Compounds for the inhibition of KIFC3, methods of making the same, and pharmaceutical compositions comprising the same

Abstract

In one aspect, the disclosure relates to compounds and pharmaceutical compositions for treating diseases or disorders associated with aberrant Kifc3 function in a subject. In a further aspect, the disease or disorder can be selected from neonatal thrombocytopenia, delayed platelet recovery following umbilical cord blood stem cell transplantation, Down syndrome-associated myeloid neoplasm, or any combination thereof. In one aspect, the subject can be a human, including a neonatal human, and performing the method increases platelet production in the subject. Also disclosed are methods for ex vivo platelet production using the disclosed compounds and pharmaceutical compositions.

Description

COMPOUNDS FOR THE INHIBITION OF KIFC3, METHODS OF MAKING THE SAME, AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 646,003, filed May 13, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number R01 HL149667 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Adult and neonatal megakaryopoiesis employ distinct morphogenesis programs. Adulttype development follows a sequence of endomitotic polyploidization, massive enlargement, and efficient elaboration of cytoplasmic processes that yield abundant platelets in the circulation. Fetal-type development, applicable to normal human neonates, comprises extensive proliferation, minimal enlargement, diminished process extension, and diminished platelet production; these features, as well as the hypoactivity of neonatal platelets, specifically serve the needs of a normal growing fetus and newborn but can cause problems in disease states. The inefficient platelet production compromises reserve capacity, leading to thrombocytopenia in conditions with increased platelet demands, and the proliferative progenitors have an increased propensity for neoplastic transformation.

[0004] Several of the molecular determinants of Mk ontogeny have been characterized, most recently Dyrk kinase antagonism of the co-activator M kl 1 as a druggable component of the fetal program. However, the generalized toxicity associated with Dyrk inhibition highlighted a need to identify downstream elements more amenable to therapeutic manipulation.

[0005] Pharmacologic interventions to induce adult-type megakaryopoiesis, i.e. ontogenic switching, do not exist but would greatly benefit several disease conditions: 1) neonatal thrombocytopenia, 2) platelet recovery post umbilical cord blood (UCB) transplant, 3) Down syndrome-associated megakaryocyte (Mk) neoplasms, and 4) large scale ex vivo platelet production. These needs and other needs are satisfied by the present disclosure.SUMMARY

[0006] In one aspect, the disclosure relates to compounds and pharmaceutical compositions for treating diseases or disorders associated with aberrant Kifc3 function in a subject. In a further aspect, the disease or disorder can be selected from neonatal thrombocytopenia, delayed platelet recovery following umbilical cord blood stem cell transplantation, Down syndrome-associated myeloid neoplasm, or any combination thereof. In one aspect, the subject can be a human, including a neonatal human, and performing the method increases platelet production in the subject. Also disclosed are methods for ex vivo platelet production using the disclosed compounds and pharmaceutical compositions.

[0007] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0009] FIG. 1 shows a schematic of the Kifc3-Cep192 pathway.

[0010] FIG. 2 shows KIFC3 is an ontogenic gene regulated by Dyrk. Analysis of RNA-seq data: Full-length KIFC3 decreases in adult (PB) vs neonatal (CB) Mk and is repressed by Dyrkl inhibitors harmine (H) and EHT 1610 (E). N = 3; ***P < 0.005, ANOVA with Tukey post hoc.

[0011] FIGs. 3A-3C show Kifc3 is an ontogenic protein regulated by Dyrkl. FIGs. 3A-3B show IBs of whole cell lysates (WCL) from Mk cultured 7d as in FIG. 2, using Sigma rabbit pAb (FIG.3A) or Santa Cruz (SC) mouse mAb (FIG. 3B). Signals normalized to tubulin. FIG. 3C: IF using SC mouse mAb, 630x. N = 3 for IBs, 4 for IF. *, ***, ****P < 0.05, 0.005, 0.001 , ANOVA with Tukey.

[0012] FIGs. 4A-4C show Kifc3 knockdown enhances morphogenesis in neonatal (CB) but not adult Mk. Neonatal and adult human progenitors underwent lentiviral shRNA knockdowns using indicated hairpins, followed by 5d Mk culture. FIG. 4A IBs of WCL. FIG. 4B: Analysis of ploidy (PI) in gated viable CD41+ cells. Flow plots indicate % of cells with 2N, 4N, and >8N DNA content. FIG. 4C: Analysis of size (FSC) in gated viable CD41+ cells. N - 5 (CB) or 4 (adult); *, **, ***P < 0.05, 0.01 , 0.005, ANOVA with Tukey. FIG. 4D shows immunoblotting on whole cell lysate using subcutaneous monoclonal antibodies.

[0013] FIGs. 5A-5B show Kifc3 knockdown enhances platelet release in neonatal (CB) Mk. Neonatal human progenitors underwent lentiviral shRNA knockdowns as described previously using indicated hairpins, followed by 13d 2-phase culture and platelet harvesting. FIG. 5A: Flow cytometric quantitation of relative platelet number / Mk. FIG. 5B: Flow analysis of platelet upregulation of CD62P in response to agonists. N = 3; *, **, ***P < 0.05, 0.01 , 0.005, ANOVA with Tukey.

[0014] FIGs. 6A-6H show Cep170 is not affected by Mk ontogeny or Kifc3. Human progenitors underwent Mk culture and IF staining as in FIGs. 3A-3B. Neonatal (FIGs. 6A-6B) and adult (FIGs. 6C-6D) Mk from 6d culture. White boxes: regions expanded in FIGs. 6B and 6D. FIGs. 6E-6H: Neonatal progenitors underwent shRNA knockdowns with sh GFP (FIGs. 6E-6F) or sh Kifc3-63 (FIGs. 6G-6H) as in FIGs. 4A-4C with 5d of Mk culture. Confocal images were with obtained 63x oil objective and fixed signal intensity gain. Maximum intensity projections of all Z slices spanning entire cells are shown.

[0015] FIGs. 7A-7G show control of Cep192 localization and levels in Mk by ontogenic stage. Culture, IF, and imaging were conducted as in FIGs. 6A-6D. FIGs. 7A-7B: Neonatal (CB) Mk from 6d culture. FIGs. 7C-7E: Adult Mk from 6d culture. FIG. 7F: IB of WCL from 7d Mk culture. FIG. 7G: Graphs from IF experiments: % cells with Cep192 dispersal and Cep192 mean fluorescence intensity (MFI). N = 3 (> 60 cells / sample); *, **P < 0.05, 0.01 , Student’s t test.

[0016] FIGs. 8A-8G show Levels of Kifc3 dictate Mk Cep192 pattern. IF and imaging as in FIGs. 7A-7G, conducted on neonatal (CB) Mk - / + lentiviral shRNA knockdown of Kifc3. Neonatal progenitors underwent transduction, selection, and Mk culture as in FIGs. 4A-4C. shRNAs targeted GFP (FIGs. 8A-8B), Kifc3-62 (FIGs. 8C-8D), and Kifc3-63 (FIGs. 8E-8F). FIG. 8G:Graphs: % cells with Cep192 dispersal and Cep192 relative mean fluorescence intensity (rel MFI). N = 3 (> 40 cells / sample); *, **P < 0.05, 0.01 , ANOVA with Dunnett post hoc.

[0017] FIG. 9 shows a comparison of isoforms for the mouse Cep192 gene. The box indicates the CRISPR-targeted exon used herein.

[0018] FIG. 10 shows the selective effect Cep192 mutation on platelet counts. Data mined from the International Mouse Phenotyping Consortium, comparing Cep192 CRISPR mutant (Jax #042356) homozygote (HOM) and wild type (WT) mice. N = 4-6 for HOM and 504-512 for WT. Statistical method consisted of a linear mixed model framework, including weight.

[0019] FIGs. 11A-11C show identification of candidate Kifc3 inhibitors. FIG. 11 A. LS5, identified in primary screen, and A1 , identified in secondary screen. FIG. 11B. Docking of LS5 (arrow on right) in a2 / L5 / a3 pocket of Kifc3 motor domain. ADP and Mg (ball) are on left. FIG. 11 C. Bioassay: neonatal progenitors cultured 7d in Mk medium - / + compounds were analyzed as in FIGs. 4A- 4C. Graph: Comparison of LS5 at 40 pM with A1 at 10 pM. N = 3; *, ***P < 0.05, 0.005 ANOVA with Tukey.

[0020] FIGs. 12A-12D show A1 treatment phenocopies Kifc3 knockdown. FIG. 12A. Neonatal progenitors were treated and analyzed as in FIGs. 11 A-11 C. N = 3; *, **, ****p < 0.05, 0.01 , 0.001 ANOVA with Tukey. FIGs. 12B-12C. Cells cultured as in A with DMSO (FIG. 12B) or 2.5 pM A1 (FIG. 12C) were analyzed as in FIGs. 7A-8G.

[0021] FIG. 13 shows potential mechanisms for Kifc3-Cep192 control of Mk morphogenesis.

[0022] FIG. 14 shows Cep192 knockdown impairs adult Mk morphogenesis. Top row, left: Neonatal Mk from transduced with control shRNA vector. Top row, middle: Neonatal Mk transduced with sh 62. Top row, right: Neonatal Mk transduced with sh 63. Bottom row: Quantification of IF (> 40 cells / sample), N = 3. Adult progenitors were treated and analyzed as in FIGs. 4A-4C. Adult CD34 cells subjected to lentiviral transduction and selection underwent 7d Mk culture followed by flow cytometry with gating for viable, singlet, CD41+ cells. N = 4. IB on WCL using Bethyl Rab Ab.

[0023] FIG. 15 shows a potential mechanism for shifting from cytokinesis complexes to morphogenesis complexes.

[0024] FIG. 16 shows Plk4 inhibition uncouples ploidy from platelet production. Adult human CD34+ cells pre-cultured 3d (left) or 6d (right) in Mk medium were treated with centrinone (CN) 3d. N = 3-4; *P < 0.05 Student’s T.

[0025] FIGs. 17A-17B shows the chiral center of an inhibitor has no effect on bioactivity of disclosed compound A1. Parent mix and enantiomers were analyzed as in FIGs. 11A-12C. Neonatal Mk from 7d culture - / + compounds A1 and LS5 were analyzed by flow cytometry. N = 3.

[0026] FIG. 18 shows an overlay of LS5 and compound A1 from the present disclosure in the a2 / L5 / a3 pocket of the Kifc3 motor domain.

[0027] FIGs. 19A-19D show Cep192 knockdown impairs adult Mk C morphogenesis. Adult human progenitors underwent lentiviral shRNA knockdowns using indicated hairpins, followed by 5d Mk culture. FIG. 19A: IBs of WCL. FIG. 19B: Analysis of ploidy (PI) in gated viable CD41 + cells. Flow plots indicate % of cells with 2N, 4N, and > 8N DNA content. FIG. 19C: Analysis of size (FSC) in gated viable CD41+ cells. N = 4; *, **, ***P < 0.05, 0.01 , 0.005, ANOVA with Dunnett.

[0028] FIGs. 20A-20E show A1-E88 phenocopies Kifc3 knockdown in Mk but does not affect other lineages. FIGs. 20A-20C: Neonatal progenitors treated with 20 pM A1-E88 (E88) underwent Mk culture followed by analysis of morphogenesis (FIGs. 20A-20B) and platelet release (FIG. 20C) as in FIGs. 4A-5B. N = 4; *, **P < 0.05, 0.01 , Students t test. FIGs. 20D-20E. Neonatal progenitors treated with 20 M E88 underwent erythroid (FIG. 20D) or granulocytic (FIG. 20E) culture and analysis.

[0029] FIGs. 21A-21B show modulation of Cep192 levels and localization in neonatal Mk by putative Kifc3 inhibitor. Culture, IB, IF, and imaging were conducted as in FIGs. 7A-8G, with treatments as indicated. FIG. 21 A Adult and neonatal (CB) Mk from 7d culture. Graph: Cep192 levels in neonatal Mk relative to DMSO control, with tubulin normalization. N = 3 ; **P < 0.05, 0.01, ANOVA with Dunnett. FIG. 21 B: Neonatal Mk, 6d culture. DMSO: 35 cells; E88: 26 cells

[0030] FIG. 22 shows Kifc3 enforcement in adult Mk impairs morphogenesis. Adult human progenitors were transduced with lentiviral expression constructs (pGenlenti backbone) using KIFC3 ENST00000541240.5 (see FIG. 2). Following 7 days Mk culture, cells underwent flow cytometry as in FIGs. 4A-4D. N = 3; **P < 0.01 , Student’s t test.

[0031] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that boththe foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0032] Studies of Dyrk inhibition highlighted a need to identify downstream elements more amenable to therapeutic manipulation and further identified Kifc3, a minus-end-directed kinesin motor involved in delivery of proteins to the centrosome. Important features from a therapeutic standpoint include a unique, structurally characterized ATP-binding pocket and the absence of abnormalities in knockout mice. Characterization of Kifc3 in neonatal and adult human Mk uncovered several key properties: 1) downmodulation in adult vs neonatal cells, 2) induction of adult morphogenesis in neonatal cells subjected to knockdown (kd), 3) strong enhancement in functional platelet production by neonatal cells subjected to kd. In silico modelling using kinesin domain structures of Kifc3 and of Kifd - / + its inhibitor enabled identification of candidate Kifc3 small molecule inhibitors that exert effects on neonatal Mk resembling those seen with Kifc3 kd. of Kifc3 in megakaryopoiesis. Cep192 acts as a scaffold for aurora (Aurk) and polo-like (Plk) kinases, recruiting these factors to centrosomes to coordinate organelle maturation. Importantly, Cep192 also recruits these kinases to the periphery to promote actin remodeling and process extension.

[0033] Elevated levels of Cep192 sequester these kinases from mitotic scaffolds that coordinate cytokinesis. In these studies, Cep192 resides predominantly at the centrosome in neonatal Mk; in adult Mk most of the Cep192 has dispersed from the centrosome and overall levels are increased. Knocking down Kifc3 in neonatal Mk causes Cep192 to disperse from the centrosome and increase in amount. In mice where CRISPR has eliminated the long Cep192 isoform-1 , which binds Plk1 and Plk4, blood counts show a decrease in platelets but not red or white cells. Herein, a model is proposed (FIG. 1) in which the diminished Kifc3 in adult Mk changes the distribution and levels of Cep192, in turn re-localizing Aurk and Plk factors. This arrangement promotes Plk / Aurk-mediated actin remodeling supporting enlargement and process extension, while simultaneously disabling cytokinesis in favor of endomitosis. Experiments in Aim 1 will delineate molecular mechanisms by which Kifc3-Cep192 signaling controls Mk morphogenesis. Aim 2 will translate these findings through development of a clinically applicable Kifc3 inhibitor.Diminished Morphogenesis in Fetal-type Megakaryocytes

[0034] As compared with adult-type megakaryocytes (Mk), fetal-type Mk display increased progenitor proliferation, decreased morphogenesis, and decreased platelet release. Mkmorphogenesis comprises a coordinated sequence of events that prime the cells for intravascular platelet release: endomitosis, polyploidization, cellular enlargement, and polarized process extension. Attenuation of this program occurs most prominently during fetal development but persists in normal infants for up to a year and strongly manifests in iPSC-derived megakaryocytes. These ontogenically controlled features ensure functional adaptation to host needs. Thus, fetal and infantile development are best supported by rapid Mk expansion and by production of hypoactive platelets. However, fetal-type megakaryopoiesis can cause clinical problems in circumstances requiring sharply augmented platelet production, i.e. “stress thrombopoiesis”, and can also undergo neoplastic transformation. Below are four clinical scenarios in which fetal-type megakaryopoiesis plays a major role.Neonatal Thrombocytopenia

[0035] Low platelet counts have been described in up to 4% of all newborns, 25% of NICU patients, and 50% of ELBW infants (weight < 1 kg). 10% of preterm infants receive platelet transfusions while in the hospital. Given the US prematurity rate of 10.7% and US birth rate of 3.7 million / year, an estimated 37,000 babies receive platelet transfusions each year. Recent clinical trials have demonstrated increased morbidity and mortality associated with liberal platelet transfusions for preterm infants in the NICU. Factors contributing to deleterious outcome include the inappropriately high pro-coagulant and pro-inflammatory properties of adult platelets, as well as volume overload. Because of these findings, guidelines recommend a restrictive platelet threshold of 25 * 109 / L, rather than the liberal one of 50 109 / L, for initiating transfusion of preterm infants; unfortunately, liberal transfusion practice remains prevalent due to concerns about intracranial hemorrhage.

[0036] A strategy to circumvent platelet transfusions using thrombopoietin receptor agonists (TRA) has received low enthusiasm from the neonatology community due to the lack of efficacy of romiplostim in newborn mice and the potential of eltrombopag for neurodevelopmental toxicity. In one aspect, the disclosed compounds can be administered to preterm infants and / or other neonates to increase the infants’ own production of platelets, thus eliminating the need for transfusions from adults, and improving outcomes for the neonates.Umbilical Cord Blood Stem Cell TransplantationUmbilical cord blood (UBC) hematopoietic stem cell transplantation (HCT) initially gained popularity because of several advantages it offered over adult donor HCT: 1) ease and rapidity of obtaining and disseminating units, 2) diminished graft-versus-host disease enabling lessrestrictive HLA matching, 3) suitability for those lacking a matched donor ensuring availability for ethnic minority recipients, 4) robust self-renewal properties enabling ex vivo expansion. Thus, >40,000 UBC HCT have been performed for a range of neoplastic and non-neoplastic diseases. However, popularity of this treatment has declined due to inferior hematopoietic recovery combined with the emergence of haplo-identical HCT. Use of UBC HCT, as compared with adult, particularly affects platelet recovery, and a large comparative trial identified delayed platelet recovery as an independent risk factor for treatment-related and overall mortality. Attempts to enhance platelet recovery with romiplostim or thrombopoietin treatment had minimal efficacy in UCB recipients, in contrast to the benefits of TRA treatment in adult HCT recipients. Recently, UCB HCT has undergone a renaissance due to advances in augmentation of stem cell numbers, previously a limiting factor of this approach; 47 different studies registered with ClinicalTrials.gov are actively recruiting patients. Most prominently, omidubicel, a nicotinamide-expanded UBC product, demonstrated in a phase III, 2-arm trial striking clinical advantages over standard UCB, with neutrophilic engraftment rates similar to those with adult HCT, decreased infection rates, and significantly decreased red cell transfusions. However, despite the infusion of 30-fold more CD34+ cells / kg in the omidubicel arm, no significant decrease in platelet transfusions resulted. The poor platelet engraftment, refractory to TRA and stem cell expansion treatments, arises from properties intrinsic to UCB-derived Mk. Analysis of marrow Mk in UCB versus adult HCT showed equivalence in numbers but a marked decrease in size in UCB-derived Mk, predicted to reduce the platelet-producing mass by >3-fold. Importantly, Mk size also has implications for hematopoietic stem cell (HSC) function, with high ploidy large cytoplasmic Mk (LCM) maintaining HSC quiescence. Mice with small Mk manifested pronounced defects in HSC reconstitution of hematopoiesis by limiting dilution and competitive transplantation, further contributing to defective platelet recovery. The HSC defects associated with small Mk may explain the increased mortality associated with delayed platelet recovery, a finding that could not be attributed to defects in hemostasis. In one aspect, disclosed herein is a method for improving outcomes of umbilical cord blood stem cell transplants in a subject, the method including administering A1 , LS5, or a disclosed compound to umbilical cord blood stem cells prior to transplanting the umbilical cord blood stem cells into the subject..Down Syndrome-associated Myeloid Neoplasms

[0037] Up to 30% of infants with Down syndrome (~5000 / year) develop transient abnormal myelopoiesis (TAM) in their first 3 months. TAM consists of a clonal megakaryoblastic proliferation in blood, marrow, and liver caused by three drivers: fetal-type megakaryopoiesis, trisomy 21 , andsomatic mutations in GATA1. -70% of patients require no therapy due to spontaneous regression of blasts as hematopoiesis naturally shifts toward an adult-type program. However, 22-42% of TAM patients display life-threatening symptoms requiring treatment with low-dose cytarabine, which is effective but dangerously myelosuppressive in this setting. In addition, TAM patients with delayed blast regression, i.e. persistence of molecular minimum residual disease 3 months from diagnosis, demonstrate a 3-fold increase in risk of progression to full blown myeloid leukemia (ML-DS). Thus, targeted therapy directed toward one of the three molecular drivers could potentially obviate cytotoxic therapy and diminish risk of leukemic progression for >1000 infants / year. Inhibitors of Dyrkla, upregulated with trisomy 21 , have been proposed to treat Downsyndrome-associated disorders, but a recent study has identified Dyrkla as a tumor suppressor in the setting of TAM.Ex Vivo Generation of Transfusion Products for Thrombocytopenia

[0038] Major forces continue to motivate development of ex vivo platelet production for clinical use. One comprises the donor platelet shortage affecting clinical care globally. In the US, platelet transfusions increased by 16% between 2017 and 2019, concurrent with a 2% drop in platelet donation. A survey from the fall of 2019, prior to Covid-19 impairment of the blood supply, indicated that 40% of US hospitals routinely confronted platelet supply challenges that affected patient care. Another force consists of the rising cost of platelets, now estimated at $1360 / unit, not including new expenses for pathogen inactivation. An additional benefit of a donorindependent platelet supply is the capacity to generate H LA-null units for heavily transfused patients who have developed antibodies. Because of their limited expandability, primary adult Mk progenitors cannot be used, despite their superior efficiency in platelet production. iPSC-derived Mk also have limited expandability and must undergo oncogene-induced immortalization to be scaled up for clinical platelet production. iPSCMk also show limited morphogenesis and platelet production due to their fetal phenotype UCB Mk represent a promising source due to their capacity for extensive expansion without the need for oncogene transduction. In addition, UCB Mk can be used for direct infusion, bypassing the platelet collection phase which damages the platelets, adds cost, and increases risk of contamination. Their main drawback is that they produce 10-fold fewer platelets / cell than adult Mk.Molecular Pathways Controlling Mk Ontooenic Stage

[0039] Over the past 15 years several groups have identified molecular differences between fetal and adult Mk using various model systems. Comparing primary human cord blood CD34+progenitor-derived Mk (CB-Mk) and adult peripheral blood CD34+ progenitor-derived Mk (PBMk), distinct transcriptional elongation modes that determined levels of Mkl1 were identified, a coactivator driving Mk morphogenesis. Subsequently, conversion of the fetal to an adult ontogenic program was demonstrated through pharmacologic induction of Mkl1. The strategy comprised inhibition of Dyrk kinase activity, known to repress Mkl 1 function and worked well with a variety of human fetal-type Mk: CB Mk, iPSC-derived Mk, conditionally immortalized Mk, and SR1- expanded CB Mk.

[0040] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0041] Disclosed herein are compounds of Formula I:Formula I wherein X is CH or N; wherein Y is CH2or NH; wherein Z is N, CH, or C; wherein n is from 0 to 4; wherein Ri is an aryl group, heteroaryl group, cycloalkyl group, or heterocycloalkyl group; wherein R2is OH or O; or wherein Ri and R2together with Z form a substituted or unsubstituted aryl group, heteroaryl group, cycloalkyl group, or heterocycloalkyl group; wherein R3 is a substituted or unsubstituted aryl or heteroaryl group; andwherein R4is a substituted or unsubstituted alkyl group, aryl group, or amide.

[0042] In some aspects, the compound is not A1 or LS5

[0043] In one aspect, when X is CH, the compound has a stereocenter at X. In another aspect, Rs can be a phenyl group substituted with hydroxamic acid such as, for example,In one aspect, n is 0; in an alternative aspect, n is 1. In one aspect, Ri is phenyl. In another aspect, X is N and R4is benzyl, or X is CH and R4

[0045] In still another aspect, the compound can be selected fromwherein * represents a stereocenter.

[0046] In some aspects, the compound has substantially S stereochemistry, has substantially R stereochemistry, has an enantiomeric excess of from about 5% to about 95% R stereochemistry, has an enantiomeric excess of from about 5% to about 95% S stereochemistry, or is a racemic mixture.

[0047] Also disclosed herein is a pharmaceutical composition including one or more of the disclosed compounds. In some aspects, the pharmaceutical composition further includes at least one carrier or excipient.

[0048] In another aspect, disclosed is a method for treating a disease or disorder associated with aberrant Kifc3 function in a subject, the method including at least the step of administering a disclosed compound or pharmaceutical composition, or a pharmaceutical composition including A1 or LS5 to the subject. In a further aspect, the disease or disorder includes comprises neonatal thrombocytopenia, delayed platelet recovery following umbilical cord blood stem cell transplantation, Down syndrome-associated myeloid neoplasm, or any combination thereof. In one aspect, the subject can be a human, such as, for example, a neonatal human. In a further aspect, the method can increase platelet production in the subject.

[0049] In still another aspect, disclosed is a method of ex vivo platelet production, the method including at least the step of administering a disclosed compound or pharmaceutical composition, or a pharmaceutical composition including A1 or LS5 to primary adult megakaryocyte progenitor cells.

[0050] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0051] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0052] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0053] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0054] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0055] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0056] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0057] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0058] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a Kifc3 inhibitor,” “a pharmaceutically acceptable salt,” or “an excipient,” include, but are not limited to, mixtures or combinations of two or more such Kifc3 inhibitors, pharmaceutically acceptable salts, or excipients, and the like.

[0059] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can beexpressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0060] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0061] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0062] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal valueindicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0063] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0064] As used herein, “neonatal” refers to a newborn infant up to 28 days of age.

[0065] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more - CO(CH2)SCO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0066] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted ( / .e., further substituted or unsubstituted).

[0067] In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0068] The term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain ( / .e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0069] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t- butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.

[0070] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyl” specifically refers to an alkylgroup that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxyal kyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0071] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0072] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0073] The term “alkanediyl” as used herein, refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, — CH2 — (methylene), — CH2CH2 — , — CH2C(CH3)2CH2 — , and — CH2CH2CH2 — are non-limiting examples of alkanediyl groups.

[0074] The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as — OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as — OA1— OA2or — OA1— (OA2)a— OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0075] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0076] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0077] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0078] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, andis included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0079] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized IT electrons above and below the plane of the molecule, where the TT clouds contain (4n+2) TT electrons. A further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “ Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

[0080] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, — NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond. For example, biaryl to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0081] The term “aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C=O.

[0082] The terms “amine” or “amino” as used herein are represented by the formula — NAW, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is — NH2.

[0083] The term “alkylamino” as used herein is represented by the formula — NH(-alkyl) and — N (-alkyl)2, where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylaminogroup, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

[0084] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0085] The term “ester” as used herein is represented by the formula — OC(O)A1or — C(O)OA1, where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula — (A1O(O)C-A2-C(O)O)a— or — (A1O(O)C-A2-OC(O))a— , where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

[0086] The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula — (A1O-A2O)a— , where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0087] The terms “halo,” “halogen” or “halide,” as used herein can be used interchangeably and refer to F, Cl, Br, or I.

[0088] The terms “pseudohalide,” “pseudohalogen” or “pseudohalo,” as used herein can be used interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanato, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0089] The term “heteroalkyl” as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptional ly quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0090] The term “heteroaryl” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1 ,2- b]pyridazinyl, imidazo[1 ,2-a]pyrazinyl, benzo[c][1 ,2,5]thiadiazolyl, benzo[c][1 ,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0091] The terms “heterocycle” or “heterocyclyl,” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle,” and “polycyclic heterocycle.” Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1 ,2,3-oxadiazole, 1 ,2,5-oxadiazole and 1 ,3,4-oxadiazole, thiadiazole, including, 1 ,2,3-thiadiazole, 1,2,5-thiadiazole, and 1 ,3,4-thiadiazole, triazole, including, 1 ,2,3-triazole, 1,3,4-triazole, tetrazole, including 1 ,2,3,4-tetrazole and 1 ,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including 1 ,2,4-triazine and 1 ,3,5-triazine, tetrazine, including 1 ,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like. Alternatively, for example,a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0092] The term “bicyclic heterocycle” or “bicyclic heterocyclyl” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1 , 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[1 ,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1 ,3-benzodioxolyl, 2,3-dihydro- 1 ,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, 1 H-pyrazolo[4,3-c]pyridin-3-yl; 1 H-pyrrolo[3,2- b]pyridin-3-yl; and 1 H-pyrazolo[3,2-b]pyridin-3-yl.

[0093] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0094] The term “hydroxyl” or “hydroxy” as used herein is represented by the formula — OH.

[0095] The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0096] The term “azide” or “azido” as used herein is represented by the formula — N3.

[0097] The term “nitro” as used herein is represented by the formula — NO2.

[0098] The term “nitrile” or “cyano” as used herein is represented by the formula — CN.

[0099] The term “silyl” as used herein is represented by the formula — SiA1A2A3, where A1, A2,and A3can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0100] The term “sulfo-oxo” as used herein is represented by the formulas — S(O)A1, — S(O)2A1, — OS(O)2A1, or — OS(O)2OA1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0101] The term “thiol” as used herein is represented by the formula — SH.

[0102] “R1,” “R2,” “R3,”... “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant ( / .e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

[0103] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stableor chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted ( / .e., further substituted or unsubstituted).

[0104] The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0105] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o-4R°; -(CH2)o-40R°; -0(CH2)o-4R°, -O- (CH2)O-4C(0)QR°; -(CH2)O-4CH(OR°)2; -(CH2)O-4SR°; -(CH2)o-4Ph, which may be substituted with R°; -(CH2)o-40(CH2)o-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o-40(CH2)o-i-pyridyl which may be substituted with R°; -NO2; -CN; - N3; -(CH2)O-4N(R°)2; -(CH2)O-4N(R°)C(0)R°; -N(R°)C(S)R°; -(CH2)O-4N(RO)C(O)NRO2; -N(RO)C(S)NR°2; -(CH2)O-4N(R°)C(0)OR°;N(R°)N(R°)C(O)R°; -N(Ro)N(R°)C(O)NRo2; -N(R°)N(R°)C(O)OR°; -(CH2)o-4C(0)R°; -C(S)R°; - (CH2)O-4C(0)OR°; -(CH2)O-4C(0)SR°; -(CH2)o-4C(0)OSiR°3; -(CH2)o-40C(0)R°; -OC(0)(CH2)o- 4SR-, SC(S)SR°; -(CH2)O-4SC(0)R°; -(CH2)O-4C(0)NR°2; -C(S)NRO2; -C(S)SR°; -(CH2)O-4OC(O)NRO2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)O-4SSR°; - (CH2)O-4S(0)2R0; -(CH2)O-4S(0)2OR°; -(CH2)O-40S(0)2R°; -S(O)2NRO2; -(CH2)O-4S(O)RO; -N(RO)S(0)2NRO2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NRO2;P(O)2RO; -P(O)RO2; -OP(O)RO2; -OP(O)(ORO)2; SiR°3; -(C1-4 straight or branched alkylene)O- N(R°)2; or -(C1-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -0(CH2)o- iPh, -CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0106] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), -(CH2)O-2OH, -(CH2)O-20R*, -(CH2)O-2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)0-SH, -(CH2)O_2NH2, -straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci_4aliphatic, - CH2Ph, -O(CH2)0-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0107] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(O)R‘, =NNHC(O)OR*, =NNHS(O)2R*, =NR‘, =NOR*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0108] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, - OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci_4aliphatic, -CH2Ph, -O(CH2)0-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0109] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R+, -NR+2, -C(O)R+, -C(O)OR+, -C(O)C(O)R+, -C(O)CH2C(O)R+, -S(O)2R+, -S(O)2NR+2, -C(S)NR+2I-C(NH)NR+2Ior -N(R+)S(O)2R+; wherein each Rt is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0- 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of Rt, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclicri ng having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0110] Suitable substituents on the aliphatic group of Rt are independently halogen, - R*, -(haloR*), -OH, -OR’, -O(haloR’), -ON, -C(O)OH, -C(O)OR*, -NH2, -NHR", -NR*2, or - NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2Ph, -O(CH2)0-iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0111] The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate.

[0112] The terms “hydrolysable group” and “hydrolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-lnterscience, 1999).

[0113] The term “organic residue” defines a carbon containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

[0114] A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4- thiazolidinedione radical in a particular compound has the structure:regardless of whether thiazolidinedione is used to prepare the compound. In some embodiments the radical (for example an alkyl) can be further modified ( / .e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” The number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary elsewhere herein.

[0115] “Organic radicals,” as the term is defined and used herein, contain one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bound to at least some of the carbon atoms of the organic radical. One example of an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthyl radical. In some embodiments, an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, monosubstituted amino, di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, wherein the terms are defined elsewhere herein. A few non-limiting examples of organic radicals that include heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals and the like.

[0116] “Inorganic radicals,” as the term is defined and used herein, contain no carbon atoms and therefore comprise only atoms other than carbon. Inorganic radicals comprise bonded combinations of atoms selected from hydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, and halogens such as fluorine, chlorine, bromine, and iodine, which can be present individually or bonded together in their chemically stable combinations. Inorganic radicals have 10 or fewer, or preferably one to six or one to four inorganic atoms as listed above bonded together. Examples of inorganic radicals include, but not limited to, amino, hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonly known inorganic radicals. The inorganicradicals do not have bonded therein the metallic elements of the periodic table (such as the alkali metals, alkaline earth metals, transition metals, lanthanide metals, or actinide metals), although such metal ions can sometimes serve as a pharmaceutically acceptable cation for anionic inorganic radicals such as a sulfate, phosphate, or like anionic inorganic radical. Inorganic radicals do not comprise metalloids elements such as boron, aluminum, gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gas elements, unless otherwise specifically indicated elsewhere herein.

[0117] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

[0118] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0119] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-lngold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0120] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically-labeled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,35S,18F, and36CI, respectively. Compounds further comprise prodrugs thereof and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. I sotopical ly labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.

[0121] The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, theinvention includes all such possible solvates.

[0122] The term “co-crystal” means a physical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework in the crystalline lattice. In certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.

[0123] It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.keto form enol form amide form imidic acid formLikewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, the invention includes all such possible tautomers.

[0124] It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.

[0125] In some aspects, a structure of a compound can be represented by a formula:which is understood to be equivalent to a formula:

[0126] wherein n is typically an integer. That is, R" is understood to represent five independent substituents, Rn<aRn<bRn(c), Rn<dand R',(e). By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance Rn(a)is halogen, then Rn(b)is not necessarily halogen in that instance.

[0127] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).Pharmaceutical Compositions

[0128] As used herein, “administering” can refer to an administration that is oral, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, or via an implanted reservoir or other device that administers, either actively or passively (e.g. by diffusion) a composition the perivascular space and adventitia. For example a medical device such as a stent can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells. The term “parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for preventionof a disease or condition.

[0129] As used herein, “therapeutic agent” can refer to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a pharmacologic, immunogenic, biologic and / or physiologic effect on a subject to which it is administered to by local and / or systemic action. A therapeutic agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. A therapeutic agent can be a secondary therapeutic agent, or in other words, the component(s) of a composition to which an additional part and / or other effect of the composition is attributed. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta- blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double-and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. The term therapeutic agent also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or prodrugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0130] As used herein, “attached” can refer to covalent or non-covalent interaction between two or more molecules. Non-covalent interactions can include ionic bonds, electrostatic interactions, van der Walls forces, dipole-dipole interactions, dipole-induced-dipole interactions, London dispersion forces, hydrogen bonding, halogen bonding, electromagnetic interactions, TT-TT interactions, cation-n interactions, anion-TT interactions, polar TT-interactions, and hydrophobic effects.

[0131] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0132] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as neonatal thrombocytopenia, myeloid neoplasms in Down syndrome, and / or another disorder impacted by insufficient platelet count. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of neonatal thrombocytopenia, myeloid neoplasms in Down syndrome, and / or another disorder impacted by insufficient platelet count in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used hereincan refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0133] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0134] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0135] As used herein, “effective amount” can refer to the amount of a disclosed compound or pharmaceutical composition provided herein that is sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term can also include within its scope amounts effective to enhance or restore to substantially normal physiological function.

[0136] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in someinstances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0137] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0138] A response to a therapeutically effective dose of a disclosed compound and / or pharmaceutical composition, for example, can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0139] In one aspect, an effective dose of a disclosed compound can result in a systemic or local concentration of the compound of from about 10 pM to about 20 pM, or of about 20 pM. In a further aspect, this concentration shows therapeutic effects in both neonatal and adult subjects. In another aspect, a concentration of a disclosed compound of from about 10 pM to about 20 pMex vivo or applied to cord blood stem cells is also therapeutically effective.

[0140] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0141] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0142] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0143] The term “pharmaceutically acceptable salts”, as used herein, means salts of the active principal agents which are prepared with acids or bases that are tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to; sodium, potassium, calcium, ammonium, organic amino, magnesium salt, lithium salt, strontium salt or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to; those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like.

[0144] The term “pharmaceutically acceptable ester” refers to esters of compounds of the present disclosure which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Examples of pharmaceutically acceptable, non-toxic esters of the present disclosure include C 1 -to-C 6 alkyl esters and C 5 -to-C 7 cycloalkyl esters, although C 1 -to-C 4 alkyl esters are preferred. Esters of disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable esters can be appended onto hydroxy groups by reaction of the compound that contains the hydroxy group with acid and an alkylcarboxylic acid such as acetic acid, or with acid and an arylcarboxylic acid such as benzoic acid. In the case of compounds containing carboxylic acid groups, the pharmaceutically acceptable esters are prepared from compounds containing the carboxylic acid groups by reaction of the compound with base such as triethylamine and an alkyl halide, for example with methyl iodide, benzyl iodide, cyclopentyl iodide or alkyl triflate. They also can be prepared by reaction of the compound with an acid such as hydrochloric acid and an alcohol such as ethanol or methanol.

[0145] The term “pharmaceutically acceptable amide” refers to non-toxic amides of the present disclosure derived from ammonia, primary C 1 -to-C 6 alkyl amines and secondary C 1 -to-C 6 dialkyl amines. In the case of secondary amines, the amine can also be in the form of a 5- or 6- membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C 1 -to-C 3 alkyl primary amides and C 1 -to-C 2 dialkyl secondary amides are preferred. Amides of disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable amides can be prepared from compounds containing primary or secondary amine groups by reaction of the compound that contains the amino group with an alkyl anhydride, aryl anhydride, acyl halide, or aroyl halide. In the case of compounds containing carboxylic acid groups, the pharmaceutically acceptable amides are prepared from compounds containing the carboxylic acid groups by reaction of the compound with base such as triethylamine, a dehydrating agent such as dicyclohexyl carbodiimide or carbonyl diimidazole, and an alkyl amine, dialkylamine, for example with methylamine, diethylamine, and piperidine. They also can be prepared by reaction of the compound with an acid such as sulfuric acid and an alkylcarboxylic acid such as acetic acid, or with acid and an arylcarboxylic acid such as benzoic acid under dehydrating conditions such as with molecular sieves added. The composition can contain a compound of the present disclosure in the form of a pharmaceutically acceptable prodrug.

[0146] The term “pharmaceutically acceptable prodrug” or “prodrug” represents those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use. Prodrugs of the present disclosure can be rapidlytransformed in vivo to a parent compound having a structure of a disclosed compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0147] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.gr, a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0148] The term “contacting” as used herein refers to bringing a disclosed compound or pharmaceutical composition in proximity to a cell, a target protein, or other biological entity together in such a manner that the disclosed compound or pharmaceutical composition can affect the activity of the a cell, target protein, or other biological entity, either directly; / .e., by interacting with the cell, target protein, or other biological entity itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the cell, target protein, or other biological entity itself is dependent.

[0149] Described herein are compounds and compositions that have therapeutic or clinical utility. Also described herein are methods of synthesizing the compounds. Also described herein are methods of administering the compounds and compositions to a subject in need thereof. In some aspects, the subject can have neonatal thrombocytopenia, myeloid neoplasm in Down syndrome, or another condition impacted by low platelet count. Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.

[0150] In various aspects, the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one disclosed compound, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof. As used herein,“pharmaceutically-acceptable carriers” means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, and adjuvants. The disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy and pharmaceutical sciences.

[0151] In a further aspect, the disclosed pharmaceutical compositions comprise a therapeutically effective amount of at least one disclosed compound, at least one product of a disclosed method, or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutically acceptable carrier, optionally one or more other therapeutic agent, and optionally one or more adjuvant. The disclosed pharmaceutical compositions include those suitable for oral, rectal, topical, pulmonary, nasal, and parenteral administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. In a further aspect, the disclosed pharmaceutical composition can be formulated to allow administration orally, nasally, via inhalation, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially and intratumorally.

[0152] As used herein, “parenteral administration” includes administration by bolus injection or infusion, as well as administration by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0153] In various aspects, the present disclosure also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, a therapeutically effective amount of a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof. In a further aspect, a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof, or any subgroup or combination thereof may be formulated into various pharmaceutical forms for administration purposes.

[0154] Pharmaceutically acceptable salts can be prepared from pharmaceutically acceptable non-toxic bases or acids. For therapeutic use, salts of the disclosed compounds are those whereinthe counter ion is pharmaceutically acceptable. However, salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are contemplated by the present disclosure. Pharmaceutically acceptable acid and base addition salts are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the disclosed compounds are able to form.

[0155] In various aspects, a disclosed compound comprising an acidic group or moiety, e.g., a carboxylic acid group, can be used to prepare a pharmaceutically acceptable salt. For example, such a disclosed compound may comprise an isolation step comprising treatment with a suitable inorganic or organic base. In some cases, it may be desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free acid compound by treatment with an acidic reagent, and subsequently convert the free acid to a pharmaceutically acceptable base addition salt. These base addition salts can be readily prepared using conventional techniques, e.g., by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they also can be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before.

[0156] Bases which can be used to prepare the pharmaceutically acceptable base-addition salts of the base compounds are those which can form non-toxic base-addition salts, i.e., salts containing pharmacologically acceptable cations such as, alkali metal cations (e.g., lithium, potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water-soluble amine addition salts such as N-methylglucamine-(meglumine), lower alkanolammonium and other such bases of organic amines. In a further aspect, derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. In various aspects, such pharmaceutically acceptable organic non-toxic bases include, but are not limited to, ammonia, methylamine, ethylamine, propylamine, isopropylamine, any of the four butylamine isomers, betaine, caffeine, choline, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, N,N'- dibenzylethylenediamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, tromethamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,quinuclidine, pyridine, quinoline and isoquinoline; benzathine, / V-methyl-D-glucamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, hydrabamine salts, and salts with amino acids such as, for example, histidine, arginine, lysine and the like. The foregoing salt forms can be converted by treatment with acid back into the free acid form.

[0157] In various aspects, a disclosed compound comprising a protonatable group or moiety, e.g., an amino group, can be used to prepare a pharmaceutically acceptable salt. For example, such a disclosed compound may comprise an isolation step comprising treatment with a suitable inorganic or organic acid. In some cases, it may be desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with a basic reagent, and subsequently convert the free base to a pharmaceutically acceptable acid addition salt. These acid addition salts can be readily prepared using conventional techniques, e.g., by treating the corresponding basic compounds with an aqueous solution containing the desired pharmacologically acceptable anions and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they also can be prepared by treating the free base form of the disclosed compound with a suitable pharmaceutically acceptable non-toxic inorganic or organic acid.

[0158] Acids that can be used to prepare the pharmaceutically acceptable acid-addition salts of the base compounds are those which can form non-toxic acid-addition salts, i.e., salts containing pharmacologically acceptable anions formed from their corresponding inorganic and organic acids. Exemplary, but non-limiting, inorganic acids include hydrochloric hydrobromic, sulfuric, nitric, phosphoric and the like. Exemplary, but non-limiting, organic acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, isethionic, lactic, maleic, malic, mandelicmethanesulfonic, mucic, pamoic, pantothenic, succinic, tartaric, p-toluenesulfonic acid and the like. In a further aspect, the acid-addition salt comprises an anion formed from hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0159] In practice, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, of the present disclosure can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired foradministration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present disclosure can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the present disclosure, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and / or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0160] It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. That is, a “unit dosage form” is taken to mean a single dose wherein all active and inactive ingredients are combined in a suitable system, such that the patient or person administering the drug to the patient can open a single container or package with the entire dose contained therein, and does not have to mix any components together from two or more containers or packages. Typical examples of unit dosage forms are tablets (including scored or coated tablets), capsules or pills for oral administration; single dose vials for injectable solutions or suspension; suppositories for rectal administration; powder packets; wafers; and segregated multiples thereof. This list of unit dosage forms is not intended to be limiting in any way, but merely to represent typical examples of unit dosage forms.

[0161] The pharmaceutical compositions disclosed herein comprise a compound of the present disclosure (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents. In various aspects, the disclosed pharmaceutical compositions can include a pharmaceutically acceptable carrier and a disclosed compound, or a pharmaceutically acceptable salt thereof. In a further aspect, a disclosed compound, or pharmaceutically acceptable salt thereof, can also beincluded in a pharmaceutical composition in combination with one or more other therapeutically active compounds. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0162] Techniques and compositions for making dosage forms useful for materials and methods described herein are described, for example, in the following references: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.).

[0163] The compounds described herein are typically to be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The deliverable compound will be in a form suitable for oral, rectal, topical, intravenous injection or parenteral administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. The compounds may be administered as a dosage that has a known quantity of the compound.

[0164] Because of the ease in administration, oral administration can be a preferred dosage form, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. However, other dosage forms may besuitable depending upon clinical population (e.g., age and severity of clinical condition), solubility properties of the specific disclosed compound used, and the like. Accordingly, the disclosed compounds can be used in oral dosage forms such as pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0165] The disclosed pharmaceutical compositions in an oral dosage form can comprise one or more pharmaceutical excipient and / or additive. Non-limiting examples of suitable excipients and additives include gelatin, natural sugars such as raw sugar or lactose, lecithin, pectin, starches (for example corn starch or amylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tylose, talcum, lycopodium, silica gel (for example colloidal), cellulose, cellulose derivatives (for example cellulose ethers in which the cellulose hydroxy groups are partially etherified with lower saturated aliphatic alcohols and / or lower saturated, aliphatic oxyalcohols, for example methyl oxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate), fatty acids as well as magnesium, calcium or aluminum salts of fatty acids with 12 to 22 carbon atoms, in particular saturated (for example stearates), emulsifiers, oils and fats, in particular vegetable (for example, peanut oil, castor oil, olive oil, sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower seed oil, cod liver oil, in each case also optionally hydrated); glycerol esters and polyglycerol esters of saturated fatty acids C12H24O2 to C18H36O2 and their mixtures, it being possible for the glycerol hydroxy groups to be totally or also only partly esterified (for example mono-, di- and triglycerides); pharmaceutically acceptable mono- or multivalent alcohols and polyglycols such as polyethylene glycol and derivatives thereof, esters of aliphatic saturated or unsaturated fatty acids (2 to 22 carbon atoms, in particular 10-18 carbon atoms) with monovalent aliphatic alcohols (1 to 20 carbon atoms) or multivalent alcohols such as glycols, glycerol, diethylene glycol, pentacrythritol, sorbitol, mannitol and the like, which may optionally also be etherified, esters of citric acid with primary alcohols, acetic acid, urea, benzyl benzoate, dioxolanes, glyceroformals,tetra hydrofurfuryl alcohol, polyglycol ethers with C1-C12-alcohols, dimethylacetamide, lactamides, lactates, ethylcarbonates, silicones (in particular medium-viscous polydimethyl siloxanes), calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, magnesium carbonate and the like.

[0166] Other auxiliary substances useful in preparing an oral dosage form are those which cause disintegration (so-called disintegrants), such as: cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose or microcrystalline cellulose. Conventional coating substances may also be used to produce the oral dosage form. Those that may for example be considered are: polymerizates as well as copolymerizates of acrylic acid and / or methacrylic acid and / or their esters; copolymerizates of acrylic and methacrylic acid esters with a lower ammonium group content (for example EudragitR RS), copolymerizates of acrylic and methacrylic acid esters and trimethyl ammonium methacrylate (for example EudragitR RL); polyvinyl acetate; fats, oils, waxes, fatty alcohols; hydroxypropyl methyl cellulose phthalate or acetate succinate; cellulose acetate phthalate, starch acetate phthalate as well as polyvinyl acetate phthalate, carboxy methyl cellulose; methyl cellulose phthalate, methyl cellulose succinate, -phthalate succinate as well as methyl cellulose phthalic acid half ester; zein; ethyl cellulose as well as ethyl cellulose succinate; shellac, gluten; ethylcarboxyethyl cellulose; ethacrylate-maleic acid anhydride copolymer; maleic acid anhydride-vinyl methyl ether copolymer; styrol-maleic acid copolymerizate; 2-ethyl-hexyl-acrylate maleic acid anhydride; crotonic acid-vinyl acetate copolymer; glutaminic acid / glutamic acid ester copolymer; carboxymethylethylcellulose glycerol monooctanoate; cellulose acetate succinate; polyarginine.

[0167] Plasticizing agents that may be considered as coating substances in the disclosed oral dosage forms are: citric and tartaric acid esters (acetyl-triethyl citrate, acetyl tributyl-, tributyl-, triethyl-citrate); glycerol and glycerol esters (glycerol diacetate, -triacetate, acetylated monoglycerides, castor oil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-, dipropylphthalate), di-(2-methoxy- or 2-ethoxyethyl)-phthalate, ethylphthalyl glycolate, butylphthalylethyl glycolate and butylglycolate; alcohols (propylene glycol, polyethylene glycol of various chain lengths), adipates (diethyladipate, di-(2-methoxy- or 2-ethoxyethyl)-adipate; benzophenone; diethyl- and diburylsebacate, dibutylsuccinate, dibutyltartrate; diethylene glycol dipropionate; ethyleneglycol diacetate, -dibutyrate, -dipropionate; tributyl phosphate, tributyrin; polyethylene glycol sorbitan monooleate (polysorbates such as Polysorbar 50); sorbitan monooleate.

[0168] Moreover, suitable binders, lubricants, disintegrating agents, coloring agents, flavoringagents, flow-inducing agents, and melting agents may be included as carriers. The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include, but are not limited to, lactose, terra alba, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol talc, starch, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0169] In various aspects, a binder can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. In a further aspect, a disintegrator can include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0170] In various aspects, an oral dosage form, such as a solid dosage form, can comprise a disclosed compound that is attached to polymers as targetable drug carriers or as a prodrug. Suitable biodegradable polymers useful in achieving controlled release of a drug include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, caprolactones, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and hydrogels, preferably covalently crosslinked hydrogels.

[0171] Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

[0172] A tablet containing a disclosed compound can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of thepowdered compound moistened with an inert liquid diluent.

[0173] In various aspects, a solid oral dosage form, such as a tablet, can be coated with an enteric coating to prevent ready decomposition in the stomach. In various aspects, enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid- methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate. Akihiko Hasegawa “Application of solid dispersions of Nifedipine with enteric coating agent to prepare a sustained-release dosage form” Chem. Pharm. Bull. 33:1615-1619 (1985). Various enteric coating materials may be selected on the basis of testing to achieve an enteric coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength (e.g., see S. C. Porter et al. “The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalate and Cellulose acetate Phthalate”, J. Pharm. Pharmacol. 22:42p (1970)). In a further aspect, the enteric coating may comprise hydroxypropyl-methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate.

[0174] In various aspects, an oral dosage form can be a solid dispersion with a water soluble or a water insoluble carrier. Examples of water soluble or water insoluble carrier include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, hydroxypropylmethyl-cellulose, phosphatidylcholine, polyoxyethylene hydrogenated castor oil, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, or hydroxypropylmethylcellulose, ethyl cellulose, or stearic acid.

[0175] In various aspects, an oral dosage form can be in a liquid dosage form, including those that are ingested, or alternatively, administered as a mouth wash or gargle. For example, a liquid dosage form can include aqueous suspensions, which contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. In addition, oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain various excipients. The pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions, which may also contain excipients such as sweetening and flavoring agents.

[0176] For the preparation of solutions or suspensions it is, for example, possible to use water, particularly sterile water, or physiologically acceptable organic solvents, such as alcohols (ethanol, propanol, isopropanol, 1 ,2-propylene glycol, polyglycols and their derivatives, fattyalcohols, partial esters of glycerol), oils (for example peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovine hoof oil), paraffins, dimethyl sulfoxide, triglycerides and the like.

[0177] In the case of a liquid dosage form such as a drinkable solutions, the following substances may be used as stabilizers or solubilizers: lower aliphatic mono- and multivalent alcohols with 2- 4 carbon atoms, such as ethanol, n-propanol, glycerol, polyethylene glycols with molecular weights between 200-600 (for example 1 to 40% aqueous solution), diethylene glycol monoethyl ether, 1 ,2-propylene glycol, organic amides, for example amides of aliphatic C1-C6-carboxylic acids with ammonia or primary, secondary or tertiary C1-C4-amines or C1-C4-hydroxy amines such as urea, urethane, acetamide, N-methyl acetamide, N,N-diethyl acetamide, N,N-dimethyl acetamide, lower aliphatic amines and diamines with 2-6 carbon atoms, such as ethylene diamine, hydroxyethyl theophylline, tromethamine (for example as 0.1 to 20% aqueous solution), aliphatic amino acids.

[0178] In preparing the disclosed liquid dosage form can comprise solubilizers and emulsifiers such as the following non-limiting examples can be used: polyvinyl pyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phosphatides such as lecithin, acacia, tragacanth, polyoxyethylated sorbitan monooleate and other ethoxylated fatty acid esters of sorbitan, polyoxyethylated fats, polyoxyethylated oleotriglycerides, linolizated oleotriglycerides, polyethylene oxide condensation products of fatty alcohols, alkylphenols or fatty acids or also 1- methyl-3-(2-hydroxyethyl)imidazolidone-(2). In this context, polyoxyethylated means that the substances in question contain polyoxyethylene chains, the degree of polymerization of which generally lies between 2 and 40 and in particular between 10 and 20. Polyoxyethylated substances of this kind may for example be obtained by reaction of hydroxyl group-containing compounds (for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid radicals) with ethylene oxide (for example 40 Mol ethylene oxide per 1 Mol glyceride). Examples of oleotriglycerides are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, corn oil. See also Dr. H. P. Fiedler “Lexikon der Hillsstoffe fur Pharmazie, Kostnetik und angrenzende Gebiete” 1971 , pages 191-195.

[0179] In various aspects, a liquid dosage form can further comprise preservatives, stabilizers, buffer substances, flavor correcting agents, sweeteners, colorants, antioxidants and complex formers and the like. Complex formers which may be for example be considered are: chelate formers such as ethylene diamine retrascetic acid, nitrilotriacetic acid, diethylene triaminepentacetic acid and their salts.

[0180] It may optionally be necessary to stabilize a liquid dosage form with physiologically acceptable bases or buffers to a pH range of approximately 6 to 9. Preference may be given to as neutral or weakly basic a pH value as possible (up to pH 8).

[0181] In order to enhance the solubility and / or the stability of a disclosed compound in a disclosed liquid dosage form, a parenteral injection form, or an intravenous injectable form, it can be advantageous to employ a-, [3- or y-cyclodextrins or their derivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-p-cyclodextrin or sulfobutyl-p-cyclodextrin. Also co-solvents such as alcohols may improve the solubility and / or the stability of the compounds according to the present disclosure in pharmaceutical compositions.

[0182] In various aspects, a disclosed liquid dosage form, a parenteral injection form, or an intravenous injectable form can further comprise liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

[0183] Pharmaceutical compositions of the present disclosure suitable for injection, such as parenteral administration, such as intravenous, intramuscular, or subcutaneous administration. Pharmaceutical compositions for injection can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

[0184] Pharmaceutical compositions of the present disclosure suitable for parenteral administration can include sterile aqueous or oleaginous solutions, suspensions, or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable form is sterile and must be effectively fluid for use in a syringe. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0185] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In some aspects, a disclosed parenteral formulation can comprise about 0.01-0.1 M, e.g. about 0.05 M, phosphate buffer. In a further aspect, a disclosed parenteral formulation can comprise about 0.9% saline.

[0186] In various aspects, a disclosed parenteral pharmaceutical composition can comprise pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include but not limited to water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. In a further aspect, a disclosed parenteral pharmaceutical composition can comprise may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. Also contemplated for injectable pharmaceutical compositions are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the subject or patient.

[0187] In addition to the pharmaceutical compositions described herein above, the disclosed compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

[0188] Pharmaceutical compositions containing a compound of the present disclosure, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0189] The pharmaceutical composition (or formulation) may be packaged in a variety of ways.Generally, an article for distribution includes a container that contains the pharmaceutical composition in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, foil blister packs, and the like. The container may also include a tamper proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container typically has deposited thereon a label that describes the contents of the container and any appropriate warnings or instructions.

[0190] The disclosed pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Pharmaceutical compositions comprising a disclosed compound formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0191] The exact dosage and frequency of administration depends on the particular disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compounds of the present disclosure.

[0192] Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0.1 to 70 % by weight, more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptablecarrier, all percentages being based on the total weight of the composition.

[0193] In the treatment conditions that require an increase in platelet differentiation by megakaryocytes, an appropriate dosage level will generally be about 0.01 to 1000 mg per kg patient body weight per day and can be administered in single or multiple doses. In various aspects, the dosage level will be about 0.1 to about 500 mg / kg per day, about 0.1 to 250 mg / kg per day, or about 0.5 to 100 mg / kg per day. A suitable dosage level can be about 0.01 to 1000 mg / kg per day, about 0.01 to 500 mg / kg per day, about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg / kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 mg of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response.

[0194] Such unit doses as described hereinabove and hereinafter can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day. In various aspects, such unit doses can be administered 1 or 2 times per day, so that the total dosage for a 70 kg adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration. In a further aspect, dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.

[0195] A typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

[0196] It can be necessary to use dosages outside these ranges in some cases as will beapparent to those skilled in the art. Further, it is noted that the clinician or treating physician will know how and when to start, interrupt, adjust, or terminate therapy in conjunction with individual patient response.

[0197] The present disclosure is further directed to a method for the manufacture of a medicament for modulating platelet activity (e.g., treatment of one or more disorders associated with Kifc3 and / or megakaryocyte dysfunction) in mammals (e.g., humans) comprising combining one or more disclosed compounds, products, or compositions with a pharmaceutically acceptable carrier or diluent. Thus, in one aspect, the present disclosure further relates to a method for manufacturing a medicament comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent.

[0198] The disclosed pharmaceutical compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological or clinical conditions.

[0199] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.

[0200] As already mentioned, the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and a pharmaceutically acceptable carrier. Additionally, the present disclosure relates to a process for preparing such a pharmaceutical composition, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound according to the present disclosure.

[0201] As already mentioned, the present disclosure also relates to a pharmaceutical composition comprising a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of diseases or conditions for a disclosed compound or the other drugs may have utility as well as to the use of such a composition for the manufacture of a medicament. The present disclosure also relates to a combination of disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and a Kifc3 inhibitor. The present disclosure also relates to such a combination for use as a medicine.The present disclosure also relates to a product comprising (a) disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and (b) an additional Kifc3 or related inhibitory therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment or prevention of a condition in a mammal, including a human, the treatment or prevention of which is affected or facilitated by the modulatory effect of the disclosed compound and the additional therapeutic agent. The different drugs of such a combination or product may be combined in a single preparation together with pharmaceutically acceptable carriers or diluents, or they may each be present in a separate preparation together with pharmaceutically acceptable carriers or diluents.

[0202] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.ASPECTS

[0203] The present disclosure can be described in accordance with the following numbered aspects, which should not be confused with the claims.

[0204] Aspect 1. A compound having Formula I:wherein X is CH or N; wherein Y is CH2or NH; wherein Z is N, CH, or C; wherein n is from 0 to 4; wherein Ri is an aryl group, heteroaryl group, cycloalkyl group, or heterocycloalkyl group; wherein R2is OH or O;or wherein Ri and R2 together with Z form a substituted or unsubstituted aryl group, heteroaryl group, cycloalkyl group, or heterocycloalkyl group; wherein R3is a substituted or unsubstituted aryl or heteroaryl group; and wherein R4is a substituted or unsubstituted alkyl group, aryl group, or amide; provided the compound is not A1 or LS5

[0205] Aspect 2. The compound of aspect 1, wherein X is CH and the compound comprises a stereocenter at X.

[0206] Aspect 3. The compound of aspect 1 or 2, wherein R3is a phenyl group substituted with hydroxamic acid.

[0207] Aspect 4. The compound of aspect 3, wherein R3is

[0208] Aspect 5. The compound of any one of aspects 1-4, wherein n is 0.

[0209] Aspect 6. The compound of any one of aspects 1-4, wherein n is 1 .

[0210] Aspect 7. The compound of any one of aspects 1-6, wherein Ri is phenyl.

[0211] Aspect 8. The compound of any one of aspects 1-7, wherein X is N and R4is benzyl.

[0212] Aspect 9. The compound of any one of aspects 1-7, wherein X is CH and R4is benzyl.

[0213] Aspect 10. The compound of any one of aspects 1-7, wherein X is

[0214] Aspect 11. The compound of any one of aspects 1-10, wherein

[0215] Aspect 12. The compound of any one of aspects 1-11 , wherein the compound is selected from:wherein * represents a stereocenter.

[0216] Aspect 13. The compound of any one of aspects 2-12, wherein the compound has substantially S stereochemistry, has substantially R stereochemistry, has an enantiomeric excess of from about 5% to about 95% R enantiomer, has an enantiomeric excess of from about 5% to about 95% S enantiomer, or is a racemic mixture.

[0217] Aspect 14. A pharmaceutical composition comprising the compound of any one of aspects 1-13.

[0218] Aspect 15. The pharmaceutical composition of aspect 14, further comprising at least one carrier or excipient.

[0219] Aspect 16. A method for treating a disease or disorder associated with aberrant Kifc3 function in a subject, the method comprising administering the compound of any one of aspects 1-14, the pharmaceutical composition of aspect 15 or 16, or a composition comprising A1 or LS5 to the subject.

[0220] Aspect 17. The method of aspect 16, wherein the disease or disorder comprises neonatal thrombocytopenia, delayed platelet recovery following umbilical cord blood stem cell transplantation, Down syndrome-associated myeloid neoplasm, or any combination thereof.

[0221] Aspect 18. The method of aspect 16 or 17, wherein the subject is a human.

[0222] Aspect 19. The method of aspect 18, wherein the human is neonatal.

[0223] Aspect 20. The method of any one of aspects 16-19, wherein performing the method increases platelet production in the subject.

[0224] Aspect 21 . A method for treating a disease or disorder associated with aberrant Kifc3 function in a subject, the method comprising administering the compound of any one of aspects1-14, the pharmaceutical composition of aspect 15 or 16, or a composition comprising A1 or LS5 to umbilical cord blood stem cells prior to transplanting the umbilical cord blood stem cells into the subject..

[0225] Aspect 22. A method for ex vivo platelet production, the method comprising administering the compound of any one of aspects 1-14, the pharmaceutical composition of aspect 15 or 16, or a composition comprising A1 or LS5 to primary adult megakaryocyte progenitor cells.EXAMPLES

[0226] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1 : Identification of a Key Downstream Target of Dyrk > Mkl1 Signaling

[0227] Major side effects including tremors, seizures, and death, limited the ability to use Dyrk inhibitors in mice. In addition, a recent report reveals a pro-oncogenic consequences of Dyrkl inhibition. Therefore, RNA-seq studies were conducted to identify elements downstream Dyrk®Mkl1 more amenable to therapeutic targeting. These studies employed primary human Mk precursors purified from short term (96-hour) cultures of CD34+ progenitors, comparing the following samples: 1) adult (PB), 2) neonatal (CB), 3) neonatal treated with the classic Dyrk inhibitor harmine (CB / H), and 4) neonatal treated with a structurally unrelated Dyrk inhibitor EHT 1610 (CB / E). This analysis identified a limited set of ontogenic genes (differing between adult and neonatal Mk) that showed congruent regulation by both Dyrk inhibitors: 26 up and 48 down. For most of these genes, foldchange and significance were near threshold (1.5 and 0.05). However, one gene, KIFC3, showed consistent and strong changes: down in adult Mk and down in neonatal Mk treated with either inhibitor (FIG. 2). Immunoblots (IBs) with two different antibodies and immunofluorescence (IF) confirmed these findings at the protein level (FIGs. 3A-3C). The juxtanuclear pattern of Kifc3 seen by IF on neonatal Mk (FIG. 3C, CB) conforms to that described in epithelial cells by Xu et al.

[0228] To determine the functional significance of Kifc3 downregulation, neonatal (CB) and adult (PB) Mk underwent lentiviral shRNA knockdowns with two different hairpins, which modestly lowered protein levels (20-50%, FIG. 4A). Both hairpins enhanced Mk morphogenesis (ploidy and size) in neonatal Mk but had no effect on adult Mk (FIGs. 4B-4C). The stronger hairpin (sh 63) induced polyploidization and enlargement to a Level characteristic of adult Mk (FIGs. 4B-4C). The neonatal-specific effect of Kifc3 knockdown on Mk supports a more focused role in ontogenic programming than that of Dyrk kinases, whose inhibition also affects adult Mk. The viability and fertility of Kifc3 ko mice further supports a more specialized function of this factor.

[0229] To determine effects of Kifc3 knockdown on platelet release by CB Mk, cells subjected to lentiviral shRNA knockdowns as in FIGs. 4A-4C, underwent extended culture, followed by flow cytometry of the particulate fraction in the supernatant. As previously described, platelets are discerned by staining with thiazole orange (TO), expression of CD41 and CD42, and light scatter properties matching those of normal donor platelets. These experiments showed a two-fold increase in platelets released per Mk with weak Kifc3 knockdown and a 3-fold increase with moderate knockdown (FIG. 5A). The platelets showed agonist responsiveness, with possible enhancement by weak Kifc3 knockdown (FIG. 5B).Control of CEP192 by Mk Ontogeny and Kifc3

[0230] Kifc3 is a widely-expressed minus end-directed microtubule motor in the Kinesin-14 family, which includes 3 mammalian paralogues, Kifc1-3. Of Kifc3 cargos identified, several consist of centrosomal components. In HeLa cells overexpressing GFP-tagged Cep170, siRNA knockdown of Kifc3 impaired centrosomal recruitment. Cep170 was therefore examined in neonatal vs adult Mk and in neonatal Mk with Kifc3 knockdown. Because Cep170 localizes to the mother centriole, cells were co-stained for centrobin, a daughter centriole marker. Cep170 retained tight centriolar localization in neonatal and adult Mk (FIGs. 6A-6D), and knockdown of Kifc3 in neonatal Mk did not alter this localization (FIGs. 6E-6H). Centrobin also remained centriolar under all conditions. However, in adult Mk, centrobin displayed multiple foci encircling a single Cep170 focus, suggestive of mother centriole clustering (FIGs. 6G-6H).

[0231] Recent studies have discovered that the centrosomal protein Cep192 undergoes release from centrosomes in response to extracellular stimuli, shifting to the cell periphery where it acts to promote actin remodeling. Molecular motors that dictate Cep192 localization remain uncharacterized. To this end, the influences of Mk ontogeny and Kifc3 on Cep192 were examined. As shown in FIGs. 7A-7G, ontogenic status affects both the localization and overall levels ofCep192. Specifically, Cep192 in neonatal Mk resided almost exclusively adjacent to centrioles (centrobin, FIGs. 7A-7B, 7G). By contrast, adult Mk expressed higher levels of Cep192, with a major fraction dispersed away from the centriole (FIGs. 7C-7G). To determine the influence of Kifc3 levels on Cep192 patterns, neonatal Mk subjected to shRNA knockdowns underwent IF microscopy as in FIGs. 7A-7G. These experiments revealed a strong influence of Kifc3 levels, with even a minor knockdown (sh62) dramatically affecting Kifc3 distribution and abundance (FIGs. 8A-8G). These findings suggest that Kifc3 could serve both as a Cep192 transporter and destabilizer. Thus, the decreased Kifc3 in adult vs neonatal Mk may underly the ontogenic regulation of Cep192 illustrated in FIGs. 7A-7G.Implication of Cep192 in Platelet Production In Vivo

[0232] The murine Cep192 gene spans ~85 kb, generates ~7 different splice variants, and utilizes 3 different in-frame start codons (FIG. 9). A CRISPR mutant generated by the Knockout Mouse Phenotyping Program carries a 391 bp deletion in exon 5 (box) (Jax strain #042356). This mutation eliminates expression of the top 5 isoforms in FIG. 9. However, the bottom 2 isoforms use downstream ATGs and encode amino terminal truncations of 230 and 632 amino acids (aa), compared with the full-length isoform (2515 aa). These two isoforms lack Cep192 binding sites for Plk1 (aa 33-55) and Plk4 (aa 190-240). The shorter isoform also lacks an intact Aurora A binding domain (aa 543-747). Peripheral blood counts showed a decrease in platelets in homozygous Cep192-mutant mice, with no alterations in red or white cell counts (FIG. 10). These findings support the possibility that Cep192 plays a specialized role in megakaryopoiesis and that amino terminal domain(s) are necessary for optimal platelet production.

[0233] To assess the role of Cep192 in adult-type Mk morphogenesis, adult progenitors transduced with lentiviral shRNAs underwent Mk cultures followed by flow cytometric assessment. Three different hairpins (1 , 3, 5) reduced Cep192 to levels similar to those in neonatal Mk (FIG. 19A). All three hairpins strongly reduced Mk polyploidization and enlargement (FIGs. 19B-19D).Design of Kifc3 Small Molecule Inhibitors

[0234] The amenability of kinesin motor domains to small molecule inhibition has fostered clinical drug development in oncology. Kifd , a paralog of Kifc3, has undergone several inhibitor screens, yielding three distinct small molecule inhibitors. Two of these, AZ82 and SR31527, were identified by high throughput screens; the third, CW069, was found by in silico screening. AZ82 showed isoform specificity, with no inhibitory activity toward Kifc3; the other two have not been tested. The Kifd and Kifc3 motor domains have 59% sequence similarity and possess three small moleculebinding pockets: 1) the a4 / a6 cleft, 2) the a2 / L5 / a3 pocket, and 3) the Mg-ADP binding site. Park et al. elucidated structural basis for AZ82 specificity, showing enhanced docking of AZ82 with the a4 / a6 cleft of Kifcl vs the a4 / a6 cleft of Kifc3. CW069 by contrast associates with the a2 / L5 / a3 pocket, which is also structurally distinct between Kifcl and Kifc3.

[0235] The screening strategy for candidate Kifc3 inhibitors has consisted of computer-aided drug design (CADD), combining both structure-based and ligand-based approaches. Because Kifcl inhibitors share a phenyl or pyridine core, the Enamine HTS library (14,062 molecules) was used to identify phenyl / pyridine-based structures lacking Pan Assay Interference properties (PAINS) (5195 molecules). After generation of 3D conformations, including stereoisomers and tautomers, compounds were screened for similarity to the Kifcl inhibitors (174 molecules). Virtual docking was then conducted using both the a4 / a6 cleft and the a2 / L5 / a3 pocket of Kifc3, applying a series of programs for optimization (GOLD suite v. 5.8.1 , LigandScout v. 4.4.3, PyRod, Desmond, and VMD v. 1.9.3). The docking yielded 28 hits, predominantly candidate ligands for the a2 / L5 / a3 pocket, which then underwent bioassay screening at a range of concentrations.

[0236] The bioassay, culture of neonatal Mk followed by flow cytometry as in FIGs. 4A-4C, identified one compound, LS5, that recapitulated the phenotype of Kifc3 knockdown. Notably, none of the Kifcl inhibitors at published effective doses had any effect in the bioassay (not shown). A secondary virtual screen interrogated pharmacophore and shape-based properties to identify 14 additional LS5 analogs, of which one, A1 , displayed enhanced activity in the bioassay (FIGs. 11A-11C). Interestingly, of the 14 LS5 analogs in the secondary screen, A1 showed the highest similarity to LS5 (FIGs. 11A-11C). Modeling to explain the increased activity of A1 , with minimal structural change, is discussed below (FIG. 18). Further characterization of A1 revealed dose-dependent activity with minimal toxicity (FIGs. 12A-12C).

[0237] In addition, low-dose A1 treatment induced dispersion of Cep192 away from centrioles, as seen with Kifc3 knockdown (FIGs. 8A-8G). These findings suggest that A1 exerts its effects through Kifc3 inhibition, to be rigorously tested.Specific Premise and Hypothesis

[0238] Kifc3 regulation of Cep192 localization and levels is linked to Mk ontogenic phenotype. Cep192 release from the centrosome occurs in adult-type Mk in association with decreased Kifc3 levels. Knockdown or inhibition of Kifc3 in fetal-type Mk induces an ontogenic shift to adult-type Mk. It is postulated that centrosomal release of Cep192 promotes actin recruitment of Aurk / PIk client kinases to enable cytoskeletal remodeling driving Mk morphogenesis (FIG. 1). Prior studieshave demonstrated that Cep192-mediated assembly of Plk4 / AurkB complexes at the cell periphery remodel cortical actin and promote process extension. It is predicted that increased Cep192 levels redirect client kinases from their functions in cytokinesis and shift cells toward endomitotic cycling. Thus, Kifc3-Cep192 signaling may coordinate sequential execution of endomitotic shift, cellular enlargement, and process extension in adult Mk morphogenesis. Lack of adverse effects in knockout mice and enhanced platelet production with shRNA knockdown (FIG. 5A) nominate Kifc3 as a safe, effective target for pharmacologic treatment of the conditions listed in Significance.Example 2: Defining the Mechanistic Basis for Kifc3-Cep192 Control of Mk Morphogenesis

[0239] Elucidating the mechanisms by which Kifc3-Cep192 dictate Mk ontogenic phenotype will enable optimal design of pharmacologic treatments for neonatal thrombocytopenia, platelet recovery post UBC HCT, Down syndrome TAM, and ex vivo platelet production. RNA-seq data indicate that the increase in Cep192 in adult vs neonatal Mk does not occur at the transcriptional level. Cep192 modulation generally occurs by ubiquitylation and degradation mediated by two E3 ligases, Trim37 and Fbxl13. Experiments will test the models proposed in FIG. 13, addressing mechanisms by which Kifc3 may promote Cep192 catabolism. Possibilities include: 1) microtubule-dependent mechanisms with Kifc3 transporting either Cep192 or its ligases or 2) microtubule independent mechanisms in which Kifc3 serves as a scaffold to assemble Cep192 and ligases. Distinguishing the specific mechanism will influence strategies for drug design. Pex5 is included in this model as a potential adapter due to its known interactions with Kifc3 and T rim37.

[0240] Cep192 participation in adult Mk morphogenesis is supported by preliminary knockdown data in human progenitors (FIG. 14) and by the decreased platelet counts in Cep192-mutant mice (FIG. 10). Multiple studies have shown that Cep192 knockdown does not affect mitotic progression. Therefore, the contribution of Cep192 to adult Mk morphogenesis likely does not reflect a generic pro-mitotic function. These experiments will employ mouse models and human progenitor cultures to identify Cep192 domains critical in promoting morphogenesis, with particular focus on cofactor recruitment. These experiments will inform on which kinases and other Cep192-associated factors participate.

[0241] Downstream mediators of Cep192 will also be determined. FIG. 15 shows how Cep192 intersects with several relevant pathways. Its recruitment of AurkB to the cell periphery induces cell protrusion through actin remodeling mediated by a formin switch. The formin switch consists of Daam2 replacement of Daaml , which has previously been shown to be downregulated inhuman subjects with thrombocythemia. Recruitment of Plk4 activates the same pathway but also can influence Arp2 / 3 and Cdc42, both of which are activated by Plk4. As a net result, Cep192 redirects these kinases away from their functions in cytokinesis and toward actin remodeling, thereby coordinating Mk endomitosis with cytoskeletal rearrangements. Plk1 , a Cep192 partner involved in mitosis and endomitosis, may also participate. Cep192 also recruits in a cell cycledependent manner the phosphatase PP1 , which could oppose the action of associated kinases. Arp2 / 3 and Cdc42 represent attractive targets because both have been implicated in Mk process extension in mice and in human hereditary thrombocytopenias.Experimental Approach

[0242] To determine how Kifc3 governs Cep192, initial experiments will examine the requirement of intact microtubules. For these experiments, neonatal Mk - / + shRNA knockdown of Kifc3 as in FIGs. 4A-4C and 8A-8G will undergo treatment on day 5 with nocodazole at 5-50 nM for 1-5 hours. The cells will then undergo IF staining for Cep192 and confocal microscopy as in FIGs. 8A-8G, with co-staining to mark centrioles, nuclei, and peripheral F-actin (centrobin, DAPI, and phalloidin). In parallel, cells will be subjected to IB of whole cell lysates to examine Cep192 expression. If microtubules are required, it will be determined whether Cep192 or the E3 ligases serve as cargo for Kifc3. In these experiments, neonatal Mk - / + shRNA of Kifc3 will undergo IF staining forTrim37 and Fbxl13 as in FIGs. 8A-8G to determine proximity to centrioles. In previous studies, Trim37 localized completely to centrosomes, while Fbxl13 localized both centrosomally and diffusely in the cytoplasm.

[0243] To assess Kifc3 cargos, neonatal progenitors will be cultured with SR1 , as has been described, which permits extended culture of undifferentiated neonatal progenitors, while preserving potential for Mk differentiation and ontogenic switching. During expansion culture, progenitors will undergo transduction with lentiviral constructs expressing a BiolD-Kifc3 fusion or BiolD alone as control. Overexpression will be avoided by using a low multiplicity of infection and immunoblot confirmation of fusion levels well below those of endogenous Kifc3. Following induction of Mk differentiation, the cells will undergo proximity labelling by addition of biotin to the medium, streptavidin pulldown, and mass spectrometry to identify candidate cargos, as described. Because of the potential for artifacts in this system, immunoprecipitation (IP) of endogenous Kifc3 on unperturbed neonatal Mk will also be conducted, using bead-conjugated, IP-validated rabbit polyclonal IgG Ab (10125-2-AP, Proteintech) vs control rabbit IgG. IBs will then probe for candidate cargos: Cep192, Trim37, Pex5, Fbxl13, and factors identified by BiolDlabeling. To verify the binding of putative cargos to the cargo-binding domain of Kifc3, SR1- expanding neonatal progenitors will undergo transduction with lentiviruses expressing Flag- tagged isolated domains-cargo-binding, coiled-coil, and motor-followed by Mk differentiation, Flag-IP, and IB.

[0244] For functional validation, neonatal Mk will undergo shRNA knockdown of verified Kifc3 partners. Cargos that participate in the Kifc3-Cep192 pathway will demonstrate a knockdown phenotype resembling that of Kifc3 (see FIGs. 4A-5B and 8A-8G). Gain of function studies will confirm domain requirements of Kifc3 by expressing domain-deletion mutants and full-length protein in adult Mk. In preliminary experiments, lentiviral expression of full-length Kifc3 suppresses adult Mk polyploidization (FIG. 22).

[0245] For in vivo analysis of Cep192 domains involved in megakaryopoiesis, the Cep192- CRISPR mice from FIGs. 9-10 (Jax strain #042356) will initially be analyzed. As described above, the two remaining Cep192 isoforms in these mice lack the only known Plk4 binding site. They also lack one Plk1 binding site but retain a second C-terminal binding site. The shorter isoform also lacks the Aurk binding site. To assess the role of Cep192 recruitment of Plk4, the Cep192- CRISPR homozygous mice and littermate controls will be analyzed as previously described: resting platelet count, Tpo-stimulated platelet count, Mk counts in marrow and spleen, Mk size and ploidy, and ex vivo culture of fetal liver Mk. In addition, In situ quantitative studies of the steps of podosome extension, sinusoidal penetration, and intravascular platelet release will be conducted, using a suite of electron microscopy (EM) techniques specifically adapted for this purpose. To determine contributions of Aurk recruitment, the fetal liver culture system used herein will compare Mk morphogenesis and platelet release using Cep192-CRISPR progenitors - / + isoform-specific shRNA knockdown to eliminate the long isoform. For human progenitors, isoformspecific knockdown will be applied to determine roles of Plk4 and Aurk recruitment, applying the approach in FIG. 14, which shows effects of global Cep192 knockdown on adult Mk morphogenesis.

[0246] Initial experiments to characterize Cep192 clients will focus on Plk4 and AurkB. Based on data from Luo et al. in breast epithelial cells, these kinases function in a redundant manner as complexes with Cep192 to drive actin remodeling and cellular protrusions. Both factors have also been implicated in control of cytokinesis, with inhibition leading to polyploidization. Findings in adult human Mk show that Plk4 antagonism with the highly specific inhibitor centrinone increases polyploidization but not platelet production (FIG. 16), thus uncoupling these normally linkedfeatures. Similar experiments will be conducted with AZD1152 HQPA, a highly specific AurkB inhibitor, at doses of 10-50 nM. Because of the potential for redundancy, adult Mk will also be cotreated with both inhibitors at their lowest effective dose. All experiments will assess Mk morphogenesis, platelet production, and effects on actin remodeling factors. Prior studies have shown Plk4 induction of invasion by direct phosphorylation of Arp2 T37 / T38; therefore total Arp2 and phospho-Arp2 (T37 / 38) in adult Mk - / + Plk4 and AurkB inhibitors will be analyzed by IB using commercially available antibodies as described. Kazazian et al. also showed strong regulation of Cdc42 by Plk4; all samples will therefore undergo PAK-PBD pulldown assays to quantify Cdc42- GTP. Plk4 and AurkB also influence actin by their recruitment of Daam2 to the cell periphery; cortical Daam2 localization will thus be measured in cells using IF, as described.

[0247] To examine the role of these kinases in the ontogenic Kifc3-Cep192 pathway, total and phosphorylated forms will be analyzed in neonatal and adult Mk by IF and IB, to determine subcellular distribution and levels. The effects on these parameters of Kifc3 knockdown in neonatal Mk and of Cep192 knockdown in adult Mk will be determined. Functional studies will examine how Plk4 and AurkB inhibitors affect neonatal Mk - / + Kifc3 knockdown (FIGs. 4A-4C) and adult Mk - / + Cep192 knockdown (FIG. 14). The actin effector pathways (Arp2 phosphorylation, Cdc42 activation, and Daam2 localization) will be assessed in neonatal versus adult Mk, in neonatal Mk - / + Kifc3 knockdown, and in adult Mk - / + Cep192 knockdown. To test the model in FIG. 15, in which shifting of kinases from INCENP to Cep192 coordinates Mk morphogenesis, adult Mk will be subjected to INCENP knockdown and overexpression to determine effects on ploidy and platelet release. If, as predicted, INCENP knockdown enhances these parameters, whether AurkB inhibition abolishes this effect will be tested.

[0248] In vivo analysis of Plk4 and AurkB will employ mice with floxed alleles for each gene (available from GemPharmatech), coupled with deleter strains for either early Mk-Ery progenitors (Gata1-CreERT2, tamoxifen inducible, from Jax) or for mid-development Mk (Pf4-Cre, from Jax). Platelet and Mk studies will use the approaches already described. Because of potential for redundancy, similar studies will be carried out on double-floxed mice if clear phenotypes do not occur with single kinase loss.Analysis of Results

[0249] All experiments undergo > 3 independent repeats, using appropriate statistical tests for significance. Primary CD34+ progenitors derive from different donors for each iteration to minimize donor-specific effects. Loss-of-function studies use lentiviral shRNA knockdowns with >2 different hairpins and, when feasible, CRISPR / Cas9 knockouts. Lentiviral gene enforcement maintains protein levels within a physiologic range by MOI adjustment. All genetic manipulations are validated by target protein quantitation. Where feasible protein quantitation employs multiple techniques (IB and IF) and multiple antibodies, including isotype matched negative controls. Whenever possible, findings with drug inhibitors undergo validation with gene-based loss-of- function. IF images are analyzed in a blinded manner using standard quantitative approaches with broadly available software.

[0250] Fiji image analysis is used to quantify IF distribution and intensity, as described. Distribution of Cep192 and Daam2 will be analyzed as in Luo et al.. Specifically, proximity to centrioles will be compared with proximity to cortical F-actin, and total MFI per cell will be measured (see FIGs. 8A-8G). If Kifc3 control of Mk morphogenesis requires microtubules, then nocodazole treatment should recapitulate the effects of Kifc3 knockdown in control cells but have negligeable effect in cells with Kifc3 knockdown. If the E3 ligase Trim37 determines the ontogenic differences in Cep192, all isoforms will show similar regulation. If Fbxl13 serves this function, then only the long Cep192 isoforms with intact Nter will be affected. Based on the lineage-selective decrease in platelets in Cep192-CRISPR mice (FIG. 10), marrow abnormalities are predicted to be restricted to Mk. However, flow cytometry will be used for comprehensive characterization of progenitor compartments and of erythroid and myeloid differentiation, as described, to assess specificity of abnormalities. Similar analysis will be applied to conditional Aurkb and Plk4 ko mice.

[0251] In primary human progenitor studies, adult Mk, as compared with neonatal Mk, is predicted to display: 1) shifting of phospho- AurkB and Plk4 away from cleavage furrow and midbody toward cell periphery, 2) enhanced phosphorylation of Arp2, 3) enhanced activation of Cdc42, and 4) enhanced localization of Daam2 at cell periphery. Knockdown of Kifc3 in neonatal Mk is predicted to recapitulate these 4 adult features; conversely, Cep192 knockdown in adult Mk should convert these 4 features to neonatal status. If Arp2, Cdc42 and / or Daam2 display predicted ontogenic changes, functional significance will be tested by applying their inhibitors to adult Mk and to neonatal Mk - / + Kifc3 knockdown: CK-666, CASIN, SMIFH2, respectively.

[0252] Human studies will use adult and neonatal progenitors from female and male donors. Mouse studies will use female and male cohorts. In both systems, the influence of sex will be analyzed in age-matched groups.

[0253] To confirm that findings with nocodazole directly result from microtubule disruption, prolonged treatment will be avoided, which can alter cell cycle-dependent factors. In addition,experiments will be conducted with vincristine as an additional microtubule disrupter. If lentiviral expression of BiolD tagged Kifc3 cannot recapitulate endogenous levels in neonatal Mk, CRISPR / Cas9 will be used to introduce a BiolD tag onto endogenous Kifc3. If the efficiency is too low to apply to SR1-expanded neonatal progenitors, imMKCL conditionally immortalized Mk will be used, which have a neonatal phenotype. An alternative for isoform-specific Cep192 knockdowns consists of CRISPR / Cas9 on imMKCL, subsequently treated with Dyrk inhibitors to induce fetal-adult switch. Findings in the Cep192-CRISPR mice may reflect non-cell-autonomous influences; marrow transplantation and ex vivo cultures will address the cellular basis for any significant phenotypes observed. If neither Plk4 nor AurkB serve as Cep192 clients in Mk morphogenesis, similar in vitro and in vivo approaches will determine participation of Plk1 and AurkA.Example 3: Harnessing of Kifc3-Cep192 Signaling for Clinical Applications

[0254] Pharmacologic induction of ontogenic switching, from fetal / neonatal to adult Mk, offers clinical benefits in four different settings: neonatal thrombocytopenia, delayed platelet engraftment post UCB HCT, Down syndrome-associated Mk neoplasms, and ex vivo generation of transfusion products. Partial knockdown of Kifc3 shifts neonatal Mk to an adult phenotype, including enhanced production of agonist-responsive platelets (FIGs. 5A-5B). Lack of pathology in Kifc3-null mice indicates feasibility for developing nontoxic inhibitors for in vivo treatment. In silico screening using the Kifc3 motor domain structure has yielded two related lead molecules, LS5 and A1 , that elicit adult-type morphogenesis in neonatal Mk (FIGs. 11A-12C). These effects do not result from cross-inhibition of Kifd because treatment of neonatal Mk with Kifcl inhibitors (AZ82, SR31527, and CW069) has no effect on morphogenesis (not shown). The most potent molecule A1 is a mixture of two enantiomers with a single chiral center. Enantiopure forms of A1 have been synthesized and tested, which show equivalent morphogenetic induction (FIG. 17). These findings indicate a flexibility for hydroxyl orientation and suggest that an enantiomerspecific scaffold will not be necessary in designing new molecules.

[0255] Third generation molecules with > 10-fold greater potency compared with A1 will be identified using the neonatal Mk bioassay in FIGs. 11A-12C. These candidates will then be taken through a series of secondary assays to determine: 1) enhancement of platelet release, 2) lack of effect on adult Mk, and 3) inhibition of purified Kifc3 but not Kifd . The third generation candidates will be compared for in silico Kifc3 docking efficiency, potency in enhancing plateletrelease from neonatal Mk, and inhibition of purified kinesins. Correlation of these findings will yield structure-activity relationships (SAR) and guide the process of rational drug design.

[0256] On a more basic level, SAR will provide new tools to probe Kifc3 function and help elucidate the molecular basis for Kifc3 regulation of Cep192 (see FIG. 13). In vivo testing of top hits will employ two different models. In the first model, newborn mice receive a single subcutaneous injection of the TRA romiplostim (ROM) followed by analysis blood, marrow, and spleen Mk at day 5. Sparger et al. showed that neonates, in contrast to adults, fail to augment Mk size and have a blunted platelet response. In this model, it will be determined whether candidate compounds can confer in neonates adult-type responsiveness to ROM. The second model will employ humanized mice stably engrafted with neonatal progenitors and determine whether candidate compounds can enhance circulating human platelet counts. The two models complement one another in that the first can be applied to genetically manipulated mice for target validation and the second can assess human applicability.

[0257] Target validation of the top candidate will employ two steps. In the first step, treated and control mice from both models will undergo analysis of Mk to determine: a) morphogenesis features, b) Cep192 localization and levels, and c) status of Cep192 clients identified in previous experiments. The second step will comprise treatment of neonatal Kifc3- / - and Cep192-CRISPR mice using the model of Sparger et al.Experimental Approach

[0258] The initial steps for SAR optimization of Kifc3 inhibitor uses current findings (FIGs. 11A- 12C, 17) to generate predictions about efficacy enhancement. Overlay of LS5 and A1 in the a2 / L5 / a3 pocket of Kifc3 motor domain provides an explanation for the 4-fold enhanced potency of A1 (FIG. 18). Specifically, A1 possesses a carbonyl oxygen (upper purple arrow) predicted to abut Mg2+complexed with ADP. This feature has potential to enhance both pocket binding and inhibition of nucleotide exchange. Therefore, alternative groups will be introduced at the position of the carbonyl oxygen to test the consequences of enhanced Mg2+ binding or of potential Mg2+- ADP displacement. In silico modeling was conducted using approaches described to predict energetic implications of introducing an array of acidic groups at this position. For the top 5-10 candidates, Organic syntheses were conducted in the same manner as she did for Kifcl inhibitors. The second site for SAR optimization consists of the aromatic ring designated by the lower purple arrow. Modeling places this ring adjacent to a hydrophobic cavity (“Could grow?”), suggesting enhanced binding with either polycyclic groups or with phenyl groups with hydrophobicextensions. A similar process of in silico modeling of candidate modifications of A1 will be followed by synthesis of top 5-10 molecular structures.

[0259] For combinatorial modification of the two sites (upper and lower purple arrows together), single-site A1 derivatives will first be subjected to the bioactivity and enzymatic assays. For bioactivity, drug effects on neonatal vs adult Mk morphogenesis and platelet release will be assessed as in FIGs. 4A-5B, looking for those compounds that most closely recapitulate the phenotype of Kifc3 knockdown. For enzyme activity, drug inhibition of purified Kifc3 and Kifd motor domains will be measured. Modifications enhancing A1 activity will then be incorporated into combinatorial modeling of energetic effects of co-modifying carbonyl (top arrow) and phenyl (bottom arrow) groups. The top 5-10 candidates will be synthesized. These double-modified candidates, as well as the top two single-modified candidates, will undergo detailed functional assessments using a range of doses to quantitate EC50 values.

[0260] The top three compounds will undergo scaled-up synthesis and purification, followed by determination of maximum tolerated doses (MTD) in adult and neonatal wild type B6 mice subjected to single intraperitoneal (IP) injections. One compound, based on efficacy and toxicity profiles, will be chosen to carry forward with in vivo screen. All neonatal mice will receive 300 ng / g subcutaneous ROM on post-natal day 1 , and then half will receive compound vs solvent control. The compound will be administered at doses of 50% of neonatal MTD by IP injection once per day on days 1 -3. On day 5 animals will be euthanized to obtain blood for CBC analysis of platelets; extruded marrow for flow analysis of Mk morphogenesis, as described; and intact femur for analysis of in situ Mk podosome formation. The humanized mice consisting of NBSGW animals engrafted with cord blood CD34+ cells will be prepared by Jackson Labs (see Jax Humanized Mice Solutions). These animals will receive doses of 50% adult MTD by IP injection once per day on 5 consecutive days. Circulating human platelets will be quantified as has been described at 6 hours, 1 day, 4 days, and 10 days after the last injection. Half of the animals will be euthanized at 4 days to obtain marrow samples to assess morphogenesis and in situ podosome formation, using human specific anti-CD41 to distinguish human Mk.

[0261] The first phase of target validation will consist of comparing drug effect in vivo on neonatal vs adult Mk morphogenesis. Along these lines, adult mice will be analyzed in the same manner as neonates to determine whether the candidate drug augments ROM-induced Mk morphogenesis. Similarly, NBSGW mice engrafted with adult human CD34+ cells will receive the same assessment as the cord blood CD34+ humanized mice.

[0262] The next phase will determine treatment effects on Kifc3-Cep192 signaling in Mk. For these experiments, marrow samples will undergo IF with co-staining for CD41 , Cep192, centrobin and F-actin, followed by analysis of Cep192 release from centrosomes within the CD41+ Mk, as well as overall Cep192 signal. A similar approach will assess localization of AurkB and Plk4 in Mk of treated animals. In the final phase of target validation, mice deficient in key pathway elements will be tested for drug responsiveness. Specifically, Kifc3- / - and Cep192-CRISPR mice will be used in the neonatal ROM model.Analysis of Results

[0263] All measures described previously will be applied. Measures specific to these experiments include use of multiple unrelated screening assays, in vivo testing with two distinct animal models, and multiphase target validation. By filtering out compounds affecting Kifcl , potential for off-target effects will be diminished. Target validation studies will further determine the ontogenic and molecular specificity of compounds using an array of in vivo assay systems.

[0264] Based on the enhanced platelet production associated with Kic3 knockdown, it is predicted that compounds with enhanced Kifc3 docking will display enhanced bioactivity and a corresponding enhancement in enzyme inhibition. In the in vivo models, effective Kifc3 inhibitors should augment platelet counts, Mk morphogenesis, and Cep192 reconfiguration, in a manner dependent in Kifc3. Both sexes will be represented in all experiments and will be compared with one another to determine influence.

[0265] One potential problem is that bio- and enzyme assays may show no correlation. Such a finding suggests a mechanism of action affecting Kifc3 conformation and / or stability. In that case, drug binding to Kifc3 and Kifcl motor domains will be measured using ITC. In addition, drug effects on Kifcl will be tested using bioassays described by Watts et al. The compounds that perform well in vitro may lack activity in vivo. In such a case, pharmacokinetic properties will also be included as part of the screening process, subjecting candidate compounds to absorption and half-life determinations from plasma measurements. If a compound works with humanized but not neonatal mice, species specificity will be examined using multiple approaches: in silico modeling, murine fetal liver Mk cultures, and enzyme or binding assays with murine protein. Low levels of human platelets in humanized mice can make their measurement challenging. Treatment of the mice with the human specific TRA eltrombopag can be used for selective augmentation of human platelets. Alternative strains to be considered for in vivo target validation include floxed Kifc3 and Cep192 with Pf4-Cre. An SAR strategy was selected because multiple, related lead compoundswith variable potency and robust bioactivity have been identified. However, an additional approach, by which structurally distinct compounds might be discovered, could consist of high- throughput enzymatic screening using Kifc3 (vs Kifcl) motor domain.

[0266] Cord blood CD34+ cells from de-identified normal donors are purchased from AllCells. Samples have been screened for pathogens using AABB guidelines. Nevertheless, the cells are always handled using Biosafety Level II precautions.Use of an Enantiopure Compound

[0267] Because A1 is a racemic mixture, an enantiopure derivative, A1-E88, was used for more extensive characterization. In these experiments, A1-E88 at 20 pM (E88) exerted effects similar to Kifc3 knockdown, enhancing neonatal Mk morphogenesis (FIGs. 20A-20B) and platelet release (FIG. 20C). Furthermore E88 did not affect differentiation of other lineages, except to enhance slightly erythroid viability (FIGs. 20D-20E). Culture and analysis of erythroid and granulocytic lineages followed a published protocol, except for using CD15 rather than CD13 as a granulocyte marker. To determine whether A1-E88 treatment exerted molecular effects similar to Kifc3 knockdown, cells cultured as in FIGs. 20A-20C were analyzed for Cep192 expression and localization. The consistent augmentation of Cep192 in neonatal Mk by A1-E88 (and racemic A1) (FIG. 21A), as well as the Cep192 dispersion observed in a preliminary experiment (FIG. 21 B), support that the compound targets Kifc3-Cep192 signaling. Virtual screening for off-target mechanisms using the program COMET from PDBind+ (web app) predicted no other motor protein ligands.Example 4: Synthesis Strategies and Exemplary Compounds

[0268] Following testing of A1 and LS5, additional molecules were designed with the aim of increasing binding to Kifc3 while retaining good drug-like properties. Some analogues include the addition of a hydroxamic acid functionality to improve binding to Zn2+in the Kifc3 active site. Further modifications include the introduction of heteroatoms on the phenyl ring that sits in the smaller binding pocket to generate additional protein residue interactions. Both S and R enantiomers of each compound will be synthesized.

[0269] An exemplary diversity oriented synthesis approach is shown in Scheme 1 :Scheme 1

[0270] Position R can be varied to include heteroatoms and non-polar groups to enhance binding in the second binding pocket, while the carbon marked by * is synthesized in both possible enantiomers. Exemplary analogues are shown in Scheme 2:Scheme 2

[0271] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.REFERENCES1. Bastian, T.W., et al., Eltrombopag, a thrombopoietin mimetic, crosses the blood-brain barrier and impairs iron-dependent hippocampal neuron dendrite development. J. Thromb. Haemost., 2017. 15: p. 565-574.2. Cacheiro, P., M.A. Haendel, and D. Smedley, New models for human disease from the International Mouse Phenotyping Consortium. Mamm. Genome, 2019. 30: p. 143-150.3. Chaikuad, A., et al., An unusual binding model of the methyl 9-anilinothiazolo[5,4-f ] quinazoline-2- carbimidates (EHT 1610 and EHT 5372) confers high selectivity for dualspecificity tyrosine phosphorylation-regulated kinases. J. Med. Chem., 2016. 59: p. 10315- 10321. Cheng, E.-c., et al., Role for MKL1 in megakaryocytic maturation. Blood, 2009. 113(12): p. 2826-2834. Christakopoulos, G.E., et al., Phase I dose-finding, safety, and tolerability trial of romiplostim to improve platelet recovery after UCB transplantation. Transplant. Cell. Then, 2021. 27: p. 497.e1-497.e6. Davenport, P., E. Soule-Albridge, and M. Sola-Visner, Hemostatic and immunologic effects of platelet transfusions in neonates. Clin. Perinatal., 2023. 50: p. 793-803. Davenport, P., Z.-J. Liu, and M. Sola-Visner, Fetal vs adult megakaryopoiesis. Blood, 2022. 139(22): p. 3233-3244. Duting, S., et al., A Cdc42 / RhoA regulatory circuit downstream of glycoprotein lb guides transendothelial platelet biogenesis. Nat. Commun., 2017. 8: p. 15838 (1-13). Eckly, A., et al., Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids. J. Thromb. Haemost., 2020. 18: p. 2987-3001. Elagib, K.E., et al., Calpain 2 activation of P-TEFb drives megakaryocyte morphogenesis and is disrupted by leukemogenic GATA1 mutation. Dev. Cell, 2013. 27: p. 607-620. Elagib, K.E., et al., Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetaladult megakaryocyte transition. J. Clin. Invest., 2017. 127(6): p. 2365-2377. Elagib, K.E., et al., Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes. J. Clin. Invest., 2022. 132(19): p. e154839 (1-14). Fotaki, V., et al., Dyrkla haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice. Mol. Cell. Biol., 2002. 22(18): p. 6636-6647. Fung, E., et al., FBXL13 directs the proteolysis of CEP192 to regulate centrosome homeostasis and cell migration. EMBO Rep., 2018. 19: p. e44799 (1-16). Gao, Y., et al. , Role of RhoA-Specific Guanine Exchange Factors in Regulation of Endomitosis in Megakaryocytes. Dev. Cell, 2012. 22: p. 573-584.Guarguaglini, G., et al., The forkhead-associated domain protein Cep170 interacts with pololike kinase 1 and serves as a marker for mature centrioles. Mol. Biol. Cell, 2005. 16: p. 1095- 1107. Gupta, A.O. and J.E. Wagner, Umbilical cord blood transplants: current status and evolving therapies. Front. Pediatr., 2020. 8: p. 570282 (1-11). Hata, S., et al., The balance between KIFC3 and EG5 tetrameric kinesins controls the onset of mitotic spindle assembly. Nat. Cell Biol., 2019. 21 : p. 1138-1151. Heazlewood, S.Y., et al., High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production. Nat. Commun., 2023. 14: p. 2099 (1-18). Hofmann, A., S. Ozawa, and A. Shander, Activity-based cost of platelet transfusions in medical and surgical inpatients at a US hospital. Vox Sang., 2021. 116: p. 998-1004. Horwitz, M.E., et al., Omidubicel vs standard myeloablative umbilical cord blood transplantation: results of a phase 3 randomized study. Blood, 2021. 138(16): p. 1429-1440. Hutchins, J.R.A., et al., Systematic analysis of human protein complexes identfies chromosome segregation proteins. Science, 2010. 328: p. 593-599. Ignatz, M., et al., Umbilical cord blood produces small megakaryocytes after transplantation. Biol. Blood Marrow Transplant., 2007. 13: p. 145-150. Ito, Y., et al., Turbulence activates platelet biogenesis to enable clinical scale ex vivo production. Cell, 2018. 174: p. 636-648. Jones, R., et al., Phase II study to assess the efficacy, safety and tolerability of the mitotic spindle kinesin inhibitor AZD4877 in patients with recurrent advanced urothelial cancer. Invest. New Drugs, 2013. 31(4): p. 1001-1007. Joukov, V. and A. De Nicolo, Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis. Sci. Signal., 2018. 11 : p. eaar4195 (1-25). Joukov, V., J.C. Walter, and A. De Nicolo, The Cep192-organized Aurora A-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly. Mol. Cell, 2014. 55: p. 578-591 . Kazazian, K., et al., Plk4 promotes cancer invasion and metastasis through Arp2 / 3 complex regulation of the actin cytoskeleton. Cancer Res., 2017. 77(2): p. 434-447.Kong, J., et al., Spatiotemporal contact beyween peroxisomes and lipid droplets regulates fasting-induced lipolysis via PEX5. Nat. Commun., 2020. 11 : p. 578 (1-16). Li, J. -J., et al., Differentiation route determines the functional outputs of adult megakaryopoiesis. Immunity, 2024. 57: p. 478-494. Liu, Y., et al., Direct interaction between CEP85 and STIL mediates PLK4-driven directed cell migration. J. Cell Sci. , 2020. 133: p. jcs238352 (1-9). Luo, Y., et al., Atypical function of a centrosomal module in WNT signalling drives contextual cancer cell motility. Nat. Commun., 2019. 10: p. 2356 (1-20). Majhail, N.S., et al., Hospitalization and healthcare resource utilization of omidubicel-only versus umbilical cord blood transplantation for hematologic malignancies: secondary analysis from a pivotal phase 3 clinical trial. Transplant. Cell. Then, 2023. 29: p. 749.e1-749.e5. Maliga, Z., et al., A genomic toolkit to investigate kinesin and myosin motor function in cells. Nat. Cell Biol., 2013. 15(3): p. 325-334. Mattia, G., et al., Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release. Blood, 2002. 99(3): p. 888-897. Meitinger, F., et al., TRIM37 controls cancer-specific vulnerability to PLK4 inhibition. Nature, 2020. 585: p. 440-446. Meng, L., et al., Bimodal interaction of mammalian Polo-like kinase 1 and a centrosomal scaffold, Cep192, in the regulation of bipolar spindle formation. Mol. Cell. Biol., 2015. 35: p. 2626-2640. Nair, J.S., et al., Aurora B kinase regulates the postmitotic endoreduplication checkpoint via phosphorylation of the Retinoblastoma protein at serine 780. Mol. Biol. Cell, 2009. 20: p. 2218- 2228. Nasa, I., et al., Recruitment of PP1 to the centrosomal scaffold protein CEP192. Biochem. Biophys. Res. Commun., 2017. 484: p. 864-870. Neunert, C.E. and M.J. Rose, Romiplostim for the management of pediatric immune thrombocytopenia: drug development and current practice. Blood Adv., 2019. 3(12): p. 1907- 1915.Osterman, M.J.K., et al., Births: final data for 2021. National Vital Statistics Reports, 2023. 72(1): p. 1-53. Park, H.-W., et al., Structural basis of small molecule ATPase inhibition of a human mitotic kinesin motor protein. Sci. Rep., 2017. 7: p. 15121 (1-12). Patel, A., et al., Pre-clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice. Transfusion, 2019. 59: p. 3698-3713. Press, M.F., et al., Role for polo-like kinase 4 in mediation of cytokinesis. Proc. Natl. Acad. Sci. USA, 2019. 116(23): p. 11309-11318. Rahman, N.-T., et al., MRTFA augments megakaryocyte maturation by enhancing the SRF regulatory axis. Blood Adv., 2018. 2(20): p. 2691-2703. Ramirez, P., et al., Delayed platelet recovery after allogeneic transplantation: a predictor of increased treatment-related mortality and poorer survival. Bone Marrow Transplant., 2011. 46: p. 981-986. Reed, F.E., et al., Structure-function analysis of the role of megakaryoblastic leukemia 1 in megakaryocyte polyploidization. Haematologica, 2022. 107(12): p. 2972-2976. Riley, W., et al., Ensuring a reliable platelet supply in the United States. N. Engl. J. Med., 2023. 388(22): p. 2017-2020. Schaller, D., et al., Exploiting water dynamics for pharmacophore screening. Methods Mol. Biol., 2021. 2266: p. 227-238. Sears, R.M., D.G. May, and K.J. Roux, BiolD as a tool for protein-proximity labeling in living cells. Methods Mol. Biol., 2019. 2012: p. 299-313. Sharma, N., et al., Computational benchmarking of putative KIFC1 inhibitors. Med. Res. Rev., 2023. 43: p. 293-318. Sit, Y.T., et al., Synergistic roles of DYRK1A and GATA1 in trisomy 21 megakaryopoiesis. JCI Insight, 2032. 8(23): p. e172851 (1-17). Sonnen, K.F., et al., Human Cep192 and Cep152 cooperate in Plk4 recruitment and centriole duplication. J. Cell Sci., 2013. 126(14): p. 3223-3233. Sparger, K.A., et al., Developmental differences beyween newborn and adult mice in response to Romiplostim. Platelets, 2018. 29(4): p. 365-372.Stanworth, S.J. and A.D. Mumford, How I diagnose and treat neonatal thrombocytopenia. Blood, 2023. 141 (22): p. 2685-2697. Sugimoto, N., et al., iPLATI: the first-in-human clinical trial of iPSC-derived platelets as a phase 1 autologous transfusion study. Blood, 2022. 140(22): p. 2398-2402. Sugimoto, N., et al., Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLATI clinical trial. Blood Adv., 2022. 6(23): p. 6056-6069. Suzuki, D., et al., iPSC-derived platelets depleted of HLA class I are inert to anti-HLA class I and natural killer cell immunity. Stem Cell Reports, 2020. 14: p. 49-59. Tang, B., et al., Recombinant human thrombopoietin promotes platelet engraftment after umbilical cord blood transplantation. Blood Adv., 2020. 4(16): p. 3829-3839. Trakala, M., et al., Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1 -deficient mice. Blood, 2015. 126(14): p. 1707-1714. Verma, A., et al., Management of Down Syndrome-associated leukemias. JAMA Oncol., 2023. 9(9): p. 1283-1290. Wang, W., et al., TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import. J. Cell Biol., 2017. 216(9): p. 2843-2858. Watts, C.A., et al., Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes. Chem. Biol., 2013. 20: p. 1399-1410. Wong, Y.L, et al., Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science, 2015. 348(6239): p. 1155-1160. Wu, J., et al., Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1 . ACS Chem. Biol., 2013. 8: p. 2201-2208. Xu, X., et al., Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms. Blood, 2012. 120(17): p. 3575-3585. Xu, Y., et al., Role of KIFC3 motor protein in Golgi positioning and integration. J. Cell Biol., 2002. 158(2): p. 293-303. Yang, Z., et al., Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3. Mol. Cell. Biol., 2001. 21(3): p. 765-770.Yeow, Z.Y., et al., Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer. Nature, 2020. 585: p. 447-452. Zhang, W., et al., Discovery of a novel inhibitor of kinesin-like protein KIFC1. Biochem. J., 2016. 473: p. 1027-1035. Zou, C., et al., Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication. J. Cell Biol., 2005. 171(3): p. 437-445.

Claims

CLAIMSWhat is claimed is:

1. A compound having Formula I:wherein X is CH or N; wherein Y is CH2or NH; wherein Z is N, CH, or C; wherein n is from 0 to 4; wherein Ri is an aryl group, heteroaryl group, cycloalkyl group, or heterocycloalkyl group; wherein R2is OH or O; or wherein Ri and R2together with Z form a substituted or unsubstituted aryl group, heteroaryl group, cycloalkyl group, or heterocycloalkyl group; wherein R3 is a substituted or unsubstituted aryl or heteroaryl group; and wherein R4 is a substituted or unsubstituted alkyl group, aryl group, or amide; provided the compound is not A1 or LS52. The compound of claim 1 , wherein X is CH and the compound comprises a stereocenter at X.

3. The compound of claim 1, wherein R3is a phenyl group substituted with hydroxamic acid.

5. The compound of claim 1 , wherein n is 0.

6. The compound of claim 1 , wherein n is 1.

7. The compound of claim 1, wherein R1 is phenyl.

8. The compound of claim 1 , wherein X is N and R4 is benzyl.

9. The compound of claim 1 , wherein X is CH and R4 is benzyl.

10. The compound of claim 1 , wherein12. The compound of claim 1 , wherein the compound is selected from:wherein * represents a stereocenter.

13. The compound of claim 2, wherein the compound has substantially S stereochemistry, has substantially R stereochemistry, has an enantiomeric excess of from about 5% to about 95% R enantiomer, has an enantiomeric excess of from about 5% to about 95% S enantiomer, or is a racemic mixture.

14. A pharmaceutical composition comprising the compound of claim 1.

15. The pharmaceutical composition of claim 14, further comprising at least one carrier or excipient.

16. A method for treating a disease or disorder associated with aberrant Kifc3 function in a subject, the method comprising administering the compound of any one of claims 1-14, the pharmaceutical composition of claim 15 or 16, or a composition comprising A1 or LS5 to the subject.

17. The method of claim 16, wherein the disease or disorder comprises neonatal thrombocytopenia, delayed platelet recovery following umbilical cord blood stem cell transplantation, Down syndrome-associated myeloid neoplasm, or any combination thereof.

18. The method of claim 16, wherein the subject is a human.

19. The method of claim 18, wherein the human is neonatal.

20. The method of claim 16, wherein performing the method increases platelet production in the subject.

21. A method for treating a disease or disorder associated with aberrant Kifc3 function in a subject, the method comprising administering the compound of any one of claims 1-14, the pharmaceutical composition of claim 15 or 16, or a composition comprising A1 or LS5 to umbilical cord blood stem cells prior to transplanting the umbilical cord blood stem cells into the subject..

22. A method for ex vivo platelet production, the method comprising administering the compound of any one of claims 1-14, the pharmaceutical composition of claim 15 or 16, or a composition comprising A1 or LS5 to primary adult megakaryocyte progenitor cells.

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