NUTRITIONAL EMULSIONS COMPRISING CALCIUM HMB AND

SOLUBLE PROTEIN

FIELD OF THE DISCLOSURE

[0001 ] The present disclosure relates to nutritional emulsions comprising calcium beta-hydroxy-beta-methylbutyrate (calcium HMB) and soluble protein.

BACKGROUND OF THE DISCLOSURE

[0002] Beta-hydroxy-beta-methylbutyrate (HMB) is a naturally occurring amino acid metabolite that is often formulated into a variety of nutritional products and supplements. HMB is commonly used in such products to help build or maintain healthy muscle mass and strength in selected individuals.

[0003] HMB is a metabolite of the essential amino acid leucine and has been shown to modulate protein turnover and inhibit proteolysis. In most individuals, muscle converts approximately 5% of available leucine to HMB, thus producing about 0.2 to 0.4 grams of HMB per day for a 70 kg male. In studies where various kinds of stress were induced in animals, HMB supplementation increased lean mass. Clinical studies also suggest that HMB has at least two functions in recovery from illness or injury including protection of lean mass from stress-related damage and enhancement of protein synthesis. It has been suggested that HMB may also be useful in enhancing immune function, reducing the incidence or severity of allergy or asthma, reducing total serum cholesterol and low density lipoprotein cholesterol, increasing the aerobic capacity of muscle, and other uses.

[0004] Since HMB is most often used in individuals to support the development and maintenance of healthy muscle mass and strength, many HMB products have been formulated with additional nutrients that may also be helpful in promoting healthy muscle. Some of these HMB products contain additional nutrients such as fat.

carbohydrate, protein, vitamins, minerals and so forth. Calcium HMB is the most

commonly used form of HMB when formulated into oral nutritional products, which products include tablets, capsules, reconstitutable powders, and nutritional liquids and emulsions. Nutritional emulsions are particularly useful in this regard because such emulsions may contain a balance of fat, protein, carbohydrates, vitamins, and minerals that is useful for helping maintain healthy muscle.

[0005] It has no been found, however, that nutritional emulsions containing calcium HMB are often not physically stable over time, that such emulsions are not readily stable with many protein systems, and that protein-containing and or other sediments form in the emulsions during or after formulation, especially when packaged and stored for extended periods of time.

[0006] It has also been discovered that these nutritional emulsions containing calcium HMB develop an undesirably bitter off flavor or after taste after the emulsion has been packaged and stored for extended periods of at least 1-3 months.

[0007] There is therefore a need for nutritional emulsions comprising calcium HMB that remain physically stable during shelf life and or do not develop a bitter flavor or after taste over time.

SUMMARY OF THE DISCLOSURE

[0008] One embodiment of the present disclosure is directed to a nutritional emulsion comprising fat, carbohydrate, protein and calcium HMB, wherein the protein comprises from about 50% to 100% by weight of soluble protein. The soluble protein is preferably selected from sodium caseinate, whey protein concentrate, and combinations thereof. Sodium caseinate is most preferred.

[0009] Another embodiment of the present disclosure is directed to nutritional emulsions comprising fat. carbohydrate, protein, and calcium HMB, wherein the protein comprises from about 50% to 100% by weight of soluble protein and the nutritional emulsion has a weight ratio of soluble protein to calcium HMB of from about 5: 1 to about [0010] Another embodiment of the present disclosure is directed to a packaged nutritional composition comprising a hermetically sealed container and a nutritional emulsion contained therein, the nutritional emulsion comprising fat, carbohydrate, protein and calcium HMB, wherein the protein comprises from about 50% to about 100% by weight of soluble protein and the packaged composition has been stored for between 3 and 24 months at 18°C to 24° C for between 3 and 24 months following formulation and packaging of the packaged composition.

[001 1 ] It has been found that the addition of calcium HMB to nutritional emulsions can result in the development of a bitter flavor or after taste, which typically does not manifest until the product is manufactured, packaged, and stored for a period of at least about 1 to about 3 months. Indeed, it has been found that nutritional emulsions comprising calcium HMB often produce little or no bitter flavor or after taste when consumed within about 1 month, including from about 1 to about 3 months after formulation, but that such bitter flavor or aftertaste surprisingly develops in the packaged product over time.

[0012] It has also been discovered that many nutritional emulsions comprising calcium HMB are physically unstable, often resulting in the collection of excessive protein-containing and or other sediments at the bottom of the emulsion container, thus reducing nutrient availability as well as the effective shelf life of the product.

[0013] It has now also been found that these instability and or flavor issues can be minimized or eliminated by formulating with soluble protein that represents from about 50% to 100% by weight of the total protein in the nutritional emulsion, especially when the nutritional emulsion contains a weight ratio of the soluble protein to the calcium HMB of from about 5: 1 to about 12: 1. Soluble proteins of particular use in this regard include sodium caseinate and or whey protein concentrate, especially sodium caseinate.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0014] The nutritional emulsions of the present disclosure comprise calcium HMB and at least one ingredient, feature, or element to inhibit the development of bitter flavor or after taste and or to improve product stability over shelf life. The essential features of the nutritional emulsions, as well as some of the many optional variations and additions, are described in detail hereafter.

[0015] The term "calcium HMB" as used herein, unless otherwise specified, refers to the calcium salt of beta-hydroxy-beta-methybutyrate (also referred to as beta-dydroxy 1-3 -methyl butyric acid, beta-hydroxy isovaleric acid, or HMB), which is most typically in a monohydrate form. All weights, percentages, and concentrations as used herein to characterize calcium HMB are based on the weight of calcium HMB

monohydrate, unless otherwise specified.

[0016] The term "nutritional emulsion" as used herein, unless otherwise specified, refers to liquid emulsions comprising fat, protein, and carbohydrate which are suitable for oral administration to a human.

[0017] The terms "fat" and "oil" as used herein, unless otherwise specified, are used interchangeably to refer to lipid materials derived or processed from plants or animals. These terms also include synthetic lipid materials so long as such synthetic materials are suitable for oral administration to humans.

[0018] The term "shelf stable" as used herein, unless otherwise specified, refers to a nutritional emulsion that remains commercially stable after being packaged and then stored at 18-24°C for at least 3 months, including from about 6 months to about 24 months, and also including from about 12 months to about 18 months.

[001 ] The term "pH stable" as used herein, unless otherwise specified, means that the pH is resistant or at least more resistant to pH reductions due to a buffering effect of HMB.

[0020] The term "plastic" as used herein, unless otherwise specified, means food grade plastics approved by the U.S. Food and Drug Administration or other suitable regulatory group, some non-limiting examples of which include polyvinyl chlorides, polyethylene terephthalate, high density polyethylene, polypropylenes, polycarbonates, and so forth.

[0021 ] The terms "sterile", "sterilized" and "sterilization" as used herein, unless otherwise specified, refer to the reduction in transmissible agents such as fungi, bacteria, viruses, spore forms, and so forth, in food or on food grade surfaces to the extent necessary to render such foods suitable for human consumption. Sterilization processes may include various techniques involving the application of heat, peroxide or other chemicals, irradiation, high pressure, filtration, or combinations or variations thereof.

[0022] All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore, do not include solvents or byproducts that may be included in commercially available materials, unless otherwise specified.

[0023] All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

[0024] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0025] The various embodiments of the nutritional emulsions of the present disclosure may also be substantially free of any optional or selected essential ingredient or feature described herein, provided that the remaining nutritional emulsion still contains all of the required ingredients or features as described herein. In this context, and unless otherwise specified, the term "substantially free" means that the selected nutritional emulsion contains less than a functional amount of the optional ingredient, typically less than about 0.5%. including less than about 0.1 %. and also including zero percent, by weight of such optional or selected essential ingredient.

[0026] The nutritional emulsions and corresponding manufacturing methods of the present disclosure can comprise, consist of. or consist essentially of the essential elements of the disclosure as described herein, as well as any additional or optional element described herein or otherwise useful in nutritional emulsion formula applications.

Product Form

[0027] The nutritional emulsions of the present disclosure are aqueous emulsions comprising fat, protein, and carbohydrate. These emulsions are flowable or drinkable liquids at from about 1 to about 25°C and are typically in the form of oil-in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.

[0028] The nutritional emulsions may be and typically are shelf-stable. The nutritional emulsions typically contain up to about 95% by weight of water, including from about 50% to about 95%. also including from about 60% to about 90%, and also including from about 70% to about 85%. of water by weight of the nutritional emulsions.

[0029] The nutritional emulsions may be formulated with sufficient kinds and amounts of nutrients so as to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional emulsion for use in individuals afflicted with specific diseases or conditions. These nutritional emulsions may thus have a variety of product densities, but most typically have a density greater than about 1.055 g/mL, including from 1.06 g/ml to 1.12 g/ml, and also including from about 1 .085 g/ml to about 1.10 g/ml.

[0030] The nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise from about 100 to about 500 kcal/240 ml. including from about 150 to about 350 kcal/240 ml, and also including from about 200 to about 320 kcal/240 ml. These nutritional emulsions also comprise calcium HMB as described hereinafter, the amount of which most typically ranges from about 0.4 to about 3.0 g/240 ml. including from about 0.75 to about 2.0 g/240 ml, including about 1.5 g/240 ml.

[0031] The nutritional emulsion may have a pH ranging from about 3.5 to about 8, but are most advantageously in a range of from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2.

[0032] Although the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size ranges from about 100 to about 300 ml, including from about 150 to about 250ml, including from about 190 ml to about 240 ml.

Macronutrients

[0033] The nutritional emulsions comprise fat, protein, and carbohydrate.

Generally, any source of fat, protein, and carbohydrate that is known or otherwise suitable for use in nutritional products may also be suitable for use herein, provided that such macronutrients are also compatible with the essential elements of the nutritional emulsions as defined herein.

[0034] Although total concentrations or amounts of the fat, protein, and carbohydrates may vary depending upon the nutritional needs of the intended user, such concentrations or amounts most typically fall within one of the following embodied ranges, inclusive of any other essential fat. protein, and or carbohydrate ingredients as described herein.

[0035] Carbohydrate concentrations most typically range from about 5% to about 40%. including from about 7% to about 30%, including from about 10% to about 25%, by weight of the nutritional emulsion; fat concentrations most typically range from about 1% to about 30%, including from about 2% to about 15%. and also including from about 4% to about 10%, by weight of the nutritional emulsion; and protein concentrations most typically range from about 0.5% to about 30%. including from about 1 % to about 15%, and also including from about 2% to about 10%, by weight of the nutritional emulsion.

[0036] The level or amount of carbohydrates, fats, and or proteins in the nutritional emulsions may also be characterized in addition to or in the alternative as a percentage of total calories in the nutritional compositions as set forth in the following table.

[0037] Non-limiting examples of suitable fats or sources thereof for use in the nutritional emulsions described herein include coconut oil, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palm olein, canola oil, marine oils, cottonseed oils, and combinations thereof.

[0038] Non-limiting examples of suitable carbohydrates or sources thereof for use in the nutritional emulsions described herein may include maltodextrin, hydrolyzed or modified starch or cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrates, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), and combinations thereof.

[0039] Non-limiting examples of suitable protein or sources thereof for use in the nutritional emulsions include hydrolyzed, partially hydrolyzed or non-hydrolyzed proteins or protein sources, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g.. soy) or combinations thereof. Non-limiting examples of such proteins include milk protein isolates, milk protein concentrates as described herein, casein protein isolates, whey protein, sodium or calcium caseinates, whole cow's milk, partially or completely defatted milk, soy protein isolates, soy protein concentrates, and so forth.

Calcium HMB

[0040] The nutritional emulsions comprise calcium HMB, which means that the emulsions are either formulated with the addition of calcium HMB, most typically as a monohydrate, or are otherwise prepared so as to contain calcium and HMB in the finished product. Any source of HMB is suitable for use herein provided that the finished product contains calcium and HMB, although such a source is preferably calcium HMB and is most typically added as such to the nutritional emulsion during formulation.

[0041] The term "added calcium HMB" as used herein means a calcium salt of HMB, most typically as monohydrate calcium salt of HMB, as the HMB source added to the nutritional emulsion.

[0042] Although calcium HMB monohydrate is the preferred source of HMB for use herein, other suitable sources may include HMB as the free acid, a salt, an anhydrous salt, an ester, a lactone, or other product forms that otherwise provide a bioavailable form of HMB from the nutritional emulsion. Non-limiting examples of suitable salts of HMB for use herein include HMB salts, hydrated or anhydrous, of sodium, potassium, magnesium, chromium, calcium, or other non-toxic salt form. Calcium HMB

monohydrate is preferred and is commercially available from Technical Sourcing

International (TSI) of Salt Lake City, Utah.

[0043] The concentration of calcium HMB in the nutritional emulsions may range up to about 10%, including from about 0.1 % to about 8%, and also including from about 0.2% to about 5.0%, and also including from about 0.3% to about 3%, and also including from about 0.4% to about 1 .5%, by weight of the nutritional emulsion.

Soluble Protein

[0044] The nutritional emulsions of the present disclosure may comprise selected amounts or ratios of soluble protein to improve product stability and minimize the development of bitter flavors and after taste during shelf life.

[0045] The soluble protein may represent from about 50% to 100%, including from 55% to 100%, including from about 60% to about 100%, including from about 60%

to about 85%, including from about 60% to about 80%, and also including from about 65% to about 75%, by weight of the total protein in the nutritional emulsion. The concentration of soluble protein may range from at least about 0.5%. including from about 1% to about 26%, and also including from about 2% to about 15%, also including from about 3% to about 10%, and also including from about 4% to about 8%, by weight of the nutritional emulsion.

[0046] The amount of soluble protein included in the nutritional emulsions may also be characterized as a weight ratio of soluble protein to calcium HMB, wherein the nutritional emulsion includes a weight ratio of soluble protein to calcium HMB of at least about 3.0, including from about 4.0 to about 12.0, also including 6.1 to about 12, also including from about 7.0 to about 1 1.0, and also including from about 8.0 to about 10.0.

[0047] The term "soluble protein" as used herein, unless otherwise specified, refers to those proteins having a solubility of at least about 90% as measured in accordance with a Protein Solubility Measurement Test that includes the following steps: ( 1 ) suspend the protein at 2.00% (w/w) in water; (2) stir vigorously for one hour at 20°C to form a suspension; (3) remove an aliquot of the suspension, and determine protein concentration as total protein; (4) centrifuge the suspension at 31,000 x g and at 20°C for one hour; (5) determine the protein concentration in the supernatant (the soluble protein); and (6) express the soluble protein as a percentage of the total protein.

[0048] Any soluble protein source is suitable for use herein provided that it meets the solubility requirement as defined herein, some non-limiting examples of which include sodium caseinate (>95% solubility as determined by the Protein Solubility Measurement Test), whey protein concentrate (>90% solubility as determined by the Protein Solubility Measurement Test), and combinations thereof. Non-soluble proteins may of course also be included in the nutritional emulsions provided that the remaining soluble protein componenl is represented in accordance with the requirements as set forth herein.

[0049] Soluble protein suitable for use herein may also be characterized by the content of phosphoserine in the protein, wherein the soluble proteins in this context are defined as those proteins having at least about 100 mmoles. including from about 150 to 400 mmoles, including from about 200 to about 350 mmoles, and also including from about 250 to about 350 mmoles, of phosphoserine per kilogram of protein.

[0050] When the soluble protein is defined in terms of phosphoserine content, it has been found that the weight ratio of the soluble protein (with the defined

phosphoserine content) to the calcium HMB may be at least about 3: 1 , including at least about 5: 1 , and also including at least about 7: 1 , and also including from about 9: 1 to about 30: 1. In this context, the proteins having the requisite content of phosphoserine are most typically in the form of monovalent caseinate salts such as sodium caseinate, potassium caseinate, and combinations thereof.

[0051 ] In one embodiment, the soluble protein may also be characterized by a mole ratio of monovalent caseinate phosphoserine to calcium HMB monohydrate of least about 0.2, including from about 0.2 to about 2.0, and also including from about 0.25 to 1.7.

[0052] It should be understood, however, that any phosphoserine-containing protein may be suitable for use herein provided that it has the requisite phosphoserine content and that the phosphoserine used in calculating the ratios are not bound, complexed, or otherwise attached to a polyvalent cation such as calcium or magnesium.

[0053] It should also be noted that alternative definitions as described herein for soluble proteins may include proteins that have little or no phosphoserine content, so that the soluble protein fraction of the compositions may include soluble protein with and/or without phosphoserine. The soluble protein for use herein may therefore be defined by any one or more of the soluble protein characterizations, separately or in combination.

[0054] The phosphoserine moieties within the protein may therefore be available for binding with the calcium released from the calcium HMB so that the above ratios of soluble protein to calcium HMB are the ratio of protein with phosphoserine moities that are unbound, unattached, or otherwise available to bind soluble calcium from the calcium HMB during formulation. It could be, for example, that a mixture of calcium caseinate and sodium caseinate are used in the composition, but the ratio of proteins

defined by a phosphoserine content to calcium HMB is calculated based on the protein fraction from the sodium caseinate and additionally any protein from the calcium caseinate fraction that is not bound to calcium.

Soluble Calcium Binding Capacity

[0055] The nutritional emulsions may comprise a selected weight ratio of a soluble calcium binding capacity (SCBC) to the total soluble calcium in the emulsion to improve product stability and minimize the development over time of bitter flavors and after taste.

[0056] The ratio of the soluble calcium binding capacity (defined herein) to total soluble calcium of the nutritional emulsions is a weight ratio of at least about 2.3, including from about 2.3 to about 12.0, also including from about 3.0 to about 8.0, and also including from about 4.0 to about 6.5, wherein the ratio is determined in accordance with the following formulas:

Ratio = SCBC / [soluble calcium]

SCBC = (0.32 x [soluble citrate] + 0.63 [soluble phosphate] + 0.013 x [soluble protein])

[0057] The weight ratio of SCBC to the concentration of total soluble calcium can be adjusted to minimize the concentration of unbound calcium in the nutritional emulsion, or to minimize the weight ratio of such unbound calcium to HMB in the emulsions, to improve product stability and reduce the development over time of bitter flavors and after tastes.

[0058] The nutritional emulsions of the present disclosure comprise calcium as a desirable ingredient in the nutritional emulsions suitable for use in developing or maintaining healthy muscle in targeted individuals. Some or all of the calcium may be provided by the addition of calcium HMB as described herein. Any other calcium source, however, may be used provided that such other source is compatible with the essential elements of the nutritional emulsions.

[0059] The concentration of calcium in the nutritional emulsions typically exceeds about 10 mg/L, and may also include concentrations of from about 25 mg/L to about 3000 mg/L, also including from about 50 mg/L to about 500 mg/L, and also including from about 100 mg/L to about 300mg/L.

[0060] To minimize the taste and stability issues in the nutritional emulsions, the calcium is formulated so as to minimize the extent to which the calcium is solubilized in the emulsions. As such, solubilized calcium concentrations in the nutritional emulsions may be less than about 900 mg/L, including less than about 700mg/L, also including from about 500mg/L to about 700mg/L, and also including from about 400mg/L to about 600 mg/L. In this context, the term "solubilized calcium" refers to free, ionized, or supernatant calcium in the nutritional emulsion as measured at 20°C.

[0061 ] The calcium in the nutritional emulsions may also be characterized by a ratio (on an equivalents basis) of solubilized citrate to solubilized calcium of not more than 5.0, including not more than 4.0, also including not more than 3.0, and also including from about 0.8 to about 3.0. In this context, the terms "solubilized citrate" and

"solubilized calcium" refers to the equivalents of citrate and calcium cations,

respectively, present in the supernatants of the nutritional emulsion as measured at 20°C.

[0062] The calcium component of the nutritional emulsion may also be characterized by a solubilized calcium level that represents less than 900 mg/L, including less than 700 mg/L, and also including less than 600 mg/L, and also including from 400 mg/L to 700 mg/L of the nutritional emulsion, wherein the weight ratio of calcium HMB to the solubilized calcium ranges from about 6 to about 15, including from about 6 to about 12, also including from about 6 to about 10, and also including from about 6 to about 8.

Vitamin D

[0063] The nutritional emulsions of the present disclosure may further comprise vitamin D to help maintain healthy muscle in the targeted user. Vitamin D forms include Vitamin D2 (ergocalciferol) and Vitamin D3 (cholecalciferol) or other forms suitable for use in a nutritional product.

[0064] The amount of Vitamin D in the nutritional emulsion most typically ranges up to about 1000 IU, more typically from about 10 to about 600 IU, and more typically from about 50 to 400 IU, per serving of the nutritional emulsion.

Optional Ingredients

[0065] The nutritional emulsions described herein may further comprise other optional ingredients that may modify the physical, chemical, hedonic or processing characteristics of the products or serve as pharmaceutical or additional nutritional components when used in the targeted population. Many such optional ingredients are known or otherwise suitable for use in other nutritional products and may also be used in the nutritional emulsions described herein, provided that such optional ingredients are safe and effective for oral administration and are compatible with the essential and other ingredients in the selected product form.

[0066] Non-limiting examples of such optional ingredients include

preservatives, antioxidants, emulsifying agents, buffers, pharmaceutical actives, additional nutrients as described herein, colorants, flavors, thickening agents and stabilizers, and so forth.

[0067] The nutritional emulsions may further comprise vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin E, vitamin , thiamine, riboflavin, pyridoxine, vitamin B12, carotenoids, niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts, and derivatives thereof, and combinations thereof.

[0068] The nutritional emulsion may further comprise minerals, non-limiting examples of which include phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, selenium, chloride, and combinations thereof.

[0069] The nutritional emulsions may also include one or more masking agents to reduce or otherwise obscure the development of any residual bitter flavors and after taste in the emulsions over time. Suitable masking agents include natural and artificial sweeteners, sodium sources such as sodium chloride, and hydrocolloids, such as guar gum, xanthan gum, carrageenan, gellan gum, and combinations thereof. The amount of masking agent in the nutritional emulsion may vary depending upon the particular masking agent selected, other ingredients in the formulation, and other formulation or product target variables. Such amounts, however, most typically range from at least about 0.1%, including form about 0.15% to about 3.0%, and also including from about 0.18% to about 2.5%, by weight of the nutritional emulsion.

Methods of Manufacture

[0070] The nutritional emulsions for use herein may be manufactured by any known or otherwise suitable method for making nutritional emulsions, including milk-based nutritional emulsions.

[0071] In one suitable manufacturing process, at least three separate slurries are prepared, including a protein-in-fat (PIF) slurry, a carbohydrate-mineral (CHO-MIN) slurry, and a protein-in- water (PIW) slurry. The PIF slurry is formed by heating and mixing the selected oils (e.g., canola oil, corn oil, etc.) and then adding an emulsifier (e.g., lecithin), fat soluble vitamins, and a portion of the total protein (e.g.. milk protein concentrate, etc.) with continued heat and agitation. The CHO-MIN slurry is formed by adding with heated agitation to water: minerals (e.g., potassium citrate, dipotassium phosphate, sodium citrate, etc.), trace and ultra trace minerals (TM/UTM premix), thickening or suspending agents (e.g. Avicel, gellan, carrageenan), and calcium HMB or other HMB source. The resulting CHO-MIN slurry is held for 10 minutes with continued heat and agitation before adding additional minerals (e.g., potassium chloride, magnesium carbonate, potassium iodide, etc.), carbohydrates (e.g., frucotooligosaccharide, sucrose, corn syrup, etc.). The PIW slurry is then formed by mixing with heat and agitation the remaining protein (e.g., sodium caseinate. soy protein concentrate, etc.) into water.

[0072] The resulting slurries are then blended together with heated agitation and the pH adjusted to the desired range, typically from 6.6-7.0, after which the composition is subjected to high-temperature short-time (HTST) processing during which the composition is heat treated, emulsified and homogenized, and then allowed to cool.

Water soluble vitamins and ascorbic acid are added, the pH is again adjusted to the desired range if necessary, flavors are added, and water is added to achieve the desired total solid level. The composition is then aseptically packaged to form an aseptically packaged nutritional emulsion, or the composition is added to retort stable containers and then subjected to retort sterilization to form retort sterilized nutritional emulsions.

[0073] The manufacturing processes for the nutritional emulsions may be carried out in ways other than those set forth herein without departing from the spirit and scope of the present invention. The present embodiments are, therefore, to be considered in all respects illustrative and not restrictive and that all changes and equivalents also come within the description of the present disclosure.

Method of Use

[0074] The nutritional emulsions described herein are useful to provide supplement, primary, or sole sources of nutrition, and or to provide individuals one or more benefits as described herein. In accordance with such methods, the emulsions may be administered orally as needed to provide the desired level of nutrition, most typically in the form of one to two servings daily, in one or two or more divided doses daily, e.g., serving sizes typically ranging from about 100 to about 300 ml, including from about 150 to about 250ml, including from about 190 ml to about 240 ml, wherein each serving contains from about 0.4 to about 3.0 g, including from about 0.75 to about 2.0 g, including about 1.5 g, of calcium HMB per serving.

[0075] Such methods are further directed to provide the individual upon administration of such products, most typically after daily use over an extended period of time of from about 1 to about 6 months, including from about 1 to about 3 months, one or more of l) to support maintenance of lean body mass, 2) to support of strength and or muscle strength, 3) to decrease protein breakdown and damage of muscle cells, and 4) to help with muscle recovery following exercise or other trauma, and 5) to reduce muscle protein breakdown following exercise.

[0076] Such methods are also helpful to achieve one or more of 1 ) to maintain and support lean body mass in elderly with sarcopenia, 2) to provide nutrition to support an active and independent lifestyle in individuals, especially in the elderly, 3) to support recovery of muscle strength, 4) to help rebuild muscle and regain strength, and 5) to improve strength, including muscle strength, and mobility.

DATA

[0077] Nutritional emulsions A, B and C as referenced below are formulated, aseptically packaged, and evaluated for flavor. The nutritional emulsions are similar in composition and are prepared by similar methods. Each sample is an oil-in-water emulsion comprising fat, protein, carbohydrates, vitamins, and minerals, and includes 1.5 g of calcium HMB per each 240 ml of emulsion. The samples vary, however, in that each contains a different percentage of soluble protein by weight of total protein. The samples are evaluated 6 months after formulation and packaging using standard sensory methods (5 point scale), the results of which are summarized below.

[0078] The data show a surprising correlation between the soluble protein fraction (e.g.. Na caseinate) and the development of bitter and soapy sensory notes over time. The data also suggest, surprisingly, that the development of such undesirable sensory notes can be minimized or reduced by formulating with a majority of the total protein as soluble protein.

EXAMPLES

[0079] The following examples illustrate specific embodiments and or features of the nutritional emulsions of the present disclosure. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present disclosure, as many variations thereof are possible without departing from the spirit and scope of the disclosure. All exemplified amounts are weight percentages based upon the total weight of the composition, unless otherwise specified.

[0080] The exemplified compositions are shelf stable nutritional emulsions prepared in accordance with the manufacturing methods described herein, such that each exemplified composition, unless otherwise specified, includes an aseptically processed embodiment and a retort packaged embodiment. These compositions are aqueous oil-in-water emulsions that are packaged in 240 ml plastic containers and remain physically stable for 12-18 months after formulation/packaging at storage temperatures ranging from 1-25°C. The packaged emulsions likewise remain pH stable over time and do not develop an excessively bitter flavor or aftertaste during the storage.

Examples 1-4

[0081 ] Examples 1 -4 illustrate nutritional emulsions of the present disclosure, ingredients of which are listed in the table below. All ingredient amounts are listed as logram per 1000 kilogram batch of product, unless otherwise specified.

Examples 5-8

[0082] Examples 5-8 illustrate nutritional emulsions of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts are listed as kg per 1000 kg batch of product, unless otherwise specified.

Examples 9-12

[0083] Examples 9-12 illustrate nutritional emulsions of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Examples 13-16

[0084] Examples 13- 16 illustrate nutritional emulsions of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Examples 17-20

[0085] Examples 17-20 illustrate nutritional emulsions of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified
CLAIMS
1. A method for designing a ligand that covalently binds a target protein, the method
comprising:
A) providing a structural model of a reversible ligand docked within, or in
proximity to, a ligand-binding site in a target protein;
B) identifying a lysine residue of the target protein in, or in proximity to, the
ligand-binding site that is less than about 15A from the reversible ligand when the reversible
ligand is docked in, or in proximity to, the ligand-binding site;
C) producing at least a structural model of at least one ligand-warhead compound
docked within, or in proximity to, the ligand-binding site wherein the ligand-warhead
compound comprises the reversible ligand in step B) or a portion thereof, a warhead
comprising a reactive chemical moiety, and optionally a Tether; and
D) identifying a ligand-warhead compound whose structural model allows the
lysine residue in step B) to readily assume a conformation that brings the side chain primary
amine group of the lysine residue within bond-forming proximity of the warhead electrophile.
2. The method of claim 1, further comprising:
E) forming, for the ligand-warhead compound identified in step D), a ligandprotein
covalent adduct by forming a covalent bond between the side chain primary amine
group of the lysine residue identified in step B) and the warhead electrophile in ligandwarhead
compound identified in step D) while substantially maintaining the non-covalent
interactions between the pharmacophore of the ligand and the ligand-binding site.
3. The method of claim 2, further comprising:
F) evaluating the conformation of the resulting ligand-protein covalent adduct
formed in step E) by analyzing the global energy of the resulting conformation, or by
analyzing the energy of the conformation of the Tether.
4. The method of claim 3, wherein steps A)-F) are iterated with changes to the Tether
and the global energy of the resulting conformation is less than the previous iteration.
5. The method of claim 2, further comprising:
F) determining whether the ligand-binding site is occluded when the covalent
bond is formed between the side chain primary amine group of the lysine residue in, or in
proximity to, the ligand-binding site and the warhead electrophile.
6. The method of claim 2, wherein the covalent bond formed in step E) is formed using a
computational method in which the warhead and the side chain of the lysine residue are
flexible and the remainder of the structures of the ligand-warhead compound and the
ligand-binding site are fixed.
7. The method of claim 1, wherein in step B) each of the lysine residues in, or in
proximity to, the ligand-binding site of the target protein, which is less than about 15A from
the reversible ligand when the reversible ligand is docked in, or in proximity to, the ligandbinding
site, is identified.
8. The method of claim 1, wherein step C) comprises providing a plurality of models of
the ligand-warhead compound, wherein the warhead is bonded to a different substitutable
position of the ligand or a portion of the ligand in each model of the ligand-warhead
compound, optionally with the Tether in between the warhead and the substitutable position.
9. The method of claim 1, wherein the target protein is an identified member of an
identified protein family and the lysine residue is not conserved across the identified
members of the protein family.
10. The method of claim 1, wherein the target protein is an identified member of an
identified protein family and the lysine residue is conserved among more than one identified
member of the identified protein family.
11. The method of claim 10, wherein the lysine residue is conserved across the identified
members of the protein family.
The method of claim 1, wherein the target protein has catalytic activity.
13. The method of claim 1, wherein the protein family is selected from the group
consisting of BCL-2, Calpains, Caspases, Cathepsins, HCV, HDAC, HSP70, HSP90, IAP,
Kinase, MDM2, MMP, NHR, PI3K, Phosphatase, PARP, and HIV Protease.
14. The method of claim 13, wherein the target protein is selected from the
consisting of XIAP, cIAPl and cIAP2, RI3Kb/g , PDPK1, and HCV-NS3.
The method of claim 1, wherein the ligand-binding site is a ligand-binding site for
The method of claim 1, wherein the lysine residue is not a catalytic residue.
The method of claim 1, wherein the ligand-warhead compound has a structure of
Warhead Tether Scaffold Tether Warhead
x y
wherein
Scaffold is
a) a radical resulting from the removal of a hydrogen of a ligand capable of
binding to, or in proximity to, the ligand-binding site; or
b) a portion of a pharmacophore of a ligand resulting from truncation of the
pharmacophore, such that the Scaffold is capable of binding to, or in proximity to, the
ligand-binding site;
Warhead is an organic moiety optionally containing one or more heteroatoms selected
from O, N, and S; the organic moiety having a molecular weight of about 14 daltons to about
200 daltons; Warhead being capable of reaction with a side chain primary amine group of a
lysine residue; and Warhead being attached to Scaffold through Tether; and
Tether is null, a bond, or a bivalent C1-C15 saturated, unsaturated, straight, branched,
cyclic, bicyclic, tricyclic alkyl, alkenyl, alkynyl; bridged bicyclic, heterocycle, heteroaryl, or
aryl moiety; wherein optionally one or more methylene units of the hydrocarbon chain are
independently replaced by -NR , -0-, -C(O)-, -S-, -SO-, -S0 2-, -C(=S)-, or C(=NRi)-;
optionally one or more hydrogens are independently replaced by heteroatoms; and optionally
one or more methine groups of the C1-C15 alkyl, when present, are independently replaced by
x is 0, 1, or 2;
y is 1, 2, or 3; and
Ri is hydrogen or Ci-Cg alkyl;
with the proviso that the compound of Fomula I is not
wortmannin:
or known analogues of wortmannin that covalently modify lysine through substantially the
same mechanism as wortmannin:
-431-
any mechanism-based irreversible inhibitors.
The method of claim 17, wherein the ligand-warhead compound has a structure of
Scaffold Tether Warhead
G
19. The method of claim 18, wherein Warhead is a radical resulting from the removal of a
hydrogen of a compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m,
I-n, I-o, I-p, I-q, I-r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, , and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
20. The method of claim 19, wherein as least one of R2 and R3 of the compounds of
Formulas I-b and I-c is hydrogen.
21. The method of claim 19, wherein the compound of Formula I-a, I-d, I-e, I-j, I-k, or I-
1is a compound of Formula Il-a, Il-b, II-c, Il-d, Il-e, Il-f, Il-g, Il-h, Il-i, II-j, Il-k, II-l, li¬
ra, Il-n, II-o, II-p, Il-q, Il-r, II-s, Il-t, II-u, II-v, II-w, II-x, Il -y, II-z, Il-aa, Il-bb, II-cc,
Il-dd, Il-ee, Il-ff, Il-gg, Il-hh, Il-ii, Il-jj, Il-kk, II-ll, Il-mm, Il-nn, II-oo, or II-pp;
wherein
each m is independently an integer from 0-4;
each is independently an integer from 0-3;
each m4 is independently an integer from 0-5;
each n2 is independently an integer from 0-2;
each R2, R3, R4, R , R6, R7, Rs, R9, Rio, R 11, R12, R13, R14, and R15 is independently
hydrogen or Ci-C 6 alkyl; R is hydrogen, Ci-C 6 alkyl, halogen, CF3, or nitro; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO2-, or -C(=S)-; one or more methine groups of the Ci-C 6
alkyl, when present, can be independently replaced by N ;
R i is hydrogen or Ci-C 8 alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, Rs, R9, Rio, R 11, R12,
R i3, Ri4, and R15 when taken together form a 3- to 8-membered carbocyclic or heterocyclic
ring or an aryl or heteroaryl group.
22. The method of claim 19, wherein the compound of Formula I-d, or I-h is a compound
of Formula Ill-a, Ill-b, III-c, Ill-d, Ill-e, Ill-f, Ill-g, Ill-h, or Ill-i;
wherein
n3 is an integer from 0-2;
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl;
each Bi, B2, B4, and B is independently CR7 or N and each B3 is NR7, O, or S;
each Rzl , R 2, R 3, R 4, and R is hydrogen, Ci-C alkyl, halogen, CF3, or nitro;
one or more methylene groups of the Ci-C 6 alkyl can be optionally replaced by -NRi-,
-0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl,
when present, can be independently replaced by
Ri is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, and R8 when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group.
23. The method of claim 19, wherein the compound of Formula I-h is a compound of
Formula IV-a, IV-b, IV-c, IV-d, IV-e, IV-f, IV-g, IV-h, or IV-i:
V-a IV-b IV-c IV-d IV-e
V-f IV-g IV-h IV-i
wherein any of the substitutable hydrogens on the nitrogen heterocycle of the compound can
be substituted with alkyl, alkoxy, amido, acyl, acyloxy, oxoacyl, halogen.
24. The method of claim 19, wherein the radical resulting from the removal of a hydrogen
of a compound of Formula I-a, I-d, I-k, or I-m is a radical of Formula V-a, V-b, V-c, V-d,
V-e V-f , V-g, V-h , V-i, or V-j;
v-f V-g V-h V-i V-J
wherein
m i and m2 are each independently an integer from 0 to 2;
each R2, R3, R4, R , R6, R7, Rs, R9, Rio, and R is independently hydrogen or Ci-
C6 alkyl;
one or more methylene groups of the Ci-C 6 alkyl can be optionally replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO2-, or -C(=S)-;
one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by N ;
R i is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, Rs, R9, Rio, and R
when taken together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or
heteroaryl group.
25. The method of claim 19, wherein the compounds of Formulae I-a, I-b, I-c, I-d, I-e, If,
I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q, I-r, I-s, and I-t are selected from the group
consisting of:
JJ kk II
OOO
wherein any substitutable hydrogen may be substituted with the substituents as those defined
by R2-R8 in formulas I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q,
I-r, I-s, and I-t.
26. The method of claim 19, wherein the radical resulting from the removal of a hydrogen
of a compound of Formula I-a, I-d, I-k, or I-m is a radical of Formula Vl-a, Vl-b, VI-c, VIl
-e, Vl-f, Vl-g, Vl-h, Vl-i, Vl-j, Vl-k, VI-1, Vl-m, VI-h, VI-o, VI-p, or Vl-q:
VI- Vl-i Vl-j Vl -k VI-1 Vl-m Vl-n
; wherein R is hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, -CH2OCH3, and
-CH2CH2OCH3.
27. The method of claim 19, wherein Scaffold is selected from the group consisting of
Formulas VII, VIII, IX-a, IX-b, XI, XII, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII,
XXXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, and XXXVII.
28. The method of claim 18, wherein Scaffold is a radical resulting from the removal of
one or more hydrogens of a compound of Formula VII:
VII
wherein
V and W are each independently -(CRi4Ri5) q 3(CRi6Ri7)r-;
q and r are each independently 0, 1, 2, 3, or 4;
X3 is -CRigRisr, or -NR 2o-; and
Rx, Ry, Ri2, Ri3, Ri4, Ri5, Ri6, R , Ri8, Ri9, and R2o are each independently hydrogen
or Ci-C 6 alkyl; wherein one or more methylene groups of the Ci-C 6 alkyl can be replaced by -
NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, -C(=S)-, optionally substituted aryl or heteroaryl groups;
one or more methine groups of the Ci-C alkyl, when present, can be independently replaced
by -N- ; and Ri is hydrogen or Ci-Cg alkyl.
The method of claim 28, wherein the compound of Formula VII is a compound of
wherein
V and W are each independently -(CRi4Ri5) q 3(CRi6Ri7)r-;
q and r are each independently 0, 1, 2, 3, or 4;
X3 is -CRi8Ri9-, or -NR 20-;
p is 0, 1, 2, 3, or 4;
Ri2, Ri3, Ri4, Ri5, Ri6, Ri7, Ri8, Ri9, and R2o are each independently hydrogen
or Ci-C alkyl; wherein one or more methylene groups of the Ci-C alkyl can be replaced by -
NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, -C(=S)-, optionally substituted aryl or heteroaryl groups;
one or more methine groups of the Ci-C6 alkyl, when present, can be independently replaced
Ri is hydrogen or Ci-Cg alkyl; and;
R23 is hydrogen, Ci-C alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-S0 2-, or -C(=S)-; one or more methine groups of the Ci-C6 alkyl, when present, can be
independently replaced by N ; and
optionally R2i and R23 taken together can form a 4- to 8-membered
carbocyclic or heterocyclic ring.
The method of claim 19, wherein the compound of Formula I ' is a compound of
wherein
V and W are each independently -(CRi4Ri5) q 3(CRi6Ri7 )r-;
q and r are each independently 0, 1, 2, 3, or 4;
X3 is -CRi8Ri9-, or -NR 20-;
p is 0, 1, 2, 3, or 4;
Ri2, Ri3, Ri4, Ri5, Ri6, Ri7, Ri8, Ri9, and R2o are each independently hydrogen
or Ci-C6 alkyl; wherein one or more methylene groups of the Ci-C6 alkyl can be replaced by -
NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, -C(=S)-, optionally substituted aryl or heteroaryl groups;
one or more methine groups of the Ci-C 6 alkyl, when present, can be independently replaced
Ri is hydrogen or Ci-Cg alkyl; and;
R23 is hydrogen, Ci-C alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ; and
optionally R2i and R23 taken together can form a 4- to 8-membered
carbocyclic or heterocyclic ring;
T is Tether; and
Rw is Warhead.
The method of claim 30, wherein the compound of Formula Vll-b is a compound of
32. The method of claim 31, wherein the compound of Formula Vll-h is a compound of
Formula Vll-j, Vll-k, VII-1, Vll-m, Vll-n, or VII-o:
33. The method of claim 31, wherein the compound is selected from the group consisting
of:
VII-7 VII-8 VII-9
-447-
-448-
-449-
VII-43 VII-44
VII-47 VII-48
an O
VII-49
34. The method of claim 19, wherein Scaffold is a radical resulting from the removal of a
hydrogen of a compound of Formula VIII:
wherein
s and p are each independently 0, 1, 2, 3, or 4;
R12, Ri3, R21, R22, R24, R25, R26, R27, R28, R29, R30, R31, R32, and R33 are each
independently hydrogen or Ci-C6 alkyl;
R23 is hydrogen, Ci-C6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-,
-SO2-, or -C(=S)-; one or more methine groups of the Ci-C alkyl, when present, can b <
independently replaced by
Ri is hydrogen or Ci-Cg alkyl; and
optionally R2i and R23 taken together can form a 4- to 8-membered carbocyclic or
heterocyclic ring.
The method of claim 18, wherein the compound of Formula I ' is a compound of
wherein
X4 is -CR33- or -N-;
s and p are each independently 0 , 1, 2, 3, or 4;
Ri2, Ri3, R2i, R22, R24, R2 5, R26, R27, R2 8, and R33 are each independently hydrogen or
Ci-C6 alkyl;
R23 is hydrogen, Ci-C6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-S0 2-, or -C(=S)-; one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-Cg alkyl; and
optionally R2i and R23 taken together can form a 4- to 8-membered carbocyclic or
heterocyclic ring; and
Warhead is a radical resulting from the removal of a hydrogen of a compound of
Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q, I-r, I-s, and
I-t:
wherein
each Xi and Xg is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR , or -NR Ry,;
each X is independently N or R ;
each R2, R3, R4, R5, Re, R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-,
-SO 2-, or -C(=S)-; one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-Cg alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, Re, R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n10 , n , and n i2 is an integer
from 0-1; and n i3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
36. The method of claim 35, wherein the compound of Formula Vlll-a or VHI-b is
selected from the group consisting of:
VIII-1 VIII-2
VIII-3 VIII-4
VIII-7
37. The method of claim 19, wherein Scaffold is a radical resulting from the removal of a
h drogen of a compound of Formula IX-a or IX-b
IX-a IX-b
wherein
X5 is -0-, -CR42R43- or -NR42-;
Ri2, Ri3, R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R37, R38, R39, R40, R41, R42, and
R43 are each independently hydrogen or Ci-C6 alkyl; wherein one or more methylene groups
of Ci-C alkyl can be optionally replaced by -NR , -0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
one or more methine groups of the Ci-C alkyl, when present, can be independently replaced
Ri is hydrogen or Ci-Cg alkyl;
D , E, F, G, and H are each independently optionally substituted aryl or heteroaryl;
wherein F and G are fused together to form a bicyclic optionally substituted aryl or
heteroaryl.
The method of claim 18, wherein the compound of Formula I ' is a compound of
wherein
Ri2, Ri3, R31, are each independently hydrogen or Ci-C alkyl; wherein one or more
methylene groups of Ci-C6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-S0 2-, or -C(=S)-; one or more methine groups of the Ci-C6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C8 alkyl;
F, G, and H are each independently optionally substituted aryl or heteroaryl; wherein
F and G are fused together to form a bicyclic optionally substituted aryl or heteroaryl;
T is Tether;
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, , and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
The method of claim 18, wherein the compound of Formula I ' is a compound of
wherein
p is an integer from 0 to 4, u is an integer from 1 to 4;
B and B are each independently CR or N;
R 9 is hydrogen, Ci-C6 alkyl, halogen, amino, nitro, or -NH(CO)NR gR 9;
R ois hydrogen, Ci-C6 alkyl, halogen, amino, nitro;
R , R i, R 2, R73 , R74, R7 5, R7 6, R77, R7g, and R are each independently hydrogen or
Ci-C6 alkyl; Ri is hydrogen or Ci-Cg alkyl; wherein one or more methylene groups of the Ci-
C6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-; one or more
methine groups of the Ci-C6 alkyl, when present, can be independently replaced by
optionally R 8, and R taken together form a 4- to 8-membered carbocyclic or
heterocyclic ring;
T is Tether; and
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
each Xi and X is independently -0-, -S-, or -NR6
each X2 is independently -R , -OR6, or -NR6R7,;
each X is independently N or R ;
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; o re methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; ¾ is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n i0, n , and n i2 is an integer
from 0-1; and n is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
40. The method of claim 39, wherein the compound of Formula XI is a compound of
F rmula Xl-a, CI-b, or XI-c:
4 1. The method of claim 40, wherein the compound of Formula Xl-a, CI-b or XI-c is a
compound of Formula Xl-d, CI-e, Xl-f, CI-g, Xl-h, CI-i, or Xl-j :
42. The method of claim 4 1, wherein the compound of Formula XI-d, Xl-e, CI-f, XI-g,
Xl-h, CI-i, or CI-j is a compound of Formula CI-k, XI-1, Xl-m, Xl-n, XI-o, XI-p, or Xl-q;
wherein
Rgs, and R8g are each independently hydrogen or Ci-C alkyl; Ri is hydrogen or Ci-Cg
alkyl;
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-,
-C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
one or more methine groups of the Ci-Ce alkyl, when present, can be independently
replaced by N ; and
¾ is an integer from 0 to 3.
43. The method of claim 42, wherein the compound of Formula Xl-d, CI-e, XI-f, Xl-g,
-h, CI-i, or CI-j is a compound of Formula CI-r, XI-s, CI-t, XI-u, XI-v, XI-w, or XI-x;
X l-x
44. The method of claim 41, wherein the compound of Formula Xl-d, Xl-e, CI-f, Xl-g,
-h, CI-i, or CI-j is a compound of Formula CI-y, XI-z, CI-aa, or Xl-bb;
45. The method of claim 41, wherein the compound of Formula Xl-d, Xl-e, XI-f, Xl-g,
Xl-h, CI-i, or CI-j is a compound of Formula XI-cc, Xl-dd, Xl-ee, or Xl-ff:
XI-cc Xl-dd
Xl-ee Xl-ff
46. The method of claim 39, wherein the compound of Formula XI is selected from the
group consisting of:
-466-
XI-29 XI-30
-468-
-469-
XI-45 XI-46

The method of claim 18, wherein the compound of Formula G is a compound of
wherein
Ri and R2 are each independently hydrogen or Ci-Cs alkyl; wherein one or more
methylene groups of the Ci-C6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-,
or -C(=S)-; one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by -NT
is Tether;
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, II-
r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, , and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
48. The method of claim 18, wherein the compound of Formula I ' is a compound of
Formula XXXVI:
wherein
R is H, optionally substituted Ci-C3 branched or straight chain alkyl, or optionally
substituted Ci-C3 branched or straight chain acyl;
T is Tether; and
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, , and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
49. The method of claim 18, wherein the compound of Formula I ' is selected from the
group consisting of:
XXXVI-1 XXXVI-2
50. The method of claim 19, wherein Scaffold is a radical resulting from the removal of a
hydrogen of a compound of Formula XVI-a, XVI-b, or XVI-c:
XVI-c
wherein
R90, R91, R92, R93, R94, R95, R 6, R97, R98, R99, R100, R102, R104, Rl05, Rl06, Rl07, Rl08,
R109, R 110, R 111, R112, R 113, and R 4 are each independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of Ci-C alkyl can be optionally replaced by -NRr, -0-, -
C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
R103 is hydrogen, Ci-C 6 alkyl, or C2-C alkenyl;
one or more methine groups of the Ci-C 6 alkyl, when present, can be independently
replaced by
R i is hydrogen or Ci-C 8 alkyl;
each R101 and R101 is independently hydrogen, Ci-C 6 alkyl, C2-C alkenyl, halogen,
amino, nitro, optionally substituted aryl or heteroaryl; and
n6 and n are each independently integer from 0 to 4; and
n is an integer from 0 to 2.
51. The method of claim 18, wherein the compound of Formula I' is a compound of
Formula XVI-d, XVI-e, or XVI-f:
XVI-d
XVI-f
wherein
R90 and R 114 are each independently hydrogen or Ci-C alkyl;
R103 is hydrogen, Ci-C 6 alkyl, or C2-C8 alkenyl;
R101 is hydrogen, Ci-C 6 alkyl, C2-C8 alkenyl, halogen, amino, nitro, optionally
substituted aryl or heteroaryl; and
n6 is an integer from 0 to 4;
n is an integer from 0 to 2;
wherein one or more methylene groups of Ci-C6 alkyl can be optionally replaced by -
NR , -0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-; and
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
each Xi and Xg is independently -0-, -S-, or -NR6
each X2 is independently -R , -OR6, or -NR6 R7,;
each X is independently ;
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; o re methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; ¾ is an integer
from 1-2; n4 is an integer from 1-3; and each one of n , n i0, n , and n i2 is an integer from 0-1;
and n is an integer from 0-2, wherein when any one of the foregoing n integers is more than
1, the adjacent carbons represented by the integer can form a single or double bond; and
T is Tether.
2. The method of claim 51, wherein the compound of Formula XVI-d, XVI-e, or XVI-f
a compound of Formula XVI-g, XVI-h, or XVI-i:
3. The method of claim 18, wherein the compound of Formula XVI-d, XVI-e, or XVI-f
selected from the group consisting of:
XVI-3 XVI-4

-484-
-485-
-486-
-487-
54. The method of claim 18 wherein the compound of Formula I' is a compound of
Formula XXII-a Formula XXII-b, or Formula XXII-c:
wherein
n, m, p, and q for Formula XXII-a and Formula XXII-b are each independently 0, 1,
2, 3; provided that n and q are not 0 at the same time, and m and q are not 0 at the same time;
T is tether;
Rw is Warthead and is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, nl l and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond;
A2 is an optionally substituted ring selected from a 4-8 membered saturated or
partially unsaturated heterocyclic ring having one or two heteroatoms independently selected
from nitrogen, oxygen, or sulfur, or a 5-10 membered saturated or partially unsaturated
bridged bicyclic heterocyclic ring having at least one nitrogen, at least one oxygen, and
optionally 1-2 additional heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
B is an optionally substituted group selected from phenyl, an 8- to 10-membered
bicyclic aryl ring, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or -T-Rwh;
and
C2 is hydrogen or an optionally substituted ring selected from a 3- to 7-membered
saturated or partially unsaturated carbocyclic ring, a 7- to 10-membered saturated or partially
unsaturated bicyclic carbocyclic ring, a 4- to 7-membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 7- to 10-membered saturated or partially unsaturated bicyclic heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, phenyl, an
8- to 10-membered bicyclic aryl ring, a 5- to 6-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-to 10-membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
5. The method of claim 54, wherein the compound of Formula XXII-a, XXII-b, XXII-c
selected from the group consisting of:
-492-
XXII-17 XXII-18
-494-
-495-
-496-
56. The method of claim 18, wherein the compound of Formula I ' is a compound of
Formula XXIII:
XXIII
wherein:
Rwh is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, Iq,
I-r, I-s, and I-t:
wherein
each Xi and Xg is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently or
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C alkyl can be replaced by -NRr, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-Cg alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; ¾ is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , nio, , and n i2 is an integer
from 0-1; and n is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond;
R201 is hydrogen or Ci_6 alkyl;
R2o2 is hydrogen or an optionally substituted group selected from Ci_6 alkyl, Ci_6
alkoxy, or (C1-6 alkylene)-R 203; or
R201 and R2o2 are taken together with the intervening carbon to form an optionally
substituted ring selected from a 3- to 7-membered carbocyclic ring or a 4- to 7-membered
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
R203 is a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, a 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or a 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; and
Ring A6 is absent or an optionally substituted group selected from a 4- to 7-membered
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, or a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected
from nitrogen, oxygen, or sulfur.
57. The method of claim 56, where the compound of Formula XXIII is selected from
the group consisting of:
XXIII-1
-500-
-501-
58. The method of claim 18, wherein the compound of Formula G is a compound of
Formula XXIV-a or Formula XXIV-b:
XXIV-a XXIV-b
wherein
Rwh is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
I-a I-b I-c I-d I-e I-f
wherein
each Xi and Xg is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently or
each R2, R3, R4, R5, R6, R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C alkyl can be replaced by -NRr, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-Cg alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R6, R7, and Rg when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; ¾ is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n10 , n , and n i2 is an integer
from 0-1; and n is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond;
R2o4 is an hydrogen or an optionally substituted group selected from Ci_6 aliphatic, -
(CH 2)m-(3- to 7-membered saturated or partially unsaturated carbocyclic ring), -(CH 2)m-(7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring), -(CH 2)m-(4- to 7-
membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur), -(CH 2)m-(7- to 10-membered
saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur), -(CH 2)m-phenyl, -(CH 2)m-(8- to 10-
membered bicyclic aryl ring), -(CH 2)m-(5- to 6-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur), or -(CH 2)m-(8- to 10-
membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur);
each R205 and R2 0 6 i independently -R", halogen, -N0 2, -CN, -OR", -SR",
-N(R") 2, -C(0)R", -C0 2R", -C(0)C(0)R", -C(0)CH 2C(0)R", -S(0)R", -S(0) 2R",
-C(0)N(R") 2, -S0 2N(R") 2, -OC(0)R", -N(R")C(0)R", -N(R")N(R") 2,
-N(R")C(=NR")N(R") 2, -C(=NR")N(R") 2, -C=NOR", -N(R")C(0)N(R") 2,
-N(R")S0 2N(R") 2, -N(R")S0 2R", or -OC(0)N(R") 2;
each R" is independently hydrogen or an optionally substituted group selected from
Ci_6 aliphatic, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, an 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; or
two R" groups on the same nitrogen are taken together with the nitrogen to which
they are attached to form an optionally substituted 5-8 membered saturated, partially
unsaturated, or aromatic ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur;
m is an integer from 0 to 6, inclusive;
each n for Formula XXIV-a or Formula XXIV-b is independently 0, 1, or 2; and
Ring A5 is an optionally substituted 6-membered heterocyclic or heteroaryl ring
having 1-2 nitrogens.
59. The method of claim 58, wherein the compound of Formula XXIV-a or Formula
-b is selected from the group consisting of
XXIV-3
60. The method of claim 18, wherein the compound of Formula I' is a compound of
Formula XXV:
wherein
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
wherein
each Xi and Xg is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently or
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C alkyl can be replaced by -NRr, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-Cg alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , nio, , and n i2 is an integer
from 0-1; and n i3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond;
each R205 and R206 is independently -R", halogen, -N0 2, -CN, -OR", -SR", -N(R") 2,
-C(0)R", -C0 2R", -C(0)C(0)R", -C(0)CH 2C(0)R", -S(0)R", -S(0) 2R", -C(0)N(R") 2,
-S0 2N(R") 2, -OC(0)R", -N(R")C(0)R", -N(R")N(R") 2, -N(R")C(=NR")N(R") 2,
-C(=NR")N(R") 2, -C=NOR", -N(R")C(0)N(R") 2, -N(R")S0 2N(R") 2, -N(R")S0 2R", or
-OC(0)N(R") 2;
each R" is independently hydrogen or an optionally substituted group selected from
Ci_6 aliphatic, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, an 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; or
optionally, two R" groups on the same nitrogen are taken together with the nitrogen to
which they are attached to form an optionally substituted 5-8 membered saturated, partially
unsaturated, or aromatic ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur;
m is an integer from 0 to 6, inclusive;
each n for Formula XXV is independently 0, 1, or 2; and
Ring A5 is an optionally substituted 6-membered heterocyclic or heteroaryl ring
having 1-2 nitrogens.
6 1. The method of claim 60, wherein the compound of Formula XXV is selected from the
group consisting of:
XXV-13 XXV-14 XXV-15
62. The method of claim 18, wherein the compoun dof Formula G is a compound of
Formula XXVII:
XXVII
wherein:
T is Tether;
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, Iq,
I-r, I-s, and I-t:
wherein
each Xi and Xg is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently or
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C alkyl can be replaced by -NRr, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-Cg alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; ¾ is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , nio, , and n i2 is an integer
from 0-1; and n is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
63. The method of claim 62, wherein the compound of Formula XXVII is selected from
th roup consisting of:
64. The method of claim 18, wherein the compound of Formula G is a compound of
Formula XXXVII:
XXXVII
wherein
T is Tether;
Rw is Warhead which is a radical resulting from the removal of a hydrogen of a
compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, Iq,
I-r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n in Warhead is an integer from 2-4; each ni and n2 is independently an integer from
0-2; n3 is an integer from 1-2; n4 is an integer from 1-3; and each one of n , n10, , and n12 is
an integer from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n
integers is more than 1, the adjacent carbons represented by the integer can form a single or
double bond; and
each n in Formula XXXVII is 0, 1, or 2.
65. The merthod of claim 64, wherein the compound of Formula XXXVII is selected
from the group consisting of:
XXXVII-1 XXXVII-2
XXXVII-5
66. A method for designing a ligand that covalently binds a lysine residue of a target
protein, the method comprising:
A) providing a structural model of a reversible ligand docked in, or in proximity
to, a ligand-binding site in a target protein, wherein the reversible ligand makes at least one
non-covalent contact with the ligand-binding site;
B) identifying a lysine residue in, or in proximity to, the ligand-binding site of the
target protein that is adjacent to the reversible ligand when the reversible ligand is docked in,
or in proximity to, the ligand-binding site;
C) producing structural models of a plurality of ligand-warhead compounds
docked in, or in proximity to, the ligand-binding site wherein each ligand-warhead compound
comprises a warhead covalently attached to a substitutable position of the reversible ligand in
step B), the warhead comprising a reactive chemical moiety and optionally a linker;
D) identifying among the structural models in step C) at least one ligand-warhead
compound whose structural model allows the side chain primary amine group of the lysine
residue in step B) to be within bonding distance of the warhead electrophile; and
E) further identifying among the structural models identified in step D) a
hydrogen-bond-donor-containing amino acid residue in, or in the proximity to, the ligandbinding
site, wherein the hydrogen-bond donor moiety is within hydrogen-bonding distance
of the warhead electrophile, and/or within Coulombic interaction distance of the side chain
primary amine group of the lysine residue identified in step D).
67. The method of claim 66, further comprising:
F) forming, for a ligand-warhead compound identified in step E), a ligandprotein
covalent adduct by forming a covalent bond between the side chain primary amine
group of the lysine residue identified in step B) and the warhead electrophile; and also
forming a hydrogen bond between the hydrogen-bond donor moiety and the warhead
electrophile; or a hydrogen bond between the hydrogen-bond donor moiety and the side chain
primary amine group of the lysine residue identified in step D) while substantially
maintaining the non-covalent interactions between the pharmacophore of the ligand and the
ligand-binding site.
68. The method of claim 67, further comprising:
G) evaluating a resulting conformation of the ligand-protein covalent adduct
by analyzing the global energy of the resulting conformation.
69. The method of claim 68, wherein steps A)-G) are iterated with changes to the linker
and the global energy of the resulting conformation is less than the previous iteration.
70. The method of claim 66, wherein the hydrogen-bond donor-containing amino acid
residue is any amino acid residue that is capable of acting as a hydrogen bond donor.
7 1. The method of claim 66, wherein the hydrogen-bond donor-containing amino acid
residue is selected from the group consisting of arginine, lysine, threonine, serine, histidine,
and tyrosine.
72. The method of claim 71, wherein the hydrogen-bond donor-containing amino acid
residue is selected from the group consisting of arginine and lysine.
73. The method of claim 66, wherein the target protein is selected from the group
consisting of XIAP, ML-IAP, PDPK1, and RI3Kb/g .
74. The method of claim 73, wherein the target protein is XIAP and the lysine identified
is K297 or K299.
75. The method of claim 66, wherein the ligand-warhead compound is selected from the
group consisting of Formulas VII, VIII, IX-a, IX-b, XI, XII, XVI, XVII, XVIII, XIX, XX,
XXI, XXII, XXIII, XXXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, and XXXVII.
76. The method of claim 66, wherein the Warhead is a radical resulting from the removal
of a hydrogen of a compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, Im
I-n, I-o, I-p, I-q, I-r, I-s, and I-t:
wherein
each Xi and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R
each X is independently N or R ;
each R2, R3, R4, R5, R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
R is hydrogen or Ci-C alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, nl l and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
77. The method of 76 wherein the Warhead is a radical resulting from the removal of a
hydrogen of a compound of Formula I- and X2 is -NR6R7.
78. A method for identifying at least one lysine residue within at least one protein,
wherein the at least one identified lysine can be modified covalently, the method comprising:
A) identifying at least one protein having a ligand-binding site;
B) providing a three-dimensional structural model for the identified protein;
C) docking a reversible ligand in, or in proximity to, the identified protein's
ligand-binding site in the structural model, wherein the reversible ligand makes at least one
non-covalent contact with the ligand-binding site, thereby creating a structural model of a
reversible ligand bound to, or in proximity to, an identified protein's ligand-binding site; and
D) identifying in the structural model of a reversible ligand bound to, or in
proximity to, an identified protein's ligand-binding site one or more lysine residues in, or in
proximity to, the ligand-binding site of the identified protein which is less than about 15A
from the reversible ligand.
79. The method of claim 78, comprising identifying a plurality of proteins having
ligand-binding sites that are structurally homologous.
The method of claim 79, wherein the method comprises
A) providing a three-dimensional structural model for at least one of the
identified proteins;
B) docking a reversible ligand in, or in proximity to, the structural model of
the ligand-binding site of at least one of the identified proteins, wherein the reversible ligand
makes at least one non-covalent contact with the ligand-binding site, thereby creating a
structural model of a reversible ligand bound to, or in proximity to, an identified protein's
ligand-binding site; and
C) identifying in the structural model of a reversible ligand bound to, or in
proximity to, an identified protein's ligand-binding site one or more lysine residues in, or in
proximity to, the ligand-binding site of the identified protein which is less than about 15A
from the reversible ligand.
81. The method of claim 80, wherein the method comprises comparing the threedimensionally
equivalent amino acid positions of the homologous ligand-binding sites of
more than one of the plurality of identified proteins and determining the prevalence of lysine
residues in, or in proximity to, the ligand binding sites of the identified proteins.
82. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in only one of the identified proteins.
83. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in more than one of the identified
proteins.
84. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in less than 10% of the identified
proteins.
85. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in less than 50% of the identified
proteins.
86. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in more than 50% of the identified
proteins.
87. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in more than 75% of the identified
proteins.
88. The method of claim 81, wherein the prevalence of lysine residues in, or in proximity
to, the ligand binding sites of the identified proteins is in all of the identified proteins.
89. The method of claim 78, wherein the protein is selected from the group consisting of
of BCL-2, Calpains, Caspases, Cathepsins, HCV, HDAC, HSP70, HSP90, IAP, Kinase,
MDM2, MMP, NHR, RI3Kb/g , Phosphatase, PARP, and HIV Protease.
90. The method of claim 89, wherein the protein is selected from the group consisting
XIAP, PI3K, PDPK1 and HCV-NS3.
9 1. A method for selecting a warhead that binds to a target lysine within a ligand-binding
site of a protein the method comprising
(a) identifying at least one protein having a ligand-binding site;
(b) providing a three-dimensional structural model for the identified protein;
(c) identifying the location of at least one lysine in, or in proximity to, the
ligand-binding site of step (a);
(d) providing at least one warhead in proximity to the at least one identified
lysine;
(e) aligning the electrophilic atom of the warhead within bonding distance of
the primary amine of the at least one identified lysine;
(f) forming a covalent bond between an electrophilic atom of the warhead and
the primary amine of the at least one lysine;
(g) docking a reversible ligand in the identified protein's ligand-binding site
within 15 Aof the covalently attached warhead of step (f), wherein the reversible ligand
maintains most of its known noncovalent interactions with the ligand-binding site;
(h) aligning the closest atom of the ligand with the covalently bound warhead
of step (f) and providing the geometric requirements for the design of a tether between the
ligand and the covalently bound warhead of step (f), that also is complementary with the
protein surface in the region between the warhead and the ligand.
92. A method of covalently modifying a lysine residue in, or in proximity to, a ligandbinding
site of a protein, the method comprising:
A) contacting a compound of Formula I :
Warhead Tether Scaffold Tether Warhead
x y
I
with a protein containing a lysine residue in, or in proximity to, a ligand-binding site of a
polyprotein;
B) forming a covalent bond between the side chain primary amine group of
the lysine residue and Warhead of the compound;
wherein
Scaffold is
a) a radical resulting from the removal of a hydrogen of a ligand capable of
binding to, or in proximity to, the ligand-binding site; or
b) a portion of a pharmacophore of a ligand resulting from truncation of the
pharmacophore, such that the Scaffold is capable of binding to, or in proximity to, the
ligand-binding site;
Warhead is an organic moiety optionally containing one or more heteroatoms selected
from O, N, and S; the organic moiety having a molecular weight of about 14 daltons to about
200 daltons; Warhead being capable of reaction with a side chain primary amine group of a
lysine residue; and Warhead being attached to Scaffold through Tether;
Tether is null, a bond, or a bivalent C1-C15 saturated, unsaturated, straight, branched,
cyclic, bicyclic, or tricyclic hydrocarbon moiety; wherein optionally one or more methylene
units of the hydrocarbon chain are independently replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -
S0 2-, -C(=S)-, or C(=NRi)-; optionally one or more hydrogens are independently replaced by
heteroatoms; and optionally one or more methine groups of the C1-C15 alkyl, when present,
are independently replaced by
x is 0, 1, or 2;
y is 1, 2, or 3; and
Ri is hydrogen or Ci-C8 alkyl;
wherein the compound of Formula I is not a naturally occurring compound that exerts
its biological effect through an inherent ability to covalently bind lysine; nor is the compound
of Formula I a compound, natural or synthetic, which is based primarily on amino acids
nucleoside/nucleotide derived drugs, simple lysine alkylating agents, anhydrides, steroid
derived lysine alkylators, or sugar based lysine alkylators,
with the proviso that the compound of Fomula I is not
wortmannin:
known analogues of wortmannin that covalently modify lysine through substantially the same
mechanism as wortmannin:
-525-
any mechanism-based irreversible inhibitors.
The method of claim 92, wherein the compound of Formula I is a compound of
Scaffold Tether Warhead
G
94. The method of claim 93, wherein Warhead is a radical resulting from the removal
of a hydrogen of a compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, Im
I-n, I-o, I-p, I-q, I-r, I-s, and I-t:
each Xi and X is independently -0-, -S-, or -NR6
each X2 is independently -R , -OR6, or -NR6R7,;
each X is independently N or R ;
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C 6 alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; o re methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each n i and n2 is independently an integer from 0-2; ¾ is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n i0, n , and n i2 is an integer
from 0-1; and n is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
95. The method of claim 94, wherein as least one of R2 and R 3 of the compounds of
Formulas I-b and I-c is hydrogen.
96. The method of claim 94, wherein the compound of Formula I-a, I-d, I-e, I-j, I-k,
or I-l is a compound of Formula Il-a, Il-b, II-c, Il-d, Il-e, Il-f, Il-g, Il-h, II-i, II-j, Il-k, II-l,
Il-m, Il-n, II-o, II-p, Il-q, Il-r, II-s, Il-t, II-u, II-v, II-w, II-x, Il -y, II-z, H-aa, Il-bb, II-cc,
Il-dd, Il-ee, Il-ff, Il-gg, Il-hh, Il-ii, Il-jj, Il-kk, II-ll, Il-mm, Il-nn, II-oo, or II-pp;
wherein
each m is independently an integer from 0-4;
each m is independently an integer from 0-3;
each m4 is independently an integer from 0-5;
each n2 is independently an integer from 0-2;
each R2, R3, R4, R5, Re, R7, Rs, R9, Rio, R11, R12, R13, R14, and R15 is independently
gen or Ci-C alkyl; R is hydrogen, Ci-C alkyl, halogen, CF3, or nitro; wherein
more methylene groups of the Ci-C 6 alkyl can be replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO2-, or -C(=S)-; one or more methine groups of the Ci
alkyl, when present, can be independently replaced by ;
Ri is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, Rs, R9, Rio,
R1 1, R12 , Ri3, Ri4, and R15 when taken together form a 3- to 8-membered carbocyclic or
heterocyclic ring or an aryl or heteroaryl group.
97. The method of claim 94, wherein the compound of Formula I-d, or I-h is a compound
of Formula Ill-a, Ill-b, III-c, Ill-d, Ill-e, Ill-f, Ill-g, Ill-h, or Ill-i;
Ill-d
III-a Ill-b III-c
Ill-e Ill-f III-g
Ill-h Ill-i
wherein
n3 is an integer from 0-2;
each R2, R3, R4, R5, Re, R7, and Rg is independently hydrogen or Ci-C 6 alkyl;
each Bi, B2, B4, and B is independently CR7 or N and each B3 is N R7, O, or S;
each Rzl , R 2, R 3, R 4, and R is hydrogen, Ci-C alkyl, halogen, CF3, or nitro;
one or more methylene groups of the Ci-C 6 alkyl can be optionally replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6
alkyl, when present, can be independently replaced by
Ri is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, and R8 when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group.
98. The method of claim 94, wherein the compound of Formula I-h is a compound of
Formula IV-a, IV-b, IV-c, IV-d, IV-e, IV-f, IV-g, IV-h, or IV-i:
IV-f IV-g IV-h IV-i
wherein any of the substitutable hydrogens on the nitrogen heterocycle of the compound can
be substituted with alkyl, alkoxy, amido, acyl, acyloxy, oxoacyl, halogen.
99. The method of claim 94, wherein the radical resulting from the removal of a
hydrogen of a compound of Formula I-a, I-d, I-k, or I-m is a radical of Formula V-a, V-b,
V-c, V-d, V-e, V-f, V-g, V-h, V-i, or V-j;
V-a V-b V-c V-e
V-f V-g V-h V-i V-j
wherein
m i and m2 are each independently an integer from 0 to 2;
each R2, R3, R4, R , R6, R7, Rs, R9, Rio, and R is independently hydrogen or Ci-
C6 alkyl;
one or more methylene groups of the Ci-C 6 alkyl can be optionally replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO2-, or -C(=S)-; one or more methine groups of the Ci-C 6
alkyl, when present, can be independently replaced by N ;
R i is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, Rs, R9, Rio, and R
when taken together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or
heteroaryl group.
100. The method of claim 94, wherein the compounds of Formula I-a, I-b, I-c, I-d, I-e,
I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q, I-r, I-s, and I-t are selected from the
group consisting of:
JJ kk II
OOO
wherein any substitutable hydrogen may be substituted with the substituents as those defined
by R2-R8 in formulas I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q,
I-r, I-s, and I-t.
101 . The method of claim 94, wherein the radical resulting from the removal of a
hydrogen of a compound of Formula I-a, I-d, I-k, or I-m is a radical of Formula Vl-a, Vl-b,
I -c , Vl-d, Vl-e, Vl-f, Vl-g, Vl-h, Vl-i, Vl-j, Vl-k, VI-1, Vl-m, VI- h, VI-o, V I -p , or Vl-q:
VI-a VVll--bb VVII--cc V l -d Vl-e V l -f Vl-g
Vl-h VVll--ii VVll--ji Vl-k VI-1 Vl-m Vl-n
wherein R is hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, -CH2OCH3, and
-CH2CH2OCH3.
102. The method of claim 94, wherein Scaffold is selected from the group consisting of
Formulas VII, VIII, IX-a, IX-b, XI, XII, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII,
XXXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, and XXXVII.
103. A compound of Formula I :
Warhead Tether Scaffold Tether Warhead
x y
wherein
Scaffold is
a) a radical resulting from the removal of a hydrogen of a ligand capable of
binding to, or in proximity to, the ligand-binding site; or
b) a portion of a pharmacophore of a ligand resulting from truncation of the
pharmacophore, such that the Scaffold is capable of binding to, or in proximity to, the
ligand-binding site;
Warhead is an organic moiety optionally containing one or more heteroatoms selected
from O, N, and S; the organic moiety having a molecular weight of about 14 daltons to about
200 daltons; Warhead being capable of reaction with a side chain primary amine group of a
lysine residue; and Warhead being attached to Scaffold through Tether;
Tether is null, a bond, or a bivalent C1-C15 saturated, unsaturated, straight, branched,
cyclic, bicyclic, or tricyclic hydrocarbon moiety; wherein optionally one or more methylene
units of the hydrocarbon chain are independently replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -
S0 2-, -C(=S)-, or C(=NRi)-; optionally one or more hydrogens are independently replaced by
heteroatoms; and optionally one or more methine groups of the C1-C15 alkyl, when present,
are independently replaced by N ;
x is 0, 1, or 2;
y is 1, 2, or 3; and
Ri is hydrogen or Ci-C8 alkyl,
with the proviso that the compound of Fomula I is not
wortmannin:
-537-

o. alkene alkene
any mechanism-based irreversible inhibitors.
The compound of claim 103, wherein the compound of Fomula I is compound of
V
105. The compound of claim 104, wherein Warhead is a radical resulting from the
removal of a hydrogen of a compound of Formula I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j,
I-k, 1-1, 1-m, I-n, I-o, I-p, I-q, I-r, I-s, and I-t:
wherein
each X i and X is independently -0-, -S-, or -NR 6-;
each X2 is independently -R , -OR 6, or -NR 6R7,;
each X is independently N or R ;
each R2, R3, R4, R , R6, R7, and Rg is independently hydrogen or Ci-C alkyl; wherein
one or more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and R when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group;
A and B are each independently an optionally substituted monocyclic, bicyclic, or
tricyclic aryl or heteroaryl; and
n is an integer from 2-4; each ni and n2 is independently an integer from 0-2; n3 is an
integer from 1-2; n4 is an integer from 1-3; and each one of n , n^, , and ni2 is an integer
from 0-1; and ni3 is an integer from 0-2, wherein when any one of the foregoing n integers is
more than 1, the adjacent carbons represented by the integer can form a single or double
bond.
106. The compound of claim 105, wherein as least one of R2 and R3 of the compounds
of Formulas I-b and I-c is hydrogen.
107. The compound of claim 105, wherein the compound of Formula I-a, I-d, I-e, I-j,
I-k, or I-l is a compound of Formula Il-a, Il-b, II-c, Il-d, Il-e, Il-f, Il-g, Il-h, II-i, II-j, Il-k,
II-l, Il-m, Il-n, II-o, II-p, Il-q, Il-r, II-s, Il-t, II-u, II-v, II-w, II-x, Il -y, II-z, Il-aa, Il-bb,
II-cc, Il-dd, Il-ee, Il-ff, Il-gg, Il-hh, Il-ii, Il-jj, Il-kk, II-ll, Il-mm, Il-nn, II-oo, or II-pp;
wherein
each m is independently an integer from 0-4;
each is independently an integer from 0-3;
each m4 is independently an integer from 0-5;
each n2 is independently an integer from 0-2;
each R2, R3, R , R , R6, R7, Rs, Rs>, Rio, Rii, R12, R13, R14, and R15 is independently
hydrogen or Ci-C6 alkyl; R is hydrogen, Ci-C6 alkyl, halogen, CF3, or nitro; wherein
one or more methylene groups of the Ci-C6 alkyl can be replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C6
alkyl, when present, can be independently replaced by N ;
Ri is hydrogen or Ci-C8 alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, Rs, R9, Rio,
R1 1, R12 , Ri3, Ri4, and R15 when taken together form a 3- to 8-membered carbocyclic or
heterocyclic ring or an aryl or heteroaryl group.
108. The compound of claim 105, wherein the compound of Formula I-d, or I-h is a
compound of Formula Ill-a, Ill-b, III-c, Ill-d, Ill-e, Ill-f, Ill-g, Ill-h, or Ill-i;
wherein
n3 is an integer from 0-2;
each R2, R3, R4, R5, R , R7, and Rg is independently hydrogen or Ci-C6 alkyl;
each Bi, B2, B4, and B is independently CR7 or N and each B3 is NR7, O, or S;
each Rzl , R 2, R 3, R 4, and R is hydrogen, Ci-C alkyl, halogen, CF3, or nitro;
one or more methylene groups of the Ci-C 6 alkyl can be optionally replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6
alkyl, when present, can be independently replaced by
Ri is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, and R8 when taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group.
109. The compound of claim 105, wherein the compound of Formula I-h is a
compound of Formula IV-a, IV-b, IV-c, IV-d, IV-e, IV-f, IV-g, IV-h, or IV-i:
IV-a IV-b IV-c IV-d IV-e
IV-f IV-g IV-h IV-i
wherein any of the substitutable hydrogens on the nitrogen heterocycle of the compound can
be substituted with alkyl, alkoxy, amido, acyl, acyloxy, oxoacyl, halogen.
110. The compound of claim 105, wherein the radical resulting from the removal of a
hydrogen of a compound of Formula I-a, I-d, I-k, or I-m is a radical of Formula V-a, V-b,
V-c, V-d, V-e, V-f, V-g, V-h, V-i, or V-j;
V-a V-b V-c V-e
V-f V-g V-h V-i V-j
wherein
mi and m2 are each independently an integer from 0 to 2;
each R2, R3, R4, R , R6, R7, Rs, R9, Rio, and R is independently hydrogen or Ci-
C6 alkyl;
one or more methylene groups of the Ci-C6 alkyl can be optionally replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-Cg alkyl; and
optionally when proper any two of R2, R3, R4, R5, R6, R7, Rs, R9, Rio, and R
when taken together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or
heteroaryl group.
111. The compound of claim 105, wherein the compounds of Formulae I-a, I-b, I-c, Id,
I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q, I-r, I-s, and I-t are selected from
the group consisting of:
JJ kk II
OOO
wherein any substitutable hydrogen may be substituted with the substituents as those defined
by R2-R8 in formulas I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, I-m, I-n, I-o, I-p, I-q,
I-r, I-s, and I-t.
112. The compound of claim 105, wherein the radical resulting from the removal of a
hydrogen of a compound of Formula I-a, I-d, I-k, or I-m is a radical of Formula Vl-a, Vl-b,
VI-c, Vl-d, l - e , Vl-f, Vl-g, Vl-h, Vl-i, Vl-j, Vl-k, VI-1, Vl-m, VI- h, VI-o, V I -p , or Vl-q:
Vl-a VVll- b VVII--cc Vl-d Vl-e Vl-f Vl-g
Vl-h VVll--ii VVll--ji Vl-k VI-1 Vl-m Vl-n
wherein R is hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, -CH2OCH3, and
-CH2CH2OCH3.
113. The compound of claim 104, wherein Scaffold is selected from the group
consisting of Formulas VII, VIII, IX-a, IX-b, XI, XII, XVI, XVII, XVIII, XIX, XX, XXI,
XXII, XXIII, XXXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, and XXXVII.
114. The compound of claim 105, wherein Scaffold is a radical resulting from the
removal of one or more hydrogens of a compound of Formula VII:
wherein V and W are each independently -(CRi4Ri5) qX3(CRi6Ri7)r-;
q and r are each independently 0, 1, 2, 3, or 4;
X3 is -CRigRisr, or -NR 2o-; and
Rx, Ry, R12, Ri3, Ri4, Ri5, Ri6, Ri7, Ri8, Ri9, and R2o are each independently
hydrogen or Ci-C 6 alkyl; wherein one or more methylene groups of the Ci-C 6 alkyl can be
replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, -C(=S)-, optionally substituted aryl or
heteroaryl groups; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by NI ; and
Ri is hydrogen or Ci-C 8 alkyl.
115. The compound of claim 114, wherein the compound of Formula VII is a
compound of Formula Vll-a:
wherein
V and W are each independently -(CRi 4Ri5)qX3(CR
p, q and r are each independently 0, 1, 2, 3, or 4;
X3 is -CRigRisr, or -NR20-; and
R21 and R22 are each independently hydrogen or Ci-C 6 alkyl;
R23 is hydrogen, Ci-C 6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-
-SO2-, or -C(=S)-; one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by
N
R i is hydrogen or Ci-Cg alkyl; and
optionally R21 and R23 taken together can form a 4- to 8-membered
carbocyclic or heterocyclic ring.
The compound of claim 105, wherein the compound of Formula I ' is a compound
wherein
V and W are each independently -(CRi4Ri5) qX3(CRi6Ri7 )r-;
p, q and r are each independently 0, 1, 2, 3, or 4;
X3 is -CRigRisr, or -NR20-; and
R21 and R22 are each independently hydrogen or Ci-C alkyl;
R23 is hydrogen, Ci-C 6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-SO2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
NI
R i is hydrogen or Ci-C 8 alkyl; and
optionally R2i and R23 taken together can form a 4- to 8-membered
carbocyclic or heterocyclic ring;
T is Tether; and
Rw is Warhead.
117. The compound of claim 116, wherein the compound of Formula Vll-b is a
compound of Formula Vll-h;
118. The compound of claim 117, wherein the compound of Formula Vll-h is a
compound of Formula Vll-j, Vll-k, VII-1, Vll-m, Vll-n, or VII-o:
wherein
each R2, R3, R4, and R is independently hydrogen or Ci-C6 alkyl;
one or more methylene groups of the Ci-C6 alkyl can be optionally replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
one or more methine groups of the Ci-C6 alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-C8 alkyl;
119. The compound of claim 117, wherein the compound of Formula Vll-h is selected
from the group consisting of:
-554-
-555-
VII-34 VII-38
VII-49
120. The compound of claim 105, wherein Scaffold is a radical resulting from the
removal of a hydrogen of a compound of Formula VIII:
wherein
s is 0, 1, 2, 3, or 4; and
R12, Ri3, R21, R22, R24, R25, R26, R27, R28, R29, R30, R31, R32, and R33 are each
independently hydrogen or Ci-C 6 alkyl;
R23 is hydrogen, Ci-C 6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-SO 2-, or -C(=S)-; one or more methine groups of the Ci-C alkyl, when present, can be
independently replaced by N ;
Ri is hydrogen or Ci-Cg alkyl; and
optionally R2i and R23 taken together can form a 4- to 8-membered carbocyclic or
heterocyclic ring.
121 . The compound of claim 104, wherein the compound of Formula I' is a compound
of Formula Vlll-a or Vlll-b:
wherein
s is 0, 1, 2, 3, or 4;
R12, Ri3, R21, R22, R24, R25, R26, R27, R28, R29, R30, R31, R32, and R33 are each
independently hydrogen or Ci-C alkyl;
R23 is hydrogen, Ci-C 6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by ;
Ri is hydrogen or Ci-C 8 alkyl; and
optionally R2i and R23 taken together can form a 4- to 8-membered carbocyclic or
heterocyclic ring;
T is Tether; and
Rw is Warhead.
122. The compound of claim 121, wherein the compound of Formula VHI-a or Vlll-b
is selected from the group consisting of:
VIII -7
123. The compound of claim 105, wherein Scaffold is a radical resulting from the
removal of a hydrogen of a compound of Formula IX-a or IX-b:
IX-a IX-b
wherein
X5 is -0-, -CR42R43- or -NR42-
Rl2, Rl3, R27, R28, R2 , R30, R31, R32, R33, R34, R35, R36, R37, R38, R3 , R40, R4I, R42,
and R43 are each independently hydrogen or Ci-C 6 alkyl; wherein one or more methylene
groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -
C(=S)-;
Ri is hydrogen or Ci-C 8 alkyl; one or more methine groups of the Ci-C alkyl,
when present, can be independently replaced by N ; Ri is hydrogen or Ci-Cg alkyl;
D, E, F, G, and H are each independently optionally substituted aryl or heteroaryl;
wherein F and G are fused together to form a bicyclic optionally substituted aryl or
heteroaryl.
124. The compound of claim 105, wherein the compound of Formula I ' is a compound
of Formula IX-c or IX-d:
wherein
Ri2, Ri3, and R31 are each independently hydrogen or Ci-C6 alkyl; wherein one or
more methylene groups of Ci-C6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-,
-SO-, -S0 2-, or -C(=S)-;
Ri is hydrogen or Ci-C8 alkyl; one or more methine groups of the Ci-C alkyl,
when present, can be independently replaced by N ; Ri is hydrogen or Ci-Cg alkyl;
F, G, and H are each independently optionally substituted aryl or heteroaryl;
wherein F and G are fused together to form a bicyclic optionally substituted aryl or
heteroaryl;
T is Tether; and
Rw is Warhead.
125. The compound of claim 105, wherein the compound of Formula I ' is a compound
of Formula XI:
wherein
p is an integer from 0 to 4, u is an integer from 1 to 4;
B and B are each independently CR or N;
R 6 is hydrogen, Ci-C 6 alkyl, halogen, amino, nitro, or -NH(CO)NR R79;
R 0 is hydrogen, Ci-C alkyl, halogen, amino, nitro;
R , R7 1, R 2, R73, R74, R75, R76, R77, R78, and R are each independently hydrogen
or Ci-C 6 alkyl; Ri is hydrogen or Ci-Cg alkyl; wherein one or more methylene groups of the
Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more
methine groups of the Ci-C alkyl, when present, can be independently replaced by ¾ ¾
optionally R , and R7 taken together form a 4- to 8-membered carbocyclic or
heterocyclic ring.
126. The compound of claim 125, wherein the compound of Formula XI is a
compound of Formula Xl-a, CI-b, or XI-c;
wherein
R70 is -F, -CI, -Br, or -I;
T is Tether; and
Rwh is Warhead.
127. The compound of claim 126, wherein the compound of Formula Xl-a, CI-b or
XI-c is a compound of Formula Xl-d, CI-e, Xl-f, CI-g, Xl-h, CI-i, or Xl-j:
wherein
R2 , R3 , R4, R 5, R6, R7, R80, Rsi, R82, R 83 R84, R R86, a d R 7 are each
independently hydrogen or Ci-C 6 alkyl; R i is hydrogen or Ci-Cg alkyl;
one or more methylene groups of the Ci-C alkyl can be replaced by -NRr, -O-
-C(O)-, -S-, -SO-, -SO2-, or -C(=S)-;
one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
128. The compound of claim 127, wherein the compound of Formula Xl-d, CI-e, Xl-f,
Xl-g, CI-h, Xl-i, or CI-j is a compound of Formula CI-k, XI-1, CI-m, Xl-n, XI-o, XI-p, or
xi-q;
wherein
X is CH2, NH, O, or S;
R88, and R8 are each independently hydrogen or Ci-C alkyl; Ri is hydrogen or
Ci-C8 alkyl;
one or more methylene groups of the Ci-C alkyl can be replaced by -NRi-, -0-,
-C(0)-, -S-, -SO-, -S0 2-, or -C(=S)s
one or more methine groups of the Ci-Ce alkyl, when present, can be
independently replaced by N ; and
n is an integer from 0 to 3.
129. The compound of claim 127, wherein the compound of Formula Xl-d, CI -e, Xl-f,
CI -g, Xl-h, CI -i, or CI -j is a compound of Formula CI -r, XI-s, CI -t, XI-u, XI-v, XI-w, or
-x:
X l-x
The compound of claim 127, wherein the compound of Formula Xl-d, Xl-e, XI-f,
CI-g, Xl-h, CI-i, or CI-j is a compound of Formula CI-y, XI-z, CI-aa, or Xl-bb:
131. The compound of claim 127, wherein the compound of Formula Xl-d, Xl-e, Xl-f,
CI-g, Xl-h, CI-i, or CI-j is a compound of Formula XI-cc, Xl-dd, Xl-ee, or Xl-ff:
XI-cc Xl-dd
Xl-ee Xl-ff
The compound of claim 126, wherein the compound of Formula XI is selected
the group consisting of:
-570-

-572-
XI-35 XI-36
XI-37 XI-38
XI-39 XI-40
XI-45 XI-46
-575-
The compound of claim 105, wherein the compound of Formula I ' is a compound
XII
wherein
Ri and R2 are each independently hydrogen or Ci-Cs alkyl; wherein one or more
methylene groups of the Ci-C alkyl can be replaced by -NRr, -0-, -C(O)-, -S-, -SO-, -S0 2-,
or -C(=S)-; one or more methine groups of the Ci-C6 alkyl, when present, can be
independently replaced by N ;
T is Tether; and
Rw is Warhead.
134. The compound of claim 105, wherein the compound of Formula I' is a compound
of Formula XXXVI:
wherein
R is H, optionally substituted C1-C3 branched or straight chain alkyl, or optionally
substituted C1-C3 branched or straight chain acyl;
T is Tether; and
Rw is Warhead.
135. The compound of claim 134, wherein the compound of Formula XXXVI is selected
from the group consisting of:
XXXVI-1 XXXVI-2
136. The compound of claim 105, wherein Scaffold is a radical resulting from the
removal of a hydrogen of a compound of Formula XVI-a, XVI-b, or XVI-c:
wherein
R90, R91, R92, R93, R94, R95, R 6, R97, R98, R99, R100, R102, R104, Rl05, Rl06, R107,
R108, R109, R 110, R11 1, R 112, R 113, and R 4 are each independently hydrogen or Ci-C6 alkyl;
wherein one or more methylene groups of Ci-C6 alkyl can be optionally replaced by -NRr,
-0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
R103 is hydrogen, Ci-C6 alkyl, or C2-C alkenyl;
one or more methine groups of the Ci-C6 alkyl, when present, can be
independently replaced by NI ;
Ri is hydrogen or Ci-C 8 alkyl;
each R101 and R101 is independently hydrogen, Ci-C6 alkyl, C2-C alkenyl,
halogen, amino, nitro, optionally substituted aryl or heteroaryl;
n6 and n are each independently integer from 0 to 4; and
n is an integer from 0 to 2.
137. The compound of claim 105, wherein the compound of Formula I' is a compound
of Formula XVI-d, XVI-e, or XVI-f.
XVI-d
XVI-f
wherein
R90 and R 114 are each independently hydrogen or Ci-C 6 alkyl;
R103 is hydrogen, Ci-C 6 alkyl, or C2-C8 alkenyl;
Ri is hydrogen or Ci-Cg alkyl;
Rioi is hydrogen, Ci-C 6 alkyl, C2-C8 alkenyl, halogen, amino, nitro, optionally
substituted aryl or heteroaryl; and
n6 is an integer from 0 to 4;
n is an integer from 0 to 2;
wherein one or more methylene groups of the Ci-C 6 alkyl can be replaced by -
NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6
alkyl, when present, can be independently replaced by and
Ri is hydrogen or Ci-Cg alkyl.
138. The compound of claim 137, wherein the compound of Formula XVI-d, XVI-e,
-f is a compound of Formula XVI-g, XVI-h, or XVI-i:
wherein
each R2, R3, R4, and R is independently hydrogen or Ci-C 6 alkyl; wherein one or
more methylene groups of the Ci-C 6 alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can be
independently replaced by
Ri is hydrogen or Ci-C 8 alkyl;
wherein optionally when proper any two of R2, R3, R4, R5, R , R7, and Rgwhen taken
together form a 3- to 8-membered carbocyclic or heterocyclic ring or an aryl or heteroaryl
group; and optionally X2 and any one of R2, R3, and R 4 when taken together form a 3- to 8-
membered carbocyclic or heterocyclic ring or an aryl or heteroaryl group.
The compound of claim 137, wherein the compound of Formula I ' is selected
the group consisting of:
XVI-1 XVI-2

-584-
-585-
-586-
XVI-23 XVI-24
XVI-25
140. The compound of claim 105 wherein the compound of Formula I ' is a compound
of Formula XXII-a Formula XXII-b, or Formula XXII-c:
wherein
n, m, p, and q for Formula XXII-a are each independently 0, 1, 2, 3; provided that n
and q are not 0 at the same time, and m and q are not 0 at the same time;
A2 is an optionally substituted ring selected from a 4-8 membered saturated or
partially unsaturated heterocyclic ring having one or two heteroatoms independently selected
from nitrogen, oxygen, or sulfur, or a 5-10 membered saturated or partially unsaturated
bridged bicyclic heterocyclic ring having at least one nitrogen, at least one oxygen, and
optionally 1-2 additional heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
B is an optionally substituted group selected from phenyl, an 8- to 10-membered
bicyclic aryl ring, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or -T-Rwh;
and
C2 is hydrogen or an optionally substituted ring selected from a 3- to 7-membered
saturated or partially unsaturated carbocyclic ring, a 7- to 10-membered saturated or partially
unsaturated bicyclic carbocyclic ring, a 4- to 7-membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 7- to 10-membered saturated or partially unsaturated bicyclic heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, phenyl, an
8- to 10-membered bicyclic aryl ring, a 5- to 6-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-to 10-membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur;
T is Tether; and
Rw is Warhead.
141 . The compound of claim 140, wherein the compound of Formula XXII-a, XXII-b,
XXII -c is selected from the group consisting of:
-591-
XXII-17 XXII-18

-594-
-595-
142. The compound of claim 105, wherein the compound of Formula I' is a compound
of Formula XXIII:
XXIII
wherein:
Rw is Warhead;
R201 is hydrogen or Ci_6 alkyl;
R202 is hydrogen or an optionally substituted group selected from Ci_6 alkyl, Ci_6
alkoxy, or (C1-6 alkylene)-R 203; or
R201 and R202 are taken together with the intervening carbon to form an optionally
substituted ring selected from a 3- to 7-membered carbocyclic ring or a 4- to 7-membered
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
R203 is a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, a 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or a 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; and
Ring A6 is absent or an optionally substituted group selected from a 4- to 7-membered
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, or a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected
from nitrogen, oxygen, or sulfur.
143. The compound of claim 142, where the compound of Formula XXIII is selected
from the group consisting of:
-598-
144. The compound of claim 105, wherein the compound of Formula I is a compound
of Formula XXIV-a or Formula XXIV-b:
XXIV-a XXIV-b
wherein
Rw is Warhead;
R2 o4 is an hydrogen or an optionally substituted group selected from Ci_ aliphatic, -
(CH2)m-(3- to 7-membered saturated or partially unsaturated carbocyclic ring), -(CH2)m-(7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring), -(CH2)m-(4- to 7-
membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur), -(CH2)m-(7- to 10-membered
saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur), -(CH2)m-phenyl, -(CH2)m-(8- to 10-
membered bicyclic aryl ring), -(CH2)m-(5- to 6-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur), or -(CH2)m-(8- to 10-
membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur);
each R205 and R206 is independently -R", halogen, -N0 2, -CN, -OR", -SR",
-N(R") 2, -C(0)R", -C0 2R", -C(0)C(0)R", -C(0)CH 2C(0)R", -S(0)R", -S(0) 2R",
-C(0)N(R") 2, -S0 2N(R") 2, -OC(0)R", -N(R")C(0)R", -N(R")N(R") 2,
-N(R")C(=NR")N(R") 2, -C(=NR")N(R") 2, -C=NOR", -N(R")C(0)N(R") 2,
-N(R")S0 2N(R") 2, -N(R")S0 2R", or -OC(0)N(R") 2;
each R" is independently hydrogen or an optionally substituted group selected from Ci_6
aliphatic, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to 10-
membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, an 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; or
two R" groups on the same nitrogen are taken together with the nitrogen to which they
are attached to form an optionally substituted 5-8 membered saturated, partially unsaturated,
or aromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
m is an integer from 0 to 6, inclusive;
each n for Formula XXIV-a or Formula XXIV-b is independently 0, 1, or 2; and
Ring A5 is an optionally substituted 6-membered heterocyclic or heteroaryl ring having 1-
2 nitrogens.
145. The compound of claim 144, wherein the compound of Formula XXIV-a or
F -b is selected from the group con isting of
XXIV-1 XXIV-2
XXIV-3
146. The compound of claim 105, wherein the compound of Formula G is a compound
of Formula XXV:
or a pharmaceutically acceptable salt thereof;
wherein
each R205 and R206 is independently -R", halogen, -N0 2, -CN, -OR", -SR", -N(R") 2,
-C(0)R", -C0 2R", -C(0)C(0)R", -C(0)CH 2C(0)R", -S(0)R", -S(0) 2R", -C(0)N(R") 2,
-S0 2N(R") 2, -OC(0)R", -N(R")C(0)R", -N(R")N(R") 2, -N(R")C(=NR")N(R") 2,
-C(=NR")N(R") 2, -C=NOR", -N(R")C(0)N(R") 2, -N(R")S0 2N(R") 2, -N(R")S0 2R", or
-OC(0)N(R") 2;
each R" is independently hydrogen or an optionally substituted group selected from
Ci_6 aliphatic, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, an 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; or
optionally, two R" groups on the same nitrogen are taken together with the nitrogen to
which they are attached to form an optionally substituted 5-8 membered saturated, partially
unsaturated, or aromatic ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur;
m is an integer from 0 to 6, inclusive;
each nis independently 0, 1, or 2;
Ring A5 is an optionally substituted 6-membered heterocyclic or heteroaryl ring
having 1-2 nitrogens; and
Rw is Warhead.
147. The compound of claim 146, wherein the compound of Formula XXV is selected
from the group consisting of:
XXV-13 XXV-14 XXV-15
148. The compound of claim 105, wherein the compound of Formula I' is a compound of
Formula XXVII:
XXVII
pharmaceutically acceptable salt thereof;
wherein:
T is Tether;
Rwh is Warhead; and
R is H, alkyl, or alkoxy.
149. The compound of claim 148, wherein the compound of Formula XXVII is selected
fr m the group consisting of:
150. The compound of claim 105, wherein the compound of Formula I ' is a compound of
Formula XXXVII:
XXXVII
or a pharmaceutically acceptable salt thereof;
wherein
T isTether; and
Rw is Warhead.
151. The compound of claim 150, wherein the compound of Formula XXXVII is
selected from the group consisting of:
XXXVII-1 XXXVII-2
XXXVII-5
152. Protein-modifier-ligand conjugates of the Formula XIII,
XIII
wherein
Scaffold is
a) a radical resulting from the removal of a hydrogen of a ligand capable of
binding to, or in proximity to, the ligand-binding site; or
b) a portion of a pharmacophore of a ligand resulting from truncation of the
pharmacophore, such that the Scaffold is capable of binding to, or in proximity to, the
ligand-binding site;
Warhead is an organic moiety optionally containing one or more heteroatoms selected
from O, N, and S; the organic moiety having a molecular weight of about 14 daltons to about
200 daltons; Warhead being capable of reaction with a side chain primary amine group of a
lysine residue; and Warhead being attached to Scaffold through Tether; and
Tether is null, a bond, or a bivalent C1-C15 saturated or unsaturated, straight,
branched, or cyclic hydrocarbon moiety, an aryl moiety or heteroaryl moiety, and, optionally,
one or more methylene units of the hydrocarbon chain are independently replaced by -NRi-, -
0-, -C(O)-, -S-, -SO-, -SO 2-, -C(=S)-, or -C(=NRi)-; and one or more methine groups of the
Ci-C 6 alkyl, when present, can be independently replaced by
x is 0, 1, or 2;
y is 1, 2, or 3;
Ri is hydrogen or Ci-Cg alkyl; and
Yi is a bivalent or trivalent moiety resulting from the removal of a hydrogen of a
radical of Formula XlV-a, XlV-b, XIV-c, XlV-d, XlV-e, XlV-f, XlV-g, XlV-h, or XlV-i,
wherein
each Xi and X2 is independently -CR2R3R4, -OR 2, or -NR 2R3;
each R2, R3, R4, R5, Re, R7, and Rg is independently hydrogen or Ci-C 6
alkyl;
optionally when proper any two of R2, R3, R4, R5, R6, R7, and Rg can
be linked together to form a 3- to 8-membered carbocyclic or heterocyclic ring;
one or more methylene groups of the Ci-C 6 alkyl can be replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6
alkyl, when present, can be independently replaced by N ; and
n is an integer from 2-4, rri4 is an integer from 1to 2;
M is connected to the position labeled as "*";
is a single or a double bond;
A is an optionally substituted aryl or heteroaryl;
a hydrogen of a radical of Formula XlV-a, XlV-b, XIV-c, XlV-d, XlV-e, XlV-f,
XlV-g, XlV-h, or XlV-i, is substituted by Tether-Scaffold; and
M is -NH- or =N-, the nitrogen atom of M being a nitrogen from the side chain
primary amine group of the lysine residue of the polypeptide.
153. The conjugate of claim 152, wherein the conjugate of Formula XIII, is a
conjugate of Formula XIII':
Scaffold Tether Y, = M Protein
XIII'
154. The conjugate of claim 153, wherein Scaffold is selected from the group
consisting of Formulas VII, VIII, IX-a, IX-b, XI, XII, XVI, XVII, XVIII, XIX, XX, XXI,
XXII, XXIII, XXXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, and XXXVII.
155. The conjugate of claim 153, wherein M(CH2 ) 4-Protein is selected from the group
consisting of M(CH2)4-K1236-HCV-NS3, M(CH2)4-K2016-HCV-NS3, M(CH2)4-K2560-
HCV-NS3, M(CH2)4-K191-(Baculoviral IAP repeat-containing protein 1), M(CH2)4-K199-
(Baculoviral IAP repeat-containing protein 1), M(CH2)4-K305-(Baculoviral IAP repeatcontaining
protein 2), M(CH2)4-K291-(Baculoviral IAP repeat-containing protein 3),
M(CH2)4-K297-(Baculoviral IAP repeat-containing protein 4), M(CH2)4-K299-(Baculoviral
IAP repeat-containing protein 4), M(CH2)4-K3 1l-(Baculoviral IAP repeat-containing protein
4), M(CH2)4-K062-(Baculoviral IAP repeat-containing protein 5), M(CH2)4-K079-
(Baculoviral IAP repeat-containing protein 5), M(CH2)4-K121-(Baculoviral IAP repeatcontaining
protein 7), M(CH2)4-K135-(Baculoviral IAP repeat-containing protein 7),
M(CH2)4-K146-(Baculoviral IAP repeat-containing protein 7), M(CH2)4-K036-(Baculo viral
IAP repeat-containing protein 8), M(CH2)4-K050-(Baculo viral IAP repeat-containing protein
8), M(CH2)4-K061-(Baculoviral IAP repeat-containing protein 8), M(CH2)4-K776-
(Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha isoform), M(CH2)4-
K802-(Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha isoform),
M(CH2)4-K777-(Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta
isoform), M(CH2)4-K805-(Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit
beta isoform), M(CH2)4-K802-(Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic
subunit gamma isoform), M(CH2)4-K807-(Phosphatidylinositol-4,5-bisphosphate 3-kinase
catalytic subunit gamma isoform), M(CH2)4-K833-(Phosphatidylinositol-4,5-bisphosphate 3-
kinase catalytic subunit gamma isoform), M(CH2)4-K890-(Phosphatidylinositol-4,5-
bisphosphate 3-kinase catalytic subunit gamma isoform), M(CH2)4-K086-(3-
phosphoinositide-dependent protein kinase 1), M(CH2)4-K163-(3-phosphoinositidedependent
protein kinase 1), M(CH2) 4-K169-(3-phosphoinositide-dependent protein kinase
1), and M(CH2)4-K207-(3-phosphoinositide-dependent protein kinase 1).
156. The conjugate of claim 153, wherein Tether is null, a bond, or a bivalent C1-C15
saturated or unsaturated, straight, branched, or cyclic hydrocarbon moiety, and, optionally,
one or more methylene units of the hydrocarbon chain are independently replaced by -NRi-, -
0-, -C(O)-, -S-, -SO-, -SO2-, -C(=S)-, or -C(=NRi)-; and one or more methine groups of the
Ci-C6 alkyl, when present, can be independently replaced by N ; and
Ri is hydrogen or Ci-Cg alkyl.
157. The conjugate of claim 153, wherein the bivalent or trivalent moiety resulting
from the removal of a hydrogen of a radical of Formula XlV-a, XlV-d, XlV-h, or XlV-i is a
moiety of Formula XV-a, XV-b, XV-c, XV-d, XV-e, XV-f, or XV-g;
wherein
m4 is an integer from 1 to 2;
each R2, R3, R4, R5 and R6 is independently hydrogen or Ci-C6 alkyl; wherein
optionally when proper any two of R2, R3, R4, R5 and R6 can be linked
together to form a 3- to 8-membered carbocyclic or heterocyclic ring; and
one or more methylene groups of the Ci-C6 alkyl can be replaced by
-NRi-, -0-, -C(O)-, -S-, -SO-, -S0 2-, or -C(=S)-;
one or more methine groups of the Ci-Ce alkyl, when present, can be
independently replaced by N ;
M is connected to the position of Yi labeled as "*"; and
Tether is connected to the position of Yi labeled as "**".
158. The conjugate of claim 157, wherein the bivalent moiety of Formula XV-a, XV-b,
XV-c, XV-d, XV-e, XV-f, or XV-g is a bivalent moiety of Formula XV-h, XV-i, XV-j, XVk,
XV-1, XV-m, XV-n, XV-o, XV-p, XV-q, XV-r, XV-s, or XV-t;
wherein
M is connected to the position of Y1 labeled as "*"; and
Tether is connected to the position of Yi labeled as "**".
159. A method of treating an XIAP-mediated disorder in a patient comprising the step of
irreversibly inhibiting XIAP by covalently modifying a lysine residue conserved at an
equivalent position of K297 of XIAP.
160. A conjugate of the formula K297-linker-inhibitor moiety, wherein the K297 is K297
ofXIAP.
161 . The conjugate of claim 160, wherein the inhibitor moiety is of the Formula A:
A
wherein
V and W are each independently -(CRi4Ri5)qX3(CRi6Ri7)rS
p, q and r are each independently 0, 1, 2, 3, or 4;
X 3 is -CRigRisr, or -NR20-; and
R21 and R22 are each independently hydrogen or Ci-C alkyl;
R23 is hydrogen, Ci-C6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-SO2-, or -C(=S)-; one or more methine groups of the Ci-C6 alkyl, when present, can be
independently replaced by NI ;
Ri is hydrogen or Ci-C8 alkyl; and
optionally R21 and R23 taken together can form a 4- to 8-membered
carbocyclic or heterocyclic ring.
The conjugate of claim 160, wherein the inhibitor moiety is of the Formula B
B
wherein
p and s are each independently 0, 1, 2, 3, or 4;
R12, Ri3, R21, R22, R24, R25, R26, R27, R28, R29, R30, R31, R32, and R33 are each
independently hydrogen or Ci-C 6 alkyl;
R23 is hydrogen, Ci-C 6 alkyl, halogen, amino, or nitro; wherein one or more
methylene groups of Ci-C alkyl can be optionally replaced by -NRr, -0-, -C(O)-, -S-,
-SO 2-, or -C(=S)-; one or more methine groups of the Ci-C 6 alkyl, when present, can b <
independently replaced by
Ri is hydrogen or Ci-Cg alkyl; and
optionally R2i and R23 taken together can form a 4- to 8-membered carbocyclic or
heterocyclic ring.
163. A method of treating an PDPKl -mediated disorder in a patient comprising the step of
irreversibly inhibiting PDPKl by covalently modifying a lysine residue conserved at an
equivalent position K169 of PDPKl.
164. A method of treating an PDPKl -mediated disorder in a patient comprising the step of
irreversibly inhibiting PDPKl by covalently modifying a lysine residue conserved at an
equivalent position K173 of PDPKl.
165. A method of treating an PDPKl -mediated disorder in a patient comprising the step of
irreversibly inhibiting PDPKl by covalently modifying a lysine residue conserved at an
equivalent position K86 of PDPKl.
166. A conjugate of the formula K169-linker-inhibitor moiety, wherein the K169 is K169
of PDPKl.
167. A conjugate of the formula K173-linker-inhibitor moiety, wherein the K173 is K173
of PDPKl.
168. A conjugate of the formula K86-linker-inhibitor moiety, wherein the K86 is K86 of
PDPKl.
169. The conjugate of claims 166-168, wherein the inhibitor moiety is of the Formula C:
C
wherein
B and B are each independently CR or N;
is hydrogen, Ci-C alkyl, halogen, amino, nitro, or -NH(CO)NR 8R79;
R o is hydrogen, Ci-C6 alkyl, halogen, amino, nitro;
R , R7 1, R 2, R73, R74, R75, R76, R77, R78, and R are each independently hydrogen or
Ci-C6 alkyl;
Ri is hydrogen or Ci-C 8 alkyl; wherein one or more methylene groups of the Ci-C
alkyl can be replaced by -NRi-, -0-, -C(O)-, -S-, -SO-, -SO2-, or -C(=S)-; one or more
methine groups of the Ci-C6 alkyl, when present, can be independently replaced by
optionally R7 , and R taken together form a 4- to 8-membered carbocyclic or
heterocyclic ring; and
p is an integer from 0 to 4, u is an integer from 1 to 4.
The conjugate of claims 166-168, wherein the inhibitor moiety is of the Formula D
D
wherein R is H, optionally substituted C1-C3 branched or straight chain alkyl, or
optionally substituted C1-C3 branched or straight chain acyl.
171 . A method of treating an HCV protease-mediated disorder in a patient comprising the
step of irreversibly inhibiting HCV protease by covalently modifying a lysine residue
conserved at an equivalent position K136 of HCV-protease subtype of NS3/4A.
172. A conjugate of the formula K136-linker-inhibitor moiety, wherein the K136 is K136
ofNS3/4A.
173. The conjugate of claim 172, wherein the inhibitor moiety is of the Formula E, F, o r
G:
F
E
wherein
R90, R94, R95, 6, R97, R98, R99, R100, R102, R104, Rl05, Rl06, R107, Rl08, R109, R 110, R 111,
R 112, R 113, and R 114 are each independently hydrogen or Ci-C alkyl; wherein one or more
methylene groups of Ci-C 6 alkyl can be optionally replaced by -NRi-, -0-, -C(O)-, -S-, -SO-,
-S0 2-, or -C(=S)-;
R103 is hydrogen, Ci-C 6 alkyl, or C2-C alkenyl;
one or more methine groups of the Ci-C 6 alkyl, when present, can be independently
replaced by
R i is hydrogen or Ci-Cg alkyl;
each Rioi is independently hydrogen, Ci-C 6 alkyl, C2-C8 alkenyl, halogen, amino,
nitro, optionally substituted aryl or heteroaryl; n6 is an integer from 0 to 4; and n is an
integer from 0 to 2.
174. A method of treating an PI3K-mediated disorder in a patient comprising the step of
irreversibly inhibiting PI3K by covalently modifying a lysine residue conserved at an
equivalent position K777 of PI3K.
175. A conjugate of the formula K777-linker-inhibitor moiety, wherein the K777 is K777
o RBKb.
176. A method of treating an PI3K-mediated disorder in a patient comprising the step of
irreversibly inhibiting PI3K by covalently modifying a lysine residue conserved at an
equivalent position K802 of PI3K.
177. A conjugate of the formula K802-linker-inhibitor moiety, wherein the K802 is K802
ofPBKy.
178. A method of treating an PI3K-mediated disorder in a patient comprising the step of
irreversibly inhibiting PI3K by covalently modifying a lysine residue conserved at an
equivalent position K890 of RI3Kg.
179. A conjugate of the formula K890-linker-inhibitor moiety, wherein the K890 is K890
ofPBKy.
180. The conjugate of claim 175, 177, or 179 wherein the inhibitor moiety is of the
formula H, J, or K :
H J K
wherein
n, m, p, and q are each independently 0, 1, 2, 3; provided that n and q are not 0 at the
same time, and m and q are not 0 at the same time;
A2 is an optionally substituted ring selected from a 4-8 membered saturated or
partially unsaturated heterocyclic ring having one or two heteroatoms independently selected
from nitrogen, oxygen, or sulfur, or a 5-10 membered saturated or partially unsaturated
bridged bicyclic heterocyclic ring having at least one nitrogen, at least one oxygen, and
optionally 1-2 additional heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
B is an optionally substituted group selected from phenyl, an 8- to 10-membered
bicyclic aryl ring, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring
having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or -T-Rwh;
and
C2 is hydrogen or an optionally substituted ring selected from a 3- to 7-membered
saturated or partially unsaturated carbocyclic ring, a 7- to 10-membered saturated or partially
unsaturated bicyclic carbocyclic ring, a 4- to 7-membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or
sulfur, a 7- to 10-membered saturated or partially unsaturated bicyclic heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, phenyl, an
8- to 10-membered bicyclic aryl ring, a 5- to 6-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-to 10-membered
bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, or sulfur.
181. The conjugate of claim 175, 177, or 179 wherein the inhibitor moiety is of Formula L
or M:
L M
wherein
R-204 is an hydrogen or an optionally substituted group selected from Ci_6 aliphatic, -
(CH 2)m-(3- to 7-membered saturated or partially unsaturated carbocyclic ring), -(CH 2)m-(7- to
10-membered saturated or partially unsaturated bicyclic carbocyclic ring), -(CH 2)m-(4- to 7-
membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, or sulfur), -(CH2)m-(7- to 10-membered
saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, or sulfur), -(CH2)m-phenyl, -(CH2)m-(8- to 10-
membered bicyclic aryl ring), -(CH2)m-(5- to 6-membered heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur), or -(CH2)m-(8- to 10-
membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur);
each R205 and R206 is independently -R", halogen, -N0 2, -CN, -OR", -SR",
-N(R") 2, -C(0)R", -C0 2R", -C(0)C(0)R", -C(0)CH 2C(0)R", -S(0)R", -S(0) 2R",
-C(0)N(R") 2, -S0 2N(R") 2, -OC(0)R", -N(R")C(0)R", -N(R")N(R") 2,
-N(R")C(=NR")N(R") 2, -C(=NR")N(R") 2, -C=NOR", -N(R")C(0)N(R") 2,
-N(R")S0 2N(R") 2, -N(R")S0 2R", or -OC(0)N(R") 2;
each R" is independently hydrogen or an optionally substituted group selected from Ci_6
aliphatic, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a 7- to 10-
membered saturated or partially unsaturated bicyclic carbocyclic ring, a 4- to 7-membered
saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, a 7- to 10-membered saturated or partially
unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, phenyl, an 8- to 10-membered bicyclic aryl ring, a 5- to 6-
membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur; or
two R" groups on the same nitrogen are taken together with the nitrogen to which they
are attached to form an optionally substituted 5-8 membered saturated, partially unsaturated,
or aromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or
sulfur;
m is an integer from 0 to 6, inclusive;
each n is independently 0, 1, or 2; and
Ring A5 is an optionally substituted 6-membered heterocyclic or heteroaryl ring having 1-2
nitrogens.

WHAT IS CLAIMED IS:

1. A nutritional emulsion comprising fat, carbohydrate, protein, and calcium HMB wherein a soluble protein represents from 50% to 100% by weight of total protein in the emulsion.

2. The nutritional emulsion of claim 1 wherein the soluble protein represents from about 55% to 100% by weight soluble protein and includes phosphoserine-containing protein having at least about 100 mmoles of phosphoserine per kilogram of phosphoserine-containing protein.

3. The nutritional emulsion of claim 2 wherein the soluble protein represents from about 60% to 85% by weight soluble protein.

4. The nutritional emulsion of claim 1 wherein the soluble protein includes at least one protein selected from the group consisting of sodium caseinate, whey protein concentrate, and combinations thereof.

5. The nutritional emulsion of claim 1 wherein the emulsion comprises from about 0.3% to about 1.5% by weight of calcium HMB.

6. The nutritional emulsion of claim 1 wherein the emulsion is packaged in a hermtically sealed container and is shelf stable at a temperature of from at 18°C to 24° C for at least about 3 months.

7. A nutritional emulsion comprising fat, carbohydrate, protein, and calcium HMB wherein soluble protein comprises from about 50% to 100% by weight of total protein and the emulsion has a weight ratio of soluble protein to calcium HMB of at least about 5.0.

8. The nutritional emulsion of claim 7 wherein the soluble protein represents from about 55% to 100% by weight soluble protein and includes phosphoserine-containing protein having at least about 100 mmoles of phosphoserine per kilogram of phosphoserine-containing protein.

9. The nutritional emulsion of claim 7 wherein the soluble protein represents from about 60% to 85% by weight soluble protein.

10. The nutritional emulsion of claim 7 wherein the soluble protein includes at least one protein selected from the group consisting of sodium caseinate, whey protein concentrate, and combinations thereof.

1 1. The nutritional emulsion of claim 7 wherein the soluble protein comprises sodium caseinate.

12. The nutritional emulsion of claim 7 wherein the concentration of soluble protein is from about 1 % to about 26% by weight of the nutritional emulsion.

13. The nutritional emulsion of claim 7 wherein the emulsion comprises from about 0.3% to about 1.5% by weight of calcium HMB.

14. The nutritional emulsion of claim 7 wherein the weight ratio of soluble protein to calcium HMB is from about 7: 1 to about 10: 1.

15. The nutritional emulsion of claim 7 wherein the emulsion is packaged in a hermtically sealed container and is shelf stable at a temperature of from at 18°C to 24° C for at least about 3 months.