Methods for the manufacture of diketopiperazine derivatives

Abstract

Methods for producing substituted diketopiperazines, including fumaryl substituted amino alkyl diketopiperazines, with improved purity and desirable isomer content are described herein. The methods provide substituted diketopiperazines that are useful as pharmaceutical drug delivery systems for delivery to the lungs of individuals. The resulting substituted diketopiperazines show reduced or undetectable amounts of certain impurities that are known to affect product purity and performance.

Description

METHODS FOR THE MANUFACTURE OF DIKETOPIPERAZINE DERIVATIVES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No.63 / 738,039, filed on December 23, 2024, the entire disclosure of which is incorporated herein by reference in full.FIELD

[0002] The present invention relates to compositions for delivering active agents, and particularly delivery of biologically active agents to the lungs for a variety of therapeutic interventions. Disclosed embodiments are in the field of chemical synthesis and more particularly are related to improved synthetic methods for making diketopiperazine (DKP) derivatives, compositions and methods of making the compositions. In particular, the DKP derivatives can be used as a delivery system for drugs or active agents in the treatment of disease or disorders as described herein.BACKGROUND

[0003] Drug delivery is an ongoing area of pharmaceutical technology that seeks to provide solutions for delivering pharmaceutical interventions to patients in need. In many cases, oral delivery of a drug will suffice as it shows benefits in ease of administration, patient compliance, and decreased cost. However, there are a variety of therapies that cannot be delivered in this manner or are not well-tolerated. For example, when the active agent is unstable under the conditions encountered in the gastro-intestinal tract when administered orally to a subject, on route to reaching its targeted location. Because of this, many compounds are ineffective or exhibit low or unpredictable efficacy when administered orally.

[0004] Due to the problems associated with oral drug delivery, other routes have been explored, including transdermal, injectable, and delivery via the lungs. Advantages of the lungs for delivery of systemic agents include the large surface area and the ease of uptake by the lung's mucosal surface. That being said, pulmonary drug delivery systems present many difficulties, for example, the use of propellants, and aerosolization of biological agents such as proteins and peptides can lead to denaturation, and excessive loss of the agent to be delivered. One other problem associated with all of these forms of pulmonary drug delivery is that it is difficult to deliver drugs into the lungs due to problems in getting the drugs past all of the natural barriers, such as the cilia lining the trachea, and in trying to administer a uniform volume and weight of drug.

[0005] One class of delivery system to the lung that has shown promise in therapeutic use is diketopiperazines (DKP). Such DKPs have been shown to effectively deliver biologically active agents across the lung to the systemic circulation. However, as is true for much in the arena of chemical synthesis, conventional methods for synthesizing DKPs often produce unwanted side products including solvates and byproducts. Removing these impurities can require efforts ranging from repurifying the materials, factory down-time, and to loss of entire batches. All of these scenarios lead to increased cost for the final product. Thus, there is an unmet need for improved processes that provide DKP delivery systems that show improved purity profiles and reduced levels of unwanted impurities.

[0006] Moreover, it is well understood that reduction or elimination of chemical impurities is a common goal of chemical synthesis generally. However, in the arena of pharmaceutical delivery systems, chemical purity is of utmost priority. Elimination of impurities has a number of benefits for pharmaceutical products including reducing waste, avoiding potential unwanted side-effects of the impurities, and reducing or eliminating the number of unwanted sideproducts or impurities that must be investigated or tracked to comply with ongoing safety requirements. Thus, while it is often a goal for chemical synthesis, achieving very high levels of purity (e.g., over 95%) is often harder than it may appear. For example, many unwanted sides-products will share commonalities with the desired target molecule, making separation from the desired compound difficult if not impossible.SUMMARY

[0007] 3,6-bis-substituted-2,5-diketopiperazines (an example of a DKP derivative) have been shown to effectively deliver biologically active agents across the lining of the lung. The general inventive concepts are based, in part, on the recognition that conventional methods for making DKP derivates result in the production of unwanted side products or an undesirable amounts of such side products. The general inventive concepts provide methods for avoiding the production of these unwanted impurities in whole or in part.

[0008] The general inventive concepts are based in part on the discovery that certain impurities such as product solvates and specific side-products (identified by their molecular weight) can be reduced or eliminated when using the methods described herein. Further, the general inventive concepts provide methods that can produce FDKP products with unexpected levels of purity above that of conventional methods.

[0009] The present disclosure provides systems, compositions and methods that allow for improved production of chemical systems for delivery to the lungs by way of improving onexisting / conventional methods for the production of DKPs, including fumaryl diketopiperazines (e.g., FDKP). Embodiments disclosed herein achieve improved product quality by providing diketopiperazine compositions with reduced amounts of known impurities, while maintaining important properties of the FDKP (e.g., suitable cis / trans isomer content). In particular, the diketopiperazine compositions are exemplified by an FDKP compound having a trans isomer content of about 45% to about 65% and a reduced level of known impurities, including to levels below detectability under conventional testing methods.

[0010] In certain embodiments, the general inventive concepts provide a method for reducing or eliminating impurities in the production of a fumaryl substituted aminoalkyl diketopiperazine, the method comprising: a cyclocondensation reaction of a N-protected lysine in the presence of a catalyst to form a N-protected alkylamino diketopiperazine, a substitution reaction to from an activated monoethyl fumarate, a deprotection reaction to remove the N-protecting group from the N-protected alkylamino diketopiperazine, a coupling reaction between the deprotected alkylamino diketopiperazine and the activated monoethyl fumarate to form ethylfumaryl amino alkyl diketopiperazine, a recrystallization of the ethylfumaryl amino alkyl diketopiperazine, a saponification reaction to remove ethyl groups form the ethylfumaryl amino alkyl diketopiperazine, and a recrystallization of the fumaryl amino alkyl diketopiperazine; wherein the fumaryl amino alkyl diketopiperazine a trans isomer content of about 45% to about 65% and is substantially free from at least one of impurities corresponding toMW396 and MW788.

[0011] In an embodiment, the general inventive concepts provide a method of making a diketopiperazine having a reduced amount of specific impurities and a trans isomer content of about 45% to about 65%. In certain embodiments, the method provides a diketopiperazine having the formula 2, 5-diketo-3,6-bis(N — X-4-aminobutyl)piperazine, wherein X is selected from the group consisting of moi eties having a carbon chain length of 1-8 carbons. In an exemplary embodiment, the diketopiperazine has the formula 2,5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine, a trans isomer content of about 45% to about 65% and is substantially free from at least one of impurities corresponding to MW396 and MW788.

[0012] In another embodiment, the FDKP is combined with a drug or active agent. The compositions are based on diketopiperazine powders for pulmonary inhalation. Various exemplary embodiments contemplate inhalable compositions comprising an active agent (such as treprostinil and insulin nintedanib), a derivative thereof or analogs thereof and a method for treating various conditions such as pulmonary arterial hypertension and / or idiopathicpulmonary fibrosis are disclosed herein via the combination of FDKP and an active agent. Methods of manufacturing pharmaceutical compositions are also disclosed.

[0013] Other aspects and features of the general inventive concepts will become more readily apparent to those of ordinary skill in the art upon review of the following description of various exemplary embodiments in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The general inventive concepts, as well as embodiments and advantages thereof, are described below in greater detail, by way of example, with reference to the drawings in which:

[0015] Figure 1 shows a general synthetic chemical pathway for producing an FDKP according to the general inventive concepts.

[0016] Figure 2 shows a chemical reaction to form a diketopiperazine according to the general inventive concepts.

[0017] Figure 3 shows a chemical reaction between a diketopiperazine and a fumaryl reactant to form a further functionalized diketopiperazine according to the general inventive concepts.

[0018] Figure 4 shows a saponification reaction of a functionalized diketopiperazine according to the general inventive concepts.

[0019] Figure 5 shows a purification / recrystallization reaction to produce a fumaryl diketopiperazine according to the general inventive concepts.

[0020] Figure 6 shows a variety of chemical impurities that can arise during production of an FDKP and their respective molecular weights.

[0021] Figure 7 shows a 'H NMR spectrum of a trifluoroacetyl protected diketopiperazine.

[0022] Figure 8 shows an ATR infrared spectrum of the trifluoroacetyl protected diketopiperazine.

[0023] Figure 9 shows a 'H NMR spectrum of a monoethylfumaryl substituted aminoalkyl diketopiperazine.

[0024] Figure 10 shows an infrared spectrum of the monoethylfumaryl substituted aminoalkyl diketopiperazine.

[0025] Figure 11 shows a1H NMR spectrum of the FDKP.

[0026] Figure 12 shows a13C NMR spectrum of the FDKP.

[0027] Figure 13 an infrared spectrum of the FDKP.

[0028] Figure 14 shows an HPLC trace of FDKP after purification.DETAILED DESCRIPTION

[0029] Unless defined otherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the various embodiments, the preferred methods and materials are described herein. All references cited herein, including published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are each incorporated by reference in their entireties, including all data, tables, figures, and text presented in the cited references. In the drawings, the thickness of the lines, layers, and regions may be exaggerated for clarity. It is to be noted that like numbers found throughout the figures denote like elements.

[0030] Fumaryl diketopiperazine (2,5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine; FDKP) is one preferred diketopiperazine suitable for the general inventive concepts and for pulmonary delivery of active agents. FDKP possesses two asymmetric centers in the diketopiperazine ring. FDKP is manufactured as a mixture of geometric isomers that are identified as “cis-FDKP” and “trans-FDKP” according to the arrangement of side chains relative to the central “ring” of the diketopiperazine. The R,R and S,S enantiomers have the propenyl(amidobutyl) “side arms” projecting from the same planar side of the diketopiperazine ring (A and B below) and are thus referred to as the cis isomers while the R,S compound has the “side arms” projecting from opposite planar sides of the diketopiperazine ring and is referred to as the trans isomer. See e.g., U.S. Pat. No. 8,227,409 entitled “Diketopiperazine microparticles with defined isomer contents” for a more detailed description of the relationship and performance of provided by specific isomer ranges for FDKP. For additional information on diketopiperazines as delivery agents, see U.S. Pat. Nos.5,352,461 entitled “Self Assembling Diketopiperazine Drug Delivery System;” 5,503,852 entitled “Method For Making Self-Assembling Diketopiperazine Drug Delivery System;” 6,071,497 entitled “Microparticles For Lung Delivery Comprising Diketopiperazine;” 6,331,318 entitled “Carbon-Substituted Diketopiperazine Delivery System.” For information on synthesis of particular diketopiperazines see U.S. Pat. Nos. 8,912,328 entitled “Formation of N-protected 3,6-bis-(4-aminoalkyl)-2,5-diketopiperazine;” 8,962,836 entitled “Formation of N-protected 3,6-bis-(4-aminoalkyl)-2,5-diketopiperazine Through a Cyclic N-Protected Amino Ester Intermediate;” and 9,193,691 entitled “Methods for the Synthesis of Ethylfumarates and their Use as Intermediates.” Each of the foregoing are incorporated herein by reference in its entirety for all that it teaches regarding diketopiperazines and diketopiperazine-mediated drug delivery, microparticles that incorporate a drug ormicroparticles onto which a drug can be adsorbed, and how the combination of a drug and a diketopiperazine can impart improved drug stability and / or absorption characteristics.

[0031] There are a variety of impurities that are produced during synthesis of the FDKP as in e.g., Fig. 1, all of which should be minimized. Fig. 6 shows some non-limiting structures of identified impurities which are reduced according to the general inventive concepts. Two specific known impurities that affect the process of producing FDKP and desired purity levels have been identified as having a MW of 788 and 396 respectively. The proposed structure of these impurities are shown below.

[0032] The general inventive concepts are based in part on the discovery that certain impurities such as product solvates and specific side-products (identified by their molecular weight, e.g., MW396 and MW788) can be reduced or eliminated when using the methods for making FDKP described herein. In certain exemplary embodiments, the general inventive concepts provide methods of the synthesis of FDKP that reduces or substantially eliminates certain impurities, including impurities identified as MW788 and MW396, among others, to levels below detectability. Reduction or elimination of these known impurities and achieving surprising overall product purity is a goal of the general inventive concepts.

[0033] In a specific embodiment, the method of making a diketopiperazine having a trans isomer content of about 45% to about 65% and a reduced level of known impurities (e.g., those associated with solvates described herein and those associated with MW396 and MW788, among others), including to levels below detectability under conventional testing methods, utilizes a diketopiperazine having the formula 2, 5-diketo-3,6-bis(N — X-4-aminobutyl)piperazine, wherein X is selected from the group consisting of fumaryl, succinyl, maleyl, and glutaryl. In other embodiments of the FDKP compositions, the trans isomer content can be from about 50% to about 65%, from about 52% to 62%, or from about 53% to about 65%. In another embodiment of the FDKP, the trans isomer content is from about 53% to about63%. In an exemplary embodiment, the diketopiperazine has the formula 2,5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine.

[0034] In particular embodiments, the general inventive concepts provide a method for reducing or eliminating impurities in the production of FDKP having a trans isomer content ranging from about 45% to about 65% comprises a cyclocondensation reaction, a functionalization reaction, a saponification reaction and a recrystallization. In certain embodiments, the general inventive concepts provide a method for reducing or eliminating impurities in the production of a fumaryl substituted aminoalkyl diketopiperazine (e.g., FDKP) having a trans isomer content ranging from about 45% to about 65%, the method comprising: a cyclocondensation reaction of a N-protected lysine in the presence of a catalyst to form a N-protected alkylamino diketopiperazine, a substitution reaction to from an activated monoethyl fumarate, a deprotection reaction to remove the N-protecting group from the N-protected alkylamino diketopiperazine, a coupling reaction between the deprotected alkylamino diketopiperazine and the activated monoethyl fumarate to form ethylfumaryl amino alkyl diketopiperazine, a recrystallization of the ethylfumaryl amino alkyl diketopiperazine, a saponification reaction to remove ethyl groups form the ethylfumaryl amino alkyl diketopiperazine, and a recrystallization of the fumaryl amino alkyl diketopiperazine; wherein the fumaryl amino alkyl diketopiperazine a trans isomer content of about 45% to about 65% and is substantially free from at least one of impurities corresponding to MW396 and MW788.

[0035] In one embodiment, the functionalization step comprises introducing a functional moiety having an ester protecting group and the method comprises a saponification reaction of the functionalized diketopiperazine compound comprising an ester protecting group, including, an alkyl ester such as ethyl ester, or methyl ester. In one embodiment, the method comprises saponifying the aforementioned ester of FDKP in a solvent such as a waterbased solvent and adding a base, including an inorganic metallic base. In a particular embodiment, the method comprises mixing the DKP such as fumaryldiketopiperazine ethyl ester in a solvent such as water: methanol at a ratio of 1:1 to about 3:1, respectively. In other embodiments, other water-miscible solvents can be used, including, other alcohols, tetrahydrofuran, dioxane, acetone, acetonitrile and the like. In this and other embodiments, the saponification reaction further comprises adding a solution comprising sodium hydroxide; holding the reaction mixture at a temperature ranging from about 20°C to about 70°C or to refluxing conditions; filtering the reaction mixture to yield a filtrate; adding an acidic solution to the filtrate, and collecting the solid material formed by filtration and washing the resulting FDKP solid material.

[0036] In a particular embodiment, the method comprises recrystallizing the FDKP material comprising heating a suspension of FDKP in trifluoroacetic acid to about 40° C. to about 85° C. for about 1 minute to about 2 hours and cooling the reaction. After cooling, adding glacial acetic acid to the solution and cooling the suspension for about 1 to about 24 hours at about 20° C. In one embodiment, the crystallization step comprises a recrystallization temperature ranging from about 15°C to about 25°C, including about 20°C, for about 1 hour to about 5 hours in a solution comprising trifluoroacetic acid:glacial acetic acid, respectively, in a ratio ranging from about 0.4 to about 0.7. In this embodiment, the method further comprises washing the reaction mixture with glacial acetic.

[0037] Fig. 1 shows an exemplary general procedure for the production of a FDKP. In an exemplary embodiment, Step 1 is a cyclo-condensation reaction of a N-protected lysine performed in A-m ethyl pyrrolidone with a catalyst (e.g., phosphorus pentoxide). In the embodiment shown the N-protected lysine is trifluoroacetyl-lysine (i.e., e-trifluoroacetyl-L-lysine, TFA-lysine). The molar ratio of reactants in step 1 is believed to be an important factor in overall purity. In certain embodiments, the molar ratio of N-protected lysine (e.g., TFA-lysine) to catalyst (e.g., P2O5) is from about 5 : 1 to 3 : 1 , including a ratio of about 4:1. In certain embodiments, the cyclo-condensation is a high temperature reaction, including a temperature of from 125°C to 165°C, and including heating to a temperature of about 150°C. In certain embodiments, the cyclo-condensation reaction is heated for a period of 1 hour to 5 hours, including a period of 2 hours to 3 hours, and including heating for a period of about 2.5 hours. In certain embodiments, the reaction is cooled after the period of heating is completed, including cooling to a temperature of about 90°C to 110°C, and including cooling to a temperature of about 100°C. The resulting trifluoroacetic acid protected DKP (i.e., TDKP) is isolated by adding water to the reaction mixture and filtering the resultant slurry to obtain the solids. In certain embodiments, the amount of water added to the cooled reaction mixture is greater than that of the reaction solvent, including more than double the amount of solvent (e.g., N-methyl pyrrolidone), and including a ratio of water to solvent of about 5:2. In certain embodiments, after cooling water is added to the reaction mixture in one batch, in certain embodiments, the water is added to the reaction mixture over a period of at least 15 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. In certain embodiments, after quenching the reaction, the quenched mixture is cooled, including cooling to a temperature of about 50°C, including cooling to a temperature of about 40°C, including cooling to a temperature of about 30°C, and including cooling to a temperature of about 25°C. In certain embodiments, the cooled mixtureis maintained at the cooled temperature for a period of at least 30 minutes, including a period of at least 45 minutes, including a period of at least an hour, and including a period of 75 minutes to 90 minutes. In certain embodiments, after quenching the reaction with water, the TDKP is isolated (e.g., by filtration) washed with water and dried. In certain aspects, this TDKP product may be used in the processes of the general inventive concepts without further purification.

[0038] In an exemplary embodiment, Step 2a of Fig. 1 comprises a reaction that deprotects (i.e., removes) the trifluoroacetyl protecting groups from the TDKP. In certain embodiments this deprotection reaction proceeds via hydrolysis, including basic hydrolysis. The result of the hydrolysis of the TDKP is free amine groups pendant on the butyl groups of the diketopiperazine (not shown in in Fig. 1). (After removal of the groups, the TDKP solution is filtered and combined with the 4-nitrophenyl ethyl fumarate solution resulting in coupling of the deblocked TDKP amines with the ethyl fumaryl moiety of EFC.) As mentioned, in one reaction, protecting groups of the TDKP are removed by hydrolysis. In an embodiment, an organic solvent selected from acetone, methanol, ethanol, isopropanol, tetrahydrofuran, dioxane, and acetonitrile, is added to a reaction vessel and TDKP is added to the organic solvent. In certain embodiments, the TDKP mixture is stirred for a period of time prior to further addition of reactants, including stirring for a period of at least 30 minutes, including a period of at least about 60 minutes. A mixture of a strong base in water is added to the TDKP reaction vessel. In certain embodiments, the strong base is an inorganic hydroxide, including sodium hydroxide. In certain embodiments, the hydroxide and TDKP are mixed at a molar ratio of at least 2:1, including a molar ratio of at least 2.1:1, including a ratio of at least 2.2:1, and including a ratio of about 2.3:1. In certain embodiments, the hydroxide is provided as a mixture of sodium hydroxide in water, including a 25% mixture of NaOH, including a mixture of 40% NaOH, and including a mixture of about 50% NaOH. In certain embodiments, the mixture of NaOH is added over a period of time, including addition over a period of at least 10 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. In certain embodiments, additional water is added to the reaction vessel in addition to the water / NaOH solution. In certain embodiments, water is added in an amount of 25% that of the acetone / water, including 50%, including 75%, and including adding water in an amount approximately equal to that of the water and acetone. In certain embodiments, the hydrolysis reaction is maintained at a set temperature for a period of at least 15 minutes, including at least 30 minutes, including at least 45 minutes, and including holding at a set temperature for a period of about 60 minutes. In certain embodiments, the reaction ismaintained at a set temperature of below 50°C, including below 40°C, and including a temperature of about 30°C after addition of the hydroxide mixture.

[0039] Also in step 2a, an activated monoethyl fumarate is formed. In a separate reaction mixture, an activated form of monoethyl fumarate is made. In certain embodiments, the activated monoethyl fumarate is 4-nitrophenyl ethyl fumarate. In certain embodiments, ethyl fumaryl chloride (EFC) is mixed with sodium 4-nitrophenolate to form the 4-nitrophenyl ethyl fumarate. To accomplish this, in certain embodiments, 4-nitrophenol is mixed with an organic solvent selected from acetone, methanol, ethanol, isopropanol, tetrahydrofuran, dioxane, and acetonitrile; and water. In certain embodiments, the organic solvent is acetone and the acetone:water are provided in a weight ratio of 1:1 to 1:4, including a ratio of about 2:3. In certain embodiments, an inorganic hydroxide is added as a mixture of sodium hydroxide in water, including a 25% mixture of NaOH, including a mixture of 40% NaOH, and including a mixture of about 50% NaOH. In certain embodiments, the mixture of NaOH is added over a period of time, including addition over a period of at least 10 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. In certain embodiments, additional water is added to the reaction vessel in addition to the water / NaOH solution already present. In certain embodiments, the reaction temperature is maintained at below 30°C, including below 25°C, and including about 20°C. To this reaction is added a solution of EFC in an organic solvent. In certain embodiments, the organic solvent is the same organic solvent used previously with 4-nitrophenol. In certain embodiments, the combined mixture is allowed to warm and is maintained at a temperature of about 30°C during addition of the EFC. In certain embodiments, the 4-nitrophenol and sodium hydroxide are provided in a molar ratio of 1:1 to the EFC. In certain embodiments, the 4-nitrophenol and sodium hydroxide are provided in a molar excess to the EFC, including an excess of about 5% to about 10%, and including an excess of about 10%. In certain embodiments, the sodium hydroxide is provided in an excess to that of the 4-nitrophenol, including an excess of about 5% to about 10%, and including an excess of about 10%. In certain embodiments, the 4-nitrophenol and EFC reaction is maintained at a temperature of about 30°C for a period of at least 10 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. After the period of heating, the reaction is diluted with another portion of the organic solvent (e.g., acetone, methanol, ethanol, isopropanol, tetrahydrofuran, dioxane, and acetonitrile) to form a diluted mixture comprising the 4-nitrophenyl ethyl fumarate).

[0040] Generally, the aim of optimizing overall yield in a multi-step chemical synthesis is accomplished by isolation and purification of each intermediate prior to subsequent reaction. This approach hopes to avoid loss of the final target due to: a) by-products of the previous steps reacting with intermediates or starting materials; and b) loss due to more complicated isolation and purification of the target molecule. Nevertheless, disclosed embodiments provide methods for the synthesis of e.g., FDKP with improved purity and reduced amounts of specific impurities via use of in situ generated intermediates (e.g., 4-nitrophenyl ethyl fumarate and deprotected TDKP). The embodiments provide results which, counter to general thought, achieve higher yield, reactor throughput, and lower impurities, than traditional, isolate-and-purify-type methods. More specifically, embodiments show methods for the generation and use of fumaryl intermediates in situ and without purification.

[0041] The diluted 4-nitrophenyl ethyl fumarate is added to the deprotected TDKP. In certain embodiments, the 4-nitrophenyl ethyl fumarate is provided in a molar excess relative to the (deprotected) TDKP (based on calculated amounts due to not isolating the intermediate). In certain embodiments, the 4-nitrophenyl ethyl fumarate and deprotected TDKP are present in a molar ratio of 1.5:1, including a ratio of 2:1, and including a molar ratio of about 2.5:1, or more. In certain embodiments, the diluted 4-nitrophenyl ethylfumarate is added over a period of time, including addition over a period of at least 10 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. Upon addition, the combined mixture is heated to a temperature of 30°C to 60°C, including a temperature of about 45°C. In certain embodiments, the heated reaction mixture is maintained at a temperature of about 30°C for a period of at least 10 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. After heating, the mixture is cooled to a temperature of about 30°C and is quenched with water. In certain embodiments, the quenched mixture is stirred for a period of at least 10 minutes, including at least 20 minutes, including at least 30 minutes at 30°C. After stirring at 30°C, the mixture is filtered and the resulting product (e.g., EFDKP) is washed, including at least one wash with an organic solvent (e.g., acetone) and one wash with water, and including two washes with an organic solvent and two washes with water.

[0042] In certain embodiments, the crude EFDKP (i.e., (E)-3,6-bis[4-(7V-ethoxycarbonyl-2-propenyloyl)aminobutyl]-2,5-diketopiperazine) is purified by recrystallization from an organic solvent. In certain embodiments, the EFDKP is purified by recrystallization from acetic acid (a.k.a., glacial acetic acid). In certain embodiments, the crude EFDKP is mixed with acetic acid in a ratio of 1:1 to 1:10, including a ratio of about 1:4 byweight. In certain embodiments, the mixture of crude EFDKP and acetic acid is heated to a temperature of at least 80°C, including at least 90°C, including at least 100°C, including heating to reflux. In certain embodiments, the mixture of crude EFDKP and acetic acid is heated for a period of less than 2 hours, including less than 1 hour, including less than 30 minutes, including about 15 minutes. In certain embodiments, the heated mixture is cooled to a temperature of less than 100°C, including a temperature of about 90°C to about 95°C. In certain embodiments, after cooling a second solvent is added to the mixture over a period of time, including at least 30 minutes, including about 45 minutes. In certain embodiments, the second solvent is water and is added such that the ratio of acetic acid to water is about 10:1, including about 5:1, including about 4:1, and including between 4:1 and 5:1. In certain embodiments, the EFDKP is isolated by filtration, including by centrifuge filtration. In certain embodiments, after isolation, the EFDKP is washed with a mixture of solvents, including a mixture of acetic acid and water. In certain embodiments, the wash mixture comprises a 3:1 mixture of acetic acid and water. In certain embodiments, the wash mixture is provided in an amount of about twice the amount of EFDKP by weight. In certain embodiments, the washed EFDKP is further washed at least once with water.

[0043] In certain embodiments, the EFDKP is mixed with an alcohol such as methanol. In certain embodiments, methanol is provided in an amount of at least 1:1 that of the EFDKP by weight, including at least 2:1 and including about 2.5:1 by weight. Water is added to the mixture. In certain embodiments, water is provided in an amount of at least 1:1 that of the methanol, including about 2:1 by weight. In certain embodiments, a metal hydroxide is added to the mixture, including sodium hydroxide in a molar ratio of at least 1 : 1 to EFDKP, including at least 2:1, and including a molar ratio of sodium hydroxide to EFDKP of between 2:1 and 2.25:1. In certain embodiments, the reaction mixture is heated prior to addition of the sodium hydroxide, including heating to a temperature of about 65°C. In certain embodiments, the sodium hydroxide is added over a period of time while maintaining a temperature of about 65°C, including addition over a period of at least 10 minutes, including a period of at least 30 minutes, including a period of at least 45 minutes, and including a period of about 60 minutes. In certain embodiments, the mixture of EFDKP and sodium hydroxide is heated for a period of at least 10 minutes, including at least 20 minutes, including at least 30 minutes. In certain embodiments, the temperature is increased to about 75°C during the period of heating. In certain embodiments, the mixture of EFDKP and sodium hydroxide is filtered and maintained at a temperature of about 60°C. In certain embodiments, acetic acid is added to the filtered mixture. In certain embodiments, acetic acid is added until the mixture achieves a pH of lessthan 8, including less than 7, including less than 6, including a pH of less than about 5.5. In certain embodiments, the reaction is then cooled, including colling to a temperature of less than 45°C, including less than 30°C, including a temperature of about 20°C. In certain embodiments, the mixture is maintained at the cooled temperature for more than 30 minutes, including more than 1 hour, including more than 1.5 hours, and including about 2 hours. In certain embodiments, the cooled mixture is filtered and washed. In certain embodiments, the filtered FDKP is washed with more than one solvent, including washing with two solvents. In certain embodiments, the FDKP is washed with water and acetone, including washing first with water followed by washing with acetone.

[0044] In certain embodiments, FDKP is purified by recrystallization, including recrystallization from trifluoroacetic acid (TFAA). In certain embodiments, FDKP is mixed with TFAA in a ratio of TFAA:FDKP of greater than about 3:1 by weight, including greater than about 4:1, and including about 5: 1. In certain embodiments, the mixture of FDKP in TFAA is heated, including heating to a temperature of greater than 50°C, including a temperature of greater than 60°C, including a temperature of greater than 70°C, and including heating to reflux. In certain embodiments, the heated mixture of FDKP and TFAA is heated to reflux for between 1 and 30 minutes, including about 10 minutes. In certain embodiments, the mixture is cooled to a temperature of about 55°C and acetic acid is added. In certain embodiments, acetic acid is added until the ratio of acetic acid to TFAA is about 1 : 1 or more. In certain embodiments, the acetic acid is added over a period of at least 15 minutes while maintaining a temperature of about 55°C, including at least 20 minutes, including at least 30 minutes, including at least 45 minutes, and including about 1 hour. In certain embodiments, the mixture is then cooled after adding the acetic acid, including cooling to a temperature of less than about 40°C, including less than about 30°C, including about 20°C. In certain embodiments, the mixture is cooled rapidly. In certain embodiments, the mixture is cooled over a period of at least 15 minutes, including at least 30 minutes, including at least 1 hour, and including cooling over a period of about 1.5 hours. In certain embodiments, the cooled mixture is filtered to isolate the FDKP and the FDKP is washed. In certain embodiments, the filtered FDKP is washed with more than one solvent, including washing with two solvents. In certain embodiments, the FDKP is washed with acetone, followed by washing with a mixture of water and acetone. In certain embodiments, the FDKP is washed with acetone, followed by washing at least once with a mixture of water and acetone, followed by washing with acetone. In certain embodiments, the mixture of water and acetone in the washing comprises water and acetone in a ratio of greater than 2:1, including about 2.5:1.

[0045] While not wishing to be bound by theory, Applicants believe that longer contact time between TFAA and FDKP during recrystallization leads to impurities, including a solvate of TFAA:FDKP. Impurity MW450 also increases with increasing exposure of FDKP to TFAA. The general inventive concepts seek to reduce or eliminate this impurity.

[0046] The general inventive concepts also relate to methods of use of the FDKP compound having a trans isomer content of about 45% to about 65% and a reduced level of known impurities, including to levels below detectability under conventional testing methods and an active agent to treat one or more conditions or diseases. In certain embodiments, the FDKP compound having a trans isomer content of about 45% to about 65% and a reduced level of known impurities is combined with the active agent and processed for delivery of the active agent to the lungs.

[0047] In certain exemplary embodiments, the active agent is a drug or pharmaceutical composition. Active agents can include, one or more endocrine hormones, including, insulin or an analog thereof, parathyroid hormone or an analog thereof, calcitonin, glucagon, glucagon-like peptide 1, oxyntomodulin, peptide YY, leptin, a cytokine, a lipokine, an enkephalin, a cyclosporin, an anti-IL-8 antibody, an IL-8 antagonist including ABX-IL-8, an LTB receptor blocker, including LY29311, BIIL 284 and CP105696; a triptan such as sumatriptan or palmitoleate, a growth hormone or analogs thereof, a parathyroid hormone related peptide (PTHrP), ghrelin, obestatin, enterostatin, granulocyte macrophage colony stimulating factor (GM-CSF), amylin, amylin analogs, clopidogrel, PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), adiponectin, cholecystokinin (CCK), secretin, gastrin, motilin, somatostatin, brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormone releasing factor (GHRF), integrin beta-4 precursor (ITB4) receptor antagonist, analgesics, nociceptin, nocistatin, orphanin FQ2, CGRP, angiotensin, substance P, neurokinin A, a pancreatic polypeptide, a neuropeptide Y, a delta-sleep-inducing peptide, or a vasoactive intestinal peptide. Additionally, the active agents can include one or more than one small molecules for treating lung diseases, or disorders, including, a prostaglandin or analog thereof, for example, Treprostinil, Iloprost, epoprostenol and the like; Nintedanib, Clofazimine, derivatives and / or analogs thereof, salts, esters, or solvates thereof. In certain exemplary embodiments, the drug is Treprostinil.

[0048] In one embodiment, the general inventive concepts further contemplate administering to a subject in need of treatment a stable pharmaceutical composition comprising, one or more active agents, for delivery to lung tissue, wherein more than one active agent can be formulated together or formulated separately to be administered separately and at different intervals duringa therapy. In a particular embodiment, the pharmaceutical composition comprises a formulation for inhalation comprising a therapeutically effective dose of a dry powder comprising one or more active agents, including, a small molecule such as pirfenidone, pyridone analogs, nintedanib, derivatives thereof, or analogs thereof; and / or combinations thereof. In certain embodiments, the pharmaceutical composition can further comprise any molecule or compound which is suitable for treating idiopathic lung disease and can be present in the composition either alone or in combination with other active agents, including, deoxyribonuclease I (Dnase I) and granulocyte macrophage colony stimulating factors (GM-CSF), anti-inflammatories, including, kinase inhibitors such as tyrosine kinase inhibitor molecules. The pharmaceutical composition comprises optionally, one or more pharmaceutically acceptable excipients and / or carriers. In this and other embodiments the pharmaceutical composition is provided to the patient in a container, capsule or cartridge for inhalation using a dry powder inhaler.

[0049] In some embodiments, the general inventive concepts contemplate treatment of interstitial lung disease, including, pulmonary fibrosis comprises, administering to a subject in need of treatment, a pharmaceutical composition comprising pirfenidone and / or nintedanib separately, sequentially or combinations thereof with one or more of a vasodilator compound. In one embodiment, the method comprises a combination therapy comprising, administering to the subject a vasodilator, including, sildenafil, tadalafil, vardenafil, a prostaglandin, a prodrug thereof, a prostaglandin derivative, a prostaglandin analog, for example, treprostinil, or a pharmaceutically acceptable salt of these compounds thereof, including, treprostinil sodium, or prodrugs thereof. In a particular embodiment, the method comprises treating interstitial lung disease and pulmonary arterial hypertension simultaneously comprising delivering to the lungs of the patient a combination therapy comprising a dry powder formulation comprising pirfenidone and / or nintedanib and / or a dry powder composition comprising a vasodilator compound, including, treprostinil, treprostinil, or a pharmaceutically acceptable salt of these compounds thereof, including, treprostinil sodium, or prodrugs thereof, and into the systemic circulation of a subject, by pulmonary inhalation using a dry powder inhaler.

[0050] In certain embodiments, the method comprises providing to a patient in need of treatment a dry powder inhaler comprising the active agent, for example, nintendanib, pirfenidone, or treprostinil in a stable dry powder formulation, and administering the active agent by oral inhalation. In one embodiment, the vasodilator can be formulated together with the pirfenidone, nintedanib in the same formulation or separately and administered separatelyin its own formulation and provided to the patient at different intervals or sequentially during a dosing session.

[0051] In certain embodiments, the drug delivery system comprises a dry powder inhaler comprising a diketopiperazine-based drug formulation for delivering small molecules, for example, pirfenidone, nintedanib, a prostaglandin, or analogs thereof, including, tresprostinil and protein-based products for treating pulmonary fibrosis and PAH. The method provides advantages over typical methods of drug delivery, such as, oral tablet and subcutaneous and intravenous injectable / infusion drug products that are sensitive to degradation and / or enzymatic deactivation.Examples

[0052] The following examples illustrate features and / or advantages of the systems and methods according to the general inventive concepts. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the general inventive concepts, as many variations thereof are possible without departing from the spirit and scope of the general inventive concepts.

[0053] A batch of FDKP was made according to a conventional method (Comparative) and a Test batch was made according to the general inventive concepts described herein. The batches were analyzed for a variety of impurities (designated by MW below) and the results of the chemical analysis is presented in Table 1. The proposed structure of many of the MW impurities are shown in Fig. 6.Table 1

[0054] As can be seen from the Table, impurities MW396 and MW 788 are below detectable levels whereas the comparative methods provided materials with undesirable levels of these impurities. Similarly, when manufactured according to the general inventive concepts, levels of e.g., MW450 are substantially lower and total impurities are likewise greatly reduced while the HPLC percentage was increased (a measure of purity). Detected levels of impurities MW466 and MW484 are inconsistent but appear to be trending lower or unchanged in the aggregate. Levels of acetic acid and TFAA (likely representing solvates) are reduced from undesirable levels. Each of these changes show improvements over conventional methods and provide benefits to the end product. While not wishing to be bound by theory, Applicant believes acetone and acetic acid residual solvent impurities in FDKP are controlled with proper drying as described herein. Residual trifluoroacetic acid is controlled by limiting the time FDKP is exposed to trifluoroacetic acid along with proper washing of the final product filter cake as described above.

[0055] FDKP was produced and purified in a method according to the general inventive concepts is as follows: Charge A-methyl-2-pyrrolidone (205.6 kg) to a glass-lined reactor. Start agitation and charge TFA-lysine (100 kg) to the reactor at ambient temperature. Charge phosphorus pentoxide (15.2 kg) to the TFA-lysine slurry. Heat the reaction mixture to 150 °C and hold at 150 °C for 150 minutes. Cool the reaction mixture to 100 °C. Transfer the reaction mixture to a larger glass-lined reactor. Rinse the transfer line with A-methyl-2-pyrrolidone (20 kg). Charge water (500 L) over 60 minutes. Cool the precipitated mixture to 25 °C and hold for 90 minutes. Isolate the precipitated solids in an auto filter dryer. Wash the isolated solids twice with water (265 L per wash). A 500 MHz 'H NMR spectrum of the trifluoroacetyl protected diketopiperazine in DMSO-de is shown in Fig. 7 and an ATR infrared spectrum of TDKP is shown in Fig. 8.

[0056] The following step is run in clean equipment with a nitrogen atmosphere containing less than 2% oxygen. Certain steps are run concurrently prior to convergence of the reactants. Charge acetone (38.2 kg) to the reactor. Start the agitator with addition of acetone, and charge TDKP (17.0 kg, real basis) to the reactor. Agitate for 60 minutes. Charge a solution of 50%sodium hydroxide (7.0 kg) and water (44.8 L) to the reactor over a 60 minute period. Maintain the reactor at 30 °C during the sodium hydroxide charge. Hold the reaction mixture at 30 °C for 60 minutes after the caustic dosing is complete. Transfer the reaction mixture through a filter. Continue holding the filtered TDKP hydrolysis mixture at 30 °C. Charge 4-nitrophenol (14.5 kg), acetone (20.8 kg), and water (32.9 L) to glass-lined reactor. Begin agitation to dissolve the 4-nitrophenol. Add a solution of 50% sodium hydroxide (8.65 kg) and water (10.4 L) to reactor over 60 minutes. Maintain reaction temperature around 20 °C during this addition. Charge a solution of EFC (15.4 kg) and acetone (13.9 kg) into the 100 gallon glass-lined reactor. This charge is begun with the contents at 20 °C. The reaction is allowed to exotherm to 30 °C and the reaction mixture held at 30 °C. Rinse in the EFC charge line with acetone (4.0 kg). Hold the contents at 30 °C for 60 minutes. Dilute the reaction mixture with acetone (16.9 kg). Transfer over 60 minutes. Hold the mixture temperature at 35 °C. Heat the contents to 45 °C and hold for 60 minutes. Cool contents to 30 °C. Quench with water (87.8 L). Stir at 30 °C for 30 minutes. Isolate the solids (EFDKP) by centrifuge filtration. Wash the filter cake twice with acetone (26.9 kg each). Wash the filter cake twice with water (34.1 L each). A 300 MHz 'H NMR spectrum of EFDKP in DMSO-de is shown in Fig. 9 and an ATR infrared spectrum of EFDKP is shown in Fig. 10.

[0057] Charge crude EFDKPC (45 kg, dry basis) and glacial acetic acid (177.0 kg) to the a glass-lined reactor and begin agitation. Heat the contents to reflux and hold at reflux for 15 minutes. Cool to 92 °C and then add water (40.5 L) over 45 minutes. Cool to 20 °C and hold for 120 minutes. Isolate product solids by centrifuge filtration. Wash the filter cake once with a 75% acetic acid solution prepared from glacial acetic acid (70.6 kg) and water (23 L). Wash the filter cake once with water (180 L). Dry the solids in a vacuum tumble dryer under full vacuum with a jacket temperature of 45-75 °C.

[0058] Charge methanol (37.4 kg) to a reactor and start agitation. Charge EFDKP (15.0 kg). Begin heating to 65 °C. Charge water (70.9 kg) to the reactor. Begin heating to 60 °C. Transfer the warm water from as quickly as possible and immediately begin the addition of 50% sodium hydroxide (5.1 kg). The caustic addition should be done over 60 minutes while maintaining the reaction mixture at 65 °C. Hold for 30 minutes after sodium hydroxide addition. Heat the reaction mixture to 75 °C as part of this hold time. Set the reactor jacket temperature to 65 °C. Transfer the reaction mixture through a heated filter. Maintain the filtered reaction mixture at about 60 °C. Add glacial acetic acid (5.5 kg). Measure the pH of the suspension. If necessary, adjust the pH to less than 5.5 with additional glacial acetic acid. Cool to 20 °C and hold for two hours. Isolate the solids. Wash the filter cake with water (25.5 L) and then acetone (20.2 kg)to give crude FDKP (i.e., it is a mixture of free acid and sodium slats in addition to identified impurities).

[0059] Charge crude FDKP (17.5 kg) and trifluoroacetic acid (86.5 kg) to a glass-lined reactor and begin agitation. Heat contents to reflux and hold at reflux for 10 minutes. Transfer the hot solution through a PTFE filter to a larger 100 gallon glass-lined reactor. Rinse the transfer line and filter with trifluoroacetic acid (5.0 kg). Cool the solution to 55 °C and then add glacial acetic acid (91.8 kg) over 60 minutes while holding the reaction mixture at 55 °C. Cool the precipitating mixture to 20 °C with a linear cooling rate over 1.5 hours. Hold at 20 °C for 1.5 hours. Transfer the mixture to a stirred filter dryer. Use nitrogen pressure up to 20 psig to aid filtration. Wash the filter cake with acetone (22.7 kg) that has passed through a 3 pm filter. Wash the filter cake with a solution of acetone (8.8 kg) and water (20.8 L) that has passed through a 3 pm filter. Again wash the filter cake with a solution of acetone (8.8 kg) and water (20.8 L) that has passed through a 3 pm filter. Again wash the filter cake with acetone (22.7 kg) that has passed through a 3 pm filter to provide FDKP according to the general inventive concepts. The assay correct recovery is about 85% and the overall recovery is about 20%. A1H NMR spectrum of FDKP is shown in Fig. 11, a13C spectrum of FDKP is shown in Fig. 12 (the pairing seen in some of the13C peaks is caused by the FDKP cis and trans isomers), an IR spectrum of FDKP is shown in Fig. 13. Figure 14 shows an exemplary HPLC trace of FDKP with the first FDKP peak as the cis isomer.

[0060] While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character. It should be understood that only the exemplary embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0061] Unless otherwise indicated, all numbers expressing quantities of elements or components used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present exemplary embodiments. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0062] Unless otherwise indicated, any element, property, feature, or combination ofelements, properties, and features, may be used in any embodiment disclosed herein, regardless of whether the element, property, feature, or combination of elements, properties, and features was explicitly disclosed in the embodiment. It will be readily understood that features described in relation to any particular aspect described herein may be applicable to other aspects described herein provided the features are compatible with that aspect. In particular: features described herein in relation to the method may be applicable to the delivery system and vice versa.

[0063] Every numerical range given throughout this specification and claims will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0064] The terms “treating” and “treatment” as used herein, unless otherwise specified, includes delaying the onset of a condition, ameliorating or reducing the severity of symptoms of a condition, or eliminating some or all of the symptoms of a condition.

[0065] The term “an effective amount” is intended to qualify the amount of an active or therapeutic agent which will achieve the goal of treating a disease, or that which will achieve the goal of decreasing the risk that the patient will suffer an adverse health event, including reducing or preventing one or more symptoms, while avoiding adverse side effects such as those typically associated with alternative therapies. The effective amount may be administered in one or more doses.

[0066] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of’ excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0067] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

[0068] The systems of the present disclosure may also be substantially free of any optional or selected element or feature described herein, provided that the remaining composition still contains all of the required elements or features as described herein. In this context, and unless otherwise specified, the term “substantially free” means that the selected composition containsless than a functional amount of the optional ingredient, typically less than 0.1% by weight, and also including zero percent by weight of such optional or selected essential ingredient.

Claims

Claims:

1. A diketopiperazine having the formula 2, 5-diketo-3,6-bis(N-fumaryl-4-aminobutyl)piperazine, wherein the diketopiperazine has a trans isomer content of about 45% to about 65% and is substantially free from at least one of impurities corresponding to MW396 andMW788.

2. The diketopiperazine of claim 1, wherein the diketopiperazine has a HPLC purity of greater than 97%, including greater than 98%, and including 99% or more.

3. The diketopiperazine of claim 1, wherein the fumaryl aminoalkyl diketopiperazine comprises acetic acid in an amount of less than 2400 ppm.

4. The diketopiperazine of claim 3, wherein the fumaryl aminoalkyl diketopiperazine comprises acetic acid in an amount of from 1350 ppm to less than 2400 ppm.

5. The diketopiperazine of claim 1, wherein the fumaryl aminoalkyl diketopiperazine comprises trifluoroacetic acid in an amount of less than 2100 ppm.

6. The diketopiperazine of claim 1, wherein the fumaryl aminoalkyl diketopiperazine comprises trifluoroacetic acid in an amount of from 1725 ppm to less than 2100 ppm.

7. The diketopiperazine of claim 1, wherein the fumaryl aminoalkyl diketopiperazine comprises reduced amounts of impurities corresponding to MW450, MW466, MW484.

8. The diketopiperazine of claim 1, wherein the trans isomer content is from about 53% to about 63%.

9. A method for reducing or eliminating impurities in the production of a fumaryl substituted aminoalkyl diketopiperazine, the method comprising:a cyclocondensation reaction of a N-protected lysine in the presence of a catalyst to form a N-protected alkylamino diketopiperazine,a substitution reaction to from an activated monoethyl fumarate,a deprotection reaction to remove the N-protecting group from the N-protected alkylamino diketopiperazine,a coupling reaction between the deprotected alkylamino diketopiperazine and the activated monoethyl fumarate to form an ethylfumaryldiketopiperazine (EFDKP), a recrystallization of the EFDKP,a saponification reaction to remove ethyl groups form the ethylfumaryl amino alkyl diketopiperazine, anda recrystallization of the fumaryldiketopiperazine;wherein the fumaryl aminoalkyl diketopiperazine has a trans isomer content of about 45% to about 65% and is substantially free from at least one of impurities corresponding toMW396 andMW788.

10. The method of claim 9, wherein the fumaryl aminoalkyl diketopiperazine is substantially free from impurities corresponding to MW396 and MW788.

11. The method of claim 9, wherein the fumaryl aminoalkyl diketopiperazine has a HPLC purity of greater than 97%, including greater than 98%, and including 99% or more.

12. The method of claim 9, wherein the fumaryl aminoalkyl diketopiperazine comprises acetic acid in an amount of less than 2400 ppm.

13. The method of claim 12, wherein the fumaryl aminoalkyl diketopiperazine comprises acetic acid in an amount of from 1350 ppm to less than 2400 ppm.

14. The method of claim 9, wherein the fumaryl aminoalkyl diketopiperazine comprises trifluoroacetic acid in an amount of less than 2100 ppm.

15. The method of claim 9, wherein the fumaryl aminoalkyl diketopiperazine comprises trifluoroacetic acid in an amount of from 1725 ppm to less than 2100 ppm.

16. The method of claim 9, wherein the fumaryl aminoalkyl diketopiperazine comprises reduced amounts of impurities corresponding to MW450, MW466, MW484.

17. The method of claim 9, wherein:the cyclocondensation comprises mixing a N-protected amino acid and a catalyst in an organic solvent and heating to cyclocondense the amino acid, quenching the reaction with water to form a N-protected aminoalkyl diketopiperazine, isolating the resulting solids and washing twice with water;the substitution comprises mixing sodium hydroxide, 4-nitrophenol, and ethyl fumaryl chloride in a mixture of water and a second organic solvent and heating to form an activated monoethyl fumarate;the deprotection comprises in a separate reaction vessel, mixing the N-protected aminoalkyl diketopiperazine with sodium hydroxide in a mixture of a second organic solvent and water and heating the mixture to form an aminoalkyldiketopiperazine; the coupling comprises mixing the aminoalkyl diketopiperazine and the activated monoethyl fumarate in the second organic solvent and heating to a temperature of a temperature of 30°C to 60°C, to form the EFDKP, quench with water and stir for a predetermined amount of time, isolating the resulting solids and washing with the second solvent (2x) and water (2x);the recrystallization of the EFDKP comprises mixing the ethylfumaryl amino alkyl diketopiperazine with glacial acetic acid, heating the mixture cool the mixture, isolate the resulting solids, wash the solids with a mixture of acetic acid and water, wash the solids with water;the saponification comprises mixing the ethylfumaryl amino alkyl diketopiperazine with methanol and water twice, heating and adding a mixture of 50% sodium hydroxide and water over about 1 hr, wherein the sodium hydroxide is provided in an amount of greater than twice the molar equivalent of the EFDKP, heating the mixture to saponify ethyl moieties and form fumaryl diketopiperazine (FDKP);the recrystallization of the FDKP comprises mixing the FDKP with trifluoroacetic acid (TFAA) and heating for a predetermined period of time, adding glacial acetic acid with cooling and mixing, filtering resulting solids, and washing the resulting solids at least twice with acetone and a mixture of acetone and water.

18. The method of claim 17, wherein the recrystallization of FDKP step comprises cooling with a liner cooling rate over a period of between 1 and 2 hours and holding at a temperature of about 20°C for less than 2 hours.

19. The method of claim 17, wherein the recrystallization of FDKP step comprises cooling the mixture to a temperature of below 60°C prior to adding glacial acetic acid.

20. The method of claim 17, wherein the recrystallization of FDKP step comprises adding until the ratio of acetic acid to TFAA is about 1:1.

Images