+

WO2017053360A1 - Bicyclic and tricyclic cap bearing mercaptoacetamide derivatives as histone deacetylase inhibitors - Google Patents

Bicyclic and tricyclic cap bearing mercaptoacetamide derivatives as histone deacetylase inhibitors Download PDF

Info

Publication number
WO2017053360A1
WO2017053360A1 PCT/US2016/052780 US2016052780W WO2017053360A1 WO 2017053360 A1 WO2017053360 A1 WO 2017053360A1 US 2016052780 W US2016052780 W US 2016052780W WO 2017053360 A1 WO2017053360 A1 WO 2017053360A1
Authority
WO
WIPO (PCT)
Prior art keywords
disease
cancer
compound
present
cell
Prior art date
Application number
PCT/US2016/052780
Other languages
French (fr)
Inventor
Alan Kozikowski
Gian Paolo VALLERINI
Mariana SEGRETTI
Wei LV
Original Assignee
The Board Of Trustees Of The University Of Illinois
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The University Of Illinois filed Critical The Board Of Trustees Of The University Of Illinois
Publication of WO2017053360A1 publication Critical patent/WO2017053360A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/04—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms
    • C07D215/08—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms with acylated ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/18—Halogen atoms or nitro radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/40—Nitrogen atoms attached in position 8
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to thiol-based HDACIs, compositions comprising the HDACIs, and therapeutic uses of the HDACIs.
  • PTMs Covalent post-translational modifications of epigenomic proteins contribute to their biological roles, and thus serve as carriers of epigenetic information from one cell generation to the next.
  • Epigenetics means on top of or above genetics, and refers to external modifications to DNA and associated histones that turn genes "on” or “off.” These modifications do not change the DNA sequence, but instead, they affect how cells "read” genes.
  • PTMs play key roles in the regulation of protein function, transcription, DNA replication, and repair of DNA damage (1).
  • the major events surrounding epigenetic control are focused on three modes of action: writers, readers, and erasers.
  • the writers are responsible for adding a variety of PTM marks to histones which include, inter alia, acetylation which is catalyzed by histone acetyltransferases
  • HATs histone deacetylases
  • HDACs that catalyze the removal of these marks.
  • HDACs are responsible for catalyzing the hydrolysis of the acetyl mark to provide the unsubstituted lysine residue.
  • the HDAC family consists of at present 18 enzymes which are classified into four subgroups according to their homology to the yeast family.
  • HDAC1, 2, 3 and 8 - categorized as class I HDACs according to their homology with yeast Rpd3 - are characterized by ubiquitous expression and localization to the nucleus.
  • Class II HDACs show tissue-specific expression and shuttle between the nucleus and cytoplasm.
  • HDAC4 Homologous to yeast Hdal, these enzymes are subdivided in class Ila (HDAC4, 5, 7 and 9) and class lib (HDAC6 and 10).
  • HDAC 11 the only member of the class IV subfamily, shows similarities to the catalytic domains of both class I and II enzymes.
  • Class I, II, and IV HDACs require Zn 2+ as a cofactor of the deacetylating activity and are also referred to as the conventional HDACs.
  • the sirtuins 1-7 are dependent on nicotinamide adenine dinucleotide for their activity and form class III of the HDACs.
  • HDACi HDAC inhibitors
  • HDAC6 has emerged as a particularly attractive target, especially in view of the fact that HDAC6 knockout animals remain viable.
  • HDAC6 has no apparent role in the PTM of histone proteins, but rather is involved in regulating the acetylation status of oc-tubulin, HSP-90, cortactin, HSF-1, and other protein targets. This enzyme also plays a role in the recognition and clearance of polyubiquitinated misfolded proteins from the cell through aggresome formation.
  • HDAC6 selective compounds has recently been reviewed (2).
  • HDACis are comprised of three main motifs: a zinc binding group (ZBG), a cap group, and a linker that bridges the previous two ( Figure 1).
  • ZBG zinc binding group
  • Figure 1 A properly optimized cap group can improve both potency and selectivity, presumably through its ability to engage in appropriate contacts with residues on the enzyme surface.
  • Many HDACIs such as trichostatin A (TSA) and SAHA contain a hydroxamic acid function as zinc-binding group (ZBG).
  • TSAHA trichostatin A
  • ZBG hydroxamic acid function as zinc-binding group
  • hydroxamates are in some cases metabolically unstable (short half-life), and their potent metal-chelating ability might lead to off- target activity at other zinc-containing enzymes(3).
  • a potential drawback of thiol-containing HDACIs is their ability to undergo oxidative dimerization to give disulfides as products. Although the disulfide bond can be reduced inside of the cells to afford the parent thiol compound, the dimerization reaction could in certain instances represent a problem and in such cases it may be advantageous to employ a pro-drug from of the thiol, namely its thioester analog (6).
  • the present invention relates to HDAC inhibitors (HDACi), pharmaceutical compositions comprising the HDACi, and methods of treating diseases and conditions wherein inhibition of HDAC provides a benefit, such as a cancer, a neurological disease, a psychiatric illness, a neurodegenerative disorder, a peripheral neuropathy, stroke, hypertension, an inflammation, traumatic brain injury, rheumatoid arthritis, allograft rejection, and autoimmune diseases, comprising administering a therapeutically effective amount of an HDACi to an individual in need thereof.
  • the present invention also relates to a method of increasing the sensitivity of a cancer cell to radiotherapy and/or chemotherapy.
  • the present invention also allows for the use of these HDAC inhibitors in combination with other drugs and/or therapeutic approaches.
  • the present HDACi exhibit selectivity for particular HDAC isozymes, such as HDAC6, over other HDAC isozymes.
  • the present invention relates to HDACi having the structural formula as illustrated below.
  • the HDACi of the present invention contain a Cap group which is generally comprised of an aromatic ring, a linker group which is comprised of, inter alia, methylene groups or substituted methylene groups, and the zinc binding group, which is in this case the mercaptoacetamide group (-NHC(0)CH 2 SH)
  • this invention is a compound of formula I
  • Cap is a group of formula:
  • Y and Z are independently selected from C and N, provided that when Y is N, represents a single bond;
  • W is selected from O, S and (CH 2 ) m ;
  • R H or Ci-C 4 alkyl;
  • m is 0, 1 or 2;
  • n is 1, 2 or 3; and
  • A is one or more substituents independently from H, Br, CI, Me, CF 3 , N3 ⁇ 4, CN, OCH 3 and OH, and A can be appended to any one or each of the fused aromatic rings.
  • Fig. 1 shows structures of SAHA, TSA, and general structure of an HDAC inhibitor.
  • FIG. 2a shows Western blots of acetylated tubulin compared to a-tubulin in E17 cells.
  • E17 cells were treated with TSA, Tubastatin A, or the thiol analogues 1, 2a, 2b, and 3a for 24 h at the indicated concentrations, and their effects on acetylated tubulin were compared with those of the DMSO control.
  • Fig. 2b shows results from the same test, but in which the increase of acetylated histone H3, compared to histone H3, was evaluated.
  • Fig. 3 shows the effects of thiol compounds on murine Treg suppressive functions in vitro. Residual CFSE+ Teff cell proliferation is shown at each ratio of Treg:Teff cell; assays were performed in triplicate and repeated at least once; mean +SD shown; *p ⁇ 0.05 compared to corresponding DMSO-treated cultures.
  • the present invention is directed to novel HDACIs of formula I and their use in therapeutic treatments of, for example, cancers, inflammations, traumatic brain injuries, neurodegenerative disorders, neurological diseases, peripheral neuropathies, strokes, hypertension, autoimmune diseases, inflammatory diseases, and malaria.
  • the present HDACIs also increase the sensitivity of a cancer cell to the cytotoxic effects of radiotherapy and/or chemotherapy.
  • the present HDACIs selectively inhibit HDAC6 over other HDAC isozymes.
  • a disease or condition wherein inhibition of HDAC provides a benefit pertains to a condition in which HDAC and/or the action of HDAC is important or necessary, e.g., for the onset, progress, expression of that disease or condition, or a disease or a condition which is known to be treated by an HDAC inhibitor (such as, e.g., TSA, pivalolyloxymethylbutane (AN-9; Pivanex), FK-228 (Depsipeptide), PXD-101, NVP-LAQ824, SAHA, MS-275, and or MGCD0103).
  • an HDAC inhibitor such as, e.g., TSA, pivalolyloxymethylbutane (AN-9; Pivanex), FK-228 (Depsipeptide), PXD-101, NVP-LAQ824, SAHA, MS-275, and or MGCD0103.
  • Such conditions include, but are not limited to, cancer, psoriasis, fibroproliferative disorders (e.g., liver fibrosis), smooth muscle proliferative disorders (e.g., atherosclerosis, restenosis), neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, Huntington's chorea, amyotropic lateral sclerosis, spino-cerebellar degeneration, Rett syndrome), peripheral neuropathies (Charcot-Marie-Tooth disease, Giant Axonal Neuropathy (GAN)), inflammatory diseases (e.g., osteoarthritis, rheumatoid arthritis, colitis), diseases involving angiogenesis (e.g., cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy), hematopoietic disorders (e.g., anemia, sickle cell anemia, thalasseimia), fungal infections, parasitic infections (e.g., malaria, trypanosomias
  • the term "second therapeutic agent” refers to a therapeutic agent different from a present HDACI and that is known to treat the disease or condition of interest.
  • the second therapeutic agent can be a known chemotherapeutic drug, like taxol, or radiation, for example.
  • HDAC refers to a family of enzymes that remove acetyl groups from a protein, for example, the ⁇ -amino groups of lysine residues at the N-terminus of a histone.
  • the HDAC can be a human HDAC, including, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC 8, HDAC9, HDAC10, and HDAC11.
  • the HDAC also can be derived from a protozoal or fungal source.
  • HDACIs HDAC inhibitors
  • M zinc binding group
  • L linker region
  • Cap capping group
  • treat refers to eliminating, reducing, relieving, reversing, and/or ameliorating a disease or condition and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated, including the treatment of acute or chronic signs, symptoms and/or malfunctions.
  • the terms “treat,” “treating,” “treatment,” and the like may include “prophylactic treatment,” which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously-controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition, "treatment” therefore also includes relapse prophylaxis or phase prophylaxis.
  • the term “treat” and synonyms contemplate administering a therapeutically effective amount of a compound of the invention to an individual in need of such treatment.
  • a treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.
  • terapéuticaally effective amount refers to an amount of the active ingredient(s) that, when administered, is (are) sufficient, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to an individual in need thereof.
  • the therapeutically effective amount of the agent may reduce (i.e., retard to some extent and preferably stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; reduce HDAC signaling in the target cells; and/or relieve, to some extent, one or more of the symptoms associated with the cancer.
  • the administered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
  • Concurrent administration means that two or more agents are administered concurrently to the subject being treated.
  • concurrently it is meant that each agent is administered either simultaneously or sequentially in any order at different points in time. However, if not administered simultaneously, it is meant that they are administered to an individual in a sequence and sufficiently close in time so as to provide the desired therapeutic effect and can act in concert.
  • a present HDACI can be administered at the same time or sequentially in any order at different points in time as a second therapeutic agent.
  • a present HDACI and the second therapeutic agent can be administered separately, in any appropriate form and by any suitable route.
  • a present HDACI and the second therapeutic agent can be administered in any order to a subject in need thereof.
  • a present HDACI can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent treatment modality (e.g., radiotherapy), to an individual in need thereof.
  • a second therapeutic agent treatment modality e.g., radiotherapy
  • a present HDACI and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
  • the components of the combination therapies are administered at 1 minute to 24 hours apart.
  • the present invention is directed to HDACIs, compositions comprising the present HDACI, and therapeutic uses of the HDACIs of formula I:
  • Cap is a group of formula:
  • Y and Z are independently selected from C and N, provided that when Y is N, represents a single bond;
  • W is selected from O, S and (CH2) m ;
  • R H or Ci-C 4 alkyl;
  • A is one or more (e.g., 1, 2, 3, or 4) substituents independently from H, Br, CI, Me, CF 3 , NH 2 , CN, OCH 3 and OH, and A can be appended to any one or each of the fused aromatic rings.
  • Z is C.
  • Z is N.
  • R is H
  • A is CI
  • W is (CH 2 ) m .
  • Cap is selected from:
  • A can be appended to any one or each of the fused aromatic rings.
  • Representative compounds of the invention include, but are not limited to:
  • HDAC H-oxidative deficiency oxidative deficiency oxidative deficiency
  • the present HDACIs are used in methods of treating a disease or condition wherein inhibition of HDAC provides a benefit, for example, cancers, neurological diseases, neurodegenerative conditions, peripheral neuropathies, autoimmune diseases, inflammatory diseases and conditions, stroke, hypertension, traumatic brain injury, autism, and malaria.
  • the methods comprise administering a therapeutically effective amount of a present HDACI to an individual in need thereof.
  • the present methods also encompass administering a second therapeutic agent to the individual in addition to a present HDACI.
  • the second therapeutic agent is selected from agents, such as drugs and adjuvants, known as useful in treating the disease or condition afflicting the individual, e.g., a chemotherapeutic agent and/or radiation known as useful in treating a particular cancer.
  • Prodrugs of the present compounds also are included in the present invention. It is well established that a prodrug approach, wherein a compound is derivatized into a form suitable for formulation and/or administration, then released as a drug in vivo, has been successfully employed to transiently (e.g., bioreversibly) alter the physicochemical properties of the compound (see, H. Bundgaard, Ed., “Design of Prodrugs,” Elsevier, Amsterdam, (1985); R.B. Silverman, "The Organic Chemistry of Drug Design and Drug Action,” Academic Press, San Diego, chapter 8, (1992); K.M. Hillgren et al., Med. Res. Rev., 15, 83 (1995)). Specific prodrugs of HDACIs are discussed in WO 2008/055068, incorporated in its entirety herein by reference.
  • the prodrug comprises an acylated derivative of the sulfur atom of formula
  • R is selected from straight or branched Ci-C 6 alkyl) aryl or heteroaryl.
  • aryl is phenyl and heteroaryl is pyridyl.
  • compositions of the invention can exist as salts.
  • Pharmaceutically acceptable salts of the present HDACIs often are preferred in the methods of the invention.
  • pharmaceutically acceptable salts refers to salts or zwitterionic forms of the present compounds. Salts of the present compounds can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation.
  • the pharmaceutically acceptable salts of the present compounds can be acid addition salts formed with pharmaceutically acceptable acids.
  • acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric.
  • Nonlimiting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pival
  • available amino groups present in the compounds of the invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
  • any reference to compounds of the present invention appearing herein is intended to include the present compounds as well as pharmaceutically acceptable salts, hydrates, or prodrugs thereof.
  • the present compounds also can be conjugated or linked to auxiliary moieties that promote a beneficial property of the compound in a method of therapeutic use.
  • Such conjugates can enhance delivery of the compounds to a particular anatomical site or region of interest (e.g., a tumor), enable sustained therapeutic concentrations of the compounds in target cells, alter pharmacokinetic and pharmacodynamic properties of the compounds, and/or improve the therapeutic index or safety profile of the compounds.
  • Suitable auxiliary moieties include, for example, amino acids, oligopeptides, or polypeptides, e.g., antibodies, such as monoclonal antibodies and other engineered antibodies; and natural or synthetic ligands to receptors in target cells or tissues.
  • Other suitable auxiliaries include fatty acid or lipid moieties that promote biodistribution and/or uptake of the compound by target cells (see, e.g., Bradley et al., Clin. Cancer Res. (2001) 7:3229).
  • the present compounds have been evaluated for their activity at HDAC6 and their selectivity for HDAC6 compared to HDAC1. It previously was shown that selective HDAC6 inhibitors are implicated in a variety of disease states including, but not limited to, arthritis, autoimmune disorders, inflammatory disorders, cancer, neurological diseases such as Rett syndrome, peripheral neuropathies such as CMT, stroke, hypertension, and diseases in which oxidative stress is a causative factor or a result thereof. It also was shown that selective HDAC6 inhibitors, when administered in combination with rapamycin, prolonged the lifespan of mice with kidney xenografts. This model was used to evaluate the immunosuppressant properties of the present compounds and serve as a model of transplant rejection.
  • selective HDAC6 inhibitors confer neuroprotection in rat primary cortical neuron models of oxidative stress. These studies identified selective HDAC6 inhibitors as nontoxic neuroprotective agents.
  • the present compounds behave in a similar manner because they also are selective HDAC6 agents.
  • the present compounds demonstrate a ligand efficiency that renders them more drug-like in their physiochemical properties.
  • the present compounds maintain the potency and selectivity observed in prior HDACIs. The present compounds therefore are pharmaceutical candidates and research tools to identify the specific functions of HDAC6.
  • the present invention relates to a method of treating an individual suffering from a disease or condition wherein inhibition of HDAC provides a benefit comprising administering a therapeutically effective amount of a claimed HDACi compound to an individual in need thereof.
  • the methods of the present invention can be accomplished by administering one of the HDACi of the present invention as the neat compound or as a pharmaceutical composition.
  • Administration of a pharmaceutical composition, or a neat HDACI of the present invention can be performed during or after the onset of the disease or condition of interest.
  • the pharmaceutical compositions are sterile, and contain no toxic, carcinogenic, or mutagenic compounds that would cause an adverse reaction when administered.
  • a present HDACi may be administered in conjunction with a second therapeutic agent useful in the treatment of a disease or condition wherein inhibition of
  • the second therapeutic agent is different from the present HDACi.
  • a present HDACi and the second therapeutic agent can be administered simultaneously or sequentially.
  • a present HDACI and second therapeutic agent can be administered from a single composition or two separate compositions.
  • a present HDACi and the second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect.
  • the second therapeutic agent is administered in an amount to provide its desired therapeutic effect.
  • the effective dosage range for each second therapeutic agent is known in the art, and the second therapeutic agent is administered to an individual in need thereof within such established ranges.
  • the present invention therefore is directed to compositions and methods of using such compounds in treating diseases or conditions wherein inhibition of HDAC provides a benefit.
  • the present invention also is directed to pharmaceutical compositions comprising a present HDACi and an optional second therapeutic agent useful in the treatment of diseases and conditions wherein inhibition of HDAC provides a benefit.
  • kits comprising a present HDACi and, optionally, a second therapeutic agent useful in the treatment of diseases and conditions wherein inhibition of HDAC provides a benefit, packaged separately or together, and an insert having instructions for using these active agents.
  • a present HDACI and the second therapeutic agent can be administered together as a single-unit dose or separately as multi-unit doses, wherein the present HDACI is administered before the second therapeutic agent or vice versa.
  • One or more dose of a present HDACI and/or one or more dose of the second therapeutic agent can be administered.
  • the present HDACIs therefore can be used in conjunction with one or more second therapeutic agents, for example, but not limited to, anticancer agents.
  • a present HDACi is a potent inhibitor of HDAC and can be used in treating diseases and conditions wherein inhibition of HDAC provides a benefit, for example, cancer, a neurological disease, a neurodegenerative condition, traumatic brain injury, stroke, an inflammation, an autoimmune disease, and autism.
  • the present invention provides methods for treating cancer, including but not limited to killing a cancer cell or neoplastic cell; inhibiting the growth of a cancer cell or neoplastic cell; inhibiting the replication of a cancer cell or neoplastic cell; or ameliorating a symptom thereof, said methods comprising administering to a subject in need thereof a therapeutically effective amount of a present HDACi.
  • the selective HDACi may be able to facilitate the killing of cancer cells through reactivation of the immune system by mechanisms relating to the PDI receptor.
  • the invention provides a method for treating cancer comprising administering to a subject in need thereof an amount of a present HDACI or a pharmaceutically acceptable salt thereof sufficient to treat the cancer.
  • a present HDACI can be used as the sole anticancer agent, or in combination with another anticancer treatment, e.g., radiation, chemotherapy, and surgery.
  • the invention provides a method for increasing the sensitivity of a cancer cell to the cytotoxic effects of radiotherapy and/or chemotherapy comprising contacting the cell with a present HDACI or a pharmaceutically acceptable salt thereof in an amount sufficient to increase the sensitivity of the cell to the cytotoxic effects of radiotherapy and/or chemotherapy.
  • the present invention provides a method for treating cancer comprising: (a) administering to an individual in need thereof an amount of a present HDACI compound; and (b) administering to the individual an amount of radiotherapy, chemotherapy, or both.
  • the amounts administered are each effective to treat cancer.
  • the amounts are together effective to treat cancer.
  • the invention provides a method for treating cancer, said method comprising administering to a subject in need thereof a pharmaceutical composition comprising an amount of a present HDACI effective to treat cancer.
  • This combination therapy of the invention can be used accordingly in a variety of settings for the treatment of various cancers.
  • the individual in need of treatment has previously undergone treatment for cancer.
  • Such previous treatments include, but are not limited to, prior chemotherapy, radiotherapy, surgery, or immunotherapy, such as cancer vaccines.
  • the cancer being treated is a cancer which has demonstrated sensitivity to radiotherapy and/or chemotherapy or is known to be responsive to radiotherapy and/or chemotherapy.
  • Such cancers include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, larynx cancer, cervical cancer, nasopharynx cancer, breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small-cell lung cancer, non-small cell lung cancer, brain tumors, or other CNS neoplasms.
  • the cancer being treated has demonstrated resistance to radiotherapy and/or chemotherapy or is known to be refractory to radiotherapy and/or chemotherapy.
  • a cancer is refractory to a therapy when at least some significant portion of the cancer cells are not killed or their cell division is not arrested in response to therapy.
  • Such a determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context.
  • a cancer is refractory where the number of cancer cells has not been significantly reduced or has increased.
  • cancers and metastases selected from the group consisting of solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
  • the present HDACIs can also be administered to prevent progression to a neoplastic or malignant state, including but not limited to the cancers listed above.
  • Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79).
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells.
  • a typical metaplasia involves a somewhat disorderly metaplastic epithelium.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where chronic irritation or inflammation exists, and often is found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a subject can indicate the desirability of prophylactic/therapeutic administration of the composition of the invention.
  • characteristics of a transformed phenotype include, for example, morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein.
  • leukoplakia a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
  • fibrocystic disease cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia) is indicative of the desirability of prophylactic intervention.
  • human papilloma virus can lead to cervical cancer (see, e.g., Hernandez- Avila et al., Archives of Medical Research (1997) 28:265-271), Epstein-Barr virus (EBV) can lead to lymphoma (see, e.g., Herrmann et al., Pathol (2003) 199(2): 140-5), hepatitis B or C virus can lead to liver carcinoma (see, e.g., El- Serag, Clin Gastroenterol (2002) 35(5 Suppl 2):S72-8), human T cell leukemia virus (HTLV)-I can lead to T-cell leukemia (see e.g., Mortreux et al., Leukemia (2003) 17(l):26-38), human herpesvirus-8 infection can lead to Kaposi's sarcoma (see, e.g., Kadow e
  • a subject exhibiting one or more of the following predisposing factors for malignancy can be treated by administration of the present HDACIs and methods of the invention: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), a first degree kinship with persons having a cancer or procancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome,
  • the present HDACIs and methods of the invention are administered to a human subject to prevent progression of breast, colon, ovarian, or cervical cancer.
  • the invention provides methods for treating cancer comprising (a) administering to an individual in need thereof an amount of a present HDACI; and (b) administering to the individual one or more additional anticancer treatment modality including, but not limited to, radiotherapy, chemotherapy, surgery or immunotherapy, such as a cancer vaccine.
  • the administering of step (a) is prior to the administering of step (b).
  • the administering of step (a) is subsequent to the administering of step (b).
  • the administering of step (a) is concurrent with the administering of step (b).
  • the additional anticancer treatment modality is radiotherapy and/or chemotherapy. In another embodiment, the additional anticancer treatment modality is surgery.
  • the additional anticancer treatment modality is immunotherapy, such as cancer vaccines.
  • a present HDACI or a pharmaceutically acceptable salt thereof is administered adjunctively with the additional anticancer treatment modality.
  • the additional anticancer treatment modality is radiotherapy.
  • any radiotherapy protocol can be used depending upon the type of cancer to be treated.
  • Embodiments of the present invention employ electromagnetic radiation of: gamma-radiation (10 " to 10 " m), X-ray radiation (10 “ to 10 “ m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1 mm), and microwave radiation (1 mm to 30 cm).
  • X-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage X-ray radiation can be used for skin cancers.
  • Gamma-ray emitting radioisotopes such as radioactive isotopes of radium, cobalt and other elements, can also be administered.
  • Illustrative radiotherapy protocols useful in the present invention include, but are not limited to, stereotactic methods where multiple sources of low dose radiation are simultaneously focused into a tissue volume from multiple angles; "internal radiotherapy,” such as brachytherapy, interstitial irradiation, and intracavitary irradiation, which involves the placement of radioactive implants directly in a tumor or other target tissue; intraoperative irradiation, in which a large dose of external radiation is directed at the target tissue which is exposed during surgery; and particle beam radiotherapy, which involves the use of fast-moving subatomic particles to treat localized cancers.
  • internal radiotherapy such as brachytherapy, interstitial irradiation, and intracavitary irradiation, which involves the placement of radioactive implants directly in a tumor or other target tissue
  • intraoperative irradiation in which a large dose of external radiation is directed at the target tissue which is exposed during surgery
  • particle beam radiotherapy which involves the use of fast-moving subatomic particles to treat localized
  • radiosensitizers activated by electromagnetic radiation, e.g., X-rays.
  • X-ray-activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5- bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cis-platin, and therapeutically effective analogs and derivatives of the same.
  • Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent.
  • photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, PHOTOFRIN ® , benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
  • Radiosensitizers can be administered in conjunction with a therapeutically effective amount of one or more compounds in addition to a present HDACI, such compounds including, but not limited to, compounds that promote the incorporation of radiosensitizers to the target cells, compounds that control the flow of therapeutics, nutrients, and/or oxygen to the target cells, chemotherapeutic agents that act on the tumor with or without additional radiation, or other therapeutically effective compounds for treating cancer or other disease.
  • radiosensitizers examples include, but are not limited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., FLUOSOLW ® -DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, antiangiogenesis compounds, hydralazine, and L-BSO.
  • 5-fluorouracil 5-FU
  • leucovorin oxygen
  • carbogen red cell transfusions
  • perfluorocarbons e.g., FLUOSOLW ® -DA
  • 2,3-DPG 2,3-DPG
  • BW12C calcium channel blockers
  • pentoxifylline e.g., antiangiogenesis compounds
  • hydralazine hydralazine
  • L-BSO L-BSO.
  • a present HDACI or a pharmaceutically acceptable salt thereof is administered prior to the administration of radiotherapy and/or chemotherapy.
  • a present HDACI or a pharmaceutically acceptable salt thereof is administered adjunctively with radiotherapy and/or chemotherapy.
  • a present HDACI and additional treatment modalities can act additively or synergistically (i.e., the combination of a present HDACI or a pharmaceutically acceptable salt thereof, and an additional anticancer treatment modality is more effective than their additive effects when each are administered alone).
  • a synergistic combination permits the use of lower dosages of a present HDACI and/or the additional treatment modality and/or less frequent administration of a present HDACI and/or additional treatment modality to a subject with cancer.
  • the ability to utilize lower dosages of a present HDACI and/or an additional treatment modality and/or to administer a compound of the invention and the additional treatment modality less frequently can reduce the toxicity associated with the administration without reducing the efficacy of a present HDACI and/or the additional treatment modality in the treatment of cancer.
  • a synergistic effect can result in the improved efficacy of the treatment of cancer and/or the reduction of adverse or unwanted side effects associated with the administration of a present HDACI and/or an additional anticancer treatment modality as monotherapy.
  • the present HDACIs may act synergistically with radiotherapy when administered in doses typically employed when such HDACIs are used alone for the treatment of cancer. In another embodiment, the present HDACIs may act synergistically with radiotherapy when administered in doses that are less than doses typically employed when such HDACIs are used as monotherapy for the treatment of cancer.
  • radiotherapy may act synergistically with a present HDACI when administered in doses typically employed when radiotherapy is used as monotherapy for the treatment of cancer.
  • radiotherapy may act synergistically with a compound of the invention when administered in doses that are less than doses typically employed when radiotherapy is used as monotherapy for the treatment of cancer.
  • the effectiveness of the HDACIs as HDAC inhibitors for sensitizing cancer cells to the effect of radiotherapy can be determined by the in vitro and/or in vivo determination of post- treatment survival using techniques known in the art.
  • in vitro determinations exponentially growing cells can be exposed to known doses of radiation, and the survival of the cells monitored. Irradiated cells are plated and cultured for about 14- about 21 days, and the colonies are stained. The surviving fraction is the number of colonies divided by the plating efficiency of unirradiated cells. Graphing the surviving fraction on a log scale versus the absorbed dose on a linear scale generates a survival curve. Survival curves generally show an exponential decrease in the fraction of surviving cells at higher radiation doses after an initial shoulder region in which the dose is sublethal.
  • a similar protocol can be used for chemical agents when used in the combination therapies of the invention.
  • Inherent radiosensitivity of tumor cells and environmental influences, such as hypoxia and host immunity, can be further assessed by in vivo studies.
  • the growth delay assay is commonly used. This assay measures the time interval required for a tumor exposed to radiation to regrow to a specified volume. The dose required to control about 50% of tumors is determined by the TCD 50 assay.
  • In vivo assay systems typically use transplantable solid tumor systems in experimental subjects. Radiation survival parameters for normal tissues as well as for tumors can be assayed using in vivo methods known in the art.
  • the present invention provides methods of treating cancers comprising the administration of an effective amount of a present HDACI in conjunction with recognized methods of surgery, radiotherapy, and chemotherapies, including, for example, chemical-based mimics of radiotherapy whereby a synergistic enhancement of the effectiveness of the recognized therapy is achieved.
  • the effectiveness of a treatment can be measured in clinical studies or in model systems, such as a tumor model in mice, or cell culture sensitivity assays.
  • the present invention provides combination therapies that result in improved effectiveness and/or reduced toxicity. Accordingly, in one aspect, the invention relates to the use of the present HDACIs as radiosensitizers in conjunction with radiotherapy.
  • the combination therapy of the invention comprises administering a present HDACI with one or more additional anticancer agents
  • the present HDACI and the additional anticancer agents can be administered concurrently or sequentially to an individual.
  • the agents can also be cyclically administered. Cycling therapy involves the administration of one or more anticancer agents for a period of time, followed by the administration of one or more different anticancer agents for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one or more of the anticancer agents of being administered, to avoid or reduce the side effects of one or more of the anticancer agents being administered, and/or to improve the efficacy of the treatment.
  • An additional anticancer agent may be administered over a series of sessions; anyone or a combination of the additional anticancer agents listed below may be administered.
  • the present invention includes methods for treating cancer comprising administering to an individual in need thereof a present HDACI and one or more additional anticancer agents or pharmaceutically acceptable salts thereof.
  • a present HDACI and the additional anticancer agent can act additively or synergistically.
  • Suitable anticancer agents include, but are not limited to, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mereaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campatheeins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5-fluorouracil (5-FU), taxanes (such as docetaxel and paclitaxel), leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas (such as car
  • the anti-cancer agent can be, but is not limited to, a drug selected from the group consisting of alkylating agents, nitrogen mustards, cyclophosphamide, trofosfamide, chlorambucil, nitrosoureas, carmustine (BCNU), lomustine (CCNU), alkylsulphonates, busulfan, treosulfan, triazenes, plant alkaloids, vinca alkaloids (vineristine, vinblastine, vindesine, vinorelbine), taxoids, DNA topoisomcrase inhibitors, epipodophyllins, 9- aminocamptothecin, camptothecin, crisnatol, mitomycins, mitomycin C, anti-metabolites, anti- folates, DHFR inhibitors, trimetrexate, IMP dehydrogenase inhibitors, mycophenolic acid, tiazofurin, ribavirin, EICAR, rib
  • anti-cancer agents include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; arnbomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelcsin; bleomycin sulfate; brequinar sodium; bropirimine; busul fan; cactinomycin; calusterone; caracemide; carbetimer; carmustine; carubicin hydrochloride; carzelesin; cedefingol
  • anti-cancer drugs that can be used in the present invention include, but are not limited to: 17-AAG; 20-epi-l,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL TK antagonists; altretamine; ambamustine; amidox; arnifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis
  • the present HDACIs can be administered in conjunction with chemical agents that are understood to mimic the effects of radiotherapy and/or that function by direct contact with DNA.
  • Preferred agents for use in combination with the present HDACIs for treating cancer include, but are not limited to cis-diamminedichloro platinum (II) (cisplatin), doxorubicin, 5-fluorouracil, taxol, and topoisomerase inhibitors such as etoposide, teniposide, irinotecan and topotecan.
  • the invention provides methods of treatment of cancer using the present HDACIs as an alternative to chemotherapy alone or radiotherapy alone where the chemotherapy or the radiotherapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated.
  • the individual being treated can, optionally, be treated with another anticancer treatment modality such as chemotherapy, surgery, or immunotherapy, depending on which treatment is found to be acceptable or bearable.
  • the present HDACIs can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants.
  • This can involve a multi-step process in which the subject's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the subject is then administered an amount of a present HDACI effective to eradicate the subject's remaining bone-marrow cell population, then the stem cell graft is infused back into the subject. Supportive care then is provided while bone marrow function is restored and the subject recovers.
  • the present methods for treating cancer can further comprise the administration of a present HDACI and an additional therapeutic agent or pharmaceutically acceptable salts or hydrates thereof.
  • a composition comprising a present HDACI is administered concurrently with the administration of one or more additional therapeutic agent(s), which may be part of the same composition or in a different composition from that comprising the present HDACI.
  • a present HDACI is administered prior to or subsequent to administration of another therapeutic agent(s).
  • the other therapeutic agent may be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine, and tropisetron.
  • the antiemetic agent is granisetron or ondansetron.
  • the other therapeutic agent may be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargrarnostim, molgramostim, and epoietin alfa.
  • the other therapeutic agent may be an opioid or nonopioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone, and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam, and sulindac.
  • the other therapeutic agent may be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirene, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the present HDACIs are used in methods of treating diseases, conditions, and injuries to the central nervous system, such as neurological diseases, neurodegenerative disorders, and traumatic brain injuries (TBIs).
  • a present HDACI is capable of crossing the blood brain barrier to inhibit HDAC in the brain of the individual.
  • the present HDACI compounds also provide a therapeutic benefit in models of peripheral neuropathies, such as CMT.
  • HDAC6 inhibitors have been found to cross the blood nerve barrier and rescue the phenotype observed in transgenic mice exhibiting symptons of distal hereditary motor neuropathy.
  • Administration of HDAC6 inhibitors to symptomatic mice increased acetylated a- tubulin levels, restored proper mitochondrial motility and axonal transport, and increased muscle re-innervation.
  • Other peripheral neuropathies include, but are not limited to, giant axonal neuropathy and various forms of mononeuropathies, polyneuropathies, autonomic neuropathies, and neuritis.
  • the present HDACI compounds also ameliorate associative memory loss following ⁇ elevation.
  • mice were infused with ⁇ 42 via cannulas implanted into dorsal hippocampus 15 minutes prior to training.
  • the test compounds are dosed ip (25 mg/kg) 2 hours before training.
  • Fear learning was assessed 24 hours later.
  • Contextual fear conditioning performed 24 hours after training shows a reduction of freezing in ⁇ -infused mice compared to vehicle-infused mice.
  • Treatment with a present compound ameliorates deficit in freezing responses in ⁇ -infused mice, and has no effect in vehicle-infused mice.
  • a test compound alone does not affect the memory performance of the mice.
  • treatment had no effects on motor, sensorial, or motivational skills assessed using the visible platform test in which the compounds are injected twice a day for two days.
  • no signs of overt toxicity including changes in food and liquid intake, weight loss, or changes in locomotion and exploratory behavior, are observed.
  • These results demonstrate that the HDACIs of the present invention are beneficial against impairment of associative memory following ⁇ elevation.
  • the present HDACIs therefore are useful for treating a neurological disease by administration of amounts of a present HDACI effective to treat the neurological disease or by administration of a pharmaceutical composition comprising amounts of a present HDACI effective to treat the neurological disease.
  • the neurological diseases that can be treated include, but are not limited to, Huntington's disease, lupus, schizophrenia, multiple sclerosis, muscular dystrophy, dentatorubralpallidoluysian atrophy (DRRLA), spinal and bulbar muscular atrophy (SBMA), and fine spinocerebellar ataxias (SCAl, SCA2, SCA3/MJD (Machado- Joseph Disease), SCA6, and SCA7), drug-induced movement disorders, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, Pick's disease, Alzheimer's disease, Lewy body dementia, cortico basal degeneration, dystonia, myoclonus, Tourette's syndrome, tremor, chorea, restless leg syndrome, Parkinson's
  • the neurological disease treated is Huntington's disease, Parkinson's disease, Alzheimer's disease, spinal muscular atrophy, lupus, or schizophrenia.
  • a present HDACI also can be used with a second therapeutic agent in methods of treating conditions, diseases, and injuries to the CNS.
  • Such second therapeutic agents are those drugs known in the art to treat a particular condition, diseases, or injury, for example, but not limited to, lithium in the treatment of mood disorders, estradiol benzoate, and nicotinamide in the treatment of Huntington's disease.
  • the present HDACIs also are useful in the treatment of TBIs. Traumatic brain injury (TBI) is a serious and complex injury that occurs in approximately 1.4 million people each year in the United States. TBI is associated with a broad spectrum of symptoms and disabilities, including a risk factor for developing neurodegenerative disorders, such as Alzheimer's disease.
  • TBI Traumatic brain injury
  • TBI produces a number of pathologies including axonal injury, cell death, contusions, and inflammation.
  • the inflammatory cascade is characterized by proinflammatory cytokines and activation of microglia which can exacerbate other pathologies.
  • proinflammatory cytokines and activation of microglia which can exacerbate other pathologies.
  • no efficacious anti-inflammatory therapies are currently available for the treatment of TBI.
  • HDAC inhibitors have been found to be protective in different cellular and animal models of acute and chronic neurodegenerative injury and disease, for example, Alzheimer's disease, ischemic stroke, multiple sclerosis (MS), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA).
  • MS multiple sclerosis
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • SMA spinal muscular atrophy
  • SBMA spinal and bulbar muscular atrophy
  • a recent study in experimental pediatric TBI reported a decrease in hippocampal CA3 histone H3 acetylation lasting hours to days after injury. These changes were attributed to documented upstream excitotoxic and stress cascades associated with TBI. HDACIs also have been reported to have anti-inflammatory actions acting through acetylation of non-histone proteins.
  • the HDAC6 selective inhibitor 4-dimethylamino-N-[5-(2- mercaptoacetylamino)pentyl]benzamide (DMA-PB), was found to be able to increase histone H3 acetylation and reduce microglia inflammatory response following traumatic brain injury in rats, which demonstrates the utility of HDACIs as therapeutics for inhibiting neuroinflammation associated with TBI.
  • the present HDACIs therefore also are useful in the treatment of inflammation and strokes, and in the treatment of autism and autism spectrum disorders.
  • the present HDACIs further can be used to treat parasitic infections, (e.g., malaria, toxoplasmosis, trypanosomiasis, helminthiasis, protozoal infections (see Andrews et al. Int. J. Parasitol. 2000, 30(6), 761-768).
  • the compound of the invention can be used to treat malaria.
  • a present HDACI can be co-administered with an antimalarial compound selected from the group consisting of aryl amino alcohols, cinchona alkaloids, 4-aminoquinolines, type 1 or type 2 folate synthesis inhibitors, 8-aminoquinolines, antimicrobials, peroxides, naphthoquinones, and iron chelating agents.
  • the antimalarial compound can be, but is not limited to, quinine, quinidine, mefloquine, halfantrine, chloroquine, amodiaquine, proguanil, chloroproquanil, pyrimethamine, primaquine, 8-[(4-amino-l-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5- [(3-trifluoromethyl)phenoxy]quinoline succinate (WR238,605), tetracycline, doxycycline, clindamycin, azithromycin, fluoroquinolones, artemether, areether, artesunate, artelinic acid, atovaquone, and deferrioxamine.
  • the antimalarial compound is chloroquine.
  • the present HDACIs also can be used as imaging agents.
  • the labeled compound can image HDACs, tissues expressing HDACs, and tumors.
  • Labeled HDACIs of the present invention also can image patients suffering from a cancer, or other HDAC-mediated diseases, e.g., stroke, by administration of an effective amount of the labeled compound or a composition containing the labeled compound.
  • the labeled HDACI is capable of emitting positron radiation and is suitable for use in positron emission tomography (PET).
  • a labeled HDACI of the present invention is used to identify areas of tissues or targets that express high concentrations of HDACs.
  • the extent of accumulation of labeled HDACI can be quantified using known methods for quantifying radioactive emissions.
  • the labeled HDACI can contain a fluorophore or similar reporter capable of tracking the movement of particular HDAC isoforms or organelles in vitro.
  • the present HDACIs useful in the imaging methods contain one or more radioisotopes capable of emitting one or more forms of radiation suitable for detection by any standard radiology equipment, such as PET, SPECT, gamma cameras, MRI, and similar apparatus.
  • Substituted HDACIs of the present invention also can contain isotopes of fluorine ( 18 F) and iodine ( 123 I) for imaging methods.
  • a labeled HDACI of the present invention contains an alkyl group having a U C label, i.e., a 11 C-methyl group, or an alkyl group substituted with 18 F, 123 I, 125 I, 131 I, or a combination thereof.
  • Fluorescently-labeled HDACIs of the present invention also can be used in the imaging method of the present invention.
  • Such compounds have an FITC,carbocyamine moiety or other fluorophore which will allow visualization of the HDAC proteins in vitro.
  • the labeled HDACIs and methods of use can be in vivo, and particularly on humans, and for in vitro applications, such as diagnostic and research applications, using body fluids and cell samples. Imaging methods using a labeled HDACI of the present invention are discussed in WO 03/060523, designating the U.S. and incorporated in its entirety herein.
  • the method comprises contacting cells or tissues with a radiolabeled, isotopically labeled, fluorescently labeled, or tagged (such as biotin tagged) compound of the invention, and making a radiographic, fluorescent, or similar type of image depending on the visualization method employed, i.e., in regared to radiographic images, a sufficient amount to provide about 1 to about 30 mCi of the radiolabeled compound.
  • Preferred imaging methods include the use of labeled HDACIs of the present invention which are capable of generating at least a 2: 1 target to background ratio of radiation intensity, or more preferably about a 5: 1, about 10: 1, or about 15: 1 ratio of radiation intensity between target and background.
  • the labeled HDACIs of the present invention are excreted from tissues of the body quickly to prevent prolonged exposure to the radiation of the radiolabeled compound administered to the individual.
  • labeled HDACIs of the present invention are eliminated from the body in less than about 24 hours. More preferably, labeled HDACIs are eliminated from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes.
  • preferred labeled HDACIs are eliminated in about 60 to about 120 minutes.
  • the present invention also embodies the use of derivatives containing tags (such as biotin) for the identification of biomolecules associated with the HDAC isoforms of interest for diagnostic, therapeutic or research purposes.
  • tags such as biotin
  • the present HDACIs also are useful in the treatment of autoimmune diseases and inflammations.
  • Compounds of the present invention are particularly useful in overcoming graft and transplant rejections and in treating forms of arthritis.
  • Tregs may be adoptively transferred to naive hosts to achieve beneficial therapeutic effects.
  • HDAC6-/- mice treated for 14 days with low-dose RPM are assessed for displaying signs of tolerance induction and resistance to the development of chronic rejection, a continuing major loss of graft function long-term in the clinical transplant population. Tolerance is assessed by testing whether mice with long-surviving allografts reject a subsequent third-party cardiac graft and accept additional donor allografts without any immunosuppression, as can occur using a non-selective HDACI plus RPM.
  • HDAC6 targeting is assessed in additional transplant models seeking readouts of biochemical significance, as is monitored clinically.
  • effects of HDAC6 in targeting in renal transplant recipients are assessed.
  • Renal transplants are the most common organ transplants performed, and the kidney performs multiple functions, e.g., regulating acid/base metabolism, blood pressure, red cell production, such that efficacy in this model indicates the utility of HDAC6 targeting.
  • islet transplantation is a major unmet need given that clinical islet allografts are typically lost after the first one or two years post- transplant.
  • the present compounds are envisioned to demonstrate anti- arthritic efficacy in a collagen-induced arthritis model in DBA1/J mice.
  • DBA1/J mice male, 7-8 weeks
  • Systemic arthritis is induced with bovine collagen type II and CFA, plus an IFA booster injection on day 21.
  • a present HDACI is dosed at 50 mg/kg and 100 mg/kg on day 28 for 2 consecutive weeks, and the effects determined from the Average Arthritic Score vs. Days of Treatment data.
  • immunosuppressive therapy is critical to the viability of the donor organ in the host.
  • immunosuppressive agents include azathioprine, methotrexate, cyclophosphamide, FK-506, rapamycin, and corticosteroids.
  • the present HDACIs are potent immunosuppressive agents that suppress humoral immunity and cell-mediated immune reactions, such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis and graft versus host disease.
  • HDACIs of the present invention are useful for the prophylaxis of organ rejection subsequent to organ transplantation, for treatment of rheumatoid arthritis, for the treatment of psoriasis, and for the treatment of other autoimmune diseases, such as type I diabetes, Crohn's disease, and lupus.
  • a therapeutically effective amount of a present HDACI can be used for immunosuppression including, for example, to prevent organ rejection or graft vs. host disease, and to treat diseases and conditions, in particular, autoimmune and inflammatory diseases and conditions.
  • autoimmune and inflammatory diseases include, but are not limited to, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, psoriasis, diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, arthritis (rheumatoid arthritis, arthritis chronic progrediente, and arthritis deformans) and rheumatic diseases, autoimmune hematological disorder (hemolytic anaemia, aplastic anaemia, pure red cell anaemia and
  • a present HDACI can be used alone, or in conjunction with a second therapeutic agent known to be useful in the treatment of autoimmune diseases, inflammations, transplants, and grafts, such as cyclosporin, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to persons skilled in the art.
  • a second therapeutic agent known to be useful in the treatment of autoimmune diseases, inflammations, transplants, and grafts, such as cyclosporin, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to persons skilled in the art.
  • HDACs include, but are not limited to asthma, cardiac hypertrophy, giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy, autonomic neuropathy, neuritis in general, and neuropathy in general. These disease and conditions also can be treated by a method of the present invention.
  • a therapeutically effective amount of one or more HDACI of the present invention is administered to a human being in need thereof. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment (diagnosis) that takes into consideration signs, symptoms, and/or malfunctions that are present, the risks of developing particular signs, symptoms and/or malfunctions, and other factors.
  • a present HDACI can be administered by any suitable route, for example by oral, buccal, inhalation, topical, sublingual, rectal, vaginal, intracisternal or intrathecal through lumbar puncture, transurethral, nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site) administration.
  • Parenteral administration can be accomplished using a needle and syringe or using a high pressure technique.
  • compositions include those wherein a present HDACI is present in a sufficient amount to be administered in an effective amount to achieve its intended purpose.
  • the exact formulation, route of administration, and dosage is determined by an individual physician in view of the diagnosed condition or disease. Dosage amount and interval can be adjusted individually to provide levels of a present HDACI that is sufficient to maintain therapeutic effects.
  • Toxicity and therapeutic efficacy of the present HDACI compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 .
  • Compounds that exhibit high therapeutic indices are preferred.
  • the data obtained from such procedures can be used in formulating a dosage range for use in humans.
  • the dosage preferably lies within a range of circulating compound concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective amount of a present HDACI required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and ultimately is determined by the attendant physician. Dosage amounts and intervals can be adjusted individually to provide plasma levels of the HDACI that are sufficient to maintain the desired therapeutic effects.
  • the desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses often are desired, or required.
  • a present HDACI can be administered at a frequency of: four doses delivered as one dose per day at four-day intervals (q4d x 4); four doses delivered as one dose per day at three-day intervals (q3d x 4); one dose delivered per day at five- day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5); or, any dose regimen determined to be appropriate for the circumstance.
  • the dosage of a composition containing a present HDACI, or a composition containing the same can be from about 1 ng/kg to about 200 mg/kg, about 1 ⁇ g/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg of body weight.
  • the dosage of a composition may be at any dosage including, but not limited to, about 1 ⁇ g/kg, 10 ⁇ g/kg, 25 ⁇ g/kg, 50 ⁇ g/kg, 75 ⁇ g/kg, 100 ⁇ g/kg, 125 ⁇ g/kg, 150 ⁇ g/kg, 175 ⁇ g/kg, 200 ⁇ g/kg, 225 ⁇ g/kg, 250 ⁇ g/kg, 275 ⁇ g/kg, 300 ⁇ ⁇ ⁇ ⁇ , 325 ⁇ ⁇ ⁇ ⁇ , 350 ⁇ ⁇ ⁇ , 375 ⁇ ⁇ ⁇ ⁇ , 400 ⁇ ⁇ ⁇ ⁇ , 425 ⁇ ⁇ ⁇ ⁇ , 450 ⁇ ⁇ ⁇ ⁇ , 475 ⁇ ⁇ ⁇ ⁇ , 500 ⁇ ⁇ ⁇ ⁇ , 525 ⁇ g/kg, 550 ⁇ ⁇ ⁇ ⁇ , 575 ⁇ ⁇ ⁇ ⁇ , 600 ⁇ ⁇
  • the above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention.
  • the physician determines the actual dosing regimen that is most suitable for an individual patient, which can vary with the age, weight, and response of the particular patient.
  • a present HDACI used in a method of the present invention typically is administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose.
  • a present HDACI can be administered, per dose, in an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams, including all doses between 0.005 and 500 milligrams.
  • the HDACIs of the present invention typically are administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • Pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the present HDACIs.
  • carrier refers to a diluent, adjuvant, or excipient, with which a present HDACI is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • the carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
  • the pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when a present HDACI is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can be manufactured, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
  • a therapeutically effective amount of a present HDACI is administered orally, the composition typically is in the form of a tablet, capsule, powder, solution, or elixir.
  • the composition additionally can contain a solid carrier, such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain about 0.01% to about 95%, and preferably from about 1% to about 50%, of a present HDACI.
  • a liquid carrier such as water, petroleum, or oils of animal or plant origin
  • the liquid form of the composition can further contain physiological saline solution, dextrose or other saccharide solutions, or glycols.
  • the composition When administered in liquid form, the composition contains about 0.1% to about 90%, and preferably about 1% to about 50%, by weight, of a present compound.
  • a therapeutically effective amount of a present HDACI is administered by intravenous, cutaneous, or subcutaneous injection
  • the composition is in the form of a pyrogen- free, parenterally acceptable aqueous solution.
  • parenterally acceptable solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains an isotonic vehicle.
  • a present HDACI can be infused with other fluids over a 10-30 minute span or over several hours.
  • the present HDACIs can be readily combined with pharmaceutically acceptable carriers well-known in the art.
  • Such carriers enable the active agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by adding a present HDACI to a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, for example, fillers and cellulose preparations. If desired, disintegrating agents can be added.
  • a present HDACI can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form.
  • suspensions of a present HDACI can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • a present composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a present HDACI also can be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases.
  • a present HDACI also can be formulated as a depot preparation.
  • Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • a present HDACI can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins.
  • a present HDACI can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.
  • excipients such as starch or lactose
  • capsules or ovules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.
  • Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents.
  • the present HDACIs also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily.
  • the present HDACIs are best used in the form of a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.
  • a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.
  • the present invention includes kits which comprise one or more compounds or compositions packaged in a manner that facilitates their use to practice methods of the invention.
  • the kit includes a compound or composition described herein as useful for practice of a method (e.g., a composition comprising a present HDACI and an optional second therapeutic agent), packaged in a container, such as a sealed bottle or vessel, with a label affixed to the container or included in the kit that describes use of the compound or composition to practice the method of the invention.
  • the compound or composition is packaged in a unit dosage form.
  • the kit further can include a device suitable for administering the composition according to the intended route of administration, for example, a syringe, drip bag, or patch.
  • the present compounds is a lyophilate.
  • the kit can further comprise an additional container which contains a solution useful for the reconstruction of the lyophilate.
  • Prior HDACIs possessed properties that hindered their development as therapeutic agents.
  • the present HDACIs were synthesized and evaluated as inhibitors for HDAC.
  • the present compounds demonstrate an increased HDAC6 potency and selectivity against HDACI and HDAC8 with improvements in BEI relative to prior compounds.
  • the improved properties of the present compounds, particularly the increase in BEI and reduced potency at HDAC8, indicate that the present compounds are useful for applications such as, but not limited to, immunosuppresssive and neuroprotective agents.
  • compounds of the present invention typically have a bonding affinity (IC50) to HDAC6 of less than ⁇ , less than 25 ⁇ , less than ⁇ , less than ⁇ ⁇ , less than 0.5 ⁇ , and less than 0.2 ⁇ .
  • the mercaptoacetamide 3a containing a tetrahydroquinoline (THQ) cap, was obtained from coupling of N-Boc-7-aminoheptanoic acid (10, Scheme 1) with ⁇ , ⁇ - dimethylhydroxylamine hydrochloride to give the Weinreb amide 11, which was reduced to aldehyde 12 with LiAlH 4 . Subsequently, reductive amination of intermediate 12 with the THQ cap afforded 13. Next, Boc deprotection, amide formation in the presence of PyBOP, and removal of the trityl group gave mercaptoacetamide 3a.
  • THQ tetrahydroquinoline
  • a microwave vessel equipped with a magnetic stir bar and a septum, was charged with a mixture of cesium carbonate (3.26 g, 10 mmol), 8-aminoquinoline (0.59 g, 5.2 mmol), and 6a (1.31 g, 5.2 mmol) in DMF (4 mL).
  • the vessel was sealed and irradiated at 120 °C for 40 min.
  • the reaction mixture was diluted with ethyl acetate, and the organic layer was washed with 10% aqueous lithium chloride. The organic fraction was dried over anhydrous Na 2 S0 4 , filtered and concentrated under reduced pressure.
  • Trifluoroacetic acid (0.67 mL, 8.9 mmol) was added to a solution of 7a (0.28 g, 0.89 mmol) in CH 2 CI 2 (5 mL). The mixture was stirred at room temperature for 2 h. After this time, the reaction mixture was cooled to 0 °C, then 1 M aqueous NaOH was added, under stirring (pH was adjusted to 10). The layers were separated and the aqueous layer was extracted with CH 2 CI 2 . The organic layers were combined, dried over anhydrous Na 2 S0 4 and filtered. Evaporation of the solvent in vacuo afforded 0.144 g (0.7 mmol, 76% yield) of target product, as a yellow oil. This was used in the following step without any further purification.
  • N,N-Diisopropylethylamine (DIPEA, 3.39 niL, 19.5 mmol) was added to a solution of 6-((ieri-butoxycarbonyl)amino)hexanoic acid (16, 3.0 g, 13.0 mmol) in DMF (13 mL).
  • PyBOP (7.4 g, 14.3 mmol) was then added and the reaction mixture was stirred at room temperature for 10 min.
  • 1,2,3,4-Tetrahydroquinoline (1.8 mL, 14.3 mmol) was then added and the resulting reaction mixture was stirred at room temperature overnight.
  • the effectiveness, or potency, of a present HDACi with respect to inhibiting the activity of an HDAC is measured by an IC 50 value.
  • the quantitative IC 50 value indicates the concentration of a particular compound that is needed to inhibit the activity of an enzyme by 50% in vitro. Stated alternatively, the IC 50 value is the half maximal (50%) inhibitory concentration of a compound tested using a specific enzyme, e.g., HDAC, of interest. The smaller the IC 50 value, the more potent the inhibiting action of the compound because a lower concentration of the compound is needed to inhibit enzyme activity by 50%.
  • a present HDACi inhibits HDAC enzymatic activity by about at least 50%, preferably at least about 75%, at least 90%, at least 95%, or at least 99%.
  • a present compound was also have been tested against HDACi, 2, 3, 4, 5, 8, 10, and 11.
  • the tested compounds showed a range of IC 50 values vs. HDAC6 of about 1 nM to greater than 1 ⁇ , and a range of IC 50 values vs. HDACI of about 1 ⁇ to greater than 20 ⁇ . Therefore, in some embodiments, a present HDACI is a selective HDAC6 inhibitor which, because of a low affinity for other HDAC isozymes, e.g., HDACi, may give rise to fewer side effects than compounds that are non-selective HDAC inhibitors.
  • the present HDACis interact with and reduce the activity of all histone deacetylases in a cell. In some preferred embodiments, the present HDACis interact with and reduce the activity of fewer than all histone deacetylases in the cell. In certain preferred embodiments, the present HDACis interact with and reduce the activity of primarily one histone deacetylase (e.g., HDAC6), but do not substantially interact with or reduce the activities of other histone deacetylases (e.g., HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-7, HDAC- 8, HDAC-9, HDAC- 10, and HDAC- 11).
  • HDAC-6 histone deacetylase
  • the present invention therefore provides HDACis for the treatment of a variety of diseases and conditions wherein inhibition of HDAC has a beneficial effect.
  • a present HDACI is selective for inhibition of HDAC6 by at least 10-fold over the other HDAC isozymes, i.e., 1-5, and 7-11.
  • HDACI, 2, 4, 5, 6, 7, 8, 9, 10, and 11 assays used isolated recombinant human protein; HDAC3/NcoR2 complex was used for the HDAC3 assay.
  • HDAC8 is fluorogenic diacyl peptide based on residues 379-382 of p53 (RHKAcKAc).
  • Acetyl- Lys(trifluoroacetyl)-AMC substrate was used for HDAC4, 5, 7, and 9 assays.
  • Compounds were dissolved in DMSO and tested in 10-dose IC50 mode with 3-fold serial dilution starting at 30 ⁇ .
  • Control Compound Trichostatin A (TSA) was tested in a 10-dose ICso with 3-fold serial dilution starting at 5 ⁇ .
  • ICso values were extracted by curve-fitting the dose/response slopes. Assays were performed in duplicate and IC 50 values are an average of data from both experiments.
  • Substrate for HDACI and HDAC 6 Acetylated peptide substrate for HDAC, based on residues 379-382 of p53 (Arg-His-Lys-Lys(Ac)), a site of regulatory acetylation by the p300 and CBP acetyltransferases (lysines 381, 382)1-6, is the best for HDAC from among a panel of substrates patterned on p53, histone H3 and histone H4 acetylation sites.
  • HDAC1 75 nM HDAC1 and 50 ⁇ HDAC substrate are in the reaction buffer and 1 % DMSO final. Incubate for 2 hours at 30°C.
  • HDAC 6 12.6 nM HDAC6 and 50 ⁇ HDAC substrate are in the reaction buffer and 1 % DMSO final. Incubate for 2 hours at 30°C.
  • HDAC inhibitory activity of some exemplary mercaptoacetamides is HDAC inhibitory activity of some exemplary mercaptoacetamides.
  • the present compounds have been evaluated for their activity at HDAC6 and their selectivity for HDAC6 compared to HDAC1. It previously was shown that selective HDAC6 inhibitors may be employed in a variety of disease states including, but not limited to, arthritis, autoimmune disorders, inflammatory disorders, cancer, neurological diseases such as Rett syndrome, peripheral neuropathies such as CMT, stroke, hypertension, dementias, and diseases in which oxidative stress is a causative factor or a result thereof. It also was shown that selective HDAC6 inhibitors, when administered in combination with rapamycin, prolonged the lifespan of mice with kidney xenografts. This model was used to evaluate the immunosuppressant properties of the present compounds and serve as a model of transplant rejection.
  • selective HDAC6 inhibitors confer neuroprotection in rat primary cortical neuron models of oxidative stress. These studies identified selective HDAC6 inhibitors as nontoxic neuroprotective agents.
  • the present compounds behave in a similar manner because they also are selective HDAC6 agents.
  • the present compounds demonstrate a ligand efficiency that renders them more drug-like in their physiochemical properties.
  • the present compounds do not contain a hydroxamate group as the zinc binding group, and as such, they are unable to undergo the Lossen rearrangement, a reaction that results in many of the hydroxamates showing activity in the Ames test, i.e., many hydroxamates prove to be genotoxic.
  • the present compounds therefore are pharmaceutical candidates and research tools to identify the specific functions of HDAC6, and to treat diseases that are linked to the expression of HDAC6.
  • THQ and 8-aminoquinoline caps generated potent and selective analogs, but it is important to note that the THQ cap is more lipophilic, suggesting that compounds such as 3a
  • Tregs murine Foxp3+ regulatory T cells
  • HDACIs have previously been shown to enhance the suppressive effects of Foxp3+ regulatory T cells.
  • the pharmacological modulation of Treg suppression is considered as a possible therapeutic approach to slow or reverse the pathogenesis of autoimmune disorders, to prevent allograft rejection, inflammatory bowel disease, and rheumatoid arthritis.
  • CFSE carboxyfluorescein succinimidyl ester

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Histone deacetylases inhibitors (HDACIs) and compositions containing the same are disclosed. Methods of treating diseases and conditions wherein inhibition of HDAC provides a benefit, like a cancer, a neurodegenerative disorder, a peripheral neuropathy, a neurological disease, traumatic brain injury, stroke, hypertension, malaria, an autoimmune disease, autism, autism spectrum disorders, and inflammation, also are disclosed.

Description

BICYCLIC AND TRICYCLIC CAP BEARING MERCAPTOACETAMIDE DERIVATIVES AS HISTONE DEACETYLASE INHIBITORS
STATEMENT OF GOVERNMENT INTEREST
[0001] This invention was made with government support under 1R01 NS079183 granted by the National Institutes of Health. The government has certain rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62/221,843, filed September 22, 2015, incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] This invention relates to thiol-based HDACIs, compositions comprising the HDACIs, and therapeutic uses of the HDACIs.
BACKGROUND OF THE INVENTION
[0004] Covalent post-translational modifications (PTMs) of epigenomic proteins contribute to their biological roles, and thus serve as carriers of epigenetic information from one cell generation to the next. Epigenetics means on top of or above genetics, and refers to external modifications to DNA and associated histones that turn genes "on" or "off." These modifications do not change the DNA sequence, but instead, they affect how cells "read" genes. PTMs play key roles in the regulation of protein function, transcription, DNA replication, and repair of DNA damage (1).
[0005] The major events surrounding epigenetic control are focused on three modes of action: writers, readers, and erasers. The writers are responsible for adding a variety of PTM marks to histones which include, inter alia, acetylation which is catalyzed by histone acetyltransferases
(HATs). Readers refer to the proteins that recognize and bind to these PTM marks thereby mediating their effects, and erasers encompass various enzymes such as the histone deacetylases
(HDACs) that catalyze the removal of these marks. In the case of acetylated histone lysine residues, HDACs are responsible for catalyzing the hydrolysis of the acetyl mark to provide the unsubstituted lysine residue. The HDAC family consists of at present 18 enzymes which are classified into four subgroups according to their homology to the yeast family. HDAC1, 2, 3 and 8 - categorized as class I HDACs according to their homology with yeast Rpd3 - are characterized by ubiquitous expression and localization to the nucleus. Class II HDACs show tissue-specific expression and shuttle between the nucleus and cytoplasm. Homologous to yeast Hdal, these enzymes are subdivided in class Ila (HDAC4, 5, 7 and 9) and class lib (HDAC6 and 10). HDAC 11, the only member of the class IV subfamily, shows similarities to the catalytic domains of both class I and II enzymes. Class I, II, and IV HDACs require Zn2+ as a cofactor of the deacetylating activity and are also referred to as the conventional HDACs. The sirtuins 1-7 are dependent on nicotinamide adenine dinucleotide for their activity and form class III of the HDACs.
[0006] Pharmacologic manipulation of the enzymes involved in regulating protein PTMs, especially those tied to very specific PTM marks, holds tremendous possibilities in better understanding the workings of the cell. The discovery of selective small molecule modulators of these enzymes would provide chemical tools to better understand the role of these PTMs at the cellular level, but may also lead to important disease modifiers. Within the HDAC field, there exists a plethora of compounds that are able to block the deacetylase enzymes, and several have made their way to the marketplace for cancer therapy. The majority of these HDAC inhibitors (HDACi), however, are not very isoform selective. Many of them inhibit across more than one class of HDAC enzymes and are thus labeled pan-selective. Of the various HDAC isoforms that appear to be promising therapeutic targets for treating humans diseases such as cancer and certain CNS disorders, HDAC6 has emerged as a particularly attractive target, especially in view of the fact that HDAC6 knockout animals remain viable. HDAC6 has no apparent role in the PTM of histone proteins, but rather is involved in regulating the acetylation status of oc-tubulin, HSP-90, cortactin, HSF-1, and other protein targets. This enzyme also plays a role in the recognition and clearance of polyubiquitinated misfolded proteins from the cell through aggresome formation. The development of HDAC6 selective compounds has recently been reviewed (2). In general, HDACis are comprised of three main motifs: a zinc binding group (ZBG), a cap group, and a linker that bridges the previous two (Figure 1). A properly optimized cap group can improve both potency and selectivity, presumably through its ability to engage in appropriate contacts with residues on the enzyme surface. [0007] Many HDACIs such as trichostatin A (TSA) and SAHA contain a hydroxamic acid function as zinc-binding group (ZBG). Unfortunately, hydroxamates are in some cases metabolically unstable (short half-life), and their potent metal-chelating ability might lead to off- target activity at other zinc-containing enzymes(3). In addition, many of the hydroxamic acid based inhibitors have been found to be Ames-positive, suggesting that these agents might present genotoxic effects. While several of the HDACis on the market are Ames positive and cause chromosomal aberrations, these are being used only for cancer, wherein this undesired side effect can to a certain extent be tolerated in a disease considered to be life threatening. Certainly, for use in diseases that would require chronic, longer term use of an HDACi, it would be preferable to have compounds that are not Ames positive/gentoxic. However, even for cancer, it is known that use of genotoxic agents can lead to a genomic instability that may be transmitted to offspring in cases where the treated adults have children(4). As such, there is a great need for the discovery of potent and selective HDAC inhibitors that bear alternative ZBGs.
SUMMARY OF THE INVENTION
[0008] We have discovered a class of thiol-based (SH containing) HDAC6i that bear a mercaptoacetamide group as the ZBG, some of which show high selectivity for the inhibition of HDAC6 (5).
[0009] A potential drawback of thiol-containing HDACIs is their ability to undergo oxidative dimerization to give disulfides as products. Although the disulfide bond can be reduced inside of the cells to afford the parent thiol compound, the dimerization reaction could in certain instances represent a problem and in such cases it may be advantageous to employ a pro-drug from of the thiol, namely its thioester analog (6).
[0010] Accordingly, the present invention relates to HDAC inhibitors (HDACi), pharmaceutical compositions comprising the HDACi, and methods of treating diseases and conditions wherein inhibition of HDAC provides a benefit, such as a cancer, a neurological disease, a psychiatric illness, a neurodegenerative disorder, a peripheral neuropathy, stroke, hypertension, an inflammation, traumatic brain injury, rheumatoid arthritis, allograft rejection, and autoimmune diseases, comprising administering a therapeutically effective amount of an HDACi to an individual in need thereof. The present invention also relates to a method of increasing the sensitivity of a cancer cell to radiotherapy and/or chemotherapy. The present invention also allows for the use of these HDAC inhibitors in combination with other drugs and/or therapeutic approaches. In some embodiments, the present HDACi exhibit selectivity for particular HDAC isozymes, such as HDAC6, over other HDAC isozymes.
[0011] More particularly, the present invention relates to HDACi having the structural formula as illustrated below. Thus, the HDACi of the present invention contain a Cap group which is generally comprised of an aromatic ring, a linker group which is comprised of, inter alia, methylene groups or substituted methylene groups, and the zinc binding group, which is in this case the mercaptoacetamide group (-NHC(0)CH2SH)
[0012] In its principal aspect, this invention is a compound of formula I
Figure imgf000005_0001
or a pharmaceutically acceptable salt or prodrug thereof wherein Cap is a group of formula:
Figure imgf000005_0002
represents a single or double bond,
Y and Z are independently selected from C and N, provided that when Y is N, represents a single bond;
W is selected from O, S and (CH2)m;
X is selected from C(=0), CHR, and NHR when Y is C, or C(=0) and CHR when Y is N; R = H or Ci-C4 alkyl; R1 is H or C(=0)Ci_4 alkyl; m is 0, 1 or 2; n is 1, 2 or 3; and
A is one or more substituents independently from H, Br, CI, Me, CF3, N¾, CN, OCH3 and OH, and A can be appended to any one or each of the fused aromatic rings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Fig. 1 shows structures of SAHA, TSA, and general structure of an HDAC inhibitor.
[0014] Fig. 2a shows Western blots of acetylated tubulin compared to a-tubulin in E17 cells. E17 cells were treated with TSA, Tubastatin A, or the thiol analogues 1, 2a, 2b, and 3a for 24 h at the indicated concentrations, and their effects on acetylated tubulin were compared with those of the DMSO control.
[0015] Fig. 2b shows results from the same test, but in which the increase of acetylated histone H3, compared to histone H3, was evaluated.
[0016] Fig. 3 shows the effects of thiol compounds on murine Treg suppressive functions in vitro. Residual CFSE+ Teff cell proliferation is shown at each ratio of Treg:Teff cell; assays were performed in triplicate and repeated at least once; mean +SD shown; *p<0.05 compared to corresponding DMSO-treated cultures.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is directed to novel HDACIs of formula I and their use in therapeutic treatments of, for example, cancers, inflammations, traumatic brain injuries, neurodegenerative disorders, neurological diseases, peripheral neuropathies, strokes, hypertension, autoimmune diseases, inflammatory diseases, and malaria. The present HDACIs also increase the sensitivity of a cancer cell to the cytotoxic effects of radiotherapy and/or chemotherapy. In some embodiments, the present HDACIs selectively inhibit HDAC6 over other HDAC isozymes. [0018] The present invention is described in connection with preferred embodiments. However, it should be appreciated that the invention is not limited to the disclosed embodiments. It is understood that, given the description of the embodiments of the invention herein, various modifications can be made by a person skilled in the art. Such modifications are encompassed by the claims below.
[0019] The term "a disease or condition wherein inhibition of HDAC provides a benefit" pertains to a condition in which HDAC and/or the action of HDAC is important or necessary, e.g., for the onset, progress, expression of that disease or condition, or a disease or a condition which is known to be treated by an HDAC inhibitor (such as, e.g., TSA, pivalolyloxymethylbutane (AN-9; Pivanex), FK-228 (Depsipeptide), PXD-101, NVP-LAQ824, SAHA, MS-275, and or MGCD0103). Examples of such conditions include, but are not limited to, cancer, psoriasis, fibroproliferative disorders (e.g., liver fibrosis), smooth muscle proliferative disorders (e.g., atherosclerosis, restenosis), neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, Huntington's chorea, amyotropic lateral sclerosis, spino-cerebellar degeneration, Rett syndrome), peripheral neuropathies (Charcot-Marie-Tooth disease, Giant Axonal Neuropathy (GAN)), inflammatory diseases (e.g., osteoarthritis, rheumatoid arthritis, colitis), diseases involving angiogenesis (e.g., cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy), hematopoietic disorders (e.g., anemia, sickle cell anemia, thalasseimia), fungal infections, parasitic infections (e.g., malaria, trypanosomiasis, helminthiasis, protozoal infections), bacterial infections, viral infections, and conditions treatable by immune modulation (e.g., multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergies, asthma, allergic rhinitis, inflammatory bowel disease; and for improving grafting of transplants). One of ordinary skill in the art is readily able to determine whether a compound treats a disease or condition mediated by HDAC for any particular cell type, for example, by assays which conveniently can be used to assess the activity of particular compounds.
[0020] The term "second therapeutic agent" refers to a therapeutic agent different from a present HDACI and that is known to treat the disease or condition of interest. For example, when a cancer is the disease or condition of interest, the second therapeutic agent can be a known chemotherapeutic drug, like taxol, or radiation, for example. [0021] The term "HDAC" refers to a family of enzymes that remove acetyl groups from a protein, for example, the ε-amino groups of lysine residues at the N-terminus of a histone. The HDAC can be a human HDAC, including, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC 8, HDAC9, HDAC10, and HDAC11. The HDAC also can be derived from a protozoal or fungal source.
[0022] HDAC inhibitors (HDACIs) typically contain three structural elements which are analogous to the structure of acetyllysine. These three structural elements are a zinc binding group (M), which is responsible for chelation of zinc in the active site, a linker region (L), which binds to the hydrophobic channel that connects the active site to the outer enzyme surface, and a capping group (Cap), which interacts with residues at the outer enzyme surface.
[0023] The terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, relieving, reversing, and/or ameliorating a disease or condition and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated, including the treatment of acute or chronic signs, symptoms and/or malfunctions. As used herein, the terms "treat," "treating," "treatment," and the like may include "prophylactic treatment," which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously-controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition, "treatment" therefore also includes relapse prophylaxis or phase prophylaxis. The term "treat" and synonyms contemplate administering a therapeutically effective amount of a compound of the invention to an individual in need of such treatment. A treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.
[0024] The term "therapeutically effective amount" or "effective dose" as used herein refers to an amount of the active ingredient(s) that, when administered, is (are) sufficient, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to an individual in need thereof. In the case of a cancer or other proliferation disorder, the therapeutically effective amount of the agent may reduce (i.e., retard to some extent and preferably stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; reduce HDAC signaling in the target cells; and/or relieve, to some extent, one or more of the symptoms associated with the cancer. To extent the administered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
[0025] "Concurrent administration," "administered in combination," "simultaneous administration," and similar phrases mean that two or more agents are administered concurrently to the subject being treated. By "concurrently," it is meant that each agent is administered either simultaneously or sequentially in any order at different points in time. However, if not administered simultaneously, it is meant that they are administered to an individual in a sequence and sufficiently close in time so as to provide the desired therapeutic effect and can act in concert. For example, a present HDACI can be administered at the same time or sequentially in any order at different points in time as a second therapeutic agent. A present HDACI and the second therapeutic agent can be administered separately, in any appropriate form and by any suitable route. When a present HDACI and the second therapeutic agent are not administered concurrently, it is understood that they can be administered in any order to a subject in need thereof. For example, a present HDACI can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent treatment modality (e.g., radiotherapy), to an individual in need thereof. In various embodiments, a present HDACI and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In one embodiment, the components of the combination therapies are administered at 1 minute to 24 hours apart.
[0026] The use of the terms "a", "an", "the", and similar referents in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein merely serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value and subrange is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as" and "like") provided herein, is intended to better illustrate the invention and is not a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0027] In particular, the present invention is directed to HDACIs, compositions comprising the present HDACI, and therapeutic uses of the HDACIs of formula I:
Figure imgf000010_0001
or a pharmaceutically acceptable salt or prodrug thereof wherein Cap is a group of formula:
Figure imgf000010_0002
represents a single or double bond,
Y and Z are independently selected from C and N, provided that when Y is N, represents a single bond;
W is selected from O, S and (CH2)m; X is selected from C(=0), CHR, and NHR when Y is C, or C(=0) and CHR when Y is N; R = H or Ci-C4 alkyl;
R1 is H or C(=0)Ci-4 alkyl; m is 0, 1 or 2; n is 1, 2 or 3; and
A is one or more (e.g., 1, 2, 3, or 4) substituents independently from H, Br, CI, Me, CF3, NH2, CN, OCH3 and OH, and A can be appended to any one or each of the fused aromatic rings.
[0028] In one embodiment of the invention, Z is C.
[0029] In another embodiment, Z is N.
[0030] In another embodiment, R is H.
[0031] In another embodiment, A is CI.
[0032] In another embodiment, W is (CH2)m.
[0033] In another embodiment, Cap is selected from:
Figure imgf000011_0001
, wherein A can be appended to any one or each of the fused aromatic rings.
[0034] Representative compounds of the invention include, but are not limited to:
Figure imgf000011_0002
Figure imgf000012_0001
Figure imgf000012_0002
[0035] Compounds of the present invention inhibit HDAC and are useful in the treatment of a variety of diseases and conditions. In particular, the present HDACIs are used in methods of treating a disease or condition wherein inhibition of HDAC provides a benefit, for example, cancers, neurological diseases, neurodegenerative conditions, peripheral neuropathies, autoimmune diseases, inflammatory diseases and conditions, stroke, hypertension, traumatic brain injury, autism, and malaria. The methods comprise administering a therapeutically effective amount of a present HDACI to an individual in need thereof.
[0036] The present methods also encompass administering a second therapeutic agent to the individual in addition to a present HDACI. The second therapeutic agent is selected from agents, such as drugs and adjuvants, known as useful in treating the disease or condition afflicting the individual, e.g., a chemotherapeutic agent and/or radiation known as useful in treating a particular cancer.
[0037] Prodrugs of the present compounds also are included in the present invention. It is well established that a prodrug approach, wherein a compound is derivatized into a form suitable for formulation and/or administration, then released as a drug in vivo, has been successfully employed to transiently (e.g., bioreversibly) alter the physicochemical properties of the compound (see, H. Bundgaard, Ed., "Design of Prodrugs," Elsevier, Amsterdam, (1985); R.B. Silverman, "The Organic Chemistry of Drug Design and Drug Action," Academic Press, San Diego, chapter 8, (1992); K.M. Hillgren et al., Med. Res. Rev., 15, 83 (1995)). Specific prodrugs of HDACIs are discussed in WO 2008/055068, incorporated in its entirety herein by reference.
[0038] In an embodiment, the prodrug comprises an acylated derivative of the sulfur atom of formula
-S-C(0)-R" wherein R" is selected from straight or branched Ci-C6 alkyl) aryl or heteroaryl. In an embodiment, aryl is phenyl and heteroaryl is pyridyl.
[0039] Compounds of the invention can exist as salts. Pharmaceutically acceptable salts of the present HDACIs often are preferred in the methods of the invention. As used herein, the term "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the present compounds. Salts of the present compounds can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of the present compounds can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric. Nonlimiting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present invention appearing herein is intended to include the present compounds as well as pharmaceutically acceptable salts, hydrates, or prodrugs thereof.
[0040] The present compounds also can be conjugated or linked to auxiliary moieties that promote a beneficial property of the compound in a method of therapeutic use. Such conjugates can enhance delivery of the compounds to a particular anatomical site or region of interest (e.g., a tumor), enable sustained therapeutic concentrations of the compounds in target cells, alter pharmacokinetic and pharmacodynamic properties of the compounds, and/or improve the therapeutic index or safety profile of the compounds. Suitable auxiliary moieties include, for example, amino acids, oligopeptides, or polypeptides, e.g., antibodies, such as monoclonal antibodies and other engineered antibodies; and natural or synthetic ligands to receptors in target cells or tissues. Other suitable auxiliaries include fatty acid or lipid moieties that promote biodistribution and/or uptake of the compound by target cells (see, e.g., Bradley et al., Clin. Cancer Res. (2001) 7:3229).
[0041] The present compounds have been evaluated for their activity at HDAC6 and their selectivity for HDAC6 compared to HDAC1. It previously was shown that selective HDAC6 inhibitors are implicated in a variety of disease states including, but not limited to, arthritis, autoimmune disorders, inflammatory disorders, cancer, neurological diseases such as Rett syndrome, peripheral neuropathies such as CMT, stroke, hypertension, and diseases in which oxidative stress is a causative factor or a result thereof. It also was shown that selective HDAC6 inhibitors, when administered in combination with rapamycin, prolonged the lifespan of mice with kidney xenografts. This model was used to evaluate the immunosuppressant properties of the present compounds and serve as a model of transplant rejection. Furthermore, it was previously shown that selective HDAC6 inhibitors confer neuroprotection in rat primary cortical neuron models of oxidative stress. These studies identified selective HDAC6 inhibitors as nontoxic neuroprotective agents. The present compounds behave in a similar manner because they also are selective HDAC6 agents. The present compounds demonstrate a ligand efficiency that renders them more drug-like in their physiochemical properties. In addition, the present compounds maintain the potency and selectivity observed in prior HDACIs. The present compounds therefore are pharmaceutical candidates and research tools to identify the specific functions of HDAC6.
[0042] Thus, in one embodiment, the present invention relates to a method of treating an individual suffering from a disease or condition wherein inhibition of HDAC provides a benefit comprising administering a therapeutically effective amount of a claimed HDACi compound to an individual in need thereof.
[0043] The methods of the present invention can be accomplished by administering one of the HDACi of the present invention as the neat compound or as a pharmaceutical composition. Administration of a pharmaceutical composition, or a neat HDACI of the present invention, can be performed during or after the onset of the disease or condition of interest. Typically, the pharmaceutical compositions are sterile, and contain no toxic, carcinogenic, or mutagenic compounds that would cause an adverse reaction when administered.
[0044] In some embodiments, a present HDACi may be administered in conjunction with a second therapeutic agent useful in the treatment of a disease or condition wherein inhibition of
HDAC provides a benefit. The second therapeutic agent is different from the present HDACi. A present HDACi and the second therapeutic agent can be administered simultaneously or sequentially. In addition, a present HDACI and second therapeutic agent can be administered from a single composition or two separate compositions. A present HDACi and the second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect.
[0045] The second therapeutic agent is administered in an amount to provide its desired therapeutic effect. The effective dosage range for each second therapeutic agent is known in the art, and the second therapeutic agent is administered to an individual in need thereof within such established ranges.
[0046] The present invention therefore is directed to compositions and methods of using such compounds in treating diseases or conditions wherein inhibition of HDAC provides a benefit. The present invention also is directed to pharmaceutical compositions comprising a present HDACi and an optional second therapeutic agent useful in the treatment of diseases and conditions wherein inhibition of HDAC provides a benefit. Further provided are kits comprising a present HDACi and, optionally, a second therapeutic agent useful in the treatment of diseases and conditions wherein inhibition of HDAC provides a benefit, packaged separately or together, and an insert having instructions for using these active agents.
[0047] A present HDACI and the second therapeutic agent can be administered together as a single-unit dose or separately as multi-unit doses, wherein the present HDACI is administered before the second therapeutic agent or vice versa. One or more dose of a present HDACI and/or one or more dose of the second therapeutic agent can be administered. The present HDACIs therefore can be used in conjunction with one or more second therapeutic agents, for example, but not limited to, anticancer agents.
[0048] Within the meaning of the present invention, the term "disease" or "condition" denotes disturbances and/or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and/or malfunctions. As demonstrated below, a present HDACi is a potent inhibitor of HDAC and can be used in treating diseases and conditions wherein inhibition of HDAC provides a benefit, for example, cancer, a neurological disease, a neurodegenerative condition, traumatic brain injury, stroke, an inflammation, an autoimmune disease, and autism.
[0049] In one preferred embodiment, the present invention provides methods for treating cancer, including but not limited to killing a cancer cell or neoplastic cell; inhibiting the growth of a cancer cell or neoplastic cell; inhibiting the replication of a cancer cell or neoplastic cell; or ameliorating a symptom thereof, said methods comprising administering to a subject in need thereof a therapeutically effective amount of a present HDACi. Additionally, it is noted that the selective HDACi may be able to facilitate the killing of cancer cells through reactivation of the immune system by mechanisms relating to the PDI receptor.
[0050] In one embodiment, the invention provides a method for treating cancer comprising administering to a subject in need thereof an amount of a present HDACI or a pharmaceutically acceptable salt thereof sufficient to treat the cancer. A present HDACI can be used as the sole anticancer agent, or in combination with another anticancer treatment, e.g., radiation, chemotherapy, and surgery.
[0051] In another embodiment, the invention provides a method for increasing the sensitivity of a cancer cell to the cytotoxic effects of radiotherapy and/or chemotherapy comprising contacting the cell with a present HDACI or a pharmaceutically acceptable salt thereof in an amount sufficient to increase the sensitivity of the cell to the cytotoxic effects of radiotherapy and/or chemotherapy.
[0052] In a further embodiment, the present invention provides a method for treating cancer comprising: (a) administering to an individual in need thereof an amount of a present HDACI compound; and (b) administering to the individual an amount of radiotherapy, chemotherapy, or both. The amounts administered are each effective to treat cancer. In another embodiment, the amounts are together effective to treat cancer.
[0053] In another embodiment, the invention provides a method for treating cancer, said method comprising administering to a subject in need thereof a pharmaceutical composition comprising an amount of a present HDACI effective to treat cancer.
[0054] This combination therapy of the invention can be used accordingly in a variety of settings for the treatment of various cancers. In a specific embodiment, the individual in need of treatment has previously undergone treatment for cancer. Such previous treatments include, but are not limited to, prior chemotherapy, radiotherapy, surgery, or immunotherapy, such as cancer vaccines. [0055] In another embodiment, the cancer being treated is a cancer which has demonstrated sensitivity to radiotherapy and/or chemotherapy or is known to be responsive to radiotherapy and/or chemotherapy. Such cancers include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, larynx cancer, cervical cancer, nasopharynx cancer, breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small-cell lung cancer, non-small cell lung cancer, brain tumors, or other CNS neoplasms.
[0056] In still another embodiment, the cancer being treated has demonstrated resistance to radiotherapy and/or chemotherapy or is known to be refractory to radiotherapy and/or chemotherapy. A cancer is refractory to a therapy when at least some significant portion of the cancer cells are not killed or their cell division is not arrested in response to therapy. Such a determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context. In a specific embodiment, a cancer is refractory where the number of cancer cells has not been significantly reduced or has increased.
[0057] Other cancers that can be treated with the compounds and methods of the invention include, but are not limited to, cancers and metastases selected from the group consisting of solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma; blood-borne cancers, including but not limited to: acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblasts leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myclomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and multiple myeloma; acute and chronic leukemias: lymphoblastic, myelogenous lymphocytic, and myelocytic leukemias; lymphomas: Hodgkin's disease and non-Hodgkin's lymphoma; multiple myeloma; Waldenstrom's macroglobulinemia; heavy chain disease; and polycythemia vera.
[0058] The present HDACIs can also be administered to prevent progression to a neoplastic or malignant state, including but not limited to the cancers listed above. Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. For example, endometrial hyperplasia often precedes endometrial cancer and precancerous colon polyps often transform into cancerous lesions. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where chronic irritation or inflammation exists, and often is found in the cervix, respiratory passages, oral cavity, and gall bladder.
[0059] Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a subject, can indicate the desirability of prophylactic/therapeutic administration of the composition of the invention. Such characteristics of a transformed phenotype include, for example, morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein.
[0060] In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
[0061] In another embodiment, fibrocystic disease (cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia)) is indicative of the desirability of prophylactic intervention.
[0062] The prophylactic use of the compounds and methods of the present invention are also indicated in some viral infections that may lead to cancer. For example, human papilloma virus can lead to cervical cancer (see, e.g., Hernandez- Avila et al., Archives of Medical Research (1997) 28:265-271), Epstein-Barr virus (EBV) can lead to lymphoma (see, e.g., Herrmann et al., Pathol (2003) 199(2): 140-5), hepatitis B or C virus can lead to liver carcinoma (see, e.g., El- Serag, Clin Gastroenterol (2002) 35(5 Suppl 2):S72-8), human T cell leukemia virus (HTLV)-I can lead to T-cell leukemia (see e.g., Mortreux et al., Leukemia (2003) 17(l):26-38), human herpesvirus-8 infection can lead to Kaposi's sarcoma (see, e.g., Kadow et al., Curr Opin Investig Drugs (2002) 3(11): 1574-9), and Human Immune deficiency Virus (HIV) infection contribute to cancer development as a consequence of immunodeficiency (see, e.g., Dal Maso et al., Lancet Oncol (2003) 4(2): 110-9).
[0063] In other embodiments, a subject exhibiting one or more of the following predisposing factors for malignancy can be treated by administration of the present HDACIs and methods of the invention: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), a first degree kinship with persons having a cancer or procancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.), and exposure to carcinogens (e.g., smoking, and inhalation of or contacting with certain chemicals).
[0064] In another specific embodiment, the present HDACIs and methods of the invention are administered to a human subject to prevent progression of breast, colon, ovarian, or cervical cancer.
[0065] In one embodiment, the invention provides methods for treating cancer comprising (a) administering to an individual in need thereof an amount of a present HDACI; and (b) administering to the individual one or more additional anticancer treatment modality including, but not limited to, radiotherapy, chemotherapy, surgery or immunotherapy, such as a cancer vaccine. In one embodiment, the administering of step (a) is prior to the administering of step (b). In another embodiment, the administering of step (a) is subsequent to the administering of step (b). In still another embodiment, the administering of step (a) is concurrent with the administering of step (b).
[0066] In one embodiment, the additional anticancer treatment modality is radiotherapy and/or chemotherapy. In another embodiment, the additional anticancer treatment modality is surgery.
[0067] In still another embodiment, the additional anticancer treatment modality is immunotherapy, such as cancer vaccines.
[0068] In one embodiment, a present HDACI or a pharmaceutically acceptable salt thereof is administered adjunctively with the additional anticancer treatment modality.
[0069] In a preferred embodiment, the additional anticancer treatment modality is radiotherapy. In the methods of the present invention, any radiotherapy protocol can be used depending upon the type of cancer to be treated. Embodiments of the present invention employ electromagnetic radiation of: gamma-radiation (10" to 10" m), X-ray radiation (10" to 10" m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1 mm), and microwave radiation (1 mm to 30 cm).
[0070] For example, but not by way of limitation, X-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage X-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered. Illustrative radiotherapy protocols useful in the present invention include, but are not limited to, stereotactic methods where multiple sources of low dose radiation are simultaneously focused into a tissue volume from multiple angles; "internal radiotherapy," such as brachytherapy, interstitial irradiation, and intracavitary irradiation, which involves the placement of radioactive implants directly in a tumor or other target tissue; intraoperative irradiation, in which a large dose of external radiation is directed at the target tissue which is exposed during surgery; and particle beam radiotherapy, which involves the use of fast-moving subatomic particles to treat localized cancers.
[0071] Many cancer treatment protocols currently employ radiosensitizers activated by electromagnetic radiation, e.g., X-rays. Examples of X-ray-activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5- bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cis-platin, and therapeutically effective analogs and derivatives of the same.
[0072] Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same. [0073] Radiosensitizers can be administered in conjunction with a therapeutically effective amount of one or more compounds in addition to a present HDACI, such compounds including, but not limited to, compounds that promote the incorporation of radiosensitizers to the target cells, compounds that control the flow of therapeutics, nutrients, and/or oxygen to the target cells, chemotherapeutic agents that act on the tumor with or without additional radiation, or other therapeutically effective compounds for treating cancer or other disease. Examples of additional therapeutic agents that can be used in conjunction with radiosensitizers include, but are not limited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., FLUOSOLW®-DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, antiangiogenesis compounds, hydralazine, and L-BSO.
[0074] In a preferred embodiment, a present HDACI or a pharmaceutically acceptable salt thereof is administered prior to the administration of radiotherapy and/or chemotherapy.
[0075] In another preferred embodiment, a present HDACI or a pharmaceutically acceptable salt thereof is administered adjunctively with radiotherapy and/or chemotherapy.
[0076] A present HDACI and additional treatment modalities can act additively or synergistically (i.e., the combination of a present HDACI or a pharmaceutically acceptable salt thereof, and an additional anticancer treatment modality is more effective than their additive effects when each are administered alone). A synergistic combination permits the use of lower dosages of a present HDACI and/or the additional treatment modality and/or less frequent administration of a present HDACI and/or additional treatment modality to a subject with cancer. The ability to utilize lower dosages of a present HDACI and/or an additional treatment modality and/or to administer a compound of the invention and the additional treatment modality less frequently can reduce the toxicity associated with the administration without reducing the efficacy of a present HDACI and/or the additional treatment modality in the treatment of cancer. In addition, a synergistic effect can result in the improved efficacy of the treatment of cancer and/or the reduction of adverse or unwanted side effects associated with the administration of a present HDACI and/or an additional anticancer treatment modality as monotherapy.
[0077] In one embodiment, the present HDACIs may act synergistically with radiotherapy when administered in doses typically employed when such HDACIs are used alone for the treatment of cancer. In another embodiment, the present HDACIs may act synergistically with radiotherapy when administered in doses that are less than doses typically employed when such HDACIs are used as monotherapy for the treatment of cancer.
[0078] In one embodiment, radiotherapy may act synergistically with a present HDACI when administered in doses typically employed when radiotherapy is used as monotherapy for the treatment of cancer. In another embodiment, radiotherapy may act synergistically with a compound of the invention when administered in doses that are less than doses typically employed when radiotherapy is used as monotherapy for the treatment of cancer.
[0079] The effectiveness of the HDACIs as HDAC inhibitors for sensitizing cancer cells to the effect of radiotherapy can be determined by the in vitro and/or in vivo determination of post- treatment survival using techniques known in the art. In one embodiment, for in vitro determinations, exponentially growing cells can be exposed to known doses of radiation, and the survival of the cells monitored. Irradiated cells are plated and cultured for about 14- about 21 days, and the colonies are stained. The surviving fraction is the number of colonies divided by the plating efficiency of unirradiated cells. Graphing the surviving fraction on a log scale versus the absorbed dose on a linear scale generates a survival curve. Survival curves generally show an exponential decrease in the fraction of surviving cells at higher radiation doses after an initial shoulder region in which the dose is sublethal. A similar protocol can be used for chemical agents when used in the combination therapies of the invention.
[0080] Inherent radiosensitivity of tumor cells and environmental influences, such as hypoxia and host immunity, can be further assessed by in vivo studies. The growth delay assay is commonly used. This assay measures the time interval required for a tumor exposed to radiation to regrow to a specified volume. The dose required to control about 50% of tumors is determined by the TCD50 assay.
[0081] In vivo assay systems typically use transplantable solid tumor systems in experimental subjects. Radiation survival parameters for normal tissues as well as for tumors can be assayed using in vivo methods known in the art.
[0082] The present invention provides methods of treating cancers comprising the administration of an effective amount of a present HDACI in conjunction with recognized methods of surgery, radiotherapy, and chemotherapies, including, for example, chemical-based mimics of radiotherapy whereby a synergistic enhancement of the effectiveness of the recognized therapy is achieved. The effectiveness of a treatment can be measured in clinical studies or in model systems, such as a tumor model in mice, or cell culture sensitivity assays.
[0083] The present invention provides combination therapies that result in improved effectiveness and/or reduced toxicity. Accordingly, in one aspect, the invention relates to the use of the present HDACIs as radiosensitizers in conjunction with radiotherapy.
[0084] When the combination therapy of the invention comprises administering a present HDACI with one or more additional anticancer agents, the present HDACI and the additional anticancer agents can be administered concurrently or sequentially to an individual. The agents can also be cyclically administered. Cycling therapy involves the administration of one or more anticancer agents for a period of time, followed by the administration of one or more different anticancer agents for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one or more of the anticancer agents of being administered, to avoid or reduce the side effects of one or more of the anticancer agents being administered, and/or to improve the efficacy of the treatment.
[0085] An additional anticancer agent may be administered over a series of sessions; anyone or a combination of the additional anticancer agents listed below may be administered.
[0086] The present invention includes methods for treating cancer comprising administering to an individual in need thereof a present HDACI and one or more additional anticancer agents or pharmaceutically acceptable salts thereof. A present HDACI and the additional anticancer agent can act additively or synergistically. Suitable anticancer agents include, but are not limited to, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mereaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campatheeins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epirubicin, 5-fluorouracil (5-FU), taxanes (such as docetaxel and paclitaxel), leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas (such as carmustine and lomustine), platinum complexes (such as cisplatin, carboplatin and oxaliplatin), imatinib mesylate, hexamethylmelamine, topotecan, tyrosine kinase inhibitors, tyrphostins herbimycin A, genistein, erbstatin, and lavendustin A.
[0087] In one embodiment, the anti-cancer agent can be, but is not limited to, a drug selected from the group consisting of alkylating agents, nitrogen mustards, cyclophosphamide, trofosfamide, chlorambucil, nitrosoureas, carmustine (BCNU), lomustine (CCNU), alkylsulphonates, busulfan, treosulfan, triazenes, plant alkaloids, vinca alkaloids (vineristine, vinblastine, vindesine, vinorelbine), taxoids, DNA topoisomcrase inhibitors, epipodophyllins, 9- aminocamptothecin, camptothecin, crisnatol, mitomycins, mitomycin C, anti-metabolites, anti- folates, DHFR inhibitors, trimetrexate, IMP dehydrogenase inhibitors, mycophenolic acid, tiazofurin, ribavirin, EICAR, ribonuclotide reductase inhibitors, hydroxyurea, deferoxamine, pyrimidine analogs, uracil analogs, floxuridine, doxifluridine, ratitrexed, cytosine analogs, cytarabine (ara C), cytosine arabinoside, fludarabine, purine analogs, mercaptopurine, thioguanine, DNA antimetabolites, 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole (inosine glycodialdehyde), macebecin II, pyrazoloimidazole, hormonal therapies, receptor antagonists, anti-estrogen, tamoxifen, raloxifene, megestrol, LHRH agonists, goserelin, leuprolide acetate, anti-androgens, flutamide, bicalutamide, retinoids/deltoids, cis-retinoic acid, vitamin A derivative, all-trans retinoic acid (ATRA-IV), vitamin D3 analogs, El) 1089, CB 1093, ICH 1060, photodynamic therapies, vertoporfin, BPD-MA, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A (2BA-2-DMHA), cytokines, interferon-a, interferon-I3, interferon-y, tumor necrosis factor, angiogenesis inhibitors, angiostatin (plasminogen fragment), antiangiogenic antithrombin UI, angiozyme, ABT-627, Bay 12- 9566, benefin, bevacizumab, BMS-275291, cartilage-derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), fibronectin fragment, Gro-beta, halofuginone, heparinases, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon inducible protein (IP- 10), interleukin-12, kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitors (UMPs), 2-methoxyestradiol, MMI 270 (CGS 27023A), MoAb IMC-I Cl l, neovastat, NM-3, panzem, Pl-88, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prinomastat, prolactin 161(D fragment, proliferin-related protein (PRP), PTK 787/ZK 222594, retinoids, solimastat, squalamine, SS 3304, SU 5416, SU 6668, SU 11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-470, transforming growth factor-beta (TGF-11), vasculostatin, vasostatin (calreticulin fragment), ZD 6126, ZD 6474, famesyl transferase inhibitors (FTI), bisphosphonates, antimitotic agents, allocolchicine, halichondrin B, colchicine, colchicine derivative, dolstatin 10, maytansine, rhizoxin, thiocolchicine, trityl cysteine, isoprenylation inhibitors, dopaminergic neurotoxins, l-methyl-4- phenylpyridinium ion, cell cycle inhibitors, staurosporine, actinomycins, actinomycin D, dactinomycin, bleomycins, bleomycin A2, bleomycin B2, peplomycin, anthracycline, adriamycin, epirubicin, pirarnbicin, zorubicin, mitoxantrone, MDR inhibitors, verapamil, Ca 'ATPase inhibitors, and thapsigargin.
[0088] Other anti-cancer agents that may be used in the present invention include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; arnbomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelcsin; bleomycin sulfate; brequinar sodium; bropirimine; busul fan; cactinomycin; calusterone; caracemide; carbetimer; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexorrnaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta- la; interferon gamma- lb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mecchlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitusper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfarnide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsornycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracit mustard; uredepa; vapreotide; verteporfln; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozolc; zeniplatin; zinostatin; zorubicin hydrochloride.
[0089] Further anti-cancer drugs that can be used in the present invention include, but are not limited to: 17-AAG; 20-epi-l,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL TK antagonists; altretamine; ambamustine; amidox; arnifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara CDP DL PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR-ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta alethine; betaclarnycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylsperrnine; bisnafide; bistratene A; bizelesin; bortezomib; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL- 2; carboxamide amino triazole; carboxyarnidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors; castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexveraparnil; diaziquone; didemnin B; didox; diethylnor spermine; dihydro 5 azacytidine; dihydrotaxol, 9; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fltidarabine; fluorodaunoruniein hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immuno stimulant peptides; insulin like growth factor 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubiein; ipomeanol, 4 ; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum complexes; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 based therapy; mustard anti-cancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N acetyldinaline; N substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06 benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum complexes; platinum triamine complex; porfimer sodium; porfiromycin; prednisone; acridones; prostaglandin J2; proteasome inhibitors; protein A based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloaeridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone BI; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0090] It is a further aspect of the invention that the present HDACIs can be administered in conjunction with chemical agents that are understood to mimic the effects of radiotherapy and/or that function by direct contact with DNA. Preferred agents for use in combination with the present HDACIs for treating cancer include, but are not limited to cis-diamminedichloro platinum (II) (cisplatin), doxorubicin, 5-fluorouracil, taxol, and topoisomerase inhibitors such as etoposide, teniposide, irinotecan and topotecan.
[0091] Additionally, the invention provides methods of treatment of cancer using the present HDACIs as an alternative to chemotherapy alone or radiotherapy alone where the chemotherapy or the radiotherapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The individual being treated can, optionally, be treated with another anticancer treatment modality such as chemotherapy, surgery, or immunotherapy, depending on which treatment is found to be acceptable or bearable.
[0092] The present HDACIs can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This can involve a multi-step process in which the subject's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the subject is then administered an amount of a present HDACI effective to eradicate the subject's remaining bone-marrow cell population, then the stem cell graft is infused back into the subject. Supportive care then is provided while bone marrow function is restored and the subject recovers. [0093] The present methods for treating cancer can further comprise the administration of a present HDACI and an additional therapeutic agent or pharmaceutically acceptable salts or hydrates thereof. In one embodiment, a composition comprising a present HDACI is administered concurrently with the administration of one or more additional therapeutic agent(s), which may be part of the same composition or in a different composition from that comprising the present HDACI. In another embodiment, a present HDACI is administered prior to or subsequent to administration of another therapeutic agent(s).
[0094] In the present methods for treating cancer the other therapeutic agent may be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine, and tropisetron.
[0095] In a preferred embodiment, the antiemetic agent is granisetron or ondansetron. In another embodiment, the other therapeutic agent may be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargrarnostim, molgramostim, and epoietin alfa.
[0096] In still another embodiment, the other therapeutic agent may be an opioid or nonopioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone, and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam, and sulindac. [0097] In still another embodiment, the other therapeutic agent may be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirene, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
[0098] In addition to treating cancers and sensitizing a cancer cell to the cytotoxic effects of radiotherapy and chemotherapy, the present HDACIs are used in methods of treating diseases, conditions, and injuries to the central nervous system, such as neurological diseases, neurodegenerative disorders, and traumatic brain injuries (TBIs). In preferred embodiments, a present HDACI is capable of crossing the blood brain barrier to inhibit HDAC in the brain of the individual.
[0099] The present HDACI compounds also provide a therapeutic benefit in models of peripheral neuropathies, such as CMT. HDAC6 inhibitors have been found to cross the blood nerve barrier and rescue the phenotype observed in transgenic mice exhibiting symptons of distal hereditary motor neuropathy. Administration of HDAC6 inhibitors to symptomatic mice increased acetylated a- tubulin levels, restored proper mitochondrial motility and axonal transport, and increased muscle re-innervation. Other peripheral neuropathies include, but are not limited to, giant axonal neuropathy and various forms of mononeuropathies, polyneuropathies, autonomic neuropathies, and neuritis.
[00100] The present HDACI compounds also ameliorate associative memory loss following Αβ elevation. In this test, mice were infused with Αβ42 via cannulas implanted into dorsal hippocampus 15 minutes prior to training. The test compounds are dosed ip (25 mg/kg) 2 hours before training. Fear learning was assessed 24 hours later.
[0100] Contextual fear conditioning performed 24 hours after training shows a reduction of freezing in Αβ-infused mice compared to vehicle-infused mice. Treatment with a present compound ameliorates deficit in freezing responses in Αβ-infused mice, and has no effect in vehicle-infused mice. A test compound alone does not affect the memory performance of the mice. In addition, treatment had no effects on motor, sensorial, or motivational skills assessed using the visible platform test in which the compounds are injected twice a day for two days. During these experiments, no signs of overt toxicity, including changes in food and liquid intake, weight loss, or changes in locomotion and exploratory behavior, are observed. [0101] These results demonstrate that the HDACIs of the present invention are beneficial against impairment of associative memory following Αβ elevation.
[0102] The present HDACIs therefore are useful for treating a neurological disease by administration of amounts of a present HDACI effective to treat the neurological disease or by administration of a pharmaceutical composition comprising amounts of a present HDACI effective to treat the neurological disease. The neurological diseases that can be treated include, but are not limited to, Huntington's disease, lupus, schizophrenia, multiple sclerosis, muscular dystrophy, dentatorubralpallidoluysian atrophy (DRRLA), spinal and bulbar muscular atrophy (SBMA), and fine spinocerebellar ataxias (SCAl, SCA2, SCA3/MJD (Machado- Joseph Disease), SCA6, and SCA7), drug-induced movement disorders, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, Pick's disease, Alzheimer's disease, Lewy body dementia, cortico basal degeneration, dystonia, myoclonus, Tourette's syndrome, tremor, chorea, restless leg syndrome, Parkinson's disease, Parkinsonian syndromes, anxiety, depression, psychosis, manic depression, Friedreich's ataxia , Fragile X syndrome, spinal muscular dystrophy, Rett syndrome, Rubinstein-Taybi syndrome, Wilson's disease, multi-infarct state, CMT, GAN and other peripheral neuropathies.
[0103] In a preferred embodiment, the neurological disease treated is Huntington's disease, Parkinson's disease, Alzheimer's disease, spinal muscular atrophy, lupus, or schizophrenia.
[0104] A present HDACI also can be used with a second therapeutic agent in methods of treating conditions, diseases, and injuries to the CNS. Such second therapeutic agents are those drugs known in the art to treat a particular condition, diseases, or injury, for example, but not limited to, lithium in the treatment of mood disorders, estradiol benzoate, and nicotinamide in the treatment of Huntington's disease.
[0105] The present HDACIs also are useful in the treatment of TBIs. Traumatic brain injury (TBI) is a serious and complex injury that occurs in approximately 1.4 million people each year in the United States. TBI is associated with a broad spectrum of symptoms and disabilities, including a risk factor for developing neurodegenerative disorders, such as Alzheimer's disease.
[0106] TBI produces a number of pathologies including axonal injury, cell death, contusions, and inflammation. The inflammatory cascade is characterized by proinflammatory cytokines and activation of microglia which can exacerbate other pathologies. Although the role of inflammation in TBI is well established, no efficacious anti-inflammatory therapies are currently available for the treatment of TBI.
[0107] Several known HDAC inhibitors have been found to be protective in different cellular and animal models of acute and chronic neurodegenerative injury and disease, for example, Alzheimer's disease, ischemic stroke, multiple sclerosis (MS), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). A recent study in experimental pediatric TBI reported a decrease in hippocampal CA3 histone H3 acetylation lasting hours to days after injury. These changes were attributed to documented upstream excitotoxic and stress cascades associated with TBI. HDACIs also have been reported to have anti-inflammatory actions acting through acetylation of non-histone proteins. The HDAC6 selective inhibitor, 4-dimethylamino-N-[5-(2- mercaptoacetylamino)pentyl]benzamide (DMA-PB), was found to be able to increase histone H3 acetylation and reduce microglia inflammatory response following traumatic brain injury in rats, which demonstrates the utility of HDACIs as therapeutics for inhibiting neuroinflammation associated with TBI.
[0108] The present HDACIs therefore also are useful in the treatment of inflammation and strokes, and in the treatment of autism and autism spectrum disorders. The present HDACIs further can be used to treat parasitic infections, (e.g., malaria, toxoplasmosis, trypanosomiasis, helminthiasis, protozoal infections (see Andrews et al. Int. J. Parasitol. 2000, 30(6), 761-768).
[0109] In certain embodiments, the compound of the invention can be used to treat malaria. A present HDACI can be co-administered with an antimalarial compound selected from the group consisting of aryl amino alcohols, cinchona alkaloids, 4-aminoquinolines, type 1 or type 2 folate synthesis inhibitors, 8-aminoquinolines, antimicrobials, peroxides, naphthoquinones, and iron chelating agents. The antimalarial compound can be, but is not limited to, quinine, quinidine, mefloquine, halfantrine, chloroquine, amodiaquine, proguanil, chloroproquanil, pyrimethamine, primaquine, 8-[(4-amino-l-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5- [(3-trifluoromethyl)phenoxy]quinoline succinate (WR238,605), tetracycline, doxycycline, clindamycin, azithromycin, fluoroquinolones, artemether, areether, artesunate, artelinic acid, atovaquone, and deferrioxamine. In a preferred embodiment, the antimalarial compound is chloroquine.
[0110] The present HDACIs also can be used as imaging agents. In particular, by providing a radiolabeled, isotopically labeled, or fluorescently-labeled HDACI, the labeled compound can image HDACs, tissues expressing HDACs, and tumors. Labeled HDACIs of the present invention also can image patients suffering from a cancer, or other HDAC-mediated diseases, e.g., stroke, by administration of an effective amount of the labeled compound or a composition containing the labeled compound. In preferred embodiments, the labeled HDACI is capable of emitting positron radiation and is suitable for use in positron emission tomography (PET). Typically, a labeled HDACI of the present invention is used to identify areas of tissues or targets that express high concentrations of HDACs. The extent of accumulation of labeled HDACI can be quantified using known methods for quantifying radioactive emissions. In addition, the labeled HDACI can contain a fluorophore or similar reporter capable of tracking the movement of particular HDAC isoforms or organelles in vitro.
[0111] The present HDACIs useful in the imaging methods contain one or more radioisotopes capable of emitting one or more forms of radiation suitable for detection by any standard radiology equipment, such as PET, SPECT, gamma cameras, MRI, and similar apparatus.
Preferred isotopes including tritium ( 3 H) and carbon ( 11 C). Substituted HDACIs of the present invention also can contain isotopes of fluorine ( 18 F) and iodine ( 123 I) for imaging methods. Typically, a labeled HDACI of the present invention contains an alkyl group having a UC label, i.e., a 11 C-methyl group, or an alkyl group substituted with 18 F, 123 I, 125 I, 131 I, or a combination thereof.
[0112] Fluorescently-labeled HDACIs of the present invention also can be used in the imaging method of the present invention. Such compounds have an FITC,carbocyamine moiety or other fluorophore which will allow visualization of the HDAC proteins in vitro.
[0113] The labeled HDACIs and methods of use can be in vivo, and particularly on humans, and for in vitro applications, such as diagnostic and research applications, using body fluids and cell samples. Imaging methods using a labeled HDACI of the present invention are discussed in WO 03/060523, designating the U.S. and incorporated in its entirety herein. Typically, the method comprises contacting cells or tissues with a radiolabeled, isotopically labeled, fluorescently labeled, or tagged (such as biotin tagged) compound of the invention, and making a radiographic, fluorescent, or similar type of image depending on the visualization method employed, i.e., in regared to radiographic images, a sufficient amount to provide about 1 to about 30 mCi of the radiolabeled compound.
[0114] Preferred imaging methods include the use of labeled HDACIs of the present invention which are capable of generating at least a 2: 1 target to background ratio of radiation intensity, or more preferably about a 5: 1, about 10: 1, or about 15: 1 ratio of radiation intensity between target and background.
[0115] In preferred methods, the labeled HDACIs of the present invention are excreted from tissues of the body quickly to prevent prolonged exposure to the radiation of the radiolabeled compound administered to the individual. Typically, labeled HDACIs of the present invention are eliminated from the body in less than about 24 hours. More preferably, labeled HDACIs are eliminated from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes. Typically, preferred labeled HDACIs are eliminated in about 60 to about 120 minutes.
[0116] In addition to isotopically labeled and fluorescently labeled derivatives, the present invention also embodies the use of derivatives containing tags (such as biotin) for the identification of biomolecules associated with the HDAC isoforms of interest for diagnostic, therapeutic or research purposes.
[0117] The present HDACIs also are useful in the treatment of autoimmune diseases and inflammations. Compounds of the present invention are particularly useful in overcoming graft and transplant rejections and in treating forms of arthritis.
[0118] Despite successes of modern transplant programs, the nephrotoxicity, cardiovascular disease, diabetes, and hyperlipidemia associated with current therapeutic regimens, plus the incidence of post-transplant malignancies and graft loss from chronic rejection, drive efforts to achieve long-term allograft function in association with minimal immunosuppression. Likewise, the incidence of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is increasing. Animal studies have shown that T regulatory cells (Tregs) expressing the forkhead transcription family member, Foxp3, are key to limiting autoreactive and alloreactive immunity. Moreover, after their induction by costimulation blockade, immunosuppression, or other strategies, Tregs may be adoptively transferred to naive hosts to achieve beneficial therapeutic effects. However, attempts to develop sufficient Tregs that maintain their suppressive functions post-transfer in clinical trials have failed. Murine studies show that HDACIs limit immune responses, at least in significant part, by increasing Treg suppressive functions, (R. Tao et al., Nat Med, 13, 1299-1307, (2007)), and that selective targeting of HDAC6 is especially efficacious in this regard.
[0119] With organ transplantation, rejection begins to develop in the days immediately post- transplant, such that prevention rather than treatment of rejection is a paramount consideration. The reverse applies in autoimmunity, wherein a patient presents with the disease already causing problems. Accordingly, HDAC6-/- mice treated for 14 days with low-dose RPM (rapamycin) are assessed for displaying signs of tolerance induction and resistance to the development of chronic rejection, a continuing major loss of graft function long-term in the clinical transplant population. Tolerance is assessed by testing whether mice with long-surviving allografts reject a subsequent third-party cardiac graft and accept additional donor allografts without any immunosuppression, as can occur using a non-selective HDACI plus RPM. These in vivo studies are accompanied by assessment of ELISPOT and MLR activities using recipient lymphocytes challenged with donor cells. Protection against chronic rejection is assessed by analysis of host anti-donor humoral responses and analysis of graft transplant arteriosclerosis and interstitial fibrosis in long- surviving allograft recipients.
[0120] The importance of HDAC6 targeting is assessed in additional transplant models seeking readouts of biochemical significance, as is monitored clinically. Thus, the effects of HDAC6 in targeting in renal transplant recipients (monitoring BUN, proteinuria) and islet allografts (monitoring blood glucose levels) are assessed. Renal transplants are the most common organ transplants performed, and the kidney performs multiple functions, e.g., regulating acid/base metabolism, blood pressure, red cell production, such that efficacy in this model indicates the utility of HDAC6 targeting. Likewise, islet transplantation is a major unmet need given that clinical islet allografts are typically lost after the first one or two years post- transplant. Having a safe and non-toxic means to extend islet survival without maintenance CNI therapy would be an important advance. Transplant studies also are strengthened by use of mice with floxed HDAC6. Using existing Foxp3-Cre mice, for example, the effects of deletion of HDAC6 just in Tregs is tested. This approach can be extended to targeting of HDAC6 in T cells (CD4-Cre) and dendritic cells (CDl lc-Cre), for example. Using tamoxifen-regulated Cre, the importance of HDAC6 in induction vs. maintenance of transplants (with implications for short- term vs. maintenance HDAC6I therapy) is assessed by administering tamoxifen and inducing HDAC6 deletion at varying periods post-transplant.
[0121] Studies of autoimmunity also are undertaken. In this case, interruption of existing disease is especially important and HDAC6 targeting can be efficacious without any requirement for additional therapy (in contrast to a need for brief low-dose RPM in the very aggressive, fully MHC-mismatched transplant models). Studies in mice with colitis indicated that HDAC6-/- Tregs were more effective than WT Tregs in regulating disease, and tubacin was able to rescue mice if treatment was begun once colitis had developed. These studies are extended by assessing whether deletion of HDAC6 in Tregs (Foxp3/Cre) vs. T cells (CD4=Cre) vs. DC (CDl lc-Cre) differentially affect the development and severity of colitis. Similarly, control of colitis is assessed by inducing HDAC6 deletion at varying intervals after the onset of colitis with tamoxifen-regulated Cre.
[0122] The present compounds are envisioned to demonstrate anti- arthritic efficacy in a collagen-induced arthritis model in DBA1/J mice. In this test, DBA1/J mice (male, 7-8 weeks) are used, with 8 animals per group. Systemic arthritis is induced with bovine collagen type II and CFA, plus an IFA booster injection on day 21. A present HDACI is dosed at 50 mg/kg and 100 mg/kg on day 28 for 2 consecutive weeks, and the effects determined from the Average Arthritic Score vs. Days of Treatment data.
[0123] Despite efforts to avoid graft rejection through host-donor tissue type matching, in the majority of transplantation procedures, immunosuppressive therapy is critical to the viability of the donor organ in the host. A variety of immunosuppressive agents have been employed in transplantation procedures, including azathioprine, methotrexate, cyclophosphamide, FK-506, rapamycin, and corticosteroids. [0124] The present HDACIs are potent immunosuppressive agents that suppress humoral immunity and cell-mediated immune reactions, such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis and graft versus host disease. HDACIs of the present invention are useful for the prophylaxis of organ rejection subsequent to organ transplantation, for treatment of rheumatoid arthritis, for the treatment of psoriasis, and for the treatment of other autoimmune diseases, such as type I diabetes, Crohn's disease, and lupus.
[0125] A therapeutically effective amount of a present HDACI can be used for immunosuppression including, for example, to prevent organ rejection or graft vs. host disease, and to treat diseases and conditions, in particular, autoimmune and inflammatory diseases and conditions. Examples of autoimmune and inflammatory diseases include, but are not limited to, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, psoriasis, diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, arthritis (rheumatoid arthritis, arthritis chronic progrediente, and arthritis deformans) and rheumatic diseases, autoimmune hematological disorder (hemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopaenia), systemic lupus erythematosus, polychondritis, sclerodoma, Wegener granulamatosis, dermatomyositis, chronic active hepatitis, psoriasis, Steven- Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (ulcerative colitis and Crohn's disease) endocrine opthalmopathy, Graves disease, sarcoidosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis, and glomerulonephritis.
[0126] A present HDACI can be used alone, or in conjunction with a second therapeutic agent known to be useful in the treatment of autoimmune diseases, inflammations, transplants, and grafts, such as cyclosporin, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to persons skilled in the art.
[0127] Additional diseases and conditions mediated by HDACs, and particularly HDAC6, include, but are not limited to asthma, cardiac hypertrophy, giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy, autonomic neuropathy, neuritis in general, and neuropathy in general. These disease and conditions also can be treated by a method of the present invention.
[0128] In the present method, a therapeutically effective amount of one or more HDACI of the present invention, typically formulated in accordance with pharmaceutical practice, is administered to a human being in need thereof. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment (diagnosis) that takes into consideration signs, symptoms, and/or malfunctions that are present, the risks of developing particular signs, symptoms and/or malfunctions, and other factors.
[0129] A present HDACI can be administered by any suitable route, for example by oral, buccal, inhalation, topical, sublingual, rectal, vaginal, intracisternal or intrathecal through lumbar puncture, transurethral, nasal, percutaneous, i.e., transdermal, or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site) administration. Parenteral administration can be accomplished using a needle and syringe or using a high pressure technique.
[0130] Pharmaceutical compositions include those wherein a present HDACI is present in a sufficient amount to be administered in an effective amount to achieve its intended purpose. The exact formulation, route of administration, and dosage is determined by an individual physician in view of the diagnosed condition or disease. Dosage amount and interval can be adjusted individually to provide levels of a present HDACI that is sufficient to maintain therapeutic effects.
[0131] Toxicity and therapeutic efficacy of the present HDACI compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from such procedures can be used in formulating a dosage range for use in humans. The dosage preferably lies within a range of circulating compound concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
[0132] A therapeutically effective amount of a present HDACI required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and ultimately is determined by the attendant physician. Dosage amounts and intervals can be adjusted individually to provide plasma levels of the HDACI that are sufficient to maintain the desired therapeutic effects. The desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more subdoses per day. Multiple doses often are desired, or required. For example, a present HDACI can be administered at a frequency of: four doses delivered as one dose per day at four-day intervals (q4d x 4); four doses delivered as one dose per day at three-day intervals (q3d x 4); one dose delivered per day at five- day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5); or, any dose regimen determined to be appropriate for the circumstance.
[0133] The dosage of a composition containing a present HDACI, or a composition containing the same, can be from about 1 ng/kg to about 200 mg/kg, about 1 μg/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg of body weight. The dosage of a composition may be at any dosage including, but not limited to, about 1 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μβ^β, 325 μβ^β, 350 μβ^β, 375 μβ^β, 400 μβ^β, 425 μβ^β, 450 μβ^β, 475 μβ^β, 500 μβ^β, 525 μg/kg, 550 μβ^β, 575 μβ^β, 600 μβ^β, 625 μβ^β, 650 μβ^β, 675 μβ^β, 700 μβ^β, 725 μβ^β, 750 μβ^β, 775 μβ^β, 800 μβ^β, 825 μβ^β, 850 μβ^β, 875 μβ^β, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, or 200 mg/kg. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention. In practice, the physician determines the actual dosing regimen that is most suitable for an individual patient, which can vary with the age, weight, and response of the particular patient.
[0134] A present HDACI used in a method of the present invention typically is administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, a present HDACI can be administered, per dose, in an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams, including all doses between 0.005 and 500 milligrams.
[0135] The HDACIs of the present invention typically are administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the present HDACIs.
[0136] The term "carrier" refers to a diluent, adjuvant, or excipient, with which a present HDACI is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. The pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when a present HDACI is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
[0137] These pharmaceutical compositions can be manufactured, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of a present HDACI is administered orally, the composition typically is in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition additionally can contain a solid carrier, such as a gelatin or an adjuvant. The tablet, capsule, and powder contain about 0.01% to about 95%, and preferably from about 1% to about 50%, of a present HDACI. When administered in liquid form, a liquid carrier, such as water, petroleum, or oils of animal or plant origin, can be added. The liquid form of the composition can further contain physiological saline solution, dextrose or other saccharide solutions, or glycols. When administered in liquid form, the composition contains about 0.1% to about 90%, and preferably about 1% to about 50%, by weight, of a present compound.
[0138] When a therapeutically effective amount of a present HDACI is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen- free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains an isotonic vehicle. A present HDACI can be infused with other fluids over a 10-30 minute span or over several hours.
[0139] The present HDACIs can be readily combined with pharmaceutically acceptable carriers well-known in the art. Such carriers enable the active agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a present HDACI to a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. If desired, disintegrating agents can be added.
[0140] A present HDACI can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
[0141] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of a present HDACI can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, a present composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0142] A present HDACI also can be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases. In addition to the formulations described previously, a present HDACI also can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, a present HDACI can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins.
[0143] In particular, a present HDACI can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents. The present HDACIs also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the present HDACIs are best used in the form of a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.
[0144] As an additional embodiment, the present invention includes kits which comprise one or more compounds or compositions packaged in a manner that facilitates their use to practice methods of the invention. In one simple embodiment, the kit includes a compound or composition described herein as useful for practice of a method (e.g., a composition comprising a present HDACI and an optional second therapeutic agent), packaged in a container, such as a sealed bottle or vessel, with a label affixed to the container or included in the kit that describes use of the compound or composition to practice the method of the invention. Preferably, the compound or composition is packaged in a unit dosage form. The kit further can include a device suitable for administering the composition according to the intended route of administration, for example, a syringe, drip bag, or patch. In another embodiment, the present compounds is a lyophilate. In this instance, the kit can further comprise an additional container which contains a solution useful for the reconstruction of the lyophilate.
[0145] Prior HDACIs possessed properties that hindered their development as therapeutic agents. In accordance with an important feature of the present invention, the present HDACIs were synthesized and evaluated as inhibitors for HDAC. The present compounds demonstrate an increased HDAC6 potency and selectivity against HDACI and HDAC8 with improvements in BEI relative to prior compounds. The improved properties of the present compounds, particularly the increase in BEI and reduced potency at HDAC8, indicate that the present compounds are useful for applications such as, but not limited to, immunosuppresssive and neuroprotective agents. For example, compounds of the present invention typically have a bonding affinity (IC50) to HDAC6 of less than ΙΟΟμΜ, less than 25μΜ, less than ΙΟμΜ, less than Ι μΜ, less than 0.5μΜ, and less than 0.2 μΜ.
Synthetic Methods and Procedures
[0146] The synthesis of mercaptoacetamide 1 was reported previously and is not covered by this patent (see: Chemistry and biology of mercaptoacetamides as novel histone deacetylase inhibitors . Chen B, Jung M, Velena A, Eliseeva E, Dritschilo A, Kozikowski AP., Bioorg Med
Chem Lett. 2005 Mar 1 ; 15(5): 1389-92) , while the mercaptoacetamides 2a-c were synthesized starting from N-protected amino alcohols of varying lengths (5a-c, Scheme 1). Their conversion into the corresponding alkyl halides (6a-c) took place under mild conditions in the presence of triphenylphosphine (PPh3) and tetrabromomethane (CBr4). 8-Aminoquinoline was alkylated with
6a-c under microwave (μ\¥) irradiation to give intermediates 7a-c, respectively. The Boc protecting groups were removed with trifluoroacetic acid (TFA), and then the free amines were subjected to a coupling reaction with 2-(tritylthio)acetic acid in the presence of benzotriazol-1- yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Removal of the trityl protecting groups in the presence of TFA and triethylsylane (Et3SiH) afforded the final mercaptoacetamides 2a-c.
[0147] The mercaptoacetamide 3a, containing a tetrahydroquinoline (THQ) cap, was obtained from coupling of N-Boc-7-aminoheptanoic acid (10, Scheme 1) with Ν,Ο- dimethylhydroxylamine hydrochloride to give the Weinreb amide 11, which was reduced to aldehyde 12 with LiAlH4. Subsequently, reductive amination of intermediate 12 with the THQ cap afforded 13. Next, Boc deprotection, amide formation in the presence of PyBOP, and removal of the trityl group gave mercaptoacetamide 3a.
[0148] Compound 3b was synthesized through coupling of the THQ cap moiety with N-Boc- 6-aminohexanoic acid (16). The resulting amide was then taken through the same three final steps described above for analog 3a.
Figure imgf000047_0001
[0149] Scheme 1. Reagents and conditions: a) PPh3, CBr4, THF, 0 °C to rt, 4 h, 78-95% yield; b) 8-aminoquinoline, Cs2C03, DMF, μψ, 120 °C, 40 min, 14-17% yield; c) TFA, CH2C12, rt, 2 h, 54-98% yield; d) 2-(tritylthio)acetic acid, PyBOP, Et3N, CH2C12, rt, 20 h, 56-89% yield; e) TFA, Et3SiH, CH2C12, 0 °C to rt, 2 h, 51-88% yield; f) N, 0-dimethylhydroxylamine hydrochloride, EDC, DMAP, Et3N, rt, overnight, 90% yield; g) LiAlH4, THF, -78 °C to 0 °C, 10 min, 94% yield; h) 1,2,3,4-tetrahydroquinoline, NaBH(OAc)3, CH3C02H, 1,2-dichloroethane, rt, overnight, 21% yield; i) 1,2,3,4-tetrahydroquinoline, PyBOP, DIPEA, DMF, rt, overnight, 61% yield.
[0150] The same synthetic path used for compound 3a led to analog 3c, introducing the 6- chloro- 1,2,3,4-tetrahydroquinoline core in the cap-linker reductive amination step (Scheme 2).
[0151] To synthesize compound 4, 4-[((ieri-butoxycarbonyl)amino)methyl]benzoic acid (25, Scheme 2) and 8-aminoquinoline were reacted in the presence of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP). The final N- deprotection, amide coupling, and S-deprotection steps were carried out as previously described.
Figure imgf000048_0001
[0152] Scheme 2. Reagents and conditions: a) N,0-dimethylhydroxylamine hydrochloride, EDC, DMAP, Et3N, rt, overnight, quantitative yield; )LiAlH4, THF, -78 °C, 1.5 h, 50% yield; c) 6-chloro- 1,2,3,4-tetrahydroquinoline, NaBH(OAc)3, 1,2-dichloroethane, rt, overnight, 66% yield; d) TFA, CH2C12, rt, 2 h, 88-93% yield; e) 2-(tritylthio)acetic acid, PyBOP, Et3N, CH2C12, rt, 20 h, 25-59% yield; ) TFA, Et3SiH, CH2C12, 0 °C to rt, 2 h, 51-73% yield; g) 8-aminoquinoline, EDC, DMAP, Et3N, DMF, rt, overnight, 88% yield. Exemplary Procedures to Prepare HDAC Inhibitors.
General remarks
[0153] All starting materials and solvents were purchased from commercial suppliers at reagent purity and, unless otherwise noted, were used as obtained without any further purification. Dry solvents used as media in moisture-sensitive reactions were purchased from Sigma- Aldrich at anhydrous grade and handled under argon. All reactions were carried out in dry conditions, under inert (argon) atmosphere. Microwave reactions were run in a Biotage Initiator microwave reactor. Reactions were monitored by thin layer chromatography on silica gel-coated glass plates (TLC LuxPlate Silica gel 60 F254, Merck), with visualization at 254 nm, and/or using appropriate dyes. Where indicated, synthetic intermediates were purified by 230-400 mesh silica gel flash chromatography on a CombiFlash system, using appropriate solvent mixtures. Final products were purified by preparative HPLC using a Shimadzu preparative liquid chromatograph [ACE 5AQ (150 x 21.2 mm) with 5 μιη particle size. Method 1: 25-100% MeOH/H20, 30 min; 100% MeOH, 5 min; 100-25% MeOH/H20, 4 min. Method 2: 8-100% MeOH/H20, 30 min; 100% MeOH, 5 min; 100-8% MeOH/H20, 4 min. Method 3: 0% MeOH, 5 min; 0-100% MeOH/H20, 25 min; 100% MeOH, 5 min; 100-0% MeOH/H20, 4 min. Flow rate = 17 mL/min], with monitoring at 254 and 280 nm. Both solvents were spiked with 0.05% TFA. 1H and 13 C NMR spectra were recorded at 400 MHz and 100.6 MHz, respectively, on Bruker DPX- 400 or AVANCE-400 spectrometers. Chemical shifts (δ scale) are reported in parts per million (ppm) relative to TMS. 1H NMR spectra are reported in this order: multiplicity and number of protons; signals were characterized as: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), bs (broad signal). HRMS spectra were recorded using ESI with an LCMS-IT-TOF (Shimadzu). Purity of all final compounds was determined by analytical HPLC [ACE 3AQ C18 column (150 x 4.6 mm, particle size 3 μΜ); 0.05% TFA in H2O/0.05% TFA in MeOH gradient eluting system; flow rate = 1.0 mL/min]. All compounds were tested at >95% purity as determined by HPLC analysis.
Products synthesis and characterization
General procedure A: synthesis of tert-Butyl (4-bromobutyl)carbamate (6a). [0154] To a solution of tert-butyl (4-hydroxybutyl)carbamate (5a) (1.0 g, 5.3 mmol) and PPh3 (2.09 g, 8 mmol) in 20 mL of THF, a solution of CBr4 (2.7 g, 8 mmol) in 10 mL of THF was added dropwise, under stirring, at 0 °C. The mixture was allowed to warm to room temperature and stirred for 4 h. The solvent was evaporated in vacuo, then the residue was purified by silica gel flash chromatography, eluting with hexanes, then hexanes/ethyl acetate, from 95/5 to 8/2, affording 1.31 g (5.2 mmol, 98% yield) of pure, target product, as a colorless oil.
1H NMR (400 MHz, CDC13): δ = 4.54 (bs, 1 H); 3.44 (f, 2 H); 3.17-3.14 (m, 2 H); 1.92-1.87 (m, 2 H); 1.69-1.63 (m, 2 H); 1.46 (s, 9 H).
Synthesis of tert-Butyl (5-bromopentyl)carbamate (6b).
[0155] Compound 6b (colorless oil, 0.856 g, 3.2 mmol, 95% yield) was prepared from tert- butyl (5-hydroxypentyl)carbamate, according to General procedure A.
[0156] 1H NMR (400 MHz, CDC13): δ = 4.53 (bs, 1 H); 3.42 (f, 2 H); 3.14-3.13 (m, 2 H); 1.91-1.87 (m, 2 H); 1.50-1.46 (m, 13 H).
Synthesis of tert-Butyl (6-bromohexyl)carbamate (6c).
[0157] Compound 6c (colorless oil, 3 g, 10.7 mmol, 78% yield) was prepared from tert-butyl (6-hydroxyhexyl)carbamate (5c), according to General procedure A.
[0158] 1H NMR (400 MHz, CDC13): δ = 4.58 (bs, 1 H); 3.38 (f, 2 H); 3.09-3.08 (m, 2 H); 1.85-1.80 (m, 2 H); 1.49-1.40 (m, 13 H); 1.33-1.27 (m, 2 H).
General procedure B: synthesis of tert-Butyl (4-(quinolin-8-ylamino)butyl)carbamate (7a).
[0159] A microwave vessel, equipped with a magnetic stir bar and a septum, was charged with a mixture of cesium carbonate (3.26 g, 10 mmol), 8-aminoquinoline (0.59 g, 5.2 mmol), and 6a (1.31 g, 5.2 mmol) in DMF (4 mL). The vessel was sealed and irradiated at 120 °C for 40 min. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, and the organic layer was washed with 10% aqueous lithium chloride. The organic fraction was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude was purified by silica gel flash chromatography, eluting with hexanes/diethyl ether, from 95/5 to 7/3, affording 0.28 g (0.9 mmol, 17% yield) of pure, target product, as a colorless oil. [0160] 1H NMR (400 MHz, CDC13): δ = 8.71 (dd, 1 H); 8.06 (dd, 1 H); 7.41-7.36 (m, 2 H); 7.05 (d, 1 H); 6.67 (d, 1 H); 6.14 (bs, 1 H); 4.57 (bs, 1 H); 3.38-3.34 (m, 2 H); 3.22-3.20 (m, 2 H); 1.86- 1.79 (m, 2 H); 1.73-1.66 (m, 2 H); 1.46 (s, 9 H).
Synthesis of tert-Butyl (5-(quinolin-8-ylamino)pentyl)carbamate (7b).
[0161] Compound 7b (colorless oil, 0.179 g, 0.5 mmol, 17% yield) was prepared from compound 6b, according to General procedure B.
[0162] 1H NMR (400 MHz, CDC13): δ = 8.72-8.71 (m, 1H), 8.06 (dd, 1 H); 7.41-7.35 (m, 2 H); 7.04 (dd, 1 H); 6.67 (d, 1 H); 4.55 (bs, 1 H); 3.76 (f, 2 H); 3.33-3.32 (m, 2 H); 3.17-3.16 (m, 2 H); 1.88-1.80 (m, 2 H); 1.60- 1.51 (m, 2 H); 1.46 (s, 9 H).
Synthesis of tert-Butyl (6-(quinolin-8-ylamino)hexyl)carbamate (7c).
[0163] Compound 7c (colorless oil, 0.515 g, 1.5 mmol, 14% yield) was prepared from compound 6c, according to General procedure B.
[0164] 1H NMR (400 MHz, CDC13): δ = 8.72 (dd, 1 H); 8.06 (dd, 1 H); 7.41-7.35 (m, 2 H); 7.04 (d, 1 H); 6.67 (d, 1 H); 6.12 (bs, 1 H); 4.54 (bs, 1 H); 3.34-3.29 (m, 2 H); 3.14-3.13 (m, 2 H); 1.83- 1.74 (m, 2 H); 1.54-1.37 (m, 15 H).
General procedure C: synthesis of N/-(Quinolin-8-yl)butane-l,4-diamine (8a).
[0165] Trifluoroacetic acid (0.67 mL, 8.9 mmol) was added to a solution of 7a (0.28 g, 0.89 mmol) in CH2CI2 (5 mL). The mixture was stirred at room temperature for 2 h. After this time, the reaction mixture was cooled to 0 °C, then 1 M aqueous NaOH was added, under stirring (pH was adjusted to 10). The layers were separated and the aqueous layer was extracted with CH2CI2. The organic layers were combined, dried over anhydrous Na2S04 and filtered. Evaporation of the solvent in vacuo afforded 0.144 g (0.7 mmol, 76% yield) of target product, as a yellow oil. This was used in the following step without any further purification.
Synthesis of N/-(Quinolin-8-yl)pentane-l,5-diamine (8b).
[0166] Compound 8b (yellow oil, 0.113 g, 0.5 mmol 91% yield) was prepared from compound 7b, according to General procedure C.
Synthesis of N/-(Quinolin-8-yl)hexane-l,6-diamine (8c). [0167] Compound 8c (yellow oil, 0.357 g, 1.5 mmol, 98% yield) was prepared from compound 7c, according to General procedure C.
General procedure D: synthesis of N-(4-(Quinolin-8-ylamino)butyl)-2-(tritylthio)acetamide (9a).
[0168] 2-(Tritylthio)acetic acid (0.074 g, 0.8 mmol), (benzotriazol- 1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 0.488 g, 0.94 mmol), and triethylamine (0.19 mL, 1.34 mmol) were added to 8a (0.144 g, 0.67 mmol) in CH2C12 (10 mL). The reaction mixture was stirred at room temperature for 20 h. The solvent was evaporated and the crude was purified by silica gel flash chromatography, eluting with hexanes/ethyl acetate, from 9/1 to 6/4, affording 0.242 g (0.5 mmol, 68% yield) of pure, target product, as a yellow oil.
[0169] 1H NMR (400 MHz, CDC13): δ = 8.71 (dd, 1 H); 8.06 (dd, 1 H); 7.50-7.37 (m, 17 H); 7.04 (d, 1 H); 6.65 (d, 1 H); 6.09 (bs, 1 H); 3.40 (f, 2 H); 3.31 (f, 2 H); 3.14 (s, 2 H); 1.82- 1.78 (m, 2 H); 1.72- 1.69 (m, 2 H).
Synthesis of N-(5-(Quinolin-8-ylamino)pentyl)-2-(tritylthio)acetamide (9b).
[0170] Compound 9b (yellow oil, 0.200 g, 0.4 mmol, 74% yield) was prepared from compound 8b, according to General procedure D.
[0171] 1H NMR (400 MHz, CDC13): δ = 8.70 (dd, 1 H); 8.06 (dd, 1 H); 7.50-7.42 (m, 8 H); 7.35-7.24 (m, 9 H); 7.04 (d, 1 H); 6.66 (d, 1 H); 6.05 (bs, 1 H); 3.40 (f, 2 H); 3.31 (f, 2 H); 3.15 (s, 2 H); 3.05 (f, 2 H); 1.81- 1.75 (m, 2 H); 1.43- 1.42 (m; 2 H).
Synthesis of N-(6-(Quinolin-8-ylamino)hexyl)-2-(tritylthio)acetamide (9c).
[0172] Compound 9c (yellow oil, 0.613 g, 1.1 mmol, 74% yield) was prepared from compound 8c, according to General procedure D.
[0173] 1H NMR (400 MHz, CDC13): δ = 8.72 (dd, 1 H); 8.07 (dd, 1 H); 7.45-7.36 (m, 8 H); 7.33-7.24 (m, 9 H); 7.05 (d, 1 H); 6.67 (d, 1 H); 6.04 (bs, 1 H); 3.31 (f, 2 H); 3.16 (s, 2 H); 2.99- 2.94 (m, 2 H); 1.81- 1.74 (m, 2 H); 1.49-1.44 (m, 2 H); 1.37-1.28 (m, 4 H).
General procedure E: synthesis of 2-Mercapto-N-(4-(quinolin-8-ylamino)butyl)acetamide (2a). [0174] Trifluoroacetic acid (0.35 mL, 4.6 mmol) and triethylsilane (0.15 mL, 0.92 mmol) were added to 9a (0.242 g, 0.46 mmol) in CH2CI2 (5 mL), at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. Volatiles were removed in vacuo and 2 mL of MeOH were added. Purification of the crude by preparative HPLC (Method 2) afforded 0.095 g (0.24 mmol, 51% yield) of pure, target product (monomer, TFA salt), as a yellow solid.
[0175] 1H NMR (400 MHz, MeOD): δ = 8.90 (d, 1 H); 8.53 (d, 1 H); Ί .10-1.69 (m, 1 H); 7.61- 7.59 (m, 1 H); 7.54-7.52 (m, 1 H); 7.26 (d, 1 H); 3.44 (f, 2 H); 3.32-3.29 (m, 2 H); 3.14 (s, 2 H); 1.87- 1.82 (m, 2 H); 1.75- 1.70 (m, 2 H).
[0176] 13C NMR (100.6 MHz, MeOD): δ = 171.7, 146.2, 138.8, 129.0, 127.7, 121.2, 118.2, 118.4, 111.5, 109.6, 44.8, 38.5, 26.4, 26.1, 24.7.
[0177] ESI-HRMS calcd for [Ci5Hi9N2OS + H]+: 290.1322 m/z, found: 290.1322 m/z. [0178] HPLC purity: 99.7%.
Synthesis of 2-Mercapto-N-(5-(quinolin-8-ylamino)pentyl)acetamide (2b).
[0179] Compound 2b (yellow solid, monomer, TFA salt, 0.097 g, 0.23 mmol, 87% yield) was prepared from compound 9b, according to General procedure E.
[0180] 1H NMR (400 MHz, CDCI3): δ = 9.69 (bs, 2 H); 9.06-9.05 (dd, 1 H); 8.62-8.60 (dd, 1 H); 7.75-7.71 (dd, 1 H); 7.67-7.63 (f, 1 H); 7.28-7.26 (d, 1 H); 7.03 (bs, 1 H); 6.99-6.97 (d, 1 H); 3.36-3.33 (m, 4 H); 3.25 (s, 2 H); 2.02- 1.84 (bs + m, 1 + 2 H); 1.69- 1.58 (m, 4 H).
[0181] 13C NMR (100.6 MHz, CDC13): δ = 169.3, 143.6, 142.2, 140.8, 130.3, 130.0, 129.8, 120.3, 114.0, 109.8, 43.4, 39.4, 28.5, 27.8, 27.5, 24.1.
[0182] ESI-HRMS calcd for [Ci6H2iN2OS + H]+: 304.1478 m/z, found: 304.1454 m/z. [0183] HPLC purity: 99.7%.
Synthesis of 2-Mercapto-N-(6-(quinolin-8-ylamino)hexyl)acetamide (2c).
[0184] Compound 2c (yellow solid, monomer, TFA salt, 0.306 g, 0.7 mmol, 88% yield) was prepared from compound 9c , according to General procedure E. [0185] 1H NMR (400 MHz, MeOD): δ = 8.87 (dd, 1 H); 8.43 (dd, 1 H); Ί .65-1.62 (m, 2 H); 7.48 (d, 1 H); 7.19 (d, 1 H); 3.42 (f, 2 H); 3.22 (f, 2 H); 3.13 (s, 2 H); 3.31 (f, 2 H); 1.85-1.77 (m, 2 H); 1.59-1.50 (m, 2 H); 1.49-1.48 (m, 2 H).
[0186] 13C NMR (100.6 MHz, MeOD) δ 169.1, 142.9, 142.6, 141.2, 130.1, 129.7, 120.3, 113.8, 109.4, 43.6, 39.4, 28.7, 27.9, 27.8, 26.5, 26.1. ESI-HRMS calcd for [C17H23N3OS + H]+: 318.1635 m/z, found: 318.1611 m/z.
[0187] HPLC purity: 97.9%.
Synthesis of tert-Butyl (7-(methoxy(methyl)amino)-7-oxoheptyl)carbamate (11).
[0188] To a solution of 7-((ieri-butoxycarbonyl)amino)heptanoic acid (10) (3.0 g, 12.2 mmol) in CH2CI2 (50 niL) was added l-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDC, 3.51 g, 18.3 mmol) and triethylamine (3.4 mL, 24.4 mmol), followed by Ν,Ο- dimethylhydroxylamine hydrochloride (1.31 g, 13.4 mmol) and 4-(dimethylamino)pyridine (DMAP, 0.147 g, 1.2 mmol). The resulting reaction mixture was stirred at room temperature overnight. Water was added, then the layers were separated and the aqueous phase was extracted with CH2CI2. The combined organic fractions were washed with water and brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. Purification of the crude by silica gel flash chromatography afforded 3.15 g (10.9 mmol, 90% yield), as a colorless oil.
[0189] 1H NMR (400 MHz, CDC13): δ = 5.28 (bs, 1 H); 3.65 (s, 3 H); 3.14 (s, 3 H); 3.07 (f, 2 H); 2.38 (f, 2 H); 1.62-1.59 (m, 2 H); 1.47-1.37 (m, 11 H); 1.32-1.23 (m, 4 H).
Synthesis of tert-Butyl (7-oxoheptyl)carbamate (12).
[0190] A 1 M THF solution of LiAlH4 (16.35 mL, 16.4 mmol) was added to a cooled (-78 °C) solution of 11 (3.15 g, 10.9 mmol) in 10 mL of THF. The mixture was allowed to warm to 0 °C and stirred at this temperature for 10 min. Saturated aqueous NH4C1 was slowly added, under stirring, at 0 °C, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous NaHC03 and brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The crude was purified by silica gel flash chromatography, affording 2.34 g (10.2 mmol, 94% yield) of pure, target product, as colorless oil. [0191] 1H NMR (400 MHz, CDC13): δ = 9.75 (s, 1 H); 4.56 (bs, 1 H); 3.10-3.09 (m, 2 H); 2.44-2.40 (m, 2 H); 1.64- 1.58 (m, 2 H); 1.47-1.43 (m, 11 H); 1.34- 1.29 (m, 4 H).
Synthesis of tert-Butyl (7-(3,4-dihydroquinolin-l(2H)-yl)heptyl)carbamate (13).
[0192] A solution of 1,2,3,4-tetrahydroquinoline (0.665 g, 5.0 mmol) in 5 mL of 1,2- dichloroethane was treated with 12 (2.29 g, 10 mmol), followed by sodium triacetoxyborohydride (2.12 g, 10 mmol) and acetic acid (1.4 mL). The resulting suspension was stirred at room temperature overnight. After this time, the mixture was cooled to 0 °C, quenched with 10 mL of 1 M NaOH and stirred for 20 minutes. The layers were separated, then the aqueous phase was extracted with CH2CI2. The organic layer was washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. Purification of the crude by silica gel flash chromatography afforded 0.360 g (1.0 mmol, 21% yield) of pure, target product, as a colorless oil.
[0193] 1H NMR (400 MHz, CDCI3): δ = 7.06 (f, 1 H); 6.95 (d, 1 H); 6.59-6.54 (m, 2 H); 4.56 (bs, 1 H); 3.29 (f, 2 H); 3.24 (f, 2 H); 3.13-3.12 (m, 2 H); 2.77 (f, 2 H); 1.99-1.93 (m, 2 H); 1.62- 1.59 (m, 2 H); 1.47 (s, 11 H); 1.39- 1.31 (m, 6 H).
Synthesis of 7-(3,4-Dihydroquinolin-l(2H)-yl)heptan-l-amine (14).
[0194] Compound 14 (colorless oil, 0.240 g, 0.97 mmol, 94% yield) was prepared from compound 13, according to General procedure C.
Synthesis of N-(7-(3,4-Dihydroquinolin-l(2H)-yl)heptyl)-2-(tritylthio)acetamide (15).
[0195] Compound 15 (yellow oil, 0.500 g, 0.9 mmol, 89% yield) was prepared from compound 14, according to General procedure D.
[0196] 1H NMR (400 MHz, CDCI3): δ = 7.45-7.43 (m, 6 H); 7.34-7.31 (m, 6 H); 7.27-7.24 (m, 3 H); 7.06 (f, 1 H); 6.95 (d, 1 H); 6.59-6.54 (m, 2 H); 6.04 (bs, 1 H); 3.28 (f, 2 H); 3.23 (f, 2 H); 3.16 (s, 2 H); 2.98-2.93 (m, 2 H); 1.99- 1.93 (m, 2 H); 1.59- 1.57 (m, 2 H); 1.47 (s, 11 H); 1.36- 1.29 (m, 6 H); 1.25- 1.23 (m, 2 H).
Synthesis of N-(7-(3,4-Dihydroquinolin-l(2H)-yl)heptyl)-2-mercaptoacetamide (3a).
[0197] Compound 3a (brown oil, dimer, di-TFA salt, 0.21 g, 0.2 mmol, 74% yield) was prepared from compound 15, according to General procedure E. [0198] 1H NMR (400 MHz, MeOD): δ = 7.25-7.21 (m, 2 H); 7.14-7.12 (m, 2 H); 3.55-3.52 (m, 2 H); 3.47-3.43 (m, 4 H); 3.23 (f, 2 H); 2.92 (i, 2 H); 2.17-2.10 (m, 2 H); 1.77-1.75 (m, 2 H); 1.58-1.54 (m, 2 H); 1.42-1.39 (m, 6 H).
[0199] 13C NMR (100.6 MHz, CDC13): δ = 168.7, 142.8, 129.1, 126.9, 123.8, 118.3, 113.4, 52.7, 48.7, 38.5, 28.9, 28.6, 27.9, 27.0, 26.5, 26.3, 25.3, 20.4.
[0200] ESI-HRMS calcd for [C36H54N402S2 + H]+: 639.3761 m/z, found: 639.3722 m/z. [0201] HPLC purity: 97.3%.
Synthesis of tert-Butyl (6-(3,4-dihydroquinolin-l(2H)-yl)-6-oxohexyl)carbamate (17).
[0202] N,N-Diisopropylethylamine (DIPEA, 3.39 niL, 19.5 mmol) was added to a solution of 6-((ieri-butoxycarbonyl)amino)hexanoic acid (16, 3.0 g, 13.0 mmol) in DMF (13 mL). PyBOP (7.4 g, 14.3 mmol) was then added and the reaction mixture was stirred at room temperature for 10 min. 1,2,3,4-Tetrahydroquinoline (1.8 mL, 14.3 mmol) was then added and the resulting reaction mixture was stirred at room temperature overnight. After this time, the mixture was diluted with ethyl acetate and washed with saturated aqueous NH4C1, 10% aqueous LiCl, and brine. The organic phase was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. Purification of the crude by silica gel flash chromatography, eluting with hexanes/ethyl acetate, from 9/1 to 4/6, afforded 2.7 g (7.9 mmol, 61% yield) of pure target product, as a yellow oil.
[0203] 1H NMR (400 MHz, CDC13): δ = 7.18-7.07 (m, 4 H); 4.65 (bs, 1 H); 3.78-3.75 (f, 2 H); 2.71-2.67 (f, 2 H); 2.50-2.47 (f, 2 H); 2.33-2.30 (f, 2 H); 1.97-1.90 (m, 2 H); 1.69-1.60 (m, 2 H); 1.52-1.33 (m, 11 H); 1.22-1.21 (m, 2 H).
Synthesis of 6-Amino-l-(3,4-dihydroquinolin-l(2H)-yl)hexan-l-one (18).
[0204] Compound 18 (yellow oil, 1.0 g, 4.2 mmol, 54% yield) was prepared from compound
17, according to General procedure C.
Synthesis of N-(6-(3,4-Dihydroquinolin-l(2H)-yl)-6-oxohexyl)-2-(tritylthio)acetamide (19).
[0205] Compound 19 (colorless oil, 0.7 g, 1.4 mmol, 56% yield) was prepared from compound 18, according to General procedure D. [0206] 1H NMR (400 MHz, CDC13): δ = 7.44-7.42 (d, 6 H); 7.29-7.27 (d, 6 H); 7.24-7.09 (m, 7 H); 6.07-6.05 (bs, 1 H); 3.79-3.76 (f, 2 H); 3.11 (s, 2 H); 2.95-2.92 (m, 2 H); 2.72-2.68 (f, 2 H); 2.50-2.47 (i, 2 H); 1.97- 1.90 (m, 2 H); 1.68-1.61 (m, 2 H); 1.34- 1.27 (m, 2 H); 1.25-1.20 (m, 2 H).
Synthesis of N-(6-(3,4-Dihydroquinolin-l(2H)-yl)-6-oxohexyl)-2-mercaptoacetamide (3b).
[0207] Compound 3b (colorless oil, monomer, TFA salt, 0.316 g, 0.7 mmol, 55% yield) was prepared from compound 19, according to General procedure E.
[0208] 1H NMR (400 MHz, CDCI3): δ = 10.51 (bs, 1 H); 7.28-7.12 (m, 4 H); 7.02 (bs, 1 H); 3.80-3.77 (f, 2 H); 3.29-3.23 (m, 4 H); 2.73-2.70 (f, 2 H); 2.54-2.50 (f, 2 H); 2.00- 1.92 (m, 3 H); 1.71- 1.63 (m, 2 H); 1.57- 1.48 (m, 2 H); 1.34-1.32 (m, 2 H).
[0209] 13C NMR (100.6 MHz, CDC13): δ = 23.7, 24.8, 25.9, 26.3, 27.7, 28.5 (2 C), 33.8, 39.3, 124.3, 125.1, 125.7, 128.2 (2 C), 138.4, 169.7, 172.8.
[0210] ESI-HRMS calcd for [Cn^NiOiS + H]+: 321.1631 m/z, found: 321.1494 m/z. [0211] HPLC purity: 97.3%.
Synthesis of tert-Butyl (6-(methoxy(methyl)amino)-6-oxohexyl)carbamate (20).
[0212] EDC (4.1 g, 21.4 mmol), triethylamine (3.98 mL, 28.5 mmol), Ν,Ο- dimethylhydroxylamine hydrochloride (1.53 g, 15.7 mmol) and DMAP (0.174 g, 1.4 mmol) were added in this order to a solution of compound 16 (3.3 g, 14.2 mmol) in dry CH2CI2 (57 mL). The resulting reaction mixture was stirred at room temperature overnight, after which time a white precipitate formed. Saturated aqueous NH4C1 was added to the reaction mixture and the phases were separated. The organic phase was washed with saturated aqueous NH4C1 and brine, dried over anhydrous Na2S04 and filtered. Evaporation of the solvent under reduced pressure afforded 3.9 g (14.2 mmol, quantitative yield) of target product, as a colorless oil. This was used in the following step without any further purification.
[0213] 1H NMR (400 MHz, CDC13): δ = 4.62 (bs, 1 H); 3.64 (s, 3 H); 3.14 (s, 3 H); 3.09-3.08 (m, 2 H); 2.40-2.35 (m, 2 H); 1.65- 1.58 (m, 2 H); 1.51-1.43 (m, 2 H); 1.40 (s, 9 H); 1.37- 1.31 (m, 2 H). Synthesis of tert-Butyl (6-oxohexyl)carbamate (21).
[0214] Asolution of compound 20 (1.9 g, 6.9 mmol) in dry THF (69 mL) was cooled to -78 °C, then LiAlH4 (0.394 g, 10.4 mmol) was added at this temperature, in one portion. The resulting reaction mixture was stirred at -78 °C for 1.5 h, then allowed to warm to 0 °C. 0.1 N aqueous HC1 was carefully added under stirring and the resulting reaction mixture was extracted with Et20. The organic phase was washed with brine, dried over anhydrous Na2S04 and filtered. Evaporation of the solvent under reduced pressure gave 1.7 g of a 1/1 mixture of starting material and target product (3.4 mmol, 50% yield), as a colorless oil. This was used in the following step without any further purification.
Synthesis of tert-Butyl (6-(6-chloro-3,4-dihydroquinolin-l(2H)-yl)hexyl)carbamate (22).
[0215] Sodium triacetoxyborohydride (1.67 g, 7.9 mmol) was added in one portion to a stirred solution of 6-chloro-l,2,3,4-tetrahydroquinoline (0.662 g, 3.95 mmol) and 21 (1.7 g, 7.9 mmol) in 1,2-dichloroethane (20 mL). The resulting reaction mixture was stirred at room temperature overnight. Saturated aqueous NaHC03 was added to the reaction mixture and it was extracted with chloroform. The organic phase was washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. Purification by silica gel flash chromatography, eluting with hexanes, then hexanes/ethyl acetate, from 95/5 to 85/15, afforded 0.95 g (2.6 mmol, 66% yield) of pure, target product, as a colorless oil.
[0216] 1H NMR (400 MHz, CDC13): δ = 9.98-6.96 (dd, 1 H); 6.90-6.89 (d, 1 H); 6.46-6.44 (d, 1 H); 4.55 (bs, 1 H); 3.27-3.24 (f, 2 H); 3.22-3.19 (f, 2 H); 3.15-3.10 (m, 2 H); 2.73-2.70 (f, 2 H); 1.96- 1.90 (m, 2 H); 1.61- 1.55 (m, 2 H); 1.53-1.46 (m + s, 2 + 9 H); 1.41-1.35 (m, 4 H).
Synthesis of 6-(6-Chloro-3,4-dihydroquinolin-l(2H)-yl)hexan-l-amine (23).
[0217] Compound 23 (yellow oil, 0.314 g, 1.2 mmol 86% yield) was prepared from compound
22, according to General procedure C.
[0218] 1H NMR (400 MHz, CDC13): δ = 6.98-6.96 (dd, 1 H); 6.90 (s, 1 H); 6.47-6.44 (d, 1 H); 3.27-3.24 (f, 2 H); 3.23-3.19 (f, 2 H); 2.73-2.70 (m, 4 H); 1.96- 1.90 (m, 2 H); 1.62-1.55 (m, 2 H); 1.48- 1.45 (m, 2 H); 1.37- 1.36 (m, 4 H); 1.12 (bs, 2 H).
Synthesis of N-(6-(6-Chloro-3,4-dihydroquinolin-l(2H)-yl)hexyl)-2-(tritylthio)acetamide (24). [0219] Compound 24 (colorless oil, 0.166 g, 0.3 mmol, 25% yield) was prepared from compound 23, according to General procedure D.
[0220] 1H NMR (400 MHz, CDC13): δ = 7.47-7.45 (d, 6 H); 7.35-7.31 (f, 6 H); 7.28-7.26 (d, 3 H); 7.01-6.98 (dd, 1 H); 6.93-6.92 (d, 1 H); 6.48-6.46 (d, 1 H); 6.05 (bs, 1 H); 3.28-3.25 (f, 2 H); 3.23-3.20 (i, 2 H); 3.16 (s, 2 H); 3.01-2.96 (dd, 2 H); 2.74-2.71 (f, 2 H); 1.97-1.91 (m, 2 H); 1.61- 1.54 (m, 2 H); 1.41- 1.28 (m, 6 H).
Synthesis of N-(6-(6-Chloro-3,4-dihydroquinolin-l(2H)-yl)hexyl)-2-mercaptoacetamide (3c).
[0221] Compound 3c (pinkish oil, 0.068 g, 0.2 mmol, 73%) was prepared from compound 24, according to General procedure E. The compound was isolated as dimer and free base, after neutralization of the collected fractions from preparative HPLC with saturated aqueous NaHC03, extraction with chloroform, and lyophilization.
[0222] 1H NMR (400 MHz, CDC13): δ = 6.98-6.95 (dd, 1 H); 6.90-6-89 (d, 1 H); 6.76 (bs, 1 H); 6.45-6.43 (d, 1 H); 3.42 (s, 2 H); 3.33-3.28 (dd, 2 H); 3.26-3.24 (f, 2 H); 3.22-3.19 (f, 2 H); 2.73-2.70 (f, 2 H); 1.95- 1.89 (m, 2 H); 1.61-1.54 (m, 4 H); 1.43- 1.31 (m, 4 H).
[0223] 13C NMR (100.6 MHz, CDC13): δ = 168.3, 143.8, 128.6, 126.6, 123.8, 119.6, 111.4, 51.4, 49.3, 42.6, 40.0, 29.4, 28.0, 26.8 (2 C), 22.0.
[0224] ESI-HRMS calcd for [C34H48C12N402S2 + H]+: 679.2669 m/z, found: 321.1494 m/z. [0225] HPLC purity: 95.2%.
Synthesis of tert-Butyl 4-(quinolin-8-ylcarbamoyl)benzylcarbamate (26).
[0226] To a solution of 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid (25, 1.0 g, 4.0 mmol) in DMF (5 mL) was added triethylamine (1.12 mL, 8.0 mmol), EDC (1.15 g, 6.0 mmol), followed by 8-aminoquinoline (0.576 g, 4.0 mmol) and DMAP 0.049 g, 0.4 mmol). The resulting mixture was stirred at room temperature overnight. Water was added to the reaction mixture, then the layers were separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. Purification of the crude by silica gel flash chromatography afforded 1.32 g (3.5 mmol, 88% yield) of pure, target product, as a white solid. 1H NMR (400 MHz, CDC13): δ = 10.67 (bs, 1 H); 8.87 (dd, 1 H); 8.80-8.79 ( , 1 H); 8.12 (dd, 1 H); 7.99 (d, 2 H); 7.54-7.48 ( , 2 H); 7.43-7.40 ( , 3 H); 5.28 (bs, 1 H); 4.38-4.37 (m, 2 H); 1.47 is, 9 H).
Synthesis of 4-(Aminomethyl)-N-(quinolin-8-yl)benzamide (27).
[0227] Compound 27 (off-white solid, 0.900 g, 3.2 mmol, 93% yield) was prepared from compound 26, according to General procedure C.
Synthesis of N-(Quinolin-8-yl)-4-((2-(tritylthio)acetamido)methyl)benzamide (28).
[0228] Compound 28 (off-white solid, 1.12 g, 1.9 mmol, 59% yield) was prepared from compound 27, according to General procedure D.
[0229] 1H NMR (400 MHz, CDC13): δ = 8.93 (dd, 1 H); 8.86 (dd, 1 H); 8.19 (dd, 1 H); 8.02 (d, 2 H); 7.62-7.56 (m, 2 H); 7.54-7.42 (m, 8 H); 7.32-7.22 (m, 10 H); 8.40 (bs, 1 H); 4.24 (d, 2 H); 3.23 (s, 2 H).
Synthesis of 4-((2-Mercaptoacetamido)methyl)-N-(quinolin-8-yl)benzamide (4).
[0230] Compound 4 (off-white solid, monomer, TFA salt, 0.340 g, 0.7 mmol, 51%) was prepared from compound 28, according to General procedure E.
[0231] 1H NMR (400 MHz, CDC13): δ = 10.74 (bs, 1 H); 8.93 (d, 1 H); 8.89 (dd, 1 H); 8.24 (d, 1 H); 8.08 (d, 2 H); 7.63-7.60 (m, 2 H); 7.54-7.48 (m, 3 H); 7.15 (bs, 1 H); 4.60 (d, 2 H); 3.36 (d, 2 H); 1.94 (f, 1 H).
[0232] 13C NMR (100.6 MHz, CDCI3): δ = 168.0, 164.7, 147.8, 141.4, 138.2, 136.4, 134.1, 133.9, 127.7, 127.6, 127.5, 127.2, 121.6, 121.3, 116.7, 43.2, 27.9.
[0233] ESI-HRMS calcd for [C19H17N3O2S + H]+: 352.1114 m/z, found: 352.1080 m/z. [0234] HPLC purity: 99.5%.
IC50 Values for HDAC6
[0235] The effectiveness, or potency, of a present HDACi with respect to inhibiting the activity of an HDAC is measured by an IC50 value. The quantitative IC50 value indicates the concentration of a particular compound that is needed to inhibit the activity of an enzyme by 50% in vitro. Stated alternatively, the IC50 value is the half maximal (50%) inhibitory concentration of a compound tested using a specific enzyme, e.g., HDAC, of interest. The smaller the IC50 value, the more potent the inhibiting action of the compound because a lower concentration of the compound is needed to inhibit enzyme activity by 50%.
[0236] In preferred embodiments, a present HDACi inhibits HDAC enzymatic activity by about at least 50%, preferably at least about 75%, at least 90%, at least 95%, or at least 99%.
[0237] Compounds of the present invention were tested for IC50 values against both HDAC6 and HDACI. In some embodiments, a present compound may also have been tested against HDACi, 2, 3, 4, 5, 8, 10, and 11. The tested compounds showed a range of IC50 values vs. HDAC6 of about 1 nM to greater than 1 μΜ, and a range of IC50 values vs. HDACI of about 1 μΜ to greater than 20 μΜ. Therefore, in some embodiments, a present HDACI is a selective HDAC6 inhibitor which, because of a low affinity for other HDAC isozymes, e.g., HDACi, may give rise to fewer side effects than compounds that are non-selective HDAC inhibitors.
[0238] In some embodiments, the present HDACis interact with and reduce the activity of all histone deacetylases in a cell. In some preferred embodiments, the present HDACis interact with and reduce the activity of fewer than all histone deacetylases in the cell. In certain preferred embodiments, the present HDACis interact with and reduce the activity of primarily one histone deacetylase (e.g., HDAC6), but do not substantially interact with or reduce the activities of other histone deacetylases (e.g., HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-7, HDAC- 8, HDAC-9, HDAC- 10, and HDAC- 11).
[0239] The present invention therefore provides HDACis for the treatment of a variety of diseases and conditions wherein inhibition of HDAC has a beneficial effect. Preferably, a present HDACI is selective for inhibition of HDAC6 by at least 10-fold over the other HDAC isozymes, i.e., 1-5, and 7-11.
[0240] The IC50 values for compounds of structural formula 1 - 3 vs. HDACI and HDAC6 were determined as follows:
[0241] The HDACI, 2, 4, 5, 6, 7, 8, 9, 10, and 11 assays used isolated recombinant human protein; HDAC3/NcoR2 complex was used for the HDAC3 assay. Substrate for HDACI, 2, 3, 6,
10, and 11 assays is a fluorogenic peptide from p53 residues 379-382 (RHKKAc); substrate for
HDAC8 is fluorogenic diacyl peptide based on residues 379-382 of p53 (RHKAcKAc). Acetyl- Lys(trifluoroacetyl)-AMC substrate was used for HDAC4, 5, 7, and 9 assays. Compounds were dissolved in DMSO and tested in 10-dose IC50 mode with 3-fold serial dilution starting at 30 μΜ. Control Compound Trichostatin A (TSA) was tested in a 10-dose ICso with 3-fold serial dilution starting at 5 μΜ. ICso values were extracted by curve-fitting the dose/response slopes. Assays were performed in duplicate and IC50 values are an average of data from both experiments.
Materials
[0242] Human HDAC1 (GenBank Accession No. NM_004964): Full length with C-terminal GST tag, MW= 79.9 kDa, expressed by baculovirus expression system in Sf9 cells. Enzyme is in 50 mM Tris-HCl, pH 8.0, 138 mM NaCl, 20 mM glutathione, and 10% glycerol, and stable for >6 months at -80 °C. Purity is> 10% by SDS-PAGE. Specific Activity is 20 /μξ, where one U = 1 pmol/min under assay condition of 25 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KC1, 1 mM MgCl2, 0.1 mg/ml BSA, 100 μΜ HDAC substrate, and 13.2 ng/μΐ HDACi, incubation for 30 min at 30°C.
[0243] Human HDAC 6 (GenBank Accession No. BC069243): Full length with N-terminal GST tag, MW= 159 kDa, expressed by baculovirus expression system in Sf9 cells. Enzyme is in 50 mM Tris-HCl, pH 8.0, 138 mM NaCl, 20 mM glutathione, and 10% glycerol, and stable for >6 months at -80°C. Purity is >90% by SDS-PAGE. Specific Activity is 50 U^g, where one U =1 pmol/min under assay condition of 25 mM Tris/Cl, pH8.0, 137 mM NaCl, 2.7 mM KC1, 1 mM MgCl2, and 0.1 mg/ml BSA, 30 μΜ HDAC substrate, and 5 ng/μΐ HDAC6, incubation for 60 min at 30 °C.
[0244] Substrate for HDACI and HDAC 6: Acetylated peptide substrate for HDAC, based on residues 379-382 of p53 (Arg-His-Lys-Lys(Ac)), a site of regulatory acetylation by the p300 and CBP acetyltransferases (lysines 381, 382)1-6, is the best for HDAC from among a panel of substrates patterned on p53, histone H3 and histone H4 acetylation sites.
[0245] References: W. Gu et al., Cell (1997) 90 595; K. Sakaguchi et al., Genes Dev., (1998) 12 2831; L. Liu et al., Mai. Cell. Biol., (1999) 19 1202; A. Ito et al., EMBO J., (2001) 20 1331; N.A. Barlev et al., Mai. Cell, (2001) 8 1243; and A. Ito et al., EMBO J., (2002) 21, 6236. Reaction Buffer: 50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/ml BSA.
Assay Conditions
[0246] HDAC1: 75 nM HDAC1 and 50 μΜ HDAC substrate are in the reaction buffer and 1 % DMSO final. Incubate for 2 hours at 30°C. HDAC 6: 12.6 nM HDAC6 and 50 μΜ HDAC substrate are in the reaction buffer and 1 % DMSO final. Incubate for 2 hours at 30°C.
IC50 Calculations
[0247] All IC50 values are automatically calculated using the GraphPad Prism version 5 and Equation of Sigmoidal dose-response (variable slope): Y = Bottom + (Top- Bottom)/(l+10A((LogEC50-X)*HillSlope)), where X is the logarithm of concentration, Y is the response, Y starts at Bottom and goes to Top with a sigmoid shape. In most cases, "Bottom" is set 0, and "Top" is set "less than 120%". This is identical to the four parameter logistic equation. IC50 curves also are drawn using the GraphPad Prism.
Table 1
HDAC inhibitory activity of some exemplary mercaptoacetamides.
Figure imgf000063_0001
3b 1.95 NA 0.0269
0 2 0
Figure imgf000064_0001
TSA 0.0172 0.0012
[a] Results were determined by CRO Reaction Biology (Mavern, PA, USA); ten-dose IC50 values were determined by using threefold serial dilutions starting at concentrations of 30μΜ. [b] ClogP values were calculated using ChemBioOffice-ChemDraw V12.
[0248] Compounds of the present invention have been found to show selectivity for the Class lib HDAC enzymes, and specifically for HDAC6. Because of the ability of HDAC6 to control the levels of acetylation of certain cytoplasmic proteins like tubulin, cortactin, HSP90, inhibitors of HDAC6 have the possibility to treat a number of diseases. These include but are not limited to Alzheimer's disease, Rett syndrome, Charcott Marie Tooth Disease, colitis, arthritis, certain cancers including melanomas. Many of these indications have been illustrated in the literature using the key compound Tubastatin A which was invented by Kozikowski et al.
[0249] The present compounds have been evaluated for their activity at HDAC6 and their selectivity for HDAC6 compared to HDAC1. It previously was shown that selective HDAC6 inhibitors may be employed in a variety of disease states including, but not limited to, arthritis, autoimmune disorders, inflammatory disorders, cancer, neurological diseases such as Rett syndrome, peripheral neuropathies such as CMT, stroke, hypertension, dementias, and diseases in which oxidative stress is a causative factor or a result thereof. It also was shown that selective HDAC6 inhibitors, when administered in combination with rapamycin, prolonged the lifespan of mice with kidney xenografts. This model was used to evaluate the immunosuppressant properties of the present compounds and serve as a model of transplant rejection. Furthermore, it was previously shown that selective HDAC6 inhibitors confer neuroprotection in rat primary cortical neuron models of oxidative stress. These studies identified selective HDAC6 inhibitors as nontoxic neuroprotective agents. The present compounds behave in a similar manner because they also are selective HDAC6 agents. The present compounds demonstrate a ligand efficiency that renders them more drug-like in their physiochemical properties. In addition, the present compounds do not contain a hydroxamate group as the zinc binding group, and as such, they are unable to undergo the Lossen rearrangement, a reaction that results in many of the hydroxamates showing activity in the Ames test, i.e., many hydroxamates prove to be genotoxic. The present compounds therefore are pharmaceutical candidates and research tools to identify the specific functions of HDAC6, and to treat diseases that are linked to the expression of HDAC6.
[0250] Prior SAR studies suggested that the chain length for HDAC6 inhibitors is optimal when n = 2 or 3. Our findings corroborate these results, and also suggest that compounds with a shorter alkyl chain, n = 1, may still exhibit very good activity (2a, HDAC6 IC50 = 7.9 nM).
[0251] Previously, our research had determined that a benzyl linker was optimal for potent HDAC6- selective hydroxamic acid derivatives. Having this in mind, we explored an aromatic linker together with the mercaptoacetamide ZBG; however, analog 4 presented very low activity. This result indicates that while the benzyl linker is appropriate for hydroxamic acids, this is not the case for thiols.
[0252] Both THQ and 8-aminoquinoline caps generated potent and selective analogs, but it is important to note that the THQ cap is more lipophilic, suggesting that compounds such as 3a
(CLogP = 4.21) are more likely to penetrate the blood brain barrier for the treatment of CNS
(central nervous system) diseases. On the other hand, replacing the aminoquinoline cap in compound 2c (n = 3) with the THQ cap, 3a (n = 3), resulted in a slight decrease in potency. This difference in potency may result from the ability of the 8-aminoquinoline cap to establish an additional hydrogen bonding interaction with the surface of the protein, due to the presence of a second nitrogen atom. A decrease in potency is observed when the amide linker of compound 1 is replaced with a secondary amine as in compound 2c, although the same pattern is not maintained for compounds with a shorter alkyl chain (2a and 2b). [0253] Next, to assess HDAC6 selectivity in cells we investigated the ability of compounds 1, 2a, 2b and 3a to induce acetylation of a-tubulin, the primary substrate of HDAC6. Low micromolar concentrations of the test compounds applied to primary cultures of cortical neurons (E17) led to a dose-dependent increase in acetylated a-tubulin (AcTub) levels without a concomitant elevation of H3 acetylation. These results are consistent with a selective action at HDAC6 isoform (Figure 3). All tested compounds induced tubulin acetylation at 1 μΜ, with compound 2b showing the greatest efficacy (more than 10-fold induction of AcTub at 1 μΜ), with results comparable to the hydroxamate containing HDACI Tubastatin A (TubA) and Trichostatin A (TSA), used as positive controls. Compound 1 produced a 5-fold induction of tubulin acetylation at 1 μΜ, while 2a and 3a had much weaker activity at the same concentration.
[0254] Next, to examine whether these thiols might have an anti-inflammatory action, these compounds were tested for their ability to enhance the immunosuppressive function of murine Foxp3+ regulatory T cells (Tregs) in vitro. HDACIs have previously been shown to enhance the suppressive effects of Foxp3+ regulatory T cells. The pharmacological modulation of Treg suppression is considered as a possible therapeutic approach to slow or reverse the pathogenesis of autoimmune disorders, to prevent allograft rejection, inflammatory bowel disease, and rheumatoid arthritis. Pharmacologic inhibition of HDAC6, using hydroxamate-based compounds such as Tubastatin A and its analogs, was previously shown to increase the suppressive functions of murine and human Foxp3+ Treg cells. Thus, CFSE (carboxyfluorescein succinimidyl ester) labeled Teffs were incubated in the presence and absence of Tregs, with or without the addition of selected HDAC6 inhibitors at multiple concentrations. Using the thiols reported herein, varying effects were observed. Direct effects of compounds on Teff proliferation in these assays was not seen, as shown by comparable Teff cell proliferation in the absence of Treg cells (0: 1 Treg:Teff ratio).
[0255] Compared to corresponding cells exposed to DMSO alone, all seven compounds, namely 2a-2c, 3a-c and 4, increased Treg function, resulting in decreased Teff cell proliferation at the 1: 1 Treg:Teff ratio, but only three compounds, namely 2a-c, were also effective at the 1:2 Treg:Teff ratio, and only compound 2b was also effective at the 1:4 Treg:Teff ratio. Summary
[0256] We have developed a small series of mercaptoacetamides two of which exhibit single digit nM HDAC6 inhibitory activity. In particular, compound 2b induced a dose-dependent increase in acetylated a-tubulin (AcTub) using cortical neurons (El 7) with activity being apparent at 0.5 μΜ and with no significant effect on H3 acetylation. These compounds were also found to enhance Treg suppression of Teff proliferation in vitro. Because there is no history of mercaptoacetamides being linked to genotoxicity, the results found herein suggest that these compounds be explored further for applications to both cancer and CNS diseases, especially for disease indications in which prolonged drug use will be required. Further efforts to generate analogs with improved LogBB values are being made in order to evaluate these compounds in various animal models of neurodegenerative diseases.
REFERENCES
1. Lakshmaiah, K. C; Jacob, L. A.; Aparna, S.; Lokanatha, D.; Saldanha, S. C, Epigenetic therapy of cancer with histone deacetylase inhibitors. Journal of cancer research and therapeutics 2014, 10 (3), 469-78.
2. Kalin, J. H.; Bergman, J. A., Development and Therapeutic Implications of Selective Histone Deacetylase 6 Inhibitors. Journal of medicinal chemistry 2013, 56 (16), 6297-6313.
3. Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez, B.; Deprez-Poulain, R., Hydroxamates: relationships between structure and plasma stability. Journal of medicinal chemistry 2009, 52 (21), 6790-802.
4. Glen, C. D.; Dubrova, Y. E., Exposure to anticancer drugs can result in transgenerational genomic instability in mice. Proceedings of the National Academy of Sciences of the United States of America 2012, 109 (8), 2984-8.
5. Kozikowski, A. P.; Chen, Y.; Gaysin, A.; Chen, B.; D'Annibale, M. A.; Suto, C. M.; Langley, B. C, Functional differences in epigenetic modulators- superiority of mercaptoacetamide-based histone deacetylase inhibitors relative to hydroxamates in cortical neuron neuroprotection studies. Journal of medicinal chemistry 2007, 50 (13), 3054-61.
6. Vesci, L.; Bernasconi, E.; Milazzo, F. M.; De Santis, R.; Gaudio, E.; Kwee, I.; Rinaldi, A.; Pace, S.; Carollo, V.; Giannini, G.; Bertoni, F., Preclinical antitumor activity of ST7612AA1: a new oral thiol-based histone deacetylase (HDAC) inhibitor. Oncotarget 2015, 6 (8), 5735-48.
7. Zhong, C; Zhao, X.; Sarva, J.; Kozikowski, A.; Neale, J. H.; Lyeth, B. G., NAAG peptidase inhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluid percussion TBI in rats. Journal of neurotrauma 2005, 22 (2), 266-76.

Claims

s claimed:
A compound of formula I, or a pharmaceutically acceptable salt or prodrug thereof.
Figure imgf000068_0001
I wherein
Cap is a group of formula:
Figure imgf000068_0002
represents a single or double bond,
Y and Z are independently selected from C and N, provided that when Y is N, represents a single bond;
W is selected from O, S and (CH2)m;
X is selected from C(=0), CHR, and NHR when Y is C, or C(=0) and CHR when Y is N;
R1 isHorC(=0)Ci_4alkyl; RisHorCi-Qalkyl; m is 0, 1 or 2; n is 1, 2 or 3; and A is one or more substituents independently from H, Br, CI, Me, CF3, N¾, CN, OCH3 and OH, and A can be appended to any one or each of the fused aromatic rings.
A compound according to claim 1 wherein Z is C.
A compound according to claim 1 wherein Z is N.
A compound according to claim 1 wherein R is H.
A compound according to claim 1 wherein A is CI.
A compound according to claim 1 wherein W is (CH2)m-
A compound according to claim 1 wherein Cap is selected from the group consisting of:
Figure imgf000069_0001
, an
A compound according to claim 1 selected from the group consisting of:
Figure imgf000069_0002
Figure imgf000070_0001
A compound according to claim 1 wherein said prodrug comprises an acylated derivative of the sulfur atom of formula -S-C(0)-R" wherein R" is selected from straight or branched Ci-C6 alkyl, aryl or heteroaryl.
10. A composition comprising one or more compounds according to claim 1 and a pharmecutically acceptable excipient or carrier.
11. A composition comprising (a) one or more compounds according to claim 1, (b) a second therapeutic agent useful in the treatment of a disease or condition wherein inhibition of HDAC provides a benefit, and (c) an optional excipient and/or pharmaceutically acceptable carrier.
12. The composition of claim 11 wherein the second therapeutic agent comprises a chemotherapeutic agent useful in the treatment of a cancer.
13. A method of treating a disease or condition wherein inhibition of HDAC provides a benefit comprising administering a therapeutically effective amount of one or more compounds according to claim 1 to an individual in need thereof.
14. The method of claim 13 wherein the HDAC is HDAC 6.
15. The method of claim 13 further comprising administering a therapeutically effective amount of a second therapeutic agent useful in the treatment of the disease or condition.
16. The method of claim 15 wherein the compound of claim 1 and the second therapeutic agent are administered simultaneously.
17. The method of claim 15 wherein the compound of claim 1 and the second therapeutic agent are administered separately.
18. The method of claim 13 wherein the disease or condition is a cancer.
19. The method of claim 15 wherein the disease is a cancer and the second therapeutic agent is one or more of a chemotherapeutic agent, radiation, and an immunotherapy.
20. The method of claim 15 wherein the second therapeutic agent comprises radiation, and the radiation optionally is administered in conjunction with radiosensitizers and/or therapeutic agents.
21. The method of claim 13 wherein the disease or condition is a neurological disease, a neurodegenerative disorder, a peripheral neuropathy, a psychiatric illness, or a traumatic brain injury.
22. The method of claim 13 wherein the disease or condition is a stroke.
23. The method of claim 13 wherein the disease or condition is an inflammation or an autoimmune disease.
24. The method of claim 23 further comprising administering a therapeutically effective amount of a second therapeutic agent useful in the treatment of the autoimmune disease or the inflammation.
25. The method of claim 13 wherein the disease or condition is autism or an autism spectrum disorder including Rett syndrome.
26. The method of claim 13 wherein the disease or condition is depression or bipolar disorder.
27. A method of increasing sensitivity of a cancer cell to cytotoxic effects of a radiotherapy and/or a chemotherapy comprising contacting the cell with a compound of claim 1 in an amount sufficient to increase the sensitivity of the cell to the radiotherapy and/or the chemotherapy.
28. The method of claim 27 wherein the cell is an in vivo cell.
29. A method of producing immunosuppression comprising administering an effective amount of a compound according to claim 1 to an individual in need thereof.
30. A compound according to claim 1 wherein the compound is labeled with a fluorescent dye, a radioisotope selected from 3 H, 11 C, 18 F, 123 I, 125 I, and 131 I, a molecular tag, or a mixture thereof.
31. The compound according to claim 30 wherein the label comprises a Cn methyl group.
32. An imaging method comprising
(a) providing a radiolabeled compound according to claim 29;
(b) contacting a cell or a tissue with the radio labeled compound; and
(c) making a radiographic image of the contacted cell or tissue.
PCT/US2016/052780 2015-09-22 2016-09-21 Bicyclic and tricyclic cap bearing mercaptoacetamide derivatives as histone deacetylase inhibitors WO2017053360A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562221843P 2015-09-22 2015-09-22
US62/221,843 2015-09-22

Publications (1)

Publication Number Publication Date
WO2017053360A1 true WO2017053360A1 (en) 2017-03-30

Family

ID=58387078

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/052780 WO2017053360A1 (en) 2015-09-22 2016-09-21 Bicyclic and tricyclic cap bearing mercaptoacetamide derivatives as histone deacetylase inhibitors

Country Status (1)

Country Link
WO (1) WO2017053360A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018200608A1 (en) * 2017-04-26 2018-11-01 The Board Of Trustees Of The University Of Illinois Nrf and hif activators/hdac inhibitors and therapeutic methods using the same
WO2024261329A1 (en) * 2023-06-21 2024-12-26 Augustine Therapeutics Heteroaryl-amine compounds and use thereof as hdac6 inhibitors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140128408A1 (en) * 2011-02-01 2014-05-08 The Board Of Trustees Of The University Of Illinois Hdac inhibitors and therapeutic methods using the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140128408A1 (en) * 2011-02-01 2014-05-08 The Board Of Trustees Of The University Of Illinois Hdac inhibitors and therapeutic methods using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PARIS ET AL.: "Histone Deacetylase Inhibitors: From Bench to Clinic", JOURNAL OF MEDICINAL CHEMISTRY, vol. 51, 2008, pages 1505 - 1529, XP002495888 *
TESSIER ET AL.: "Diphenylmethylene hydroxamic acids as selective class IIa histone deacetylase inhibitors", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 19, 2009, pages 5684 - 5688, XP026586307 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018200608A1 (en) * 2017-04-26 2018-11-01 The Board Of Trustees Of The University Of Illinois Nrf and hif activators/hdac inhibitors and therapeutic methods using the same
US10934255B2 (en) 2017-04-26 2021-03-02 The Board Of Trustees Of The University Of Illinois Nrf and HIF activators/HDAC inhibitors and therapeutic methods using the same
WO2024261329A1 (en) * 2023-06-21 2024-12-26 Augustine Therapeutics Heteroaryl-amine compounds and use thereof as hdac6 inhibitors

Similar Documents

Publication Publication Date Title
AU2017202477B2 (en) HDAC inhibitors and therapeutic methods using the same
US8748451B2 (en) HDAC inhibitors and therapeutic methods of using same
AU2018258338B2 (en) Nrf and HIF activators/HDAC inhibitors and therapeutic methods using the same
US11739064B2 (en) HDAC inhibitors and therapeutic methods using the same
US20240343697A1 (en) Isoxazole hydroxamic acids as histone deacetylase 6 inhibitors
WO2017053360A1 (en) Bicyclic and tricyclic cap bearing mercaptoacetamide derivatives as histone deacetylase inhibitors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16849461

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16849461

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载