Carbon monoxide releasing molecules and uses thereof

Abstract

Provided herein are novel carbon-monoxide releasing molecules (CO-RMs) of the Formula (I): and esters, amides, salts, solvates and hydrates thereof; wherein R1 and R2 are as described herein. Also provided are pharmaceutical compositions comprising these compounds, methods of their preparation, and their use in the treatment of liver disease and inflammation.

Description

FIELD OF THE INVENTION

The instant disclosure relates to carbon monoxide releasing molecules (CO-RMs) and uses thereof to treat liver diseases and inflammatory conditions.

BACKGROUND OF THE INVENTION

Carbon monoxide (CO) is the most commonly encountered environmental poison. Paradoxically, more than half a century ago, it was found that carbon monoxide is constantly formed in humans in small quantities, and that under certain pathophysiological conditions this endogenous production of carbon monoxide may be increased (Sjostrand, Scan J Clin Lab Invest. (1949) 1: 201-214). Thus, although it has been known for a long time that carbon monoxide is generated in the human body, only in recent years have scientists begun to explore the possible biological activities of this gaseous molecule. The main endogenous source of carbon monoxide is heme oxygenase, which exists in constitutive (HO-2 and HO-3) and inducible (HO-1) isoforms. Heme serves as substrate for HO-1 and HO-2 in the formation of carbon monoxide, free ferrous iron, and biliverdin, the latter being rapidly converted to bilirubin by biliverdin reductase (see, e.g., Maines, Annu Rev Pharmacol Toxicol. (1997) 37:517-554). It is generally believed that HO-1 represents a pivotal inducible defensive system against stressful stimuli, including UVA radiation, carcinogens, ischemia-reperfusion damage, endotoxic shock, and several other conditions characterized by production of oxygen-derived free radicals (see, e.g., Abraham et al., Cell Physiol Biochem. (1996) 6: 129-168). As part of its physiological and cytoprotective actions, heme oxygenase-derived carbon monoxide appears to play a major role as neurotransmitter, regulator of sinusoidal tone, inhibitor of platelet aggregation, and suppressor of acute hypertensive responses. Exogenously applied carbon monoxide has been a very useful experimental procedure to reveal the beneficial effects of carbon monoxide in animal disease models (see, e.g., US 2002155166, US 2003039638, US 2003219496, US 2003219497, US 2004052866, WO 03/103585, WO 04/043341). Thus, consistent findings reveal a series of important cellular functions that support a versatile role for carbon monoxide.

Carbon monoxide administration by inhalation is not practical for clinical applications, as it requires special delivery devices such as ventilators, face masks, tents, or portable inhalers. Moreover, carbon monoxide delivery to therapeutic targets by inhalation is inefficient, because it involves transport of carbon monoxide by hemoglobin. Hemoglobin binds carbon monoxide reversibly, but with very high affinity. Therefore, the doses required to deliver carbon monoxide to therapeutic targets in diseased tissues are likely to be associated with adverse effects. Carbon monoxide releasing molecules (CO-RMs) is a potential therapeutic alternative that can deliver carbon monoxide directly to therapeutic targets without the formation of intermediate CO-hemoglobin complexes (see, e.g., Johnson et al., Angew Chem Int Ed Engl (2003) 42:3722-3729). The advantages of carbon monoxide delivery by CO-RMs over carbon monoxide delivery by inhalation is generally recognized. However, CO-RMs should be able to deliver carbon monoxide selectively to diseased tissues. The identification of CO-RMs that are best suited for the treatment of a particular disease remains a major challenge of CO-RM development. Thus, there continues to remain a need for CO-RMs which, upon administration in vivo, selectively target a particular disease or organ with therapeutic benefit.

SUMMARY OF THE INVENTION

The present application provides inventive molybdenum CO-RM compounds, pharmaceutical compositions thereof, and methods of preparation, use, and treatment.

In one aspect, provided is a compound of the Formula (I):

wherein:

each instance of R¹ is independently hydrogen, an unsubstituted C_1-3alkyl, or a C_1-alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂, wherein each instance of R^A1 is independently hydrogen or C_1-10alkyl;

each instance of R² are independently hydrogen, an unsubstituted C_1-3alkyl, or a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, wherein each instance of R^A2 is independently hydrogen or C_1-10alkyl;

or R¹ and R² and the carbon to which they are both attached are independently joined to form a C_3-4 carbocyclyl;

provided that each instance of R¹ and R² attached to the same carbon are not both hydrogen.

In certain embodiments, each instance of R² is hydrogen. In certain embodiments, each instance of R² is hydrogen and each instance of R¹ is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is C_1-3alkyl substituted with —CO₂R^A.

In certain embodiments, each instance of R² is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R¹²)₂, and each instance of R¹ is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂.

In certain embodiments, each instance of R¹ is independently an unsubstituted C_1-3 alkyl; and each instance of R² is independently an unsubstituted C_1-3alkyl.

In certain embodiments, each instance of R¹ is independently an unsubstituted C_1-3alkyl; and each instance of R² is independently a C_1-3alkyl substituted with —CO₂R^A2.

In certain embodiments, each instance of R¹ is independently a C_1-3alkyl substituted with —CO₂R^A1; and each instance of R² is independently a C_1-3alkyl substituted with —CO₂R^A2.

In certain embodiments, R¹ and R² and the carbon to which they are both attached are independently joined to form a C_3-4 carbocyclyl.

Exemplary compounds of the Formula (I) include, but are not limited to:

and salts, esters, amides, solvates, and hydrates thereof, and combinations thereof.

Also provided are pharmaceutical compositions comprising a compound of the Formula (I), or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof, and a pharmaceutically acceptable excipient.

In another aspect, provided are esters of the Formula (I), e.g., of the Formula (II):

or a salt, solvate or hydrate thereof, or a combination thereof;wherein R¹ and R² are as defined herein; and each instance of R³ is independently C_1-6alkyl.

In yet another aspect, provided are amides of the Formula (I), e.g., of the Formula (III):

or a salt, solvate or hydrate thereof, or a combination thereof;wherein R¹ and R² are as defined herein; and each instance of R⁴ is independently hydrogen or C_1-6alkyl.

In still yet another aspect, provided are methods or use of treating liver disease and/or an inflammatory disease in a subject. In certain embodiments, the method comprises administering an effective amount of a compound of Formula (I), or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof. In other embodiments, the method comprises instructing the subject to take an effective amount of a compound of Formula (I), or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount.

Also provided are methods of preparing compounds of the present invention.

The details of one or more embodiments of the disclosure are set forth in the accompanying Figures and the Detailed Description. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1C. FIG. 1A depicts the CO release from Compound 1b (50 μM) in 50 mM HEPES buffer (pH 7.4) in the dark or under light. FIG. 1B depicts the CO released from Compound 1b (10 μM) in 0.5 M K-phosphate buffer (pH 7.4) in the presence or absence of rat liver microsomes. FIG. 1C depicts the tissue CO distribution experiments performed in CD-1 female mice. Compound 1b was administered i.v. (50 mg/kg in 150 μL PBS pH 7.5). 25 minutes after dosing, the animal was euthanized, the heart, kidneys, liver and lung were collected, and CO quantization was done. Samples of the freshly collected blood were transferred to AVOXimeter 4000 cuvettes (ITC) to measure the levels of carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb) and methemoglobin (MetHb) using a portable AVOXimeter 4000 CO-oximeter. The results are shown as mean percentage of total hemoglobin species in circulation.

FIGS. 2A-2E. FIG. 2A depicts the CO release from Compound 2b (50 μM) in 50 mM HEPES buffer (pH 7.4) in the dark or under light. FIG. 2B depicts the CO released from Compound 2b (10 μM) in 0.5 M K-phosphate buffer (pH 7.4) in the presence or absence of rat liver microsomes. FIGS. 2C-2E depict three identical tissue CO distribution experiments performed in CD-1 female mice. Compound 2b was administered i.v. (50 mg/kg in 150 μL PBS pH 7.5). 25 minutes after dosing, the animal was euthanized, the heart, kidneys, liver and lung were collected, and CO quantization was done. Samples of the freshly collected blood were transferred to AVOXimeter 4000 cuvettes (ITC) to measure the levels of carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb) and methemoglobin (MetHb) using a portable AVOXimeter 4000 CO-oximeter. The results are shown as mean percentage of total hemoglobin species in circulation.

FIGS. 3A-3B. FIG. 3A depicts the ORTEP drawing of Compound 3a; the labeling scheme for all non-hydrogen atoms is shown. Thermal elipsoids are at the 30% probability level. FIG. 3B depicts the x-ray powder diffraction (XRPD) pattern of Compound 3a.

FIG. 4A-4G. FIG. 4A depicts the ORTEP drawing of Compound 3b; the labeling scheme for all non-hydrogen atoms is shown. Thermal elipsoids are at the 30% probability level. FIG. 4B depicts the x-ray powder diffraction (XRPD) pattern of Compound 3b, Type I product. FIG. 4C depicts the x-ray powder diffraction (XRPD) pattern of Compound 3b, Type II product. FIG. 4D depicts CO release from Compound 3b (50 μM) in 50 mM HEPES buffer (pH 7.4) in the dark or under light. FIG. 4E depicts CO release from Compound 3b (10 μM) in 0.5 M K-phosphate buffer (pH 7.4) in the presence or absence of rat liver microsomes. FIGS. 4F-4G depict two identical tissue CO distribution experiments performed in CD-1 female mice. Compound 3b was administered i.v. (50 mg/kg in 150 μL PBS pH 7.5). 25 minutes after dosing, the animal was euthanized, the heart, kidneys, liver and lung were collected, and CO quantization was done. Samples of the freshly collected blood were transferred to AVOXimeter 4000 cuvettes (ITC) to measure the levels of carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb) and methemoglobin (MetHb) using a portable AVOXimeter 4000 CO-oximeter. The results are shown as mean percentage of total hemoglobin species in circulation.

FIGS. 5A-5B. FIG. 5A depicts the CO released from Compound 4b (10 μM) in 0.5 M K-phosphate buffer (pH 7.4) in the presence or absence of rat liver microsomes. FIG. 5B depicts the tissue CO distribution experiments performed in CD-1 female mice. Compound 4b was administered i.v. (50 mg/kg in 150 μL PBS pH 7.5). 25 minutes after dosing, the animal was euthanized, the heart, kidneys, liver and lung were collected, and CO quantization was done. Samples of the freshly collected blood were transferred to AVOXimeter 4000 cuvettes (ITC) to measure the levels of carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb) and methemoglobin (MetHb) using a portable AVOXimeter 4000 CO-oximeter. The results are shown as mean percentage of total hemoglobin species in circulation.

FIGS. 6A-6B. FIG. 6A depicts the CO released from Compound 5b (10 M) in 0.5 M K-phosphate buffer (pH 7.4) in the presence or absence of rat liver microsomes. FIG. 6B depicts the tissue CO distribution experiments performed in CD-1 female mice. Compound 5b was administered i.v. (50 mg/kg in 150 μL PBS pH 7.5). 25 minutes after dosing, the animal was euthanized, the heart, kidneys, liver and lung were collected, and CO quantization was done. Samples of the freshly collected blood were transferred to AVOXimeter 4000 cuvettes (ITC) to measure the levels of carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb) and methemoglobin (MetHb) using a portable AVOXimeter 4000 CO-oximeter. The results are shown as mean percentage of total hemoglobin species in circulation.

FIG. 7 depicts the pharmacokinetics of Compound 3b in CD-1 mice. Compound 3b was administered intravenously (i.v.), at a dose of 100 mg/kg. Blood samples were collected after 1, 5, 10, 20 and 30 minutes after administration. The concentration of Compound 3b in serum was assayed by HPLC. The experimental curve represents elimination from circulation and decay through CO release.

FIG. 8 depicts the amount of CO released from Compound 3b, expressed in equivalents of CO, in phosphate buffer pH 7.4 (“buffer”), phosphate buffer pH 7.4 in the presence of human liver microsomes (“human”) or rat liver microsomes (“rat”). The assay was performed in closed vials during 1 hour at 37° C. A gas aliquot was removed from the air space of the vials and CO concentration was determined by GC-RCP at different time points.

FIG. 9 depicts the percentage of carboxyhemoglobin (CO-Hb) measured in CD-1 female mice following Compound 3b administration at 300 mg/kg, intravenously. The CO-Hb was quantified over 80 min by using an oximeter (Avoximeter 4000 from A-vox Instruments). The base line of CO-Hb measured with this instrument is 3-4%.

FIGS. 10A-10B depict the kinetics of alanine aminotransferase (ALT) production and clearance. FIG. 10A depicts the kinetics of ALT production after 300 mg/kg administration of acetaminophen (APAP) by intraperitoneal (i.p.) injection. ALT is expressed in U/L over time (hours). FIG. 10B depicts the kinetics of APAP clearance after administration of 300 mg/kg by i.p. injection. APAP is expressed in g/L over time (hours).

FIGS. 11A-11B. FIG. 11A depicts the effect of treatment with Compound 3b on serum ALT in an APAP-induced acute liver failure (ALF) model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. One hour after APAP injection, the animals were treated with 0.3, 3, 30 or 60 mg/kg doses of Compound 3b. ALT was measured 22 h after APAP injection (n=5 mice for each group). FIG. 11B depicts the effect of Compound 3b in the liver damage induced by APAP. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. Compound 3b (0.3, 3, 30, 60 mg/kg) or Compound 1b (60 mg/kg) were administered to mice 1 h after APAP. Twenty-two hours after APAP injection, serum ALT was measured (results in FIG. 11A), centrilobular sections of mouse livers were cut and stained with hematoxylin and eosin, and the percentage of liver necrosis was determined (FIG. 11B).

FIGS. 12A-12C. FIGS. 12A-12B depict the effect of treatment with NAC or Compound 3b on serum ALT in an ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. Animals were treated with NAC(N-acetyl-cysteine; 300 mg/kg) or Compound 3b (60 or 120 mg/kg) administered at one and three hours after APAP injection. ALT was measured 22 h after APAP injection (n=4 or n=5 mice for each group). FIG. 12B is a zoom of FIG. 12A to help visualize the differences in the treated groups. FIG. 12C confirms the results obtained with ALT serum determinations (FIGS. 12A-12B), and depicts the effect of treatment with NAC or Compound 3b on liver damage in an APAP-induced ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. Animals were treated with NAC (300 mg/kg) or Compound 3b (60 or 120 mg/kg) administered at one and three hours after APAP injection. Twenty-two hours after APAP injection, serum ALT was measured (indicated in FIGS. 12A-12B), centrilobular sections of mouse livers were cut and stained with hematoxylin and eosin, and the percentage of liver necrosis was determined.

FIG. 13 depicts the effect of treatment with NAC or Compound 3b on serum ALT in an APAP-induced acute liver failure (ALF) model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. At five hours and seven hours after APAP injection, the animals were treated with NAC (150 mg/kg) or Compound 3b (120 mg/kg) or a combination of both compounds. Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group).

FIG. 14 depicts the effect of late treatment with Compound 3b on serum ALT in ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. At sixteen hours after APAP injection, the animals were treated with different doses of Compound 3b (0.3, 3, 30 or 60 mg/kg). Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group).

FIGS. 15A-15B depict the effect of combination treatment with NAC and Compound 3b on serum ALT in ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. NAC (300 mg/kg) and Compound 3b (120 mg/kg, FIG. 15A or 60 mg/kg, FIG. 15B) were administered to mice, separately or in combination, one hour and three hours after APAP injection. Experiments 1 and 2 represent two independent experiments. Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group).

FIG. 16 depicts the effect of NAC and Compound 3b in the liver damage induced by Acetaminophen. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. NAC and Compound 3b were administered at one and three hours after APAP administration. Twenty-two hours after APAP injection, serum ALT was measured and centrilobular sections of mouse livers were cut and stained with hematoxylin and eosin. A: Naïve mouse; B and C: APAP control mouse; D and E: APAP plus Compound 3b 2×100 mg/kg (duplicate experiments); F and G: APAP plus Compound 3b 2×300 mg/kg (duplicate experiments); H and I: APAP plus NAC 2×300 mg/kg (duplicate experiments).

FIG. 17 depicts the survival curve of C57BL/6 mice injected (i.p.) with 500 mg/kg of APAP. Mice were fasted overnight and APAP was administered afterward (n=15). Mice survival was monitored for 4 days.

FIGS. 18A-18B depicts the survival curve of C57BL/6 mice injected (i.p.) with 500 mg/kg of APAP and treated with Compound 3b. Mice were fasted overnight and APAP was administered afterward (n=15). Compound 3b was administered at a dose of 120 mg/kg at 1 h and 3 h (A) or at 3 h and 5 h (B) after APAP administration. Mice survival was monitored for 4 days.

FIG. 19 depicts the survival curve of C57BL/6 mice injected (i.p.) with 500 mg/kg of APAP and treated with Compound 3b, NAC or both administered at different times. Mice were fasted overnight and APAP was administered afterward (n=15). Compound 3b was administered at a dose of 120 mg/kg (ip) at +3 h or +4 h after APAP administration. Another group of mice received NAC (150 mg/kg, ip) at +1 h after APAP. The remaining two groups are administered in NAC+ Compound 3b combination mode; one group of mice was administered with NAC (150 mg/kg, ip) at +1 h plus Compound 3b (120 mg/kg, ip)+3 h and other with Compound 3b (150 mg/kg, ip) at +1 h plus Compound 3b (120 mg/kg, ip)+4 h. Mice survival was monitored for 4 days.

FIG. 20 depicts the effect of Compound 3b in the Ischemia-Reperfusion model. In anesthetized C57BL/6 mice, the hepatic artery and portal vein were clamped for 30 min; 24 h later, serum ALT levels were determined. Compound 3b was administered by i.p. injection at 30 mg/kg, 1 hour before and 1 h after surgery.

FIGS. 21A-21B depict the effect of Compound 3b in an ex-vivo model of apoptosis. Apoptosis of primary hepatocytes from C57BL/6 mice was induced with Actinomycin D (ActD; 200 ng/mL) and Tumour Necrosis Factor-α (TNF-α; 10 ng/mL). Compound 3b was added to the cultures 1 hour before and 1 hour after the addition of the apoptosis inducers ActD/TNF-α. Compound 3b was tested at concentrations of 25, 50, 100 and 150 μg/0.5 mL well. Hepatocytes survival was determined 24 hours after the addition of the apoptosis inducers. FIG. 21A: Percentage of increase in survival ActD/TNF-α treated cells in the presence of Compound 3b, relative to cells treated with only the apoptosis inducers. FIG. 21B: Toxicity of the Compound 3b doses on murine hepatocytes (not treated with apoptosis inducers). Data are the mean±S.D. of 3-5 independent experiments (3-5 livers from different mice).

FIG. 22 depicts the effect of Compound 3b in a model of liver regeneration. Animals were anesthetized and 70% of the liver was resected, corresponding to the left and median liver lobes. Compound 3b was administered (2×100 mg/kg) 1 h before and 1 h after surgery. Two days later, animals were sacrificed, livers harvested, weighed, and stained for phosphohistone H3 (PH3, a specific marker for cell division).

FIGS. 23A-23C depict the effect of Compound 3b in a model of TNBS (2,4,6-trinitrobenzenesulfonic acid) induced colitis. Colitis was induced in Balb/C mice by a single intracolonic administration of 100 ml of 40% ethanol containing 4 mg of TNBS. Dexamethasone (Dex; 0.3 mg/kg), Compound 3b (120 mg/kg) or its vehicle were administered daily for 3 consecutive days starting from 1 hour before colitis induction (day 1). On day 4 after colitis induction, mice were sacrificed, colon was isolated, cleaned and a 7 cm segment of the distal colon was resected and weighed (FIG. 23A). Stool consistency was determined using the score: 0, well-formed pellets; 1, loose stool; 2, liquid stool or bloody stool (FIG. 23B). Animals were weighed throughout the entire study (FIG. 23C).

FIG. 24 depicts the effect of Compound 4b in the ALF model in mice, administered (i.p.) at a dose of 0.3, 3, 10 or 30 mg/kg given 3 hours and 5 hours (+3 h, +5 h) after APAP (300 mg/kg) administration. Serum ALT levels were evaluated 22 hours after APAP administration. Compound 4b was able to reduce ALT levels induced by APAP in a dose-dependent manner; at a dose of 30 mg/kg the ALT levels were 75% reduced relative to untreated control animals.

FIG. 25 depicts the effect of Compound 5b in the ALF model in mice, administered (i.p.) at a dose of 10, 30 or 120 mg/kg given 3 hours and 5 hours (+3 h, +5 h) after APAP (300 mg/kg) administration. Serum ALT levels were evaluated 22 hours after APAP administration. Compound 5b was able to reduce ALT levels induced by APAP in a dose-dependent manner; at a dose of 120 mg/kg the ALT levels were 70% reduced relative to untreated control animals.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present disclosure is based, at least in part, on the surprising discovery that particular carbon monoxide releasing molecules (CO-RMs) compounds of the Formula (I):

are therapeutic CO-releasing molecules with specificity for the liver, and have been found to reduce liver necrosis stimulate liver regeneration, and reduce inflammation.

As is understood from the following disclosure, compounds of the Formula (I) require that each instance of R¹ and R² attached to the same carbon are not both hydrogen. That requirement is important aspect of the present discovery. For example, a structurally similar CO-releasing molecule wherein each instance of R¹ and R² is hydrogen is inactive in an in vivo model of liver failure.

Thus, the compounds of the present disclosure are considered particularly useful for the treatment of diseases of the liver, but are also generally contemplated for use in the treatment of inflammatory diseases.

Compounds

In one aspect, the present disclosure provides a compound of the Formula (I):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof;wherein:

each instance of R¹ is independently hydrogen, an unsubstituted C_1-3alkyl, or a C_1-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂, wherein each instance of R^A1 is independently hydrogen or C_1-10alkyl;

each instance of R² are independently hydrogen, an unsubstituted C_1-3alkyl, or a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, wherein each instance of R^A2 is independently hydrogen or C_1-10alkyl;

or R¹ and R² and the carbon to which they are both attached are independently joined to form a C_3-4 carbocyclyl;

provided that each instance of R¹ and R² attached to the same carbon are not both hydrogen.

The specific chemical terms are described below. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987. General principles of organometallic chemistry is described in S. W. Kirtley in Comprehensive Organometallic Chemistry I (G. Wilkinson, F. G. A. Stone, W. Abel Eds, Vol 3, 1080, Pergamon, Oxford 1982; M. J. Winter in Comprehensive Organometallic Chemistry II (W. Abel, F. G. A. Stone, G. Wilkinson Eds), Vol 5, 163, Pergamon, Oxford 1995; and M. Tamm, R. J. Baker, in Comprehensive Organometallic Chemistry III (R. H. Crabtree and D. M. P. Mingos Eds), Vol 5, 391, Elsevier, Oxford 2007.

When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C_1-6 alkyl” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C_2-6, C_2-5, C_2-4, C_2-3, C_3-6, C_3-5, C_3-4, C_4-6, C_4-5, and C_5-6 alkyl.

“Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C_1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C_1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C_1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C_1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C_1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C_1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C_1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C_1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C_1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C_2-6alkyl”). Examples of C_1-6 alkyl groups include methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆). Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈), n-nonyl (C₉), n-decyl (C₁₀), and the like.

“Carbocyclyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 4 ring carbon atoms (“C_3-4 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Exemplary C_3-4 carbocyclyl groups include, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl (C₄), and cyclobutenyl (C₄). In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 4 ring carbon atoms (“C_3-4 cycloalkyl”).

“Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (“C_2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C_2-9alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C_2-8alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C_2-7alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C_2-6alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C_2-5alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C_2-4alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C_2-3alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C_2-4 alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂— alkenyl groups include the aforementioned C_2-4 alkenyl groups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and the like.

“Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (“C_2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C_2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C₂— alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C_2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C_2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C_2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C_2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C_2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C_2-4 alkynyl groups include, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C₂— alkenyl groups include the aforementioned C_2-4 alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and the like. Additional examples of alkynyl include heptynyl (C₇), octynyl (C₈), and the like.

“Heterocyclyl” refers to a radical of a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic or bicyclic, and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.

In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, and the like.

“Aryl” refers to a radical of a monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10π electrons shared in a cyclic array) having 6-10 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C_6-10 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆ aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

Alkyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, may be optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, i.e., a non-hydrogen substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds that results in the formation of a stable compound. Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, substituted or unsubstituted hydroxyl (e.g., —OH, alkoxy), substituted or unsubstituted thiol (e.g., —SH, alkylthiooxy), substituted or unsubstituted amino (e.g., —NH₂, alkyl amino, dialkyl amino), cyano, nitro, halo (i.e., —F, Br, —Cl, —I), ester, amide, imino, —CO₂H, —CHO, and the like.

The term “salt” or “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, see Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19, and P. Heinrich Stahl and Camille G. Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH 2002. Pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts (i.e., a salt formed from the compound upon addition of an acid) and pharmaceutically acceptable base addition salts (i.e., a salt formed from the compound upon addition of a base). Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate salts. Pharmaceutically acceptable base addition salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and quaternary amine salts.

An “ester” of a compound of the present disclosure refers to that compound wherein one or more of the acidic hydrogens of the carboxylic acid (—CO₂H) groups provided in the molecule are replaced with a non-hydrogen group (e.g., an alkyl group). Exemplary esters of compounds of the present disclosure include, but are not limited to, compounds which comprise one or more of the following groups:

wherein R³, R^A1, and R^A2 are as described herein, provided that R³, R^A1, and R^A2 are not hydrogen.

An “amide” of a compound of the present disclosure refers to that compound wherein one or more of the —OH groups of the carboxylic acid (—CO₂H) provided in the molecule are replaced with a substituted or unsubstituted amino group. Exemplary amides of compounds of the present disclosure include, but are not limited to, compounds which comprise one or more of the following groups:

wherein R⁴, R^A1, and R^A2 are as described herein.

The term “hydrate” refers to a compound of the present disclosure non-covalently associated with one or more molecules of water. Likewise, a “solvate” refers to a compound of the present disclosure non-covalently associated with one or more molecules of an organic solvent.

(i) Various Embodiments of R¹ and R²

In certain embodiments, at least one instance of R¹ and R² and the carbon to which they are both attached are joined to form a C₃ carbocyclyl. In certain embodiments, at least one instance of R¹ and R² and the carbon to which they are both attached are joined to form a cyclopropanyl (C₃) ring. In certain embodiments, at least one instance of R¹ and R² and the carbon to which they are both attached are joined to form a cyclobutanyl (C₄) ring.

In certain embodiments, at least two instances of R¹ and R² and the carbon to which they are both attached are independently joined to form a C_3-4 carbocyclyl. In certain embodiments, at least two instances of R¹ and R² and the carbon to which they are both attached are joined to form a cyclopropanyl (C₃) ring. In certain embodiments, at least two instances of R¹ and R² and the carbon to which they are both attached are joined to form a cyclobutanyl (C₄) ring.

In certain embodiments, each instance of R¹ and R² and the carbon to which they are both attached are independently joined to form a C_3-4 carbocyclyl. In certain embodiments, each instance of R¹ and R² and the carbon to which they are both attached are joined to form a cyclopropanyl (C₃) ring. In certain embodiments, each instance of R¹ and R² and the carbon to which they are both attached are joined to form a cyclobutanyl (C₄) ring.

In certain embodiments, at least one instance of R¹ and R², wherein R¹ and R² are attached to the same carbon, comprise R¹ independently selected from hydrogen, an unsubstituted C_1-3alkyl, and a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂, wherein each instance of R^A1 is independently hydrogen or C_1-10 alkyl; and R² independently selected from hydrogen, an unsubstituted C_1-3alkyl, and a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, wherein each instance of R^A2 is independently hydrogen or C_1-10alkyl.

In certain embodiments, at least two instances of R¹ and R², wherein R¹ and R² are attached to the same carbon, comprise R¹ independently selected from hydrogen, an unsubstituted C_1-3alkyl, and a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂, wherein each instance of R^A1 is independently hydrogen or C_1-10alkyl; and R² independently selected from hydrogen, an unsubstituted C_1-3alkyl, and a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, wherein each instance of R^A2 is independently hydrogen or C_1-10alkyl.

In certain embodiments, each instance of R¹ and R², wherein R¹ and R² are attached to the same carbon, comprise R¹ independently selected from hydrogen, an unsubstituted C_1-3alkyl, and a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂, wherein each instance of R^A1 is independently hydrogen or C_1-10 alkyl; and R² independently selected from hydrogen, an unsubstituted C_1-3alkyl, and a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, wherein each instance of R^A2 is independently hydrogen or C_1-10alkyl.

As used herein, wherein R¹ is an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂, it is understood that each instance of R¹ may encompass a variety of different groups, e.g., for example, each instance of R¹ may be independently selected from unsubstituted C_1-2alkyl, C_1-2alkyl substituted with —CO₂R^A1, C_1-2alkyl substituted with —C(═O)N(R^A1)₂, unsubstituted C_2-3alkyl, C_2-3alkyl substituted with —CO₂R^A1, C_2-3alkyl substituted with —C(—O)N(R^A1)₂, unsubstituted C₁alkyl, C₁alkyl substituted with —CO₂R^A1, C₁alkyl substituted with —C(═O)N(R^A1)₂, unsubstituted C₂alkyl, C₂alkyl substituted with —CO₂R^A1, C₂alkyl substituted with —C(═O)N(R^A1)₂, unsubstituted C₃alkyl, C₃alkyl substituted with —CO₂R^A1, or C₃alkyl substituted with —C(═O)N(R^A1)₂. For example, in certain embodiments, each instance of R¹ is independently selected from —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, —CH(CO₂R^A1)CH₃, —CH₂CH₂CH₂CO₂R^A1, —CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), —C(CO₂R^A1)(CH₃)₂, —CH₂C(═O)N(R^A1)₂, —CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(C(═O)N(R^A1)₂)(CH₃)₂.

As generally described above, each instance of R^A1 is independently hydrogen or C_1-10alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen or C_1-8alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen or C_1-6alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen or C_1-4alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen or C_1-3alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen or C_1-2alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen or C₁alkyl. In certain embodiments, each instance of R^A1 is independently hydrogen.

Furthermore, as used herein, wherein R² is an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, it is understood that each instance of R² may encompass a variety of different groups, e.g., for example, each instance of R² may be independently selected from unsubstituted C_1-2alkyl, C_1-2alkyl substituted with —CO₂R^A2, C_1-2alkyl substituted with —C(═O)N(R^A2)₂, unsubstituted C_2-3alkyl, C_2-3alkyl substituted with —CO₂R^A2, C_2-3alkyl substituted with —C(═O)N(R^A2)₂, unsubstituted C₁alkyl, C₁alkyl substituted with —CO₂R^A2, C₁alkyl substituted with —C(═O)N(R^A2)₂, unsubstituted C₂alkyl, C₂alkyl substituted with —CO₂R^A2, C₂alkyl substituted with —C(═O)N(R^A2)₂, unsubstituted C₃alkyl, C₃alkyl substituted with —CO₂R^A2, or C₃alkyl substituted with —C(═O)N(R^A2)₂. For example, in certain embodiments, each instance of R² is independently selected from —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A2, —CH₂CH₂CO₂R², —CH(CO₂R^A2)CH₃, —CH₂CH₂CH₂CO₂R^A2, —CH₂CH(CO₂R^A2)CH₃, —CH(CO₂R^A2)CH₂CH₃, —CH(CH₃)(CHCO₂R^A2), —C(CO₂R^A2)(CH₃)₂, —CH₂C(═O)N(R^A2)₂, —CH₂CH₂CH₂C(═O)N(R^A2)₂, —CH(C(═O)N(R)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A2)₂, —CH₂CH(C(═O)N(R^A2)₂)CH₃, —CH(C(═O)N(R^A2)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R²)₂), and —C(C(═O)N(R^A2)₂)(CH₃)₂.

As generally described above, each instance of R^A2 is independently hydrogen or C_1-10alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen or C_1-8alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen or C_1-6alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen or C_1-4alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen or C_1-3alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen or C_1-2alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen or C₁alkyl. In certain embodiments, each instance of R^A2 is independently hydrogen.

(ii) Embodiments Wherein One of R¹ and R² is Hydrogen

In certain embodiments, each instance of R² is hydrogen. In this instance, in certain embodiments, each instance of R¹ is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂. In certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, at least one instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, at least two instances of R¹ is an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least two instances of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least two instances of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, at least two instances of R¹ is an unsubstituted C_1-3alkyl.

In certain embodiments, R² is hydrogen and each instance of R¹ is independently unsubstituted C_1-3alkyl or C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_1-2alkyl or C_1-2alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_2-3alkyl or C_2-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₁alkyl or C₁alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₂alkyl or C₂alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₃alkyl or C₃alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, —CH(CO₂R^A1)CH₃, CH₂CH₂CH₂CO₂R^A1, —CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), —C(CO₂R^A1)(CH₃)₂, —CH₂C(═O)N(R^A1)₂, —CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(C(—O)N(R^A1)₂)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CO₂R^A1 or —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is —CH₃. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1. In certain embodiments, each instance of R¹ is —CH₂CO₂H. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂.

In certain embodiments, each instance of R² is hydrogen and each instance of R¹ is independently C_1-3alkyl substituted with —CO₂R^A1. In this instance, in certain embodiments, each instance of R¹ is independently C_1-2alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is independently C_2-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is independently C₁alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is independently C₂alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is independently C₃alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, —CH(CO₂R^A1)CH₃, —CH₂CH₂CH₂CO₂R^A1, —CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), or —C(CO₂R^A1)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1. In certain embodiments, each instance of R¹ is —CH₂CO₂H.

In certain embodiments, each instance of R² is hydrogen and each instance of R¹ is independently C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In this instance, in certain embodiments, each instance of R¹ is independently C_1-2alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently C_2-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently C₁alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently C₂alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently C₃alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂, —CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(═O)N(R^A1)₂)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is —CH₂C(═O)NH₂.

In certain embodiments, each instance of R² is hydrogen and each instance of R¹ is independently unsubstituted C_1-3alkyl. In this instance, in certain embodiments, each instance of R¹ is independently unsubstituted C_1-2alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C_2-3alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C₁alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C₂alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C₃alkyl. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, or —CH(CH₃)₂. In certain embodiments, each instance of R¹ is —CH₃.

(iii) Embodiments Wherein Neither R¹ Nor R² are Hydrogen

In certain embodiments, each instance of R² is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂, and each instance of R¹ is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In this instance, in certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, at least one instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, at least two instances of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_1-2alkyl or C_1-2alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_2-3alkyl or C_2-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₁alkyl or C₁alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₂alkyl or C₂alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₃alkyl or C₃alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, —CH(CO₂R^A1)CH₃, —CH₂CH₂CH₂CO₂R^A1, —CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), —C(CO₂R^A1)(CH₃)₂, —CH₂C(═O)N(R^A1)₂, —CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(C(═O)N(R^A1)₂)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CO₂R^A1 or —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is —CH₃. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1. In certain embodiments, each instance of R¹ is —CH₂CO₂H. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)NH₂.

In certain embodiments, each instance of R² is independently an unsubstituted C_1-3alkyl, and each instance of R¹ is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂. In this instance, in certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, at least one instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, at least two instances of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(—O)N(R^A1)₂. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C_1-2alkyl or C_1-2alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_2-3alkyl or C_2-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₁alkyl or C₁alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₂alkyl or C₂alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₃alkyl or C₃alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, CH(CO₂R^A1)CH₃, —CH₂CH₂CH₂CO₂R^A1, CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), —C(CO₂R^A1)(CH₃)₂, —CH₂C(═O)N(R^A1)₂—CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(C(═O)N(R^A1)₂)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CO₂R^A1 or —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is —CH₃. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1. In certain embodiments, each instance of R¹ is —CH₂CO₂H. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)NH₂.

In certain embodiments, each instance of R² is independently a C_1-3alkyl substituted with —CO₂R¹², and each instance of R¹ is independently an unsubstituted C_1-3 alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In this instance, in certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, at least one instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, at least two instances of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(O)N(R^A1)₂. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C_1-2alkyl or C_1-2alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_2-3alkyl or C_2-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₁alkyl or C₁alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₂alkyl or C₂alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₃alkyl or C₃alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, CH(CO₂R^A1)CH₃, —CH₂CH₂CH₂CO₂R^A1, CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), —C(CO₂R^A1)(CH₃)₂, —CH₂C(═O)N(R^A1)₂, —CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(═O)N(R^A1)₂)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CO₂R^A1 or —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is —CH₃. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1. In certain embodiments, each instance of R¹ is —CH₂CO₂H. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)NH₂.

In certain embodiments, each instance of R² is independently a C_1-3alkyl substituted with —C(═O)N(R^A2)₂, and each instance of R¹ is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A1 or —C(—O)N(R^A1)₂. In this instance, in certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, at least one instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, at least one instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, at least two instances of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R¹ is a C_1-3alkyl substituted with —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is an unsubstituted C_1-3alkyl. In certain embodiments, each instance of R¹ is independently unsubstituted C_1-2alkyl or C_1-2alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C_2-3alkyl or C_2-3alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₁alkyl or C₁alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₂alkyl or C₂alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently unsubstituted C₃alkyl or C₃alkyl substituted with —CO₂R^A1 or —C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CO₂R^A1, —CH₂CH₂CO₂R^A1, CH(CO₂R^A1)CH₃, —CH₂CH₂CH₂CO₂R^A1, —CH₂CH(CO₂R^A1)CH₃, —CH(CO₂R^A1)CH₂CH₃, —CH(CH₃)(CHCO₂R^A1), —C(CO₂R^A1)(CH₃)₂, —CH₂C(═O)N(R^A1)₂, —CH₂CH₂C(═O)N(R^A1)₂, —CH(C(═O)N(R^A1)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A1)₂, —CH₂CH(C(═O)N(R^A1)₂)CH₃, —CH(C(═O)N(R^A1)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A1)₂), and —C(C(═O)N(R^A1)₂)(CH₃)₂. In certain embodiments, each instance of R¹ is independently —CH₃, —CH₂CO₂R^A1 or —CH₂C(═O)N(R^A1)₂. In certain embodiments, each instance of R¹ is —CH₃. In certain embodiments, each instance of R¹ is independently —CH₂CO₂R^A1. In certain embodiments, each instance of R¹ is —CH₂CO₂H. In certain embodiments, each instance of R¹ is independently —CH₂C(═O)N(R^A1)₂.

In any of the above embodiments, R² is independently an unsubstituted C_1-3alkyl or a C_1-3alkyl substituted with —CO₂R^A2 or —C(═O)N(R^A2)₂.

In any of the above embodiments, each instance of R² is independently C_1-3alkyl substituted with —CO₂R^A2. In this instance, in certain embodiments, each instance of R² is independently C_1-2alkyl substituted with —CO₂R^A2. In certain embodiments, each instance of R² is independently C_2-3alkyl substituted with —CO₂R^A1. In certain embodiments, each instance of R² is independently C₁alkyl substituted with —CO₂R^A2. In certain embodiments, each instance of R² is independently C₂alkyl substituted with —CO₂R^A2. In certain embodiments, each instance of R² is independently C₃alkyl substituted with —CO₂R^A2. In certain embodiments, each instance of R² is independently —CH₂CO₂R^A2, —CH₂CH₂CO₂R^A2, —CH(CO₂R^A2)CH₃, —CH₂CH₂CH₂CO₂R^A2, —CH₂CH(CO₂R^A2)CH₃, —CH(CO₂R^A2)CH₂CH₃, —CH(CH₃)(CHCO₂R^A2), or —C(CO₂R^A2)(CH₃)₂. In certain embodiments, each instance of R² is independently —CH₂CO₂R². In certain embodiments, each instance of R² is —CH₂CO₂H.

In any of the above embodiments, each instance of R² is independently C_1-3alkyl substituted with —C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is independently C_1-2alkyl substituted with —C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is independently C_2-3alkyl substituted with —C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is independently C₁alkyl substituted with —C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is independently C₂alkyl substituted with —C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is independently C₃alkyl substituted with —C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is independently —CH₂C(═O)N(R^A2)₂, —CH₂CH₂C(═O)N(R^A2)₂, —CH(C(═O)N(R^A2)₂)CH₃, —CH₂CH₂CH₂C(═O)N(R^A2)₂, —CH₂CH(C(═O)N(R^A2)₂)CH₃, —CH(C(═O)N(R^A2)₂)CH₂CH₃, —CH(CH₃)(CHC(═O)N(R^A2)₂), and —C(C(═O)N(R^A2)₂)(CH₃)₂. In certain embodiments, each instance of R² is independently —CH₂C(═O)N(R^A2)₂. In certain embodiments, each instance of R² is —CH₂C(═O)NH₂.

In any of the above embodiments, each instance of R² is independently unsubstituted C_1-3alkyl. In this instance, in certain embodiments, each instance of R² is independently unsubstituted C_1-2alkyl. In certain embodiments, each instance of R² is independently unsubstituted C_2-3alkyl. In certain embodiments, each instance of R² is independently unsubstituted C₁alkyl. In certain embodiments, each instance of R² is independently unsubstituted C₂alkyl. In certain embodiments, each instance of R² is independently unsubstituted C₃alkyl. In certain embodiments, each instance of R² is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, or —CH(CH₃)₂. In certain embodiments, each instance of R² is —CH₃.

(iv) Additional Embodiments

It should be understood from the above discussion that the compound of Formula (I), as described above, and subsets thereof (e.g., compounds of Formula (II)), encompass compounds wherein each instance of the ligand (i):

may be the same or different. For example, in certain embodiments, each instance of the ligand (i) is the same. In certain embodiments, at least one instance of the ligand (i) is different.

In certain embodiments, each instance of R¹ is the same. In certain embodiments, each instance of R² is the same. For example, in certain embodiments, R¹ and R² attached to the same carbon are the same. In certain embodiments, each instance of R¹ and R² is —CH₃. In certain embodiments, each instance of R¹ and R² is —CH₂CH₃. In certain embodiments, each instance of R¹ and R² is —CH₂CH₂CH₃. In certain embodiments, each instance of R¹ and R² is —CH₂CO₂H. In certain embodiments, each instance of R¹ and R² is —CH₂CO₂CH₃.

However, in certain embodiments, at least one instance of R¹ is different from R² attached to the same carbon or from another instance of R¹. In certain embodiments, at least one instance of R² is different from R¹ attached to the same carbon or from another instance of R². In certain embodiments, R¹ and R² attached to the same carbon are different groups. For example, in certain embodiments, R² is hydrogen and R¹ is —CH₃. In certain embodiments, R² is hydrogen and R¹ is —CH₂CH₃. In certain embodiments, R² is hydrogen and R¹ is —CH₂CH₂CH₃. In certain embodiments, R² is hydrogen and R¹ is —CH₂CO₂H. In certain embodiments, R² is hydrogen and R¹ is —CH₂CO₂CH₃.

As understood from the above discussion, the groups R¹ and R² attached to the same carbon each may be different, e.g., for example wherein R² is hydrogen and R¹ is not hydrogen. In this instance, the ligand of the formula (I) is a chiral ligand, i.e., having (R) or (s) stereochemistry. In certain embodiments, the chiral ligand has (R) stereochemistry. In certain embodiments, the chiral ligand has (S) stereochemistry.

Various combinations of the above described embodiments are further contemplated herein.

For example, in certain embodiments of Formula (I), neither R¹ nor R² is hydrogen.

In other embodiments of Formula (I), each instance of R² is hydrogen, i.e., to provide a compound of the Formula (I-a):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein R¹ is as described herein.

In other embodiments, wherein R¹ is an unsubstituted C_1-3alkyl, the compound of Formula (I) is a compound of the Formula (I-b):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein R² is as described herein and each instance of m is independently 0, 1, or 2. In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, R² is hydrogen. In certain embodiments R² is not hydrogen.

For example, in certain embodiments of Formula (I-b), wherein R² is hydrogen, the compound of the Formula (I) is a compound of the Formula (I-c):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein each instance of m is independently 0, 1, or 2. In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2.

In certain embodiments of Formula (I-b), wherein R² is an unsubstituted C_1-3alkyl, the compound is of the Formula (I-d):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein each instance of r is independently 0, 1, or 2. In certain embodiments, r is 0. In certain embodiments, r is 1. In certain embodiments, r is 2.

In yet other embodiments, wherein R¹ is a C_1-3alkyl substituted by —CO₂R^A1, the compound of Formula (I) is a compound of the Formula (I-e):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein R² and R^A1 are as described herein, and each instance of p is independently 1, 2, or 3. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, R² is hydrogen. In certain embodiments, R² is not hydrogen. In certain embodiments, R^A1 is hydrogen.

For example, in certain embodiments of Formula (I-e), wherein R² is hydrogen, the compound is of the Formula (I-f):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein R^A1 is as described herein, and each instance of p is independently 1, 2, or 3. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, R^A1 is hydrogen.

In yet other embodiments, wherein R¹ is a C_1-3alkyl substituted by —CO₂N(R^A1)₂, the compound of Formula (I) is a compound of the Formula (I-g):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein R^A1 is as described herein, and each instance of p is independently 1, 2, or 3. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, R^A1 is hydrogen.

For example, in certain embodiments of Formula (I-g), wherein R² is hydrogen, the compound is of the Formula (I-h):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein R^A1 is as described herein, and each instance of p is independently 1, 2, or 3. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, R^A1 is hydrogen.

In still yet other embodiments of Formula (I), wherein each instance of R¹ and R² and the carbon to which they are both attached are independently joined to form a C_3-4 carbocyclyl, the compound is of the Formula (I-i):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof; wherein each instance of q is independently 1 or 2. In certain embodiments, q is 1. In certain embodiments, q is 2.

Exemplary compounds of Formula (I) include, but are not limited to:

and salts, esters, amides, solvates, and hydrates thereof, and combinations thereof.

In certain embodiments, the compound of Formula (I) is compound (2-b):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

The present disclosure further provides a compound of the molecular formula C₁₅H₁₅N₃O₉Mo or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

In certain embodiments, the compound of Formula (I) is compound (3-b):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

The present disclosure further provides a compound of the molecular formula C18H₂₁N₃O₉Mo or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

In certain embodiments, the compound of Formula (I) is compound (4-b):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

The present disclosure further provides a compound of the molecular formula C18H₁₅N₃O₁₅Mo or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

In certain embodiments, the compound of Formula (I) is compound (5-b):

or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

The present disclosure further provides a compound of the molecular formula C18H₁₅N₃O₉Mo or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof.

It is understood that the compound of Formula (I) encompasses salts, esters, amides, solvates, hydrates and any combination thereof, of the compound. Salts, esters, amides, solvates, and hydrates are described herein.

In certain embodiments, the compound of Formula (I) comprises a salt. In certain embodiments, the compound of Formula (I) comprises a mixture of the fully protonated compound and one or more salts. Specific salt forms of the compound of Formula (I) are contemplated herein since the compound contains multiple acidic groups which may form a salt upon contact with a base (“base addition salt”).

For example, in certain embodiments, the salt of the compound of Formula (I) is a salt provided from the deprotonation of one, two, three, or more carboxylic acids groups attached thereto, e.g., such as the salts depicted in Formula (I-j), (I-k), (I-l), or (I-m):

or a solvate or hydrate thereof, or a combination thereof, wherein x is 1, 2, 3, 4, 5, or 6, and Q is a cation, i.e., which renders the compound neutral.

It is clear from the depiction of the compound of Formula (I-m) that the present disclosure contemplates salt formation from the deprotonation of one or more —CO₂H substituents attached to the ligand (i). For the sake of brevity, the compound of Formula (I-m) is depicted as fully deprotonated. However, all intermediate salt forms, wherein 1, 2, 3, 4, or 5 of the carboxylic acid groups are deprotonated, are also contemplated.

Furthermore, as understood from the above, Q can be any cation, i.e., any atom or group of atoms that bears a positive charge. In certain embodiments, each instance of the cation Q is independently Na⁺, K⁺, Li⁺, Mg²⁺, Ca²⁺, Zn²⁺, Al³⁺, or a quaternary amine of the formula [NR^B₄]⁺, wherein each R^B is independently hydrogen or substituted or unsubstituted C_1-10alkyl, or two R^B groups are joined to form a substituted or unsubstituted 5-10 membered heteroaryl or substituted or unsubstituted 5-10 membered heterocyclyl ring.

In certain embodiments, at least one instance of Q is Na⁺, K⁺, Li⁺, Mg²⁺, Ca²⁺, Zn²⁺, or Al³⁺. In certain embodiments, at least one instance of Q is Na⁺, K⁺, or Li⁺. In certain embodiments, at least one instance of Q is Na⁺. In certain embodiments, at least one instance of Q is K⁺. In certain embodiments, at least one instance of Q is Li⁺. In certain embodiments, each instance of the cation Q is independently Na⁺, K⁺, or Li⁺. In certain embodiments, each instance of the cation Q is Na⁺ (i.e., to provide a sodium salt). In certain embodiments, each instance of the cation Q is K⁺ (i.e., to provide a potassium salt). In certain embodiments, each instance of the cation Q is Li⁺ (i.e., to provide a lithium salt).

In certain embodiments, at least one instance of the cation Q is a quaternary amine of the formula [NR^B₄]⁺ or [(R^B)₂N═R^C]⁺ wherein R^C is substituted or unsubstituted C_1-10alkyl (e.g., substituted or unsubstituted C_1-8alkyl, substituted or unsubstituted C_1-6alkyl, substituted or unsubstituted C_1-4alkyl, substituted or unsubstituted C_1-3alkyl, or substituted or unsubstituted C_1-2alkyl), or R^C and R^B are joined to form a substituted or unsubstituted 5-10 membered heteroaryl or substituted or unsubstituted 5-10 membered heterocyclyl ring; and each instance of R^B is independently hydrogen or substituted or unsubstituted C_1-10alkyl (e.g., substituted or unsubstituted C_1-8alkyl, substituted or unsubstituted C_1-6alkyl, substituted or unsubstituted C_1-4alkyl, substituted or unsubstituted C_1-3alkyl, or substituted or unsubstituted C_1-2alkyl), or two R^B groups and the nitrogen to which they are attached are joined to form a substituted or unsubstituted 5-10 membered heteroaryl, a substituted or unsubstituted 5-10 membered heterocyclyl ring.

Exemplary quaternary amines of the formula [NR^B₄]⁺ include, but are not limited to, choline, histidine, lysine, and arginine:

In certain embodiments, a salt of the compound (2-b) is selected from any one of the following mono-, di-, or tri-salt forms:

or a solvate or hydrate thereof, or a combination thereof, wherein x is 1, 2, or 3, and Q is a cation, as described herein.

In certain embodiments, a salt of the compound (3-b) is selected from any one of the following mono-, di-, or tri-salt forms:

or a solvate or hydrate thereof, or a combination thereof, wherein x is 1, 2, or 3, and Q is a cation as described herein.

In certain embodiments, the compound of Formula (I) is an ester and/or an amide, as described herein. In certain embodiments, the ester and/or the amide hydrolyzes in vivo to the carboxylic acid compound of the Formula (I). In certain embodiments, the ester and/or the amide is a prodrug. A “prodrug” refers to an ester and/or an amide of a compound of Formula (I) that can react under biological conditions (e.g., in vitro or in vivo enzymatic conditions) to provide the parent carboxylic acid compound. In certain embodiments, a prodrug has improved physical and/or delivery properties over the parent compound. Prodrugs are typically designed to enhance pharmacologically, pharmaceutically and/or pharmacokinetically based properties associated with the parent compound. The advantage of a prodrug can lie in its physical properties, such as enhanced water solubility for parenteral administration at physiological pH compared to the parent compound, or it enhances absorption from the digestive tract, or it may have enhanced stability for long-term storage.

In certain embodiments, the ester is a prodrug, i.e., hydrolyzes in vivo to the carboxylic acid compound of the Formula (I).

In certain embodiments, the ester of the compound of Formula (I) is a compound of Formula (II):

or a salt, solvate, or hydrate thereof, or a combination thereof; wherein R¹ and R² are as described herein; and each instance of R³ is independently C_1-6alkyl, provided that each instance of R¹ and R² attached to the same carbon are not both hydrogen.

In certain embodiments, each instance of R³ is independently C_1-5alkyl, C_1-4alkyl, C_1-3alkyl, C_1-2alkyl, C_2-6alkyl, C_2-5alkyl, C_2-4alkyl, C_2-3alkyl, C_3-6alkyl, C_3-5alkyl, C_3-4alkyl, C_4-6alkyl, C_5-6alkyl, C₆alkyl, C₅alkyl, C₄alkyl, C₃alkyl, C₂alkyl, or C₁alkyl. In certain embodiments, each instance of R³ is independently —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CH₂CH₂CH₃, —CH₂CH(CH₃)₂, —C(CH₃)₃, —CH₂CH₂CH₂CH₂CH₃, —CH₂CH₂CH(CH₃)₂, or —CH₂CH₂CH₂CH₂CH₂CH₃. In certain embodiments, each instance of R³ is —CH₃. In certain embodiments, each instance of R³ is —CH₂CH₃.

Exemplary compounds of Formula (II) include, but are not limited to:

and salts, solvates, and hydrates thereof, and combinations thereof.

In certain embodiments, the compound of Formula (I) is an amide, as described herein. In certain embodiments, the amide is a prodrug, i.e., hydrolyzes in vivo to the carboxylic acid compound of the Formula (I).

In certain embodiments, the amide of the compound of Formula (I) is a compound of Formula (III):

or a salt, solvate, or hydrate thereof, or a combination thereof; wherein R¹ and R² are as described herein; and each instance of R⁴ is independently hydrogen or C_1-6alkyl, provided that each instance of R¹ and R² attached to the same carbon are not both hydrogen.

In certain embodiments, R⁴ is C_1-5alkyl, C_1-4alkyl, C_1-3alkyl, C_1-2alkyl, C_2-6alkyl, C_2-5alkyl, C_2-4alkyl, C_2-3alkyl, C_3-6alkyl, C_3-5alkyl, C_3-4alkyl, C_4-6alkyl, C_5-6alkyl, C₆alkyl, C₅alkyl, C₄alkyl, C₃alkyl, C₂alkyl, or C₁alkyl. In certain embodiments, R⁴ is —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CH₂CH₂CH₃, —CH₂CH(CH₃)₂, —C(CH₃)₃, —CH₂CH₂CH₂CH₂CH₃, —CH₂CH₂CH(CH₃)₂, or —CH₂CH₂CH₂CH₂CH₂CH₃. In certain embodiments, each instance of R⁴ is —CH₃. In certain embodiments, each instance of R⁴ is —CH₂CH₃.

Exemplary compounds of Formula (III) include, but are not limited to:

and salts, solvates, and hydrates thereof, and combinations thereof.Methods of Preparation

Provided are methods of making compounds of the present disclosure, i.e., compounds of the Formula (I), (II), and (III).

For example, in one aspect, provided is a method of preparing a compound of the Formula (II):

or a salt, solvate, or hydrate thereof, or a combination thereof; wherein R¹, R², and R³ are as defined herein;

the method comprising reacting a molybdenum tri-CO complex with an isocyanide of the formula:

wherein R¹, R², and R³ are as described herein; to provide a compound of the Formula (II).

In certain embodiments, the molybdenum tri-CO complex is of the formula:Mo(CO)₃L₃ wherein L₃ represents either three monodentate ligands, one bidentate ligand and one monodentate ligand, or one tridentate ligand.

Exemplary monodentate ligands include, but are not limited to, CO, organonitriles (e.g., CH₃CN, CH₃CH₂CN), monosubstituted amines, disubstituted amines, trisubstituted amines, heterocyclyls (e.g., pyridine, piperidine), dialkylcyanamides, triphenylphosphine oxide, THF, DMF, or NMF.

Exemplary bidentate ligands include, but are not limited to, 1,5-cyclooctadiene, norbornadiene, 1,2-ethylenediamine, tetramethylethylenediamine, 1,2-dimethoxyethane, diglyme, or 2,5-dithiahexane.

Exemplary tridentate ligands include, but are not limited to, conjugated cyclic triene (e.g., cycloheptatriene), conjugated acyclic triene, arenes (e.g., benzene, toluene, xylene, mesitylene, naphthalene), tetraazamacrocyles (e.g., tetraazacyclododecane), polyamines (e.g., diethylenetriamine), and trithiocylononane.

In certain embodiments, the molybdenum tri-CO complex is of the formula:Mo(CO)₃L₃ wherein L₃ represents either three monodentate ligands. In certain embodiments, the three monodentate ligands are CO ligands. In certain embodiments, the three monodentate ligands are organonitrile ligands.

In certain embodiments, the molybdenum tri-CO complex is of the formula:Mo(CO)₃L₃ wherein L₃ represents one bidentate ligand and one monodentate ligand.

In certain embodiments, the molybdenum tri-CO complex is of the formula:Mo(CO)₃L₃ wherein L₃ represents one tridentate ligand. In certain embodiments, the tridentate ligand is a cyclic triene. In certain embodiments, the cyclic triene is cycloheptatriene.

In certain embodiments, the molybdenum tri-CO complex is:

In certain embodiments, the molybdenum tri-CO complex is:

In yet another aspect, provided is a method of preparing a compound of the Formula (I):

or a solvate or hydrate thereof, or a combination thereof; wherein R¹ and R² are as defined herein; the method comprising hydrolyzing a compound of Formula (II) to provide a compound of the Formula (I).

In certain embodiments, the step of hydrolyzing comprises an acid. In certain embodiments, the acid is an acid catalyst.

In certain embodiments, the step of hydrolyzing comprises a base. In certain embodiments, the base is a base catalyst. In certain embodiments, the base is an inorganic base. In certain embodiments, the base is a hydroxide. Exemplary hydroxides include NaOH, KOH, and LiOH.

In certain embodiments the step of hydrolyzing comprises an enzyme. In certain embodiments the enzyme is a carboxyesterase. In certain embodiments the enzyme is a lipase.

Chemically coupling the carboxylic acid with an amine (e.g., of the formula HN(R⁴)₂) or alcohol (e.g., of the formula HOR³) may employ methods well-known in the art; see, e.g., Smith and March March's Advanced Organic Chemistry, 5^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^th Edition, Cambridge University Press, Cambridge, 1987, for examples of reaction conditions useful in these types of chemical conversions.

For example, in one aspect, provided is a method of preparing a compound of the Formula (III):

or a solvate or hydrate thereof, or a combination thereof; wherein R¹, R², and R⁴ are as defined herein; the method comprising coupling an amine of the formula HN(R⁴)₂ and a compound of Formula (I) to provide a compound of the Formula (III).

In certain embodiments, the step of coupling comprises contacting the compound of the Formula (I) and the amine of the formula HN(R⁴)₂ with a peptide coupling agent.

Pharmaceutical Compositions

In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure or a salt, ester, amide, solvate, or hydrate thereof, or combination thereof, and a pharmaceutically acceptable excipient. In certain embodiments, the compound of the present disclosure or a pharmaceutically acceptable salt thereof is provided in an effective amount in the pharmaceutical composition.

Pharmaceutically acceptable excipients include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and/or manufacture of pharmaceutical compositions agents can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the compound of the present disclosure (the “active ingredient”) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and/or combinations thereof.

Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the compounds of the disclosure are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the compounds of the instant disclosure with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active ingredients can be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compound of the instant disclosure may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the disclosure can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the disclosure formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition of the disclosure. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the disclosure can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the disclosure can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this disclosure.

Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease, disorder, or condition being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.

The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

In certain embodiments, the compounds of the disclosure may be administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 150 mg/kg, 0.001 mg/kg to about 100 mg/kg, 0.001 mg/kg to about 50 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

It will be also appreciated that a compound or composition, as described herein, can be administered in combination with one or more additional therapeutically active agents. The compounds or compositions can be administered in combination with additional therapeutically active agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.

The compound or composition can be administered concurrently with, prior to, or subsequent to, one or more additional therapeutically active agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the inventive compound with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved. In general, it is expected that additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

Still further encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise an inventive pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of an inventive pharmaceutical composition or compound. In some embodiments, the inventive pharmaceutical composition or compound provided in the container and the second container are combined to form one unit dosage form.

Optionally, a single container may comprise one or more compartments for containing an inventive pharmaceutical composition or compound, and/or a pharmaceutically acceptable excipient for suspension or dilution. In some embodiments, a single container can be appropriate for modification such that the container may receive a physical modification so as to allow combination of compartments and/or components of individual compartments. For example, a foil or plastic bag may comprise two or more compartments separated by a perforated seal which can be broken so as to allow combination of contents of two individual compartments once the signal to break the seal is generated. A kit may thus comprise such multi-compartment containers providing an inventive pharmaceutical composition or compound and one or more pharmaceutically acceptable excipients.

Optionally, instructions for use are additionally provided in such kits of the disclosure. Such instructions may provide, generally, for example, instructions for dosage and administration. In other embodiments, instructions may further provide additional detail relating to specialized instructions for particular containers and/or systems for administration. Still further, instructions may provide specialized instructions for use in conjunction and/or in combination with an additional therapeutic agent.

Methods of Treatment and Use

The present disclosure is based at least in part on the discovery that compounds of the Formula (I) release an effective amount of carbon monoxide (CO) preferentially in the liver. These compounds also display anti-inflammatory activity and regenerative activity in the liver.

Thus, in one aspect, provided is a method of treating liver disease in a subject in need thereof, the method comprising administering an effective amount of a compound of Formula (I), or a salt, ester, amide, solvate, or a hydrate thereof, or a combination thereof, to treat the liver disease.

In another aspect, provided is a method of treating liver disease in a subject in need thereof, the method comprising instructing the subject to take an effective amount of a compound of Formula (I), or a salt, ester, amide, solvate, or a hydrate thereof, or a combination thereof, to treat the liver disease.

In another aspect, provided is a compound of Formula (I) for use in treating liver disease.

In certain embodiments, the method or use is a therapeutic treatment, and the effective amount is a therapeutically effective amount. In other embodiments, the method or use is a prophylactic treatment, and the effective amount is a prophylactically effective amount.

Exemplary liver diseases include, but are not limited to, drug-induced liver injury (e.g., acetaminophen-induced liver injury), hepatitis (e.g., chronic hepatitis, viral hepatitis, alcohol-induced hepatitis, autoimmune hepatitis, steatohepatitis), non-alcoholic fatty liver disease, alcohol-induced liver disease (e.g., alcoholic fatty liver, alcoholic hepatitis, alcohol-related cirrhosis), liver cirrhosis, liver cancer, primary biliary cirrhosis, cholestatis, cystic disease of the liver, and primary sclerosing cholangitis.

In certain embodiments, the liver disease is drug-induced liver injury. In certain embodiments, the drug induced liver injury is acetaminophen-induced liver injury.

In certain embodiments, the liver disease causes liver cell death in the subject. In certain embodiments, the administration of the compound stimulates liver cell regeneration in the subject.

In certain embodiments, the method further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is N-acetylcysteine (NAC).

In certain embodiments the liver disease is associated with inflammation.

In another aspect, provided is a method of treating an inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of a compound of Formula (I), or a salt, ester, amide, solvate, or a hydrate thereof, or a combination thereof, to treat the inflammatory disease.

In another aspect, provided is a method of treating an inflammatory disease in a subject in need thereof, the method comprising instructing the subject to take an effective amount of a compound of Formula (I), or a salt, ester, amide, solvate, or a hydrate thereof, or a combination thereof, to treat the inflammatory disease.

In another aspect, provided is a compound of Formula (I) for use in treating an inflammatory disease.

In certain embodiments, the method or use is a therapeutic treatment, and the effective amount is a therapeutically effective amount. In other embodiments, the method or use is a prophylactic treatment, and the effective amount is a prophylactically effective amount.

Exemplary inflammatory diseases include, but are not limited to, inflammation associated with asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type I diabetes mellitus, type 2 diabetes mellitus), a skin condition (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), endometriosis, Guillain-Barre syndrome, infection, ischaemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine headaches, tension headaches), ileus (e.g., postoperative ileus and ileus during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorder [e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD), Crohn's disease, Behcet's syndrome, colitis (e.g., ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, indeterminate colitis, microscopic colitis, chemical colitis, infectious colitis, fulminant colitis), and inflammatory bowel syndrome (IBS)], lupus, multiple sclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, reperfusion injury, regional enteritis, rheumatic fever, systemic lupus erythematosus, schleroderma, scierodoma, sarcoidosis, spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantation rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scarring, burns, physical injury), vasculitis, vitiligo and Wegener's granulomatosis. In some preferred embodiments, the inflammatory disorder is colitis.

A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other non-human animals, for example mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys), reptiles, amphibians, and fish. In certain embodiments, the non-human animal is a mammal. The non-human animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

The terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from a disease, disorder or condition which reduces the severity of the disease, disorder or condition or retards or slows the progression of the disease, disorder or condition (“therapeutic treatment”), and also contemplates an action that occurs before a subject begins to suffer from the disease, disorder or condition and which inhibits or reduces the severity of the disease, disorder or condition (“prophylactic treatment”).

In general, the “effective amount” of a compound refers to an amount sufficient to elicit the desired biological response, i.e., treating the disease, disorder or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the disclosure may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and condition of the subject. An effective amount encompasses therapeutic and prophylactic treatment.

A “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of disease, disorder or condition or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

A “prophylactically effective amount” of a compound is an amount sufficient to prevent the disease, disorder or condition, or one or more symptoms associated with the disease, disorder or condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of the compound, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease, disorder or condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

EXAMPLES

In order that the disclosure described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this disclosure in any manner.

Example 1

Preparation of Mo Complexes

Synthesis of Mo(CO)₃(η⁶-C₇H₈)

The preparation of the molybdenum tri-CO complex Mo(CO)₃(η⁶-C₇H₈) is described in the literature (see, e.g., W. A. Herrmann and A. Salzer, Synthetic Methods of Organometallic and Inorganic Chemistry, volume 1, Georg Thieme Verlag, New York, 1996, p 129; and Abel et al., J. Chem. Soc. (1958) 4559).

Preparation of Tricarbony[tris(isocyanoacetic acid)]Mo(0) (1-b)

Preparation of tricarbonyl[tris(isocyanoacetic acid ethyl ester)]Mo(0) (1-a): Mo(CO)₃(η⁶-C₇H₈) (2.1 g; 7.72 mmol; 272.1117 g/mol) was dissolved in 40 mL of MeOH to give a red, slightly turbid solution. CNCH₂CO₂Et (3 eq.; 2.53 mL; 23.15 mmol; 113.11 g/mol; 1.035 g/mL) was dissolved in 20 mL MeOH and added to the previous solution. The red solution immediately turned darker, greenish, and gradually became lighter. The solution was stirred at room temperature for 45 min, when a TLC analysis (hexane:ethyl acetate 1:3) showed that (Mo(CO)₃(η⁶-C₇H₈)) had been completely consumed. The mixture was filtered to remove some fine black powder giving a dark red solution that was taken to dryness yielding a dark oil. The oil was immediately loaded onto a silica column equilibrated in hexane. The column was eluted with hexane (approx 1-2 column volumes) then the product was eluted with hexane:ethyl acetate (6:4). TLC analysis of the product fraction showed it to be a mixture of two well separated compounds. The mixture was again chromatographed using the same eluent and collecting smaller fractions. The major product eluted second; it was collected and taken to dryness affording an off-white powder, compound (1-a). C₁₈H₂₁N₃O₉Mo (519.31 g/mol). Yield: 55% (Batch No. 2) and 64% (Batch No. 4).

Characterization of (1-a): Elemental Analysis: Calculated for C₁₈H₂₁N₃O₉Mo: % C, 41.63; % H, 4.07; % N, 8.09; Batch No. 2 Found: % C, 41.00; % H, 4.08; % N, 8.24. IR (KBr): Bands (C≡O): 1941(s); 1873(s); Bands (C═O): 1749(s); Bands (C≡N): 2181(s); 2135(s). ¹H-NMR (CDCl₃, 400 MHz, rt, δ in ppm): δ=4.37 (s, 2H), 4.27 (q, 2H), 1.32 (t, 3H). ¹³C-NMR (CDCl₃, 100 MHz, rt, δ in ppm): δ=211.9 (C≡O), 167.72 (CN), 164.87 (C═O), 62.74 (CNCH₂CO), 45.53 (COCH₂CH₃), 14.15 (COCH₂CH₃).

Preparation of tricarbonyl[tris(isocyanoacetic acid)]Mo(0) (1-b): Compound (1-a) (0.250 g, 0.48 mmol, 519.31 g/mol) was dissolved in anhydrous THF (20 mL), and an aqueous solution of sodium hydroxide (16 equivalents, 8.0 mmol, 0.32 g, 8 mL) was added dropwise. The solution became turbid and was left stirring at room temperature under nitrogen. After 24 hours, TLC (hexane:ethyl acetate, 6:4) indicated complete consumption of starting material (R_f 0.2). The mixture was concentrated and re-dissolved in water (20 mL). Hydrochloric acid (1 M) was then added dropwise (˜6 mL-until pH˜3 and beginning of precipitation) and a white precipitate formed. The precipitate was filtered and washed with cold water. The off-white compound was dried in vacuo to provide compound (1-b). C₁₂H₉O₉N₃Mo (435.1583 g/mol). Yield: 100%.

Characterization of (1-b): Elemental Analysis: Calculated for C₁₂H₉O₉N₃Mo: % C, 33.12; % H, 2.08; % N, 9.66; Batch No. 2 Found: % C, 32.40; % H, 2.12; % N, 9.86; Batch No. 3 Found: % C, 30.50; % H, 2.12; % N, 9.13; Calculated for MoC₁₂H₉N₃O₉.(NaCl)_0.6% C, 30.65; % H, 1.93; % N, 8.94. IR (KBr): Bands (C≡O): 1941(s); 1849(s); Bands (C═O): 1720(s); Bands (C≡N): 2195(s); 2159(s). ¹H-NMR (D₂O, 400 MHz, rt, δ in ppm): δ=4.52 (s, 2H). ¹H-NMR (d⁶-DMSO, 400 MHz, rt, δ in ppm): δ=4.64 (s, 2H). ¹³C-NMR (d⁶-DMSO, 100 MHz, rt, δ in ppm): δ=213.9 (C≡O), 166.25 (CN), 159.9 (CO), 45.90 (CNCH₂). ¹³C-NMR (d⁶-acetone, 100 MHz, rt, δ in ppm): δ=214.4 (C≡O), 167.52 (CN), 163.81 (CO), 46.10 (CNCH₂).

Preparation of Tricarbonyl[tris(2-isocyanopropionic acid)]Mo(0) (2-b)

Preparation of tricarbonyl[tris(2-isocyanopropionic acid methyl ester)]Mo(0) (2-a): (η⁶-C₇H₈)Mo(CO)₃ (0.383 g; 1.41 mmol; 272.11 g/mol) was dissolved in 30 mL of MeOH and CNCH(CH₃)CO₂Me (3 equiv.; 0.4784 g; 4.23 mmol; 113.12 g/mol), dissolved in 10 mL of MeOH, was slowly added. The dark red suspension turned orange-brown and was stirred at room temperature during 1 h. The solution was taken to dryness, giving an orange oil, which was applied onto a silica column equilibrated in hexane. It was eluted with hexane and then ethyl acetate/hexane 4:6. Fractions were pooled based on TLC analysis. The product solution was taken to dryness giving a green oil, compound (2-a). C₁₈H₂₁N₃O₉Mo (519.3186 g/mol). Yield: 0.3677 g, (50%). Batch No. 2 was prepared analogously giving a yield of 63%.

Characterization of (2-a): IR(CHCl₃): Bands (C≡O): 1948(s); 1887(br, s); Bands (C═O): 1746(s); Bands (C≡N): 2103(s). ¹H-NMR (CDCl₃, 400 MHz, rt, δ in ppm): δ=4.49 (q, 1H), 3.82 (s, 3H), 1.66 (d, 3H).

Preparation of tricarbonyl[tris(2-isocyanopropionic acid)]Mo(0) (2-b): Compound (2a) (0.3677 g; 7.08×10⁻⁴ mol; 519.3186 g/mol) was dissolved in 20 mL of THF and placed in an ice bath. NaOH (10 equiv.; 0.283 g; 7.08 mmol; 40 g/mol) was dissolved in 5 mL of H₂O and slowly added to the previous solution. The solution was stirred for 4 h while it slowly warmed to room temperature. It was then taken to dryness giving a white powder. The solid was dissolved in water and hydrochloric acid (1 M) was added until the pH reached 1 to provide a white precipitate, compound (2-b) which was collected by filtration and washed with cold water. C₁₅H₁₅N₃O₉Mo (477.2382 g/mol). Yield: 96%.

Characterization of (2-b): Elemental Analysis: Calculated for C₁₅H₁₅N₃O₉Mo.0.5NaCl: % C: 35.57; % H: 2.98; % N: 8.30; Batch No. 1 Found: % C, 35.35; % H, 3.19; % N, 8.55. IR (KBr): Bands (C≡O): 1920(s); 1867(br, s); Bands (C═O): 1729(s); Bands (C≡N): 2170(s); 2123(s). ¹H-NMR (CD₃COCD₃, 400 MHz, rt, δ in ppm): δ=4.78 (q, 1H), 1.67 (d, 3H).

Preparation of Tricarbonyl[tris(2-isocyano-2-methyl-propionic acid methyl ester)]Mo(0) (3-a)

Method A

Methyl 2-isocyano-2-methylpropanoate (3.068 g; 24.127 mmol; 127.14 g/mol) was dissolved in 65 mL of THF. (η⁶-C₇H₈)Mo(CO)₃ (⅓ equiv.; 2.2 g; 8.085 mmol; 272.11 g/mol) was slowly added, in portions as a solid. An orange-red, slightly turbid solution was obtained. This was stirred at room temperature (22-23° C.) and 500 rpm. TLC analysis in ethyl acetate:hexane (1:1) after 30 min showed one main spot with Rf 0.65 (revealed with ceric ammonium molybdate), and another small spot with Rf 0.9 that didn't color with ceric ammonium molybdate. After 1 h reaction time, TLC analysis showed only one major spot with Rf 0.65. The solution was concentrated and a precipitate was formed when half of the solvent was evaporated. The solution was further concentrated almost to dryness (ca. 5 mL remaining) and Et₂O (45 mL) was added. The resulting precipitate was filtered and washed with 2×15 mL of Et₂O to provide a beige powder, compound (3-a). Yield: 4.22 g (93.0%) (MW=561.3939 g/mol).

Characterization of compound (3-a): Elemental Analysis: Calculated for MoC₂₁H₂₇N₃O₉: % C, 44.92; % H, 4.85; % N, 7.48. Found: % C, 44.70; % H, 5.05; % N, 7.61. ¹H-NMR (CDCl₃, 400 MHz, rt, δ in ppm; 10 mg/600 μL): δ=3.82 (s, 3H)OC H₃, 1.68 (s, 6H) C(CH₃)₂. ¹³C-NMR (100.6 MHz, CDCl₃, rt, δ in ppm, 10 mg/600 μL): 212.66 ( C≡O); 170.82 ( C≡N); 166.68 (C═O); 61.16 (C_quat); 53.52 (OCH₃); 27.81 (2× CH₃). IR (0.4 mg/280 mg KBr): Bands (C≡O): 1932(s), 1873(sh), 1860(s); Bands (C═O): 1749 and 1742(m, split); Bands (C≡N): 2168(m), 2124 and 2111(m, split). IR (0.5 mg/mL CHCl₃): Bands (C≡O): 1944(s), 1888(s); Bands (C═O): 1744(m); Bands (C≡N): 2152(w), 2090(m).

Method B

The following alternative preparation was adapted from Achatz et al., Anorg. Allg. Chem (2005) 631:2339-2346, where Mo(CO)₃(NCMe)₃ is formed and used in situ.

Mo(CO)₆ (0.6 g; 2.27 mmol; 264 g/mol) was heated to reflux in CH₃CN for 20 hours. After cooling to room temperature, 1 g (3.5 eq.) CNC(CH₃)₂CO₂Me was added and the reaction was stirred at room temperature for another 20 hours and at 55° C. for 5 hours. The solvent was evaporated under vacuum and the residue was washed with ethyl acetate/hexane (2:3) and the black residue was chromatographed over silica gel with dichloromethane to give 250 mg of compound (3-a). Yield: 21%.

TABLE 1
X-ray Powder Diffraction data for Compound 3a
(spectra provided in FIG. 3B)
Pos.		FWHM	d-spacing	Rel. Int.	Tip width
[°2Th.]	Height [cts]	[°2Th.]	[Å]	[%]	[°2Th.]
7.0301	1086.94	0.2319	12.57423	4.97	0.2783
7.3100	4729.63	0.1181	12.09333	21.62	0.1417
9.7404	842.89	0.0634	9.08068	3.85	0.0761
10.2522	21880.08	0.1181	8.62851	100.00	0.1417
10.5961	1578.50	0.4069	8.34923	7.21	0.4883
11.1690	803.13	0.0748	7.92216	3.67	0.0897
11.5561	5718.57	0.1949	7.65770	26.14	0.2339
11.5823	4312.47	0.2362	7.64044	19.71	0.2834
11.9561	65.64	0.0900	7.40239	0.30	0.1080
12.2441	301.11	0.1181	7.22888	1.38	0.1417
12.8761	196.44	0.0900	6.87548	0.90	0.1080
13.2278	1664.17	0.1181	6.69341	7.61	0.1417
14.1561	1221.10	0.0010	6.25654	5.58	0.0012
14.5561	14614.87	0.1423	6.08550	66.80	0.1707
14.5913	6264.68	0.2362	6.07086	28.63	0.2834
15.8761	2170.69	0.0010	5.58238	9.92	0.0012
16.1858	4203.96	0.1181	5.47624	19.21	0.1417
16.4267	1503.52	0.3431	5.39646	6.87	0.4117
16.5692	1233.75	0.1181	5.35039	5.64	0.1417
16.7961	918.22	0.4154	5.27862	4.20	0.4984
17.2361	836.42	0.0010	5.14485	3.82	0.0012
17.5515	2770.11	0.1181	5.05308	12.66	0.1417
18.1161	803.92	0.0010	4.89687	3.67	0.0012
18.6117	10181.39	0.1181	4.76756	46.53	0.1417
18.9242	3006.51	0.1181	4.68954	13.74	0.1417
19.6593	4402.52	0.1181	4.51582	20.12	0.1417
20.0361	1744.89	0.1026	4.43174	7.97	0.1231
20.1394	1868.29	0.1181	4.40923	8.54	0.1417
20.5782	1082.94	0.0617	4.31262	4.95	0.0740
20.5961	1956.44	0.0010	4.31963	8.94	0.0012
20.8445	3487.36	0.1574	4.25812	15.94	0.1889
21.1916	1379.14	0.1181	4.18917	6.30	0.1417
21.8365	1274.91	0.1181	4.06688	5.83	0.1417
22.1161	358.84	0.0900	4.01609	1.64	0.1080
22.4411	804.84	0.1574	3.95866	3.68	0.1889
22.6781	928.98	0.3323	3.91782	4.25	0.3988
23.0189	2058.35	0.1181	3.86058	9.41	0.1417
23.5656	379.02	0.1181	3.77224	1.73	0.1417
23.9315	225.32	0.1181	3.71538	1.03	0.1417
24.3088	1283.09	0.1574	3.65856	5.86	0.1889
25.1256	966.15	0.1574	3.54145	4.42	0.1889
25.8356	801.00	0.1574	3.44572	3.66	0.1889
26.2350	863.64	0.1181	3.39415	3.95	0.1417
26.5574	1002.35	0.1181	3.35367	4.58	0.1417
27.7020	511.31	0.1574	3.21765	2.34	0.1889
27.9561	352.11	0.0900	3.18899	1.61	0.1080
28.4762	368.84	0.1574	3.13191	1.69	0.1889
28.8820	800.58	0.1574	3.08882	3.66	0.1889
29.3887	1651.43	0.1574	3.03671	7.55	0.1889
29.8361	146.46	0.0900	2.99219	0.67	0.1080
30.2165	1040.25	0.1574	2.95537	4.75	0.1889
30.8992	1213.52	0.1574	2.89161	5.55	0.1889
31.1561	969.14	0.0010	2.86836	4.43	0.0012
31.4427	1953.63	0.1574	2.84286	8.93	0.1889
31.9903	257.05	0.1574	2.79543	1.17	0.1889
32.3561	193.07	0.0900	2.76467	0.88	0.1080
32.6777	1161.21	0.1574	2.73818	5.31	0.1889
33.3123	2979.60	0.1181	2.68746	13.62	0.1417
33.5452	2099.09	0.5730	2.66933	9.59	0.6876
33.9961	258.12	0.0900	2.63495	1.18	0.1080
34.7439	892.15	0.2362	2.57993	4.08	0.2834
34.7961	1482.26	0.0010	2.58258	6.77	0.0012
36.1400	187.16	0.1574	2.48340	0.86	0.1889
36.9215	285.89	0.1968	2.43262	1.31	0.2362
37.5161	553.76	0.0900	2.39542	2.53	0.1080
37.7521	1047.46	0.1574	2.38098	4.79	0.1889
38.1617	310.89	0.1574	2.35636	1.42	0.1889
39.3561	135.20	0.0900	2.28756	0.62	0.1080
39.7513	928.26	0.1181	2.26572	4.24	0.1417
39.9754	618.59	0.1181	2.25353	2.83	0.1417
40.5647	358.12	0.1574	2.22214	1.64	0.1889
40.8789	527.11	0.1574	2.20579	2.41	0.1889
41.6261	857.01	0.1181	2.16790	3.92	0.1417
41.9554	1396.25	0.1574	2.15164	6.38	0.1889
42.3427	568.09	0.1181	2.13286	2.60	0.1417
42.9455	868.03	0.1574	2.10430	3.97	0.1889
43.5161	273.05	0.0900	2.07803	1.25	0.1080
43.8132	281.04	0.1968	2.06462	1.28	0.2362
44.3961	699.17	0.0900	2.03886	3.20	0.1080
44.6649	2250.52	0.1574	2.02721	10.29	0.1889
46.1282	117.20	0.1181	1.96625	0.54	0.1417
46.5849	80.29	0.2362	1.94803	0.37	0.2834
48.3071	1791.64	0.1440	1.88253	8.19	0.1728
49.0361	1.00	0.0900	1.85623	0.00	0.1080

Preparation of Tricarbonyl[tris(2-isocyano-2-methyl-propionic acid ethyl ester)]Mo(0) (3-c)

(η⁶-C₇H₈)Mo(CO)₃ (0.644 g; 2.37 mmol; 272.11 g/mol) was dissolved in 30 mL of MeOH and CNC(CH₃)₂CO₂Et (3 equiv.; 0.903 g; 6.40 mmol; 141.17 g/mol), dissolved in 10 mL of MeOH was slowly added. The dark red solution turned orange-brown and was stirred at room temperature for 2.5 hours. The solution was concentrated and diethyl ether was added, but no precipitate formed, therefore the solution was evaporated to dryness. IR analysis of the crude product mixture indicated that the desired product was contaminated with Mo(CO)₄(CNC(CH₃)₂CO₂Et)₂ (band at 2019 cm⁻¹). Therefore the mixture was separated by silica gel column chromatography. The column was first washed with hexane and then eluted with a mixture of hexane and ethyl acetate (8:2) to provide compound (3-c) as a greenish oil. Yield: 0.94 g (73%).

Characterization of compound (3-c): Elemental Analysis: Calculated for C₂₄H₃₃N₃O₉Mo: % C, 47.77; % H, 5.51; % N, 6.96. Found: % C, 47.70; % H, 5.20; % N, 6.63. IR (KBr): Bands (C≡O): 1939(s); 1868(br, s); Bands (C═O): 1744(s); Bands (C≡N): 2154(s); 2104 (s). ¹H-NMR (CDCl₃, 400 MHz, rt, δ in ppm): δ=4.25 (q, 6H), 1.66 (s, 18H), 1.34 (t, 9H).

Preparation of Tricarbonyl[tris(2-isocyano-2-methyl-propionic acid)]Mo(0) (3b)

Following the procedure described by Beck and coworkers (Achatz et al., Anorg. Allg. Chem (2005) 631:2339-2346) saponification of compound (3-a) or (3-c) with NaOH followed by protonation with an acid (e.g., HCl or H₂SO₄) in aqueous solution led to the formation of tricarbonyl[tris(2-isocyano-2-methyl-propionic acid)]Mo(0) (3-b). Compound (3-b) is stable when stored in brown vials at ambient temperature under N₂ for over 6 months or more.

It was observed after the preparation of several batches that two sets of spectroscopic data were obtained for compound (3-b). The Type I product corresponds to the fully protonated tricarboxylic acid complex and the Type II product contains minor amounts of carboxylic acid sodium salt arising from a small degree of deprotonation (variable amounts 0.4-0.6 mol Na per mol Mo). Type II product can be converted into Type I by adding excess acid during the synthesis or through washing with acid at the end of the synthesis. The biological activity of the Type I and Type II products are the same. Type I and Type II products are recognized by the differences in their IR spectra (KBr) and ¹³C NMR spectra. The overall composition and the facial stereochemistry of Compound (3-b) was confirmed by X-ray crystallography.

Preparation of Tricarbonyl[tris(2-isocyano-2-methyl-propionic acid)]Mo(0), (3-b) Type I

Method A

Compound (3-a) (1.914 g; 3.41 mmol; 561.3939 g/mol) was dissolved in 36 mL of THF and placed in an ice bath. NaOH (10 equiv.; 1.364 g; 34.1 mmol; 40 g/mol) was dissolved in 9 mL of H₂O and slowly added to the previous yellow solution. A yellow, turbid solution was obtained, which was stirred for 1 hour while it slowly warmed to room temperature. A TLC analysis in hexane:ethyl acetate (1:1) showed only one spot with Rf 0 at the end of this time. The solvents were concentrated until all the THF was gone. Then, 27 mL of water were added, followed by 17.05 mL of 1M H₂SO₄ acid. A white precipitate formed, which then turned into a yellow oil. The water was evaporated until a white powder was reformed. After filtration, the pH of the colorless solution was 1. The solid was washed with 5×20 mL portions of water. The pH of the washing solutions was successively 3, 4, 4, 4 and 4. The final washed product was dried under vacuum. After drying at 1.0×10⁻² Torr for 30 h and at 2.0×10⁻⁵ Torr for 9 h the IR spectrum (1.2 mg of compound (3-b) in 840 mg of KBr) and NMR (10 mg/600 μL of acetone-d⁶) showed the complex was pure. Yield: 1.600 g (90.3%). C₁₈H₂₁O₉N₃Mo (519.31 g/mol).

Characterization of compound (3-b), Type I. Elemental Analysis: Calculated for C₁₈H₂₁O₉N₃Mo: % C, 41.63; % H, 4.08; % N, 8.09. Found: C, 41.50; % H, 4.43; % N, 8.31. ¹H-NMR (CD₃COCD₃, 400 MHz, rt, δ in ppm, 10 mg/600 μL): δ=1.71 (s, CH₃). ¹³C-NMR (CD₃COCD₃, 100.6 MHz, rt, δ in ppm, 10 mg/600 μL): 214.65 (C≡O); 171.37 (C≡N); 164.34 (C═O); 62.08 (C_quat); 27.85 (2×CH₃). IR (KBr): Bands (C≡O): 1925(s), 1875(s); 1841(sh), Bands (C═O): 1747(w, sh) and 1719(m), Bands (C≡N): 2157(m), 2110(s). IR(CHCl₃): Bands (C≡O): 1942(s), 1882(s); Bands (C═O): 1737(w); Bands (C≡N): 2152(w), 2098(m).

Method B

A second method was developed to obtain type I, fully protonated complexes.

Compound (3-a) (1.01 g; 1.800 mmol; 561.3939 g/mol) was dissolved in 20 mL of THF giving a transparent yellow solution. This solution was placed in an ice bath and stirred for about 10 minutes. NaOH (10 equiv.; 0.722 g; 18.00 mmol; 40 g/mol) was dissolved in 5 mL of H₂O (miliQ) and added dropwise. The yellow solution was stirred without removing the ice-bath. The solution became light-yellow and turbid. After 20 minutes, TLC analysis performed in hexane:ethyl acetate (1:1) showed complete consumption of starting material (Rf 0.8, stains blue with ceric ammonium molybdate) and a new spot at the starting point (stains strongly blue with ceric ammonium molybdate). After another hour, Dowex 50WX8-200 ion exchange resin (Ref. 217506; Exchange Capacity 1.7 meq/mL; 6 g) was added and the solution stirred for 10 minutes (the Dowex resin had been activated prior to use by dissolving it in 1M aqueous HCl and stirring for two hours, followed by filtration and washing with water until pH neutral). The Dowex resin was filtered off and the solution evaporated (bath temperature 30° C.) giving a white material and a gummy brown residue (the NMR in acetone showed some THF contamination). The next day, the crude (white material) was washed with water (2×30 ml). The suspension was allowed to rest and then filtered. The final pH of the washing solution was 2.6 (pH electrode). The white powder, compound (3b), Type I product, was dried in vacuum. Yield: 0.66 g (70%).

Characterization of compound (3-b), Type I. Elemental Analysis: Calculated for C₁₈H₂₁O₉N₃Mo: % C, 41.63; % H, 4.08; % N, 8.09. Found: % C, 41.30; % H, 4.23; % N, 8.05. ¹H-NMR (CD₃COCD₃, 400 MHz, rt, δ in ppm, 10 mg/600 μL): δ=1.71 (s, CH₃). ¹³C-NMR (CD₃COCD₃, 100.6 MHz, rt, δ in ppm, 10 mg/600 μL): 214.70 (C≡O); 171.46 (C≡N); 164.22 (C═O); 62.10 (C_quat); 27.86 (2×CH₃). IR (KBr): Bands (C≡O): 1924(s), 1871(s); 1840(sh); Bands (C═O): 1747(w, sh) and 1721(m); Bands (C≡N): 2158(m), 2108(s). IR(CHCl₃): Bands (C≡O): 1941(s), 1882(s); Bands (C═O): 1737(w); Bands (C≡N): 2153(w), 2099(m).

TABLE 2
X-ray Powder Diffraction data for Compound 3b, Type I
(spectra provided in FIG. 4B)
Pos.		FWHM	d-spacing	Rel. Int.	Tip width
[°2Th.]	Height [cts]	[°2Th.]	[Å]	[%]	[°2Th.]
7.3071	104.21	0.2362	12.09818	5.15	0.2834
8.5643	570.16	0.1574	10.32490	28.16	0.1889
9.9976	2025.02	0.1181	8.84764	100.00	0.1417
11.1631	225.27	0.6884	7.92639	11.12	0.8261
11.3636	403.87	0.1574	7.78695	19.94	0.1889
12.5320	1651.83	0.1574	7.06346	81.57	0.1889
14.5147	290.27	0.1181	6.10275	14.33	0.1417
14.9564	728.89	0.1181	5.92349	35.99	0.1417
15.5593	242.84	0.1732	5.69531	11.99	0.2078
15.9707	350.89	0.1574	5.54950	17.33	0.1889
17.1217	474.25	0.1574	5.17896	23.42	0.1889
17.5108	231.68	0.1181	5.06474	11.44	0.1417
18.0265	467.34	0.1181	4.92100	23.08	0.1417
18.3523	524.18	0.1181	4.83437	25.88	0.1417
18.8952	235.57	0.1968	4.69666	11.63	0.2362
19.5631	1443.96	0.3012	4.53781	71.31	0.3615
19.6857	1861.41	0.1574	4.50981	91.92	0.1889
20.0428	332.44	0.1856	4.43026	16.42	0.2227
20.3432	299.89	0.1181	4.36551	14.81	0.1417
20.7693	181.47	0.1181	4.27691	8.96	0.1417
22.2149	1436.61	0.1968	4.00177	70.94	0.2362
22.8270	189.65	0.1181	3.89583	9.37	0.1417
23.0031	351.89	0.0010	3.86640	17.38	0.0012
23.3512	500.66	0.1181	3.80953	24.72	0.1417
23.8140	349.40	0.1968	3.73654	17.25	0.2362
24.3728	344.81	0.1181	3.65213	17.03	0.1417
24.6863	472.11	0.1574	3.60645	23.31	0.1889
25.1231	84.73	0.0900	3.54474	4.18	0.1080
26.0031	39.50	0.0900	3.42674	1.95	0.1080
27.8772	167.00	0.1574	3.20048	8.25	0.1889
28.3631	60.99	0.0900	3.14675	3.01	0.1080
28.6635	102.42	0.1181	3.11444	5.06	0.1417
29.4031	258.48	0.1672	3.03777	12.76	0.2006
29.7607	240.64	0.1181	3.00208	11.88	0.1417
30.3092	448.03	0.1574	2.94899	22.12	0.1889
30.7590	639.70	0.1968	2.90688	31.59	0.2362
30.9231	375.19	0.0010	2.89183	18.53	0.0012
31.8625	407.11	0.1574	2.80868	20.10	0.1889
32.9911	132.45	0.1574	2.71513	6.54	0.1889
33.3765	242.32	0.1574	2.68466	11.97	0.1889
34.6343	209.74	0.2755	2.58999	10.36	0.3306
35.4600	133.72	0.1181	2.53155	6.60	0.1417
36.6431	131.56	0.0900	2.45249	6.50	0.1080
36.9291	234.25	0.1181	2.43415	11.57	0.1417
37.3231	128.05	0.0900	2.40935	6.32	0.1080
38.1379	245.64	0.3149	2.35973	12.13	0.3779
38.6431	89.09	0.0900	2.33004	4.40	0.1080
39.5231	72.92	0.0900	2.28016	3.60	0.1080
40.0232	133.72	0.2362	2.25282	6.60	0.2834
40.9283	148.51	0.1181	2.20506	7.33	0.1417
42.2431	68.44	0.0900	2.13943	3.38	0.1080
42.4031	76.70	0.0900	2.13172	3.79	0.1080
42.5631	62.75	0.0900	2.12408	3.10	0.1080
43.0418	90.09	0.9446	2.10156	4.45	1.1336
44.0031	98.50	0.0900	2.05786	4.86	0.1080
44.6400	144.25	0.2755	2.02996	7.12	0.3306
45.6313	75.09	0.3936	1.98814	3.71	0.4723
46.8031	22.71	0.0900	1.94106	1.12	0.1080
48.5231	37.91	0.0900	1.87621	1.87	0.1080
50.0381	82.16	0.3149	1.82289	4.06	0.3779
50.5231	54.18	0.0900	1.80653	2.68	0.1080
52.1631	26.97	0.0900	1.75353	1.33	0.1080
53.3498	62.23	0.6298	1.71729	3.07	0.7557
54.9231	56.59	0.0900	1.67176	2.79	0.1080
55.9430	69.71	0.4723	1.64368	3.44	0.5668
57.3994	34.49	0.4723	1.60539	1.70	0.5668
60.1631	33.11	0.0900	1.53808	1.63	0.1080
61.2152	49.98	0.9446	1.51415	2.47	1.1336
62.7421	35.86	0.7680	1.47970	1.77	0.9216
65.2831	2.62	0.0900	1.42930	0.13	0.1080
66.5631	19.62	0.0900	1.40489	0.97	0.1080
67.1631	26.13	0.0900	1.39379	1.29	0.1080

Preparation of Tricarbonyl[tris(2-isocyano-2-methyl-propionic acid)]Mo(0), (3-b) Type II

Compound (3-a) (1.06 g; 1.888 mmol; 561.3939 g/mol) was dissolved in 40 mL of THF and placed in an ice bath. NaOH (10 equiv.; 0.755 g; 18.88 mmol; 40 g/mol) was dissolved in 10 mL of H₂O and added dropwise to the previous solution over 10 min. The light-yellow, turbid solution was stirred for 2 h while it slowly warmed to room temperature, without removing the ice-bath. After 2 h, TLC analysis performed in hexane:ethyl acetate (1:1) showed only hydrolyzed product with Rf=0. The light-yellow, turbid solution was taken to dryness giving a large amount of a white powder. The solid was redissolved in water (10 mL) and 1 M H₂SO₄ (9.4 mL) was added dropwise. The solution became blue and had a final pH of 1-2. The solid was filtered and washed with 5×10 mL of water. Each time the solution was stirred for 2 min and allowed to rest. The final pH of the washing solution was between 4 and 5. The off-white powder was dried in vacuum to provide compound (3-b) containing sodium (Type II). Yield: 0.920 g (93.8%).

Characterization of compound (3-b), Type II. Elemental Analysis: Calculated for C₁₈H₂₁O₉N₃Mo: % C, 41.63; % H, 4.08; % N, 8.09. Found: C, 40.60; % H, 3.80; % N, 7.82. The compound was further dried in high-vacuum (2×10⁻⁵ Torr) during 1 week. Found: % C, 41.20; % H, 4.15; % N, 8.24. ¹H-NMR (CD₃COCD₃, 400 MHz, rt, 8 in ppm, 10 mg/600 μL): δ=1.68 (s, CH₃) ¹³C-NMR (CD₃COCD₃, 100.6 MHz, rt, δ in ppm, 10 mg/600 μL): 214.93 (C≡O); 172.59 (C≡N); 163.48 (C═O); 62.60 (C_quat); 27.92 (2×CH₃); IR (KBr): Bands (C≡O): 1927(s), 1859(s); Bands (C═O): 1755(m) and 1685(w); Bands (C≡N): 2166(m), 2105 (m). IR(CHCl₃): Bands (C≡O): 1942(s), 1882(s); Bands (C═O): 1735(w) and 1677(w); Bands (C≡N): 2157(w), 2099(m).

TABLE 3
X-ray Powder Diffraction data for Compound 3b, Type II
(spectra provided in FIG. 4C)
Pos.		FWHM	d-spacing	Rel. Int.	Tip width
[°2Th.]	Height [cts]	[°2Th.]	[Å]	[%]	[°2Th.]
8.4046	693.12	0.1181	10.52069	10.77	0.1417
9.0220	1116.38	0.1181	9.80208	17.34	0.1417
10.4406	897.42	0.1181	8.47318	13.94	0.1417
11.1898	718.15	0.1181	7.90751	11.15	0.1417
12.0642	4146.69	0.1181	7.33625	64.40	0.1417
12.3598	558.34	0.1181	7.16147	8.67	0.1417
12.7161	352.12	0.0010	6.96163	5.47	0.0012
13.1525	1852.12	0.1181	6.73158	28.77	0.1417
14.1853	1751.55	0.1181	6.24372	27.20	0.1417
14.4761	855.55	0.5774	6.11895	13.29	0.6929
14.9150	1108.39	0.0557	5.93985	17.21	0.0668
15.2754	6245.08	0.1574	5.80052	97.00	0.1889
16.8191	3219.27	0.1181	5.27143	50.00	0.1417
17.5629	2561.96	0.1181	5.04984	39.79	0.1417
18.1561	770.77	0.0798	4.88617	11.97	0.0957
18.4106	3739.81	0.1754	4.81919	58.08	0.2104
18.5642	6438.53	0.1181	4.77965	100.00	0.1417
18.8668	1896.91	0.1181	4.70368	29.46	0.1417
19.3886	548.12	0.1181	4.57825	8.51	0.1417
19.5770	754.05	0.0319	4.53461	11.71	0.0383
19.9962	3012.25	0.1181	4.44048	46.78	0.1417
20.3682	4161.26	0.1181	4.36023	64.63	0.1417
21.0361	758.19	0.0842	4.22327	11.78	0.1010
21.0361	758.19	0.0842	4.22327	11.78	0.1010
21.0361	758.19	0.0842	4.22327	11.78	0.1010
21.1445	1109.60	0.1181	4.20186	17.23	0.1417
21.4743	715.52	0.1181	4.13806	11.11	0.1417
22.4230	461.94	0.1181	3.96508	7.17	0.1417
22.9853	1320.11	0.1181	3.86934	20.50	0.1417
23.2480	1361.50	0.1181	3.82621	21.15	0.1417
23.7168	251.03	0.1574	3.75163	3.90	0.1889
24.2239	1107.55	0.1181	3.67423	17.20	0.1417
24.5635	880.09	0.0386	3.62421	13.67	0.0463
24.6166	875.68	0.0728	3.61651	13.60	0.0874
24.6285	596.28	0.1968	3.61479	9.26	0.2362
25.2742	964.10	0.1574	3.52388	14.97	0.1889
25.8361	89.65	0.0900	3.44851	1.39	0.1080
26.2646	694.73	0.1181	3.39320	10.79	0.1417
26.6375	726.26	0.1574	3.34655	11.28	0.1889
27.8654	342.90	0.1574	3.20181	5.33	0.1889
28.0361	193.62	0.0900	3.18270	3.01	0.1080
28.3296	584.86	0.1181	3.15039	9.08	0.1417
28.5032	708.22	0.0202	3.13159	11.00	0.0242
28.9163	1335.22	0.1574	3.08779	20.74	0.1889
29.5701	385.66	0.1181	3.02099	5.99	0.1417
29.9695	667.36	0.1181	2.98164	10.37	0.1417
30.5576	891.48	0.1574	2.92558	13.85	0.1889
30.9107	335.55	0.1181	2.89296	5.21	0.1417
31.2307	343.16	0.1181	2.86405	5.33	0.1417
31.4361	365.48	0.0900	2.84580	5.68	0.1080
31.7528	1213.96	0.1574	2.81814	18.85	0.1889
31.9561	222.28	0.0900	2.80067	3.45	0.1080
32.3161	162.00	0.0900	2.77029	2.52	0.1080
32.6753	1762.53	0.1574	2.74065	27.37	0.1889
33.0260	752.80	0.0603	2.71234	11.69	0.0723
33.5161	385.22	0.0900	2.67380	5.98	0.1080
33.5408	471.86	0.1574	2.66967	7.33	0.1889
33.5961	851.84	0.0010	2.67203	13.23	0.0012
33.9812	400.50	0.1181	2.63607	6.22	0.1417
34.4694	309.84	0.1181	2.59985	4.81	0.1417
34.7561	169.83	0.0900	2.57906	2.64	0.1080
35.4745	110.00	0.1181	2.52845	1.71	0.1417
36.1065	181.14	0.1968	2.48563	2.81	0.2362
36.5241	255.60	0.2362	2.45817	3.97	0.2834
37.1576	763.34	0.1574	2.41770	11.86	0.1889
37.9056	445.34	0.1574	2.37169	6.92	0.1889
38.3937	1081.37	0.0721	2.34265	16.80	0.0866
38.6178	928.72	0.2362	2.32957	14.42	0.2834
39.2817	314.81	0.1181	2.29172	4.89	0.1417
39.7236	132.84	0.1181	2.26723	2.06	0.1417
40.2360	366.37	0.1574	2.23953	5.69	0.1889
40.6669	254.37	0.1181	2.21679	3.95	0.1417
40.8361	189.89	0.0900	2.20800	2.95	0.1080
41.6361	173.21	0.0900	2.16741	2.69	0.1080
41.8622	277.62	0.1968	2.15622	4.31	0.2362
42.3961	164.70	0.0900	2.13030	2.56	0.1080
42.5561	163.70	0.0900	2.12266	2.54	0.1080
42.8155	302.26	0.1181	2.11039	4.69	0.1417
43.1695	609.30	0.1968	2.09390	9.46	0.2362
43.8690	202.44	0.1181	2.06213	3.14	0.1417
44.2503	175.16	0.1574	2.04524	2.72	0.1889
44.6012	267.21	0.1181	2.02996	4.15	0.1417
44.9961	151.84	0.0900	2.01306	2.36	0.1080
46.0376	178.86	0.1968	1.96991	2.78	0.2362
46.6165	140.91	0.1181	1.94678	2.19	0.1417
47.4015	102.08	0.1181	1.91636	1.59	0.1417
47.8761	121.07	0.0900	1.89847	1.88	0.1080
48.3561	208.06	0.0900	1.88074	3.23	0.1080
48.4788	138.59	0.5760	1.87626	2.15	0.6912
48.5961	164.94	0.0900	1.87666	2.56	0.1080

Preparation of Tricarbonyl[tris(lithium 2-isocyanosuccinate)]Mo(0) (4-b)

Preparation of tricarbonyl[tris(2-isocyanosuccinate methyl ester)]Mo(0) (4-a): (η⁶-C₇H₈)Mo(CO)₃ (0.281 g; 1.032 mmol; 272.11 g/mol) was dissolved in 20 mL of MeOH and CNCH(COOMe)CH₂COOMe (3 equiv.; 0.5303 g; 3.1 mmol; 171.14 g/mol), dissolved in 10 mL of MeOH was slowly added. The dark-red solution slowly turned orange-brown. The reaction was stopped after 1.5 h and taken to dryness to provide compound (4-a). Yield: 100%. C₂₄H₂₇N₃O₁₅Mo (693.4284 g/mol)

Characterization of Compound (4-a): Elemental Analysis: Calculated for MoC₂₄H₂₇N₃O₁₅: % C, 41.57; % H, 3.92; % N, 6.06. Found: % C, 41.3; % H, 3.94; % N, 6.06. IR (KBr): Bands (C≡O): 1943(s); 1876(s); Bands (C═O): 1740(s); Bands (C≡N): 2165(sh); 2107(s). ¹H-NMR (CDCl₃, 400 MHz, rt, δ in ppm): δ=4.82 (t, 3H), 3.86 (s, 9H), 3.77 (s, 9H), 3.04 (m, 6H).

Preparation of tricarbonyl[tris(lithium 2-isocyanosuccinate)]Mo(0) (4-b): Compound (4-a) (0.6669 g; 9.62×10⁻⁴ mol; 693.4224 g/mol) was dissolved in 20 mL of THF at 0° C. LiOH.H₂O (6 equiv.; 242 mg; 5.77 mmol), dissolved in 5 mL of H₂O also at 0° C. The solution of lithium hydroxide was added, the reaction was kept overnight, warming to room temperature then evaporated to dryness to provide compound (4-b) as the hexylithium salt. Yield of compound (4-b): 100%.

Characterization of compound (4-b): Elemental Analysis: Calculated for MoC₁₈H₉N₃O₁₅Li₆: % C, 33.53; % H, 1.41; % N, 6.52. Found: % C, 33.42; % H, 3.06; % N, 4.94. IR (KBr): Bands (C≡O): 1944(s); 1872(s); Bands (C═O): 1586(s); Bands (C≡N): 2144(s) ¹H NMR (D₂O, 400 MHz, rt, δ in ppm): δ=4.64-4.60 (m, 3H), 2.85-2.69 (m, 6H).

Preparation of Tricarbonyl[tris(isocyanocyclopropylcarboxylic acid]Mo(0 (5-b)

Preparation of tricarbonyl[tris(isocyanocyclopropylcarboxylic acid ethyl ester)]Mo(0) (5-a): Mo(CO)₃(η⁶-C₇H₈) (0.521 g; 1.92 mmol; 272.1117 g/mol) was suspended in 25 mL of MeOH to give a red suspension. Ethyl isocyanocyclopropylcarboxylate (3 eq.; 0.8 g; 5.75 mmol; 139.16 g/mol) was dissolved in 20 mL of MeOH and was added slowly to the previous suspension. A light orange solution was immediately obtained. The solution was stirred at room temperature for 1 hour, after which the solvent was concentrated and a white powder started to fall down. The solid, slightly contaminated with traces of an oil, was filtered and washed with 5 mL of MeOH. It was then transferred to an activated silica-gel chromatographic column. Fractions were collected in test tubes and the elution was followed by TLC. The eluent mixture (hexane (7)/ethyl acetate (3)) was always the same, however the first 6 test tubes eluted corresponded to trace mixtures of impurities and were discarded. The clean fractions were gathered and evaporated to dryness to give a green oil. Yield: 54% (0.6194 g).

Characterization for compound (5a): Calculated for C₂₄H₂₇N₃O₉Mo: % C, 48.25; % H, 6.24; % N, 7.03. Found: % C, 47.60; % H, 4.80; % N, 7.05. IR (KBr): Bands (C—O): 1942 (s); 1875 (s); Bands (C═O): 1738 (m); Bands (C≡N): 2154 (m); 2106 (s). ¹H-NMR(C₆D₆, 400 MHz, rt, δ in ppm): δ=3.86 (q, 2H, OCH₂CH₃), 1.08 (m, 2H, cycle), 0.97 (t, 3H, OCH₂CH₃), 0.87 (m, 2H, cycle). ¹³C-NMR(C₆D₆, 100 MHz, rt, δ in ppm): δ=212.5 (C—O), 168.3 (CN), 166.8 (C═O), 62.5 (CCH₂CH₂), 36.5 (CCH₂CH₂), 19.3 (COCH₂CH₃), 14.15 (COCH₂CH₃).

Preparation of tricarbonyl[tris(isocyanocyclopropylcarboxylic acid)]Mo(0) (5-b): Compound (5-a) (0.619 g, 1.04 mmol, 597.432 g/mol) was dissolved in anhydrous THF (20 mL) and placed in an ice bath. An aqueous solution of sodium hydroxide (10 equivalents, 10.37 mmol, 0.4148 g) in 5 mL of water was added dropwise to the yellow solution. A yellow emulsion was obtained, which stirred for 4 hours while slowly coming to room temperature. THF was evaporated, and when only water was left, 15 mL more of this solvent were added. The solution was filtered to remove traces of a solid, and 5.2 mL of 1M H₂SO₄ aqueous solution (37 mmol) were added dropwise. After all the acid was added and a brownish oil precipitated, the solution's pH was 2. The solvent was concentrated, more oil separated, and after a certain point the oil turned into a dark yellow powder. It was filtered and washed 4 times with 20 mL of water each (pH 5). The solid was dried under vacuum. Yield: 79% (0.420 g).

Characterization of (5b): Calculated for C₁₈H₁₅N₃O₉Mo: % C, 42.12; % H, 2.95; % N, 8.19. Found: % C, 42.40; % H, 3.32; % N, 8.48. IR (KBr): Bands (C≡O): 1941 (s); 1874 (s); Bands (C═O): 1705 (m); Bands (C≡N): 2153 (m); 2109 (s). ¹H-NMR (CD₃OD, 400 MHz, rt, δ in ppm): δ=1.66, 1.63 (m, 4H, cycle). ¹³C-NMR (CD₃OD, 100 MHz, rt, 6 in ppm): δ=213.7 (C—O), 171.5 (CN), 164.2 (C═O), 37.2 (CCH₂CH₂), 20.2 (CCH₂CH₂).

Example 2

CO-Release Kinetics

The CO release kinetics of Compounds 1b, 2b, 3b, 4b, and 5b was performed in vitro in HEPES buffer (pH 7.4) or in phosphate buffer (pH 7.4) and in the presence of liver microsomes. The quantization of the released CO was performed according to Vreman et al. Anal. Biochem. (2005) 341: 280-289 using a Gas Chromatograph with a Reducing Compound Photometer detector (GC-RCP; Peak Laboratories, Mountain View, Calif.), which allows quantifying CO in gas mixtures at concentrations as low as 1-2 parts per billion (ppb).

General Method for the Determination of CO Release in HEPES Buffer

The CO release kinetics of all compounds was evaluated in 50 mM HEPES buffer (pH 7.4) in a sealed 8-mL vial. Stock solutions (5 mM) of the compounds were prepared in PBS buffer (each compound was soluble in PBS after the addition of 3 equivalents of NaOH) and 10 μL were added to 990 μL of 50 mM HEPES buffer (final concentration in buffer was 50 μM). Since light activation can release CO from molybdenum carbonyl compounds, CO release was determined with solutions in closed, clear glass vials kept under light (on the laboratory bench under regular laboratory lighting) or in the dark (wrapped in aluminum foil and kept inside a cardboard box). Gas samples (from 10 μL up to 500 μL) of the vial headspace (7 ml volume) were removed with a gas-tight syringe at 15, 30, 60, 120, 240, 360 min, and 24 hours after start of the incubation. The gas samples were injected into sealed vials containing air (8 mL) for dilution. The entire gas volume of the vials (8 mL) was transferred with carrier gas to the GC-RCP and analyzed for CO. The GC-RCP had been calibrated with gas containing a known amount of CO. The calibration curve had been established starting with gas from a cylinder which contained synthetic air with 30 ppm CO (Linde, Cat. No. 14960013) and preparing dilutions in 8-mL vials as described above.

General Method for the CO Release in the Presence of Liver Microsomes

The CO release kinetics of Compounds 1b, 2b, 3b, 4b, and 5b were also evaluated in the presence of rat or human liver microsomes. In a 8-mL vial, the following reagents were combined:

713 μL of purified water

200 μL of 0.5 M potassium phosphate buffer, pH 7.4

50 μL of NADPH regenerating system solution A (BD, Cat. No. 451220)

10 μL of NADPH regenerating system solution B (BD, Cat. No. 451200)

2 μL of 5 mM solution of the compound in DMSO (10 μM final concentration)

The vials were closed and after mixing, the samples were warmed to 37° C. for 5 minutes before an aliquot (25 μL, 0.5 mg protein content) of rat liver microsomes (BD, Cat. No. 452511) or human liver microsomes (BD, Cat. No. 452161) was added to each vial. In parallel analogous vials with all the reagents except the microsomes were prepared. Due to the light-sensitivity of the molybdenum compounds, the reactions were performed in the dark. The samples were incubated at 37° C. and gas samples of the headspace (from 100 μL to 500 μL) of the vials were removed with a gas-tight syringe after 5, 10, 15, 30, and 60 minutes (in some cases samples were also taken at 1, 2, 3 or 4 minutes) upon microsomes addition. The gas samples were then injected into empty sealed vials (8 mL volume) and the amount of CO present in the vials was measured using GC-RCP.

CO Calibration Curve for the GC-RCP

A calibration curve of CO was established in order to be able to extrapolate the peak areas obtained in the GC-RCP to the known CO concentrations. Different gas volumes of a 30 ppm CO gas bottle (Linde, Cat. No. 14960013) were injected into 8 ml vials. The peak area values obtained in the GC-RCP and the corresponding ppm or pmol of CO in each sample was plotted in a graph. A linear response curve was obtained for CO concentrations of up to 0.857 ppm (or 268 pmol). Above these values the readings of the GC-RCP are not accurate.

Table 4 provides the volume of gas injected from a 30 ppm CO gas bottle (Linde, Cat. No. 14960013) into an 8-mL vial and the corresponding CO concentrations. Values of the CO peak areas from the different samples analyzed in the GC-RCP.

	TABLE 4
	Volume
	from CO	CO	CO
	bottle (μl)	(ppm)	(pmol)	Peak area
	0	0	0	4854782
	25	0.107	33	9682321
	50	0.214	67	13791770
	75	0.321	100	19163149
	100	0.429	134	27436859
	200	0.857	268	49579021
	250	1.071	335	1085495
	350	1.5	469	29003989

The CO equivalents released at each time point from each compound were determined by the following equation: (CO released (pmol)×number of CO ligands in the molecule)/([CO] in the compound (pmol)). A 1 mL solution of a tri-carbonyl compound, at 50 μM concentration contains a total of 150,000 pmol of CO.

CO Release Kinetics of Compound 1b

The CO release kinetics of Compound 1b in 50 mM HEPES (pH 7.4) buffer are presented in FIG. 1A. The half-life of CO release is approximately 4 hours in the dark and about 1 h under light. In 24 hours in 50 mM HEPES buffer, Compound 1b releases 1.6 CO equivalents under light and 1.4 CO equivalents in the dark, respectively.

In an in vitro metabolism study of Compound 1b, it was determined that 60% of Compound 1b decayed in a one-hour reaction with liver microsomes. Sixty percent decay could release up to 1.8 molar equivalents of CO. This decay rate is faster than what was observed in pH 7.4 buffer alone, where 0.5 molar equivalents of CO were released in about 4 h. Therefore liver microsomes accelerate the decomposition of the compound. In order to investigate if this accelerated decomposition of Compound 1b in the presence of microsomes was inducing a fast CO release from the compound, the CO release profile of Compound 1b in the presence of liver microsomes was determined. Microsomes were incubated with Compound 1b using closed vials to enable quantitative measurement of the released CO. As a control, Compound 1b was incubated under similar conditions in the absence of microsomes. The results are presented in FIG. 1B and show an increased rate of CO release from Compound 1b in the presence of microsomes after 10 minutes of reaction.

CO Release Kinetics of Compound 2b

The CO release kinetics of Compound 2b in 50 mM HEPES (pH 7.4) buffer are presented in FIG. 2A. The half-life of CO release in the case of Compound 2b is approximately 4 hours in the dark but under light it takes about 90 minutes to release 0.5 CO equivalents. After 24 hours incubation in 50 mM HEPES buffer, Compound 2b releases 1.5 CO equivalents under light and 1.2 CO equivalents in the dark, respectively.

The CO release kinetics of Compound 2b in the presence of rat liver microsomes were also investigated. Microsomes were incubated with Compound 2b using closed vials to enable the quantitative measurement of released CO. As a control, Compound 2b was incubated in similar conditions in the absence of microsomes. The results are presented in FIG. 2B. Microsomes accelerated the decay of Compound 2b.

CO Release Kinetics of Compound 3b

The CO release kinetics of Compound 3b in 50 mM HEPES (pH 7.4) buffer are presented in FIG. 4D. The half-life of CO release is approximately 2 hours in the dark and about 1 h under light. In 24 hours in 50 mM HEPES buffer, Compound 3b releases 1.8 CO equivalents under light and 1.3 CO equivalents in the dark, respectively.

In an in vitro metabolism study of the CO release kinetics of Compound 3b, it was determined that 80% of Compound 3b decayed in 60 minutes incubation with liver microsomes. In order to investigate if this accelerated decomposition of Compound 3b in the presence of microsomes was inducing a faster CO release from the compound, the CO release profile of Compound 3b in the presence of liver microsomes was determined. Microsomes were incubated with Compound 3b using closed vials to enable quantitative measurement of the released CO. As a control, Compound 3b was incubated under similar conditions in the absence of microsomes. The results are presented in FIG. 4E and show an increased rate of CO release from Compound 3b in the presence of microsomes. The initial rate of CO release is 0.0297 CO equivalents (297.22 pmol) per minute in the presence of microsomes and 0.0072 CO equivalents (72.265 pmol) per minute in the absence of microsomes. These results indicate that the initial CO release rate is approximately 4 times faster in the presence of microsomes.

CO Release Kinetics of Compound 4b

The CO release kinetics of Compound 4b in 50 mM HEPES (pH 7.4) buffer are presented in FIG. 5A. The stability of compound 4b was determined by measuring the release of CO in vitro in 50 mM Hepes buffer (pH 7.4) using a RCP-equipped GC. Compound 4b concentration was 50 μM. The half-life for CO release is defined as the time necessary to release 0.5 molar equivalents of CO and was found to be about 1 h for compound 4b (in the absence of light). The half-life for CO release was also measured in sheep blood using an oximeter. A concentration of 632.1 μg/mL of compound 4b was used. This concentration corresponds to the calculated Cmax in the mouse for a dose of 50 mg/kg. The half-life for CO release in blood in vitro was found to be about 2.5 hours.

CO Release Kinetics of Compound 5b

The CO release kinetics of Compound 5b in 50 mM HEPES (pH 7.4) buffer are presented in FIG. 6A. The stability of compound 5b was determined by measuring the release of CO in vitro in 50 mM Hepes buffer (pH 7.4) using a RCP-equipped GC. Compound 5b concentration was 50 μM. The half-life for CO release is defined as the time necessary to release 0.5 molar equivalents of CO and was found to be 6 h for compound 5b (in the absence of light).

The half-life for CO release was also measured in sheep blood using an oximeter. A concentration of 632.1 μg/mL of compound 5b was used (this concentration corresponds to the calculated Cmax in the mouse for a dose of 50 mg/kg). Compound 5b released 0.12 molar equivalents of CO in 3 hours.

Summary

Table 5 summarizes the CO release half-lives of molybdenum carbonyl isocyano compounds in 50 mM HEPES buffer (pH 7.4) or in potassium phosphate buffer (pH 7.4) in the presence of microsomes. Compound 4b is the least stable, with a half-life of 1 hour for spontaneous CO release in solutions of pH 7.4. Compound 1b and 2b have half-lives of spontaneous CO release of 4 hours.

	TABLE 5
	CO release Half-life in the dark
	(time to release 0.5CO equivalents)
	HEPES buffer	Microsomes
Compound	(pH 7.4)	K-phosphate buffer (pH 7.4)
1b	4 h	20 min
		(1.1CO eq. in 1 h)
2b	4 h	30 min.
		(0.9CO eq. in 1 h)
3b	2 h	13 min
		(1.5CO eq. in 1 h)
4b	1 h	—
5b	6 h	—

The CO release kinetics of these compounds were evaluated in the presence of liver microsomes. For all compounds evaluated, microsomes accelerated the release of CO. These results suggest that the compounds might be activated in vivo by metabolism in the liver. In fact, tissue CO distribution experiments performed in mice showed that the administration of Compound 3b or Compound 2b increased CO levels in the liver, when compared with blood, kidney, heart and lung tissues (see Example 3). However, there was no higher CO accumulation in the liver of animals treated with Compound 1b even though this compound was also activated by microsomes in vitro.

Example 3

Accumulation of Carbon Monoxide (CO) in Tissues after Administration of Carbon-Monoxide Releasing Molecule

Carbon monoxide-releasing molecules (CO-RMs) are carriers of carbon monoxide (CO) and can release CO in vivo. CO can bind to hemoglobin in the blood and to various heme proteins in cells. Vreman et al. Anal Biochem 341:280-289 describes a method to release CO bound to tissues into the gas phase, and quantify it using GC-RCP chromatography. We applied this methodology to assay CO in various tissues of mice treated with CO-RMs (see Example 2, herein). However, since the CO release method of Vreman (in vitro incubation with sulfosalicylic acid) also releases CO from the CO-RM, the CO released from tissue samples represents CO bound to tissue proteins and CO from CO-RM compound accumulated in tissue. For CO-RM compounds with slow CO release rates in vivo, the CO measured at the earliest sampling point (5 min after CO-RM administration) may therefore mostly be a measure for the tissue distribution of the intact CO-RM.

Methods

The compound was dissolved in PBS and the acids neutralized with 3 eq. NaOH (from a NaOH stock solution 2.5M). The pH was brought down to about 7.5 by adding HCl (from a stock solution 1 M). The compound was then administered i.v. (50 mg/kg in 150 μL) to two CD-1 female mice (Charles River). The mice were warmed for ten minutes under an infrared lamp to facilitate the i.v. administration into a tail vein. Five and twenty minutes after the dosing, the two mice were anesthetized with IsoFluorane, and the blood was collected from the retro-orbital plexus using a Pasteur pipette containing ˜20 μL of heparin solution. The animal was opened; the portal vein was cut and the organs were perfused by injecting 10-15 mL of cold PBS in the left ventricle of the heart. Then the heart, kidneys, liver and lung were collected, briefly washed with potassium phosphate buffer, dried with a paper towel and snap frozen in liquid nitrogen. Samples of the freshly collected blood were transferred to AVOXimeter 4000 cuvettes (ITC) to measure the levels of carboxyhemoglobin (COHb), oxyhemoglobin (O2Hb) and methemoglobin (MetHb) using a portable AVOXimeter 4000 CO-oximeter. The results are shown as mean percentage of total hemoglobin species in circulation. The organs and blood were conserved at −20° C. until the CO quantization was done.

For CO quantization, the protocol described by Vreman et al was followed. The organs were cut into small pieces, which were weighed and 4 volumes of water (a volume corresponding to four times the weight of the tissue sample) were added to each organ sample. The tissues were homogenized using a tissue tearer. Aliquots of homogenate (30 L) were placed into vials to which were added water (25 L) and sulfosalicylic acid (SSA, 5 μL, 30% [wt/vol]) immediately before they were closed with a gas-tight septum cap. The vials were incubated on ice for at least 20 min before being analyzed by GC-RCP (in later experiments, the incubation time was extended to 45 minutes). The amount of CO was calculated using a calibration curve prepared from CO standards. The results for Compound 1b (one experiment, FIG. 1C), Compound 2b (three repeated experiments, FIGS. 2C-2E), Compound 3b (two repeated experiments, FIGS. 4F-4G), Compound 4b (FIG. 5B), and Compound 5b (FIG. 6B), are presented as pmol CO per mg of fresh wet tissue (pmol CO/mg FW). COHb was determined using an oximeter (AVOXimeter 4000, USA).

Results: Tissue CO distribution experiments performed in mice showed that the administration of compound 3b or compound 2b increased CO levels in the liver, when compared with blood, kidney, heart and lung tissues. In contrast, there is no preferential accumulation of CO in the liver of animals treated with compound 1b, relative to other organs, even though this compound was also activated by microsomes in vitro. The results show that Compounds 2b and 3b distribute preferentially to the liver shortly after administration, while compound 1b does not. Interestingly, all these three compounds are decomposed by liver microsomes to liberate CO at relatively similar rates, although somewhat faster in the case of compound 3b. Compound 3b, which has a preferential accumulation in the liver and also a faster CO release rate induced by liver microsome, has a better CO delivery profile to this organ than both 2b and 1b. The latter releases CO faster than 2b but its liver tropism is low enough to fully compromise therapeutic efficacy. Compound 2b strikes an intermediate balance and still retains therapeutic activity although lower than that of Compound 3b. Taken together these two sets of results suggest that the release of CO in the liver is not enough to produce therapeutic action and that some preferential accumulation in the liver is also needed (see below Example 4 “Therapeutic benefit of Compound 3b in APAP-induced acute liver failure (ALF) model”).

Example 4

Carbon-Monoxide Releasing Molecules for the Treatment of Liver Disease

Preliminary Safety Studies in Mice

Preliminary safety studies in mice demonstrated that doses up to 1 g/kg showed no toxicity. FIG. 7 depicts the pharmacokinetics of Compound 3b in CD-1 mice. Compound 3b was administered intravenously (i.v.), at a dose of 100 mg/kg. Blood samples were collected after 1, 5, 10, 20 and 30 minutes after administration. The concentration of Compound 3b in serum was assayed by HPLC. The experimental curve represents elimination from circulation and decay through CO release.

In Vivo Distribution

It was found that Compound 3b rapidly distributes to the liver. Five minutes after i.v. injection of Compound 3b, CO concentration is five times higher in the liver than in blood; twenty minutes later, among the four non-blood tissues assayed, CO concentration is still highest in the liver.

Table 6 provides the CO distribution in blood, heart, liver, lung and kidney following Compound 3b administration at 50 mg/kg by i.p. injection. CO was assayed by GC-RCP, 5 and 20 minutes after administration of the compound. The CO concentration in the several organs is normalized to the amount of CO quantified in the blood (CO concentration as fraction of concentration in blood 50 mg/kg Compound 3b injected i.v. at t=0; CO assayed in tissues by GC at t=5 min and t=20 min).

	TABLE 6
	5 min	20 min
	Blood	1	1
	Heart	0.046 ± 0.012	0.072 ± 0.055
	Liver	5.26 ± 3.18	0.163 ± 0.062
	Lung	0.049 ± 0.020	0.060 ± 0.046
	Kidney	0.419 ± 0.295	0.074 ± 0.028

Human and Rat Microsomes Accelerate CO Release from Compound 3b

FIG. 8 depicts the amount of CO released from Compound 3b, expressed in equivalents of CO, in phosphate buffer pH 7.4 (“buffer”), phosphate buffer pH 7.4 in the presence of human microsomes (“human”) or rat microsomes (“rat”). The assay was performed in closed vials during 1 hour at 37° C. A gas aliquot was removed from the air space of the vials and CO concentration was determined by GC-RCP at different time points.

It was found that in pH 7.4 buffer Compound 3b releases 0.25 mol equiv. of CO in 1 hour. It was also found that microsomes accelerate CO release by a factor of about 4.0-5.5. The tissue distribution to the liver and enhanced CO release by liver microsomes suggest specific CO delivery to the liver.

COHb Levels in Compound 3b-Treated Mice

FIG. 9 depicts the percentage of carboxyhemoglobin (CO-Hb) measured in CD-1 female mice following Compound 3b administration at 300 mg/kg, intravenously. The CO-Hb was quantified over 80 min by using an oximeter (Avoximeter 4000 from A-vox Instruments). The base line of CO-Hb measured with this instrument is 3-4%. It was found that mice treated i.v with Compound 3b at a dose of 300 mg/kg did not exceed 7.5% COHb in blood and that therapeutic doses of Compound 3b did not cause symptomatic levels of COHb in blood of mice.

Therapeutic Benefit of Compound 3b in APAP-Induced Acute Liver Failure (ALF) Model

Compound 3b was studied in a sublethal model for acetaminophen (APAP)-induced Liver Injury.

FIGS. 10A-10B depict the kinetics of alanine aminotransferase (ALT) production and clearance. FIG. 10A depicts the kinetics of ALT production after 300 mg/kg administration of APAP by intraperitoneal (i.p.) injection. ALT is expressed in U/L over time (hours). FIG. 10B depicts the kinetics of APAP clearance after administration of 300 mg/kg by i.p. injection. APAP is expressed in g/L over time (hours).

FIG. 11A depicts the effect of treatment with Compound 3b on serum ALT in an APAP-induced acute liver failure (ALF) model. Male C57BL/6 mice fasted overnight to increase CyP450 activity. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. One hour after APAP injection, the animals were treated with 0.3, 3, 30 or 60 mg/kg doses of Compound 3b. Serum alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=5 mice for each group). FIG. 11B depicts the effect of Compound 3b in the liver damage induced by APAP. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. Compound 3b (0.3, 3, 30, 60 mg/kg) or Compound 1b (60 mg/kg) were administered to mice 1 h after APAP. Twenty-two hours after APAP injection serum ALT was measured (indicated in FIG. 11A), centrilobular sections of mouse livers were cut and stained with hematoxylin and eosin, and the percentage of liver necrosis was determined. The data indicates a dose-dependent reduction of ALT levels of 9% to 83% relative to APAP control, and a dose-dependent reduction of necrosis of 0% to 56% relative to APAP control. The data also indicates that Compound 1b does not reduce necrosis relative to APAP control, even at the highest dose of 60 mg/kg. These data unequivocally show a strong therapeutic activity of Compound 3b in the treatment of the acetaminophen induced ALF and the lack of activity of Compound 1b at similar doses. This difference in therapeutic activity strongly correlates with the profiles of CO delivery to the liver by accumulation and CO release, both of which favor Compound 3b over Compound 1b as discussed in Example 3.

Therapeutic Benefit of Compound 3b at Higher Doses in ALF Model

Compound 3b was also studied to see if there was therapeutic benefit at higher doses. Three groups of 5 mice were treated with 2 doses of compound given at 1 h and 3 h after APAP challenge: (1) Compound 3b, 2×60 mg/kg; (2) Compound 3b, 2×120 mg/kg; and (3) NAC, 2×300 mg/kg, and the effect evaluated 22 h post APAP.

FIGS. 12A-12B depict the effect of treatment with NAC or Compound 3b on serum ALT in an ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. Animals were treated with NAC(N-acetyl-cysteine; 300 mg/kg) or Compound 3b (60 or 120 mg/kg) administered at one and three hours after APAP injection. Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group). FIG. 12B is a zoom of FIG. 12A to help visualize the differences in the treated groups.

FIG. 12C confirms the results obtained with ALT serum markers (FIGS. 12A-12B), and depicts the effect of treatment with NAC or Compound 3b on liver damage in an APAP-induced ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. Animals were treated with NAC (300 mg/kg) or Compound 3b (60 or 120 mg/kg) administered at one and three hours after APAP injection. Twenty-two hours after APAP injection, serum ALT was measured (indicated in FIGS. 12A-12B), centrilobular sections of mouse livers were cut and stained with hematoxylin and eosin, and the percentage of liver necrosis was determined.

The results indicate that at both doses used Compound 3b is highly therapeutic, and at 2×120 mg/kg, Compound 3b is as effective as NAC.

Dosing Compound 3b Late after APAP Injury in ALF Model is More Effective than Dosing NAC

FIG. 13 depicts the effect of treatment with NAC or Compound 3b on serum ALT in an APAP-induced acute liver failure (ALF) model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. At five hours and seven hours after APAP injection, the animals were treated with NAC (150 mg/kg) or Compound 3b (120 mg/kg) or a combination of both compounds. Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group). *NAC administration to mice mimics human dose, which is 150 mg/kg (loading dose) over 60 min; 50 mg over 4 hours (2^nd dose); 100 mg over 16 hours (3^rd dose); for a total of 300 mg over 21 hours.

FIG. 14 depicts the effect of late treatment with Compound 3b on serum ALT in ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. At sixteen hours after APAP injection, the animals were treated with different doses of Compound 3b (0.3, 3, 30 or 60 mg/kg). Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group).

It was found that NAC given late after APAP injury is no longer effective and increases serum ALT levels (is toxic). Compound 3b is therapeutic when administered late after APAP injury when NAC has lost activity. Furthermore, NAC does not interfere with the activity of Compound 3b.

It was also found that treatment with a single dose of Compound 3b administered 16 hours after APAP injury produces a dose-dependent therapeutic effect. Serum ALT levels reach maximum at about 22 h post APAP administration, when the measurements were taken, and return to near normal at 48 h post APAP. Thus later treatments in this model are not indicative of drug activity.

Dosing Compound 3b Plus NAC Early after APAP Injury Shows Additive Effect in ALF Model

FIGS. 15A-15B depict the effect of combination treatment with NAC and Compound 3b on serum ALT in ALF model. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. NAC (300 mg/kg) and Compound 3b (120 mg/kg, FIG. 15A or 60 mg/kg, FIG. 15B) were administered to mice, separately or in combination, one hour and three hours after APAP injection. Experiments 1 and 2 represent two independent experiments. Alanine aminotransferase (ALT) was measured 22 h after APAP injection (n=4 or n=5 mice for each group). The ALT levels and the respective percentage of ALT increase, relative to the increase observed in mice injected with APAP alone, are presented in Table 7.

Table 7 provides the measured ALT levels in mice serum 22 hours after APAP administration and the percentage of ALT increase relative to the increase observed in mice, which were injected with APAP alone.

TABLE 7
	Measured ALT values	% ALT increase relative to increase
Treatment at 1 h and 3 h	(mean ± S.D.)	with APAP alone
post APAP	Experiment 1	Experiment 2	Experiment 1	Experiment 2
APAP, 300 mg/kg at t = 0	9050	12220	100 (=9050 ALT	100 (=12220 ALT
(no treatment)			U/L)	U/L)
NAC (2 × 300 mg/kg)	1745 ± 738	6620 ± 2142	19.3 ± 8.7	54.2 ± 17.5
3b (2 × 120 mg/kg)	4480 ± 630	4360 ± 926	49.5 ± 7.0	35.7 ± 7.6
NAC plus 3b (2 × 120 mg/kg)	123 ± 59	352 ± 59	1.4 ± 0.6	2.9 ± 3.1
3b (2 × 60 mg/kg)	5180 ± 1312	5500 ± 987	57.2 ± 14.5	45.0 ± 8.1
NAC plus 3b (2 × 60 mg/kg)	132 ± 156	189 ± 103	1.5 ± 1.7	1.5 ± 0.8
The data provides the experimental serum ALT values for each animal.
Results: 2 × 60 mg/kg Compound 3b is nearly as effective as 2 × 120 mg/kg Compound 3b. The therapeutic effects of NAC and Compound 3b are additive. In combination, the low and higher doses of Compound 3b are equally effective.

Liver Histology Analysis in ALF Model

FIG. 16 depicts the effect of NAC and Compound 3b in the liver damage induced by Acetaminophen. ALF was induced in C57Bl/6 male mice with a single dose of acetaminophen (300 mg/kg) by i.p. injection. NAC and Compound 3b were administered at one and three hours after APAP administration. Twenty-two hours after APAP injection, serum ALT was measured and centrilobular sections of mouse livers were cut and stained with hematoxylin and eosin. A: Naïve mouse; B and C: APAP control mouse (duplicate experiment); D and E: APAP plus Compound 3b 2×100 mg/kg (duplicate experiment); F and G: APAP plus Compound 3b 2×300 mg/kg (duplicate experiment); H and I: APAP plus NAC 2×300 mg/kg (duplicate experiment).

Results: a dose of 300 mg/kg acetaminophen causes strong necrosis in the mouse liver. Necrosis is centrilobular and perivascular as described in the literature. Livers from Compound 3b or NAC treated animals have very little if any necrosis. Livers from Compound 3b and NAC treated animals looked different, which suggested to the pathologist different drug mechanisms in this model. Therefore one might expect additivity for the therapeutic effects when NAC and Compound 3b are given in combination.

Hypothesis: NAC given early (<3-5 h) in this model, prevents liver injury by reacting with the toxic acetaminophen metabolite, NAPQI. Compound 3b given early or late (<16 h) accelerates healing by limiting inflammation and promoting regeneration. This hypothesis of the mechanism of action of Compound 3b is supported by the observation that perivascular areas had large numbers of mitotic figures.

Dosing Compound 4b in the ALF Model

FIG. 24 depicts the effect of Compound 4b in the ALF model in mice, administered (i.p.) at a dose of 0.3, 3, 10 or 30 mg/kg given 3 hours and 5 hours (+3 h, +5 h) after APAP (300 mg/kg) administration. Serum ALT levels were evaluated 22 hours after APAP administration. Compound 4b was able to reduce ALT levels induced by APAP in a dose-dependent manner, at a dose of 30 mg/kg the ALT levels were 75% reduced relative to untreated control animals.

Dosing Compound 5b in the ALF Model

FIG. 25 depicts the effect of Compound 5b in the ALF model in mice, administered (i.p.) at a dose of 10, 30 or 120 mg/kg given 3 hours and 5 hours (+3 h, +5 h) after APAP (300 mg/kg) administration. Serum ALT levels were evaluated 22 hours after APAP administration. Compound 5b was able to reduce ALT levels induced by APAP in a dose-dependent manner, at a dose of 120 mg/kg the ALT levels were 70% reduced relative to untreated control animals.

Lethal APAP Liver Failure Model

Compound 3b was tested in a lethal APAP liver failure model (see, e.g. Imaeda et al. J Clin Invest (2009) 119:305-14).

Male C57BL/6 mice fasted overnight to increase CyP450 activity. APAP dosed i.p. at 500 mg/kg. Survival monitored over 3 to 5 days. Survival in APAP-treated control animals is 40-60% at the end of the experiment.

FIG. 17 depicts the survival curve of C57BL/6 mice injected (i.p.) with 500 mg/kg of APAP. Mice were fasted overnight and APAP was administered afterward (n=15). Mice survival was monitored for 4 days.

FIGS. 18A-18B depicts the survival curve of C57BL/6 mice injected (i.p.) with 500 mg/kg of APAP and treated with Compound 3b. Mice were fasted overnight and APAP was administered afterward (n=15). Compound 3b was administered at a dose of 120 mg/kg at 1 h and 3 h (A) or at 3 h and 5 h (B) after APAP administration. Mice survival was monitored for 4 days.

FIG. 19 depicts the survival curve of C57BL/6 mice injected (i.p.) with 500 mg/kg of APAP and treated with Compound 3b and NAC separately and the respective combinations using different administration times. Mice were fasted overnight and APAP was administered afterward (n=15). Compound 3b was administered at a dose of 120 mg/kg (ip) at +3 h or +4 h after APAP administration. Another group of mice received NAC (150 mg/kg, ip) at +1 h after APAP. The remaining two groups are administered in NAC+ Compound 3b combination mode; one group of mice was administered with NAC (150 mg/kg, ip) at +1 h plus Compound 3b (120 mg/kg, ip)+3 h and other with Compound 3b (150 mg/kg, ip) at +1 h plus Compound 3b (120 mg/kg, ip)+4 h. Mice survival was monitored for 4 days.

Results: Compound 3B dosed at 2×120 mg/kg, 1 h and 3 h or 3 h and 5 h after APAP administration, causes 100% survival, whereas 53% (8 of 15) of animals die in the APAP control groups. NAC dosed at 2×300 mg/kg using the same schedules effected also 100% survival in both experiments (data not shown). The survival profile of mice treated with APAP and NAC+Compound 3b combinations as described above, is increased compared to individual treatments with NAC or Compound 3b, indicating a possible additive therapeutic effect of these molecules in the ALF model.

Ischemia-Reperfusion Model

Liver ischemia-reperfusion model in C57BL/6 mice. In anesthetized C57BL/6 mice, the hepatic artery and portal vein were clamped for 30 min; 24 h later, serum ALT levels were determined. Compound 3b was dosed i.p. at 30 mg/kg each 1 h before and 1 h after surgery.

FIG. 20 depicts the effect of Compound 3b in the Ischemia-Reperfusion model. In anesthetized C57BL/6 mice, the hepatic artery and portal vein were clamped for 30 min; 24 h later, serum ALT levels were determined. Compound 3b was administered by i.p. injection at 30 mg/kg, 1 hour before and 1 h after surgery.

Results: Compound 3b-treated animals had baseline levels (29 U/L) of serum ALT, whereas untreated control animals had 5-fold (149 U/L) elevated ALT levels. Compound 3b demonstrates prevention of ischemia/reperfusion damage in livers of mice. Compound 3b demonstrates anti-inflammatory activity in the liver.

Ex-Vivo Model of Apoptosis

FIGS. 21A-21B depict the effect of Compound 3b in an ex-vivo model of apoptosis. Apoptosis of primary hepatocytes from C57BL/6 mice was induced with Actinomycin D (ActD; 200 ng/mL) and Tumour Necrosis Factor-α (TNF-α; 10 ng/mL). Compound 3b was added to the cultures 1 hour before and 1 hour after the addition of the apoptosis inducers ActD/TNF-α. Compound 3b was tested at concentrations of 25, 50, 100 and 150 μg/0.5 mL well. Hepatocytes survival was determined 24 hours after the addition of the apoptosis inducers. FIG. 21A: Percentage of increase in survival ActD/TNF-α treated cells in the presence of Compound 3b, relative to cells treated with only the apoptosis inducers. FIG. 21B: Toxicity of the Compound 3b doses on murine hepatocytes (not treated with apoptosis inducers). Data are the mean±S.D. of 3-5 independent experiments (3-5 livers from different mice).

Results: Compound 3b has strong anti-apoptotic activity in the liver. ActD/TNF-α treatment alone reduces survival to 24.8±5.5% (n=23; data not shown). Compound 3b also prevents hepatocyte death induced by ActD/TNF-α. It was found that survival of hepatocytes increased in a dose dependent manner, and that this increase is more than 125% at 2×150 μg/0.5 mL well (2×600 μM) treatment with Compound 3b.

Liver Regeneration Model

Liver regeneration in the murine partial hepatectomy model to test for regenerative activity. Model: partial (70%) liver resection in mice and follow rate of regeneration.

FIG. 22 depicts the effect of Compound 3b in a model of liver regeneration. Animals were anesthetized and 70% of the liver was resected, corresponding to the left and median liver lobes. Compound 3b was administered (2×100 mg/kg) 1 h before and 1 h after surgery. Two days later, animals were sacrificed, livers harvested, weighed, and stained for phospho histone H3 (PH3, a specific marker for cell division). The data demonstrates increased mitotic activity of the treated group relative to control.

Results: Liver weight data 48 h after resection hints at faster regeneration with Compound 3b (89% increase in control vs. 102% in Compound 3b-treated animals). Compound 3b increases PH3 staining which indicates that there is an increased frequency of cell division. NAC has no effect and may be detrimental (see, e.g., Yang et al., Crit. Care (2009) 13: R55).

TNBS-Induced Colitis Model

FIGS. 23A-23C depict the effect of Compound 3b in a model of TNBS induced colitis. Colitis was induced in Balb/C mice by a single intracolonic administration of 100 ml of 40% ethanol containing 4 mg of TNBS (2,4,6-trinitrobenzenesulfonic acid). Dexamethasone (Dex; 0.3 mg/kg), Compound 3b (120 mg/kg) or its vehicle were administered daily for 3 consecutive days starting from 1 hour before colitis induction (day 1). On day 4 after colitis induction, mice were sacrificed, colon was isolated, cleaned and a 7 cm segment of the distal colon was resected and weighed (FIG. 23A). Stool consistency was determined using the score: 0, well-formed pellets; 1, loose stool; 2, liquid stool or bloody stool (FIG. 23B). Animals were weighed through out the entire study (FIG. 23C).

Other Embodiments

All patents, patent applications, and literature references cited herein are incorporated herein by reference.

The foregoing has been a description of certain non-limiting embodiments of the disclosure. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

1. A compound of Formula:

2. A pharmaceutical composition comprising a compound of claim 1, or a salt, ester, amide, solvate, or hydrate thereof, or a combination thereof, and a pharmaceutically acceptable excipient.

3. A method of preparing a compound of claim 1, the method comprising reacting a molybdenum tri-CO complex with an isocyanide of the formula:

4. A compound of the Formula (II):

5. A compound of the Formula (III):