A focal plane shutter for digital cameras includes a first driving member for a first blade and a second driving member for the first blade, mounted to the same shaft; arms for the first blade on which a set spring is hooked; and a set member for restraining the first driving member for the first blade in the proximity of the position where the exposure operation is completed, at a set position by rotating a restraining member, and actuating the first driving member for the first blade to the starting position of the exposure operation in the restoration to the initial position immediately after the release.

1. Field of the Invention
This invention relates to a focal plane shutter for digital cameras.
2. Description of Related Art
In focal plane shutters for digital cameras, like focal plane shutters for silver-halide film cameras, there are types in which two shutters, a first blade and a second blade, are provided. The first blade and the second blade are actuated successively in the same direction in photography so that the imaging surface of an image sensor, such as a CCD, is continuously exposed from one end to the other. These first and second blades are connected to a driving member for the first blade and a driving member for the second blade, and after the release of a camera, power to an electromagnet for the first blade and an electromagnet for the second blade is disconnected in turn so that the above driving members are rotated successively by biasing forces of their driving springs and thereby an exposure operation is performed.
The first blade and the second blade are such that after the exposure operation is completed, a set member is actuated from its initial position to rotate the driving members against the biasing forces of the driving springs and thereby the first blade and the second blade are actuated toward their set positions. The set member remains at the set position without returning to the initial position until the release button of the camera is pushed in the next photography. After the release button of the camera is pushed in the next photography and the driving members are securely retained by attractive forces of the electromagnets, the set member is restored to the initial position. In a digital camera equipped with a focal plane shutter of this type, therefore, the imaging surface of the image sensor is covered by the first blade in a set condition, and thus an optical finder, as in the case of the silver-halide film camera, becomes necessary for a finder observing an image of an object to be photographed.
However, when the shutter is constructed so that, in the set operation by the set member mentioned above, the second blade is actuated toward the set position, while the first blade is restrained at the position where the exposure operation is completed, it becomes possible to cause light from the object to strike the imaging surface of the image sensor in the set condition. Hence, the image of the object formed on the imaging surface can be displayed and observed on a monitor such as a liquid crystal display device. However, in the case where the shutter is constructed as mentioned above, it is necessary that when the release button is pushed in photography, the restraint of the first blade is first released so that the first blade is actuated toward the starting position of the exposure operation and after the imaging surface of the image sensor is once brought into a covered state, the first blade and the second blade start the exposure operation as described above.
An example of a focal plane shutter for digital cameras constructed as mentioned above is disclosed in Japanese Patent Kokai No. 2001-222059. According to the structure of this shutter, a conventional driving member for the first blade is designed to include a first driving member for the first blade connecting the first blade and a second driving member for the first blade rotated by the driving spring in the exposure operation. In the set operation, the set member keeps the restraint of the first driving member for the first blade in the proximity of the position where the exposure operation is completed and moves only the second driving member for the first blade toward the set position. After that, when the release button is pushed and the set member is restored to the initial position, the set member releases its restraint so that the first driving member for the first blade and the first blade can be actuated toward starting positions of the exposure operation by biasing forces of set springs.
The focal plane shutter set forth in Kokai No. 2001-222059, however, is constructed so that when the set member is restored to the initial position after the release button of the camera is pushed, the restraint of the first driving member for the first blade is released immediately before the set member reaches the initial position, and the first blade is actuated from the proximity of the position where the exposure operation is completed toward the starting position of the exposure operation. Consequently, the first driving member for the first blade and the first blade, after bounding at the starting positions of the exposure operation, are brought into fixed states after the set member is restored to the initial position. Hence, after the set member is restored to the initial position, the exposure operation of the first blade cannot be immediately started and after the release button is pushed, time required until the first blade starts the exposure operation becomes long. This causes the problem that when the object is moving, there is a high possibility of missing a shutter chance.
Thus, a special restraining member is rotatably mounted on the same shaft as the set member so that when the set operation is performed, the restraining member, instead of the set member, restrains the first driving member for the first blade immediately in the proximity of the position where the exposure operation is completed. When the release button is pushed and the set member is restored to the initial position, the restraint is released at the initial stage of this restoration operation, while when the set member has been restored to the initial position, the first driving member for the first blade and the first blade, after bounding at the starting positions of the exposure operation, are brought into the fixed states. A focal plane shutter for digital cameras constructed in this way is disclosed in Japanese Patent Kokai No. 2003-222928.
In the structure of the focal plane shutter set forth in Kokai No. 2003-222928, in addition to the fact that the set member and the restraining member are mounted on the same shaft, as mentioned above, the set member is provided with a hook section that has flexibility and is configured with an engaging part at its top, and the restraining member is configured with two parts to be engaged on the periphery. When the set operation is performed by the set member, the engaging part of the hook section engages one part to be engaged and the restraining member is rotated in such a way that it is pulled. After the restraining member abuts on a stopper at the position where it is capable of restraining the rotation of the first driving member for the first blade, its engagement is released by the flexure of the hook section and only the set member continues to rotate so that, at the set position, the hook section is restored to the original by its own resilience and then is stopped. When the operation is carried out so that the set member is restored to the original position, the engaging part of the hook section engages the other part to be engaged of the restraining member to rotate the restraining member in such a way as to push it, and thereby the restraint of the first driving member for the first blade is released. After that, when the restraining member abuts on the stopper, its engagement is released by the flexure of the hook section, and immediately before reaching the set position, the hook section is restored to the original by its own resilience and then is stopped.
The focal plane shutter described in Kokai No. 2003-222928 is constructed so that the set member reciprocates and rotates the restraining member through the engagement of the engaging part of the hook section with two parts to be engaged of the restraining member and this engagement is released by the flexure of the hook section. However, it is extremely difficult to actually mass-produce the focal plane shutter constructed in this way. This is because it is very difficult to fabricate the shutters so that the resilience of the hook section is uniformly provided or so that the friction surface of the engaging part and those of the two parts to be engaged have a preset arrangement relationship.
Consequently, in the case where such focal plane shutters are mass-produced, even though the restraining member can be rotated by the engagement of the engaging part with the parts to be engaged, sometimes the hook section is not favorably flexed and the engagement of the engaging part with the parts to be engaged is not smoothly released when the rotation of the restraining member is stopped. Further, even though the engagement of the engaging part with the parts to be engaged can be smoothly released when the rotation of the restraining member is stopped, the engagement of the engaging part with the parts to be engaged may be released before the restraining member, when rotated, abuts on the stopper at a preset position of rotation. In particular, when the set member is restored to the initial position, the part to be restrained of the first driving member for the first blade is pressed against the restraining part of the restraining member by the biasing force of the set spring, and thus before the restraint of the first driving member for the first blade is released the hook section may be flexed to release the engagement of the engaging part with the parts to be engaged. Moreover, cases sometimes occur in which, while the set member is repeatedly rotated, the hook section, after being flexed, ceases to be restored to a preset original shape, the engagement of the engaging part with the parts to be engaged is not favorably carried out, and the restraining member cannot be securely rotated by the set member.


A method of making a thin film transistor device, including forming and patterning a semiconductor film to form first and second semiconductor films in, respectively, low-voltage driven and high-voltage driven thin film transistor formation regions. The method also includes forming a first insulating film on the first and second semiconductor films, and forming a first gate electrode on the first insulating film in the low-voltage driven thin film transistor formation region. Additionally, a second insulating film is formed on the entire surface of the resultant structure above the substrate, and a second gate electrode is formed on the second insulating film in the high-voltage driven thin film transistor formation region. The method also includes etching the first and second insulating films, thus forming first and second gate insulating films below, respectively, the first and second gate electrodes, with the second gate insulating film being wider than the second gate electrode.

1. Field of the Invention
The present invention relates to a thin film transistor device having at least two kinds of thin film transistors, each having a gate insulating film, a thickness of which is different from that of the other, and to a method of manufacturing the same. More particularly, the present invention relates to a thin film transistor device which can be applied to a liquid crystal display apparatus united with peripheral circuits and an organic EL display apparatus and to a method of manufacturing the same.
2. Description of the Prior Art
As display apparatuses of electronic instruments such as notebook type personal computers and portable terminals, a liquid crystal display panel and an organic electro luminescence (EL) display panel have been used. A plurality of pixels are arranged in horizontal and vertical directions in these display panels, and a desired image is displayed by controlling a voltage applied to each pixel. In an active matrix type liquid crystal display panel, one or a plurality of thin film transistors (TFTs) are provided for each pixel.
In recent years, development of a display panel united with peripheral circuits such as a driver (driving circuit), which are formed on a substrate thereof, has proceeded, and such products of the display panel have been put into practical use. Moreover, development of a so-called system-on-glass device, in which a memory, an image correction arithmetic circuit, and an arithmetic circuit having a high data processing function are formed on a display panel, has proceeded.
FIG. 1 is a section view showing an example of a TFT (n-type) used for a conventional liquid crystal display apparatus united with a peripheral circuit.
An underlayer insulating film 11 made of a silicon oxide film or the like is formed on a substrate 10, and a polysilicon film 12 serving as an operational layer of the TFT is selectively formed on the underlayer insulating film 11. In the polysilicon film 12, a pair of high concentration impurity regions (source/drain) 13 formed by doping n-type impurities thereinto at a high concentration are formed so as to sandwich a channel region therebetween. In tip portions of these high concentration impurity regions 13 closer to the channel region, low concentration impurity regions, lightly doped drain (hereinafter referred to as LDD region) 14, which are formed by doping n-type impurities thereinto at a low concentration, are respectively formed.
A gate insulating film 15 made of a silicon oxide film or the like is formed on the underlayer insulating film 11 and the polysilcon film 12, and a gate electrode 16 is formed on the gate insulating film 15. Moreover, an interlayer insulating film 17 made of a silicon oxide film or the like is formed on the gate insulating film 15 and the gate electrode 16.
Electrodes (source electrode and drain electrode) 18 are formed on the interlayer insulating film 17. These electrodes 18 are electrically connected to the respective high concentration impurity regions 13 via contact holes formed in the interlayer insulating film 17 and the gate insulating film 15.
As shown in FIG. 1, in order to suppress deterioration of an ON characteristic due to hot carriers and to decrease an OFF current, the TFT of the liquid crystal display apparatus united with peripheral circuits generally has an LDD structure, in which the low concentration impurity regions 14 are formed in the tip portions of the high concentration impurity regions 13 closer to the channel region. When viewed from above, an edge of each low concentration impurity region 14 is located approximately just below the gate electrode 16. Note that the portions corresponding to the low concentration impurity regions 14 may be used also as an offset region without doping the impurities into those portions.
FIGS. 2A and 2B are section views showing other examples of conventional TFTs. FIG. 2A shows a structure of a low-voltage driven TFT, and FIG. 2B shows a structure of a high-voltage driven TFT. These low and high-voltage driven TFTs are formed on the same substrate 20.
An underlayer insulating film 21 is formed on the substrate 20, and a polysilicon film 22 serving as an operational layer of the TFT is formed on the underlayer insulating film 21. The underlayer insulating film 21 is made of, for example, a silicon oxide film (SiO2), and has a thickness of about 80 nm. Moreover, a thickness of the polysilicon film 22 is about 50 nm.
As shown in FIG. 2A, a pair of high concentration impurity regions 23 serving as a source/drain are formed in the polysilicon film 22 of the low-voltage driven TFT so as to sandwich a channel region. A thin gate insulating film 25a is formed on the channel region of the low-voltage driven TFT, which is formed in the polysilicon film 22, and a gate electrode 26a is formed on the gate insulating film 25a. The gate insulating film 25a is made of, for example, a silicon oxide film, and has a thickness of about 30 nm.
On the other hand, as shown in FIG. 2B, in a polysilicon film 22 of the high-voltage driven TFT, a pair of high concentration impurity regions 23 are formed so as to sandwich a channel region. Moreover, in this polysilicon film 22, low concentration impurity regions (LDD regions) 24 are formed so that each is located between the respective high concentration impurity regions 23 and the channel region. A thick gate insulating film 25b is formed on the channel region and the low concentration impurity regions 24 in the polysilcon film of the high-voltage driven TFT, and a gate electrode 26b is formed on the gate insulating film 25b. The gate insulating film 25b is made of, for example, a silicon oxide film, and has a thickness of about 130 nm. Moreover, the gate electrodes 26a and 26b of the low and high-voltage driven TFTs are made of, for example, Cr (chromium), and have a thickness of about 400 nm.
The polysilicon film 22 and the gate electrodes 26a and 26b are covered with an interlayer insulating film 27 made of a silicon nitride film (SiN) or the like. A thickness of the interlayer insulating film is, for example, 300 nm. Electrodes (source electrodes and drain electrodes) 28, which are electrically connected to the respective high concentration impurity regions 23 via contact holes formed in the interlayer insulating film 27, are formed on the interlayer insulating film 27.
Since the low-voltage driven TFT shown in FIG. 2A has a thin gate insulating film and no LDD region showing a high resistivity, this TFT can perform a high speed operation with a low voltage. Since the high-voltage driven TFT shown in FIG. 2B has a thick gate insulating film and the low concentration impurity regions (LDD region) 24 provided therein, which prevent occurrence of hot carriers, this TFT can prevent deterioration of characteristics even when this TFT is driven with a high voltage.
In Japanese Patent Laid-open No. 10(1998)-27909, No. 10(1998)-170953, No. 5(1993)-142571 and No. 7(1995)-249766, technologies for forming transistors having gate insulating films of different thickness in the same semiconductor substrate are disclosed.
In Japanese Patent Laid-open No. 8(1996)-220505, a technology is proposed, in which of a high and low-voltage driven transistors used for a liquid crystal display apparatus, only the high-voltage driven transistor is constructed of an LDD structure.
The inventors of this application consider that there are the following problems in the above described conventional thin film transistor device.
Since the TFT shown in FIG. 1 has the LDD structure, this TFT can suppress deterioration of characteristics due to hot carriers. An ON characteristic is restricted by a resistivity of the low concentration impurity regions (LDD layer) 14. Therefore, a high speed operation of the TFT is disturbed, thus producing obstacles in incorporating high function circuits such as an image data memory and an image processing arithmetic circuit, of which a high speed operation is required, into a display panel.
In the TFT shown in FIG. 2A, the thickness of the gate insulating film 25a is small, and the gate insulating film 25a is formed so as to have the same width as that of the electrode 26a. Accordingly, a gap between the gate electrode 26a and the source/drain (high concentration impurity region 23) is very small. Impurities, contaminant ions and the like adhere to a side wall of the gate insulating film 25a in a step such as etching, and a leak current is apt to occur when a gap between the gate electrode 26a and the source/drain is small.
When the conventional low and high-voltage driven TFTs shown in FIGS. 2A and 2B are formed on the same substrate, the gate insulating film 25a and the gate electrode 26a of the low-voltage driven TFT are formed, and then the gate insulating film 25b and the gate electrode 26b of the high-voltage driven TFT are formed. In this case, a surface of the polysilicon film 22 is treated by solution containing hydrofluoric acid to purify an interface between the polysilicon film 22 and the gate insulating film 25b. At this time, the gate insulating film 25a of the low-voltage driven TFT is corroded by the hydrofluoric acid solution, and the leak current is more apt to occur.

Method of manufacturing a thin film transistor device


With respect to reduced coenzyme Q10, there has been no report about the presence of crystal polymorphism, and it has been considered that a conventionally obtained crystal form is only one form. The present invention relates to a reduced coenzyme Q10 crystal having an endothermic peak indicating melting at 54±2° C. during temperature rise at a rate of 5° C./min by differential scanning calorimetry (DSC), and/or to a reduced coenzyme Q10 crystal showing characteristic peaks at diffraction angles (2θ±0.2°) of 11.5°, 18.2°, 19.3°, 22.3°, 23.0° and 33.3° by powder X-ray (Cu—Kα) diffraction. The crystal form is a novel reduced coenzyme Q10 crystal which has a higher melting point and a lower solubility in a solvent, and is more excellent in stability than the conventionally known reduced coenzyme Q10 crystal.

Coenzyme Q is an essential component widely distributed in living organisms from bacteria to mammals, and is known as a component of mitochondrial electron transfer system in cells in the living body. Coenzyme Q serves as a transfer component in the electron transfer system by the repetition of oxidation and reduction in mitochondria, and, further, reduced coenzyme Q is known to have antioxidant activity. The major component in humans is coenzyme Q10 which is one having 10 isoprenoid repeating structures in its side chain, and, usually, about 40 to 90% thereof is present in the living body as the reduced form. The physiological activity of coenzyme Q includes activation of energy production by mitochondrial activation, activation of cardiac function, an effect of stabilizing cell membranes, and an effect of protecting cells by antioxidant activity.
While coenzyme Q10 currently produced and sold is, in large part, oxidized coenzyme Q10, reduced coenzyme Q10 which exhibits higher oral absorbability than that of oxidized coenzyme Q10 has also been commercially available and has come to be used in recent years.
Patent Literature 1 discloses a common method for obtaining reduced coenzyme Q10. Furthermore, several methods for obtaining reduced coenzyme Q10 as a crystal are also known. For example, in Patent Literature 2, reduced coenzyme Q10 is produced as a crystal by crystallization in an alcohol solution and/or a ketone solution. In Patent Literature 3, reduced coenzyme Q10 is crystallized by adding the high concentration liquid phase thereof to a poor solvent.
In addition, Patent Literature 4 discloses that reduced coenzyme Q10 is dissolved in oil and fat and then cooled, thereby making it possible to yield a crystal which has a different X-ray diffraction pattern from that of a usual reduced coenzyme Q10 crystal and is excellent in stability.
Reduced coenzyme Q10 is known to usually have a property such that it is easily oxidized in the presence of molecular oxygen to be converted into oxidized coenzyme Q10. In response, for example, Patent Literature 5 discloses, as a method for stabilizing reduced coenzyme Q10, a method for allowing reduced coenzyme Q10 to contact and coexist with ascorbic acids or citric acids. In addition, the conventional reduced coenzyme Q10 crystal has the property of being very easily electrostatically charged.
By the way, it has been reported for many compounds, whether organic compounds or inorganic compounds, that a plurality of crystal forms having different crystal structures are generally present, which are called "crystal polymorphs". A plurality of crystal forms in a crystal polymorphism each show different patterns in analysis such as X-ray diffraction or infrared spectroscopic analysis, as well as have different physical properties such as melting point and solubility. In general, there is a tendency that a more energetically stable crystal form under the defined conditions has a higher melting point and a lower solubility, and a crystal form having the highest melting point and the lowest solubility is usually called "stable form". In the case of a crystal form other than the stable form, transition to the stable form can occur during operation such as crystallization, drying, or pulverization. The transition is a very natural phenomenon that a substance changes toward an energetically stable state, but physical properties of the resulting crystal also change due to such a phenomenon, thereby possibly causing the crystal or a formulation containing such a crystal as an active ingredient to have problems in terms of quality. The crystal in the stable form not only causes no such transition but also has a high melting point as described above, thereby making it possible to be dried at a higher temperature during drying thereof, and has a low solubility, thereby making it possible to yield a crystal in a larger amount during crystallization, and, therefore, has an advantage of increasing efficiency at the time of production. For such reasons, in the case where a compound whose crystal polymorphs are present is utilized for, in particular, medical applications or the like, it is important to select an optimal crystal form such as the stable form.
It has also been reported that different crystal forms have different electrostatic charges. If a crystal takes an electrostatic charge, its sticking to equipment at the time of production, or the like, not only decreases the efficiency at the production but also causes problems in terms of safety, such as dust explosion and contamination of facilities/workers. In the case of a compound having crystal polymorphs, selecting the optimal crystal polymorph can be one effective measure against the above problems. For example, in Patent Literature 6, it has been reported that a new crystal form (type IV) of a 1,2-dihydropyridine compound has a lower electrostatic charge than other crystal forms.

Reduced coenzyme Q10 crystal having excellent stability


Neurotransmission by excitatory amino acids (EAAs) such as glutamate is mediated via membrane-bound surface receptors. This neurotransmission has been found to be modulated by certain modulatory proteins. DNA coding for a family of such modulatory proteins has now been isolated and the modulatory proteins have been characterized. Herein described are recombinant cell lines which produce these modulatory proteins as heterologous membrane-bound products. Also described are related aspects of the invention, which are of commercial significance, including the use of cell lines which express the modulatory proteins either homomerically, or heteromerically in a complex with an NMDA receptor, as a tool for discovery of compounds which affect the function of the modulatory proteins.

This invention relates to the application of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for proteins which modulate the function of glutamate receptors.
In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the xe2x80x9csendingxe2x80x9d neuron which then binds to a surface receptor on the xe2x80x9creceivingxe2x80x9d neuron, to cause excitation thereof. L-glutamate is the most abundant neurotransmitter in the CNS, and mediates the major excitatory pathway in vertebrates. Glutamate is therefore referred to as an excitatory amino acid (EAA) and the receptors which respond to it are variously referred to as glutamate receptors, or more commonly as EAA receptors.
Members of the EAA receptor family can be grouped into three main types based on differential binding to certain glutamate analogs. One type of EAA receptor, which in addition to glutamate also binds the compound NMDA (N-methyl-D-aspartate), is referred to as the NMDA type of EAA receptor. Two other glutamate-binding types of EAA receptor, which do not bind NMDA, are named according to their preference for binding with two other EAA receptor agonists, namely AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate (2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate). Accordingly, receptors which bind glutamate but not NMDA and which bind with greater affinity to kainate than to AMPA, are referred to as kainate-type EAA receptors. Similarly, those EAA receptors which bind glutamate but not NMDA, and which bind AMPA with greater affinity than kainate are referred to as AMPA-type EAA receptors.
The glutamate-binding EAA receptor family is of great physiological and medical importance. Glutamate is involved in many aspects of long-term potentiation (learning and memory), in the development of synaptic plasticity, in epileptic seizures, in neuronal damage caused by ischemia following stroke or other hypoxic events, as well as in other forms of neurodegenerative processes. The development of therapeutics which modulate these processes is being slowed by the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of an appropriate EAA receptor. The brain derived tissues currently used to screen candidate drugs are heterogeneous receptor sources, possessing on their surface many receptor types which interfere with studies of the EAA receptor/ligand interface of interest. The search for human therapeutics is further complicated by the limited availability of brain tissue of human origin. It would therefore be desirable to obtain cells that are genetically engineered to produce only the receptor of interest. With cell lines expressing cloned receptor cDNA, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs.
Non-human cDNAs which appear to encode the NMDA-type of EAA receptor have recently been identified and isolated. A cDNA encoding a subunit polypeptide of an NMDA receptor in rat, designated NR1, has been isolated as described by Moriyoshi et al. in Nature 354: 31, 1991. An extension of this work has revealed seven isoforms of NR1, presumably generated by combinations of alternative RNA splicing in the amino- and carboxy-terminal regions of NR1 (Anantharam et al. FEBS Lett. 305: 27, 1992; Durand et al. Proc. Natl. Acad. Sci. USA 89: 9359, 1992; Nakanishi et al. Proc. Natl. Acad. Sci. USA 89: 8552, 1992; Sugihara et al. Biochem. Biophys. Res. Commun. 185: 826, 1992; Hollmann et al. Neuron 10: 943, 1993; Kusiak and Norton. Mol. Brain. Res. 20: 64, 1993). DNA encoding NR1 and one of its isoforms have also been cloned from mouse brain by Yamazaki et al. as described in FEBS Lett. 300: 39, 1 992. Other rat NMDA receptor subunits, designated NR2A, NR2B, NR2C and NR2D, have also been identified (Monyer et al. Science 256: 1217, 1992; Ishii et al. J. Biol. Chem. 268: 2836, 1993), as well as mouse NMDA receptor subunits which have been designated xcex51, xcex52, xcex53 and xcex54 (Meguro et al. Nature 357: 70, 1992; Kutsuwada et al. Nature 358: 36, 1992; Ikeda et al. FEBS Lett. 313: 34, 1992).
There has emerged from these molecular cloning advances, a better understanding of the structural features of NMDA receptors and their subunits, as they exist in the non-human brain. According to the current model, each NMDA receptor is heteromeric, consisting of individual membrane-anchored subunits, each comprising transmembrane regions and extracellular domains that dictate ligand-binding properties and contribute to the ion-gating function served by the receptor complex.
In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable to obtain knowledge of human EAA receptors, and proteins which modulate the activity of these receptors. Such an understanding would provide a means to screen for compounds that selectively interact with this activity, i.e. to stimulate or inhibit receptor activity, thereby providing a means to identify compounds having potential therapeutic utility in humans. Non-human mammalian models are not suitable for this purpose despite significant protein homology due to the fact that minute sequence discrepancies have been found to cause dramatic pharmacological and functional variation between species homologues of the same protein (Oksenberg et al., Nature, 360:1 61, 1992; Hall et al. Trends Pharmacol. Sci. 14: 376, 1993). It is therefore particularly desirable to provide cloned cDNA encoding human EAA receptor proteins or modulatory proteins thereof, and cell lines expressing these proteins, in order to generate a screening method for compounds therapeutically useful in humans. These, accordingly, are objects of the present invention.
Human cDNAs encoding a family of NMDA receptor modulatory proteins have been identified and characterized. This family of modulatory proteins, herein referred to as the human NR2A modulatory protein family, comprises a parent protein, designated the human NR2A-1 protein, as well as functional sequence-related variants of the human NR2A-1 protein and functional fragments of the NR2A-1 protein.
In one of its aspects, thus, the present invention provides an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human NR2A protein, or functional fragments thereof.
In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a human EAA receptor modulatory protein belonging to the herein-defined NR2A family. In related aspects of the present invention, there are provided recombinant DNA constructs and methods useful to obtain substantially homogeneous sources of the human NR2A protein, comprising the steps of culturing the genetically engineered cells, and then recovering the cultured cells.
In another aspect of the present invention, there is provided a method for evaluating interaction between a candidate ligand and a human EAA receptor modulatory protein, which comprises the steps of incubating the candidate ligand with a genetically engineered cell as described above, or with a membrane preparation derived therefrom, and then assessing said interaction by determining the extent of protein/ligand binding, or by determining the ligand-induced electrical current across said cell.
In yet another aspect of the present invention, a cell that has been engineered genetically to produce a human heteromeric NR2A/receptor complex comprising an NR2A protein and an NMDA receptor subunit is provided.
In a further aspect of the present invention, there is provided a method for evaluating interaction between a candidate ligand and a human heteromeric ; NR2A/receptor complex comprising an NR2A protein and an NMDA receptor subunit, said method comprising the steps of incubating the candidate ligand with a cell line engineered to produce said receptor complex, or with a membrane preparation derived therefrom, and then assessing the interaction therebetween by determining the extent of protein/ligand binding, or by determining the ligand-induced electrical current across said cell.
Other aspects of the present invention include a human NR2A protein, in a form essentially free from other proteins of human origin, functional and immunogenic fragments of the protein, antibodies which bind to the protein, and oligonucleotides which hybridize to nucleic acid encoding the protein.


A flashing motorcycle brake light enhancement system including a plurality of super bright light emitting diode (LED) modules which signal the driver of a following vehicle to slow down to avoid a rear-end collision. The application of the motorcycle's brake supplies input current to a plurality of (LED) modules via a flasher unit and connected wiring. The flashing circuit is controlled by a single, blinking light emitting diode, which is operated in combination with a resistor matched to a relay coil, to actuate a mechanical relay, which passes full battery voltage to the (LED) modules. In one embodiment the flashing (LED) modules are contained within weather-resistant housings which are attached in symmetrical arrangement and externally of the rear-mounted motorcycle brake light housing. In an alternative embodiment, the flashing (LED) modules and related components of the brake light enhancement system are mounted internally of the stock motorcycle brake light housing so as to be directed rearwardly and visibly to a driver in a following vehicle. The (LED) modules use very little electrical power and do not overload the pre-existing motorcycle brake light circuit. Further, this minimal use of electrical current causes no power variation and has no effect on the anti-lock braking system of motorcycle so equipped. The brake light enhancement system is provided in kit form so as to be adaptable to motorcycles of various manufacturers.

1. Field of Invention
This invention relates generally to the operation of vehicle brake lights and, more particularly, to a brake light enhancement system for motorcycles.
Brake lights are one of the most important safety features included on any vehicle. Brake lights indicate when a vehicle's brakes are being applied to alert the driver of a following vehicle to slow down in order to avoid a rear end collision.
Despite the use of brake lights, the incidence of rear end collisions remains relatively high among motorcyclists. When motorcycles are stopped, especially when stopped behind other traffic, they are less visible than larger vehicles. Since the tail light is constantly illuminated on most motorcycles due to so-called "lights on laws", the illumination of the brake light may be hard to recognize when the motorcycle is near other vehicles with bright brake lights. Further, the transition of light brightness during braking may not be noticed if a following driver is not looking directly at the motorcycle as the brakes are applied.
Various alternative brake light systems have been developed in an effort to solve this problem. Such systems often cause brake lights to flash automatically. However, such alternatively brake light systems have numerous disadvantages which compromise rather than improve safety.
For example, many alternative systems utilize unreliable mechanical components such as incandescent bulbs, mechanical relays having physical contacts, cams, levers, and other mechanical parts which can cause the entire vehicle brake light system to fail. Thus, while such conventional flashing brake light systems provide an increased measure of safety when working properly, any improvement is clearly overshadowed by the possibility of a dangerous total brake light failure.
In addition, such alternative brake light systems may require extensive modification of the stock equipment for proper installation and function. Further, conventional flashing brake light systems impose an additional load on the electrical circuitry which can cause a power variation and result in failure of the anti-lock brake system found on many late model motorcycles.
The present invention solves these problems by providing a reliable flashing brake light enhancement system that may be added to the stock motorcycle brake light without any significant modification and without effecting the operation of the stock equipment thereby eliminating the risk of a total failure of brake lights and other related systems.
2. Description of Related Art
U.S. Pat. No. 5,345,218 to Daniel S. Woods discloses a flashing brake light system wherein a center high-mounted stop lamp flashes when the brakes are initially applied. After a predetermined duration, the center high-mounted stop lamp remains continuously activated until the brakes are removed. This system is intended for automobile use and requires additional components and complex circuitry not required by the present invention.
U.S. Pat. No. 4,290,048 to David S. Cutlip, et al. discloses a vehicle turn signaling apparatus which signals the direction of a turn by a simultaneous initiation of a right-turn indicator lamp and a left-turn lamp, one in a flashing mode to signal the turn direction, the other in a steady mode of illumination to provide a positional reference for the flashing lamp.
U.S. Pat. No. 4,860,177 to John B. Simms discloses a bicycle safety light which improves the visibility of the bicycle and rider in low light conditions by a constantly moving and converging pattern of light rows which visually direct a following driver to the center position of the rider and the bicycle. However, this system is neither brake actuated nor intended for motor vehicle use.
U.S. Pat. No. 5,264,826 to Steven Henderson, et al. discloses an emergency flasher circuit for use with motorcycles and bicycles for selectively applying the signal to one or both of the left and right turn indicator lights and a headlight of a motorcycle or bicycle, for alerting drivers of motor vehicles to the presence of the motorcycle or bicycle. This system is not utilized in conjunction with the brake light system.
U.S. Pat. No. 5,298,882 to Ching-Yuan Tsai discloses an LED display collision warning circuit which displays different warning indications in response to signals from a door-actuated circularly lighting and extinguishing circuit, a brake-actuated alternately flashing circuit, or a headlight-actuated night distance detection circuit so as to effectively prevent collisions and accidents.
U.S. Pat. No. 5,166,672 to Andre Duneau discloses an multiple indicator display having a plurality of miniature lamps for a rear light of an automotive vehicle comprising a number of compartments each of which is associated with a particular function such braking, fog lamp operation, direction indication, etc.
U.S. Pat. No. 4,928,084 to Steven M. Reiser discloses a vehicle brake light and display system utilizing a display screen having an array of light emitting diodes arranged to transmit a selected pattern or message in red light. Upon actuation of the brake of the vehicle in which the display screen is mounted, display of the message is disabled and all of the light emitting elements are energized to provide a solid display of red over the entire screen.
Finally, U.S. Pat. Nos. 5,175,528 and 5,313,188 to Robert S. Choi, et al. disclose battery powered flashing super luminescent light emitting safety warning lights for alerting drivers to the presence of bicyclists, joggers, and pedestrians along roadways during darkness. This system is not intended for vehicular use.

Motorcycle brake light enhancement system


The present invention relates to 16-membered macrocyclic compounds. In one aspect of the present invention, compounds of the formula

Epothilone A (Rxe2x95x90H) and Epothilone B (Rxe2x95x90CH3) are produced by Sorangium cellulosum strain So ce 90, the structures of which are shown below, and were the first of several epothilones to be isolated and characterized. Hofle et al., 1996, Angew. Chem. Int. Ed. Engl. 35(13/14): 1567-1569.
Epothilone A and epothilone B possess many of the advantageous properties of taxol. As a result, there is significant interest in these and structurally related compounds as potential chemotherapeutic agents. The desoxy counterparts of epothilones A and B are known as epothilone C (Rxe2x95x90H) and epothilone D (Rxe2x95x90CH3), and also exhibit similar anti-tumor activity but with less cytotoxicity. The structures of epothilones C and D are shown below.
Although other naturally occurring epothilones have been described in the literature, these compounds are produced in exceedingly small amounts. For example, PCT publication WO 99/65913 describes 39 naturally occurring epothilones obtained from Sorangium cellulosum So ce 90 of which epothilones A, B, C, and D together account for approximately 98.9% of the total epothilones produced. The 35 other naturally occurring epothilone compounds together account for the remaining 1.1% and include epothilone C6 (which may also be referred to as 10,11-dehydroepothilone C) and whose structure is shown below
Due to the increasing interest in epothilones as anti-cancer agents, novel derivatives of these compounds are needed and desired to more fully develop their therapeutic potential.
The present invention relates to 16-membered macrocyclic compounds. In one aspect of the present invention, 16-membered macrocyclic compounds having a conjugated diene are provided. In another aspect of the present invention, compounds of the following formula
are provided wherein:
R1, R2, R3, and R5 are each independently hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl or alkylaryl;
R4 is hydrogen, C1-C10 alkyl, C1-C10 hydroxyalkyl, C1-C10 haloalkyl, aryl, xe2x80x94C(xe2x95x90O)R6, xe2x80x94C(xe2x95x90O)OR6, xe2x80x94NR6R7 where R6 and R7 are each independently hydrogen, C1-C10 aliphatic, aryl or alkylaryl;
W is O, NR8 where R8 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl or alkylaryl;
X is O, CH2 or a carbon-carbon double bond;
Y is absent or a C1-C10 alkyl, C2-C10 alkenyl, or C2-C10 alkynyl; and
Ar is aryl; provided that 10,11-dehydroepothilone C is excluded. In another aspect of the present invention, methods for using the inventive compounds are provided.
The present invention relates to novel compounds that are useful for the treatment of cancer and other conditions characterized by abnormal cellular proliferation in a subject in need thereof.
Definitions
Statements regarding the scope of the present invention and definitions of terms used herein are listed below. The definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
All stereoisomers of the inventive compounds are included within the scope of the invention, as pure compounds as well as mixtures thereof. Individual enantiomers, diastereomers, geometric isomers, and combinations and mixtures thereof are all encompassed by the present invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also encompassed within the scope of this invention.
Protected forms of the inventive compounds are included within the scope of the present invention. A variety of protecting groups are disclosed, for example, in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley and Sons, New York (1999), which is incorporated herein by reference in its entirety. For example, a hydroxy protected form of the inventive compounds are those where at least one of the hydroxyl groups is protected by a hydroxy protecting group. Illustrative hydroxyl protecting groups include but not limited to tetrahydropyranyl; benzyl; methylthiomethyl; ethylthiomethyl; pivaloyl; phenylsulfonyl; triphenylmethyl; trisubstituted silyl such as trimethyl silyl, triethylsilyl, tributylsilyl, tri-isopropylsilyl, t-butyldimethylsilyl, tri-t-butylsilyl, methyldiphenylsilyl, ethyldiphenylsilyl, t-butyldiphenylsilyl and the like; acyl and aroyl such as acetyl, pivaloylbenzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryl and the like. Keto groups in the inventive compounds may similarly be protected.
The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs are functional derivatives of the compounds that are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term xe2x80x9cadministeringxe2x80x9d shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to a subject in need thereof. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d, H. Bundgaard ed., Elsevier, 1985.
As used herein, the term xe2x80x9caliphaticxe2x80x9d refers to saturated and unsaturated straight chained, branched chain, cyclic, or polycyclic hydrocarbons that may be optionally substituted at one or more positions. Illustrative examples of aliphatic groups include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. The term xe2x80x9calkylxe2x80x9d refers to straight or branched chain saturated hydrocarbon substituent. xe2x80x9cAlkenylxe2x80x9d refers to a straight or branched chain hydrocarbon substituent with at least one carbon-carbon double bond. xe2x80x9cAlkynylxe2x80x9d refers to a straight or branched chain hydrocarbon substituent with at least one carbon-carbon triple bound.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or polycyclic groups having at least one aromatic ring structure that optionally include one or more heteroatoms and preferably include three to fourteen carbon atoms. Aryl substituents may optionally be substituted at one or more positions. Illustrative examples of aryl groups include but are not limited to: furanyl, imidazolyl, indanyl, indenyl, indolyl, isooxazolyl, isoquinolinyl, naphthyl, oxazolyl, oxadiazolyl, phenyl, pyrazinyl, pyridyl, pyrimidinyl, pyrrolyl, pyrazolyl, quinolyl, quinoxalyl, tetrahydronaphththyl, tetrazolyl, thiazolyl, thienyl, and the like.
The aliphatic (i.e., alkyl, alkenyl, etc.) and aryl moieties may be optionally substituted with one or more substituents, preferably from one to five substituents, more preferably from one to three substituents, and most preferably from one to two substituents. The definition of any substituent or variable at a particular location in a molecule is independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein. Examples of suitable substituents include but are not limited to: alkyl, alkenyl, alkynyl, aryl, halo; trifluoromethyl; trifluoromethoxy; hydroxy; alkoxy; cycloalkoxy; heterocyclooxy; oxo; alkanoyl (xe2x80x94C(xe2x95x90O)-alkyl which is also referred to as xe2x80x9cacylxe2x80x9d)); aryloxy; alkanoyloxy; amino; alkylamino; arylamino; aralkylamino; cycloalkylamino; heterocycloamino; disubstituted amines in which the two amino substituents are selected from alkyl, aryl, or aralkyl; alkanoylamino; aroylamino; aralkanoylamino; substituted alkanoylamino; substituted arylamino; substituted aralkanoylamino; thiol; alkylthio; arylthio; aralkylthio; cycloalkylthio; heterocyclothio; alkylthiono; arylthiono; aralkylthiono; alkylsulfonyl; arylsulfonyl; aralkylsulfonyl; sulfonamido (e.g., SO2NH2); substituted sulfonamido; nitro; cyano; carboxy; carbamyl (e.g., CONH2); substituted carbamyl (e.g., xe2x80x94C(xe2x95x90O)NRRxe2x80x2 where R and Rxe2x80x2 are each independently hydrogen, alkyl, aryl, aralkyl and the like); alkoxycarbonyl, aryl, substituted aryl, guanidino, and heterocyclo such as indoyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where applicable, the substituent may be further substituted such as with, alkyl, alkoxy, aryl, aralkyl, halogen, hydroxy and the like.
The terms xe2x80x9calkylarylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d refer to an aryl group with an aliphatic substituent that is bonded to the compound through the aliphatic group. An illustrative example of an alkylaryl or arylalkyl group is benzyl, a phenyl with a methyl group that is bonded to the compound through the methyl group (xe2x80x94CH2Ph where Ph is phenyl).
The term xe2x80x9cacylxe2x80x9d refers to xe2x80x94C(xe2x95x90O)R where R is an aliphatic group, preferably a C1-C6 moiety.
The term xe2x80x9calkoxyxe2x80x9d refers to xe2x80x94OR wherein O is oxygen and R is an aliphatic group.
The term xe2x80x9caminoalkylxe2x80x9d refers to xe2x80x94RNH2 where R is an aliphatic moiety.
The terms xe2x80x9chalogen,xe2x80x9d xe2x80x9chaloxe2x80x9d, or xe2x80x9chalidexe2x80x9d refer to fluorine, chlorine, bromine and iodine.
The term xe2x80x9chaloalkylxe2x80x9d refers to xe2x80x94RX where R is an aliphatic moiety and X is one or more halogens.
The term xe2x80x9chydroxyalkylxe2x80x9d refers to xe2x80x94ROH where R is an aliphatic moiety.
The term xe2x80x9coxoxe2x80x9d refers to a carbonyl oxygen (xe2x95x90O).
In addition to the explicit substitutions at the above-described groups, the inventive compounds may include other substitutions where applicable. For example, the lactone or lactam backbone or backbone substituents may be additionally substituted (e.g., by replacing one of the hydrogens or by derivatizing a non-hydrogen group) with one or more substituents such as C1-C5 aliphatic, C1-C5 alkoxy, aryl, or a functional group. Illustrative examples of suitable functional groups include but are not limited to: acetal, alcohol, aldehyde, amide, amine, boronate, carbamate, carboalkoxy, carbonate, a carbodiimide, carboxylic acid, cyanohydrin, disulfide, enamine, ester, ether, halogen, hydrazide, hydrazone, imide, imido, imine, isocyanate, ketal, ketone, nitro, oxime, phosphine, phosphonate, phosphonic acid, quaternary ammonium, sulfenyl, sulfide, sulfone, sulfonic acid, thiol, and the like.
The term xe2x80x9cpurifiedxe2x80x9d as used herein to refer to a compound of the present invention, means that the compound is in a preparation in which the compound forms a major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more by weight of the components in the composition.
The term xe2x80x9csubjectxe2x80x9d as used herein, refers to an animal, preferably a mammal, who has been the object of treatment, observation or experiment and most preferably a human who has been the object of treatment and/or observation.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
The term xe2x80x9ccompositionxe2x80x9d is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is a salt of one or more of the inventive compounds. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include but are not limited to:
acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like.
The term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is a medium that is used to prepare a desired dosage form of the inventive compound. A pharmaceutically acceptable carrier includes solvents, diluents, or other liquid vehicle; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents, preservatives; solid binders; lubricants and the like. Remington""s Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe, ed. (Amer. Pharmaceutical Assoc. 2000), both of which are incorporated herein by reference in their entireties, disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
The term xe2x80x9cpharmaceutically acceptable esterxe2x80x9d is an ester that hydrolzyes in vivo into a compound of the present invention or a salt thereof. Illustrative examples of suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids such as formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.
Compounds of the Present Invention
In one aspect of the present invention, the compounds are a 16 membered macrocycle having a conjugated diene. The macrocycle may be a cyclic ester or lactone, a cyclic amide or lactam, or a cyclic thioester.
In another aspect of the present invention, compounds of the following formula
are provided wherein:
R1, R2, R3, and R5 are each independently hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl or alkylaryl;
R4 is hydrogen, halogen, C1-C10 alkyl, C1-C10 hydroxyalkyl, C1-C10 haloalkyl, aryl, xe2x80x94C(xe2x95x90O)R6, xe2x80x94C(xe2x95x90O)OR6, xe2x80x94NR6R7 where R6 and R7 are each independently hydrogen, C1-C10 aliphatic, aryl or alkylaryl;
W is O, NR8 where R8 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl or alkylaryl;
X is O, CH2 or a carbon-carbon double bond;
Y is absent or a C1-C10 alkyl, C2-C10 alkenyl, or C2-C10 alkynyl; and
Ar is aryl; provided that 10,11-dehydroepothilone C is excluded.
In one embodiment, compounds of formula I are provided wherein:
R1, R2, R3, and R5 are each independently hydrogen or C1-C5 alkyl;
R4 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 haloalkyl, xe2x80x94C(xe2x95x90O)R6, xe2x80x94C(xe2x95x90O)OR6, xe2x80x94NR6R7 where R6 and R7 are each independently hydrogen or C1-C5 alkyl;
W is O or NR8 where R8 is hydrogen or C1-C5 alkyl;
X is O, CH2 or a carbon-carbon double bond;
Y is absent or C2-C5 alkenyl; and
Ar is a heteroaryl.
In another embodiment, compound of formula I are provided wherein:
R1, R2, R3, and R5 are each independently hydrogen, methyl or ethyl;
R4 is hydrogen, methyl, ethyl, hydroxymethyl, hydroxyethyl, fluoromethyl or fluoroethyl;
W is O or NH;
X is O, CH2 or a carbon-carbon double bond;
Y is absent or C2-C5 alkenyl; and
Ar is an aryl selected from the group consisting of phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, quinolyl, isoquinolyl, quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl, benzoimidazolyl, and benzopyrazolyl.
In another embodiment, compound of formula I are provided wherein:
R1, R2, R3, and R5 are each independently hydrogen or methyl;
R4 is hydrogen, methyl, ethyl, or fluoromethyl;
W is O or NH;
X is O, CH2 or a carbon-carbon double bond;
Y is absent or C2-C3 alkenyl; and
Ar is an aryl selected from the group consisting of thiazolyl, oxazolyl, quinolyl, isoquinolyl, quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl, benzoimidazolyl, and benzopyrazolyl.
In another aspect of the present invention, compounds of the following formula
are provided wherein:
W is O or NR8 where R8 is hydrogen or C1-C5 alkyl;
X is O, CH2 or a carbon-carbon double bond;
R4 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 haloalkyl, xe2x80x94C(xe2x95x90O)R6, xe2x80x94C(xe2x95x90O)OR6, xe2x80x94NR6R7 where R6 and R7 are each independently hydrogen or C1-C5 alkyl; and,
R9 is aryl or R11CHxe2x95x90C(R10)xe2x80x94 wherein R11 is aryl and R10 is hydrogen, halide, C1-C5 alkyl, C1-C5 hydroxylalkyl, or C1-C5 haloalkyl.
In another embodiment, compounds of formula II are provided wherein:
W is O or NH; and,
X is CH2 or a carbon-carbon double bond;
R4 is hydrogen, methyl, ethyl, hydroxymethyl, hydroxyethyl, fluoromethyl or fluoroethyl; and,
R9 is a bicyclic heteroaryl or R11CHxe2x95x90C(R10)xe2x80x94 wherein R11 is 2-methyl-1,3-thiazolinyl, 2-methyl-13, oxazolinyl, 2-hydroxymethyl-1,3-thiazolinyl, or 2-hydroxymethyl-1,3-oxazolinyl and R10 is hydrogen or methyl.
In another embodiment, compounds of formula II are provided wherein:
W is O or NH; and,
X is CH2 or a carbon-carbon double bond;
R4 is hydrogen, methyl, ethyl, hydroxymethyl, hydroxyethyl, fluoromethyl or fluoroethyl; and,
R9 is selected from the group consisting of
In another aspect of the present invention, compounds of the following formulas
are provided wherein:
R1, R2, R3, and R5 are each independently hydrogen or C1-C5 alkyl; and,
R4 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 haloalkyl, xe2x80x94C(xe2x95x90O)R6, xe2x80x94C(xe2x95x90O)OR6, xe2x80x94NR6R7 where R6 and R7 are each independently hydrogen or C1-C5 alkyl;
R12 is hydrogen, halide, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 haloalkyl, xe2x80x94(CH2)mC(xe2x95x90O)R6, xe2x80x94(CH2)mC(xe2x95x90O)OR6, xe2x80x94(CH2)mNR6R7 where m is 0, 1 or 2, and R6 and R7 are each independently hydrogen or C1-C5 alkyl; provided that 10,11-dehydroepothilone C is excluded.
In one embodiment, compounds are of formulas III or IV wherein
R1, R2, R3, and R5 are each independently hydrogen, methyl or ethyl;
R4 is methyl, ethyl, hydroxymethyl, fluoromethyl, hydroxyethyl, or fluoroethyl; and,
R12 is hydrogen, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 haloalkyl, xe2x80x94(CH2)mC(xe2x95x90O)R6, xe2x80x94(CH2)mC(xe2x95x90O)OR6, xe2x80x94(CH2)mNR6R7 where m is 0 or 1, and R6 and R7 are each independently hydrogen or methyl.
In another embodiment, compounds are of formulas III or IV wherein
R1, R3, and R5 are each hydrogen;
R2 is methyl;
R4 is methyl, ethyl, or fluoromethyl; and,
R12 is methyl, hydroxymethyl, fluoromethyl, and aminomethyl.
In another aspect of the present invention, the following compounds are provided:
The compounds of the present invention are cytotoxic agents and may be used in any suitable manner including but not limited to as anti-cancer agents. An illustrative assay for assessing the degree of cytotoxicy and tubulin polyermization is described in Example 1.
Synthetic Methods
General principles of organic chemistry including functional moieties and reactivity and common protocols are described by for example in Advanced Organic Chemistry 3rd Ed. by Jerry March (1985) which is incorporated herein by reference in its entirety. In addition, it will be appreciated by one of ordinary skill in the art that the synthetic methods described herein may use a variety of protecting groups whether or not they are explicitly described. A xe2x80x9cprotecting groupxe2x80x9d as used herein means a moiety used to block functional moiety such as oxygen, sulfur, or nitrogen so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. General principles including specific functional groups and their uses are described for example in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons, New York (1999).
In one aspect of the present invention, two fragments are coupled together and subsequently cyclized to form 16 membered macrocycles of the present invention. In one embodiment, fragments A and B are joined together under Heck coupling conditions (a palladium catalyst such as (diphenylphosphineferrocenyl)dichloropalladium; a base such as cesium carbonate or 9-borabicyclo[3.3.1]nonane; and triphenylarsine). In another embodiment, fragments A and Bxe2x80x2 (an alkyne form of fragment B) are joined together under Suzuki coupling reaction conditions. Fragment Bxe2x80x2 is treated with a borane such as catechol borane or 9-borabicyclo[3.3.1]nonane, and then fragments Bxe2x80x2 and A, a palladium catalyst such as (diphenylphosphineferrocenyl)dichloropalladium, a base such as cesium carbonate, and triphenylarsine are reacted together. Both methods are illustrated schematically by Scheme 1 where Ar, W, Y, R1, R2, R3, R4, R5 are as described previously and Z is a protected hydroxy group or a protected amino group that is capable of becoming W upon deprotection and cyclization.
Fragment A is made using a number of methods. Scheme 2A illustrates one embodiment where R5 is hydrogen and Z is a protected hydroxy group and Arxe2x80x94Y, and R4 are as described previously.
Briefly, (2R)-N-acetyl-2,10-camphorsultam is treated with a dialkylboron triflate such as diethylborontriflate and a base such as diisopropyl ethylamine and then reacted with Arxe2x80x94Yxe2x80x94CHO in an Oppolzer aldol condensation. The resulting alcohol is protected by reacting the compound with triethylsilyl triflate and lutidine and reduced to form the aldehyde. Fragment A is formed by extending the aldehyde in a Wittig reaction by treating the aldehyde with a phosphonium salt such as iodoethyltriphenylphosphonium iodide prepared in situ from ethyltriphenylphosphonium iodide.
Scheme 2B illustrates another embodiment where Arxe2x80x94Y, R4 and R5 are as described previously. This method is preferred where R5 is a non-hydrogen moiety.
Aldehyde Arxe2x80x94Yxe2x80x94CHO is treated with diisopinocampheyl-allylborane in a Brown asymmetric allylation. The resulting alcohol is protected with triethylsilyl triflate and lutidine and the alkene is oxidized to an aldehyde. The aldehyde is extended as in Scheme 2A in a Wittig reaction with a phosphonium salt such as iodoethyltriphenylphosphonium iodide prepared in situ from ethyltriphenylphosphonium iodide.
Scheme 3 illustrates another embodiment where Z is a protected amino group and Arxe2x80x94Y, R4 and R5 are as described previously.
Briefly, the hydroxy protected form of fragment A of the opposite configuration as that described in Schemes 2A and 2B is deprotected and treated with diphenylphosphoryl azide and diazabicycloundecene to form the azido-version of fragment A.
Fragment B where R1 is hydrogen is prepared as described by Scheme 4A.
A 1,1-diisopropoxy-2,2-dimethyl-3-alkanone is extended in an aldol condensation reaction. The resulting hydroxyl group is protected with trichloroethoxycarbonyl chloride, and the acetal protecting group is removed to yield a C3-C11 Intermediate of fragment B. This intermediate aldehyde is extended in another aldol reaction and protected to yield fragment B.
Fragment Bxe2x80x2 (where R3 is hydrogen) and where R1 is hydrogen is prepared as described by Scheme 4B.
The method is similar to that described by Scheme 4A except that the first aldol reaction is performed using the alkynal
instead of the alkenal
Fragments B and Bxe2x80x2 where R1 is a non-hydrogen is prepared as described by Scheme 5.
The alkene and alkyne versions of the C3-C11 Intermediate are prepared as described by Schemes 4A and 4B respectively. The C3-C11 Intermediate is treated with N-propionyl-benzyloxazolidinone in an Evans aldol reaction. The resulting alcohol is protected with the silyl group and then benzyloxazlidinone is hydrolyzed. Esterification with with tert-butanol yields fragment B.
Fragments A and B can be joined together in a Heck coupling reaction to form a diene as shown by Scheme 6A.
Alternatively, Fragments A and Bxe2x80x2 can be joined together in a Suzuki coupling reaction to form the same diene as shown by Scheme 6B.
Compounds of the present invention that are cyclic lactones are made from the coupling of fragment A (where Z is a protected hydroxyl group) and fragment B (or Bxe2x80x2) as illustrated by Scheme 7A.
The diene product of either the Heck coupling of fragments A and B or the Suzuki coupling of fragments A and Bxe2x80x2 is subjected to an ester exchange and then partially deprotected. The resulting product is lactonized and deprotected to yield compounds corresponding to formula I where W is O.
Compounds of the present invention that are cyclic lactams are made from the coupling of fragment A (where Z is a protected amino group such as N3) and fragment B (or Bxe2x80x2) as illustrated by Schemes 7B and 7C.
The diene product of either the Heck coupling of fragments A and B or the Suzuki coupling of fragments A and Bxe2x80x2 is subjected to a Staudinger reduction and the resulting amine is protected. The resulting product is treated with trifluoroacetic acid and then cyclized to form the cyclic lactam. Deprotection of the Troc protecting group yields compounds corresponding to formula I where W is NH.
Methods for making N-alkyl lactam derivatives are described by Scheme 7C.
The amino-carboxyacid is made as described by Scheme 7B and then subjected to reductive amination. The resulting product is then cyclized and deprotected to yield compounds corresponding to formula I where W is NR8.
In another aspect of the present invention, the inventive compounds are made from modified versions of fragments A and B (designated as Axe2x80x3 and Bxe2x80x3) using Stille coupling.
As shown by Scheme 8, the Stille coupling reaction of fragments Axe2x80x3 and Bxe2x80x3 makes the same intermediate as that produced from the previously described Heck coupling or Suzuki coupling reactions. This intermediate can be cyclized under conditions similar to that described by Schemes 7A, 7B, and 7C to make the compounds of the present invention.
Fragment Axe2x80x3 can be made as described by Scheme 9 by stannylation of fragment A.
Fragment Bxe2x80x3 where R3 is hydrogen is made by can be made starting with fragment Bxe2x80x2.
As shown by Scheme 10A, fragment Bxe2x80x2 is treated with catechol borane in a hydroboration reaction and then iodinated to yield fragment Bxe2x80x3 where R3 is hydrogen.
Fragment Bxe2x80x3 where R3 is a non-hydrogen is also made starting from fragment Bxe2x80x2
As shown by Scheme 10B, fragment Bxe2x80x2 is treated wtih zirconocene dichloride and trialkylaluminum in a Schwartz reaction to yield fragment Bxe2x80x3 where R3 is alkyl.
Biological Methods
In another aspect of the present invention, a subset of the inventive compounds is made using biological methods. In one embodiment, 10,11-dehydroepothilone D, whose structure is shown below,
is isolated from a strain of Myxococcus xanthus, K111-40-1. This strain expresses the epothilone polyketide synthase but not an active epoK gene product and so produces primarily epothilone D and lesser amounts of epothilone C and 10,11-dehydroepothilone D. Strain K111-40-1 (PTA-2712) was deposited with the American Type Culture Collection (xe2x80x9cATCCxe2x80x9d), 10801 University Blvd., Manassas, Va., 20110-2209 USA, on Nov. 21, 2000. In another embodiment, 10,11-dehydroepothilone D may be isolated from M. xanthus strain K111-72-4.4 that expresses the epothilone polyketide synthase and contains an epoK gene with an inactivating in frame deletion. Strain K111-72-4.4 (PTA-2713) also was deposited with the ATCC on Nov. 21, 2000. Methods for fermentation of these strains, purification of 10,11-dehydroepothilone D produced by these strains, and recombinant strains that make 10,11-dehydroepothilone D are described in related application U.S. Ser. No. 09/825,876 filed on Apr. 3, 2001, by inventors Robert Arslanian, John Carney and Brian Metcalf entitled EPOTHILONE COMPOUNDS AND METHODS FOR MAKING AND USING THE SAME.
Recombinant techniques can be used to make subset of the compounds of the present invention. These compounds include those with substituents at the C-2 and/or C-6 and/or C-8 and/or C-10 and/or C-14 positions that differ from the naturally occurring epothilones A-D. Procedures for making these kinds of changes in heterologous hosts such as Myxococcus xanthus, Steptomyces lividans, and Pseudomonas fluorescens are described in U.S. Pat. No. 6,303,342, entitled RECOMBINANT METHODS AND MATERIALS FOR PRODUCING EPOTHILONE AND EPOTHILONE DERIVATIVES, which is incorporated herein by reference. Among other things, the patent provides the nucleotide sequence of the epothilone PKS and modification enzyme genes cloned from Sorangium cellulosum SMP44; cosmids containing overlapping fragments of the epothilone PKS and modification enzyme genes; plasmids having the full complement of epoA, epoB, epoC, epoD, epoE, epoF, epoK, and epoL genes; and heterologous host cells for making epothilones and epothilone derivatives. Cosmids, pKOS35-70.1A2 (ATCC 203782), pKOS35-70.4 (ATCC 203781), pKOS35-70.8A3 (ATCC 203783), and pKOS35-79.85 (ATCC 203780); plasmid pair, pKOS039-124R (PTA-926) and pKOS039-126R (PTA-927); and strain K111-32.25 (PTA-1700) derived from Myxococcus xanthus containing all the epothilone genes and their promoters, have been deposited with the ATCC on April 14, 2000. Additional procedures for making epothilones in Myxococcus are described in: U.S. Ser. No. 09/560,367 filed Apr. 28, 2000; No. 60/232,696, filed Sep. 14, 2000, now lapsed; No. 60/257,517 filed Dec. 21, 2000, now lapsed; and No. 60/269,020, filed Feb. 13, 2001, all of which are entitled PRODUCTION OF POLYKETIDES and are also incorporated herein by reference. Illustrative examples of compounds that may be made using recombinant techniques include: 2-methyl-10,11-dehydroepothilone C or D; 6-desmethyl-10,11-dehydroepothilone C or D; 8-desmethyl-10,11-dehydroepothilone C or D; 10-methyl-10,11-dehydroepothilone C or D; and 14-methyl-10,11-dehydroepothilone C or D.
In another aspect of the present invention, biologically derived strategies are used to modify certain compounds of the present invention regardless of whether the compounds are made biologically or by de novo chemical synthesis. In one embodiment, a microbially-derived hydroxylase is used to hydroxylate a terminal alkane, particularly an alkyl substituent of the thiazole moiety of the inventive compounds. Protocols for effectuating such a transformation are described for example by PCT Publication No. WO 00/39276 which is incorporated herein in its entirety by reference. Example 26 describes in greater detail the hydroxylation of the C-20 methyl of 10,11-dehydroepothilone D to 21-hydroxy-10,11-dehydroepothilone D. This general method can be readily adapted for making corresponding 21-hydroxy derivatives from other compounds of the invention.
In another embodiment, Epo K, a P450 epoxidase that performs the epoxidation reaction in host cells that naturally produce epothilones or another epoxylase may be used to make 12,13-epoxy versions of the compounds of the present invention. A general method for using EpoK for epoxidation is described by Example 5 of PCT publication WO 00/31247 which is incorporated herein by reference. Example 27 describes in greater detail the epoxidation of 10,11-dehydroepothilone D to 10,11-dehydroepothilone B, the general method which can be readily adapted for making corresponding 12,13-epoxide derivatives from other compounds of the invention.
Alternatively, the epoxidation reaction can occur by contacting an epothilone compound containing a double bond at a position that corresponds to the bond between carbon-12 and carbon 13 to a culture of cells that expresses a functional Epo K. Such cells include the myxobacterium Sorangium cellulosum. In particularly preferred embodiments, the Sorangium cellulosum expresses Epo K but does not contain a functional epothilone polyketide synthase (xe2x80x9cPKSxe2x80x9d) gene. Such strains may be made by mutagenesis where one or more mutations in the epothilone PKS gene render it inoperative. Such mutants can occur naturally (which may be found by screening) or can be directed using either mutagens such as chemicals or irradation or by genetic manipulation. A particularly effective strategy for making strains with an inoperative epothilone PKS is homologous recombination as described by PCT publication WO 00/31247.
Formulation
A composition of the present invention generally comprises an inventive compound and a pharmaceutically acceptable carrier. The inventive compound may be free form or where appropriate as pharmaceutically acceptable derivatives such as prodrugs, and salts and esters of the inventive compound.
The composition may be in any suitable form such as solid, semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th edition, Lippicott Williams and Wilkins (1991) which is incorporated herein by reference. In general, the pharmaceutical preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use. The carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
In one embodiment, the compositions containing an inventive compound are Cremophor(copyright)-free. Cremophor(copyright) (BASF Aktiengesellschaft) is a polyethoxylated castor oil which is typically used as a surfactant in formulating low soluble drugs. However, because Cremophor(copyright) can case allergic reactions in a subject, compositions that minimize or eliminate Cremophor(copyright) are preferred. Formulations of epothilone A or B that eliminate Cremophor(copyright) are described for example by PCT Publication WO 99/39694 which is incorporated herein by reference and may be adapted for use with the inventive compounds.
Where applicable, the inventive compounds may be formulated as microcapsules and nanoparticles. General protocols are described for example, by Microcapsules and Nanoparticles in Medicine and Pharmacy by Max Donbrow, ed., CRC Press (1992) and by U.S. Pat. Nos. 5,510,118; 5,534,270; and 5,662,883 which are all incorporated herein by reference. By increasing the ratio of surface area to volume, these formulations allow for the oral delivery of compounds that would not otherwise be amenable to oral delivery.
The inventive compounds may also be formulated using other methods that have been previously used for low solubility drugs. For example, the compounds may form emulsions with vitamin E or a PEGylated derivative thereof as described by WO 98/30205 and 00/71163which are incorporated herein by reference. Typically, the inventive compound is dissolved in an aqueous solution containing ethanol (preferably less than 1% w/v). Vitamin E or a PEGylated-vitamin E is added. The ethanol is then removed to form a pre-emulsion that can be formulated for intravenous or oral routes of administration. Another strategy involves encapsulating the inventive compounds in liposomes. Methods for forming liposomes as drug delivery vehicles are well known in the art. Suitable protocols include those described by U.S. Pat. Nos. 5,683,715; 5,415,869, and 5,424,073 which are incorporated herein by reference relating to another relatively low solubility cancer drug taxol and by PCT Publication WO 01/10412 which is incorporated herein by reference relating to epothilone B. Of the various lipids that may be used, particularly preferred lipids for making epothilone-encapsulated liposomes include phosphatidylcholine and polyethyleneglycol-derivitized distearyl phosphatidylethanolamine. Example 28 provides an illustrative protocol for making liposomes containing 10,11-dehydroepothilone D, the general method which can be readily adapted to make liposomes containing other compounds of the present invention.
Yet another method involves formulating the inventive compounds using polymers such as polymers such as biopolymers or biocompatible (synthetic or naturally occurring) polymers. Biocompatible polymers can be categorized as biodegradable and non-biodegradable. Biodegradable polymers degrade in vivo as a function of chemical composition, method of manufacture, and implant structure. Illustrative examples of synthetic polymers include polyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolic acids and copolymers thereof, polyesters polyamides polyorthoesters and some polyphosphazenes. Illustrative examples of naturally occurring polymers include proteins and polysaccharides such as collagen, hyaluronic acid, albumin, and gelatin.
Another method involves conjugating the compounds of the present invention to a polymer that enhances aqueous solubility. Examples of suitable polymers include polyethylene glycol, poly-(d-glutamic acid), poly-(1-glutamic acid), poly-(1-glutamic acid), poly-(d-aspartic acid), poly-(1-aspartic acid), poly-(1-aspartic acid) and copolymers thereof. Polyglutamic acids having molecular weights between about 5,000 to about 100,000 are preferred, with molecular weights between about 20,000 and 80,000 being more preferred and with molecular weights between about 30,000 and 60,000 being most preferred. The polymer is conjugated via an ester linkage to one or more hydroxyls of an inventive epothilone using a protocol as essentially described by U.S. Pat. No. 5,977,163 which is incorporated herein by reference, and by Example 29. Preferred conjugation sites include the hydroxyl off carbon-21 in the case of 21-hydroxy-derivatives of the present invention. Other conjugation sites include the hydroxyl off carbon 3 and the hydroxyl off carbon 7.
In another method, the inventive compounds are conjugated to a monoclonal antibody. This strategy allows the targeting of the inventive compounds to specific targets. General protocols for the design and use of conjugated antibodies are described in Monoclonal Antibody-Based Therapy of Cancer by Michael L. Grossbard, ed. (1998) which is incorporated herein by reference.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. For example, a formulation for intravenous use comprises an amount of the inventive compound ranging from about 1 mg/mL to about 25 mg/mL, preferably from about 5 mg/mL to 15 mg/mL, and more preferably about 10 mg/mL. Intravenous formulations are typically diluted between about 2 fold and about 30 fold with normal saline or 5% dextrose solution prior to use.
Methods to Treat Cancer
In one aspect of the present invention, the inventive compounds are used to treat cancer. In one embodiment, the compounds of the present invention are used to treat cancers of the head and neck which include tumors of the head, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas. In another embodiment, the compounds of the present invention are used to treat cancers of the liver and biliary tree, particularly hepatocellular carcinoma. In another embodiment, the compounds of the present invention are used to treat intestinal cancers, particularly colorectal cancer. In another embodiment, the compounds of the present invention are used to treat ovarian cancer. In another embodiment, the compounds of the present invention are used to treat small cell and non-small cell lung cancer. In another embodiment, the compounds of the present invention are used to treat breast cancer. In another embodiment, the compounds of the present invention are used to treat sarcomas which includes fibrosarcoma, malignant fibrous histiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma, neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part sarcoma. In another embodiment, the compounds of the present invention are used to treat neoplasms of the central nervous systems, particularly brain cancer. In another embodiment, the compounds of the present invention are used to treat lymphomas which include Hodgkin""s lymphoma, lymphoplasmacytoid lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, B-lineage large cell lymphoma, Burkitt""s lymphoma, and T-cell anaplastic large cell lymphoma.
The method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from cancer. The method may be repeated as necessary either to contain (i.e. prevent further growth) or to eliminate the cancer. Clinically, practice of the method will result in a reduction in the size or number of the cancerous growth and/or a reduction in associated symptoms (where applicable). Pathologically, practice of the method will produce at least one of the following: inhibition of cancer cell proliferation, reduction in the size of the cancer or tumor, prevention of further metastasis, and inhibition of tumor angiogenesis.
The compounds and compositions of the present invention can be used in combination therapies. In other words, the inventive compounds and compositions can be administered concurrently with, prior to, or subsequent to one or more other desired therapeutic or medical procedures. The particular combination of therapies and procedures in the combination regimen will take into account compatibility of the therapies and/or procedures and the desired therapeutic effect to be achieved.
In one embodiment, the compounds and compositions of the present invention are used in combination with another anti-cancer agent or procedure. Illustrative examples of other anti-cancer agents include but are not limited to: (i) alkylating drugs such as mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide; (ii) antimetabolites such as methotrexate; (iii) microtubule stabilizing agents such as vinblastin, paclitaxel, docetaxel, and discodermolide; (iv) angiogenesis inhibitors; (v) and cytotoxic antibiotics such as doxorubicon (adriamycin), bleomycin, and mitomycin. Illustrative examples of other anti-cancer procedures include: (i) surgery; (ii) radiotherapy; and (iii) photodynamic therapy.
In another embodiment, the compounds and compositions of the present invention are used in combination with an agent or procedure to mitigate potential side effects from the inventive compound or composition such as diarrhea, nausea and vomiting. Diarrhea may be treated with antidiarrheal agents such as opioids (e.g. codeine, diphenoxylate, difenoxin, and loeramide), bismuth subsalicylate, and octreotide. Nausea and vomiting may be treated with antiemetic agents such as dexamethasone, metoclopramide, diphenyhydramine, lorazepam, ondansetron, prochlorperazine, thiethylperazine, and dronabinol. For those compositions that includes polyethoxylated castor oil such as Cremophorg(copyright), pretreatment with corticosteroids such as dexamethasone and methylprednisolone and/or H1 antagonists such as diphenylhydramine HCl and/or H2 antagonists may be used to mitigate anaphylaxis. Illustrative formulations for intravenous use and pretreatment regiments are described by Examples 30 and 31 respectively.
Methods of Treating of Non-cancer, Cellular Hyperproliferative Disorders
In another aspect of the present invention, the inventive compounds are used to treat non-cancer disorders that are characterized by cellular hyperproliferation. In one embodiment, the compounds of the present invention are used to treat psoriasis, a condition characterized by the cellular hyperproliferation of keratinocytes which builds up on the skin to form elevated, scaly lesions. The method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from psoriasis. The method may be repeated as necessary either to decrease the number or severity of lesions or to eliminate the lesions. Clinically, practice of the method will result in a reduction in the size or number of skin lesions, diminution of cutaneous symptoms (pain, burning and bleeding of the affected skin) and/or a reduction in associated symptoms (e.g., joint redness, heat, swelling, diarrhea. abdominal pain, Pathologically, practice of the method will result in at least one of the following: inhibition of keratinocyte proliferation, reduction of skin inflammation (for example, by impacting on: attraction and growth factors, antigen presentation, production of reactive oxygen species and matrix metalloproteinases), and inhibition of dermal angiogenesis.
In another embodiment, the compounds of the present invention are used to treat multiple sclerosis, a condition characterized by progressive demyelination in the brain. Although the exact mechanisms involved in the loss of myelin are not understood, there is an increase in astrocyte proliferation and accumulation in the areas of myelin destruction. At these sites, there is macrophage-like activity and increased protease activity which is at least partially responsible for degradation of the myelin sheath. The method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from multiple sclerosis. The method may be repeated as necessary to inhibit astrocyte proliferation and/or lessen the severity of the loss of motor function and/or prevent or attenuate chronic progression of the disease. Clinically, practice of the method will result in in improvement in visual symptoms (visual loss, diplopia), gait disorders (weakness, axial instability, sensory loss, spasticity, hyperreflexia, loss of dexterity), upper extremity dysfunction (weakness, spasticity, sensory loss), bladder dysfunction (urgency, incontinence, hesitancy, incomplete emptying), depression, emotional lability, and cognitive impairment. Pathologically, practice of the method will result in the reduction of one or more of the following, such as myelin loss, breakdown of the blood-brain barrier, perivascular infiltration of mononuclear cells, immunologic abnormalities, gliotic scar formation and astrocyte proliferation, metalloproteinase production, and impaired conduction velocity.
In another embodiment, the compounds of the present invention are used to treat rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that sometimes leads to destruction and ankyiosis of affected joints. Rheumatoid arthritis is characterized by a marked thickening of the synovial membrane which forms villous projections that extend into the joint space, multilayering of the synoviocyte lining (synoviocyte proliferation), infiltration of the synovial membrane with white blood cells (macrophages, lymphocytes, plasma cells, and lymphoid follicles; called an xe2x80x9cinflammatory synovitisxe2x80x9d), and deposition of fibrin with cellular necrosis within the synovium. The tissue formed as a result of this process is called pannus and, eventually the pannus grows to fill the joint space. The pannus develops an extensive network of new blood vessels through the process of angiogenesis that is essential to the evolution of the synovitis. Release of digestive enzymes (matrix metalloproteinases (e.g., collagenase, stromelysin)) and other mediators of the inflammatory process (e.g., hydrogen peroxide, superoxides, lysosomal enzymes, and products of arachadonic acid metabolism) from the cells of the pannus tissue leads to the progressive destruction of the cartilage tissue. The pannus invades the articular cartilage leading to erosions and fragmentation of the cartilage tissue. Eventually there is erosion of the subchondral bone with fibrous ankylosis and ultimately bony ankylosis, of the involved joint.
The method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from rheumatoid arthritis. The method may be repeated as necessary to accomplish to inhibit synoviocyte proliferation and/or lessen the severity of the loss of movement of the affected joints and/or prevent or attenuate chronic progression of the disease. Clinically, practice of the present invention will result in one or more of the following: (i) decrease in the severity of symptoms (pain, swelling and tenderness of affected joints; morning stiffness, weakness, fatigue, anorexia, weight loss); (ii) decrease in the severity of clinical signs of the disease (thickening of the joint capsule, synovial hypertrophy, joint effusion, soft tissue contractures, decreased range of motion, ankylosis and fixed joint deformity); (iii) decrease in the extra-articular manifestations of the disease (rheumatic nodules, vasculitis, pulmonary nodules, interstitial fibrosis, pericarditis, episcleritis, iritis, Felty""s syndrome, osteoporosis); (iv) increase in the frequency and duration of disease remission/symptom-free periods; (v) prevention of fixed impairment and disability; and/or (vi) prevention/attenuation of chronic progression of the disease. Pathologically, practice of the present invention will produce at least one of the following: (i) decrease in the inflammatory response; (ii) disruption of the activity of inflammatory cytokines (such as IL-I, TNFa, FGF, VEGF); (iii) inhibition of synoviocyte proliferation; (iv) inhibition of matrix metalloproteinase activity, and/or (v) inhibition of angiogenesis.
In another embodiment, the compounds of the present invention are used to threat atherosclerosis and/or restenosis, particularly in patients whose blockages may be treated with an endovascular stent. Atheroschlerosis is a chronic vascular injury in which some of the normal vascular smooth muscle cells (xe2x80x9cVSMCxe2x80x9d) in the artery wall, which ordinarily control vascular tone regulating blood flow, change their nature and develop xe2x80x9ccancer-likexe2x80x9d behavior. These VSMC become abnormally proliferative, secreting substances (growth factors, tissue-degradation enzymes and other proteins) which enable them to invade and spread into the inner vessel lining, blocking blood flow and making that vessel abnormally susceptible to being completely blocked by local blood clotting. Restenosis, the recurrence of stenosis or artery stricture after corrective procedures, is an accelerated form of atherosclerosis.
The method comprises coating a therapeutically effective amount of an inventive compound on a stent and delivering the stent to the diseased artery in a subject suffering from atherosclerosis. Methods for coating a stent with a compound are described for example by U.S. Pat. Nos. 6,156,373 and 6,120, 847. Clinically, practice of the present invention will result in one or more of the following: (i) increased arterial blood flow; (ii) decrease in the severity of clinical signs of the disease; (iii) decrease in the rate of restenosis; or (iv) prevention/attenuation of the chronic progression of atherosclerosis. Pathologically, practice of the present invention will produce at least one of the following at the site of stent implanataion: (i) decrease in the inflammatory response, (ii) inhibition of VSMC secretion of matrix metalloproteinases; (iii) inhibition of smooth muscle cell accumulation; and (iv) inhibition of VSMC phenotypic dedifferentiation.
Dosage Levels
In one embodiment, dosage levels that are administered to a subject suffering from cancer or a non-cancer disorder characterized by cellular proliferation are of the order from about 1 mg/m2 to about 200 mg/m2 which may be administered as a bolus (in any suitable route of administration) or a continuous infusion (e.g. 1 hour, 3 hours, 6 hours, 24 hours, 48 hours or 72 hours) every week, every two weeks, or every three weeks as needed. It will be understood, however, that the specific dose level for any particular patient depends on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the condition being treated.
In another embodiment, the dosage levels are from about 10 mg/m2 to about 150 mg/m2, preferably from about 10 to about 75 mg/m2 and more preferably from about 15 mg/m2 to about 50 mg/m2 once every three weeks as needed and as tolerated. In another embodiment, the dosage levels are from about 1 mg to about 150 mg/m2, preferably from about 10 mg/m2 to about 75 mg/m2 and more preferably from about 25 mg/m2 to about 50 mg/m2 once every two weeks as needed and as tolerated. In another embodiment, the dosage levels are from about 1 mg/m2 to about 100 mg/m2, preferably from about 5 mg/m2 to about 50 mg/m2 and more preferably from about 10 mg/m2 to about 25 mg/m2 once every week as needed and as tolerated. In another embodiment, the dosage levels are from about 0.1 to about 25 mg/m2, preferably from about 0.5 to about 15 mg/m2 and more preferably from about 1 mg/m2 to about 10 mg/m2 once daily as needed and tolerated.
A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the present invention and shall not be construed as being a limitation on the scope of the invention or claims.

Epothilone derivatives and methods for making and using the same

Abstract: are provided wherein: