Patent Application
20140242046
The field of invention relates to methods and compositions for the production of stem cells.
A variety of methods are available for reprogramming adult cells to obtain induced pluripotent stem cells. However, the methods currently available suffer from low efficiency and incomplete programming. In addition, some of the methods currently available result in the upregulation of oncogenes, thereby increasing the risk of tumor formation. New methods for the production of induced pluripotent stem cells are needed therefore.
Aspects of the disclosure provide compositions and methods for reprogramming differentiated cells to produce pluripotent cells (e.g., stem cells). In some embodiments, certain enzyme inhibitors enhance cellular reprogramming by accelerating the reprogramming process and/or by simplifying the process (for example, by reducing the number and type of factors required for cellular reprogramming). For example, in some embodiments, methods and compositions described herein can be used to reprogram differentiated cells without using transcription factors that can have unwanted side effects such as oncogenesis. In some embodiments, inhibiting one or more of Dot1L (a histone H3 methyltransferase), YY1 (a transcriptional repressor protein), and/or SUV39H1 (a histone-lysine N-methyltransferase) helps promote the reprogramming of differentiated cells. One or more of these enzymes can be inhibited using any appropriate technique, including, for example, by inhibiting expression (e.g., transcription, translation, and/or modification) and/or activity of the enzyme(s).
Pluripotent (e.g., induced pluripotent stems cells—iPSCs) produced methods and compositions described herein are useful for therapeutic and research applications.
Accordingly, in some embodiments the disclosure provides methods and compositions for the production of stem cells. In certain embodiments, the disclosure provides stem cells (for example induced pluripotent stem cells) and methods and compositions for their use.
In some embodiments, a method of producing induced pluripotent stem cells includes inhibiting of Dot1L, YY1, and/or SUV39H1 in a differentiated cell (e.g., in a preparation containing one or more differentiated cell types). In some embodiments, the method also includes culturing the differentiated cell(s) under one or more reprogramming conditions. In some embodiments, the inhibition of Dot1L, YY1, and/or SUV39H1 occurs under the cell reprogramming conditions. However, in some embodiments, inhibition of Dot1L, YY1, and/or SUV39H1 is initiated prior to exposing the cell(s) to reprogramming conditions.
In some embodiments, the act of inhibiting includes inhibiting the activity of Dot1L, YY1, and/or SUV39H1 (e.g., inhibiting the methyltransferase activity of Dot1L). In some embodiments, the activity is inhibited by contacting a differentiated cell with a composition comprising one or more enzyme inhibitors (e.g., one or more enzyme inhibitors specific for Dot1L, YY1, and/or SUV39H1).
In some embodiments, the act of inhibiting includes knocking down the expression of Dot1L, YY1, and/or SUV39H1 (e.g., by inhibiting transcription and/or translation of the Dot1L, YY1, and/or SUV39H1 gene). In some embodiments, a differentiated cell is contacted with one or more RNAi (e.g., shRNA) molecules that specifically inhibit Dot1L, YY1, and/or SUV39H1 expression.
In some embodiments, the reprogramming conditions include using a cocktail containing one or more reprogramming factors (e.g., one or more reprogramming transcription factors). In some embodiments, the reprogramming cocktail includes Oct4 and/or Sox2. In some embodiments, the reprogramming cocktail consists essentially of Oct4 and Sox2. In some embodiments, the reprogramming cocktail includes Klf4 and/or c-Myc (e.g., in addition to Oct4 and/or Sox2). However, in some embodiments, the reprogramming cocktail does not include Klf4 or c-Myc. In some embodiments, an inhibitor (e.g., a Dot1L inhibitor) is added to the transcription factor(s) in the reprogramming cocktail.
In some embodiments, the differentiated cell is a somatic cell (e.g., a somatic cell obtained from a subject) or a cultured cell. In some embodiments, the differentiated cell is a fibroblast (e.g., an adult fibroblast). In some embodiments, the differentiated cell is a human cell (e.g., dH1fs, IMR-90 or MRC-5), or a mouse cell.
In some embodiments, the presence of induced pluripotent stem cells is determined (e.g., by detecting one or more markers characteristic of an induced pluripotent stem cell) after cellular reprogramming. In some embodiments; the presence of induced pluripotent stem cells is determined by evaluating the presence of one or more markers selected from the group consisting of SSEA4, SSEA3, Tra-1-81, Oct4, Sox2 and Nanog. In some embodiments, induced pluripotent stem cells are isolated after reprogramming of one or more differentiated cell types.
In some embodiments, the production of induced pluripotent stem cells is accelerated by inhibiting Dot1L, YY1, and/or SUV39H1 in a differentiated cell. In some embodiments, the production of induced pluripotent stem cells is more efficient when Dot1L, YY1, and/or SUV39H1 are inhibited in a differentiated cell. Inhibition can occur (e.g., be initiated) before, during, or after culturing the differentiated cell under reprogramming conditions. A reduction in time and/or increase in efficiency can be obtained relative to a reprogramming of the differentiated cell in which Dot1L, YY1, and/or SUV39H1 are not inhibited.
In some embodiments, a method of producing induced pluripotent stem cells includes upregulating the expression of Nanog and/or Lin28 in a differentiated cell. In some embodiments, these-cells are cultured (e.g., before, concurrently, and/or subsequently) under reprogramming conditions to produce induced pluripotent stem cells. In some embodiments, the expression of Nanog and Lin28 is upregulated by inhibiting Dot1L.
In some embodiments, a preparation of induced pluripotent stem cells is provided wherein the stem cells were produced as described herein. In some embodiments, a preparation of induced pluripotent stem cells includes one or more inhibitors of Dot1L, YY1, and/or SUV39H1 (e.g., one or more expression and/or activity inhibitors). In some embodiments, the inhibitors are present in trace amounts. In some embodiments, the induced pluripotent stem cells are human cells.
In some embodiments, the induced pluripotent stems cell can differentiate (e.g., be differentiated using appropriate factors and/or conditions) into ectoderm, mesoderm and/or endoderm cells.
In some embodiments, a preparation of induced pluripotent stem cells is administered to a subject. In some embodiments, a preparation of differentiated stem cells (e.g., induced pluripotent stem cells that were differentiated ex vivo) is administered to a subject. In some embodiments, the subject is a patient (e.g., a human or animal patient) in need of treatment with pluripotent stem cells and/or differentiated cells. In some embodiments, a subject in need of treatment is patient having brain damage (e.g., associated with a neurodegenerative disorder such as Parkinson's disease), cancer, spinal cord injury, heart damage, baldness, deafness, diabetes, neuronal defects, blindness, amyotrophic lateral sclerosis, a genetic disorder, infertility, and/or unhealed wounds. It should be appreciated that the pluripotent and differentiated cell populations described herein may be used in a variety of in vivo methods including but not limited to therapeutic or cosmetic applications.
In some embodiments, one or more inhibitors of Dot1L, YY1, and/or SUV39H1 is administered to a subject to promote stem cell growth or development (e.g., locally at or near the site of local administration, or systemically). In some embodiments, one or more inhibitors may be administered in combination with one or more other factors, including but not limited to, one or more transcription factors (e.g., 2, 3, 4 or more transcription factors such as those described herein), for example in the form of a transcription factor expressing vector (e.g., for inducing local stem cell populations).
In some aspects, the disclosure provides kits, compositions, and methods for identifying enhancers and/or inhibitors of cell reprogramming. In some aspects, the disclosure provides kits, compositions, and methods for producing or isolating induced pluripotent stem cells and/or differentiated cells obtained from the stem cells.
These and other aspects and embodiments of the invention are described in greater detail below.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The figures are illustrative only and are not required for enablement of the invention disclosed herein.
In one aspect, the disclosure provides methods and compositions that are useful in the production of stem cells (e.g., induced pluripotent stem cells—iPSC) from differentiated cells. In some embodiments, the inhibition of Dot1L, SUV39H1, and/or YY1 during at least a portion of a cell reprogramming technique increases the efficiency of and/or accelerates the reprogramming process. In some embodiments, the inhibition of one or more of these proteins also can be used to reduce the number of transcription factors required for a cell reprogramming procedure.
Stem cells produced using compositions and methods described herein are useful in therapeutic and/or research applications. In addition, methods and compositions of promoting stems cell production can be used for therapeutic and/or research purposes.
Methods and compositions described herein can simplify the process of reprogramming a somatic cell. In general, the production of induced pluripotent stem cells (iPSCs) from differentiated cell types by somatic cell reprogramming involves resetting the epigenetic states of the differentiated cells. Typical reprogramming techniques involve exposing cells to several transcription factors, some of which can have undesirable side-effects (e.g., they can be oncogenic). Surprisingly, inhibiting one or more of Dot1L, SUV39H1, and/or YY1 during at least a portion of a reprogramming technique allows for the use of fewer transcription factors, thereby reducing the risks associated with certain transcription factors, in addition to enhancing the efficiency and speed of the overall reprogramming procedure.
In some aspects, the disclosure also provides compositions and methods for identifying genes and proteins that are negative or positive regulators in the reprogramming of differentiated cells. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, how chromatin-modifying proteins influence the reprogramming process remains largely unknown. By identifying negative or positive regulators of iPSC generation, techniques for controlling the process of iPSC generation can be further developed or refined by inhibiting or increasing the expression and/or activity of one or more of the negative or positive regulators.
In some embodiments, inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency. However, surprisingly, inhibition of SUV39H1, YY1, and Dot1L, increased reprogramming. In contrast to genes whose functions appear to be required for reprogramming, inhibition of these three genes enhanced reprogramming (see
In some embodiments, inhibition of Dot1L, the H3K79 histone methyl-transferase, either by RNAi or by a small molecule inhibitor accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc in a reprogramming cocktail. In some embodiments, inhibition of Dot1L functions early in the reprogramming process can be used to markedly induce two alternative reprogramming factors, Nanog and Lin28. Furthermore, in loss-of-function experiments, it was shown that Nanog and Lin28 play essential functional roles in the enhancement of reprogramming by Dot1L-inhibition in some embodiments.
As shown herein, suppression of Dot1L expression using shRNAs or inhibition of its catalytic activity using a small molecule both accelerates and increases the yield of iPSCs and substitutes for both Klf4 and Myc in the reprogramming process. These effects are primarily mediated through induction of two key pluripotency factors, Nanog and Lin28, whose activation normally occurs during the later stages of reprogramming20,21,22,23. Accordingly, in some embodiments, cell reprogramming can be promoted (e.g., ex vivo) by inhibiting Dot1L and/or stimulating Nanog and/or Lin28 expression and/or activity.
Genome-wide analysis of K79me2 distribution revealed that fibroblast-specific, epithelial to mesenchymal transition-associated genes start to lose K79me2 in the initial phases of reprogramming and Dot1L inhibition facilitates the loss of this mark from such genes that eventually get repressed in the pluripotent state. Accordingly, in some embodiments these marks can be used to evaluate and/or monitor the effectiveness of Dot1L inhibition in a cell reprogramming procedure that is used to generate induced pluripotent stems cells.
It should be appreciated that in some embodiments, stem cells can be generated from cultured somatic cell lines. However, in some embodiments, methods and compositions described herein can be used to generate “personalized” stem cells (e.g., for autologous cell therapy) by obtaining one or more somatic cells from a subject and reprogramming the cell(s) ex vivo as described herein. These personalized stem cells can be useful to produce cell preparations (e.g., containing undifferentiated stem cells and/or differentiated stem cells) that can be reintroduced to the subject (e.g., to treat a disease or disorder) with a low risk of host rejection of the reimplanted cells.
In some embodiments, inhibition of Dot1L can be used to prepare cells for a reprogramming procedure by inhibiting Dot1L prior to exposing the cells to a reprogramming cocktail (e.g., containing one or more transcription factors that are useful to promote reprogramming). However, in some embodiments, Dot1L inhibition can be initiated during reprogramming. In some embodiments, one or more Dot1L inhibitors are provided along with one or more transcription factors in a reprogramming cocktail. In some embodiments, one or more Dot1L inhibitors are added to a reprogramming cocktail that contains one or more transcription factors. Accordingly, it should be appreciated that cells being reprogrammed can be contacted with (e.g., incubated with) one or more Dot1L inhibitors (and/or one or more YY1 inhibitors and/or one or more SUV39H1 inhibitors) prior to, along with, and/or after incubation with one or more transcription factors in an incubation cocktail. The presence of the inhibitor(s) can be useful to increase the efficiency of the reprogramming, accelerate the reprogramming (e.g., reduce the incubation time with the reprogramming cocktail), and/or reduce the number of transcription factors required in the incubation cocktail.
Stem cells described herein can be used for therapeutic and research applications. In some embodiments, stem cells can be administered to a subject (e.g., a human subject). In some embodiments, stem cells can be differentiated into one or more somatic cell types of interest prior to administration to a subject. In some embodiments, stem cells can be used to produce artificial tissue or organs ex vivo (e.g., for organ transplantation purposes). Techniques for differentiating stem cells into different types of cell types (e.g., prior to administration to a subject or for use in generating artificial tissue or organs) are known in the art.
Although a cell reprogramming technique typically occurs ex vivo using isolated cells that are obtained from a cell bank or a subject, methods and compositions described herein also can be used in vivo to promote stem cell production and or maintenance in a subject. For example, in some embodiments one or more inhibitors described herein can be administered to a subject (e.g., using any suitable route) to promote or maintain stem cell populations in the subject. In some embodiments, the inhibitor(s) may be administered systemically. In some embodiments, the inhibitor(s) may be administered locally (e.g., to provide local stimulation or maintenance of a stem cell population). In some embodiments, one or more inhibitors may be administered in combination with one or more other factors, including but not limited to, one or more transcription factors (e.g., 2, 3, 4 or more transcription factors such as those described herein). In some embodiments, the transcription factors are provided in the form of a transcription factor expressing vector that expresses one or more transcription factors in vivo after administration. In some embodiments, a vector can include one or more expression regulators to limit expression of the transcription factor(s) to particular tissue or cell types. In some embodiments, the inhibitor(s) and transcription factor(s) are administered locally. Accordingly, methods and compositions described herein can be used to induce local stem cell populations.
In some embodiments, one or more small molecule inhibitors of Dot1L, YY1 and/or SUV39H1 are provided to cells (e.g., ex vivo, or in vivo) in an amount sufficient to inhibit protein function. In some embodiments, the cells are exposed to 0.01 uM, 0.1 uM, 1 uM or 10 uM of inhibitor for a time sufficient to inhibit the protein during a reprogramming procedure. In some embodiments, the cells are exposed to the inhibitor for several hours or several days (e.g., 1-10 days, for example about 5 days) before, during, or after incubation with a reprogramming cocktail.
In some embodiments, one or more of the inhibitors described herein (for example one or more inhibitors of Formula I, II, III, or IV) may be used to inhibit Dot1L in vivo or in vitro. In some embodiments, an inhibitor of structure:
or a pharmaceutically acceptable salt thereof (e.g.,
may be used to inhibit Dot1L in vitro (e.g., contacted to differentiated cells in vitro, for example alone or in combination with one or more transcription factors or other agents described herein, to promote iPSC generation in vitro) or in vivo (e.g., administered to a subject in vivo, for example alone or in combination with one or more transcription factors or other agents described herein, to promote iPSC generation in vivo, for example to promote local stem cell production or to support local stem cell growth or maintenance).
In some embodiments, Dot1L, YY1 and/or SUV39H1 is inhibited by contacting a differentiated cell with one or more nucleic acids that prevent production of the protein(s). In some embodiments, Dot1L, YY1 and/or SUV39H1 is inhibited by an shRNA that knocks down expression. Non-limiting embodiments of shRNAs that knock down expression are provided in the Examples section.
Nucleic acids that prevent Dot1L, YY1 and/or SUV39H1 expression can be provided to the cells in a variety of formats including dsRNA, siRNA and shRNA. “RNA interference (RNAi)” is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene thereby inhibiting expression of the target gene. In one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. “Short interfering RNA” (siRNA), also referred to herein as “small interfering RNA” is defined as a nucleic acid-comprising agent which functions to inhibit expression of a target gene, by RNAi. An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell. In one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and may contain a 3′ and/or 5′ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA). siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs). In one embodiment, these shRNAs are composed of a short (e.g., about 19 to about 25 nucleotide) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be encoded by plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al., RNA April; 9(4):493-501 (2003), incorporated by reference herein in its entirety).
It is envisioned that inhibitors of Dot1L, YY1 and/or SUV39H1 are used in effective amounts. In some embodiments, an effective amount of Dot1L inhibitor (e.g., small molecule or RNAi molecule) is an amount sufficient to inhibit the transferase activity (e.g., by at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more) in the targeted cells (e.g., cells growing in vitro, or targeted cells types in a subject). In some embodiments, an effective amount of YY1 inhibitor is an amount sufficient to inhibit the activity (e.g., by at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more) in the targeted cells (e.g., cells growing in vitro, or targeted cells types in a subject). In some embodiments, an effective amount of SUV39H1 inhibitor is an amount sufficient to inhibit the activity (e.g., by at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more) in the targeted cells (e.g., cells growing in vitro, or targeted cells types in a subject).
In some embodiments, a subject may be evaluated to determine whether Dot1L is overexpressed. Certain genetic abnormalities associated with Dot1L (e.g., the Dot1L regulatory pathway) result in Dot1L overexpression. Assays for detecting genetic abnormalities associated with Dot1L overexpression are known in the art and can include, but are not limited to, molecular assays (e.g., hybridization or sequencing) or in situ or chromosomal assays (e.g., FISH) or other chromosomal assays. Depending on the level of Dot1L in the subject (or in cells obtained from the subject) the amount of Dot1L inhibitor that is used (e.g., in vivo or in vitro) may be adjusted. One of ordinary skill can perform assays to determine suitable levels of Dot1L inhibitor to use in order to inhibit Dot1L levels that are present in a subject (for example in vivo or in vitro in cells isolated from the subject).
In some embodiments, inhibiting Dot1L, YY1 and/or SUV39H1 can be used to promote cellular reprogramming (e.g., ex vivo or in vivo). During development, individual cells become specified to adopt distinct fates through the activation of lineage-specific gene expression programs. Under normal circumstances, these gene expression programs are stably inherited throughout mitotic divisions, thereby maintaining cell identity. At the molecular level, this cellular identity is largely controlled at the chromatin level whereby genes appropriate for a given lineage are embedded in a chromatin structure that supports their transcriptional activity, whereas genes specifying other lineages are sequestered in repressive chromatin structures resulting in transcriptional silencing1. Differentiated cells such as fibroblasts can be converted into induced pluripotent stem cells (iPSCs) upon overexpression of critical transcriptional regulators of embryonic stem cells (ESCs)2,3,4.
In some embodiments, the effect of inhibition of one or more proteins on reprogramming was evaluated in dH1fs cells. The dH1fs cells were transfected with shRNA pools (at high multiplicity of infection to ensure all cells received an shRNA vector) followed by super-infection with reprogramming vectors expressing Oct4, Sox2, Klf4 and c-Myc (OSKM), and the resulting iPSCs were identified by Tra-1-60 staining.
In one aspect, the disclosure provides a method of producing induced pluripotent stem cells, by inhibiting Dot1L (and/or YY1 and/or SUV39H1) in a differentiated cell and culturing the differentiated cell under reprogramming conditions to produce induced pluripotent stem cells. In some embodiments, the reprogramming conditions comprise the presence of a reprogramming cocktail. In some embodiments, the reprogramming cocktail comprises Oct4 and/or Sox2. In some embodiments, the reprogramming cocktail includes Klf4 and/or c-Myc. In some embodiments, the reprogramming cocktail does not include Klf4 and/or c-Myc. In some embodiments, the reprogramming cocktail consists essentially of Oct4 and Sox2. In some embodiments, Dot1L (and/or YY1 and/or SUV39H1) is inhibited at the same time as providing the reprogramming cocktail.
Reprogramming, when relating to cells, generally refers to the process of changing a cell from a first phenotype to a second phenotype. In particular, and as used herein, reprogramming refers to the process of changing a differentiated cell into an induced pluripotent stem cell.
Reprogramming conditions as used herein refers to the conditions that allow a differentiated cell to transform into an induced pluripotent stem cell. Reprogramming conditions include culture media, culture components (e.g., buffer, pH, salt) and the duration of the culturing of the cells.
In some embodiments, the reprogramming conditions comprise the presence of a reprogramming cocktail. A reprogramming cocktail as used herein refers to the combination of specific agents (e.g., Sox2, Oct 4, Klf4, c-Myc, or a combination of any 2 or 3 thereof, or all 4, and/or one or more additional transcription factors) that is required to reprogram a cell from a differentiated cell into an induced pluripotent stem cell. In some embodiments, the reprogramming cocktail comprises Oct4 and Sox2. In some embodiments, the reprogramming cocktail consists essentially of Oct4 and Sox2. A reprogramming cocktail that consists essentially of Oct4 and Sox2, refers to a reprogramming cocktail that does not include any other specific agents (e.g., transcription factors) that could be used to reprogram a differentiated cell. However, in some embodiments, a reprogramming cocktail (e.g., a cocktail that consists essentially of specified transcription factors) may include buffers, salts, sugars, and other components that may be useful to support the growth and reprogramming of the differentiated cells.
The production of induced pluripotent stem cells is generally achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into a somatic cell. In general, these nucleic acids are introduced using retroviral vectors and expression of the gene products results in cells that are morphologically and biochemically similar to pluripotent stem cells (e.g., embryonic stem cells). This process of altering a cell phenotype from a somatic cell or progenitor cell phenotype to a stem cell-like phenotype is termed “reprogramming”.
It was surprisingly found herein that inhibiting DotL1 provides an alternative route for reprogramming cell, eliminating the need for one or more cell transforming factors (e.g., c-Myc) that are associated with oncogenesis.
In some embodiments, reprogramming can be achieved by introducing a combination of stem cell-associated genes including, for example Oct3/4 (Pouf51), Sox 1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4, Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In general, successful reprogramming is accomplished by introducing Oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic or progenitor cell. In some embodiments, the nucleic acid sequences of Oct-4, Sox2, c-MYC, and Klf4 are delivered using a viral vector, such as an adenoviral vector, a lentiviral vector or a retroviral vector. However, while it is understood that reprogramming is usually accomplished by viral delivery of stem-cell associated genes, it is also contemplated herein that reprogramming can be induced using other delivery methods.
In one aspect, the disclosure provides a method of accelerating the production of induced pluripotent stem cells, the method comprising inhibiting Dot1L (and/or YY1 and/or SUV39H1) in a differentiated cell and culturing the differentiated cell under reprogramming conditions to accelerate the production of induced pluripotent stem cells, wherein the production of induced pluripotent stem cells is accelerated compared to a differentiated cell in which Dot1L is not inhibited. Thus, as provided herein, in some embodiments, a differentiated cell can be reprogrammed into an induced pluripotent stem cells by using a known combination of cell-associated genes (e.g., Oct-4, Sox2, c-MYC, and Klf4 or a subset thereof). It was unexpectedly shown herein that the addition of Dot1lL to the combination of cell-associated genes resulted in the acceleration of the production of induced pluripotent stem cells.
The efficiency of reprogramming (e.g., the number of reprogrammed cells) can be enhanced by the addition of various small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell. 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology 26(7):795-797, Marson, A., et al (2008) Cell-Stem Cell 3:132-135, which are incorporated herein by reference in their entirety. It is contemplated that the methods described herein can also be used in combination with a single small molecule (or a combination of small molecules) that enhances the efficiency of induced pluripotent stem cell production. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), trichostatin (TSA), and inhibitors of the TGF-β signaling pathway, among others. It is also contemplated herein that inhibitors can be used alone or in combination with other small molecule(s) to replace one or more of the reprogramming factors used for the production of induced pluripotent stem cells.
In some embodiments, a method of producing pluripotent stem cells includes upregulating the expression of Nanog and/or Lin28 in a differentiated cell and culturing the differentiated cell under reprogramming conditions to produce induced pluripotent stem cells. In some embodiments, Nanog and/or Lin28 are upregulated by inhibiting Dot1L. In some embodiments, Nanog and/or Lin28 are upregulated by introducing agents that stimulate expression of Nanog and/or Lin28, and/or by introducing agents that suppress removal of Nanog and/or Lin28 from the cell (e.g., by protease action). In some embodiments, promoters are introduced into the genome of the cell that result in the upregulation of the expression of Nanog and/or Lin28.
In some embodiments, a method of producing pluripotent stem cells includes inhibiting SUV39H1 in a differentiated cell and culturing the differentiated cell under reprogramming conditions to produce induced pluripotent stem cells. Histone-lysine N-methyltransferase SUV39H1 is a member of the suppressor of variegation 3-9 homolog family and encodes a protein with a chromodomain and a C-terminal SET domain. This nuclear protein moves to the centromeres during mitosis and functions as a histone methyltransferase, methylating Lys-9 of histone H3 Histone-lysine N-methyltransferase SUV39H1. It was surprisingly shown herein that inhibiting SUV39H1 provides a novel pathway for producing induced pluripotent stem cells. In some embodiments, SUV39H1 is inhibited by providing the cell with an siRNA, shRNA (or other RNAi) against SUV39H1. In some embodiments, SUV39H1 is inhibited by providing the cell with an SUV39H1 inhibitor (e.g., a small molecule inhibitor such as Chaetocin, see Greiner et al. Nat Chem Biol. 2005 August; 1(3):143-5).
In some embodiments, a method of producing pluripotent stem cells includes inhibiting YY1 in a differentiated cell and culturing the differentiated cell under reprogramming conditions to produce induced pluripotent stem cells. Transcriptional repressor protein YY1 is a ubiquitously distributed transcription factor belonging to the GLI-Kruppel class of zinc finger proteins. The protein is involved in repressing and activating a diverse number of promoters. YY1 may direct histone deacetylases and histone acetyltransferases to a promoter in order to activate or repress the promoter, thus implicating histone modification in the function of YY1. It was surprisingly shown herein that inhibiting YY1 provides a novel pathway for producing induced pluripotent stem cells. In some embodiments, YY1 is inhibited by providing the cell with an siRNA, shRNA (or other RNAi) against YY1. In some embodiments, YY1 is inhibited by providing the cell with an YY1 inhibitor (e.g., a small molecule inhibitor).
Methods described herein embrace the use of any differentiated cell (e.g., somatic cell). In some embodiments, the differentiated cell is a fibroblast, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, immune cells, hepatic, splenic, lung, circulating blood, gastrointestinal, renal, bone marrow, or pancreatic cell. The differentiated cell can be a primary cell isolated from any somatic tissue including, but not limited to brain, liver, lung, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc., Methods for isolating differentiated cells from a body are known in the art.
In some embodiments, the differentiated cell is a fibroblast. In some embodiments, the differentiated cell is an adult fibroblast.
The differentiated cell used according to the methods provided herein can be from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell.
In some embodiments, the differentiated cell is a human cell. In some embodiments, the differentiated cell is dH1fs, IMR-90 or MRC-5.
In some embodiments, the differentiated cell is a mouse cell.
The differentiated cells used in the methods provided herein, and prior to being reprogrammed, can be maintained under in vitro conditions using conventional tissue culture.
In some embodiments, one or more differentiated cells are obtained from a subject that will subsequently be treated by reimplanting induced pluripotent stem cells obtained from the differentiated cells and/or by reimplanting redifferentiated cells, tissue, or organs obtained from the stem cells.
In one aspect, the disclosure provides methods for producing stem cells. In some embodiments, the cells are induced pluripotent stem cells. The term “pluripotent,” as used in the context of cells, describes the developmental capacity of a cell or cell population and refers to cells that are capable of self-renewal and have the capacity to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent cells are embryonic stem cells (ES-cells), embryonic carcinoma cells (EC cells), and induced pluripotent stem cells (iPS cells). A pluripotent cell has the potential to give rise to any fetal or adult cell type. However, pluripotent cells cannot contribute to extraembryonic tissue, such as, the placenta. This distinguishes pluripotent cells from totipotent cells, which can give rise to all fetal, adult, and extraembryonic cell types.
In one aspect of the methods provided herein, the presence of induced pluripotent stem cells is determined. In some embodiments, the presence of induced pluripotent stem cells is determined by evaluating the presence of one or more markers selected from the group consisting of SSEA4, SSEA3, Tra-1-81, Oct4, Sox2 and Nanog.
In humans and mice, the transcription factors Oct-4, Sox2, and Nanog are biomarkers that are specific for pluripotent cells (see, e.g., Looijenga, L. H. et al. (2003) Cancer Res 63, 2244-50; Peske, M. and Schaler, H. R. (2001) Stem Cells 19, 271-8; Pan, G. and Thomson, J. A. (2007) Cell Res 17, 42-9; the entire contents of each of which are incorporated herein by reference). Additional biomarkers specific for human pluripotent cells include stage specific embryonic antigens SSEA3 and SSEA4, as well as the podocalyxin antigens TRA 1-81 and TRA 1-60 (see, e.g., Schopperle et al. (2007) Stem Cells 25(3):723-30. Epub 2006 Nov. 22; Henderson, J. K. et al. (2002) Stem Cells 20, 329-37; and Draper, J. S. et al. (2002) J Anat 200, 249-58; the entire contents of each of which are incorporated herein by reference). Additional biomarkers for mouse pluripotent stem cells include SSEA1, which is specific for pluripotent cells, and the less stringent marker alkaline phosphatase, which is indicative, but not specific for pluripotent cells (see, e.g., Brambrink, T. et al. (2008) Cell Stem Cell 2(2): 151-9; the entire contents of which are incorporated herein by reference). Expression of these pluripotency markers decreases and is eventually lost during differentiation, corresponding to a restriction in developmental potential and, thus, a loss of pluripotency (see, e.g., Schopperle, W. M. and DeWolf, W. C. (2007) Stem Cells 25, 723-30; the entire contents of which are incorporated herein by reference). Table 1 below lists some additional biomarkers useful for the identification of pluripotent stem cells from mouse, human, and other mammals, as well as their expression spectrum among pluripotent cell types.
In some embodiments, methods provided herein include a step of isolating the produced induced pluripotent stem cells. In some embodiments, the induced pluripotent stem cells are isolated from a mixed population cells comprising pluripotent cells and cells that have not yet or only partially been reprogrammed. Methods for isolating induced pluripotent stem cells are known in the art and are generally based on moieties that bind to markers that are uniquely expressed on induced pluripotent stem cells (e.g., SSEA-1).
In some embodiments, methods described herein include a step of evaluating the developmental capacity of the induced pluripotent stem cells. In some embodiments, the compositions of induced pluripotent stem cells provided herein can be evaluated for their developmental capacity. In some embodiments, the induced pluripotent stem cells are evaluated for their capacity to differentiate into ectoderm, mesoderm and endoderm cells. In some embodiments, the developmental capacity of the induced pluripotent stem cells is evaluated in a teratoma assay.
The developmental capacity of a cell or a cell population can be tested by assays well known to those of skill in the art. One test for pluripotency of a mouse cell or a population of mouse cells, is diploid blastocyst complementation, in which one or more of the cells in question are injected into a host blastocyst, which is then transferred to a foster mouse. If the injected cell(s) are pluripotent, contribution to all three germ layers, and to the germ line, will be observed in the resulting pup, which will typically be a chimera comprising cells derived from the host blastocyst and from the injected cell(s). The most stringent test for the developmental capacity of a pluripotent mouse cell, e.g., an ES or iPS cell is the tetraploid blastocyst complementation assay, which is well known to those of skill in the art (see, e.g., Eggan et al., PNAS, 2001 (May) 6209-6214; and Li et al., Reproduction, 2005 130:53-59; the entire contents of which are incorporated herein by reference). In this assay, a cell or a plurality of cells is injected into a tetraploid host blastocyst. The cells of the tetraploid host blastocyst can contribute to extra-embryonic tissues, e.g., the placenta, but cannot give rise to fetal or adult cell types. Accordingly, a tetraploid blastocyst alone cannot give rise to a live pup at birth, because it cannot generate the required fetal tissues. However, if a pluripotent cell or a plurality of such cells is injected into a tetraploid blastocyst, thus creating a complemented tetraploid blastocyst, the pluripotent cell(s) can give rise to the cell types required for embryonic development, if the injected cells exhibit a high developmental capacity. Tetraploid blastocyst complementation is a more stringent test for developmental capacity than the derivation of chimeric mice after diploid blastocyst injection, because any defect in developmental capacity of the injected cell(s) cannot be compensated by cells of the host blastocyst. A pup resulting from a complemented tetraploid blastocyst typically consists of cells derived from the injected cell(s).
Additional tests for developmental capacity or pluripotency include teratoma formation assays, also sometimes referred to as teratocarcinoma assays (see. e.g., Wesselschmidt, R. L. (2011) Methods Mol Biol. 767:231-41, the entire contents of which are incorporated herein by reference). Typically, a population of cells to be tested is injected subcutaneously into an immunocompromised host animal, e.g., a SCID mouse. If the cell population comprises pluripotent cells, a solid tumor will form at the site of injection. Teratocarcinomas derived from pluripotent cells will contain differentiated cell types of all three germ layers. This assay is an in vivo assay for differentiation capacity that can be used for all pluripotent cells, including cells that cannot be tested by blastocyst complementation (e.g., human cells).
In some embodiments, methods described herein include a step of promoting the development and/or differentiation one or more of the induced pluripotent stem cells.
In some embodiments, the iPSCs produced as described herein may be differentiated in vitro, partially or completely. Methods of promoting the development or differentiation of stem cells are known in the art. For example, differentiation protocols are known in the art and include those described in U.S. Pat. No. 7,326,572 (endoderm differentiation), U.S. Pat. No. 7,282,366 (hepatocyte differentiation), U.S. Pat. No. 7,250,294 (neural differentiation), and U.S. Pat. No. 7,033,831 (islet cell differentiation). In some embodiments, Embryoid Body differentiation, or in vitro directed differentiation using cytokines, growth factors, and/or co-culture with mature cell types may be used. A variety of differentiation factors that can act on pluripotent stem cells and their precursor progeny are known in the art. For example, members of the BMP family of factors have been used to differentiate pluripotent stem cells such. These include the use of BMP-4 and BMP-7 to generate endoderm-like differentiation. (Xu et al. Nat Biotechnol 20:1261-1264, 2002; Pera et al. J Cell Sci 117:1269-1280, 2004.) Activin A can be used to differentiate pluripotent stem cells into definitive endoderm using monolayers or three dimensional (e.g., EB) culture systems. (D'Amour et al. Nat Biotechnol 23:1534-1541, 2005.) Nervous system cells have been observed as a result of culture with epidermal growth factor and fibroblast growth factor (resulting in the generation of neurospheres that comprise neural stem cells), subsequent removal of these factors (resulting in the generation of astrocyte-like cells) or supplementation with nerve growth factor (resulting in the generation of neurons and glial cells). (Kim et al. Nature 418:50-6, 2002; Lee et al. Nat Biotechnol 18:675-9, 2000.) Dopaminergic neurons, useful in Parkinson's disease, may be formed through culture or contact with FGF20 and FGF2. Bjorklund et al. (PNAS 2002, 99:2344-2349) provides additional methods for differentiating ES cells into dopaminergic neurons. Hepatic cell differentiation may be induced through contact and/or culture with an insulin, dexamethasone, and collagen type I (via EB formation) combination; a sodium butyrate and DMSO combination; an FGF4, HGF and collagen type I combination; an aFGF, HGF, oncostatin M, dexamethasone and collagen type I combination; and a bFGF, variant HGF, DMSO and dexamethasone combination in the presence of poly-amino-urethane coated non-woven polytetrafluoroethylene fabric. (Shirahashi et al. Cell Transplant 13:197-211, 2004; Rambhatla et al. Cell Transplant 12:1-11, 2003; Schwartz et al. Stem Cells Dev 14:643-655, 2005; Baharvand et al. Int J Dev Biol 50:645-652, 2006; Soto-Gutierrez et al. Cell Transplant 15:335-341, 2006.) Hepatic differentiation may also occur spontaneously. (Lavon et al. Differentiation 72:230-238, 2004.) Pancreatic differentiation, including differentiation towards beta-islet cells, can be induced using Activin A, retinoic acid, FGF2 and FGF10, betacellulin, HGF, Exendin 4, DKK1 and DKK3. (Gu et al. Mech Dev 120:35-43, 2003; Grapin-Botton et al. Trends Genet 16:124-130, 2000; D'Amour et al. Nat Biotechnol 23:1534-1541, 2005a; D'Amour et al. published US application US2005-0266554A1.) Endothelial differentiation may be induced in the presence of ECM proteins such as collagen type IV, optionally in the presence of VEGF and bFGF. (Gerecht-Nir et al. Lab Invest 83:1811-1820, 2003.) Further reference may be made to published PCT application WO2009/007852 for a review of various differentiative procedures known in the art and applicable to the differentiation of the immature and precursor cells of the invention. Such teachings, and in particular those found on pages 57-61 (under the subheading “Cell Differentiation”) of WO2009/007852, are incorporated by reference herein. Still other references include West and Daley, 2004, Curr Opin Cell Biol 16:688-692; U.S. Pat. No. 6,534,052 B1; Kehat and Gepstein, 2003, 8:229-236; Nir et al., 2003, 58:313-323; and U.S. Pat. Nos. 6,613,568 and 6,833,269.
In some embodiments, the disclosure provides compositions comprising a population of induced pluripotent stem cells (e.g., human induced pluripotent stem cells) produced according to the methods provided herein. In some embodiments, the disclosure provides compositions comprising a population of differentiated stem cells (e.g., human differentiated stem cells) obtained from stem cells produced according to the methods provided herein. In some embodiments, a composition, in addition to the population of induced pluripotent stem cells and/or differentiated stem cells, may include cell culture and cell culture components needed for cell viability. In some embodiments, the compositions include pharmaceutical excipients allowing for the administration of the induced pluripotent stem cells.
It should be appreciated that differentiated cells (e.g., obtained from a subject), induced pluripotent stem cells, and/or differentiated iPSCs referred to herein may be isolated cells (e.g., in the form of preparations of isolated cells). As used herein, isolated cells are cells which have been physically separated from their environment. If the cells are naturally occurring, then isolation implies that the cells are physically separated from the naturally occurring environment from which they derive. In some instances, isolated cells are additionally or alternatively physically separated, in whole or in part, from an in vitro environment. Thus, as used herein, the term isolated means that a molecule, cell, cell population and the like is physically separated from an environment in which it normally exists, or in which it originally or previously existed. Isolation may refer to physical separation of cells from a culture condition (e.g., a differentiation culture), from a naturally occurring environment or source, and the like. A differentiated cell population may be isolated from a differentiation culture condition, for example, by harvesting the cells and removing the culture medium (e.g., by centrifugation). Isolating may also involve washing the cells. Typically, the cells are resuspended in fresh medium. Isolation of the differentiated cell population from the differentiation culture therefore can serve to remove factors or stimuli used to differentiated the pluripotent stem cells towards one or more lineages.
In some embodiments, cells described herein (e.g., original differentiated cells, iPSCs, and or subsequently differentiated cells) may be genetically manipulated (e.g., they may be transfected) or they may not be genetically manipulated. Transfection refers to genetic manipulation of cells to introduce and typically express an exogenous nucleic acid. The exogenous nucleic acid may be a reporter such as green fluorescent protein (GFP) or it may be a selection marker such as thymidine kinase or it may be a transcription factor or other factor that is used to promote reprogramming or subsequent redifferentiation. It will be understood that in some instances reporters such as GFP may also serve as selection markers, particularly if their expression is controlled by pluripotent gene promoters such as an Oct4 promoter.
It should be appreciated that techniques described herein may be used with cells obtained from different species, including for example, human, and various animal species including household species such as dogs and cats, agricultural species such as cows, pigs, and horses, laboratory species such as mice and rats, and the like, or other species.
In some embodiments, the disclosure provides methods of treatment using the induced pluripotent stem cells or differentiated cells (e.g., obtained from iPSCs) provided herein. In some embodiments, the methods of treatment comprise administering to a person in need thereof a composition comprising induced pluripotent stem cells and/or differentiated stems cells produced according to the methods provided herein.
In some embodiments, a person in need of treatment with a composition comprising induced pluripotent stem cells or differentiated cells is a person having brain damage, cancer, spinal cord injury, heart damage, baldness, deafness, diabetes, neuronal defects, blindness, amyotrophic lateral sclerosis, a genetic disorder, infertility, or unhealed wounds. In some embodiments, cells obtained as described herein also may be used to treat hematological malignancies, immunodeficiencies, age related macular degeneration, and other conditions. For example cell populations (e.g., differentiated cell populations) can be used in transplant settings in the treatment or prevention of various conditions including but not limited to Parkinson's disease (dopaminergic neurons), Alzheimer's disease (neural precursors), Huntington's disease (GABAergic neurons), blood disorders such as leukemia, lymphoma myeloma and anemia (hematopoietic cells), side-effects of radiation e.g., in transplant patients (hematopoietic precursors), cardiovascular disease, myocardial infarction, ischemic cardiac tissue or heart-failure (partially- or fully-differentiated cardiomyocytes), muscular dystrophy (skeletal muscle cells), liver cirrhosis or failure (hepatocytes), chronic hepatitis (hepatocytes), diabetes including type I diabetes (insulin-producing cells such as islet cells), ischemic brain damage (neurons), spinal cord injury (glial progenitor cells and motor neurons), amyotrophic lateral sclerosis (ALS) (motor neurons), orthopedic tissue injury (osteoblasts), kidney disease (kidney cells), corneal scarring (corneal stem cells), cartilage damage (chondrocytes), bone damage (osteogenic cells including osteocytes), osteoarthritis (chondrocytes), myelination disorders such as Pelizaeus-Merzbacher disease, multiple sclerosis, adenoleukodystrophies, neuritis and neuropathies (oligodendrocytes), and hair loss.
Pluripotent or differentiated cell populations may be provided as pharmaceutical compositions that are sterile and appropriate for in vivo use, optionally together with a pharmaceutically acceptable carrier. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Cell populations may be formulated for local or systemic administration including as part of an implant. Cells may be used alone or together with another agent, whether active or inactive, including but not limited to a scaffold, a matrix, and the like. These cells may further be included in a kit that additionally comprises at a minimum instructions for use of the cells, and optionally comprises one or more other agents whether active or inactive. The cells may be provided as a frozen aliquot of cells, a culture of cells, or a liquid suspension of cells.
Cells may be administered in numbers effective to produce a desired result, including but not limited to a short-term or long-term therapeutic result. Such result may include an improvement in or complete eradication of symptoms associated with a particular condition.
The cell numbers to be administered will depend on a number of factors including the weight and age of the subject, the type of condition being treated, the desired effect (e.g., short-term or long-term), and the like. Some treatments therefore may require as few as 103 cells, while others may require 104, 105, 106, 107, 108, 109 or more cells.
Cell populations produced as described herein may be studied for gene expression profiles and/or responses to various external stimuli in order to understand differentiation or other processes more fully. In some embodiments, disease-specific iPS cells may be used to model diseases (e.g., ALS or other diseases). In some embodiments, cells may be used in methods for screening and/or identifying agents (e.g., therapeutic agents such as inhibitors) being used or to be used clinically. These assays may measure the therapeutic efficacy and/or toxicity of the candidate agent, among other things. The readouts from such in vitro assays are correlative of the in vivo toxicity or efficacy such agents would exhibit in subjects. Thus, the effect of the agent on the differentiated cells generated according to the invention in vitro is a form of surrogate marker or readout for how the agent will function in vivo in a subject. The agents to be tested include those used clinically as well as experimental agents. In some more common embodiments, such testing will focus on the toxicity of agents including drugs in particular differentiated progeny. Accordingly, in these assays, the readout would be cell death (or conversely cell viability). These in vitro assays may employ suspensions of pluripotent or differentiated cells, adherent populations of pluripotent or differentiated cells, or three dimensional structures comprised of pluripotent or differentiated cells (e.g., in vitro organ tissues, matrices and architectures).
In one aspect, the disclosure provides methods for administering induced pluripotent stem cells or differentiated cells produced according to methods provided herein.
Cells can be administered to hosts by a variety of methods as discussed elsewhere herein. In certain embodiments the cells are administered by injection, such as by intravenous injection. In some embodiments cells are encapsulated for administration. In some embodiments the cells are administered in situ. In some embodiments of the invention, cells are administered in doses measured by the ratio of cells to body mass (weight). Alternatively, iPSCs can be administered in doses of a fixed number of cells.
In some embodiments the purity of cells for administration to a subject is about 100%. In other embodiments it is 95% to 100%. In some embodiments it is 85% to 95%. Particularly in the case of admixtures with other cells, the percentage of IPSCs or differentiated cells can be 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%.
The number of iPSCs or differentiated cells in a given volume can be determined by well known and routine procedures and instrumentation. The percentage of iPSCs or differentiated cells in a given volume of a mixture of cells can be determined by much the same procedures. Cells can be readily counted manually or by using an automatic cell counter. Specific cells can be determined in a given volume using specific staining and visual examination and by automated methods using specific binding reagent, typically antibodies, fluorescent tags, and a fluorescence activated cell sorter.
The choice of formulation for administering cells for a given application will depend on a variety of factors. Prominent among these will be the species of subject, the nature of the disorder, dysfunction, or disease being treated and its state and distribution in the subject, the nature of other therapies and agents that are being administered, the optimum route for administration of the cells, survivability of cells via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art. In particular, for instance, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, for example, liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).
Examples of compositions comprising iPSCs and/or differentiated cells include liquid preparations, including suspensions and preparations for intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may comprise an admixture of cells with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE,” 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
Typically, the compositions will be isotonic, for example, they will have the same iosmotic pressure as blood and lacrimal fluid when properly prepared for administration. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
A pharmaceutically acceptable preservative or cell stabilizer can be employed to increase the life of cellular compositions. If such preservatives are included, it is well within the purview of the skilled artisan to select compositions that will not affect the viability or efficacy of the cells.
Sterile injectable solutions can be prepared by incorporating the cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.
For any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodent such as mouse or rat; and, the dosage of the composition(s), concentration of components therein, and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure, and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.
Order of administration, formulations, doses, frequency of dosing, and routes of administration of factors (such as the cytokines discussed herein) and IPSCs generally will vary with the disorder or disease being treated, its severity, the subject, other therapies that are being administered, the stage of the disorder or disease, and prognostic factors, among others. General regimens that have been established for other treatments provide a framework for determining appropriate dosing in cell-mediated direct or adjunctive therapy. These, together with the additional information provided herein, will enable the skilled artisan to determine appropriate administration procedures in accordance with embodiments of the invention, without undue experimentation.
It should be appreciated that iPSCs or differentiated cells described herein can be administered to a subject by any of a variety of routes known to those skilled in the art that may be used to administer cells to a subject.
Among methods that may be used in this regard in embodiments of the invention are methods for administering cells by a parenteral route. Parenteral routes of administration useful in various embodiments of the invention include, among others, administration by intravenous, intraarterial, intracardiac, intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial, intracutaneous, intradermal, subcutaneous, and/or intramuscular injection. In some embodiments intravenous, intraarterial, intracutaneous, intradermal, subcutaneous and/or intramuscular injection are used. In some embodiments intravenous, intraarterial, intracutaneous, subcutaneous, and/or intramuscular injection are used.
Cells may be administered to the subject through a hypodermic needle by a syringe in some embodiments of the invention. In various embodiments, cells are administered to the subject through a catheter. In a variety of embodiments, cells are administered by surgical implantation. Further in this regard, in various embodiments, cells are administered to the subject by implantation using an arthroscopic procedure. In some embodiments cells are administered to the subject in or on a solid support, such as a polymer or gel. In various embodiments, cells are administered to the subject in an encapsulated form.
In additional embodiments of the invention, cells are suitably formulated for oral, rectal, epicutaneous, ocular, nasal, and/or pulmonary delivery and are administered accordingly.
Compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the formulation that will be administered (e.g., solid vs. liquid). Doses for humans or other mammals can be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.
In some embodiments, the disclosure provides kits for the production of induced pluripotent stem cells. In some embodiment, the kits include a Dot1L inhibitor. In some embodiments, the kits include a Dot1L inhibitor and a reprogramming cocktail (e.g., Sox2 and/or Oct4). In some embodiments, the kits include instructions for the production of induced pluripotent stem cells. In some embodiment, the kits include a YY1 inhibitor. In some embodiment, the kits include an SUV39H1 inhibitor.
In some embodiments, the disclosure provides a kit for the production of induced pluripotent stem cells. In some embodiments, the kit includes one or more Dot1L inhibitors. In some embodiments, the Dot1L inhibitor is a compound of Formula I, II, III or IV (for example one or more of the embodiments described herein), or a pharmaceutically acceptable salt thereof. In some embodiments, the Dot1L inhibitor is an expression inhibitor such as an RNAi inhibitor, for example one or more of the shRNA molecules provided herein.
In some embodiments, the kit includes one or more YY1 inhibitors and/or one or more SUV39H1 inhibitors. In some embodiments, the YY1 and/or SUV39H1 inhibitors are small molecule inhibitors or expression inhibitors such as RNAi inhibitors, for example one or more of the shRNA molecules provided herein.
In some embodiments, the kits include a reprogramming cocktail, or one or more moieties that can be used to generate a reprogramming cocktail. In some embodiments, the kit includes nucleic acids for the introduction of one or more stem cell-associated genes into cells, including, for example Oct3/4 (Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4, Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In some embodiments, the kit includes nucleic acids for the introduction of Oct-4, Sox2, c-MYC, and Klf4 into a cell. In some embodiments, the kit includes nucleic acids for the introduction of Oct-4, and Sox2 into a cell. In some embodiments, the nucleic acids are in delivery vehicle such as a viral vector, such as an adenoviral vector, a lentiviral vector or a retroviral vector.
In some embodiments, this kit includes one or more agents that enhance reprogramming efficiency including, for example, soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), trichostatin (TSA), and inhibitors of the TGF-β signaling pathway.
In some embodiments, this kit includes one or more agents that can bind markers to detect the presence of induced pluripotent stem cells. Such markers include SSEA4, SSEA3, Tra-1-81, Oct4, Sox2 and Nanog. Agents that bind such markers include antibodies and compounds that selectively bind the markers.
In some embodiments, the kit includes one or more elements useful in establishing a reprogramming cocktail, including buffers, salts, sugars, and other components that may be useful to support the growth and reprogramming of the differentiated cells.
In some embodiments, the kit includes one or more components for administering the induced pluripotent stem cells or differentiated cells. These components include pharmaceutical carriers for systemic and/or local administration of the cells.
In some embodiments, the kit includes separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
In one aspect, the disclosure provides methods of promoting the production of induced pluripotent stem cells. In some embodiments, the methods of producing induced pluripotent stem cells include inhibiting Dot1L in a differentiated cell. In some embodiments, the differentiated cell is cultured under reprogramming conditions to produce induced pluripotent stem cells. In some embodiments, the methods of producing induced pluripotent stem comprise inhibiting the methyltransferase activity of Dot1L.
In some embodiments, the methods of producing induced pluripotent stem comprise inhibiting Dot1L in a differentiated cell. Dot1L is a histone methyl transferase (HMT) known to methylate lysine 79 of histone H3 (“H3K79”) in vivo (Feng et al. (2002) Curr. Biol. 12: 1052-1058). Similar to other HMTs, Dot1L contains a 5-adenosylmethionine (SAM) binding site and uses SAM as a methyl donor. Dot1L nucleic acid and polypeptides have previously been described (see, e.g., U.S. Patent Application Publication No. 2005-0048634 A1 (incorporated by reference); Feng et al. (2002) Curr. Biol. 12: 1052-1058; and Okada et al. (2005) Cell 121: 167-78). The human Dot1L homolog has been cloned, isolated, and has been designated as hDot1L (human Dot1-like protein). The sequences of the human nucleic acid and protein have been deposited under GenBank Accession No. AF509504, while the mouse homolog is GenBank Accession No. XP125730). Additional hDot1L homologs are known as well (See e.g., WO2012075500). The 2.5 angstrom resolution structure of a fragment of the hDot1L protein containing the catalytic domain (amino acids 1-416) has been solved; and the atomic coordinates for amino acids 1-416 of hDot1L have been determined and deposited in the RCSB database under ID code 1NW3 and described in the scientific literature (see Min, et al. (2003) Cell 112:711-723).
In some embodiments, Dot1L is inhibited by contacting Dot1l, or a cell expressing Dot1L with one or more of the compounds provided herein. In some embodiments, Dot11 is inhibited by contacting a cell expressing Dot1l with a nuclei acid that “knocks down” Dot1L, thereby inhibiting Dot1L. It should be appreciated that inhibiting Dot1L activity as used herein included both complete (i.e., about 100%) and partial inhibition. In some embodiments, partial inhibition results in 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less up to complete inhibition of Dot1L. Dot1L inhibition in a cell can be achieved both by suppressing one more biological functions of Dot1L (e.g., by contacting the cell with a compound that binds the active site of Dot1L), or by “knocking down” Dot1L (e.g., by contacting the cell with a nucleic acid that prevents production of Dot1L in the cell. In some embodiments, Dot1L is inhibited by inhibiting the catalytic function of Dot1L.
In one aspect, the disclosure provides methods of producing induced pluripotent stem comprise inhibiting Dot1L in a differentiated cell. In some embodiments inhibiting Dot1L comprises inhibiting the methyltransferase activity of Dot1L. In some embodiments, Dot1L is inhibited by contacting the cell with one or more small molecules that inhibit Dot1L activity. Small molecules that inhibit Dot1L are described for instance in WO2012/075500, WO2012/082436, WO2012/075381, and WO2012/075492.
In some embodiments, Dot1L is inhibited by contacting the differentiated cell with a composition comprising a compound of formula I:
or a pharmaceutically acceptable salt, hydrate, enantiomer or stereoisomer thereof, wherein independently for each occurrence,
X is
R1 is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl, haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,
or (C2-C4) alkyl substituted with
except that when X is
R10 is hydrogen or alkyl;
R11a is hydrogen, alkyl, or alkyl-cycloalkyl;
R11b is hydrogen or alkyl; or taken together with R11a and the nitrogen to which it is attached forms a 4- to 8-membered heterocyclyl comprising 0 or 1 additional heteroatoms;
R13 is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or silyl;
R14 is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R15 is alkyl, cycloalkyl, or cycloalkylalkyl;
R20 is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl;
A is
R2 is
Y is —NH—, —N(alkyl)-, —O—, or —CR62—;
R22a is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl, biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl, or heteroaryloxyheteroaryl;
R22b is hydrogen or alkyl;
R24 is hydrogen or alkyl;
R25a, R25b, R25c, and R25d independently are -M2-T2, in which M2 is a bond, SO2, SO, S, CO, CO2, O, O—C1-C4 alkyl linker, C1-C4 alkyl linker, NH, or N(Rt), Rt being C1-C6 alkyl, and T2 is H, halo, or RS4, RS4 being C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C1-C4 alkyl linker, C1-C4 alkyl linker, Rt, and Rs4 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
R3 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxy, or silyloxy;
R4 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxy, or silyloxy;
R41 is hydrogen, alkyl, or alkynyl;
Z is hydrogen or
R5a is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl, aralkylthioalkyl, or heteroaralkylthioalkyl; or alkyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino, amido, aryl, and heteroaryl;
R5b is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl, aralkylthioalkyl, or heteroaralkylthioalkyl; or alkyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino, amido, aryl, and heteroaryl; or taken together with R5a and the nitrogen to which it is attached forms a 4- to 8-membered heterocyclyl comprising 0 or 1 additional heteroatoms;
R6 is hydrogen, alkyl, or halo; or two geminal R6 taken together are ethylene, propylene, or butylene;
R7a is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, lower alkoxy, or C3-C5 cycloalkyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of cyano, lower alkoxy, and halo;
R7b is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, lower alkoxy, or C3-C5 cycloalkyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of cyano, lower alkoxy, and halo; and
R7c is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, lower alkoxy, or C3-C5 cycloalkyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of cyano, lower alkoxy, and halo.
Compounds of formula I, methods of making thereof, and methods of use thereof can be found in WO 2012/075500, which is incorporated herein by reference.
In certain embodiments, X is
In certain embodiments, X is
In certain embodiments, R2 is
In certain embodiments, R2 is
In certain embodiments, R24 is hydrogen or alkyl. In certain embodiments, R24 is hydrogen.
In certain embodiments, R25a is hydrogen, alkyl, —O-alkyl, halogen, trifluoroalkyl, —O-trifluoromethyl, or —SO2-trifluoromethyl. In certain embodiments, R25b is hydrogen, alkyl, halogen, or trifluoroalkyl. In certain embodiments, R25c is hydrogen, alkyl, or halogen. In certain embodiments, R25c is hydrogen or halogen.
In certain embodiments, R2 is
In certain embodiments, Y is —NH— or —N(alkyl)-. In certain embodiments, Y is —NH—.
In certain embodiments, Y is —N(CH3)—. In certain embodiments Y is —O—. In certain embodiments, Y is —CH2—.
In certain embodiments, R22a is aryl or aralkyl. In certain embodiments, R22a is substituted phenyl or substituted benzyl. In certain embodiments, R22a is one of the following:
In certain embodiments, R22a is one of the following:
In certain embodiments, R22b is hydrogen. In certain embodiments, R22b is methyl.
In certain embodiments, R1 is hydrogen. In certain embodiments, R1 is alkyl. In certain embodiments, R1 is —CH3, —CH2CH3, —CH2CH(CH3)2 or —CH2CH2CH(CH3)2. In certain embodiments, R1 is C3-C7 cycloalkyl. In certain embodiments, R1 is cyclopropyl, cyclopropylmethyl, 2-cyclopropylethyl, cyclobutyl, cyclobutylmethyl, 2-cyclobutylethyl, cyclopentyl, cyclopentylmethyl, or 2-cyclopentylethyl. In certain embodiments, R1 is
In certain embodiments, R1 is —CH2CF3. In certain embodiments, R1 is —CH2Ph. In certain embodiments, R1 is —C(═O)H. In certain embodiments, R1 is —C(═O)CH3. In certain embodiments, R1 is heterocyclyl or heterocyclylalkyl. In certain embodiments, R1 is
In certain embodiments, R1 is
In certain embodiments, R15 is alkyl. In certain embodiments, R15 is cycloalkyl.
In certain embodiments, R15 is cycloalkylalkyl.
In certain embodiments, R1 is (C2-C4) alkyl substituted with
In certain embodiments, R1 is
In certain embodiments, R11a is hydrogen, alkyl, or alkyl-cycloalkyl. In certain embodiments, R11a is hydrogen, methyl, or i-propyl. In certain embodiments, R1 is
In certain embodiments, R13 is hydrogen.
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
and R6 is alkyl. In certain embodiments, A is
and R6 is methyl, ethyl, or isopropyl.
In certain embodiments, R3 is hydroxyl. In certain embodiments, R3 is hydrogen. In certain embodiments, R4 is hydroxyl. In certain embodiments, R4 is hydrogen. In certain embodiments, R41 is hydrogen. In certain embodiments, R41 is methyl. In certain embodiments, R3 is hydroxyl; and R4 is hydroxyl. In certain embodiments, R3 is hydroxyl; R4 is hydroxyl; and R41 is hydrogen. In certain embodiments, R3 is hydroxyl; R4 is hydroxyl; and R41 is methyl. In certain embodiments, R3 is hydrogen; and R4 is hydroxyl. In certain embodiments, R3 is hydrogen; R4 is hydroxyl; and R41 is hydrogen. In certain embodiments, R3 is hydrogen; R4 is hydroxyl; and R41 is methyl. In certain embodiments, R3 is hydroxyl; and R4 is hydrogen. In certain embodiments, R3 is hydroxyl; R4 is hydrogen; and R41 is hydrogen. In certain embodiments R3 is hydroxyl; R4 is hydrogen; and R41 is methyl.
In certain embodiments, Z is hydrogen or
In certain embodiments, Z is hydrogen. In certain embodiments, Z is
In certain embodiments, R5a is hydrogen, alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain embodiments, R5a is hydrogen, aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl. In certain embodiments, R5a is —H, —CH2CH2OCH2Ph, —CH2CH3, —CH(CH3)2, -Ph, —CH2CH(CH3), —CH3, —CH2Ph, —CH2CH2NH2, —CH2(cyclohexyl) or —CH2CH2OH. In certain embodiments, R5b is hydrogen, alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain embodiments, R5b is hydrogen, aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl. In certain embodiments, R5b is hydrogen. In certain embodiments, R5a is —H, —CH2CH2OCH2Ph, —CH2CH3, —CH(CH3)2, -Ph, —CH2CH(CH3), —CH3, —CH2Ph, —CH2CH2NH2, —CH2(cyclohexyl) or —CH2CH2OH; and R5b is —H.
In certain embodiments, R7a is hydrogen or lower alkyl. In certain embodiments, R7a is hydrogen. In certain embodiments, R7b is hydrogen or lower alkyl. In certain embodiments, R7b is hydrogen. In certain embodiments, R7c is hydrogen or lower alkyl. In certain embodiments, R7c is hydrogen.
In some embodiments, Dot1L is inhibited by contacting the differentiated cell with a composition comprising a compound of formula II:
or a pharmaceutically acceptable salt, hydrate, enantiomer or stereoisomer thereof, wherein independently for each occurrence,
X is
R1 is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl, alkylaryl, haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,
or (C2-C4)alkyl substituted with
except that when X is
R10a is hydrogen, alkyl, or alkyl-cycloalkyl;
R11b is hydrogen or alkyl; or taken together with R11a and the nitrogen to which it is attached forms a 4- to 8-membered heterocyclyl comprising 0 or 1 additional heteroatoms;
R13 is hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl or silyl;
R14 is hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;
R15 is alkyl, cycloalkyl or cycloalkylalkyl;
R20 is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;
A is
R2 is
Y is —NH—, —N(alkyl)-, —O—, or —CR62;
R22a is aryl, heteroaryl, aralkyl, heteroaralkyl, fused bicyclyl, biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl or heteroaryloxyheteroaryl;
R22b is hydrogen or alkyl;
R24 is hydrogen or alkyl;
R25a, R25b, R25c, R25d are independently -M2-T2, in which M2 is a bond, SO2, SO, S, CO, CO2, O, O—C1-C4 alkyl linker, C1-C4 alkyl linker, NH, or N(Rt), Rt being C1-C6 alkyl, and T2 is H, halo, or Rs4, Rs4 being C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C1-C4 alkyl linker, C1-C4 alkyl linker, Rt, and Rs4 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
R3 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxy or silyloxy;
R4 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy, alkylcarbonyloxy or silyloxy;
R41 is hydrogen, alkyl or alkynyl;
Z is hydrogen or
R5a is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl, alkylcarbonylarhinoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl, aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino, amido, aryl and heteroaryl;
R5b is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl, aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy, nitro, amino, amido, aryl and heteroaryl; or taken together with R5a and the nitrogen to which it is attached forms a 4- to 8-membered heterocyclyl comprising 0 or 1 additional heteroatoms;
R6 is hydrogen, alkyl or halo; or two geminal R6 taken together are ethylene, propylene or butylene; and
R7a is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, lower alkoxy, or C3-C5 cycloalkyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of cyano, lower alkoxy and halo; and
R7b is hydrogen, lower alkyl, lower haloalkyl, cyano, halo, lower alkoxy, or C3-C5 cycloalkyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of cyano, lower alkoxy and halo.
Compounds of formula II, methods of making thereof, and methods of use thereof can be found in WO 2012/082436, which is incorporated herein by reference.
In certain embodiments, X is
In certain embodiments, X is
In certain embodiments, R2 is
In certain embodiments, R2 is
In certain embodiments, R24 is hydrogen or alkyl. In certain embodiments, R24 is hydrogen. In certain embodiments, R25a is hydrogen, alkyl, —O-alkyl, halogen, trifluoroalkyl, —O-trifluoromethyl, or —SO2-trifluoromethyl. In certain embodiments, R25b is hydrogen, alkyl, halogen, or trifluoroalkyl. In certain embodiments, R25c is hydrogen, alkyl, or halogen. In certain embodiments, R25c is hydrogen or halogen.
In certain embodiments, R2 is
In certain embodiments, Y is —NH— or —N(alkyl)-. In certain embodiments, Y is —NH—. In certain embodiments, Y is —N(CH3)—. In certain embodiments, Y is —O—. In certain embodiments, Y is —CH2—. In certain embodiments, R22a is aryl or aralkyl. In certain embodiments, R22a is substituted phenyl or substituted benzyl. In certain embodiments, R22a is one of the following:
In certain embodiments, R22a is one of the following:
In certain embodiments, R22b is hydrogen. In certain embodiments, R22b is methyl.
In certain embodiments, R1 is hydrogen. In certain embodiments, R1 is alkyl. In certain embodiments, R1 is —CH3, —CH2CH3, —CH2CH(CH3)2 or —CH2CH2CH(CH3)2. In certain embodiments, R1 is
In certain embodiments, R1 is —CH2CF3. In certain embodiments, R1 is —CH2Ph. In certain embodiments, R1 is —C(═O)H. In certain embodiments, R1 is —C(═O)CH3. In certain embodiments, R1 is heterocyclyl or heterocyclylalkyl. In certain embodiments, R1 is
In certain embodiments, R1 is
In certain embodiments, R1 is
In certain embodiments, R1 is
In certain embodiments, R15 is alkyl. In certain embodiments, R15 is methyl. In certain embodiments, R15 is cycloalkyl. In certain embodiments, R15 is cycloalkylalkyl.
In certain embodiments, R1 is (C2-C4)alkyl substituted with
In certain embodiments, R1 is
In certain embodiments, R11a is hydrogen, alkyl, or alkyl-cycloalkyl. In certain embodiments, R11a is hydrogen, methyl, or i-propyl. In certain embodiments, R22a is heteroaryl. In certain embodiments, R22a is substituted phenyloxyphenyl, substituted 4-(phenyl)phenyl or optionally substituted 4-(heteroaryl)phenyl.
In certain embodiments, R22a is one of the following:
In certain embodiments, R22a is one of the following:
In certain embodiments, R11a is hydrogen. In certain embodiments, R11b is hydrogen. In certain embodiments, R11b is methyl.
In certain embodiments, R1 is
In certain embodiments, R1 is
In certain embodiments, R13 is hydrogen.
In certain embodiments, R1 is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
In certain embodiments, A is
and R6 is alkyl. In certain embodiments, A is
and R6 is methyl, ethyl, or isopropyl.
In certain embodiments, R3 is hydroxyl. In certain embodiments, R3 is hydrogen. In certain embodiments, R4 is hydroxyl. In certain embodiments, R4 is hydrogen. In certain embodiments, R41 is hydrogen. In certain embodiments, R41 is methyl. In certain embodiments, R3 is hydroxyl; and R4 is hydroxyl. In certain embodiments, R3 is hydroxyl; R4 is hydroxyl; and R41 is hydrogen. In certain embodiments, R3 is hydroxyl; R4 is hydroxyl; and R41 is methyl. In certain embodiments, R3 is hydrogen; and R4 is hydroxyl. In certain embodiments, R3 is hydrogen; R4 is hydroxyl; and R41 is hydrogen. In certain embodiments R3 is hydrogen; R4 is hydroxyl; and R41 is methyl. In certain embodiments, R3 is hydroxyl; and R4 is hydrogen. In certain embodiments, R3 is hydroxyl; R4 is hydrogen; and R41 is hydrogen. In certain embodiments, R3 is hydroxyl; R4 is hydrogen; and R41 is methyl.
In certain embodiments, Z is hydrogen or
In certain embodiments, Z is hydrogen. In certain embodiments, Z is
In certain embodiments, R5a is hydrogen, alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain embodiments, R5a is hydrogen, aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl or hydroxyalkyl. In certain embodiments, R5a is —H, —CH2CH2OCH2Ph, —CH2CH3, —CH(CH3)2, -Ph, —CH2CH(CH3), —CH3, —CH2Ph, —CH2CH2NH2, —CH2(cyclohexyl) or —CH2CH2H. In certain embodiments, R5b is hydrogen, alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain embodiments, R5b is hydrogen, aralkyloxyalkyl, alkyl, aryl, aralkyl, aminoalkyl or hydroalkyl. In certain embodiments; R5b is hydrogen. In certain embodiments, R5a is —H, —CH2CH2OCH2Ph, —CH2CH3, —CH(CH3)2, -Ph, —CH2CH(CH3), —CH3, —CH2Ph, —CH2CH2NH2, —CH2(cyclohexyl) or —CH2CH2OH; and R5b is —H.
In certain embodiments, R7a is hydrogen or lower alkyl. In certain embodiments, R7a is hydrogen. In certain embodiments, R7b is hydrogen or lower alkyl. In certain embodiments, R7b is hydrogen.
In some embodiments, Dot1L is inhibited by contacting the differentiated cell with a composition comprising a compound of formula III:
or pharmaceutically acceptable salt or ester thereof, wherein:
A is O or CH2;
each of G and J, independently, is H, halo, C(O)OH, C(O)O—C1-C6 alkyl or ORa, Ra being H, C1-C6 alkyl or C(O)—C1-C6 alkyl, wherein C(O)O—C1-C6 alkyl, C1-C6 alkyl or C(O)—C1-C6 alkyl is optionally substituted with one or more substituents selected from the group consisting of halo, cyano, hydroxyl, carboxyl, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and C3-C8 cycloalkyl;
Q is H, NH2, NHRb, NRbRc, Rb, or ORb, in which each of Rb and Rc independently is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 7-membered heterocycloalkyl, 5 to 10-membered heteroaryl, or -M1-T1 in which M1 is a bond or C1-C6 alkyl linker optionally substituted with halo, cyano, hydroxyl or C1-C6 alkoxyl and T1 is C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, or Rb and Rc, together with the N atom to which they attach, form 4 to 7-membered heterocycloalkyl having 0 or 1 additional heteroatoms to the N atom optionally substituted with C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C1-C6 alkyl, OC(O)—C1-C6 alkyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and each of Rb, Rc, and T1 is optionally substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
X is N or CRX, in which Rx is H, halo, hydroxyl, carboxyl, cyano, or Rs1, Rs1 being amino, C1-C6 alkoxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and RS1 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
L1 is N(Y), S, SO, or SO2;
L2 is CO or absent when L1 is N(Y) or is absent when L1 is S, SO, or SO2, in which Y is H, Rd, SO2Rd, or CORd when L2 is absent, or Y is H or Rd when L2 is CO, Rd being C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and Rd being optionally substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, C1-C6 alkylsulfonyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl and with C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl further optionally substituted with C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C1-C6 alkyl, OC(O)—C1-C6 alkyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl; each of R1, R2, R3, R4, R5, R6, and R7, independently, is H, halo, hydroxyl, carboxyl, cyano, Rs2, Rs2 being amino, C1-C6 alkoxyl, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, and each Rs2 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
R8 is H, halo or Rs3, Rs3 being C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, and Rs3 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano amino, C1-C6 alkoxyl, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and C3-C8 cycloalkyl;
R9 is
in which each of Re, Rf, Rg, and Rh, independently is -M2-T2, in which M2 is a bond, SO2, SO, S, CO, CO2, O, O—C1-C4 alkyl linker, C1-C4 alkyl linker, NH, or N(Rt), Rt being C1-C6 alkyl, and T2 is H, halo, or RS4, Rs4 being C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C1-C4 alkyl linker, C1-C4 alkyl linker, Rt, and RS4 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl, Ri is H or C1-C6 alkyl optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl, D is O, NRj, or CRjRk, each of Rj and Rk independently being H or C1-C6 alkyl, or Rj and Rk taken together, with the carbon atom to which they are attached, form a C3-C10 cycloalkyl ring, and E is -M3-T3, M3 being a bond or C1-C6 alkyl linker optionally substituted with halo or cyano, T3 being C3-C10 cycloalkyl, C6-C10 aryl, 5 to 10-membered heteroaryl, or 4 to 10-membered heterocycloalkyl, and T3 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, C1-C6 haloalkyl, C1-C6 haloalkoxyl, C1-C6 alkylthio, C1-C6 alkylsulfonyl, C1-C6 haloalkylsulfonyl, C1-C6 alkylcarbonyl, C1-C6 alkoxycarbonyl, oxo, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C4-C12 alkylcycloalkyl, C6-C10 aryl, C6-C10 aryloxyl, C7-C14 alkylaryl, C6-C10 aminoaryloxyl, C6-C10 arylthio, 4 to 6-membered heterocycloalkyl optionally substituted with halo, C1-C4 alkyl, C1-C4 haloalkyl, 5 to 6-membered heteroaryl optionally substituted with halo, C1-C4 alkyl, and C1-C6 alkyl that is substituted with hydroxy, halo, C1-C6 alkoxycarbonyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl optionally further substituted with halo, hydroxyl, or C1-C6 alkoxyl;
q is 0, 1, 2, 3, or 4;
m is 0, 1, or 2; and
n is 0, 1, or 2.
Compounds of formula III, methods of making thereof, and methods of use thereof can be found in WO 2012/075500, which is incorporated herein by reference.
In certain embodiments, the sum of m and n is at least 1. In certain embodiments, m is 1 or 2 and n is 0. In certain embodiments, m is 2 and n is O.
In certain embodiments, A is CH2. In certain embodiments, A is O.
In certain embodiments, L1 is N(Y). In certain embodiments, L1 is SO or SO2.
In certain embodiments, Y is Rj.
In certain embodiments, Rd is C1-C6 alkyl.
In certain embodiments, L2 is absent.
In certain embodiments, each of G and J independently is ORa. In certain embodiments, Ra is hydrogen.
In certain embodiments, R9 is
In certain embodiments, R9 is
In certain embodiments, at least one of Re, Rf, Rg, and Rh is halo (such as F, Cl, and Br), C1-C6 alkoxyl optionally substituted with one or more halo (such as OCH3, OCH2CH3, O-iPr, and OCF3), C1-C6 alkylsulfonyl optionally substituted with one or more halo (such as SO2CF3), or C1-C6 alkyl optionally substituted with one or more halo (such as CH3, i-propyl, n-butyl, and CF3). In certain embodiments, Rj is H or C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl).
In certain embodiments,
is unsubstituted benzimidazolyl or one of the following groups:
In certain embodiments, R9 is
In certain embodiments, D is O. In certain embodiments, D is NRj. In certain embodiments, Rj is H. In certain embodiments, D is CRjRk. In certain embodiments, each of Rj and Rk is hydrogen. In certain embodiments, E is -M3-T3, in which M3 is a bond or C1-C3 alkyl linker, T3 is phenyl, naphthyl, thienyl, cyclopropyl, or cyclohexyl, and T3 is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, C1-C6 haloalkyl, C1-C6 haloalkoxyl, C1-C6 alkylthio, C1-C6 alkylsulfonyl, C1-C6 alkylcarbonyl, C1-C6 alkoxycarbonyl, oxo, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C4-C12 alkylcycloalkyl, C6-C10 aryl, C6-C10 aryloxyl, C7-C14 alkylaryl, C6-C10 aminoaryloxyl, C6-C10 arylthio, 4 to 6-membered heterocycloalkyl optionally substituted with C1-C4 alkyl, 5 to 6-membered heteroaryl optionally substituted with C1-C4 alkyl, and C1-C6 alkyl that is substituted with hydroxy, C1-C6 alkoxycarbonyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl. In certain embodiments, T3 is phenyl optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, nitro, C1-C6 alkyl (e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl), C1-C6 alkoxyl, C1-C6 haloalkyl, C1-C6 haloalkoxyl, C1-C6 alkylsulfonyl, C6-C10 aryl (e.g., phenyl or naphthyl), and C6-C10 aryloxyl, and C7-C14 alkylaryl.
In certain embodiments, E is
In certain embodiments, X is N. In certain embodiments, X is CRX. In certain embodiments, X is CH.
In certain embodiments, Q is NH2 or NHRb, in which Rb is -M1-T1, M1 being a bond or C1-C6 alkyl linker and T1 being C3-C8 cycloalkyl. In certain embodiments, Q is H.
In certain embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are each H.
In certain embodiments, when R8 is halo and is attached to the same carbon atom as J, then J is not hydroxyl. In certain embodiments, when R8 is halo and is attached to the same carbon atom as G, then G is not hydroxyl. In certain embodiments, T2 is not halo when M2 is SO2, SO, S, CO or O.
In certain embodiments, T2 is a 4-8 membered heterocycloalkyl which is bound to M2 via a heteroatom. In certain embodiments, T2 is a 4-8 membered heterocycloalkyl which is bound to M2 via a N atom. In certain embodiments, T2 is a 4-8 membered heterocycloalkyl which is bound to M2 via a C atom.
In certain embodiments, the compound is of formula III-a:
or pharmaceutically acceptable salt or ester thereof, wherein A, Q, X, L1, L2, R1, R2, R3, R4, R5, R6, R7, R8, R9, q, m, and n are as described herein for formula III.
In certain embodiments, the compound is of formula III-b or III-c:
or pharmaceutically acceptable salt or ester thereof, wherein A, Q, X, L1, L2, R1, R2, R3, R4, R5, R6, R7, R8, Re, Rf, Rg, Rh, Ri, q, m, and n are as described herein for formula III.
In certain embodiments, the compound is of formula III-d:
or pharmaceutically acceptable salt or ester thereof, wherein A, Q, X, G, J, L1, L2, R1, R2, R3, R4, R5, R6, R7, R8, D, E, q, m, and n are as described herein for formula III.
In certain embodiments, the compound is of formula III-e:
or its N-oxide, or a pharmaceutically acceptable salt or ester thereof, wherein A, Q, X, Y, R1, R2, Re, Rf, Rg, Rh, and m are as described herein for formula III.
In certain embodiments, A is O. In certain embodiments, A is O and m is 2. In certain embodiments, X is N. In certain embodiments, Q is NH2 or NHRb, in which Rb is -M1-T1, M1 being a bond or C1-C6 alkyl linker and T1 being C3-C8 cycloalkyl. In certain embodiments, R1 and R2 are each H. In certain embodiments, Y is Rd. In certain embodiments, Rd is C1-C6 alkyl optionally substituted with C3-C8 cycloalkyl or halo. In certain embodiments, Rd is C3-C8 cycloalkyl optionally substituted with C1-C6 alkyl or halo. In certain embodiments, at least one of Re, Rf, Rg, and R is halo, C1-C6 alkoxyl optionally substituted with one or more halo; C1-C6 alkylsulfonyl optionally substituted with one or more halo; C1-C6 alkyl optionally substituted with one or more substituents selected from CN, halo, C3-C8 cycloalkyl, hydroxy, and C1-C6 alkoxyl; C3-C8 cycloalkyl optionally substituted with one or more C1-C6 alkyl or CN; or 4 to 8-membered heterocycloalkyl optionally substituted with one or more substituents selected from CN, halo, hydroxy, C1-C6 alkyl and C1-C6 alkoxyl. In certain embodiments, at least one of Re, Rf, Rg, and Rh is selected from F; Cl; Br; CF3; OCF3; SO2CF3; oxetanyl optionally substituted with one or more substituents selected from CN, halo, hydroxy, C1-C6 alkyl and C1-C6 alkoxyl; C3-C8 cycloalkyl optionally substituted with one or more substituents selected from C1-C4 alkyl; and C1-C4 alkyl optionally substituted with one or more substituents selected from halo, C3-C8 cycloalkyl, hydroxy and C1-C6 alkoxyl. In certain embodiments, at least one of Rf and Rg is alkyl, optionally substituted with hydroxyl. In certain embodiments, at least one of Rf and Rg is i-butyl substituted with hydroxyl.
In some embodiments, Dot1L is inhibited by contacting the differentiated cell with a composition comprising a compound of formula IV:
or a pharmaceutically acceptable salt or ester thereof, wherein:
each of G and J, independently, is H, halo, C(O)OH, C(O)O—C1-C6 alkyl or ORa, Ra being H, C1-C6 alkyl or C(O)—C1-C6 alkyl, wherein C(O)O—C1-C6 alkyl, C1-C6 alkyl or C(O)—C1-C6 alkyl is optionally substituted with one or more substituents selected from the group consisting of halo, cyano hydroxyl, carboxyl, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and C3-C8 cycloalkyl;
Q is H, NH2, NHRb, NRbRc, Rb, or ORb, in which each of Rb and Rc independently is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 7-membered heterocycloalkyl, 5 to 10-membered heteroaryl, or -M1-T1 in which M1 is a bond or C1-C6 alkyl linker optionally substituted with halo, cyano, hydroxyl or C1-C6 alkoxyl and T1 is C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, or Rb and Rc, together with the N atom to which they attach, form 4 to 7-membered heterocycloalkyl having 0 or 1 additional heteroatoms to the N atom optionally substituted with C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C1-C6 alkyl, OC(O)—C1-C6 alkyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and each of Rb, Rc, and T1 is optionally substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
X is N or CRX, in which Rx is H, halo, hydroxyl, carboxyl, cyano, or RS1, Rs1 being amino, C1-C6 alkoxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and Rs1 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
L1 is N(Y), S, SO, or SO2;
L2 is CO or absent when L1 is N(Y) or is absent when L1 is S, SO, or SO2, in which Y is H, Rd, SO2Rd, or CORd when L2 is absent, or Y is H or Rd when L2 is CO, Rd being C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl, and Rd being optionally substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, C1-C6 alkylsulfonyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl and with C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl further optionally substituted with C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH, C(O)O—C1-C6 alkyl, OC(O)—C1-C6 alkyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl;
each of R1, R2, R4, R5, R6, and R7, independently, is H, halo, hydroxyl, carboxyl, cyano, RS2, Rs2 being amino, C1-C6 alkoxyl, C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, and each Rs2 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
R8 is H, halo or Rs3, Rs3 being C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, and Rs3 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano amino, C1-C6 alkoxyl, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and C3-C8 cycloalkyl;
R9 is
in which each of Re, Rf, Rg, and Rh, independently is -M2-T2, in which M2 is a bond, SO2, SO, S, CO, CO2, O, O—C1-C4 alkyl linker, C1-C4 alkyl linker, NH, or N(Rt), Rt being C1-C6 alkyl, and T2 is H, halo, or RS4, Rs4 being C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, and each of O—C1-C4 alkyl linker, C1-C4 alkyl linker, Rt, and RS4 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl, Ri is H or C1-C6 alkyl optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl; D is O, NRj, or CRjRk, each of Rj and Rk independently being H or C1-C6 alkyl, or Rj and Rk taken together, with the carbon atom to which they are attached, form a C3-C10 cycloalkyl ring, and E is -M3-T3, M3 being a bond or C1-C6 alkyl linker optionally substituted with halo or cyano, T3 being C3-C10 cycloalkyl, C6-C10 aryl, 5 to 10-membered heteroaryl, or 4 to 10-membered heterocycloalkyl, and T3 being optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, C1-C6 haloalkyl, C1-C6 haloalkoxyl, C1-C6 alkylthio, C1-C6 alkylsulfonyl, C1-C6 haloalkylsulfonyl, C1-C6 alkylcarbonyl, C1-C6 alkoxycarbonyl, oxo, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C4-C12 alkylcycloalkyl, C6-C10 aryl, C6-C10 aryloxyl, C7-C14 alkylaryl, C6-C10 aminoaryloxyl, C6-C10 arylthio, 4 to 6-membered heterocycloalkyl optionally substituted with halo, C1-C4 alkyl, C1-C4 haloalkyl, 5 to 6-membered heteroaryl optionally substituted with halo, C1-C4 alkyl, and C1-C6 alkyl that is substituted with hydroxy, halo, C1-C6 alkoxycarbonyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl optionally further substituted with halo, hydroxyl, or C1-C6 alkoxyl;
m is 1 or 2; and
n is 1 or 2.
Compounds of formula IV, methods of making thereof, and methods of use thereof can be found in WO 2012/075492, which is incorporated herein by reference.
In certain embodiments, at least one of m and n is 2.
In certain embodiments, each of G and J independently is ORa. In certain embodiments, each of G and J is OH.
In certain embodiments, L1 is N(Y). In certain embodiments, L1 is SO or SO2.
In certain embodiments, Y is Rd. In certain embodiments, Rd is C1-C6 alkyl.
In certain embodiments, L2 is absent.
In certain embodiments, R9 is
In certain embodiments, R9 is
In certain embodiments, Ri is H or C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl).
In certain embodiments, at least one of Re, Rf, Rg, and Rh is halo (such as F, Cl, and Br), C1-C6 alkoxyl optionally substituted with one or more halo (such as OCH3, OCH2CH3, O-iPr, and OCF3), C1-C6 alkylsulfonyl optionally substituted with one or more halo (such as SO2CF3), or C1-C6 alkyl optionally substituted with one or more halo (such as CH3, i-propyl, n-butyl, and CF3). In certain embodiments, at least one of Re, Rf, Rg, and Rh is selected from the group consisting of F, Cl, CF3, OCF3, C1-C4 alkyl, and C1-C4 alkoxyl.
In certain embodiments,
is unsubstituted benzimidazolyl or one of the following groups:
In certain embodiments, R9 is
In certain embodiments, R9 is hydrogen.
In certain embodiments, D is O. In certain embodiments, D is NRj. In certain embodiments, Rj is H. In certain embodiments, D is CRjRk. In certain embodiments, each of Rj and Rk is H.
In certain embodiments, E is -M3-T3, in which M3 is a bond or C1-C3 alkyl linker, T3 is phenyl, naphthyl, thienyl, cyclopropyl, or cyclohexyl, and T3 is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxyl, C1-C6 haloalkyl, C1-C6 haloalkoxyl, C1-C6 alkylthio, C1-C6 alkylsulfonyl, C1-C6 alkylcarbonyl, C1-C659 alkoxycarbonyl, oxo, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C4-C12 alkylcycloalkyl, C6-C10 aryl, C6-C10 aryloxyl, C7-C14 alkylaryl, C6-C10 aminoaryloxyl, C6-C10 arylthio, 4 to 6-membered heterocycloalkyl optionally substituted with C1-C4 alkyl, 5 to 6-membered heteroaryl optionally substituted with C1-C4 alkyl, and C1-C6 alkyl that is substituted with hydroxy, C1-C6 alkoxycarbonyl, C3-C8 cycloalkyl, C6-C10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl. In certain embodiments, T3 is phenyl optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, nitro, C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl), C1-C6 alkoxyl, C1-C6 haloalkyl, C1-C6 haloalkoxyl, C1-C6 alkylsulfonyl, C6-C10 aryl (e.g., phenyl or naphthyl), and C6-C10 aryloxyl, and C7-C14 alkylaryl. In certain embodiments, E is
In certain embodiments, X is N. In certain embodiments, X is CRX. In certain embodiments, X is CH.
In certain embodiments, Q is NH2 or NHRb, in which Rb is -M1-T1, M1 being a bond or C1-C6 alkyl linker and T1 being C3-C8 cycloalkyl. In certain embodiments, Q is H.
In certain embodiments, R1, R2, R4, R5, R6, R7, and R8 are each H.
In certain embodiments, when R8 is halo and is attached to the same carbon atom as J, then J is not hydroxyl. In certain embodiments, when R8 is halo and is attached to the same carbon atom as G, then G is not hydroxyl. In certain embodiments, T2 is not halo when M2 is SO2, SO, S, CO or O.
In certain embodiments, T2 is a 4-8 membered heterocycloalkyl which is bound to M2 via a heteroatom. In certain embodiments, T2 is a 4-8 membered heterocycloalkyl which is bound to M2 via a N atom. In certain embodiments, T2 is a 4-8 membered heterocycloalkyl which is bound to M2 via a C atom.
In certain embodiments, the compound is of formula III-a:
or a pharmaceutically acceptable salt or ester thereof, wherein G, J, Q, X, L1, L2, R1, R2, R4, R5, R6 R7, R8, R9, m, and n are as described herein for compounds of formula IV.
In certain embodiments, the compound is of the formula IV-b or IV-c:
pharmaceutically acceptable salt or ester thereof, wherein G, J, Q, X, L1, L2, R1, R2, R4, R5, R6, R7, R8, Re, Rf, Rg, Rh, Ri, m and n are as described herein for compounds of formula IV.
In certain embodiments, the compound is of formula IV-d:
pharmaceutically acceptable salt or ester thereof, wherein G, J, Q, X, L1, L2, R1, R2, R4, R5, R6, R7, R8, D, E, m, and n are as described herein for formula IV.
Exemplary compound useful in methods described include:
or pharmaceutically acceptable salts thereof.
In some embodiments, Dot1L is inhibited by contacting the differentiated cell with a composition comprising a compound of formula:
or a pharmaceutically acceptable salt thereof.
In some embodiments, Dot1L is inhibited by contacting the differentiated cell with a composition comprising a compound of formula:
As used herein, “alkyl”, “C1, C2, C3, C4, C5 or C6 alkyl” or “C1-C6 alkyl” is intended to include C1, C2, C3, C4, C5 or C6 straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5 or C6 branched saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl is intended to include C1, C2, C3, C4, C5 and C6 alkyl groups. Examples of alkyl include moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl. In certain embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.
As used herein, the term “cycloalkyl” refers to a saturated or unsaturated nonaromatic hydrocarbon mono- or multi-ring system having 3 to 30 carbon atoms (e.g., C3-C10). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl. The term “heterocycloalkyl” refers to a saturated or unsaturated nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, or Se). Examples of heterocycloalkyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl.
The term “optionally substituted alkyl” refers to unsubstituted alkyl or alkyl having designated substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an aryl substituted with an alkyl (e.g., methylphenyl).
As used herein, “alkyl linker” is intended to include C1, C2, C3, C4, C5 or C6 straight chain (linear) saturated divalent aliphatic hydrocarbon groups and C3, C4, C5 or C6 branched saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl linker is intended to include C1, C2, C3, C4, C5 and C6 alkyl linker groups. Examples of alkyl linker include, moieties having from one to six carbon atoms, such as, but not limited to, methyl (—CH2—), ethyl (—CH2CH2—), n-propyl (—CH2CH2CH2—), i-propyl (—CHCH3CH2—), n-butyl (—CH2CH2CH2CH2—), s-butyl (—CHCH3CH2CH2—), i-butyl (—C(CH3)2CH2—), n-pentyl (—CH2CH2CH2CH2CH2—), s-pentyl (—CHCH3CH2CH2CH2—) or n-hexyl (—CH2CH2CH2CH2CH2CH2—).
“Alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. In certain embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term “C2-C6” includes alkenyl groups containing two to six carbon atoms. The term “C3-C6” includes alkenyl groups containing three to six carbon atoms.
The term “optionally substituted alkenyl” refers to unsubstituted alkenyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
“Alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, “alkynyl” includes straight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. In certain embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term “C2-C6” includes alkynyl groups containing two to six carbon atoms. The term “C3-C6” includes alkynyl groups containing three to six carbon atoms.
The term “optionally substituted alkynyl” refers to unsubstituted alkynyl or alkynyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both the unsubstituted moieties and the moieties having one or more of the designated substituents.
“Aryl” includes groups with aromaticity, including “conjugated,” or multicyclic systems with at least one aromatic ring and do not contain any heteroatom in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, etc.
“Heteroaryl” groups are aryl groups, as defined above, except having from one to four heteroatoms in the ring structure, and may also be referred to as “aryl heterocycles” or “hetero aromatic s.” As used herein, the term “heteroaryl” is intended to include a stable 5- or 6-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g. 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)p, where p=1 or 2). It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.
In the case of multicyclic aromatic rings, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline). The second ring can also be fused or bridged.
The aryl or heteroaryl aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or hetero aromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl).
As used herein, “carbocycle” or “carbocyclic ring” is intended to include any stable monocyclic, bicyclic or tricyclic ring having the specified number of carbons, any of which may be saturated, unsaturated, or aromatic. For example, a C3-C14 carbocycle is intended to include a monocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are also included in the definition of carbocycle, including, for example, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and [2.2.2]bicyclooctane. A bridged ring occurs when one or more carbon atoms link two non-adjacent carbon atoms. In one embodiment, bridge rings are one or two carbon atoms. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro rings are also included.
As used herein, “heterocycle” includes any ring structure (saturated or partially unsaturated) which contains at least one ring heteroatom (e.g., N, O or S). Examples of heterocycles include, but are not limited to, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazine and tetrahydrofuran. Examples of heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.
The term “substituted,” as used herein, means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms on the atom are replaced. Keto substituents are not present on aromatic moieties. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula.
Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.
When any variable (e.g., R*) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R* moieties, then the group may optionally be substituted with up to two R* moieties and R* at each occurrence is selected independently from the definition of R*. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.
The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O″.
As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo and iodo. The term “perhalogenated” generally refers to a moiety wherein all hydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or “haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or more halogen atoms.
The term “carbonyl” includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. Examples of moieties containing a carbonyl include, but are not limited to, aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.
The term “carboxyl” refers to —COOH or its C1-C6 alkyl ester.
“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or a carbonyl group. “Substituted acyl” includes acyl groups where one or more of the hydrogen atoms are replaced by, for example, alkyl groups, alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkyl groups, as described above, wherein oxygen, nitrogen, or sulfur atoms replace one or more hydrocarbon backbone carbon atoms.
The term “alkoxy” or “alkoxyl” includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.
The term “ether” or “alkoxy” includes compounds or moieties which contain an oxygen bonded to two carbon atoms or heteroatoms. For example, the term includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to an alkyl group.
The term “ester” includes compounds or moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group. The term “ester” includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
The term “thioalkyl” includes compounds or moieties which contain an alkyl group connected with a sulfur atom. The thioalkyl groups can be substituted with groups such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
The term “thiocarbonyl” or “thiocarboxy” includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.
The term “thioether” includes moieties which contain a sulfur atom bonded to two carbon atoms or heteroatoms. Examples of thioethers include, but are not limited to alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” include moieties with an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group. Similarly, the term “alkthioalkenyls” refers to moieties wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkenyl group; and alkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.
As used herein, “amine” or “amino” refers to unsubstituted or substituted —NH2. “Alkylamino” includes groups of compounds wherein nitrogen of —NH2 is bound to at least one alkyl group. Examples of alkylamino groups include benzylamino, methylamino, ethylamino, phenethylamino, etc. “Dialkylamino” includes groups wherein the nitrogen of —NH2 is bound to at least two additional alkyl groups. Examples of dialkylamino groups include, but are not limited to, dimethylamino and diethylamino. “Arylamino” and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively. “Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxy substituted with amino. “Alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which is bound to at least one alkyl group and at least one aryl group.
“Alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group. “Acylamino” includes groups wherein nitrogen is bound to an acyl group. Examples of acylamino include, but are not limited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
The term “amide” or “aminocarboxy” includes compounds or moieties that contain a nitrogen atom that is bound to the carbon of a carbonyl or a thiocarbonyl group. The term includes “alkaminocarboxy” groups that include alkyl, alkenyl or alkynyl groups bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. It also includes “arylaminocarboxy” groups that include aryl or heteroaryl moieties bound to an amino group that is bound to the carbon of a carbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”, “alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bound to a nitrogen atom which is in turn bound to the carbon of a carbonyl group. Amides can be substituted with substituents such as straight chain alkyl, branched alkyl, cycloalkyl, aryl, heteroaryl or heterocycle. Substituents on amide groups may be further substituted.
Compounds of the present invention that contain nitrogens can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to afford other compounds of the present invention. Thus, all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N→O or N+—O″). Furthermore, in other instances, the nitrogens in the compounds of the present invention can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m-CPBA. All shown and claimed nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R is substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.
In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present invention includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like. In addition, a crystal polymorphism may be present for the compounds represented by the formula. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present invention. Furthermore, so-called metabolite which is produced by degradation of the present compound in vivo is included in the scope of the present invention.
“Isomerism” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture.”
A carbon atom bonded to four nonidentical substituents is termed a “chiral center.” “Chiral isomer” means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture.” When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).
“Geometric isomer” means the diastereomers that owe their existence to hindered rotation about double bonds or a cycloalkyl linker (e.g., 1,3-cylcobutyl). These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.
It is to be understood that the compounds of the present invention may be depicted as different chiral isomers or geometric isomers. It should also be understood that when compounds have chiral isomeric or geometric isomeric forms, all isomeric forms are intended to be included in the scope of the present invention, and the naming of the compounds does not exclude any isomeric forms.
Furthermore, the structures and other compounds discussed in this invention include all atropic isomers thereof. “Atropic isomers” are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.
“Tautomer” is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.
Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs. Ring-chain tautomerism arises as a result of the aldehyde group (—CHO) in a sugar chain molecule reacting with one of the hydroxy groups (—OH) in the same molecule to give it a cyclic (ring-shaped) form as exhibited by glucose.
Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings (e.g., in nucleobases such as guanine, thymine and cytosine), amine-enamine and enamine-enamine. Benzimidazoles also exhibit tautomerism, when the benzimidazole contains one or more substituents in the 4, 5, 6 or 7 positions, the possibility of different isomers arises. For example, 2,5-dimethyl-1H-benzo[d]imidazole can exist in equilibrium with its isomer 2,6-dimethyl-1H-benzo[d]imidazole via tautomerization.
It is to be understood that the compounds of the present invention may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present invention, and the naming of the compounds does not exclude any tautomer form.
The term “crystal polymorphs”, “polymorphs” or “crystal forms” means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.
Compounds of the invention may be crystalline, semi-crystalline, non-crystalline, amorphous, mesomorphous, etc.
Compounds described herein include the compounds themselves, as well as their N-oxides, salts, their solvates, and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a substituted purine or 7-deazapurine compound. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate.
Likewise, a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a substituted purine or 7-deazapurine compound. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. The substituted purine or 7-deazapurine compounds also include those salts containing quaternary nitrogen atoms. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active substituted purine or 7-deazapurine compounds.
Additionally, the compounds described herein, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include hemihydrates, monohydrates, dihydrates, trihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.
“Solvate” means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O. A hemihydrate is formed by the combination of one molecule of water with more than one molecule of the substance in which the water retains its molecular state as H2O.
As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
As defined herein, the term “derivative” refers to compounds that have a common core structure, and are substituted with various groups as described herein. For example, all of the compounds represented by Formula (I) are substituted purine compounds or substituted 7-deazapurine compounds, and have Formula (I) as a common core.
The term “bioisostere” refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms. The objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound. The bioisosteric replacement may be physicochemically or topologically based. Examples of carboxylic acid bioisosteres include, but are not limited to, acyl sulfonimides, tetrazoles, sulfonates and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.
All isotopes of atoms occurring in the compounds described herein are intended to be encompassed. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include C-13 and C-14.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove.
shRNA Cloning
shRNAs were designed using the RNAi Codex26. 97-mer oligos (Table 2) These were amplified with the following primer pair (SEQ ID NOs: 1 and 2)
PCR products were gel purified, digested with EcoRI and XHoI and ligated into the MSCV-PM (Openbiosystems) vector. Clones were verified by sequencing. shRNA targeting the firefly luciferase was used as a control27. Nanog shRNA was previously described28.
293T cells were plated at a density of 2.5×106 cells per 10-cm dish. The next day, cells were transfected with 2 μg viral vector, 2 μg Gag-Pol vector and 0.22 μg VSV-G plasmid using 20 μl Fugene 6 (Roche Applied Science #1181509001) in 400 μl DMEM per plate. Supernatant was collected 48 h and 72 h post-transfection and filtered through 45 μm pore size filters. For concentration, viral supernatants were mixed with PEG3350 solution (Sigma P3640, dissolved in PBS, 10% final concentration) and left overnight at 4 C. The next day, supernatants were centrifuged at 2500 rpm for 20 minutes, and the pellets were re-suspended in PBS. Titering was performed on 293 Ts. For shRNA infections, 500 ul of viral supernatant was used to infect 25,000 cells in the presence of 10 ug/ml protamine sulfate. For fluorescent labeling of dh1fs, we used lentiviruses PRRL-GFP (Addgene #12252) and FUdGW-Tomato (Addgene #22771).
dH1f cells were first infected with shRNA viruses at high MOI to ensure all cells received at least one vector (Gauged by puromycin resistance of parallel infected wells). 25,000 shRNA-infected dH1f cells were then plated per well in 12-well plates and infected overnight with either retroviral (MOI 2.5)7 or lentiviral (Addgene #21162, 21164; 100-200 μl supernatant)29 reprogramming factors. For 2-factor reprogramming, Oct4 and Sox2 viruses were used at an MOI of 5. 6 days later, cells were trypsinized and re-plated 1:4 to 1:6 onto 6-well plates. Medium was changed to hES medium daily until Day 21 when plates were fixed. Small molecule inhibitor of Dot1L, EPZ004777 (a gift from Epizyme, Inc., Cambridge, Mass.) was dissolved in DMSO as a 10 mM stock and was added at the indicated concentrations. For Dot1L rescue experiments, an MSCV-based retroviral vector encoding human Dot1L with or without mutations in the SAM binding site (gifts of Y. Zhang) were mutagenized at the shRNA target site using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). In certain experiments, Nanog and Lin28 expression was achieved using lentivirus (Addgene #21163). IMR-90 and MRC5 human diploid fibroblasts were purchased from ATCC and 50000 cells were used in reprogramming experiments. Statistical analysis was performed using a Student's t-test.
Total RNA was extracted from three independent culture plates for each conditions with an RNeasy Mini kit (Qiagen). Synthesis of cRNA from total RNA and hybridization/scanning of microarrays were performed with Affymetrix GeneChip products (HGU133A) as described in the GeneChip manual. Normalization of the raw gene expression data, quality control checks, and subsequent analyses were done with the open-source R-project statistical software (R Development Core Team, 2007)(http://www.r-project.org/) together with Bioconductor packages. Raw data files (.CEL) were converted into probe set values by RMA normalization. Genes were selected at a threshold of Log Ratio 0.4. The microarray data have been deposited in National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE29253.
Total RNA was extracted using RNeasy Mini kit coupled with RNase-free DNase set (Qiagen) and reverse transcribed with Hexanucleotide Mix (Roche). The resulting cDNAs were used for PCR using SYBR-Green Master PCR mix (Applied Biosystem) in triplicates.
All quantitations were normalized to an endogenous Beta-Actin control. The relative quantitation value for each target gene compared to the calibrator for that target is expressed as 2-(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to Beta-Actin). List of primers can be found in Table 3.
Immunostaining of reprogramming plates were performed as described8. Briefly, cells were fixed with 4% p-formaldehyde and stained with biotin-anti-Tra-1-60 (eBioscience, #13-8863-82, 1:250) and streptavidin horseradish peroxidase (Biolegend, #405210, 1:500) diluted in PBS (3%), FCS (0.3%) Triton X-100. Staining was developed with the Vector labs DAB kit (#SK-4100), and iPSC colonies quantified with ImageJ software. For the characterization of shDot1l-iPS cells, single colonies were put onto MEF coated 96-well plates. The plates were fixed for 20 min with 4% p-formaldehyde/PBS (+/+), washed several times with PBS (+/+) and incubated overnight at 4 OC with primary antibody and Hoechst diluted in 3% donkey serum/3% BSA Fraction VII/0.01% Triton X-100/PBS (+/+); Hoechst, Invitrogen #H3570 (1:20,000), Tra-1-81/A488 (BD #560174), SSEA-4/A647 (BD #560219), Tra-1-60/A647 (BD #560122), Nanog, rabbit polyclonal (Abcam #ab21624), Oct4, rabbit polyclonal (Abcam #ab19857). For Nanog and Oct4, donkey anti-rabbit IgG/A555 (Molecular Probes #A31572) secondary antibody was used. After several washes with PBS (+/+), images were acquired using a BD Pathway 435 imager equipped with a ×10 objective.
iPSCs grown on MEFs were harvested with Collagenease IV (1 mg/ml in DMEM/F12). Cell clumps from one 6-well plate were resuspended in 50 μl DMEM/F12, 100 μl collagen I (Invitrogen-#A1064401) and 150 μl hESC-qualified matrigel (BD Biosciences-#354277). Cell clumps were then injected into the hind limb femoral muscles (100 μl suspension per leg) of Rag2 γ/c mice. After 6-8 weeks, teratomas were harvested and fixed in Bouin's solution overnight. Samples were then embedded in paraffin, and sections were stained with hematoxylin/eosin (Rodent Histopathology Core, Harvard Medical School, Boston, Mass., USA).
Embryoid body differentiation was performed as described (Loewer). To check for the presence of the reprogramming transgenes, genomic DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen) and PCR was performed with specific primers to the endogenous or the viral trangenes3.
ChIP-seq was performed as described with slight modifications25. 300 000 cells were fixed at room temperature in PBS 1% formalin (v/v) for 10 minutes with gentle agitations. Fixation was stopped by the addition of glycine (125 mM final concentration) and agitation for 5 min at room temperature. Fixed cells were washed twice in ice-cold PBS, resuspended in 100 ul of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1). Chromatin was sheared by sonication to about 100-500 bp fragments using bioruptor (diagenode, Denville, N.J.) and diluted tenfold with dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). Antibodies against specific histone modifications was added to sonicated chromatin solution and incubated at 4 degree overnight with gentle agitation. The antibodies used were anti-H3K27me3 (Millipore 07-449) and anti-H3K79me2 (abcam 3594). Immune complexes were collected by incubation with 20 ul of Protein A/G agarose beads (Millipore) for an hour at 4 degree with gentle agitation. Precipitates were washed sequentially with ice cold low salt wash (0.1% SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 150 mM NaCl), high salt wash (0.1% SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl wash (0.25M LiCl, 1% IGEPAL CA-630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and TE wash (1 mM EDTA, 10 mM Tris-HCl, pH 8.1) for 5 mins each at 4 degree with gentle agitation. Samples were centrifuged briefly in between washes to collect the beads. Immunoprecipitated DNA was eluted by incubating beads with 150 ul elution buffer (1% SDS, 0.1 M NaHCO3) with gentle agitation for 15 mins at room temperature. Elution was repeated once and eluates were combined, sodium chloride (final concentration of 0.2M) were added to the eluate and eluates were incubated at 65 degree overnight to reverse crosslinking. DNA was purified using PCR purification spin column (Qiagen). For ChIP sequencing, ChIP DNA libraries were made following Illumina ChIP-seq library preparation kit and subjected to Solexa sequencing (Illumina) at Center for Cancer Computational Biology, Dana Faber Cancer Institute.
To examine the influence of modifiers on somatic cell reprogramming, a loss-of-function approach was employed to interrogate the role of 22 select genes in DNA and histone methylation pathways.
In one example, a pool of 3 hairpins was tested for each of 22 target genes and knockdown efficiencies were observed of >60% for 21 out of 22 targets by qRT-PCR (
Eight shRNA pools negatively affected reprogramming (
This is of particular significance given the recent finding that PRC2 component Ezh2 is necessary for fusion-based reprogramming9. Thus, interference with chromatin-modifying enzymes that mediate repressive chromatin domains reduces reprogramming and is consistent with the importance of gene silencing of the somatic cell program during generation of iPSC.
In contrast to genes whose functions appear to be required for reprogramming, inhibition of three genes enhanced reprogramming: YY1, Suv39H1, and Dot1L (
Following infection with OSKM, fibroblasts expressing Dot1L shRNA formed significantly more iPSC colonies than control fibroblasts, and the degree of enhancement in reprogramming correlated with the degree of Dot1L knockdown (
To further document the competitive advantage of Dot1L shRNA-expressing fibroblasts (shDot1L) during reprogramming, experiments were performed in which fibroblasts infected with Dot1L shRNA or a control vector were differentially labeled with either tdTomato or GFP and co-cultured prior to superinfection with OSKM (
The emerging iPSC colonies were then scored for tdTomato or GFP fluorescence, which indicated their cell of origin. Co-mixture of tdTomato- and GFP-labeled control cells resulted in a 1:1 ratio of red and green iPSC colonies indicating that labeling per se did not affect relative reprogramming efficiency. In contrast, wells containing co-mixtures of tdTomato-shDot1L and GFP-shCntrl cells generated 3-fold more red colonies than green colonies, indicating that Dot1L-inhibited cells reprogram more efficiently under identical conditions (
Additionally, these cells were able to differentiate into all three embryonic germ layers in vitro and in teratomas (
To assess whether Dot1L inhibition could replace any of the 4 exogenous reprogramming factors, shDot1L and shCntrl fibroblasts were infected with 3 factors, omitting one factor at a time. In the absence of Oct4 or Sox2 no iPSC colonies emerged in both types of fibroblasts. When either Klf4 or Myc was omitted, shDot1L fibroblasts were able to give rise to robust numbers of Tra-1-60 positive colonies while control cells yielded very few colonies as reported previously3. It was examined whether Dot1L suppression would suffice to reprogram fibroblasts in the absence of both Klf4 and c-Myc. Indeed, shDot1L fibroblasts infected with only Oct4 and Sox2 gave rise to Tra-1-60-positive colonies, whereas control fibroblasts did not (
The OS-shDot1L-iPS cells had all of the hallmarks of pluripotency as gauged by endogenous pluripotency factor expression and the ability to form all three embryonic germ layers in vitro and in teratomas (
It was examined whether the cellular mechanisms by which Dot1L inhibition promotes reprogramming. It was observed that in established human iPS clones derived from shDot1L fibroblasts, Dot1L inhibition was no longer evident, reflecting the known silencing of retroviruses that occurs during reprogramming (
Examination of Dot1L knockdown levels during the course of reprogramming revealed that silencing occurred by day 15 after OSKM transduction (
There was also no difference in the number of senescent cells, as gauged by senescence-associated β-gal staining following OSKM infection (
Previous studies indicated that Dot1L null cells have increased apoptosis and accumulation of cells in G2 phase13. However, a significant increase in apoptosis or change in the cell cycle profile of Dot1L knock-down fibroblasts was not observed (
It was also examined whether Dot1L knockdown, in addition to increasing reprogramming efficiency, accelerated the kinetics of reprogramming.
When the reprogramming experiments were extended by 10 more days (at which time the already-formed iPSC colonies begin to differentiate), shDot1L cells still yielded more iPSC colonies than controls (
In addition to the shRNA mediated knockdown of Dot1L, a small molecule inhibitor of Dot1L catalytic activity was used to further validate the findings15. This inhibitor (EPZ004777, referred to as iDot1L and shown below) abrogated H3K79 methylation robustly at 1 uM to 10 uM concentration range and led to 3-4 fold enhancement of reprogramming of human fibroblasts (
Knockdown of Dot1L protein in combination with inhibitor treatment did not result in a further increase of reprogramming efficiency thereby reinforcing the previous observation that inhibition of catalytic activity of Dot1L is key to reprogramming (
It was assessed whether these observations can be extended to the murine system. It was observed that, similar to human fibroblasts, iDot1L treatment led to 3-fold enhancement of reprogramming of Oct4-GFP MEFs (
The colonies resulting from Dot1L inhibition were GFP-positive indicating the activation of the endogenous Oct4 gene. Furthermore, reprogramming of tail-tip fibroblasts (TTFs) derived from a conditional knockout Dot1L mouse strain yielded significantly more iPS colonies upon Cre mediated excision of Dot1L (
Taken together these results demonstrate that Dot1L inhibition enhances reprogramming of mouse cells as well.
To further dissect out the crucial time window for Dot1L inhibition, human fibroblasts undergoing reprogramming were treated with iDot1L at 1 week intervals. It was observed that Dot1L inhibition either in the first or the second week was sufficient to enhance reprogramming whereas pretreatment for 5 days prior to OSKM transduction had no effect (
Since the effects of Dot1L inhibition were evident early in the reprogramming process, it was investigate if gene expression changes in Dot1L-inhibited cells soon after transduction with reprogramming factors could reveal insights into the molecular mechanisms involved. A global gene-expression analyses on control and shDot1L fibroblasts prior to and 6 days after OSKM transduction was performed along with cells that were treated with iDot1L. Although thousands of genes were induced or repressed upon OSKM expression, relatively few genes were differentially expressed in shDot1L cells on Day 6 of reprogramming (22 up, 23 down). At this time point, inhibitor treated cells exhibited broader gene expression changes (405 up and 175 down), presumably due to more complete inhibition of K79me2 levels.
To understand the mechanism by which Dot1L inhibition substitutes for Klf4, gene expression analyses on control and shDot1L cells were performed upon 3-factor infection with OSM. While 94 genes were differentially upregulated in shDot1L cells in the absence of Klf4, the intersection of this set of genes with the set differentially upregulated in 4-factor reprogramming of shDot1L and inhibitor treated cells yielded only 5 common genes (
The possibility was explored that Nanog and Lin28 upregulation was responsible for the enhanced reprogramming observed following Dot1L inhibition and validated their upregulation in shDot1L fibroblasts compared to control fibroblasts 6 days after transduction with reprogramming factors (
It was hypothesized that if Nanog and Lin28 upregulation is the major mechanism by which Dot1L inhibition enhances reprogramming, then inclusion of Nanog and Lin28 in the OSKM reprogramming cocktail would not confer any additional enhancement to shDot1L cells. In fact, no significant difference in the number of colonies generated between 4-factor (OSKM) and 6-factor (OSKMNL) reprogramming of shDot1L cells was observed, although the colonies were larger in the latter conditions (
To gain insight into the genome-wide chromatin changes that are facilitated by Dot1L inhibition during reprogramming, ChIP-seq for H3K79me2 and H3K27me3 in human ES cells as well as fibroblasts undergoing reprogramming with or without the iDot1L treatment were performed (
These observations support the notion that Dot1L inhibition collaborates with OSKM in facilitating the loss of K79me2 from fibroblast specific regulators.
shRNAs were designed using the RNAi Codex26. 97-mer oligonucleotides (Table 2) were PCR amplified and cloned into MSCV-PM27 vector. Control shRNA targeting the firefly luciferase27 and the Nanog shRNA were previously described28. Reprogramming assays were carried out with either retroviral7 or lentiviral29 reprogramming factors. dH1f cells were previously described3. IMR-90 and MRC5 human diploid fibroblasts were purchased from ATCC. Immunostaining of reprogramming plates were performed as described8. For gene expression analyses, total RNA was extracted from three independent culture plates for each condition and transcriptional profiling was performed using Affymetrix U133A microarrays. Primers used for quantitative real-time PCR can be found in Table 3. ChIP-seq was performed as described with slight modifications25.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.
The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety, particularly for the use or subject matter referenced herein.
This application claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 61/547,404 filed Oct. 14, 2011, the disclosure of which is incorporated by reference herein in its entirety.
This invention was made with government support under Grant No. R01 DK70055 from the National Institutes of Health. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/000514 | 10/14/2012 | WO | 00 | 4/14/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61547404 | Oct 2011 | US |
