅heteroaryl. 8. The compound of any one of claims 1 to 5, wherein R is selected from the group consisting of -N(R^5a)R^5b;

wherein R^5a is selected from the group consisting of Cearyl, C₃-₅heteroaryl,

C₃_₈cycloalkyl, and C₃_₇heterocyclyl, wherein the Cearyl, C₃-₅heteroaryl, C₃_₈cycloalkyl, or C₃_₇heterocyclyl group substituent is -L²R⁶; and

wherein R^5b is R^c. 9. The compound of any one of claims 1 to 5, wherein R is selected from the group consisting of -N(R^5a)R^5b;

wherein R^5a and R^5b together with the amino atom to which they are attached form a C₄_₈heterocyclyl ring with a substituent that is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c ,-C(0)N(R^c)OR^c, and -L²R⁶. 10. The compound of any one of claims 1 to 5, wherein R is -Ci_₆alkyleneR^5c; and wherein R^5c is selected from the group consisting of -C(0)OR^c, -C(0)NHR^c, and -C(0)N(R^c)OR^c. 11. The compound of claim 10, wherein R³ is -Ci_₅alkyleneR^5c; and wherein R^5c is -C(0)N(H)OH.

The compound of of any one of claims 1 to 11, having Formula la

(la).

13. The compound of any one of claims 1 to 12, wherein C is selected from the group consisting of

, and N

The compound of claim 13, wherein C is selected from the group consisting of

2

15. The compound of any one of claims 1 to 14, wherein C is selected from the group consisting of

The compound of any one of claims 1 to 15, having Formula lb

(lb).

The compound of any one of claims 1 to 15, having Formula Ic

(Ic). 18. The compound of any one of claims 1 to 15, wherein R 1 and R 2 are each independently selected from the group consisting of H, halogen, Ci_₃alkyl, haloCi_₃alkyl, heteroCi_₃alkyl, hydroxyl, amino, and thio; or R 1 and R 2 together with the carbon atoms to which they are attached form a ring-fused pyrrole or imidazole group. 19. The compound of claim 18, wherein R 1 and R 2 are each H. 20. The compound of claim 18, wherein R 1 is H and R 2 is CH₃. 21. The compound of any one of claims 1 to 20, wherein R is selected from the group consisting of

from the group consisting of

The compound of any one of claims 1 to 20, wherein R is selected from the group consisting of

25. The compound of any one of claims 1 to 20, wherein R is selected from the group consisting of

26. The compound of any one of claims 1 to 25, wherein R comprises a -C(0)N(H)OH group or a salt thereof.

27. The compound of any one of claims 1 to 25 , having a molecular weight of 700 or less when not in salt form. 28. The compound of any one of claims 1 to 25 , having a molecular weight of 600 or less when not in salt form.

The compound of claim 1, having the structure

31. A method of treating a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of a compound of any one of claims 1 to 30 to a subject in need thereof. 32. The method of claim 31, wherein the disease or disorder is selected from the group consisting of cancer and inflammation. 33. The method of claim 32, wherein the disease or disorder is graft vs. host disease.

34. The method of claim 32, wherein the disease or disorder is selected from the group consisting of a hematologic cancer, a hyperproliferative condition, a

gynecologic cancer, a gastrointestinal tract cancer, a urinary tract cancer, a skin cancer, a brain tumor, a head and neck cancer, a respiratory tract cancer, an ocular cancer, and a musculoskeletal cancer 35. The method of claim 34, wherein the hyperproliferative condition is psoriasis or restenosis. 36. The method of claim 32, wherein the disease is cancer. 37. The method of claim 36, wherein the cancer is selected from the group consisting of idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia, acute erythroblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasma cell disorder, hairy cell leukemia, Kaposi's sarcoma, lymphoma, breast carcinoma, ovarian cancer, cervical cancer, vaginal or vulva cancer, endometrial hyperplasia, colorectal carcinoma, polyps, liver cancer, gastric cancer, pancreatic cancer, gall bladder cancer, prostate cancer, kidney or renal cancer, urinary bladder cancer, urethral cancer, penile cancer, melanoma, glioblastoma, neuroblastoma, astrocytoma, ependynoma, brain- stem glioma, meduUoblastoma, menigioma, astrocytoma, oligodendroglioma, nasopharyngeal carcinoma, laryngeal carcinoma, small-cell lung carcinoma, non-small-cell lung carcinoma,

mesothelioma, retinoblastoma, osteosarcoma, musculoskeleletal neoplasm, squamous cell carcinoma, and a fibroid tumour. 38. The method of any one of claims 31 to 37, wherein administration of the compound dually inhibits JAK-STAT and HDAC pathways. 39. The method of claim 38, wherein administration of the compound dually inhibits JAK2 and HDAC pathways. 40. The method of claim 39, wherein administration of the compound dually inhibits JAK2 and HDAC 6.